effect of an industrial chemical waste on the uptake j. serb. chem. soc. 87 (0) 1–17 (2022) original scientific paper jscs–12062 published 14 december 2022 1 screening the binding affinity of bile acid derivatives for the glucocorticoid receptor ligand-binding domain srđan bjedov1*, sofija bekić2, maja marinović2, dušan škorić1, ksenija pavlović1, anđelka ćelić2, edward petri2 and marija sakač1 1department of chemistry, biochemistry and environmental protection, faculty of sciences, university of novi sad, trg dositeja obradovića 3, 21000, novi sad, serbia and 2department of biology and ecology, faculty of sciences, university of novi sad, trg dositeja obradovića 2, 21000, novi sad, serbia (received 12 september, revised 22 september, accepted 28 september 2022) abstract: the necessity of anti-inflammatory drugs such as glucocorticoids has been evident during the covid-19 pandemic. glucocorticoids, are the standard therapy for the treatment of moderate and severe covid-19 patients. however, serious side effects limit the use of these drugs, and anti-inflammatory drugs with better pharmacological properties are urgently required. bile acids are of interest, because of their anti-inflammatory and immunomodulatory properties, facilitated through an unclear mechanism involving transmembrane and nuclear receptors. in this work, we screened the binding activity of a number of bile acid derivatives, for the ligand-binding domain of glucocorticoid receptor (gr-lbd), the most important receptor for anti-inflammatory processes. tested compounds include oximes, lactones, lactams, tetrazoles, dienones, c-24 alcohols and cholic acid amides. cholic acid oxime, deoxycholic acid dienone, 3-keto-24-cholic alcohol and cholic acid amide showed best binding affinities for gr-lbd among tested compounds. the in silico molecular docking explanation is provided. sar analysis showed that expansion of b and c steroid rings or attachment of heterocycle to c ring is not beneficial for binding; side chain should contain hydrogen donor group; the gr-lbd tolerate well different functionalities on c-3 position. these results provide valuable information toward synthesis of the new glucocorticoids based on bile acids. keywords: organic synthesis; docking studies; molecular modelling. introduction glucocorticoids (gcs) are a class of steroid hormones that are among the most commonly prescribed drugs used for the treatment of allergic, inflam * corresponding author. e-mail: srdjan.bjedov@dh.uns.ac.rs  serbian chemical society member. https://doi.org/10.2298/jsc220912078b https://doi.org/10.2298/jsc220912078b 2 bjedov et al. matory, and immune disorders such as rheumatoid arthritis, asthma, brain edema, shock, and various blood cancers.1,2 more recently, they were also the first drugs shown to reduce deaths from covid19.3 gcs are the most effective, cost-efficient, and necessary anti-inflammatory and immunomodulatory drugs available. however, the use of gcs is limited by serious adverse effects, such as diabetes, osteoporosis, muscle wasting, hypertension, and glaucoma. also, certain groups of patients do not respond well to gc therapy.4 thus, there is an urgent medical need for new molecules with both enhanced gc therapeutic activities, and fewer or less severe side effects. gcs mediate their effects via the glucocorticoid receptor (gr), a ligand-activated transcription factor. gc activated gr performs its anti-inflammatory functions through a number of mechanisms, among which the most important are transrepression and transactivation.5,6 as steroid molecules, bile acids (bas) play important roles as hormones that regulate a large number of metabolic processes, including inflammation. bas are enzymatically synthesized in hepatocytes from cholesterol, conjugated with glycine or taurine, and stored in the gallbladder. when food is ingested, liver-synthesized primary bas are secreted in the small intestine where they emulsify dietary lipids and lipid-soluble vitamins, enabling their absorption. a fraction of primary bas is converted to secondary bas by intestinal microbiota. secreted bas are reabsorbed and returned to the liver by a very efficient process known as enterohepatic circulation. the hormonal role of bas is mediated through the ba -activated nuclear receptor farnesoid x receptor (fxr), which controls gene transcription in ba homeostasis and via g-protein coupled receptor tgr5. tgr5 is broadly expressed in humans and is involved in various physiological and pathological processes, including energy expenditure, glucose homeostasis, obesity, and bile acid homeostasis.7 the anti-inflammatory properties of bas were reported by hench in the 1930s. he observed alleviation of rheumatic symptoms in patients with the onset of severe jaundice, a side effect associated with increased ba serum concentrations.8 bas were first used as starting compounds for the synthesis of cortisol, which resulted in the 1950 nobel prize in physiology or medicine for hench, kendall and reichstein.9 fxr is involved in the pathophysiology of several inflammatory diseases, including non-alcoholic fatty liver diseases, inflammatory bowel disease and atherosclerosis.10,11 activation of tgr5 in macrophages and monocytes suppresses lipopolysaccharide-induced production of cytokines and prevents liver damage. the beneficial effects of trg5 activation were noticed in multiple inflammatory diseases, including diet-induced obesity, atherosclerosis, colitis and steatohepatitis. more information about tgr5 and immunometabolism can be found in an excellent review by perino and schoonjans.12 although the hormonal and anti-inflammatory activity of bas via fxr and tgr5 pathways is well established, there is reasonable evidence that some of the bile acid derivatives for the glucocorticoid receptor 3 anti-inflammatory activities of bas is also mediated through grs. it was found that 5β-cholanic acid can bind to gr and modulate gr signaling in cell models of parkinson’s disease.13 taurochenodeoxycholic acid also exhibited anti-inflammatory and immunomodulatory properties by inhibiting transcription and expression of ap-1 via stimulation of the gr.14 ursodeoxycholic acid exerts immune-suppressive effects by reducing ifn-γ production by liver lymphocytes, such as nk and nkt cells, in a gr-dependent manner, which may be an important immunological mechanism.15 the promiscuity of bile acids for fxr, tgr5 and gr could be exploited in an anti-inflammatory and immunomodulatory manner. in the present work, we explored the gr binding affinity of ba derivatives as a screen for unexplored ba structural features that may be important for gr binding. this work is aimed at the development of anti-inflammatory compounds that could lead to drugs with fewer side effects than current gcs. experimental general synthetic methods 1hand 13c-nmr spectra were recorded on a bruker avance iii hd 400 (400 mhz 1h, 101 mhz 13c) apparatus using tetramethylsilane as an internal standard. hrms spectra (tof) were recorded on a 6210 time-of-flight lc/ms agilent technologies (esi+) instrument. ir spectra were recorded on a perkin elmer spectrum two ft-ir spectrometer, and thermo nexus 670 ft-ir spectrometer, and melting points were determined on stuart mp-10 apparatus. flash chromatography was performed on silica gel 60 (0.04–0.063 mm, merck). synthesis of compounds 28 and 29 was done according to a procedure described in our earlier publication,16 and compound 27 was obtained following the iqbal and elliott protocol.17 for synthesis of 25, we used a different route then leppik,18 and 1 was obtained by a method described by hüttenrauch.19 spectral data of the compounds are given in the supplementary material to this paper. (3ez, 7z, 12z)-3,7,12-trioximino-5β-cholan-24-oic acid (1) naoac (1.09 g, 13.3 mmol) and nh2oh×hcl (0.45 g, 7.09 mmol) were added to a suspension of dehydrocholic acid (dca, 0.63 g, 1.6 mmol) in meoh (20 ml). the mixture was refluxed for 40 min, and the resulting suspension filtered and washed with cold water. compound 1 was obtained as a pure mixture of geometric isomers, as a white powder with a yield of 0.6541 g (91 %; thermal decomposition before melting started at 240 °c). methyl (7z,12z)-3,3-dimethoxy-7,12-dioximino-5β-cholan-24-oate (2) naoac (0.80 g, 9.8 mmol) and nh2oh×hcl (0.25 g, 3.9 mmol) were added to a solution of 3 (0.44 g, 1 mmol) in meoh (20 ml). the mixture was refluxed for 1 h, and the resulting suspension filtered and washed with cold water and dried. pure compound 2 was obtained as a white powder at a yield of 0.35 g (72 %; thermal decomposition before melting started at 240 °c). methyl 3,3-dimethoxy-7,12-dioxo-5-cholan-24-oate (3) tsoh (0.16 g, 0.9 mmol) and 2,2-dimethoxypropane (7 ml, 57 mmol) were added to a solution of dca (1 g, 2.5 mmol) in meoh (50 ml). the mixture was refluxed for 3 h, evaporated under vacuum, dissolved in etoac, washed with cold water (2×15 ml) and dried. 4 bjedov et al. pure compound 3 was obtained as a white powder at a yield of 1.1 g (93 %; thermal decomposition before melting started at 225 °c). 12α-hydroxy-3-oxo-5β-chola-4,6-dien-24-oic acid (25) compound 27 (0.4985 g; 1.05 mmol) was dissolved in methanolic koh (1 g; 17.8 mmol in 50 ml meoh) and refluxed for 30 min. after reaction completion, the reaction mixture was poured into water (200 ml) and acidified with hcl (1:2) to ph 1. the resulting precipitate was filtered, washed with brine to neutral ph and dried. the raw mixture was purified by flash chromatography (ch2cl2/etoac 94:6). compound 25 was obtained as white needle-like crystals (after recrystallisation from acetone) at a yield of 0.3642 g (90 %); mp 251 °c, mp lit.20 249–252 °c. 12α-hydroxy-3-oxo-5β-chola-4,6-dien-24-oic acid (25) and ethyl 12α-hydroxy-3-oxo-5β -chola-4,6-dien-24-oate (26) compound 27 (0.4872 g; 1.02 mmol) was dissolved in ethanolic koh (0.19 g; 3.4 mmol in 70 ml etoh) and refluxed for 1 h. after reaction completion, reaction mixture was poured in water (200 ml) and acidified with hcl (1:2) to ph 1. the resulting precipitate was filtered, washed with brine to neutral ph and dried. the raw mixture was purified by flash chromatography (ch2cl2/etoac 94:6). compound 25 was obtained at a yield of 0.1116 g (28 %) and compound 26 (mp 136 °c) at a yield of 0.2872 g (67 %). 2-(5β-chol-3-ene-7α,12α,24-triol)-n-(1-hydroxy-2-methylpropan-2-yl)acetamide (30) triethylamine (0.3 ml), 2-amino-2-methyl-1-propanol (0.2 ml, 1.1 mmol) and eedq (0.23 g, 0.9 mmol) were added to a suspension of compound 29 (0.299 g, 0.69 mmol) in etoac (10 ml). the reaction mixture was refluxed for 5 h. after cooling to room temperature, the reaction mixture was washed successively with 3 m hcl (2×4 ml), h2o (4 ml), 10 % nahco3 (2×10 ml) and then with water to neutrality (3×5 ml). the organic layer was dried and evaporated in vacuum to give an oily residue, which was further purified by flash column chromatography (ch2cl2/meoh 96:4). pure 30 was obtained at a yield of 0.2612 g (75 %). fluorescent assay in yeast a yeast-based fluorescent screen was applied for testing the relative binding affinities of ba derivatives 1, 2, 4–23 and 25–31 for the gr ligand binding domain (lbd). assays were conducted following our previously published procedure; optimized for identification of steroidal ligands of the gr.21,13 saccharomyces cerevisiae fy250 strain (matα, ura3-52, his32δ00, leu2δ1, trp1δ6) and expression vector prf4-6-rgr lbd-eyfp were kindly provided by dr. blake peterson from the university of kansas.22 yeast cells were transformed with plasmid dna by treatment with lithium acetate and polyethylene glycol, to improve the efficiency of exogenous dna uptake, following the procedure of gietz et al.23 transformed yeast cells were then incubated at 30 c until the appearance of transformant colonies on agar plates. selection medium supplemented with 2 % raffinose was then inoculated with recombinant yeast cells grown to saturation in a biosan orbital shaker-incubator es-20/60. saturated yeast cells were diluted in fresh medium and grown to mid-logarithmic phase (od600 nm  0.5), monitored using a nicolet evolution 100 uv–vis spectrophotometer. protein expression was induced by addition of galactose to a final concentration of 2 %. bile acid derivatives, prednisolone or estradiol (highand low-affinity gr ligands) were added to a final concentration of 100 μm. stock solutions of all tested compounds were freshly prepared in dmso. incubation was continued for 15 h at 25 c. resulting fluorescence intensity was detected by bile acid derivatives for the glucocorticoid receptor 5 fluorimeter (fluoroskan ascent fl) and fluorescence microscopy (olympus bx51) using a fitc filter. for fluorimetry, 150 μl of cell suspension was added to a microplate in triplicate and fluorescence was recorded using an excitation and emission filter set of 485 and 538 nm. growth medium without cells served as a blank. to normalize fluorescence intensity to cell number, the optical density of yeast cells was measured at 600 nm using a thermo lab systems multiscan ex spectrophotometer. ligand binding affinity was calculated as the fold fluorescence change between cells exposed to test compounds and those treated with negative control ligand, estradiol. histograms showing the relative binding affinity of bile acid derivatives and control ligands for gr-lbd were created in origin pro 8 with included error bars, obtained by propagation of standard error of the mean. additionally, the fluorescence intensity distribution of recombinant yeast cells expressing gr lbd-yfp treated with bile acid derivatives or control compounds, was visualized by fluorescence microscopy. molecular docking coordinates for the gr-lbd receptor were converted to pdbqt format in the program vegazz using the available “receptor.c” script.24 structural coordinates for compounds 1, 25, 28 and 31 were created in the program avogadro 1.0.3;25 avogadro: an open-source molecular builder and visualization tool based on the structure of dexamethasone, taken from pdb 1m2z.26 ligand geometries were optimized in avogadro 1.0.3 using an mmff94 force field and 500 steps of conjugate gradient minimization with a convergence setting of 10e-7. non -polar hydrogens were merged and gasteiger partial charges added in vegazz 3.1.0,27 using the “ligand.c” script, and resulting ligand coordinates were converted to pdbqt format for autodock. grid maps for atoms present in the tested compounds were created using the program autogrid, with a grid center of 31.04, 7.76, 12.52, grid spacing of 0.0375 nm and dielectric of –0.1465. docking simulations in autodock 4.2,29 were conducted using the lamarckian genetic algorithm with the following parameters: ga population size 150. ga num evaluations 250000. ga num generations 27000. autogrid calculations and autodock simulations were conducted using the pyrx virtual screening tool (version 0.8).27 results were visualized in the program pymol (v0.99).28 results and discussion chemistry compounds used in the present study were chosen to examine the influence of easy-to-achieve modifications of the ba molecule on gr binding affinity. structures of compounds investigated in the present work can be categorized into roughly seven categories: oximes, lactones, lactams, tetrazoles, dienones, c-24 alcohols and cholic acid amides. structures of oxime derivatives are shown in fig. 1. synthesis of oximes 1 and 2 is shown in scheme 1. the reaction of dehydrocholic acid (dca) with hydroxylamine hydrochloride afforded a mixture of geometric isomers 1, at the c-3 oxime group. the mixture of isomers 1 was obtained with a yield of 91 % and individual isomers were not separated. the isomer ratio of 1 could be speculated to be 3:2 based on the intensity of c-3 signals (156.87 and 156.67 ppm) present in 13c-nmr spectra. compound 2 was obtained in a two-step sequence. first, the carbonyl group at c-3 was regioselectively trans6 bjedov et al. formed into acetal, while the carboxyl group was esterified which afforded 3 in excellent yield (93 %). this reaction represents a very good way for regioselective protection of the c-3 bile acid carbonyl group. next, c-7 and c-12 carbonyl groups were converted into oxime to obtain dioxime 2. fig. 1. structures of oximes. scheme 1. reagents and conditions: a) nh2oh×hcl, naoac, meoh, 40 min, reflux; b) meoh, 2,2-dimethoxypropane, tsoh, 3 h, reflux. unlike 1, compound 2 was obtained as a stereochemically pure compound. the stereochemistry of oxime groups at c-7 and c-12 was determined as z-based on the absence of cross-peaks in noesy nmr spectra that originate from, the through space, interaction of oxime hydrogens with any steroid skeleton hydrogens. only the z configuration for the oxime groups could provide enough dis bile acid derivatives for the glucocorticoid receptor 7 tance to explain the lack of noe interactions. stereochemistry could be explained by the larger volume available for hydroxyl groups if the oximes have a z configuration (fig. 2). oximes 4,29 530 and 631 were prepared by procedures found in the corresponding literature. fig. 2. different perspectives of 2 show the available space for z-oriented oxime groups at c-7 and c-12. bile acid derivatives with a lactone or lactam moiety in b or c steroid ring (fig. 3) 7–14 were synthesized according to published procedures,31–33 while the stereochemistry and physicochemical properties of these compounds were described by poša et al.34 fig. 3. structures of bile acid lactone and lactam derivatives 7–14. the structures of tetrazoles 15–23 used in the present work are shown in fig. 4. tetrazole rings are fused on b and c steroid rings. the general synthesis of tetrazole compounds is shown in scheme 2, using the synthesis of 16 as an example. bile acid derivative 24, with c-3 acetoxy, c-24 ethoxycarbonyl groups, and a carbonyl group at c-12, was subjected to schmidt reaction condition (scheme 2) with trimethylsilyl azide as an azide source and tri8 bjedov et al. methylsilyl trifluoromethanesulfonate as an amide intermediate activator. details of the synthesis of the tetrazole compounds used in the present study have been published by our group.35 fig. 4. structures of the bile acid tetrazole derivatives 15–23. scheme 2. the general synthesis of tetrazole compounds 15–23 is illustrated using the synthesis of 16 as an example. reagents and conditions: tmsn3, tmsotf, acn, rt, 3 h (a). structures of dienones 25 and 26, alcohols 28 and 29 and amides 30 and 31 are shown in fig. 5. dienones 25 and 26 were obtained by the following procedures: when compound 27 (scheme 3) was refluxed in methanolic potassium hydroxide solution, elimination and hydrolysis occurred to afford 25 at a yield of 90 % (78 % reported by leppik et. al. 18). reaction samples were collected every 5 min and since bile acid derivatives for the glucocorticoid receptor 9 only one product was detectable by tlc, it appears that both elimination and hydrolysis reactions occur simultaneously. the same result was obtained upon treatment of 27 with methanolic koh solution for five days at room temperature, or 2 m naoh solution for five hours at room temperature, or by reflux with hcl solution (1:3) in water/acetone 5/7 for five hours. when ethanol was used instead of methanol, in addition to 25 (28 %), transesterification occurs to afford ethyl ester 26 (67 %). signals corresponding to vinylic protons in 25 are easily visible by 1h-nmr as overlapping singlet peaks of h-4 and h-6 at 6.16 ppm, and a singlet from h-7 at 5.61 ppm. an additional quartet at 4.03 and a triplet at 1.16 in the 1h-nmr of 26 suggests the presence of an esterified carboxyl group. fig. 5. structures of dienone, alcohol and amide derivatives 25, 26, 28–31. scheme 3. reagents and conditions: a) koh, meoh, 0.5 h, reflux; b) koh, etoh, 1 h, reflux; c) hcl, h2o. easily obtainable and well-known 3-dehydrocholic acid (3dca) was transformed into the bile alcohol 28 using acetal protection as seen in 3, and subsequent reduction (scheme 4). the same bile alcohol 28 was transformed into 29 10 bjedov et al. using a wittig–horner–emmons reaction. details concerning the synthesis of 28 and 29 are available in our previous publication.16 scheme 4. synthesis of bile alcohol 28. compound 30 was obtained in the good yield (75 %) by reaction of 29 with 2-amino-2-methyl-1-propanol using eedq as a coupling agent (scheme 5). scheme 5. reagents and conditions: 2-amino-2-methyl-1-propanol, eedq, tea, etoac, 5 h, reflux (a). the cholic acid amide 31 (fig. 5) was synthesized according to a procedure reported by miljković et al.36 fluorescent assay in yeast in order to test the relative binding affinities of the synthesized ba derivatives, we used a yeast-based fluorescent screen that is optimized for identification of steroidal ligands of the glucocorticoid receptor, as previously described.13,21 briefly, the fluorescence intensity of yeast cells expressing the ligand-binding domain (lbd) of the glucocorticoid receptor (gr) fused to a yellow fluorescent protein (yfp) has been shown to respond in a dose-dependent manner to treatment with gr agonists such as prednisolone.13,21 because the assay is measured in yeast, the concentration of ligand required to elicit a response may not necessarily be the same concentration that would affect gr activity in vivo. during optimization of the assay, we measured dose response for a positive control ligand (prednisolone, fig. s-1 of the supplementary material). as assay sensitivity was highest at a final prednisolone concentration of 100 μm, bile acid derivatives were tested at 100 μm. in the present study, bile acid derivatives 1, 2, 4–23 and 25–31 were tested against gr-lbd, by mea bile acid derivatives for the glucocorticoid receptor 11 suring fold fluorescence changes between cells exposed to test compounds compared with those treated with a negative control ligand, e2 (estradiol), or a positive control gr agonist (prednisolone). it can be seen in fig. 6, that bile acid derivatives 1, 25, 28 and 31 displayed fold fluorescence changes indicative of moderate binding affinity for gr-lbd compared with prednisolone or estradiol. fig. 6. bile acid derivatives 1, 25, 28, and 31 showed fold fluorescence changes consistent with moderate binding affinity for gr-lbd-yfp based on a fluorescent screen in yeast following 15 h exposure at a final concentration of 100 μm. ligand binding affinity was calculated as the fold fluorescence change between cells exposed to test compounds compared with those treated with negative control ligand, e2 (estradiol), measured by fluorimetry. prednisolone (p) was tested as a positive control gr agonist. to validate these results, yeast cells treated with the same compounds were also visualized by fluorescence microscopy. as can be seen in fig. 7, treatment of recombinant yeast expressing gr-lbd-yfp with the gr agonist prednisolone resulted in a strong increase and relocalization of fluorescence intensity, while treatment with compounds 1, 25, 28 and 31 resulted in a more moderate increase in overall fluorescence intensity compared to cells treated with negative control estradiol. fig. 7. recombinant yeast cells expressing gr-lbd-yfp treated with 100 μm estradiol (e2, negative control gr ligand), prednisolone (p, positive control gr ligand) or test ba derivatives 1, 25, 28 and 31 for 15 h, visualized using a fluorescence microscope. the intensity of cell fluorescence is proportional to gr ligand binding affinity. 12 bjedov et al. molecular docking based on fluorescent screening in yeast, compounds 1, 25, 28 and 31 have moderate binding affinity for gr-lbd compared with prednisolone, a strong gr agonist. to visualize these results in a molecular framework, binding poses and binding energies were predicted for compounds 1, 25, 28 and 31 by molecular docking in the program autodock.37 because the structure of gr in complex with prednisolone has not been published, coordinates for gr-lbd in complex with another gr agonist, dexamethasone (pdb 1m2z), were used as ‘receptor’ for docking.26 for all docking simulations, the receptor was kept rigid and the ligand was allowed to rotate around flexible bonds. to validate the autodock protocol for our system, control redocking simulations were conducted using dexamethasone as a positive control “ligand”. autodock correctly reproduced the x-ray structure with an rmsd of < 0.06 nm2 and a strong binding energy of – 57.20 kj/mol. based on the x-ray structure, dexamethasone (dex) is in part held oriented in the gr ligand binding site via polar contacts with arginine 611, glutamine 570 and asparagine 564. as can be seen in fig. 8, very similar binding poses and intermolecular contacts are formed by test compounds 28, 25 and 31. fig. 8. molecular docking poses and predicted binding affinities for test compounds 25, 28 and 31 compared with dexamethasone (dex), a positive control gr ligand. the x-ray structure of gr ligand binding domain in complex with dexamethasone (pdb 1m2z) was used as receptor for autodock simulations. top ranking poses for the following compounds are shown: a) dexamethasone, b) 28, c) 25 and d) 31. all three compounds bind in the same orientation, forming interactions with asn564, thr739 and gln642 from the d-ring side, and arg611 and gln570 from the a-ring side of these ba derivatives. consistent with experimental results, moderate binding energies of <−41.84 kj/mol were predicted for each of these bile acid derivatives for the glucocorticoid receptor 13 test compounds: for compound 25, −46.36 kj/mol; compound 28 −45.94 kj/mol, and compound 31 −42.16 kj/mol. none of the test compounds had a predicted binding affinity greater than prednisolone, as expected. interestingly, although compounds 1 displayed moderate affinity for gr-lbd in fluorescence experiments, docking of c-3 z-isomer of 1 failed to predict binding to gr under the simulation conditions used. in general, autodock is capable of correctly predicting the binding affinity and geometry for a set of compounds if ligand binding is not associated with significant backbone and side chain conformational changes.37 thus, binding by compound 1 likely induces conformational changes to the gr active site that are different from the gr conformation present in complex with dexamethasone, preventing autodock from correctly estimating the binding pose and affinity for compound 1. for compounds 25, 28 and 31, molecular docking results could be used in future studies as a starting point to design molecules with increased binding affinity, and/or specificity for gr-lbd. conclusion compounds used in the present work were designed to examine the influence of specific functionality modifications of bas on their gr binding properties. bile acids have a suitably functionalized steroid skeleton for modification in strategic regions for gr interactions. our glucocorticoid receptor binding assay provided insight into structural features important for binding of the tested molecules to gr. the results of the sar analysis are shown in fig. 9. fig. 9. results of sar analysis. four differently functionalized compounds 1, 25, 28 and 31 showed moderate binding affinity for gr-lbd compared with prednisolone or estradiol. the oxime geometric isomers in 1 showed the best relative binding affinity, not just among 14 bjedov et al. tested oximes but among all tested compounds. although we were not able to separate the geometric isomers in mixture 1 and molecular docking did not enable prediction of possible interactions with the enzyme, binding affinity could be attributed to the c-24 carboxylic group and oxime groups. the hydroxyl group at c-11 with β orientation (11β-oh) has been previously shown to be very important for gr-glucocorticoid interactions,38 and the relative binding affinities measured for 1 suggest that the c-12 oxime group could be exploited as an alternative hydrogen bond donor to the 11β-oh group. lactone, lactam, and tetrazole derivatives did not show any significant binding affinity. it seems that the b or c steroid ring expansion or attachment of heterocycle at the c ring is not beneficial for binding affinities. compounds 28, 25 and 26 have a keto or enone moiety, which is known to be involved in anchoring steroids in the receptor pocket.39 however, it seems that the hydrogen bond donating group at c-24 is very important for binding to gr. this was observed for all of the tested compounds. ethyl ester 26 has a much smaller gr binding affinity compared to free acid 25. compound 28 with the c-3 keto and c-24 hydroxyl group has a gr binding affinity similar to 1, among the best of the tested compounds. there is a degree of plasticity in the gr-lbd for the steroid a-ring and c-3 functional groups, since compounds 1 with c-3 oxime e and z groups; 29, with carboxymethyl group at c-3, and even compound 30 with the corresponding amide at c-3 have moderate or less than moderate gr-lbd binding affinity. this work gives important insight into structural modification of bile acid steroid skeleton that could be used for development of new small molecule drug for treatment of inflammatory diseases. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/12062, or from the corresponding author on request. acknowledgements. the authors acknowledge financial support from the ministry of education, science and technological development of the republic of serbia (grant no. 451-03-9/2021-14/ 200125). и з в о д испитивање афинитета деривата жучних киселина за везивање за лиганд-везујући домен глукокортикоидног рецептора срђан бједов1, софија бекић2, маја мариновић2, душан шкорић1, ксенија павловић1, анђелка ћелић2, едвард петри2 и марија сакач1 1департман за хемију, биохемију и заштиту животне средине, природно–математички факултет, универзитет у новом саду, трг доситеја обрадовића 3, 21000, нови сад и 2департман за биологију и екологију, природно–математички факултет, универзитет у новом саду, трг доситеја обрадовића 3, 21000, нови сад антиинфламаторни лекови као што су глукокортикоиди били су неопходни током пандемије covid-19 за лечење пацијената са умереним и тешким облицима covid-19. међутим, озбиљни нежељени ефекти ограничавају употребу ових лекова и хитно су потhttps://www.shd-pub.org.rs/index.php/jscs/article/view/12062 bile acid derivatives for the glucocorticoid receptor 15 ребни антиинфламаторни лекови са бољим фармаколошким својствима. жучне киселине привлаче све више интереса, због својих антиинфламаторних и имуномодулаторних својстава, испољених засад нејасним механизмом који укључује трансмембранске и нуклеарне рецепторе. у овом раду смо испитали афинитет везивања низа деривата жучних киселина за лиганд-везујући домен глукокортикоидног рецептора (gr-lvd), најважнијег рецептора за антиинфламаторне процесе. испитивана једињења укључују оксиме, лактоне, лактаме, тетразоле, диеноне, c-24 алкохоле и амид холне киселине. међу тестираним једињењима: оксим холне киселине, диенон деоксихолне киселине, 3кето-24-алкохол и амид холне киселине показали су најбоље афинитете везивања за grlvd. објашњење везивања поменутих деривата помогли су in silico доковање. анализа односа структуре и активности је показала да експанзија b и c стероидних прстенова или везивање хетероцикла за c прстен није од користи за везивање; бочни ланац треба да садржи водоник-донорску групу; и gr-lvd добро толеришу различите функционалности на позицији c-3. ови резултати пружају вредне информације за прављење нових антиинфламаторних молекула базираних на жучним киселинама. 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(2022) https://doi.org/10.2298/jsc22 1103089v this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc221103089v https://doi.org/10.2298/jsc221103089v j. serb. chem. soc.00(0)1-16 (2022) original scientific paper jscs–12125 published dd mm, 2022 1 the influence of nanoclays on the mechanical and thermal properties of rigid pir and pur foams ruslan r. vlasov1*, daria i. ryabova1, sakina z. zeynalova2, dmitry v. sokolov1 and sergei a. ryabov1 1lobachevsky state university – faculty of chemistry, department of macromolecular compounds and colloidal chemistry, gagarina av. 23, 603098 nizhny novgorod, russia and 2tre tau engineering srl, pietro colletta 85, 10153 torino, italy (received 3 november; revised 8 december; accepted 21 december 2022) abstract: the effect of small amounts of chemically modified nanosized clays (from 0.05 to 1 %) on the morphological, physical-mechanical and thermophysical characteristics of rigid polyurethane-polyisocyanurate (pir) and polyurethane (pur) foams has been studied. the effect of these additives on the structure of the resulting material, the change in its compressive strength, young's modulus, mass loss during combustion, and thermal conductivity are evaluated. based on the results obtained, it is noted that the addition of small amounts (up to 0.2 %) of chemically modified cloisite 30b nanoclay effectively reduces the average cell size of nanocomposite foams, which leads to an improvement in their performance. keywords: polyurethane; polyurethane-polyisocyanurate foam; nanosized fillers; compressive strength; cell morphology; thermal conductivity introduction currently, one of the largest tonnage polymers is polyurethane a polymeric material, traditionally obtained by polycondensation (polyaddition) of aromatic or aliphatic diand polyisocyanates with polyol resins.1 due to the distinctive features of the material, such as high heat and sound insulation characteristics in the case of polyurethane foams, and high physical and mechanical properties, polyurethanes have now become an integral part of everyday life. one of the main industrially significant types of polyurethane are rigid polyurethane (pur) and polyurethane-polyisocyanurate (pir) foam. pir foams are obtained using isocyanate trimerization catalysts as a result of technological processes similar to those in the case of pur foams. it should be noted that pir foams have improved thermal insulation characteristics and are less flammable than conventional polyurethane foams. the formation reactions of urethane and isocyanurate groups *corresponding author e-mail: vlasovruslan.hs@yandex.ru https://doi.org/10.2298/jsc221103089v ac ce pt ed m an us cri pt mailto:vlasovruslan.hs@yandex.ru https://doi.org/10.2298/jsc221103089v 2 vlasov et al. are shown in figure 1.2 it is important to note that in the framework of the synthesis of both polyurethane and, mainly, polyurethane-polyisocyanurate foams, a number of secondary chemical processes occur, the consideration of which is beyond the scope of this publication.3,4 figure 1. reactions of urethane and isocyanurate formation it is important to note that the annually growing demand for polymeric materials inevitably leads to the presentation of ever greater requirements in terms of functional (physical-mechanical, thermophysical, etc.) characteristics of the resulting polymers, which the latter, in the case of a very wide range of applications, no longer meet. one of the most promising ways to solve this problem is the synthesis of composite materials based on these polymers. today, filled polyurethane systems are used everywhere, while the mass fraction of the filler in the formulation can traditionally vary from 1 to 50 % by weight of the composition. the most common fillers currently used in the polyurethane industry are chalk, carbon black,5 sand,6 expandable graphite,7 titanium dioxide,8 and a wide range of other oxides and inorganic salts. it should be noted that the introduction of such inorganic additives in acceptable amounts into systems for producing foams contributes to a decrease in the average cell size of the formed foam,9 since fillers act as a nucleating agent, creating many smaller cells.10,11 in addition, these additives prevent the additional growth of bubbles due to an increase in the viscosity of the reaction medium.12 thus, the use of nanosized fillers to a large extent contributes to the improvement of the morphological characteristics of foams and, as a result, to an increase in a number of performance characteristics, increasing strength indicators,13,14 to a significant extent, preventing a gradual increase in the thermal conductivity of foams during aging.15,16 ac ce pt ed m an us cri pt nanoclays-pir/pur foam composites 3 the trend towards the addition of micro-sized clays (mainly montmorillonite and vermiculite) in polyurethane foam compositions arose about 20 years ago.17-19 nanoclays, on the other hand, are finely dispersed material that improves the mechanical and thermal insulation characteristics of polyurethane composite foams due to a significantly increased specific surface area.20 it is extremely necessary to select the optimal amount of nanofiller, since at an excessively high content of these additives, a significant deterioration in the morphology of the material and degradation of performance characteristics are noted.21,22 this deterioration in morphology can manifest itself both as an increase in the average cell size,13 and a decrease in their density,23 and an increase in the anisotropy coefficient of the foam.24,25 thus, in the case of polyurethane and polyurethane-polyisocyanurate foams, nanoclays are of significant interest. in this regard, it is expedient to develop formulations of new nanocomposite materials based on polyurethanepolyisocyanurate and polyurethane foams using the additives described above, which have improved performance characteristics. experimental materials for the synthesis of polyurethane and polyurethane-polyisocyanurate foams, a mixture of polyether polyols (h6007 (hongbaoli pu, china) and hf-310 (zhejiang hengfeng new material, china)), tris(2-chloropropyl)phosphate (tcpp, shijiazhung hejia chemical products, china) as a flame retardant, a mixture of 1,1,4,7,7-pentamethyldiethylenetriamine (pmdeta, evonik, germany) and dibenzylamine (evonik, germany) as catalysts for urethane and urea formation, a solution of potassium acetate in diethylene glycol in the assessment of 30:70 (evonik, germany) as trimerization catalyst, as well as water and n-pentane (for synthesis, ekos-1, russia) as blowing agents, were used. a highly functional polymeric mdi lupranat m50 (basf, germany) with dynamic viscosity of 557 mpa s, determined at 25 °c in accordance with iso 3219-2:2021,26 and nco content of 31.5 %, determined in accordance with iso 148696:2009,27 was used as the isocyanate component. the composition of the systems used is shown in table 1. table 1. the composition of pir/pur foam systems. component amount, g pur foam pir foam polyether polyols blend 50.0 50.0 tcpp 7.0 7.0 pmdeta 0.8 0.8 dibenzylamine 2.1 2.1 potassium acetate in diethylene glycol (30:70) 2.5 water 2.4 2.4 n-pentane 2.7 2.7 polymeric mdi (lupranat m 50) 62.3 155.8 ac ce pt ed m an us cri pt 4 vlasov et al. commercially available chemically modified organoclays cloisite 30b (surface layer modified with a quaternary salt of bis-2-hydroxyethylmethylammonium, modifier content <90 meq / 100 g) and cloisite 25a (surface layer modified with a quaternary salt of diethylmethylammonium, content modifier <95 meq / 100 g) manufactured by byk additives (germany). nanoclay suspensions the insertion of fillers into the isocyanate component to obtain pur and pir foams was carried out by ultrasonic dispersion using an inlab i100-6/4 unit equipped with an i10-2.0 ultrasonic generator at a frequency of 22.5 khz with an output power of 2 kw. dynamic viscosity measurements the dynamic viscosity of the suspensions obtained was measured using a lamy rm200 cp4000 plus rotary rheometer using r-6 disc (ϕ14.62 mm) at shear rate of 60 rpm in accordance with iso 3219-2:202126 at 25 °c. further in the work, the average viscosity values determined by analyzing three samples are given. pur and pir foams preparation the isocyanate and polyol components were thermostated at 20 °c. to obtain foam samples, the required weights of the blended polyether component were placed in plastic cylinders with a volume of 1000 cm3, the required amount of n-pentane was added, after which it was actively mixed in the component for two minutes. after adding the required weight of isocyanate (or nanoclay suspensions), the system was mixed for five seconds using an automatic mixer at a speed of 3000 rpm. foaming was carried out in the above-described plastic containers or wooden cubes with a volume of 15625 cm3. 10 minutes after mixing the components, fragments were cut out from the core of the formed foams, which were subsequently used as analyzed samples. gel time the so called “gel time” was tested as the time elapsed from the start of mixing polyol and isocyanate components to the point at which soft threads are formed when an ordinary wire yarn is touched to the surface of the foaming material and withdrawn. isocyanate group content measurements the content of isocyanate groups in the polyisocyanate used was determined using the potentiometric titration method on an 848 titrino plus metrohm automatic titrator in accordance with iso 148696:2009.27 apparent density the determination of the apparent density of the synthesized foams was carried out in accordance with astm d1622-20,28 for which cubic samples of 5050mm in size were cut from the obtained materials. further in the work, the average apparent density values determined by analyzing five samples are given. mechanical tests the determination of compressive strength and young's modulus was carried out in the direction of foaming on roell/zwick z005 universal testing machine at a strain rate of 10 mm min-1 at 10 % linear strain in accordance with the en 826:2013 method.29 within this work, only the relative compressive strength and young's modulus are considered, numerically equal to the ratio of the compressive strength and young's modulus to the square of the apparent density of the foam sample. the data shown in the work are averages calculated from analyzes ac ce pt ed m an us cri pt nanoclays-pir/pur foam composites 5 of five samples of each of the investigated formulations of polyurethane-polyisocyanurate and polyurethane foams. open cell content the content of open cells (oc / %) in the analyzed polyurethane and polyurethanepolyisocyanurate foams was analyzed using an accupyc ii 1340 gas pycnometer in accordance with astm d6226-05.30 further, the paper presents the average value of the content of open cells in the studied samples, determined from the results of three independent measurements. thermal conductivity the thermal conductivity of the analyzed polyurethane-polyisocyanurate and polyurethane foams was determined at 24 °c using lasercomp fox600 hfm and fox200 sn instruments in accordance with en 12667:2001.31 the size of each sample was 20020025 mm. further, the paper presents the average values of thermal conductivity determined by analyzing three samples. morphological characteristics analysis the morphological characteristics of the closed-cell structure of the foam samples were studied using the method of scanning electron microscopy (sem). a hitachi tm4000 plus ii instrument was used as a scanning electron microscope. using a blade, 0.5 mm thick layers of the analyzed foam were cut out, which were subsequently placed in the sample compartment of the above-described photomicrograph instrument. the average cell size (ф) and the anisotropy coefficient (ay/x) were estimated using the imagej software with the help of the intersection method, in accordance with the methods proposed by brondi et al.32 each analyzed micrograph was overlaid with a grid composed of equidistant perpendicular lines (m vertical lines of length h and l horizontal lines of length w). for each of the lines, the number of crossed cells (ni, nj) was counted. dividing the length of each individual line by a certain number of intersections, we found the lengths of the chords of the cells (фi and фj, i.e. one-dimensional values). this operation was carried out for each of the lines of the constructed grid, after which the average cell size was determined in accordance with equation (1) and the anisotropy coefficient in accordance with equation (2). further in the paper, the averaged values of these quantities determined by analyzing five samples are given. , , , 1 m l i j i j m l   = = +  (1) where ф is average cell size, фi,j are the lengths of cell chords, m is the number of vertical grid lines, l is the number of horizontal grid lines. 11 / lm ji ji y x a m l ==  =  (2) where ay/x is the anisotropy coefficient, фi,j are the lengths of cell chords, m is the number of vertical grid lines, l is the number of horizontal grid lines. cell density was estimated using imagej software according to kumar's theoretical approximation principle.33,34 after opening the microphotograph in the imagej program, the area for analysis was selected. the number of cells and the square of the analyzed area of the micrograph were determined. the density of cells in the volume was estimated in accordance with the equation (3) proposed by kumar, which can be expressed in terms of the number of ac ce pt ed m an us cri pt 6 vlasov et al. cells located per cubic centimeter of the analyzed foam (cells cm-3). further in the work, the average values of cell density determined by analyzing five samples are given. 3 2 f n n a =       (3) where nf is the density of cells in the foam, n is the number of cells in the analyzed area of the microphotograph, a is the square of the analyzed area, cm2. combustibility in order to assess the change in the combustibility of the analyzed polyurethanepolyisocyanurate foams, samples (30 mm*30 mm*15 mm) were vertically fixed in a tripod, after which the edge (30 mm*30 mm) was blown over for 30 seconds by the flame of a gas burner located at a distance of 5 cm from the sample, so that the entire surface of the facet was covered in flames. the gas flow rate was 0.36 ml s-1. further, the paper presents the average values of the degree of damage by mass (sm) determined by analyzing five samples, calculated taking into account the change in the mass of the analyzed samples before and after the test. results and discussion in accordance with the iso 3219-2:2021 method,26 the dynamic viscosity of the resulting organoclay suspensions in the polyisocyanate component used was determined. the trend in dynamic viscosity versus filler content is shown in figure 2. figure 2. dependence of the dynamic viscosity of the isocyanate component on filler content thus, there is a gradual increase in the dynamic viscosity of the isocyanate component as the content of the filler in it increases. ac ce pt ed m an us cri pt nanoclays-pir/pur foam composites 7 in order to study the effect of small additions of organoclays on the physicalmechanical, thermophysical, and morphological properties, samples of rigid closed-cell polyurethane and polyurethane-polyisocyanurate foams were obtained using the suspensions of the filler in polyisocyanate. it is noted that the addition of the considered fillers into the composition slightly shifted the parameters of the system, slowing down the so called “gel time” by 1–2 seconds in the case of organoclay concentration of 0.05 and 0.1 % and 2–3 seconds in the case of using these additives in higher concentrations. these minor changes in the technological parameters of the system are the result of a clear effect exerted by the additives on the physics of the foaming process. the values of the physical-mechanical, morphological and thermophysical characteristics of the obtained composite foams determined using the methods described above are presented in tables 2 and 3. table 2. physical and mechanical characteristics of pur and pir foams foam sample ω / wt.% γ / kg m-3 σ / kpa σrel. / kpa m 6 kg-2 е / kpa еrel. / kpa m6 kg-2 p u r f o a m standard 0.00 40.35 209.71 0.129 1974.10 1.21 cloisite 30b 0.05 40.15 229.51 0.142 1981.01 1.23 0.10 39.95 238.80 0.150 1990.68 1.25 0.20 39.81 246.51 0.156 1993.45 1.26 0.50 39.72 245.13 0.155 1979.59 1.25 1.00 39.49 242.05 0.155 1976.67 1.27 cloisite 25a 0.05 40.17 224.79 0.139 1983.54 1.23 0.10 40.02 231.46 0.145 1981.21 1.24 0.20 39.79 235.02 0.148 1981.60 1.25 0.50 39.71 234.58 0.149 1977.65 1.25 1.00 39.54 232.07 0.148 1992.46 1.27 p ir f o a m standard 0.00 40.32 266.09 0.164 3319.21 2.04 cloisite 30b 0.05 40.12 295.49 0.184 3328.50 2.07 0.10 39.92 309.30 0.194 3333.48 2.09 0.20 39.78 320.13 0.202 3331.49 2.11 0.50 39.69 318.70 0.202 3350.41 2.13 1.00 39.51 316.05 0.202 3325.85 2.13 cloisite 25a 0.05 40.14 288.41 0.179 3374.97 2.09 0.10 39.99 298.31 0.187 3360.37 2.10 0.20 39.76 303.58 0.192 3328.50 2.11 0.50 39.68 302.11 0.192 3339.46 2.12 1.00 39.61 301.00 0.192 3334.81 2.13 ac ce pt ed m an us cri pt 8 vlasov et al. table 3. morphological and thermophysical characteristics of pur and pir foams foam sample ω / wt.% ф / μm ay/x / a.u. nf / cell cm-3 103 λ / mw m-1 k-1 oc / % sm / % p u r f o a m standard 0.00 568.32 1.12 5.58 26.41 5.62 cloisite 30b 0.05 528.54 1.04 16.13 25.58 5.97 0.10 500.12 1.00 20.26 25.34 6.01 0.20 454.66 0.97 33.87 25.13 6.13 0.50 471.71 0.92 30.79 25.13 6.24 1.00 511.49 0.88 29.46 25.19 6.41 cloisite 25a 0.05 539.90 1.06 15.23 25.61 6.01 0.10 505.80 0.99 18.47 25.40 6.13 0.20 471.71 0.98 32.48 25.22 6.18 0.50 488.76 0.91 29.46 25.23 6.29 1.00 522.85 0.86 27.44 25.27 6.52 p ir f o a m standard 0.00 681.07 1.07 3.27 24.17 6.24 57.03 cloisite 30b 0.05 627.20 1.02 9.78 23.40 6.63 48.35 0.10 588.72 0.95 12.36 23.17 6.68 46.22 0.20 527.15 0.93 20.79 22.96 6.81 42.03 0.50 550.24 0.88 18.89 22.97 6.93 42.05 1.00 604.11 0.85 18.06 23.03 7.12 44.78 cloisite 25a 0.05 642.59 1.03 9.25 23.41 6.68 49.35 0.10 596.41 0.94 11.23 23.23 6.81 47.01 0.20 550.24 0.93 19.93 23.07 6.87 44.27 0.50 573.32 0.88 18.06 23.06 6.99 44.61 1.00 619.50 0.82 16.82 23.11 7.24 47.04 as a result of the introduction of the considered additives into the composition for the synthesis of pir and pur foams, there is a slight decrease in the apparent density of the resulting materials. similar results confirming the decrease in the foam density with the introduction of small amounts of carbon filler were obtained by pikhurov et al.35 it is noted that the proven decrease in the apparent density of the materials under consideration can be explained by the increasing degree of stabilization of the three-dimensional polymer network.36 figure 3 shows the dependence of the change in relative compressive strength on the content of organoclays in the composition. thus, a gradual increase in the compressive strength index of the obtained composite foams, which takes place until the mass concentration of the additive is equal to 0.2 %, subsequently this index only slightly decreases. in the case of young's modulus, no such trend is observed: this value remains almost unchanged when the fillers considered in this work are introduced, regardless of their concentration. this pattern was previously revealed by gibson et al.37, who proved that young's modulus does not change significantly even with a significant decrease or increase in the average size of the foam cells (the trends of parameter is described below). on the contrary, the compressive strength index is closely related to the morphology of the cellular structure, increasing with an increase of homogeneity of the spatially cross-linked polymer network. ac ce pt ed m an us cri pt nanoclays-pir/pur foam composites 9 figure 3. dependence of relative compressive strength on filler content the observed difference between the relative strengths of composite foams obtained using cloisite 30b and cloisite 25a can be explained by the presence of hydroxyl groups on the surface of cloisite 30b particles, which contribute to a better distribution of nanosized filler particles over the emerging polymer matrix. it is known that the strength characteristics of both pir and pur foams largely depend on the morphology of the cellular structure of the material.38 within the framework of this work, the change in the morphological characteristics of the obtained foams was analyzed using the method of scanning electron microscopy. sem microphotographs examples are presented in figure 4: the standard (nonfilled) polyurethane and polyurethane-polyisocyanurate foams are shown in figures a and c, respectively, microphotographs of nanocomposite foam samples (comprising 0.2 % cloisite 30b) are shown in figures b and d. it should be noted that the introduction of the additives under consideration significantly affects the uniformity of the structure of the final polymer. trends in the average cell size and cell density in the synthesized pir and pur foams from the content of a small amount of organoclays are shown in figures 5 and 6. it is important to note that the introduction of a filler significantly affects both the average cell size and their density, contributing to the formation of the cells closest in size. it is assumed that such a modification of the cellular structure of the emerging foam makes a significant contribution to the identified increase in the functional characteristics of the foam. ac ce pt ed m an us cri pt 10 vlasov et al. figure 4. microphotographs of the obtained non-composite and composite pur (a,b) and pir (c,d) foams figure 5. dependence of the change in the average cell size of the composite foam on the content of the filler ac ce pt ed m an us cri pt nanoclays-pir/pur foam composites 11 figure 6. dependence of cell density change in composite foams on filler content based on the information presented in table 3, we failed to identify a clear dependence of the anisotropy coefficient of pir and pur foams on the type and concentration of the introduced fillers. nevertheless, it is known that the introduction of such additives can lead to an increase in the anisotropy coefficient of composite foams,39 however, an increase or decrease in this index has only a small effect on the performance characteristics of composite foams, which was proved in literature.35 it is important to note that in a number of studies, for example,13,35 as a consequence of the introduction of a filler into the system, an increase in the average cell size is noted. apparently, this trend is associated with an insufficient degree of dispersion of the solid phase during the preparation of the initial suspensions. the dependence of the thermal conductivity of the obtained pir and pur foams on the content of the filler is shown in figure 7. thus, when small amounts of the considered additives are introduced into the composition, a noticeable decrease in their thermal conductivity index is noted. the most noticeable decrease in this characteristic was observed in the case of pir foams, which may be due to the better distribution of the filler in the polymer matrix. such an effect is quite natural, since in the framework of this work, the filler was dispersed directly in the isocyanate component, the excess of which occurs during the synthesis of pir foams. espadas-escalante et al.40 noted an increase in the thermal conductivity of composite foams, probably associated with an insufficient degree of homogeneity of the final suspension used for the synthesis of foams. ac ce pt ed m an us cri pt 12 vlasov et al. figure 7. the dependence of thermal conductivity of composite foams from filler content since one of the most important characteristics of pir foams is combustibility, the effect of fillers introduced into the composition on the stability of the final foams was evaluated by assessing the degree of damage by mass during combustion. the results obtained are shown in figure 8. figure 8. the dependence of damage by mass of composite foams from filler content ac ce pt ed m an us cri pt nanoclays-pir/pur foam composites 13 based on the data presented in figure 8, the combustibility of the analyzed pir foams significantly decreases when small additives of the considered fillers are introduced into the composition. the dependence of the content of open cells in pir and pur foams on the content of the filler is shown in figure 9. figure 9. the dependence of the content of open cells in composite foams on the content of the filler thus, the content of open cells in the analyzed composite foam plastics naturally increases with an increase in the content of filler particles. however, there is an extremely low influence of this parameter on the other properties of pir and pur foams considered and described above. it is expected that such an increase in the content of open cells will primarily negatively affect the thermal conductivity of the synthesized foams, however, based on the above information (see table 3, fig. 7), the thermal insulation characteristics of the obtained foams only increase, which allows us to conclude that the degree of influence of such a small content of opened cells on other properties of the analyzed materials. figure 10 shows the dependence of the relative compressive strength on the average cell size of the obtained composite foams. thus, the relative compressive strength directly depends on the average cell size of the foams, increasing with a decrease in average cell size of the resulting foamed material. we conclude that in order to improve the strength characteristics of foam plastics, it is extremely advisable to obtain foam plastics with the best morphology of the cellular structure, including the lowest average cell size. ac ce pt ed m an us cri pt 14 vlasov et al. figure 10. dependence of relative compressive strength from the average cell size of composite foams conclusion thus, when using small amounts of chemically modified nanoclays (cloisite 30b, cloisite 25a), two series of composite polyurethane and polyurethanepolyisocyanurate foams were obtained. the main physical-mechanical, thermophysical and morphological characteristics of the synthesized materials have been studied. it is shown that the introduction of small additives of the considered fillers leads to an improvement in the main performance characteristics of pir and pur foams: an increase in relative compressive strength, a decrease in thermal conductivity and damage by mass during combustion. based on the data obtained, it is expected that the use of the nanosized fillers described in the work will be appropriate in the development of new industrially used heat-insulating foams. извод утицај наноглине на механичка и топлотна својства крутих пир и пур пена ruslan r. vlasov1, daria i. ryabova1, sakina z. zeynalova2, dmitry v. sokolov1 и sergei a. ryabov1 1lobachevsky state university – faculty of chemistry, department of macromolecular compounds and colloidal chemistry, gagarina av. 23, 603098 nizhny novgorod, russia и 2tre tau engineering srl, pietro colletta 85, 10153 torino, italy проучн је ефекат малих током деведесетих глина нано сноге (од 0,05 до 1 %) на морфологију, максо-механичке и термофизичке стазе крутих полиуретан-полиизоцијанурата (pir) и полиуретанске (pur) пена. процењени су ефекти ових адитива на структуру добијеног материјала, промену његове компресивне чврстоће, јангов модул, губитак масе током сагоревања и топлотну проводљивост. на основу добијених резултата, констатује се да додавање малих количина (до 0,2 %) хемијски модификоване cloisite 30b ac ce pt ed m an us cri pt nanoclays-pir/pur foam composites 15 наноглине ефикасно смањује просечну величину ћелија нанокомпозитних пена, што доводи до побољшања њихових перформанси. 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technology and metallurgy – national institute of the republic of serbia, belgrade, serbia (received 25 march, revised 6 may, accepted 9 may 2022) abstract: molecularly imprinting technology was applied for preparing selective sorbents for benzophenone-4 (bp4), an organic uv filter used in sunscreens and cosmetics. several imprinted polymers were prepared by bulk polymerization, using bp4 as template. combination of stability (mechanical and chemical), selectivity and robustness of the imprinted polymers with bp4 properties resulted in a successful imprinting process (imprinting factors in range 1.05–2.60). the prepared polymers were characterised by infrared spectroscopy, elemental analysis, conductometric titrations and nitrogen physisorption at 77 k. adsorption capacities and selectivity towards 7 other organic uv filters (benzophenone-3, benzophenone-8, homosalate, butyl methoxydibenzoylmethane, ethyl hexyl salicylate, ethyl hexyl p-dimethylamino benzoate and ethyl hexyl p-methoxycinnamate) were determined, proving high adsorption capacity and high selectivity for bp4 binding. the highest adsorption capacity was observed for 4-vinylpyridine/ethylene glycol dimethacrylate co-polymer prepared in dimethyl sulfoxide (1.108 mmol g-1). the imprinted polymer with the highest binding capacity was applied to solid phase extraction of bp4 from aqueous solutions with 98.5 % efficiency. keywords: uv filters; imprinting factor; binding selectivity. introduction molecularly imprinted polymers (mips) are smart synthetic materials which can be used for several applications:1–4 as sorbents for solid phase extraction (spe),5–9 as stationary phase for chromatography,10–15 as sensors,16–18 artificial enzymes,19–21 as systems for controlled drug delivery and release,22–24 etc. molecular imprinting technology (mit) is based on interactions between template molecule and functional monomer in the presence of porogen. the chosen * corresponding author. e-mail: tatjanad@chem.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc22032540p 2 pešić, krstić and verbić monomer should have at least one functional group which is likely to interact with the template. during the polymerization process monomer molecules, as present in excess, surround the template molecule, forming a pre-polymerization complex. then, in the presence of a cross-linker, a rigid polymer network is formed usually by free radical polymerization. after the template is washed out, selective binding sites complementary to template molecules in size, shape and chemical characteristics remain in the polymer, enabling the template rebinding.1 in some examples, only one monomer is used for the preparation of mips.25,26 the above-described approach is known as non-covalent imprinting and presents the most commonly used approach in mit. interactions like van der waals, hydrogen and ionic bonding are involved in non-covalent imprinting process and, later on, in template rebinding. thus, the higher the variety and strength of monomer-template interactions, the more selective polymer with higher binding capacity is obtained.1 imprinted polymers have good selectivity, chemical and mechanical stability; therefore, these materials can be universally applied in various fields of chemistry and biosciences.2 although extensive research is being done within the field, and many advances have been achieved in mit in recent decades, mips are generally still lacking high binding efficiency. most of the non-covalent mips described in the literature can bind only a small percent of the template amount initially used during polymerization.25 therefore, optimizing and fine tuning of polymerization conditions and the process itself to obtain highly selective polymers with high template binding efficiency is an on-going demand in mit.2 the growing need for skin protection from the sun radiation has established uv filters as one of the emerging environmental contaminants.27,28 regularly used sunscreen cosmetic products contain a high amount of uv filters (up to 15 %). uv filters can be divided into inorganic (zinc oxide and titanium oxide) and organic (benzophenones, cinnamates, dibenzoylmethanes, triazole derivatives, salicylates, etc).28 benzophenone-type uv-filters are major ingredients of sunscreen products nowadays. according to eu regulations, bp4 or sulisobenzone (fig. 1) and its sodium salt, bp5, can be used in sunscreens up to 5%.30 when released in the environment, several adverse effects have been noticed such as coral bleaching, estrogenic activity in fish and even endocrine disrupting effects are suspected.27,31,32 it is estimated that 40 % of coastal coral colonies worldwide are exposed to uv-filter pollution.28,33 bp3 can cause oxidative stress in zooxanthellae, organisms that live in symbiosis with corals. the decrease of zooxanthellae causes the lightening of the brown colour of the corals.27,32–34 bp4 was found in river, lake and sea water;35 the ec50 value (half maximal effective concentration) of molecularly imprinted polymers for benzophenone-4 3 bp4 is near 10 mg dm–3 in the isochrysis galbana, scorpaenopsella armata, paracentrotus lividus and mytilus galloprovincialis.27,36 ooh o so3h fig. 1. structure of benzophenone-4. although uv filters gained high attention with highly increased research interest in methods for pre-concentration and extractions from various samples with subsequent quantitation, only few papers describe mit applied for development of uv filter imprinted polymers. sun et al. described the application of mip imprinted with bp2 for solid phase extraction (spe) of four benzophenones from tap and river water. 4-vinylpyridine (4vp)/ethylene glycol dimetachrylate (edma) based polymer imprinted with benzophenone-2 (bp2) was successfully applied for the extraction of benzophenone-1 (bp1), bp2, benzophenone-4 (bp6) and benzophenone-8 (bp8).37 ayadi et al. described molecularly imprinted polyaniline on silica support for the selective adsorption of bp4 from aqueous media.38 to the best of our knowledge, no other examples of polymers imprinted using bp-type uv-filters as templates were published so far. within this study, we used bp4 as the template molecule to prepare several imprinted polymers by bulk polymerization.39 different polymer constituents were used: monomers (n,n-dimethylaminoethyl methacrylate-dmaem and 4vp), cross-linkers (divinylbenzene-dvb and edma), porogenic solvents (acn and dmso). polymers were characterized using ft-infrared spectroscopy (ftir), nitrogen adsorption and adsorption isotherms. binding capacity and selectivity towards other uv-filters was determined. elemental analysis (ea) and conductometric titrations were used in order to estimate total and accessible binding places. the goal was to prepare selective sorbents for bp4 with high binding capacity, which can be used as potential sorbents for bp4 extraction and pre-concentration during sample preparation. experimental apparatus infrared spectra were recorded using thermo scientific nicolet 6700 ft-ir spectrometer, atr technique. hplc measurements were done using agilent technologies hplc instrument series 1260 with quaternary pump, on-line degasser, auto sampler and diode array detector. conductometric titrations were done using wtwcond330i with tetracon325 conductometric cell. elemental analysis was performed by combustion analysis on a vario el iii c,h,n,s/o elemental analyzer (elementar analysesysteme, gmbh, hanau, germany). chromabond vacuum manifold (macherey-nagel) was used for spe extraction. for nitrogen physisorption sorptomatic 1990 thermo finnigan analyzer was used. multibio rs24 (biosan) rotator and microspin 12 centrifuge (biosan) were used for conducting binding experiments. 4 pešić, krstić and verbić reagents benzophenone-4 (bp4), benzophenone-3 (bp3), ethyl hexyl salicylate (os), ethyl hexyl p-dimethylamino benzoate (paba-o), n,n-dimethylaminoethyl methacrylate (dmaem), divinylbenzene (dvb), azobisisobutyronitrile (aibn) were purchased from sigma aldrich. hplc grade methanol and acetonitrile were purchased from fisher scientific. benzophenone8 (bp8) was purchased from the tokyo chemical industry. 4-vinylpyridine (4vp) and ethylene glycol dimethacrylate (edma) were purchased from acros organics. butyl methoxydibenzoylmethane (avb) and homosalate (hms) were purchased from merck. inhibitor remover (sigma aldrich) was used to remove polymerization inhibitors from 4vp, dmaem and edma (5 ml of monomer/cross-linker was passed over 250 mg of inhibitor remover). dvb was purified using alumina, in the same way as edma. commercially available bp4 is declared to have some percent of water bound. it was dried at 60 °c under vacuum, until no further change in mass was observed (color change from pale to clear lemon yellow was observed during drying). other reagents were of analytical grade and used as received. preparation of imprinted and non-imprinted polymers the imprinted polymers were prepared using bulk polymerization: template, monomer, cross-linker, porogen and polymerization initiator were placed in a glass vial. upon dissolution, argon was purged through a pre-polymerization mixture for 5 min. aibn was used as initiator (30 mg). vials were tightly closed and left in an oil bath at 60 °c for 24 h. after polymerization was completed, the glass vials were broken and the polymer grounded in a mortar with pestle. the non-imprinted polymers were prepared as the imprinted ones, omitting the template presence. composition of the prepared polymers is given in table i. table i. composition of prepared polymers polymer label mbp4 template / mg (n / mmol) vmonomer / l (n / mmol) vcross linker / ml vporogen / ml nip1 – dmaem 337 (2.0) edma 1.888 acn 2.730 mip1 154.0 (0.5) dmaem 337 (2.0) edma 1.888 acn 2.730 nip2 – dmaem 337 (2.0) dvb 1.424 acn 2.730 mip2 154.0 (0.5) dmaem 337 (2.0) dvb 1.424 acn 2.730 nip3 – 4vp 213 (2.0) edma 1.888 dmso 2.730 mip3 154.0 (0.5) 4vp 213 (2.0) edma 1.888 dmso 2.730 nip4 – 4vp 213 (2.0) dvb 1.424 dmso 2.130 mip4 154.0 (0.5) 4vp 213 (2.0) dvb 1.424 dmso 2.130 nip5 – 4vp 106.4 (1.0) dvb 1.424 dmso 2.000 mip5 308.3 (1.0) 4vp 106.4 (1.0) dvb 1.424 dmso 2.000 template was removed from the polymer by exhaustive washing using 2 % ammonia in methanol/water 1:1 volume ratio (process was monitored by hplc supernatant analysis). the polymers were then washed several times with methanol and dried in an oven at 60 °c. binding experiments batch binding experiments were performed as follows: polymer 10.0±0.2 mg was weighed in a micro tube, and 2.000 ml of template solution in acetonitrile was added. the concentrations of template solution were 0.1–10 mmol dm-3 (0.1, 0.2, 0.5, 1.0, 2.0, 4.0, 6.0, molecularly imprinted polymers for benzophenone-4 5 8.0 and 10.0). micro tubes were shaken using a rotator for 60 min, centrifuged (10 min, 14,500 rpm) and template concentration in supernatant was determined by hplc. the adsorption capacity was determined according to: 0 f( )c c v q m − = (1) where q stands for adsorption capacity (μmol g-1), c0 for total concentration of template at the beginning (mm), cf – free concentration of template after binding (mmol dm -3), v – volume of template solution used for binding experiment (ml) and m – mass of the polymer used for binding experiment (g)25. the imprinting factors were determined according to:40 ( ) ( ) bound free mip bound free nip / / n n if n n = (2) where if stands for imprinting factor, nbound / μmol – amount of the template bound to nip/ /mip and nfree / μmol – amount of the template free after binding. selectivity studies 10.0±0.2 mg of the imprinted polymer was weighed in a micro tube and 2.000 ml of 5.0 mmol dm-3 solution of selected uv filter was added. after 1 h of mixing, the polymer was removed by centrifugation, and uv filter concentration in supernatant was determined by hplc according to procedure described in section hplc analysis. hplc analysis bp4 was analyzed on purospher star rp18e 55-4 column, particle size was 3 μm, flow rate 1 ml min-1, solvent a (1 % acoh in water), solvent b (acn), solvent volume ratio 80:20. injection volume was 1.00 μl, column temperature 25 °c, detection wavelength 286 nm, retention time 3.30 min. total method duration time was 4 min. bp3 and bp8 were analyzed using waters symmetry c18 column, 3 mm×100 mm, 5 μm particle size. flow rate was 0.6 ml/min, solvent a (water), solvent b (acn), solvent volume ratio 30:70. injection volume was 1.00 μl, column temperature 25 °c, total method duration time – 4 min. retention times for bp3 and bp8 were: 2.27 and 1.46 min, respectively. for other uv filters, detection wavelengths were: 310 nm (oct and paba-o), 360 nm (avb), 238 nm (os and hms). retention times were oct – 2,25 min, avb – 2.09 min, os – 2.64 min, hms – 2.85 min and paba-o – 2.32 min. solid phase extraction 100.0 mg of mip4 was placed in an empty spe cartridge with a frit on the bottom, ptfe filter (0.45 μm) was placed on the top of the polymer. packed sorbent was washed with 1.000 ml of acn, then 3 times with hplc water (3×0.500 ml). 50 ml of bp4 solution (5.00 mg l-1) was passed through the spe cartridge at flow rate of approx. 1 ml min-1. the sorption was repeated using the same solution (once already passed). bp4 concentration was determined in filtrate using hplc. conductometric titrations 30–50 mg of polymer was placed in a titration cell, 20.0 ml of acn added, suspension was stirred using a magnetic stirrer and titrated with 0.1024 m hcl solution at 25.0±0.5 °c. the amount of the n-containing group was calculated according to end-point hcl volume. 6 pešić, krstić and verbić ftir ftir spectra were recorded for nips, washed mips and mips that were washed and subsequently tested for template re-binding. samples of polymers with bound template were obtained after binding experiments when a 10.0 mm template solution was used. after centrifugation, polymer was washed with a minimal volume of acn, and dried in oven at 60 °c and atmospheric pressure for 24 h. nitrogen physisorption nitrogen physisorption was determined at 77 k. samples were degassed for 1 h at room temperature and kept at 50 °c under vacuum for 24 h. the value of specific surface area of samples was determined using braunauer, emmet, teller method (bet). results and discussion benzophenone 4 (fig. 1) is a diprotic acid containing the sulfonic (–so3h) and the phenolic –oh group. acting as a strong acid, sulfonic group can form ionic pairs with basic monomers. hydroxyl group in ortho position is forming a pseudo six-membered ring with benzophenone group and, as such, it most probably doesn’t significantly contribute to the stability of the pre-polymerization complex. methoxy (–och3) group in the position 4 can act as h-bond acceptor (hba). two basic monomers were used in our study – dmaem and 4vp. both were expected to form ionic pairs and hydrogen bonds with template molecules, as well as to establish π–π stacking interactions (in case of 4vp). edma and dvb were used as cross-linkers, expecting that through π–π interactions dvb would contribute more to the binding capacity of the polymers than edma. if so, polymers prepared with dvb would have higher adsorption capacity than edma polymers. this was confirmed by binding experiments (section binding isotherms). acn is a commonly used solvent for mip preparation because it favours non-covalent interactions.40 thus, acn was our first choice for porogenic solvent, but was not applicable to all polymers preparation. when a combination of 4vp and edma/dvb was used, not all components were soluble in acn. it seems that the ionic pair between 4vp and bp4 (or complex in general) is not soluble in edma/acn or dvb/acn. monomer/template complex forms immediately upon mixing of these two components, which is easily observed by the colour change – mixture turns red (fig. 2), 4vp is colourless to pale yellow liquid and bp4 is yellow solid. fig. 2. mip2, mip4 and mip5: a) pre-polymerization complexes and b) prepared polymers. molecularly imprinted polymers for benzophenone-4 7 as this complex was not soluble in acn, acn/toluene mixtures, nor in acetone, other solvent had to be used. therefore, dry dmso was used even though it doesn’t enable hydrogen bonding and is not the best choice for porogen. pre-polymerization mixtures and obtained monolith polymers are shown in fig. 2. binding isotherms binding of bp4 for nips and mips was conducted by batch binding experiments.25 binding isotherms were obtained as q = f(cf) 40 where q stands for the adsorption capacity (μmol g–1) and cf for the free concentration of template after binding (mmol dm–3), as defined in eq. (1). polymers showed very high capacities for binding bp4. as it can be seen on fig. 3, bp4 binding capacity for each nip is much lower than for the corresponding mips. polymer mip4 showed the highest binding capacity. 0 2 4 6 8 0 200 400 600 800 1000 1200 nip2 mip2 nip4 mip4 nip5 mip5 q /  m o l g -1 c f / mmol dm -3 fig. 3. binding isotherms for selected polymers (table i). obtained results imply that the imprinting process was successful. if template – monomer ionic pairs are formed, the formation of high-affinity binding sites is expected, as ionic interactions prevail as the dominant ones. each available nitrogen-containing part of the polymer will bind the template. so, the only difference between the imprinted and the non-imprinted polymer is that mip has more available monomer units than nip because the template was present during polymerization. imprinting effect imprinting factor is calculated as ratio of the amount of template bound to imprinted vs. non-imprinted polymer. the imprinting factors for polymer pairs 8 pešić, krstić and verbić mip/nip 2, 4 and 5, in acn/water mixtures and acn are shown in table ii. it is interesting that the presence of water in rebinding medium has a more prominent effect on imprinted polymers. table ii. imprinting factors (if) for mip/nip pairs in acn (ifacn) and in aqueous solutions containing 20 vol. % of acn (ifacn-water) polymer ifacn ifacn-water (20 vol. % acn) improvement (ifacn-water/ifacn) mip1:nip1 1.05 1.14 1.10 mip2:nip2 2.53 2.64 1.04 mip3:nip3 1.40 1.75 1.25 mip4:nip4 2.60 3.17 1.22 mip5:nip5 2.10 3.43 1.63 the imprinting factors in aqueous medium, for dvb polymers, are higher than in acn, but the binding percentage (table iii) of the template to the polymer is lower. if water is present in porogen, some interactions between the template and the monomer, like hydrogen bonding, are disrupted. this leads to lower binding efficiency, but to higher imprinting factor. however, as the imprinted polymers have specific (imprinted) binding sites, the effect of water is lower than in non-imprinted ones. the improvement of imprinting factors in aqueous media compared to acn is also shown in table ii (calculated as ifacn-water/ifacn). high imprinting factors in aqueous medium are of considerable importance when the application of these polymers in aqueous samples is considered. table iii. binding of bp4 to studied polymers calculated as the amount of bound bp4 relative to starting bp4 concentration (10 mmol dm-3) polymer binding of bp4 (acn) / % binding of bp4 (20 % acn, v/v) / % nip1 29.54 26.64 mip1 30.52 29.38 nip2 15.57 11.05 mip2 31.94 24.69 nip3 30.88 18.89 mip3 38.61 28.96 nip4 32.25 16.31 mip4 55.41 38.22 nip5 16.89 10.70 mip5 29.54 29.10 adsorption capacities the measured adsorption capacities for imprinted polymers were in the range 0.591–1.108 mmol g–1 of bp4. these capacities are much higher than those described in literature for “regular” mips for other templates. ayadi et al.39 molecularly imprinted polymers for benzophenone-4 9 described polymers for bp uv filters binding which had a capacity of 27.90 or 96.06 μmol g–1. the adsorption capacities obtained from binding experiments were compared to those calculated from the polymer composition (based on monomer composition), but also those obtained from elemental analysis and conductometric titrations (fig. 4). 0.0 0.3 0.6 0.9 1.2 1.5 4 3 2 1 mip5nip5mip4nip4mip2 q /  m o l g -1 nip2 polymer fig. 4. 1: calculated content of n – based on monomer composition, 2: n content determined by ea, 3: adsorption capacity – determined from adsorption isotherms, 4: monomer content determined by conductometric titration – hcl consumption. according to the elemental analysis results (supplementary material to this paper, table s-v), polymers have adsorption capacity in the range 0.714 mmol g–1 (mip5) up to 1.454 mmol g–1 (mip4). capacity was calculated according to the content of nitrogen (%) in the polymer, as all nitrogen in the polymer originates from the functional monomer. as can be seen in fig. 4, all polymers have lower experimentally determined adsorption capacity than obtained from elemental analysis or based on the monomer and cross-linker composition. the only exception is polymer mip4, where the adsorption capacity is very close to the determined content of n (ea data) and similar to the calculated nitrogen content, proving the most successful imprinting process for mip4. to the best of our knowledge, this represents a unique example in the literature published so far. conductometric titrations were done to check the accessibility of monomer units in the polymer. accessible binding sites were calculated according to 0.1 mol dm–3 hcl titration volume (titration graphs shown in supplementary material, figs. s-1 and s-2). the values obtained by conductometric titrations are 10 pešić, krstić and verbić in between the calculated and the determined n, on one side, and the adsorption capacity on the other side. this can be explained as follows: not all monomer units are available for the template binding, but also, there are units not available for titration with hcl during the applied equilibration time. again, there is one exception – mip4. bet surface (supplementary material, table s-viii) areas are larger for mips compared to nips. however, it can be seen that binding capacity is not in direct correlation with specific surface area, i.e., mip4 (663 m2 g–1) and mip5 (634 m2 g–1) have very similar bet areas, but the binding capacity for these two polymers differ almost twice (1.108 mmol g–1 for mip4, 0.591 mmol g–1 for mip5). mip2/nip2 are found to be mesoporous materials, while mip4/nip4 and mip5/nip5 seem to be microporous. selectivity studies selectivity of the prepared polymers was studied by comparison of the binding of uv filters from few groups of organic uv filters. in fig. 5, the adsorption capacities are shown for template and other studied uv filters when binding was done using 5 mm solutions. 0 200 400 600 800 bp4bp8paba-ohmsosavboct q /  m o l g -1 mip2 mip4 mip5 bp3 uv filter fig. 5. selectivity of imprinted polymers towards other uv filters. as it can be seen, the binding of bp4 (55.4 %) compared to other uv filters (<5.5 %) is much higher due to different type of interactions between template and polymer: the template is bound to the polymer by strong ionic interactions between sulfonic group of the template and pyridine core (or amino group of molecularly imprinted polymers for benzophenone-4 11 dmaem) in the polymer. stronger binding is also a consequence of hydrogen bonding between the –oh group of the template and pyridine core, even though the –oh group is at least partially bound in pseudo-six-membered ring with benzophenone oxygen. none of other uv filters have the acidic group as bp4 has, so the rest of the examined compounds bind to polymers mainly by hydrogen bonding. solid-phase extraction of bp4 from aqueous solutions with mip4 as a sorbent, 95.0 % of bp4 was adsorbed after the first elution. if the same bp4 solution was passed over mip4 for the second time, the total of 98.5 % of bp4 adsorption was achieved. the obtained results indicate that the prepared imprinted polymer can be successfully applied for bp4 removal from aqueous solutions. the possibility of extraction from aqueous solutions is crucial for the application in real samples analysis as bp4 can be found in water ecosystems.36 infrared spectroscopy there is no difference between mip4 (fig. 6a) and nip4 (fig. 6c) in ftir spectra, indicating that the removal of the template molecule from the imprinted polymer was fully achieved by the applied washing procedure. 4000 3500 3000 2500 2000 1500 1000 e) d) c) b)  / cm -1 a) t / % fig. 6. infrared spectra for selected polymers: a) mip4; b) mip4 with re-bound bp4; c) nip4; d) nip4 with bound bp4; e) bp4. the difference within 1500–1700 cm–1 region between polymers with re -bound template (fig. 6b and d) and unbound ones (fig. 6a and c) confirms the 12 pešić, krstić and verbić presence of the template when compared to the spectra of pure template (fig. 6e). furthermore, the presence of the re-bound template in spectra of fig. 6b and d is visible within hydrogen bonds region (3300–3500 cm–1). this is probably due to moisture traces present in bp4. conclusion benzophenone 4 (bp4) is one of the most commonly used organic uv filter in sunscreen lotions and other cosmetic products. as an emerging environmental contaminant, the need for quantitative determination upon environmental sample preconcentration has pointed out the need for bp4 selective sorbents development. five polymer pairs of molecularly imprinted (mips) and nonimprinted polymers (nips) were prepared within this study. the polymers were characterized by template binding studies, infrared spectroscopy, elemental analysis, conductometric titrations and nitrogen adsorption analysis. all mips were successfully imprinted with bp4. however, the imprinting factor was higher in polymers containing dvb (2.10–2.60) compared to edma (1.05–1.40) polymers. the prepared polymers have high adsorption capacity for template binding – up to 1.108 mmol g–1 (mip4). binding of the template to prepared mips was compared to binding of 7 other organic uv filters, proving high bp4 mips selectivity. the nitrogen physisorption showed that differences in adsorption capacity are not the consequence of different specific surface areas. finally, mip4 was used as a sorbent for solid phase extraction of bp4 from aqueous solution, with 98.5 % sorption efficiency. the obtained results are promising for the application of mips as solid phase extraction sorbents for bp4 preconcentration from real samples, which is the subject of our ongoing study. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/11718, or from the corresponding author on request. acknowledgement. the authors would like to thank the ministry of education, science and technological development of republic of serbia (grant no: 451-03-68/2022-14/200168 and 451-03-68/2022-14/200026) for financial support. и з в о д високо селективни водокомпатибилни молекулски обележени полимери за бензофенон-4 милош п. пешић1, југослав б. крстић2 и татјана ж. вербић1 универзитет у београду – хемијски факултет, београд и 1универзитет у београду – институт за хемију, технологију и металургију – институт од националног значаја за републику србију, београд технологија молекулског обележавања примењена је у синтези селективних сорбената за бензофенон-4 (bp4), органски uv филтер који се користи у кремама за сунчање и другим козметичким производима. полимери су добијени полимеризацијом у маси, https://www.shd-pub.org.rs/index.php/jscs/article/view/11718 molecularly imprinted polymers for benzophenone-4 13 користећи (bp4) као матрицу. комбинацијом стабилности (механичке и хемијске), селективности и робусности молекулски обележених полимера са својствима bp4 извршено је успешно обележавање (фактор обележавања 1,05–2,60). карактеризација добијених полимера је извршена применом инфрацрвене спектроскопије, елементалне анализе, кондуктометријских титрација и физисорпције азота на 77 к. адсорпциони капацицети и селективност испитани су за 7 других органских uv филтера (бензофенон-3, бензофенон-8, хомосалат, бутилметоксидибензоилметан, етилхексил-салицилат, етилхексил-р-диметиламинобензоат и етилхексил-р-метоксицинамат), потврђујући велики адсорпциони капацитет и високу селективност за везивање bp4. највећи адсорпциони капацитет показао је ко-полимер 4-винилпиридина и дивинилбензена добијен коришћењем диметил-сулфоксида као порогена (1,108 mmol/g). полимер са највећим капацицетом за везивање bp4 примењен је као сорбент за екстракцију чврстом фазом bp4 из водених раствора са ефикасношћу од 98,5 %. 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samira sahi1, nadia zaidi2 and laid makhloufi2 1laboratoire de valorisation des energies fossiles (lavalef), ecole nationale polytechnique d'alger, algeria, 2laboratoire d'electrochimie, corrosion et de valorisation energétique (lecve), faculté de technologie, université de bejaia, algeria and 3military academy, university of defence, veljka lukića kurjaka 33, belgrade, serbia (received 13 may, revised 6 july, accepted 1 august 2022) abstract: in this study, an agricultural waste product was used to prepare a green corrosion inhibitor based on olive leaves (olea europaea var. syslvestris). firstly, an optimization study of antioxidant activity of o. europaea leaves monitored by the dpph free radical trapping method, was carried out using full factorial design. in the second step, the extract obtained under optimal conditions was tested as a green corrosion inhibitor for steel in 0.5 mol dm-3 hcl, using gravimetric and electrochemical methods. the results obtained by various techniques showed that the extract acted as a mixed-type inhibitor. the adsorption of the inhibitor was spontaneous (δgads = −12.443 kj mol -1), through the mechanism of physical adsorption, and it obeyed the langmuir adsorption isotherm. the highest corrosion inhibition efficiency of 92 % was obtained for 2.8 10-3 g cm-3of inhibitor, as measured by gravimetric method. keywords: antioxidant activity; extraction; green inhibitor. introduction in industry, acid solutions are widely used, and the aggressiveness of these solutions causes metals degradation, either through chemical or electrochemical reactions. the most effective method of preventing or decreasing the bare metal dissolution in such acidic solutions, is the use of corrosion inhibitors, that are nowadays widely applied. corrosion inhibitors are chemical compounds used to reduce the corrosion rate of metals and alloys in contact with aggressive environ * corresponding author. e-mail: souad.touazi@g.enp.edu.dz # serbian chemical society member. https://doi.org/10.2298/jsc220513064t 2 touazi et al. ments at low concentrations,1 thereby delaying and minimizing the corrosion process and thus avoiding economic losses due to metallic corrosion. numerous synthetic compounds show good corrosion inhibiting action, but many of them are extremely toxic to humans and environment. therefore, the development of non-toxic and biodegradable corrosion inhibitors is becoming a key issue as new global guidelines for industrial discharges are becoming increasingly ecologically strict.2−6 plants are one of the sources for these inexpensive and clean inhibitors. plant parts contain many compounds that satisfy the required criteria. in the mediterranean coastal zone, olive leaves are one of the by-products in the olive grove farming; they occur in high quantities in olive oil industry. they are also collected during the pruning of the olive trees.7 olive leaves are a cheap raw material known for its antioxidant properties.8 following this line, our work focuses on the inhibitory action of the olive leaf-based, green and natural substance, from olea europaea var. sylvestris, in the corrosion process of steel in 0.5 mol dm−3 hcl. the present investigation was divided into two parts: in the first part, a full factorial design (ffd) optimization study of antioxidant activity of the extract from o. europaea leaves was conducted, using the dpph free radical method. in the second part, the extract obtained under optimal conditions was tested for steel corrosion inhibition in hydrochloric acid by means of weight loss measurement, potentiodynamic polarization and impedance spectroscopy. experimental samples and reagents plant material consisting of leaves of olea europea var. sylvestris was harvested in the algiers region, situated in the north of algeria, during the flowering period between february and march 2018. the collected samples were identified by the national high school of agronomy. the leaves were washed in distilled water, dried at room temperature, and subsequently dried in an oven at 40 °c. they were grounded to obtain the powder, and then stored in a closed plastic box. methanol, ethanol, 2,2-diphenyl-1-picrylhydrazyl (dpph) and hcl (37 %) of analytical grade, were purchased from sigma–aldrich. plant material extraction the effects of different variables such as extraction temperature, solid/solvent ratio, water/ethanol ratio and extraction time, on the antioxidant activity, were studied using ffd. the conditions used in each experiment were settled according to the 24 ffd, as presented in table i. the powdered sample of o. europea, which had been previously weighed, was placed in a bicol flask, in contact with the extracting solvent. the mixture was kept in a thermostatic water bath with shaking. after a determined extraction time, the mixture was cooled in an ice bath, and then separated. the obtained filtered extracts were stored in the dark for future use olive leaf extract as corrosion inhibitor 3 for the determination of their antioxidant activity. the concentration of extracts (g cm-3) was determined by evaporating 10 cm3 of the filtrate and then weighing the obtained residue.9 table i. range of coded and actual values for ff independent variable coded variable level (−1) (0) (+1) extraction temperature, °c x1 55 68 81 solid/solvent ratio, g 100 cm-3 x2 08 12 16 water/ethanol ratio, vol. % x3 25 50 75 extraction time, min x4 45 60 75 estimation of antioxidant activity the effect of each antioxidant on dpph-radical was estimated according to the procedure described by brand-williams et al.10 briefly, 1 cm3 of the extract solution was added to 9 cm3 of dpph (40µg cm-3 prepared in methanol), and at the same time, a negative control (the blank) was prepared by mixing 1 cm3 of methanol with 9 cm3 of the methanol solution of dpph. the samples were kept in the dark for 30 min. the absorbance measurements were carried out at the wavelength of 517 nm, using a uv–vis spectrophotometer. the antioxidant activity (aa / %) was calculated as:11 aa = (ablank – asample)/ablank (1) where ablank represents the absorbance of negative control and asample is the absorbance of the sample (diluted extract). experimental design in this study, ffd methodology has been carried out to optimize the antioxidant activity of the samples. two levels and four variables were used in this investigation, requiring 16 (24) experiments plus four experiments performed at the center point to evaluate the experimental error variance. the four selected factors were studied between a lower level coded (−1), a higher level coded (+1) and the center point coded (0). the actual and coded levels of the independent variables are presented in table i. the regression equation which includes all interaction terms was given by the following expression: 4 3 4 0 1 3 1 i i i ij i j i i j i y b b x b x x  = = = + = + + +   (2) where yi is the predicted response, xi represents the independent variables coded (1 or −1), b0 is the average value of the response of sixteen assays, bi represents the principal effect, whereas bij represents the interaction effect and ε is a random error. corrosion test the working electrode used in the present study was steel, with the chemical composition presented in table ii. prior to each experiment, the working electrode was polished with various emery paper grades (280, 360, 800 and 1000), degreased with acetone and rinsed in distilled water before being placed into the aggressive solution. for weight loss measurements, the steel coupons with a surface area of 16.5 cm2, were immersed in 100 cm3 of 0.5 mol dm-3 hcl at 25 °c, without and with different concentrations of the olive leaf extract. corrosion rate (cr), inhibition efficiency () and the degree of sur4 touazi et al. face coverage () were calculated from the weight loss measurements, determined at different immersion times for a total duration of 8 days, by using the equations:12,13 cr = (wb – wa)/a (3)  = 100(cr(free) – cr(inh))/cr(free) (4)  = /100 (5) where wb and wa are the coupon weight measured before and after immersion in the aggressive solution, a is the exposed area, and cr(free) and cr(inh) are the corrosion rates of steel in the absence and presence of the inhibitor in the aggressive solution, respectively. table ii. composition of steel element c si mn p s mo fe content, wt. % 0.15–0.26 <0.35 <1.5 <0.035 <0.040 0.4–0.6 97–98 open circuit potential (ocp) and potentiodynamic polarization measurements were carried out using a voltamaster software piloted potentiostat pgz100. this potentiostat was connected to the three electrodes: steel panel as a working electrode, a platinum panel used as a counter electrode and k2so4-saturated sulphate electrode (sse) as the reference. tafel plots were obtained at a scan rate of 1 mv s-1, over the respective ocp range between −250 and 250 mv (from cathodic to anodic end), after the constant ocp was established. the inhibition efficiency () was determined as:14  = 100(jcorr(free) – jcorr(inh))/jcorr(free) (6) where jcorr(free) and jcorr(inh) are the corrosion current densities in free and inhibited acid solution, respectively. electrochemical impedance spectroscopy (eis) tests were carried out at ocp in an aerated solution using an autolab potentiostat/galvanostat (pgstat30) under fra software. the response of the electrochemical system to ac excitation was measured with a frequency ranging from 100 khz to 10 mhz and peak to peak ac amplitude of 10 mv. for the electrochemical tests, the exposed area of the steel was 1 cm2. results and discussion optimization of extraction parameters four variables and two levels were chosen to build the ffd in order to find the optimum combination of conditions for the extraction of active substances from the olive leaves. variables such as extraction temperature (x1), solid/solvent ratio (x2), water/ethanol ratio (x3) and extraction time (x4) were selected as the independent variables, and antioxidant activity was taken as the response of the design experiments (y). the results collected to build the ffd model are given in table s-i of supplementary material to this paper. to obtain the mathematical model that represents the antioxidant activity with the first order polynomial (eq. (2)), the regression analysis was applied on the experimental data using statistica software 7.0. various effects of factors and their interactions were calculated using the analysis of variance (anova) and other adequacy measures. olive leaf extract as corrosion inhibitor 5 fig. 1 shows the pareto chart, which represents the analysis of the calculated standardized effects of the independent variables and their interactions, on the response value of antioxidant activity. as can be seen in the pareto charts, the solid/solvent ratio (x2) presented the most influencing parameter on the response. whereas the interaction effects of extraction time with water/ethanol ratio (x3, x4) and extraction time with solid/solvent ratio (x2,x4) were insignificant, and therefore were eliminated from the regression equation. the negative value of the standardized effect indicates that the response decreases with increase of the value of this variable, and the opposite is the case for the positive value. fig. 1. pareto chart of the response value of antioxidant activity on various independent variables and their interactions. table s-ii of the supplementary material shows the anova analysis, used to verify the adequacy of the models at confidence level of 95 %. the regression p-value and f-value reveal that the model was highly significant and represented the data satisfactorily. in other words, the value of pure error is very low (0.84) suggesting strong data reproducibility. a good adjustment of the regression equation to the experimental data was verified through the determination of coefficient value r2 (0.954) which is close to 1, indicating that only 4.6 % of the variations in observed data could not be explained by the model (fig. 2a). the value of the adjusted r2 is equal to 0.905 which was also of very high importance to the model. the comparison of experimental data and predicted values is shown in fig. 2b. it can be seen that the predicted responses are in agreement with the experimental data and the difference between them does not exceed 0.055, indicating that the mathematical model is adequate. the final polynomial equation (eq. (7)) 6 touazi et al. representing the antioxidant activity as a function of the significant effects, was written as: yi = 66.26 – 1.51xi + 9.73x3 – 2.74x4 – 1.18x1x2 + 1.73x1x3 + + 1.14x1x4 + 2.83x2x3 (7) fig. 2. quality analysis of the model: a) experimental versus predicted responses, b) residual versus predicted value for antioxidant activity (yi). the numerical optimization of the desirability function gave optimal conditions to obtain olive leaf extract with a maximum antioxidant activity. with the analysis of the prediction profiler at maximal desirability (fig. s-1 in the supplementary material), it can be deduced that the best response of antioxidant activity was obtained at the following conditions: extraction temperature (55 °c), solid/ /solvent ratio (8 g 100 cm−3), water/ethanol ratio (75 %) and extraction time (45 min), with 0.99 as the value of desirability. corrosion tests weight loss measurements. different concentrations of the olive leaf extract obtained under optimum conditions deduced from antioxidant capacity measurements, were added to 0.5 mol dm−3 hcl. corrosion of steel in the solutions prepared in this manner, was monitored by weight loss experiments, i.e., by measuring the mass of the metal converted into corrosion products per unit of surface area, per unit of time. fig. 3a and b represent the variation in corrosion rate and inhibitory efficiency, calculated from the weight loss measurements. the fig. 3 inspection shows clearly that the rate of steel corrosion is significantly reduced, i.e., the inhibition efficiency is increased, with the addition of olive leaf extract. this behavior represents the extract’s inhibiting effect against the acid corrosion of steel. the maximum inhibition efficiency of 92 % was achieved for 2.8 10−3 g cm−3 of the added extract. open circuit potential measurements. it is well known that the formation of protective corrosion products or a surface protective film in general, can be eva olive leaf extract as corrosion inhibitor 7 luated by monitoring the ocp of the corroding electrode. the effect of various concentrations of olive leaf extract on the steel electrode ocp in 0.5 mol dm−3 hcl, is shown in fig. 4. fig. 3. a) variation of the weight loss data with the immersion time recorded for steel electrode in 0.5 mol dm-3 hcl solution, in the absence and presence of various concentrations of extract; b) inhibition efficiency versus the concentration of the inhibitor, deduced from a). fig. 4. ocp curves for steel in 0.5 mol dm-3 hcl solution as a function of time and inhibitor concentration. the obtained results indicate that, as the immersion time increased, the ocp of the steel samples in the solution with the extract became more positive as compared to the ocp of the steel in the blank solution, which more rapidly shifted to negative values. the increase in the ocp, once the olive leaf extract was added, may be attributed to the adsorption of the inhibitor on the active metal sites.15 potentiodynamic polarization curves. the inhibitive effect of olive leaf extract, on steel specimens immersed in 0.5 mol dm−3 hcl, was further studied by evaluating changes in cathodic and anodic behavior by recording the polarization curve from cathodic to anodic potential limit over the ocp at a scan rate of 1 mv s−1. 8 touazi et al. firstly, the effect of the extraction solvent (ethanol) on the typical anodic and cathodic polarization curves of steel in aggressive solution was studied as presented in fig. 5a. the comparison of steel polarization in 0.5 mol dm−3 hcl blank solution and in 0.5 mol dm−3 hcl with ethanol, does not indicate any influence of ethanol. therefore, the strong inhibitory activity of the extract observed at tafel plots in fig. 5b is primarily due to the presence of olive leaf compounds. fig. 5. potentiodynamic polarization of steel in 0.5 mol dm−3 hcl blank solution and in the presence of: a) ethanol; b) different concentrations of olive leaf extract. steel corrosion parameters determined from polarization curves in 0.5 mol dm−3 hcl blank solution and in the solution containing olive leaves extract, are shown in table iii. inspection of the obtained results revealed that the values of corrosion potential (ecorr) and corrosion current density (jcorr) were influenced by the presence of the inhibitor. as shown in fig. 5b, the increase in the extract concentration results in lower current densities recorded from the potentiostatic polarization curves. besides, the ecorr slightly shifts to a more positive value (noble direction of potential) in the presence of olive leaf extract. table iii. electrochemical parameters obtained from polarization curves at 25 °c. c / mg cm-3 ecorr / mv vs.sse jcorr / ma cm -2 ba / mv dec -1 bc / mv dec -1 η / % 0 −932 1.13 65 143 – 1.4 −905 0.48 39 266 57.5 2.1 −898 0.40 58 159 64.6 2.8 −882 0.22 56 151 80.5 3.6 −884 0.29 63 166 74.3 4.4 −890 0.30 63 156 73.4 since the difference between the ecorr of the steel electrode in the blank solution and in the solution containing inhibitor is lower than 85 mv, this behavior suggests that the extract functions as a mixed-type inhibitor.16,17 olive leaf extract as corrosion inhibitor 9 the highest inhibition efficiency of 80.53 % was obtained for the extract concentration value of 2.810−3 g cm−3. as can be seen, the inhibition efficiency, both determined by gravimetric tests (fig. 3) and polarization curves (table iii), increases with increasing the olive leaf extract concentration until reaching a peak value at optimal extract concentration. the values of inhibition efficiency calculated by gravimetric measurements were higher than those calculated by the electrochemical method. a similar finding between the two approaches has previously been recorded.18 the disparity observed may be due to the fact that the gravimetric method provides average corrosion rates, while electrochemical techniques give instantaneous corrosion rates. indeed, the development of a protective film during the immersion time of 8 days, in the case of gravimetric measurement, may explain the difference between the obtained values. adsorption isotherms. the adsorption of the olive leaf extract compounds at the steel surface is assumed to be responsible for the inhibitory action of the extract against the corrosion of steel in acid solution. the adsorbed layer functions as a barrier between the steel surface and the aggressive solution, resulting in a reduction of the rate of corrosion.19 consequently, the inhibition efficiency (η) derived from the weight loss curves is directly proportional to the fraction of the surface covered by the adsorbed molecules (θ).20 the mode of variation of (θ) with the extract concentration (c) determines the adsorption isotherm describing the system.21 several adsorption isotherms (langmuir, temkin and frumkin) were evaluated to determine the effective adsorption isotherm, according to the following equations:22,23 langmuir: c/ = 1/k + c (8) temkin: e–2 = kc (9) frumkin: 1  − e–2 = kc (10) where  is the adsorbate interaction parameter and k the equilibrium constant of the adsorption process (l kg−1), related to the adsorption free energy change (g0ads) by the following equation: g0ads = –rt ln (55.5k) (11) where r is the gas constant (j °k−1mol−1) and t the temperature (k). according to the experimental data, the relationship of (θ) versus c was observed to follow a linear pattern (fig. 6), which can be described by langmuir equation (eq. (8)) with r2-value equal to 0.99. the other equations (eqs. (9) and (10)) were evaluated but did not indicate a good fit (for temkin and frumkin isotherms, the r2-values obtained were 0.37 and 0.005 respectively). the calculated values of adsorption equilibrium constant (k) and free energy change of adsorption (g0ads) were found to be 2.73 l kg −1 and −12.443 kj 10 touazi et al. mol−1, respectively. the negative sign shows that the adsorption of olive leaf extract onto steel surface is a spontaneous process. the values of g0ads of −20 kj mol−1 or more positive, are well known to suggest physical adsorption, while values of −40 kj mol−1 or more negative, involve charge sharing, i.e., the charge transfer from the inhibitor molecules to the metal surface, to form the chemisorption related coordinate type of bond.9,24 the acquired value of g0ads in our work shows a strong physical adsorption of the olive leaf extract components onto the steel surface in 0.5 mol dm−3 hcl solution. fig. 6. langmuir isotherm for adsorption of olive leaf extract onto steel surface in 0.5 mol dm-3 hcl solution at 25 °c. r2 = 0.99. electrochemical impedance spectroscopy. the electrochemical impedance spectroscopy is used as a powerful technique to characterize the corrosion parameters at metal/electrolyte interface. fig. 7 highlights the nyquist plots of steel samples immersed in 0.5 mol dm−3 hcl solution in the absence and presence of various concentrations of the olive leaf extract at the ocp. the impedance plots obtained are similar in shape and consist of a single capacitive semicircle at highto-medium frequency and an inductive loop at low frequency range. the capacitive loop is related to the charge transfer resistance process, while the appearance of the pseudo-inductive loop can be explained by the relaxation of adsorption species such as (cl−)ads and (h +)ads on the surface of the electrode. 25 the inductive loop can also be related to the re-dissolution of the passivated surface.26,27 also, one can see that the addition of the olive leaf extract inhibitor, increases the size of the semi-circle in comparison to the blank solution, and the maximum circle diameter is obtained for 2.810−3 g cm−3 of inhibitor. this shows that there is an increase in the charge transfer resistance, which is in agreement with the literature,25,26,28 and previous corrosion tests in this work. the improvement in the resistance by increasing the inhibitor concentration is due to an adsorption of inhibitor molecules on the surface of the steel, indicating the effective blocking of the surface.29 the impedance data were analyzed using zsimpwin® software and fitted to the equivalent circuit model shown in fig. 8. this equivalent circuit is exten olive leaf extract as corrosion inhibitor 11 sively reported in the literature.30,31 the elements related to the circuit are: re representing the electrolyte resistance, rct as the charge transfer resistance, l as the inductance element, rl as an inductive resistance, q as the pseudocapacitance and cpe as the constant phase element. fig. 7. nyquist plots for mild steel in 0.5mol dm-3 hcl solution in the absence and presence of olive leaf extract at ecorr and t = 25 °c. fig. 8. electrical equivalent circuit model used to fit the impedance parameters for steel electrode in 0.5 mol dm−3 hcl solution. due to the roughness and homogeneity of the metal surface, as well as adsorption effects, cpe can be used to replace the double-layer capacitance, cdl. 26 the electrochemical impedance parameters obtained from the equivalent circuit are presented in table iv. table iv. eis parameters for the corrosion of steel immersed in 0.5 mol dm−3 hcl without and with different concentrations of olive leaf extract c / mg cm-3 re / ω m 2 q / 10-5 ω-1 sn cm-2 n rct / ω cm 2 rl / ω cm 2 l / h cm2 0 1.14 36.64 0.80 99.22 6.63 9.20 1.4 0.87 29.69 0.82 135.50 13.37 16.77 2.1 1.23 25.33 0.81 187.20 14.01 32.95 2.8 0.96 26.41 0.84 247.50 45.44 377.70 3.6 0.95 28.93 0.82 218.30 56.11 1552 4.4 1.44 20.48 0.87 208.20 2.371010 0.10 from table iv, a significant increase in the charge transfer resistance (rct) was observed in the presence of the inhibitor, providing a high anticorrosive efficiency. the largest effect was observed at 2.810−3 g cm−3 of olive leaf extract, 12 touazi et al. which gave rct value equal to 247 ω cm 2. this behavior is due to an adsorption of inhibitor molecules on the metal surface, leaving less electroactive sites available for corrosion, i.e., the inhibitor builds up a protective film on the metallic surface that isolates it from the corrosive environment, thereby impeding further charge and mass transfer.31,32 in addition, the increased n value after the addition of extract to the hydrochloric acid solution (0.82−0.87) when compared to that obtained in the blank solution (0.80), indicates the reduction in the surface in homogeneity due to the adsorption of inhibitor molecules on the active adsorption sites at steel surface.32,33 also, from table iv, one can notice that the q value is lower in the presence of olive leaf extract compared to the blank solution. the diminishing values of q can be related to a lower local dielectric constant due to the replacement of water molecules with inhibitor molecules. this process increases the thickness of the electrical double layer and reduces the surface area exposed to the aggressive media, implying an adsorption of inhibitor molecules on the surface of the steel.31,32 conclusion in the present work, an optimization study of the antioxidative matter extraction from olea europaea var. syslvestris leaves was performed using full factorial design. the statistical analysis of the experimental results showed that the final polynomial equation, representing the antioxidant activity as a function of the significant effect, gave a reasonably good fit with an r2 value of 0.955. by response optimizer graph plot, the best response value of antioxidant activity was acquired at the following extraction parameters: extraction temperature 55 °c, solid/solvent ratio 8 g 100 cm−3, water/ethanol ratio 75 %, and extraction time 45 min. afterwards, the olive leaf extract obtained under the final optimized conditions was tested as a green corrosion inhibitor for steel in 0.5 mol dm−3 hcl. results of the electrochemical performance and weight loss analysis of steel samples, in hcl with the addition of o. europaea leaf extract, indicated that the extract: − is a successful corrosion inhibitor for steel in 0.5 mol dm−3 hcl solution, with a maximum inhibition efficiency of 92 %, achieved at extract concentration of 2.810−3 g cm−3, − acts as a mixed-type inhibitor and − acts as an adsorption inhibitor, where the adsorption phenomenon is spontaneous, following the mechanism of physical adsorption related to the langmuir adsorption isotherm. olive leaf extract as corrosion inhibitor 13 supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/11851, or from the corresponding author on request. и з в о д антикорозионо дејство супстанци екстрахованих из лишћа маслине под оптималним параметрима, претходно одређеним применом методологије experimental design souad touazi1,2, михаел m. бучко3, radouane maizia2, samira sahi1, nadia zaidi2 и laid makhloufi2 1laboratoire de valorisation des energies fossiles (lavalef), ecole nationale polytechnique d'alger, algeria, 2laboratoire d'electrochimie, corrosion et de valorisation energétique (lecve), faculté de technologie, université de bejaia, algeria и 3војна академија, универзитет одбране, вељка лукића курјака 33, београд у овом истраживању, лишћe маслине (olea еuropaea var. sylvestris) je искоришћенo за добијање зеленог инхибитора корозије. прво су одређени оптимални параметри екстракције активних материја из лишћа, мерењем њихове антиоксидантне активности на слободним органским радикалима. оптимизација параметара је извршена full factorial design методом. у другој фази истраживања, екстракт добијен при оптималним параметрима је испитиван као инхибитор корозије челика у 0,5 mol dm-3 hcl, гравиметријском и електрохемијским методама. резултати показују да се екстракт понаша као мешовити инхибитор. адсорпција инхибитора на супстрату је спонтана (δg0ads = −12,443 kj mol-1), одиграва се према механизму физичке адсорпције и може се описати лангмировом адсорпционом изотермом. највећи степен инхибирања корозије од 92 % мерено гравиметријском методом, се постиже при концентрацији инхибитора у киселини од 2,810-3 g cm-3. 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five types of surfactants alcohol polyoxyethylene ether sodium sulfate, coconut oil diethanolamine, sodium lauryl sulfate, polyacrylamide and polyether-modified silicone resin emulsion were selected as foaming agents. through laboratory experiments, the effect on the expansion ratio and foam stability of the surfactant type/concentration, fly ash particle concentration/size and ph were investigated. the foaming condition was determined by numerical optimization. the results of this study may serve as a reference for understanding the preparation of a novel threephase foam. it is hoped that this work could provide useful guidance for the preparation of efficient three-phase fire-extinguishing foam for the safe guarding of process safety in the field of chemical production, transportation, and storage suitable for drug delivery than al12p12 and al12n12 based on their recovery times. keywords: fighting fire foam; expansion ratio; half-life; surfactant; fly ash; ph. introduction coal accounts for 60 % of the fuel used for power generation in china at present,1 and for a long time into the future, the main body of china’s energy consumption will remain coal.2 however, a gradually expanding demand has brought more risks, such as coal field fire accidents,3 this usually brings heavy losses and danger in the field of mining, transportation and storage. therefore, effective and efficient fire-extinguishing material is particularly important in the process safety of firefighting and safeguarding. foam mainly includes solidified foam, a type of dispersed gas-liquid/solid system and is an effective environmentally friendly and promising material in extinguishing fires, mineral float * corresponding author. e-mail: wb6314005@126.com https://doi.org/10.2298/jsc220205060w 2 wang et al. ation, and enhanced oil recovery.4,5 foam two-phase foam and three-phase foam, screening, cooling and insulating are the three main effects to extinguish fires.6 two-phase foam is a dispersion system composed of an aqueous surfactant solution and gas (air or nitrogen), which has an important role in fighting fires. used properly, two-phase foam has previously effectively controlled mine fires.7 however, its high specific surface area and surface energy lead to thermodynamic instability.8,9 moreover, the gravity and laplace pressure in the aqueous foam cause foam drainage to coalesce and coarsen, which leads to foam decay.10 the efficiency of the fire extinguishing foam is mainly determined by its stability and heat insulation performance. therefore, stabilized two-phase foams have limited applications in large-scale coal and oil fields.11 three-phase foam is a system for dispersing gas and solid particles in a liquid phase,12 in which particles stabilize the foam by adsorbing onto the gas/ /water interface.13 compared with a traditional gas-liquid two-phase foam, the addition of solid particles will inevitably affect the precipitation rate of water in the liquid film and the stability of the foam. researchers have tried to add a burning-resistant powder to two-phase foam to form three-phase foam that was composed of water, air (or n2) and solid. 14,15 fly ash (fa) is a massive waste residue produced as a by-product of coal combustion in power stations, out of nearly a billion tons produced in sample amount, not more than 25 % is utilized and the remaining is left untreated.16,17 using fa produced by coal-fired boilers as solid particles to form fa three -phase foam system could reduce the cost of the system. at the same time, it could reduce the pollution of solid waste by reducing fa discharge. preground fa particles are commonly used to stabilize the three-phase foam and enhance oil recovery.18 previous studies have reported that fa particles produced by ballmilling could significantly improve the stability and resistance factor of the foam system. although fa particles can effectively improve foam stability, grinding causes a significant increase in cost and work load. a water-based foam can produce an efficient fire prevention performance because it has good cooling and a filling effect in the fire extinction stage. therefore, a water-based and non-ground fa stabilized foam system could promote the development of fire extinguishing materials. currently, three-phase foams stabilized by unground fa have been applied to try and prevent and control spontaneous combustion in coal.15,19,20 the stability of three-phase foam is determined by the synergistic effect between surfactant and particles.21–23 the synergistic effect is associated with the physicochemical properties, concentrations of surfactants and solid particles.24 mxed surfactants show a stronger synergistic interaction compared with a single surfactant.25 however, whether it help to improve the properties of fire fighting foam, and the influence of fa/surfactant on the stability and expansion ratio of three-phase foam still require clarification.26 influencing factors of preparing three-phase foam 3 in this study, because the used fly ash was negatively charged, cationic foam agents are unsuitable for the fly ash-based foam, anionic foamers, i.e., fatty alcohol polyoxyethylene ether sodium sulfate (aes) and sodium lauryl sulfate (k12), and nonionic foamer, i.e., polyacrylamide (pam), coconut oil diethanolamine (6501), foam stabilizer: modified silicone resin polyether emulsion (fm-550), were first evaluated for the foaming agent system. here air as gas phase and nonground fa particles as solid phase were used to generate water-based three-phase foam. the formulation of foaming agent was optimized by orthogonal experiment. the effects of the fa particle size/content, ph on improving the expansion ratio and foam stability were investigated. as a result, the fa supported foam exhibited better stability and expansion ratio. the design in this work could be used to study the fire fighting efficiency of different three-phase foams and serve as a prototype to develop better generators for both lab research and practical applications. experimental materials and instrumentations all chemicals including aes, k12, 6501, pam and fm-550 (active ingredient: 55 %) were industrial grade purchased from lvsen chemical reagent co., ltd., china and used without further purification. deionized water was used in all experiments. the fa used was obtained from huainan tianjiaan power co., ltd., china. air compressor (det750-30 l, purchased from shenba compressor manufacturing co. zhejiang, china): air pressure of 0.8 mpa, and air speed of 80 l min-1. the foam gun delivered foam with a high-pressure spray by an air compressor. preparation of foam the three-phase foam was prepared using a self-made designed system, which was mainly composed of an air compressor and a foam gun (shown in fig. 1). first, a foam solution is prepared in a polyvinyl chloride (pvc) jar according to the ratio of ingredients, then fa particles were added as the solid phase, and fully stirred until a homogeneous slurry was obtained. the foam gun was provided with an air compressor to provide gas and pressure, then air was introduced through an air inlet on the tube and mixed with the slurry. the pressure then enables spray through the nozzle, whereby uniform and stable three-phase foams were jetted by the air through the outlet for fire extinguishing. after the foam was formed, the initial foam volume was immediately recorded, which was used to describe foaming capacity and stability. all the foam tests were performed at room temperature. a study of the pro fig. 1. preparation process of the three-phase foam. https://fanyi.baidu.com/#en/zh/investigate 4 wang et al. perties of the foam in different foaming agent, different fa content/size and ph enabled the optimal parameters to be found. characterizations and calculation the particle size distribution of fa sample was measured by a laser particle sizer (marvin mastersizer 2000, uk). the fa was investigated by x-ray diffraction (xrd, lab xrd, shimadzu, japan) test in the range of 10–60 with monochromatic cukα radiation at 40 kv and 3 nm. foaming ability is one of the most fundamental properties of foam, which is expressed by the expansion ratio (f) and was calculated based on eq. (1) according to the current chinese national standards (gb/t 1966–1996): 2 1 f m m  = − (1) : the density of the foaming solution (assumed to be equal to the density of water, 1 kg dm3); : volume of foam collector, dm-3; m1: the mass of the empty foam collector, kg; m2: the mass of the foam collector was filled with foam, kg. every experiment was repeated 5 times, and three values of good precision were averaged as the measurement result. the relative change in foam volume as a function of time can be considered an indication of foam stability. generally, foam stabilization performance is expressed by the defoaming time (decay half-life, t1/2), t1/2 for which the foam volume decreases by 50 %. results and discussion characterization of fa fa is a highly heterogeneous material usually consisting of silicon dioxide and aluminum oxide as the major mineral components.27 laser particle analyzer is commonly used to measure the particle size distribution. as shown in fig. 2a, the used fa particle size distribution range was 1–500 m, and most fa diameter of particles were about 300 μm. the fa was also characterized by xrd, with fig. 2b showing the spectra of the sample particles, the primary diffraction peaks at the 2θ values of the particles were located. these results indicate that the fig. 2. particle size distribution (a) and xrd analysis (b) of fa. influencing factors of preparing three-phase foam 5 used fa contained mainly sio2 and al2o3, the major absorptive capacity of water was low and was relatively resistant to dissolution. factors influencing of foam properties optimization of foaming agent. the surfactant in the foam could be adsorbed at the air/liquid interface to form and stabilize the foam. the orthogonal test is a method used in multiple factors analysis, which is the method of partial factor design by selecting the representative points from the comprehensive test. to determine the optimal foaming condition of the generator, the foaming agents were optimized by means of orthogonal experiment and extremum difference analysis. a five-factors four-levels including aes, 6501, k12, pam and fm-550 were designed and a total of 16 tests were performed to analyze their foam expansion (f) and half-life (t1/2). 28 in the experiment, the basic system was 200 g of water. factor level values are shown in table i and the scheme of the orthogonal test are shown in table ii. each group of experiments was repeated five times, and the average value of the three with good precision was recorded. table i. factors and levels of foaming agent compounding system level content, g aes 6501 k12 pam fm-550 1 0.0 0.0 0.0 0.0 0.0 2 3.0 1.0 3.0 0.1 1.0 3 4.5 2.0 4.5 0.15 1.5 4 6.0 3.0 6.0 0.2 2.0 table ii. orthogonal experiment of foaming agent compound system number content, g f t1/2 / min aes 6501 k12 pam fm-550 1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2 0.0 1.0 3.0 0.1 1.0 38.4 224 3 0.0 2.0 4.5 0.15 1.5 30.0 294 4 0.0 3.0 6.0 0.2 2.0 25.6 246 5 3.0 0.0 3.0 0.15 2.0 32.7 335 6 3.0 1.0 0.0 0.2 1.5 19.2 426 7 3.0 2.0 6.0 0.0 1.0 42.5 223 8 3.0 3.0 4.5 0.1 0.0 53.4 42 9 4.5 0.0 4.5 0.2 1.0 32.2 327 10 4.5 1.0 0.0 0.15 0.0 38.8 37 11 4.5 2.0 6.0 0.1 2.0 26.8 280 12 4.5 3.0 3.0 0.0 1.5 28.2 256 13 6.0 0.0 6.0 0.1 1.5 29.2 294 14 6.0 1.0 4.5 0.0 2.0 25.2 310 15 6.0 2.0 3.0 0.2 0.0 41.4 56 16 6.0 3.0 0.0 0.15 1.0 23.2 471 https://fanyi.baidu.com/#en/zh/multiple%20factors%20analysis 6 wang et al. in order to systematically analyze the degree of influence of different foaming agents, the range analysis method was introduced according to the orthogonal test. the results showed that the sequence of the influence of surfactant on the f and t1/2 were as follows: k12>aes>pam>6501>fm-550 (table iii), fm-550 >pam>aes>k12>6501 (table iv). the best array was a2b3c3d2e2 and a4b4c4d3e3, respectively. these results suggested that the surfactant plays the main role in the process of fire-retardant foam. based on the range analysis, the best proportion of the foaming agent compounding system is aes:6501:k12: :pam:fm-550 = 6:6:9:0.3:2 (mass ratio, 200 g water, which was recorded as the foaming agent in the follow-up experiments). table iii. analysis of foam expansion ratio range; k is the sum of the foaming multiples in parallel experiments, k is the average foaming multiples, a is the experimental results of aes, b is the experimental results of 6501, c the experimental results of k12, d the experimental results of pam, e the experimental results of fm-550 parameter f a (aes) b (6501) c (k12) d (pam) e (fm-550) k1 94.0 94.1 81.2 95.9 133.6 k2 147.8 121.6 140.7 147.8 136.3 k3 126.0 140.7 140.8 124.7 106.6 k4 119.0 130.4 124.1 118.4 110.3 k1 23.5 23.5 20.3 24.0 33.4 k2 37.0 30.4 35.2 37.0 34.1 k3 31.5 35.2 35.2 31.2 26.6 k4 29.8 32.6 31.0 29.6 27.6 range 13.5 11.7 14.9 13.0 8.5 primary and secondary sequence order c>a>d>b>e optimization a2 b3 c3 d2 e2 table iv. analysis of foam half-life range parameter t1/2 / min a (aes) b (6501) c (k12) d (pam) e (fm-550) k1 764 956 934 789 135 k2 1026 997 871 840 1245 k3 900 853 973 1137 1270 k4 1131 1015 1043 1035 1171 k1 191 239 233 197 34 k2 256 249 218 210 311 k3 225 213 243 284 318 k4 282 254 261 264 293 range 91 41 43 87 284 primary and secondary sequence order e>d>a>c>b optimization a4 b4 c4 d3 e3 influencing factors of preparing three-phase foam 7 foaming agent and water ratio (foaming liquid) the best proportion of foaming agent were mixed with water in the mass ratio of 1:10, 1:15, 1:20, 1:25, 1:30 (recorded as the foaming liquid) to study the foaming capacity and foam stability. as shown in fig. 3, with other condition remaining unchanged, the f and t1/2 of the foam first increase and then decreases with decreaseing water, when 1:20 was added, both had the maximum at 34.2 and 7.5 h respectively, which depicts that the foaming agent and water ratio play other main roles for the foaming capacity and foam stabilization. fig. 3. optimal ratio of foaming agent and water. the ratio of foaming liquid/fa fa particles are added to the foaming liquid (1:20 ratio of foaming agent and water) to form a three-phase foam system. comparing the f and t1/2, the performance of three-phase foam is evaluated under different solid–liquid ratios. as observed in fig. 4, with increasing fa mass fraction, the t1/2 values of the foam increased, but foam volume decreased continuously, thereby indicating that the fa can increase foam stability, but has an inhibitory effect on foam volume. the enhancement of foam stability of three-phase foam was attributed to the existence of fa particles. the attached particles decrease the pressure difference between the gas/liquid interface, slowing down the drainage process. foam fire extinguishing mainly depends on covering the surface of burning objects to prevent them from contacting oxygen and continuing burning. therefore, f has a greater impact on the foam extinguishing property than its t1/2 period. according to experiment results, when the solid to liquid ratio is lower than 1:3, the expansion ratio below 10, limits its practical application as a fire extinguisher. when choosing the proportion with the solid to liquid ratio of 1:4 for the fire-retardant foam, not only ensures comprehensive properties such as f and stability are the best, but the use of stabilizer can also be reduced at the same time, and the f meets the current requirements of coal mine fire-extinguishing materials. by focusing on its f and t1/2, a solid–liquid ratio of 1:4 was determined. based on 8 wang et al. this, the optimized fa three-phase foam system has excellent stability with a foam t1/2 that can reach more than 28.7 h. fig. 4. influence of solid-liquid ratio on the performance of three-phase foams. fa particle size fa particle is widely applied in the physical property test. to avoid particle size effect, specific graded particles were selected for physical parameter measurement experiments.29 in lab research, there are some particle size distribution testing such as sieving, laser particle analyzer, etc. sieving is the simplest powder particle size test method, the size of the lower and upper standard sieves as the lower and upper limits of the particle size. that is, broken fa samples can be separate by sieves of different sizes, and the fa particles after sieving were divided into different particle groups.: <38, 38–75, 75–106, 106–150 and 150– –380 μm. fig. 5 shows that with increasing particle size, the foam t1/2 tends to stabilize and then decrease. fig. 5. influence of particle size on the properties of three-phase foam. no significant differences were observed in values of the f among smaller particle fractions (<75 μm). thus, smaller particles can keep good dispersion in influencing factors of preparing three-phase foam 9 the dispersion liquid, and hence the results can be grained large area. the larger particles are not suitable for stabilization because they are dispersed difficultly and the gravity in the aqueous foam leads to foam decay. ph in actual application in coal mines or oil fields, foam is often used in areas where coal or oil are prone to spontaneous combustion. in such places, the ph value differs from respective levels in the laboratory. therefore, it is necessary to study the influence of ph value factors on the characteristics of the foam, and hence, the ph value of the foaming system was adjusted to about 4, 5, 6, 7, 8, 9, and 10. fig. 6 shows that with increasing ph value, both the foaming ability and foam stabilization first increase and then decrease. this is mainly because at a low ph value, the foaming solution is acidic, and the h+ in the solution will react with the anionic foaming agent (such as: –so3 –), resulting in a decreased effective content of the foaming agent and weakened foaming ability. fig. 6. influence of ph on the performance of three-phase foam. with increasing ph value, the relative effective content of the foaming agent increases, and foaming capacity is increased. when the ph value exceeds 7, the oh– combines with the hydrophilic group in the foaming agent, which affects the foaming effect. however, changes of the f and t1/2 were not obvious, so they can be used for wider application fields. conclusions this study combines the advantages of foam fire-extinguishing technologies, and a new type of mine fire-extinguishing foam was prepared. firstly, the optimum ratio of foaming agents was obtained by orthogonal experiment and variance analysis for different factors and levels. then, fa particles were used as the solid phase to generate three-phase foams. the effects of solid to liquid ratio, fa size distribution and the system ph on the foaming multiple and stabilization time of the foam were assessed. under the optimized operating conditions of 1:4 10 wang et al. solid–liquid ratio, smaller particle fractions (<75 μm) and from weak acid to weak base, the fly ash-based three-phase foam showed satisfactory stability and foaming ability. these investigations provided a novel route for preparing high -efficiency fire-extinguishing foams applicable in the confined space of underground coal mines. acknowledgment. the study was financially supported by the key research and development projects in anhui province (grant number 202004h07020022). и з в о д експериментално испитивање фактора који утичу на припрему трофазне пене bin wang1,2, sheng zuo1, xixi zuo1 и xiangmei ma1 1school of chemical engineering, anhui university of science and technology, huainan, anhui, 232001, china и 2disaster prevention and control in deep coal mines, anhui university of science and technology, huainan, anhui, 232001, china трофазна пена се сматра једним од перспективних напредних материјала за гашење пожара. међутим, услови припреме, цена и ефекат су кључни фактори за индустријску примену. у овој студији предложен је нови трофазни систем пене са летећим пепелом и комплексним сурфактантом. пет типова сурфактаната: алкохол полиоксиетилен-етар, натријум-сулфат, диетаноламин кокосово уље, натријум-лаурил-сулфат, полиакриламид и емулзија силиконске смоле модификоване полиетаром су одабрани као средства за пењење. кроз лабораторијске експерименте, испитан је утицај на степен експанзије и стабилност пене типа/концентрације сурфактанта, концентрације/величине честица летећег пепела и ph. стање пене је одређено нумеричком оптимизацијом. резултати ове студије могу послужити као референца за разумевање припреме нове трофазне пене. надамо се да овај рад може да пружи корисне смернице за припрему ефикасне трофазне пене за гашење пожара за безбедно чување безбедности процеса у области хемијске производње, транспорта и складиштења. 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(2022) https://doi.org/10.2298/jsc220404052s this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/‌jsc220404052s j. serb. chem. soc. 00(0) 1-13 (2021) original scientific paper jscs–11744 published dd mm, yyy 1 π–π interactions in structural stability: role in superoxide dismutases srđan đ. stojanović1 and mario v. zlatović,2* 1university of belgrade-institute of chemistry, technology and metallurgy, department of chemistry, belgrade, serbia and 2faculty of chemistry, university of belgrade, belgrade, serbia (received 4 april; revised 25 may; accepted 11 june 2022) abstract: in the present work, the influences of π–π interactions in superoxide dismutase (sod) active centers were analyzed. the majority of the aromatic residues are involved in π–π interactions. predominant type of interacting pairs is his–his and his–trp pairs. in addition to π–π interactions, π residues also form πnetworks in sod proteins. the π–π interactions are most favorable at the pair distance range of 5–7 å. we observed that most of the π–π interactions shows stabilization energies in the range −4.2 to −12.6 kj mol-1, while the metal assisted π–π interactions showed an energy in the range −83.7 to −334.7 kj mol-1. most of the π–π interacting residues were evolutionary conserved and thus probably important in maintaining the structural stability of proteins through these interactions. a high percentage of these residues could be considered as stabilization centers, contributing to the net stability of sod proteins. keywords: superoxide dismutase; dispersive forces; catalytic site introduction interaction between the arene systems (π–π) has been recognized as a key stabilizing force in supramolecular chemistry, drug design, biochemistry, crystal engineering and molecular science.1-6 interactions between aromatic amino acid side chains are abundant in proteins, it has been reported and gained widespread acceptance that majority (about 60 %) of all the aromatic residues in proteins are involved in aromatic interactions and among them more than 80 % are involved in imparting stability to proteins.7,8 the nature of π–π interaction was primarily thought to be dispersive with notable electrostatic contribution depending on the system in question.9 at the supramolecular level, the aromatic rings can interact in different ways: stacked arrangement (face-to-face, perfect alignment, offset, slipped, parallel displaced) and edge-to-face, t-shaped conformation.10 although π–π interactions are accepted as a weak, they still play an important role in the *corresponding author: mario@chem.bg.ac.rs https://doi.org/10.2298/jsc220404052s ac ce pt ed m an us cri pt mailto:mario@chem.bg.ac.rs https://doi.org/10.2298/jsc220404052s 2 stojanović and zlatović folding and the thermal stability of proteins.11,12 the calculated π–π interaction energies of the parallel, edge–face (t-shaped) and offset stacked are –6.2, –10.3 and –10.4 kj mol–1, respectively,13 and the major source of attraction is not short range (such as charge-transfer), but long-range interactions (quadrupole–quadrupole electrostatic and dispersion).14 it has been suggested that the perpendicular and the parallel-displaced configurations are more common than the sandwich geometry as these, especially as the former one exposes three aromatic faces to the outside, offering greater possibility for additional interactions with other groups.15 aromatic residues show a high tendency towards forming clusters beyond the dimer, having a significant influence on protein folding, structure, and stability.2,16 the presented study expands on our previous work on the anion–π and cation– π interactions of sod crystal structures by analyzing the same protein group with respect to π–π interactions, in order to better understand their stabilizing role.17,18 we have focused our study at the sod active centers and hence the π–π interactions within a protein are not considered. results from this study might be used for understanding of structure-function relationships and can provide a new dimension of molecular recognition and self-assembly. experimental dataset for this study, we used the protein data bank (pdb), accessed on may 10th, 2021, at that moment listing 183,118 resolved structures.19 the selection criteria for superoxide dismutase to be included in the dataset were as follows: (1) crystal structures of proteins containing e.c. number 1.15.1.1 (superoxide dismutase) with metal were accepted; (2) theoretical model structures and nmr structures were not included (these structures were not accepted as it was difficult to define the accuracy of the ensemble of structures in terms of displacement that was directly comparable to the x-ray diffraction studies); (3) only crystal structures with the resolution of 2.0 å or better and a crystallographic r-factor of 25.0 % or lower were accepted; and (4) we included only representatives having at least 30 % sequence identity. after assembling the dataset, several structures containing ligands and mutant amino acids were rejected, leaving 43 proteins that were actually used as the dataset in our analysis. hydrogen atoms were added and optimized, where needed, using the program reduce,20 with default settings. reduce software adds hydrogen atoms to protein and/or dna structures in standardized geometry, optimizing them to the orientations of oh, sh, nh3 +, met methyls, asn and gln sidechain amides, and his rings. software determines best hydrogen positions by selecting the best overall score from all of the possible combinations, taking into the account single scores assigned for each individual residue and for groups containing movable protons partitioned in closed sets of local interacting networks. the pdb ids of selected protein chain structures were as follows: 1ar5:a, 1cbj:a, 1d5n:a, 1hl5:a, 1ids:a, 1isa:a, 1kkc:a, 1luv:a, 1my6:a, 1qnn:a, 1srd:a, 1to4:a, 1unf:x, 1xre:a, 1xuq:a, 1y67:a, 1yai:a, 1yso:a, 2aqn:a, 2cw2:a, 2goj:a, 2rcv:a, 2w7w:a, 3ak2:a, 3ce1:a, 3dc6:a, 3evk:a, 3f7l:a, 3h1s:a, 3js4:a, 3lio:a, 3lsu:a, 3mds:a, 3pu7:a, 3tqj:a, 4br6:a, 4c7u:a, 4f2n:a, 4ffk:a, 4yet:a, 5a9g:a, 5vf9:a, and 6bej:a. ac ce pt ed m an us cri pt π–π interactions in sod 3 π–π interaction analysis a computer program discovery studio visualizer 202021 was used for the calculation of various types π–π interactions and their geometrical features with default settings (fig. 1). π–π interactions are determined following the methodology of mcgaughey.10 this method finds stacked and staggered π–π interactions by performing the following tests: (1) the distance between the centroid of each pair of π rings is determined to find those which fall within the π– π centroid (rcen) cutoff distance (rcen < 7 å). for these, an atom from each ring should be within the closest atom distance (rclo) cutoff distance (rclo < 7 å). the angle θ between the normal of one or both rings and the centroid–centroid vector must fall between 0° and ± the theta angle cutoff (θ < 90°), and the angle λ between the normal to each ring must fall between 0° and ± the lambda angle cutoff (λ < 90°). the aromatic systems include the aromatic side chains of the residues tryptophan (trp), tyrosine (tyr), phenylalanine (phe), and histidine (his). however, as his can act either as cation or as an aromatic moiety depending on its protonation state, in our study, both the possibilities are considered. fig. 1. parameters for π–π interactions: (rcen) the distance between the centroid of each pair of π rings; (rclo) the distance between the closest atom of each π ring; () the angle between the normal of one or both rings and the centroid–centroid vector; and (λ) the angle between the normal to each ring computation of π–π interaction energy in order to apply ab initio methods in determining the energies of π–π pairs on desired level of theory, with sufficient level of accuracy and still in satisfactory time frame, calculations were performed on structurally reduced model systems: phenylalanine was simplified to toluene (1), histidine to 5-methyl-1h-imidazole (2), tryptophan to 3-methyl-1h-indole (3) and tyrosine was reduced to 4-methylphenol (4) (fig. 2).17 using of reduced model of large systems in calculations of specific intramolecular interaction is well known and already proved methodology,22 producing results accurate enough, and still significantly reducing computation times and strength needed for them. larger models, like whole amino acids, or parts of protein chain, would unnecessary complicate ac ce pt ed m an us cri pt 4 stojanović and zlatović calculations and probably even bring in the errors. numerous interactions mechanisms are possible in a larger protein structure, and a single binding energy computation cannot always correctly determine which of these interactions are present and to what amount they contribute to overall stabilization. as a result, separating the involvement of the π–π interaction and their energy contributions from the interacting pair residues involved in other noncovalent interactions is difficult. fig. 2. structurally reduced structures used for calculations of π–π interaction energy: (1) instead of phe; (2) instead of his; (3) instead of trp; (4) instead of tyr. ab initio calculations were performed using jaguar from schrödinger suite 2018-1,23 using lmp2 method with triple zeta dunning’s correlation consistent basis set24 and ++ diffuse functions.25 all calculations were performed in vacuum. the lmp2 method applied to the study of π–π interactions, showed to be considerably faster than the mp2 method, while the calculated interaction energies and equilibrium distances were almost identical for both methods.26 several authors found that lmp2 represents an excellent method for calculation of interaction energies in proteins.27,28 sometimes, ab initio calculation results can be largely influenced by bsse, and considering it is mandatory, making the calculation times significantly longer. local correlation methods (such as lmp2) not only reduce the cost of the calculations, but the local møller– plesset second-order method lmp2 is well known for reducing intramolecular bsse.29-31 geometries of interacting structures were optimized using lmp2/cc-pvtz(-f)++ level of theory and their single point energies calculated at lmp2/cc-pvtz++ level. for transition metal atoms, we used the lmp2/lacvp** for geometry optimization and lmp2/lacv3p** for energy evaluation with effective core potentials (ecps). the lacv3p basis set is a triplecontraction of the lacvp basis set,32 developed and tested at schrödinger, inc.23 optimized geometries were placed in space to match corresponding complexes by superimposing heavy atoms onto their respective coordinates from crystal structures and then the energies of dimeric structures produced in that way were calculated. the π–π interaction energies in dimers (π–π pairs) were calculated as the difference between the energy of the complex and the sum of the energies of the monomers in their ac ce pt ed m an us cri pt π–π interactions in sod 5 optimized geometries. the π–π interaction energies in the ternary complex with metal were calculated using equation (1): e = em – (em + e) (1) where, emππ, emπ and eπ are the total energies of the ternary (metal–π–π), binary (metal– π) and monomeric systems (π).33 computation of stabilization centres stabilization centres (sc) are defined as the clusters of residues making cooperative, noncovalent long-range interactions.34 measured as individual interactions, stabilisation forces resulting from noncovalent long-range interactions are not very strong, but since they are cooperative by their nature, in regions where they act in a group (sc), they could play an important role in maintaining the overall stability of protein structures. in order to analyse sc of interaction-forming residues, we used the scide program.35 the criteria scide uses for determining sc are as follows: (1) two residues are in contact if there is, at least, one heavy atom–atom distance smaller than the sum of their van der waals radii plus 1 å. (2) a contact is recognized as “long-range” interaction if the interacting residues are, at least, ten amino acids apart. (3) two residues form a stabilization centre if they are in long-range interaction and if it is possible to select one–one residue from both flanking tetrapeptides of these two residues that make, at least, seven contacts between these two triplets.34 computation of conservation of amino acid residues the conservation of amino acid residues in each protein was computed using the consurf server.36 this server computes the conservation based on the comparison of the sequence of given pdb chain with the proteins deposited in swiss–prot database37 and identifies ones that are homologous to the pdb sequence. the number of psi–blast iterations and the e-value cut-off used in all similarity searches were 1 and 0.001, respectively. all the sequences, evolutionary related to each one of the proteins in the dataset, were used in the subsequent multiple alignments. based on these protein sequence alignments, the residues were classified into nine categories, from highly variable to highly conserved. residues with a score of 1 are considered to be highly variable and residues with a score of 9 are considered to be highly conserved. results and discussion in this study, we have investigated the structural stability patterns of π–π interactions in active centres of sod proteins in relation to other environmental preferences like preference of π–π interaction forming residues, interaction geometries and energetic contribution of π–π interactions, stabilization centres and conservation patterns. the analyzed protein set contains 43 protein chain crystal structures and 1116 π–π interactions, there is an average of 26 π–π interactions per active center in sod. preference of aromatic residues for forming π–π interactions we have analyzed the frequency of occurrence of aromatic amino acid residues which are involved in π–π interactions. the results are given in table 1. it can be seen that the contribution of his residue exceeds those of other three aromatic residues. the reason for this could be because, of all the aromatic amino ac ce pt ed m an us cri pt 6 stojanović and zlatović acids, his occurs most frequently in both coordination spheres of sod active centres.17,38 the number of interactions involving other aromatic residues is similar. we compared the occurrence of interacting pairs to find the preference by sod proteins (table 1). the highest percentage of interactions are seen between his–his pairs. among the hetero-pairs, the occurrences of his–trp pair are more frequent than other interacting pairs. hence, these interactions may be quite important in the structural stability of sod proteins. table i. frequency of occurrence of π–π interaction-forming residues in active centers of superoxide dismutase residue number of occurrence a occurrence, %b his 1067 47.80 phe 256 11.47 trp 510 22.85 tyr 399 17.88 total 2232 100 interacting pair his–his 344 30.82 his–phe 64 5.73 his–trp 204 18.28 his–tyr 111 9.95 phe–phe 28 2.51 phe–trp 93 8.33 phe–tyr 43 3.85 trp–trp 69 6.18 trp–tyr 75 6.72 tyr–tyr 85 7.63 total 1116 100 athe number of times a particular amino acid occurs in an appropriate interaction; b percent of amino acid occurs in an appropriate interaction a larger π-network will add more stability and play an important role in understanding the structure of proteins.39 we analyzed the π–π networks in these proteins as well. the analysis showed that about 73 % of the total π–π interactions in the dataset are involved in the formation of multiple π interactions. the connectivity of π-ring is found to increase along the length of a network from 2π to 7π. a large π-network can enhance the stability of a protein conformation and can have a considerable influence on protein–ligand interactions. it has also been shown that addition of an aromatic pair on the protein surface increases its stability.40 an illustrative example of a typical 7π-network of cambialistic sod from propionibacterium shermanii is shown in fig. 3. ac ce pt ed m an us cri pt π–π interactions in sod 7 fig. 3. example of a multiple π interactions (π-network) for the cambialistic sod from propionibacterium shermanii (pdb code 1ar5); the interactions are marked with a dashed lines (color version in supplementary material sm) interaction geometries and energetic contribution of π–π interactions on the basis of orientation of the aromatic rings, the π–π interactions between two aromatic species have been broadly classified into three categories: edge to face (t-shaped), parallel displaced, and parallel stacked.41 for example, mcgaughey et al. analyzed 505 proteins and determined that an offset parallelstacked conformation was on average 4.2 kj mol-1 more stabilizing than a t-shaped geometry.10,42 we have also analyzed the frequency distribution of the distance and angle parameters of π–π interacting pairs. these results are shown in figs. 4 and 5. the distribution of the centroid–centroid distance (rcen) for π–π interactions was found to be most favorable in the distance range of 5–7 å (fig. 4a). this is because of t-shaped orientations having a longer rcen than parallel orientations. at separation distances below 4.5 å, aromatic pairs are rarely observed, a result of obvious physical constraints. the plot of rclo distance distribution derived from π– π interaction pairs (fig. 4b), shows distribution mainly below 5.0 å. an analysis of the plane–plane angles () indicate that coplanarity, capable to maximizing π– π stacking and packing,43 was observed in relatively high number of cases (fig. 4c). an analysis of angle λ showed a preference for t-shaped orientations with angles above 30° (fig. 4d). the native structure is the compromise of a large number of noncovalent interactions that exist in proteins and the geometrical features relating two residue-types are expected to be rather broad. overall, there was no clear overall preference for either “stacked” or “t-shaped” arrangements. for the latter, a clear orientational preference has not been determined experimentally.44,45 ac ce pt ed m an us cri pt 8 stojanović and zlatović fig. 4. interaction geometries of π–π interactions in sod: a) rcen distance distribution, b) rclo distance distribution c)  angle distribution, d) λ angle distribution. to estimate the stabilization energy of the different π–π pairs, energy calculations were carried out. to avoid calculation of more than 1000 interactions, we carefully selected 200 structures representing almost all the interactions which had been found. the results of calculations of the interaction energies for all possible interacting pairs are presented in fig. 5. the calculated energies range between –33.5 and +16.7 kj mol-1, with a most populated bin in the range −4.2 to −12.6 kj mol−1. the energies calculated for many of the π–π interactions are substantially stabilizing, with 16 % of the total showing positive (repulsive) predicted interaction energies. the repulsive nature of those interactions emerges from the unfavourable geometries of π–π interactions in the crystal structures and is usually counterbalanced by other interactions.17 the strongest attractive interaction (–32.7 kj mol−1) arises for the his27–tyr11 pair in mnsod structure from escherichia coli (pdb code 1d5n; fig. 6a). the energies associated with π–π interactions may be important contributors to the overall stability of biomolecular structures and complexes and to their function through substrate binding and protein–protein interactions. ac ce pt ed m an us cri pt π–π interactions in sod 9 fig. 5. interaction energies of π–π interactions in sod fig. 6. details of π–π interactions: a) the strongest attractive π–π interaction of escherichia coli mnsod (pdb code 1d5n). the interaction is marked with a dashed line: a:his27-a:tyr11; rcen = 5.70 å, rclo = 3.61 å,  = 59.77, λ = 51.92, e = –32.7 kj mol -1; b) interaction energy of π–π interaction in the presence of metal cation (zn2+) in cu/zn tomato chloroplast sod (pdb code 3pu7): a:zn2+-a:his27—a:tyr11; rcen = 3.97 å, rclo = 2.98 å,  = 26.01, λ = 41.21, e = –327.7 kj mol -1 (color version in sm) as support for the context of the π–π interactions in the protein structure affecting the energetics of the system, we have analyzed the occurrence of m–π–π (m = zn2+) interactions in the dataset and found 40 cation–π interactions between zn2+ metal cations and the π systems of surrounding amino acids. for these ternary complexes the interaction energies are large (at least ten times larger than those calculated for individually interactions). the π–π interaction energies are large and negative, ranging from –83.7 to –334.7 kj mol−1 due to the strong electrostatic effect caused by the proximity of the metal centre, thus revealing a ac ce pt ed m an us cri pt 10 stojanović and zlatović synergistic effect between the different interactions. quantum chemical calculations indicate that the metal ion assisted π–π interaction strengths may become comparable in magnitude to that of the hydrogen bonding interaction. from our results on the interplay between cation–π and π–π interactions we suggest that these interactions can provide additional stability to the sod proteins. due to the presence of a great number of cation–π and π–π interactions in biological systems, this effect is important and helps to understand some biological processes where the interplay between both interactions exist. it also should be taken into account in supramolecular chemistry and crystal engineering fields.39 in fig. 6b, we showed structural details of the π–π interaction involving transition metal ion of bovine cu/zn tomato chloroplast sod (pdb code 3pu7). the degree of cooperativity of cation–π and π–π interaction may be quantified by comparing interaction energies in the absence of zn2+ (–19.3 kj mol-1) and in the presence of this cation (–327.7 kj mol-1). stabilization centres and conservation of amino acid residues proteins should have well-balanced stability allowing structural fluctuations and concomitantly ensuring the long-lasting equilibrium structure. residues can be considered part of stabilization centers if they are involved in medium or longrange interactions.34 we have computed the stabilization centers for all π–π interaction forming residues in sod active centers. considering the whole data set, 45.2 % of all stabilizing residues are involved in building π–π interactions. it was interesting to note that all residues involved in π–π interactions were included in at least one stabilization center. these observations strongly reveal that these residues may contribute significantly to the structural stability of these proteins in addition to participating in π–π interactions. the level of evolutionary conservation was often used as an indicator for the importance of certain position in maintaining the protein’s structure and/or function.46 among the π–π interacting residues, 74.6 % of them showed a conservation score of higher or equal to 6. from our results we are able to infer that most of the amino acid residues involved in π–π interactions might be evolutionarily conserved and might have a significant contribution to the stability of sod proteins. conclusion in the present study, the analysis of the role of π–π interactions in sod proteins indicate that most of the aromatic residues are involved in π–π interactions and contribute significantly to the structural stability of sod proteins. considering the individual contribution of aromatic residues towards π–π interactions, his residues are found to have exceeded the other three aromatic amino acids. among the interacting pairs, the his–his and his–trp pairs have the highest frequency of occurrence than other pairs. the significant number of π–π interacting residues ac ce pt ed m an us cri pt π–π interactions in sod 11 identified in the dataset is involved in the formation of πnetworks. we also find that all these interacting pairs are favorable in the distance range of 5–7 å. an analysis of the plane–plane angles indicate no clear overall preference for either the “stacked” or “t-shaped” arrangements. the analysis of the energetic contribution of the protein interacting residues has revealed that most of the π–π interactions have an energy in the range −4.2 to −12.6 kj mol-1. the strongest interactions (from –83.7 to –334.7 kj mol-1) arise for the metal assisted π–π interactions. we found that, all the residues found in π– interactions are important in locating one or more stabilization centers, 45.2 % of all stabilizing residues are involved in building π–π interactions, providing an additional stabilization of the sod proteins. moreover, the majority of the residues (74.6 %) involved in π– interactions were evolutionarily conserved. in conclusion, the results obtained from this study will be very helpful in further understanding the structural stability and functions of sod proteins. acknowledgements: the authors would like to thank the ministry of education, science and technological development of republic of serbia (grant no: 451-03-68/2022-14/200026 and 451-03-68/2022-14/200168) for financial support. извод испитивање улоге катјон–π интеракција у активним центрима супероксид-дисмутаза срђан ђ. стојановић1 и марио в. златовић2 1универзитет у београду – институт за хемију, технологију и металургију, београд, србија и 2хемијски факултет, универзитет у београду, београд, србија у овом раду анализирани су утицаји π–π интеракција у активним центрима супероксид дисмутазе (сод). већина ароматичних остатака је укључена у ππ интеракције. парови his–his и his–trp су доминантни тип парова у интеракцији. поред π–π интеракција, π остаци такође формирају π-мреже у сод протеинима. ππ интерагујући парови су најповољнији у опсегу дистанци од 5–7 å. приметили смо да већина π–π интеракција има енергију у опсегу од −4.2 до −12.6 kj mol-1, док су π–π интеракције уз асистенцију метала показале енергију у опсегу −83.7 до −334.7 kj mol1. већина π–π интерагујућих остатака били су еволутивно конзервирани и могли би бити важни у одржавању структурне стабилности кроз ове интеракције. висок проценат ових остатака може се сматрати стабилизационим центрима који доприносе нето стабилности сод протеина. 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https://doi.org/10.1093/nar/gkq399 https://doi.org/10.1093/nar/gkg095 https://doi.org/10.2298/jsc210321042s https://doi.org/10.1021/cr500344e https://doi.org/10.1073/pnas.1030237100 https://doi.org/10.1021/jp809062x https://doi.org/10.1016/j.pbiomolbio.2007.03.016 https://doi.org/10.1039/b718519g https://doi.org/10.1021/jp960267d https://doi.org/10.1021/acs.cgd.8b01857 https://doi.org/10.1093/nar/gki370 ac ce pt ed m an us cri pt j. serb. chem. soc. 00(0) s1-s2 (2021) supplementary material s1 supplementary material to π–π interactions in structural stability: role in superoxide dismutases srđan đ. stojanović1 and mario v. zlatović,2* 1university of belgrade-institute of chemistry, technology and metallurgy, department of chemistry, belgrade, serbia and 2faculty of chemistry, university of belgrade, belgrade, serbia fig. s-1. example of a multiple π interactions (π-network) for the cambialistic sod from propionibacterium shermanii (pdb code 1ar5); the interactions are marked with a pink dashed lines *corresponding author: mario@chem.bg.ac.rs ac ce pt ed m an us cri pt mailto:mario@chem.bg.ac.rs s2 stojanović and zlatović fig. s-2. details of π–π interactions: a) the strongest attractive π–π interaction of escherichia coli mnsod (pdb code 1d5n). the interaction is marked with a pink dashed line: a:his27-a:tyr11; rcen = 5.70 å, rclo = 3.61 å,  = 59.77, λ = 51.92, e = –32.7 kj mol -1; b) interaction energy of π–π interaction in the presence of metal cation (zn2+) in cu/zn tomato chloroplast sod (pdb code 3pu7): a:zn2+-a:his27—a:tyr11; rcen = 3.97 å, rclo = 2.98 å,  = 26.01, λ = 41.21, e = –327.7 kj mol -1 ac ce pt ed m an us cri pt accepted manuscript this is an early electronic version of an as-received manuscript that hasbeen accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as r. v. kapustin, i. i. grinvald, a. v. vorotyntsev, a. n. petukhov, v. m. vorotyntsev, s. s. suvorov, a. v. baryscheva, j. serb. chem. soc. (2022) https://doi.org/10.2298/jsc211003087k this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc211003087k j. serb. chem. soc.00(0)1-13 (2022) original scientific paper jscs–11415 published dd mm, 2022 1 formation of intermediate gas-liquid system in aromatics’ thin layers rostislav v. kapustin1, iosif i. grinvald1*, andrey v. vorotyntsev2, anton n. petukhov2,3, vladimir m. vorotyntsev1, sergey s. suvorov2 and alexandra v. baryscheva2 1alekseev state technical university of nizhny novgorod, nizhny novgorod, russia; 2lobachevsky state university of nizhny novgorod, nizhny novgorod, russia and 3dmitry mendeleev university, moscow, russia (received 3 october; revised 25 november; accepted 9 december 2022) abstract: the present work discusses the ir spectroscopic experiments and quantum-chemical dft study of structure and intermolecular binding in the intermediate gas-liquid systems of aromatics, namely, benzene, furane, pyridine and thiophene. these systems can be generated in thin layers near a solid surface by two different methods, depending on the physical properties of the sample. the first method includes evaporation with a subsequent compression of a sample in a variable thickness optical cell is applied to volatile components: benzene, furane, thiophene. for benzene and pyridine, the second method is used, which involves a heating-initiated evaporation into a closed inter-window space with an aftercooling of a sample. it was shown that the formed layer is not an adsorbate or a condensate. the ir data obtained by these two methods allow to conclude that the revealed systems of the considered aromatics manifest dual gas-liquid spectral properties which can pass into each other in case of varying external conditions. according to the dft calculation results, the spatial arrangement in the aromatic thin layers can be described as a combination of πand σbonded clusters, which simulate the gas and the liquid phase state properties. keywords: fluid-like; intermediate phase; ir spectroscopy; dft calculations introduction the concept of thin layers in the near-surface area was mentioned in the works of ananikov’s group, devoted to the mechanisms of heterogeneous catalysis. 1 this term primarily refers to catalytically active metal particles, and it can mean both a nanoscale layer and molecular clusters. however, later it was shown that the very concept of the thin layer has a much wider application. the thin-layer effect was observed in different phase states of organic liquids: in a solid-film-encapsulated *corresponding author e-mail: grinwald@mts-nn.ru https://doi.org/10.2298/jsc211003087k ac ce pt ed m an us cri pt mailto:grinwald@mts-nn.ru https://doi.org/10.2298/jsc211003087k 2 kapustin et al. form, in a thin-layer liquid on a solid surface, in dense vapours near a solid surface, and also in a low-temperature matrix. 2 various theoretical and experimental methods for studying transient thin-layer systems have been suggested, including ab initio dft calculations in the gaussian software package (b3lyp 6–311++g [2d, 2p] basis set), which make it possible not only to optimize the geometry of molecular clusters, but also to compare theoretical ir vibrational spectra with experimental ones directly. 3 however, the available computing schemes does not allow to calculate the entire supramolecular system in all its complexity and heterogeneity, being limited only to isolated clusters. therefore, it is only experimental in situ ir spectral methods for studying organic-compound thin layers that make it possible to reveal their unique phase properties and make assumptions about their spatial structure. 4 the structure and properties of the thin layers can vary and manifest themselves differently depending on the method of generation, the state of aggregation, and the class of the compound, as well as the types of intermolecular interaction prevailing in it. for example, in liquid chlorocarbons, chlorine acts as the main binding particle for gaseous clusters’ and condensed supramolecular structures. accordingly, tetrachloromethane forms the most stable thin-layer gasphase system among them. the unusual intermolecular properties of tetrachloromethane were predicted in the spectral study. 5 there was shown that gas-phase tetrachloromethane exists not only in the single molecular shape that has td symmetry, but also in the transformed shape having c3v symmetry. this supposition was confirmed by the spectra of gaseous tetrachloromethane at various temperatures. it was resumed that the pyramidal structure relates to the cluster shape, where the chlorine atom provides the binding between molecules. the chlorine atom shift in the cluster can occur owing to the association of the molecules. it leads to the transformation of the molecular geometry to the almost planar d3h symmetry, in which a1 stretching band is forbidden in ir spectra. the above interpretation of the liquid chlorocarbons’ ir spectral data based on the symmetry point groups’ theory showed the appearance of two structural modifications – the pyramidal c3v and the biplanar d2h or d2v symmetry. the dft calculations also predicted the transformation of the initial isomer into biplanar and pyramidal ones. thus, it was concluded that the phase characteristics in chlorocarbons can be combined: in the gas phase, some interactions resembling liquid ones may retain, and vice versa. the most unusual and chemically resistant form of the studied thin layers are solid-phase chlorosilane films with an encapsulated liquid, generated in an argon atmosphere – they are able to selectively protect the internal component, preventing its air hydrolysis (which could be explosive), 6 but allowing slow oxidation over months, which was repeatedly confirmed by ir spectroscopy and electron microscopy data. 7 ac ce pt ed m an us cri pt formation of intermediate aromatic systems 3 systems that combine gaseous and liquid physicochemical properties are known as supercritical fluids (scf). 8 they can dissolve large volumes of gases and mix indefinitely with each other. in addition, a slight adjustment of pressure and temperature within the limits of supercritical values makes it possible to influence the density of the scf and its other parameters to such an extent that, in the phase diagram, the scf is even conditionally divided into gas-like and liquidlike states. although there is actually no clear boundary between these two states, many researchers conditionally draw it along the so-called widom line, the transition through which is described by the term pseudo-boiling, which once again emphasizes the variability degree of the scf physicochemical properties under changing external conditions. one of the most reliable methods for tracking such phase transformations is considered to be ir spectroscopy, since it allows one to unambiguously assign individual characteristic bands to the gas or liquid phase and thus clearly distinguish between the gas and liquid properties of the sample. 9 in the thin layers of chloromethanes and chloroethanes, it was possible to reproduce the main properties of scf. specifically, in the ir spectra, two separate phases – gas-like and liquid-like – were distinguished by their manifestation in the characteristic bands that change a shape depending on the phase state of the substance. similar to the scf, in the thin layers these states smoothly and continuously pass into each other with a slight adjustment of pressure and temperature. depending on the boiling point of the component, under ambient conditions it may appear in the thin layers more as a gas-like or liquid-like one. 10 it is important to note that the terms ‘gas-like’ and ‘liquid-like’ used in the thinlayer system description are not accidental or intended to emphasize the similarity of a given state with a fluid. the states observed by ir spectroscopy are in fact neither a real gas nor a real liquid, but only exhibit some of their spectral properties, which is easy to prove: it is almost impossible to obtain the ir spectrum of a real gas at an equilibrium vapor pressure in the thin spectral cell (1–6 mm). as already mentioned, the properties of thin-layer gas-liquid systems are largely determined by the types of intermolecular bonding. for this reason, the compounds with aromatic properties, both proper hydrocarbons and heterocycles, are of particular interest for study since they are distinguished from the other previously studied organic compounds in terms of intermolecular interactions. back in the 70s shakhparonov, who predicted the existence of the specific interactions in the non-polar hydrogen-bonded liquids, suggested the formation of molecular π-complexes (molecular stacks) between aromatic rings. 11 later, this concept was confirmed and developed by abramovich’s group 12–14 . the single benzene molecule geometry is usually taken as a planar ring. this geometry corresponds to d6h symmetry point group; according to the selection rules, only one stretching c–h band (e-specie) should be active in its ir spectra. nevertheless, in the real liquid benzene spectra there are three bands, and in the ac ce pt ed m an us cri pt 4 kapustin et al. solid benzene there are even four bands in c–h stretching region. three bands that have isotopic h/d shift close to the theoretical prediction also are observed in benzene-d6 spectrum. this spectral picture is assigned to the existence of two molecular forms in the liquid phase: a planar shape, in which one ir band is active (d6h symmetry), and a shape with two ir bands (a1and e-species), corresponding to the c3v symmetry. assumedly, the benzene molecule exists in the liquid state as a cluster system, where c–h bonds deviate from the ring plane to the neighbouring molecule, which leads to a distortion of the c–h stretching vibrations’ symmetry. 15 in solution of dichlorobenzene, there are strong neighbouring-molecule interactions between chlorine atom and carbon in the aromatic ring, and between carbon atoms as well. since the arrangements of molecules in a crystal always correspond to the potential energy minima of the system, it can be used to verify the results of molecular light scattering experiments combined with modelling procedure and consequently reveal the structure and characteristics of the mutual arrangement in molecules as well as the intermolecular binding types in the liquid phase. 16 a structural element that determines the molecular arrangement in the benzene liquid phase, was earlier presented as a set of dimers that have several geometry configurations. later, the trimers were considered as a formed element in the ‘stack model’ of the benzene liquid phase. the conducted dft calculations with different transformations of the initial molecular geometry predict that the optimal configuration is the stack with a ‘chair’ shape of the central and two planar rings; the trimers are bonded in a spatial structure by the hydrogen bridges. 17 this concept explained the ir data outside the scope of traditional assignment. in the trimer spectrum, a c–h stretching band assigning to both planar rings should be observed while the c–h central-ring stretching exhibits two bands: the first one assigns to a pair of equivalent (c1–h1) and (c4–h4) bonds, and the second one – to a quartet: (ci–hi), i = 2.3, 5.6. the stretching bands of the hydrogen bridges shift in the middle ir region due to the mixing of the c–h and c–c stretching. consequently, there is a pair of bands that corresponds to two bridging c–h bonds’ stretching in the stacks bound in two mutually perpendicular plains, as it was shown earlier. 18 based on the data presented above, it can be stated that the formation mechanism of the thin-layer systems with dual gas-liquid phase characteristics is caused and provided by the intermolecular binding. using the example of chlorocarbons and chlorosilanes, one can see how the properties of the thin layers change depending on the supramolecular geometry. the formation of certain stable structures with unusual phase properties indirectly confirms the presence of nonstandard intermolecular binding realized through such structures. thus, the experimental studies of the thin aromatic layers considered in the present work are intended to find a new and more reliable confirmation of the above assumptions about the supramolecular geometry of aromatics. if the intermolecular binding is ac ce pt ed m an us cri pt formation of intermediate aromatic systems 5 indeed the thin-layer systems’ basis, then the properties of such systems for aromatics should differ from chlorides to the same extent as their supramolecular characteristics do. pure benzene, a common organic solvent that can be directly compared with liquid chlorocarbons, is studied as a typical representative of aromatics. moreover, it is very promising to study heterocycles, which are known to have some aromatic properties as well. below we consider the question of whether the heterocyclic intermolecular interactions, and hence the characteristics of their thin-layer systems, differ from those of benzene. in order to clearly distinguish between the proper aromatic properties, which manifest themselves to the same extent in various heterocycles, from the intermolecular interactions of the heteroatom itself, which change depending on the compound, we have selected three aromatic heterocycles with different heteroatoms: thiophene, furane, and pyridine. the main objective of the work is to study the four selected substances under conditions as close as possible to those in which the chlorides were considered, for the most accurate comparison. as a part of the presented work, we have concluded an extensive quantum-chemical study based on the dft calculations is also expected to complement the earlier theoretical studies of chlorocarbons with the data on intermolecular binding in the aromatics and allow the quantum chemical model of the gas-liquid thin-layer system structure to be evaluated for the varied classes of compounds in terms of dft study. experimental in the present work, as mentioned above, four components were studied: benzene, thiophene, furane, and pyridine. the optimal parameters for thin-layer gas-liquid systems’ generation (evaporation time τ, cell type, heating temperature t and boiling point tb) depend on the individual properties of each component, primarily its boiling point, as shown in the table i below. table i. the optimal parameters for the selected compounds’ transient state formation compound τ / min cell type t / k tb / k benzene 60 vtoc/mcoh 295/323 353 thiophene 60 vtoc 295 357 furane 25 vtoc 295 304 pyridine 10 mcoh 363 389 for the selected components, whose intense evaporation do not require additional heating, a perkin-elmer variable-thickness optical cell was applied (here we use the abbreviation vtoc). this method was described in detail in the earlier paper.10 the formation of the dual gas-liquid thin-layer system occurred under the compression-extension procedure of the cell inter-window distance from 6 to 1 mm and in reverse. the optimal evaporation time required for the gas-liquid state formation depends on the boiling point of compound and varies from 25 to 60 minutes. the effect of the thin gas-liquid layers generation for benzene and low-volatile pyridine has been achieved by the method of evaporation of liquid sample placed between optical ac ce pt ed m an us cri pt 6 kapustin et al. window in following way. the sample drops (fig. 1, 1) on the edge of a kbr optical window (fig. 1, 2) so that to prevent its falling into the ir optical beam (fig. 1, 3) under a teflon 1-mmthickness gasket (fig. 1, 4), covering them from above with another optical window (fig. 1, 5). then both windows with a gasket between them are placing and clamping in a manual collapsible optical holder (here we use the abbreviation mcoh) for subsequent heating up to optimal for this experiment’s temperature (table i). figure 1. the scheme of the thin-layer system heating-accelerated generation and detection for the spectral detection and study of the generated state, ir spectra recorded in the vtoc and the mcoh were compared with those obtained in a gas cell (for a gas phase) and for a liquid phase between optical windows at ambient conditions. the effectiveness and accuracy of the two methods used for the study of the thin-layer systems generation was previously demonstrated in our works.7,10 it was proved that the revealed layer is not an adsorbate or a condensate because the ir spectra of substances, evaporated into the vtoc at 6 mm thickness, disappeared after the cell thickness compression to 0.05 mm. besides, the layer origin was tested by the heating method (see above). the layers have been formed by the heating-accelerated evaporation into the cell with 1 mm gasket while at the same procedure conducted with 0.2 mm gasket, the intermediate shape was not detected. ir-spectra were recorded by ir-fourier spectrometers ir affinity1 (shimadzu co. inc.) and fsm 1202 (infraspec co. ltd.) in 500 – 4000 cm–1 range with 2 cm–1 resolution and 60 scans. the spectra fragments are given in the figures with the wavenumber axis expansion according to the original record. the vtoc used was manufactured by perkin-elmer co. ltd. the purity of the components was not less than 99.9 % as it was confirmed by chromatomass spectrometry data obtained by gcms–qp2010 plus spectrometer (shimadzu co. inc.). results and discussion in this section, the ir results for selected aromatics, including benzene and heterocycles, are presented. we highlighted the c–h out-of-plane bending vibration (800 – 600 cm –1 ) and high frequencies’ (3000 – 2000 cm –1 ) regions. the ac ce pt ed m an us cri pt formation of intermediate aromatic systems 7 main manifestations of the thin layer formation could be expected in these ranges, because the first of them is sensitive to the intermolecular “stack” binding and the second one can display the e‧‧‧h (e=c, o, s or n) intermolecular binding in “chains”. 17 the most valuable results for benzene were obtained by heating of the sample between optical windows and subsequent evaporation into vtoc (see section “experimental”). in the experiments with furane and thiophene, the gas-liquid systems were generated in vtoc, while for pyridine, they were obtained by heating between optical windows. gas-liquid system in the thin layers of benzene the band of c–h out-of-plane bending vibration with maximum at 673 cm –1 , resembling the band in a gas phase (fig. 2, spectrum a) with poorly resolved p-, q-, r-branches, was revealed at the sample evaporation procedure in vtoc by thickness compression from 6 to 1 mm (fig. 2, spectrum c). at the minimal cell thickness of 0.05 mm, the c band disappears. at the thin-layer generation by heating of the sample between optical windows without a gasket, the c band of spectrum was not observed as well. therefore, we can conclude that this band is not assigned to the adsorbed molecular layer. as the spectrum of the equilibrium gas phase cannot be observed at 1 mm optical cell thickness, the observed band can be attributed to the transient shape that appears in the thin layers and combines gas and liquid properties. the band of liquid benzene can be considered as the gas-phase band transformation through the transitional phase state band (fig. 2, spectrum a). figure 2. fragment of benzene spectra in c–h out-of-plane bending vibration range. spectra: a – liquid phase; b – gas phase; c – transient phase in the thin layer in the spectra obtained by evaporation of the sample, placed between optical windows, with the heating to 323 k, the new bands at 654, 685 and 730 cm –1 were revealed (fig. 3, spectrum b). the first two of them locate near the pand r-branac ce pt ed m an us cri pt 8 kapustin et al. ches of gaseous benzene, at 659 and 687 cm –1 , respectively (fig. 3, spectrum a), while the third band is located closely to the position of the similar mono-substituted arenes’ band. the spectrum “b” after cooling transforms into the spectrum of the transitional shape, presented in fig. 2 (spectrum c). these data can be interpreted as a manifestation of the benzene thin layer formation with the structure and binding that differ from the ones obtained in the method above by evaporation into vtoc. figure 3. fragment of benzene spectra in c–h out-of-plane bending vibration range. spectra: a – gaseous benzene; b – benzene transient shape in the thin layer. gas-liquid systems in the thin layers of thiophene and furane in the spectra of furane, obtained at the evaporation into vtoc and subsequent lowing of the cell thickness from 6 to 1 mm, the bands at 759, 749 and 727 cm –1 were detected (fig. 4a, spectrum c). the position of the central band is very close to one in the spectrum of liquid (fig. 4a, spectrum a) and gaseous furane (fig. 4a, spectrum b). as the spectrum of a real gas phase cannot be recorded at 1 mm cell thickness, the spectrum c can be assigned to the transient gas-liquid system of furane forming in the thin layer, as well as in the benzene spectrum above. the new bands of the thin layer system are detected at 2598 cm –1 , unlike the benzene gas-liquid system (fig. 4b, spectrum c). in the spectra of liquid and gaseous furane, these bands are absent (fig. 4b, spectra a and b). since this region (3600 – 3000 cm –1 ) is characteristic for the o–h stretching vibrations, the revealed band can be assigned to the intermolecular o‧‧‧h stretching that arises in the thinlayer gas-liquid shape. the similar spectral picture was observed for thiophene in the c–h out-ofplane bending and the high-frequency regions (fig. 5a). the band at 2151 cm –1 can be assigned to the s‧‧‧h intermolecular stretching in the thin-layer gas-liquid system of thiophene (fig. 5b). ac ce pt ed m an us cri pt formation of intermediate aromatic systems 9 a b figure 4. furane spectra in c–h out-of-plane bending vibration (a) and high-frequency (b) ranges. spectra: a – liquid phase; b – gas phase, c – transient shape in the thin layer a b figure 5. thiophene spectra in c–h out-of-plane bending vibration (a) and high-frequency (b) ranges. spectra: a – liquid phase; b – gas phase; c – transient phase in the thin layer gas-liquid system in the thin layers of pyridine for pyridine, the generation of the thin layer system can be realized by heating to 373 k in the inter-window space with a 1 mm cell gasket. the observed ir absorption (fig. 6a, spectrum b) is presented in comparison with the liquid phase (fig. 6a, spectrum a). the spectrum of pyridine equilibrium gas phase spectrum cannot be recorded due to low volatility of the substance. however, in this case, the transition shape with dual gas-liquid spectral properties was also observed. in the high-frequency region, a new band in the ir spectrum of the generated thin layer at 3420 cm –1 was observed (fig. 6b, spectrum b). this band can be assigned to an intermolecular n‧‧‧h hydrogen bond forming within an intermediate shape. ac ce pt ed m an us cri pt 10 kapustin et al. a b figure 6. pyridine spectra in c–h out-of-plane bending vibration (a) and high-frequency (b) ranges. spectra: a – liquid phase; b – transient phase in the thin layer dft modelling of intermolecular binding in the thin layers the description of the intermolecular binding in a condensed state can be considered in terms of dft study only as a quantitative model. it is well-known that ab initio quantum chemical calculations simulate the condensed phase for organic molecules as a set of dimers, trimers and rarely as polymolecular clusters. 3,17,18 therefore, our effort to evaluate the intermolecular binding in the gasliquid system of the thin layers is based on the similar conception, including the calculation of the dimer structure parameters for the different molecular shapes. the aromatics’ system can exist in spatial arrangements of so called “stack” shape – with parallel and perpendicular oriented aromatic rings. 18 however, the ir data indicate the formation of the c‧‧‧h interstack σ-binding as well. 17,18 the results of dft study, in terms of the similar concept, are presented in this section. the calculations were caried out in terms of dft technique with b3lyp and gd3bj functionals 19,20 with 6–311++g (2d, 2p) basis set. for geometry optimization procedure we have used the algorithm 21 . the intermolecular distances calculated by the b3lyp functional are given on figs. 7 to 10 in brackets. the additional criterium of a system geometry correct calculation is the absence of negative vibration frequencies in the final data set. benzene system. two types of clusters, which represent the benzene spatial arrangement, are given in fig. 7. dft calculations of the optimized geometry for structure a (“stack” shape) in the variant with the b3lyp functional gives a geometry with aromatic rings arranged at an angle, so the comparison of intermolecular bond lengths with the gd3bj functional can be taken rather conditionally, hence the bond lengths obtained by the b3lyp functional are not given. the calculated spectral data for these structures generally agree with the experimental ir picture in ch stretching region, in which three bands are observed instead of one ch stretching band prognosticated for the planar isolated ring (this ac ce pt ed m an us cri pt formation of intermediate aromatic systems 11 problem was discussed earlier). 18 the calculations predict two bands assigning to the shape with parallel (fig. 7a) and one band for the “t” shape (fig. 7b). the combination of these arrangement variants leads to the appearance of three bands in the spectra (table ii). figure 7. elements of spatial arrangement with optimized geometry parameters of the benzene clusters(the distances are given in å) the calculated ir frequencies correctly predict the existence of three bands in the ch stretching region arising due to the bands’ combination of two molecular shapes – a and b. 17 table ii. the experimental and calculated frequencies data of ch stretching bands wavenumber, cm-1 experimental 3092, 3071, 3036 calculated 3122, 3053 (shape a) 3179 (shape b) furane and thiophene systems for the furan molecule, the geometry optimization procedure gives two variants of σ-bonded dimers – one with the at-an-angle aromatic rings’ arrangement and the intermolecular (c–c) distance of about 3.1 å (fig. 8a) and with the distance of about 2.7 å (fig. 8b). the thin-layer transient gas-liquid system, in terms of the similar model, can form under the o‧‧‧h intermolecular bonded clusters with two molecular arrangement types. the structure of thiophene gas-liquid system in the thin layer predicted in dft calculation can be presented as a state with two different shapes π-bonded (“stack” shape) and σ-bonded s‧‧‧h shape (fig. 9). in this case, dft calculations with the b3lyp potential do not give an optimized geometry for the “stack” shape, so these values are not shown in figure 9. ac ce pt ed m an us cri pt 12 kapustin et al. figure 8. elements of spatial arrangement with optimized geometry parameters of the furan clusters (the distances are given in å) figure 9. elements of spatial arrangement with optimized geometry parameters of the thiophene clusters (the distances are given in å) the calculated ir frequencies in ch out-of-plane bending (q-branch only) and ch stretching ranges, where the manifestation of intermediate phase shape formation was observed, in table iii are presented (for the gd3bj functional only). the calculated and relative experimental intensities were not listed in table iii, because these values strongly depend on the experimental conditions and do not give any useful information in terms of the considered problem. table iii. the calculated and experimental ir frequencies of heterocyclic aromatics in ranges of ch out of plane bending – ρ(ch) and ch‧‧‧x stretching – q(ch) frequencies wavenumber, cm-1 c5h5o c5h5s c6h6n ρ(ch) q(ch‧‧‧o) ρ(ch) q(ch‧‧‧s) ρ(ch) q(ch‧‧‧n) experimental 749 2598 709 2151 698 3420 calculated shape a 756 3259 723 3253 712 3156 shape b 757 3259 723 3253 716 3174 ac ce pt ed m an us cri pt formation of intermediate aromatic systems 13 significant differences for the calculated and experimental frequencies for the ch‧‧‧x stretching vibrations are probably caused by the hydrogen atom shift to the heteroatoms in the real systems. 22 pyridine system the cluster structure of pyridine system predicted in the dft study with the gd3bj functional can be presented as a state with combination of “stack” shape (fig. 10a) and σone formed under intermolecular hydrogen n‧‧‧h bond (fig. 10b). figure 10. elements of spatial arrangement with optimized geometry parameters of the pyridine clusters (distances are given in å) conclusion the presented experimental and computational data allow to make the following main conclusions. 1. the gas-liquid systems of aromatics including heterocyclic aromatic compounds can be generated in the thin layers by evaporation and subsequent compression of the substances in vtoc or by heating of the sample placed between optical windows and evaporation into the small inter-window space. 2. these systems manifest the dual gas-liquid spectral properties of the considered aromatics. in terms of the presented concept, their structure can be interpreted as a transient phase state arising at the liquid state transformation into the gas. 3. the spatial arrangement of the thin layers can be described as a combination of πand σbonded clusters, which can simulate the gas and the liquid phase state properties. therefore, the thin layers can be considered as an intermediate state of organic liquids. acknowledgements: the study was supported by ministry of science and higher education of the russian federation as part of the scientific project fssm-2021-0013 of the laboratory of smart materials and technology (lsmt). ac ce pt ed m an us cri pt 14 kapustin et al. извод формирање интермедијарног система гас-течно у танким слојевима ароматичних једињења rostislav v. kapustin1, iosif i. grinvald1, andrey v. vorotyntsev2, anton n. petukhov2,3, vladimir m. vorotyntsev1, sergey s. suvorov2 и alexandra v. baryscheva2 1alekseev state technical university of nizhny novgorod, nizhny novgorod, russia; 2lobachevsky state university of nizhny novgorod, nizhny novgorod, russia и 3dmitry mendeleev university, moscow, russia у овом раду разматрани су подаци добијени иц спектроскопским експериментима и кванто-хемијским dft испитивањем структуре и интермолекулских веза у интермедијерним системима гас-течно ароматичних једињења бензена, фурана, пиридина и тиофена. ови системи могу настати у танким слојевима у близини чврсте површине применом две различите методе, у зависности од физичких особина узорка. прва метода која укључује исправање а затим компресију узрока у оптичкој ћелији променљиве дебљине је примењена на испарљива једињења: бензен, фуран и тиофен. за бензен и пиридин, коришћена је друга метода, која подразумева испаравање иницирано загревањем у затвореном простору између прозора са накнадним хлађењем узорка. показано је да настали слој није адсорбат или кондензат. на основу иц података добијених овим двема методама може се закључити да добијени системи кондензованих ароматичних једињења показују дуалне гас-течно спектралне особине које могу прелазити из једних у друге у случају варирања спољних услова. резултати dft прорачуна показаују да се просторно уређење у танким слојевима ароматичних једињења може описати као комбинација πи σповезаних кластера, које симулирају особине гасне и течне фазе. 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pt ed m an us cri pt https://dx.doi.org/10.1070/rcr4932 https://dx.doi.org/10.1063/1.5079232 https://dx.doi.org/10.2298/jsc210426048g https://dx.doi.org/10.1007/s11172-017-1814-8 https://dx.doi.org/10.1134/s003602441709014x https://dx.doi.org/10.1007/s11224-019-01293-1 https://dx.doi.org/10.1007/s11224-019-01293-1 https://dx.doi.org/10.1134/s0036024419130107 https://dx.doi.org/10.1134/s0036024419110025 https://dx.doi.org/10.5772/intechopen.100429 https://dx.doi.org/10.1351/pac-con-12-03-06 https://dx.doi.org/10.1063/1.3382344 http://dx.doi.org/10.1021/acs.jpclett.6b00780 https://doi.org/10.1063/1.5144603 https://dx.doi.org/10.1021/ar100093z ac ce pt ed m an us cri pt {retracting note} j. serb. chem. soc. 86 (9) 899 (2021) note jscs–5470 published 17 august, 2021 899 retracting note paper entitled "bioleaching of copper, zinc and gold from a polymetallic ore flotation concentrate from čoka marin deposite (serbia)", by j. avdalović et al. https://doi.org/10.2298/jsc210127016a, published on august 1, 2021 in the online first sections of the journal of the serbian chemical society has been retracted and withdrawn since the authors (corresponding author) did not approve galley proof prepared by editorial office. retracted manuscript was deposited in the doiserbia quarantine, repository service of the national library of serbia: http://doiserbia.nb.rs/issue.aspx? issueid=1981 https://doi.org/10.2298/jsc210817062r https://doi.org/10.2298/jsc210127016a http://doiserbia.nb.rs/issue.aspx?‌issueid=1981 http://doiserbia.nb.rs/issue.aspx?‌issueid=1981 https://doi.org/10.2298/jsc210817062r effect of an industrial chemical waste on the uptake volume 87 (2022) 1 2022 list of referees editorial board of the journal is grateful to the following referees for reviewing the manuscripts during 2022: biljana abramović, faculty of science, university of novi sad, serbia marija ajduković, institute of chemistry, technology and metallurgy, department of catalysis and chemical engineering, university of belgrade, serbia tatjana anđelković, department of chemistry, faculty of sciences and mathematics, university of niš, serbia maasoud arabueh, nuclear science and technology research institute (nstri), tehran, iran jelena arsеnijević, faculty of pharmacy, department of pharmacognosy university of belgrade, serbia kuzhivelil joseph arun, sree kerala varma college,thrissur, kerala, india jelena bajat, faculty of technology and metallurgy, university of belgrade, serbia sabina begić, faculty of technology, university of tuzla, tuzla, bosnia and herzegovina amina benazzouz-touami, département de chimie, facultédes sciences, université mouloud mammeri, tizi ouzou, algeria dejan bezbradica, faculty of technology and metallurgy, university of belgrade, serbia lucian m. birsa, department of chemistry,“al. i. cuza” university of iasi, romania stevan blagojević, institute of general and physical chemistry, university of belgrade, serbia žanka bojić-trbojević, institute for the application of nuclear energy-inep, university of belgrade, serbia aleksandra bondžić, vinča institute of nuclear sciences, university of belgrade, serbia joaquin maria campos rosa, departamento de química farmacéutica y orgánica, facultad de farmacia, granada, spain vesna cvetković institute of chemistry, technology and metallurgy, university of belgrade, serbia ilija cvijetić, inovation center of faculty of chemistry, university of belgrade, serbia nikola cvjetićanin, faculty of physical chemistry, university of belgrade, serbia željko čupić, institute of chemistry, technology and metallurgy, university of belgrade, serbia aleksandra daković, institute for technology of nuclear and other mineral raw materials, belgrade, serbia ljiljana damjanović vasilić, faculty of physical chemistry, university of belgrade, serbia pablo a. denis. universidad de la república de uruguay, montevideo, uruguay vesna n. despotović, faculty of sciences, university of novi sad, serbia dušan dimić, faculty of physical chemistry, university of belgrade, serbia ana dobrota, faculty of physical chemistry, university of belgrade, serbia vladimir dodevski, vinča institute of nuclear sciences, university og belgrade, serbia 2 journal of the serbian chemical society svitlana dolenko, dumanski institute of colloid and water chemistry, national academy of sciences of ukraine, kiev, ukraine rajendra dongre, department of chemistry, r.t.m. nagpur university, nagpur, india snežana dragović, institute for the application of nuclear energy, university of belgrade, belgrade, serbia yaraslau dzichenka, institute of bioorganic chemistry nasb, minsk, belarus stojan đokić, department of chemical and materials engineering, university of alberta, edmonton, canada veljko r. đokić, ic of faculty of technology and metallurgy, university of belgrade, serbia ivana đorđević, institute of chemistry, technology and metallurgy, university of belgrade, serbia jelena đuriš, faculty of phrmacy, university of belgrade, serbia nataša džunuzović, institute for multidisciplinary research, university of belgrade, serbia elemike elias emeka, federal university of petroleum resources, effurun warri, delta state, nigeria slavica erić, faculty of pharmacy, university of belgrade, serbia mihajlo etinski, faculty of physical chemistry, university of belgrade, serbia gordana đ. gajica, faculty of chemistry, university of belgrade, serbia marija gizdavic nikolaidis, the university of auckland., auckland, new zealand delia gligor, universitatea babeș-bolyai, cluj napoca, romania nikola gligorijević, institute for the application of nuclear energy, university of belgrade, serbia biljana đ. glišić, department of chemistry, faculty of science, university of kragujevac, serbia slobodan glišić, university of niš, faculty of technology, leskovac, serbia gordana gojgić-cvijović, institute of chemistry, technology and metallurgy, university of belgrade, serbia kristina gopčević, school of medicine, university of belgrade, serbia sonja grubišić, faculty of chemistry, university of belgrade. serbia maja gruden, faculty of chemistry, university of belgrade. serbia snežana grujić, faculty of technology and metallurgy, university of belgrade, serbia milica gvozdenović, faculty of technology and metallurgy, university of belgrade, serbia karoly heberger, istitute of excellence, hungarian academy of sciences, budapest, hungary saša horvat, department of chemistry, biochemistry and environmental protection, faculty of science, university of novi sad ljubiša ignjatović, faculty of physical chemistry, university of belgrade, serbia janez ilaš, university of ljubljana, faculty of pharmacy, ljubljana, slovenia nataša ilić, institute for application of nuclear energy-inep, university of belgrade, serbia goran janjić, institute of chemistry, technology and metallurgy, university of belgrade, serbi sanja jevtić, faculty of technology and metallurgy, university of belgrade, serbia kristina joksimović, institute of chemistry, technology and metallurgy, university of belgrade, serbia marija jovanović, faculty of pharmacy, university of belgrade, serbia vesna jovanović, faculty of chemistry, university of belgrade, serbia branko jovčić, faculty of biology, university of belgrade, serbia ivan juranić, faculty of chemistry, university of belgrade, serbia yacine kerchich, national polytechnic school of algiers, algeria vesna kojić, oncology institute of vojvodina, sremska kamenica, serbia anton kokalj, jozef stefan institute, department of physical and organic chemistry, ljubljana, slovenia 2021 list of referees 3 jelena korać jačić, institute for multidisciplinary research, university of belgrade, serbia darko kosanović, institute of technical sciences of the serbian academy of sciences and arts, belgrade, serbia slađana kostić-rajačić, institute of chemistry, technology and metallurgy, university of belgrade, serbia slavica lazarević, faculty of technology and metallurgy, university of belgrade, serbia tamara lazarević pašti, vinča institute of nuclear sciences, university of belgrade, serbia sadullah levent kuzu, department of environmental engineering, faculty of civil engineering, yildiz technical university, istanbul, turkey chen lifang, tianjin university of science and technology, tianjin, china davor lončarević, department of catalysis and chemical engineering, institute of chemistry, technology and metallurgy, university of belgrade, serbia marija lučić škorić, innovation center of the faculty of technology and metallurgy, belgrade, serbia zoran maksimović, faculty of pharmacy, university of belgrade, serbia shweta malode, kle technological university, karnataka, india nikola maraković, institute for medical research and occupational health, zagreb, croatia silvija markic, university og bremen, germany aleksandar marinković, faculty of technology and metallurgy, university of belgrade, serbia bratislav marinković, institute of physics, university of belgrade, zemun, serbia sanja marinović, institute of chemistry, technology and metallurgy, university of belgrade, serbia branislav marković, institute for technology of nuclear and other mineral raw materials, belgrade, serbia satwinder marok, photochemistry department, chemical industries researches institute, national research centre, cairo, egypt manuel martínez, departament de quımica inorganica, universitat de barcelona, spain gordana medunić, faculty of science, university of zagreb, croatia ivana mihajlović, department of environmental engineering and occupational safety and health, faculty of technical sciences, university of novi sad, serbia daniel mijailović, inovation center of faculty of technology and metallurgy, university of belgrade, sebia vesna milanović, faculty of chemistry, university of belgrade, serbia nikola milašinović, department of forensics, academy of criminalistic and police studies, belgrade, serbia miloš milčić, faculty of chemistry, university of belgrade, serbia milica milenković, faculty of chemistry, university of belgrade, serbia marija milovanović, center for molecular medicine and stem cell research, faculty of medicine university of kragujevac, serbia ana miltojević, department of chemistry, faculty of sciences and mathematics, university of niš, serbia djenana miodragovic, chemistry of life processes institute, northwestern university, evanston, il, usa aleksandra mitrović, faculty of chemistry, university of belgrade, serbia abdelmalek khorief nacereddine, higher normal school of technological education-skikda, algeria takeshi nakano, research center for environmental preservation, osaka university, japan 4 journal of the serbian chemical society jovan nedeljković, institute of nuclear sciences vinča, belgrade, serbia jasmina nikolić, faculty of technology and metallurgy, university of belgrade, serbia katarina nikolić, faculty of chemistry, university of belgrade, serbia marija nikolić, faculty of technology and metallurgy, university of belgrade, serbia nebojša nikolić, institute of chemistry, technology and metallurgy, department of electrochemistry, university of belgrade, serbia branislava nikolovski, faculty of technology, university of novi sad, serbia hany nour, photochemistry department, chemical industries research institute, national research centre, cairo, egypt antonije onjia, vinča institute of nuclear sciences, university of belgrade, serbia dejan opsenica, faculty of chemistry, university of belgrade, serbia metehan erdoğan, ankara yıldırım beyazıt university, turkey biljana otašević, faculty of pharmacy, university of belgrade, serbia aleksandar pavić, institute of molecular genetics and genetic engineering, university of belgrade, serbia miroslav pavlović, institute of chemistry, tehnology and metallurgy, university of belgrade, serbia mirjana perišić, institute of physics belgrade, university of belgrade, zemun, serbia milena petković, faculty of physical chemistry university of belgrade, serbia biljana petrović, department of chemistry, faculty of science, university of kragujevac, serbia rada petrović, faculty of technology and metallurgy, university of belgrade, serbia branka pilić, university of novi sad, faculty of technology, novi sad, serbia marijana ponjavić, faculty of technology and metallurgy, university of belgrade, serbia dragan m. popović, department of chemistry, institute of chemistry, technology and metallurgy, university of belgrade, serbia marko popović, school of life sciences weihenstephan, technische universität münchen, germany velimir popsavin, faculty of science, university of novi sad, serbia umesh prasad, arizona state university, usa ralph puchta, department of chemistry and pharmacy, friedrich alexander university erlangennürnberg, erlangen, germany katarina putica, innovation center of faculty of chemistry, serbia vladislav rac, faculty of agriculture, university of belgrade, zemun, serbia dušanka radanović, institute of chemistry, technology and metallurgy, university of belgrade, serbia mirjana radanović, faculty of sciences, university of novi sad, serbia jelena radić-perić, faculty of physical chemistry, university of belgrade, serbia vuk radmilović, faculty of technology and metallurgy, university of belgrade, serbia bojana radojković, institute of chemistry, technology and metallurgy, university of belgrade, serbia jelena radosavljević, faculty of chemistry, university of belgrade, serbia ksenija radotić, institute for multidisciplinary research, university of belgrade, serbia milan radovanović, tehnical faculty in bor, university of belgrade, bor, serbia ivona radović, faculty of technology and metallurgy, university of belgrade, serbia muhammad khalid, department of chemistry, khwaja fareed university of engineering and information technology, pakistan 2021 list of referees 5 danijela ranđelović, institute of chemistry, technology and metallurgy, university of belgrade, serbia slavica ražić, faculty of pharmacy, university of belgrade, serbia dubravka relić, faculty of chemistry, university of belgrade, serbia ivan ristić, faculty of technology, university of novi sad, serbia miroslav ristić, faculty of physical chemistry, university of belgrade, serbia ritu raval, manipal academy of higher education, manipal, india augusto rocha, federal institute of education, science and technology of pará, brazil tamara rončević, faculty of sciences, university of novi sad, serbia vladislav sadykov, boreskov institute of catalysis, novosibirsk, russian federation abubakar siddiq salihu, umaru musa yar'adua university, katsina, nigeria victoria samanidou, aristotle university of thessaloniki, greece muniyandi sankaralingam, department of chemistry, national institute of technology calicut, kerala, india jasmina savić, vinča institute of nuclear sciences, university of belgrade, serbia sanja savić, institute of chemistry, technology and metallurgy, university of belgrade, serbia milica selaković, innovative centre, faculty of chemistry, belgrade, serbia milan senćanski, vinča institute of nuclear sciences, university of belgrade, serbia fahimeh shojaie, university of advanced technology, kerman, iran sonja smiljanić, faculty of technology and metallurgy, university of belgrade, serbia pavle spasojević, innovation centre of faculty of technology and metallurgy, university of belgrade, serbia milica sredojević, innovative centre of faculty of chemistry, university of belgrade, serbia vojislav stanić, vinča institute of nuclear sciences, university of belgrade, serbia zoran stević, technical faculty bor, school of electrical engineering belgrade, university of belgrade, serbia sanja stojadinović, institute of chemistry, technology and metallurgy, university of belgrade, serbia zorica stojanović, faculty of technology, university of novi sad, serbia gordana stojanović, department of chemistry, faculty of science and mathematics, university of niš, serbia zoran šaponjić, vinča institute of nuclear sciences, belgrade, serbia sanja šešlija, institute of chemistry, technology and metallurgy -department of electrochemistry, university of belgrade, serbia irena škorić, faculty of chemical engineering and technology, university of zagreb, zagreb, croatia biljana šmit, faculty of science, university of kragujevac, serbia bo tang, school of pharmacy, nantong university, jiangsu province, china nikola tasić, national institute of chemistry, ljubljana, slovenia vele tešević, faculty of chemistry, university of belgrade, serbia tamara todorović, faculty of chemistry, university of belgrade, serbia biljana tomasević, faculty of chemistry, university of belgrade, serbia miloš tomić, faculty of technology and metallurgy, university of belgrade, serbia vesna vasić, vinča institute of nuclear sciences, belgrade, serbia đenđi vaštag, faculty of sciences, university of novi sad, serbia tatjana ž. verbić, faculty of chemistry, university of belgrade, serbia 6 journal of the serbian chemical society gorica veselinović, institute of chemistry, technology and metallurgy, department of chemistry, university of belgrade, serbia željko vitnik, department of chemistry, institute of chemistry, technology and metallurgy, university of belgrade, serbia ladislav vrsalović, faculty of chemistry and technology, university of split, croatia jelena vujančević, institute of technical sciences of sasa, belgrade, serbia marija vlkčević, faculty of technology and metallurgy, university of belgrade, serbia pamela j. weathers, department of biology and biotechnology, worcester polytechnic institute, worcester, ma, usa erdal yabalak, faculty of arts and science, mersin university, mersin, turkey hyusein yemendzhiev, burgas university, department of chemical technology, burgas, bulgaria constatinos zacharis, department of chemistry, aristotle university of thessaloniki, greece george a. zachariadis, aristotle university of thessaloniki, greece snežana zarić, faculty of chemistry, university of belgrade, serbia marija živković, faculty of science, university of kragujevac, serbia nikola živković, vinča institute of nuclear sciences, belgrade, serbia suzana žunec, institute for medical research and occupational health, zagreb, croatia accepted manuscript this is an early electronic version of an as-received manuscript that has been accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as i. a. kirigiti, n. s. aminah and s. thomas, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc230201049k this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230201049k j. serb. chem. soc.00(0)1-12 (2023) original scientific paper jscs–12261 published dd mm, 2023 1 identification of organic compounds using artificial neural networks and refractive index innocent abel kirigiti1, nanik siti aminah1*, samson thomas2 1department of chemistry, faculty of science and technology, universitas airlangga, surabaya 60115, indonesia and 2department of chemistry, faculty of mathematics and natural sciences, universitas indonesia, depok 16424, indonesia (received 1 february; revised 15 february; accepted 4 august 2023) abstract: identification of chemical compounds has many applications in science and technology. however, this process still relies heavily on the knowledge and experience of chemists. thus, the development of techniques for faster and more accurate chemical compound identification is essential. in this work, we demonstrate the feasibility of using artificial neural networks to accurately identify organic compounds through the measurement of refractive index. the models were developed based on refractive index measurements in different wavelengths of light, from uv to the far-infrared region. the models were trained with about 250,000 records of experimental optical constants for 60 organic compounds and polymers from published literature. the models performed with accuracies of up to 98%, with better performance observed for refractive index measurements across the visible and ir regions. the proposed models could be coupled with other devices for autonomous identification of chemical compounds using a single-wavelength dispersive measurement. keywords: machine learning; anns; classification, deep learning, materials identification. introduction organic material identification is a vital process across various industries such as pharmaceuticals, food, agriculture, and environmental science. the ability to identify organic compounds quickly and accurately is imperative for the development of new products, monitoring environmental pollutants, and detecting contaminants in food and drugs.1–3 however, traditional methods of material identification, such as gas chromatography and mass spectrometry, can be tedious and costly.4,5 therefore, it is crucial to explore new ways to facilitate organic * corresponding author e-mail: nanik-s-a@fst.unair.ac.id https://doi.org/10.2298/ jsc230201049k a cc ep te d m an us cr ip t mailto:nanik-s-a@fst.unair.ac.id 2 kirigiti et al. materials identification. one such approach that has gained attention in recent times is the use of machine learning techniques. machine learning (ml) is a branch of computer science that focuses on developing algorithms that can learn from and make decisions based on complex data.6 one recent development in ml is deep learning, a cutting-edge field that uses artificial neural networks (anns) to improve the performance of traditional ml models.7 anns are artificial systems that are modelled after biological neural networks and are able to learn and perform tasks without pre-programmed rules by being exposed to various datasets and examples.8 deep learning is among the most effective, efficient, and cost-effective approaches to ml.9 additionally, anns have the advantage of being able to increase their accuracy in production. unlike traditional ml models like random forests, anns don’t need to be fully re-trained as more data becomes available; this can lead to significant cost savings in terms of computational resources. therefore, anns are a suitable approach to ml. anns have found applications in various fields such as environmental science, where they are used to predict the percentage of water pollutant removal based on experimental variables such as temperature and treatment time.10–12 moreover, raman spectroscopy imaging has been widely used in combination with machine learning (ml) techniques to identify the properties and structures of organic compounds. raman spectroscopy is a non-destructive imaging method that provides information about the vibrational modes of a compound, which can be used to determine its chemical structure and composition. one of the key benefits of using raman spectroscopy imaging in conjunction with ml algorithms is its ability to accurately identify the chemical structure and composition of various organic compounds.13,14 studies have reported ml models based on raman spectra that were able to classify materials like biomolecules, organics, and inorganics.15–18 despite the higher performance of ml models using spectra images as input, the setup and equipment for obtaining associated spectra data is more complex and expensive. therefore, a simple measurement like the refractive index (n) can offer alternatives. refractive index of a sample is defined as the ratio of the speed of light in a vacuum to its speed in the sample medium. the chemical composition of a sample can also affect its refractive index through the presence of certain functional groups or atoms that can interact with light in specific ways.19 for example, refractive index has been used for detection of components with low chromophoric activities such as sugars, triglycerides, organic acids, pharmaceutical excipients, and polymers.20 so, the refractive index is an optical property that carries enough information related to chemical composition. a cc ep te d m an us cr ip t identififying organic compounds by machine learning 3 machine learning models for predicting refractive indices of polymers have been previously reported.21,22 in another work, refractive index was used as input to ml models to differentiate normal and malignant tissues in biomedical.23 this work was inspired by a study attempting to apply random forests (rf), a traditional ml algorithm and refractive index to identify organic compounds.24 in order to train the machine learning models, we use data from a public database of refractive indices for organic compounds and polymers. the database contains data from literature gathered over a long period of time.25 to the best of our knowledge, this is the first work to report the use of refractive index for classification of organic compounds with artificial neural networks (anns). experimental database version 1.0.0 of a web scrapper built using python was run on the refractive index website, which is a database for experimental optical constants from published literature since 1940.25 the scrapper targeted 60 organic compounds and polymers. the scrapped data was stored as comma-separated values (csv). the file contains four columns: organic compound (book), wavelength (λ), refractive index (n), and extinction coefficient (k). the scrapped database has a total of 248,756 entries and 9645 missing values of k. the database was split into five categories as follows (fig 1); ultraviolet (0-0.4 µm), visible (0.4 – 0.75µm), near ir (0.75 – 1.5 µm), ir (1.5 4µm), and far ir (>4µm). fig. 1. raw scrapped data from the refractive index website a cc ep te d m an us cr ip t 4 kirigiti et al. data pre-processing all the missing values for extinction coefficients in the raw database were replaced by zero. since the uv and visible data was too small for training the models, data augmentation was performed on these regions using the sellmeier equation.26 this is a mathematical formula that can be used to predict the refractive index of a material as a function of wavelength. data augmentation was required to artificially synthesize more data using domain knowledge;27 this technique has been previously used with raman spectra-based organic classifiers.16–18 the sellmeier equation is given by the following equation: ‚‚ 𝑛2(λ) = 1 + 𝐵1λ2 λ2−𝐶1 + 𝐵2λ2 λ2−𝐶2 +. .. (1) where n(λ) is the refractive index at wavelength λ. b₁, b₂, ... and c₁, c₂, ... are sellmeier coefficients that are specific to the material. a custom python script was used to estimate the missing sellmeier coefficients by curve fitting (see the supporting information). artificial neural network classifiers (anns) for organic compounds scikit-learn, tensorflow, and keras python libraries were used for training and evaluating the accuracy of the ann classifiers. this was done in a google collaboratory environment.28,29 seven different models were developed according to available categories (fig. 4). each model contains three main layers: an input layer, hidden layers, and an output layer. the input layer takes in three independent variables: the wavelength of light (λ), refractive index (n), and the extinction coefficient (k). hidden layers contain neurons; they extract and represent features from the input data, allowing the network to learn. the output layer performs the final compound classification, which is based on voting among 60 possible compounds. the compound with a high probability is considered the output of the model.7,28 an overview of the model design is shown in fig. 2 below: fig. 2. ann organic classifier model architecture to evaluate the model’s performance, monitoring was performed during the training and testing stages. in the training stage, the loss and accuracy of all models were monitored by a validation data set. if the model's prediction is perfect, the loss is zero. this tells how poorly or well a model behaves after each iteration of the optimization.29,30 on the other hand, the testing stage was performed using a test data set; the test data serves as an estimate of its performance on new, unseen data. accuracy, precision, recall, and the f1 score were used as evaluation metrics for the classifier. precision tells us how many of the positive predictions were correct; recall tells us how many of the actual positives were identified while the f1 score gives a single metric to a cc ep te d m an us cr ip t identififying organic compounds by machine learning 5 evaluate the overall performance of the model by balancing precision and recall.28,31 these evaluation metrics are mathematically defined as follows: 𝑝𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛 = 𝑇𝑃 𝑇𝑃+𝐹𝑃 (2) 𝑟𝑒𝑐𝑎𝑙𝑙 = 𝑇𝑃 𝑇𝑃+𝐹𝑁 (3) 𝐹1 𝑠𝑐𝑜𝑟𝑒 = 2×𝑝𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛×𝑟𝑒𝑐𝑎𝑙𝑙 𝑝𝑟𝑒𝑐𝑖𝑠𝑖𝑜𝑛+𝑟𝑒𝑐𝑎𝑙𝑙 (4) where the term true positives (tp) refer to the number of samples correctly predicted as positive, while false positives (fp) indicate the number of samples incorrectly predicted as positive. similarly, true negatives (tn) refer the number of samples correctly predicted as negative, and false negatives (fn) represents the number of samples incorrectly predicted as negative. results and discussion data pre-processing fig. 3. missing extinction coefficient values in raw data analysis of the raw data revealed the percent of missing extinction coefficient values in each region as follows: uv (58.99%), visible (82.67%), near ir (7.28%), ir (0.32%), and far ir (0.25%). the final database with data augmentation contains seven categories (fig 4). uv region data was increased from 1807 to 132314, and visible region data was increased from 6268 to 120455. the amount of data in other regions was left unaltered. a cc ep te d m an us cr ip t 6 kirigiti et al. fig. 4. augmented refractive index data performance of artificial neural network classifiers table 1. performance evaluation for ann classifiers ann model precision (%) recall (%) f1 score (%) accuracy (%) uv 79.00 81.00 79.00 59.00 uv augmented data 82.00 82.00 81.00 81.49 visible 73.00 69.00 67.00 69.22 visible – augmented data 86.00 87.00 85.00 86.60 near ir 99.00 98.00 98.00 98.44 ir 98.00 98.00 98.00 97.72 far ir 85.00 84.00 84.00 84.09 a cc ep te d m an us cr ip t identififying organic compounds by machine learning 7 fig. 5. testing accuracies for the ann classifiers the accuracies of the ann models are listed in the following order: near ir (98.44%) > ir (97.72%) > visible-augmented (86.60%) > far ir (84.09%) > uvaugmented (81.49) > visible (69.22%) > uv (59.00%). it is observed that models in the near ir and ir regions outperform other regions (fig. 5). moreover, from figs. 6 and 7, the losses of the near ir and ir regions converge to low and stable values, while the accuracy reaches high and stable values. this indicates that the models in the near ir and ir regions are generalizing well and not overfitting.28,32 the precision, recall, and f1 scores of the near ir and ir models shows high performance of above 98% (table 1). meanwhile, the performance of models in the far ir, uv, and visible regions is unsatisfactory. the recall, precision, and f1 scores in these regions are low (table 1). their loss–accuracy plots show unstable values, and bumpy nonconverging lines, indicating their unreliability (figs. s1–s5). for uv and visible regions, more than 58% of extinction coefficient values (k) were missing in their datasets (fig 3), which may provide the model with less information to accurately learn the relationships between input and output variables. as a result, overfitting or poor generalization performance may be observed.33 nevertheless, the accuracies for uv and visible models were observed to increase by more than 17% with data augmentation (fig 5); this suggests the possibility of improving the performance by increasing the amount of training data in these regions. a cc ep te d m an us cr ip t 8 kirigiti et al. fig. 6. training and validation for ann model in near ir region fig. 7. training and validation for ann model in ir region the accuracy and loss plots obtained from the near ir (fig. 6) and ir (fig. 7) models are smoother, and the lines converge well, while plots for models containing augmented data show convergence but with bumpy lines (fig. s1s5). this implies that the validation dataset is not a good representation of the training data set; this may be due to artificially synthesized data from the data augmentation process, which introduced noise.28,32 a cc ep te d m an us cr ip t identififying organic compounds by machine learning 9 comparison with other machine learning organic classifiers the developed models are comparable with models from previous studies (table 2), indicating the potential of using refractive index measurement to facilitate the identification of organic compounds using machine learning. table 2. comparison of this work with some previous studies using raman spectra data with machine learning. resnet = residual neural network, drcnn = deeply-recursive convolutional neural network, ann= artificial neural network, cnn = convolutional neural network, knn= k-nearest neighbor, ml = machine learning method dataset accuracy (%) reference resnet organic biomolecules 100.00 15 cnn organic and inorganic compounds 100.00 16 drcnn organic compounds and minerals 98.10 17 knn organic biomolecules 93.90 15 knn edible oils (fatty acids) 88.90 18 ann classifier organic compounds and polymers 81.49 (uv) 86.60 (vis) 98.44 (near ir) 97.72 (ir) 84.09 (far ir) this work conclusion in this study, artificial neural network classifiers (anns) for identifying organic compounds were developed and tested successfully. the models rely on refractive index measurements across the uv and far ir spectral regions. information related to the refractive index of an organic compound and the wavelength of light used facilitates its accurate identification by artificial neural networks. anns in the near ir and ir regions showed better performance, with accuracy levels above 97%, suggesting the potential of refractive index measurements in these regions. the observed performance is comparable to models using raman spectra as inputs. although the accuracies for the uv, visible, and far ir regions are slightly lower, ranging from 81% to 86%, additional data and hyperparameter optimizations showed the possibility of improving performance in the future. this study demonstrates the feasibility of using artificial neural networks to identify organic compounds using a single wavelength dispersive measurement. a cc ep te d m an us cr ip t 10 kirigiti et al. supplementary material supplementary materials are available electronically from https://www.shdpub.org.rs/index.php/jscs/article/view/12261, or from the corresponding authors on request. и з в о д идентификација органских једињења коришћењем вештачких неуронских мрежа и индекса преламања innocent abel kirigiti1, nanik siti aminah1 и samson thomas2 1department of chemistry, faculty of science and technology, universitas airlangga, surabaya 60115, indonesia and 2department of chemistry, faculty of mathematics and natural sciences, universitas indonesia, depok 16424, indonesia идентификација хемијских једињења има много примена у науци и технологији. међутим, овај се процес још увек много ослања на знање и искуство хемичара. тако је од суштинске важности развој техника за брже и поузданије идентификовање хемијских једињења. у овом раду, представићемо изводљивост коришћења неуронских мрежа за поуздано идентификовање органских једињења мерењем индекса преламања. развијени су модели засновани на мерењима индекса преламања на различитим таласним дужинама светлости, од uv до далеке инфрацрвене области. модели су тренирани са око 250,000 записа експерименталних оптичких константи за 60 органских једињења и полимера из публиковане литературе. модели су извођени са поузданошћу до 98%, са бољим резултатом опаженим за мерења индекса преламања у видљивој и иц области. предложени модели се могу спрегнути са другим уређајима за аутономну идентификацију хемијских једињења дисперзивним мерењем на једној таласној дужини. 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m. s., 1023 bojilov, d. g., 139 bojović, s., 1177 bokhari, t. h., 591 borić, i., 639 bouakkadia, a., 673 bouarra, n., 63, 725 boudjemaa, a., 63 bui, v. b., 615 bukvički, d. r., 1139, 1249 butt, s. a., 195 calhelha, r. c., 1291 cao, g., 247 cao, j., 917 chaurasia, b., 269 chayrov, r. l., 625 chehrazi, m., 313 choudhary, a., 165 cvetković, d. d., 367, 533 cvetković, m., 355, 1177 cvetković, n. m., 819 cvijetić, i., 561 čabarkapa, i., 355 čobeljić, b. r., 459 čomor, m. i., 495 ćirić, a., 1291, 1205 dabai, f. n., 283 dabić, p. z., 663 danac, r., 901 danalev, d. l., 625 das, b. k., 327 destanoğlu, o., 77 dilmaghani, k. a., 1013 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 1326 journal of the serbian chemical society dimitrijević, m., 927 dimitrijević-branković, s. i., 651 djurković djaković, o., 115 dojčinović, b., 885 driouche, y., 673 đajić, n., 125 đorđević, a. s., 257 đorđević, d., 927 đorđević, i., 1177 đorđević, m. m., 1259 đorđević, n., 1113 đukić, m. g., 1271 đurišić-mladenović, n., 739 elhleli, h., 25 el-yakubu jibril, b., 283 eren, b., 955 erić, s., 561, 639 esmaeilzadeh, s., 1039 espinoza, e. m., 983 faramarzi, z., 685 feng, y., 469 fernandes, â., 1291 ferreira, i. c.f.r., 1291 filipović, n. r., 393 frolova, i., 299 gagić, t., 603 gaur, p., 269, 941 gavrilović, m., 1281 ghomsheh, s. m. t., 871 ghorbannejad, s., 1013 glamočlija, j., 1291 gligorijević, n., 795 gođevac, d., 1195 grinvald, i., 1067 grishenkova, o. v., 1075 gros, n., 1089 grushko, i., 103 guo, h., 917 gurkan, y. y., 955 halilović, dž., 1023 hamidi, n., 725 hatipoglu, a., 955 hidayat, a. t., 971 hidayat, i. w., 971 honarasa, f., 381 huang, g., 917 hussein, h., 299 ickovski, j. d., 1259 ignjatović, j., 125 ignjatović, lj. m., 753 ignjatović, n. l., 555 imran, m., 195 isakov, a. v., 1075 isernia, l. f., 983 ivanov, i. i., 139 ivanova, v., 1229 ivanović, s. g., 1205, 1249, 1195 ivanovska, a., 885 ivković, i. m., 1249 jabua, z., 181 jadranin, m. b., 1177, 1219,1241 jahromi, h. j., 685 janaćković, đ., 1103 janaćković, p., 1281 jegdić, b. v., 407 jelić, m., 699 jiang, f., 859 jiang, g.-y., 469 jin, x., 917 joksović, lj. g., 585 jovanović, a. z., 699 jovanović, d., 1023 jovanović, j. d., 819 jovanović, o. p., 1259 jovanović, s. č., 1271 jović, b. d., 367 ju, x.-h., 171 juliandri, j., 971 kamyab moghadas., b., 313 kapustin, r., 1067 kekutia, s., 181 kertiou, n., 673 keskin, e., 711 khalil, s. m., 165 khan, a., 195 khiari, r., 25 khosa, m. k., 591 khudorozhkova, a. o., 1075 kiec-kononowicz, k., 639 knez, ž., 603 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ vol. 86: author index 1327 knežević, a. z., 809 kolašinac, r., 1023 kop, t. j., 1023 kostić, a., 1219 kostić, m., 885 kovač, s. z., 663 kovačina, j. n., 407, 555 kremenović, a. s., 663 kriechbaum, m., 181 krishnankutty, k., 39 krmar, j., 125 krstić, g. b., 1219 krstić, n. m., 459 krstić, n., 927 kuljanin, a., 1089 kumar shah, s., 483 kumar, i., 507 kumar, r., 327 kuz'min, v. e., 625 lan, x.-b., 247 laptev, m. v., 1075 latacz, g., 639 lazarević pašti, t. d., 699 liang, m., 171 lopukhova, m., 299 lubelska, a., 639 ludwiczuk, a., 1139 ljujić, j., 1205 ljupković, r. b., 1259 ma, j., 859 ma, x., 859 mahmoud, y., 521 mallick, s., 51, 571 mandić, b., 1195 mandrone, m., 1195 mannai, f., 25 manolov, s. p., 139 maqsood, h. r., 195 marin, p. d., 1281 markhulia, j., 181 markov, s. l., 533 marković, b., 1113 marković, d., 1291 marković, t., 1291 marunkić, d. d., 407 matić, i. z., 459 matloob, a., 195 mayanti, t., 971 mehmood, a., 195 mehrotra, r., 269 mentus, s., 209 messadi, d., 63, 673 mićić, r., 739 mihajlovski, k. r., 651, 1103 mikelashvili, v., 181 milanovic, v. d., 997 milić, d. r., 1023 milić, m., 651 milosavljević, s. m., 809 miljuš, g., 795 minić, s., 795 mirza, b., 547 mitić, d. m., 393 mitić, v. d., 257, 927 mojović, z. d., 495 moussaoui, y., 25 mrmošanin, j. m., 1271 nadji, n., 63 naik, r. m., 507 nedić, o., 795 nedjhioui, m., 725 nikodinović-runić, j., 639 nikolić, i. c., 1249 nikolić, j., 927 nikolić, m., 1291 nikoo, a., 1013 noei, m., 685, 871 nouri, l., 63 novaković, i. t., 459 novaković, j., 1281 novaković, m. m., 809, 1139, 1219, 1249 novaković, t. b., 495 ognichenko, l. n., 625 omari, s., 725 omel’chuk, a., 845 onjia, a. e., 753 opsenica, i., 115 otašević, b., 125 oyegoke, t., 283 özok, h. i̇., 711 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 1328 journal of the serbian chemical society pallikkavil, r., 39 panić, v. v., 555 pantović pavlović, m. r., 555 pantović spajić, k., 1113 pašti, i. a., 699 patel, a., 51 patidar, k., 429 patil, p., 153 pavlenko, t., 845 pavlović, a. n., 1271 pavlović, m. m, 555, 1113 pavlović, v. b., 367 pavun, l., 885 pecarski, d., 651 penezić, a., 795 pešić, m., 1219 petrović, g. m., 257 petrović, r., 1103 petrović, z. z., 781 pezo, l. l., 355, 533 pham, t. h., 615 pohorenko, y., 845 popova, s., 299 popović, g., 927 potkonjak, n., 699 protić, a., 125 pshenychnyi, r., 845 radojković, b. m., 407 radovanović, l., 1103 radovanović, ž., 1103 radulović, j. m., 753 radulović, m., 1281 rahim, a., 971 rahmatulloh, a., 831 raidas, m. l., 941 rajčević, n., 1281 ralevic, l. r., 997 ranitović, a. s., 367, 533 rankov šicar, m., 739 ražić, s., 1127 redžepović, i., 445 rehan ullah, s. m., 767 ristić, m., 1195 ristić, p. g., 393 rodić, m. v., 393 rosca, i., 901 rukh, s., 195 saberi, m., 341, 871 safidine, z., 521 sahoo, r. n., 51, 571 saičić, r. n, 1305 saif, m. j., 591 samadizadeh, m., 1053 saneblidze, l., 181 satapathy, b. s., 51, 571 savic, v. m., 639 savić, t. d., 495 schmidtke, m., 625 segundo, m. a., 1127 selaković, m., 115 setamdideh, d., 1039 shahi, n., 483 shakir, m. s., 591 shapkin, v. a., 625 sharaf el-din, n. a. e.-s., 213 shiraz, n. z., 1053 shova, s., 901 shukla, s. n., 269, 941 simić, k., 1177, 1205, 1195 simić, m. r., 639 simić, z., 585 singh, m., 327 sirajudeen, a. a. o., 1 sladić, d. m., 459 sofrenić, i. v., 809, 1177, 1205 sokić, m., 1113 soković, m., 1291, 1205 srbljanović, j., 115 sredović ignjatović, i. d., 753 staleva, p., 1229 stamenković, j. g., 257 stanić, p. b., 585 stankov jovanović, v., 927 stankova, i. g., 625 stanković, j., 355, 1205 stanković, m. b., 809 stanojević, b. p., 555 stanojević, o. j., 1249 stanojković, j., 1023 stepić, k. d., 1259 stešević, d., 1281, 1241 stevanović, j. s., 555 stevanović, s. i. 407 stiković, s., 1241 stojanović, g. s., 257, 1271, 1259 stojanović, k., 1113 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ vol. 86: author index 1329 stojanović, s. đ., 781 stojković, d., 1291 šeregelj, v. n., 367 škerget, m., 603 šmit, b. m., 585 šolaja, b., 115 šovljanski, o. lj., 367, 533 štajner, t., 115 štrukelj, b., 125 šukalović, v., 795 tešević, v. v., 809 than, h. a. q., 615 todorova, m., 1229 todorović, t. r., 393 todorović, ž. n., 753 todosijević, m., 1195 tomasevic, b. i., 997 tomić, a. m., 533 tomić, m., 739 tong, j.-b., 469 torabi, s., 381 tot, m., 115 trachevskyi, v., 845 trendafilova, a., 1229 trifunović, s., 1241 trivic, d. d., 997 trmčić, m., 1305 ummathur, m. b., 39 uzairu, a., 283 vajs, v. v., 1219 vashishtha, m., 429 vaya, d., 327 veljić, m. m., 1249 verbić, t., 115 vogel, m., 1127 vojnović, s., 639 vučković, i., 1241 vujisić, lj., 809 vulić, j. j., 367 wang, b., 859 wang, q., 917 wang, t.-h., 469 xia, q.-y., 171 xia, l.-j., 247 xu, j., 247 yadav, a. p., 483 yadav, r., 507 yang, y., 247 yardim, y., 711 yousefinejad, s., 381 yunusoğlu, o., 711 yusuf, m., 971 zahir, e., 767 zaikov, y. p., 1075 zangade, s., 153 zhao, q., 917 zhou, s.-q., 171 zhuk, s. i., 1075 zia, k. m., 591 zildžović, s., 1113 zlatković, b. k., 1271, 1259 zlatović, m. v., 781 zloh, m., 561 zubair, a., 165 zuo, x., 859 živković, m. b., 459 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 1330 journal of the serbian chemical society subject index of vol. 86 and list of referees in 2021 are given in the electronic form at the internet address of the journal of the serbian chemical society: http://www.shd.org.rs/jscs end of volume 86. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice volume 85 (2020) 1 subject index 1,2-diamine, 601 1,4-dihydropyridine, 79 1,8-naphthalimide derivatives, 1253 2-diketone, 601 3d qsar, 335, 979 4-dicarboxylic acid, 427 4-nitrophenol, 1463 acetylcholinesterase, 1405 acid treated adsorbent, 1083 acidity, 1345 acrylic acid hydrogels, 939 actinomycetes, 279 activation energy, 651 adsorption capacity, 1083 adsorption layer, 781 adsorption model, 265 adsorption, 111, 251, 651 advance materials, 661 advanced oxidation processes, 547 agricultural waste, 177 aim, 37, 1033 allelochemicals, 141 alzheimer’s disease, 1405 ammonium, 395 analgesics, 711 anionic surfactant, 661 ann, 1149 antibacterial activity, 291 anticorrosion, 1317 antiferromagnetic coupling, 1577 antifungal activity, 203, 291 antifungal, 1137 anti-inflammatory drug, 1223 antileishmanial drug, 53 antimicrobial activity, 637, 979, 1475, 1591 antimicrobial agents, 155 antimicrobial potential, 1149 antimycotics, 897 antioxidant, 37 antioxidative tests, 623 antiproliferative activity, 637 antiradical activity, 517 aops, 125 aqueous solution, 1463 aromatic aldehydes, 79 artemisia ketone, 1565 artemisia, 575 artificial neural networks, 9 azido-bridged, 1279 azo dye, 125 azo dyes, 547, 1253 battery cathodes, 1047 benzofuran, 1405 binary mixtures, 227 binuclear complex, 1429 bioadsorbent, 265 biodegradation, 1067, 1507 bioenergetics, 1429 biological activity, 1 biological treatment, 395 biological, 215 biomass, 967 biomolecular interaction, 1265 bio-nano, 1383 bioplastics, 1507 biopolymer, 353, 369 biopolymers, 1507 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 2 journal of the serbian chemical society biosorbent, 1371 bisazaheterocycle, 601 boron-doped diamond electrode, 923 bovine, 1429 breakthrough curve, 953 bs-dft, 1577 building material, 1605 capillene, 1565 carbon molecular sieve (cms), 1083 carbon nanotubes, 1095 carbon quantum dots, 1095 carbon-carbon coupling, 1405 carrier, 857 cellulose, 845 ceramics, 1329 characterization, 481, 609 charge transfer, 651 chemical composition, 1001 chemical oxygen demand, 1357 chemistry education, 559 chemo-biosynthetic, 353 choke correlation, 687 choke flowrate, 687 citrus peel, 1371 cloud point extraction, 89 cluster analysis, 1605 co(ii), 215 coagulation activity, 1643 cobalt, complexes, 453 combined bioremediation, 111 complexing, 671 conceptual change, 559 conductivity, 67, 1303 confluence shape, 381 contaminated soil, 421 continuous adsorption, 953 controlled-release, 493, 1223 conversion, 967 coordination complexes, 885 copper(ii), 89, 1033 copper(ii) complexes, 321 copper, 795, 885 corn flour, 191 corrosion inhibition, 481 critical flow, 687 crown ether, 909 crystal structure, 751, 885 cu(ii) and co(ii) complexes, 637 cu(ii) complex, 1591 cu(ii), 215 current density, 1357 cyclic voltammetry, 1185 cytotoxic, 1 cytotoxicity, 291, 321, 721, 1539 d2ehpa, 1055 degradation of dyes, 547 degradation, 857 degraded habitats, 141 denitrification capacity, 1417 denitrification, 395 denitrifying soil bacteria, 1417 density functional theory, 1021 density, 237, 1303 despić, 1237 dess, 1303 dft, 37, 215, 481, 1033, 1445 diayangambin, 575 differential pulse voltammetry, 505 diffusion, 531 diimine, 321 diketonitrile, 1163 diluent effect, 909 dipole moment, 1445 dna interaction, 751 dna, 1137 dna-binding, 203 docking, 335, 1137, 1291 dopaminergic, 711 doped polyaniline, 251 double end-on, 1279 double-hybrid functionals, 1577 dpph, 845 dražić, 1237 drug release, 531 dye adsorption, 661 e/z isomers, 979 eae, 163 ediation, 821 edxrf, 1605 efficiency of mixers, 381 electrochemical reaction, 505 electrodeposited polypyrrole, 1317 electrodeposition, 795 electrodes, 1383 electrolysis, 347 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. volume 85: subject index 3 electron localization function, 765 electronic spectral patterns, 661 electrosynthesis, 79 ellagic acid, 867 ellagitannins, 867 enantiomer, 1 enantioselectivity, 279 encapsulation, 369 endothermic, 251 enhanced in situ bioremediation, 1067 enterobacter, 609 entropy, 251 environment, 967 enzyme activity, 177 enzyme inhibition, 721 epiyangambin, 575 equation of state, 237 equilibrium, 967 error analysis, 251 ethylene, reaction mechanism, 1175 experimental verification, 1417 extraction kinetics, 1055 extracts, 439 fastness properties, 1253 fe(iii), 1055 fe3+, 1463 fepo4⋅2h2o, 671 ferromagnetic coupling, 1577 ferrous sulfate, 547 fertilizer, 493 ffh, 1237 filtration, 1357 flavonoids, 141, 623 fluconazole, 1137 fluorescence sensing, 1463 fluorescence, 1021 fouling minimization, 1629 frap, 845 fruška gora, 517 ftir, 421 fukui function, 1163 fukui, 1291 fullerenes, 1095 functional groups, 1047 fungal, 111, 857 fungi, 177 furanolactones, 1539 gallic acid, 867 gas chromatography–mass spectrometry, 1565 genetic algorithm, 1149 glazes, 1329 global reactivity descriptors, 53 glycosidic bonds, 97 goethite immobilization, 125 goniofufurone mimics, 1539 g-quadruplex, 1021 graphene, 1095 graphitized carbon black, 781 green technology, 97 hemicelluloses, 1621 herbal dust, 9 herbs, 1001 heterobivalent, 711 heterocyclic compounds, 321 high level liquid waste, 909 high-pressure, 237 high-temperature, 237 histidine ligands, 1429 hot water extracts, 721 hs-gc–ms/fid, 1001 human cancer, 1265 humic acid, 421 humification degree, 406 humification, 821 hyaluronic acid, 1197 hybrid material, 1223 hydrazones, 453 hydrocarbon-contaminated groundwater, 1067 hydrogen peroxide scavenge, 517 hydrogen peroxide, 547 hydrolytic enzymes, 177 hysteresis, 67 icp-oes, 1605 ihtm, 1237 imidazolium, 291 imide of pyridine-3, 427 immobilization, 111, 857 impregnated adsorbents, 1083 index of atherogenicity, 305 index of thrombogenicity, 305 infrared spectra, 885 inhibition effect, 885 inhibition, 609 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 4 journal of the serbian chemical society inhibitor, 1265 in-house validation, 1605 interactions, 1137 intercalation compounds, 1223 intermolecular interactions, 781 iodide/iodate test experiment, 381 ionic liquid, 237, 291 ionisable organic pollutants, 697 isoelectric point, 1621 isoxaflutole, 1163 kinetic characterisation, 25 kinetic models, 953 kmno4, 601 laminar flow regime, 381 layered double hydroxide, 1223 lead removal, 953 lead-acid battery, 953 lignan, 575 lignin, 1621 lignocellulosic feedstock, 177 li-ion, 1047 linear poisson–boltzmann equation, 1429 lipid oxidation, 1553 lipid peroxidation, 721 lipophilicity, 897 lithium-ion batteries, 1383 long-chain organic acids, 305 macrodiolide, 1129 magneto-structural correlations, 1577 map, 1055 marc, 305 mathematical models, 737 mechanism, 427 medt, 765 membrane filtration, 1629 mg substrate, 1317 micro-channel, 381 microencapsulation, 531 microorganism, 421 microplastics, 697 micro-raman spectroscopy, 1329 migraine, 1291 mineral additives, 1605 missense mutation, 1265 mitsunobu reaction, 1129 modelling, 809 moisture content, 191 molecular descriptors, 467 molecular docking, 751 molecular modeling, 1265 molecular orbital, 1175 molecular weight, 897 molybdate, 1317 molybdenum dioxide, 1185 morphology, 795 multi-component, 79 multiple sclerosis, 163 multi-walled carbon nanotubes, 1185 murzuq basin, 1489 mutant proteins, 25 nanoclay, 1197 nanomaterials, 1475 nanomedicine, 1383 nanosized, 79 nano-technologies, 1383 naphthols, 1253 natural antioxidants, 1553 nbo, 1445 nci, 765 nitrification, 395 nitrogen cycle, 1417 nutritional value, 737 octaazamacrocycle, 637 octanol/water partition coefficient, 467 oil-oil correlation, 1489 opioids, 711 organic load, 1643 oxa-michael ring-closure, 1539 oxidation, 831 oxidation–reduction, 279 oxidative stability, 1553 oxime esters, 1 oxovanadium, 37 particles, 347 passive mixers, 381 pcm, 1445 pe, 697 peony, 845 pessap, 687 pesticide, 857 pesticides residue, 191 pesticides, 809 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. volume 85: subject index 5 pet, 697 ph influence, 279 pharmaceutical formulation, 923 pharmacophore, 335 phenolic acids, 141 phenolics, 517, 623 phosphacyclopropenylidene, 1175 photocatalysis, 1475 photoluminescence, 1475 physicochemical properties, 1345 pinene, 1565 piperazine, 711 piperidine, 711 pka calculations, 1429 plasma discharge, 831 platinum, 1185 polar surface area, 897 polarography, 517 polyhydroxyalkanoates, 1507 polylactic acid, 1507 polymeric cu(ii) complex, 203 polyphenolics, 1011 polyphenols, 439, 845 polysaccharides, 439 post-synthesis al-grafting, 1345 powder, 347 powders, 439 preferential solvation, 227 preparation, 671 principal component analysis, 1001 probiotics, 163 process parameters, 531 protein enriched product, 737 protein purification, 25 proteins, 1643 pulsating overpotential, 795 punica granatum, 1553 purification, 609 pyrazole, 885 pyrazoline, 155 pyridoxal, 37, 1033 pyrimidine, 481 qspr methods, 467 qsrr, 9, 1149 quantum chemical study, 53 quantum-chemical method, 427 quercetin, 227 radical grafting, 353 radical growth inhibition, 141 reaction and protein field, 1429 reactivity, 1291 refractive index, 1303 response surface methodology, 1629 revegetation, 406 review, 1383 reviewnologies, 1383 rosa canina, 1553 rosin, 369 rsa, 1011 salicylate, 291 salt reduction, 737 salycilate, 1317 sar analysis, 1539 scanning electron microscope (sem), 347 biogel, 353 schiff base, 37, 215, 1033, 1279 science process skills, 559 sedimentation, 1357 self-assembled monolayers, 67 sem-eds, 1329 semi-ipn hydrogels, 1197 sensitivity analysis, 1629 sertraline, 505 sesamin, 575 sesartemin, 575 sharpless asymmetric dihydroxylation, 1129 shellac, 369 silica sba-15, 1345 single crystal x-ray analysis, 453, 1591 sio2 nanoparticles, 939 sirte basin, 1489 smo inhibitors, 335 sno2, 427 sodium dodecyl sulphate, 923 soil, 809 solanum pimpinellifolium, 623 solubility, 897 soluble carbohydrates, 305 solvatochromic effects, 1253 solvatochromism, 227 solvent effects, 979, 1445 solvent evaporation method, 531 sorption, 809, 1371 spectrophotometry, 89 spectroscopic characterization, 1591 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 6 journal of the serbian chemical society spectroscopy, 53 spent materials, 671 spice, 1001 standardization, 439 statistical analysis, 177 stereoselective, 1 stereoselectivity, 1129 strontium extraction, 909 structure simplification-based drug design, 1539 structure, 203 subcritical water, 97, 867 substituent effects, 979 sugar degradation products, 97 sulfonamide, 155 supercritical fluid extraction, 9 surfactant rich phase, 89 surfactant vesicles, 493 surfactants, 67, 781 sustainability 1507 sweet chestnut, swelling/deswelling kinetics, 1197 syngas, 967 synthesis, 155 tartrazine, 251 tautomerization, 1033 td-dft, 979 technosols, 406 temperature, 191 terpenoids, 9 textile dyes, 831, 939 the krafft temperature, 67 theoretical computations, 1445 therapeutic success, 897 thermal stability, 1553 thermal treatment, 265 thermodynamic, 967 thermo-responsive hydrogels, 1197 thiazole, 1445 thiazolidinone, 155 thin films, 1095 thiosemicarbazone, 321 tkx-50, 651 tmf, 1237 top soil restoration, 406 total dissolved solids, 1357 total petroleum hydrocarbons, 821 tpc, 1011 tramadol, 923 transient state, 651, 765 triptanes, 1291 triton x, 781 turbulence promoter, 1629 tutundžić, 1237 uhplc-ltq orbitrap ms, 1011 ultrasound irradiation,125 ultrasounds-assisted extraction, 845 urine samples, 923 v2o5, 427 vinyl pyrrolidone, 939 viscosity, 1303 vitis vinifera, 305 volume, 897 wastewater treatment, 395, 939 wastewater, 1371 water analysis, 89 water clarification, 1643 wittig olefination, 1539 woody species, 141 xrpd, 1329 yeast, 25 zein, 369 zeta potential, 1621 zro2, 427 α-amino acids, 1 β-galactosidase, 609 γ-aminobutyric acid (gaba), 163 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 404 not found 404 not found accepted manuscript this is an early electronic version of an as-received manuscript that hasbeen accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as g. andrejić, m, kovačević, ž, dželetović, u. aleksić, i. grdović and t. rakić, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc230113028a this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230113028a j. serb. chem. soc.00(0)1-11 (2023) original scientific paper jscs–12228 published dd mm, 2023 1 potentially toxic element accumulation in two equisetum species spontaneously grown in the flotation tailings gordana andrejić 1 * milijana kovačević 2 , željko dželetović 1 , uroš aleksić 1 , isidor grdović 2 and tamara rakić 2 1 university of belgrade, institute for the application of nuclear energy, banatska 31b, 11080 belgrade, serbia and 2 university of belgrade, faculty of biology, studentski trg 16, 11000 belgrade, serbia (received 13 january; revised 21 june; accepted 1 july 2023) abstract: decades of mining activity have resulted in the accumulation of significant amounts of tailings that are deposited over the natural vegetation, forming deposits tens of meters thick. the tailings are poor in organic matter and macronutrients and contain a high concentration of potentially toxic elements (pte). their surface remains unvegetated for long periods of time and is susceptible to fluvial and wind erosion. equisetum arvense and e. telmateia appear to be the first colonizers in the tailings of the pb-cu-zn mine in serbia. each plant was sampled along with its associated substrate. pseudototal and available metals in the substrate, as well as total as, cd, cu, fe, mn, ni, pb and zn concentrations in the plant parts were determined by atomic absorption spectrophotometry. the findings show that both species have high bioaccumulation capacity and tolerance to otherwise toxic concentrations due to efficient accumulation, immobilization, and detoxification of these elements in their underground parts. it is expected that the long-term presence of metal-tolerant horsetail species would increase the organic matter content of flotation residues, thus gradually improving their physical, chemical, and biological properties. this, in turn, would promote the natural succession of other metal-tolerant plant species and soil microorganisms. keywords: potentially toxic elements, pollution, phytoremediation. introduction as a result of the substantial global demand for mineral raw materials and their extensive use in various industries, mining and ore processing remain prominent sectors within the primary industry of certain countries, yielding significant financial profits. despite advancements in mining technology and waste reduction strategies, mining activities continue to pose a significant threat to *corresponding author. e-mail: gordanaa@inep.ac.rs https://doi.org/10.2298/jsc230113028a a cc ep te d m an us cr ip t https://doi.org/10.2298/jsc230113028a andrejić et al.. the environment due to the generation of spoils and effluents containing exceedingly high concentrations of potentially toxic elements (ptes). it is worth noting that only a small fraction of the total processed material is extracted as a concentrated metal product, while a substantial proportion, up to even 97-99% of the mined ore, becomes flotation tailings. flotation tailings share a similar composition to the original ore, and containing significant amounts of unrecovered minerals, contribute to the environmental pollution.1-3 flotation tailings consists of fine particles (ø=<75 μm) that remain after the technological processing and extraction of desired elements from the polymetallic ore, and are discarded with water into the tailing pond. further release of ptes from these particles occurs through various processes of chemical and physical particle disintegration. eventually, pte enter the food web, often leading to adverse effects on the health and biodiversity of surrounding ecosystems, as well as posing risks to human health.4 this issue is particularly prominent in countries where regulatory framework to mitigate environmental pollution risks is not effectively enforced. furthermore, the flotation tailings are also abundant in process chemicals, including organic solvents like xanthate, which are utilized for the effective separation of metals during mineral flotation processes. it is noteworthy that a significant portion, nearly half, of the xanthates employed in the mineral flotation process is ultimately discharged into the flotation tailings.5 the presence of these process chemicals within the tailings, further emphasizes the complex composition and potential environmental impact of these waste materials. additionally, flotation tailings surface layers are prone to fluvial erosion and the dispersion of fine particles through wind, further exacerbating their environmental impact.4,6-7 therefore, pte contamination can extend several kilometres beyond the mining site, significantly affecting local and regional land use and posing health risks to nearby human communities. restoring large areas covered by flotation tailings presents significant challenges due to unfavourable physical and chemical properties of these technosols, which strongly hinder plant growth and result in consequent long-term lack of vegetation cover.8-10 vascular plants that establish themselves as the initial colonizers of flotation tailings are uncommon and exhibit remarkable adaptability to the challenging environmental conditions found in such areas. among these rare plant species that pioneer the colonization of polymetallic flotation tailings, several belong to the genus equisetum. equisetum is an ancient genus of vascular plants with a long evolutionary history dating back to the upper devonian period.11 all species within the equisetum genus are perennial plants, possessing erect herbaceous stems that emerge from an extensive rhizome system bearing adventitious roots.11-12 horsetails have demonstrated a remarkable ability to tolerate unfavourable physical and chemical properties of substrates, even allowing for the accumulation of metals at higher concentrations.13-14 this a cc ep te d m an us cr ip t elemental analysis of equisetum sp. 3 resilience in the face of challenging soil conditions is thought to be linked to their archaic adaptation to geothermal environments characterized by high levels of potentially toxic metals and metalloids harmful to most plant species, such as arsenic (as) or mercury (hg).15 in the specific context of this study, the two horsetail species, equisetum arvense and e. telmateia, were observed as the initial colonizers in the flotation tailings of a lead-copper-zinc mine in serbia. the objectives of this research were twofold: (i) to analyse the concentrations of potentially toxic elements (ptes) in the flotation tailings as well as in the underground and aerial parts of the two equisetum species, and (ii) to evaluate their capacity for bioaccumulation and their potential for utilization in the initial stages of bioremediation efforts. experimental site description plants and their belonging substrates were collected from two different parcels of the flotation tailings of the active pb-cu-zn mine on mt. rudnik (44,11 n; 20,495 e; 500 m asl), located in central serbia (fig. 1a). equisetum arvense and e. telmateia populations are distributed across two distinct sections within the peripheral area of the flotation tailings, adjacent to the surrounding natural vegetation. these sections benefit from a sufficient water supply as a result of the presence of a nearby stream (fig. 1b). it is worth noting that no deposition of waste material has occurred in either of these tailing parcels for the past four years preceding the sampling. the climate in this region and at this altitude is temperate. fig. 1 location of the mine (a) and strands of equisetum arvense and e. telmateia in the flotation tailings dump (b). substrate and plant sampling each plant was sampled together with the substrate surrounding the rhizome and its adventitious roots, from the depth 0-20 cm, in august 2017. each composite substrate sample b a cc ep te d m an us cr ip t andrejić et al.. (fea, e. arvense flotation tailings substrate; fet, e. telmateia flotation tailings substrate) was composed of well mixed substrate samples collected from the rhizosphere of 15 individuals. substrate analysis the substrate samples were cleaned from stones and biotic material. after air drying, the samples were ground and sieved (pore diameter 200 µm). ten grams of a homogenized substrate was mixed with 25 ml of distilled water or with 25 ml of 1m kcl to determine active (phh2o) and exchangeable (phkcl) substrate acidity, respectively. 16 the samples were stirred for 30 min and the ph was measured directly in the suspension (iskra ma 5730). for determination of the pseudo-total pte concentration (as, cd, cu, fe, mn, ni, pb, zn) in the substrate, samples were dried to constant weight at 100°c, and digested in 65% hno3 at 150°c in kjeldaltherm® digestion block (gerhardt, germany), according to the us epa method 3051. 17 solutions for determining available pte concentrations were made by extraction for 2 h in 1 m ammonium acetate and 0.01 m edta mixture (ph 7). 18 the concentrations of pte were determined by atomic absorption spectrophotometer (shimadzu aa-7000, japan) comparing sample absorption values with those of known standards. the precision of the procedure for pte was evaluated by analyzing the certified soil material (soil 90-0115-0106, bipea-bureau interprofessionnel d ´etudes analytiques). plant analysis to avoid contamination by residual substrate particles, the collected plant samples were carefully washed in tap water and then thoroughly rinsed with deionized water. the plant material was divided into (vegetative) shoots and underground parts, air-dried, powdered, ovendried at 100°c for 24h, and completely digested in 65% hno3 at 150°c in kjeldaltherm® digestion block, according to the us epa method 3051. 17 concentrations of pte (as, cd, cu, fe, mn, ni, pb, zn) in plant samples were determined by atomic absorption spectrophotometer (shimadzu aa – 7000, japan), comparing the sample absorption values with those of known standards. analytical procedure accuracy was assessed by analysis of the standard reference plant material nist 1515 (apple leaves). phytoremediation potential the phytoremediation potential of two horsetail species was determined on the basis of bioconcentration factor (bcf) and translocation factor (tf). 19 bcf provides information on the accumulation of pte in the underground plant parts and their efficiency of pte removal from the substrate. translocation factor (tf) is the ratio of the pte concentration in the aboveground part of the plant and its concentration in the undergound parts, and provides information on the plant’s efficiency in tranlocating a certain pte from underground to its aboveground parts. bcf and tf values were calculated using the following formulas: bcf = cunderground plant parts / csubstrate (1) tf = cshoot / cunderground plant parts (2) where cunderground plant parts and cshoot represent pte concentrations in plant underground parts and shoots, respectively, whereas csubstrate is the available pte concentration in the belonging substrate. statistical analysis data are expressed by the mean ± standard deviation of eight replicates. all data were initially tested by the shapiro-wilk test for normality. statistically significant differences between two independent groups were calculated using the non-parametric mann-whitney u a cc ep te d m an us cr ip t elemental analysis of equisetum sp. 5 test because of the non-normality of the data. statistical analyses were performed in r (v3.5.1; r core team 2018). results and discussion flotation tailings chemical properties as indicated in table i, both the active (phh2o) and exchangeable soil acidity (phkcl) were within the neutral range. the phh2o was statistically significantly lower in e. arvense substrate compared to that of e. telmateia, whereas phkcl was lower again in the e. arvense substrate, but without statistical significance (table i). at a soil ph around neutral, it is assumed that all the studied elements are well dissolved in the soil water solution, although their availability would increase with decreasing ph, except for as. both substrates exhibited elevated pseudo-total concentrations of several elements (as, cd, cu, pb, zn) that surpassed the maximum allowable concentrations (mac) specified in international soil guidelines (table i). these concentrations were several-fold higher (ni, pb, zn, cd, cu) and approximately 25-fold higher (as) than the mac values. since the substrates studied were solid waste materials from extractive metallurgical processes, it was expected that such high concentrations of potentially toxic elements (ptes) would be observed. although detected pseudo-total pte concentrations were very similar between the substrate samples, there were statistically significant differences regarding all elements, with exception of as. this heterogeneity is a common characteristic of the flotation tailings resulting from the deposition of waste material originating from different mined ores, which can vary in their metal content.20 the portion of edta-available elements in the pseudo-total content in fet and fea substrate is listed in descending order, respectively, and was as follows: pb (45%, 44%) > cd (36%, 37%) > mn (16%, 20%) > ni (13%, 8%) > zn (8%, 10%) > cu (7%, 10%) > fe (0.2%). the presence of elevated metal availability in flotation tailings has been previously established in the literature for various metals. for instance, kasowska et al.21 highlighted the considerable presence of readily available copper (cu) in copper ore flotation tailings, which constituted approximately 41% of its pseudo-total concentration. additionally, karczewska and milko22 reported that edta exhibited the highest effectiveness as a chelating agent, enabling the release of up to 16% of edta-available lead from the pseudototal metal content. the prominent metal availability in the investigated flotation tailings may be attributed to the presence of residual metal xanthates and their degradation products in the tailings. the highest pb and cd edta-availability can be attributed to very high edta efficiency for desorption of listed metals from xanthates.23-24 while it is commonly known that metal availability is moderate at neutral and tends to be higher under acidic conditions, it is important to consider that in substrates already colonized by plants the metal bioavailability can be a cc ep te d m an us cr ip t andrejić et al.. further increased in plant rhizosphere. this enhanced bioavailability of metals can be attributed to some microbiome and plant roots exudates, such as organic acids and hydrogen ions (h+), that acidify the root microenvironment and desorb metals at the negatively charged reactive sites of soil particles making them more available for uptake by roots.25 table i. ph values and pte concentrations (mg kg -1 ) in the flotation tailings fet fea mac (mg/kg)* phh2o 7.6 ± 0.05 b 7.5 ± 0.03 a phkcl 7.4 ± 0.1 a 7.0 ± 1.3 a pseudo-total as 484 ± 243 a 533 ± 36 a 15-20 cd 9.6 ± 0.2 b 8 ± 0.1 a 1-5 cu 349 ± 12 b 300 ± 4 a 60-150 fe 25848 ± 95 b 25399 ± 145 a mn 2003 ± 25 b 1759 ± 61 a 1500-3000 ni 68 ± 1.3 a 75 ± 1 b 20-60 pb 448 ± 43 a 558 ± 6 b 20-300 zn 1823 ± 24 b 1445 ± 30 a 100-300 edta available cd 3.5 ± 1.6 a 3.0 ± 0.2 a cu 25 ± 18 a 33 ± 0.8 a fe 49 ± 4.6 a 62 ± 7 b mn 328 ± 24 a 348 ± 11 a ni 8.5 ± 7 a 6 ± 4 a pb 200 ± 7 a 247 ± 21 a zn 153 ± 37 a 149 ± 40 a *mac maximum allowable concentration. 26 values with no letter in common are significantly different (mann-whitney u, p<0.05) potentially toxic element accumulation by horsetails the analysis of concentrations of potentially toxic elements in the investigated species, e. arvense and e. telmateia, reveals a significant accumulation of pte in their underground parts. notably, the concentrations of the investigated elements, except for as and mn, were considerably higher in the underground plant parts (table ii) compared to the corresponding edta-available element concentrations in the substrates (table i). consequently, this disparity led to their bcfs > 1 (table iii). both e. arvense and e. telmateia exhibited the highest bioconcentration factors for fe and ni, followed by zn and cu. generally, e. arvense demonstrated higher bcfs compared to e. telmateia, with cu and ni showing a 1.7-fold and 4.5fold higher bcfs in e. arvense. both equisetum species efficiently retained and immobilized accumulated pte in their underground parts, manifesting low transfer rates to shoots. this was detected through significantly lower concentrations of the elements in the shoots, accompanied by tfs < 1 (table iii). however, an exception was noted only for mn (tf = 1.24) in e. telmateia. the a cc ep te d m an us cr ip t elemental analysis of equisetum sp. 7 overall results indicate that both species possess a substantial bioaccumulation capacity and tolerance to elevated concentrations of pte in their underground parts. their metal tolerance is attributed to complex and efficient pte detoxification systems in rhizomes that could be also associated with typically high silicon content in equisetum that is not simply beneficial, but essential mineral element.27-28 it is well documented that si alleviates heavy metal stress in horsetails and some other plants by being involved in external and internal mechanisms of si-mediated alleviation of metal toxicity, as a part of their biochemistry.14,29-33 previous studies have shown that horsetails are often behaving as extremophiles regarding their substrate properties. thus, e. arvense was found to tolerate nitrogen deficiency and specific elemental composition in volcanic tephra and flotation tailings, therefore being the most or among the most successful and dominant herbaceous species in such specific disturbed habitats.34-36 also, e. palustre, e. ramosissimum and e. ramosisti were among the first plant colonizers in natural succession of the mine and flotation tailings.37-40 furthermore, owing to its metal tolerance e. hyemale was efficiently used in the removal of pb and cr from the leachate in the wastewater treatment biotechnology.41 table ii. pte concentrations in underground parts and shoots of e. telmateia and e. arvense grown in the flotation tailings (mg kg -1 ) e. telmateia e. arvense upper concentration limits 20 u n d e rg ro u n d p a rt s as 18 ± 1 a 23 ± 0.7 b / cd 5.0 ± 0.1 a 9.3 ± 0.3 b / cu 51.6 ± 3.3 a 117 ± 8 b / fe 711 ± 6 a 842 ± 15 b / mn 93.9 ± 3.3 a 318 ± 15 b / ni 13.8 ± 0.5 a 44.2 ± 2.8 b / pb 65.4 ± 2.4 a 232 ± 24 b / zn 389 ± 14 a 633 ± 33 b / sh o o ts as 35.5 ± 2.6 b 10 ± 0.4 a 5-20 cd 1.3 ± 0.1 a 3.1 ± 0.1 b 5-30 cu 10.5 ± 0.7 b 8.0 ± 0.3 20-100 fe 618 ± 4 b 380 ± 4 a / mn 116 ± 3.6 a 270 ± 6 b 400-1000 ni 6.5 ± 0.3 b 5.9 ± 0.4 10-100 pb 26.2 ± 0.8 b 24.3 ± 0.6 30-300 zn 317 ± 14 b 235 ± 4 100-400 a cc ep te d m an us cr ip t andrejić et al.. table iii. bioconcentration (bcf) and translocation factor (tf) for two horsetail species grown in flotation tailings bcf tf e.telmateia e. arvense e.telmateia e. arvense cd 1.43 2.66 0.26 0.33 cu 2.06 4.68 0.20 0.07 fe 14.51 17.18 0.87 0.45 mn 0.29 0.91 1.24 0.85 ni 1.62 5.20 0.47 0.13 pb 0.33 1.16 0.40 0.10 zn 2.54 4.14 0.81 0.37 even though equisetum species typically form extensive colonies on wetlands, they have some properties that can be helpful in substrates that appear dry on the surface. one such trait is the presence of an extensive underground rhizome system that can penetrate up to a depth of one meter, allowing them to access water from deeper soil layers.12 this morphological adaptation proves particularly valuable in colonising substrates with unfavourable granulometric structure and water conditions, such as flotation tailings. conclusion the current study demonstrates the ability of wetland species equisetum arvense and e. telmateia to successfully establish themselves in heavily metalpolluted flotation tailings. despite absorbing substantial concentrations of various potentially toxic elements (as, cd, cu, pb and zn) in their underground parts, these plants exhibit no adverse effects from the elevated levels of these elements. remarkably, the concentrations of pte in the shoots remain consistently low, indicating that the pte tolerance of the two investigated horsetail species relies primarily on their capacity to exclude these elements. the findings provide further evidence that the pte tolerance observed in both species is supported by efficient adaptive mechanisms, enabling the accumulation, effective immobilization, and detoxification of pte within the underground plant parts. furthermore, the significant availability of potentially toxic elements (pte) within the flotation tailings, containing multiple metals, highlights the potential for secondary extraction of residual metals from this type of waste material. reprocessing the tailings would not only reduce metal leaching that are precious for industry, but also mitigate their detrimental environmental impact. although pte-tolerant horsetail plants could be suitable candidates for metal extraction from the rhizosphere, the collection of their underground parts from flotation tailings presents technical challenges. however, their vital ecological role in such technosols primarily lies in increasing organic matter content through the decomposition of old underground and aerial parts. this, in turn, enhances the substrate's capacity to retain pte. over the long term, this process facilitates gradual improvements in the physical, chemical, and biological properties of the a cc ep te d m an us cr ip t elemental analysis of equisetum sp. 9 substrate and promotes natural ecological succession by other metal-tolerant plant species and soil microorganisms. acknowledgements: this work was supported by the serbian ministry of education, science and technological development (grant no. 451-03-68/2022-14/ 200178 and 451-0368/2022-14/ 200019). compliance with ethical standards: conflict of interest: the authors declare that they have no conflict of interest. и з в о д акумулација потенцијално токсичних елемената код две самоникле врсте рода equisetum на одлагалишту флотационе јаловине гордана андрејић1, милијана ковачевић2, жељко џелетовић1, урош алексић1, исидор грдовић2 и тамара ракић2 1универзитет у београду, инстиут за примену нуклеарне енергије, банатска 31б, 11080 београд, србија и 2универзитет у београду, биолошки факултет, студентски трг 16, 11000 београд, србија резултат вишедеценијских рударских активности су знатне количине флотационе јаловине која се одлаже на велике површине стварајући наносе дебљине неколико десетина метара. јаловина се карактерише дефицитом органске материје и макронутријената, садржи хемикалије пореклом из технолошког процеса и високу концентрацију потенцијално токсичних елемената (пте). последично, површина јаловишта изузетно дуго остаје без вегетације и подложна је флувијалној и еолској ерозији. equisetum arvense и e. telmateia се појављују као пионирске врсте на одлагалишту флотационе јаловине рудника pb-cu-zn у србији који је истраживан у овој студији. свака биљка је узоркована заједно са супстратом из зоне ризосфере. псеудоукупне и приступачне концентрације аѕ, cd, cu, fe, mn, ni, pb, zn у супстрату и концентрације у биљним ткивима одређене су атомском апсорпционом спектрофотометријом. резултати показују да су механизми толеранције засновани на ефикаснној акумулацији, имобилизацији и детоксификацији пте у подземним биљним деловима. ове две врсте раставића имају неизоставну улогу у повећању садржаја органске материје што постепено побољшава физичка, хемијска и биолошка својстава супстрата, а тиме и подржава природне еколошке сукцесије другим биљним врстама и земљишним микроорганизмима отпорним на метале. 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http://dx.doi.org/10.4067/s0717-97072019000104298 https://doi.org/10.1034/j.1399-3054.1998.1040413.x https://doi.org/10.1016/j.envexpbot.2007.10.024 https://doi.org/10.1155/2015/396010 https://doi.org/10.3390/plants8030071 https://doi.org/10.1007/s13205-019-1613-z https://doi.org/10.1080/00380768.1992.10416712 https://doi.org/10.1046/j.1469-8137.2002.00363.x https://doi.org/10.1016/j.envpol.2004.03.030 https://doi.org/10.1111/j.1526-100x.2012.00913.x https://doi.org/10.1051/matecconf/201710004030 https://doi.org/10.1080/15226514.2018.1556590 404 not found 404 not found accepted manuscript this is an early electronic version of an as-received manuscript that has been accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as t. vitomirov, b. čobeljić, a. pevec, d. radanović, i. novaković, m. savić, k. anđelković, m. šumar-ristović, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc230623044v this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230623044v j. serb. chem. soc.00(0)1-13 (2023) original scientific paper jscs–12258 published dd mm, 2023 1 binuclear azide-bridged hydrazone cu(ii) complex: synthesis, characterization and evaluation of biological activity teodora vitomirov1, božidar čobeljić1, andrej pevec2, dušanka radanović3, irena novaković3, milica savić3, katarina anđelković1, maja šumar-ristović1* 1university of belgrade – faculty of chemistry, studentski trg 12–16, 11000 belgrade, serbia, 2faculty of chemistry and chemical technology, university of ljubljana, večna pot 113, 1000 ljubljana, slovenia, and 3university of belgrade, institute of chemistry, technology and metallurgy, department of chemistry, njegoševa 12, 11000 belgrade, serbia (received 23 june; revised 27 june; accepted 21 july 2023) abstract: the condensation product of 7-acetyl-6-azaindole and girard's t reagent ((e)-2-(2-(1-(1h-pyrrolo[2,3-c]pyridin-7-yl)ethylidene)hydrazineyl)n,n,n-trimethyl-2-oxoethan-1-aminium, hl ligand) was used as a ligand in the reaction with cu(bf4)2·6h2o and nan3. the reaction led to the formation of a binuclear cu(ii) complex containing two end-to-end (di--1,3-n3) azide bridges, as well as two nno-donor hydrazone ligands, forming an axially elongated square pyramidal geometry around each cu(ii) center. this end-to-end (di--1,3n3) azide bridge binding mode has not yet occurred in cu(ii) complexes containing the nno-donor hydrazone ligands, which makes the structure of the complex even more interesting for further studies. the complex was characterized by elemental analysis, ir spectroscopy and x-ray crystallography, and it was found that it crystallizes in the triclinic space group p–1 with the asymmetric unit comprising one cu(ii) centre, zwitterionic ligand l, one azide (n3 −) ligand, and bf4 − counter anion. examination of antimicrobial activity of the complex shows higher antifungal and antibacterial activity towards tested gram-positive bacteria in comparison to the hydrazone ligand, with the antifungal activity of the complex even being comparable to the activity of amphotericin b. keywords: girard's t reagent; x-ray crystallography; antibacterial activity; antifungal activity. *corresponding authors e-mail: majas@chem.bg.ac.rs https://doi.org/10.2298/jsc230623044v a cc ep te d m an us cr ip t mailto:majas@chem.bg.ac.rs https://doi.org/10.2298/jsc230623044v 2 vitomirov et al. introduction azide-bridged binuclear copper(ii) complexes have been the subject of considerable research in the field of coordination chemistry due to their interesting structural and magnetic properties.1–3 these complexes consist of two copper(ii) ions bridged by an azide group via either end-on (eo) or end-to-end (ee) coordination mode, forming single or double ligand bridges: µ1,1-n3 (eo) and µ1,3n3 (ee).4 the azide group provides a unique building block for the construction of complex architectures and is a versatile bridging ligand that can adopt different coordination modes, such as mono-, bior tri-dentate,1 depending on the nature of the metal ion and the reaction conditions. furthermore, it also acts as a strong magnetic coupler facilitating ferromagnetic and antiferromagnetic coupling between metal ions within a binuclear complex.2 on the other hand, the binuclear copper(ii) complexes are known to exhibit either antiferromagnetic or ferromagnetic behavior, depending on the nature of the bridging ligand, the cu– x–cu angle and the geometry of the complex.3,5–9 in recent years, there has been growing interest in the biological activity of binuclear copper(ii) complexes due to their potential applications as anticancer10,11 and antibacterial12–14 аgents. the ability of these complexes to catalyze the formation of reactive oxygen species (ros) and to interact with dna and proteins makes them attractive candidates for the development of new therapeutic agents. when discussing the azide-bridged binuclear cu(ii) complexes, several studies have reported the potential antibacterial activity of these complexes, all containing a tridentate nno-donor hydrazone ligand, against various strains of bacteria.10,15,16 the mechanism of action is believed to be the disruption of the bacterial cell membrane due to the reduced polarity of the metal ion and increased lipophilicity of the formed complex, compared to lone metal ions and ligands.17 in addition to their biological applications, azide-bridged binuclear copper(ii) complexes have also shown promising catalytic properties under sustainable and user-friendly conditions. these complexes have been reported to exhibit catalytic activity towards various reactions, including the n-arylation of imidazole and benzimidazole18 as well as the synthesis of 1,2,3-triazoles.19 the catalytic activity of these complexes is attributed to the redox properties of copper(ii) ions and the cu-n3-cu bridge, which can facilitate electron transfer processes. in this study, we have synthesized and fully characterized a new binuclear copper(ii) complex containing double end-to-end (di-µ1,3-n3) azide bridge, along with nno-donor hydrazone ligands, and investigated its antibacterial and antifungal activities. the results of our study provide a detailed characterization of a binuclear copper(ii) complex, as well as an overview of the potential of this complex as a new therapeutic agent. a cc ep te d m an us cr ip t binuclear azide-bridged hydrazone cu(ii) complex 3 experimental materials and methods all chemicals and solvents (reagent grade) were obtained from commercial suppliers (nan3 from riedel-de haën; all other chemicals from sigma-aldrich) and used without further purification. elemental analyses (c, h, and n) were performed by standard micro-methods using the elementar vario eliii c.h.n.s.o analyzer. ir spectra were recorded on a nicolet 6700 ft-ir spectrometer using the atr technique from 4000 – 400 cm-1 (strong–s, medium–m, weak–w). nmr spectra were recorded with a varian 400/54 ps spectrometer in deuterated dimethyl sulfoxide (dmso-d6). ligand synthesis the ligand synthesis was carried out in two steps – the first step was obtaining 7-acetyl-6azaindole using 7-bromo-6-azaindole as a starting compound. the reaction was performed by adding 0.75 mmol (18.2 mg) of mg to 0.75 mmol (147 mg) of 7-bromo-6-azaindole in anhydrous diethyl ether and making a grignard’s reagent, which then reacted with equimolar amount (0.75 mmol, 58.9 mg) of acetyl chloride and a small amount (2 mol %) of fecl3 as a catalyst. this reaction was carried out at -60 °c using dry ice as a cooling bath. the next step was to synthesize the condensation product of 7-acetyl-6-azaindole and girard's t reagent, ((e)-2-(2-(1-(1h-pyrrolo[2,3-c]pyridin-7-yl)ethylidene)hydrazineyl)n,n,n-trimethyl-2-oxoethan-1-aminium). the reaction was carried out by dissolving 0.5 mmol (80 mg) of 7-acetyl-6-azaindole in methanol and adding 0.5 mmol (83.8 mg) of girard’s t reagent to the reaction mixture, which was then refluxed for 3 hours. after cooling down to room temperature, the bright yellow precipitate was filtered and rinsed with ethanol. the reaction yield was 81 % (125.2 mg) and the ligand was then characterized by elemental analysis, ir and nmr spectroscopy. labelling of c and h atoms described with nmr spectroscopy is presented in fig. 1. elemental analysis for c14h20cln5o (%), calculated: c 54.28, h 6.51, n 22.61. found (%): c 54.68, h 7.09, n 22.13. ir (atr, cm-1) selected peaks: 3377.4 (m), 3068.5 (m), 3019.4 (m), 2976.5 (m),1703.4 (s), 1618.3 (w), 1571.8 (m), 1550.4 (m), 1491.2 (m), 1431.9 (m), 1398.4 (s), 1333.1 (m), 1280.2 (m), 1231.0 (m), 1164.0 (m), 1125.8 (m), 987.3 (m), 947.3 (m), 919.3 (m), 858.8 (w), 818.5 (m), 800.1 (m), 710.9 (m), 655.4 (m), 609.1 (w). 1h nmr (400 mhz, dmso-d6), δ (ppm): 11.66 (s, 1h, n2-h), 11.50 (s, 1h, n4-h), 8.15 – 7.72 (4h, c1-h, c2-h, c4-h, c5-h),4.91 (s, 2h, c11-h), 3.34 (t, 9h, c12-h), 2.32 (s, 3h, c9-h). 13c nmr (125 mhz, dmso-d6), δ (ppm):167.24 (c10), 161.48 (c8), 156.29 – 120.21 (c1, c2, c3, c4, c5, c6, c7), 63.21 (c11), 53.74 (c12),12.73 (c9). complex synthesis the synthesis of the complex was performed by dissolving 0.25 mmol (77.3 mg) of ligand in 20 ml of methanol and then adding 0.25 mmol (86.3 mg) of cu(bf 4)2·6h2o, previously dissolved in 5 ml of h2o, and 1 mmol (65 mg) of nan3 directly into the reaction mixture. the mixture was refluxed for 2 hours, and after 10 days dark green monocrystals of complex were obtained and filtered from the solution. the reaction yield was 73 % (169.6 mg) and the complex was characterized by elemental analysis, ir spectroscopy and x-ray crystallography. elemental analysis for c28h38b2cu2f8n16o2(%), calculated: c 36.11, h 4.11, n 24.06. found (%): c 35.74, h 4.25, n 24.58. a cc ep te d m an us cr ip t 4 vitomirov et al. ir(atr, cm-1) selected peaks:3350.3 (w), 3098.4 (w), 3044.3 (w), 2988.8 (w), 2063.4 (s), 1583.1 (s), 1557.8 (s), 1530.8 (s), 1501.3 (m), 1483.2 (s), 1441.4 (m), 1400.5 (m), 1347.7 (m), 1319.7 (m), 1296.1 (m), 1248.7 (m), 1185.3 (m), 1118.1 (m), 1034.8 (s), 997.2 (m), 972.1 (m), 926.3 (m), 910.6 (m), 815.7 (m), 733.2 (m), 655.2 (w). x-ray structure determination the crystal structure of compound [cu2l2(1,3-n3)2](bf4)2 was determined by singlecrystal x-ray diffraction methods. crystallographic data and refinement details are given in table i. diffraction data were collected with agilent supernova dual source diffractometer using an atlas detector and equipped with mirror-monochromated mokα radiation (λ = 0.71073 å). the data were processed by using crysalis pro.20 the structure was solved using sir-9221 and refined against f2 on all data by full-matrix least-squares with shelxl–2016.22 all nonhydrogen atoms were refined anisotropically. the nitrogen n2 bonded hydrogen atom was located in the difference map and refined with the distance restraints (dfix) with d(n–h = 0.86 å and with uiso(h) = 1.2ueq(n). all other hydrogen atoms were included in the model at geometrically calculated positions and refined using a riding model. the f4 fluorine atom in bf4 – is disordered over two orientations and was refined with the use of part instruction. the occupancy of f4a and f4b refined to the ratio of 55 and 45%, respectively. ccdc 2271001 contains the supplementary crystallographic data for this paper. these data can be obtained free of charge from the cambridge crystallographic data centre via www.ccdc.cam.ac.uk/data_request/cif. table i. crystal data and structure refinement details for [cu2l2(1,3-n3)2](bf4)2 formula c28h38b2cu2f8n16o2 fw (g mol–1) 931.44 crystal size (mm) 0.500.400.10 crystal color green crystal system triclinic space group p –1 a (å) 7.8476(3) b (å) 9.8765(6) c (å) 13.2990(9) α (º) 110.341(6) β (º) 103.742(5) γ (º) 92.524(4) v (å3) 929.70(10) z 1 calcd density (g cm-3) 1.664 f(000) 474 no. of collected reflns 8891 no. of independent reflns 4259 rint 0.0430 no. of reflns observed 3639 no. parameters 279 r[i> 2σ (i)]a 0.0595 wr2(all data) b 0.1815 goof, sc 1.051 δρmax/δρmin (eå 3) +0.88/–0.80 a cc ep te d m an us cr ip t http://www.ccdc.cam.ac.uk/data_request/cif binuclear azide-bridged hydrazone cu(ii) complex 5 a r = ∑||fo| – |fc||/∑|fo|. b wr2 = {∑[w(fo2 – fc2)2]/∑[w(fo2)2]}1/2. cs = {∑[(fo 2 – fc 2)2]/(n/p}1/2 where n is the number of reflections and p is the total number of parameters refined. antimicrobial activity in vitro antibacterial and antifungal activity was tested against four gram-positive bacteria(bacillus subtilis atcc 6633, clostridium sporogenes atcc 19404, kocuria rhizophila atcc 9341, staphylococcus aureus atcc 6538), four gram-negative bacteria (proteus hauseri atcc 13315, escherichia coli atcc 25922, pseudomonas aeruginosa atcc 9027,salmonella enterica atcc 13076), and three fungal strains (aspergillus brasiliensis atcc16404, candida albicans atcc 10231, saccharomyces cerevisae atcc 9763), by the double dilution method in microtiter plates 23. antibacterial activity was determined using mueller hinton broth, whereas antifungal activity was determined using sabouraud dextrose broth. one hundred microliters of fresh mueller hintonor sabouraud dextrose broth were added to each well of the plate. then, 100 µl of the compounds stock solution (10 mg/ml) prepared by dissolving compounds in dmso was added. each well was inoculated with 10 µl (106 cells per ml) of bacterial cultures and 10 µl (105 spores per ml) of fungal strains for antibacterial and antifungal determination, respectively. bacterial strains were incubated at 37 °c for 24 h. erythromycin was used as a positive control, while water served as a negative control. fungal strains were incubated at 28 °c for 48 h. amphotericin b was used as a positive control, while dmso was used as a negative control. the bacterial growth was visualized by adding 20 μl of 0.5% 2,3,5-triphenyltetrazolium chloride (ttc) aqueous solution.24 the mic was determined as the lowest concentration that resulted in inhibition of visible microbial growth. the brine shrimp test the brine shrimp test was performed against freshly hatched nauplii of artemia salina.25 the compounds were dissolved in dmso and various amounts (0.01–1 mg) were added to artificial sea water containing 10–20 nauplii. after 24 h illumination at room temperature, the number of dead and surviving nauplii were counted and statistically analyzed. all samples were tested in triplicate. lc50 was defined as concentration of compounds that caused the death of 50% of nauplii. assessment of radical-scavenging activity antioxidative activity of initial cu(ii) salt, appropriate ligand and the synthesized complex was determined using a dpph (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay. all tested compounds were dissolved in dmso (stock concentrations were 10 mg/ml). foreach tested compound, two rows of the 96-well microplate were used: one for measuring the absorbance of the compounds themselves and the other for antioxidant activity. 50 μl of stock solutions of tested compounds were loaded into plate and double diluted by using multi-channel pipette. the rows used to measure the absorbance of the compounds were supplemented with 100 μl of pure methanol, while the other rows were supplemented with 100 μl of freshly prepared methanolic dpph solution (6.58 × 10-5 m). in the control, 50 μl pure dmso was loaded. final concentrations of the compounds ranged from (0.5 mg per well) to (2.4∙10-4 mg per well). after 30 min of incubation at 37 °c in the dark, the absorption was measured at 517 nm. all measurements were done in triplicate. free radical scavenging activity of compounds was measured using the equation: a cc ep te d m an us cr ip t 6 vitomirov et al. activity = 100×(𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙− (𝐴𝑠𝑎𝑚𝑝𝑙𝑒−𝐴0)) 𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 (1) where acontrol is the absorbance of dpph in the control probe, asample is the absorbance of dpph in the samples, and a0 is the absorbance of solutions of complexes 1 and 2 in dmso, due to their intensive green colors. ic50 is defined as the concentration of antioxidant agent necessary to reduce the starting amount of dpph by 50% and is calculated from the concentration-dependent free radical scavenging activity graph. ascorbic acid was used as a control probe. results and discussion the ligand synthesis was carried out in two steps, as presented in fig. 1. after acetylation of 7-bromo-6-azaindole, the obtained 7-acetyl-6-azaindole reacted with girard’s t reagent in a molar ratio 1:1 and the ligand was formed. the complex was then obtained in the reaction of cu(bf4)2·6h2o, ligand and nan3 in a molar ratio 1:1:4, as described in fig. 2. fig. 1. step by step ligand synthesis fig. 2. complex synthesis ir spectra ir spectroscopy confirmed the coordination of the ligand via the pyrrole nitrogen of 7-acetyl-6-azaindole ring. the stretching vibrational mode of the n–h group appears in the ir spectrum of the ligand as a wide band at 3377.4 cm-1 and disappears in the spectrum of the complex. the ligand spectrum also shows a very intense band at 1703.4 cm-1, corresponding to the c=o stretching vibration of the carbonyl group. this peak does not appear in the spectrum of the complex, instead, a cc ep te d m an us cr ip t binuclear azide-bridged hydrazone cu(ii) complex 7 an intense peak appears at 1034.8 cm-1, coming from the c–o stretching vibration. these all indicate the delocalization of the electron pair of the carbonyl group towards oxygen, which then favours the coordination of the ligand via the now negatively charged oxygen atom. the strong band appearing in the ir spectrum of the complex at 2063.4 cm-1 points out the presence of the n3– group in the complex structure. x-ray crystal structure determination complex crystallizes in the triclinic space group p–1, with the asymmetric unit comprising one cu(ii) centre, zwitterionic ligand l, one azide (n3−) ligand, and bf4− counter anion. the crystal structure displays a centrosymmetric binuclear complex with the crystallographicaly independent cu1 centre being coordinated to three donor atoms (n1, n3 and o1) of l and two n atoms (n6 and n8a where a = −x,−y,−z+1) from two azide ligands which bridge two cu(ii) centres by adopting a double end-to-end (di--1,3-n3) coordination mode. the cu1cu1a separation is 4.8232(6) å. the molecular structure of the dimeric cationic moiety in the [cu2l2(1,3-n3)2](bf4)2 complex and the atom-labelling scheme are shown in fig. 3. selected bond lengths and angles for the structure are given in table s-i in the supplementary material. the coordination polyhedron around cu(ii) is described as an axially elongated square pyramid with an index of trigonality (5) 26 of 0.05 [5 = (−)/60, where  and  are the two largest angles around the central atom]. the 5 is 0 for regular square based pyramidal geometry and 1.00 for regular trigonal bipyramidal geometry. the four in-plane bond distances are: cu1–n1 1.972(3), cu1–n3 1.975(3), cu1–o1 1.930(3) and cu1–n6 1.957(3) å, and the apical distance cu1–n8a (symmetry code a =−x,−y,−z+1)is 2.426(4) å. the azide anion bridges in an asymmetric (basal–apical) fashion so that the in-plane and axial cu–n(n3−) bond distances are significantly different. the tridentate nno coordination of l to cu(ii) ion generates one six-membered chelate ring (cu–n– c–c–c–n) and one five-membered chelate ring (cu–n–n–c–o) fused along the cu1–n3 bond. the chelate rings are non-coplanar, as indicated by the dihedral angle of 3.8 å. in this complex the cu–nar and cu–nimine bonds are comparable in length (cu1–n1 1.972(3) and cu1–n3 1.975(3) å, respectively). however, in cu(ii) complexes with two fused five-membered chelate rings generated by chelation of tridentate nno donor hydrazone ligands the cu–nar bonds are longer than the cu–nimine bonds.27,28 complex cations and bf4− anions generate a threedimensional structure by means of intermolecular n–hf, c–hf and c–hn hydrogen bonds given in table s-ii. in addition, the complex cations are connected by means of intermolecular  bonds extending in [110] direction (table s-iii, fig. s-1). a search of the cambridge structural database (csd)29 for the binuclear cu(ii)-azido complexes with hydrazone-based nno-donor ligands revealed 12 crystal structures with double end-on (di--1,1-n3) coordination mode of bridging a cc ep te d m an us cr ip t 8 vitomirov et al. azide anions. no crystal structure of binuclear cu(ii)-azido complex with tridentate nno-donor hydrazone ligand having double end-to-end (di--1,3-n3) coordination mode of bridging azides has been observed. the structure presented here is the first case. details of csd search are given in table s-iv in the supplementary material. crystallographic programs ortep-3 for windows30 and mercury31 were used to prepare the drawings. fig. 3. ortep presentation of the complex cation [cu2l2(1,3-n3)2] 2+ in [cu2l2(1,3-n3)2](bf4)2. thermal ellipsoids are drawn at the 30% probability level. the unlabeled part of the dimeric molecule is generated by symmetry operation –x, –y, –z+1. antimicrobial activity antimicrobial activity of the complex and the starting compounds (cu(bf4)2·6h2o and the ligand) was studied in dmso solution by examining their minimum inhibitory concentration (mic) on gram-positive and gram-negative bacteria and fungal strains. erythromycin and amphotericin b were used as the standard drugs to compare the minimum inhibitory concentration values and the results are presented in table ii. comparing the activities of the complex and the a cc ep te d m an us cr ip t binuclear azide-bridged hydrazone cu(ii) complex 9 ligand itself, the complex was shown to have higher antibacterial activity against all tested gram-positive bacteria and half of the tested gram-negative bacteria. the most significant difference between the antimicrobial activities of the complex and the ligand is the one against s. aureus, where the activity of the complex is 3 times higher than that of the ligand alone. the antifungal activities of the complex are significantly higher than those of the ligand, especially in the case of s. cerevisiae, where the antifungal activity of the complex is even comparable to the activity of amphotericin b. table ii. antimicrobial activity of the complex and the starting compounds (mic values in mm) microorganism complex ligand cu(bf4)2·6h2o standard a,b e. coli 0.671 1.011 1.821 0.038 p. aeruginosa 0.671 1.011 3.642 0.076 p. hauseri 1.342 1.011 3.642 0.038 s. enterica 0.671 0.505 3.642 0.038 s. aureus 0.671 2.022 3.642 0.076 c. sporogenes 0.671 1.011 3.642 0.076 k. rhizophila 1.342 2.022 3.642 0.076 b. subtilis 0.671 1.011 3.642 0.076 s. cerevisiae 0.084 2.022 3.673 0.011 c. albicans 0.168 4.044 7.346 0.022 a. brasiliensis 0.356 4.044 3.673 0.044 astandard used for bacterial strains was erythromycin. bstandard used for fungal strains was amphotericin b. the brine shrimp test the brine shrimp lethality bioassay is an excellent predictive tool for the toxic potential of new bioactive compounds. the results of this test can be extrapolated to cell-line toxicity and anti-tumor activity.25,32 the results presented in table iii showed high toxicity of the ligand, compared to moderate toxicities of the complex and the cu(ii) salt. this moderate toxicity of the synthesized complex may indicate its potential as a new active drug. table iii. results of the brine shrimp test (lc50, in mm) for the complex and the starting compounds compound lc50(mm) complex 0.613±0.051 ligand 0.238±0.092 cu(bf4)2·6h2o 1.174±0.155 k2cr2o7 0.077±0.016 assessment of radical-scavenging activity assessment of radical-scavenging capacity was determined using a dpph free radical scavenging assay and the results are presented in table iv. according to a cc ep te d m an us cr ip t 10 vitomirov et al. the obtained results, the basic salt and ligand did not possess dpph radical scavenging capacity, whereas the complex showed moderate antioxidant activity. table iv. dpph radical scavenging (ic50, in mm) of starting compounds and the complex compound ic50 (mm) complex 1.450±0.036 ligand 7.834±0.126 cu(bf4)2·6h2o >9.651 ascorbic acid 0.079±0.003 conclusion in this paper we have thoroughly described the synthesis and characterization of a new binuclear azide-bridged cu(ii) complex containing nno-donor hydrazone ligands obtained in a condensation reaction between 7-acetyl-6azaindole and girard's t reagent. the x-ray crystallographic analysis of the complex revealed a binuclear structure, in which two cu(ii) ions are bridged by two end-to-end (di-µ1,3-n3) azide ligands, while the hydrazone ligands coordinate to each cu(ii) center in a tridentate manner forming an axially elongated square pyramidal geometry around each metal ion. the complex crystallizes in the triclinic space group p–1. detailed research of the cambridge structural database revealed there are no crystal structures of binuclear cu(ii)-azido complexes with tridentate nno-donor hydrazone ligands having double end-to-end (di--1,3-n3) coordination mode of bridging azides, making this complex structure unique and rather interesting. antimicrobial activity of the complex was also examined, and it was shown that the complex exhibits higher antibacterial activity towards all tested gram-positive bacteria than the ligand itself, while the antifungal activity of the complex towards all tested fungal strains was not only higher than that of the ligand, but also comparable to the activity of the standard drug. results obtained in the evaluation of antimicrobial activity of the complex may indicate its potential as an antifungal agent. acknowledgment. this research has been financially supported by the ministry of science, technological development and innovation of republic of serbia, contract numbers: 451-0347/2023-01/200026, 451-03-47/2023-01/200168 and 451-03-47/2023-01/200288, as well as by the science fund of the republic of serbia, #7750288, tailoring molecular magnets and catalysts based on transition metal complexes – tmmagcat, and by the slovenian research agency (arrs), grant number p1-0175. we thank the en-fist centre of excellence, ljubljana, slovenia, for the use of the supernova difractometer. supplementary material supplementary materials are available electronically from https://www.shdpub.org.rs/index.php/jscs/article/view/12452, or from the corresponding authors on request. a cc ep te d m an us cr ip t https://www.shd-pub.org.rs/index.php/jscs/article/view/12452 https://www.shd-pub.org.rs/index.php/jscs/article/view/12452 binuclear azide-bridged hydrazone cu(ii) complex 11 и з в о д динуклеарни хидразонски комплекс cu(ii) са азидним мостом: синтеза, карактеризација и евалуација биолошке активности теодора витомиров1, божидар чобељић1, андреј певец2, душанка радановић3, ирена новаковић3, милица савић3, катарина анђелковић1, маја шумар-ристовић1,* 1универзитет у београду – хемијски факултет, студентски трг 12–16, 11000 београд, србија, 2факултет за хемију и хемијску технологију, универзитет у љубљани, вечна пот 113, 1000 љубљана, словенија, и 3универзитет у београду, институт за хемију, технологију и металургију, центар за хемију, његошева 12, 11000 београд, србија кондензациони производ 7-ацетил-6-азаиндола и жираровог т реагенса (лиганд hl) коришћен је као лиганд у реакцији са cu(bf4)2·6h2o и nan3. реакција је довела до формирања бинуклеарног cu(ii) комплекса који садржи два азидна моста у „end-to-end“ (di--1,3-n3) моду, као и два nno-донорска хидразонска лиганда који заједно формирају аксијално издужену квадратно-пирамидалну геометрију око сваког централног металног јона. овај „end-to-end“ (di--1,3-n3) азидни мост се до сада није појављивао у структурама бакар(ii) комплекса који садрже nno-донорске хидразонске лиганде, што чини структуру комплекса још интересантнијом за будућа испитивања. овај комплекс је окарактерисан елементалном анализом, иц спектроскопијом и рендгенском структурном анализом и пронађено је да кристалише у триклиничној просторној групи p–1 са асиметричном јединицом која се састоји из једног cu(ii) центра, цвитер-јонског лиганда (l), једног азидног лиганда (n3−) и bf4− контра-јона. испитивање антимикробне активности комплекса показало је вишу антифунгалну активност, као и вишу антибактеријску активност према грам-позитивним бактеријама, у односу на сам хидразонски лиганд, док је антифунгална активност комплекса чак упоредива са активношћу амфотерицина б који је коришћен као стандард. 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(https://doi.org/10.1002/pca.2800020303) a cc ep te d m an us cr ip t https://doi.org/10.1107/s2052520616003954 https://doi.org/10.1107/s0021889812029111 https://doi.org/10.1107/s002188980600731x https://doi.org/10.1002/pca.2800020303 404 not found enhancement of amylase production by aspergillus sp. using carbohydrates mixtures from triticale j. serb. chem. soc. 80 (10) 1279–1288 (2015) udc 582.282.123.4:547.454:664.644.5+ jscs–4796 663.142/.143:577.15.002.2+633.11’13 original scientific paper 1279 enhancement of amylase production by aspergillus sp. using carbohydrates mixtures from triticale biljana dojnov1*, marica grujić2, bojana perčević2 and zoran vujčić2 1department of chemistry, institute of chemistry, technology and metallurgy, university of belgrade, njegoševa 12, belgrade, serbia and 2department of biochemistry, faculty of chemistry, university of belgrade, studentski trg 12–16, belgrade, serbia (received 17 march, revised 7 may, accepted 12 may 2015) abstract: with the purpose of finding a suitable available inducer in combination with starvation, carbohydrate mixtures from triticale were used and compared with well-known amylase inducers in fungi. carbohydrate mixtures from triticale induced the production of an amylase cocktail (α-amylase and glucoamylase) in aspergillus niger, unlike induction with well-known inducers that induce only glucoamylase, shown by zymography and tlc analysis of the carbohydrate mixtures before and after fermentation. glucoamylase production by a. niger was the highest in the presence of the extract obtained after autohydrolysis of starch from triticale (95.88 u ml-1). carbohydrate mixtures from triticale induced the production of α-amylase in a. oryzae. more α-amylase isoforms were detected when using a complex carbohydrate mixture, compared to induction with maltose or starch. a 48-h induction was the most efficient using a triticale extract (101.35 u ml-1). carbohydrates from triticale extracts could be used as very good cheap amylase inducers. triticale, still not fully utilized, could be taken into consideration as an inducer in amylase production by aspergillus sp, and in such a way, it could be used as the sole substrate in fermentation. keywords: α-amylase; glucoamylase; maltose; starch; enzyme production; fungi. introduction the fungi aspergillus sp. are well-known producers of amylases, which are industrially important enzymes. filamentous fungi produce hydrolytic enzymes in the form of enzymes mixtures – cocktails. glucoamylase and α-amylase are produced concomitantly in fungal fermentations.1–4 maximal production of the enzymes may be achieved by using appropriate inducer molecules. induction is the main controlling mechanism in the pro * corresponding author. e-mail: bdojnov@chem.bg.ac.rs doi: 10.2298/jsc150317043d _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1280 dojnov et al. duction of amylases in fungi. the effects of small molecules on amylase production were examined even when using complicated bioreactors.5 it is known that α-amylase and glucoamylase are inducible enzymes in aspergillus sp.6 the induction mechanism of amylase production in aspergillus sp. has been successfully studied.7,8 induction study of α-amylase production in a. oryzae began in 1961.9 starch and its hydrolysis products, mostly studied is maltose, are known inducer of α-amylase in a. oryzae.10–12 maltose is also a good glucoamylase inducer in a. niger.13 starvation launches a special mechanism leading to increased amylase production in aspergillus sp.14 considering all the above known, the induction mechanisms and the fact that aspergillus sp. produce concomitantly α-amylase and glucoamylase, a cost -effective fermentation could be optimized by using inducers for the production of special amylase cocktail. this brought about the idea to examine the possibility of using a carbohydrate mixture from triticale (x triticosecale, wittmak). triticale is an important industrial crop insufficiently utilized yet. triticale cultivation has many benefits compared to other crops and its production and use have been intensively studied.5,15 triticale contains high amounts of starch (about 60 %) and protein (from 12 to 15 %).16 it also contains higher amounts of major mineral elements (k, p and mg) and nutritionally important minor elements (na, mn, fe, cu and zn) than wheat.17 the opportunity of using cost effective and available carbohydrate mixtures from triticale as inducers of amylase in the two most important fungal producers of amylase, a. niger and a. oryzae, were examined in this study. two kinds of triticale extracts, starchy extract and the extract obtained after starch hydrolysis by endogenous amylases were used as the sole fermentation substrates in submerged fermentation (smf) and were compared with synthetic media containing the known inducers maltose and starch. the induction mechanisms were combined with mycelial starvation to cover all known methods of induction. experimental reagents all used reagents and solvents were of the highest purity and purchased from merck and sigma–aldrich. triticale (x triticosecale sp.) “rtanj” line was obtained from the “center for small grains kragujevac”, kragujevac, serbia. microorganisms and fermentation conditions aspergilus niger atcc 10864 and a. oryzae atcc 56747 strains were cultivated while obtaining matured spores. spore suspensions were prepared in a 0.1 % tween 80 solution at a concentration of 5.9×105 spores ml-1. fermentations two parallel fermentations were performed with a. niger and a. oryzae. submerged fermentations (smf) were performed for 73 h at 30 °c and 210 rpm. the spore suspensions (10 vol. %) were inoculated in czapec solution with 0.5 % yeast extract. fungal mycelia _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ enhancement of amylase production using triticale 1281 obtained in 18 h (5 %) were transferred into induction or non-induction media, as shown in experiments scheme in fig. 1. fungal mycelia were washed twice with water and dried with filter paper between the experimental phases shown in schematic diagram. fermentations were stopped after scheduled time (fig. 1) by centrifugation of the biomass for 15 min at 5000×g. fig. 1. schematic presentation of amylase induction in a. niger and a. oryzae. mg – medium with glycerol, sm – starvation medium, mm – medium with maltose, at – autohydrolysate of triticale extract, t – triticale extract, ms – medium with starch. compositions of specified substrates are given in table i. table i. the compositions of the media used in the fermentations by a. niger and a. oryzae following the scheme shown in fig. 1 medium composition mg medium with glycerol peptone 20 g l-1; glycerol 30 g l-1; kh2po4 5 g l-1; mgso4 2.5 g l-1 sm starvation medium kh2po4 5g l-1; mgso4 2.5 g l-1 mm medium with maltose peptone20 g l-1; maltose 30 g l-1; kh2po4 5g l-1; mgso4 2.5 g l-1 at autohydrolysate of triticale extract decanted extract obtained after incubation of milled triticale (x triticosecale sp.) and water in 1:3 ratio, autohydrolysis at 60 °c for 3 ha t triticale extract decanted extract obtained from mixing milled triticale (x triticosecale sp.) and water in 1:3 ratio without auto hydrolysis of starch ms medium with starch peptone 20 g l-1; raw starch 30 g l-1; kh2po4 5 g l-1; mgso4 2.5 g l-1 apreparation of autohydrolysate of the triticale extract is described in the experimental, media preparation _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1282 dojnov et al. media preparation the compositions of the media used in the examination of the induction of amylase production by aspergillus sp. are given in table i. triticale was finely ground to flour using a “bragal” mill. the triticale flour was suspended in water (1:3 w/v ratio), mixed and strained through a strainer to obtain the triticale extract (t). the autohydrolysate of triticale (at) was prepared by incubation of suspension of triticale flour and water (1:3 w/v ratio) for 3 h at 60 °c.18 the obtained suspension was strained through a strainer to separate the liquid from solid part. the liquid phase was used as the at. the other media were prepared by mixing the individual components. all media were autoclaved under standard conditions prior to use. amylase activity assay the amylase activity was assayed at ph 5.0 according to the dinitrosalicylic acid (dns) procedure19 using of 1.0 % (w/v) soluble starch as substrate, for 30 min at 35 °c. maltose was used as the standard. each data point represents the mean of three independent assays (standard error, se, values were less than 5 % of the means). one unit of α-amylase activity was defined as the amount of enzyme required to produce 1 μmol of maltose in 1 min at 35 °c. glucoamylase activity assay glucoamylase activity was assayed at ph 5.0 using 1.0 % (w/v) soluble starch as the substrate in 30 min at 35 °c. glucose (final product of the reaction) was detected in the reaction mixture by coupled reaction with glucose oxidase and horseradish peroxidase (hrpo, trinder reagent). each data point represents the mean of three independent assays (se values were less than 5 % of the means). one unit of glucoamylase activity was defined as the amount of enzyme required to produce 1 μmol of glucose in 1 min at 35 °c. zymographic detection of α-amylase and glucoamylase α-amylase and glucoamylase were detected simultaneously using zymography.4 α-amylase was detected in a paa (polyacrylamide) gel with copolymerized β-limit dextrins, stained by iodine solution. the α-amylase activity appeared as clear bands on a purple background. both amylases were detected as clear bands on a blue background, using soluble starch as substrate and iodine solution for staining, in the native ef (electrophoresis) paa gel after printing. glucoamylases were detected on an nc (nitrocellulose) membrane using a substrate solution (1.0 % (w/v) starch in buffer) and a reaction mixture for glucose detection (glucose oxidase, hrpo and 4-cl-α-naphthol as substrate). specific reaction product, purple and insoluble, appeared on the nc in bands corresponding to glucoamylase. starch, reducing sugar and glucose concentrations the concentrations of starch in triticale extract and triticale autohydrolysate were determined by the iodine dextrine color (idc) method by measuring the absorbance at 590 nm.20 reducing sugars were determined by the 3,5-dinitrosalicylic acid (dns) method19 using maltose as the standard, while glucose concentration was measured by trinder reagent. tlc analysis of carbohydrates in triticale extract and triticale autohydrolysate carbohydrates were detected by thin layer chromatography (tlc) on silica plates, 4.5 cm×6 cm (silica gel 60 f-254, merck, darmstadt, germany), using a camag development chamber in the tank configuration. the plates were developed by the double-ascending method in a solvent system consisting of butan-1-ol, ethanol, water and glacial acetic acid (5:3:2:0.5 volume ratio). standard solution of the oligosaccharides mixtures (1.0 mg ml-1 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ enhancement of amylase production using triticale 1283 each) was prepared in water and they consisted of: glucose (c1), maltose (c2), maltotriose (c3), maltotetraose (c4), maltopentaose (c5), maltohexaose (c6) and maltoheptaose (c7) (across and sigma aldrich, usa). all separations were performed at ambient temperature (22±2 °c). the carbohydrates were detected by spraying the plates with an ethanolic solution containing 0.5 % (w/v) α-naphthol and 5 vol. % h2so4, followed by heating for 10 min at 120 °c. results and discussion induction of amylase cocktails in a. niger and a. oryzae were examined by submerged fermentations (smf) according to scheme showed in fig. 1. the impact of two kinds of triticale extracts were compared with the impacts of known amylase inducers using various media, the compositions of which are given in table i. carbohydrate composition of triticale extract and triticale autohydrolysate triticale extract (t) and triticale autohydrolysate (at) differed in their carbohydrate contents, especially in their starch contents, table ii. triticale grains contain more than 60 % starch, classifying it as a starchy cereal.16,21 the concentration of starch in the triticale extract used in this research was 10 mg ml–1, which represents the quantity of starch available to the fungi during fermentation. only a trace of starch was detected in triticale autohydrolysate because of starch hydrolysis during the autohydrolysis process by the α-amylase contained in the triticale. the amount of reducing sugars was increased 12.5 times after autohydrolysis, which corresponds to the decreased starch content. table ii. starch, reducing sugar and glucose contents in triticale extract and triticale autohydrolysate sample starch, mg ml-1 reducing sugars, mm glucose, mm triticale extract (t) 10.24 16.60 11.04 triticale autohydrolysate (at) 0.75 201.09 146.18 the tlc analysis revealed that the triticale extract contained a wide range of carbohydrates, fig. 2c, lane t. maltose was the most abundant carbohydrate, apart from glucose and maltotriose, in triticale extract after autohydrolysis, fig. 2c, lane at. these differences suggest potential different induction of α-amylase and glucoamylase in aspergillus sp. induction of amylase production in aspergillus sp. non-growing mycelia of aspergillus sp. are a good model system of amylase induction.11 for this reason, 20-h cultures of both aspergillus species were used. starvation before addition of carbohydrates is well known as good method for enzyme induction.11,14,22 starvation of aspergillus mycelia for 5 h was applied before adding the inducers to the medium, fig. 1. this enables fungi to meta_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1284 dojnov et al. bolize all ingredients present in growth medium and to maximalize the uptake of new molecules added after starvation. all inductions were monitored after 2 h (rapid induction) to compare the impact of maltose and starch11 with the impact of the triticale extract and of the triticale autohydrolysate. the inductions were further monitored and production levels were determinate in 48 h, which is actually 72 h after the start, because it is commonly used fermentation time for fungi smf. fig. 2. glucoamylase and α-amylase production by a. niger depending on the type of inducers (carbohydrate source) after 2 and 48 h. a) enzymatic activities, u ml-1, after 2 h of induction; b) enzymatic activities, u ml-1, after 48 h of induction; c) tlc analysis of the carbohydrates in the smf samples; s – standard carbohydrates: 1 – glucose, 2 – maltose, 3 – maltotriose, 4 – maltotetraose, 5 – maltopentaose, 6 – maltohexaose and 7 – maltoheptaose; d) zymographic detection of α-amylase and glucoamylase in the smf samples. the arrows indicate the positions of the α-amylase isoforms (α-a1 to α-a4) and the glucoamylase isoform (g-a1). g – glycerol, mm – medium with maltose, at– autohydrolysate of the triticale extract, t – triticale extract and ms – medium with starch. induction of glucoamylase and α-amylase production in a. niger the impact of all inducers on a. niger amylases production were monitored by enzymatic assays, tlc analysis of the obtained carbohydrates, and zymogram detection of α-amylase and glucoamylase in the fermentation extracts and the results are presented in fig. 2. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ enhancement of amylase production using triticale 1285 rapid induction (after 2 h) with triticale extract and triticale autohydrolysate led only to a noticeable increase in the α-amylase production, as evidenced by the enzymatic assay and zymogram, fig. 2a and d. low levels of both amylases were detected in all the examined extracts, indicating that 2 h was too short for production. the glucoamylase level was lower in the medium with maltose than in medium with glycerol, which is contrary to literature results.11 however, as this was not the case after the 48 h of fermentation with induction (fig. 2b), it could be because the mycelia had not started to express the induced enzymes within 2 h. levels of enzymes detected after 2 h in medium with glycerol originated from the standard enzyme pool. the results obtained for a. oryzae confirmed this assumption, fig. 3a and b. the observation indicated that it is necessary to monitor the fermentation for 48 h. fig. 3. production of α-amylase by a. oryzae in dependence on the type of inducer (carbohydrate source) after 2and 48-h fermentation. a) and b) enzymatic activities, u ml-1, after 2 and 48 h of induction, respectively; c) tlc analysis of carbohydrates in the smf samples; s – standard carbohydrates: 1 – glucose, 2 – maltose, 3 – maltotriose, 4 – maltotetraose, 5 – maltopentaose, 6 – maltohexaose and 7 – maltoheptaose; d) zymographic detection of α-amylase and glucoamylase in the smf samples. the arrows indicate the positions of the α-amylase isoforms (α-a1 to α-a5). g – glycerol, mm – medium with maltose, at– autohydrolysate of the triticale extract, t – triticale extract and ms – medium with starch. the fact that glucoamylase and α-amylase were produced concomitantly is often ignored and a. niger was shown as a producer of glucoamylase solely.23–25 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1286 dojnov et al. the results prove that a. niger produce glucoamylase and α-amylase in different ratio, depending on the applied inducers. maltose was a strong inducer of glucoamylase after 48 h, fig. 2b and d, which was shown by using maltose and at as inducers. maltose was the most abundant carbohydrate in at according to tlc analysis, fig. 2c. the high level of glucoamylase in the fermentation with at was confirmed by enzymatic assay, specific zymogram detection and tlc, as well the detection of a high quantity of glucose. maltose is a well-known glucoamylase inducer.13,26 the obtained results confirmed this, and showed that maltose was a better glucoamylase inducer when used in a mixture with the other carbohydrates (maltotriose and glucose) in at. a. niger produced glucoamylase when cultivated on starch as a carbon source – control medium, and with t. this proves that starch is a good amylase inducer and the starch hydrolysis products formed during fermentation are especially good amylase inducers. the use of t as an inducer favors the production of α-amylase in a. niger, figs. 2b and d. the choice of the carbohydrate mixture as inducer affects various amylase complexes leading to enrichment with glucoamylase if at was used or α-amylase if t was used. induction of α-amylase production by a. oryzae the impacts of all the examined carbohydrates as inducers on the production of amylases by a. oryzae were monitored in the same way as for a. niger and the results are shown in fig. 3. a. oryzae produced only α-amylase isoforms in all the examined fermentations, fig. 3d. the period of 2 h was too short for production according to the obtained low level of amylase, fig. 3a. t and at proved to be better α-amylase inducers than maltose and starch after 48 h induction. the triticale extract, containing a mixture of carbohydrates c1 to c7 (fig. 3c lane t), induced the highest amount of α-amylase production after 48 h (fig. 3b). carbohydrate profile of starch hydrolysis products corresponded to typical fungal α-amylase profiles after 48 h fermentation with t and at inducers (fig. 3c).14 starch and its hydrolysis products are well known inducers of α-amylase.10,11 this was also shown in the presented results obtained using starch as the carbon source (control medium) and, particularly, the triticale extract in the fermentation. the best-known and most studied inducers of α-amylase in aspergillus sp. are maltose and isomaltose, arising from maltose during fermentations.7,9,11 moreover, the obtained results confirmed that maltose is a good inducer for α-amylase in a. oryzae, using maltose and at, which contained a high amount of maltose (fig. 3c line at). lead to increase in α-amylase production after 48 h, of which the triticale extract was the most effective, fig. 3b. the tlc analysis (fig. 3c) showed that the t extract contained a spectrum of carbohydrates from c1 to c5, responsible _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ enhancement of amylase production using triticale 1287 for the highest level of α-amylase production. both types of triticale extracts induced as many as five α-amylase isoforms, fig. 3d. major α-amylase isoform (α-a2) was presented in all tested samples. however, only α-a1 and α-a2 were present when a. oryzae was cultivated on starch. this further favors the usage of the triticale extracts as the α-amylase inducer in a. oryzae because the presence of more enzyme isoforms in an enzyme preparation provides easier adaptation to the required industrial conditions of starch hydrolysis. conclusions the presented results satisfied the aims set out in the introduction section, i.e., improving amylase production levels and allowing the use of a single fungal strain and a cheap and accessible inducer for the production of specific amylase complexes that might give different product profiles of starch hydrolysis depending on the industrial requirements. this could open a new chapter in triticale utilization. it could be considered as a universal means, as was proven for the two most widely used fungal amylase producer strains. the benefits derived from the consequences of the presented results might be a greater use of triticale, otherwise insufficiently used, as well as higher fungal amylase production. acknowledgments. this study was supported by a grant from the ministry of education, science and technological development of the republic of serbia (project no. 172048). we are grateful to olga reljić, professor of english, for the english corrections. и з в о д повећање продукције амилаза смешом угљених хидрата из тритикала коришћењем aspergillus sp. биљана дојнов1, марица грујић2, бојана перчевић2 и зоран вујчић2 1центар за хемију, институт за хемију, технологију и металургију универзитета у београду, његошева 12, београд и 2катедра за биохемију, хемијски факултет, универзитет у београду, студентски трг 12–16, београд у циљу проналажења одговарајућег лако доступног индуктора гљивичних амилаза у комбинацији са гладовањем, смеша угљених хидрата из тритикала је испитана и упоређена са већ описаним и познатим индукторима. смеша угљених хидрата из тритикала је код aspergillus niger индуковала продукцију амилазног коктела (α-амилазе и глукоамилазе), за разлику од индукције са добро познатим индукторима који индукују само глукоамилазу, што је показано зимограмом и tlc анализама угљених хидрата смеша пре и после ферментације. продукција глукоамилазе a. niger је била највећа у присуству екстракта добијеног после аутохидролизе скроба из тритикала (95,88 u/ml). смеша угљених хидрата из тритикала је код a. oryzae индуковала продукцију α-амилазе. значајно више α-амилазних изоформи је детектовано коришћењем комплексних смеша угљених хидрата као индуктора, у поређењу са малтозом или скробом. индукција у трајању од 48 h је најефикаснија када се користи екстракт тритикала (101,35 u ml-1). угљени хидрати из екстраката тритикала могу да се користе као веома добри и јефтини индуктори амилазе. тритикале, житарица која још увек није у потпуности искоришћенa, може се узети за разматрање као индуктор у производњи амилаза коришћењем aspergillus sp., и то тако да _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1288 dojnov et al. се користи као једини супстрат у подлози за ферментације без додатка других нутритивних елемената. 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(oxford, u.k.) 43 (2008) 280 26. m. i. rajoka, a. yasmeen, world j. microbiol. biotechnol. 21 (2005) 179. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice synthesis and antimicrobial evaluation of some novel thiomorpholine derived 1,4-disubstituted 1,2,3-triazoles j. serb. chem. soc. 81 (3) 233–242 (2016) udc 547.869.2+547.79:544.478–034.3+ jscs–4842 66.095.252.091.7:547.21.024’437:615.28–188 original scientific paper 233 synthesis and antimicrobial evaluation of some novel thiomorpholine derived 1,4-disubstituted 1,2,3-triazoles kumaraswamy battula1, sirassu narsimha1, vasudevareddy nagavelli1*, priyanka bollepelli2 and mutheneni srinivasa rao2 1department of chemistry, kakatiya university, warangal-506009, india and 2chemical biology laboratory, indian institute of chemical technology, hyderabad-500007, india (received 18 august, revised 13 october, accepted 21 october 2015) abstract: a convenient synthesis of novel 1,4-disubstituted 1,2,3-triazoles (4a–j and 5a–j) is reported via copper(i)-catalyzed one pot [3+2] cycloaddition of various alkyl halides, sodium azide with 4-(prop-2-yn-1-yl)thiomorpholine and 4-(prop-2-yn-1-yl)thiomorpholine 1,1-dioxide. all the synthesized compounds were investigated for their antimicrobial activity. compounds 4a, 4b, 4c, 4g, 5a and 5j against staphylococcus epidermidis, 4a, 5a and 5d against pseudomonas aeruginosa, 4a, 4b and 4g against klebsiella pneumoniae, 4b, 5a and 5d against s. aureus and 5b, 5e and 5j against bacillus subtilis showed excellent antibacterial activity compared to the standard drugs penicillin and streptomycin. compounds 4c, 4e, 4f, 4j, 5c, 5d, 5g and 5j registered moderate antifungal activity as compared with the standard drug amphotericin b. keywords: one pot synthesis; copper-catalyzed; azide alkyne cycloaddition; alkyl azides; antimicrobial activity. introduction nowadays, bacterial infection remains a serious threat to human lives due to their increasing resistance towards current antibiotics. thus, there is a big scope for the invention of new antimicrobial agents. 1,4-disubstituted 1,2,3-triazoles are known to possess many activities, such as anti-hiv,1,2 antimicrobial,3 anticancer,4–6 anti-inflammatory7 and fluorescent,8 as well as inhibitors of kinase-3β9,10 and other enzyme inhibitors.11–13 the 1,2,3-triazole moiety-containing drug molecules, such as tazobactam,14 cefatrizine15 and carboxyamidotriazole (cai),16 are available (fig. 1). there are various methods available for the one-pot synthesis of 1,4-disubstituted-1,2,3-triazoles.17–19 however, the copper(i)-catalyzed one-pot three -component cycloaddition of alkyl halides with sodium azide and terminal * corresponding author. e-mail: vasujac3@gmail.com doi: 10.2298/jsc150818088b _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 234 battula et al. alkynes is one of the best methods for the multicomponent regioselective synthesis of 1,4-disubstituted-1,2,3-triazoles.20 this method was independently pioneered by fokin et al.21 in 2004 through dramatic modification of the huisgen 1,3-dipolar cycloaddition reaction.22 fig. 1. some 1,2,3-triazole ring-containing drugs on the market. previously, the synthesis and antibacterial activity of 1,2,3-triazole derivatives of morpholine-3-carboxylic acid ester were reported.23 the morpholine moiety has played a significant role in medicinal chemistry.24 some of the morpholine ring-containing drugs are shown in fig. 2. thiomorpholine derivatives are known to exhibit biological activities including antimycobacterial,25 anticancer,26 anti-inflammatory and antioxidant agents27 and dipeptidyl peptidase iv (dpp-iv) inhibitors.28 in view of the above considerations and in continuation of ongoing research on the synthesis of 1,4-disubtituted-1,2,3-triazole derivatives, the synthesis of thiomorpholine derived 1,4-disubstituted-1,2,3-triazoles and their antimicrobial activity are reported herein. fig. 2. some of morpholine ring-containing drugs on the market. results and discussion chemistry in the present work, a series of novel thiomorpholine derived 1,4-disubtituted-1,2,3-triazoles was synthesized employing the copper-catalyzed azide alkyne cycloaddition (cuaac) reaction, scheme 1. the 1,4-disubstituted-1,2,3 -triazole derivatives were synthesized in a one-pot reaction, starting from alkyne, alkyl halide and sodium azide in the presence of cu(i) catalyst. 4-(prop-2-yn-1 -yl)thiomorpholine (2) was synthesized by reacting thiomorpholine with propargyl bromide in the presence of cs2co3 in acetone at room temperature. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. thiomorpholine derivatives as antimicrobial agents 235 4-(prop-2-yn-1-yl)thiomorpholine 1,1-dioxide (3) was obtained by oxidation of the sulfur group in 4-(prop-2-yn-1-yl)thiomorpholine with 3-chloroperbenzoic acid (mcpba) in dichloromethane at room temperature.29 cycloaddition of compounds 2 and 3 with in situ prepared alkyl azides in the presence of cu(i) catalyst yielded 1,4-disubstituted-1,2,3-triazole derivatives 4a–j and 5a–j in good yields (table i). the structures of the newly synthesized compounds were confirmed by spectral techniques, such as ir, 1h-nmr, 13c-nmr and esi-ms. scheme 1. synthetic route to the 1,4-disubstituted-1,2,3-triazoles 4a–j and 5a–j. reagents and conditions: i) propargyl bromide / cs2co3, acetone, r.t., 6 h; ii) mcpba/dcm, r.t., 12 h; iii) r-br / nan3, cui, thf–h2o (1:1), r.t.–50 °c, 8–12 h. table i. the 1,4-disubstituted-1,2,3-triazoles 4a–j and 5a–j synthesized from different alkyl bromides entry r1-br product entry product 4a c7h15br 5a n s n n n 6 o o 4b c8h17 br 5b 4c c10h21 br 5c _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 236 battula et al. table i. continued entry r1-br product entry product 4d c13h27 br 5d 4e c14h29 br 5e 4f c15h31 br 5f 4g c17h35 br 5g 4h c6h13br 5h 4i c4h7o2br 5i 4j c12h25br 5j spectral analysis the analytical and spectral data of the synthesized compounds are given in the supplementary material to this paper. in general, compounds containing the 1,2,3-triazole ring show absorption in the region 3000–3100 cm–1 in ir spectra. the newly synthesized compounds 4a–j and 5a–j exhibited a strong absorption band in the region 3018–3037 cm–1, which confirmed the formation of the 1,2,3 -triazole ring. for convenience, compound 4a was selected for the nmr spectral _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. thiomorpholine derivatives as antimicrobial agents 237 discussion. the presence of a singlet peak that appeared in the downfield region at 7.76 ppm (1h, s) confirmed the formation of the 1,2,3-triazole ring. the triplet that appeared at 4.35 ppm (2h t, j = 7.32 hz) corresponded to an –n–n–ch2– group attached to a triazole ring. the singlet peak that appeared at 4.03 ppm (2h, s) corresponded to a –ch2–triazole group attached to thiomorpholine nitrogen. two sets of multiplet signals that appeared in the upfield region 3.20–2.78 ppm (8h, m) corresponded to the thiomorpholine –n–ch2 and –s–ch2 groups. the multiplet peak present in the upfield region at 1.90–1.80 ppm (2h, m) corresponded to the –n–n–ch2–ch2– group. the presence of one multiplet in the region 1.40–1.20 (8h, m) ppm confirmed the four methylene (–ch2–) groups in the n-heptyl chain. the triplet peak that appeared in the upfield region at 0.87 ppm (3h, t, j = 6.71 hz) corresponded to the methyl (–ch3) group. in the 13c-nmr spectrum of compound 4a, two carbon signals that appeared at 139.4 and 124.4 ppm confirmed the formation of the triazole ring. in the upfield region, the methyl group, thiomorpholine –s–ch2– and –n–ch2– carbons were observed at 13.7, 25.8 and 53.2 ppm, respectively. five methylene (–(ch2)5–) group carbons were observed in the region 31.2–22.2 ppm. thiomorpholine-n attached (–n–ch2–) and triazole attached (–n–n–ch2–) carbons were observed at 51.9 and 50.3 ppm, respectively. the esi-mass spectra showed a 283 (m+h) peak, which confirmed the molecular weight of compound 4a. antibacterial activity all the synthesized compounds 4a–j and 5a–j were investigated for their in vitro antibacterial activity against various gram-positive microorganisms, i.e., bacillus subtilis, staphylococcus aureus and s. epidermidis, and gram-negative microorganisms, i.e., escherichia coli, pseudomonas aeruginosa and klebsiella pneumoniae. penicillin and streptomycin were used as standard drugs for comparison. the results of the antibacterial activity screening (table s-i of the supplementary material to this paper) revealed that compound 4a, possessing an 1-heptyl group on the triazole ring, showed excellent inhibition against s. epidermidis, p. aeruginosa and k. pneumoniae microorganisms with minimal inhibitory concentration (mic) values of 2.34, 2.34 and 9.37 µg ml–1, respectively. compound 4b having a 1-octyl group exhibited excellent inhibition against s. epidermidis, s. aureus and k. pneumoniae with mic values of 1.17, 2.34 and 2.34 µg ml–1, respectively. compound 4c, bearing 1-decyl substitution, showed very good inhibition against s. epidermidis as compared with the standard drugs. compounds 4d, 4e and 4f, possessing longer alkyl carbon chains on the triazole ring, showed no inhibition of any of the bacterial strains even at the highest concentration of 150 µg ml–1. whereas, compound 4g, derived from the 1-heptadecyl group, exhibited excellent inhibition against s. epidermidis and k. pneumoniae strains with mic values of 1.17 and 4.68 µg ml–1, respectively. surpris_________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 238 battula et al. ingly, compound 4h, derived from the 1-hexyl group, exhibited no inhibition against any of the bacterial strains even at the highest concentration of 150 µg ml–1. compound 5a, possessing 1-heptyl substitution on the triazole ring, showed equipotent inhibition against s. aureus, s. epidermidis and p. aeruginosa strains with mic values of 2.34, 2.34 and 9.37 µg ml–1, respectively, in comparison with the standard drugs. compound 5b, derived from the 1-octyl group, exhibited excellent inhibition of b. subtilis and s. aureus strains at lower concentrations (2.34 and 4.68 µg ml–1). moreover, compound 5d against p. aeruginosa and 5e against b. subtilis exhibited very good inhibition with mic values of 4.68 and 9.37 µg ml–1, respectively. compounds 5f–i exhibited no inhibition of any of the tested strains even at the highest concentration of 150 µg ml–1. however, compound 5j, carrying an n-dodecyl group, showed very good inhibition against b. subtilis and s. epidermidis strains with mic values of 2.34 and 9.37 µg ml–1, respectively. among all the synthesized compounds, 4a, 4b and 5a showed equipotent to better antibacterial activity when compared with the activities of the thiomorpholine derivatives reported by several authors.30,31 the better activity of compounds 4a, 4b, 5a and 5b might be attributed to the presence of the 1-heptyl and 1-octyl groups on the 1,2,3-triazole ring. antifungal activity all the synthesized compounds 4a–j and 5a–j were investigated for their in vitro antifungal activity against the fungal strains candida albicans, saccharomyces cerevisiae, aspergillus niger and a. flavus. amphotericin b was used as the standard drug for comparison. the results of the in vitro antifungal activity screening (table s-ii of the supplementary material) revealed that compounds 4c, 4e and 5j exhibited moderate antifungal activity against c. albicans, a. niger and a. flavus strains with zones of inhibition ranging from 9 to 16 mm. compounds 4f, 4j, 5c and 5d exhibited moderate inhibition of c. albicans and a. niger strains with zones of inhibition ranging from 8 to 14 mm. furthermore, compound 5g registered moderate inhibition against c. albicans and a. flavus organisms. compounds 4a, 4b, 4d, 4g, 4h, 4i, 5a, 5b, 5e, 5f, 5h and 5i exhibited no inhibition against all fungal strains, even at the highest concentration of 150 µg ml–1. from the above observations, it is obvious that replacement of the 1-octyl group with 1-decyl, 1-dodecyl, 1-tetradecyl and 1-pentadecyl groups on the triazole ring resulted in better activity against all the tested organisms (except s. cerevisiae) as compared with the other synthesized thiomorpholine derivatives. materials and methods all the reagents and solvents were purchased from sigma–aldrich/merck chemicals, india and used without further purifications. thin-layer chromatography (tlc) was performed using merck silica gel 60 f254 precoated plates (0.25 mm) and silica gel (100–200 mesh) was used for column chromatography. the progress of the reactions as well as the _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. thiomorpholine derivatives as antimicrobial agents 239 purity of the compounds was monitored by thin layer chromatography using ethyl acetate/ /hexane (7/3) as eluent. phosphomolybdic acid (pma) stain was used for detection. melting points were determined using a cintex apparatus and are uncorrected. 300 mhz and 500 mz spectrometers were used for 1h-nmr spectroscopy and the latter for 13c-nmr spectroscopy of 4a–j. coupling constant (j) are presented in hertz and spin multiples are given as s (singlet), d (doublet), t (triplet) and m (multiplet). mass spectra were recorded by using the esi-ms method. synthesis of 4-(prop-2-yn-1-yl)thiomorpholine (2) the title compound was prepared according to a known literature procedure.32 synthesis of 4-(prop-2-yn-1-yl)thiomorpholine 1,1-dioxide (3) to a stirred solution of 4-(prop-2-yn-1-yl)thiomorpholine (3 g, 0.021 mol) in dichloromethane (100 ml) was added mcpba (10.9 g, 0.0638 mol) at 0 °c. the reaction mixture was stirred at room temperature for 12 h. after completion of the reaction, the solvent was removed under reduced pressure to afford the crude compound. the crude product was partitioned between ethyl acetate and aqueous nahco3 solution. then, the organic layer was separated, washed with brine, dried over anhydrous na2so4 and evaporated under reduced pressure to afford 2.8 g (77 %) of 4-(prop-2-yn-1-yl)thiomorpholine 1,1-dioxide (3). general procedure for the synthesis of 1,4-disubstituted-1,2,3-triazoles (4a–j and 5a–j) to a stirred solution of alkyl bromide (1.67 mmol, 1 eq.) in aqueous thf solution (h2o:thf, 1:1), was added sodium azide (2.01 mmol, 1.2 eq.) and the resulting mixture was stirred at room temperature for 2 h. then, alkyne (1.67 mmol, 1 eq.) and 10 mol % cu(i) were added to the reaction mixture and stirred at ambient temperature for 8–12 h. after completion of the reaction, the mixture was diluted with water, extracted with ethyl acetate, dried over na2so4 and evaporated under reduced pressure to afford the crude compounds. the crude compounds were purified by column chromatography using silica gel (100–200 mesh) and (20–30 %) ethyl acetate in n-hexane as eluent. evaporation of the solvent afforded compounds 4a–j and 5a–j in good yields. antibacterial activity the minimum inhibitory concentrations (mic) of the synthesized compounds were tested against the gram-positive organisms bacillus subtilis (mtcc 441), staphylococcus aureus (mtcc 96) and s. epidermidis (mtcc 2639) and the gram-negative organisms escherichia coli (mtcc 443), pseudomonas aeruginosa (mtcc 741), and klebsiella pneumoniae (mtcc 618) using the broth dilution method.33-37 penicillin and streptomycin were also screened under identical conditions for comparison. antifungal activity the in vitro antifungal activities of the synthesized compounds were determined against the fungal strains candida albicans (mtcc 227), saccharomyces cerevisiae (mtcc 36), aspergillus niger (mtcc 282) and aspergillus flavus (mtcc 8654) by the agar well diffusion method.38 ready-made potato dextrose agar (pda) medium (hi-media, 39 g) was suspended in distilled water (1000 ml) and heated to boiling until it dissolved completely, the medium and petri dishes were autoclaved at a pressure of 4.4 g m-2 for 20 min. the medium was poured into sterile petri dishes under aseptic conditions in a laminar air flow chamber. when the medium in the plates had solidified, 0.5 ml of week-old culture of the test organism was inoculated and uniformly spread over the agar surface with a sterile l-shaped rod. solutions were prepared by dissolving the compound in dimethyl sulfoxide (dmso) and _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 240 battula et al. different concentrations (100 and 150 µg ml-1) were made. after inoculation, the wells were scooped out with a 6 mm sterile cork borer and the lids of the dishes were replaced. different concentrations of the test solutions were added to each well and controls were maintained. the treated samples and the controls were kept at 27 °c for 48 h. inhibition zones were measured and the diameters were calculated in mm. three to four replicates were maintained for each treatment. amphotericin b was used as the standard drug for comparison. conclusions in summary, some novel thiomorpholine derived 1,4-disubstituted-1,2,3 -triazoles were synthesized and their antimicrobial activities investigated. the majority of the compounds were found to possess interesting antibacterial activities against the tested bacterial strains when compared to the standard drugs penicillin and streptomycin. some of the synthesized compounds also registered moderate antifungal activity. the antimicrobial activities of the compounds were modulated by structural modifications in the alkyl group attached to the triazole ring. thus, these active compounds could be very good candidates for further antimicrobial investigations. supplementary material analytical and spectral data of the synthesized compounds and the results of the antimicrobial screening (tables s-i and s-ii) are available electronically from http:// //www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgments. financial assistance from university grants commission, new delhi, for major research project to v. r. nagavelli is highly acknowledged. narsimha is thankful to the council of scientific and industrial research new delhi, for the award of senior research fellowship. и з в о д синтеза и испитивање антимикробне активности нових деривата 1,4-дисупституисаних 1,2,3-триазола и тиоморфолина kumaraswamy battula1, sirassu narsimha1, vasudevareddy nagavelli1, priyanka bollepelli2 и mutheneni srinivasa rao2 1 department of chemistry, kakatiya university, warangal-506009, india и 2chemical biology laboratory, indian institute of chemical technology, hyderabad-500007, india приказана је погодна синтеза нових 1,4-дисупституисаних 1,2,3-триазола 4a–j и 5a–j, бакар (i)-катализованом [3+2] циклоадицијом различитих халогеналкана и натријум-азида са 4-(проп-2-ин-1-ил)тиоморфолином и 4-(проп-2-ин-1-ил)тиоморфолин -1,1-диоксидом. испитана је антимикробна активност свих синтетисаних једињења. најбољу активност показују једињења 4a, 4b, 4c, 4g, 5a и 5j према staphylococcus epidermidis, 4a, 5a и 5d према pseudomonas aeruginosa, 4a, 4b и 4g према klebsiella pneumoniae, 4b, 5a и 5d према s. aureus и 5b, 5e и 5j према bacillus subtilis, када се упореде са стандардним лековима пеницилинoм и стрептомицином. једињења 4c, 4e, 4f, 4j, 5c, 5d, 5g и 5j имају умерену антифунгалну активност када се упореде са стандардним леком aмфотерицином б. (примљено 18. августа, ревидирано 13. октобра, прихваћено 21. октобра 2015) _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. thiomorpholine derivatives as antimicrobial agents 241 references 1. r. alvarez, s. velazquez, a. s. felix, s. aquaro, e. de clercq, c.-f. perno, a. karlsson, j. balzarini, m. j. camarasa, j. med. chem. 37 (1994) 4185 2. s. velazquez, r. alvarez, c. perez, f. gago, e. de clercq, j. balzarini, m. j. camarasa, antiviral chem. chemother. 9 (1998) 481 3. b. s. holla, m. mahalinga, m. s. karthikeyan, b. poojary, p. m. akberali, n. s. kumari, eur. j. med. chem. 40 (2005) 1173 4. y. chen, m. l. sanchez, d. n. savoy, d. d. billadeau, g. s. dow, a. p. kozikowski, j. med. chem. 51 (2008) 3437 5. c. j. carroux, g. m. rankin, j. moeker, l. f. bornaghi, k. katneni, j. morizzi, s. a. charman, d. vullo, c. t. supuran, s. a. poulsen, j. med. chem. 56 (2013) 9623 6. j. a. stefely, r. palchaudhuri, p. a. miller, r. j. peterson, g. c. moraski, p. j. hergenrother, m. j. miller, j. med. chem. 53 (2010) 3389 7. s. shafi, m. m. alam, m. naveen, m. chaitanya, g. vanaja, a. m. kalle, r. pallu, m. s. alam, eur. j. med. chem. 49 (2012) 324 8. c. dyrager, k. borjesson, p. diner, a. elf, b. albinsson, l. m. wilhelmsson, m. grotli, eur. j. org. chem. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {evaluation of antitumor potential of cu(ii) complex with hydrazone of 2-acetylthiazole and girard's t reagent} j. serb. chem. soc. 87 (2) 181–192 (2022) original scientific paper jscs–5514 published 2 february 2022 181 evaluation of antitumor potential of cu(ii) complex with hydrazone of 2-acetylthiazole and girard’s t reagent nevena stevanović1#, mima jevtović2#, dragana mitić2#, ivana z. matić3, marija đorđić crnogorac3, miroslava vujčić4#, dušan sladić1#, božidar čobeljić1# and katarina anđelković1#* 1university of belgrade-faculty of chemistry, studentski trg 12–16, 11000 belgrade, serbia,2innovative centre of the faculty of chemistry, studentski trg 12–16, 11000 belgrade, serbia, 3institute of oncology and radiology of serbia, 11000 belgrade, serbia and 4university of belgrade-institute of chemistry, technology and metallurgy, department of chemistry, njegoševa 12, 11000 belgrade, serbia (received 3 december, revised 17 december, accepted 20 december 2021) abstract: in this paper, the previously synthesized cu(ii) complex ([cul1(n3) (ch3oh)]bf4) with n,n,n-trimethyl-2-oxo-2-(2-(1-(thiazol-2-yl)ethylidene)hydrazinyl)ethan-1-aminium chloride, has been characterized and its biological activity has been studied in detail. the cu(ii) complex consists of ligand coordinated in a deprotonated, formally neutral zwitter-ionic form, via nno atoms, one azido ligand and one methanol molecule. the cu(ii) complex was selected due to results of the cytotoxic activity, the brine shrimp test and dpph radical scavenging activity, which were previously performed. the effects of cu(ii) complex on cell cycle phase distribution of cervical adenocarcinoma hela cells were investigated in order to examine the mechanisms of its anticancer activity. the measurement of intracellular ros levels in hela and hacat cell lines were evaluated in order to explore their possible generation and the role in cytotoxic activity. the possible anti-invasive and anti-angiogenic properties of cu(ii) complex were evaluated. dna binding experiments, including fluorescence displacement study and dna cleavage experiments, were performed in order to obtain information on the type of dna–metal complex interactions. keywords: n-acylhydrazone; cell cycle analysis; gene expression analyses; uv–vis measurements. introduction in recent years, schiff base complexes, especially hydrazone based ones, represent an interesting group of complexes, due to its easy preparation and * corresponding author. e-mail: kka@chem.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc211203114s ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 182 stevanović et al. structural diversity of ligands and wide spectrum of biological activities. n-acylhydrazones contain a carbonyl group that enables the electron delocalization and can act as an additional coordination site. many hydrazones show different biological activities, but in many cases their metal complexes show higher activity then the free ligand.1,2 copper (ii) complexes have d9 geometries (jahn–teller effect) and therefore show distorted octahedral and tetrahedral symmetries. the distortion is observed as axial elongation so that cu(ii) complexes can have square planar or square pyramidal geometries, but some can also possess trigonal bipyramidal geometry.3 most biologically active n-acylhydrazone copper complexes synthesized so far possess tridentate ligands, with ono4–8 and nno9–12 sets of donor atoms. considering the structure of the complex, there are no recorded structures of copper complexes with n-acylhydrazones containing a five-membered ring in the part of the molecule that originate from aldehyde/ketone, however the analogous structures of thiosemicarbazone cu(ii) complexes have been reported.13,14 in all reported complexes, the geometry around copper(ii) ion is square pyramidal. biological studies of these complexes showed moderate antibacterial13 and cytotoxic activities.14 by biological investigation of complexes of copper with n-acylhydrazone ligands it was observed that they can show wide spectra of biological activity, such as cytotoxic,4,6,7,12 antibacterial5,8–11 and antifungal,5,8 with higher activity than referent compounds.3 in continuation of our previous investigations of synthesis and characterrization of cu(ii) complexes with girard’s reagent t-based hydrazones,15–17 biological properties of a previously synthesized cu(ii) complex were investigated in this paper. in previous biological studies cu(ii) complex with n,n,n-trimethyl-2-oxo-2-(2-(1-(thiazol-2-yl)ethylidene)hydrazinyl)ethan-1-aminium chloride showed moderate antibacterial activity against all tested bacterial strains (best activity towards escherichia coli strain and very weak selectivity towards gram-negative bacteria) and moderate antifungal activity.19 also, this complex exhibited high radical-scavenging activity which is not surprising given the existence of a redox-active cu2+ in structure. another significant feature is the pronounced cytotoxic activity against tested human cancer cell lines. bearing that in mind, in this paper the previously synthesized cu(ii) complex has been subjected to further biological examination that includes: investigation of mechanisms of anticancer activity, ros generation in mediating cytotoxic effects, in vitro anti-angiogenic activity, effects on gene expression levels and dna interaction studies. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ biological activity of a copper(ii) compound 183 experimental chemistry 2-acetylthiazole (99 %) was obtained from acros, and girard’s t reagent (99 %) from aldrich. ir spectra were recorded on a nicolet 6700 ft-ir spectrometer using the atr technique in the region 4000−400 cm-1 (vs – very strong, s – strong, m – medium, w – weak). elemental analyses (c, h and n) were performed by standard micro-methods using the elementar vario eliii c.h.n.s.o analyzer. general procedure for the synthesis of the ligand and cu(ii) complex (1). the ligand hlcl was synthesized by the reaction of girard’s t reagent and 2-acetylthiazole according to the previously described method.18 the cu(ii) complex 1 was synthesized according to the previously described method,19 equimolar amounts of ligand hlcl and cu(bf4)2·6h2o were briefly dissolved in methanol. after complete dissolution of cu(bf4)2·6h2o in the reaction mixture, 4 eq. of nan3 were added and mixture was refluxed. biology cell cycle analysis. the changes in cell cycle distribution of hela cells treated with cu(ii) complex were analyzed by flow cytometry. hela cells were exposed to ic50 and 2ic50 concentrations of cu(ii) complex for 24 h. afterwards, the cells were collected by trypsinization, washed with phosphate buffered saline (pbs) and fixed in 70 % ethanol on ice, according to standard protocol.20 the cell samples were stored at –20 °c for at least one week before analysis. at the day of analysis, the cell samples were washed, resuspended in pbs containing rnase a and incubated for 30 min at 37 °c. subsequently, the propidium iodide solution was added to cell samples. percentages of cells within subg1, g1, s, and g2/m phases of the cell cycle were assessed using a bd facs calibur flow cytometer. the analyses of acquired data (10000 events collected for each gated cell sample) were performed by bd cellquest software. cell cycle data are presented as mean ± s.d. of three independent experiments. endothelial cell tube formation assay. the possible in vitro anti-angiogenic activity of tested cu(ii) complex was investigated on human umbilical vein ea.hy926 cells using endothelial cell tube formation assay.21,22 the ea.hy926 cells were seeded on the surface of corning® matrigel® basement membrane matrix and incubated with subtoxic ic20 concentration of the complex for 24 h (the applied concentration was 20 µm, as determined by mtt test for 24 h treatment). after the incubation of 20 h, the photomicrographs of control and treated ea.hy926 cells were captured under the inverted phase-contrast microscope. gene expression analyses. hela cells were seeded into 25 cm2 cell culture flasks (1.5×106 cells per flask). after 24 h, the cells were treated with subtoxic ic20 concentration of cu(ii) complex (the applied concentration was 15 µm, as determined by mtt test for 24 h treatment). control cell sample was incubated in nutrient medium only. after 24 h treatment, hela cells were collected by trypsinization, washed with pbs, and stored at –80 °c. total rna was isolated from hela cells using tri reagent® (sigma aldrich) according to the manufacturer’s protocol. the concentration and quality of isolated rna for each sample was determined spectrophotometrically (biospec-nano, shimadzu). high-capacity cdna reverse transcription kit (thermo fisher scientific) was used for reverse transcription of 1 µg of total rna into single-stranded cdna. measurement of mrna expression levels of selected genes was done by real-time quantitative pcr (rt-qpcr) and taqman®gene expression assays (mmp2-hs01548727_m1, mmp9-hs00957562_m1 and vegfa-hs00900055_m1) using lightcycler® 480 ii system (roche diagnostics gmbh, mannheim, germany). gene expres________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 184 stevanović et al. sion values were normalized to gapdh (hs02758991_g1) and were obtained by comparative δδct method, analyzed with lightcycler® 480 software. uv–vis measurements experiments for dna binding experiments, calf thymus dna (ct-dna, lyophilized, highly polymerized, obtained from serva, heidelberg, germany) was dissolved in 40 mm bicarbonate buffer, ph 8.4) and left overnight at 4 c. this stock solution was stored at 4 c and was stable for several days. a solution of ct-dna in water gave a ratio of uv absorbance at 260 and 280 nm, a260/a280 of 1.89–2.01, indicating that dna was sufficiently free of protein. the concentration of dna (3 mg/ml, 9.98 mm) was determined from the uv absorbance at 260 nm using the extinction coefficient ε260 = 6600 m -1 cm-1.23 the stock solution of complex [cul1(n3) (ch3oh)]bf4 (1) was freshly prepared by dissolving the compound in dimethyl sulfoxide at concentrations of 10 mm (5.28 mg/ml). reaction mixtures (1 ml in 40 mm bicarbonate buffer, ph 8.4) consisting of different concentrations of the complex 1 (1, 5 and 10 µm) and 49.9 µm of ct-dna (calculated per phosphate) were incubated at 37 c for 60 min with occasional vortexing. the absorbance titration was performed at a fixed concentration of the compound (10 µm) with gradual increase of the concentration of double stranded ct-dna (4.99, 5.98, 6.98, 7.98, 8.98, 9.98, 10.98, 11.97, 12.97, 13.97 and 14.97×10–5 m). the absorbance at 258 nm was monitored for each concentration of dna. the binding constant kb was determined using: 24 cdna(εa–εf) -1 = cdna(εb–εf) -1 + kb -1(εb–εf) -1 (1) where εa, εf, εb are absorbance/[compound], extinction coefficient of the free compound and extinction coefficient of the bound compound, respectively. fluorescence measurements the competitive interactions of 1 and the fluorescence probe, either ethidium bromide (eb) or hoechst 33258 (h), with ct-dna were studied by measuring the change of fluorescence intensity of the probe-dna solution after addition of the complex. reaction mixtures containing 50 µm of ct-dna (calculated per phosphate) in 1 ml of 40 mm bicarbonate solution (ph 8.4) were pretreated with 1.5 µl of 1 % h probe solution (28 µm final concentration) or 1 µl of 1 % eb solution (25 µm final concentration, in separate experiments) for 20 min and the mixture was analyzed by fluorescence measurement. then the increasing concentrations of the complexes (0.5, 1, 2, 3, 5, 7, 9, 11, 13 and 15×10–5 m for displacement of eb and 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4×10-5 m for displacement of h) were successively added and the changes in the fluorescence intensity were measured using a thermo scientific lumina fluorescence spectrometer (finland) equipped with a 150 w xenon lamp. the slits on the excitation and emission beams were fixed at 10 nm. all measurements were performed by the excitation at 350 nm for hoechst 33258, and by the excitation at 500 nm for eb in the range of 390−600 nm. the control was probe-ct-dna solution. the obtained fluorescence quenching data were analyzed according to the stern–volmer equation: i0/i = 1 + kc1/cct-dna (2) where i0 and i represent the fluorescence intensities of probe-ct-dna in absence and presence of 1, respectively, where k is the quenching constant. the k value was calculated from the ratio of the slope to the intercept from the plot of i0/i versus c1/cct-dna. the primary spectra of all spectrometric measurements were imported into origin 2018. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ biological activity of a copper(ii) compound 185 results and discussion the reaction of 2-acetylthiazole and girard’s t reagent was performed according to the previously reported method18 and yielded the ligand n,n,n-trimethyl-2-oxo-2-(2-(1-(thiazol-2-yl)ethylidene)hydrazinyl)ethan-1-aminium chloride (hlcl), which was used for the synthesis of cu(ii) complex (scheme 1). the reaction of the ligand hlcl with metal salt cu(bf4)‧6h2o and nan3 in mole ratio 1:1:4 in methanol resulted in formation of mononuclear cu(ii) complex (1) with the composition [cul1(n3)(ch3oh)]bf4 (scheme 1). the ligand is coordinated in a deprotonated formally neutral zwitter-ionic form via thiazole nitrogen, azomethine nitrogen and carbonyl oxygen atoms. ligand and complex 1 were characterized by ir spectroscopy (figs. s-3 and s-4 of the supplementary material to this paper) and elemental analysis. scheme 1. synthesis of cu(ii) complex (1). effects of cu(ii) complex on cell cycle phase distribution since the cu(ii) complex 1 exerted a strong cytotoxic activity against cancer cell lines further examination of mechanisms of its anticancer activity was undertaken. the effects of 1 on cell cycle phase distribution of cervical adenocarcinoma hela cells were investigated after incubation with ic50 and 2ic50 concentrations of the compound for 24 h. these results are presented in fig. 1. cu(ii) complex 1 applied at ic50 concentration caused a small increase in the percentages of hela cells in subg1 and s phases of the cell cycle in comparison with untreated hela cell sample as a control. in addition, the pronounced increase in the percentage of cells in g2/m cell cycle phase was observed after the treatment of hela cells with ic50 concentration of cu(ii) complex 1, when compared with control cells. these changes were accompanied with a decrease of percentage of hela cells in g1 phase. similar changes in the cell cycle phase distribution were found in hela cells exposed to higher (2ic50) concentration of complex – a small increase of the percentage of cells in subg1 phase and a prominent increase of the percentage of cells in g2/m phase. the only exception was the absence of changes in the percentage of cells in s phase of the cell cycle. these results suggest that the antiproliferative activity of cu(ii) complex 1 might be attributed to a pronounced arrest of cells in g2/m cell cycle phase. the g2/m cell cycle arrest induced by the complex in hela cells might be explained by the ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 186 stevanović et al. effects on cell cycle progression from g2 phase to mitosis at g2/m checkpoint, or by the effects on cell progression through mitosis – m phase arrest. the observed g2/m phase arrest might be associated with possible dna damage effects of the copper complex. fig. 1. changes in the cell cycle phase distribution of hela cells treated with ic50 (a) and 2ic50 concentrations (b) of cu(ii) complex for 24 h. effect of cu(ii) complex on in vitro angiogenesis the possible in vitro anti-angiogenic activity of the novel cu(ii) complex 1 on human umbilical vein endothelial ea.hy926 cells was investigated using tube formation assay. as it could be seen in fig. 2 the incubation of ea.hy926 cells with subtoxic ic20 concentration of cu(ii) complex 1 for 24 h notably decreased angiogenesis in vitro. the complex suppressed the extensive sprouting of elongated ea.hy926 cells and the formation of capillary-like tubes, in comparison with the untreated ea.hy926 cells as control. the in vitro anti-angiogenic activity of investigated 1 is in agreement with the findings about inhibitory effects of other cu(ii) complexes on angiogenesis in vitro.25,26 fig. 2. photomicrographs of control ea.hy926 cells and ea.hy926 cells treated with subtoxic ic20 concentration of cu(ii) complex for 20 h. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ biological activity of a copper(ii) compound 187 effects of cu(ii) complex on gene expression levels in order to further explore the possible anti-invasive and anti-angiogenic properties of cu(ii) complex (1), its effects in hela cells on the expression levels of genes whose protein products are implicated in cancer cell invasion and angiogenesis: matrix metalloproteinase-2 (mmp2), matrix metalloproteinase-9 (mmp9) and vascular endothelial growth factor a (vegfa) were evaluated. a slight decrease in mmp9 gene expression level was detected in hela cells exposed for 24 h to subtoxic ic20 concentration of the complex in comparison with this level in control hela cells (fig. 3). however, the increase in the mmp2 gene expression level was observed in hela cells exposed to the tested complex. the cu(ii) complex 1 notably downregulated expression level of vegfa in hela cells, when compared with this level in control cell sample. since vegfa gene encodes growth factor, which is involved in activating angiogenesis, lowering its level in cancer cells confirms the anti-angiogenic effect of this complex in vitro. the ability of copper complex to suppress angiogenesis in vitro represents an important anticancer property of this novel compound because targeting angiogenesis, which is essential for the expansion of malignant tumor growth, can inhibit further cancer progression.27 fig. 3. changes in gene expression levels of mmp2 (a), mmp9 (b), and vegfa (c), in hela cells treated with subtoxic ic20 concentration of cu(ii) complex for 24 h. dna interaction studies dna is one of the medicinal therapeutic targets of anticancer agents. transition metal complexes interact with dna non-covalently or by covalent binding. non-covalent binding usually occurs either in the minor groove by combination of hydrogen bonding, van der waals contacts and electrostatic interactions, or by intercalation of a planar aromatic moiety between two base pairs of dna via π-stacking take place.28,29 covalent modifications of dna usually occur at n7 -positions on easily accessible purines in the major groove of the helix.30 a combination of these interactions is possible and the consequences of such interactions might be reversible or irreversible.31 in this work, spectroscopic methods were employed to investigate the interaction modes of complex 1. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 188 stevanović et al. the electronic absorption spectra of 1, recorded at different concentrations with fixed concentration of ct-dna or without it, are shown in fig. s-1a of the supplementary material. it was found that the absorption maximum of the complex was centered at 358 nm. after the interaction with ct-dna, the changes in spectra were detected. analysis of the absorption changes induced upon binding of 1 to ct-dna is shown in the inset in fig. s-1a. by comparison of the sum of absorbances at 258 nm of free complex 1 and free ct-dna and the observed absorbance of the 1-ct-dna, it can be concluded that there was a concentrationdependent difference between them. the bands which correspond to ct-dna showed the broadening of the peak at 258 nm in concentration-dependent way, and the hypochromism of 1.87, 6.67 and 8.58 % for 1, 5 and 10 µm of the complex, respectively (fig. s-1a, inset). significant changes in spectra of 1 after the interaction with ct-dna were observed for bands at 358 nm, as evidenced by hypochromism of 42.31, 23.26 and 18.79 % (for 1, 5 and 10 µm of the complex, respectively). hypochromism and red shift are common evidence for intercalation. given that no shifts in the spectrum were detected and considering the positive charge on molecule of the complex, the obtained results indicate that the electrostatic interaction is the probable mode of binding. in order to obtain more information on interaction of the complex with dna, spectroscopic titration of the solution of the complex the concentration of which was kept constant (10 µm) with the solutions of increasing concentrations of ct-dna was performed. the results are shown in fig. s-1b. a quantitative determination of the binding strength of the compound to dna is made by calculation of binding constants kb of the compound from eq. (1). 24 the intrinsic binding constant kb of 1 (inset in fig. s-1b) was calculated as 2.9×10 3 m–1. the obtained value was lower than the value reported for a classical intercalator (ranged 106 to 107 m–1)32. the value of kb was comparable to those for metal complexes binding in the minor groove of dna.33 however, non-linearity of the curve showed that at least two types of binding sites are available for the interaction of complex 1 with dna, suggesting different binding modes. in order to further investigate the binding mode, fluorescence spectroscopy was performed by the fluorescence displacement experiments carried out with two different dyes: ethidium bromide (eb), a typical intercalator, and hoechst 33258 (h), a minor groove binder. while eb fluoresces weakly, its fluorescence yield increases significantly in the presence of ct-dna (fig s-2a). binding of eb to ct-dna was followed by the excitation at 500 nm with fluorescence maximum at 600 nm. the addition of increasing concentrations of 1 to eb–ct -dna system caused reduction in the fluorescence intensity of the band at 600 nm by 32 % with the maximal applied concentration of the complex. the criterion for fluorescence quenching by a typical strong intercalator is the fluorescence value reduction of more than 50 %.34–36 this means that that the mech________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ biological activity of a copper(ii) compound 189 anism of fluorescence of eb quenching by complex 1 is different from intercalation. the extent of this quenching of eb by 1 was estimated using stern– volmer equation (2). the fluorescence quenching constant (k) was calculated from the ratio of the slope to the intercept from the plot i0/i versus c1 as shown in the inset in fig. s 2a. the plot is linear and gives a k of 3.07×103 m–1. hoechst 33258 recognizes at least four at base pairs and binds strongly and selectively with high affinity to double-stranded b-dna structure by the combination of hydrogen bonding, van der waals contacts with the walls of the minor groove, and the electrostatic interactions between its cationic structure and the dna.37 as the emission spectra showed (fig. s-2b), the fluorescence of h increased significantly upon the interaction with ct-dna. a successive addition of 1 to h-ct-dna solution caused appreciable reduction in the fluorescence intensity of the band at 443 nm (about 60 % with maximal applied concentration of 1), accompanied by a small blue shift of emission maximum to 441 nm. the quenching constant calculated by the linear regression of a plot i0/i against c1 (inset in fig. s-2b) was k = 4.16×104 m–1. the plot displayed a good linear relationship for the investigated concentration ranges of 1, indicating the displacement of h33258 from h−ct-dna. by comparing the results, it can be concluded that the competition of 1 with h 33258 was more efficient than in case of ethidium bromide in binding to dna. in view of fluorescence and uv–vis absorption spectral results, it can be deduced that the interaction of the complex 1 with dna occurred via minor groove binding and electrostatic interactions between a positively charged quarternary ammonium group and negatively charged phosphate groups of dna backbone. for effects of cu(ii) complex on intracellular ros levels, bsa fluorescence measurements and dna cleavage experiments, see supplementary material. conclusions the ligand hlcl and cu(ii) complex 1 have been characterized by elemental analysis and ir spectroscopy. the five-coordination geometry of the cu(ii) ion (mononuclear complex 1) can be described as distorted square-based pyramidal. cu(ii) ion is coordinated through nno set of donor atoms, of formally neutral zwitter-ionic form, of hlcl ligand, one nitrogen atom of the azide ligand, while fifth remaining coordination site is occupied by the oxygen atom from methanol. the effects of cu(ii) complex on cell cycle phase distribution showed a small increase in the percentages of hela cells in subg1 and s phases and also a pronounced increase in the percentage of cells in g2/m phase of the cell cycle. the results of effects of cu(ii) complex on ros generation suggested the antioxidant properties of the tested complex at lower concentration. the results obtained via dna interaction studies indicate that minor groove binding and electrostatic interactions are the probable mode of binding for complex 1. the ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 190 stevanović et al. lower value of intrinsic binding constant kb of 1 and nonlinearity of the curve indicates that at least two types of binding sites are responsible for the interaction of the complex with dna. of a particular interest is anti-angiogenetic activity of the complex, since the formation of blood vessels is crucial for tumor growth. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/11447, or from the corresponding author on request. acknowledgement. the authors are grateful to the ministry of education, science and technological development of the republic of serbia for the financial support (grant numbers: 451-03-9/2021-14/200043, 451-03-9/2021-14/200026, 451-03-9/2021-14/200168 and 451-03-9/2021-14/200288). и з в о д испитивање антитуморског потенцијала комплекса cu(ii) са хидразоном 2-ацетилтиазола и жираровог т-реагенса невена стевановић1, мима јевтовић2, драгана митић2, ивана з. матић3, марија ђорђић црногорац3, мирослава вујчић4, душан сладић1, божидар чобељић1 и катарина анђелковић1 1 универзитет у београду-хемијски факултет, студентски трг 12–16, 11000 београд, 2 иновациони центар, хемијски факултет, студентски трг 12–16, 11000 београд, 3 универзитет у београду-институт за онкологију и радиологију србије, 11000 београд и 4 универзитет у београду-институт за хемију, технологију и металургију, центар за хемију, његошева 12, 11000 београд у овом раду урађена је карактеризација и детаљно испитивање биолошке активности претходно синтетисаног комплекса cu(ii) са n,n,n-триметил-2-оксо-2-(2-(1-(тиазол-2-ил)етилиден)хидразинил)етан-1-аминијум-хлоридом. комплекс cu(ii) састоји се из депротонованог, формално неутралног, лиганда координованог преко nno атома у цвитер-јонском облику, једног азидо лиганда и једног молекула метанола. комплекс cu(ii) одабран je на основу резултата претходно испитиване цитотоксичне активности, урађених тестова на рачићима artemia salina и dpph теста. у циљу одређивања механизма антитуморског дејства, урађена је анализа ћелијског циклуса аденокарцинома грлића материце. мерењем продукције реактивних оксидативних врста проучаван је њихов утицај и улога у цитотоксичној активности. испитивана су и потенцијална антиинвазивна и анти-ангиогена својства комплекса cu(ii). ради одређивања типа интеракција између комплекса метала и днк урађена су испитивања: интеракција са днк, флуоресценције и цепања днк ланца. 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________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ http://dx.doi.org/10.1080/00958972.2017.1405262 http://dx.doi.org/10.1006/bbrc.2000.2707 http://dx.doi.org/10.1021/acs.jmedchem.5b00651 http://dx.doi.org/10.1021/acs.jmedchem.5b00651 http://dx.doi.org/10.1016/j.ejmech.2016.03.003 http://dx.doi.org/10.3390/jcm9010084 http://dx.doi.org/10.1016/j.mrfmmm.2007.03.008 http://dx.doi.org/10.1016/j.ccr.2009.01.004 http://dx.doi.org/10.1021/ja00230a017 http://dx.doi.org/10.2174/156802611798040787 http://dx.doi.org/10.1021/ja00294a054 http://dx.doi.org/10.1016/j.jinorgbio.2004.12.018 http://dx.doi.org/10.1039/c1dt10714c http://dx.doi.org/10.1021/ic300739x http://dx.doi.org/10.1021/ic502271k http://dx.doi.org/10.1016/s0166-1280(02)00026-x 404 not found accepted manuscript this is an early electronic version of an as-received manuscript that has been accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as m. šokarda slavić, v. ralić, b. nastasijević, m. matijević, z. vujčić, and a. margetić, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc221116011s this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc‌221116011s j. serb. chem. soc.00(0)1-13 (2023) original scientific paper jscs–12032 published dd mm, 2023 1 a novel pga/tio2 nanocomposite prepared with poly(γ-glutamic acid) from the newly isolated bacillus subtilis 17b strain marinela šokarda slavić1§*, vanja ralić2§, branislav nastasijević3, milica matijević2, zoran vujčić4, aleksandra margetić1 1university of belgrade – institute of chemistry, technology and metallurgy – national institute of the republic of serbia, department of chemistry, belgrade, republic of serbia, 2center for light-based research and technologies, coherence, department of atomic physics, vinča institute of nuclear sciences, national institute of the republic of serbia, university of belgrade, belgrade, republic of serbia, 3vinča institute of nuclear sciences, national institute of the republic of serbia, university of belgrade, belgrade, republic of serbia, and 4university of belgrade – faculty of chemistry, department of biochemistry, belgrade, republic of serbia (received 16 november 2022; revised 23 january 2023; accepted 3 march 2023) abstract: poly(γ-glutamic acid) (pga), naturally produced by bacillus species, is a biodegradable, non-toxic, biocompatible, and non-immunogenic negatively charged polymer. due to its properties, it has found various applications in the food, cosmetic and pharmaceutical industries. in this work, bacillus subtilis 17b was selected as the best pga producer among fifty wild-types bacillus strains tested and characterized as a glutamate-independent producer. the production of pga by the newly identified strain was optimized and increased tenfold using the box-behnken experimental design. the purity of pga after recovery and purification from the fermentation broth was confirmed by sds-page followed by methylene blue staining. pga was characterized by esi ms and used for the preparation of a new nanocomposite with tio2. the synthesis of pga/tio2 nanocomposite, its structural analysis, and cytotoxic effect on the cervical cancer cell line (hela cell) was investigated to determine the potential anti-cancer usage of this newly prepared material. encouraging, pga/tio2 nanocomposite showed an increased cytotoxic effect compared to tio2 alone. keywords: pga production; wild-type bacillus strain; pga characterization; cytotoxicity. *corresponding author e-mail: marinela.sokarda@ihtm.bg.ac.rs https://doi.org/10.2298/jsc221116011s §authors contributed equally to this work a cc ep te d m an us cr ip t mailto:marinela.sokarda@ihtm.bg.ac.rs https://doi.org/10.2298/jsc‌221116011s 2 slavić et al. introduction polyglutamic acid (pga) is a biodegradable naturally occurring biopolymer that consists of land d-glutamic acid. pga is a negatively charged water-soluble polymer. unlike amino acids in polypeptides, glutamic acid monomers in pga create amide bonds between α-amino and γ-carboxyl groups.1 pga was first discovered in 1937. as a major component of the b. anthracis capsule.2 owing to its biodegradability, pga has various current and potential applications including drug carrier, anti-cancer drug delivery treatment of rheumatoid arthritis, genes, protein and peptide delivery, water treatment polluted by heavy metal and basic dye adsorber, thickener, and bitterness relieving agent, cryoprotectant, etc.3 pga proved to be an efficient drug delivery matrix as a nontoxic, biodegradable, biocompatible, and non-immunogenic polymeric material for water-soluble and insoluble drugs and other molecules as nanoparticles.3 negatively charged serum proteins and erythrocytes were reported to not aggregate with anionic polymeric carriers (e.g. pga), which overcame serum inhibitory effects.4 it is also known that polymeric nanoparticles can collect specifically in cancer tissues,5 making them effective anticancer drug carriers. studies of titanium dioxide nanoparticles and their various organic compound hybrid materials are becoming increasingly important due to their potential use in novel medical therapies. furthermore, to improve efficient anticancer and antimicrobial therapies, many other approaches utilizing tio2 have been tested.6 the importance of the development of new formulations is also indicated by the fact that increasing the therapeutic efficiency of tio2 can be achieved by using nanocomposites. currently, the majority of commercial γ-pga is made by cost-effective microbial fermentation from biomass instead of chemical synthesis, peptide synthesis, or biotransformation. polyglutamic acid is mainly produced by bacteria of the genus bacillus. most common species include b. licheniformis, b. subtilis, b. megaterium, b. pumilis, b. mojavensis, and b. amyloliquefaciens.1 a few unusual pga producers include the halophilic archaebacterium natrialba aegyptiaca7 and the gram-negative bacterium fusobacterium nucleatum.8 these microorganisms produce pga as an extracellular viscous material during fermentation that can then be isolated and purified.9 bacillus species are widely used industrial organisms due to their high growth rates, short fermentation cycle times, and relatively inexpensive nutritional requirements.10 the types and characteristics of a pga-producing bacterial strain dictate the medium composition. firstly, all pga-producing bacteria can be divided into glutamic acid-dependent and independent.11 glutamic acid-dependent strains require the presence of l-glutamic acid in the fermentation broth, usually, in the range of 20-30 g l-1.12 for successful pga production, it is necessary to take into account the individual but also synergistic impact of the above factors for the design of bacterial growth and production medium. according to the literature, the a cc ep te d m an us cr ip t production of pga and pga/tio2 nanocomposite 3 doe approach has successfully been used for carbon and nitrogen source selection for pga production.13 to find optimal production conditions, it is necessary to use a statistical method that takes into account the analysis of the synergistic effect of at least three important factors, such as box-behnken design (bbd). there are examples in the literature where a high level of production of a significant product was obtained by using bbd to optimize the microbiological process.14 as the possibility of preparation of pga/tio2 nanocomposite and the potential of its anticancer activity has not been investigated so far, this was set as the goal of this work. in this study, the best pga producer among 50 different natural bacillus isolates originating from the soil, and different locations in serbia, was selected and identified. in order to achieve the highest level of pga production influence of carbon source was analyzed using the doe approach (bbd) where concentrations of glucose, glycerol and glutamate were varied. purified pga was used for the preparation of nanocomposite with tio2. both, pga and pga/tio2 were structurally characterized by esi ms. pga/tio2 cytotoxicity was examined using cervical cancer (hela) cells. experimental chemicals unless otherwise stated, all reagents and solvents were purchased from merck (darmstadt, germany) and sigma-aldrich (st. louis, mo, usa). all chemicals were of the best available purity and used without further purification. screening of γ-pga producers pga production was investigated in 50 isolated wild-type strains of bacillus sp. an overnight culture of each strain was prepared by incubation of a single colony in 5 ml of sterilized luria–bertani (lb) medium and grown for 18 h at 37 ℃ and 150 rpm. the screening medium was prepared according to the medium most commonly used for pga production and contains per liter:15 glucose, 100 g; sodium glutamate, 0.5 g; citric acid, 0.5 g; nh4cl, 10 g, kh2po4, 0.34 g; na2hpo4 x 12h2o, 0.895 g; mgso4 x 7h2o, 0.5 g; fecl3 x 6h2o, 0.04 g; cacl2 x 2h2o, 0.15 g; mncl2 x 4h2o, 0.26 g. the fermentation broth was prepared by mixing 2 ml of an overnight culture of each strain with 48 ml of the screening medium. fermentation was carried out for 5 days at 37 ℃ and 150 rpm. the best producer strain was selected and used in further experiments. identification of selected strain the selected bacillus 17b strain was identified by 16s rrna gene sequence analysis. total dna from bacillus 17b strain was isolated using the phenol-chloroform extraction method previously described by hopwood and co-authors with minor modifications.16 logarithmic phase cells were treated with lysozyme (4 mg ml-1, for 15 min at 37°c) prior to treatment with 2 % sds. the 16s rrna gene was amplified using 27f (5’aga gtt tga tcc tgg ctc ag 3’) and 1492r (5’-ggt tac ctt gtt acg act t-3’) universal primers.17 the amplified pcr fragment was sequenced by a macrogen sequencing service (macrogen europe, amsterdam, netherlands). sequence annotation and a database search for a cc ep te d m an us cr ip t 4 slavić et al. sequence similarities were completed using the blast program of the national center for biotechnology information – ncbi.18 optimization of pga production media bbd (box behnken design) was used to identify optimal concentrations of three different carbon sources using design-expert software (version 11, stat-ease, inc, usa). three analyzed factors were glucose (a), glycerol (b), and glutamate (c). the evaluated response y was the level of γ-pga production (g l-1). all three factors (a, b and c) were studied at three different levels (concentrations) (table s-i) through sets of 14 experiments (table s-ii). the other components of the medium were constant (citric acid, 0.5 g; nh4cl, 10 g, kh2po4, 0.34 g; na2hpo4 x 12h2o, 0.895 g; mgso4 x 7h2o, 0.5 g; fecl3 x 6h2o, 0.04 g; cacl2 x 2h2o, 0.15 g; mncl2 x 4h2o, 0.26 g). the fermentation broth was prepared by mixing 2 ml of an overnight culture with 48 ml of medium. production fermentations were carried out for 5 days at 37 ℃ and 150 rpm in a thermostat shaker (ks 4000i, ika). the experimental data of the bbd was represented in the general form of the two-factor interaction (2fi) model as shown in equation 1, to develop an empirical model which will be used to analyze the effect of factor interactions. 𝑦 = 𝛽0 + ∑ 𝛽𝑖 𝑥𝑖 + ∑ 𝛽𝑖𝑗 𝑥𝑖 𝑥𝑗 𝑞 1≤𝑖≤𝑗 𝑞 𝑖=1 + 𝜀 (1) where y is the predicted response (pga yield), β0 is a constant coefficient, q is the number of variables, βi is the linear coefficient, βij represents the interaction coefficient and xi and xj are the process variables and ε is the residual. analysis of variance (anova) was used to assess the significance of the model and the impact of coefficients in regression analysis. isolation of γ-pga from the culture broth the culture broth was centrifuged at 10000 g for 20 minutes at 25 ℃. the supernatant was adjusted to ph 3 using 1 m h2so4 and stored at 4 ℃ for 12 h. the precipitate was removed by centrifugation at 10000 g for 20 minutes at 25 ℃. the resulting supernatant was mixed with ice-cold ethanol in a ratio of 1:3 (v:v). the pga precipitate formed was separated after centrifugation at 5000 g for 20 minutes at 4 ℃. the obtained pga precipitate was dissolved in distilled water and purified using a sephadex g-25 column. the remaining proteins in the pga solution were hydrolyzed with 50 µg ml-1 proteinase k,19 and the resulting mixture was ultrafiltered through a microcon membrane with a 30 kda cut-off to protein hydrolysis products. the retentate (pga) was air-dried and used for further analysis. to confirm the purity of the isolated pga, sds page was performed using a hoefer™ mighty small™ ii mini vertical electrophoresis system with a 10 % polyacrylamide gel.20 the gel was stained by the modified method of yamaguchi et al.21 to verify the presence of proteins, the gel was first stained with coomassie brilliant blue (g-250) and rinsed with 7 % acetic acid/ 5 % ethanol. after a short rinse with distilled water, pga in the gel was stained with 0.5 % methylene blue dissolved in 3 % acetic acid. the gel was rinsed with distilled water. fourier-transform infrared spectroscopy fourier-transform infrared spectroscopy (ftir) of pga and tio2 was done using thermo electron corporation nicolet 380 spectrometer in attenuated total reflection (atr) mode. spectra resolution was 4 cm-1 in the range of 4000-400 cm-1. a cc ep te d m an us cr ip t production of pga and pga/tio2 nanocomposite 5 electrospray ionization mass spectrometry an isolated polymer of glutamic acid and acidic hydrolyzed pga was analyzed by electrospray ionization mass spectrometry (esi ms) on a waters quattro micro api mass spectrometer. all samples were previously diluted with 0.1 % water solution of formic acid to the concentration of 0.1 mg ml-1, whereas the hydrolysate was diluted 1000 times, to avoid the influence of the high concentration of hcl used for its hydrolysis. pga was hydrolyzed by using a microwave digestor (milestone start d, sk-10t) in the presence of 6 m hcl. the process was performed according to the manufacturer´s instructions and it was finished within 20 minutes. the spectra acquisition was done in the positive ion mode in the mass range from 50 to 4000 da. the temperature of the ionization source was 125 ℃, of a gas carrier 380 ℃, the capillary voltage was 3 kv, whereas the conus voltage was in the range from 20 to 90 v, depending on the sample. pga/tio2 nanocomposite formation and characterization of nanocomposite colloidal spherical tio2 nanoparticles (average diameter, d~5 nm) were synthesized by the modified method of rajh et al.22 nanoparticle concentration was determined after dissolving the particles in concentrated h2so4 by the concentration of the peroxide complex, as previously described.23 pga and tio2 were mixed in different ratios (present in table s-iii) and incubated for 2 hours at 25 ℃ with agitation at 150 rpm. 24 a formed white pga/tio2 nanocomposite (nc) precipitate was recovered by centrifugation at 10000 g for 30 minutes and air-dry. cytotoxic effect of nanocomposite hela cells were cultured in dulbecco's modified eagle's medium supplemented with glucose, l-glutamine, fetal bovine serum, penicillin, and streptomycin solution, according to the supplier’s instructions. cells were seeded in flat-bottomed 96-well microtiter plates (2000 per well) and incubated overnight with varying concentrations of pga, tio2 nanoparticles and an nc. samples were prepared in distilled water and incubation terminated after 48 h. cell viability was determined using the sulphorhodamine b (srb) assay.25 the absorbance was measured at 550 nm with a reference wavelength of 690 nm in a microplate reader (wallac, victor2 1420 multilabel counter, perkinelmer, turku, finland). the results were presented as a percent of cell viability determined according to the following equation: 𝐶𝑒𝑙𝑙 𝑣𝑖𝑎𝑏𝑖𝑙𝑖𝑡𝑦 (%) = 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝐴𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 × 100 (2) the measurements were made in quintuplicate and the results were presented as the mean ± standard deviation. results and discussion pga producer screening pga is an extracellular polymer produced by certain bacillus species including bacillus paralicheniformis atcc 9945a,26 which was used as a standard pga producer strain for comparisons with wild-type isolates. the pga-producing ability of 50 different bacillus sp. strains originating from serbia, deposited in a laboratory bank ub483, was investigated. screened bacillus isolates produced pga in the range of 0.40 to 4.07 mg ml-1. the strain indicated as 17b exhibited a cc ep te d m an us cr ip t 6 slavić et al. the highest yield of pga (4.07 mg ml-1), higher even than b. paralicheniformis atcc 9945a (2.27 mg ml-1) under the conditions used for fast selection of strains. selected isolate (17b) was identified as b. subtilis according to the highest identity (99 %) with the 16s rrna gene of the species bacillus subtilis from the ncbi database. strain b. subtilis 17b was selected for medium optimization, as it showed the highest pga-producing capabilities. optimization of culturing conditions for pga production the box-behnken experimental design was set using the concentrations of carbon sources (glucose, glutamate, and glycerol) as variables and pga yield was measured as a response (table i). response surface methodology (rsm) was used to determine the synergistic effect of glucose, glycerol, and glutamate concentrations on pga yield, and the results are given in table i. table i. box-behnken experimental design pga yield. run cglucose /g l -1 (a) cglycerol /g l -1 (b) cglutamate /g l -1 (c) yieldpga, mgml -1 (y) actual value* predicted value 1 10 0 2.75 13.9 13.7 2 80 0 2.75 28.0 28.3 3 10 50 2.75 38.6 37.0 4 80 50 2.75 34.0 32.9 5 10 25 0.5 18.8 23.6 6 80 25 0.5 28.4 32.6 7 10 25 5 28.0 27.1 8 80 25 5 30.0 28.6 9 45 0 0.5 20.6 17.7 10 45 50 0.5 40.0 38.5 11 45 0 5 21.6 24.3 12 45 50 5 27.3 31.4 13 45 25 2.75 37.4 27.9 14 45 25 2.75 25.2 27.9 *all data are significantly different from each other according to tukey’s test (p<0.05). the regression analysis has produced a two-factor interaction (2fi) model that describes pga yield as a function of independent variables and their interactions in terms of coded factors: 𝑃𝐺𝐴 𝑦𝑖𝑒𝑙𝑑 (𝑚𝑔𝑚𝐿−1) = 27.99 + 2.64𝐴 + 6.98𝐵 − 0.113𝐶 − 4.68𝐴𝐵 − 1.90𝐴𝐶 − 3.42𝐵 (3) the coefficients are obtained based on the least-squares method in such a way that the sum of the squares of the errors, ε, is minimized. a positive coefficient value indicates that this variable has a positive effect on pga yield, while a minus indicates a negative impact. statistical analysis was performed to assess the significance of the model used and the coefficients in the 2fi regression equation. anova parameters are shown in table s-iv. models f and p values, 3.88 and a cc ep te d m an us cr ip t production of pga and pga/tio2 nanocomposite 7 0.049 respectively, indicate that the model is significant. both glucose and glycerol have a positive impact on pga production, while glutamate has a negative impact. based on anova, only glycerol shows the most significant impact on the increase of pga yield. the two-factor interactions of these components have insignificant negative impacts. response surface 3d graphs show the two-interactive effects of independent variables on pga yield (figure s-1). figure s-1a represents the combined effects of glucose and glycerol concentrations, while figure s-1b shows the effects of glycerol and glutamate concentrations on pga production. glycerol has a higher linear coefficient and steeper rise than glucose (figure s-1a), thus positively impacting pga production. even though glutamate has a negative effect on pga yield with its linear coefficient being -0.113, its impact is barely detectable. neither graph reveals a finite plateau, implicating a possibility of further medium and yield improvement. according to the results, a minimal concentration of glutamate (0.5 g l-1) is optimal. also, it is necessary to provide a high concentration of glucose and glycerol (80 g l-1 and 50 g l-1, respectively) for the production of 38 g l-1 of pga. the obtained result indicates that b. subtilis 17b used in this study is a glutamate-independent strain, which in the literature implies a potentially lower production cost. 27 in recent years, studies on the synthesis of pga have mostly concentrated on glutamate-dependent strains.28,29 however, the main limitation for large-scale production of pga is the high production costs due to the addition of significant amounts of expensive exogenous l-glutamate.30 since glutamateindependent strains could significantly lower the cost of pga synthesis and simplify the fermentation process, more attention is given to them nowadays.31 glutamate-independent strains were considered ineligible for industrial γ-pga production, limited by their low γ-pga productivity,31 but based on the results shown in this study this state could be modified. in a screening study, b. subtilis 17b strain showed two times higher productivity of pga than b. paralicheniformis atcc 9945a and after optimization of the cultivation, productivity was improved tenfold. isolation and purification of pga pga was purified from the fermentation broth from accompanying small molecules including yellow pigment and proteins (using proteinase k) and analyzed by sds-page after standard cbb staining and methylene blue staining.32 sds page electropherogram is given in figure 1. samples before proteinase k treatment are rich in proteins (line 1). after the treatment with proteinase k, most proteins were removed from samples (line 2). detection of pga in sds page was not possible by staining with cbb, therefore the basic dye methylene blue was used for pga’s visualization (figure 1, mb). this is most likely because polyglutamic acid is a negatively charged polymer with no hydrophobic regions that could be stained using cbb.33 the polymer (1-2 mb) a cc ep te d m an us cr ip t 8 slavić et al. showed multiple wide blue bands. methylene blue staining of pga was better after proteinase treatment, presumably because of the stronger interaction of pga and methylene blue in absence of proteins. this phenomenon could be due to the formation of electrostatic interactions between pga and proteins from the sample, thus reducing the number of free functional groups available for interaction with methylene blue. furthermore, successful basic dye staining proved the presence of a negatively charged polymer. fig. 1. sds page electropherograms of pga produced by b. subtilis 17b stained with cbb and methylene blue (mb). 1 – before; 2 after proteinase k treatment. mass spectrometry of isolated pga detection of the number of glu units in the pga was determined by esi ms, and the identity of individual signals was confirmed by comparison with hydrolyzed polymer and standard (glu). the sample ionization has been assisted by the addition of formic acid, in order to increase the ion yield, and the positive ion esi mass spectra are given in figure s-2, whereas the signal identity is listed in table s-v. esi ms is, a so-called, “soft” ionization technique,34 and the extent of fragmentation are low. mostly, the loss of h2o, co or co2 from the -cooh group could be detectable. a cc ep te d m an us cr ip t production of pga and pga/tio2 nanocomposite 9 in summary, all detected ions imply that isolated polymer contains glu and that the highest number of glu units is 15 which is in agreement with the literature.35 characterization of pga-tio2 nanocomposite systems and potential application the potential application of pga produced by b. subtilis 17b was tested by the formation of pga/tio2 nc for the possibility to increase the cytotoxic effect of tio2 nanoparticles against hela cells. to examine nanocomposite formation, we recorded the spectra of purified pga, pga/tio2 nanocomposite, and tio2 nanoparticles (figure s-3). the band at 1650-1660 cm-1 in the pga ftir spectrum can be identified as the amide i band. the signal at 1079-1116 cm-1 is the result of a c-n bond. the low-intensity signal at 1450-1457 cm-1 originates from weak carbonyl absorption and is consistent with the literature.36 the over 3000 cm-1 region is of lower intensity, presumably because the sample was isolated from the basic medium making it mostly deprotonated. this reduces the number of hydrogen bonds that otherwise amplify the o-h and n-h bond signals. to this broad region belong signals of aliphatic n-h stretching that have no clear bands. however, region 3409-3442 cm-1 can be distinguished and may correspond to oh group absorption.37 all the assigned signals indicate a glutamic acid polymer, while the absence of a c-o bond confirms the absence of polysaccharide and that pga was successfully purified. although the pga/tio2 nanocomposite ftir spectrum shows a significant difference compared to the pga spectrum, the problem might arise from the concentration ratios, as the tio2 signals dominate. it seems that all of the main signals from the pga spectrum disappear in the nc spectrum, implying the existence of pga-tio2 interaction. the high-intensity signals at 2321-2365 cm-1 for the pga spectrum and 2312-2355 cm-1 for the nanocomposite spectrum originate from the asymmetric stretching of co2 that was present in both samples and it is stemming from the air.38 however, the interaction between these two components is demonstrated by slower sedimentation of tio2 nanoparticles in the presence of pga in the solution. the experiment that was performed by centrifugation of suspensions containing varying pga: tio2 mass ratio at 12 000 × g was done. whereas only a short impulse was required to sediment tio2 nanoparticles from a physiological solution without pga (ph 7), at the pga: tio2 ratios at 1:25 to 1:2, more than 30 minutes of centrifugation was necessary, which indicates stabilization of tio2 in the physiological solution. since the electrostatic interactions between an organic polymer (alginate) and tio2 nanoparticles resulting in their stabilization has already been demonstrated,39 we assume that in a similar manner, a -coogroup interacts with the surface of tio2 nanoparticles. in the next step, we preliminarily tested the cytotoxicity of all components and the nc system against hela cells. namely, colloidal tio2 nanoparticles form a a cc ep te d m an us cr ip t 10 slavić et al. stable colloidal solution at ph 2, which is not in a physiological range and limit their application in biological systems.40 therefore, our hypothesis was that the addition of pga will stabilize the system and enable its application in the physiological range. fig. 2. hela cells viability after treatment with three different concentrations of pga isolated from b. subtilis 17b, tio2 nanoparticles, and pga/tio2 ncs. as presented in figure 2, the addition of pga produced by bacillus sp. 17b increased the cytotoxic effect of tio2, whereas pga alone showed no cytotoxicity, as compared to control/untreated cells. although the tio2 nanoparticles demonstrated a light-induced cytotoxic effect, there are also data that show that these nanoparticles can also be cytotoxic in the dark,41 and our results are in line with these findings. these preliminary results demonstrated the potential application of pga as stabilizers for tio2 nanoparticles that have various biological applications,42 but the mechanism of its action and other physicochemical properties of the system need to be further investigated in more detail. conclusion the newly isolated strain of b. subtilis 17b, a natural isolate from the soil, a promising pga producer (initially showing twice the pga production compared to the commercially used strain b. paralicheniformis atcc 9945a) produced a high level of pga after applying statistical optimization methods (pga production increased tenfold). b. subtilis 17b was characterized as glutamate-independent a cc ep te d m an us cr ip t production of pga and pga/tio2 nanocomposite 11 which candidate it as a promising strain for pga production due to the costeffectiveness process. the pga-tio2 nanocomposite showed better cytotoxicity toward hela cells than tio2, which opened up the possibility of application in biological systems considering that tio2 nanoparticles are not stable in a physiological ph range. acknowledgments: this work was financially supported by the ministry of education, science and technological development of the republic of serbia (grant no. 451-03-68/202214/200026; 451-03-68/2022-14/200168; 450-03-68/2022-14/200017). the authors are thankful to dr marijana petković, coherence center, for their critical reading of the manuscript, and dr jelena zakula for the cell cytotoxicity assay. supplementary material additional data are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/12132, or from the corresponding author on request. и з в о д нови пгк/tio2 нанокомпозит добијен од поли(γ -глутаминске киселине) из новоизолованог соја bacillus subtilis 17б маринела шокарда славић1, вања ралић2, бранислав настасијевић3, милица матијевић2, зоран вујчић4, александра маргетић1 универзитет у београду – институт за хемију, технологију и металургију – институт од националног значаја за републику србију, београд, србија, 2центар за конверзију светлосне енергије coherence, лабораторија за атомску физику, 3институт за нуклеарне науке „винча" – институт од националног значаја за републику србију, универзитет у београду, београд, србија i 4универзитет у београду хемијски факултет, београд, србија поли(γ-глутаминска киселина) (пгk), коју производе бактерије рода bacillus, је биоразградив, нетоксичан, биокомпатибилан и неимуноген негативно наелектрисани полимер. због својих својстава нашао је разноврсну примену у прехрамбеној, козметичкој и фармацеутској индустрији. у овом раду, bacillus ѕubtilis 17б је изабран као најбољи пгк продуцер међу педесетак тестираних природних изолата бактерија из овог рода и окарактерисан као глутамат независтан продуцер. производња пгк овим новоидентификованим сојем је оптимизована и десетоструко увећана коришћењем box-behnken експерименталног дизајна. чистоћа пгк након изоловања и пречишћавања из ферметационе течности је потврђена електрофорезом (sds-page) након бојења метиленским плавим. пгк је окарактерисана масеном спекроскопијом (esi ms) и коришћена за добијање новог нанокомпозита са тiо2. синтеза пгк/тiо2 нанокомпозита, његова структурна анализа и цитотоксични ефекат на ћелијску линију рака грлића материце (hela ћелије) је испитан да би се утврдила потенцијална употреба овог новодобијеног материјала у борби против ћелија рака. нанокомпозит пгк/тiо2 показао је повећан цитотоксични ефекат на поменуте ћелије рака у поређењу са самим тiо2. 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janfaza, t. gholikhani, l. tayebi, int. j. nanomedicine 15 (2020) 3447 (https://doi.org/10.2147/ijn.s249441). a cc ep te d m an us cr ip t https://doi.org/10.1021/ic00181a003 https://doi.org/10.1016/j.envpol.2017.05.025 https://doi.org/10.1093/jnci/82.13.1107 https://doi.org/10.1016/0141-8130(94)90032-9 https://doi.org/10.1080/07388550601166458 https://doi.org/10.1016/j.procbio.2003.09.025 https://doi.org/10.1016/j.procbio.2019.11.034 https://doi.org/10.1186/s13568-017-0512-0 https://doi.org/10.1016/j.biortech.2010.12.065 https://doi.org/10.1006/bbrc.1999.1298 https://doi.org/10.1271/bbb.60.255 https://pubmed.ncbi.nlm.nih.gov/18568044 https://doi.org/10.1007/s13361-017-1731-y https://doi.org/10.1166/jnn.2010.2614 https://doi.org/10.1186/2191-0855-3-36 https://doi.org/10.1366/000370206778397371 https://doi.org/10.1021/acs.est.6b03684 https://doi.org/10.1016/0021-9797(75)90270-2 https://doi.org/10.1016/j.tiv.2012.12.018 https://doi.org/10.2147/ijn.s249441 http://dx.doi.org/10.1021/acs.orglett.5b00067 http://dx.doi.org/10.1007/s00044-020-02522-7 http://dx.doi.org/10.1007/s00044-020-02522-7 accepted manuscript this is an early electronic version of an as-received manuscript that has been accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as s. stamenković stojanović, i. karabegović, b. danilović, s. mančić and m. lazić, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc230407036s this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230407036s j. serb. chem. soc.00(0)1-16 (2023) original scientific paper jscs–12350 published dd mm, 2023 1 high cell density cultivation of bacillus subtilis ncim 2063: modeling, optimization and a scale-up procedure sandra stamenković stojanović*, ivana karabegović, bojana danilović, stojan mančić and miodrag lazić faculty of technology, university of nis, bulevar oslobodjenja 124, 16000 leskovac, serbia (received 7 april; revised 3 may; accepted 8 july 2023) abstract: bacillus subtilis is a non-pathogenic, sporulating, gram-positive bacteria with pronounced antimicrobial and metabolic activity and great potential for wide application in various fields. the aim of this paper was to determine the optimum b. subtilis ncim 2063 growth conditions and to scale up biomass production from shake flasks to a bioreactor level. the critical growth parameters and their interaction effects were studied using box-bekhen experimental design and response surface methodology. developed model equations were statistically significant with good prediction capability. it was found that during shake flask cultivation glucose should be added in concentration up to 5 g l-1 in dsm medium, otr at 10 mol m-3h-1 and temperature of 33 °c, to achieve the maximum number of viable cells and spores. to scale up the process from shake flasks to the bioreactor level kla was used as a main criterion. scale up effect was evaluated by comparing the growth kinetics in the shake flasks and in a laboratory bioreactor. the total number of cells obtained in the bioreactor was 4.57·109 cfu ml-1 which is 1.41 times higher than the number of cells in the shake flasks (3.24·109 cfu ml-1), proving that the scale-up procedure was conducted successfully. keywords: shake flask, bioreactor, microbial biomass. introduction bacillus subtilis is a gram-positive, rod-shaped bacteria with a unique ability to rapidly multiply and to form endospores, thus being resistant to adverse environmental conditions. this strain has the ability to produce industrially important metabolites such as antibiotics, polysaccharides and proteins1–3 and to degrade different pollutants in the environment.4 a great diversity of previously published papers has showed that b. subtilis is capable of producing various important biotechnological products, such as different enzymes5,6 (akcan 2012, božić, 2011), polysaccharides7 and surfactants7,8. many *corresponding authors e-mail: sandra.stamenkovic@live.com https://doi.org/10.2298/jsc230407036s a cc ep te d m an us cr ip t mailto:sandra.stamenkovic@live.com https://doi.org/10.2298/jsc230407036s 2 stamenković stojanović et al. of them, including iturin a and surfactin, have antibiotic, antiviral and immunomodulatory effect when applied on humans. additionally, b. subtilis has wide application in the environmental protection, as it can degrade different trace organic compounds9 and organoclorine insecticides10. it is also attributed to probiotic properties and "gras" (generally recognized as safe) status, which makes it safe for human use. thanks to the fact that it has exceptional physiological properties, it can easily adapt to new environmental conditions and has the ability to produce a large number of metabolites, this species is very attractive for industrial application and commercial biomass production.3,11 microbial biomass production implies cultivation in bioreactors in liquid, semi-liquid or solid nutrient media. at the beginning of the research, cultivation is carried out in shake flasks of smaller volumes, in order to establish optimal production procedures at minimum costs. different optimization techniques can be applied to determine the optimum medium composition, carbon and nitrogen source concentration, mixing speed, temperature, or ph value. based on such experiments, valuable data are obtained about the microorganism, it's growth kinetics and nutrient requirements. if modern multifactorial optimization techniques are applied, experimental data are used to develop a predictive model that will describe the further behavior of the system.12 after that, the scale of the process is gradually increased to a laboratory bioreactor, and further on to bioreactors of larger volumes, to determine whether the same or similar results will be achieved on larger-scale equipment.13,14 the transition from shake flask to laboratory bioreactor is a critical point of the highest importance in the whole scale-up process. if done properly, it ensures the smooth further continuation of the scale-up procedure from laboratory to pilot and industrial scale bioreactor.15 when transitioning from a shake flask to laboratory bioreactor particular changes in the vessel geometry and size occur, which cause changes in mixing efficiency, affect oxygen supply, and increase the possibility of creating "dead zones" and uneven nutrient distribution. for that reason, the shake flask-bioreactor transition needs to be carefully optimized and designed. modeling the complex hydrodynamic behavior is one of the most difficult numerical problems that has fundamental importance in many aspects of engineering. the key parameters are related to mass and heat transfer, mixing and aeration.16 in order to optimize the performance of the bioreactor, it is necessary to know the local fluid dynamics in the bioreactor, i.e. the relation between hydrodynamics and the mass transfer coefficient.17 volumetric oxygen mass transfer coefficient (kla) and oxygen transfer rate (otr) are the key parameters used to describe the efficiency of oxygen utilization. kla is most commonly used as a scale-up criterion in aerobic processes used to estimate the efficiency of the bioreactor.18,19 this practice is supported by the fact that the main problem in aerobic systems is the adequate supply of oxygen from the gas to the liquid phase. dimensional analysis helps to a cc ep te d m an us cr ip t bacillus subtilis ncim 2063 cultivation 3 develop correlations that will ensure a constant value of kla corresponding to the desired otr. the success of the scale-up process is confirmed experimentally when it is shown that the same or similar results can be achieved in a bioreactor under the same conditions as in shake flasks.17 therefore, the aim of this work was to produce good-quality biomass of desired high cell and spore density, and to: • assess the individual and combined effect of critical b. subtilis ncim 2063 growth parameters: oxygen transfer rate, mixing speed, temperature and glucose concentration; • determine the optimum growth conditions for b. subtilis ncim 2063 on a shake flasks level using response surface methodology (rsm); • provide -statistically significant model equations for the shake flasks level; • scale-up the cultivation of b. subtilis ncim 2063 from shake flasks to batch bioreactor using kla as scale-up criterion; • evaluate the success of the scale-up procedure by bioreactor cultivation with additional analysis of kinetic and stoichiometric parameters. experimental inoculum preparation sporogenic gram-positive bacterium b. subtilis 2063 from the ncim collection was provided by a private company fertico d.o.o. located in nis, serbia. the bacterial culture was stored at -80 °c in vials and at 4 °c on agar plates. 300 ml nutrient broth was inoculated with a single loop of b. subtilis ncim 2063 and incubated at 37 °c for 24 h in shake flasks at 150 rpm. a 1% (v/v) overnight culture was used as inoculum for further cultivation. shake flask cultivation study to optimize the b. subtilis growth conditions in the shake-flasks, the box-benken experimental design (bbd) was used (table 1). 1 % b. subtilis 2063 overnight culture was used to inoculate sterilized nutrient dsm medium (8g nutrient broth, 10 ml 10% (w/v) kcl, 10 ml 1.2% (w/v) mgso4, 1 ml 1 m ca(no3)2, 1 ml 0.01 m mncl2, 1 ml 1 mm feso4) in a 500 ml erlenmayer vessel. dsm is a commonly used medium for bacillus cultivation, owing its popularity to its simplicity, high biomass yield and sporulation efficiencies 20–24. the three factors varied on three levels were: temperature (25-37 °c), otr (2-10 mol m-3h-1) and glucose concentration (0-20 g l-1). the factors and their levels were selected based on the preliminary experiments and available literature data 22,25–27. cultivation was performed for 24 h on a rotary shaker at 150 rpm according to the design matrix. the total number of viable vegetative cells and the number of spores were chosen as dependent variables. the viable cell count was determined using the spread plate method. spores were counted using the same method, but the samples were previously heated at 80 °c for 10 min. a cc ep te d m an us cr ip t 4 stamenković stojanović et al. table 1 coded and non-coded values of process factors according to bbd design symbol factor low level (-1) middle level (0) high level (+1) a temperature, °c 25 31 37 b otr, mol/m3h 2 6 10 c glucose concentration, g/l 0 10 20 bioreactor cultivation 1% b. subtilis ncim 2063 inoculum was used to inoculate the laboratory bioreactor containing a sterile dsm medium. the cultivation was performed at the optimum conditions determined in the shake flasks and after the scale-up procedure. the bioreactor cultivation was performed in a bioreactor klfm, bioengineering, wald, switzerland (working volume: 2.5 l, total volume 3.7 l), equipped with two rashtan impellers with 6 blades (48 mm diameter) and 4 baffles. sterile air supplied from an external compressor was used for aeration with a specific air flow of 0.3 v.v.m. the bioreactor is equipped with a ph (bioengineering 4695) and an oxygen electrode (mettler toledo 3420036) and connected to the bioscada software system, which monitors the process parameters. kla and otr determination kla values in the bioreactor were determined using the absorption method, while the otr values in shake flasks and in the bioreactor were calculated according to equations 1 and 2, respectively 28: 𝑂𝑇𝑅 = 7.23 ⋅ 10−4 ⋅ ( 𝑉𝐿 𝑉𝐹 ) −0.845 ⋅ 𝐶 ⋅ 𝑁 (1) 𝑂𝑇𝑅 = 𝑘𝐿 𝑎 ⋅ (𝐶 ∗ − 𝐶) (2) where: vl is a liquid volume (ml), vf is erlenmayer flask volume (ml), c is dissolved oxygen concentration (mg l-1), n is shaking speed (s-1), and c* oxygen solubility in the medium at a given temperature. kinetic and stoichiometric parameters the yield coefficients and were calculated according to the following equations: 𝑌𝑥/𝑜 = 𝑋 𝑂𝑇𝑅⋅𝑡 (4) 𝑌𝑥/𝑠 = 𝑠0−𝑠 𝑥−𝑥0 (5) where: s0 is initial glucose concentration (g l -1), s is final glucose concentration, x is final biomass concentration (g l-1), x0 is initial biomass concentration (g l -1), is the biomass concentration at the moment t, otr is oxygen transfer rate (mol m3h-1), and is time (s). yx/0 was calculated under the assumption that all of the oxygen that was transferred to the medium was also consumed by the microorganism. specific growth rate µm and generation time td were calculated using the following equations29: 𝑙𝑛 𝑥 𝑥0 = 𝜇𝑚 ⋅ 𝑡 (6) a cc ep te d m an us cr ip t bacillus subtilis ncim 2063 cultivation 5 𝑡𝑑 = 𝑙𝑛 2 𝜇𝑚 (7) where is the biomass concentration at the moment t, and is the initial biomass concentration (g l-1). statistical analysis each experiment was run three times in parallel, and the findings were reported as the mean value of three repetitions ± standard deviation. the program origin 6.0. excel 2013 and expert design 7.0 were used for the statistical processing of experimental data. rsm and derringer's desirability function were used for modeling and optimization. the adequacy of the response surface model was assessed using the analysis of variance (anova). results and discussion the reliability and durability of microbial biomass formulation largely depend on the number of living cells, particularly the spores, which is why it is important to enabling the microorganism to reach a high cell density and to sporulate. three independent factors (temperature, oxygen transfer rate and initial glucose concentration) were varied at three levels according to the box-benken experimental design, and their effect on total vegetative cell and spore count after 24 h of cultivation is shown in table s1 (supplementary file). by applying the nonlinear regression method to the obtained experimental data the following mathematical models were proposed for the total number of b. subtilis vegetative cells and spores, respectively: 𝑋 = 9.04 + 0.01 ⋅ 𝐴 − 0.29𝐵 + 0.02𝐶 + 6.04 ⋅ 10−3𝐴𝐵 − 8.75 ⋅ 10−4𝐴𝐶 + 0.01𝐵𝐶 − 81.87 ⋅ 10−4𝐴2 + 0.01𝐵2 − 5.36 ⋅ 10−3𝐶2 (8) 𝑌 = 7.05 + 0.06𝐴 − 0.18𝐵 + 0.105𝐶 + 0.02𝐵2 − 7.43 ⋅ 10−3𝐶2 (9) where x is vegetative cell count, y is spore count, a is the temperature (°c), b is otr (mol m-3h-1) and c is initial glucose concentration (g l-1). the significance and reliability of the models were assessed by comparing the predicted and experimental values and by conducting anova analysis (table 2). it can be observed that each of the individual factors in the model that predicts the number of viable vegetative cells is statistically significant with a degree of reliability of 99 %. the calculated p-value for the lack of fit (0.219) for this model is statistically insignificant and indicates a good fit of the experimental data for both of the models. the highest significance was recorded for initial glucose concentration and otr (in both their individual and quadratic form). individual temperature term, as well as temperature otr interaction term are also found to significantly influence the number of b. subtilis vegetative cells. since otr and the initial glucose concentration are significant on a quadratic level, a small variation in their values will greatly affect the growth rate and the final number of cells21. a cc ep te d m an us cr ip t 6 stamenković stojanović et al. table 2 anova for the models obtained for b. subtilis ncim 2063 viable cell and spore count parameter viable cell count spore count f value p-value f value p-value model 41.32 < 0.0001 20.86 < 0.0001 a 13.62 0.0078 5.84 0.0363 b 109.04 <0.0001 0.99 0.3425 c 126.23 < 0.0001 91.22 < 0.0001 ab 5.03 0.0599 ac 0.66 0.4436 bc 40.2 0.0004 9.26 0.0124 a2 0.011 0.9177 b2 6.5 0.0381 4.13 0.0696 c2 75.52 <0.0001 14.52 0.0034 lack of fit 2.3 0.2186 3.59 0.1182 r2 0.982 0.926 adj r2 0.958 0.882 pred r2 0.802 0.697 c.v., % 1.47 5.10 press 1.25 6.59 mrpd, % 0.1 1.52 values of p less than 0.05 (p < 0.05) indicate model terms are significant. on the other hand, sporulation was highly affected by initial glucose concentration (individual and quadratic term) which is the most significant factor. apart from glucose, temperature, quadratic term of otr and interaction between otr and initial glucose concentration were also significant. the spore-predicting model was also statistically significant (with an f value of 21.01 and 0.0002 for the p-value), with an insignificant lack of fit. the reliability of the models was additionally assessed by analyzing the values of r 2 , adj r 2 and pred r 2 , which are in accordance with the requirements that r 2 and adj r 2 should be reasonably close to 1, and that the difference between adj r 2 and pred r 2 should not exceed 0.2.30 obtained values imply that both developed regression equations have a good fit and that they can successfully predict system responses. the regression equation is also graphically represented in two-dimensional contours, visualizing the relationship between the response and each of the independent variables in the system (figure 1). a cc ep te d m an us cr ip t bacillus subtilis ncim 2063 cultivation 7 a) b) fig 1 contour plots representing the total number of b. subtilis ncim 2063 viable cells as a function of glucose concentration, g l-1, temperature (°c), and otr 2 mol m-3h-1 (a), 10 mol m-3h-1 (b) it can be concluded that at higher initial glucose concentrations, an increase in otr has a positive effect on the total number of viable vegetative cells, while temperature has no significant effect. by reducing the concentration of glucose, the effect of temperature becomes more pronounced, with the largest number of cells being achieved in a medium with 10 g l-1 glucose. in contrast, in a glucosefree medium, increasing the otr has little effect on the cell number. this effect is diminished by an increase in temperature. such moderate interaction of otr and temperature was also previously confirmed through the calculated p-value. similarly, the significance of this interaction effect was observed in a study optimizing the growth of bacillus coagulans using the rsm method, with maximum biomass yield obtained by a combination of high temperatures and specific airflow.31 the graphic analysis confirmed the importance of oxygen availability, which is explained by the fact that lack of oxygen reduces the ph of the substrate, leading to rapid lysis of the cell and the initiation of new metabolic pathways.32 design-expert® software viable cell count design points 9.92 7.1 x1 = c: glucose x2 = a: temperature actual factor b: otr = 2.00 0.00 5.00 10.00 15.00 20.00 25.00 28.00 31.00 34.00 37.00 viable cell count c: glucose a : t e m p e ra tu re 8.88318 9.0207 9.05671 9.0977 8.92181 8.95783 8.78066 8.82888 8.30145 7.80329 8.64518 8.04074 design-expert® software viable cell count design points 9.92 7.1 x1 = c: glucose x2 = a: temperature actual factor b: otr = 10.00 0.00 5.00 10.00 15.00 20.00 25.00 28.00 31.00 34.00 37.00 viable cell count c: glucose a : t e m p e ra tu re 8.88318 9.0207 9.0207 9.15821 9.15821 9.29572 9.29572 9.43324 9.05671 9.05671 9.0977 9.0977 8.92181 8.95783 9.73352 9.60996 9.8606 9.23384 9.23384 a cc ep te d m an us cr ip t 8 stamenković stojanović et al. a) b) fig 2 contour plots for the number of b. subtilis ncim 2063 spores as a function of otr, temperature and initial glucose concentration 0 g l-1 (a), 20 g l-1 (b) figure 2 shows the visualized relationship between dependent and independent variables for the spore-predicting model. the maximum number of spores was obtained in a glucose-free medium at otr = 10 mol m-3h-1, while the effect of temperature was negligible. as expected, nutirent deprevation inititated sporulation, which allows b. subtilis to enter a dormant state, preserving its genetic material and resistance to harsh conditions until favorable growth conditions are restored24. the effect of temperatures higher than 30 °c is most pronounced at the maximum applied initial glucose concentration. in a glucose-free medium, increasing the otr increases the number of sporulated cells. as the initial glucose concentration increases, the influence of otr on the sporulation is reduced with the increase in the initial glucose concentration in the medium, although cultivation at higher temperatures reduces the effect of glucose. it can be explained by the fact that increasing the initial glucose concentration increases the viscosity of the medium, which creates greater resistance of the liquid film and thus reduces the real otr value. increasing the temperature increases the solubility of the components of the nutrient medium, which diminishes the negative effect of glucose in this interaction. the literature data agree with the experimental results achieved here. it was found that the maximum yield of spores is achieved at low initial glucose concentrations and that bacterial culture begins to sporulate when the cell density is about 108 cfu ml-1.33 the reason for this is the characteristic of cells to sporulate in unfavorable environmental conditions, ie. in conditions when nutrients are not available in excess.34 as a result of that, the microorganism can design-expert® software spore count design points 9.33 5.1 x1 = a: temperature x2 = b: otr actual factor c: glucose = 0.00 25.00 28.00 31.00 34.00 37.00 2.00 4.00 6.00 8.00 10.00 spore count a: temperature b : o t r 8.38958 8.62514 8.62514 8.86069 8.45831 8.36041 9.31996 design-expert® software spore count design points 9.33 5.1 x1 = a: temperature x2 = b: otr actual factor c: glucose = 20.00 25.00 28.00 31.00 34.00 37.00 2.00 4.00 6.00 8.00 10.00 spore count a: temperature b : o t r 5.25458 5.68167 6.10875 6.53583 6.96292 6.96292 a cc ep te d m an us cr ip t bacillus subtilis ncim 2063 cultivation 9 undergo metabolic shifts, activating alternative metabolic pathways, or emploing strategies such as gluconeogenesis to sustain growth using non-carbohydrate carbon sources. the gluconeogenesis pathway involves a series of enzymatic reactions that convert non-carbohydrate precursors into glucose-6-phosphate, which are metabolized through glycolysis for energy production or used as a precursor for biosynthesis 35. multicriteria optimization using derringer's desirability function derringer's desirability function is used in complex multivariate processes in which variables that need to be optimized are influenced by multiple factors simultaneously. based on multicriteria optimization (table s2, supplementary file), several combinations of process conditions have been proposed to obtain the maximum value of the desirability function as well as of both response variables. taking into account the response surface analysis and knowing that the increase in temperature does not decisively affect the increase in the total number of viable cells, it was decided to maximize the cell growth and provide the necessary conditions for sporulation, while achieving energy savings. according to that, the following optimum conditions for the cultivation of b. subtilis ncim 2063 were proposed: t = 33 °c, otr =10 mol m-3h-1, and initial glucose concentration 4.89 g l-1. under these conditions, the model predicts a maximum viable vegetative cell concentration of 9.66 log (cfu ml-1) and spores of 9.19 log (cfu ml-1) with a high desirability function value (0.931). the experimental cell density for the total number of cells and spores obtained under the given conditions was 9.51 ± 0.09 log (cfu ml-1) and 9.08 ± 0.06 log (cfu ml-1), respectively. the experiment was performed in triplicate, and the relative error between predicted and obtained values for vegetative cells and spores was 1.5% and 1.2%, respectively, which confirms that there is a good agreement between predicted and experimental values. a scarce number of previously published studies is dedicated to the topic of multicriteria optimization of b. subtilis growth conditions. most of the available research deals with optimizing the media composition,36–40 while a small number of them optimize growth conditions. a group of authors conducted a screening of the influence of volumetric airflow and mixing rate on cell density and sporulation of b. subtilis ea-cb0575 using a central composite experimental design.31 as optimal conditions, a mixing speed of 432 rpm and a volumetric airflow of 12 l/min at a temperature of 30 °c are given.41 the same experimental design was used to optimize the sporulation of b. subtilis in a solid medium. it was found that temperature and volumetric air flow have an impact on sporulation, with 27 °c and 1.2 l min-1 being recommended as optimal values for solid medium, respectively.42 a cc ep te d m an us cr ip t 10 stamenković stojanović et al. scale up from shake flask to laboratory bioreactor kla is a key parameter for scaling and optimization in mechanical mixing systems, where the rate of oxygen mass transfer between the gas and liquid phases is an essential phenomenon for process control.43 hence, kla was chosen as the basic criterion for the scale-up procedure to the bioreactor level. the main goal of the scale-up process was to define the values of process conditions at the bioreactor level that will enable the same value of kla established for shake flasks: namely to define the mixing speed that will provide the desired oxygen transfer from gas to a liquid phase. firstly, kla was measured at different mixing speeds in the dsm medium at the bioreactor level (table 3). as expected, reducing the mixing speed also affects the reduction of the oxygen mass transfer rate. an increase in the stirrer speed from 100 rpm to 400 rpm, causes an exponential increase in the kla value. such a result is in accordance with the literature data, since in the medium with 10 g l-1 of glucose at a specific air flow of 1 v.v.m, an increase in the value of kla from 25.2 h-1 to 104.4 h-1 was detected when the mixing speed was increased from 300 rpm to 600 rpm.44 at higher mixing speeds the air bubbles break into small bubbles, which increases the gas-liquid interfacial surface to transfer the oxygen in the medium, thus increasing the kla.45 table 3 influence of mixing speed on the kla values in dsm medium at bioreactor level mixing speed, rpm 100 200 300 400 kla, h-1 5.26±0,02 6.51±0,11 11.88±0,15 45.35±0,25 otr, mmol m-3h-1 1.15 1.42 2.59 9.88 after applying regression analysis to experimental data obtained by the absorption method, the following empirical equation was developed to describe the relationship between the mixing speed n and kla: 𝑘𝐿 𝑎 = exp (7.1 ∙ 10 −3𝑁 + 0.7) (9) based on the obtained correlation it was calculated that at given conditions in a laboratory bioreactor containing dsm medium (33 °c and 0.3 v.v.m air flow rate) the mixing speed should be set to 452 rpm in order to achieve the required kla value (45.35 h-1). bioreactor cultivation the success of the scale-up procedure was evaluated after the cultivation of b. subtilis ncim 2063 at determined optimum conditions at the bioreactor level. it was concluded that, at the end of the cultivation period, the total number of viable cells in the bioreactor was 9.65 ± 0.05 log (cfu ml-1), which is 1.4 times more than the number of cells achieved in shake flasks (9.51 ± 0.09 log (cfu ml-1) under the same conditions. growth kinetic and stoichiometric analysis lead to the same conclusion (fig 3, table 4). after 24 h cultivation, the biomass concentration was a cc ep te d m an us cr ip t bacillus subtilis ncim 2063 cultivation 11 higher in the bioreactor than in the shake flasks, although similar values of specific growth rate were recorded in both systems. the higher cell density at the end of cultivation in the bioreactor can be explained by better oxygen saturation of the medium, which is confirmed by a higher biomass yield on oxygen (yx/o) in the bioreactor (table 4). namely, in shake flasks, gas induction is based only on surface aeration. initially, the substrate is saturated with air and the amount of oxygen is sufficient for microbe growth. after the exponential phase, a sharp drop in the oxygen concentration occurs in shake flasks. in the case of bioreactors, mixing and a constant supply of fresh air provide sufficient levels of oxygen, which can contribute to greater multiplication of cell mass, or lead to prolongation of the exponential phase.46 this once again confirms that aeration and mixing play a very important role in the metabolic activity of microorganisms. given that a satisfactory number of cells was achieved in the bioreactor at the end of the cultivation period, it is concluded that the scale-up process was successfully implemented, which created the basic condition for further scale-up to the semiindustrial level. table 4 specific growth rate (μm ), generation time (td ), final biomass concentration (x) and biomass yield from oxygen consumption (yx/o) during shake flask and bioreactor cultivation of b. subtilis ncim 2063 cultivation μm, h -1 td, h x, g l -1 yx/o, g g -1 bioreactor 0.44±0.08 1.57±0.58 7.24±0.00 0.81±0.00 shake flask 0.41±0.03 1.71±0.26 5.6±0.51 0.71±0.31 the success of the kla-based scale-up process strategy has been shown earlier in the literature. kla was used as a criterion for increasing the scale of phenyl acetyl carbinol production using the yeast saccharomyces cerevisiae. in that research, the kla value was first estimated by the absorption method in shake flasks, based on which appropriate correlations were developed. similar kla values and higher yield of the desired product were achieved in a 5 l bioreactor.46 an analogous scale-up strategy was applied for the cultivation of corynebacterium glutamicum and the production of lactic acid using adapted empirical models obtained by the sulfite method.47 the optimal value of kla (31 h-1) was also the leading parameter for adjusting the mixing speed and air volume flow in order to obtain a quality azospirillum brasilense-based product for pathogen biocontrol at the semiindustrial level.18 a cc ep te d m an us cr ip t 12 stamenković stojanović et al. fig. 3. b. subtilis ncim 2063 growth kinetics at bioreactor and shake flask level at dsm medium containing 4.89 g l-1 glucose at bioreactor level under the optimum conditions: 33 °c and 452 rpm conclusion in this study, the conditions for batch cultivation of b. subtilis ncim 2063 were optimized to maximize viability and sporulation. the individual and combined effects of kla, temperature and glucose concentration were assessed and explained. glucose and kla have the greatest statistical significance (both as an individual and as a quadratic term) for the number of viable cells, followed by the interaction factor of these two terms, the individual temperature factor and the interaction of temperature and kla. when it comes to the total number of spores, the concentration of glucose (individual and quadratic term), temperature, and the interaction of kla and glucose have the greatest influence on this response. statistically significant quadratic models were developed with an insignificant lack of fit, which is confirmed by a good agreement between experimentally obtained and predicted data. using derringer's desirability function the following optimum conditions were proposed for a dsm medium: t=33 °c, kla =50 mol m-3h-1 and glucose concentration 4.89 g l-1. scale-up from shake flasks to a batch bioreactor was performed using kla as a scale-up criterion. an empirical equation was developed to calculate the exact stirring speed needed to achieve the desired kla. the success of the scale-up procedure was evaluated by bioreactor cultivation with additional analysis of kinetic and stoichiometric parameters. given that a satisfactory number of cells has been achieved in the bioreactor and that the scalea cc ep te d m an us cr ip t bacillus subtilis ncim 2063 cultivation 13 up process was successfully implemented, a prerequisite is created to further scale up the process to semi-industrial and industrial levels in further research. acknowledgment: ministry of science, technological development and innovation, republic of serbia, project no: 451-03-47/2023-01/200133 supplementary material supplementary materials are available electronically from https://www.shdpub.org.rs/index.php/jscs/article/view/12350, or from the corresponding authors on request. и з в о д култивација bacillus subtilis ncim 2063: моделовање, оптимизација и процедура повећања размере сандра стаменковић стојановић, ивана карабеговић, бојана даниловић, стојан манчић и миодраг лазић технолошки факултет у лесковцу, универзитет у нишу, булевар ослобођења 124, 16000 лесковац, србија bacillus subtilis је непатогена, грам-позитивна бактерија која спорулише и има изражену антимикробну и метаболичку активност, а самим тим и велики потенцијал за примену у различитим областима. циљ овог рада био је одредити оптималне услове раста за b. subtilis ncim 2063 и повећати размере процеса са ерленмајера на ниво биореактора. критични параметри раста и ефекти њихове интеракције су изучавани применом боксбенкеновог експерименталног дизајна и методе одзивних површина. развијене једначине модела биле су статистики значајне. током култивације у ерленмајерима са мешањем, глукозу треба додати у концентрацији до 5 g l-1 при концентрацији otr од 10 mol m-3h-1 и на 33 °c, како би се постигао максималан број ћелија и спора. за повећање размере процеса са нивоа ерленмајера на ниво биореактора kla је коришћен као главни критеријум. ефекат повећања размере утврђен је поређењем кинетике раста у ерленмајерима и у биореактору. укупан број ћелија добијен у биореактору био је 4,57·109 cfu ml-1 што је1,41 пута више у одосу на број ћелија добијен у ерленмајеру, 3,24·109 cfu ml-1, доказујући да је процедура повећања размере успешно спроведена. 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http://dx.doi.org/10.1002/bit.21359 bactericidal effects of copper-polypyrrole composites modified with silver nanoparticles against gram-positive and gram-negative bacteria accepted manuscript this is an early electronic version of an as-received manuscript that has been accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as p. l. marucci, m. g. sica, l. i. brugnoni and m. b. gonzález, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc230213047m this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230213047m j. serb. chem. soc.00(0)1-18 (2023) original scientific paper jscs–12276 published dd mm, 2023 1 bactericidal effects of copper-polypyrrole composites modified with silver nanoparticles against gram-positive and gram-negative bacteria patricia l. marucci1, maria g. sica1,4, lorena i. brugnoni1, 2 and maría b. gonzález3,* 1department of biology, biochemistry and pharmacy, national university of the south, bahía blanca, argentina, 2institute of biological and biomedical sciences, national university of the south, conicet, bahía blanca, argentina, 3chemical engineering department, institute of electrochemistry and corrosion engineering, national university of the south, conicet, bahía blanca, argentina, and 4department of health sciences, national university of the south, bahía blanca, argentina (received 13 february; revised 26 march; accepted 2 august 2023) abstract: the aim of this research is to study the bactericidal effects of copperpolypyrrole composites deposited onto 316l ss modified with silver nanoparticles. the antimicrobial properties were evaluated against twenty-four strains of gram-positive and gram-negative bacteria. among the twenty-four strains studied, isolates included reference strains (e. coli atcc 25922, e. coli 0157:h7 edl 933, s. aureus atcc 25923 and l. monocytogenes atcc 7644), as well as strains isolated from food and clinical samples. the antimicrobial activity of the composites demonstrated that all ppy-modified films had antibacterial properties. notably, cu-ppyagnp500 exhibited the strongest inhibitory activity against both gram-negative and gram-positive bacteria. surface modification of 316l ss with these films is a promising and viable alternative for the development of novel antibacterial composites that can inhibit the growth of a significant number of bacteria. keywords: conducting polymer, antibacterial applications, metallic nanoparticles, stainless steel. introduction antibiotic resistance is considered by the world health organization as a critical problem of global concern, which causes higher medical costs, prolonged hospital stays, and increased mortality.1 this threat is caused by the overuse and abuse of antibiotics, coupled with the natural evolutionary processes of bacteria. there is a significant interest from all nations in developing a global action plan to *corresponding author e-mail: belen.gonzalez@uns.edu.ar https://doi.org/10.2298/jsc230213047m a cc ep te d m an us cr ip t mailto:belen.gonzalez@uns.edu.ar https://doi.org/10.2298/jsc230213047m 2 marucci et al. prevent and control the growth of antibiotic resistance.2 in the biomedical field, bacterial infections at the site of implanted medical devices such as catheters and artificial prostheses are a serious and persistent problem. although medical implants greatly improve the patients’ quality of life, implant-related infections are recognized as a tragic problem, often requiring revision surgery for individuals with severe infections.3 to overcome this problem, surface modifications are used to improve the antibacterial properties of materials. 4,5 some antibacterial strategies for metal implants may include inhibiting the adhesion, the colonization, the biofilm formation and the proliferation of bacteria.6,7 recently, many organic compounds, including conducting polymers or biopolymers, have demonstrated their potential as antibacterial and antiviral agents to combat infections caused by harmful bacteria and viruses. 8,9 in recent years, the research on conducting polymers and their composites as antibacterial agents has gained momentum.8,10 in particular, polypyrrole (ppy) has been successfully explored for new antibacterial systems due to its easy preparation, low cost, low toxicity and biocompatibility.11 in a recent study, the antibacterial properties and the synergistic behavior of a composite consisting of silver nanoparticles, single-walled carbon nanotubes, and ppy were reported. the prepared ternary composite exhibits the following order of performance within 24 hours at a concentration of 0.048 mg/ml: b. cereus > e. coli > p. aeruginosa > methicillin-resistant s. aureus.12 another noteworthy composite consisting of ppy and zinc-doped copper oxide microparticles showed remarkable antimicrobial effects against e. coli and s. aureus. the authors postulated that the mechanism of cell death was mainly induced by the release of reactive oxygen species (ros), which damage bacterial membranes, dna and proteins.13 recent studies conducted in our laboratory have demonstrated that hybrid antimicrobial coating materials containing polypyrrole combined with copper14,15, silver16 or zinc17 species have also shown great bactericidal effects. silver has a long history of use in pharmacology and medicine as an antibacterial agent. in the field of orthopedics, silver nanoparticles (agnps) have yielded excellent results in modifying implant surfaces to prevent implant-related infections18. furthermore, the antibiotic activity of agnps has been studied in various bacteria, yielding positive results.19 however, the exact mechanisms by which ag acts as antimicrobial agent have not been fully clarified. three hypotheses have been proposed: (1) ag+ are taken up by bacteria, triggering a cascade of intracellular reactions that disrupt atp production and dna replication; (2) silver ions promote the generation of reactive oxygen species (ros) both inside and outside bacterial cells, causing oxidative stress and subsequent damage to bacterial membrane lipids and dna; (3) ) in the case of agnps, it is believed that these particles can penetrate cell membranes and activate one or all of the aforementioned mechanisms.20 a cc ep te d m an us cr ip t polypyrrole composite with bactericidal properties. 3 the incorporation of copper as antibacterial agent in metallic implants has also been considered.21 the interaction between copper and bacteria cells involves several important events that can be listed as follows: the first event (a) is the accumulation of cu ions on the bacteria membrane or within the cell. this accumulation not only causes membrane damage through depolarization, but also leads to the leakage of intracellular components; (b) the generation of ros by copper ions may lead to further cell damage; (c) cu ions inside bacterial cells may bind with dna molecules, resulting in the disruption of the helical structure and biochemical processes.22 on the other hand, 316l stainless steel (316l ss) is the most commonly used alloy in medical implants due to its good corrosion performance, notable biocompatibility, high mechanical properties, and low cost accessibility.23 according to this, in our laboratory a microstructured ppy film was electrosinthetized onto 316l ss from a solution containing copper species. the results indicated that ppy/cu-modified electrodes are effective for water disinfection contaminated with e. coli.14 furthermore, it has been demonstrated that a a salicylate-doped ppy film is an effective matrix for immobilizing ag species, and the resulting composites exhibit excellent performance in inhibiting the activity of staphylococcus aureus bacteria. these findings suggest that the composite holds promise for biomedical applications.16 considering the advantages of silver and copper species, as well as the synergistic effect of these metals with ppy in antibacterial studies, the aim of this research is to study the bactericidal effect of copper-polypyrrole composites modified with silver nanoparticles deposited on 316l ss. according to the bibliography consulted, this study is the first to evaluate the antibacterial properties of a ppy composite against a total of twenty-four strains of both gram-positive and gram-negative bacteria. these isolates include reference strains (e. coli atcc 25922, e. coli 0157:h7 edl 933, s. aureus atcc 25923 and l. monocytogenes atcc 7644) and strains isolated from food and clinical samples. given the extensive range of bacteria examined, the resulting composite shows promise as a surface modification material for 316l ss, with potential applications in various fields such as medical instruments, water treatment devices, and food processing. experimental chemicals pyrrole (99.9 % purity), silver nitrate (99.9 % purity) and sodium salicylate (99.9 % purity) were obtained from sigma (sigma chemicals, munich, germany). copper sulphate pentahydratade (98 % purity) and potassium nitrate (98 % purity) were obtained from cicarelli laboratorios (santa fe, argentina). pyrrole (sigma–aldrich) was freshly distilled under reduced pressure prior to use. a cc ep te d m an us cr ip t 4 marucci et al. bacterial strains the antibacterial properties of composites were tested against four reference strains (escherichia coli atcc 25922, e. coli 0157:h7 edl 933, staphylococcus aureus atcc 25923 and listeria monocytogenes atcc 7644), four e. coli strains (named 6, 17, 19 and 28) isolated from recreational waters24, thirteen s. aureus strains (named 1 and 13) obtained from the nose of asymptomatic volunteers, p. aeruginosa isolated from drinking water and salmonella spp. and l. innocua isolated from meat products. electrosynthesis and characterization of composite the composites were electrosynthesized onto 316l ss sheets (wt. % is 17.47 cr, 10.32 ni, 1.88 mn, 1.90 mo, 0.39 si, 0.025 c, and fe balance) with an exposed area of 0.25 cm2. prior to each experiment, the exposed 316l ss surface was abraded with a 1200-grit finish using sic, then degreased with acetone, and finally washed with triply distilled water. for the electrochemical experiments, a conventional three-electrode system and a 20 cm3 metrohm cell were used. a pt sheet served as the counter electrode, while a commercially available ag/agcl/3m kcl electrode (metrohm) was used as the reference electrode. as described in our earlier work, ppy films and copper-ppy (cu-ppy) composites were obtained potentiostatically at 0.90 v from a solution containing 0.25 m py + 0.50 m nasa. in the case of the cu-ppy composite, a concentration of 0.10 m cuso4·5h2o was also incorporated in the electrosynthesis solution.15 silver nanoparticles were electrodeposited onto ppy and cu-ppy using a solution containing agno3 + kno3, following the potentiostatic double-pulse technique introduced by scheludko and todorova25. the process is based onthe following parameters: nucleation potential e1= -0.8 v for nucleation time t1=0.5 s and growth potentials of e2= 0.1 v for growth time t2=30 s. the size of the nanoparticles was controlled by the solution concentration. electrochemical experiments were performed using a potentiostat–galvanostat autolab/pgstat128 n. morphological studies of gold metallized films were conducted using a scanning electron microscopy (sem), specifically the leo 1450 vp system, which was equipped with an energy-dispersive scanning (eds) probe. additionally, the electrical conductivity of the films was measured using a homemade device through the two-probe method. disk diffusion assay the antimicrobial effectiveness of the coatings was tested using the kirby-bauer disk diffusion method, following the model of antibiogram execution.26 briefly, cultures of the strains under study were cultivated on muller–hinton agar (mha) plates from britania laboratories s.a., argentina, for 24 h at 37 °c. the resulting colonies were suspended in muller-hinton broth from the same manufacturer. the turbidity (expressed as optical density; od) of the bacterial suspensions was measured using an optical spectrophotometer (λ = 600 nm). the suspensions were adjusted to a turbidity of 0.5 based on the mcfarland standard (106 cfu ml−1). a sterilized cotton swab was dipped into the resulting suspension, and used to apply a bacterial lawn on mha plates. petri plates were left to dry for 10 minutes, after which the composites were distributed, using the bare alloy and the ppy covered electrode as control samples. incubation was carried out at 37 °c, and after 24 hr, the plates were examined to identify the presence or absence of zones of inhibition. when zones of inhibition were observed, their diameter was measured with a ruler with a resolution of up to 1 mm. for each type of composite and microbial strain, the mean and standard deviation (sd) were calculated based on data obtained from two independent replicates. a cc ep te d m an us cr ip t polypyrrole composite with bactericidal properties. 5 results and discussion electrosynthesis and characterization of composites the formation and characterization of ppy and cu-ppy coatings on 316l ss were previously discussed in our prior work.15 as mentioned before, both films have a morphology composed of hollow rectangular-sectioned microtubes. the proposed mechanism for the formation of these microtubes consists in the early crystallization of salicylic acid (hsa) on a granular ppy. these crystals are formed due to the rapid production of h+ ions, which protonate the salicylate anions, constituting the building blocks for the formation of the rectangular microtubes.27 firstly, the electrodeposition of silver nanoparticles onto ppy was performed using the potentiostatic double-pulse technique. the initial pulse is applied to facilitate the formation of nuclei, while the subsequent pulse, with a more positive potential than the first one, regulates the growth of the nuclei deposited during the preceding pulse.25 to control the nanoparticles size, two solutions with different concentrations were used: for smaller nanoparticles, a solution with a concentration of 5 mm agno3 + 50 mm kno3 was utilized. sem/eds analysis was performed to characterize the composite. in fig. 1(a), it can be observed that the surface of the ppy film is effectively decorated with agnps, approximately 100 nm in size. eds spectra [fig. 1 (b)] were used to demonstrate the presence of metallic silver, with the signal typically appearing between 3-3.6 kev.28 the weight percentage registered for this element was 6.8 %. the spectrum also displayed signals of c and o associated with organic compounds.29 in order to increase the size of the deposits on the ppy films, the concentration of the electrosynthesis solution was raised to 10 mm agno3 + 100 mm kno3. as seen in fig. 1(c), nanoparticles of approximately 500 nm were obtained. in this case, the eds analysis [fig. 1 (d)] shows a signal signal indicating the presence of metallic silver, with a weight percentage of 13.8 %. the results demonstrated a direct correlation between the increase in ag weight percentage and the increment in the particle size. to facilitate comprehension, the obtained composites were named ppy/agnp100 and ppy/agnp500, according to the nanoparticle size. regardless of the agnps size, a rosette-like structure characterizes most of the deposits. these preliminary findings indicate that, when applying the same electrochemical procedure, a rise in the concentration of the electrosynthesis solution, would not only leads to a growth in nanoparticle size but also to an increase in the amount of metallic silver in the composites. a cc ep te d m an us cr ip t 6 marucci et al. fig.1. sem images and eds spectra of ppy/agnp100 (a, b) and ppy/agnp500 (c, d). to verify the amount of metallic silver deposited in the composites, a cyclic voltammetry experiment was performed. the procedure involved cycling the electrodes, which were covered with ppy/agnp100 and ppy/agnp500 in a 0.25 m nasa solution between the potentials of 0.60 and 1.20 v at 0.01 v s-1. the polymer without nanoparticles was also cycled. figure 2 illustrates that the electrochemical behavior of the composites differs significantly depending on the size of the silver nanoparticles. the strong and well-defined anodic peaks at 0.50 v are a result of the oxidation process of the deposited metal.16 for the ppy/agnp500 composite, the oxidation peak is associated with a current density of 6.5 ma/cm² (see curve b), while for the ppy/agnp100 composite, the oxidation peak is associated with a current density of 2.5 ma/cm² (see curve a). the peak area analysis of the oxidative peaks reveals that, as the nanoparticle size increases, the peak area also increases, indicating a higher concentration of metallic silver in the composite. moreover, at approximately 1.0 v, a peak associated with the a cc ep te d m an us cr ip t polypyrrole composite with bactericidal properties. 7 overoxidation of the polymer is observed in both composites.30 in the reverse scan, cathodic peaks appear at 0.0 v, indicating the reduction of silver ions. based on the comparison of the current density of reduction peaks, it can be concluded that a higher amount of silver ions redeposited back into the composites is associated with a larger size of agnps. in contrast, regarding unmodified ppy, no peak is observed in this potential range (curve c). fig. 2. cyclic voltammograms registered in a 0.25 m nasa solution at 0.01 v s−1 for 316l ss electrode coated with: (a) ppy/agnp100 and (b) ppy/agnp500. the data for unmodified ppy coated-316l ss is also provided (curve c). with the aim of determining the amount of silver deposited in the composites, conductivity was measured. the values obtained were 28 m-1 for ppy/agnp100 and 42 s m-1 for ppy/agnp500. these values fall within the range already reported for conductive polymers modified with nanoparticles.31 in the case of unmodified ppy, the conductivity value was 3.8 s m-1. these values were estimated based on a film thickness of 7 m.32 firstly, it can be observed that the incorporation of silver nanoparticles contributes to the improvement of the electrical conductivity of the ppy films.28 the difference between the conductivity values of ppy/agnp100 a cc ep te d m an us cr ip t 8 marucci et al. and ppy/agnp500 could be attributed to the size of agnps deposited in the composites. it is worth noting that an increase in the conductivity of the composites is associated with an augment of metallic silver deposited on the polymer. indeed, diantoro et al. proved conclusively that increased quantities of agnps integrated into a conducting polymer enhance the conductivity of the composite.33 considering the excellent results obtained in water disinfection with cu-ppy films15, we decided to deposit agnps onto cu-ppy to study the antibacterial influence of both metals. in this process, silver nanoparticles were electrodeposited on cu-ppy films, using the previously described double-pulse technique. the silver nanoparticles were obtained from a solution containing 10 mm agno3 + 100 mm kno3. sem micrograph of the polymer surface confirms the presence of ppy microtubes decorated with approximately 500 nm-sized agnps [fig. 3 (a)]. the magnified image shows that most of the nanoparticles have rosette-like structures [fig. 3 (b)]. in this case, the composite was named cu-ppy/agnp500. eds spectra confirm the presence of ag and cu in the composite, with respective weight percentages of 4.7% and 4.9%. the signals of c and o correspond to the polymer composition, as expected. fig. 3. sem images (a, b) and eds spectra (c) of cu-ppy/agnp500. a cc ep te d m an us cr ip t polypyrrole composite with bactericidal properties. 9 to evaluate the chemical state of copper in the cu-ppy and cu-ppy/agnp500 composites, a cyclic voltammetry experiment was performed in a 0.25 m nasa solution. in figure 4, the incorporation of cu2+ is evidenced, in both films, by the presence of cathodic peaks at a potential of about 0.10 v vs. ag/agcl, which are attributable to ion reduction.15 in the case of cu-ppy/agnp500, an overlap of ag+ and cu2+ reduction peaks is registered. the inset in figure 4 shows the anodic peaks of copper metal dissolution in both composites. as it was expected, the presence of a strong and well-defined anodic peak at 0.50 v is showed for cuppy/agnp500 as a result of the oxidation process of the agnps (curve b). in this case, the current density associated with the oxidation peak of the silver nanoparticles is 6.0 ma/cm², a similar value to the one obtained previously. on the other hand, no peak is observed for the cu-ppy composite (curve a) in this potential range. fig. 4. cyclic voltammograms registered in a 0.25 m nasa solution at 0.01 v s−1 for 316l ss electrode coated with: (a) cu-ppy and (b) cu-ppy/agnp500. the inset shows the curves amplified. xps analysis was used to determine the specific electron binding energies of the elements on the surface of the composites. figure 5 displays the survey spectra of the composites ppy/agnp100, ppy/agnp500 and cu-ppyagnp500. the elements a cc ep te d m an us cr ip t 10 marucci et al. detected in all composites are c 1s, o 1s, n 1s, since they are the main components of the polypyrrole matrix and the dopant (sa/hsa).22 in figure 6(a), the highresolution spectrum of ag 3d is presented. as can be seen, this element is also present in all samples. the two significant xps signals, located at approximately 374.2 ev and 368.2 ev and separated by a distance of 6.0 ev, correspond to the ag 3d3/2 and ag 3d5/2 binding energies of ag°, respectively.34 the results indicate the successful reduction of silver ions to zero-valent silver nanoparticles during the double pulse technique. on the other hand, the existence of cu2+ is detected solely in the xps survey of cu-ppyagnp500 (fig. 5, curve c), which is consistent with the cu 2p signal. in figure 6(b), the peaks at approximately 935.3 ev and 955.3 ev are assigned to cu 2p3/2 and cu 2p1/2, respectively, while the peaks in the range of 940-945 ev and 964.1 ev correspond to the shake-up satellite peaks of cu 2p, as supported by previous research.35,36 the xps characterization supports the hypothesis that the polymer matrix contains cu2+, as postulated in our previous study, where the presence of cathodic peaks during potentiodynamic polarization (fig. 4) was attributed to ion reduction.15 a cc ep te d m an us cr ip t polypyrrole composite with bactericidal properties. 11 fig. 5. xps survey spectra of a) ppy/agnp100, b) ppy/agnp500 and c) cuppyagnp500. a cc ep te d m an us cr ip t 12 marucci et al. fig. 6. xps spectra of cu-ppyagnp500: ag 3d (a) and cu 2p (b). agar diffusion assay in order to assess the in vitro antibacterial efficacy of the resulting composites, the agar diffusion method was employed. the diameter of inhibition zones (diz) after 24 h is shown in figure 7. overall, cu-ppy/agnp500 exhibited antibacterial activity against the the majority of the species tested (23/24). this composite proved effective against all s. aureus strains and all gram-negative bacteria under investigation. while several studies suggest that gram-positive s. aureus is more resistant to silver nanoparticles compared to gram-negative e. coli 37-39, this result does not consistently hold true.40 ppy/agnp500 and cu-ppy/agnp500 had a significant impact on the cellular viability of s. aureus, e. coli, s. enterica, l. innocua and p. aeruginosa. however, no activity was observed on the growth of listeria monocytogenes atcc 7644, which was only inhibited by ppy/agnp100. in particular, compared to ppy/agnp100 and ppy/agnp500, cu-ppy/agnp500 exhibited higher antimicrobial activity against s. aureus strains 3, 8 and 12; e. coli atcc 25922; e. coli strain 19; s. enterica; p. auruginosa; and l. innocua. given the nature of the agar diffusion method, which involves measuring the diameter of inhibition zones (diz) on agar plates using a ruler with a resolution of 1 mm, it is important to acknowledge the possibility of measurement errors. however, this method demonstrates the potential antibacterial effect of composites against various microbial strains. in the agar diffusion method, the diz surrounding a composite serves as an indicator of the microorganisms' sensitivity to the composite. while studies reporting diz values offer a quantitative measure of antibacterial activity, there is still significant non-uniformity in the experimental a cc ep te d m an us cr ip t polypyrrole composite with bactericidal properties. 13 methods employed. the diz can exhibit variability, even within the context of the same microbial strain exposed to silver/copper composites. these variations may be attributed to several factors, including variations in the growth medium, differences in the initial concentration of microorganisms, and the size, shape and composition of nanoparticles. thus, comparing the sensitivity of different strains to copper or silver nanocomposites is often not feasible, despite the wealth of available literature on the topic.41 fig. 7. growth inhibition of different microbial species by copper and silver ions released from a coated specimen. most research on bactericidal effects of silver and cooper has been limited to a small number of microbial strains or, in some cases, only a single strain. however, this study sought to compare the antibacterial effects of silver and copper compounds across a wider spectrum of twenty-four bacterial strains. these strains included four reference strains, five strains isolated from water sources, thirteen strains obtained from human clinical samples and two strains isolated from food sources. the findings of this study revealed strain-specific characteristics, which can eventually contribute to the improved and effective utilization of these compounds for specific applications. in recent years, numerous interconnected investigations have been conducted to enhance the utilization of agnps for bacteria inhibition. however, an ongoing debate persists regarding the antibacterial action mechanism of agnps. previous studies have indicated that the bactericidal properties of agnps are primarily attributed to a two-step mechanism. firstly, ag+ ions are released from the surfaces of nanoparticles. subsequently, these released ag+ ions interact with cellular targets, resulting in their bactericidal effect.42 a cc ep te d m an us cr ip t 14 marucci et al. the cellular uptake of agnps is another mechanism associated with physical interaction, and occurs when nps are small enough to cross the cell membrane.43 mukha et al. showed that the antibacterial activity of agnps smaller than 10 nm is attributed to both membrane damage and their ability to penetrate into the cell.44 similarly, dong et al. evaluated nps of varying sizes and reached the conclusion that agnps of smaller dimensions are more effective due to their ability to pierce the cell membrane.45 in a separate study, oves et al. synthesized agnps with bacterial exopolysaccharides, spherical shape, and a size of about 35 nm.46 they demonstrated that the antimicrobial activity of these nps against b. subtilis and methicillin-resistant staphylococcus aureus (mrsa) is attributed to ros production within bacterial cells. it has been suggested that agnps smaller than the pore size can easily penetrate the pore networks and efficiently depolarize the inner membrane, causing a bactericidal effect.47 in general, bacterial cells have a size in the micrometer range, whereas their outer cell membranes feature pores measuring in the nanometer range (<50 nm). since agnps used in this study are larger than the pores of the outer cell membrane, they are unable to penetrate the cell membrane. conversely, the higher antibacterial efficacy of ppy/agnp500, in contrast with ppy/agnp100, can be attributed to the larger amount of ionic silver released as a result of the larger nanoparticle size. the antibacterial activity of cu-ppy/agnp500 composites encompasses a series of steps. one of these steps involves the dissolution of agnps to release silver ions, along with the diffusion of copper ions from the ppy matrix. the release of silver ions leads to the disruption of the cytoplasm and the cell wall, while also triggering the production of ros. this ros production deactivates respiratory enzymes and inhibits the release of adenosine triphosphate.48 it is possible that the mechanism underlying the antibacterial action of cooper ions may be attributed to the generation of ros, which induces oxidative stress and/or damage in the bacteria.49 in addition to ros production, the cationic interaction of ag+ and cu2+ with negatively charged components of the bacteria cell membrane results in improved bactericidal efficacy at higher concentrations, achieved through the processes of cell lysis and bacteria collapse.50 hence, it is expected that both cu and agnps are able to interact with the entire surface of the bacterium. as a result, the copper and silver ions, which are toxic to bacteria, penetrate the cell wall, initiating a series of reactions that ultimately result in cell death.51,22 drawing on these findings, it can be suggested that the antimicrobial activity primarily stems from the presence of silver in agnp, while the addition of copper ions further enhances its antibacterial efficacy. consistent with this, mujeeb et al. found that silver-copper nanocomposites (ag-cuncs), synthetized using an olax scandens leaf extract, exhibited a greater antimicrobial activity than monometallic agnps. importantly, this enhanced antimicrobial activity was associated eith an increase in the production of ros.52 a cc ep te d m an us cr ip t polypyrrole composite with bactericidal properties. 15 conclusions in the present study, ppy and cu-ppy composites underwent electrochemical modification using two different sizes of agnps. the results demonstrated that a higher concentration of metallic silver in the composite corresponded to a larger size of nanoparticles. furthermore, it was found that the size of agnps deposited on the ppy film influenced the antibacterial activity of the modified composites. it was proposed that the higher antibacterial efficacy of ppy/agnp500, compared to ppy/agnp100, can be attributed to a greater release of ionic silver resulting from the larger nanoparticle size. although all ppy-modified films exhibited antibacterial properties, cu-ppy/agnp500 emerged as the composite that exhibited the strongest antibacterial activity against most of the species tested. this heightened antibacterial activity can be largely ascribed to the synergistic potential of both metals for eradicating bacteria. the surface modification of 316l ss with these films holds promise as a viable alternative for the development of novel antibacterial composites capable of inhibiting the proliferation of large amounts of bacteria. supplementary material supplementary materials are available electronically from https://www.shdpub.org.rs/index.php/jscs/article/view/12276, or from the corresponding authors on request. acknowledgements: the financial support provided by conicet (2021-2023 gi11220200102064co), anpcyt (pict-2019-02758), and universidad nacional del sur (pgiuns 24/m159), in bahía blanca, argentina are gratefully acknowledged. и з в о д бактерицидни ефекат композита бакар-полипирол модификованих наночестицама сребра на грам-позитивне и грам-негативне бактерије patricia l. marucci1, maria g. sica1,4, lorena i. brugnoni1, 2 and maría b. gonzález3,* 1department of biology, biochemistry and pharmacy, national university of the south, bahía blanca, argentina, 2institute of biological and biomedical sciences, national university of the south, conicet, bahía blanca, argentina, 3chemical engineering department, institute of electrochemistry and corrosion engineering, national university of the south, conicet, bahía blanca, argentina, and 4department of health sciences, national university of the south, bahía blanca, argentina у раду је испитиван бактерицидни ефекат композита бакар-полипирол (cu-ppy) таложених на нерђајући челик 316l и модификованих сребром. процењена су антимикробна својства према двадесет четири соја грам-позитивних и грам-негативних бактерија. међу двадесет четири проучавана соја, изолати су укључивали референтне сојеве (e. coli atcc 25922, e. coli 0157:h7 edl 933, s. aureus atcc 25923 and l. monocytogenes atcc 7644), као и сојеве изоловане из хране и клиничке узорке. антимикробна активност композита показала је да сви филмови модификованог полипирола имају антибактеријска својства. значајно је да је cu-ppyagnp500 показао најјачу инхибиторну активност против грам-негативних и грам-позитивних бактерија. модификација површине челика 316l a cc ep te d m an us cr ip t https://www.shd-pub.org.rs/index.php/jscs/article/view/12276 https://www.shd-pub.org.rs/index.php/jscs/article/view/12276 16 marucci et al. овим филмовима је обећавајућа и одржива алтернатива за развој нових антибактеријских композита који могу инхибирати раст значајног броја бактерија. 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(https://doi.org/10.3389/fchem.2020.00103). a cc ep te d m an us cr ip t https://doi.org/10.1016/j.actbio.2007.11.006 https://doi.org/10.1007/978-3-642-24428-5_3 https://doi.org/10.1007/978-3-642-24428-5_3 https://doi.org/10.1021/la303370d https://doi.org/10.3109/17435390.2013.870243 http://doi.org/10.1134/s0003683813020117 https://doi.org/10.1371/journal.pone.0222322 https://doi.org/10.3389/fphar.2019.00801 https://doi.org/10.1126/scitranslmed.3006276. https://doi.org/10.2147/ijn.s189295 https://doi.org/10.1002/smll.201200772 https://doi.org/10.1039/c5ra03633j https://doi.org/10.1016/j.matdes.2021.109672 https://doi.org/10.3389/fchem.2020.00103 {synthetic route towards 1,2,3,4-tetrahydroquinoxaline/ /piperi¬dine combined tricyclic ring system} j. serb. chem. soc. 87 (2) 169–179 (2021) original scientific paper jscs–5513 published 1 october 2021 169 synthetic route towards 1,2,3,4-tetrahydroquinoxaline/ /piperidine combined tricyclic ring system mihajlo j. krunić#, ivana i. jevtić*#, jelena z. penjišević# and slađana v. kostić-rajačić# university of belgrade-institute of chemistry, technology and metallurgy, department of chemistry, njegoševa 12, 11000 belgrade, serbia (received 16 april, revised 2 august, accepted 20 august 2021) abstract: the synthetic route toward novel tricyclic, nitrogen-containing system is disclosed. three novel compounds possessing structural features of 1,2,3,4-tetrahydroquinoxaline and decahydropyrido[3,4-b]pyrazine are synthesized starting from readily available precursors in six or seven steps, of which the last three or four steps respectively are diastereoselective. key reaction steps include n-acylation, hofmann rearrangement and ring-closing buchwald–hartwig reaction. compounds trans-8, cis-12 and trans-12 are synthesized in order to prove that this novel, tricyclic system can be functionalized with various groups. synthetic significance of this heterocyclic system lies in the possibility for the orthogonal functionalization of three different amino groups, allowing fine structural tuning. keywords: heterocycles; 1,2,3,4-tetrahydroquinoxaline; piperazine; anilidopiperidine; buchwald–hartwig reaction. introduction nitrogen-containing heterocycles represent very important structural moieties occurring in many pharmacologically active compounds.1,2 almost 75 % of all food and drug administration (fda) approved drugs possess nitrogen heterocycles as pharmacophores.3 the possibilities for the derivatization of nitrogen functional groups make these heterocycles significant as intermediates in the organic synthesis. moreover, nitrogen heterocycles can form versatile noncovalent interactions with the protein target including electrostatic interactions, hydrogen bonding, dipole– dipole interactions, etc., which makes them desirable as pharmacophores in medicinal chemistry.4 due to their significance for organic chemistry in general, * corresponding author. e-mail: ivana.jevtic@ihtm.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc210416068k ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 170 krunić et al. the design of new nitrogen heterocycles and the development of new, more efficient ways for their synthesis has been a subject of many research projects, for several decades now.5 both quinoxalines and their derivatives 1,2,3,4-tetrahydroquinoxalines represent an important class of nitrogen-containing heterocycles that can be found in many pharmacologically active natural and synthetic compounds (fig. 1).6,7 1,2,3,4-tetrahydroquinoxalines are structural motifs present in compounds that act as anticancer agents,8 prostaglandin d2 receptor antagonists,9 cholesteryl ester transfer protein inhibitors,10 and others.11–13 fig. 1. structures of the novel tricyclic system of general structure a and similar known heterocycles. many synthetic procedures for the synthesis of functionalized 1,2,3,4-tetrahydroquinoxalines have been reported so far. among the most common approaches are the catalytic hydrogenation of quinoxaline,14–16 and reactions including aniline derivatives such as cyclization/reduction or oxidation/cyclization reactions, including reactions of o-phenylenediamines or o-nitroanilines,17 with various ketones,18,19 dihydroxy compounds20 or butanedione.17 majumdar et al. proposed iodocyclization as a method for constructing 1,2,3,4-tetrahydroquinoxalines among the other heterocycles, starting from o-substituted aniline derivatives possessing an isolated, unsaturated c–c bond.21 tandem reduction/reductive aminations,22 and reduction/michael addition,23 have been reported by bunce et al. to produce functionalized 1,2,3,4-tetrahydroquinoxalines, starting from o-nitroaniline. organometallic catalyzed intramolecular couplings have also been reported. yang et al. reported the synthesis of 1,2,3,4-tetrahydro-2-vinylquinoxalines via palladium (ii)-catalyzed tandem allylation of o-phenylenediamines with cis-1,4-diacetoxy-2-butene.24 palladium-catalyzed regioand stereoselective tandem arylation/heteroannulation reaction afforded 2-alkyl(aryl)idenequinoxalines,25 while intramolecular buchwald–hartwig reaction of 2-haloanilines with sulfonamido group has also been reported.26 krchnak et al. reported tin (ii)-catalyzed solid-phase cyclization of functionalized o-nitroanilines, via intramolecular nucleophilic substitution.27 microwave assistant reductive cyclization of substituted o-nitroaniline afforded 1,2,3,4-tetrahydroquinoxalines, as reported by merisor et al.28 however, limitations in most cases include low yields, complicated reaction procedures and expensive catalysts. cyclization/ /acylation of o-phenylenediamines with α-chloroketones29 and diboronic acid ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1,2,3,4-tetrahydroquinoxaline/piperidine heterocyclic system 171 mediated cyclization/cascade transfer hydrogenation of o-phenylenediamines and o-nitroanilines with 1,2-dicarbonyl compounds,30 are some recent examples towards more efficient synthesis of 1,2,3,4-tetrahydroquinoxalines. on the contrary to the well-known 1,2,3,4-tetrahydroquinoxalines, there are only a few reports on decahydropyrido[3,4-b]pyrazines (fig. 1). those include several indole derivatives investigated for their antihypertensive31,32 and analgesic33 activity more than three decades ago, among which the most notable example was the antihypertensive agent atriposin, albeit it was never marketed. the value of polycyclic nitrogen-containing heterocycles for synthetic and medicinal chemistry has already been disclosed. as a part of our ongoing research on functionalized nitrogen heterocycles, we envisaged the synthesis of general structure a possessing 1,2,3,4-tetrahydroquinoxaline and decahydropyrido[3,4-b]pyrazine moieties fused in a novel tricyclic system (fig. 1). the presence of three secondary amino groups that can be selectively functionalized in different reaction steps, thus allowing the formation of orthogonally protected or highly functionalized derivatives, is a valuable asset in synthetic chemistry. that can be especially important for the synthesis of bifunctional or bidentate ligands in multi-target drug design. the tricyclic system presented in this paper is practically unknown, except for the single lactam structure.34 experimental general information unless stated otherwise all solvents were freshly distilled under argon prior to being used. all reagents that were purchased from a commercial vendor were used as supplied. 1hand 13c-nmr spectra were recorded on bruker avance iii spectrometer, at 500 mhz for the proton (1h) and at 126 mhz for the carbon (13c), and varian/agilent, at 400 mhz for the proton (1h) and at 101 mhz for the carbon (13c). chemical shifts are given in ppm from tetramethylsilane (tms) as internal standard in deuterated chloroform (cdcl3). 2d nmr spectra (hsqc) were recorded at 400 and 500 mhz. coupling constants (j) are rep.orted in hz. unless stated otherwise, all spectra were recorded at 25 °c. high resolution mass spectra (hrms) were obtained with a heated esi (hesi)-ltq orbitrap xl spectrometer. all reactions were monitored by thin layer chromatography (tlc). dry-column flash chromatography was carried out using silica gel (10–18 or 18–32 μm, icn-woelm). melting points were obtained at a heating rate of 4 °c/min, and are uncorrected. ir spectra were recorded by using a thermo scientific nicolet 6700 fourier-transform spectrometer operated in the atr mode. structures of all new compounds were determined by methods of 1d, 2d nmr and ir spectroscopy. structures of the final two compounds were additionally confirmed by high resolution mass spectrometry (hrms). analytical and spectral data of the compounds are given in supplementary material to this paper. syntheses general procedure for the synthesis of enamines 3a and 3b.35 to a magnetically stirred solution of methyl 1-benzyl-4-oxopiperidine-3-carboxylate 1 (20.0 mmol) in acetic acid ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 172 krunić et al. (acoh, 20 ml), 2-bromoaniline 2a or 2-iodoaniline 2b (28.0 mmol) was added, and the mixture was stirred at room temperature. the reaction was monitored by tlc (sio2 plates; petroleum ether/ethyl acetate (etoac), 8:2). after 24 h, the mixture was concentrated by rotary evaporator, and then neutralized with 1.5 m solution of na2co3 (ph ~11). the mixture was extracted with etoac (2×50 ml), and collected organic layers were concentrated by rotary evaporator. the product was purified by recrystallization from isopropyl alcohol (i-proh). general procedure for the synthesis of anilinoesters 4a and 4b.35 to a magnetically stirred solution of enamine 3a or 3b (9.4 mmol) in methanol (meoh, 65 ml), nabh3cn (13.2 mmol) and acoh (28.2 mmol) were added and the mixture was stirred at room temperature. the reaction was monitored by tlc (sio2 plates; petroleum ether/etoac, 8:2). after 24 h, conc. hcl was added to ph ~1, and the mixture was concentrated by rotary evaporator. the mixture was neutralized with 1.5 m solution of na2co3 (ph ~11) and extracted with etoac (3×50 ml). the combined organic layers were concentrated by rotary evaporator providing the crude product (mixture of cisand trans-diastereomers). diastereomers were separated by dry -column flash chromatography (sio2; petroleum ether/etoac, 10:0 to 7:3) for the spectroscopic analysis; however, the mixture of cis/trans diastereomers was used in the next step, without separation. general procedure for the synthesis of anilinocarboxamides 5a and 5b.35 to a magnetically stirred solution of anilinoester cis/trans-4a or cis/trans-4b (4.4 mmol) in n,n-dimethylformamide (dmf, 8 ml), lih (8.8 mmol) and formamide (hconh2, 17.6 mmol) were added. the mixture was stirred at room temperature. the reaction was monitored by tlc (sio2 plates; ch2cl2/meoh, 95:5). after 24 h, the mixture was concentrated by rotary evaporator and partitioned between water (100 ml) and ch2cl2 (3×30 ml). the combined organic layers were concentrated by rotary evaporator, affording the crude product as a mixture of cisand trans-diastereomers. diastereomers were separated by dry-column flash chromatography (sio2; ch2cl2/meoh, 100:0 to 95:5) and used in the next step as separate diastereomers. general procedure for the synthesis of anilidocarboxamides 9a and 9b. to a magnetically stirred solution of anilinocarboxamide 5a and 5b (1.1 mmol) in dmf (8 ml), pyridine (3.3 mmol), 4-(dimethylamino)pyridine (dmap, 0.33 mmol) and propionyl bromide (etcobr, 11.0 mmol) were added and the mixture was stirred at room temperature. the reaction was monitored by tlc (sio2 plates; ch2cl2/meoh, 95:5). the mixture was concentrated by rotary evaporator, and a 1.5 m solution of na2co3 was added (ph ~11). the mixture was extracted with ch2cl2 (3×30 ml), and the combined organic layers were concentrated by rotary evaporator. the product was dissolved in methanolic ammonia (7–9 m, 30 ml) and the mixture was stirred at room temperature. the reaction was monitored by tlc (sio2 plates; ch2cl2/meoh, 95:5). after 24 h, the mixture was concentrated by rotary evaporator and the crude product was purified by dry-column flash chromatography (sio2; ch2cl2/meoh, 100:0 to 95:5). general procedure for the synthesis of carbamates 6a, 10a and 10b.36 to a magnetically stirred solution of carboxamide trans-5a, trans-9a, cis-9b or trans-9b (1.0 mmol) in meoh (8 ml), lioh·h2o (8.0 mmol) was added at room temperature. n-bromoacetamide (nba, 3.0 mmol) was added in three 1mmol aliquots while the mixture was stirred at 60 °c in the dark. the reaction was monitored by tlc (sio2 plates; petroleum ether/etoac, 6:4). after 5 min, the mixture was concentrated by rotary evaporator, and mixed with a 1 m solution of naoh. the mixture was extracted with ch2cl2 (3×30 ml), and the collected organic layers were concentrated by rotary evaporator. the crude product was purified by dry-column flash chromatography (sio2; petroleum ether/etoac, 10:0 to 6:4). ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1,2,3,4-tetrahydroquinoxaline/piperidine heterocyclic system 173 general procedure for the synthesis of compounds 7a, 11a and 11b.35 to a magnetically stirred solution of carbamate trans-6a, trans-10a, cis-10b or trans-10b (0.22 mmol) in dichloroethane (2 ml), me3sii (0.77 mmol) was added, and the mixture was stirred at room temperature in the dark. the reaction was monitored by tlc (sio2 plates; ch2cl2/meoh, 95:5). after 24 h, excess of meoh was added and the mixture was allowed to stir for 15 min. the mixture was then concentrated by rotary evaporator and a 1 m hcl was added in excess, followed by the neutralization with 1.5 m na2co3. the mixture was extracted with ch2cl2 (3×15 ml) and the combined organic layers were concentrated by rotary evaporator affording the crude product. crude products were used in the next step without further purification. general procedure for the synthesis of compounds trans-8, cis-12 and trans-12. to a magnetically stirred solution of compound trans-7a, cis-11b or trans-11a (0.17 mmol) in 1,4 -dioxane (2 ml), tert-butyl alkoxide (t-buona, 0.26 mmol) or cs2co3 (0.34 mmol) was added. after 5 min, pd(oac)2 (0.0034 mmol) and 2,2′-bis(diphenylphosphino)-1,1′-binaphthalene (binap, 0.01 mmol) were added, and the mixture was stirred at reflux under argon atmosphere. the reaction was monitored by tlc (sio2 plates; ch2cl2/meoh, 95:5). after 8 h, the mixture was concentrated by rotary evaporator. the mixture was partitioned between brine (30 ml) and ch2cl2 (3×10 ml). the combined organic layers were concentrated, and the product was further purified by dry-column flash chromatography (sio2; ch2cl2/meoh, 100:0 to 95:5). results and discussion previously, we disclosed the synthesis of 3-amino-anilidopiperidines, also known as 3-aminofentanyls.35,36 these compounds are represented by the general structure b, where x is hydrogen (scheme 1). however, we envisaged that the analogues, where x represents bromine or iodine, may provide access to the novel tricyclic system of general structure a. the retrosynthetic approach was based on our previously optimized synthesis of 3-aminofentanyls, starting from β-ketoester d (scheme 1).35,36 therefore, the synthetic protocol was expected to mirror the previous one, except for the use of 2-bromoaniline or 2-iodoaniline that that could possibly influence the reaction conditions and outcomes. the key step, intramolecular buchwald–hartwig amination, if successful, would secure the novel heterocyclic system a, possessing the combined 1,2,3,4-tetrahydroquinoxaline and decahydropyrido[3,4-b]pyrazine moiety. scheme 1. the proposed retrosynthetic route to the novel tricyclic system a. the synthetic approach is presented in scheme 2. the starting β-ketoester 1, while available commercially, was readily prepared on a multi-gram scale, acc. to ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 174 krunić et al. our previously published procedure.37 the n-benzyl moiety was chosen since it can be selectively cleaved by various reagents, thus allowing nitrogen functionalization.38 condensation of 1 with 2-haloaniline 2a or 2b afforded stable enamines 3a and b, respectively, followed by the quantitative reduction to cis/trans anilinoesters 4a and b. while separable by chromatography, the cis/trans mixtures were used directly in the next step. in parallel to the earlier results, formamide-mediated aminolysis of the methoxycarbonyl group provided quantitative conversion to carboxamides 5a and b. the procedure typically results in an extensive, base-catalyzed epimerization at c-3,35 although cis/trans ratio may vary, depending on the particular diastereomer. the obtained cis/trans mixtures were quantitatively separated by chromatography or crystallization, furnishing pure cis-5a, trans-5a, cis-5b and trans-5b. the relative stereochemistry was tentatively assigned via 2d nmr spectroscopy only, since no x-ray crystal structures are currently available. scheme 2. reagents and conditions: a) 2a, 2b, acoh, r.t., 24 h; b) nabh3cn, acoh, meoh, r.t., 24 h; c) hconh2, lih, dmf, r.t., 16 h; d) nba, lioh.h2o, meoh, reflux, 5 min; e) me3sii, (ch2)2cl2, r.t., 24 h; f) pd(oac)2, binap, t-buona or cs2co3, dioxane, reflux, 8 h; g) 1. etcobr, pyridine, dmap, dmf, r.t., 24 h, 2. nh3/meoh. the hofmann rearrangement was then examined on carboxamides 5a (scheme 2). we chose our recently published protocol due to good chemoselectivity and generally high yields, compared to the related methods.39 as found in the earlier research, the formation of cyclic ureas was expected, via intramolecular addition of aromatic nitrogen to the intermediate isocyanate. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1,2,3,4-tetrahydroquinoxaline/piperidine heterocyclic system 175 surprisingly, trans-5a furnished carbamate trans-6a. apparently, the presence of ortho bromine precluded cyclization, and the normal carbamate formed instead. however, cis-5a failed to react analogously, yielding a complex mixture only. after the removal of the carbamate group and subsequent intramolecular buchwald–hartwig reaction of amine trans-7a, the cyclization product trans-8 was obtained, although in low yields. compound trans-8 represents essentially a novel class of heterocycles on its own, not readily accessible by alternative approaches. however, since both piperazine nitrogens are unprotected and the selective functionalization is practically not possible, this particular cyclization appears to be of a limited significance. nonetheless, a potential improvement of the hofmann rearrangement and the use of aryl iodides instead of bromides could substantially improve the usefulness of the transformation. to increase the synthetic significance of this cyclization, and the novel heterocyclic system, the protection of one piperazine nitrogen had to be achieved earlier in the synthetic pathway. in our previous research,35 we found that aromatic amines analogous to 5 (having phenylamino group), were unusually difficult to acylate, likely due to the interference of carboxamido group, rather than the typical steric hindrance. in the case of all four carboxamides 5, the acylation completely failed with propionyl chloride, under any attempted conditions. apparently, the presence of ortho halogens drastically reduced the nucleophilicity of the aromatic nitrogen. after numerous experiments, it was found that the use of propionyl bromide, in the presence of pyridine and dmap in dmf, was the only effective reagent for the acylation. comparable to the previous findings,35 acyl-amido group formed concomitantly (not shown in the scheme 2), and then was selectively cleaved by methanolic ammonia. the four obtained carboxamides 9 were used as substrates for the hofmann rearrangement.35 surprisingly, only trans-9a and trans-9b reacted as anticipated, providing carbamates trans-10a and trans-10b respectively, albeit in modest yields, with high amount of recovered carboxamides. carboxamides cis-9a and cis-9b yielded complex mixtures with no expected carbamates. upon further examination of the reaction conditions, we discovered that gradual addition of the small amounts of nba, not only increase the yields of trans carbamates, but also it allowed the formation of cis-10b carbamate from the carboxamide cis-9b. presumably, decomposition of nba under these conditions occurred more rapidly than the reaction with carboxamides. carboxamide cis-9a however, yielded complex mixtures with no expected carbamates, under any attempted conditions, similarly as in the case of cis-5a. we did not further investigate this phenomena, since we had both diastereomers of 10b to proceed with the next reaction steps. subsequent removal of the carbamate group using me3sii, furnished amines trans-11a, cis-11b and trans-11b, in nearly quantitative yields. with the required amines at hand, we ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 176 krunić et al. were able to examine the intramolecular buchwald–hartwig amination, a key transformation of this synthesis. the reaction conditions were mainly chosen from the numerous literature reports, while the phosphine ligand was limited to binap, as the only one available.40 also, having fairly sensitive substrates, strong bases were to be avoided, with the preference to alkaline earth carbonates. despite numerous attempts, trans-11a did not produce the expected product. the use of cs2co3 in dioxane mainly resulted in the recovered reactant, while in diglyme, at 120–140 °c, it decomposed. the reaction also resulted in the decomposition with t-buona, probably because it is a much stronger base than alkali metal carbonates. changing molar ratios of pd(oac)2 and binap had no significant effect. fortunately, both diastereomers of 11b, gave the piperazines cis-12 and trans-12 diastereoselectively, and in moderate yields. the higher yield in the case of cis-12 can be due to the more favorable orientation of the primary amino group and aryl iodide in cis-11b. compounds cis-12 and trans-12 represent another two examples of the novel class of tricyclic, nitrogen-containing system. obtaining both diastereomers is of significance, since it demonstrates the adaptability of the synthetic route, especially the cyclization process. also, these heterocycles are suitably functionalized for further elaboration, and as such are potential precursors of pharmacologically active compounds. the piperidine nitrogen, protected by benzyl group, can be readily deprotected and used as a reactive, secondary amino function. one of the piperazine nitrogens is free and can be acylated or alkylated independently, while the other nitrogen is protected as carboxamide. thus, we were able to prove that the initial synthetic plan was viable, although, at present, with some limitations. aryl bromides are apparently insufficiently reactive substrates in these particular aminations, limiting the choice to the iodides. there is a plethora of phosphine ligands, some of which known to be more active than binap, permitting additional variations.26,40 on the other hand, cs2co3 as a base is effective, with the potential alternatives including k2co3, k3po4, and other reagents less basic than alkoxides. as already mentioned, the formation of 1,2,3,4-tetrahydroquinoxaline core by buchwald–hartwig cyclization is known from literature.26 however, this cyclization is significantly different from ours. the former presents n-arylation of a sulfonamido group, whereas in our case, the nucleophile is the primary amino group. from our experience and findings in the literature, the reactivity of amides and amines in buchwald–hartwig reaction is different, often requiring very different reaction conditions. conclusion the synthetic route toward novel, tricyclic nitrogen containing system is presented herein. three novel compounds, possessing 1,2,3,4-tetrahydroquinoxaline ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1,2,3,4-tetrahydroquinoxaline/piperidine heterocyclic system 177 and decahydropyrido[3,4-b]pyrazine moieties, are synthesized starting from readily available precursors in six or seven steps, of which the last three or four steps respectively, are diastereoselective. synthetically challenging n-acylation of the secondary arylamino group in the presence of adjacent, primary carboxamide function, is optimized. optimized, nba-mediated hofmann rearrangement, gave the desired carbamates in the case of trans-5a and trans-9a, cis-9b and trans-9b. carbamate cleavage, followed by the intramolecular buchwald–hartwig reaction as the cyclization step, afforded novel tricyclic compounds trans-8, cis-12 and trans-12 in moderate yields. such nitrogen containing heterocyclic systems can be of interest for organic synthesis and as building blocks in medicinal chemistry, since the orthogonal functionalization of three different amino groups allows the fine structural tuning. however, an extensive further investigation would be necessary to confirm the potential scope and usefulness of the present results. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/10671, or from the corresponding author on request. acknowledgement. the authors would like to thank the ministry of education, science and technological development of the republic of serbia (grant no: 451-03-9/2021-14/ /200026) for financial support. и з в о д синтетски пут за добијање 1,2,3,4-тетрахидрохиноксалинскo/пиперидинског трицикличног система михајло ј. крунић, ивана и. јевтић, јелена з. пењишевић и слађана в. костић-рајачић универзитет у београду-институт за хемију, технологију и металургију, центар за хемију, његошева 12, 11000 београд у овом раду представљена је синтеза новог трицикличног система који садржи азот. три нова једињења код којих су комбиноване структурне карактеристике 1,2,3,4-тетрахидрохиноксалина и декахидропиридо[3,4-b]пиразина, добијена су полазећи од лако доступних прекурсора, у шест или седам фаза од којих су последње три или четири, редом, диастереоселективне. кључне синтетичке трансформације укључују n-ациловање, hofmann премештање и интрамолекулску buchwald–hartwig реакцију, као фазу у којој долази до циклизације. једињења trans-8, cis-12 и trans-12 су синтетисана како би се представила могућност функционализације новог трицикличног система. синтетички значај новог хетероцикличног система представљен је у могућности ортогоналне функционализације три различите амино групе, чиме се може постићи фино подешавање структуре. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 404 not found accepted manuscript this is an early electronic version of an as-received manuscript that hasbeen accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as o. babatunde, s. hameed, k. a. mbachu, f. saleem, s. chigurupati, a. wadood, a. ur rehman, v. venugopal, k. m. khan, m. taha, o. ekundayo, m a. khan, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc 211106005b this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing, and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc211106005b https://doi.org/10.2298/jsc211106005b j. serb. chem. soc.00(0)1-18 (2023) original scientific paper jscs–11370 published dd mm, 2023 1 evaluation of derivatives of 2,3-dihydroquinazolin-4(1h)-one as inhibitors of cholinesterases and their antioxidant activity: in vitro, in silico, and kinetics studies oluwatoyin babatunde1, shehryar hameed1, kingsley adibe mbachu1,2, faiza saleem1, sridevi chigurupati3, abdul wadood4, ashfaq ur rehman4, vijayan venugopal5, khalid mohammed khan1,7* muhammad taha7, olusegun ekundayo2 and maria aqeel khan6 1h. e. j. research institute of chemistry, international center for chemical and biological sciences, university of karachi, karachi-75270, pakistan; 2department of chemistry, university of ibadan, nigeria; 3department of medicinal chemistry and pharmacognosy, college of pharmacy, qassim university, buraydah-52571, saudi arabia; 4department of biochemistry, computational medicinal chemistry laboratory, ucss, abdul wali khan university, mardan, pakistan; 5faculty of pharmacy, aimst university, kedah-08100, malaysia; 6third world center for science and technology, h. e. j. research institute of chemistry, international center for chemical and biological sciences, university of karachi, karachi-75270, pakistan and 7department of clinical pharmacy, institute for research and medical consultations (irmc), imam abdulrahman bin faisal university, p. o. box 1982, dammam, 31441, saudi arabia (received 6 november 2021; revised 17 may 2022; accepted 2 february 2023) abstract: in search of potent inhibitors of cholinesterase enzymes and antioxidant agents, synthetic derivatives dihydroquinazolin-4(1h)-one 1-38 were evaluated as potential anti-alzheimer agents through in vitro acetylcholinesterase (ache) and butyrylcholinesterase (bche) inhibitions and radical (dpph and abts) scavenging activities. the (sar) was mainly based on the different substituents at the aryl part which showed a significant effect on the inhibitory potential of enzymes and radical scavenging activities. the kinetic studies of most active compounds showed a noncompetitive mode of inhibition for ache and a competitive mode of inhibition for the bche enzyme. additionally, molecular modeling studies were carried out to investigate the possible binding interactions of quinazolinone derivatives with the active site of both enzymes. keywords: quinazolinone; dual inhibitors; acetylcholinesterase; butyrylcholinesterase *corresponding author e-mail: khalid.khan@iccs.edu, drkhalidhej@gmail.com; tel.: +922134824910 https://doi.org/10.2298/jsc211106005b a cc ep te d m an us cr ip t mailto:khalid.khan@iccs.edu mailto:drkhalidhej@gmail.com https://doi.org/10.2298/jsc211106005b 2 babatunde et al. introduction in the central nervous system (cns), one of the preeminent neurotransmitters is acetylcholine (ach) which is related to memory and cognition. insufficient ach levels in the cns can lead to diseases such as alzheimer’s disease (ad).1 ad is the most common cause of dementia in elderly people and is characterized by several impaired cortical functions, including judgment, memory loss, comprehension, orientation, language deficit, and learning capacity.2 the predominant symptoms of all types of dementia are thought to be associated with the gradual decline of broad and compact cholinergic innervation of the human cerebral cortex. this decline contributes to the behavioral and cognitive deficits in ad and is also linked with the reduced levels of neurotransmitters, choline acetyltransferase, acetylcholinesterase (ache), and ach.3 ache and butyrylcholinesterase (bche) enzymes are hydrolytic enzymes that act on the neurotransmitter ach by cleaving it into choline and acetate, thereby stopping their action in the synaptic cleft.4 both enzymes are found in amyloid plaques and neurofibrillary tangles in the brain.5 ache is the most important enzyme that regulates the level of acetylcholine in a healthy brain, while bche plays an insignificant role. in ad patients, the ache activity decreases, bche activity increases and the ratio between ache and bche varies from normal to high levels (0.6-11) in the cortical regions of the brain that affect the disease.6,7 these observations lead to the concept of dual inhibition, and the most effective treatment approach has been suggested to increase ach levels and limit cholinergic function by inhibiting ache and bche enzymes. quinazolinones are extensively explored and are considered important as bioactive synthetic molecules for the development of novel therapeutic agents.8 quinazolinone belongs to the n-containing fused heterocyclic compounds and is a quinazoline with a carbonyl group in the c4n2 ring. there are two isomers possible: 4-quinazolinone and 2-quinazolinone, however, the 4-quinazolinone isomer is more common.9 these compounds have raised universal concerns due to their broad and pronounced biopharmaceutical activities.10 many substituted quinazolinones have a broad range of bioactivities such as antimicrobial, antimalarial, antifungal, antiprotozoal, anticancer, antiviral, anti-inflammatory, anti-tubercular, anticonvulsant, diuretic, acaricidal, muscle relaxant, antidepressant, weedicide, and many other biological activities.11 quinazolinone compounds are also used in the syntheses of a variety of functional substances for synthetic chemistry and are also present in various drugs (figure 1). 12 a cc ep te d m an us cr ip t synthesis of 2,3-dihydroquinazolin-4(1h)-one and their derivatives 3 figure 1. pharmacological importance of quinazolinone-based drugs antioxidant compounds exhibit an important part as a health protection factor.13 free radicals are ions, atoms, or molecules possessing an unpaired electron such as hydroxyl, nitric oxide, and superoxide which are called reactive oxygen species (ros) 14. ros are generated in the human body and can damage dna, proteins, and lipids thus may lead to different complications such as inflammation, toxicity, and carcinogenesis. plants-derived antioxidants include carotenes, phytoestrogens, vitamin c, vitamin e, and phytates.15 furthermore, chronic diseases which are life-limiting, such as diabetes, cancer, arteriosclerosis, ad, and aging, are developed by radical reactions.16 natural or synthetic antioxidant compounds terminate the chain reactions by interacting with free radicals before essential molecules are damaged.17 thus, the synthesis of new potent antioxidant compounds is of vital importance for rapidly quantifying the effectiveness of antioxidants in disease prevention. our research group is continuously doing efforts in search of lead compounds for two decades to discover new enzyme inhibitors.18-21 previously, we have explored a large number of potent inhibitors based on quinazoline derivatives, including α-amylase, α-glucosidase,22,23 β-glucuronidase,24, and antileishmanial activities.25 these heterocycles are reported to possess various significant biological activities. derivatives of dihydroquinazolin-4(1h)-one, in particular, has drawn more and more attention for synthesizing pharmaceuticals and in the field of agrochemicals. herein we are going to report dihydroquinazolin-4(1h)ones as a new class of inhibitors against acetylcholinesterase, butyrylcholinesterase enzymes, and with its antioxidant potential (figure 2). a cc ep te d m an us cr ip t https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/heterocyclic-compound https://www.sciencedirect.com/topics/chemistry/agrochemical 4 babatunde et al. figure 2. rationale of the current study in this study, dihydroquinazolin-4(1h)-ones 1-38 (table i) have been reported as antioxidant agents and potent cholinesterase inhibitors which may improve clinical outcomes for developing anti-ad agents. table i. in vitro acetylcholinesterase, butyrylcholinesterase activity, and antioxidant activity after using dihydroquinazolin-4(1h)-one derivatives 1-38 comp. no. r ache activity bche activity dpph radical activity abts radical activity ic50 ± sema / µm) 1 4-cl (c6h4) 35.04 ± 0.20 37.13 ± 0.18 41.7 ± 0.06 42.97 ± 0.19 2 2-cl (c6h4) 23.08 ± 0.03 26.08 ± 0.43 17.65 ± 0.23 19.47 ± 0.03 3 2,6-cl (c6h3) 24.94 ± 0.12 27.13 ± 0.08 30.7 ± 0.06 32.97 ± 0.19 4 2,4-cl (c6h3) 24.57 ± 0.07 27.57 ± 0.07 16.33 ± 0.02 18.01 ± 0.12 5 2-oh, 3,5-cl (c6h2) 61.89 ± 0.12 67.91 ± 0.18 57.33 ± 0.02 58.01 ± 0.12 6 2-cl, 6-no2 (c6h3) nab nab 70.7± 0.06 71.97± 0.19 7 5-cl, 2-oh (c6h3) 81.94 ± 0.12 82.13 ± 0.08 83.57 ± 0.17 83.68 ± 0.36 8 3,5-och3 (c6h3) nab nab 96.65 ± 0.03 94.47 ± 0.13 9 2,5-och3 (c6h3) 88.15 ± 0.12 87.15 ± 0.12 84.04 ± 0.02 85.99 ± 0.09 10 2,6-och3 (c6h3) 26.94 ± 0.12 27.99 ± 0.09 24.33 ± 0.02 25.01 ± 0.12 11 3,4-och3 (c6h3) 87.27 ± 0.18 86.08 ± 0.43 87.57 ± 0.08 89.27 ± 0.18 a cc ep te d m an us cr ip t synthesis of 2,3-dihydroquinazolin-4(1h)-one and their derivatives 5 12 2-br, 4,5-och3 (c6h3) 67.91 ± 0.18 69.02 ± 0.11 51.65 ± 0.03 52.47 ± 0.13 13 2,4-och3 (c6h3) 89.7 ± 0.16 85.97 ± 0.19 82.17 ± 0.14 82.01 ± 0.09 14 3,4,5-och3 (c6h2) nab nab 86.65 ± 0.23 87.47 ± 0.03 15 2,3,4-och3 (c6h2) nab nab 83.33 ± 0.02 85.01 ± 0.12 16 3-oc2h5, 4-och3 (c6h3) 27.57 ± 0.07 29.13 ± 0.18 30.04 ± 0.02 31.99 ± 0.09 17 3-och3, 4-oc2h5 nab nab 92.7± 0.06 94.97± 0.19 18 3,5-och3, 4-oh (c6h2) 87.27± 0.18 89.7± 0.16 83.46± 0.03 84.61± 0.11 19 4-br, 3,5-och3 (c6h2) 83.08 ± 0.03 84.94 ± 0.12 76.33 ± 0.02 79.01 ± 0.12 20 4-f, 3-och3 (c6h3) 51.94 ± 0.12 53.33 ± 0.02 48.65 ± 0.23 49.47 ± 0.03 21 3-br, 2-och3 (c6h3) 89.17 ± 0.16 88.33 ± 0.12 81.7 ± 0.06 85.97 ± 0.19 22 2-f, 4-och3 (c6h3) 27.91 ± 0.18 29.02 ± 0.11 31.33 ± 0.12 32.01 ± 0.12 23 2-cl, 3-och3 (c6h3) 88.15 ± 0.12 87.13 ± 0.12 83.04 ± 0.02 84.99 ± 0.09 24 3-oc2h5, 2-oh (c6h3) 61.01 ± 0.17 64.57 ± 0.07 49.84 ± 0.03 52.71 ± 0.11 25 2-och2(c6h5) (c6h4) nab nab 72.7 ± 0.06 74.97 ± 0.19 26 3-och2(c6h5) 4-och3 (c6h3) nab nab 88.89 ± 0.10 89.09 ± 0.09 27 4-och2(c6h5) (c6h4) nab nab 84.89 ± 0.20 89.09 ± 0.19 28 4-br (c6h4) 25.33 ± 0.02 26.27 ± 0.18 27.33 ± 0.02 28.01 ± 0.12 29 4-cf3 (c6h4) nab nab 92.13 ± 0.08 92.79 ± 0.17 30 2-thiophene 43.08 ± 0.03 46.08 ± 0.43 47.65 ± 0.23 49.47 ± 0.03 31 3-bromo, 4-oh (c6h3) 85.33 ± 0.02 87.47 ± 0.13 83.01 ± 0.07 83.11 ± 0.15 32 4-och3, 3-oh (c6h3) 77.27 ± 0.18 75.04 ± 0.52 71.7 ± 0.06 72.97 ± 0.19 33 3-oh (c6h4) 47.17 ± 0.15 48.15 ± 0.12 42.33 ± 0.12 43.01 ± 0.12 34 2-oh (c6h4) 27.57 ± 0.07 29.02 ± 0.11 28.46 ± 0.03 30.71 ± 0.11 35 4-oh (c6h4) 37.7 ± 0.16 38.94 ± 0.12 39.7 ± 0.16 40.97 ± 0.14 36 3,4-oh (c6h3) 45.04 ± 0.52 47.7 ± 0.16 48.46 ± 0.03 52.71 ± 0.11 37 2,5-oh (c6h3) 77.33 ± 0.02 79.7 ± 0.16 76.65 ± 0.03 77.47 ± 0.13 a cc ep te d m an us cr ip t 6 babatunde et al. 38 2,3-oh (c6h3) 81.94 ± 0.12 83.33 ± 0.02 82.7 ± 0.06 84.97 ± 0.19 standard= asc. acidc 15.08 ± 0.03 16.09 ± 0.17 standard = donepezild 15.08 ± 0.03 15.08 ± 0.03 sema (standard error of the mean); nab (not active); ascorbic acidc (standard for dpph and abts activities); donepezild (standard for ache and bche inhibitions). experimental materials and methods all enzymes were purchased from sigma-aldrich and used without further purification. the acetylcholinesterase enzyme from electrophorus electricus (electric eel) supplied by sigma-aldrich (gmbh, usa) whereas butyrylcholinesterase from equine serum procured from sigma-aldrich, sre020, missouri, usa); 5,5-dithio-bis-nitrobenzoic acid (dtnb), acetylthiocholine iodide 99 % (atchi), donepezil hydrochloride was obtained from sigma-aldrich (united kingdom). all reagents were purchased from merck (germany) and sigma-aldrich (usa). thin-layer chromatography was carried out on precoated silica gel, gf-254 (merck, germany). spots were visualized under ultraviolet light at 254, 366 nm or iodine vapors. eiand hrei-ms spectra were recorded on mat 312 and mat 113d mass spectrometers. the 1h-, 13c-nmr were recorded on bruker am spectrometers, operating at 300 and 400 mhz. the chemical shift values are presented in ppm (δ), relative to tetramethylsilane (tms) as an internal standard, and the coupling constant (j) is in hz. cholinesterase enzyme activity the in vitro ache and bche inhibitory activity were measured using the methods described earlier.26 briefly, stock solutions (1 mg/ml) of test compounds were prepared using 0.01 % dmso. working solutions (0.01 – 100 μg/ml) were prepared by serial dilutions. the various concentrations of test compounds (10 μl) were pre-incubated with sodium phosphate buffer (0.1 m; ph 8.0; 150 μl); ache solution/ bche (0.1 u/ml; 20 μl) for 15 min at 25 ˚c and addition of dtnb (10 mm; 10 μl). the reaction was initiated by the addition of atchi (14 mm; 10 μl). the reaction mixture was mixed using a cyclomixer and incubated for 10 min at room temperature. the absorbance was measured using a microplate reader at 410 nm wavelength against the blank reading containing 10 μl dmso instead of the test compound. the inhibition was calculated using the formula described in eq. (1) and the ic50 was calculated. donepezil (0.01–100 μg/ml) was used as the positive control. inhibition= ((1-absorbance sample)/absorbance control)100 (1) a cc ep te d m an us cr ip t synthesis of 2,3-dihydroquinazolin-4(1h)-one and their derivatives 7 kinetic study assay in derivatives of 2,3-dihydroquinazolin-4(1h)-one, seven compounds 2, 3, 4, 10, 16, 28 and 34 were selected for kinetic studies, based on their lower ic50 values (23.08 to 27.57 μm). in kinetic studies, we used acetyl thiocholine iodide (atci)/butyrylthiocholine iodide (btci) as a substrate at various concentrations (0.175, 0.35, 0.7, and 1.40 mm) and different concentrations of ache/bche inhibitors (0, 0.625, 1.25 and 2.5 μm) were used. enzyme inhibition kinetic mechanisms were determined by using sigma plot 14.0 software. the rate of substrate and inhibitor reactions was calculated. based on the rate of reactions, the software showed the type of enzyme kinetics mechanism. kinetic studies have shown all the compounds followed as non-competitive type inhibitors (table i). the types of inhibition of ache/bche were determined by lineweaver burk plots. the reciprocal of the rate of the reaction was plotted against the reciprocal of substrate concentration to monitor the effect of the inhibitor on both km (michaelis constant the substrate concentration at which the reaction rate is 50% of the vmax) and vmax (in enzyme kinetics, vmax is the maximum velocity of an enzymatically catalyzed reaction when the enzyme is saturated with its substrate. values. radical scavenging assay dpph (2,2-diphenyl-1-picrylhydrazyl) radical activity preparation of the dpph solution was adopted from molyneux 27 and blois 28 with minor modifications. all the test compounds were dissolved in 95 % ethanol. briefly, 0.5 ml of test compounds were added (0 blank control, 10, 25, 50, 100, 250, 500 and 1000 g/ml) to 0.5 ml of dpph (2 µm in 95 % ethanol) and the mixture was incubated at room temperature for 30 min. the absorbance was measured at 517 nm,29 and the percentage inhibition of test compounds was calculated using the following equation using microsoft excel software (version 2010). ascorbic acid was used as the positive control. scavenging = ((1-absorbance sample)/absorbance control)100 (2) the ic50 (half maximal inhibitory concentration) was calculated by constructing a non-linear regression graph between inhibition vs. concentration, using graph pad prism software (version 5).34 abts (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) free-radical cation activity the abts free radical cation scavenging ability of the synthesized compounds was determined according to the procedure described earlier.30 abts was dissolved in distilled water (7 mm) and potassium persulphate (2.45 mm) was added. this reaction mixture was left overnight (12 to 16 h) in the dark, at room temperature. various concentrations of test substances (1000, 500, 250, 100, 50, 25, and 10 µg/ml) were incubated with the abts+ solution for 30 min. the absorbance was measured at 734 nm, the inhibition was calculated using the a cc ep te d m an us cr ip t 8 babatunde et al. formula described in eq. (1) and the ic50 was calculated. ascorbic acid was used as the positive control. molecular docking protocol acetylcholinesterase and butyrylcholinesterase molecular docking (md) was performed using molecular operating environment (moe)31 to explore the binding mode of the synthetic compounds against acetylcholinesterase (ache) and butyrylcholinesterase (bche) enzymes. first, the 3d structures for all the compounds were generated using the moebuilder module. next, the compounds were protonated, and energy was minimized using the default parameters of the moe. the structural coordinates for ache and bche were retrieved from the protein databank (pdb code; 1acl and 1p0p). all the structure was subjected to moe for preparation. further, the protonation was done using the default parameters of the structure preparation module of moe. next, the energy was minimized for both coordinates to get minimal energy conformation. finally, refined structures were used for the docking study using the default parameters of moe. before running the docking protocol, we selected a total of often conformations for each compound. the top-ranked conformations based on docking score (s) were selected for protein-ligand interaction (pl) analysis. results and discussion chemistry dihydroquinazolin-4(1h)-ones 1-38 were synthesized by treating isatoic anhydride, substituted aldehyde, and aniline under reflux for 3 h. the reaction was carried out in acetic acid as a solvent at 80-90 °c in scheme 1. after reaction completion, it was cooled to room temperature. the solution was added to ice water to form a precipitate. the mixture was filtered, and the crude product was washed continuously with an excess of water. the obtained crude product was washed with different solvents to remove impurities, on crystallization from ethanol gave the corresponding pure products having 60-85 % yields.23 molecular structures of all compounds 1–38 were identified by ei-ms, hrei-ms, 1h-, and 13c-nmr. scheme 1. synthesis of dihydroquinazolin-4(1h)-ones 1-38 a cc ep te d m an us cr ip t synthesis of 2,3-dihydroquinazolin-4(1h)-one and their derivatives 9 in vitro ache, bche inhibitions, and antioxidant activities: all synthetic dihydroquinazolin-4(1h)-ones 1-38 were screened for in vitro acetylcholinesterase and butyrylcholinesterase inhibitions, and antioxidant activities. all compounds exhibited good to moderate inhibitory activities in the range of ic50 values 23.08-89.7 and 26.01-89.7 µm against ache, and bche inhibitions and 16.33-96.65 and18.01-94.97 µm against dpph and atbs activities when compared to the donepezil (ic50 = 15.08 ± 0.07 µm) and ascorbic acid as the standards (ic50 = 15.08 ± 0.07 and 16.09 ± 0.17 µm), respectively (table i). the structure-activity relationship proposed that all structural features such as benzene ring, carbonyl group, quinazoline moiety, phenyl ring, and aryl ring “r” are taking part in the activity, and due to the presence of different groups “r” at the aryl part significant fluctuation in the activity was observed (figure s1 in supplementary material). sar for ache and bche inhibitions and antioxidant activities structure-activity relationship (sar) was discussed for all synthetic compounds which were screened for in vitro acetylcholinesterase, butyrylcholinesterase inhibitions, and antioxidant (dpph and abts) activities. structure-activity relationship (sar) for ache and bche inhibitory activities compounds 1-7, 28, and 29 were halogen-substituted including f, cl, and br. these compounds displayed inconsistent inhibitory activities against acetylcholinesterase (ache) and butyrylcholinesterase (bche) enzymes. of these, compound 2 with the ortho-chloro substitution was found to be the most potent ache and bche inhibitor with ic50 values of 23.08 ± 0.03, and 26.08 ± 0.43 µm, respectively. a comparison of the inhibitory activities of compound 2 and compound 1 showed a positional effect on the inhibitory potential. such in compound 1 the presence of chloro group at para-position reduces the inhibitory activity as shown by the ic50 values 35.04 ± 0.20 µm for ache and 37.13 ± 0.18 µm for bche enzymes. correspondingly, in compounds 3 (ic50 = 24.94 ± 0.12, 27.13±0.08 µm) and 4 (ic50 = 24.57 ± 0.07, 27.57 ± 0.07µm), a slight decrease in the inhibitory potential was seen by the addition of chloro groups at the ortho-, paraand di-ortho-positions against ache and bche enzymes, respectively. however, the presence of chloro groups in compounds 5-7, along with other groups such as no2 and oh, demonstrated lower potential against ache and bche enzymes. para-bromo substituted compound 28 (ic50 = 25.33 ± 0.02, 26.27 ± 0.18 µm), exhibited pronounced activity against both ache and bche enzymes, respectively. however, compound 29 with trifluoromethyl substitution was found to be inactive against both enzymes which indicates that the trifluoromethyl group is not actively involved in the binding interaction to the active site of the enzyme (figure s-2). a cc ep te d m an us cr ip t 10 babatunde et al. in a recently published quinazolinone derivative 33, cholinesterase inhibitors showed superior inhibitory activity compared to the standard drug tacrine. among them, halogenated compounds showed potential activity against ache and bche enzymes. these results showed similarity with our work in which halogenated compounds showed potential activities as compared to the standard donepezil. it has been found that the incorporation of methoxy substitutions in compounds 8-17 at different positions of aryl moiety (r) has a varying degree of inhibition. among them, ortho-dimethoxy substituted compound 10 was found significantly active with ic50 = 26.94 ± 0.12, 27.99 ± 0.09 µm for ache and bche enzymes as compared to its ortho, meta-dimethoxy derivative compound 9. surprisingly, it was found that its meta-dimethoxy substituted positional isomer 8 was completely inactive. there might be a possibility that compound 8 attained such a conformation that does not fit well into the active site of the enzyme. however, when methoxy groups are present at the adjacent positions in compound 11 (ic50= 87.27 ± 0.02, 86.08 ± 0.43 μm) a noticeable decline in the activity was observed as compared to compound 12 (ic50= 67.91 ± 0.18, 69.02 ± 0.11 μm), where an additional bromo group is present at ortho-position. the positional isomer of 11 i.e., compound 13 demonstrated weak inhibitory potential against both enzymes. in the case of trimethoxy substituted derivatives (compounds 14 and 15), a complete loss of activity was observed. this might be due to the steric hindrance and bulkiness of the groups. compounds 16 with para-methoxy and meta-ethoxy substitutions displayed considerable inhibitory potential with ic50 = 27.57 ± 0.07; 29.13 ± 0.18 μm against acetylcholinesterase and butyrylcholinesterase enzymes, respectively. in contrast, compound 17 was found to be inactive against both enzymes (figure-s-3). compounds 18-24 and 32 with the combinations of ethoxy/methoxy and other substitutions such as oh, cl, f, and br, exhibited moderate inhibition activities against both enzymes. ortho-fluoro and para-methoxy substituted compound 22 was found to have relatively good activity in comparison to its other positional analogs. compound 18 displayed ic50 values of 27.91 ± 0.18, and 27.91 ± 0.18 µm against ache and bche enzymes, respectively. in contrast, its positional isomer (compound 20) exhibited low inhibitory potential with ic50 = 51.41 ± 0.12, 53.33 ± 0.02 µm against acetylcholinesterase and butyrylcholinesterase enzymes. the activity of the combination of cl, br, and oh with methoxy-substituted compounds 18, 19, 21, 23, 24, and 32, displayed moderate to weak inhibitory activities which indicates that these groups are creating steric hindrance and less binding interaction in the enzyme’s active site or their positive mesomeric effect is negatively contributing in the activity (figure s-4). surprisingly, ortho, meta, and para benzyloxy substituted derivatives 25-27 were found to be inactive against acetylcholinesterase and butyrylcholinesterase enzymes. it might be due to bulky groups that do not favorably fit in the active site a cc ep te d m an us cr ip t synthesis of 2,3-dihydroquinazolin-4(1h)-one and their derivatives 11 of the enzyme, which displayed that the presence of hydrophobic groups on the aryl part more specifically the presence of the benzyloxy group, resulted in the loss of activity profile of compounds 25, 26 and 27, respectively. exceptionally, thiophene-substituted analog 30 showed moderate activity against ache and bche enzymes with ic50 values of 43.08 ± 0.03 and 46.08 ± 0.43 µm, respectively (figure s-5). mono-hydroxyl substituted compounds 31-35 showed good to moderate results against acetylcholinesterase and butyrylcholinesterase enzymes. the activity of five hydroxy-substituted derivatives such as 31-35 was different from each other against both enzymes. however, the structure of all five derivatives is very similar to each other but differ only in the position of hydroxyl at aryl part “r”. amongst them, compound 34 (ic50 = 27.57 ± 0.07, 29.02 ± 0.11 µm) has ortho-hydroxyl group exhibited better activity against ache and bche enzymes as compared with compounds 33 and 35, respectively, which indicate that groups and position displayed significant role in the enzyme inhibition. however, compounds 31 and 32 with the combination of bromo and methoxy with a hydroxyl group, respectively, exhibited weak inhibitory activities against ache and bche enzymes. this activity pattern demonstrated the involvement of disubstituted hydroxy compounds 36-38, which also displayed moderate to weak inhibitory activities. compound 36 (ic50 = 45.04 ± 0.52, 47.7±0.16 µm) with meta, para di-hydroxy substitution showed better activity as compared to compounds 37 and 38 against acetylcholinesterase and butyrylcholinesterase enzymes (figure s6). sar for dpph and abts radical scavenging activities based on (sar), the variations observed in dpph and atbs activities of quinazolinones 1-38 were discussed and compared against standard ascorbic acid with ic50 = 15.08 ± 0.03 and 16.09 ± 0.17 µm, respectively. dichloro-substituted compound 4 showed dpph (ic50 = 16.33 ± 0.02 µm) and abts (ic50 =18.01 ± 0.12 µm) radical scavenging activities, respectively, and was found to be most active in the series. its positional isomer (compound 3) displayed a decline in activity against both radicals. however, mono-substituted compound 2 having chloro group at meta position (ic50 = 17.65 ± 0.23, 19.47 ± 0.03 µm), showed better dpph and abts radical scavenging activities as compared to its positional isomer 1. antiradical activity depends on proton and electron transfer between the radical and the scavenging agent. here 1,4 disubstituted chloro compounds seem to involve electron transfer and free radical scavenging compared to monosubstituted and 1,3 disubstituted chloro compounds. the addition of hydroxyl and nitro substitution at the aryl ring in compounds 5, 6, and 7, respectively, showed moderate to weak potential against dpph and abts radical scavenging activities. the activity of di-methoxy substituted compounds 8, 9, and 11-13 showed a further decrease in the activity as compared to ortho-dimethoxy substituted compound 10 which showed enhanced dpph and abts radical a cc ep te d m an us cr ip t 12 babatunde et al. scavenging activities. the addition of the methoxy group in compounds 14 and 15 further reduced the activity (figure s-7). in the case of compound 16 (ic50 = 30.04 ± 0.02, 31.99 ± 0.99 µm) para-methoxy and meta-ethoxy groups showed better activities as compared to compound 17 (figure s-7). another combination of methoxy with oh, br, f, and cl substitutions in compounds 18-23 showed weak potential against dpph and atbs activities. compounds 25, 26, and 27 bearing benzyloxy substitution displayed decreased radical scavenging activities against dpph and atbs. the incorporation of the bromo group as “r” in compound 28 with ic50 values 27.33 ± 0.02, 28.01 ± 0.12 µm, showed better potential than compound 31. mono-hydroxy and di-hydroxy substituted compounds 32, 33, 35, and 38 demonstrated good potential against dpph and abts radical scavenging activities as compared to compound 34. compounds 23, 29, and 30 showed a further decline in the activities as compared to the standard ascorbic acid (figure s-7). kinetic studies on acetylcholinesterase inhibitors kinetic studies on the most active ache enzyme inhibitors (compounds 2-4, 10, 16, 28, and 34) were analyzed to interpret the enzyme inhibition mechanisms by using graph fitting analysis in the sigma-plot enzyme kinetic software (figures s-8a-b). in 2,3-dihydroquinazolin-4(1h)-ones all the seven compounds (2, 3, 4, 10, 16, 28, and 34) vmax and km (michelis-menton constant) were in the range of 60.5 to 79.8 (µm/min)/mg and 3.0 to 3.6 mm respectively (figure s-8a). the ki (dissociation constant) values were confirmed from the dixon plot by plotting the reciprocal of the rate of reaction against different concentrations of compounds, where ki values of all eight compounds were in the range of 5.0 to 5.9 µm (figure s-8b). in the uncompetitive type of inhibition, only vmax values are affected, and no changes in the km value. the low vmax and no effect in the km value of these compounds indicated an uncompetitive type of inhibition (table ii). table ii. kinetic studies of active compounds for acetylcholinesterase inhibition (uncompetitive type of inhibition) compound no vmax (µm/min)/mg km / mm ki / µm 2 79.8 ± 1.2 3.2 ± 0.01 5.2 ± 0.1 3 70.4 ± 1.0 3.6 ± 0.02 5.4 ± 0.2 4 60.5 ± 2.2 3.0 ± 0.01 5.5 ± 0.5 10 66.8 ± 1.8 3.3 ± 0.02 5.8 ± 0.1 16 71.0 ± 1.2 3.1 ± 0.01 5.0 ± 0.2 28 65.4 ± 1.0 3.2 ± 0.02 5.3 ± 0.1 34 53.2 ± 2.2 3.4 ± 0.01 5.9 ± 0.2 donepezil 62.0 ± 1.0 3.0 ± 0.01 5.1 ± 0.1 a cc ep te d m an us cr ip t synthesis of 2,3-dihydroquinazolin-4(1h)-one and their derivatives 13 kinetic studies on butyrylcholinesterase inhibition kinetic studies on the most active ache enzyme inhibitors compounds 2-4, 10, 16, 28, and 34 were analyzed to interpret their inhibition mechanisms (figure s-9). in 2,3-dihydroquinazolin-4(1h)-ones the vmax and km of all the seven compounds were in the range of 80.3 to 85.4 (µm/min)/mg and 3.1 to 31.8 mm respectively (figure s-9a). the ki values were confirmed from the dixon plot by plotting the reciprocal of the rate of reaction against different concentrations of compounds, where ki values of all the five compounds were in the range of 10.3 to 10.9 µm (figure s-9b). in the competitive type of inhibition, only km values are affected and there are no changes in the vmax value. the high km and no effect in the vmax of these compounds indicated a competitive type of inhibition (table iii). table iii. kinetic studies of active compounds for butyrylcholinesterase inhibition (competitive type of inhibition) compound no vmax (µm/min)/mg km / mm ki / µm 2 82.0 ± 2.2 3.1 ± 0.2 10.6 ± 0.5 3 80.3 ± 2.7 9.2 ± 0.1 10.4 ± 0.3 4 82.2 ± 5.3 20.2 ± 0.2 10.7 ± 0.2 10 85.4 ± 1.2 2.1 ± 0.1 10.6 ± 0.4 16 82.0 ± 1.4 3.7 ± 0.2 10.4 ± 0.1 28 84.1 ± 2.4 31.8 ± 0.1 10.3 ± 0.1 34 82.5 ± 2.9 4.7 ± 0.1 10.9 ± 02 donepezil 80.1 ± 1.6 13.5 ± 0.1 10.2 ± 0.1 molecular docking studies ache and bche molecular docking study md was performed to explore the binding mode of the synthesized compounds against the targeted enzyme (ache and bche). md results are in good agreement with experimental results. we have noticed that compounds bearing the electronwithdrawing groups (ewgs) showed the best inhibitory activity against both targets. interestingly, as compared with the other activity (α-amylase and α-glucosidase),23 we have noted that the compounds bearing 1,3-dichlorobenzene showed high inhibitory potency as compared to 1-chlorobenzene. similarly, the following compounds showed invert phenomena in the activity against both targets. those compounds bearing 1-chlorobenzene/1-bromobenzene substitution were found to be active. the pli profile was enlisted for all docked compounds in tables s-i and sii in the supplementary material. a cc ep te d m an us cr ip t 14 babatunde et al. acetylcholinesterase (ache) molecular docking study the docking results for most active compound 2 against ache revealed that the 3-methyl-tetrahydro pyrimidine-4(1h)-one moiety of the compound adopted several favorable interactions with catalytic residues (figure 3a surface representation) including acidic residue glu72, hydrophobic side chain tyr334, trp279, and phe331, respectively (figure 3b). figure 3a-c. the pli profile for synthesized compounds against the acetylcholinesterase (ache) enzymes. (a) the surface representation of the enzyme, (b) the binding mode of the most potent compound 2 in the series, and (c) compound 4. a double-sided arrow represented the π-stacking the reason for high potency might include the high number of adopted favorable interactions with catalytic residues. in the case of the 2nd ranked active compound 4, where the substitution groups are the 2,3-dichloro, a similar interaction was observed. but the only difference so far found is: the active compound adopted π-stacking interaction with the 1-chloro moiety, whereas it lacks in the 2nd active compound (figure 3c). this might be one of the reasons for reduced activity in compound 4. the pli profile was enlisted for all docked compounds in (table s-ii). butyrylcholinesterase (bche) molecular docking study in the case of the docking results for most active compounds against bche (figure 4a), activity revealed that the compound bearing electron-withdrawing groups (ewg), i.e. 1-chlorobenzene (figure 4b) and 1-bromobenzene (figure-4c), showed best inhibitory activity against the bche enzyme. the protein-ligand interaction (pli) profile for the most active compound 2 and 2nd ranked active compound 28 revealed an interesting observation that both the compound shared similar interaction with the hydrophobic residue phe329. a cc ep te d m an us cr ip t synthesis of 2,3-dihydroquinazolin-4(1h)-one and their derivatives 15 figure 4a-c. the pli profile for synthesized compounds against the butyrylcholinesterase (bche) enzymes. (a) the surface representation of the enzyme, (b) the binding mode of the most potent compound 2 in the series, and (c) compound 28. the π-stacking was represented by a double-sided arrow more interestingly, the most active compound 2 adopted interaction with the acidic residue glu70 while compound 28 with glu197, which suggested that might be these two residues play a vital role in enhancing the enzymatic activity. the hydrophobic residue trp82, which is an active residue in the active site and plays a vital role in the enzymatic activity, adopted two π-stacking interactions with the substituted benzene ring while the compound 28 does not attempt to adapt interaction even though this residue is found in proximity with the 6-ring of the compound. overall, these results describe that the compounds bearing the ewg either at orthoor meta-position displayed good inhibitory potential against the enzyme while others bearing both orthoand metaor orthoand para-positions showed less activity. the pli profiles were enlisted for all docked compounds in (table s-ii). conclusion in the present study, compounds showed moderate to good inhibition against ache, bche, and antioxidant activities as compared with the standards donepezil and ascorbic acid, respectively. a structure-activity relationship was also established. in silico modeling studies revealed the binding mode of the quinazolinone derivatives. the kinetic studies on the seven most active compounds 2, 3, 4, 10, 16, 28, and 34 were carried out. the compounds 2, 3, 4, 10, 16, 28, and 34 were found to have an uncompetitive mode for acetylcholinesterase enzyme and the compounds 2, 3, 4, 10, 16, 28, and 34 were found to be the competitive mode for butyrylcholinesterase enzymes. acknowledgments: the authors acknowledge the financial support of the sindh higher education commission (shec), pakistan vide letter no. no.dd/shec/1-14/2014, project code shec/srsp/med-3/15/2021-21. a cc ep te d m an us cr ip t 16 babatunde et al. supplementary material additional data are available electronically at the pages of the journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/11370, or from the corresponding author on request. извод изучавање деривата 2,3-дихидрохиназолин-4(1h)-он као инхибитора холинестераза и њихове антиоксидативне активностиактивности: in vitro, in silico, и кинетичка испитивања oluwatoyin babatunde1, shehryar hameed1, kingsley adibe mbachu1,2, faiza saleem1, sridevi chigurupati3, abdul wadood4, ashfaq ur rehman4, vijayan venugopal5, khalid mohammed khan1,7, muhammad taha7, olusegun ekundayo2 и maria aqeel khan6 1h. e. j. research institute of chemistry, international center for chemical and biological sciences, university of karachi, karachi-75270, pakistan; 2department of chemistry, university of ibadan, nigeria; 3department of medicinal chemistry and pharmacognosy, college of pharmacy, qassim university, buraydah-52571, saudi arabia; 4department of biochemistry, computational medicinal chemistry laboratory, ucss, abdul wali khan university, mardan, pakistan; 5faculty of pharmacy, aimst university, kedah-08100, malaysia; 6third world center for science and technology, h. e. j. research institute of chemistry, international center for chemical and biological sciences, university of karachi, karachi-75270, pakistan и 7department of clinical pharmacy, institute for research and medical consultations (irmc), imam abdulrahman bin faisal university, p.o. box 1982, dammam, 31441, saudi arabia током истраживања нових активних инхибитора холинестераза и антиоксидативних агенаса, испитивани су синтетички деривати дихидрохиназолин-4(1h)-он 1-38 као потенцијални агенси за третман алцхајмерове болести инхибицијом ацетилхолин естеразе (ache), бутирлихолин естеразе (bche) и као хватачи слободних радикала (dpph и abts). доминантан утицај на инхибицију ензима и способност хватања слободних радикала имају супституенти на ароматичном језгру. на основу резултата испитивања кинетике закључено је да једињења делују некопетентивним механизмом инхибиције. молекулским моделовањем су испитане могуће интеракције током везивања киназолинских деривата у активним местима оба ензима. 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(https://doi.org/10.1016/j.sjbs.2021.05.072) a cc ep te d m an us cr ip t https://www.researchgate.net/publication/237620105_the_use_of_the_stable_radical_diphenylpicrylhydrazyl_dpph_for_estimating_antioxidant_activity https://www.researchgate.net/publication/237620105_the_use_of_the_stable_radical_diphenylpicrylhydrazyl_dpph_for_estimating_antioxidant_activity https://doi.org/10.1016/s0891-5849(98)00315-3 https://doi.org/10.1016/s0891-5849(98)00315-3 http://www.sciepub.com/reference/263157 https://doi.org/10.1016/j.ejps.2007.10.004 https://doi.org/10.1016/j.ijbiomac.2020.12.121 https://doi.org/10.1016/j.sjbs.2021.05.072 {examination and optimization of lignocellulolytic activity of stereum gausapatum f28 on beechwood sawdust supplemented with molasses stillage} j. serb. chem. soc. 87 (4) 437–450 (2021) original scientific paper jscs–5532 published 12 november 2022 437 examination and optimization of lignocellulolytic activity of stereum gausapatum f28 on beechwood sawdust supplemented with molasses stillage jelena jović1*, jian hao2 and ljiljana mojović1# 1university of belgrade, faculty of technology and metallurgy, department of biochemical engineering and biotechnology, karnegijeva 4, 11000 belgrade, serbia and 2laboratory of biorefinery, shanghai advanced research institute, chinese academy of sciences, pudong, shanghai 201210, people’s republic of china (received 1 june, revised 26 august, accepted 16 september 2021) abstract: this study provides a detailed analysis of the lignocellulolytic activity of a new isolate stereum gausapatum f28, a serbian autochthonous fungi, on beechwood sawdust supplemented with cheap waste, sugar beet molasses stillage. advanced multiple response optimization techniques were applied to improve ligninolytic and reduce hydrolytic activity as a requirement for potential biorefinery use. the applied techniques were supposed to select cultivation conditions that would give manganese peroxidase and laccase activities above 0.84 and 0.12 u g-1 substrate, respectively, and cellulase and xylanase activities below 1.12 and 1.4 u g-1 substrate. the optimal cultivation conditions that met the set requirements included molasses stillage concentration of 10 %, substrate moisture content of 53 %, incubation temperature of 23.5 °c, and ph 5.2. the research showed that the addition of molasses stillage had a positive effect on enzyme production and that the optimal stillage concentration differed depending on the enzyme type (for laccase it was <5 %, manganese peroxidase ≈12 %, cellulase ≈21 % and xylanase ≈16 %), which should be taken into consideration when optimizing the desired process. keywords: response surface method; genetic algorithm; fungi; biomass; enzymes. introduction the growth of enzyme usage in industry has increased the demand for novel microbial strains that could be used for the productions of various enzymes to meet the current requirements.1 wood-decay fungi are recognized as organisms suitable for the production of the industrially valuable enzymes laccase (ec * corresponding author. e-mail: yowitch@gmx.com # serbian chemical society member. https://doi.org/10.2298/jsc200601075j ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 438 jović, hao and mojović 1.10.3.2), manganese-dependent peroxidase (mnp; ec 1.11.1.13), versatile peroxidase (vp; ec 1.11.1.16), lignin peroxidase (lip; ec 1.11.1.14), cellulase (ec 3.2.1.4), xylanase (ec 3.2.1.8), and other hemicellulases. these fungi have been extensively examined for application in wastewater treatment, in the pretreatment of biomass to facilitate its conversion to value-added chemicals, and production of lignocellulose-degrading enzymes, which can be used in various processes in the pulp and paper industry, food and beverage, personal care, healthcare, pharmaceutical and other industries.2,3 the application of fungi in the production of enzymes and other industrially and commercially valuable products from lignocellulosic waste and by-products from various industries has been emphasized in recent years. lignocellulose has attracted attention as it is a renewable and inedible material that is generated in large amounts every year; the agro-industry alone produces about 5 billion metric tons of lignocellulosic waste per year.1 by-products from various industries pose a danger to the environment; the demands for proper management of this waste have increased, thus enforcing the development of new technologies based on natural systems or processes to remove, purify, or convert this material into value-added products. however, for some agents, such as molasses stillage (ms) – a by-product of the alcohol industry that generates 8–15 l of ms per l of produced alcohol – adequate applications and disposal are still being sought.4 the potential solutions include its use as a substrate or a supplement in a lingocellulosic substrate for enhancement of fungal enzyme production (laccase, amylase, xylanase).5–7 still, to recommend the use of this agent in the industry, new fungal isolates have to be examined, and particularly the influence of this agent on their enzyme production/activity. enzyme production and product quality can be improved using proper optimization techniques in the production process or system. biological processes contain a large number of natural variations.8 different parts of a complex network of microbial reactions are affected by various environmental/cultivation factors and factor interactions, making the formulation of appropriate conditions difficult. increased knowledge about the examined system or process and the reliability of experimental data are prerequisites for overcoming the mentioned difficulties, which can be accomplished by appropriate experimental design and statistical evaluation of the collected results.8 the optimization methods can be divided into traditional (one factor at a time) and advanced (statistical/mathematical).8 traditional methods are still used in the initial research to determine the composition of the appropriate medium for the production of a new metabolite or when a new organism is used in the production of an already known metabolite. advanced methods involve the application of experimental design, in which the experimental phase enables determination of potentially influential factors and development of a predictive model that can be used to optimize factor settings ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. optimization of fungal lignocellulolytic activity 439 that give the best value of the response variable.9 the advanced methods can be used for the optimization of single or multiple responses. optimization of a single response usually involves the application of response surface methods (rsm), such as central composite design (ccd) or box–behnken design (bbd), while for multiple response optimization, often used methods include desirability function and/or evolutionary algorithms (among which genetic algorithm (ga) is the most often used), or artificial neural networks.10 this research used ccd of rsm, desirability function, and ga to evaluate the influence of factors and factor interactions on the laccase, manganese peroxidase (mnp), cellulase, and xylanase activity of a fungal isolate, create predictive models, and optimize the cultivation conditions. the aim of the present research was a detailed analysis of the lignocellulolytic activity of s. gausapatum f28, a novel serbian autochthonous fungal isolate, with the purpose of application in various biotechnological processes – from enzyme production to the pretreatment of lignocellulose. the study examined the effect of ms on the lignocellulolytic activity of this fungus to assess the possibility of using this agent as a supplement to improve the production of ligninolytic or hydrolytic enzymes. the research also examined the possibility of steering the lignocellulolytic activity of this isolate into the desired direction using advanced optimization methods. experimental organism and substrate this research examined an isolate stereum gausapatum f28 collected in southern serbia (previously identified using molecular methods,11 its dna sequence was deposited in the ncbi genbank database under the accession number ky26475311). the lignocellulosic substrate was beechwood sawdust obtained from a local sawmill. sugar beet molasses stillage (ms) was a waste obtained from the local alcohol industry. inoculum preparation and cultivation conditions the active culture was maintained at 30 °c on malt extract agar and every 2–3 weeks transferred to a fresh agar. the inoculum was prepared on an inoculum agar medium (the precise composition was given in a previous research5). three mycelial discs, approximately 10 mm in diameter each, were taken from the edge of 7-day-old inoculum culture and transferred to 100-ml erlenmeyer flasks containing 4.5 g dry substrate mass of the lignocellulosic substrate. the dry substrate mass was determined using the nrel/tp-510-42621 protocol.12 the substrate moisture was adjusted using diluted ms (ms concentrations and moisture content are given in table s-i of the supplementary material to this paper) or distilled water. the substrate was autoclaved for 30 min before inoculation. enzyme extraction and assays the enzymes were extracted with 50 ml of distilled water, which was added to erlenmeyer flasks and subsequently shaken for 40 min at 220 rpm and 25 °c, and then filtered through a whatman no. 1 filter paper and centrifuged at 4185g using a z-206-a high capacity, compact centrifuge (hermle labortechnik gmbh, wehingen, germany). the extracts ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 440 jović, hao and mojović were stored at 4 °c until use. enzyme activity assays were conducted within 24 h of the extraction on a ultrospec 3300 pro spectrophotometer, amersham biosciences ltd., little chalfont, uk. the laccase activity was determined using the guaiacol assay, the mnp and vp activities using the phenol red assay and the lip activity using azure b. these assays were performed according to the procedures provided in the research of jović at al.11 cellulase (cmcase) and xylanase activity were measured using a modified dinitrosalicylic acid (dns) reagent method proposed by miller following the procedure described in detail in the research by jović at al.5 experimental design and optimization response surface method. a central composite design (ccd) of the response surface method (rsm) was used to examine the influence of four factors (ms concentration, substrate moisture, temperature and ph) and factor interactions on the ligninolytic and hydrolytic activity of s. gausapatum f28. the factors were selected using fractional factorial design before rsm. the ccd of rsm had two blocks, i.e., a factorial block that contained 24 full factorial design (16 star points) and four replicates in the center point, and an axial block with eight axial points and three replicates in the center point. the response values were obtained in 31 experimental runs. the studied system was described using a second-order polynomial equation (eq. (1)) for each examined response: y = β0 + σβixi + σβijxiixjj + σβiix2ii (1) where i = 1,2,…,k and j = 1,2,…,k. in eq. (1), y is a response, xi/xj are coded value of the ith factor, k is the number of examined factors, and β0, βi, βii and βij are the second order regression coefficients. each factor was examined at five different levels (–2.098, –1, 0, 1, 2.098). the actual and coded values of the examined factors are given in table s-i. linear regression analysis and diagnostics (r2 value and diagnostic plots), anova, and “adequate precision” (ap, calculated by eq. (2)) were used to examine the model quality: ap = (max(ŷ) − min(ŷ))/(√(pσ2/n)) (2) where ŷ is prediction at the run settings, p is the number of terms in the model, σ2 is the mean square of residuals, and n is the number of runs in the design. optimization using desirability function and genetic algorithm. a combination of desirability function and ga was used for the optimization of the lignocellulolytic activity of s. gausapatum f28. two types of individual desirability functions were used, i.e., the transformation of maximization for ligninolytic activities and transformation of minimization for hydrolytic activities. software and statistical analysis. the software used for the design analysis was r studio version 1.3.959, and r language version 3.6.3,13 and r packages rsm,14 desirability15 and ga.16 the values were expressed as mean ± standard deviation. anova was used for comparison of the mean values. differences were considered significant at p < 0.05. results and discussion s. gausapatum is the primary colonizer of oak,17 capable of producing various lignocellulose degrading enzymes. the enzymes of its lignocellulolytic system have been largely identified to date. still, changes in its lignocellulolytic system have been insufficiently studied, both under different cultivation conditions and different incubation durations, and the information about the utilization of ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. optimization of fungal lignocellulolytic activity 441 waste material in lignocellulolytic enzyme production is limited. understanding these changes could facilitate as well as improve the precision of the application of s. gausapatum in specific industrial processes, such as biomass conversion or enzyme production. this research examined the mnp, laccase, cellulase and xylanase activity on beechwood sawdust supplemented with ms. the lip and vp activities were excluded as less relevant components of the lignocellulolytic system of s. gausapatum f28 for lignin degradation based on the screening analysis (fractional factor design), which showed that these activities were at the statistical error level, or under certain conditions completely undetectable. response surface method, model design, and model quality analysis the influences of the examined factors and factor interactions on the activity of laccase, mnp, cellulase and xylanase were investigated using ccd of rsm. the ccd matrix and the predicted and measured response values of general laccase, mnp, cellulase, and xylanase activities and activities per dry substrate mass are given in tables s-ii, s-iia, s-iii and s-iiia of the supplementary material. to explain the correlation between the response variables and factors, second -order polynomial equations were created (eqs. (s1)–(s4) in the supplementary material) – laccase activity model (eq. (s1)), mnp activity model (eq. (s2)), cellulase activity model (eq. (s3)), and xylanase activity model (eq. (s4)). the applied analysis of regression coefficients and model quality revealed that the generated models needed improvements, and that the effect of blocking was statistically significant in the case of the hydrolytic activities, but not in the case of ligninolytic activities. statistically insignificant terms were removed gradually until the minimal adequate models with an improved quality and predictability were gained (eqs. (s1)–(s4)). after each term removal, a model quality check was performed. a particular statistically-not-significant term would be retained in the equation only if the analysis revealed that its removal impaired the model quality and predictability (tables s-iv–s-vii of the supplementary material). the term removal reduced the r2-value of the models to some extent (table s-viii) and lowered the difference between the r2 and the adjusted r2 of the obtained minimal models (table s-ix). diagnostic plots confirmed the assumptions of the regression (linearity, normal distribution, homoscedasticity and presence/absence of influential data points). the plots are presented in figs. s-1–s-4 of the supplementary material, while the calculated ap-values confirmed good signal (table s-ix), which indicated a good quality of all four minimal adequate models. influence of the examined factors on the ligninolytic and hydrolytic activity the influence of factors and factor interactions on the ligninolytic and hydrolytic activities of the fungal isolate was examined and visualized using contour plots. the contour plots were created by plotting the response values against any ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 442 jović, hao and mojović two independent variables while keeping the other variables at their central (0) level. it was revealed that the ms concentration and substrate moisture had the greatest influence on the ligninolytic and hydrolytic activity. the laccase model is a min–max function with stationary points at an ms concentration of 16.6 %, a substrate moisture of 67.9 %, a temperature of 24.5 °c, and a ph of 6.4. the contour plots show that high laccase activity could be obtained at higher temperature (above 25 °c), low ms concentration (below 5 %), ph around 5.5, and substrate moisture between 70 and 80 % (figs. s-6a and c and s-7 of the supplementary material). stationary points obtained for the mnp activity – an ms concentration of 12.62 %, a substrate moisture of 70 %, a temperature of 27.5 °c, and a ph of 5.6 – are points of maximum; hence, they represent the optimal cultivation conditions for mnp activity (figs. s-6b and d and s-8). in general, lower values of temperature and substrate moisture are expected for mnp production.5 however, the optimal temperature depends on the fungal species or strain, and it can vary from 25 (recorded for some species such as trametes hirsuta5,18) to 35 °c (for species such as trametella trogii18). according to the studies conducted by humphrey and siggers, and herrick,19,20 s. gausapatum is a species that grows at lower temperatures. its optimal growth temperature is around 24–25 °c, but it can grow at temperatures between 5 and 33 °c, although at maximum or minimum values of this range, it grows very little, while at 38 °c, its growth is completely inhibited. in this research, s. gausapatum f28 grew poorly at 33 °c, while at 37 °c, its growth was completely inhibited. therefore, the optimal temperature for laccase production could be expected at temperatures up to 30 °c. the optimal substrate moisture for the production of ligninolytic enzymes also depends on the fungal species. different studies reported good ligninolytic enzyme production at a substrate moisture content between 65 to 90 %,21,22 while the optimal moisture content for fungal growth and biomass utilization can range from 3022 to 90 %.21 the level of the initial substrate moisture influences the production of ligninolytic enzyme, more specifically, the type of ligninolytic enzyme. a higher moisture level (80–90 %) is often reported as more suitable for laccase production,5,11,21 while a lower moisture level (50–65 %) for mnp production.5,21 a good production of ligninolytic enzymes is often reported at ph 5.22 ms is not extensively examined as a supplement for the production of ligninolytic enzymes. a previous study with trametes hirsuta f13 showed that a high concentration of this agent (above 20 %)5 was required for laccase production. in the case of s. gausapatum f28, examined in the current research, the laccase production requires a low concentration of this agent (below 5 %), while the optimal ms concentration for mnp production obtained for s. gausapatum f28 (12.62 %) was close to that obtained for t. hirsuta f13 (14.36 %).5 the ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. optimization of fungal lignocellulolytic activity 443 optimal concentration of ms required for the production of laccase may depend on the fungal species. according to the performed rsm analysis, only two factors had a statistically significant influence on the cellulase activity, i.e., the ms concentration and the substrate moisture. the stationary points obtained for the cellulase activity model – ms concentration of 21.58 % and substrate moisture of 82.76 % – were the points of maximum and represent the optimal conditions for cellulase production (fig. s-5d). three factors had a statistically significant influence on the xylanase activity, i.e., the ms concentration, the substrate moisture, and ph (fig. s-5a–c). the stationary points obtained for the xylanase model were also the points of maximum, which indicates optimal cultivation conditions. these conditions included an ms concentration of 16.26 %, a substrate moisture of 70 %, and a ph of 5.6. the moisture content is a critical parameter for the production of cellulases and xylanases, and depending on the fungal species and origin of the lignocellulosic substrate, the optimal moisture content could vary from 60 to 80 %.23 the optimal ph value also depends on the fungal species; ph values between 4 and 6.5 are often reported as optimal for the production of hydrolytic enzymes.23,24 studies that examined the use of stillage in the production of fungal cellulase or xylanase are still rare. in a study conducted by acharya et al.,7 an increase in the concentration of anaerobically treated distillery spent wash improved cellulase production by aspergillus ellipticus. in the study conducted by shahryari,6 the addition of tin stillage from a residual stream of the whole-wheat ethanol process positively affected the production of xylanase by neurospora intermedia. the influence of ms on the fungal production of xylanase has not hitherto been reported. according to the results obtained in the current research with s. gausapatum f28, the addition of the appropriate ms concentration can improve the production of xylanase and cellulase. however, a higher ms concentration is required for the production of cellulase (≈21 %) than for xylanase production (≈16 %). optimization of the lignocellulolytic activity the optimization process took into consideration changes in the enzyme content under different cultivation conditions and the role and importance of certain lignocellulolytic enzymes in biomass degradation. the aim was to determine cultivation conditions that would promote ligninolytic and reduce hydrolytic activities, bearing in mind the potential application in the pretreatment of lingocellulosic biomass. variations that exist among lignocellulose of different origins and the complex structure of this material make selection and optimization of a proper pretreatment method difficult. however, some requirements, such as efficient lignin removal and recovery of a high percentage of holocellulose, apply to all pretreatment methods.25 in the case of the biological process, the production ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 444 jović, hao and mojović organism would need a certain amount of consumable carbohydrates to survive during the pretreatment, which further complicates process optimization. the dominant ligninolytic activity of s. gausapatum f28 on beechwood sawdust supplemented with ms was mnp activity. this enzyme is crucial for the initial lignin degradation.26 the optimization goal was to improve ligninolytic activity; yet, the research showed that different cultivation conditions promoted laccase or mnp activity, but not both simultaneously. however, the differences observed with s. gausapatum f28 were less extreme than in a previous study with t. hirsuta f13.5 the optimization took the next best approach by prioritizing mnp production and adjusting laccase production accordingly. the laccase activity values varied between 0.04 and 0.24 u g–1 of dry substrate, but the most common activity values ranged between 0.11 and 0.17 u g–1. under the conditions that yielded moderate laccase activity (0.12–0.17 u g–1), a fairly high mnp activity was observed (≥0.84 u g–1). the cultivation conditions that yielded laccase activity below 0.06 u g–1 were hostile for ligninolytic and hydrolytic enzymes. it was also noticed that, under the conditions that yielded laccase activity above 0.17 u g–1, the recorded mnp activity was extremely low (the values were below 0.34 u g–1, see table s-ii). laccase and mnp activity values of 0.12 and 0.84 u g–1, respectively, were chosen as the desired minimum activities and set as lower limits in the function of maximization. the values were selected based on the obtained results, observations, and the enzyme importance for lignin degradation.26 the chosen upper limits of mnp and laccase activities were the values that, when transformed by the function of maximization, gave the highest possible value below 1 to enable the selection of a single and objective solution. the scale factor for the laccase model was set at 0.1 to slow growth toward 0.17 u g–1. the scale factor for the mnp model was set at 3 to enable rapid achievement of high mnp activities. the function of minimization was used to lower the cellulase and xylanase activity. a cellulase activity of 1.12 u g–1 and a xylanase activity of 1.4 u g–1 were set as the upper limits. each of them makes about 40 % of the highest achieved corresponding activity value (table s-ii). these values were selected based on the rsm analysis as the values that would not jeopardize achieving an mnp activity above 0.84 u g–1. the lower limits were chosen as values that, when transformed by the function of minimization, give the highest possible value below 1. as the production organism requires some sugar from the substrate to survive, the fast achievement of too low activity values could be counterproductive, therefore, the scale factors were set at 0.1. the cultivation conditions selected using a ga were an ms concentration of 10 %, a substrate moisture of 53 %, a temperature of 23.5 °c, and a ph of 5.2. these conditions were expected to yield a laccase activity of 0.13 u g–1, an mnp ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. optimization of fungal lignocellulolytic activity 445 of 1.28 u g–1, a cellulase value of 0.93 u g–1, and an xylanase value of 1.27 u g–1. the overall desirability value was 0.568 (fig. s-9 of the supplementary material). validation of the predicted optimum and importance of molasses stillage the optimal cultivation conditions gave the following activity values: laccase 0.17±0.01, mnp 1.05±0.05, cellulase 1.11±0.05 and xylanase 1.40±0.12 u g–1. the differences between experimental and predicted values were not statistically significant (p > 0.05; fig. 1). fig. 1. data validation, comparison of the experimental and predicted laccase, mnp, cellulase, and xylanase activity values after seven days of incubation under the optimal cultivation conditions. the same optimal conditions, but without the addition of ms, were applied to examine the contribution of ms to the enzyme production. the results showed that the addition of ms improved laccase activity by about 18 % and mnp activity by about 55 %, which confirmed that ms could be used as a supplement for improved production of ligninolytic enzymes of s. gausapatum f28. the improvement in the laccase activity was not statistically significant (p = = 0.811 > 0.05), while the improvement in the mnp activity, which was prioritized in the optimization, was significant (p = 0.022 < 0.05). the achieved hydrolytic activities were low and in accordance with the set optimization conditions; although, the activity values produced in the presence of ms were similar to those obtained in its absence (fig. 2). cultivation conditions change over time, which directly affects the production of enzymes of the fungal lignocellulolytic system and indirectly affects the processes in which these enzymes participate, such as biomass decomposition. in previous research, s. gausapatum f28 showed high efficiency in biomass decomposition but low selectivity in comparison to t. hirsuta f13.11 that research did not investigate fluctuations and profile of lignocellulolytic activities involved in a more than 30-day long biomass treatment. however, current research examined changes in the lignocellulolytic system of s. gausapatum f28 to provide more ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 446 jović, hao and mojović insights for a better understanding of previous results, which could help to develop methods to improve a process during an extended course of cultivation. the laccase and mnp activities decreased drastically after 18 and 33 days of incubation (fig. 2). the laccase activities measured in the absence of ms were similar to those obtained in the presence of ms (the values were below 0.03 u g–1 in both cases). by the 18th day of incubation, in the presence of ms, the mnp activity dropped to 0.39±0.02 u g–1, and then, by the 33rd day, it had increased to 0.68±0.01 u g–1. these activities were higher than the corresponding activities obtained without ms supplementation by ≈44 and ≈66 %, respectively. the cellulase activity increased after 18 days of incubation to 1.58±0.11 u g–1 on a substrate supplemented with ms, and by the 33rd day, it had decreased by 12 % to the value of 1.41±0.22 u g–1. without ms supplementation, the cellulase activity changed only slightly, it first rose to 1.15 u g–1 (18th day) and then decreased to 1.11 u g–1 (33rd day). after 18 days of cultivation on the substrate supplemented with ms, the xylanase activity decreased only slightly (≈6 %), and by the 33rd day, it had dropped by an additional 19 % to 1.05 u g–1. after 18 days of incubation, without ms supplementation, the xylanase activity decreased by ≈42 %, and by the 33rd day by an additional 3 % to the final value of 0.71 u g–1. fig. 2. influence of ms and changes in lignocellulolytic activity during 33 days of incubation. potential for industrial use fungi that are capable to produce highly active enzymes and to withstand harsh conditions are targeted for industrial use. most of the fungi already used in industry have been genetically modified to meet these requirements, but new organisms able to produce enzymes in higher amounts or with new characteristics are still being sought. fungi often examined for industrial use belong to genera pleurotus, daedaleopsis, ganoderma, irpex, polyporus, pycnoporus, bjerkandera, lentinus, etc. they are examined for the production of industrial enzymes or other industrial applications such as biomass conversion to valuable chemicals, dye removal and for degradation of organic phenolic and non-phenolic compounds, or wastewater treatment. for example, tripathi et al.27 examined the ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. optimization of fungal lignocellulolytic activity 447 production of ligninolytic enzymes by bjerkandera adusta and lentinus squarrosulus and reported laccase activities of ≈5.5 and 64 u l–1, and mnp activities of ≈215 and 370 u l–1, respectively. a study conducted by eichlerová and baldrian.28 examined potential candidates for industrial use that belong to the genera pleurotus, daedaleopsis, ganoderma, irpex, polyporus, pycnoporus, etc. this study was focused on laccase and mnp enzyme production and fungal application in dye degradation. the enzyme activities differed depending on the fungal species and ranged from 0.04–106 u l–1 for laccase, and 0.09–20.36 u l–1 for mnp enzymes with the best laccase activity values obtained with p. ostreatus (≈106 u l–1), cyclocybe erebia (≈114 u l–1), and abortiporus biennis (≈103 u l–1), and the best mnp values were recorded for omphalina mutila (≈20 u l–1), hericium erinaceus (≈17 u l–1), and mycetinis alliaceus, phellinus robustus, inonotus obliquus and fomitiporia mediterranea each with an activity of about 16 u l–1. s. gausapatum used in the current research showed the maximum laccase activity of 0.23 u g–1 (20.1 u l–1) and maximum mnp activity of 1.38 u g–1 (123.3 u l–1; see table s-iia) which puts this isolate in the group of good mnp and laccase producers, and as a candidate for industrial application. production of enzymes depends on the substrate type and fungal isolate. substrates with a lower amount of lignin and a higher amount of polysaccharides are more suitable for the production of hydrolytic enzymes than substrates with a higher share of lignin, such as beechwood sawdust. a study conducted by namnuch et al.29 examined cmcase and xylanase production by a. flavus kub2 on various substrates. the best activity was obtained on sugarcane bagasse (cmcase 1.04 u ml–1 and xylanase 258.38 u ml–1) while the lowest values were reported for production on sawdust (cmcase 0.06 u ml–1 and xylanase 14.07 u ml–1). the maximum cmc and xylanase activities obtained with s. gausapatum f28 used in the current research were 1.87 u g–1 (166.54 l–1) and 1.66 u g–1 (148.09 u l–1), respectively, showing potential for cmc production; however, more investigation of various substrates to find the most suitable substrate for hydrolytic enzyme production is needed. conclusions this research examined and optimized the lignocellulolytic system of serbian autochthonous isolate s. gausapatum f28 on beechwood sawdust supplemented with ms. advanced multiple response optimization methods were used to enhance ligninolytic and reduce hydrolytic activity. based on the results, s. gausapatum f28 can grow and produce enzymes laccase, mnp, cellulase and xylanase on waste biomass (beechwood sawdust), while cheap waste, ms, can be used to improve the production of these enzymes. the optimal ms concentration is different for the production of each enzyme type. another important cultivation factor, substrate moisture content, also differed depending on the enzyme ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 448 jović, hao and mojović type. variations in the optimal ph and temperature were less distinct and therefore less influential. s. gausapatum is an insufficiently researched species, but based on the results obtained for the isolate f28, its characteristics are promising for use in biorefinery or enzyme production. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/10808, or from the corresponding author on request. acknowledgements. the research presented in this paper was funded by the ministry of education, science and technological development of the republic of serbia (project #i-1). it is a part of scientific and technological collaboration of republic of serbia and the people’s republic of china и з в о д испитивање и оптимизација лигноцелулолитичке активности s. gausapatum f28 на пиљевини букве са додатком меласне џибре јелена jовић1, jian hao2 и љиљана мојовић1 1 универзитет у београду, технолошко-металуршки факултет, катедра за биохемијско инжењерство и биотехнологију, карнегијева 4, 11000 београд и 2 laboratory of biorefinery, shanghai advanced research institute,chinese academy of sciences, pudong, shanghai 201210, people’s republic of china ово истраживање детаљно анализира лигноцелулолитичку активност изолата s. gausapatum f28, аутохтоне гљиве србије, на пиљевини букве суплементисане јефтиним отпадом – меласном џибром пореклом од шећерне репе. примењене су напредне технике оптимизације вишеструких одговора ради унапређења лигнинолитичке и смањења хидролитичке активности фунгалног изолата као услова за потенцијалну примену у биорафинеријским поступцима. примењеном техникама требало је да се одаберу услови култивације који би дали активности ензима манган пероксидаза и лаказа изнад 0,84 u g-1, односно 0,12 u g-1 супстрата, и активности целулаза и ксиланаза испод 1,12 u g-1, односно 1,4 u g-1 супстрата. оптимални услови којима су испуњени постављени захтеви укључују концентрацију џибре од 10 %, влажност супстрата од 53 %, температуру инкубације од 23,5 °c и ph 5,2. истраживање је показало да је додатак меласне џибре позитивно утицао на производњу ензима, али и да се оптимална концентрација џибре разликује за различиту врсту ензима (лаказа <5 %, манган пероксидаза ~12 %, целулаза ~21 %, ксиланаза ~16 %), на шта треба обратити пажњу приликом оптимизације жељеног процеса. 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(praha) 58 (2013) 641 (https://dx.doi.org/10.1007/s12223-0130253-7) 22. p. a. geethanjali, h. g. gowtham, m. jayashankar, bull. natl. res. cent. 44 (2020) 173 (https://dx.doi.org/10.1186/s42269-020-00426-5) 23. a. kumar, int. j. microbiol. 2020 (2020) e8894215 (https://dx.doi.org/https://doi.org/10.1155/2020/8894215) 24. l. m. legodi, d. la grange, e. l. j. van rensburg, i. ncube, enzyme res. 2019 (2019) e1390890 (https://dx.doi.org/https://doi.org/10.1155/2019/1390890) 25. m. galbe, o. wallberg, biotechnol. biofuels 12 (2019) 294 (https://dx.doi.org/10.1186/s13068-019-1634-1) ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 450 jović, hao and mojović 26. r. reina, h. kellner, j. hess, n. jehmlich, i. garcía-romera, e. aranda, m. hofrichter, c. liers, plos one 14 (2019) e0212769 (https://dx.doi.org/10.1371/journal.pone.0212769) 27. a. tripathi, r. c. upadhyay, s. singh, indian j. microbiol. 52 (2012) 381 (https://dx.doi.org/10.1007/s12088-011-0232-0) 28. i. eichlerová, p. baldrian, j. fungi 6 (2020) 301 (https://dx.doi.org/10.3390/jof6040301) 29. n. namnuch, a. thammasittirong, s. n.-r. thammasittirong, mycology 12 (2021) 119 (https://dx.doi.org/10.1080/21501203.2020.1806938). ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. @article{jovic2022, author = {jovi{\'{c}}, jelena and hao, jian and mojovi{\'{c}}, ljiljana}, journal = {journal of the serbian chemical society}, title = {{examination and optimization of lignocellulolytic activity of stereum gausapatum f28 on beechwood sawdust supplemented with molasses stillage}}, year = {2022}, issn = {1820-7421}, month = {apr}, number = {4}, pages = {437--450}, volume = {87}, abstract = {this study provides a detailed analysis of the lignocellulolytic act­ivity of a new isolate stereum gausapatum f28, a serbian autochthonous fungi, on beechwood sawdust supplemented with cheap waste, sugar beet molasses still­age. advanced multiple response optimization techniques were applied to imp­rove ligninolytic and reduce hydrolytic activity as a requirement for poten­tial biorefinery use. the applied techniques were supposed to select cultivation conditions that would give manganese peroxidase and laccase activities above 0.84 and 0.12 u g-1 substrate, respectively, and cellulase and xylanase activities below 1.12 and 1.4 u g-1 substrate. the optimal cultivation conditions that met the set requirements included molasses stillage concentration of 10 %, substrate moisture content of 53 %, incubation temperature of 23.5 °c, and ph 5.2. the research showed that the addition of molasses stillage had a positive effect on enzyme production and that the optimal stillage concentration differed depend­ing on the enzyme type (for laccase it was <5 %, manganese peroxidase ≈12 %, cellulase ≈21 % and xylanase ≈16 %), which should be taken into consider­ation when optimizing the desired process.}, doi = {10.2298/jsc200601075j}, file = {::;:02_10808_5532.pdf:pdf}, keywords = {biomass, enzymes, fungi, genetic algorithm}, publisher = {serbian chemical society}, url = {https://www.shd-pub.org.rs/index.php/jscs/article/view/10808}, } effect of an industrial chemical waste on the uptake j. serb. chem. soc. 87 (0) 1–14 (2022) original scientific paper jscs–11596 published november 23 2022 1 electrochemical properties of carbonized bentonite nataša p. jović-jovičić1, danica v. bajuk bogdanović2#, tatjana b. novaković1, predrag t. banković1, aleksandra d. milutinović-nikolić1# and zorica d. mojović1* 1university of belgrade – institute of chemistry, technology and metallurgy, department of catalysis and chemical engineering, njegoševa 12, 11000 belgrade, serbia and 2faculty of physical chemistry, university of belgrade, studentski trg 12–16, 11000 belgrade, serbia (received 27 january, revised 22 march, accepted 26 march 2022) abstract: organomodified bentonites were obtained by modification of bentonite clay from local mine bogovina, with four different alkylammonium ions in the amounts that correspond to cation exchange capacity. carbonized bentonites, obtained by pyrolyzing the organomodified bentonites in the flow of nitrogen, were characterized using xrd, low-temperature n2 physisorption and raman spectroscopy. structural and textural properties of carbonized bentonites depended on the arrangement of alkylammonium cations in the paternal organomodified bentonite, while the raman spectroscopy confirmed the presence of amorphous carbon. the obtained carbonized bentonites were used for modification of the carbon paste electrode. the modified electrodes were investigated using cyclic voltammetry and electrochemical impedance spectroscopy. the electrosorption of chloride and sulfate anions on carbonized bentonites was studied by chronocoulometry. the results were interpreted in the terms of surface groups and textural properties of the carbonized bentonites. keywords: fe(cn)6 3-/4-; electrosorption; chlorides; sulfates. introduction clays are materials with a wide range of application, present in plentiful deposits on or near the surface of the earth. the smectite type of clays, such as bentonite, is of particular interest. because of the properties such as high cation exchange capacity, surface area, and hydration characteristics, smectites can easily undergo a vast variety of modifications.1 clay-modified electrodes have been investigated for a couple of decades to alter the electrode surface, consequently facilitating the reactions that occur on that surface.2–5 differently treated clays were used for electrode modification. * corresponding author. e-mail: zorica.mojovic@ihtm.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc220127030j mailto:zorica.mojovic@ihtm.bg.ac.rs https://doi.org/10.2298/jsc220127030j 2 jović-jovičić et al. the most used are acid activation,6 pillaring7 and modification by organic compounds.8 each of these modifications brought new properties to the composite material broadening the range of their applications (as electrochemical sensors for a variety of analytes,9 electrodes for energy storage applications, ion-selective electrodes). among these applications, the electrochemical sensors for pharmaceutical10 and food quality11 can be singled out as the most advanced ones. further improvement of clay electrodes has been attempted through the synthesis of carbon-clay composites.12–14 the carbon-clay composites combined a relatively high specific surface area of the clay with a good electrical conductivity of carbon and can open new possible applications of these hybrid materials. carbon electrodes are used for capacitive deionization of water, among other applications. this area of investigation acquired great attention in the last years due to worldwide problems regarding the lack of clean freshwater.15 this work aims to establish the potential usability of carbonized bentonites for capacitive deionization. in the previous publication of this group of authors16 carbon-clay composites were obtained by carbonizing clays modified with different amounts of hexadecyltrimethylammonium (hdtma) ion. the performance of carbonized samples and parenting organomodified samples was compared. the results showed that the amount of present organic precursor had a significant influence on the electrochemical properties of the resulting carbonized clay. it was concluded that the electroactivity of carbonized clay-based electrodes depended on the porosity, ion-exchange capacity, and carbon-clay interface contact. one of the important parameters for efficient electrosorption is the behaviour of the electrode/electrolyte interface. the pore size distribution, rather than specific surface area, is one of the main factors influencing the formation of an electric double layer, and therefore the efficiency of electrosorption.17 in this work the influence of different organic precursors on the electrochemical properties of resulting carbonized bentonite was investigated. bentonite clay from a local deposit (bogovina, serbia) was modified with four different alkylammonium anions. the response of the obtained carbonized bentonites was tested toward negatively charged redox probe as well as for the electrosorption of chloride and sulfate ions. experimental bentonite (clay from bogovina, coal and bentonite mine, serbia) fraction with a particle size of up to 74 µm, enriched with sodium using the ion-exchange procedure, was employed for modification with different organic cations. the procedure for organo-bentonite preparation was adopted according to baskaralingam.18 four alkylammonium ions (hexadecyltrimethylammonium (hdtma), dodecyltrimethylammonium (ddtma), benzyltrimethylammonium (btma) and trimethylammonium (tma), all acquired from sigma–aldrich) were introduced in the amounts which correspond to the value of cation exchange capacity (cec) of the clay. the cec value was previously estimated by ammonium acetate procedure (epa, method 9080)19 to be 63.3 mmol of monovalent cation per 100 g of dry clay.20 the obtained organomodified bentonites were heated at the rate of 5 °c min-1 until 400 °c was electrochemical properties of carbonized bentonite 3 reached. the temperature was held at 400 °c for 30 min. subsequently, the samples were let to cool down to room temperature. the cycle of heating and cooling was performed in the stream of nitrogen. the samples were designated as c-hdtma-b, c-ddtma-b, c-btma-b and c-tma-b, per the applied parental material. the xrd analysis was performed using a rigaku smart lab automatic multipurpose x-ray diffractometer (equipped with low background si sample holder support; 1d d/tex 250 ultra detector in xrf mode) and cu anode (λ = 0.1542 nm). the diffractograms of the samples were obtained in the 2θ range from 2 to 45°, with the scanning rate of 0.3° min−1, and scanning step of 0.01°. textural properties were assessed from nitrogen adsorption-desorption isotherms recorded at −196 °c and relative pressure in the range 0.05 < p/p0 < 0.98 using a sorptomatic 1990, thermo finnigan. samples were degassed at 150 °c for 10 h under vacuum. the specific surface area of the samples was calculated according to the brunauer, emmett and teller (bet) method from the linear part of the nitrogen adsorption isotherms.21–23 the pore size distribution has been computed from the desorption branch of the isotherms using barrett, joyner and halenda (bjh) method.21 the raman spectra were recorded using a thermo dxr raman microscope and laser excitation at the wavelength of 532 nm. measurements were performed using the constant laser power of 9 mw, exposure time of 20 s, repetition of 10, grating with 900 lines mm-1, and spectrograph aperture of 50 μm pinhole. the thermo scientific omnic 9 software was used for the spectra acquisition and analysis. after the acquisition, the fluorescence background was subtracted from the raman spectra using the fifth-order polynomial fit. the electrochemical investigation of the samples was performed using modified a carbon paste electrode as the working electrode. carbon pastes with the carbonized bentonite samples as modifiers were prepared manually by mixing 500 mg of a sample, 25 mg of carbon black (cb, vulcan-xc 72r), and 300 mg of paraffin oil. the paste was packed into a hollow (2 mm diameter) teflon tube while the electrical contact was provided using a copper wire. the reference electrode was ag/agcl in 3 m kcl, while a platinum rod served as the counter electrode. the electrochemical measurements were performed using an autolab electrochemical workstation (autolab pgstat302n, metrohm-autolab bv, netherlands). impedance measurements were performed in 1 mm k4[fe(cn)6] in 0.2 m kcl, at open circuit potential using a 5 mv rms sinusoidal modulation in the 100 khz–0.1 hz frequency range. cyclic voltammetry measurements were performed in the same electrolyte, at the scan rate of 50 mv s-1. the electrosorption properties of carbonized clays were tested in 0.2 m nacl and 0.2 m na2so4 solutions using chronocoulometry. chronocoulometric curves were obtained by applying a potential of 0.5 v vs. ag/agcl for 20 s and recording the transient current. results and discussion xrd analysis the results of the xrd analysis for the carbonized samples are presented in fig. 1. the crystalline phases expected for bentonite were observed: smectite, quartz, feldspar and calcite.24 it is well known that the incorporation of quaternary ammonium cations (qac) into interlamellar space results in the increase of d001 values of smectite. 1 the previous studies of this group have shown that the incorporation of qac in the different amounts corresponding to the cation exchange 4 jović-jovičić et al. capacity (cec) of smectite leads to the formation of different molecular arrangements, depending on the structure and qac alkyl chain length.16,25–27 short-chain qac dominantly forms monolayers with characteristic d001  1.4 nm, while longer chain qac make bilayers structure (d001  1.8 nm) with the alkyl chain axes parallel to the silicate layers.1 pseudo-trimolecular arrangement structures of kinked alkyl chains are observed with highly charged smectites and/or long surfactant cations and exhibit d001  2.2 nm. 1 the specific arrangements of organic cations in samples with the amount of qac equal to cec are summarized in table i. fig. 1. x-ray diffractograms of carbonized bentonites (sm – smectite, q – quartz, f – feldspar and c – calcite). table i. basal spacing values previously obtained for the samples with qac incorporated in the amount equal to cec sample d001 / nm qac arrangement ref. hdtma-b 2.00 pseudo-trimolecular layer 13 ddtma-b 1.78 bimolecular layer 22 btma-b 1.46 monomolecular layer 23 tma-b 1.40 monomolecular layer 24 c-hdtma-b, obtained by the carbonization of 1.0 hdtma-b, showed d001 = 1.40 nm which is in agreement with the previous findings. 28 taken into account that the elemental silicate sheet of bogovina smectite is approx. 1.01 nm28 the interlamellar distance of 0.40 nm is almost the same as reported by ruiz-garcía et al.29 for sucrose carbonated smectite. the value of 0.40 nm is in good agreement with the formation of a carbon monolayer between smectite lamellae.29,30 according to literature data, the type electrochemical properties of carbonized bentonite 5 of organic precursor and clay mineral had a strong impact on the formation of carbon structure and thickness.29–31 the c-ddtma-b besides the presence of d001 of 1.40 nm also showed the reflection that corresponded to collapsed structure with the characteristic basal spacing of 1.0 nm.1 only the collapsed structure was detected for c-tma-b and c-btma-b samples derived from the short -chain and the aromatic qac precursors, respectively. based on the obtained results, it seems that the samples with lower carbon content, either due to the smaller number of carbon atoms present in qac precursor or lower loading of qac,16 led to the formation of the collapsed structure during the applied carbonization procedure at elevated temperature. textural properties nitrogen adsorption/desorption isotherms for carbonized bentonites (fig. 2a) exhibited similar profiles. fig. 2. a) nitrogen adsorption/desorption isotherms recorded for carbonized bentonites; b) corresponding pore size distributions obtained by the barrett–joyner–halenda (bjh) method. all investigated samples were of type iib, according to the iupac classification, with the h3 type hysteresis loop.21 the h3 loop is characteristic of powders and aggregates of plate-shaped particles, clays, and pigments. hysteresis loops at higher relative pressures are the result of capillary condensation. narrower hysteresis loops, such as those obtained for c-ddtma-b and c-btma-b samples, usually indicate irregularly shaped aggregates. the specific surface area and total pore volume (table ii) of the carbonized bentonites depended on the organic precursor used for the synthesis of organobentonites. the lowest value was obtained for c-hdtma-b and the highest for 6 jović-jovičić et al. c-tma-b reflecting the amount of carbon generated in the pores of bentonite upon the pyrolysis of the organic precursor. table ii. textural properties calculated from n2 adsorption-desorption isotherms; sbet is specific surface area; v0.98 is total pore volume estimated from the amount of nitrogen adsorbed at the relative pressure of 0.98; dmax is the maximum pore diameter sample sbet / m 2 g-1 v0.98 / cm 3 g-1 dmax1 / nm dmax2 / nm dmax3 / nm c-hdtma-b 27 0.093 4.0 7.0 – c-ddtma-b 47 0.130 3.9 8.7 20.7 c-btma-b 34 0.102 4.1 6.7 – c-tma-b 89 0.127 4.0 6.1 – the pore size distribution in the mesoporous region obtained by the bjh method (fig. 2b) exhibits two peaks in the mesoporous region. the first peak had maxima at about 4.0 nm and is characteristic of clays rich in smectite.32 the second peak had the maxima within the range from 6.1–8.7 nm and can be considered as the indicator of precursor influence. the third maximum at 20.7 appeared only for the c-ddtma-b sample. raman spectroscopy raman spectra (fig. 3) were recorded in the raman shift range from 2200 to 800 cm–1. fig. 3. raman spectra of c-hdtma-b, c-ddtma -b, c-btma-b and c-tma-b in the range between 2200 and 800 cm-1. the raman spectra of the investigated carbonized clays were similar. the typical d and g bands can be observed at 1580 and 1380 cm−1 representing electrochemical properties of carbonized bentonite 7 the graphitized and disordered sp2 structure. the g band corresponds to the in -plane, doubly degenerated e2g phonon at the graphene brillouin zone centre. the d peak is linked to the breathing modes of the sp2 rings and requires a defect for its activation.33 in addition, the band at 1180 cm–1 observed in all spectra could be attributed to the contribution of sp3 carbon vibrations.34 the appearance of the d peak is correlated to the presence of aromatic rings or the formation of clusters in the amorphous carbon.35 the noticeable difference of the d peak in the c-hdtma-b and c-ddtma-b spectra are the result of different arrangements of the organic precursor in the smectite interlamellar space. hdtma was arranged in a pseudo-trimolecular layer enabling the formation of more clusters. according to ferrari and robertson,35 unlike in graphite, the development of the d peak in amorphous carbons indicates ordering. the same peak in the c-btma -b spectrum arose because of the presence of an aromatic ring. electrochemical properties of carbonized bentonite the electrochemical properties of the carbonized bentonite were first tested using cyclic voltammetry in 1 mm potassium hexacyanoferrate (ii) in 0.2 m kcl (fig. 4a). all recorded cyclic voltammograms (cvs) showed the characteristic pair of peaks corresponding to the oxidation and the reduction of [fe(cn)6] 3−/4−. the response of anionic probe on cationic clay greatly depends on the modification of the clay. smectite sheets have a permanent negative charge introduced by isomorphic substitution. it is expected that these charges would repel the anionic redox probe. however, the cationic sites present at the edges of clay particles36 and/or introduced by clay modification provide the sites for interaction with the anionic probe. cvs recorded using c-hdtma-b and c-ddtma-b containing electrodes were similar, as expected, because of the similarity of molecules of the used organic precursors. the peak-to-peak separation was 0.43 v, ia/ic ratio was 1.05 and half-wave potential was 0.31 v. cv of c-btma-b electrode exhibited lower peak-to-peak separation of 0.23 v. the ia/ic ratio was 0.9 and the half-wave potential was 0.26 v. c-tma-b electrode showed the highest current, although the peak corresponding to the oxidation of [fe(cn)6] 4– was not well resolved. the estimated peak-to-peak separation and the half-wave potentials were 0.30 and 0.26 v, respectively. the shift of the half-wave potentials and peak-to-peak separation toward lower values for c-btma-b and c-tma-b showed that [fe(cn)6] 4– was more easily oxidized at these electrodes with faster electron transfer. the results of the cv of c-btma-b the ia/ic ratio to be below unity indicating better retention of the formed [fe(cn)6] 3– at the electrode surface. the presence of [fe(cn)6] 3– on the electrode surface additionally contributed to the repulsion, thus increasing the charge transfer resistance. the response of the carbon paste electrode also depends on the source of carbon. in our previous publication,37 we have investigated the influence of gra8 jović-jovičić et al. phite and carbon black on the response of carbon clay-based electrodes. better results were obtained with carbon black, although the graphite-based electrodes showed improvement in comparison to the electrodes based only on carbon clay, without the addition of an external carbon source. fig. 4. carbon paste electrodes modified with different clay samples: a) cyclic voltammograms recorded in 1 mm k4[fe(cn)6] + 0.2 m kcl at a scan rate of 50 mv s -1; b) nyquist plot recorded in the same solution in ocp; c) bode plot recorded in the same solution in ocp; d) the equivalent electric circuit used to fit eis data. further investigation was performed using the electrochemical impedance spectroscopy (eis) in the same solution. the nyquist plots obtained for the c -hdtma-b and c-ddtma-b electrodes (fig. 4b) were similar to each other and consisted of two depressed semi-circles. the accompanying bode plots (fig. 4c) showed two relaxation time constants. hence, these spectra were fitted with an equivalent circuit consisting of the series combination of an internal resistance rs, a parallel rct1–cpe1 and a parallel rct2–cpe2 (equivalent circuit a). the electrochemical properties of carbonized bentonite 9 first rct–cpe pair corresponds to the charge transfer resistance and the capacitance accompanied with the charge transfer resistance. the second rct–cpe pair describes the second semi-circle at lower frequencies and corresponds to the ionic resistance in the electrode and the diffusion impedance. the nyquist plots obtained for the c-btma-b electrode and the c-tma-b electrode consisted of a semi-circle and the linear part. the accompanying bode plots (fig. 4c) exhibited one relaxation time constant. hence, these spectra were fitted with a modified randles circuit (equivalent circuit b) consisting of internal resistance (rs) in a series with a parallel combination of a double-layer capacitance represented with a constant phase element (cpe2) and an impedance of a faradaic reaction (serial combination of charge transfer resistance, rct, and a constant phase element (cpe1)). the constant phase element (cpe) is often used to replace the double -layer capacitance (cdl) and the warburg impedance in the original randles circuit. the capacitance element cpe will become the pure capacitance, pure resistance, and warburg impedance when n = 1, n = 0 and n = 0.5, respectively. the resistor (r), capacitor (c) and warburg impedance element (w) can, therefore, be considered to be a special case of cpe.38 the results obtained using fitting with these circuits are presented in table iii. the highest value of charge transfer resistance was obtained for c-btma-b. table iii. the electrochemical parameters obtained by fitting the eis data recorded using the electrodes with the cb/clay ratio of 1:20 in 1 mm k4[fe(cn)6] in 0.2 m kcl electrode rs / kω rct1 / kω q1 a / µs sn1 n1 rct1 / kω q2 /µs s n2 n2 c-hdtma-b 3.8 7.8 0.9 0.7 388 5.7 0.4 c-ddtma-b 5.3 21.5 0.6 0.7 345 4.3 0.4 c-btma-b 7.0 105.0 25.7 0.6 4.1 – 0.5 c-tma-b 9.8 12.5 29.3 0.5 9.1 – 0.4 athe impedance of constant phase element: z = 1/y = 1/(iω)nq; where y is admittance, i is the imaginary unit, ω is the angular frequency, n is the exponent associated with the system inhomogeneity and q has the numerical value of the admittance 1/|z| at ω = 1 rad/s the behaviour of the redox probe [fe(cn)6] 3–/4– is influenced by the electronic properties, microstructure, and surface properties of the electrode surface.39 the investigated composites contained bentonite, and its negative surface charge repulses the negatively charged redox probe.8 however, the positively charged ph-dependent sites enable the interaction of the clay-modified electrodes with the negatively charged redox probe. at the ph of the used electrolyte (ph 5.5), >aloh and >sioh groups are present at the clay surface because of the deprotonation of >aloh2 + and >sioh2 + at lower ph values.40 the pzc measurement16 revealed that the contribution of a variable charge was less pronounced at carbonized clays, especially for those with higher carbon content. on the other hand, the carbonized ben10 jović-jovičić et al. tonites showed to have a different distribution of surface groups in comparison with that of the starting bentonite. besides that, they contain additional surface groups, such as >c=o, >cooh, >c-oh, >nh2, 41,42 and some of them (>cooh groups) are shown to have an inhibitory effect on the electrochemistry of fe(cn)6 3–/4–.43 the intercept of the semicircle with the real axis at higher frequencies represents the internal resistance, rs. the internal resistance mainly represents the resistance of the bulk electrolyte. the value of rs increased in the following order: c-hdtma-b < c-ddtma-b < c-btma-b < c-tma-b. the observed shift of the rs to higher values can be ascribed to the increase of the ionic resistance of the electrode surface,44 i.e., the electrode layer adjacent to the bulk electrolyte. the increase of the thickness of this layer, i.e., the increase of the depth of the electrolyte penetration into the electrode,45 led to the increase of rs. the electrosorption properties of investigated carbonized bentonites were tested from 0.2 m nacl and 0.2 m na2so4. the anodic polarization was applied to test the sorption of anions and the obtained chroncoulometric curves were normalized for the sake of comparison (fig. 5). fig. 5. normalized chronocoulometric curves of carbonized bentonites in: a) 0.2 m nacl and b) 0.2 m na2so4. the time constant ( = rc) can be obtained by applying:46 0 ln 1 q t rcq    − − =     (1) where q0 is the electrode’s charge at the equilibrium, t is the time of the pulse, r is the resistance and c is the electrode's capacitance. the time constant, obtained as a slope of the linear part of the plot, is presented in table iv. the electrosorption depends on the average pore dimensions,47 the ionic charge and the hydrated radius.48 the electrosorption was hindered by the pre electrochemical properties of carbonized bentonite 11 sence of smaller pores resulting in the higher values of time constants. the values of the obtained time constants correlate with the values of the second maxima obtained from the n2 adsorption-desorption isotherms: the higher the constant the smaller pore diameter. the shorter time constants were obtained for chlorides than for sulfates. the slower kinetics of electrosorption recorded for the sulfate ions is the consequence of their higher charge that caused stronger electrostatic interactions. table iv. time constants (s) of the carbonized bentonite electrodes calculated from the chronocoulometric curves recorded in 0.2 m nacl solution and 0.2 m na2so4 solution electrode solution nacl na2so4 c-hdtma-b 9.80 10.39 c-ddtma-b 8.73 9.11 c-btma-b 10.29 11.33 c-tma-b 10.75 11.40 conclusion organomodified bentonite composite, prepared by the use of different alkylammonium salts (hdtma, ddtma, btma and tma), was pyrolyzed and carbon-bentonite hybrid composites were obtained. the carbonized bentonites were characterized using the xrd, low-temperature n2 physisorption and raman spectroscopy. the structure with the defined d001 basal spacing was found only for hdtma-derived sample, while the samples derived from organoclays with shorter chains showed the presence of collapsed structures. extreme values of the specific surface area and pore volume were obtained for the carbonized clay originating from the bentonite modified with the surfactant with the highest and lowest numbers of carbon atoms. the raman spectroscopy confirmed the formation of amorphous carbon in the carbonized bentonites. electrochemical impedance spectroscopy and cyclic voltammetry showed that the carbonized bentonite sample derived from tma-clay exhibited the highest electrochemical activity toward the fe(cn)6 3-/4redox couple. the internal resistance of the samples increased with the decrease of the chain length of the used organic precursor. the chronocoulometry was used to study the electrosorption properties of carbonized bentonites toward chloride and sulphate ions. the electrosorption was hindered at the samples with smaller pores. the type of organic precursor influenced the pore size of the carbonized bentonite and their electrosorption properties. acknowledgment. this work was financially supported by the ministry of education, science and technological development of the republic of serbia (grant no. 451-03-9/2021 -14/200026). 12 jović-jovičić et al. и з в о д електрохемијска својства карбонизованих бентонита наташа п. јовић-јовичић1, даница в. бајук богдановић2, татјана б. новаковић1, предраг т. банковић1, александра д. милутиновић-николић1 и зорица д. мојовић1 1универзитет у београду институт за хемију, технологију и металургију, центар за катализу и хемијско инжењерство, његошева 12, 11 000 београд и 2факултет за физичку хемију, универзитет у београду, студентски трг 12–16, 11 000 београд органомодификовани бентонити су добијени модификацијом бентонита из локалног рудника боговина са са четири различита алкиламонијум јона у количини која је једнака његовом капацитету јонске измене. карбонизовани бентонити, добијени пиролизом органомодификованих бентонита у струји азота, су окарактерисани помоћу дифракције х-зрачења (xrd), нискотемпературском физисорпцијом азота и раманском спектроскопијом. структурна и текстурална својства карбонизованих бентонита зависe од уређења алкиламонијум катјона у одговарајућем полазном органомодификованом бентониту. резутлати карактеризације су показали да особине узорака зависе од распореда алкиламонијум катјона у органомодификованом бентониту од ког су настали, док је раманска спектроскопија потврдила присуство аморфног угљеника. добијени карбонизовани бентонити су коришћени за модификацију електроде од пасте угљеника. модификоване електроде су испитане помоћу цикличне волтаметрије и електрохемијске импедансне спектроскопије. електросорпција хлорида и сулфата је проучавана помоћу хронокулометрије. резултати су протумачени на основу присутних површинских група и текстуралних својстава карбонизованих бентонита. 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(9) 969–981 (2022) original scientific paper jscs–5571 published 1 august 2022 969 apoptosis induction in hela cervical cancer cell line by steroidal 16,17-seco-16,17a-dinitriles lidija d. aleksić1, andrea r. nikolić2#, vesna v. kojić1, marija n. sakač2 and suzana s. jovanović-šanta2*# 1oncology institute of vojvodina, faculty of medicine, university of novi sad, put doktora goldmana 4, 21204 sremska kamenica, serbia and 2university of novi sad, faculty of sciences, department of chemistry, biochemistry and environmental protection, trg dositeja obradovica 3, novi sad, serbia (received 23 july 2021, revised 3 april, accepted 18 april 2022) abstract: steroids are good candidates for drug development, thanks to their low general toxicity and possibility for structure modifications connected with change of their activity. several 16,17-secoandrost-4-ene-16,17a-dinitrile compounds were synthesized and screened for anticancer effect previously, including 6-oxo and 6-hydroxyimino compounds. this research is continued with the attempts for different synthetic strategy and evaluation of anticancer effect mechanism. synthesis of 3-hydroxyimino compounds was successful, but inseparable mix of isomers was excluded from biological tests. tested secodinitriles expressed cytotoxic effect on hela cervix cancer cells as a model system, with submicromolar to molar ic50 values, where 6-substituted derivatives were more effective. after 72 h treatment with equitoxic concentrations equal ic50 values of test compounds the mechanism of this effect was studied using flow cytometry and specific fluorescent dyes. modest change in both g0/g1 and g2/m resting phases and change in mitochondrial membrane potential were noticed, while the most pronounced effect was apoptosis induction. total apoptosis was in range 50.72–58.31 % in all cell samples treated with secodinitriles, compared to 7.44 % in control samples. total percent of dead cells, including both apoptotic and necrotic, ranged from 55.24 to 65.34 %, compared to 10.68 % in control. selectivity towards cancer cells is very important feature of these compounds indicating their potential use as lead compounds in the drug development for the treatment of cancers of steroid hormone-dependent tissues. keywords: secosteroids; anticancer effect; cell cycle; mitochondrial membrane potential; proapoptotic effect; flow cytometry. * corresponding author. e-mail: suzana.jovanovic-santa@dh.uns.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc210723035a ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 970 aleksić et al. introduction cancer incidence and mortality are rapidly growing worldwide. cervical cancer, with an estimated 570,000 cases and 311,000 deaths in 2018 worldwide, ranks as fourth most frequently diagnosed cancer, fourth leading cause of cancer death in women and second in incidence and mortality, behind breast cancer. it is the most commonly diagnosed cancer in 28 countries and the leading cause of cancer death in 42 countries.1 such scary data are very stimulating for many researchers to participate actively in the discovery of new, more active anticancer drugs. in the last period, increasing advances in cancer research have enhanced the understanding of cancer biology and mechanism, so it is established that anticancer effects of cytotoxic drugs or compounds under test can be exhibited through selective cytotoxicity, anti-proliferative action, induction of apoptosis or other changes in the cancer cells, which would lead to cancer cells death.2 the design and development of effective anticancer drugs with low or no side effects are the main topic of research in many scientific groups worldwide.3 steroids are among the substances of interest, thanks to their huge structural diversity and, accordingly, huge ability of modified steroids to interact with various biological targets, as well as to take roles in signalling pathways. many synthetic steroids are pharmacologically active compounds. they can be used either for hormonal therapy, one of the main approaches for the medical treatment of hormone-dependent cancers, including inhibition of specific steroid-converting enzymes or steroid hormone receptors signaling,4 or for treating of cancer thanks to their cytotoxic effects through non-hormonal targets or pathways. there are many steroids, natural5 or synthetic, expressing antiproliferative effect against cancer cells.6 6-hydroxyimino cholestane compounds, isolated from marine sponges, expressed cytotoxicity against several types of cancer cells.7 based on that, many 6or 3-hydroxyiminosteroid derivatives, mostly cholestane-based, were synthesized as analogs of natural steroids, where some of them expressed biomedical potential, including cytotoxic or antiviral activity.8–11 based on these facts, as well as on our previous results, here we present an attempt to synthesize 3-hydroxyimino and 6-hydroxyimino-d-secoandrostane derivatives through modified synthetic steps. besides, the present study aimed to investigate the mechanism of anticancer effect of a group of steroidal 16,17-seco -dinitrile against hela human cervical cancer cell line, as a model system for steroid hormone-dependent cancer of reproductive tissue. the proportion of the apoptotic and necrotic cells, cell cycle distribution and change in mitochondrial membrane potential (δψm) of hela cells after treatment with selected secosteroids, recognized previously to express antiproliferative effect,12,13 was evaluated using flow cytometry (fc), powerful technique in drug discovery and medicine. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ apoptotic effect of steroidal secodinitriles 971 experimental general melting points are reported as uncorrected (electrothermal apparatus 9100). infrared (ir) spectra were recorded on a spectrometer (nexus 670), with wave numbers in cm-1. nuclear magnetic resonance (nmr) spectra were obtained using a spectrometer (bruker ac 250e) operating at 250 (1h) and 62.5 mhz (13c) and are reported in ppm (δ-scale) downfield from the tetramethylsilane internal standard, with coupling constants (j) given in hz. high resolution mass spectra were recorded using time-of-flight liquid chromatography–mass spectrometry (6210; agilent technologies), operated in positive electrospray ionization mode. experimental conditions, yields and spectral characterisation of novel compounds are given in supplementary material to this paper. test compounds. steroidal 16,17-seco-16,17a-dinitriles (5–8), for which antiproliferative effect and/or potential for induction of apoptosis of hela cells were noticed after 48 h treatment, were synthesized as previously described,12,13 or via alternative pathways, and purified to analytical grade. reference compounds used in study – exemestane (exe), cisplatin (cis) and doxorubicin (dox) – were dissolved in dmso and diluted with pbs to prepare working solutions. cell-based experiments cell lines and treatment. antiprolifertive activity of the steroidal compounds was tested against cervix adenocarcinoma hela cell line (atcc ccl2), as well as against normal fetal lung fibroblasts, mrc-5 cell line (atcc ccl 171), used for testing selectivity of cytotoxicity. cells were grown in dulbecco’s modified eagle’s medium (dmem), supplemented with 4.5 % glucose, 10 % of fetal calf serum (sigma-aldrich) and antibiotic/antimycotic solution (sigma–aldrich). cell lines were cultured in flasks (costar, 25 cm2) or 6-well plates at 37 °c in high humidity and 5 % co2 atmosphere. only viable cells were used in the assay, while viability was measured by det with trypan blue.14 antiproliferative assay. antiproliferative activity of selected modified steroids was evaluated by mtt assay,15,16 where number of viable cells was measured after 72 h exposure to test compounds in concentration range from 0.01 to 100 µm (0.01; 0.1; 1; 10 and 100 µm). reference compounds used in this assay were cisplatin (cis) and doxorubicin (dox), as nonselective anticancer agents,17,18 and steroidal aromatase inhibitor, exemestane (exe), used to test general steroidal toxicity (fig. s-1, supplementary material).19 cells were seeded into 96 -well plates at a density of 5000 cells/well, stood overnight in complete medium at 37 oc, after which the solutions containing the test compounds were added (10 µl/well) in all wells except controls. after incubation during 72 h, 10 µl of mtt solution (5 mg/ml) and acidified 2-propanol were added to each well. absorbance was read after few minutes incubation at room temperature on a spectrophotometric plate reader (multiscan mcc340, labsystems) at 540/690 nm. wells without cells containing complete medium only were used as a blank. flow cytometry analysis. hela cells were seeded into 6-well plates at a densities from 3×105 to 4×105 cells/well. treatment of hela cells for all flow cytometry analyses (cell cycle analysis, detection of apoptosis and necrosis and quantification of mitochondrial membrane potential change) was performed with equitoxic concentration of test compounds, corresponding ic50 values of each compound after 72 h treatment.20 for each analysis treated cells were stained with specific dye (or dyes) and prepared for the analysis in specific manner. flow cytometry was performed using facscalibur becton dickinson (bd) bioscience immunocytometry system, while results were analysed using cellquest software;21 in each analysis ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 972 aleksić et al. 10000 (apoptosis detection) or 20000 (cell cycle and mmp change) events were recorded from samples containing 106 of cells, and percentage of cells in each population was determined. cell cycle analysis. distribution of cells in various phases of cell cycle was analysed by measuring cellular dna content after 72 h exposure to modified steroids, when cells were harvested with trypsin and centrifuged at 1500 rpm for 10 min, then washed twice with cold phosphate-buffered saline (pbs). cells were fixed with 70 % ethanol for 1 h at 20 °c, washed with pbs, re-suspended in a solution of pi (50 μl/ml, invitrogen) and rnase a (250 μg/ml, thermo fisher scientific) at a density of 106 cells /ml and incubated in the dark for 30 min at room temperature.22 such ethanol-fixed cells were analysed by flow cytometry. after recording 20000 events, the content of the cells in the different cell cycle phases (subg1, g0/g1, s and g2/m) were determined by using cellquest software.21 the subg1 fraction was regarded as the apoptotic cell population. detection of apoptosis and necrosis. apoptosis induction of hela cells was measured by flow cytometry, using annexin v-fluorescein isothiocyanate propidium iodide (fitc/pi; bd pharmingen) apoptosis detection assay. in brief, hela cells were exposed to test substances during 72 h at 37 °c, then harvested, washed twice with ice-cold pbs and re-suspended in annexin v binding buffer at a density of 106 cells /ml. subsequently, 100 μl of cell suspension was transferred into a flow cytometry tube, and 5 μl of annexin v-fitc and 10 μl of pi were added to the cell suspension. the sample was gently mixed by vortex. the stained samples were incubated for 15 min at room temperature in the dark. an additional 400 μl annexin v binding buffer was added to each tube. samples with 106 of cells were analysed within 1 h on flow cytometer. the percentages of the cells in the different populations (normal, early apoptotic, late apoptotic and necrotic cells) were determined in each analysis by using cellquest software. quantification of mitochondrial transmembrane potential. mitochondrial transmembrane potential (δψm) was measured using a cationic fuorochrome rhodamine 123 (rh123; sigma– –aldrich), as described previously.23 briefly, 106 cells, resuspended in 200 μl of pbs, were stained with rh123 (2.5 μg/ml) for 30 min at 37 °c. after washing with pbs, samples were analysed by flow cytometry using cell quest software (becton dickinson, heidelberg, germany). m1 region represents cells with intact mytochondrial membrane integrity, while m2 region includes cells with membrane integrity loss. data analysis. all experiments in this study were performed twice. in the antiproliferative assay two independent experiments were conducted in quadruplicate for each concentration of test compound. antiproliferative activity was determined using the mtt assay after exposure to 0.01, 0.1, 1, 10 and 100 µm test compound for 48 or 72 h. mean values and standard deviations (sd) were calculated for each concentration, and ic50 values were determined by median effect analysis. all statistical processing were calculated in microsoft office excel program. results and discussion steroidal compounds are known for their specific physiological effects. on the other hand, modified steroids can express effects more or less different from their natural analogues, despite large similarity in their structures. design of novel compounds can be based on structural features of known physiologically or pharmacologically active compounds. in such chemistry-driven medicinal chem________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ apoptotic effect of steroidal secodinitriles 973 istry a series of steroidal 16,17-seco-16,17a-dinitriles were synthesized,12,13 possessing both steroidal core, important for expressing some specific biological effects, and nitrile functions, present in structures of some drugs and responsible for their activity.24 biomedical potential of hydroxyiminosteroids was recognized, while androstane-derived hydroxyimino compounds are among the topics in our research. based on these facts and anticancer properties of our previously synthesized androstane-derived compounds, we synthesized two novel 3-hydroxyimino16,17-secoandrost-4-en-16,17a-dinitrile compounds and attempted to apply alternative synthetic approach in the synthesis of (6e)-hydroxyimino derivative12 in order to obtain substances for biomedical tests and potentially improve synthetic procedures (scheme 1). novel compounds and those, recognized earlier for their cytotoxic effect against cervical adenocarcinoma hela cell line, were planned to be tested for their pharmacological effect and an attempt was made to find the mechanism of activity against cancerous cell lines. cn cn ro cn cn ho n oh cn cn o n oh 1 r=ac 2 r=h 6 7 a j b i cn cn o 3 cn cn n ho 4a 3e-isomer 4b 3z-isomer cn cn o 5 c d cn cn o o 8 cn cn ho o 9 k e-h l 12 3 4 5 6 7 8 910 11 12 13 14 15 16 1718 19 17a scheme 1. a) etona, etoh, 50–55 °c, 1 h; b) cro3, h2so4, h2o, acetone, 0 °c, 25 min; c) nh2oh×hcl, etoh, acona, 70 °c, 2 h; d) ddq, toluene, tfa, bstfa, reflux, 6 h; e) 1. m-cpba, ch2cl2, rt, 90 min; 2. cro3, h2o, acetone, rt, 70 min; f) socl2, pyridine, 0 °c, 90 min; g) nh2oh×hcl, pyridine, rt, 24 h; h) etona, etoh, rt, 1 h; i) cro3/py, rt, 75 min; j) cro3, h2so4, h2o, acetone, 0 °c, 25 min; k) nabh4, cocl2×6h2o, meoh, rt, 15 min; l) nh2oh×hcl, acona, etoh, rt, 20 h. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 974 aleksić et al. starting compounds in these syntheses (1 and 2) were obtained by known procedure.13 oppenauer oxidation of compound 2 afforded enone 3.13 oxyimination of compound 3 with hydroxylamine hydrochloride in ethanol in the presence of sodium-acetate afforded mixture of (3e) and (3z) isomers, namely 4a and b, in a total yield of 90 %. isomers could not be separated by crystallization or column chromatography, so the mixture of isomers was characterized. the presence of both (3e) and (3z) hydroxyimino derivatives in the obtained final product was confirmed by analysis of nmr spectra. in the 1h-nmr spectrum in dmso-d6 two clearly differentiated singlets at 10.27 and 10.54 ppm were observed, corresponding to the protons of two hydroxyl groups of hydroxyimino functions of two isomers. singlets on 5.73 and 6.36 ppm originate from h-4 atoms of two isomers. (3e) and (3z) isomers were obtained in ratio 7.5:1, where (3e) isomer 4a was identified as dominant isomer, based on noe nmr experiment. namely, by irradiation of h-4 proton (signal at 6.36 ppm), an increase of the signal intensity at 10.27 ppm was observed, suggesting distance less than 4 å between h-4 and the hydrogen of hydroxyimino group. compound 3 served also as starting compound in the synthesis of dienone 5, synthesized by known procedure.13 in order to improve total yield of hydroxyiminoenone 7, synthesized by oxidation of c-3 hydroxyl group of 6 with chromium trioxide/pyridine complex and reported previously13 (scheme 1, e–i), an attempt to synthesize its precursor hydroxyiminoenol 6, via alternative synthetic pathway, has been made (scheme 1 a, j–l). previously we synthesized compound 6 from 1 in four steps (“3-acetyloxy pathway”). rapid one-pot procedure was used for the synthesis of 5α-hydroxy-6 -oxo derivative starting from 1. after dehydration of this compound, resulting 3β-acetoxy-4-en-6-one derivative was treated with hydroxylamine hydrochloride in pyridine to obtain 3β-acetoxy-4-en-6-hydroxyimino compound. the cleavage, of the acetate from c-3 with sodium ethoxide in ethanol, has occurred giving desired hydroxyiminoalcohol 6. retro-synthetic analysis indicated that the (6e)-3β-hydroxy-hydroxyimino16,17-secoandrost-4-ene-16,17a-dinitrile (6) could be obtained by oxyimination of 3β-hydroxy-6-oxo-16,17-secoandrost-4-ene-16,17a-dinitrile (9). this key intermediate (9) we synthesized by regioselective reduction of the oxo group at position c-3 of 4-ene-3,6-dione 8. the reaction was performed with sodium borohydride in methanol, in the presence of cobalt (ii) chloride hexahydrate, where after 15 min at room temperature product was obtained in satisfactory yield (64 %). the structure of the compound 9 was confirmed based on nmr spectral data. the presence of a hydroxyl group in the structure of compound 9 was determined based on the doublet at 5.03 ppm in the proton nmr spectrum, derived from the hydrogen atom of that group, while the presence of the oxo group was confirmed by 13c-nmr spectral signal for the c-6 atom at 200.19 ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ apoptotic effect of steroidal secodinitriles 975 ppm. in the mass spectrum dominant peak corresponds to [m+h]+ signal. in a final step of the synthesis, oxyimination of compound 9 with hydroxylamine hydrochloride in ethanol at room temperature in the presence of sodium acetate, was attempted, but product 9 was not obtained even after 20 h. all synthesized compounds were tested for their antiproliferative effects against the spectrum of cancer cell lines, except (3e)and (3z)-4-ene-3-oximes 4a and b, whose mix could not be separated. namely, separation and purification of compounds are of particular importance in drug development, where interactions of drug or drug candidate with receptors in the living cells are based on the compound’s purity.25 some of tested secodinitriles previously were recognized as antiproliferative agents in in vitro experiments against cancer cell lines of reproductive tissues.12,13 since non-hormonal mechanism was proposed,13 while apoptosis was visually noticed after 48 h treatment for couple of compounds,12 more extensive research has been undertaken as an attempt to define the mechanism by which several of these compounds reduce the number of cancer cells, using hela cervix cancer cell line as a model system, which was the most sensitive cell line on these compounds. in the previous studies antiproliferative effect of the series of androstane -derived secodinitriles after 48 h treatment was tested against the palette of carcinoma cell lines. secodinitrile compounds which expressed significant cytotoxicity (with ic50 lower than 10 µm) against hela cervical adenocarcinoma cell lines (5–8) and novel compound 9 were included in this experiment, where their antiproliferative effects were measured against the same cell line after 72 h treatment. 3-hydroxyimino derivatives 4a and b were excluded from this research, because they could not be separated and thus fully purified, while purity of compounds is crucial for their binding to the biological target molecules. antiproliferative effects of the reference compounds against hela cell line were tested in the same conditions (fig. s-1). all androstane-derived and reference compounds were also tested against normal fetal lung fibroblasts cell line mrc-5. secodinitrile compounds expressed very good, submicromolar to micromolar cytotoxicity after 72 h treatment of hela cells (ic50 values 0.46–8.23 µm), similar to widely used cytotoxic drug cisplatin, though cisplatin was more toxic against healthy cells, mrc-5 cell line. opposite, secosteroids were not toxic on healthy cells (table i). exemestane, aromatase inhibitor, the drug of choice in the treatment of estrogen-dependent diseases, decreased number of hela cells only after longer treatment (72 h). the most effective in reducing number of hela cells were 16,17-seco-16,17a-dinitriles with hydroxyimino function in position 6 (6 and 7), with submicromolar ic50 values, then 6-oxo derivative 8, while secodinitrile with no substituent at c-6 (5) was less active. 16,17-seco-16,17a-dinitrile compound, possessing a/b ring moiety similar as exemestane (6-methylene2-en-3-one), was not effective in reducing numbers of hela cells, although sub________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 976 aleksić et al. stitution at position 6 seems to be important for their biological effect, so this compound was not included in the further study.13 doxorubicin, very often used cytotoxic drug, expressed high toxicity, even equal, against both cervix cancer and normal cell lines. table i. comparison of antiproliferative effect (ic50±sd / μm) of the tested androst-5-ene16,17-seco-16,17a-dinitriles and the reference compounds, exemestane (exe), cisplatin (cis) and doxorubicin (dox) obtained for 48 and 72 h of action; n/a – ic50 value was not available due to very low effect compound cell line hela mrc-5a 48 h13 72 h 48 h13 72 h 5 12.49±1.23 8.23±2.04 >100 >100 6 0.48±0.007 0.46±0.11 >100 >100 7 2.64±0.51 0.79±0.36 >100 >100 8 4.31±0.96 2.83±0.06 >100 >100 9 >100 – >100 – exeb >100 18.18±1.25 >100 n/a cisc 1.77±0.11 2.10±0.78 0.48±0.08 0.24±0.01 doxc 1.17±0.09 0.07±0.001 0.12±0.04 0.10±0.04 anon-cancerous control; bcontrol steroidal compound in clinical use against breast cancer; ccontrol antiproliferative compound starting from the cytotoxicity data the mechanism of action of compounds, that showed to be good antiproliferative agents against treated hela cervical cancer cells (5–8), was studied. exemestane was used further as the reference compound, since active secosteroids are c-6 substituted. cell cycle of hela cells was examined, after 72 h of treatment with secodinitrile compounds in equitoxic doses, equal ic50 values, where cells after treatment were stained with propidium iodide (pi). there was modest influence of secosteroids noticed in fc analysis (figs. s-2 and s-3, and table s-i of the supplementary material): decrease in cell number in g0/g1 cell cycle phase arrest (about 10 % for all steroids, and the highest for compound 7, 14.5 %) and increase of cell numbers in g2/m phase (about 10%, for all steroidal compounds, including reference exemestane), while number of cells in synthetic (s) phase was slightly higher when cells were treated with compound 7. there was no significant change in subg1 fractions, referred hypodiploid cells. induction of apoptosis and necrosis was examined next. cells treated for 72 h with secodinitrile compounds were stained with annexin v-fitc and pi in order to distinguish and count earlyand late-apoptotic, necrotic from normal cells using flow cytometry. previously visually noticed apoptosis during shorter treatment12 was now quantified after longer treatment, since cell number in early and late apoptosis was counted. namely, annexin is dye that is binding to phospholipid constituents of cell membrane, indicating plazma membrane permeabil________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ apoptotic effect of steroidal secodinitriles 977 ity, while pi is binding to dna of cells with disturbed plazma membrane (cells in late apoptosis or necrotic cells). combination of these two dyes gives information about integrity of the cells and availability of dna binding. all steroids used in the treatment of hela cells induced apoptosis in high extent, where more cells were in late apoptosis than in early stage. total apoptotic cell number ranged from 50.72 (8) to 58.31 % (5) in all samples of cells treated with secosteroids, while in case of exemestane this effect was a little bit less manifested, 42.16 %, compared to control samples with 7.44 % of total apoptosis, i.e., spontaneous apoptosis. necrosis was noticed in all samples as well, where in the highest extent was noticed in the samples with hydroxyimino compounds 6 and 7 (about 10 %). in all cases apoptosis was more common cause of cell deaths, while total percent of dead cells ranged from 55.24 (8) to 65.34 % (5), figs. 1 and s-4, and table s-2 of the supplementary material). fig. 1. induction of apoptosis of hela cells after 72 h treatment with steroidal 16,17-seco-16,17a-dinitriles and exemestane. a distinctive feature of apoptosis is the disruption of normal mitochondrial function, especially changes that affect the mitochondrial membrane potential (mmp). mitochondrial membrane potential change (δψm) is crucial for maintaining the physiological function of the respiratory chain to generate atp, necessary for cell survival. one of the earliest changes that occurs in apoptosis is a change in the transmembrane potential, so δψm is usually associated with mitochondrial or intrinsic apoptosis, although it can cause increase in inner mytochondrial membrane permeability via death receptors or protein channels activation, associated with extrinsic apoptosis.26 to test if change in mitochondrial membrane potential included in apoptosis of hela cells, after treatment with steroids cells were stained with rhodamine, which accumulates in mitochondrial matrix if permeability of inner mitochondrial membrane raised, and analysed by fc. as can be seen in fig. s-6 (based on fig. s-5 and table s-3) of the sup________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 978 aleksić et al. plementary material, there is no big change in mmp of hela cells after treatment with secodinitriles or exe. percent of influenced cells (m2 fraction of cells), i.e., cells with more permeable mytochondrial inner membrane, is quite small in all samples (about 8 % for secosteroids and 4 % for exe). apoptosis can be induced with one or more events, which can start a series of further changes, leading to apoptotic death of the cell, via intrinsic or extrinsic pathway. in some cases, these pathways can be combined or crossed. each signalling pathway consists of many components, so it is quite difficult to precise which compound is sensitive on certain treatment. for first screening, to test antiproliferative activity of compounds, viability test is used. for recognizing the mechanism underlying cytotoxic effect many methods are available, the most of them based on using fluorescent dyes, specific for tracking certain changes in the cells. the technique the most used and useful in tracking changes in the treated cells, using specific dyes, is flow cytometry. flow cytometric analysis gives answers and/or directs further experiments, in order to find a way by which compounds express their biological or pharmacological effects. based on all mentioned, namely generally accepted facts and obtained results, apoptosis induction in androstane compounds-treated hela cells is the main cause of cell death, with no significant evidence of disturbed cell cycle or change in inner mitochondrial membrane potential. still, to have a complete insight into the mechanism and target molecules in the signalling pathways causing apoptosis, other methods should be included in the study. conclusion modified steroids can express similar or different, sometimes even opposite, biological or pharmacological effects, comparing to the endogen steroids, i.e., their parent molecules. strength of such compounds can be found during extensive biological tests. accordingly, alternative or simplified routes for the synthesis of biologically effective compounds and synthetic strategies for the synthesis of novel derivatives are always needed. 16,17-seco-16,17a-dinitriles, derivatives of androst-4-ene, were synthesized and recognized to express strong antiproliferative effect against hela cervix cancer cells, with submicromolar to molar ic50 values. an attempt to synthesize these active compounds via alternative synthetic pathway is performed, as well as synthesis of novel androstane-derived compounds. apoptosis of hela cells treated with these compounds, observed in morphological study, initiated further research to discover the mechanism of cell toxicity of these compounds. apoptosis was recognized as the most important cause cell number lowering, while certain cell cycle and mitochondrial membrane potential changes were detected, with no direct correlation with apoptosis. all changes were detected and quantified by using flow cytometry and dyes, specific for certain changes in the cells. substitution in position 6 of stero________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ apoptotic effect of steroidal secodinitriles 979 idal core seems to be important for the expressing of anticancer effect: polar 6-hydroxyimino derivatives were the most cytotoxic, with submicromolar ic50 values, while 6-methylene analogue or the compounds with no substituents at c-6 were less or no active. in the cells treatment with equitoxic doses, all tested compounds induced apoptosis in high extent (more than 47 % of the cells were apoptotic), while total percent of dead cells, both apoptotic and necrotic, was in the range of 55–65 %. further experiments are needed to define target molecules in the apoptosis promoting, while novel compounds could be designed, based on the structural features of effective and selective anticancer agents, presented here. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/10993, or from the corresponding author on request. acknowledgments. the authors acknowledge financial support of the ministry of education, science and technological development of the republic of serbia (grant no. 451-03 -68/2022-14/200125) . и з в о д индукција апоптозе hela ћелијске линије стероидним 16,17-секо-16,17а-динитрилима лидија алексић1, андреа николић2, весна којић1, марија. н. сакач2 и сузана јовановић-шанта2 1 универзитет у новом саду, медицински факултет, институт за онкологију војводине, пут др голдмана 4, 21204 сремска каменица и 1 универзитет у новом саду, природно–математички факултет, департман за хемију, биохемију и заштиту животне средине, tрг доситеја обрадовића 3, 21000 нови сад стероиди су добри кандидати за развој лекова, захваљујући њиховој ниској општој токсичности и могућим структурним модификацијама, повезаним са променом њихове активности. раније је синтетисано више 16,17-секоандрост-4-ен-16,17а-динитрила, укључујући 6-оксо и 6-оксиимино деривате и прелиминарно је испитана њихова антиканцерска активност. ова истраживања су настављена покушајима различите синтетичке стратегије и евалуације механизма антиканцерског деловања. синтеза 3-хидроксиимино једињења је била успешна, али нераздвојива смеша изомера није укључена у биолошке тестове. тестирани секодинитрили су испољили цитотоксични ефекат према hela ћелијама карцинома грлића материце, одабраном за модел систем, са субмикромоларним до моларним вредностима ic50, при чему су 6-супституисани деривати били ефикаснији. после 72-часовног третмана ћелија еквитоксичним концентрацијама испитиваних једињења, једнаким ic50 вредностима испитиваних једињења, механизам овог деловања је проучаван применом проточне цитометрије и специфичних флуоресцентних боја. уочене су извесне промене у g0/g1 и g2/m фазама мировања, као и промене у мембранском потенцијалу митохондрија, док је најизраженији ефекат био индукција апоптозе. укупна апотоза била је у опсегу 50,72 до 58,31 % у свим узорцима третираним секостероидима, у поређењу са 7.44 % у контролном узорку. проценат мртвих ћелија, укључујући апоптотичне и некротичне, био је у опсегу 55,24 до 65,34 %, у поређењу са 10,68 % у контролном узорку. веома важна карактеристика ових једињења је селектив________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 980 aleksić et al. ност према ћелијама канцера, без детектованог ефекта на здраве ћелије mrc-5 линије, што их сврстава у водећа једињења у развоју лекова против канцера ткива зависних од стероидних хормона. 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________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ @article{aleksic2022, author = {aleksi{\'{c}}, lidija d and {nikoli{\'{c}} 2#}, andrea r and koji{\'{c}}, vesna v and saka{\v{c}}, marija n and jovanovi{\'{c}}-{\v{s}}anta, suzana s}, journal = {journal of the serbian chemical society}, title = {{apoptosis induction in hela cervical cancer cell line by steroidal 16,17-seco-16,17a-dinitriles}}, year = {2022}, issn = {1820-7421}, month = {aug}, number = {9}, pages = {969--981}, volume = {87}, abstract = {steroids are good candidates for drug development, thanks to their low general toxicity and possibility for structure modifications connected with change of their activity. several 16,17-secoandrost-4-ene-16,17a-dinitrile com­pounds were syn­thesized and screened for anticancer effect previously, inc­luding 6-oxo and 6-hy­droxyimino compounds. this research is continued with the attempts for dif­fer­ent synthetic strategy and evaluation of anticancer effect mechanism. syn­thesis of 3-hydroxyimino compounds was successful, but inseparable mix of isomers was excluded from biological tests. tested seco­di­nitriles expressed cyto­toxic effect on hela cervix cancer cells as a model sys­tem, with submicromolar to molar ic50 values, where 6-substituted derivatives were more effective. after 72 h treatment with equitoxic concentrations equal ic50 values of test com­pounds the mechanism of this effect was studied using flow cytometry and specific fluorescent dyes. mod­est change in both g0/g1 and g2/m resting phases and change in mito­chondrial membrane potential were noticed, while the most prono­unced effect was apoptosis induction. total apoptosis was in range 50.72–58.31 % in all cell samples treated with secodi­nit­riles, compared to 7.44 % in control samples. total percent of dead cells, including both apoptotic and necrotic, ranged from 55.24 to 65.34 %, com­pared to 10.68 % in control. selectivity towards cancer cells is very important feat­ure of these compounds indicating their potential use as lead compounds in the drug development for the treatment of cancers of steroid hormone-dependent tissues.}, doi = {10.2298/jsc210723035a}, file = {:d\:/onedrive/mendeley desktop/aleksi{'{c}} et al. 2022 apoptosis induction in hela cervical cancer cell line by steroidal 16,17-seco-16,17a-dinitriles.pdf:pdf;:01_10993_5571.pdf:pdf}, keywords = {anticancer effect, cell cycle, flow cytometry, mitochondrial membrane potential, proapoptotic effect}, publisher = {serbian chemical society}, url = {https://www.shd-pub.org.rs/index.php/jscs/article/view/10993}, } effect of an industrial chemical waste on the uptake j. serb. chem. soc. 87 (0) 1–11 (2022) original scientific paper jscs–11570 published 18 november 2022 1 remediation of chemistry teachers’ misconceptions about covalent bonding using cognitive conflict interviews: a case study syahrial syahrial1, mashfufatul ilmah1, yahmin yahmin2, munzil munzil2 and muntholib muntholib2* 1chemistry education program, postgraduate program, universitas negeri malang, indonesia and 2department of chemistry, universitas negeri malang, indonesia (received 17 january, revised and accepted 12 september 2022) abstract: research has shown that most chemistry teachers have misconceptions about covalent bonding. this study investigates whether the cognitive conflict interview technique could persuade teachers to revise their possible misconceptions of covalent bonding. eight chemistry teachers from different schools participated in this study. two validated instruments, cognitive conflict technique interview guidelines and the open-ended covalent bonding test, were employed for the data collection. the results showed that the cognitive conflict interviews could facilitate respondents to overcome their misconceptions about covalent bonding. five of the eight respondents experienced a conceptual change from misconceptions to scientific conceptions, and three others experienced a partial conceptual change. six concepts which previously caused misconceptions were eliminated and turned into a scientific concept instead. of the 46 cases of misconceptions, 41 cases turned into scientific conceptions. the result of this study serves as an initial perspective for exploring the effectiveness of cognitive conflict interviews more broadly. keywords: chemistry teacher; conceptual changes; contradicts phenomena. introduction the concepts of covalent bonding underlie other chemistry concepts1 such as molecular structure and chemical and physical properties of compounds.2 however, various studies have shown that this subject generates many misconceptions.3–6 misconception refers to an individual concept different from the concept accepted by the scientific community,7 that must be adequately addressed because it can cause learning problems.8 * corresponding author. e-mail: muntholib.fmipa@um.ac.id https://doi.org/10.2298/jsc220117073s mailto:muntholib.fmipa@um.ac.id https://doi.org/10.2298/jsc220117073s 2 syahrial et al. various studies revealed that secondary school,2 university students9 and chemistry teachers demonstrated the misconception of covalent bonding.10,11 regardless of those results, limited evidence has been found regarding how to consider these misconceptions. how teachers explain chemical bonding may lead to students' difficulties with comprehending and learning.12 therefore, teachers’ misconceptions must be corrected to avoid transferring their misconceptions to students. the effort to solve students’ misconceptions must be initiated by ensuring that teachers hold clear scientific conceptions. cognitive conflict strategies emphasize the instability of people’s beliefs in their existing conceptions through contradictory experiences. it then allows them to replace their misconception with scientifically accepted concepts.13 this teaching strategy involves demonstrating phenomena in which students’ observations contradict what they expected.14 the contradiction causes a mental imbalance that encourages students to change their conceptions to fit the new facts they observe.15 in other words, the cognitive conflict strategies facilitate the elimination of students’ misconceptions after experiencing the counter-scientific conceptions. the cognitive conflict strategies can be carried out in various ways or methods,16 including cognitive conflict interviews.17,18 the strategy of cognitive conflict interview allows researchers to ask in-depth questions to obtain broader and more profound answers.19,20 the cognitive conflict interviews can also elaborate on students’ cognitive structures18 to uncover their misconceptions.21 studies have shown that the cognitive conflict interviews effectively revised students’ misconceptions.19,22 various chemical phenomena of covalent bonding can be used to challenge the existing teachers’ misconceptions, leading them to replace those with scientifically accepted conceptions.13–15 for example, some respondents consider the electron configuration of an element in a molecule is simply the same as its configuration in a free atom. presenting a configuration of cl as a single atom and its configuration in hcl will challenge them to rethink of their initial conception. experimental this study applied a case study and followed the interpretive paradigm23 concerning the remediation of eight chemistry teachers’ misconceptions about chemical bonding. this study was intended to explain the conceptual change phenomena occurring in chemistry teachers who held misconceptions about chemical bonding.24 the uncovered misconceptions were corrected using the cognitive conflict interview technique. respondents the respondents for this research were eight chemistry teachers from several schools in banten province, indonesia. all respondents hold bachelor’s degrees in chemical education with more than three years of teaching experience and offered their consent to participate. persuading teachers to revise their misconceptions 3 instruments two instruments, given in supplementary material to this paper, cognitive conflict interview guidelines (supplement 1) and an open-ended covalent bonding test (supplement 2), were used for data collection. the cognitive conflict interview guidelines were used to correct the teachers’ misconceptions. the open-ended test on covalent bonding consisting of 18 items was used to assess the changes in the teachers’ conceptions. the content validity and reliability of the instruments were assessed based on feedback from two experienced chemistry lecturers. content validity describes how an instrument has an appropriate sample of items for the construct being measured,25 whereas the reliability of the measurement result is its consistency and reproducibility.26 the content validity was analyzed based on its content validity index (cvi), while the reliability was analyzed based on their inter-rater agreement (ira).25,27,28 the cvi was calculated by dividing the number of experts who gave a positive rating (i.e., 3 or 4) and the total number of experts. the ira was calculated by dividing the number of items with ira items greater than 0.80 and the number of total items. the acceptable cut-off value for the cvi and the ira was 0.80.27-30 however, landis and koch31 and regier et al.32 set cvi and ira with different criteria, i.e., poor if <0.00, low or unacceptable if 0.00–0.20, fair or questionable if 0.21–0.40, moderate or good if 0.41–0.60, substantial or very good if 0.61–0.80, and almost perfect or excellent if 0.81–1.00. the validity and the reliability of each secondary instrument are shown in table i. table i. the cvi and ira of secondary instruments used in this research secondary instrument cvi ira value agreement strength value agreement strength the cognitive conflict interview guide 1.0 excellent 0.75 very good open-ended test on covalent bonding 1.0 excellent 0.69 very good table i shows that all secondary instruments are feasible and can be used in data collection. in addition, this study also employed a tape recorder to record the interviews. procedure the respondents were separately interviewed without a time limit. respondents were free to use pictures or other schematic representations to express their opinions. the open -ended test on covalent bonding was carried out one week after the interview. this test was intended to identify teachers' understanding changes after interviews. a cognitive conflict interview consists of 4 steps: 1) confirming the pre-conception, 2) creating cognitive conflicts by providing evidence, anomalies, and contradictions to promote mental imbalance that encourages teachers to change their conceptions,15 3) stimulating the equilibration process using the relevant questions to help them understand the new concept and 4) confirming the scientific concept. data analysis the responses to the covalent bonding test were analyzed using descriptive analysis and categorized into six degrees of understanding. the degrees are sound understanding, partial understanding, partial understanding with misconceptions, misconceptions, lack of knowledge and no response.33 this study simplified these six degrees of understanding into three categories (table ii). 4 syahrial et al. table ii. the scoring criteria of teachers’ responses (modified from abraham et al.33) scoring criteria the degree of understanding category example from interviews the response includes all components of the valid responses sound understanding sound understanding atoms bond to achieve a stable state or lower energy levels the response includes at least one of the components of valid response, but not all components partial understanding the atoms bond together to be stable, i.e., obey the octet or duplet rule the response shows understanding and consists of a statement that demonstrates a misconception partial understanding with misconception misconception the bond polarity of hf > hcl > hbr is due to the atomic size of f < cl < br the response includes illogical or incorrect information misconception hf, hcl, and hbr have the same bond polarity the response repeats questions only or is irrelevant or unclear lack of knowledge lack of knowledge i don’t know how the formal charge relates to stability no responses or just stated “i have no idea” no response – partial understanding is an incomplete description of chemical phenomena. example: they correctly explained that atoms bond each other to achieve stability. however, they explained, “obeying the octet or duplet rule is the root of stability.” partial understanding with misconceptions is a correct understanding but contains misconceptions. example: bond polarity of hf > hcl > hbr. however, the respondents explained that the difference in the atomic size caused the difference in the bond polarity, where the atomic size of f < cl < br. the misconception implied by this statement is the difference in the bond polarity of hf > hcl > hbr is caused by the variability in the atomic size of f < cl < br only. the difference in atomic electronegativity causes the difference, f > cl > br, rather than the difference in atomic size, f < cl < br. each response was carefully read, compared, and tentatively assigned to one of the scoring criteria. the responses that were difficult to classify were separated and discussed in focus groups involving all research members until a consensus occurred. results and discussion our previous study showed that all respondents (before receiving cognitive conflict interviews) had misconceptions about the covalent bonding concepts.11 the cognitive conflict interview guideline (supplement 1) was developed based on the misconceptions identified through this preliminary research. after the intervention (cognitive conflict interview), only 3 of 8 respondents still demonstrated misconceptions in 2 concepts. the two misconceptions are: 1) the o–h bond of the water molecule resulting from the reaction of acid-base neutralization persuading teachers to revise their misconceptions 5 is a covalent bond and 2) both n–o covalent bonds in the no2 molecule are double bonds or single bonds (table iii). table iii. the two misconceptions identified after the intervention concept misconceptions respondent no. the coordinate covalent bonding the o–h bond of the h2o molecule formed by the reaction of h+ and ohare a covalent bonding because the electron pair between the h and o atoms are shared by the h and o atoms 1 3 lewis structure the lewis structure of the no2 molecule is: because both n–o covalent bonds are identical 1 the lewis structure of the no2 molecule is: because both n–o covalent bonds are identical 3 7 table iii shows that after the intervention, the respondents who experienced misconceptions were 3, including respondents no. 1, no. 3 and no. 7. in contrast, the other five respondents understood the covalent bonding concepts well. overall, the misconceptions about the covalent bonding concepts before and after the intervention are shown in table iv. table iv. the respondents’ misconceptions about the covalent bonding concepts before and after the intervention no. concept number of respondents experiencing misconceptions before intervention after intervention 1 the purpose of bond-forming atoms 8 0 2 the coordinate covalent bonding 2 2 3 types of atoms that can form covalent bonds 7 0 4 polar and nonpolar covalent bonds 4 0 5 the polarity order of covalent bonds 4 0 6 lewis structure 8 3 7 octet rule 8 0 8a the length of c–c bonds in ethane, ethene, ethane 2 0 8b the length of the covalent bonding in hydrogen halides 3 0 total cases 46 5 table iv shows that the cognitive conflict interviews: 1) decrease the number of respondents who experience misconceptions from 8 people to 3 people; 2) reduce the number of concepts in which respondents experience misconceptions from 8 to 2 concepts; 3) reduce the number of misconception cases from 46 to 5 cases. 6 syahrial et al. previous researchers have also reported the effectiveness of cognitive conflict interviews in correcting misconceptions.19,22 cognitive conflict is closely related to conceptual change.13–15 cognitive conflict helps a person realize that there is a problem with their conception by showing phenomena, data, or evidence that contradicts their initial conception. this awareness will lead them to replace their initial conception with the scientific one. several factors could explain the effectiveness of this strategy in correcting misconceptions. firstly, oneon-one interviews provide a convenient environment leading to a productive discussion between interviewer and respondent. secondly, confirmation or supporting questions make respondents realize that their initial conception is scientifically incorrect. confirmatory questions22 can facilitate and describe the conceptual change process. thirdly, respondents’ prior knowledge was sufficient to recognize the phenomena, data, and/or evidence presented in the interviews. the plenty of experience in teaching covalent bonds leads them to quickly realize their misconceptions when facing the facts, phenomena, data, or evidence that contradicts their conceptions. however, the one-on-one interview approach is time-consuming.34 the following interview script illustrates the conceptual change regarding “atoms that form covalent bonds”. 1. researcher: what types of atoms are forming covalent bonds? metal atoms? non-metal atoms? both of them? 2. respondent: non-metal atoms. 3. researcher: are metal atoms unable to form covalent bonds? 4. respondent: yes, right. 5. researcher: if so, what type of bond is formed between the be and the cl in becl2 and between the al and the br in albr3? 6. respondent: an ionic bond because be and al are metals while cl and br are non-metals. 7. researcher: (indicating a reference that for binary compounds, a covalent bond will be formed when the two atoms forming the bond have an electronegativity difference of <1.7 according to the pauling scale) now, pay attention to the following data! (showing the data on the difference in electronegativity between be with cl and al with br) count for a moment! what is the difference in electronegativity between be and cl and al and br? smaller or greater than 1.7 on the pauling scale? 8. respondent: smaller than 1.7 on the pauling scale. 9. researcher: well, it is below 1.7 on the pauling scale. therefore, what kind of bond is formed between be and cl as well as between al and br? 10. respondent: if, based on the data, both of the bonds are covalent. persuading teachers to revise their misconceptions 7 11. researcher: well! let's go back to the becl2 and albr3. are these two compounds composed of non-metallic atoms only? 12. respondent: no. be and al are metals, while cl and br are non-metals. both compounds contain metal and non-metal atoms. 13. researcher: what does it mean? is the type of bond in a binary compound determined only by the type of atom that composes it? 14. respondent: ermmm ... it means that the kind of bond of binary compounds cannot be seen from the type of atoms composing it. is it right? 15. researcher: yes, that’s right. if so, what is your inference regarding the covalent bonding in binary compounds? 16. respondent: ummm ... the type of bond in binary compounds can’t be seen from the type of atoms forming it, metal or non-metal. however, it can be seen from the difference in electronegativity of the two types of atoms forming it. if the difference in electronegativity of the two types of atoms on the pauling scale is less than 1.7, the bond is covalent, but if it is greater than 1.7, the bond is ionic. the interview script above shows that initially, the respondent highly believed that: 1) covalent bonds only formed between non-metal atoms and 2) be–cl bond in becl2 and al–br bond in albr3 were ionic bonds (lines 2, 4 and 6). this belief or conception raises a question for the respondent when the interviewer showed the pauling scale parameter in determining the type of chemical bond, which led to a conclusion different from the respondent's conception (lines 7 and 8). this parameter made the respondent doubt their initial understanding when the interviewer pointed out that the difference in electronegativity between be and cl and al and br was below 1.7 on the pauling scale, indicating a covalent bonding, not an ionic bond, as the respondents perceived before. this misconception changed after the interviewer asked the respondent to determine the type of atoms composing the becl2 and albr3 compounds (lines 11 and 12). at the end of the interview, the respondent believed that the type of bond (covalent or ionic) formed in binary compounds was not dependent on whether the atoms were metal or non-metal but on the difference in electronegativity between the atoms (line 16). this final script indicates a conceptual change from misconception to scientific conception. regardless of the positive conceptual change, as shown in the interview script example, three respondents still experienced two misconceptions (table iii). this implies that some misconceptions has persisted, as reported by previous researchers.35 the first persistent misconception is the coordinate covalent bonding in the water molecule produced by the acid-base neutralization reaction. the two respondents (no. 1 and 3) only considered the molecular structure of h2o and 8 syahrial et al. ignored how the water molecules were formed. they did not realize that in acidbase neutralization, oh– acts as an electron-pair donor, and h+ acts as an electron pair acceptor. therefore, the bond is a coordinate covalent bonding. the lack of awareness regarding the formation of water molecules,6,24 leads to the misconception. the second persistent misconception is related to the lewis structure of the molecule or polyatomic ion. the three respondents (no. 1, 3 and 7) keep determining the lewis structure of a molecule according to the following steps: 1) determining the electron configuration of each atom; 2) determining the central atom; 3) drawing lewis symbols for all atoms; 4) pairing the electrons of each atom until the octet or duplet rule was obeyed. this procedure makes them difficult when dealing with molecules that do not obey the octet rule. the correct steps to write the lewis structure are identifying the central atom, calculating coordination numbers, bonding and lone pairs, writing a frame of lewis structure, adding electrons to all the substituents to fulfil the octet or duplet rules, calculating the formal charges of all the atoms, and making the formal charge of all atoms equal to zero.36 the lewis structure and coordinate covalent bonding concepts require a good understanding of the definitions and the rules associated with these definitions.37 to write the lewis structure of a species correctly, we need to understand the species well, the rules for writing lewis structures, and the terms associated with these rules like coordination number, bonding electron pair, lone pair, formal charge, octet, duplet36 and unpaired electrons.38 a conventional example of a coordinate covalent bonding is [cu(h2o)6] 2+, while an example of a coordinate covalent bonding that does not involve a transition element is nh4 +. the lone pairs of the o atom of the neutral molecule h2o and the n atom of the neutral molecule nh3 are critical examples of a coordinate covalent bonding. the source of the electron pair in a coordinate covalent bonding does not always come from the lone pair of an atom of the neutral molecule,39 for example, oh– in an acid–base neutralization reaction. lack of knowledge related to these concepts is a root of misconceptions about the concepts of coordinate covalent bonding and lewis structure. the issues of resistant misconceptions have been uncovered in the previous study.40 according to piaget, the incomplete conceptual change is caused by the inability to reach the equilibration phase in acquiring new concepts.1 conclusion and implication after applying the cognitive conflict interview, the number of misconception cases held by the teachers decreased. this phenomenon indicates that the cognitive conflict interviews can potentially encourage teachers to overcome misconceptions about the concepts of covalent bonding. some persistent misconceptions persuading teachers to revise their misconceptions 9 were still found, such as the o–h bond in the water molecule produced by the acid-base neutralization is a covalent bond. we realize that the sample size is insufficient to provide a robust conclusion. still, the results of this study could be a pilot to implement the cognitive conflict interviews for overcoming misconceptions and establishing a conceptual understanding of chemical bonding and other chemical topics. therefore, the effectiveness of this method in overcoming misconceptions needs to be proven by further research. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/11570, or from the corresponding author on request. acknowledgements. we would like to express our deepest gratitude to our previous adviser, prof. effendy, phd, for his guidance in conducting this research. our prayers accompany him in the barzakh realm. may allah forgive his sins, have mercy on him and gather him with the souls of pious people. we also thank habiddin, phd, for restructuring the sentences and for valuable language feedback. и з в о д отклањање заблуда о ковалентном везивању код наставника хемије помоћу интервјуа базираног на когнитивном конфликту: студија случаја syahrial syahrial1, mashfufatul ilmah1, yahmin yahmin2, munzil munzil2 и muntholib muntholib2 1chemistry education program, postgraduate program, universitas negeri malang, indonesia и 2department of chemistry, universitas negeri malang, indonesia истраживања су показала да већина наставника хемије има неке погрешне представе о ковалентном везивању. у оквиру ове студије истражено је да ли техника интервјуа базирана на когнитивном конфликту може да подстакне наставнике да ревидирају те погрешне представе о ковалентном везивању. у истраживању је учествовало осам наставника хемије из различитих школа. за прикупљање података коришћена су два валидирана инструмента, упутство за интервју, технике базиране на когнитивном конфликту и тест с питањима отвореног типа о ковалентном везивању. резултати су показали да примењени интервју може помоћи испитаницима да превазиђу своје заблуде о ковалентној вези. пет од осам испитаника направило је концептуалну промену од мисконцепција (заблуда) ка научним појмовима, док је код три испитаника концептуална промена била делимична. шест концепата који су претходно били извор заблуда елиминисани су и преведени у научне концепте. од 46 случајева заблуда, 41 случај је преведен у научне концепте. резултат ове студије служи као почетна перспектива за истраживање ефективности интервјуа базираног на когнитивном конфликту у ширем опсегу. 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oncology institute of vojvodina, put dr goldmana 4, 21204 sremska kamenica, serbia, 2university of novi sad, faculty of sciences, department of chemistry, biochemistry and environmental protection, trg dositeja obradovića 3, 21000 novi sad, serbia and 3serbian academy of sciences and arts, kneza mihaila 35, 11000 belgrade, serbia (received 30 november 2022, accepted 11 january 2023) abstract: new thiazole hybrids were synthesized and evaluated for their in vitro cytotoxicity against a panel of human malignant cell lines. the key steps in the synthesis of hybrids 3–7 involved the initial condensation of appropriate aldononitriles with cysteine ethyl ester hydrochloride, followed by subsequent treatment of resulting thiazolines with diazabicycloundecene to form the thiazole ring. bioisosteres 8 and 14 have been prepared after the stereoselective addition of 2-(trimethylsilyl)thiazole to the hemiacetals obtained by periodate cleavage of terminal diol functionality in the suitably protected d-glucose derivatives. the obtained analogues showed various antiproliferative activities in the cultures of several tumour cell lines. hybrid 6 was the most potent in hela cells, exhibiting more than 10 and 4 times stronger activity than both leads 1 and 2, respectively. the most active compound in raji cells was hybrid 12, which was nearly 2-fold more potent than the clinical antitumour drug doxorubicin. all analogues were more potent in a549 cells with respect to lead 1, while compounds 6 and 7 were slightly more active than doxorubicin. preliminary structure–activity relationship analysis revealed that the presence of a cinnamate group at the c-3 position in analogues of type 7 increases the activity of resulting molecular hybrids. keywords: molecular hybridization; pseudo-c-nucleosides; goniofufurone; tiazofurin; analogues; antiproliferative activity. * corresponding author. e-mail: velimir.popsavin@dh.uns.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc221130002k ________________________________________________________________________________________________________________________ (cc) 2023 scs. available on line at www.shd.org.rs/jscs/ 468 kojić et al. introduction molecular hybridization is a strategy of rational drug design based on the combination of pharmacophoric moieties of different bioactive substances to produce a new hybrid compound with improved affinity and efficacy, when compared to the parent drugs. in addition, this strategy may provide access to compounds with modified selectivity profiles, different or dual modes of action, and reduced unfavourable side effects.1–3 thiazole ring is a pharmacophore nucleus with various pharmaceutical applications. its derivatives have a wide range of biological activities including anticancer activity.4,5 we have recently reported on the synthesis of several thiazole bioisosteres of goniofufurone that exhibited in vitro antitumour activity against some human tumour cell lines.6 goniofufurone (1, fig. 1) is natural styryl lactone with [3.3.0]furofuranone core,7 which was isolated from the stem bark of tropical plant goniothalamus giganteus (annonaceae), and showed potent antiproliferative activity against several tumour cell lines.8 this work describes the synthesis and in vitro antitumour screening of several new thiazole hybrids with furofuranone or tetrahydrofuran scaffolds. compounds 3–5 might be considered pseudo-c-nucleosides related to tiazofurin (2), the oncolytic c-nucleoside with potent antileukaemic activity.9,10 pseudo-c -nucleosides are nucleoside analogues having a c–c bond between c-4 of the carbohydrate moiety and the heterocyclic aglycone.11 compound 8 represents a goniofufurone analogue with a thiazole replacing the phenyl ring at the c-7 position. 4 r = h 5 r = me 6 o oho n s o h2n o o ho n s o h2n or oh o o ho 8 ho o n s o o ho (+)-goniofufurone (1) ho o o ho oh n s tiazofurin (2) nh2 o ho 3 r = h 7 r = cinnamoyl o o o ro n s o h2n fig. 1. structures of (+)-goniofufurone (1), tiazofurin (2) and the corresponding analogues 3–8. experimental general procedures melting points were determined on a büchi 510, or a hot stage microscope nagema phmk 05 apparatus, and were not corrected. optical rotations were measured on a rudolph ________________________________________________________________________________________________________________________ (cc) 2023 scs. available on line at www.shd.org.rs/jscs/ thiazole hybrids with [3.3.0]furofuranone scaffold 469 research analytical automatic polarimeter, autopol iv. ir spectra were recorded on a ftir nexus 670 (thermo-nicolet) spectrophotometer. 1hand 13c-nmr spectra were recorded on a bruker ac 250 e (at 250 and 62.5 mhz, respectively) or a bruker avance iii spectrometer (at 400 and 100 mhz, respectively) employing indicated solvents (vide infra) using tms as the internal standard. chemical shifts were expressed in ppm (δ) values and coupling constants in hz (j). high-resolution mass spectra were taken on a micromass lct ka111 spectrometer or ltq orbitrap xl (thermo fisher scientific inc.) mass spectrometer. tlc was performed on dc alufolien kieselgel 60 f254 (e. merck). flash column chromatography was performed using kieselgel 60 (0.040–0.063, e. merck). all organic extracts were dried with anhydrous na2so4. organic solutions were concentrated in a rotary evaporator under reduced pressure at a bath temperature below 35 °c. the purity of tested compounds was determined by hrms and they were found to be > 95 % pure (errors were less than 5 ppm). synthetic procedures (e,z)-1,2-o-isopropylidene-α-d-xylo-pentodialdo-1,4-furanose-5-oxime (10). to a stirred and cooled (0 °c) solution of triol 9 (2.032 g, 9.23 mmol) in a mixture of 2:1 meoh/h2o (54 ml) was added naio4 (1.795 g, 10.49 mmol) in one portion. after 5 min, the cooling was stopped, and the reaction continues at room temperature for the next 4.5 h. the mixture was filtered through a celite pad, the adsorbent was washed with meoh, the filtrate was evaporated, and the residue was suspended in h2o (10 ml) and extracted with etoac (3×50 ml). the extract was dried (na2co3 and na2so4), filtered, and evaporated to give the crude aldehyde 9a (1.13 g) which was dried under a high vacuum overnight. suspension of crude aldehyde 9a (1.631 g), sodium acetate (1.677 g, 20.40 mmol), and hydroxylamine hydrochloride (2.236 g, 32.10 mmol) in etoh (40.75 ml) was vigorously stirred at room temperature for 24 h. the reaction mixture was evaporated, and the residue was purified by flash column chromatography (1:1 toluene/etoac). a mixture of eand z -oximes 10 (1.463 g, 78 % from 9) was obtained as an amorphous powder, rf = 0.27 (12:1 chcl3/ /meoh). the ratio of isomers (from 1h-nmr): e/z = 1:0.8. 3-o-acetyl-1,2-o-isopropylidene-α-d-xylo-furanoseurononitrile (11). a solution of compound 10 (1.463 g, 7.20 mmol) in acetic anhydride (29 ml) was stirred at reflux temperature for 1 h, and then evaporated. the residue was purified by flash column chromatography (19:1 toluene/etoac) to afford pure 11 (1.502 g, 92 %), as a colourless syrup, [α]d = = +7.7 (c 1.0, chcl3), rf = 0.53 (9:1 toluene/etoac). 3-o-acetyl-1,2-o-isopropylidene-4-c-(4′-ethoxycarbonylthiazol-2′-yl)-α-d-xylo-tetrofuranose (12). to a stirred solution of 11 (0.999 g, 4.39 mmol) in absolute ethanol (85 ml) l -cysteine ethyl ester hydrochloride (1.217 g, 6.56 mmol) and anhydrous et3n (0.91 ml, 6.55 mmol) were added. the reaction mixture was stirred at room temperature for 3.5 h and then evaporated. the residue was dissolved in ch2cl2 (50 ml), the organic phase was washed with water (15 ml), a saturated solution of nahco3 (15 ml), and a saturated solution of nacl (15 ml) then dried, filtered, and evaporated. a mixture of crude thiazoline derivatives 11a (1.3279 g) was obtained. to a solution of crude thiazolines 11a (1.328 g, 3.70 mmol) in dry ch2cl2 (27 ml) was added dbu (1.11 ml, 7.44 mmol). to the cooled solution (0 °c) was added brccl3 (0.31 ml, 3.14 mmol), the reaction mixture was stirred at 0 °c for 2.5 h and then left at 4 °c for another 43 h and then evaporated. the residue was purified on a column of flash silica (9:1 → 4:1 toluene/etoac) to give pure product 12 (1.100 g, 87 % based on reacted 11) as a yellow syrup. recrystallization from ch2cl2/hexane gave white needles, mp 141 °c, [α]d = −23.0 (c 0.1, chcl3), rf = 0.50 (7:3 toluene/etoac). ________________________________________________________________________________________________________________________ (cc) 2023 scs. available on line at www.shd.org.rs/jscs/ 470 kojić et al. 4-c-(4′-(carbamoyl)thiazol-2′-yl)-1,2-o-isopropylidene-α-d-xylo-tetrofuranose (3). a solution of protected thiazole 12 (1.100 g, 3.08 mmol) in saturated methanolic ammonia (25 ml) was kept at room temperature for 7 days. the reaction mixture was then evaporated and purified by flash column chromatography (chcl3 → 12:1 chcl3/meoh), to give pure 3 (0.498 g, 93 %) as a colourless syrup, [α]d = −46.5 (c 0.2, meoh), rf = 0.30 (12:1 chcl3/meoh). 4-c-(4′-(carbamoyl)thiazol-2′-yl)-d-xylo-tetrofuranose (4). a solution of 3 (0.312 g, 1.09 mmol) in 90 % aq tfa (18 ml) was stirred at 0 °c for 0.5 h and then at room temperature for 4.5 h. the reaction mixture was evaporated by azeotropic distillation with toluene. the remaining oily mixture was treated with etoac (2 ml) and saturated nahco3 (2 ml) and evaporated again whereby a mixture of anomeric lactols 4 was obtained as a syrup. the residue was purified on a column of flash silica (5:1 → 25:6 → 10:3 chcl3/meoh) to give pure product 4 (0.240 g, 89 %) in the form of pale yellow syrup, rf = 0.34 (5:1 chcl3/ /meoh). anomeric ratio (from 1h-nmr): α/β = 1:1. methyl 4-c-(4′-(carbamoyl)thiazol-2′-yl)-d-xylo-tetrofuranoside (5). a solution of 3 (0.100 g, 0.35 mmol) in 90 % aq tfa (5.80 ml) was stirred at 0 °c for 0.5 h and then at room temperature for 4.5 h. the reaction mixture was evaporated by azeotropic distillation with toluene and methanol. the residue was purified by preparative thin-layer chromatography (2 preparative plates, 5:1 chcl3/meoh, eluted with 7:3 etoac/iproh), whereby a mixture of anomeric glycosides 5 (0.048 g, 52 %) was obtained, in the form of white powder, rf = 0.38 (5:1 chcl3/meoh). anomeric ratio (from 1h-nmr): α/β = 2:1. 3,6-anhydro-2-deoxy-6-c-(4′-(carbamoyl)thiazol-2′-yl)-d-ido-hexono-1,4-lactone (6). a) to a solution of compound 4 (0.202 g, 0.82 mmol) in anhydrous dmf (3.5 ml) was added meldrum’s acid (0.394 g, 2.73 mmol) and dry et3n (0.36 ml, 2.583 mmol). the reaction mixture was stirred at 46 °c for 69 h and then evaporated. the crude product was purified by preparative thin-layer chromatography (10 preparative plates, 6:1 chcl3/meoh, eluted with 12:1 chcl3/meoh), whereby impure 6 was obtained. after additional purification on a column of flash silica (20:1 → 12:1 chcl3/meoh) and then by preparative thin-layer chromatography (2 preparative plates, 6:1 chcl3/meoh, eluted with 12:1 chcl3/meoh), pure product 6 was obtained as a white powder (0.015 g, 7 %), rf = 0.22 (12:1 chcl3/meoh). analytical sample 6 was obtained by crystallization from meoh in the form of white needles, m.p. 143 °c. b) to a cooled (0 °c) solution of 4 (0.204 g, 0.83 mmol) in dry meoh (23 ml) was added mcmp (0.7923, 2.37 mmol) and the resulting solution was stirred at room temperature for 1 h. the reaction mixture was evaporated, and the residue was purified on a column of flash silica (20:1 → 12:1 chcl3/meoh to give pure 6 as a yellow oil (0.050 g, 22 %), [α]d = = −9.4 (c 0.13, dmso), rf = 0.22 (12:1 chcl3/meoh). analytical sample 6 was obtained by crystallization from meoh as colourless needles, m.p. 143 °c. 4-c-(4′-carbamoyl)thiazol-2′-yl)-3-o-cinnamoyl-1,2-o-isopropylidene-α-d-xylo-tetrofuranose (7). to a stirred solution of compound 3 (0.0757 g, 0.2644 mmol) in a mixture of anhydrous mecn (2 ml) and anhydrous ch2cl2 (14.5 ml) was added cinnamic acid (0.088 g, 0.59 mmol), dcc (0.132 g, 0.64 mmol) and dmap (0.130 g, 1.07 mmol). after stirring at room temperature for 24 h, the reaction mixture was filtered through a pad of quartz sand, the filtrate concentrated and purified by preparative thin-layer chromatography (5 preparative plates, 12:1 chcl3/meoh, eluted with 7:3 etoac/iproh), to give pure 7 as a white powder (0.103 g, 94 %). analytical sample 7, obtained by crystallization from a mixture of meoh/ /ipr2o showed mp 180 °c, [α]d = −157.1 (c 0.40, chcl3), rf = 0.66 (12:1 chcl3/meoh). 3,6-anhydro-2-deoxy-7-c-(thiazol-2′-yl)-d-glycero-d-ido-heptono-1,4-lactone (8). to a stirred solution of compound 1012 (0.145 g, 0.71 mmol) in anhydrous mecn (15 ml) was ________________________________________________________________________________________________________________________ (cc) 2023 scs. available on line at www.shd.org.rs/jscs/ thiazole hybrids with [3.3.0]furofuranone scaffold 471 added h5io6 (0.146 g, 0.64 mmol). after stirring at room temperature for 22 h, the reaction mixture was evaporated with silica gel and purified by flash chromatography (9:1 ch2cl2/ /me2co). this gave pure 7a (0.108 g, 75 %). to the solution of 7a (0.069 g, 0.34 mmol) in anhydrous thf (3 ml) 2-tst solution (0.081 ml, 0.51 mmol) in thf (1 ml) is added dropwise. the reaction mixture was stirred at room temperature for 48 h and then evaporated. the residue was dissolved in thf (3 ml) and treated with 1 m tetrabutylammonium fluoride in thf (0.4 ml), while stirring at room temperature for 2 h. the reaction mixture was evaporated, and the oily residue was purified by preparative thin-layer chromatography (10 preparative plates, 17:3 ch2cl2/me2co, second development 4:1 ch2cl2/me2co) to afford pure 8 (0.007 g, 7.5 %) in the form of an oil, [α]d = = +10.0 (c 0.1, chcl3), rf = 0.30 (9:1 ch2cl2/me2co, three successive developments). 1,2-o-isopropylidene-5-c-(thiazol-2′-yl)-α-d-gluco-pentofuranose (14). to a solution of compound 13 (2.166 g, 8.32 mmol) in anhydrous etoac (80 ml) was added h5io6 (3.103 g, 13.61 mmol). the reaction mixture was stirred at room temperature for 6 h, then filtered and evaporated, leaving a light-pink reaction mixture. the residue was purified on a column of flash silica (11:9 et2o/light petroleum), whereby a mixture of alcohols 13a was obtained (0.876 g, 56 %) in the form of a colourless syrup, rf = 0.37 (1:1 et2o/light petroleum). ir (film): νmax 3371 cm-1 (oh). (+)esi-hrms (m/z): calculated for [c10h9o5 + nh4+] 236.11286, observed 236.11285. to a solution of purified compound 13a (0.161 g, 0.74 mmol) in anhydrous ch2cl2 (6 ml) a 2-tst (0.174 g, 1.09 mmol) solution in ch2cl2 (3 ml) was added dropwise at room temperature. after stirring at room temperature for 12 h, the solvent was evaporated and to the residue was added thf (10 ml) and tetrabutylammonium fluoride (0.886 mmol in 8.86 ml thf). after stirring at room temperature for 2 h, the reaction mixture was concentrated to a smaller volume, and after the addition of aq. nahco3 solution, extracted with etoac. the combined extracts were dried and evaporated, and the remaining crude product 14 was purified on a column of flash silica (light petroleum/etoac 1:1), whereby pure product 14 (0.048 g, 24 %) was obtained, which crystallized from a mixture of ch2cl2/hexane as white crystals, m.p. 120 °c, [α]d = −12.5 (c 0.2, acetone). cytotoxic activity test cells. the in vitro cytotoxicities of test compounds were evaluated against seven human malignant cell lines: k562 (atcc ccl-243, chronic myeloid leukaemia), hl-60 (atcc ccl-240, promyelocytic leukaemia), jurkat (atcc ccl-1435, t cells leukaemia), raji (atcc ccl-86, burkitt’s lymphoma), mcf-7 (atcc htb-22, er+ breast adenocarcinoma), hela (atcc ccl2, human cervix adenocarcinoma) and a549 (atcc htb-38, lung carcinoma). cytotoxic activity against one normal human cell line, mrc-5 (atcc ccl -185, foetal lung fibroblasts), was also estimated. mtt test. cytotoxic activity was evaluated by using standard mtt assay,13 after exposure of cells to the tested compounds for 72 h. crystal structure determination diffraction experiments were performed on an oxford diffraction gemini s diffractometer. crystal structures were solved and refined as reported previously14. all hydrogen atoms are introduced in idealized positions are refined using a riding model. pertinent crystallographic and refinement data are listed in table s-iii of the supplementary material to this paper. ccdc 2218113 and ccdc 2218112 contain supplementary crystallographic data for ________________________________________________________________________________________________________________________ (cc) 2023 scs. available on line at www.shd.org.rs/jscs/ 472 kojić et al. this paper. these data can be obtained free of charge from the cambridge crystallographic data centre via http://www.ccdc.cam.ac.uk/structures. results and discussion chemistry synthesis of compounds 3–7 is shown in scheme 1 and commenced from the commercially available monoacetone-d-glucose (9). o o o ho ho ho 9 o o o ho o 9a a o o o r1o r 10 r = ch:noh, r1 = h 11 r = cn, r1 = ac b e 4 r = h 5 r = me o ho n s o h2n or oh i or j g or h o o oaco n s 11a o eto c 12 o o oaco n s o eto 3 r = h 7 r = cinnamoyl o o o n s o h2n k d f 6 o oho n s o h2n o (from 3) ro scheme 1. a) naio4, meoh/h2o, rt, 4.5 h; b) nh2oh×hcl, naoac, etoh, rt, 24 h, 78 % from 9; c) ac2o, reflux, 1 h, 92 %; d) l-cysteine ethyl ester hydrochloride, et3n, etoh, ch2cl2, rt, 3.5 h; e) brccl3, dbu, ch2cl2, 0 °c, 2.5 h, then 4 °c, 43 h, 87 % from 11; f) saturated nh3, meoh, rt, 7 days, 93 %; g) 90 % aq tfa, 0 °c, 0.5 h, then rt, 4.5 h, 89 %; h) 90% aq tfa, meoh, 0 °c, 0.5 h, then rt, 4.5 h, 52 %; i) meldrum’s acid, et3n, dmf, 46 °c, 69 h, 7 %; j) mcmp, meoh, rt, 1 h, 22 %; k) cinnamic acid, dcc, dmap, mecn, ch2cl2, rt, 24 h, 94 %. terminal diol cleavage in 9 was achieved with sodium periodate in aqueous meoh to afford the unstable aldehyde 9a. the resulting aldehyde 9a was not purified but was rather immediately treated with hydroxylamine hydrochloride to yield the expected oxime 10 as a mixture of the corresponding eand z-isomers. the mixture was not separated but was further treated with refluxing acetic anhydride to give the corresponding nitrile 11 in 92 % yield. nitrile 11 was allowed to react with ethyl ester of cysteine hydrochloride, in the presence of triethylamine at room temperature, to afford thiazoline 11a as an inseparable mixture of c-4 epimers. the crude mixture was not separated but was immediately oxidized with bromotrichloromethane and dbu to give the thiazole 12 in an overall yield of 87 % from two steps. treatment of 12 with methanolic ammonia provided the amide 3 (93 %) as a result of successive ester ammonolysis and o-deacetylation at the c-3 position. hydrolytic removal of the isopropylidene protective group in 3 gave the expected lactol 4, which upon treatment with meldrum’s acid in the presence of triethylamine gave a low yield (7 %) of target 6. a better yield of 6 (22 %) was obtained by using the z-selective wittig olefination ________________________________________________________________________________________________________________________ (cc) 2023 scs. available on line at www.shd.org.rs/jscs/ thiazole hybrids with [3.3.0]furofuranone scaffold 473 of 4 with a stabilized c2-ylide (ph3p=chco2me, mcmp).15 apart from spectroscopic methods, the structure of compound 6 was confirmed by x-ray analysis (see supplementary material for details). finally, compound 3 was esterified with cinnamic acid, under the steglich conditions,16 to afford the corresponding 3-o -cinnamoyl derivative 7 in 94 % yield. the reason for the preparation of cinnamate 7 lies in the fact that a significant number of cinnamic acid hybrids show antitumour activity.17–19 the preparation of bioisostere 8 is shown in scheme 2. d-glucose was first converted to the protected aldehyde 7a using the procedure recently developed in our laboratory20 (see the supplementary material for details). d-glucose o o o o o ho 7a o o ho (s) 8 oh o n s sime3 n s ao ho ho ho oh oh b tst o o ho o o o 13 o o o o o ho 13a c d o o o ho (s) oh n s 14 scheme 2. a) see supplementary material and/or ref.20; b) (i) tst, thf, rt, 48 h, (ii) tbaf, thf, rt, 2 h, 7.5 %; c) ref.21; d) (i) tst, ch2cl2, rt, 12 h, (ii) tbaf, thf, rt, 2 h, 24 %. the addition of 2-(trimethylsilyl)thiazole (tst) to hemiacetal 7a (scheme 2) in thf using the adopted procedure developed by dondoni et al.22 occurred with high diastereofacial selectivity affording, after desilylation with tetrabutylammonium fluoride, a low yield of thiazole 8 (7.5 %). this two-step transformation involves the initial unmasking of hemiacetal function with the subsequent addition of reagents to the liberated aldehyde group. given that compound 8 showed relatively weak antiproliferative activity against tumour cells, the yield of this reaction was not optimized. to resolve the stereochemistry at the c-7 position in product 8, the above-described addition reaction was repeated with the known23 hemiacetal derivative 13a. the corresponding thiazole derivative 14 was obtained in a yield of 24 %. the stereochemistry of 14 was unambiguously established by x-ray crystallographic analysis (see supplementary material for details). based on this result, as well as the observations of dondoni et al.,22 we concluded that the newly introduced stereocenter of product 8 has (7s)-stereochemistry. ________________________________________________________________________________________________________________________ (cc) 2023 scs. available on line at www.shd.org.rs/jscs/ 474 kojić et al. antiproliferative activity table i shows in vitro cytotoxicities of synthesized compounds against a panel of human cell lines (k562, hl-60, jurkat, raji, mcf-7, hela, a549 and mrc-5), using the standard mtt assay. apart from the final products (5–8), intermediates, 3, 4 and 12 were also included in the assay since they can be considered pseudo-c-nucleosides related to tiazofurin. table i. in vitro cytotoxicity (ic50* / μm; values are means of three independent experiments. coefficients of variation were less than 10 %) of (+)-goniofufurone (1), tiazofurin (2), dox and analogues 3–8 and 12 after 72 h compound cell line k562 hl-60 jurkat raji mcf-7 hela a549 mrc-5 (+)-goniofufurone (1) 0.41 201.32 32.45 18.45 16.59 8.32 35.21 >100 tiazofurin (2) 2.06 0.67 0.09 5.28 2.03 3.26 5.92 0.36 3 21.01 7.64 7.09 15.64 10.52 4.36 18.21 >100 4 2.55 8.51 11.36 14.32 8.65 8.31 24.64 >100 5 17.50 7.79 11.36 7.63 18.36 8.64 5.46 >100 6 1.63 1.02 18.52 9.02 2.61 0.75 4.64 97.12 7 3.54 12.63 4.32 12.64 10.02 1.25 3.45 >100 8 3.05 3.54 25.02 25.41 7.62 9.06 11.59 >100 12 3.47 9.10 7.52 1.58 15.20 3.70 10.35 >100 dox 0.25 0.92 0.03 2.98 0.20 0.07 4.91 0.10 the results in table i show that five compounds exhibited micromolar activity in the culture of k562 cells, although only compound 6 was more potent than tiazofurin (2). almost all synthesized compounds were more active than 1 against mcf-7 and hl-60 cells, with lactone 6 being the most potent. it is noteworthy that analogue 6 exhibited a prominent potency (ic50 = 1.02 µm) against promyelocytic leukaemia cells (hl-60) with activity similar to dox. among all synthesized molecules, which showed moderate activity in jurkat and raji cell cultures where they were more active than 1 (except 8 against raji cells), the isopropylidene derivative 12 stands out, which was almost twice as active as dox and 3 times as active as tiazofurin (2) against raji cells. against alveolar basal adenocarcinoma cells (a549), all compounds were more active than 1, while two compounds (6 and 7) were slightly more active than dox. molecules 6 and 7 showed higher potency than both leads 1 and 2 against hela cells of which compound 6 showed submicromolar activity (ic50 = 0.75 µm), the best activity recorded in this assay. like natural product 1, none of the synthesized analogues were active against normal mrc-5 cells, in contrast to tiazofurin (2) and dox, which showed high potencies against these cells in the submicromolar range. * ic50 is the concentration of compound required to inhibit the cell growth by 50 % compared to an untreated control. ________________________________________________________________________________________________________________________ (cc) 2023 scs. available on line at www.shd.org.rs/jscs/ thiazole hybrids with [3.3.0]furofuranone scaffold 475 in an attempt to determine the structural features important to the activity of this series of compounds, we compared the activities of: a) compound 7 with a cinnamoyl ester group at the c-3 position, with 3 (which has an oh group at c-3); b) the activity of lactol 4, with free oh groups at c-1 and c-2, with the activity of compounds 6 and 3 with a lactone or isopropylidene ring; c) the activity of lactol 4 with the activity of the methyl glycoside 5; d) the activity of the c-7 thiazole hybrid 8 with the natural product 1 having a phenyl group at the c-7 position (fig. s-1 of the supplementary material). the results of this brief sar analysis showed that the presence of the cinnamoyl group at c-3 is beneficial for the activity of this type of compound; the absence of oh groups at c-1 and c-2 and the structural architecture of a five-membered lactone or isopropylidene ring (the analysis also showed that pseudo-c-nucleoside 5 is more active against 50 % of the tested cell lines) and that the introduction of a thiazole ring at the c-7 position of the natural product 1 instead of the phenyl ring, increases the activity against four of seven cell lines. crystal structure of pseudo-c-nucleoside 6 the molecular structure of 6 is depicted in fig. 2. absolute configuration of all stereocenters is determined both from resonant scattering effects, and findings are in line with assumed absolute configurations of stereocenters whose stereochemistries remain unchanged during the synthetic route. fig. 2. molecular structure of 6 (ccdc 2218113) with the atom numbering scheme. from the structural point of view, 6 is the first structurally characterized compound that bears a furofuranone ring core coupled to a thiazole ring. the search of the csd24 resulted in only ten structures that contain a thiazole ring coupled to the c1′ atom of a tetrahydrofuran ring substructure depicted in fig. 3a. all these structures can be regarded as tiazofurin analogues. since 6 can also be regarded as a tiazofurin analogue, where a furanose ring is fused to a lactone ring, it is of interest to compare the furanose ring conformation in 6 and these tiazofurin analogues. for this purpose, atom numbering nomenclature established for furanose rings in nucleotides is used,25,26 as shown in fig. 3a. ________________________________________________________________________________________________________________________ (cc) 2023 scs. available on line at www.shd.org.rs/jscs/ 476 kojić et al. fig. 3. a) substructure fragment used in csd search. substituents at c4′ were unspecified. b) pseudorotational circle for furanose ring conformations found in csd hits. conformation of 6 is indicated by a filled circle, and that of yihcat with a star. preferred conformational ranges are shaded. conformations of the furanose rings are analysed via cremer–pople formalism.27 it is found that in seven structures furanose rings adopt conformations that spread in pseudorotation regions demarcated by 2t1 and 2t3 conformations, while for two structures the range is enclosed between 3t2 and 3t4 conformations. these conformational ranges have been established as preferred for ribose and deoxyribose in nucleosides.28,29 structure yihcat,30 with the furanose ring in conformation between ot4 and oe is the only outlier. the conformation of the furanose ring in the 6 is very close to e2, which is also an aberration. what separates yihcat and 6 from other structures is the presence of fused rings – isopropylidene in yihcat and furofuranone in 6, which may explain their different conformations. notably, for all investigated structures, including 6, ring puckering amplitude falls in the range from 0.30 to 0.45 å. a graphical representation of ring conformation space for the investigated structures is given in fig. 3b, while details are summarized in table s-iii of the supplementary material. relative to the sugar moiety, the aglycone fragment of the nucleoside can adopt two main orientations about the glycosyl c1′–n link called syn and anti.25,26 in analogy to that, for c-nucleosides such as tiazofurin and its derivatives, a torsion angle χ (o–c1′–c–s) can be defined to assess thiazole ring orientation. it is found that the thiazole ring orients in such a way that sugar o and thiazole s atoms are in syn orientation, with a restricted range of |χ| (0–60°), and the peak of the distribution at ca. 30° (see fig. s-3 of the supplementary material). the cor________________________________________________________________________________________________________________________ (cc) 2023 scs. available on line at www.shd.org.rs/jscs/ thiazole hybrids with [3.3.0]furofuranone scaffold 477 responding torsion angle for 6 amounts to −24.3(2)°, indicating that the mutual disposition of the studied rings in 6 is in line with the literature data. two intermolecular hydrogen bonds were found in the crystal structure of 6. hydroxyl o5–h5 group is bonded to carboxamide oxygen o6′ of the neighbouring molecule. interestingly, only one of the carboxamide hydrogen atoms is involved in hydrogen bonding, with carbonyl oxygen o1 of the lactone ring being the hydrogen bond acceptor. details of hydrogen bonding are listed in table s-v of the supplementary material. conclusion in conclusion, seven new thiazole hybrids with furofuranone or tetrahydrofuran scaffolds have been synthesized and evaluated for their in vitro cytotoxicity against a panel of human malignant cell lines (k562, hl-60, jurkat, raji, mcf -7, hela and a549), as well as toward a single normal cell line (mrc-5). the key steps in the synthesis of pseudo-c-nucleosides 3–7 and 12 involved the initial cyclocondensation of the corresponding aldononitriles with cysteine ethyl ester hydrochloride, followed by subsequent treatment of the resulting c-4′ epimeric thiazolines with dbu to form the thiazole ring. goniofufurone bioisosteres 8 and 14 have been prepared by stereoselective addition of 2-(trimethylsilyl)thiazole to partially protected hemiacetals, obtained by periodate cleavage of the terminal diol function in the appropriate d-glucose derivatives. the synthesized analogues showed moderate to strong antiproliferative activity in cultures of several malignant cell lines. the strongest activity was shown by hybrid 6 (hela cells, ic50 0.75 µm) which was more than 10 or 4 times more active than both control compounds 1 and 2, respectively. the most active compound in raji cell culture was hybrid 12, which was nearly two times more potent than the commercial antitumour drug doxorubicin (dox). lung adenocarcinoma cells (a549) were the most sensitive against the synthesized compounds. all were more active than lead 1, while two compounds (6 and 7) were slightly more active than dox. a brief sar study revealed that the presence of the cinnamoyl group at c-3 may enhance the activity of this type of analogues. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/12157, or from the corresponding author on request. acknowledgments. this work was supported by the ministry of education, science and technological development of the republic of serbia (grant no. 451-03-47/202301/200125). the work was also funded by the serbian academy of sciences and arts through a research grant, number f-130. ________________________________________________________________________________________________________________________ (cc) 2023 scs. available on line at www.shd.org.rs/jscs/ 478 kojić et al. и з в о д синтеза и антипролиферативна активност нових тиазолних хибрида са [3.3.0]фурофуранонским или тетрахидрофуранским скелетом весна којић1, милош свирчев2, сања ђокић2, ивана ковачевић2, марко в. родић2, бојана срећо зеленовић2, велимир попсавин2,3 и мирјана попсавин2 1универзитет у новом саду, медицински факултет, институт за онкологију војводине, пут др голдмана 4, 21204 сремска каменица, 2универзитет у новом саду, природно–математички факултет, департман за хемију, биохемију и заштиту животне средине, трг доситеја обрадовића 3, 21000 нови сад и 3српска академија наука и уметности, кнеза михаила 35, 11000 београд синтетизовани су нови тиазолни хибриди и одређена је њихова in vitro цитотоксичност према панелу хуманих малигних ћелијских линија. кључни кораци у синтези хибрида 3–7 пoдразумевали су иницијалну кондензацију одговарајућих алдононитрила са хидрохлоридом етилестра цистеина, након чега је уследио третман резултујућих тиазолина са dbu при чему је формиран тиазолни прстен. биоизостерe 8 и 14 су добијене након стереоселективне адиције 2-(триметилсилил)тиазола на хемиацетале добијене перјодатним раскидањем терминалне диолне функције погодних деривата d-глукозе. добијени тиазолни аналози су показали различите антипролиферативне активности у културама појединих туморских ћелијских линија. најјачу активност према hela ћелијама показао је хибрид 6, који је био више од десет, односно четири пута активнији од контролних молекула 1 и 2, редом. најактивније једињење према raji ћелијaма био је хибрид 12, који је скоро два пута активнији од клиничког антитуморског лека доксорубицина. сви аналози су били активнији према а549 ћелијама у односу на контролу 1, док су једињења 6 и 7 била нешто активнија од доксорубицина. прелиминарна sar анализа је открила да присуство цинаматне групе на положају c-3, у аналозима типа 7, повећава активност резултујућих хибрида. 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biopolymers 7 (1969) 821 (https://doi.org/10.1002/bip.1969.360070602) 26. w. seanger, principles of nucleic acid structure, springer-verlag, new york, 1984 27. d. cremer, j. a. pople, j. am. chem. soc. 97 (1975) 1354 (https://doi.org/10.1021/ja00839a011) 28. c. altona, m. sundaralingam, j. am. chem. soc. 94 (1972) 8205 (https://doi.org/10.1021/ja00778a043) 29. h. p. m. de leeuw, c. a. g. hasnoot, c. altona, isr. j. chem. 20, (1980) 108 (https://doi.org/10.1002/ijch.198000059) 30. а. dondoni, m.-c. scherrmann, j. org. chem. 59 (1994) 6404 (https://doi.org/10.1021/jo00100a050). ________________________________________________________________________________________________________________________ (cc) 2023 scs. available on line at www.shd.org.rs/jscs/ @article{, author = {vesna kojić and miloš svirčev and sanja djokić and ivana kovačević and marko v. rodić and bojana srećo zelenović and velimir popsavin and mirjana popsavin}, journal = {journal of the serbian chemical society}, title = {synthesis and antiproliferative activity of new thiazole hybrids with [3.3.0]furofuranone or tetrahydrofuran scaffolds}, year = {2023}, issn = {1820-7421}, month = {4}, number = {5}, pages = {467–479}, volume = {88}, abstract = {new thiazole hybrids were synthesized and evaluated for their in vitro cytotoxicity against a panel of human malignant cell lines. the key steps in the synthesis of hybrids 3–7 involved the initial condensation of appropriate aldo­nonitriles with cysteine ethyl ester hydrochloride, followed by subsequent treatment of resulting thiazolines with diazabicycloundecene to form the thiaz­ole ring. bioiso­steres 8 and 14 have been prepared after the stereoselective addition of 2-(tri­methylsilyl)thiazole to the hemiacetals obtained by periodate cleavage of terminal diol functionality in the suitably protected d-glucose der­ivatives. the obtained analogues showed various antiproliferative activities in the cultures of several tumour cell lines. hybrid 6 was the most potent in hela cells, exhibiting more than 10 and 4 times stronger activity than both leads 1 and 2, respectively. the most active compound in raji cells was hybrid 12, which was nearly 2-fold more potent than the clinical antitumour drug doxo­rubicin. all analogues were more potent in a549 cells with respect to lead 1, while compounds 6 and 7 were slightly more active than doxorubicin. prelim­inary structure–activity relationship analysis revealed that the presence of a cinnamate group at the c-3 pos­ition in analogues of type 7 increases the act­ivity of resulting molecular hybrids.}, doi = {10.2298/jsc221130002k}, file = {:01_12157_5639.pdf:pdf}, issue = {5}, keywords = {c,analogues,antiproliferative activity,goniofufurone,nucleosides,pseudo,tiazofurin}, publisher = {national library of serbia}, url = {https://www.shd-pub.org.rs/index.php/jscs/article/view/12157}, } effect of an industrial chemical waste on the uptake j. serb. chem. soc. 87 (0) 1–14 (2022) original scientific paper jscs–11454 published 30 september 2022 1 synthesis of activated carbons from water hyacinth biomass and its application as adsorbents in water pollution control ahmad hakky mohammaad* and mirjana kijevčanin# university of belgrade faculty of technology and metallurgy, karnegijeva 4, 11000 belgrade, serbia (received 21 december 2021, revised 10 february, accepted 11 february 2022) abstract: the water hyacinth biomass was used for the synthesis of activated carbons in a process of chemical activation with zncl2, followed by controlled pyrolysis. the applied impregnation weight ratios zncl2 and dry hyacinth biomass were in the range of 0.5–3.5. the carbonization was conducted at four different temperatures (400–700 °c) under an inert atmosphere. the highest yield of activated carbon was obtained for the impregnation ratio of 0.5 and carbonization temperature of 400 °c. the samples were characterized using elemental analysis, adsorption–desorption isotherms of nitrogen and sem analysis. the activated carbon obtained with an impregnation ratio 2.0 and carbonization temperature of 500 °c (2.0ac500) showed the highest values of specific surface area and total pore volume of 1317 m2 g-1 and 0.697 cm3 g-1, respectively. the adsorption of glyphosate, pesticide with a strong negative environmental impact, was a fast process, with the equilibrium time of 120 min. the adsorption isotherms were fitted with langmuir and freundlich model. the langmuir adsorption capacity of qmax = 240.8 mg g -1 for 2.0ac500 classified the selected adsorbent as a very efficient one. the tested adsorption process followed the kinetics of the pseudo-second-order model. keywords: carbonization; characterization; pyrolysis; pesticide removal; adsorption; modelling. introduction one of the leading environmental problems in africa and asia is the presence of water hyacinth (eichhornia crassipes) in natural waters (mostly rivers and lakes). water hyacinth forms dense and impenetrable floating mats on water surfaces, causing considerable problems in aquatic ecosystems. this plant has shown a strong negative impact on the biodiversity of the aquatic system in many * corresponding author. e-mail: ahmadhakky59@gmail.com # serbian chemical society member. https://doi.org/10.2298/jsc212121006m 2 mohammaad and kijevčanin ways: its presence leads to a significant reduction in the amount of light in the water, prevents access to wildlife (birds in particular) thereby disrupting the normal functioning of fauna and representing a suitable environment for mosquitoes breeding.1 utilization of water hyacinth biomass as source of lignocellulose for the activated carbon production can be one of the strategies in water pollution control. using activated carbon obtained from lignocellulose biomass instead of fossil coal will reduce the production of greenhouse gasses and therefore represents a green approach in the synthesis of materials that can be used in process of pollutant removal.2 the water hyacinth is characterized by a high content of lignocellulose biomass, including 48 % hemicellulose as the major component, along with 20 % cellulose and with 10 % of average lignin content, so it can be potentially employed as a proper carbon source.3–5 the activated carbons have been used as adsorbents of a wide range of contaminants such as pharmaceuticals, metallic and non-metallic pollutants and dyes from aqueous solutions.2,6 in comparison with other adsorbents (zeolites, clays and polymers) activated carbons show better performance and stability in terms of adsorption.7 the chemical activation of raw lignocellulose precursor material is usually a one-step method for the activated carbons preparation. among many chemical agents, the zncl2 is one of the most effective chemicals used for producing activated carbons with highly developed porosity.8,9 this activation agent has a high activating capability, and it is relatively expensive. in process of chemical activation, the zncl2 contributes to the pore development by localized decomposition of organic matter, inhibiting tar formation and enhancing the carbon yield.8,9 water hyacinth has already been used as a precursor for activated carbon synthesis. the activation processes were dominantly performed by koh10 or h3po4 11 under various and well-studied experimental approach. on the other hand, zncl2 activation of water hyacinth has been applied, 4,12 but the impact of amount of the activation agent has not been sufficiently studied. pesticides are chemical substances used for the increase of the agricultural production. as artificial organic compounds, pesticides can remain in the environment for many years and may be transported over a long distance.13 among many pesticides, glyphosate-based herbicides as systemic, broad-spectrum herbicides are widely used, therefore contributed to concerns about their environmental impact.14 international agency for research on cancer and world health organization classify glyphosate as substance that is “probably carcinogenic to humans” (group 2a).15 according to the author’s best knowledge, there is lack in the existing literature about application of activated carbons obtained from water hyacinth bio waste as adsorbents of glyphosate. activated carbons for glyphosate removal 3 the main objective of the present work was to obtain activated carbons with a high surface area from water hyacinth biomass, using different amounts of zncl2 and applying different carbonization temperatures. the selected activated carbon was further evaluated as adsorbent for glyphosate removal. experimental materials the water hyacinth (wh) plant (karbala, iraq) was used as raw material for obtaining the activated carbon. the raw wh was washed with distilled water. the roots and stalks without leaves were chopped and dried in an oven for 24 h. the dried wh was boiled in 0.25 m hydrochloric acid to remove metallic oxides, rinsed with distilled water, and finally dried in vacuum freeze dryer for 24h. the dry wh was crushed and ground in rotary mill, and finally sieved in order to obtain particles sized 1.4–2.0 mm. the zncl2 (≥98 %), supplied from sigma–aldrich, was used as activating agent in process of chemical activation during the activated carbons synthesis. the herbicide glyphosate – gph (≥99 %), used in adsorption study, was purchased from merck. activated carbons synthesis the activated carbon (ac) based on dry water hyacinth biomass was prepared by chemical activation of dry wh with zncl2 according to the procedure described in literature. 16 the impregnation ratio was calculated as the ratio of the weight of zncl2 in solution to the weight of the dry wh. the impregnation ratio was 0.0, 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0. the 40 g of dry wh sample was added to 150 ml of solution with the appropriate mass of zncl2 and stirred at 60 °c for 4 h. the solid and liquid phases were separated by filtration through buchner funnel and dried at 105 °c during 24 h. the drying process was applied prior to carbonization in order to avoid the loss of sample caused by rash steam development. the carbonization of activated wc was carried out in electrical furnace with nitrogen flowing (150 cm3 min-1) and at heating rate of 15 °c min-1. the carbonization during 80 min was conducted at following temperatures: 400, 500, 600 and 700 °c. the obtained activated carbon was rinsed with 0.5 m hcl in order to remove the activating agent, washed with hot distilled water until neutral ph and finally dried at 110 °c for 12 h. the dry samples were weighted in order to calculate the yield. the synthetized activated carbons were denoted according to impregnation ratio and carbonization temperature, e.g., 0.5ac400 means that the impregnation ratio and the carbonization temperature were 0.5 and 400 °c, respectively. the yield of activated carbons was calculated from mass ratio between activated carbon and starting wh after drying process: ac drywhac 100 m y m = (1) where y / % is yield of the synthesis, mac / g is the mass of activated carbon and mdrywh / g is the mass of dry wh. characterization methods the synthetized acs were characterized using elemental analysis, nitrogen adsorptiondesorption isotherms and scanning electron microscopy (sem). 4 mohammaad and kijevčanin the elemental analysis was used in order to determinate the content of carbon, hydrogen, nitrogen and sulphur in raw materials and activated carbons. the analysis was performed using elemental analyser instrument (thermo scientific – flashea1112 automatic elemental analyzers). prior to analysis, the samples were dried in an oven at 110 °c. the textural properties of activated carbons were obtained from adsorption-desorption nitrogen isotherms at –196 °c (micromeritics’ asap® 2020). prior to analysis the samples were outgassed at 110 °c during 12 h. the specific surface area (sbet) was calculated according to brunner–emmett–teller method,17 the total pore volume (vt) was estimated from n2 adsorption isotherm according to gurvich rule, and represents the liquid molar volume adsorbed at pressure p/p0 of 0.999. 18,19 the volumes of micropores and mesopores were calculated using dubinin–radushkevich method20 and barrett, joyner, halenda (bjh) method,21 respectively. the morphology of activated carbons was characterized by scanning electron microscopy (sem-jeol, jsm 6360 lv). adsorption study the herbicide glyphosate (gph) was used as a model of pesticide pollutant. during adsorption study, the gph concentration was determinated by uv–vis spectrophotometer (uv– –vis 1800 shimadzu) at λmax = 264 nm. the volume of gph solution of 75 cm3 was introduced into the glass flasks and mixed with 20 mg of adsorbents. after the adsorption process, the gph concentration in the supernatant was analyzed, with previous separation of the solid phase by centrifugation at 12,000 rpm for 20 min. the effect of surface development of activated carbons on the adsorption efficiency was performed using gph starting concentration of 100 mg dm-3 for the adsorption time of 240 min. the adsorption was performed from a solution with ph 3.55, which represents the unbuffered ph value of gph solution for the investigated concentration. the effect of contact time on the gph adsorption was monitored at predetermined time intervals between 5 min and 240 min, at 25 °c, with starting gph concentration of 100 mg dm-3. the adsorption isotherms were constructed at equilibrium adsorption time at 25 °c with gph initial concentration in range 50–250 mg dm-3. the amount of the adsorbed herbicide (qt / mg g -1) was calculated according to: ( )i t t c c v q m − = (2) where ci is the initial concentration of gph /mg dm -3, ct / mg dm -3 is the concentration of gph in time t, v / dm-3 is the volume of gph solution and m / g is the mass of the adsorbent. adsorption data analysis two well-known isotherm models langmuir22 and freundlich23 were used for modelling the adsorption data, while the adsorption kinetics data were fitted with both, pseudo-first24 and pseudo-second order25 kinetics’ models. the applied isotherm and kinetics model have been frequently used for heterogeneous adsorption systems that consist of solid adsorbent and dissolved adsorbate molecule.26 activated carbons for glyphosate removal 5 results and discussion results of characterization the yield of activated carbons obtained after chemical activation with zncl2 and carbonization process is given in fig. 1. fig. 1. the influence of impregnation ratio and carbonization temperature on yield of activated carbons prepared from water hyacinth dry material. it was observed that activated carbons obtained with the impregnation ratio 0 (i.e., without applied zncl2) and the temperature range 400–700 °c had relatively low yield in the range of 19.6–24.9 %. this fact can be related to a high content of volatile matter and relatively low lignin content in raw wh material used for the preparation of activated carbons. according to the literature, the activated carbons obtained by pyrolisis without prior activation showed significant weight loss attributed to gasses extraction (co, co2 and ch4). 27 the activation agent and applied carbonization temperature have a significant impact on the yield of activated carbons (fig. 1). generally, the best yields were obtained for the lowest carbonization temperature (400 °c). with the temperature increase the yield of activated carbon decreased regardless the amount of applied zncl2, which was explained by the promotion of tar volatilization by higher temperature.28 for each carbonization temperature it was observed that the amount of the activation agent has a similar impact on yield, i.e., the activated carbon yields continually decreased with the impregnation ratio higher than 0.5 (fig. 1). this observation can be explained by larger evolution of volatiles compounds affected by dehydration agent – zncl2. 29 in order to estimate the effect of the amount of activation agent – zncl2, on surface development, the specific surface area (sbet) of samples obtained on the carbonization temperature of 400 °c was correlated with the impregnation ratio (from 0.0 to 3.0) and shown in fig. 2. the activated carbon obtained without impregnation showed the lowest value of sbet. this result was expected since the zncl2 works as a dehydration reagent during the carbonization process, which leads to carbon charring, form6 mohammaad and kijevčanin ations of the aromatic, porous structure, and restricts the formation of the tar.29 the introduction of zncl2 led to development of specific surface and this trend continued up to impregnation ratio 2.0, and after that decreased for higher impregnation ratios. these results are in accordance with literature data.27,29,30 fig. 2. the influence of impregnation ratio (mzncl2/mdry wh) on specific surface area of carbons obtained by carbonization at 400 °c. the more detailed textural properties analysis was applied on the activated carbon samples with impregnation ratio 2.0, since the carbons with highest sbet values (fig. 2) were obtained using zncl2 in this impregnation ratio. the effect of temperature (400–700 °c) on the specific surface area (sbet), micropore (vmic) and mesopore volume (vmeso) as well as on total pore volume (vtot) of the activated carbons with impregnation ratio 2.0 are presented in table i. table i. surface area and pore volumes of activated carbons with impregnation ratio 2.0 obtained at different carbonization temperatures t / °c sbet / m 2 g-1 vtot / cm 3 g-1 vmic / cm 3 g-1 vmeso / cm 3 g-1 400 1154 0.602 0.301 0.298 500 1317 0.697 0.152 0.541 600 1284 0.670 0.135 0.527 700 1163 0.605 0.113 0.485 the most of the investigated textural properties increased with the rise of final carbonization temperature from 400 to 500 °c, while further increase of temperature led to surface development decreasing. increasing temperature from 400 to 500 °c had strong positive impact on mesoporosity development, while temperatures higher than 500 °c led to slight decrease of mesopore volume. besides that, all investigated carbonization temperatures above 400 °c reduced microporosity of activated carbons. similar trends can be found in literature,27,28,31 according to rodriguez-reinoso and molina-sabio,27 zncl2 has an important role in the development of microand mesoporosity in the carboniz activated carbons for glyphosate removal 7 ation process up to 500 °c, but at higher temperatures the reaction of zncl2 with the char is negligible. the decrease in textural properties at temperatures higher than 500 °c can also be attributed to a sintering effect at high temperature, followed by the shrinkage of the char, and the realignment of the carbon structure.32 sentilkumar et al.12 applied the weight of zncl2 that corresponded to 10 % of raw water hyacinth and the synthetized activated carbon with sbet = 579.94 m2 g–1 at high temperature of 900 °c. boonpoke4 produced the microporous activated carbon with a specific surface area of 1066 m2 g–1 at 600 °c using an equal amount of zncl2 and raw water hyacinth (impregnation ratio 1:1). the present study applied different amounts of zncl2 and found that the impregnation ratio 2:1 leads to obtaining the activated carbons with higher values of sbet than those in previous studies at lower carbonization temperatures of 400 and 500 °c (table i). wu et al.10 activate the raw water hyacinth with koh and synthetized activated carbon with sbet = 1380 m 2 g–1, but on 800 °c. yang and qiu33 showed that the activated carbons with the specific surface area of even 2000 m2 g–1 could be produced from pharmaceutics’ herb residue, but synthesis required chemical activation with booth naoh and zncl2. the elemental analysis was performed in order to evaluate the effect of temperature on the chemical composition of activated carbons. the result of the elemental analysis of dry wh and the activated carbons prepared with impregnation ratio of 2.0 are presented in table ii. table ii. the results of the elemental analysis sample content of elements, wt. % c h oa n ash dry wh 41.22 6.23 47.07 1.54 3.94 ac400 80.31 3.37 15.16 0.32 0.84 ac500 81.57 3.15 14.20 0.27 0.81 ac600 83.24 2.98 12.79 0.24 0.75 ac700 84.15 2.75 12.23 0.19 0.68 athe oxygen content is calculated from the difference up to 100 % the major organic elements in all investigated samples are carbon and oxygen. the wh has higher content of ash, consisting mainly of silica and metal oxides.16 the treatment with hcl after activation process led to leaching of metal cations and therefore the ash content was reduced. during the carbonization process, with temperature rise the content of carbon increased, which was expected.16 the scanning electron microscopy (sem) was employed to show the difference in the morphology between the raw wh material and the activated carbons with impregnation ratio 2:1, obtained in the temperature range from 400–700 °c. 8 mohammaad and kijevčanin the sem images were recorded using magnification of 3000 and presented in fig. 3. in fig. 3 significant difference in surface morphology can be observed between raw wh and acs. the surface of the raw wh is moderately developed with parts of a smooth area, but after impregnation and carbonization, the raw wh biomass turns to be more porous with more open structures (fig. 3). the increase of the carbonization temperature led to a reduction in small cracks in the activated carbon surfaces which could be responsible for the reduce of textural properties. according to the textural analysis the mesopore formation was dominantly responsible for the surface development (table i). since the mesopores are those with diameter from 2–50 nm, they are not visible in fig. 3, where macroporous structure can be noticed. fig. 3. the sem images of: a) raw wh; b) 2.0ac400; c) 2.0ac500; d) 2.0ac600; e) 2.0ac700. adsorption study the effect of surface development of activated carbons on adsorption efficiency. the aim of this adsorption study was to select the activated carbon with the best adsorption properties toward gph. the effect of surface development of the activated carbons (impregnation ratio 2.0, tcarb = 400 to 700 °c) on the amount of adsorbed gph for 240 min is presented in fig. 4. although the specific surface area shows an impact on the amount of the adsorbed gph, the difference in q240 for the samples with relatively close values activated carbons for glyphosate removal 9 of sbet was negligible. for example, the samples with sbet values of 1317 and 1284 m2g–1 adsorbed almost the same amount of gph with values 153 and 151 mg g–1, respectively. the obtained results suggested that the increasing sbet values of ~30 m2 g–1 did not lead to a significant increase in the efficiency of gph removal. herath et al34 found that both physisorption and chemisorption mechanisms affected the adsorption of glyphosate onto the activated carbon, but that physical interactions dominantly increase with the rise of surface development. fig. 4. the influence of specific surface area (sbet) on the amount of the adsorbed glyphosate (q240) for 240 min. although 2.0ac500 and 2.0ac600 showed almost the same amount of adsorbed gph for the adsorption time of 240 min, the 2.0ac500 was selected for further adsorption study, since its synthesis requires lower carbonization temperature. kinetic study the effect of contact time on gph adsorption on selected adsorbent 2.0ac500 was performed in order to estimate the equilibrium time of adsorption (fig. 5). the uptake of gph increased gradually up to 120 min and after this time the amount of adsorbed gph was almost constant. for the investigated process, the time of 120 min can be considered as equilibrium, since there is no significant change in the amount of the adsorbed gph for longer times. the amount of adsorbed gph in equilibrium time was found to be qe = 151.87 mg g –1. in order to describe the kinetics of the process, the experimental data (fig. 5) were fitted with pseudo-first and pseudo-second-order kinetic models. the calculated kinetic parameters for both models are given in table iii. the results presented in table iii revealed that the experimental data show better fit with pseudo-second order kinetic model than with pseudo-first order model. the amount of adsorbed gph in equilibrium calculated from pseudo-sec10 mohammaad and kijevčanin ond order kinetic model was qe = 156.3 mg g –1 which is very close to the experimental value of qe = 151.87 mg g –1. according to many authors, pseudo-second order model indicates that the possible mechanism of investigated process included chemisorption of pollutants on adsorbent surface.35 fig. 5. the effect of contact time on gph adsorption using activated carbon 2.0ac500. table iii. the parameters of pseudo-first and pseudo–second order kinetic models pseudo-first order qe / mg g -1 k1 / min -1 r2 214.9 0.0773 0.944 pseudo-second order qe / mg g -1 k2 / g mg -1 min r2 156.3 0.0011 0.994 adsorption isotherm models the experimental isotherm data together with nonlinear fits of langmuir and freundlich models are presented in fig. 6, while the calculated isotherm’s parameters are listed in table iv. fig. 6. the adsorption isotherm for gph adsorption on 2.0ac500 on 25 °c, fitted with langmuir and freundlich isotherm model. activated carbons for glyphosate removal 11 table iv. calculated isotherm parameters for langmuir and freundlich model for gph adsorption on 2.0ac500 langmuir kl / dm 3 mg-1 qmax / mg g -1 r2 0.0284 240.8 0.995 freundlich kf / (mg g -1)(dm3 mg-1)1/n n r2 41.15 3.23 0.965 both investigated models generally could be applied to describe the gph adsorption onto 2.0ac500 process, since coefficients of determination are r 2 > > 0.900 (table iv). however, the langmuir model showed better fitting with the experimental data, having r2 = 0.995. the agreement of adsorption data with langmuir model indicated that surface of investigated adsorbent is energetically homogenous and the binding sites are uniformly distributed with the same affinity. the adsorption process occurs until monolayer surface coverage and after saturation there is no additional interaction between adsorbate molecules. the monolayer adsorption capacity (qmax) according to the langmuir model was 240.8 mg g–1. the literature review of glyphosate adsorption on carbons derived from different type of biomass and other adsorbents is presented in table v. table v. comparison of glyphosate adsorption capacity of different adsorbents adsorbent adsorption parmeters qmax / mg g -1 references 2.0ac500 sbet = 1317 m 2 g-1;ph 3.55; t = 25 °c, cgph, 50–250 mg dm -3 240.8 current study rice husk char sbet = 229 m 2 g-1; ph < 4; cgph, 0–100 mg dm -3 123.03 34 carbon derived from waste newspapers sbet = 535 m 2 g-1;ph 2.5; t = 28 °c; cgph, 5–100 mg dm -3 48.4 36 eucalyptus camaldulensis bark‑mediatedchar ph 10.18, cgph = 20.28 mg l -1, contact time 78.42 min; t = 303.23 k 66.76 37 carbon obtained from sugar cane bagasse cgph, 0.338–2.704 g l -1 161.3 38 zr-mof ph 3–6; cgph, 20–70 mg l -1; contact time: 1–180 min, t, 308–3018 k 256.54 39 resin d301 t, 303.15–318.15 k; cgph, 5–50 mg/l; ph 4 833.33 40 although chen et al.40 found that resin d301 as adsorbent showed extraordinary efficiency for glyphosate removal, the adsorption capacity for 2.0ac500 of 240.8 mg g–1 is close to the adsorption capacity of zr–mof adsorbent39 and still higher than qmax of the most reported carbon-based adsorbents. therefore the 2.0ac500 could be classified as efficient and tested in real wastewaters treatments. 12 mohammaad and kijevčanin conclusion the water hyacinth biomass was used as starting material for the production of activated carbons. the activated carbons were synthetized using chemical activation with zncl2 followed by controlled carbonization. on carbonization at various carbonization temperatures: 400, 500, 600 and 700 °c the different impregnation ratios of zncl2 in range of 0.5–3.5 were applied. the chosen synthesis parameters showed significant impact on activated carbons yield and surface development. the impregnation ratio of 0.5 and temperature of 400 °c led to the highest yield of activated carbons. on the other hand, the textural properties showed that the most developed surface of 1317 m2 g–1 and the total pore volume of 0.697 cm3 g–1 has the activated carbon obtained with the impregnation ratio 2.0 and the carbonization temperature of 500 °c. this activated carbon with the best textural properties was used as an adsorbent for glyphosate, pesticide with strong negative environmental impact. experiments showed that the adsorption takes place very fast and the equilibrium time was estimated at 120 min. the adsorption isotherms were fitted with langmuir and freundlich model, and langmuir model showed better fit indicating that adsorption occurs in the form of monolayer on energetically equal and homogenously distributed adsorption sites. the langmuir adsorption capacity of qmax=240.8 mg g –1 classified selected adsorbent as very efficient one. the adsorption kinetics study revealed that glyphosate adsorption follows the pseudo-second order kinetics, which indicates possible chemisorption mechanism. и з в о д синтеза активног угља из биомасе воденог зумбула и његова примена као адсорбената у контроли загађења воде ahmad hakky mohammaad и мирјана кијевчанин универзитет у београду технолошко–металуршки факултет, карнегијева 4, 11000 београд биомаса воденог зумбула је коришћена за синтезу активног угља у процесу хемијске активације са zncl2, након чега је уследила контролисана пиролиза. примењени масени односи импрегнације zncl2 и суве биомасе зумбула били су у распону од 0,5–3,5. карбонизација је спроведена на четири различите температуре (400–700 c) у инертној атмосфери. највећи принос активног угља добијен је за однос импрегнације 0,5 и температуру карбонизације 400 c. узорци су карактерисани применом елементалне анализе, адсорпционо–десорпционих изотерми азота и сем анализе. активни угаљ добијен за однос импрегнације 2,0 и температуру карбонизације 500 c (2.0ac500) показао је вредности специфичне површине и укупне запремине пора од 1317 и 0,697 cm3 g-1, редом. адсорпција глифосата, пестицида са јаким негативним утицајем на животну средину, била је брз процес, са равнотежним временом од 120 min. изотерме адсорпције су корелисане langmuir и freundlich моделом. langmuir адсорпциони капацитет qmax = 240,8 mg g-1 за 2.0ac500 класификовао је одабрани адсорбент као веома ефикасан. тестирани процес адсорпције пратио је кинетику модела псеудо-другог реда. 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2university of belgrade, faculty of physics, p. o. box 44, 11000 belgrade, serbia (received 10 july, revised 5 august, accepted 8 august 2022) abstract: rate coefficients for electron impact dissociation of the n2 molecule under the influence of crossed radio-frequency (rf) electric and magnetic fields were calculated for field frequencies of 13.56, 100 and 200 mhz and for root mean square values of the reduced electric field strength of 300 and 500 td. the root mean square values of the reduced magnetic field were varied from 0 to 2000 hx. тhe effects of the strength of the rf fields and their frequency on the rates for the dissociation to neutral fragments and for the dissociative ionization are discussed. the temporal evolution of the rate coefficients during one period of the rf field is shown and discussed. keywords: nitrogen molecule; neutral fragments; dissociative ionization. introduction in addition to being the main component of the earth’s atmosphere, nitrogen is abundantly present in the atmospheres of pluto, titan and triton, and in smaller quantities on mars and venus. nitrogen is also used in many rf discharge-based technologies, such as nitriding of materials,1 plasma polymerization,2 production of nanomaterials,3 medical sterilizations,4 doping of graphene and many others.5 in all these environments, the nitrogen molecules are exposed to collision with electrons. from the aspect of chemical reactivity of nitrogen plasma, one of the most important elementary processes in these collisions is certainly dissociation. dissociation can occur to form two neutral nitrogen atoms and this reaction is known as the dissociation into neutral fragments: e– + n2 → n2 * → n + n + e– (1) * corresponding author. e-mail: ristic@ffh.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc220710066r mailto:ristic@ffh.bg.ac.rs https://doi.org/10.2298/jsc220710066r 2 ristić et al. the threshold energy for reaction (1) is 9.75 ev and the majority of the electronic states of the n2 molecule lie above this energy. excess energy during dissociation is transferred to atoms. zipf and mclaughlin recognized the importance of dissociation of excited n2 molecules over radiative relaxation via a manifold of singlet valence and rydberg states.6 тhey came to the conclusion that nitrogen molecules that were excited to various 1πu and 1σu + states, whether by electron impact or uv photon absorption, mostly follow the predissociation path (i.e., the radiationless transition of a molecule from a stable excited state to an unstable excited state that leads to dissociation). photon relaxations from these singlet states via dipole -allowed transitions to the singlet ground state were expected to be dominant,7 but it turned out that they last much longer than the dissociation. namely, dissociation to neutrals occurs in a time interval of 10−13 s, while photon relaxation lasts 10−8 s.8 the main contribution to reaction (1) for electron energies lower than 100 ev stems from a family of 1πu states, which predissociate with almost 100 % efficiency. the contributions of 1σu + states to dissociation vary depending on the vibrational level of the state. at higher values of the electron energy (above 100 ev), the probability increases that the n atom and n+ are formed during dissociation, instead in reaction of two n atoms. this process can be presented by the following reaction: e– + n2 → n2 * → n+ + n + 2e– (2) and it is called dissociative ionization. it is competitive with reaction (1) and it has a threshold value of 24.29 ev.9 in this manuscript, the rate coefficients for the dissociation to neutral fragments and for dissociative ionization, under the presence of crossed rf electric and rf magnetic field, which is a situation that corresponds to the one that exists in inductively coupled plasmas, are presented. the rate coefficients were calculated for values of fields frequencies of 13.56, 100 and 200 mhz and for different strength values of electric and magnetic fields. for this purpose, the electron energy distribution functions (eedfs) that were obtained by monte carlo simulation, and the appropriate cross sections for processes (1) and (2) were used. the integral cross sections for dissociation into neutral fragments that were measured and rescaled by cosby were adopted.10 cosby obtained integral cross section (ics) by directly detecting the correlated fragment pair n + n, by a time and position sensitive detector.10 the measured ics were further adjusted by cosby according to the results of winters and as such are generally recommended and used in this work.11,12 cross sections for reaction (2) were taken from straub et al.13 in their experiment, a time-of-flight mass spectrometer was used to distinct charged particles according to their mass-to-charge ratios. for this reason, n+ generated in the dissociative ionization process were detected together with n2 2+, dissociation of n2 in e×b rf fields 3 that can also be created by electron impact. however, n2 2+ can practically be produced only in trace amounts under the considered conditions, because the highest electron energies within this work were approximately 60 ev, while the threshold for the second ionization energy of n2 is 42.88 ev. 9 the high-energy tail of the eedf barely crosses the threshold for the second ionization energy of n2 (even under conditions for which the highest energies are achieved) and very small amount of electrons are capable of performing the second ionization of the n2 molecule. therefore, it was consider appropriate to refer to the cross sections of straub et al. as the cross sections for dissociative ionization within this work.13 these cross sections were supplemented by including the threshold value for dissociative ionization of 24.29 ev.9 the cross-section values between the threshold and the first electron energy reported by straub et al.,13 which was 30 ev, were linearly interpolated. the integral cross sections used for reactions (1) and (2) in the present manuscript are shown in fig. 1. fig. 1. integral cross sections for the dissociation of n2 to neutral fragments of cosby 10 (circles) and for dissociative ionization of straub et al.13 (squares) vs. electron energy. method monte carlo simulation in order to simulate the motion of electrons through the n2 gas under the presence of time-varying electric and magnetic fields, the monte carlo (mc) code used in earlier works of our group,14,15 was modified and used.16 the motion of the electron is described by the following differential equation: 2 2 d d ( ) ( ) d d m e t t t t   = +    r r e b (3) 4 ristić et al. in eq. (3), r is the radius vector of the electron, m and e are the mass and charge of the electron, e and b are the electric and magnetic field vectors, respectively, and t is time. the time-varying electric and magnetic field vectors periodically oscillate at fixed frequency are described by eqs. (4) and (5): e(t) = 2ercos(2πft)k (4) b(t) = 2brsin(2πft)j (5) where k and j are the unit vectors in the cartesian coordinate system that characterize the direction of electric and magnetic fields, while f is the frequency of their oscillation. er and br are the root mean square values of the electric and magnetic field, respectively. absolute values of electric and magnetic field within this work will be replaced with reduced electric field and reduced magnetic field, defined as quotients er/n and br/n, n being the gas number density. the electric and magnetic fields are mutually perpendicular, and phase shift among them is /2 rad. the numerical solutions of the eq. (3) were used to calculate the position and velocity of the electron in each small time step of the simulation (much smaller than the period of oscillation) by the numerical iterative runge–kutta method.17 electrons were generated with a certain value of the kinetic energy and were simulated one by one under the defined conditions (er/n, br/n and f values specified). the simulation procedures follow every electron until it reaches the quasi-steady state, i.e., the state where energy received from the electric field is balanced with the energy that is lost in non-elastic collisions with the n2 molecules. when the quasi-steady state was reached, the ensemble of electrons obtains stable oscillations of mean electrons energy (m) over time and at this point the eedfs were sampled over one period of the fields oscillation. these eedfs are essential for obtaining the rate coefficients, as shall be explained further. the nitrogen gas in which the electrons were simulated was considered to be at 133.3 pa (n = 3.221022 m-3) and the zero-temperature gas approximation was used, meaning that every n2 molecule was in its ground electronic, vibrational and rotational state. the procedures of the simulation included the possibilities of all real electron-molecule interactions under the conditions assumed: elastic collisions, non-elastic collisions (rotational, vibrational and electronic excitations) as well as ionization. the database of the simulations containing the cross sections for all relevant processes was carefully composed. the probability of a certain scattering process at a given electron energy  depended on the corresponding effective cross section (). the decision as to which collision process will occur was left to а pseudo -random generated number, as were the scattering angles of the electron after the collision. electrons that were created in the ionization process were also simulated. the mc code was successfully tested on model gases,18,19 by comparing the transport parameters of the electrons with the benchmark values from the literature.20 for other details about the mc code, see ristić et al. and ristić et al.15,16 cross-section database finding an optimal set of cross-sections for the electron-n2 molecule interaction was crucial for a successful simulation. for elastic scattering, the recent cross-section data of allan in the electron energy range from 0 to 5.5 ev was used.21 for electron energies from 6 to 10 ev, the data of sun et al. was used,22 while in the energy region from 10 to 70 ev, the data of gote and ehrhardt was used.23 in the high-energy region from 70 to 90 ev, the data of nickel et al.24 was used. cross-section values for rotational excitations were adopted from itikawa and mason for j = 0 → 2 and j = 0 → 4 excitations.25 for vibrational excitations, the integral cross-sections dissociation of n2 in e×b rf fields 5 that were measured in our laboratory were used.26 these measurements include excitations into the first ten vibrational levels of the ground electronic state of the n2 molecule. the cross-sections for a’1σu −, w1∆u and a ’’1σg + singlet electronic states were adopted from itikawa,27 compilation proposed by brunger et al.,28 based on the experiments of trajmar et al. and campbell et al.,29,30 and the theoretical calculations of ohmori et al. and gillan et al.31,32 the cross-section values were also taken from brunger et al,28 for the excitation of a1πg state on the basis of electron energy loss measurements of finn and doering, 33 direct detection of the excited molecule of mason and newell and swarm experiments.34,35 for the c4 ’1σu + and b’1σu + states, values from ajello et al. were taken.35 the b1πu state cross-section was adopted from james et al.,36 and recent cross-section measurements of the c3 1πu and o3 1πu states were adopted from malone et al. 37 the values of the cross sections for the excitations of the a3σu +, b3πg, w 3∆u and b ’3σu − triplet electronic states were used from itikawa,27 which were recommended by brunger et al. based on previously mentioned studies.28-32 cross-section proposed by brunger et al.,28 for e3σg + state (based on several beam experiments)29,30,38,39 and for c3πu state (based also on beam experiments) 29,30,39,40 were used. f3πu and g 3πu electronic state cross sections were used from malone et al. 37 ionization cross sections were adopted from itikawa,27 based on lindsay and mangan41 and straub et al. measurements.13 dissociation was included implicitly by taking into account all significant electronic states that contribute to it, as was explained in the introduction section. validation of the present cross-section database for nitrogen was performed by comparing basic transport properties of electrons (drift velocities and diffusion coefficients) obtained by the present simulation with the experimentally measured ones. in a previous works we presented these comparisons for drift velocity, longitudinal diffusion coefficient and ionization coefficient, whereby excellent agreements were obtained.14,42 comparison was also made with the boltzmann equation-based software bolsig+ (version 1.1).43 by including the present cross-section database in both our monte carlo simulation and bolsig+ oscillating field routine, we obtained the agreement of period averaged mean electron energy within 2 % only. rate coefficient calculation the eedfs and corresponding mean electron energies were sampled within one phase of the electric field oscillation after the quasi-steady state was reached in the simulation. the eedfs obtained were then normalized by the relation: ( ) 0 , , d 1mf t     = (6) in eq. (6), the normalized eedf at the specific time t is denoted by f(m, , t), while  represents the actual kinetic energy of the electron. for every f(m,,t) in the specific moment in time (that is, in the specific phase of the electric field oscillation), the corresponding rate coefficient, k(m, t) was calculated by the following relation: 44 ( ) ( ) th 2 , ( ) , , dm mk t f t m           =  (7) where () is the effective cross-section for a given process with a threshold energy th. results and discussion according to eq. (7), the rate coefficients for dissociation processes (1) and (2) were calculated by implementing the eedfs obtained after reaching the quasi-steady state by the mc code for given input parameters. the input parameters imply er/n values of 300 and 500 td (1 td = 10 −21 v m2), br/n values 6 ristić et al. of 0, 1000 and 2000 hx (1 hx = 10−27 t m3) and values of frequency 13.56, 100 and 200 mhz. as stated earlier, the integral cross sections for the calculation of the rate coefficients for the dissociation of n2 to neutral fragments were adopted from cosby,10 while the rates for dissociative ionization were calculated based on integral cross sections measured by straub et al. with implemented energy threshold for process (2).9,13 background physics of electrons motion in crossed electric and magnetic rf fields is well known and will only be slightly addressed in this paper.16,20 since the mc simulation records many eedfs within one period of the field’s oscillation, a fine time evolution of the rate coefficients on the nanosecond scale was obtained. the rate coefficients for dissociation of n2 to neutral fragments are presented on fig. 2. for all input parameters. first, it should be noticed that the rate coefficients are oscillating at twice the frequency of the applied rf fields. the reason is that the energy of the electron is independent of the absolute direction of the electric field. the rate coefficients depend on all the varied parameters. it should be noted that when discussing the dependence of the rate coefficient on one specific parameter, the other parameters are considered as constant. by observing the coefficients at the lowest considered frequency of 13.56 mhz for given er/n and br/n, it was noticeable that the highest and lowest (zero) amplitude values of the coefficients were reached, which is expected because at that frequency, the electrons efficiently track the electric field changes and lag only slightly behind them. therefore, when the electric field (which determines the energy of the electron) reaches a maximum or minimum, the eedf also has the largest share of high-energy electrons (for given er/n and br/n) and consequently, the rate coefficient is the largest. on the other hand, when the electric field passes through the zero value, after which the electrons are slowed down, the share of fast electrons in the eedf is the smallest and the rate coefficient reaches its lowest value. at 13.56 mhz, this lowest value is zero, as can be seen from fig. 2. at higher frequencies, the eedfs have a narrowed oscillation range due to a more pronounced electron delay behind the electric field. for this reason, the rates also oscillate over a narrower range: they do not reach the maximum amplitude values they had at lower frequencies, but they do not fall to zero either. one of the characteristics of the time evolution of the rate coefficients is the existence of phase delay with respect to the electric field. this delay is also a consequence of the inertia of the motion of electrons through the n2 gas and is more pronounced at higher frequencies.14 regarding the influence of the strength of the electric and magnetic fields on the appearance of the rate coefficients, they are also noticeable in fig. 2. both electric and magnetic field strongly affect the value of the rates. at stronger electric fields, the share of high-energy tail of eedf is higher, and thus the overlap dissociation of n2 in e×b rf fields 7 integral in eq. (7) is higher, which leads to a higher values of the rate coefficients. fig. 2. rate coefficients for the dissociation of n2 to neutral fragments vs. the phase of the rf electric field at the indicated pair of f and er/n and for br/n = 0 hx (full line), br/n = 1000 hx (dashed line) and br/n = 2000 hx (dotted line). at higher br/n values, the profile of the rate coefficient with time is narrowed and at the same time reduced in height, due to the complex action of the magnetic field on the electrons. hence the overall effect of increasing magnetic field on the rate coefficients shown here is their reduction. for most practical applications in which nitrogen dissociation may be of interest in plasma chemistry and plasma technology of nitrogen-containing systems, an important fact is the time-averaged rate coefficient during one period of rf field oscillation. to this end, these period averaged rates for the dissociation of n2 into neutral fragments are given table i for all considered parameters. in the ranges of varied input parameters, the average rate coefficient changed its value from 0.341×10−9 to 3.929×10−9 cm3 s−1. by analyzing the data in table i, it could be concluded that an increase of er/n leads to larger values of the rate coefficient, while an increase of br/n leads to their lowering. increasing the fre8 ristić et al. quency does not have much effect on the value of the period averaged rate coefficients, nor can its effect be easily generalized. table i. period averaged rates (10-9 cm3 s-1) for the dissociation of n2 into neutral fragments under various conditions f / mhz er/n = 300 td er/n = 500 td (br/n) / hx 0 1000 2000 0 1000 2000 13.56 1.156 0.786 0.492 3.929 2.828 1.972 100 1.064 0.693 0.391 3.778 2.871 1.926 200 0.971 0.641 0.341 3.796 2.948 1.923 the rate coefficients for dissociative ionization of n2 are presented in fig. 3. for all input parameters. most of what has been said about the rate coefficients of dissociation into neutral fragments also applies here: er/n, br/n and f values fig. 3. rate coefficients for dissociative ionization of n2 vs. phase of the rf electric field at the indicated pair of f and er/n and for br/n = 0 hx (full line), br/n = 1000 hx (dashed line) and br/n = 2000 hx (dotted line). dissociation of n2 in e×b rf fields 9 have qualitatively the same effect on the shape of the rates. the period averaged rates of the dissociative ionization for all considered parameters are given in table ii. however, in the case of dissociative ionization (unlike the previous case), the effect of frequency on the rates is evident. raising the f significantly lowers the rate coefficients for dissociative ionization. the rates for dissociative ionization were about 10 to 15 times lower than those for the dissociation into neutral fragments. this was expected since the cross-section for dissociative ionization is lower, while the energy threshold is higher. from table ii, it could be seen that the rate values ranged from 0.022×10−10 to 3.020×10−10 cm3 s−1. nevertheless, dissociative ionization remains an important source of n+ in plasmas, in addition to direct ionization of the n atom. table ii. period averaged rates (10−10 cm3 s−1) for dissociative ionization under various conditions f / mhz er/n = 300 td er/n = 500 td (br/n) / hx 0 1000 2000 0 1000 2000 13.56 0.3493 0.2153 0.1275 3.020 1.939 1.271 100 0.2364 0.1112 0.0391 2.494 1.614 0.858 200 0.1699 0.0713 0.0225 2.181 1.309 0.604 recently, sode et al. developed a new model for the calculation of ion and radical densities in inductively coupled nitrogen plasmas.45 they proposed the equations for the calculation rate coefficients for numerous elementary processes in plasma, among them the dissociation of n2 to neutral fragments and for the dissociative ionization of n2 (see table ii of their work, reactions 1.6 and 1.9). by implementing the mean electron energy obtained from the present mc simulation in their equations, significant difference in the values of the rate coefficients were obtained. for example, at 13.56 mhz, 300 td and 0 hx, the mean electron energy obtained by the present mc code was 6.47 ev and the rate for dissociation to neutral fragments was 3.6×10−9 cm3 s−1 (the present value was 1.1×10−9 cm3 s−1), while for dissociative ionization, the rate was 1.1×10−10 cm3 s−1 (the present value was 0.35×10−10 cm3 s−1). sode et al. stated that they assumed maxwellian eedf in their equations,45 but here it could be seen that the dissociation rates in that case were roughly three times higher than the ones presented here, based on non-equilibrium eedfs. in addition, the rates obtained in that way were independent of the magnetic field or frequency values. note that the cross sections that sode et al. used were practically the same as those used here. the described example shows that the dissociation coefficients presented here could significantly improve the kinetic calculations in nitrogen plasma chemistry. 10 ristić et al. conclusions the rate coefficients for electron impact dissociation of the n2 molecule to neutral fragments, as well as for the dissociative ionization process were calculated under the presence of rf electric and magnetic fields. тhe choice of parameters included three frequencies: 13.56, 100 and 200 mhz, two er/n values: 300 and 500 td and br/n values: 0, 1000 and 2000 hx. therefore, the nitrogen molecule dissociation processes, as one of the most important processes in nitrogenous rf plasma chemistry, were quantified for the stated conditions. fine temporal evolution of the dissociation rate coefficients within the rf field phase was presented with an adequate explanation. the time -averaged values of the rate coefficients were also shown. dissociative ionization is by an order of magnitude a rarer process compared to dissociation into neutral fragments, which is expected due to its lower cross-section values and higher threshold. acknowledgements. this work was supported in part by the ministry of education, science and technological development of the republic of serbia under contracts no. 451-03 -68/2022-14/200146 and 451-03-9/2022-14/200162. the authors gratefully acknowledge the assistance of muna aoneas. и з в о д дисоцијација n2 ударом електрона у електромагнетним rf пољима мирослав ристић1, радомир ранковић1, мирјана м. војновић2, виолета в. станковић2 и горан б. попарић2 1универзитет у београду – факултет за физичку хемију, студентски трг 12-16, београд и 2универзитет у београду – физички факултет, студентски трг 16, београд коефицијенти брзине дисоцијације молекула n2 ударом електрона под утицајем укрштеног радиофреквентног (rf) електричног и магнетног поља израчунати су за фреквенције од 13.56, 100 и 200 mhz и за средње квадратне вредности редукованог електричног поља од 300 и 500 td. средње квадратне вредности редукованог магнетног поља вариране су од 0 до 2000 hx. дискутовани су eфекти јачина rf поља и њихове фреквенције на коефицијенте брзине за дисоцијацију на неутралне фрагменте и за дисоцијативну јонизацију. приказана је и продискутована временска еволуција коефицијената брзине у току једног периода rf поља. 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l. c. pitchford, plasma sources sci. technol. 14 (2005) 722 (https://doi.org/10.1088/0963-0252/14/4/011) 44. d. s. belić, chem. phys. 130 (1989) 141 (https://doi.org/10.1016/0301-0104(89)87043-0) 45. m. sode, w. jacob, t. schwarz-selinger, h. kersten, j. appl. phys. 117 (2015) 083303 (http://dx.doi.org/10.1063/1.4913623). https://doi.org/10.1007/b83711 https://doi.org/10.1016/0370-1573(83)90071-6 https://doi.org/10.1088/0953-4075/34/7/303 https://doi.org/10.1088/0022-3727/21/5/009 https://doi.org/10.1088/0953-4075/29/8/017 https://doi.org/10.1063/1.432075 https://doi.org/10.1088/0022-3700/20/15/035 https://doi.org/10.1088/0022-3700/20/15/035 https://doi.org/10.1103/physreva.40.3524 https://doi.org/10.1088/0953-4075/23/12/015 https://doi.org/10.1103/physreva.85.062704 https://doi.org/10.1103/physreva.37.3570 https://doi.org/10.1088/0953-4075/27/12/019 https://doi.org/10.1088/0953-4075/27/12/019 https://doi.org/10.1016/s0301-0104(98)00383-8 https://doi.org/10.1007/b83711 https://doi.org/10.1016/j.chemphys.2015.09.014 https://doi.org/10.1088/0963-0252/14/4/011 https://doi.org/10.1016/0301-0104(89)87043-0 http://dx.doi.org/10.1063/1.4913623 volume 87 (2022) 1 subject index 1,2,3,4-tetrahydroquinoxaline, 169 acetyl-cholinesterase (ache), 293 activation, 1395 active pocket, 293 adsorption, 939, 1395 advanced oxidation processes, 57 agricultural activities, 121 agricultural by-product, 247 air pollution, 157 alanine, 451 aldol condensation, 813 aliphatic c, 761 alkali-treated carbon materials, 867 alkane branching, 41 ammonium bifluoride, 925 anilidopiperidine, 169 anionic clays, 1011 anniversary, 1439 anova, 589 anthropogenic impact, 519 antibacterial activity, 629, 813, 1245 anticancer effect, 969 anticorrosive method, 845 antifungal activity, 813 antimicrobial activity, 1349 antimicrobial and cytotoxic activity, 451 antioxidant activity, 1245 apoptosis, 545 aromatic c, 761 aromaticity, 1439 artificial neural networks, 615 bacteria, 775 “belgrade issues”, 27 bet, 1285 bi2o3 polymorphs, 1367 bifunctional molecules, 785 binding free energy calculations, 693 bioaccessibility study, 723 bioadsorbent, 589 bioavailability of cu and cd in sediment fractions, 133 biodegradation, 7 biological activity, 193 biomarkers, 7 biomass ash, 1091 biomass, 437 biopolymer, 657 biosynthesis, 401 bisoprolol, 1185 bisphenol a, 109 bjh, 1285 blood analytes, 1025 bluetooth, 603 bombyx mori, 331 bor river, 775 boric acid, 749 boundary layer, 911 breast cancer cell line, 1109 breast cancer, 193 bubble counting procedure, 1049 buchwald–hartwig reaction, 169 butyrylcholine-esterase (bche), 293 calcium and sodium chlorides, 879 calculation of dendrites’ life duration, 1049 cancer, 1381 cancerous and normal cell lines, 1125 carbene, 857 carbohydrate, 641 carboxyl group, 761 carboxymethyl cellulose synthesis, 247 ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 2 journal of the serbian chemical society catalytic pyrolysis, 1219 catalytic site, 465 cation-π interactions, 465 cell cycle analysis, 181 cell cycle, 969 cellulose, 247 central composite design, 589 chalcopyrite, 925 characterization, 575, 1327 chemical mechanical planarization, 219 chemical warfare agents, 233 cheminformatics, 321 chemistry, 275 children toys, 723 chitinolytic enzymes, 677 chromatography, 983 clay nanofiller, 1203 clinical candidates, 785 cobwebs structure, 891 comet test, 1245 comfa, 1381 community health groups, 1025 composition-dependent properties, 1203 computational chemistry, 845 computational drug design, 1381 computational model, 1025 comsia, 1381 conducting polymer, 1297 consensual docking, 693 cooperative learning, 275 cooperativity, 785 copper tetraammine sulphate, 925 copper(i), 307 copper(ii) complexes, 451 copper, 775 corrosion current, 219 corrosion potential, 219 covalent, 1157 crystal structure, 545, 561, 1259 crystallization region, 263 crystallography, 321 current efficiency vs. electrolysis conditions, 1049 cyclam, 451 cyclic condensation, 1117 cyclic voltammetry, 345, 603 cytotoxic activity, 1349 cytotoxicity assessment, 1125 cytotoxicity, 629, 1075 čukarički rukavac, 95 dachs-eisenreich model, 157 decolorization, 57 defunctionalization, 669 degree of microphase separation, 1203 dependent variables, 1025 deployable laboratory, 233 depollution, 1327 deposition flux, 953 derivatization, 109 desulfurization, 575, 1219 dft, 307, 575, 845 dibenzoazepine-based compound, 1171 digital materials, 531 dihydroxyanthraquinone, 1171 dipole moment, 193 dissociative excitation, 479 dissociative ionization, 479 dma method, 355 dmso effect, 1143 docking, 321, 545 dot blotting, 677 doxorubicin, 641 drug carriers, 1075 drug delivery, 641, 1157 drug discovery, 321 dta, 1395 dynamic viscosity, 355 ec50, 657 ecotoxicity, 657 edta, 561 educational video, 531 effluent, 1327 electrochemical capacitance distribution, 867 electrochemical impedance spectroscopy, 219 electrochemical study, 219 electrochemical, 775 electrochemistry, 1409 electrodeposition, 899 electron localization function, 707 electron-molecule collisions, 479 electrophilic reagent, 857 emerging contaminants, 1425 emulsion, 641 ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ volume 87: subject index 3 encapsulation, 641 enrichment factor, 953 environmental pollution, 1425 environmental protection, 377 environmental variables, 389 enzyme for fat hydrolysis, 997 enzymes, 437 esterification, 761 e-teaching, 531 eucalyptus, 983 ferrier rearrangement, 629 film theory, 911 fire resistance, 363 flow cytometry, 969 fluorescent assay, 677 forced degradation study, 1185 fractionation, 761 fragmentation pathways, 1185 free radical polymerization, 629 freundlich adsorption isotherm, 83 fukui functions, 845 fungi, 437 gas chromatography, 233 gc-ms, 109, 145, 1117 gene expression analyses, 181 genetic algorithm, 437 geochemical correlation, 7 gibbs energy, 1157 gis, 389 glass transition temperature, 629 global descriptors, 845 glycine, 451 glycolipid, 1327 glycosylation, 677 gradient elution, 615 granulation, 57 graphite, 57 green analytical chemistry, 939 green chemistry, 669 green synthesis, 401 growth inhibition, 657 guanylhydrazone, 1259 hardness, 899 health risk, 723 heavy metals, 1313 herbal product, 1063 heterocycles, 169 heterogeneous catalysis, 669 high performance liquid chromatography, 615 high volume sampling, 157 history, 1439 homopolymer, 629 hpmc, 829 hydrogen peroxide, 57 hydrothermal synthesis, 205, 749 ibuprofen, 1075 icp-ms, 69 icp-oes determination, 1313 icp-oes, 69, 83, 723 igmh, 707 impurities, 1185 in vitro antimicrobial analysis, 1359 in vitro antioxidant analysis, 1359 in vitro, 1109 inaa, 69 industrial waste, 997 infrared spectroscopy, 41 inhibitiоrs, 1409 interaction, 1157 invariant point, 263 ion concentration, 953 irradiation, 813 isolate, 1327 isoxazole, 707 la (ⅲ), 505 lamiaceae, 1359 laser, 939 ldh derived mixed oxides, 1011 leaching, 145, 377 lfdft, 561 ligand hydrophobicity, 1143 limiting apparent molar volume, 1171 liver cancer cell line, 1109 lotus, 983 lower secondary school, 275 lung cancer cell line, 1109 marker substance, 1063 mas spectrometry, 233 mass transfer, 505 mechanical activation, 491, 1367 medt, 707 ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 4 journal of the serbian chemical society melissopalynology, 983 melts, 879 mesoporous zeolite, 1219 meta and para-substituted benzylidene thiosemicarbazides, 1125 metal complexes, 1259 metallic ions, 83 metals adsorption, 133 method development, 1063 method optimization, 205 method validation, 1063 methyl orange removal, 1011 michaelis addition, 813 microextraction, 109, 505 microporous zeolite, 1219 microtubes, 1297 microwave-assisted extraction, 1359 milk protein, 1273 mitochondrial membrane potential, 969 model, 911 modified electrodes, 401 molecular cloning, 677 molecular conformation, 331 molecular docking, 293 molecular dynamics, 545, 693 molecular electrostatic potential (mep), 1157 molecular hybrids, 1349 molecular modeling, 1273 monitoring, 121 mössbauer spectroscopy, 1367 mtt assay, 1245 multi-element determination, 69 multifunctional coatings, 1035 multivariate analyses, 389 n-acylhydrazone, 181 nanomaterials, 401 nanostructure, 1157 nanotechnology, 401 natural fibre composites, 363 nbo, 561 nitrogen mustard, 233 non-steroidal anti-inflammatory drug, 829 ns2b/ns3pro, 693 ocular delivery, 829 oil degradation, 997 oil genetic types, 41 opls-da, 1237 orca software, 845 organic coatings, 363 organic matter, 7, 775 organic pollutants analysis, 205 ovalbumin model system, 1143 oxamides, 545 ozone cation, 479 pannonian basin, 41 pca, 983, 1237 pcl microspheres, 1075 persistent organic pollutants (pops), 95 petroleum pollutants, 7 pfcs occurrence, 1425 pfoa, 1425 pfos, 1425 phenolic group, 761 philately, 27 photodegradation, 1285 phthalates, 355 physicochemical parameters, 121, 983 physicochemical properties, 1259 piperazine, 169 plant metabolomics, 1237 plasma electrolytic oxidation, 1035 plasticizers, 145 point-of-care testing, 603 pollution indices, 519 polyethylene, 891 polyubiquitination, 785 polyurethane, 1203 polyvinyl chloride, 355 potentiodynamic polarization, 1409 potentiostatic electrolysis, 879 powder xrd, 1367 pr, 735 prince michael, 27 proapoptotic effect, 969 prostate cancer cell line, 1109 protein aggregation, 1143 protein structure, 321 proton-coupled electron transfer, 345 prototype, 775 pulping, 247 pvc, 145 pyrazolone, 813 ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ volume 87: subject index 5 quantum chemical studies, 575 raman analysis, 749 raman spectroscopy, 331, 1367 raman, 1285 reactive blue 52, 57 reactivity, 845 recovery time, 1157 recycle of lipid waste, 997 reduction of wo3, 879 relative indentation depth, 899 remediation, 95 renewable energy, 345 response surface method, 437 response surface, 589 review, 401 rheologic properties of coating suspensions, 491 rhodium, 561 river sediments, 519 river water, 109 schiff bases, 307, 1259 schreinmakers method, 263 secosteroids, 969 sediment maturation mechanism, 133 sensitive water bodies, 121 sequential extraction, 83, 133, 1091 serbian oil fields, 41 side reactions, 1117 silk fibroin, 331 silylene, 857 simulation, 505 smartphone, 603 sodium azide, 575 solid compounds, 735 solid state synthesis, 1367 solubility, 263 solute-solvent and solute-solute interactions, 1171 specific surface area, 749 spectrochemistry, 939 spectroscopy, 1273 splp test, 1091 srk, 735 stainless steel, 1297 stamps of serbia, 27 standardless analysis, 69 star-shaped pcls, 1075 statin, 1273 student centred-learning, 275 submerged fermentation, 997 submicroscopic level, 531 sucrose-derived carbons, 867 sugar-derived carbons, 867 supercritical extraction, 735 superoxide dismutase, 465 surface oxygen groups, 749 surface properties, 205, 1203 surface, 1409 swelling and erosion, 829 tablets, 1185 talc-based fillers, 491 tank test, 1091 tbq, 1245 tclp test, 1091 ternary complex, 785 tg, 1395 the pulsating current, 899 thermal performance, 1203 thermal pyrolysis, 1219 thermodynamic modeling, 735 topčider river, 95 topography, 899 torsional free energy, 193 toxic elements, 723 toxicity, 1245 trajectory analysis, 953 tridentate coordination mode, 307 triple nature of chemical concepts, 275 tungsten oxide, 879 ultrasound-assisted extraction, 1359 undruggable proteome, 785 usnic acid, 1063 uv–vis measurements, 181 “vienna issues”, 27 viscosity b-coefficient, 1171 viscosity, 41, 263 waste sawdust, 205 waste valorisation, 997 wastewater treatment, 939, 1011 water leaching, 925 water quality, 389 wd-xrf, 69 ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 6 journal of the serbian chemical society wst-1 assay, 1125 x-ray crystallography, 307 xrd, 1285, 1395 xrf, 1395 zeolites, 1035, 1395 zinc ferrite, 377 zinc oxide, 377 zinc recovery, 377 γ-irradiation, 891 ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice effect of an industrial chemical waste on the uptake j. serb. chem. soc. 87 (0) 1–13 (2022) original scientific paper jscs–11831 published 18 november 2022 1 controlling reactions during heavy metal leaching from municipal solid waste incineration fly ash weifang chen*, yegui wang, minzhu hu, yonglun li and guilin fang school of environment and architecture, university of shanghai for science and technology, 516 jun gong road, shanghai 200093, china (received 5 may, revised 26 july, accepted 1 august 2022) abstract: this research aimed to characterize the physical and chemical properties of municipal solid waste (msw) incineration fly ash, as well as to investigate the leaching of heavy metals during toxicity characteristics leaching procedure (tclp) process. to understand the leaching behaviour, concentrations of heavy metals, chloride, sulphate and calcium at various leaching, time was monitored together with ph of the leaching solution. results showed that the chemical compositions of fly ashes led to differences in leaching environment. for the two fly ashes under study, one resulted in a leaching environment with ph of 10–12 while the other had ph about 6–7. based on ph, anions and cations concentrations, minteq software was employed to investigate the speciation of heavy metals. results showed that the shift in precipitation/dissolution balance of carbonate and hydroxides of heavy metals could explain the fluctuation in metal concentrations during the leaching process, which indicates that leaching was probably controlled by these reactions. on the other hand, addition of edta changed the controlling reactions. chelating reactions between heavy metals and edta led to much higher leaching toxicity due to the presence of heavy metals, showing that the presence of chelating organics in natural environment may facilitate heavy metal leaching. keywords: characterization; toxicity characteristics leaching procedure; minteq; precipitation/dissolution. introduction industrialization and urbanization in recent decades had led to alarming rises in the generation of municipal solid waste. it was reported that, in 2020, china produced 242 million tons of msw which were mainly disposed of by landfill and incineration.1 the disposal by landfill was intensive and the leaching of heavy metals into groundwater was problematic.2 incineration, on the other hand, * corresponding author. e-mail: chenweifang@usst.edu.cn https://doi.org/10.2298/jsc220505065c mailto:chenweifang@usst.edu.cn https://doi.org/10.2298/jsc220505065c 2 chen et al. was effective in reducing both volume (up to 90 %) and mass (up to 70 %) while generating electricity.3,4 therefore, it was considered a promising method and had witnessed rapid developments. however, air pollution and generation of residues are some of the concerns related to incineration. solid residues from msw incineration include bottom ash and fly ash.5 between them, fly ash is considered hazardous as it contains large amount of heavy metals and toxic organics.6 besides residues from msw incineration, fly ash also contained reagents and products from flue gas neutralization. therefore, the main elements in fly ash include o, cl, s ca, si, al, fe, mg, na and k, as well as toxic elements such as pb, cd, cr, ni, zn, cu, etc.7 msw fly ash accounted for about 3–15 % of the msw depending on the incineration technology.8 this means that, in china, millions of tons of fly ash need to be processed annually. due to the continuous growth in fly ash generation, many countries have called for reuse and recycling. fan et al. believed that msw fly ash is basically an aluminosilicate material.9 therefore, it could be reused as cementitious materials, soil amendment or light-weight aggregate in construction and road pavement materials.10 however, it is important to remove harmful elements before fly ash can be reused. luo et al. classified the fly ash treatment into three categories, that is, chemical and physical separation, stabilization/solidification and thermal treatment.11 the purposes of these treatments are to re-configure ashes (e.g., vitrification or sintering) to remove pollutants (e.g., washing), or to immobilize pollutants (e.g., solidification, geo-polymerization).12–15 toxicity leaching tests were often conducted to compare the effects of heavy metal removal or immobilization. the most commonly used method was toxicity characteristics leaching procedure (tclp) recommended by us epa. leaching of heavy metal was a complicated process. many reactions, involving precipitation/dissolution, diffusion, sorption and surface coating, etc., could occur during leaching.16 that is why physical and chemical factors could all influence the leaching of heavy metals. the former includes particle size, shape and porosity, temperature, time, etc., while the later includes ph, redox, complexation or sorption conditions and leaching kinetics, etc.17,18 therefore, the conditions of leaching tests have to be specified so that the results are comparable. sample preparation, composition of leachant, mixing method, liquid to solid ratio, time, temperature and filtration method were all elucidated in the tclp method. still, researchers have argued that tclp may not be able to truly reflect the leaching behaviour of ashes of high alkalinity. lu et al. compared the leaching potentials of raw and cement-solidified bottom ashes via tclp, multi-ph tclp and edta-mediated tclp and found that tclp underestimated the leaching of heavy metal.19 they recommended that edta-mediated tclp may be more fly ash heavy metal leaching 3 suitable for the evaluation the heavy metal leaching by alkaline materials, especially in the environment where organic acid may be present. this research intended to study the properties of two fly ashes and investigate the heavy metal leaching characteristics during tclp tests. ph, heavy metal, chloride, sulphate, carbonate and calcium concentrations in leaching solutions were monitored. software minteq 3.1 was used to calculate the metal speciation and solubility under the leaching conditions. at the same time, the leaching of heavy metal with addition of edta was also studied for comparison. our ultimate goal was to study the feature of heavy metal leaching in fly ashes and advance the understanding of interaction between fly ash and leachant in order to pave the way for a better prediction of heavy metal impact on the environment. experimental fly ashes fly ashes employed in this research were from municipal solid waste incineration plants in zhejiang and guangdong provinces of china. the samples were designated as fa1 and fa2, respectively. fly ash characterization the moisture content was determined according to the national standard method of china (gb/t212-2008). the proximate analysis was conducted according to astm standard method e1131-08. the chemical compositions of the samples were determined by x-ray fluorescence spectroscopy (xrf-1800, shimadzu, japan) and the crystalline phases were characterized by x-ray diffraction (xrd, rigaku uitima iv, japan) with a cukα radiation at 40 kv and 30 ma, as 2θ ranging from 5 to 90°, while ftir analysis was carried out via nicolet is10 (thermo scientific, germany). ph and the acid neutralization capacity (anc) of fly ash were measured according to the methods proposed by liu et al.20 specifically, 1 g of fly ash and 100 ml of deionized water were put into a 300 ml conical flask and stirred at 200 r/min for 15 min and ph was measured. half of the mixture was next titrated with 1 mol/l acetic acid to ph 7.0. the amount of acetic acid consumed was the anc. analysis of heavy metal contents the contents of heavy metals in fly ash were measured based on a standard method of china (hj803-2016) which was designed specifically for the analysis of metal in soil/sediments, via digestion with nitric acid/chloric acid. heavy metals in the digested solution were determined by inductively coupled plasma optical emission spectromet ry (icp-oes, optima8000, perkinelmer, us) after pretreatment. heavy metal leaching toxicity tests fly ash was dried and sieved with a 200-mesh sieve before use. tclp (usepa method 1311) and an edta-modified tclp were adopted to analyse the leaching toxicity potentials of fly ashes for comparison. with edta-modified tclp, the concentration of edta in the leachant was set at 1 mmol/l. all other procedures were conducted as stipulated by the tclp method. to study the leaching behaviour of heavy metal during tclp tests, instead of the 18 h recommended by the standard method, leaching time was set at 2, 4, 6, 8, 10, 12, 14, 16 and 18 h, respectively. after mixing for the pre-determined amount of time, the mixture was fil4 chen et al. tered and the filtrate analyzed for ph, chloride, sulfate, calcium, carbonate and heavy metal concentrations. results and discussion fly ash characterization the proximate analysis was carried out on both fly ashes to understand their basic properties as shown in table i. 78 and 89 % of fa1 and fa2 were ash. this is reasonable as fly ashes were the results of high temperature incineration and most of the volatiles or organics were burned off. the high ash contents could be attributed to ca, si compounds and other residual metals.21 according to the proximate analysis, about 22 and 11 % of the fly ash were volatile and fixed carbon indicated the residual organics. table i. physicochemical properties of fa1 and fa2 sample proximate contenta, wt. % moisture content, % ph anc / ml g-1 volatiles fixed carbon ash fa1 9 13 78 0.34 12.7 7.3 fa2 3 8 89 0.34 12.4 3.8 acontent of fixed carbon = 100 – content of ash – content of volatiles besides the proximate analysis, table i also listed the moisture content, ph and anc of these ashes. both ashes had low moisture contents (only 0.34 %) but high in ph and acid neutralization capacity (anc). the ph measured here was the natural ph in contact with water. fly ashes in this research originated from bag filters which were used to treat flue gas. lime was sprayed to neutralize acidic gases. therefore, the high ph was most likely caused by the neutralization products or cao and ca(oh)2 that remained. anc was measured to evaluate the presence of alkaline compounds and their availability and reactivity.22 although both fly ashes showed similar phs as shown in table i, their anc values differed. fa1 had a much higher anc indicating that the alkalinity in fa1 may be more readily neutralized by acetic acid. xrf and xrd analysis were next performed to further clarify the chemical compositions and crystalline structures of fly ashes. the xrf results are presented in table ii. xrd results in fig. 1 proved the presence of chloride salts, i.e., nacl and kcl. other crystals observed include portlandite (ca(oh)2), anhydrite (caso4), calcium hydrochloride (cacl(oh)), calcite (caco3) and silica (sio2). xrf and xrd results were consistent with the high ph and anc values of both fly ashes in table ii. due to the presence of these calcium-containing compounds, raw fly ashes manifested strong alkalinity.21 ftir spectrograms were obtained to investigate the surface functional groups as shown in fig. 2. the wideband at 3445 cm–1 corresponded to the fly ash heavy metal leaching 5 stretching vibration of –oh in hydration water, while the band at 1634 cm–1 was its bending vibrations.24 absorbances centred at 2510, 2360, 1795, 1451 and 875 cm–1 were caused by vibrations of co3 2– (carbonates).25 peaks at 1120 and 660 cm–1 indicated vibration stretching of s–o bonds and so3 2–, respectively.26 besides, si–o bonds were observed at 560 cm–1.27 it appears that that sio2, so4 2– and co3 2– salts were all found to be present in these fly ashes. table ii. element compositions of fa1 and fa2 via xrf (wt. %) item ash fa1 fa2 ca 55.74 50.97 cl 14.27 11.44 o 11.41 12.70 k 7.01 5.68 na 4.22 4.32 mg 1.79 0.64 s 1.32 2.38 si 0.68 1.70 fe 1.31 2.65 al 0.36 0.78 ti 0.33 0.76 br 0.18 1.02 p 0.13 0.20 10 20 30 40 50 60 b e e g g e e e b a a e e e e d in te n si ty , a .u . d d a a f f g g d d a a df e e c c a fa2 2 / ° fa1 a a: ca(oh)2 b: caso4 c: sio2 d: cacloh e: caco3 f: nacl g: kcl b e fig. 1. xrd pattern of fa1 and fa2. overall, the characterizations showed that both ashes were high in alkalinity with an abundance of chemicals such as ca-containing compounds, salts of chloride, sulphate and carbonate, and oxides (e.g., sio2). however, the exact amount of these elements differed as their origins varied. this variability could be added to the difficulty in fly ash treatment. 6 chen et al. 4000 3500 3000 2500 2000 1500 1000 500 660 2360 1795 2510 560 875 1120 1451 1634 3445 fa2 t r a n sm it ta n c e , % wavenumber, cm -1 fa1 fig. 2. ftir spectra of fa1 and fa2. heavy metal contents heavy metals were of the most concern for their toxicity. digestion+icp -oes analysis was employed to analyse the heavy metal contents in fly ashes. this method was able to detect even trace amount of heavy metals in solids. table iii lists the contents of 6 major heavy metals in fly ashes. contents of other metals such as as and sb, were also detected, but their contents were extremely low and were not of concern here. studies have shown that many heavy metals were transported in the form of volatile chloride and condensed into fly ash particles.28,29 fa1 had 1.08 g/kg of pb and 7.90 g/kg of zn. by comparison, the contents of cu, cr and cd were much smaller. however, the heavy metals contents also varied with fly ash. fa2 showed a much higher metal contents than fa1, probably due to the difference in solid wastes compositions in different city. the municipality where fa1 came from implemented a more stringent garbage sorting practice. table iii. contents of major heavy metals in fa1 and fa2 sample heavy metal content, g kg-1 cr ni cu zn pb cd fa1 0.25 0.06 0.53 7.90 1.08 0.18 fa2 2.22 0.69 1.98 26.74 2.46 0.25 heavy metal leaching toxicity the main purpose of this research was to investigate the leaching behaviour of fly ashes by studying the leaching of heavy metals at different time point and exploring the important factors affecting the leaching behaviour. fly ash heavy metal leaching 7 tclp leaching. fig.3a shows the changes of ph and heavy metals concentrations at different leaching time. as shown in fig. 3a, ph of the fa1 leaching solution rose quickly from the original 2.88 to about 10 after just 2 h. it continued to climb gradually, from 10 at 2 h to about 12 at 10 h. the changes in ph were caused by the consumption of acetic acid by the alkalinity in fly ash. for fa1, cr, ni, cu, zn and cd concentrations remained low and stable during the whole leaching process. on the contrary, there is an obvious increase in pb leaching as time was extended. 0 2 4 6 8 10 12 14 16 18 20 0.0 0.2 0.4 0.6 0.8 1.0 c o n c e n tr a ti o n , m g l -1 time, h cr ni cu zn pb cd 0 2 4 6 8 10 12 14 ph p h (a) 0 2 4 6 8 10 12 14 0.0 0.2 0.4 0.6 0.8 c o n c e n tr a ti o n , m g l -1 ph pb 2+ pbco3 pb(oh)3 pb(hco3) + pb(oh)2 (b) fig. 3. leaching of heavy metals fa1 during tclp test (a); speciation of metals in leaching environment via minteq (b). to understand the different leaching phenomena of metals, besides heavy metals, other anion and cation concentrations in leaching solution were also monitored. as shown by xrd, xrf and ftir analysis, fly ashes contained a variety of carbonate, sulphate and chloride salts and metal oxides. the release of these compounds into leaching solution could change the leaching environment thus affecting the release of heavy metals. results showed that high concentrations of potassium, sodium, chloride, sulphate, carbonate and calcium were detected in leaching liquid. for instance, the leaching solution of fa1 contained a 8 chen et al. total inorganic carbon (tic) of about around 123.3 mg/l with a sulphate concentration about 120 mg/l. the release of carbonate would not only change the acidity of leaching solution but also reacting with heavy metals to form different compounds thus affecting the solubility of metals. software minteq 3.1 was used to elaborate the speciation of metals in the leaching environment. concentrations of heavy metals, calcium, chloride, sulphate and carbonate and ph were entered into the software. minteq results showed that cr, ni, cu, zn and cd mainly existed as carbonate or hydroxide precipitates at the phs of the leaching solution (10–12). for example, for cd, cdco3 and cd(oh)2 precipitates dominated at high ph. this is in accordance with the low leaching of these metals shown in fig. 3a. that is, the high alkalinity of fa1 resulted in a very alkaline leaching solution. metals cr, ni, cu, zn and cd were bound up in carbonate or hydroxides precipitates. the only exception is pb, which has an obvious trend of increasing leaching. this can also be explained by the reactions occurring during leaching process. fig. 3b represents the speciation of pb-containing compounds at different phs at the presence of carbonate, calcium, sulphate and other heavy metals. only major species were presented here. concentrations of other species such as pboh+, pb(hco3) + and pbso4, were negligible. unlike cr, ni, cu, zn and cd, pb existed mainly as pbco3 at ph 6–10, while pb(oh)3 – became dominant as ph rose to higher than 10. it seems that as the ph of leaching solution increased, pb was dissolved gradually to form species the like of pb(oh)3 –. fig. 4a shows the leaching concentrations of heavy metals and ph changes during leaching of fa2. ph of the leaching solution reached 5.2 after 2 h, then rose to 6.0 at 6 h and fluctuated between 6.5 and 7.2 after that. fa2 had a much lower alkalinity and acid neutralization capacity. thus, the leaching solution remained acidic to near neutral. 0 2 4 6 8 10 12 14 16 18 20 0 5 10 15 20 25 30 c o n c e n tr a ti o n , m g l -1 time, h cr ni cu zn pb cd 0 1 2 3 4 5 6 7 8 9 10 ph p h (a) 0 2 4 6 8 10 12 14 0 5 10 15 20 25 c o n c e n tr a ti o n , m g l -1 ph pb 2+ cu 2+ pbco3 cu(oh)2 pb(oh)3 cuco3 (b) fig. 4. leaching concentrations of heavy metal against leaching time (a) and speciation pb and cu at different ph via minteq (b). fly ash heavy metal leaching 9 the characterization of fa2 (table iii) showed that fa2 contained much higher amount of heavy metals. therefore, higher leaching was observed for all metals. for instance, the leaching of zn at 18 h from fa1 was 0.022 mg/l, while that from fa2 reached as high as 8.21 mg/l. however, the behaviour of metal leaching also was different from that of fa1. cr, ni, zn and cd leaching stabilized after 4 h. as with fa1, minteq software was employed to calculate the predominant species. results showed that cr3+, ni2+, zn2+ and cd2+ were the dominant species in the whole range of ph during the fa2 leaching. the formation of carbonate precipitate was very low at the ph range. cr, ni, zn and cd compounds that could be dissolved by acetic acid in tclp were leached out at the first 4 h, after which time the leaching stabilized. however, for cu and pb, at the first stage of leaching (2–10 h), ph fluctuated between 5 and 6. according to the calculation via minteq, the predominant species at this stage were cu2+ and pb2+, respectively (fig. 4b). as leaching progressed, ph increased to about 7, and pbco3 and cuco3 precipitates became predominant. this is consistent with the decline in pb and cu leaching in fig. 4a after 10 h. overall, it appears that the heavy metals leaching was a complicated process. besides heavy metals, other chemicals from fly ash were also released, which changed the leaching environment. of the two fly ashes under investigation, one was much more alkaline that the other, which resulted in two different leaching environments. however, the analysis via minteq showed that regardless of the differences, the precipitation/dissolution of metal carbonate and hydroxides seemed to be the controlling reactions of heavy metal leaching, as the changes in metals containing compounds affected the extent of leaching. edta-modified tclp. as shown by tclp leaching, the fly ash compositions could greatly affect leaching behaviour by changing the leaching environment where chemical reactions (mainly precipitation/dissolution) occurred. for the two fly ashes under study, their original difference in chemical composition, and other characteristics such as alkalinity, led to difference in conditions of leaching solutions. thus, heavy metals behaved differently. in their study of the merit and demerit of tclp for the leaching toxicity evaluation, lzquierdo and querol believed high alkalinity in fly ash led to a rise in ph of the leaching solution.30 the leaching of metal was often repressed. fly ash in natural landfill environment came into contact with an environment with a much lower ph and presence of organics. thus, tclp test may underestimate the real leaching potentials. studies above already showed that the precipitation/dissolution reactions were related to the ph of leaching environment. to investigate the leaching behaviour when organics were present, edta was used to represent chelating organics and its concentration was set at 1 10 chen et al. mmol/l. fig. 5 shows the heavy metal leaching by fa1 and fa2 via edta -modified tclp. the addition of edta did not change ph of the leaching solution but increased the leaching of all metals. cu and cd leaching at 18 h for fa1 almost doubled. other metals also observed various degree of increase. this proved that the chelating capability of edta facilitated the release of metals. another proof of edta’s chelating effects is the leaching of pb and cu from fa2. without edta, the leaching of pb and cu declined as leaching progress (fig. 4a). with the presence of edta, pb leaching continued to increase from 2–8 h and stabilized after 10 h. these results showed that the chelating effects from edta changed the behaviour of metals. when acetic acid alone was used as leachant, the leaching process was controlled by the precipitation/dissolution of carbonate and hydroxides of metals. with the addition of merely 1 mmol/l of edta, chelating reactions also played a significant role. this shows that the presence of chelating organics in natural condition may have significant effects on the leaching of metals and had to be taken into consideration. 0 2 4 6 8 10 12 14 16 18 20 0.0 0.2 0.4 0.6 0.8 1.0 c o n c e n tr a ti o n , m g l -1 time, h cr ni cu zn pb cd 0 2 4 6 8 10 12 14 ph p h (a) 0 2 4 6 8 10 12 14 16 18 20 0 5 10 15 20 25 30 c o n c e n tr a ti o n , m g l -1 time, h cr ni cu zn pb cd (b) 0 1 2 3 4 5 6 7 8 9 10 ph p h fig. 5. heavy metal leaching via edta-modified tclp: a) fa1; b) fa2. conclusions fly ashes had high alkalinity because of the presence of various ca-bearing compounds, though the strength of alkalinity and their acid neutralization capacity varied. other major chemical composition in ashes include nacl, kcl, sio2 and heavy metals such as cr, ni, cu, zn, pb and cd, etc. the exact contents of these chemicals could differ based on the compositions of municipal solid waste, incineration conditions and flue gas treatment method. study of heavy metals (cr, ni, cu, zn, pb and cd) leaching behaviour via tclp showed that the process was complicated. the precipitation/dissolution reactions had significant effects on leaching. because of the alkalinity in fly ash, ph of the leaching solution was raised. calculations based on minteq software fly ash heavy metal leaching 11 showed that the shifts in the precipitation/dissolution equilibrium of carbonate and hydroxides compound of heavy metals could explain the leaching of metals over time. the differences in characteristics of fly ash (alkalinity and heavy metal content, etc.) resulted in the variation of leaching environment and the concentrations of heavy metals. but it seems that the precipitation/dissolution reactions controlled the release of heavy metals from fly ash in tclp test. on the other hand, the addition of edta introduced chelating effects. chelating of pb with edta resulted in continuous leaching of pb for 10 h. the amount of leaching was greatly increased, indicating that tclp may lead to underestimation of the metal concentrations in natural environment. acknowledgements. this work was supported by shanghai natural science foundation (14zr1428900) and national natural science foundation of china (21707090). и з в о д контролисање реакција током испирања тешких метала из летећег пепела за спаљивање комуналног чврстог отпада weifang chen, yegui wang, minzhu hu, yonglun li и guilin fang school of environment and architecture, university of shanghai for science and technology, 516 jun gong road, shanghai 200093, china ово истраживање је имало за циљ да окарактерише физичка и хемијска својства летећег пепела за спаљивање комуналног чврстог отпада и истражи испирање тешких метала током процеса tclp (енгл. toxicity characteristics leaching procedure – поступак испирања са карактеристикама токсичности). да би се разумело понашање током испирања, праћене су концентрације тешких метала, хлорида, сулфата и калцијума у различитим временима испирања, заједно са ph вредности раствора за испирање. резултати су показали да је хемијски састав летећег пепела довео до разлика у окружењу за испирање. од два летећа пепела која су проучавана, један је резултирао ph вредношћу од 10–12 у окружењу за испирање, док је други дао ph од око 6–7. софтвер minteq је коришћен за истраживање специјације тешких метала на основу ph вредности, и концентрације анјона и катјона. резултати су показали да промена у равнотежи таложења/растварања карбоната и хидроксида тешких метала може објаснити флуктуацију концентрација метала током процеса испирања, што указује да је испирање вероватно контролисано овим реакцијама. с друге стране, додавање edta је променило контролне реакције. реакције хелирања између тешких метала и edta довеле су до много веће токсичности испирања тешких метала, показујући да присуство хелирајућих органских материја у природном окружењу може олакшати испирање тешких метала. 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velimir popsavin1,2 and vesna v. kojić3 1university of novi sad, faculty of sciences, department of chemistry, biochemistry and environmental protection, trg dositeja obradovića 3, 21000 novi sad, serbia, 2serbian academy of sciences and arts, kneza mihaila 35, 11000 belgrade, serbia and 3university of novi sad, faculty of medicine, oncology institute of vojvodina, put dr goldmana 4, 21204 sremska kamenica, serbia (received 13 june, revised 5 august, accepted 18 august 2022) abstract: four novel conformationally restricted (–)-muricatacin analogues, bearing a methoxy group at the c-5 position and with an alkoxymethyl group аs the c-7 side chain, have been synthesised and their in vitro antiproliferative activity was evaluated against a panel of seven human tumour cell lines, as well as a single normal cell line. all analogues (9–12) showed diverse antiproliferative effects against all tested human malignant cell lines, but were devoid of any significant cytotoxicity towards the normal foetal lung fibroblasts (mrc-5). a structure–activity relationship study reveals that the introduction of tetrahydrofuran ring, the replacement of c-8 methylene group in the side chain of muricatacin analogues with the o-8 ether functionality, as well as the length of side chain may be beneficial for the antiproliferative effects of these lactones. all novel analogues were more potent than lead compound, (–)-muricatacin, against hl-60 cell line. keywords: d-glucose; antitumour agents; muricatacin mimics; furanolactones; cytotoxicity; sar analysis. introduction (–)-muricatacin (1) is a naturally occurring acetogenin derivative, which has been isolated by mclaughlin and co-workers1 from the seeds of annona muricata l. together with its enantiomer (+)-muricatacin (ent-1). both natural products (1 and ent-1) have received a great deal of attention due to their similar biological profiles: remarkable antiproliferative activities towards various human tumour cells,2,3 antimalarial as well as pesticidal activities.1 * corresponding author. e-mail: bojana.sreco@dh.uns.ac.rs  serbian chemical society member. https://doi.org/10.2298/jsc220613069s mailto:bojana.sreco@dh.uns.ac.rs https://doi.org/10.2298/jsc220613069s 2 stanisavljević et al. many syntheses of 1 from various precursors have been reported.4–16 also, several muricatacin analogues have been synthesised3,7,17–20 and some of them were evaluated for their antitumour activity.7,19,20 as a part of our ongoing program in the synthesis of oxygenated lactones as potential antitumour agents from abundant monosaccharides, the synthesis of four novel 8-oxa analogues of (–)-muricatacin (9–12, fig. 1) with furano-furanone ring system and methoxy group at the c-5 position was achieved from d -glucose. these molecules represent conformationally constrained oxa-analogues of lactone 1, with methoxy group at the c-5 position. analogue 9 is a heteroannelated mimic of 1 with the restricted geometry of the c4–c6 segment, due to the presence of the tetrahydrofuran (thf) ring. the molecule 10 represents a onecarbon lower homologue of 9, while the molecules 11 and 12 are twoand threecarbon lower homologues of lactone 9. fig. 1. design of (–)-muricatacin analogues with a methoxy group (9–12), a benzyl group (13–16) and with a hydroxyl group at c-5 position (17–20): i) thf-ring closure; ii) 5-o-methylation; iii) exchange of c8 methylene group with o8 ether function; iv) substitution of methyl with benzyl group at c-5; v) debenzylation at c-5. our recent results on the antiproliferative activity of analogues 17–20 showed that they exhibit moderate to submicromolar cytotoxicity.21 that led us to prepare c-5 methoxy derivatives 9–12, and to examine their cytotoxic activity, as well as the cytotoxicity of previously synthesised analogues 13–16,21 for a detailed structure–activity relationship (sar) analysis. experimental general procedures melting points were determined on büchi 510, or on hot stage microscope nagema phmk 05 apparatus and were not corrected. optical rotations were measured on autopol iv (rudolph research) automatic polarimeter. ir spectra were recorded with a ftir nexus 670 (thermo-nicolet) spectrophotometer. nmr spectra were recorded on a bruker ac 250 e or a bruker avance iii 400 mhz instrument and chemical shifts are expressed in ppm downfield from tetramethylsilane. low resolution mass spectra were recorded on finnigan-mat 8230 (ci) mass spectrometer. high-resolution mass spectra were taken on a micromass lct ka111 spectrometer or on ltq orbitrapxl (thermo fisher scientific inc.) mass spectrometer. tlc was performed on dc alufolien kieselgel 60 f254 (e. merck). flash column chromatography was performed using kieselgel 60 (0.040–0.063, e. merck). all organic ext (–)-muricatacin analogues 3 racts were dried with anhydrous na2so4. organic solutions were concentrated in a rotary evaporator under reduced pressure at a bath temperature below 35 °c. synthesis procedures methyl (z)(4) and (e)-3-o-methyl-5,6-dideoxy-1,2-o-isopropylidene-α-d-xylo-hept-5 -enofuranuronate (5). to a solution of compound 2 (1.923 g, 7.01 mmol), in dry etoac (193 ml), h5io6 (2.008 g, 8.76 mmol) was added. the mixture was stirred at room temperature for 3 h, then filtered and evaporated to afford crude aldehyde 3. to a stirred and cooled (0 c) solution of 3 (1.530 g, 7.56 mmol) in dry meoh (35 ml) mcmp (2.558 g, 7.56 mmol) was added and the mixture was stirred for 0.5 h at 0 °c and then for 2.5 h at room temperature. the reaction mixture was evaporated and the residue was purified by flash chromatography (3:2 light petroleum/et2o). the pure product 4 (1.240 g, 69 %) was first eluted, isolated as a colourless oil. further elution gave compound 5 which was additionally purified (1:1 ipr2o/ /light petroleum) to give the pure e-olefin 5 (0.133 g, 7 %). dimethylacetal 2,5-anhydro-6-deoxy-3-o-methyl-l-ido-hepturono-4,7-lactone (6). a solution of 4 (0.245 g, 0.95 mmol) in dry meoh (7 ml) which contains 2.5 % h2so4, was refluxed for 2 h. the mixture was cooled to 35 c and alkalized (ph 9) by adding solid nahco3 (0.917 g, 10.92 mmol, 11.5 eq) in three portions every 5 min. after adding the entire amount of base, the suspension was stirred at 35 c for 1 h, then filtered and evaporated. the residue was purified by flash column chromatography (3:2 cyclohexane/etoac) to give pure 6 (0.174 g, 79 %). 3,6-anhydro-2-deoxy-5-o-methyl-l-ido-heptono-1,4-lactone (8). dimethylacetal 6 (0.769 g, 3.31 mmol) was dissolved in 9:1 tfa/h2o (15.5 ml) and stirred at room temperature for 1 h. after completion of the reaction, the solution was evaporated by co-distillation with toluene. the crude aldehyde 7 was dissolved in dry meoh (39 ml) and treated with a first portion of nabh4 (0.094 g, 2.49 mmol, 3 eq). after stirring the mixture at room temperature for 0.5 h, an additional amount of nabh4 (0.063 g, 1.67 mmol, 2 eq) was added. the mixture was stirred at room temperature for additional hour. the reaction mixture was neutralized with acoh and evaporated. the residue was purified by flash chromatography (4:1 ch2cl2/etoac) to give pure alcohol 8 (0.399 g, 64 %). general procedure for the synthesis of analogues 9–12 to a solution of compound 8 (1 equiv.) in dry et2o (2 ml) ag2o (2.5 equiv.), agotf (0.3 eq) and the corresponding alkyl bromide rbr (2.5 equiv.) were added. the mixture was stirred under reflux for 16–27 h (table i). after completion of the reaction, which was detected by thin layer chromatography (tlc), the mixture was purified by flash column chromatography (7:3 light petroleum/et2o). the characterization data for all synthesised compounds (4–6 and 8–12) are given in the supplementary material to this paper. table i. preparation of final products 9–12 entry r reaction time, h product yield, % 1 c9h19 22.5 9 72 2 c8h17 16 10 86 3 c7h15 23 11 80 4 c6h13 27 12 73 4 stanisavljević et al. cytotoxic activity test cells. the in vitro cytotoxicity of test compounds was evaluated against seven human malignant cell lines: myelogenous leukaemia (k562), promyelocytic leukaemia (hl -60), t cell leukaemia (jurkat), burkitt’s lymphoma (raji), er+ breast adenocarcinoma (mcf-7), erbreast adenocarcinoma (mda-mb 231) and cervix carcinoma (hela). cytotoxic activity against normal foetal lung fibroblasts (mrc-5) was also estimated. mtt test. cytotoxic activity was evaluated by using standard mtt assay,22 after exposure of cells to the tested compounds for 72 h. results and discussion chemistry the syntheses of (–)-muricatacin analogues 9–12 are presented in scheme 1. starting compound 2 was prepared from d-glucose in two synthetic steps as previously reported by us.23 methyl derivative 2 was treated with periodic acid in dry ethyl acetate and the crude aldehyde 3 was obtained. compound 3 reacted with stabilized ylide, ph3p=chco2me, in anhydrous meoh and gave the expected z-olefin 4 (69 %) as the major product of the wittig olefination. a minor amount of corresponding e-olefin 5 (7 %) was also obtained in this reaction. scheme 1. reagents and conditions: a) h5io6, etoac, rt, 3 h; b) ph3p=chco2me, meoh, 0 °c, 0.5 h, then rt 1.5 h, 69 % for 4, 7 % for 5 (from 2); c) 2.5 % h2so4/meoh, reflux, 2 h, nahco3, rt, 1 h, 79 %; d) 9:1 tfa/h2o, rt, 1 h; e) nabh4, meoh, rt, 1.5 h, 64 % (from 6); f) c9h19br for 9, c8h17br for 10, c7h15br for 11, c6h13br for 12, ag2o, agotf, ch2cl2, reflux, 22.5 h (for 9), 16 h (for 10), 23 h (for 11), 27 h (for 12), 72 % (for 9), 86 % (for 10), 80 % (for 11), 73 % (for 12). an acid-catalyzed methanolysis of 4, in the presence of a catalytic amount of sulphuric acid gave furano-lactone 6 in 79 % yield. hydrolytic removal of the dimethyl acetal protective group in 6 followed by a subsequent nabh4 reduction of the resulting aldehyde 7 gave the corresponding primary alcohol 8 in 64 % yield. the stereochemistry of compound 8 was confirmed by single crystal x-ray diffraction analysis (for selected crystallographic and refinement details see the (–)-muricatacin analogues 5 table s-ii of the supplementary material). the molecular structure of 8 is depicted in fig. 2. all structural parameters of the molecule are within limits found in structures with similar fragments. the furano-lactone ring core is fused in cis manner and both five-membered rings are puckered, details of which are analysed by cremer–pople formalism.24 the furanose ring (counting clockwise o3→c3→c4→c5→c6) is moderately puckered (q2 = 36.05(17) pm), and its conformation is close to twisted at c5–c6 bond. the pseudorotational phase angle φ2 = 130.5(3)° indicates its deformation towards the envelope form, as the ring formally traversed 25 % along 6t5→ 6e pseudorotational pathway. ring substituents c2 (δ = 29.58(13)°), o4 (δ = 16.44(11)°), and o5 (δ = 9.82(11)°) can be classified as axial, while c7 (δ = 65.96(13)°) is equatorial; δ is the angle subtended between cremer–pople ring mean plane normal and substituent bond vector.25 the lactone ring (counting clockwise o4→c1→c2→c3→c4) is less puckered (q2 = 14.0(2) pm) and its conformation is closer to the envelope form, with c3 as the flap. its exact conformation is determined by φ2 = 130.5(3)°, which means that the ring formally traversed 37 % along e4→ 3t4 pseudorotational pathway. fig. 2. molecular structure of 8 determined by single crystal x-ray diffraction. ellipsoids are drawn at 50 % probability level. hydrogen atoms are shown as spheres of arbitrary radii. the key intermediate, alcohol 8, readily reacted with an excess of nonyl bromide in ether, in the presence of silver(i)-oxide and a catalytic amount of silver(i)-triflate, to give the expected 7-o-nonyl derivative 9 in 72 % yield. under similar experimental conditions, the primary alcohol 8 reacted with different alkyl bromides (c8–c6) to afford the corresponding ether derivatives 10– –12 in good yields (scheme 1). in vitro antiproliferative activity after completion of the synthesis, analogues 9–12 were evaluated for their in vitro cytotoxicity against a panel of seven human tumour cell lines (human myelogenous leukaemia, k562, promyelocytic leukaemia, hl-60, t cell leukaemia, jurkat, burkitt’s lymphoma, raji, er+ breast adenocarcinoma, mcf-7, er− breast adenocarcinoma, mda-mb 231, and cervix carcinoma, hela) and one normal cell line (foetal lung fibroblasts, mrc-5). cell growth inhibition was evaluated after 72 h of cells treatment by using the mtt test.22 (–)-muricatacin (1) 6 stanisavljević et al. and the commercial antitumour agent doxorubicin (dox) were used as positive controls in this assay. according to the recorded ic50 (table ii), all tumour cell lines were sensitive to all of the synthesised analogues (9–12). all four (–)-muricatacin mimics (9– –12) demonstrated diverse antiproliferative effects toward mda-mb 231 and jurkat cells, in contrast to the lead 1, which was completely inactive against these cell lines. table ii. in vitro cytotoxicity ( ic50 * / μm) of (–)-muricatacin (1), dox and analogues 9– 20 after 72 h compound cell line k562 hl-60 jurkat raji mcf-7 mda-mb 231 hela mrc-5 1 0.04 25.85 >100 0.1 21.35 >100 0.17 >100 9 10.25 17.70 15.40 21.75 4.85 11.32 13.50 >100 10 18.12 13.68 7.36 35.84 1.11 28.33 9.12 >100 11 5.60 24.54 22.97 28.49 12.31 25.33 11.51 >100 12 7.69 21.18 25.34 27.03 18.33 15.81 15.22 >100 13 8.76 6.12 9.71 15.95 22.18 39.48 68.32 >100 14 9.09 13.92 5.47 16.85 18.77 28.26 18.02 >100 15 8.87 5.67 8.86 17.33 22.87 34.59 10.90 >100 16 5.65 7.42 5.25 11.82 25.31 8.50 33.79 >100 17a 5.66 4.75 6.97 7.25 >100 >100 6.39 >100 18a 0.74 0.68 19.78 4.25 0.34 28.70 3.41 >100 19a 1.02 1.10 11.53 5.98 2.38 9.76 0.56 >100 20a 0.70 4.91 8.87 1.11 12.34 15.62 3.54 >100 dox 0.25 0.92 0.03 2.98 0.20 0.09 0.07 0.10 ataken from reference21 also, all novel analogues (9–12) were more potent than lead 1 against hl-60 cell line. the most active compound against the mcf-7 cells was analogue 10. this molecule exhibited strong cytotoxicity (ic50 = 1.11 μm) although its potency was 5.5 times lower than the activity of dox (ic50 = 0.20 μm), but 19 times higher than that of the control compound 1 (ic50 = 21.35 μm). the analogue 9 also showed very good activity (ic50 = 4.85 μm) against this cell line, which was 4 times higher than that of lead 1. the conformationally restricted benzyl analogues of 1, compounds 13–16, exhibited cytotoxic activity against all seven malignant cell lines. against the hl-60 cell line the previously synthesised analogues 13–16 showed from 1.85 * ic50 is the concentration of compound required to inhibit the cell growth by 50 % compared to an untreated control. values are means of three independent experiments. coefficients of variation were less than 10 %. (–)-muricatacin analogues 7 times (analogue 14, ic50 = 13.92 μm) to 4.5 times (analogue 15, ic50 = 4.85 μm) better cytotoxicity than natural product 1 (ic50 = 25.85 μm). against jurkat cell line benzyl derivatives 13–16 exhibited strong antiproliferative effects (ic50 values in the range of 5.25−9.71 μm). however, the parent compound 1 was completely inactive against this cell line. against the mcf-7 cell line analogues 13–16 demonstrated similar cytotoxicity (ic50: 18.77−25.31 μm) as parent compound 1 (ic50 = 21.35 μm). all synthesised compounds showed diverse growth inhibitory effects against the tested malignant cells, but were devoid of any significant cytotoxicity toward the normal foetal lung fibroblasts (mrc-5), as well as the natural product 1, in contrast to the commercial antitumour agent doxorubicin (dox) that exhibited potent cytotoxic activity (ic50 = 0.10 μm) against this cell line. sar analysis our previous findings showed that the introduction of an oxygen atom in the side chain of (–)-muricatacin analogues increases the antiproliferative activity.21 in this work we compared (–)-muricatacin analogues with this structural feature and the changes were based on the position c-5, so we compared three series of analogues: with ome-group (9–12), with obn-group (13–16) and with oh-group (17–20) at that position. analogues 9–12 demonstrated better cytotoxicity than the parent compound 1 against most of the cell lines tested in this study (fig. s-17a of the supplementary material). the corresponding benzyl derivatives (13–16) performed better antiproliferative effects in comparison to the analogues 9–12 (fig. s-17b). finally, the analogues with oh-group at c-5 position (17–20) were more potent than benzyl analogues (13–16, fig. s-17c). so, our further work will be focused on the preparation of a larger number of similar analogues and then we will be able to make a reliable conclusion that the free oh-group at position c-5 increases the cytotoxic activity of conformationally restricted (–)-muricatacin analogues. conclusion in conclusion, four novel (−)-muricatacin analogues (9–12) were designed and synthesised from d-glucose as a starting compound. the newly synthesised molecules, as well as the previously synthesised benzyl analogues (13–16), were evaluated for their in vitro cytotoxic activity against seven human malignant cell lines. a sar study showed that the presence of additional tetrahydrofuran ring, o-8 ether functionality, as well as the length of alkyl chain, may improve the cytotoxicity of analogues toward the majority of cell lines under evaluation. all synthesised compounds demonstrated diverse antiproliferative effects against the human malignant cell lines but were devoid of any significant cytotoxicity towards the normal foetal lung fibroblasts (mrc-5). hence, we believe 8 stanisavljević et al. that this approach may be of use in the search for novel, more potent and selective anticancer agents, derived from the natural product 1. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/11924, or from the corresponding author on request. acknowledgements. this work was supported by research grants from the ministry of education, science and technological development of the republic of serbia (contract no. 451-03-68/2020-14/200125). this work has also received funding from the serbian academy of sciences and arts under the strategic projects programme (grant agreement no. 01-2019 -f65), as well as from a research project from the same institution (grant no. f-130). и з в о д дивергентна синтеза и антитуморска активност нових конформационо крутих аналога (–)-мурикатацина слађана м. станисављевић1, бојана м. срећо зеленовић1, мирјана попсавин1, марко в. родић1, велимир попсавин1,2 и весна в. којић3 1универзитет у новом саду, природно–математички факултет, департман за хемију, биохемију и заштиту животне средине, трг доситеја обрадовића 3, 21000 нови сад, 2српска академија наука и уметности, кнеза михаила 35, 11000 београд и 3универзитет у новом саду, медицински факултет, онколошки институт војводине, пут др голдмана 4, 21204 сремска каменица синтетизована су четири нова конформационо крута аналога (−)-мурикатацина са метокси-групом у положају с-5 и са алкоксиметил-групом у бочном низу и испитана је њихова in vitro антипролиферативна активност према седам хуманих туморских и једној здравој ћелијској линији. сви аналози (9–12) су показали различите антипролиферативне ефекте према свим испитиваним малигним ћелијским линијама, а изостала је цитотоксична активност према ћелијској линији нормалних феталних фибробласта плућа (mrc-5). sar анализа показује да увођење тетрахидрофуранског прстена, замена с-8 метиленске групе са етарском функцијом у бочном низу, као и дужина бочног ланца, могу бити од значаја за цитотоксичне ефекте ових лактона. сви новодобијени аналози су били потентнији од водећег једињења (−)-мурикатацина према hl-60 ћелијској линији.. 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https://doi.org/10.1016/j.bmcl.2008.08.097 https://doi.org/10.1016/j.tet.2008.10.020 https://doi.org/10.1016/j.tetlet.2011.08.160 https://doi.org/10.3987/com-11-12397 https://doi.org/10.1055/s-0032-1318113 https://doi.org/10.1016/j.tetasy.2014.11.001 https://doi.org/10.1016/j.tetasy.2016.01.003 https://doi.org/10.3998/ark.5550190.0011.a15 https://doi.org/10.1002/hlca.201400356 https://doi.org/10.1055/s-0036-1590858 https://doi.org/10.1055/s-0036-1590858 https://doi.org/10.1002/chin.199923295 https://doi.org/10.1016/s0957-4166(01)00044-1 https://doi.org/10.1016/j.tet.2006.09.054 https://doi.org/10.1016/j.tet.2011.09.132 https://doi.org/10.2298/jsc190912104s https://pdfs.semanticscholar.org/3299/2997d7d34c82c2ce34937b25c5a770dbd735.pdf https://doi.org/10.1039/c8md00431e https://dx.doi.org/10.1021/ja00839a011 https://dx.doi.org/https:/doi.org/10.1002/ijch.198000048 accepted manuscript this is an early electronic version of an as-received manuscript that has been accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as: n. b. nguyen, t. q. p. phan, c. t. t. pham, h. n. nguyen, s. n. pham, q. k. a. nguyen, d. t. nguyen, j. serb. chem. soc. (2022) https://doi.org/10.2298/jsc220802080n this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc220802080n j. serb. chem. soc. 00(0) 1-12 (2022) original scientific paper jscs–12026 published dd mm, yyy 1 performance of carbon-coated magnetic nanocomposite in methylene blue and arsenate treatment from aqueous solution ngoc bich nguyen1,2*, thi que phuong phan3, cao thanh tung pham1,4, huu nghi nguyen2, sy nguyen pham5, quoc khuong anh nguyen6** and dinh thanh nguyen1,3*** 1graduate university of science and technology, viet nam academy of science and technology, hanoi city, 100000, vietnam; 2dong thap university, cao lanh city, 870000, vietnam; 3institute of applied materials science, viet nam academy of science and technology, ho chi minh city, 700000, vietnam; 4institute of chemical technology, viet nam academy of science and technology, ho chi minh city, 700000, vietnam; 5ho chi minh city university of natural resources and environment, vietnam and 6institute of applied technology and sustainable development, nguyen tat thanh university, ho chi minh city, 70000, vietnam (received 2 august; revised 5 october; accepted 3 november 2022) abstract: herein, carbon-coated magnetic nanocomposite fabricated by a lowtemperature hydrothermal method was used for methylene blue and arsenate treatment in aqueous solution. the langmuir model fits the experimental data with a calculated maximum adsorption capacity of 110.63 and 2.31 mg g-1 for methylene blue and arsenate adsorption, respectively. furthermore, the adsorption mechanisms of methylene blue as well as arsenate are physical adsorption and a combination of physical adsorption and chemisorption, respectively. gibbs free energy change with negative values indicates that methylene blue and arsenate adsorption on magnetic materials occurs naturally. this research demonstrated a simple, efficient, and reliable method for removing methylene blue and arsenate. keywords: hydrothermal carbonization; rice straw; adsorption introduction the rapid development of industries has posed many potentially serious issues in relation to ecosystems such as heavy metal and dye contamination.1 several technologies have been introduced for wastewater treatment, including oxidation, photocatalytic degradation, ultrafiltration, adsorption/ precipitation process, and coagulation.2 adsorption has been regarded as one of the most common and effective techniques for contaminant removal from wastewater.3 recently, carbon material derived from low-cost biomass residuals such as rice straw has received corresponding authors e-mail: (*)nnbich@dthu.edu.vn; (**)nqkanh@ntt.edu.vn; (***)dinhthanhng53@gmail.com https://doi.org/10.2298/jsc220802080n ac ce pt ed m an us cri pt mailto:nnbich@dthu.edu.vn mailto:nqkanh@ntt.edu.vn mailto:dinhthanhng53@gmail.com https://doi.org/10.2298/jsc220802080n 2 nguyen et al. a lot of attention due to its potential environmental benefits.4 moreover, magnetic modifications of the low-cost adsorbents can result in novel adsorbents that can be rapidly removed from the treated solution using a magnetic separator.5 methylene blue (mb) is used in many fields, so a significant amount of mb ends up in wastewater, posing a risk to aquatic life. in addition, due to its high toxicity, arsenic can cause serious health problems such as lung, liver, kidney, and skin cancers.6-11 as a result, it is critical to investigate efficient technologies for extracting mb and as(v) from aqueous solutions.12-16 rice straw (rs) is an inexpensive and abundant carbon-containing lignocellulose in vietnam. in the paper industry, biomass is usually pre-alkali-treated to remove lignin. as a result, this process emits a large amount of black liquor containing lignin, which can be utilized for carbon-containing materials.17 in this study, we simultaneously carbonize and magnetize the lignin liquor obtained from rice straw to synthesize carbon-coated magnetic nanocomposite (cmc) by a hydrothermal method. the primary goal of this work is to study the potential adsorption of low-cost cmc for mb and as(v) from wastewater. aside from that, another goal is to evaluate the effect of concentration, contact time, temperature, and ph solution. this work also discusses the langmuir and freundlich adsorption isotherm models, as well as various thermodynamic parameters like heat of adsorption (ho), entropy change (so), and gibbs free energy change (go) in mb and as(v) adsorption. experimental materials rs was collected in vietnam and washed several times with distilled water before being milled into powder and filtered through a 250 mesh sieve. potassium hydroxide (koh, ≥ 85 %), sodium hydroxide (naoh, ≥ 97 %), hydrochloric acid (hcl, 37 %), iron (iii) nitrate nonahydrate (fe(no3)3 9h2o, ≥ 99 %), sodium chloride (nacl, ≥ 99.5 %), h3aso4 in hno3 0.5 mol l -1 1000 mg l-1 purchased from merck. mb (c16h18n3scl.xh2o, 99.5 %), was obtained from sigma aldrich. all chemicals used were of analytical grade and were used as received without any further purification. synthesis of carbon-coated magnetic nanocomposite firstly, 15 g of rs was combined with 150 ml koh 5 %. the mixture was hydrothermally treated in an autoclave at 120 °c for 4 h. after slowly mixing 50 ml of 0.125 mol fe(no3)3 into 125 ml of the above-solution for 2 h, hydrothermal treatment was carried out at 180 oc for 14 h. the cmc is then collected by filtration and washed several washes with distilled water until the ph value reached neutral. finally, the remaining solid was dried in an oven at 40 °c for 12 h. in comparison, a blank sample (bs) was fabricated under the same condition but without the addition of fe(no3)3. characterization of cmc x-ray diffraction (xrd) was carried out on x d8 advance bruker with cu kα radiation (λ = 0,15418 nm). the morphology was observed with s4800 hitachi scanning electron microscope (sem) and jem1400 – jeol transmission electron microscopy (tem). energyac ce pt ed m an us cri pt removal of methylene blue, arsenate 3 dispersive x-ray spectrum (eds) was recorded on h7593 horiba. the fourier transform infrared (ft-ir) spectroscopy was measured on ir affinity-1s spectrophotometer (shimadzu). the specific surface area (bet) was determined by n2 adsorption–desorption isotherms at liquid nitrogen temperature (77 k) using quantachrome tristar 3000 v6.07a adsorption instruments. magnetization measurements were carried out using a vibrating sample magnetometer (vsm) 7307, lake shore, usa. the uv-vis spectrometry was recorded on spectro uv-2650, labomed, usa, at a wavelength of 664 nm. residual as(v) was detected by thermo scientific icap q icp-ms. the point of zero charge (phpzc) of cmc was investigated by the solid addition method.18 a series of 45 ml of 0.5 m nacl solutions were prepared in 100 ml flasks. the initial ph value (phi) of the solution was adjusted from 2 to 12 using either 0.1 m naoh or 0.1 m hcl solutions. the total volume of solution in each flask was precisely 50 ml by adding distilled water. then, 0.1 g of cmc was added to each flask and kept on shaker at 180 rpm for 24 h. the final ph (phf) of the solutions was recorded. the difference between the initial and final ph (δph = phi – phf) was plotted against phi. the point of intersection of the curve with the abscissa, where δph = 0, presented phpzc. adsorption experiment adsorption experiments were carried out using 0.1 g cmc in 100 ml of solution. variable parameters including initial concentration, contact time, temperature, and ph of the medium were thoroughly investigated. the initial ph value of the solution was adjusted using either 0.1 m naoh or 0.1 m hcl solutions. all adsorption experiments were carried out in duplicate. at predetermined time intervals, the adsorbent and solution were separated, and the residual mb and as(v) concentrations in the solution were measured using uv vis and icp ms, respectively. the removal rate, r / % were calculated using equation (1): 0 e 0 100 c c r c − = (1) where c0 and ce are the initial and equilibrium concentrations of mb or as(v) solution. we listed different kinetic models, thermodynamic equations, and adsorption isotherms in table si. non-linear chi-square test optimization is indispensable in order to identify the suitable kinetic and isotherm models to the obtained experimental results. for the present study, apart from correlation coefficient (r2), a non–linear regression model is chi–square test was performed for data optimization process. the chi–square (χ2) can be expressed as equation (2): ( ) 2 e,exp e,cal2 e,cal q q q  − =  (2) where qe,exp is the experimental value of adsorption capacity and qe,cal is the calculated value from the model. if experimental data is analogous to that from the model, 2 will be small, otherwise, it will be large. reusability 0.1 g cmc was added to 100 ml of a solution (120 mg l-1 for mb and 2.5 mg l-1 for as(v) and stirred for 60 and 90 min, respectively, for saturated adsorption. following the magnetic separation, the supernatant solution was discarded, and only adsorbed cmc was ac ce pt ed m an us cri pt 4 nguyen et al. collected. the adsorbed cmc in the case of mb was then added to ethanol and a 0.1 m hcl solution in the case of as(v) for the desorption process.13-14 the experiments were repeated 5 times in sequence to estimate the potentially regenerable property of cmc. results and discussion characterization of materials as shown in fig. 1a, regarding bs, the broad peak at 2θ = 22o represented the characteristic reflection of carbon.19 in cmc, the diffractions at 2θ = 30.46; 35.86; 43.58; 57.25 and 62.65o correspond to crystalline magnetite fe3o4 (jcpds no. 19-0629), which agree with the literature data.20 this demonstrated that fe(iii) is reduced into fe3o4 by carbon, which is formed under hydrothermal conditions by the reactions (3-6).2 fecl3 + 3koh → fe(oh)3 + 3kcl (3) fe(oh)3 → feooh + h2o (4) 2feooh → fe2o3 + h2o (5) 2fe2o3 + c → 2fe3o4 + co (6) in fig. 1b, ft-ir spectra revealed that both bs and cmc contained functional groups at 3413-3422 cm-1 (-oh stretching vibrations), 1627-1630 cm-1 (c=o stretching vibration), 1110-1114 cm-1 (c-o stretching vibration), 799-818 cm-1 and 450-474 cm-1 (si-o-si stretching vibration) and 1451-1456 cm-1 (-o-ch3 deformation vibration).1,18, 20. in general, the intensity of all peaks in cmc is lower than that of bs and has a slight shift, indicating that chemical reactions occurred when fe3+ was added to the solution. the peak near 560 cm-1, assigned to the fe-o stretching vibration, was only visible in cmc, which is consistent with xrd result.12 fig. 1. a) xrd patterns of bs, cmc, and standard fe3o4 (jcpds no. 19-0629) b) ft-ir spectra of bs and cmc according to figures s-1 and s-2, cmc is made up of c (19.21 %), o (34.38 %), fe (42.24 %) and si (4.16 %). in addition, eds elemental mapping also shows that fe is uniformly dispersed on the surface of the material, proving that ac ce pt ed m an us cri pt removal of methylene blue, arsenate 5 iron oxide was formed in cmc. fig. 2a and 2b depicts typical tem and sem images of cmc containing fe3o4 with sizes ranging from 50 to 120 nm and carbon as a shell with a thickness ranging from 30 to 50 nm. at room temperature, fig. s-3 shows the saturation magnetisation value of 33.7 emu g-1, which allows for the rapid separation and redistribution of cmc from aqueous solution and leads to costeffective and reusable applications.21 table s-ii compares the magnetization of cmc with various biochar. table s-iii displayed specific surface area, total pore volume, and mean pore size for rs, bs, and cmc. the specific surface area of cmc (171.4 m2 g-1) is significantly greater than that of bs (6.6 m2 g-1) thanks to the combination of carbon and magnetic particles.22 furthermore, the mean pore size of cmc (6 nm) is smaller than that of bs (33 nm), attributed to the covered micropores in carbon.23 fig. 2. tem (a) and sem (b) images of cmc effect of initial solution ph the ph of the solution plays an important role in the adsorption process, particularly in terms of adsorption capacity.24 because of the changing surface of cmc on mb and as(v), the ph value can alter its performance.25 investigating the influence of initial ph solutions from 3 to 11 was carried out while keeping other parameters constant such as initial concentration (120 and 2.5 mg l-1), equilibrium time (60 and 90 min at 303 k) for mb and as(v), respectively. the effect of ph on the adsorption of mb and as(v) on cmc is depicted in fig. 3a and 3b. the adsorption capacity of mb increases from 3 to 7 and changes slightly when solution ph exceeds 7. when ph < phpzc, the surface charge is positive, and when ph > phpzc, the surface charge is negative. the phpzc of cmc is approximately 6.32 (fig. s-4). ac ce pt ed m an us cri pt 6 nguyen et al. fig. 3. influence of ph value on the adsorption of (a) mb and (b) as(v) at low ph, low adsorption capacity resulted from electrostatic repulsion between the cationic ion mb and positively charged active sites on cmc. electrostatic attraction occurs between negatively active sites on cmc and the cationic ion mb at higher ph levels, facilitating adsorption capacity. arsenic acid exists in anionic forms (h2aso4 -, haso4 2-, aso4 3-).26 moreover, cmc with positively charged active sites can attract arsenate ions, increasing adsorption capacity from 84.12 to 86.6 %. in contrast, at ph ranging 7 to 11, cmc with negatively charged active sites inhibited as(v) adsorption due to ohcompeting with arsenate ions, resulting in a decrease in yield to 83.54 %.27 hence, the initial ph solution for the following experiments is 7. adsorption thermodynamics a linear van’t hoff plot (fig. 4a and 4b) of ln kd versus 1/t gives slope and intercept to determine the value of ∆h° and ∆s°, respectively. the calculated thermodynamic parameters for mb and as(v) adsorption onto cmc are summarized in table i at different temperatures. as temperature rises, the value of go becomes more negative, resulting in more spontaneous adsorption with high affinity of mb and as(v) to cmc. the value of ho for absolute physical adsorption is typically less than 20 kj mol-1, whereas chemisorption is in the range of 80 to 200 kj mol-1.28, 29 table i. thermodynamic parameters for adsorption of adsorbates onto cmc adsorbate temperature, k ∆s o / j mol-1 k-1 ∆h o / kj mol-1 ∆g o / kj mol-1 mb 303 67.41 15.06 -5.38 313 -6.01 323 -6.73 as 303 89.10 22.32 -4.70 313 -5.51 323 -6.49 ac ce pt ed m an us cri pt removal of methylene blue, arsenate 7 fig. 4. the plot of ln kd vs. 1/t for a) mb and b) as(v) adsorption onto cmc ho of mb on cmc is 15.06 kj mol-1 indicates physical adsorption while the value of ho (22.32 kj mol-1) for as(v) on cmc should be regarded as a mixture of physical adsorption and chemisorption, but dominated by physical adsorption, since the ho was a slightly higher than 20 kj mol-1. with positive values of so, there is an affinity adsorbent for adsorbate. effect of contact time and adsorption kinetics for both mb and as(v), contact intervals of 0 to 105 min and 0 to 120 min are used to evaluate the adsorption process as a function of contact time, respectively. the adsorption of mb and as(v) occurs in three stages. firstly, the adsorption rate for mb increases significantly in 10 min and 30 mins for as(v). the reason for this is that at the start, many vacant sites are available for adsorption. then, it will gradually rise until it reaches the equilibrium value of 30 min for mb and 75 min for as(v) (fig. 5a and 5b), resulting from the fewer vacant sites and repulsive forces between the occupied sites and bulk phases. fig. 5. kinetic modeling for adsorption of a) mb and b) as(v) onto cmc ac ce pt ed m an us cri pt 8 nguyen et al. therefore, we determined that the adsorption time for the next experiment will be 60 min for mb and 90 min for as(v). adsorption of mb on the surface of cmc is physical whereas adsorption of as(v) is both physical and chemical, resulting in as(v) adsorption being slower than that of mb.30, 31 to investigate the experimental data, different kinetic models including pseudo-first-order and pseudo-second-order were used to understand the adsorption process. the kinetic parameters, correlation coefficient (r2) and non-linear chi-square (χ2) were listed in table ii. the calculated qe values (qe,cal) of both models are comparable to the experimental ones (qe,exp). however, the r 2 of the pseudo-second-order kinetic model (approximately 0.99 for r2) is significantly higher than that of pseudo-first-order kinetic model (approximately 0.90 for r2), conversely, (χ2) of the pseudo-secondorder kinetic model is significantly lower than that of pseudo-first-order kinetic model, implying that the pseudo-second-order kinetic model is better for adsorption kinetics of mb and as(v) onto cmc. table ii. kinetic parameters for adsorption of a) mb; b) as(v) onto cmc at 303 k adsorbate c0 / mg l-1 first-order kinetic model second-order kinetic model qe,exp / mg g-1 k1 / min-1 qe,cal / mg g-1 r2 χ2 k2 / g mg-1 min-1 qe,cal / mg g-1 r2 χ2 mb 80 79.74 0.3357 76.94 0.864 3.043 0.0071 80.87 0.987 0.274 100 98.03 0.2972 94.56 0.875 4.403 0.0050 99.72 0.986 0.459 120 108.35 0.2870 104.55 0.900 3.950 0.0042 110.51 0.994 0.207 140 110.91 0.2830 106.58 0.899 4.120 0.0041 112.78 0.995 0.193 160 112.57 0.2796 107.75 0.893 4.473 0.0039 114.12 0.994 0.244 as(v) 1.5 1.482 0.3325 1.442 0.867 0.059 0.3774 1.507 0.987 0.006 2.0 1.929 0.3084 1.880 0.915 0.053 0.2608 1.971 0.998 0.001 2.5 2.179 0.2784 2.119 0.902 0.089 0.2049 2.227 0.993 0.006 3.0 2.257 0.2752 2.189 0.888 0.104 0.1961 2.302 0.991 0.009 3.5 2.312 0.2713 2.239 0.886 0.108 0.1855 2.362 0.989 0.010 effect of initial concentration and adsorption isotherms fig. 6a and 6b indicated that the adsorption capacity of mb and as(v) onto cmc significantly increases with increasing ranges of 80 to 120 mg l-1 and 1.5 to 2.5 mg l-1, respectively. when the concentrations of mb and as(v) exceed 120 and 2.5 mg l-1, the adsorption capacity increases insignificantly and reaches a maximum of 110.63 mg l-1 (c0 = 160 mg l -1) and 2.312 mg l-1 (c0 = 3.5 mg l -1), respectively. we can assume three main reasons to explain this phenomenon 1) a large number of available active sites are used at higher concentrations of mb and as(v) 2) improved mass transfer 3) the increased ability of mb and as(v) to collide with cmc. ac ce pt ed m an us cri pt removal of methylene blue, arsenate 9 fig 6. effect of initial concentration on adsorption capacity and removal efficiency of a) mb and b) as(v) onto cmc furthermore, as the initial concentration increases from 80 to 160 mg l-1 and from 1.5 to 3.5 mg l-1, the removal of mb and as(v) decreases from 98.79 to 69.14 % and 98.60 to 65.97 %, respectively. when using higher concentrations of adsorbates with the same weight of cmc, the percentage removal of mb and as(v) is reduced because the number of active sites on cmc remains constant. the langmuir and freundlich equations are the most used isotherms equation for modelling the adsorption data. the r2 obtained from langmuir model is significantly higher than that obtained from freundlich model, indicating that the langmuir isotherm better fits the experimental data (fig. 7a and 7b, table iii). table s-iv compares the adsorption capacity of cmc with various adsorbents. the previously reported capacity of mb and as(v) onto cmc is greater than that of many other previously reported adsorbents, implying that the as-prepared cmc has a high potential for use in wastewater treatment. a high kl value indicates the high affinity of adsorbent for mb and as(v) adsorption.32, 33 the rl values in the range of 0 and 1 indicate favourable adsorption. fig. 7. analyses of adsorption isotherm for a) mb and b) as(v) onto cmc by langmuir and freundlich models at 303 k ac ce pt ed m an us cri pt 10 nguyen et al. table iii. isotherm parameters for adsorption of mb and as(v) onto cmc at different concentration adsorbate qe,exp / mg g-1 langmuir isotherm model freundlich isotherm model qmax / mg g-1 kl/ l mg-1 rl r2 χ2 kf /mg g (l/ mg)1/n nf r2 χ2 mb 110.63 110.64 2.518 0.003 0.996 0.027 84.138 12.546 0.884 24.427 as(v) 2.31 2.285 81.919 0.003 0.985 0.003 2.325 10.950 0.904 0.608 reusability cmc regeneration and recycling are critical for practical application. as shown in fig. 8a and 8b, after five cycles, there is only a very slight decrease in removal from 107.32 to 98.73 mg g-1 for mb and from 2.165 to 1.992 mg g-1 for as(v), indicating that cmc has excellent performance and application for mb and as (v) treatment. fig. 8. regeneration for (a) mb and (b) as(v) adsorption onto cmc conclusion cmc was prepared in a straightforward and efficient manner. they also have a fast adsorption rate, high adsorption efficiency, and fast magnetic separation from treated water, making them excellent materials for environmentally treated purposes. the maximum adsorption is 110.63 mg g-1 for mb and 2.31 mg g-1 for as(v). the kinetics of adsorption can be described using a pseudo-second-order equation, and the cmc adsorption isotherm agreed well with the langmuir sorption equation. furthermore, through the desorption process, the product could be regenerated and reused multiple times. supplementary material additional data are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/12026, or from the corresponding author on request. ac ce pt ed m an us cri pt https://www.shd-pub.org.rs/index.php/jscs/article/view/12026 removal of methylene blue, arsenate 11 извод карактеризација магнетног нанокомпозита превученог угљеником за уклањање метиленско плавог и арсената из воденог раствора ngoc bich nguyen1,2, thi que phuong phan3, cao thanh tung pham1,4, huu nghi nguyen2, sy nguyen pham5 и dinh thanh nguyen1,3 1graduate university of science and technology, viet nam academy of science and technology, hanoi city, 100000, vietnam; 2dong thap university, cao lanh city, 870000, vietnam; 3institute of applied materials science, viet nam academy of science and technology, ho chi minh city, 700000, vietnam; 4institute of chemical technology, viet nam academy of science and technology, ho chi minh city, 700000, vietnam и 5ho chi minh city university of natural resources and environment, vietnam магнетни нанокомпозит превучен угљеником произведен ниско-температурском хидротермалном методом је употребљен за уклањање метиленско плавог и арсената из воденог раствора. лангмиров модел је добро описао експерименталне податке са израчунатим максималним адсорпционом капацитетом од 110,63 и 2,31 mg g 1 за метиленско плаво и арсенат, респективно. такође, одређени адсорпциони механизами су физисорпција за метиленско плаво и комбинација физисорпције и хемисорпције за арсенат. промена гибсове слободне енергије има негативне вредности што указује да се адсорпција метиленско плавог и арсената на магнетним материјалима дешава спонтано. ово истраживање показује једноставну, ефикасну и поуздану методу за уклањање метиленско плавог и арсената. 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2dong thap university, cao lanh city, 870000, vietnam; 3institute of applied materials science, viet nam academy of science and technology, ho chi minh city, 700000, vietnam; 4institute of chemical technology, viet nam academy of science and technology, ho chi minh city, 700000, vietnam and 5ho chi minh city university of natural resources and environment, vietnam fig. s-1. eds analysis (a) and elementals map (b) of cmc corresponding authors e-mail: (*)nnbich@dthu.edu.vn; (**)dinhthanhng53@gmail.com ac ce pt ed m an us cri pt mailto:nnbich@dthu.edu.vn mailto:dinhthanhng53@gmail.com s2 stanisavljević et al. fig. s-2. elemental maps of c (a), o (b), si (c) and fe (d) of cmc fig. s-3. magnetization curves and illustration of the magnetic separability of cmc ac ce pt ed m an us cri pt supplementary material s3 fig. s-4. plot of point of zero charge of cmc table s-i. different kinetic models, thermodynamic equations and adsorption isotherms model parameter equation adsorption kinetic models pseudo first-order qe / mg g -1 = equilibrium adsorption capacity qt = qe qee -k1t (1) qt / mg g -1 = adsorption capacity at time t k1 / min -1 = rate constant pseudo second-order k2 / g mg -1 min-1 = rate constant 2 2 2 e 1 e t k q t q k q t = + (2) thermodynamic equations van’t hoff equation so/ j mol-1 = entropy change o o d ln h k rt s r  =  + (3) ho/ j mol-1 = enthalpy change r / j mol-1 k-1 = 8.314 (universal gas constant) t / k = absolute temperature kd / l g -1 = qe/ce thermodynamic equilibrium constant go/ j mol-1 = gibbs free energy change go = -rt ln kd (4) adsorption isotherms langmuir qm / mg g -1 = maximum monolayer adsorption capacity of the adsorbent e e e a m m 1 k q c q c q = + (5) ka = energy constant rl = separation factor which gives an idea about langmuir isotherm a l 0 1 1 k c r = + (6) freundlich kf / mg g -1 l1/n mg-1/n = freundlich constant n = intensity of adsorption, n > 1 indicates a favourable and heterogeneous adsorption e f e ln ln 1 ln= +q k c n (7) ac ce pt ed m an us cri pt s4 stanisavljević et al. table s-ii. the comparison of the magnetization of cmc with various biochar precursors of magnetic biochar method magnetization, emu g-1 reference rice straw, fe(no3)3, koh hydrothermal 33.7 this work coconut shells, fecl3 pyrolysis, microwave 6.0 1 corn stalk, feso4, na2s2o3, naoh hydrothermal 11.2 2 corn stalk, feso4, na2s2o3, naoh pyrolysis 20.4 2 palm fiber, feso4, fecl3, nh3 pyrolysis 19.4 3 firwood, α-feooh pyrolysis 20.8 4 oleyl amine, fecl2, fecl3, naoh hydrothermal 21.7 5 rice husk, fe(no3)3, kmno4 pyrolysis 27.5 6 table s-iii. the porous parameters of rs, bs, cmc samples sample sbet / m 2 g-1 vt / cm 3 g-1 dp / nm rs 1.3 0.01 30.6 bs 6.6 0.04 33.0 cmc 171.4 0.15 6.0 table s-iv. the comparison of the maximum adsorption capacity of mb and as(v) on cmc with various adsorbents. adsorbent capacity, mg g-1 mb as(v) ref. cmc 110.63 2.31 this study fe2o3-zro2/bc 38.1 1.01 7 m-mwcnts 48.06 8 fe3o4 /mwcnt 74 9 fe3o4@c nps 117 10 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of chemistry and technology, sheremetievskiy avenue 7, 153000 ivanovo, russia (received 17 august, revised 11 october, accepted 9 november 2021) abstract: flotator oxal, a mixture of dioxane ethers and alcohols, was studied as a plasticizer for suspension pvc in comparison with the well-known dibutyl phthalate (dbp) and di-(2-ethylhexyl) phthalate (dop). the rheological parameters and gelation ability of the plasticizers were determined, and the values of the storage modulus and tangent of mechanical loss angle in the glassy and rubbery states were measured by the dma method, and the glass transition temperatures were determined. the deformation-strength properties and rigidity of polymer films were tested before and after light-thermal aging. oxal was shown to reveal a fairly low viscosity and high gelation properties in relation to pvc. at the same time, its ability to reduce the glass transition temperature and elasticize the polymer in the glassy and rubbery state is somewhat lower than that of phthalate plasticizers. pvc samples plasticized with dbp have the highest resistance to light-thermal aging. keywords: phthalates; dynamic viscosity; polyvinyl chloride; dma method. introduction plasticizers play an irreplaceable role in the manufacture of flexible materials based on rigid-chain polymers.1 initially, cellulose ethers, poly(vinyl acetate) and its copolymers2 were used as plasticizable polymers. currently, the main consumer of plasticizers is the production of flexible pvc materials, such as films, linoleum, artificial leather, awning materials, etc.3 it is customary to distinguish between internal plasticization associated with the addition of plasticizing fragments into macromolecules of rigid-chain polymers due to covalent bonding4–6 and external – by introducing special plasticizers without chemical interaction. among pvc plasticizers, the most common are esters of organic acids (phthalic, adipic, trimellitic, sebacic, azelaic), as well as phosphoric acid.7,8 to reduce volatility, polymer plasticizers are utilized, in * corresponding author. e-mail: trifonova@isuct.ru https://doi.org/10.2298/jsc210817093t ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 356 trifonova et al. particular polyesters.9 in addition a large group is made up of the so-called secondary plasticizers (extenders), used in conjunction with primary ones. the most common among them are chlorinated hydrocarbons.3 in recent decades, much attention has been paid to the search for biodegradable plasticizers of various nature,10–13 which enables a number of environmental protection and health safety problems to be solved14 and in some cases to combine plasticizing and the antipyretic function of a biodegradable additive.15 the most widespread plasticizers based on phthalic acid esters have been used for almost 100 years to change the elastic properties of poly(vinyl chloride).1 phthalate plasticizers effectively reduce the pour point and the glass transition temperature of pvc.6 nevertheless, there is a permanent search for new plasticizers that, with high efficiency, would have a lower tendency to diffuse onto the product surface, less volatility at elevated temperatures, and a lower cost.1 over the past several decades, the use of a high-boiling by-product edos (tu 2493-003-13004749-93) as pvc plasticizer has been actively discussed.16–21 it is formed in the manufacture of 4,4-dimethyl-1,3-dioxane by condensation of isobutylene with formaldehyde. in one turn, flotator oxal is the by-product of edos (technical requirements 2452-015-48158319-2009). this product contains about 50 % dioxane ethers and alcohols and about 50 % mixtures of 1, 2 and 3 atomic alcohols.20 the successful application of flotator oxal as a plasticizer in the production of plastisols based on emulsion pvc e was described.16–18 the influence of oxal on the properties of composites based on suspension pvc has practically not been studied. therefore, the estimation of the plasticizing action of oxal performed in this work seems to be very actual. experimental materials suspension pvc ongrovil s-5070 was obtained from borsodchem (hungary). dibutyl phthalate (dbp) and di-(2-ethylhexyl) phthalate (dop) were purchased from “ural plasticizers plant” (russia) and used without purification. flotator oxal t-92 (technical requirements: 20.59.59-029-05766801-2016) was purchased from “nizhnekamskneftekhim” (russia), stabilizer baerostab ubz 751 – from “baerlocher gmbh” (germany). film preparation to study the mechanical and thermophysical properties of the composites, films were produced from pvc melts. the compositions contained 70.2 % pvc, 1.7 % baerostab ubz 751 and 28.1 % plasticizer. dibutyl phthalate (dbp) and di-2-ethylhexyl phthalate (dop) were chosen for comparison with oxal. pvc, plasticizer and stabilizer were mixed, then heated for 30 min at 120 °c. the final mixing and formation of the film were performed on rollers at 150 °c for 5–6 min. to achieve a fixed thickness, the films were pressed at 160 °c and 5 mpa for 4 min, followed by cooling in a mold. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ flotator oxal as the plasticizer for pvc 357 measurements rheological tests of plasticizers were carried out in accordance with astm d2983-09 on a brookfield dv-ii+ rotary viscometer equipped with thermosel, in the range of shear rates from 0 to 186 s-1 and temperatures 22–45 °c. the size of pvc particles was determined using a polarizing microscope polam r-211 (magnification 75.6 times). dma was performed on a dma eplexor 25n (netzsch-gerätebau gmbh) in the tensile-film mode. the specimens 70.0 mm× 7.0 mm× 0.4 mm were measured from –30 to 80 °с with a frequency of 1 hz and a temperature ramp of 1 °c min-1. the storage modulus e' and tan δ were recorded as a function of temperature. the glass transition temperature was determined according to astm d7028-07(2015). the tensile strength and elongation at break were measured using a testing machine 2099 -р-5 (“tochpribor”, russia) at 25 °c before and after light-heat aging according to iso527 -2:2012. the length of the films was equal to 170 mm and the stretching speed was 25 mm min-1. the measurements of the mechanical properties of the composite films were made with 5 replicates of each composition. accelerated aging was realized in accordance with astm g151-19 and d3045-18. ultraviolet irradiation was performed with mercury-quartz lamps drt-400, exposure time 36 h at 70 °c. the hardness of films before and after uv light-thermal aging was determined on a pmzh-12 m device. the hardness was assumed to be equal to the load required for deflection of an elementary sample bent into a ring by 1/3 of the diameter. test conditions corresponded to gost 8977 (ball weight 0.86 g, sample size 20 mm× 95 mm). results and discussion rheological characteristics of plasticizers essentially influence the technological processing parameters of flexible pvc, especially in the case of using plastisols.16–18 in this regard, the rheology of the product oxal was studied in comparison with the most common phthalate plasticizers: dibutyl phthalate (dbp) and di-(2-ethylhexyl) phthalate (dop) (see experimental). the values of dynamic viscosity were measured in the shear rate range 0–186 s–1. all plasticizers studied were found to behave like newtonian liquids.22 the simulation of temperature semilogarithmic dependencies (fig. 1) according to the frenkel–eyring equation:23 # # vf vf a s h r rtn e e v η −δ δ =  (1) where η is the viscosity, v the mole volume, δs≠vf and δh≠vf the changes in activation entropy and enthalpy, respectively, of viscous flow, allowed the flow activation parameters to be calculated, which together with the viscosity at 25 °с are presented in table i. an analysis of these data indicates a slightly lower viscosity of oxal (25 °c) than that of dop, but higher than that of dbp. the free activation energy, δg≠vf, of viscous flow follows the same trends. simultaneously, the ratio of the activation parameters of oxal are completely different than those of the phthalate plasticizers (table i) – lower enthalpy and practically zero entropy of activation. this testifies ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 358 trifonova et al. to differences in the flow mechanism of the studied fluids. it is known that the value of δh≠vf is associated with the size of the molecule or its fragment involved in the momentum transfer in viscous flow.24 therefore, the activation enthalpy of dop is slightly higher than that of dbp. thus, a low oxal value indicates a weaker effect of temperature on the viscosity (fig. 1) and a smaller volume of the momentum transfer element. fig. 1. temperature dependence of the logarithm of the dynamic viscosity for plasticizers: 1 – dop, 2 –oksal, 3 – dbp. table i. rheological parameters of the considered plasticizers plasticizer η / mpa s (γ = 27.9 s-1, t = 25 °c) δh≠vf kj·mol-1 δs≠vf j·mol-1·k-1 δg≠vf kj·mol-1 (t = 25 °с) dop 70.5 25.1 29.8 16.2 oxal 65.4 16.1 0.2 16.1 dbp 39.4 23.9 30.7 14.8 one of the most important properties of a plasticizer is its gelation ability for a given polymer.6 the combination of suspension pvc with plasticizers begins with their mechanical mixing and is accompanied by swelling of the polymer particles and an increase in the particle size.3 the ratio of the size of the swollen pvc particles to the size of the initial particles allows the effective absorption of the plasticizer during hot mixing to be evaluated and, consequently, its gelation properties (table ii). table ii. average particle size of pvc and swelling degree after combining with plasticizer at 120 °c within 30 min pvc composite average particle size, μm swelling degree of the pvc particles initial pvc 79.6 1 pvc + dbp 143.9 1.81 pvc + dop 104.1 1.31 pvc + oxal 149.0 1.87 according to the data in table ii, the gelation properties of oxal is at the level of dbp – one of the most effective pvc plasticizers.3,6 thus, significant ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ flotator oxal as the plasticizer for pvc 359 absorption at the mixing stage allows “dry” blends based on suspension pvc and oxal to be produce, which could be used in calender and extrusion technological schemes. the main function of the primary plasticizer is to lower the glass transition temperature and increase the elongation and softness of the polymer compositions.6 the most informative method for evaluating the plasticizing effect is dynamic mechanical analysis (dma), which permits fundamentally important physical-mechanical and thermo-physical parameters to be measured. in this regard, the temperature dependences of the storage modulus (fig. 2a) and the tangent of the mechanical loss angle tan δ (fig. 2b) were obtained by the dma method. the values e′ in the glassy and rubbery states, as well as the glass transition temperatures tg corresponding to the maxima on the curves tan δ = f(t) are presented in table iii. a b fig. 2. temperature dependences of the storage modulus e′ (a) and tan δ (b) of pvc composites with plasticizers: 1 – oxal, 2 – dop, 3 – dbp. table iii. efficiency of pvc plasticization; plasticizer content: 28.1 % plasticizer tg / °с δtg / °с e′ / mpa at –8 °с e′ / mpa at 60 °с without plasticizer 80 – – – dbp 20.9 –59.1 2087 2.6 dop 24.9 –55.1 1513 3.4 oxal 32.9 –47.1 2620 4.5 analysis of these data shows that the rigidity of pvc films plasticized with oxal are higher than those of phthalate plasticized films both in the glassy and in the rubbery state (table iii). at the same time, the efficiency of elasticization of the latter in different relaxation states is different. thus, dop provides greater elasticity to the glassy polymer, while dbp provides highly soft for rubbery pvc. the efficiency of plasticizers, estimated by the decrease in the glass transition temperature (δtg) of the plasticized polymer relative to the original poly________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 360 trifonova et al. mer, decreases in the series dbp > dop > oxal simultaneously with a lowering in the modulus (table iii) in the rubbery state. along with the characteristics of plasticizers presented above, the deformation and strength parameters of plasticized samples and their behavior during accelerated aging are essential. therefore, in this work, the tensile strength, elongation, and hardness of plasticized pvc films before and after light-thermal aging (see experimental part) were measured. the results of these tests are listed in table iv. table iv. deformation and strength properties of pvc films before and after light-thermal aging composite properties before aging properties after aging tensile strength, mpa elongation % hardness сн tensile strength, mpa elongation % hardness сн pvc+dbp 22.7 220 18.1 22.3 190 23.2 pvc+dop 25.6 280 16.3 23.4 243 24.1 pvc+ oxal 33.7 215 55.9 28.6 143 89.4 the data (table iv) showed a higher rigidity and lower elasticity (elongation) of pvc films plasticized with oxal, in comparison with those phthalate plasticized. the highest resistance to accelerated light-thermal aging was demonstrated by the samples plasticized with dbp. conclusions the dynamic viscosity of flotator oxal was shown to be lower than that of di-(2-ethylhexyl) phthalate (dop), but higher than that of dibutyl phthalate (dbp). analysis of the activation parameters of the flow indicates a weaker dependence of the oxal viscosity on temperature compared to the phthalate plasticizers. size estimation of pvc particles swollen in the studied plasticizers revealed the high gelation ability of oxal comparable with that of dbp. a comparative study of the plasticizing effect by dynamic mechanical analysis (dma) showed that the lowering the pvc glass transition temperature under plasticizing (δtg) decreases in the series dbp > dop > oxal, simultaneously with a decrease in the storage modulus in the rubbery state. the data on the mechanical properties exhibit a higher rigidity and lower elasticity of pvc films plasticized with oxal, in comparison to those with phthalate plasticizers. the highest resistance to accelerated light-thermal aging was demonstrated by the samples plasticized with dbp. thus, the complete replacement of phthalate plasticizers with oksal makes it impossible to obtain materials with similar working properties. acknowledgements. the work was performed within the framework of the state assignment for the implementation of research work (topic no. fzzw-2020-0008). the dynamic ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ flotator oxal as the plasticizer for pvc 361 mechanical properties survey was conducted using the resources of the center for the collective use of scientific equipment by ivanovo state university of chemistry and technology. и з в о д флотациони реагенс „оксал“ као пластификатор за суспензију pvc irina p.trifonova, julia a.rodicheva, anna e.sheveleva, vladimir a. burmistrov и oscar i. koifman research institute of macroheterocycles, ivanovo state university of chemistry and technology, sheremetievskiy avenue 7, 153000 ivanovo, russia флотациони реагенс „оксал“, мешавина етара на бази диоксана и алкохола, проучаван је као пластификатор за суспензију pvc у поређењу са познатим дибутил-фталатом (dbp) и ди-2-етилхексил)-фталатом (dop). одређени су реолошки параметри и способност желирања пластификатора, мерене су вредности модула сачуване енергије и тангенса угла механичких губитака у стакластом и гумоликом стању методом динамичко-механичке анализе (dma) и одређене температуре остакљивања. својства отпорности на деформацију и крутост полимерних филмова тестирани су пре и после излагања светлости и температури. показало се да „оксал“ показује прилично низак вискозитет и висока својства желирања код pvc. истовремено, његова способност да смањи температуру остакљивања и еластичност полимера у стакластом и гумоликом стању је нешто нижа од способности фталатних пластификатора. pvc узорци пластифицирани помоћу dbp имају највећу отпорност на старење после излагања светлости и температури. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice volume 86 (2021) i subject index 1,2,3-triazole, 917 1,2,4-triazole, 1013 1,3,4-oxadiazole, 1013 1,3-benzoxazines, 213 1,3-diaryl-2-propene-1-one derivatives, 153 1,3-dipolar-cycloaddition, 901 19f-nmr spectroscopy, 845 1h nmr, 1249 2-pyrrolidinone, 1039 3d nucleation/growth, 1075 3d-qsar, 469 3-glicydyloxypropyltrimetoxy silane, 831 3-nitrobenzaldehyde, 1013 3-oxo-α,β-unsaturated steroids, 459 ace inhibitors, 469 acidic leaching, 1113 acidity, 983 acrylonitrile, 591 activated carbon, 983 adamantine derivatives, 625 adhesion potential, 533 adsorbent, 767 adsorption isotherm, 327 adsorption, 429, 699 afm, 407 agricultural wastes, 521 agrochemicals, 673 ahc, 1271 alkali rare-earth silicates, 663 alumina monolith, 615 aluminum hydrides, 299 amantadine, 625 ambipolar, 171 ames, 639 amine-based additive, 739 amino acids, 625 amorphous calcium phosphate, 555 amphoricarpolides, 1177 analytical toxicology, 77 anode, 1 antibacterial activity, 125, 139, 1291 antibacterial assay, 257 antibiofilm, 1291 anticancer activity, 153 anticandidal activity, 1291 anti-epileptic, 51 antifungal compounds, 639 anti-inflammatory, 1139 antimalarials, 115 antimicrobial activity, 153, 459, 901, 1139, 1249 antimicrobial properties, 1103 antioxidant activity, 257, 603 antitryptic activity, 139 antiviral, 1139 apiaceae (umbelliferae), 1241 apricot shell, 521 aquapentacyanoruthenate (ii) ion, 507 arsenic adsorption, 195 artemisia absinthium l., 1259 artemisia alba turra, 1259 artemisia annua l., 1259 artemisia scoparia waldst. et kit., 1259 artemisia vulgaris l., 1259 artificial neural networks, 355 ash removal, 1113 azahetarenes, 901 azeotropic mixture, 685 barium titanium oxide system, 415 bcs class ii, 51 benzimidazole, 859 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ ii journal of the serbian chemical society benzocoumarin, 971 bibenzyls, 1139 binding constant, 483 bioaccumulation factor, 927 biodiesel, 739 bioethanol, 651 biological activities, 213 biomaterials, 1103 bis-bibenzyls, 1139, 1249 bogovina coal, 1113 boron-doped diamond electrode, 711 boscalid, 247 bromobenzyl derivatives, 115 bryophytes, 1139 calibration equations, 507 candida albicans, 639 carboxyl groups, 885 carotenoids, 367 cascade reaction, 1053 catalyst deactivation, 283 catalytic site, 781 catechin, 367 cathode, 1 cbmn assey, 809 chelate complexes, 393 chemical composition, 257, 885 chemical reaction, 699 chemometrics, 1281 chemosystematics, 1177 chitosan oligolactate, 555 chqf, 971 chromatography, 1195 chronopotentiometry, 299 co-deposition, 1075 coking, 283 colorimetric test with tetrazolium salts, 555 composites, 591 condensation reaction, 1053 conductivity, 725 constant-current electrolysis, 585 cooling, 103 co-precipitation, 415 corona discharge, 615 correlation analysis, 927 cotinus coggygria, 1195 coumarin glycosides, 603 cpcm, 955 cross-coupling reaction, 269 crystal structure, 663 crystal violet, 327 crystallinity index, 521 cyanide poisoning, 77 cyclic voltammetry, 711, 1023 cyclization, 1305 cycloaddition, 1053 cyp enzymes, 639 cytotoxicity, 153, 459, 555, 1291, 1139 deep eutectic solvents, 547 degree of a vertex, 445 delayed pet-coke, 983 density functional theory, 171 density, 91 descriptors, 381, 673 dft method, 165, 955, 971 diffuse function basis set, 165 diffusivity, 341 dimethylformamide, 1013 discriminative potential, 445 dislocation density, 571 dispersion coefficient, 1089 distribution of distances, 781 diterpenoid, 917, 1229 dmso solvent, 165 drinking water treatment, 195 drug delivery, 1103 dual phase state, 1067 dye exclusion test, 555 efficiency, 313 electrochemistry, 407 electrosynthesis, 585 element composition, 927 emission factors, 753 endophytes, 125 energy carrier, 299 energy efficiency, 103 energy recovery, 313 ensemble multiple linear regression analysis, 685 epicatechin, 367 escins, 603 esterification, 983 euphorbiaceae, 1219 evidence, 77 extraction, 25, 603 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ volume 86: subject index iii extracts, 1249 finite element method, 103 first principle, 283 fluorescence quenching, 859 fluorescence, 901, 971 flux synthesis, 663 fomitopsis betulina, 809 forensic chemistry, 77 formative evaluation, 997 fractal dimension, 819 ft-ir spectra, 941 fullerene, 1023 gas separation, 341, 871 gc/ms, 257, 355, 1281, 1259 genetic algorithm, 63, 673 graph invariants, 445 graphene, 699 green chemistry, 547, 1039 h2o2 scavenging activity, 139 heavy metals, 767 hemicelluloses, 885 heterogenous catalysis, 955 heterovalent substitution, 845 hopping mechanism, 171 hydrazones, 459 hydrocarbon volume, 533 hydrodistillation, 355, 1281 hydrogen sorption, 299 hydrogen, 299 icp-oes, 1271 imine–enamine form, 39 impedance spectroscopy, 845 in silico drug discovery, 125 in vitro solubility, 571 inhibition of albumin denaturation, 139 in-service teacher training, 997 interactions, 561 interfacial tension, 725 inula, 1229 iron oxide nanoparticles, 181 iron(ii), iron(iii) oxide, 1039 isocoumarins, 639 isotherm, 429 kinetics, 429, 819, 1075 langmuir isotherm, 195, 885 lanostane triterpenoid derivatives, 809 lattice strain, 51, 571 lead and tin fluorides, 845 ligand substitution reaction, 507 lignin, 885 low-cost adsorbent, 195 ljoskavac, 1271 macro-and microelements, 1271 malachite green, 327 manganese chloride, 91 manganese hypophosphite, 91 marchantin a, 1249 math test, 533 mcm-41, 871 mcr, 1039 mdr, 1219 membrane, 341 memorized ant colony optimization, 685 metabolomics, 1177 metal complexes, 153 metal salt, 591 methylene blue, 429 microbial fuel cell, 1 microcapsules, 25 microcrystalline cellulose, 521 micronucleus, 809 microwave synthesis, 139 mif, 561 mixture descriptors, 685 modelling, 725 modified electrode, 495 molar absorptivity, 483 molecular descriptors, 63, 445 molecular docking, 469, 795 molecular modeling, 269 molecular simplex, 625 molten salts, 1075 mucilage cactus, 25 multi-component reaction, 547 multi-oxide catalysts, 615 multiple linear regression, 63 myrtus communis, 1195 nano-composite catalyst, 1039 nanomaterials, 1103 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ iv journal of the serbian chemical society natural amino acids, 381 natural products, 1305 new drug design, 469 n-heteroaromatic schiff base, 393 non-covalent interactions, 393 o-aminophenol, 39 olea europaea, 1195 olefins, 283 onset of action, 51 open-code program, 1089 opls, 1195 optimization, 651 opuntia ficus indica, 25 organic acids, 753 organic semiconductor, 171 organic synthesis, 1305 organic titanate, 415 origanum onites, 1195 origanum vulgare, 1195 oxidation of carbon monoxide, 615 oxidation stability, 739 oxidizing agents leaching, 1113 oxygen binding study, 941 p. aeruginosa, 1205 parallel accumulation serial fragmentation ion mobility mass spectrometer, 1205 pca, 753, 1229, 1195 pd-peppsi-iprdtbu-an synthesis, 247 peak overlapping area, 1089 peak processing, 1089 pentacle, 561 pesticide, 699 pet, 971 p-glycoprotein, 1219 pharmaceutical formulation, 711 pharmaceutical samples analysis, 507 phase separation time, 533 phases transition, 1067 phenanthrolinium-n-ylides, 901 phenolic antioxidants, 739 phenolic compounds, 603 phenolics, 367, 495 phenylpropanoids, 1241 photocatalytic activity, 327 physicochemical properties, 445 phytochemical stability, 367 phytochemistry, 1177 plasticization, 341 pls, 561 pollutants, 767 pollution sources, 753 poly(vinyl chloride), 591 polyacetylenes, 1241 polymer, 725 polymeric nanocomposites, 1 polymolybdate phosphate, 299 poplar propolis, 1205 prediction, 63 predictive models, 445 propofol, 711 protecting groups, 1305 protein stability, 795 protein structure, 795 protein–ligand interaction, 795 proton conductivity, 831 proton exchange membrane fuel cell, 1, 831 pseudo-second-order, 885 psf, 871 pulsed arc discharge, 181 pyranoside, 1305 qsar study, 625 qspr modelling, 685 qsrr, 381 quinoline, 115, 859 quorum sensing, 1205 rancimat induction period, 739 rate-determining step, 283 reactions, 213 remediation, 767 removal, 699 response surface methodology, 651 rimantadine, 625 ring closure, 585 schiff base pd(ii) derivative, 269 schreinmakers method, 91 scission, 283 selenium substitution, 171 selenylation, 585 sem/eds, 407, 1291 sequential injection analysis, 1089 sesquiterpene lactones, 1229 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ volume 86: subject index v silane coupling agent, 871 silica hydrogel, 819 silicates, 103 single crystal x-ray diffraction, 393, 663 single-oxide catalysts, 615 sio2/ppa, 139 slags, 103 solid electrolytes, 845 solubility, 341 solvent evaporation method, 51 solvent exchange, 819 sonochemical coprecipitation, 181 spectral analysis, 795 spectral data, 39 spectroscopic characterization, 269 sporosarcina pasteurii, 533 square–planar complexes, 459 stabilization of the sod proteins, 781 standard gibbs energy, 483 statistical methods, 673 stepwise leaching, 1113 steroidal anti inflammatory drug, 571 strain, 103 structural modification, 917 structural property, 381 substituents’ flexibility, 1023 sulfur assimilation pathway, 561 sulfur removal, 1113 summative evaluation, 997 surface functionalization, 181 surface modification, 871 suzuki reaction, 247 swcnt/water-based nanofluid, 313 synthesis, 213 tandem mass spectrometry, 1205 ternary system, 91 terpenoids, 1139, 1219 test methods, 407 tetradentate coordination, 39 theoretical study, 1053 thermal resistance, 313 thermodynamic parameters, 941 thermogravimetric analysis, 269 thin layer chromatography, 381 time-series, 753 tio2, 955 tnp, 859 tpct, 313 translocation factor, 927 triterpenoids, 1229 turbidity, 725 uv-light, 955 uv–vis spectra, 941 viscosity, 725 volatiles, 1291 vortex time, 533 walnut shell, 521 waste bread, 651 waste brewer’s yeast, 651 wastewater, 767 wet-impregnation, 615 x-ray diffraction, 415 yeast, 367 zro2, 495 β-amido-aroyl carbonyl compounds, 547 β-hematin inhibitory activity, 115 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice modern green approaches for obtaining satureja kitaibelii wierzb. ex heuff extracts with enhanced biological activity j. serb. chem. soc. 87 (12) 1359–1365 (2022) short communication jscs–5599 published 1 august 2022 1359 short communication modern green approaches for obtaining satureja kitaibelii wierzb. ex heuff extracts with enhanced biological activity vanja šeregelj1, olja šovljanski1*, jaroslava švarc-gajić1, teodora cvanić1, aleksandra ranitović1, jelena vulić1 and milica aćimović2# 1faculty of technology novi sad, university of novi sad, bulevar cara lazara 1, 21000 novi sad, serbia and 2institute of field and vegetable crops novi sad, maksima gorkog 30, 21000 novi sad, serbia (received 14 march, revised 14 may, accepted 16 may 2022) abstract: modern trends in phytochemical extraction from alternative crops support the use of alternative technologies, such as ultrasoundand microwave -assisted extractions. considering the reduction of toxic solvents, rapid and effective extraction process, the above-mentioned technologies have become the foundation of green chemistry approaches in a wide range of applications. these extractions have not been studied on satureja kitaibelii wierzb. ex heuff, which is a highly potent plant when considering its aromatic and medicinal properties. this preliminary study presents an in vitro evaluation of biological activities of ultrasoundand microwave-assisted extracts of s. kitaibelii, for the first time. furthermore, it offers a totally green, modern, fast and reproducible method for extraction of phytochemicals from s. kitaibelii herba (rtanj mountain, serbia). this short communication suggests that the applied microwave-assisted extraction, using only water as the solvent, can be a promising approach for obtaining green products with commercial potential. keywords: ultrasound-assisted extraction; microwave-assisted extraction; in vitro antioxidant analysis; in vitro antimicrobial analysis; lamiaceae. introduction satureja kitaibelii wierzb. ex heuff. or rtanj tea is an endemic lamiaceae species, mainly spread across the balkan peninsula.1 satureja kitaibelii is well known for its aromatic and medicinal properties; hence it is used as a culinary herb in mediterranean dishes, in aromatherapy, or in traditional medicine to treat various ailments.2 the extraction process is a crucial step in the valorisation of * corresponding author. e-mail: vanjaseregelj@tf.uns.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc220314043s ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1360 šeregelj et al. the plant sources; different extraction techniques and extracting solvents can influence the final phytochemical composition and bioactive potential of the obtained extracts. after reviewing scientific-relevant literature it was found that the articles regarding s. kittaibelii usually include conventional extraction approaches, essential oil, and with two non-conventional method (subcritical water extraction and ultrasound-assisted extraction).1,3–5 conventional extraction techniques have disadvantages like thermal degradation of bioactive compounds, or the use of a large quantity of organic solvents with toxic impact on the environment or on human health. recent trends in phytochemical extraction from plants recommend exploring the use of modern technologies; ultrasound-assisted (ua) and microwave-assisted extractions (ma) which become popular due to the fact that these techniques reduce the consumption of toxic solvents, increase the speed and extraction efficiency, thus being compliant to the principles of green chemistry.6 ua extracts (uae) and ma extracts (mae) of s. kittaibelii have not been studied until now. thus, the aim of this study was to investigate the effects of uae and mae on the extraction of phytochemicals from this plant, in order to obtain valuable information regarding possible application in food and pharmaceutical industries. the impact of these modern extraction technologies on the phytochemical composition and biological activity (antioxidant and antimicrobial) was evaluated by varying different green extraction solvents. experimental plant material the satureja kitaibelii herb was collected on the rtanj mountain, serbia (43◦46′34′′ n; 21◦53′36′′ e) in july 2020. voucher specimens (buns 2-1373) were used for the identification of species. the collected aboveground flowering parts were dried naturally in shade at ambient temperature. constant weight was gained after one week of drying. dry plant material was placed in a soft paper bag until further analysis. ultrasound-assisted extraction ultrasound-assisted (ua) extraction was carried out in an ultrasonic bath (iskra, slovenia) by placing samples in the proximity of the ultrasound source. ground sample (5 g) was extracted at room temperature with 100 ml of solvent (70% methanol or distilled water) for 30 min. the extracts were filtered (whatman paper no. 1) and stored at 4 °c until further analysis. microwave-assisted extraction microwave-assisted (ma) extraction was carried out in an adapted microwave oven described previously by švarc-gajić et al.7 ground sample was extracted maintaining the same sample-to-solvent ratio, extraction time, and solvent type as in the case of ua extraction, for comparison reasons. the extraction was carried for 30 min applying magnetron power of 450 w. after completing the extraction process, the extracts were filtered (whatman paper no. 1) and stored at 4 °c until further analysis. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ alterntive extractions of s. kitaibelii 1361 phytochemical analysis phenolic quantification was performed using shimadzu prominence hplc, connected to an spd-20av uv/vis detector (shimadzu, kyoto, japan). separation was performed on a luna c-18 rp column, 5 µm, 250 mm×4.6 mm (phenomenex, torrance, ca, usa) with a c18 guard column, 4 mm×9 mm×30 mm (phenomenex, torrance, ca, usa). the filtered extracts were examined by hplc reverse phase analysis as described by aćimović et al.4 in vitro antioxidant analysis antioxidant activity of extracts was investigated using four in vitro assays, as outlined by aćimović et al.:4 2,2-diphenyl-1-picrylhydrazyl (dpph), 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (abts), superoxide anion (soa) and reducing power (rp). the antioxidant activities were expressed as μmol of trolox equivalents per g of dry plant material. in vitro antimicrobial analysis antimicrobial activity of s. kitaibelii extracts was determined against the following attc referent strains: escherichia coli, pseudomonas aeruginosa, salmonella typhimurium, bacillus cereus, staphylococcus aureus, enterococcus faecalis and listeria monocytogenes (bacteria), saccharomyces cerevisiae, candida albicans (yeasts), and aspergillus brasiliensis (fungi). disk diffusion and microdilution methods for in vitro evaluation of antimicrobial activity as well as for testing minimal inhibitory concentration were performed by methods defined by mićić et al.8 statistical analysis statistical analyses were carried out using origin v. 8.0 sro software. significant differences were calculated by anova (p < 0.05). results are presented as mean value ± standard deviation (n = 3). results and discussion the phenolics profiles of the analyzed extracts are presented in table i. they possess obviously different phenolics contents depending on the applied technique and solvent, which could be related to different extraction mechanisms and polarity of the presented compounds. the highest concentration of phenolic compounds was found in the extracts prepared by mae; in terms of used solvent, significantly higher phenolic content was noted in water extract. in extracts prepared by uae, the concentration of phenolic compounds was lower, but not their number. in this case, 70 % methanol exhibited better efficiency for phenolic extraction. the obtained results were in correlation with the literature data; mašković et al.6 reported that mae ethanol extract of summer savory (satureja hortensis l.) was richer in phenolic compounds than uae. in general, microwaves induce a sudden increase in temperature inside the cellular structure, which leads to rupturing of cell walls and fast release of phytochemicals into extracting medium. the efficiency of microwave-assisted extraction lies in the fact that the energy of microwaves is directly converted to heat, by instantaneous absorption, i.e., by rapid alignment of sample dipoles with the frequency of microwaves, thus generating heat inside the matrix.10 consequently, an induced sudden increase in temperature inside cells causes rupture of cell walls and fast release of phyto________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1362 šeregelj et al. chemicals into the extracting medium. apart from solvent selectivity towards the analyte, the dielectric constant is a significant factor for obtaining high-quality extracts as well. according to vladić et al.,9 the chosen solvent should possess a high dielectric constant and strongly absorb the microwave energy; water has the highest dielectric constant, followed by methanol and ethanol. conversely, in the case of the uae, the cavitation phenomenon and free-radical formation can cause degradation of phytochemicals.10 table i. hplc analysis of phenolic compounds in extracts (c / mg g-1) obtained by ultrasound-assisted (uae) and microwave-assisted extraction (mae); values in rows with different superscripts are significantly different at p < 0.05 compound uae 70 % methanol uae water mae 70 % methanol mae water vanilic acid 3.15±0.01b – 1.13±0.00a – epicatechin gallate 7.94±0.01d 0.15±0.01a 0.40±0.00b 7.31±0.05c syringic acid 31.03±0.03b 5.17±0.04a 58.62±0.07c 59.76±0.02d coumarin acid 1.65±0.00c 0.36±0.01a 0.81±0.00b 2.50±0.02d caffeic acid 4.26±0.03d 0.55±0.00a 0.96±0.00b 2.45±0.01c gentisic acid – 1.61±0.01a 2.95±0.03b – sinapic acid 4.01±0.02c 1.14±0.02a 1.45±0.01b 4.65±0.02d rosmarinic acid 4.44±0.03c 1.45±0.00a 2.23±0.06b 8.63±0.03d ferulic acid 4.01±0.01b 3.12±0.02a 7.97±0.03c 17.96±0.1d rutin 0.04±0.00a 0.45±0.00b 0.95±0.00c 4.25±0.02d luteolin – – 0.05±0.00a – total phenolic compounds 60.53±0.14b 14.00±0.11a 77.52±0.20c 107.51±0.28d hplc analysis showed that the dominant compound in all extracts was syringic acid, which ranged from 5.17 m/g (uae water extract) to 59.76 mg/g (mae water extract). the highest content of syringic acid was earlier confirmed in s. kitaibelii subcritical water extract.4 ćetković et al.3 have also classified syringic acid among the most main phenolic compounds in s. kitaibelii extracts obtained by a conventional extraction technique and different organic solvents. in general, a single assay method is not sufficient for in vitro assessment of antioxidant activity of endogenous phytochemicals. antioxidant molecules differ in polarities, thus they can act by different mechanisms. antioxidant activity of s. kitaibelii extracts was challenged by four methods (table ii); significant antioxidant potential was found in water and 70 % methanol extracts, obtained by mae. more precisely, the antioxidant potential of tested samples decreased respectively: mae water > mae 70 %methanol > uae 70 % methanol > uae water. there were considerable differences noted in antimicrobial effects against tested microorganisms between uae and mae extracts (table iii). the uae extracts did not show antimicrobial effect, with the exception of water extract which showed low inhibition potential against a. brasiliensis. consequently, the ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ alterntive extractions of s. kitaibelii 1363 defined minimal inhibitory concentration is above the initial concentration of extract, and further antimicrobial potential of the concentrated extracts is required. however, the antimicrobial effect was observed in the case of mae extract, especially in the one prepared by using water as a solvent. table ii. in vitro antioxidant activity (μmol te/g) of satureja kitaibelii extracts obtained by ultrasound (uae)and microwave-assisted extraction (mae); values in rows with different superscripts are significantly different at p < 0.05 antioxidant uae 70 % methanol uae water mae 70 % methanol mae water dpph 78.99±14.33b 33.64±2.07a 225.63±11.45c 385.38±16.56d abts 744.66±9.74b 685.30±17.73a 1757.86±82.45c 2571.12±76.58d rp 153.20±2.99b 133.50±1.64a 336.71±5.68c 414.93±20.07d soa 3730.81±20.57b 1993.66±45.94a 4417.17±15.30c 4506.69±0.53d table iii. in vitro antimicrobial activity of satureja kitaibelii extracts obtained by ultrasound (uae)and microwave-assisted extraction (mae) test organism uae 70 % methanol uae water mae 70 % methanol mae water inhibition zone, mma e. coli atcc 25922 nd nd 13.33± 0.57 40.00±0.00 p. aeruginosa atcc 27853 nd nd nd 29.00±0.00 s. typhimurium atcc 13311 nd nd nd nd b. cereus atcc 11778 nd nd nd 28.00±0.00 s. aureus atcc 25923 nd nd 27.33±0.57 27.00±1.00 e. faecalis atcc 19433 nd nd nd nd l. monocytogenes atcc 35152 nd nd 24.00±1.00 17.33±0.57 s. cerevisiae atcc 9763 nd nd nd nd c. albicans atcc 10231 nd nd nd 18.33±0.57 a. brasiliensis atcc 16404 nd 11.00±0.0 nd nd minimal inhibitory concentration, mg/mlb e. coli atcc 25922 > 50 > 50 > 50 0.78 p. aeruginosa atcc 27853 > 50 > 50 > 50 1.56 s. typhimurium atcc 13311 > 50 > 50 > 50 > 50 b. cereus atcc 11778 > 50 > 50 > 50 0.78 s. aureus atcc 25923 > 50 > 50 12.5 0.78 e. faecalis atcc 19433 > 50 > 50 > 50 > 50 l. monocytogenes atcc 35152 > 50 > 50 25 > 50 s. cerevisiae atcc 9763 > 50 > 50 > 50 > 50 c. albicans atcc 10231 > 50 > 50 > 50 > 50 a. brasiliensis atcc 16404 > 50 > 50 > 50 > 50 a< 22 mm – low; 22–26 mm – intramedier; >26 mm – high antimicrobial activity, nd – not detected; baccording to the initial concentration of the extracts both mae extracts showed the antimicrobial effect against e. coli, s. aureus and l. monocytogenes, while water extract expressed a similar effect against p. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1364 šeregelj et al. aeruginosa, b. cereus and c. albicans. these differences can be explained by the phenolic compounds profile of the tested samples. unlike the uae samples, the methanolic mae sample contains syringic acid, while the water mae sample contains syringic acid, ferulic acid and rutin. all three mentioned phenolic compounds have previously been reported as antimicrobial agents; ferulic acid is an inhibition factor for p. aeruginosa, s. aureus, e. coli and l. monocytogenes growth,11 syringic acid inhibits the growth of s. aureus, while e. coli, p. aeruginosa, b. cereus and c. albicans are sensitive to the presence of rutin.12 the minimal inhibitory concentrations of the s. kitaibelii extracts varied according to strain level in the range of 0.78 to 25 mg/ml (table iii). the lower mic values were obtained for the water extract compared with methanolic, which also lead to differences in chemical compositions. conclusion in summary, this preliminary study indicates that the extract prepared with mae and water as solvent exhibited the highest biological activity. special significance of the presented approach is reflected in a totally green, modern, fast and reproducible process technology. according to the bioactivity screening, this research suggests that the mae water extract of s. kitaibelii could be used as a natural source of antioxidants for developing a wide range of safe and functional products which will be investigated further. acknowledgement. this work was supported by the ministry of education, science and technological development of the republic of serbia, contract no. 451-03-68/2022-14/ /200134 and 451-03-68/2022-14/200032. и з в о д модерни „зелени“ начини за добијање satureja kitaibelii wierzb. ex heuff екстракта са израженом биолошком активношћу вања шерегељ1, оља шовљански1, јарослава шварц-гајић1, теодора цванић1, александра ранитовић1, јелена вулић1 и милица аћимовић2 1технолошки факултет нови сад, универзитет у новом саду, бул. цара лазара 1, 21000 нови сад и 2институт за ратарство и повртарство нови сад, максима горког 20, 21 000 нови сад савремени трендови у фитохемијској екстракцији из алтернативних усева подржавају истраживање употребе алтернативних технологија као што су екстракције уз помоћ ултразвука и микроталаса. с обзиром на редукцију токсичних растварача и брз и ефикасан процес екстракције, поменуте технологије су постале темељ приступа “зеленој” хемији у широком спектру примена. с друге стране, ове екстракције нису проучаване на satureja kitaibelii wierzb. ex heuff, која је веома значајна ендемична биљка с обзиром на ароматична и лековита својства. ова прелиминарна студија је по први пут представила in vitro процену биолошке активности ултразвучних и микроталасних екстраката s. kitaibelii. нудећи апсолутно “зелену”, модерну, брзу и поновљиву методу за екстракцију фитокемикалија из s. kitaibelii (планина ртањ), овo истраживање сугерише да примењена екстракција уз помоћ микроталаса, користећи само воду као растварач, може бити ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ alterntive extractions of s. kitaibelii 1365 перспективан приступ за добијање “зелених” производа са комерцијалним потенцијалом. (примљено 14. марта, ревидирано 14. маја, прихваћено 16. маја 2022) references 1. m. aćimović, l. pezo, v. tešević, i. čabarkapa, m. todosijević, ind. crops prod. 154 (2020) 112752 (https://dx.doi.org/10.1016/j.indcrop.2020.112752) 2. a. đorđević, i. palić, g. stojanović, n. ristić, r. palić, int. j. food properties 17 (2014) 2157 (https://doi.org/10.1080/10942912.2013.784333) 3. g. ćetković, j. čanadanović-brunet, s. djilas, v.tumbas, s. markov, d. cvetković, int. j. mol. sci. 8 (2007) 1013 (https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3871840/) 4. m. aćimović, v. šeregelj, o. šovljanski, v. tumbas šaponjac, j. švarc-gajić, t. brezoborjan, l. pezo, ind. crops prod. 169 (2021) 113672 (https://doi.org/10.1016/j.indcrop.2021.113672) 5. k. gopčević, s. grujić, j. arsenijević, i. karadžić, l. izrael-živković, z. maksimović. plant foods hum. nutr. 74 (2019) 179 (https://doi.org/10.1007/s11130-019-0716-3) 6. p. mašković, v. veličković, m. mitić, s. đurović, z. zeković, m. radojković, a. cvetanović, j. švarc-gajić, j. vujić, ind. crops prod. 109 (2017) 875 (https://doi.org/10.1016/j.indcrop.2017.09.063) 7. j. švarc-gajić, z. stojanović, a. segura carretero, d. arráez román, i. borrás, i. vasiljević, j. food eng. 19 (2013) 525 (https://doi.org/10.1016/j.jfoodeng.2013.06.030) 8. d. micić, s. đurović, p. riabov, a. tomić, o. šovljanski, s. filip, t. tosti, b. dojčinović, r. božović, d. jovanović, s. blagojević, foods 10 (2021) 2734 (https://doi.org/10.3390/foods10112734) 9. j. vladić, t. janković, j. živković, m. tomić, g. zdunić, k. šavikin, s. vidović, s. plant foods hum. nutr. 75 (2020) 553 (https://doi.org/10.1007/s11130-020-00848-6) 10. j. švarc-gajić, samples and sample preparation in analytical chemistry, nova publishers, new york. 2011, pp. 47–49 (https://books.google.rs/books?id=vtkhyaa acaaj&dq=jaroslava+svarc+gajic&hl=en&sa=x&redir_esc=y) 11. a. pernin, v. bosc, m. maillard, f. dubois-brissonnet, front. microbiol. 10 (2019) 137 (https://doi.org/10.3389/fmicb.2019.00137) 12. s. dubey, a. ganeshpurkar, d. bansal, n. dubey, chron. young sci. 4 (2013) 153 (https://doi.org/10.4103/2229-5186.115556). ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ @article{seregelj2022, author = {{\v{s}}eregelj, vanja and {\v{s}}ovljanski, olja and {\v{s}}varc-gaji{\'{c}}, jaroslava and cvani{\'{c}}, teodora and ranitovi{\'{c}}, aleksandra and vuli{\'{c}}, jelena and a{\'{c}}imovi{\'{c}}, milica}, journal = {journal of the serbian chemical society}, title = {{modern green approaches for obtaining satureja kitaibelii wierzb. ex heuff extracts with enhanced biological activity}}, year = {2022}, issn = {1820-7421}, month = {aug}, number = {12}, pages = {1359--1365}, volume = {87}, abstract = {modern trends in phytochemical extraction from alternative crops support the use of alternative technologies, such as ultrasoundand micro­wave-assisted extractions. considering the reduction of toxic solvents, rapid and effective extraction process, the above-mentioned technologies have become the foundation of green chemistry approaches in a wide range of applications. these extractions have not been studied on satureja kitaibelii wierzb. ex heuff, which is a highly potent plant when considering its aromatic and medi­cinal properties. this preliminary study presents an in vitro evaluation of bio­logical activities of ultrasoundand microwave-assisted extracts of s. kitaibelii, for the first time. furthermore, it offers a totally green, modern, fast and repro­ducible method for extraction of phytochemicals from s. kitaibelii herba (rtanj mountain, serbia). this short communication suggests that the applied micro­wave-assisted extraction, using only water as the solvent, can be a promising approach for obtaining green products with commercial potential.}, doi = {10.2298/jsc220314043s}, file = {:d\:/onedrive/mendeley desktop/{v{s}}eregelj et al. 2022 modern green approaches for obtaining satureja kitaibelii wierzb. ex heuff extracts with enhanced biological ac.pdf:pdf;:02_11688_5599.pdf:pdf}, keywords = {lamiaceae, assisted extraction, in vitro antimicrobial analysis, in vitro antioxidant analysis, microwave}, publisher = {serbian chemical society}, url = {https://www.shd-pub.org.rs/index.php/jscs/article/view/11688}, } accepted manuscript this is an early electronic version of an as-received manuscript that hasbeen accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as l. radovanović, ž. radovanović, b. simović, m. v. vasić, b. balanč, a. dapčević, m. dramićanin, j. rogan, j. serb. chem. soc. (2022) https://doi.org/10.2298/jsc221102090r this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc221102090r j. serb. chem. soc.00(0)1-13 (2022) original scientific paper jscs–12126 published dd mm, 2022 1 structure and properties of zno/znmn2o4 composite obtained by thermal decomposition of terephthalate precursor lidija radovanović1*, željko radovanović1, bojana simović2, milica v. vasić3, bojana balanč1, aleksandra dapčević4, miroslav dramićanin5 and jelena rogan4 1innovation centre of the faculty of technology and metallurgy, university of belgrade, karnegijeva 4, belgrade, serbia; 2institute for multidisciplinary research, university of belgrade, kneza višeslava 1, belgrade, serbia; 3institute for testing of materials ims, university of belgrade, bulevar vojvode mišića 43, belgrade, serbia; 4faculty of technology and metallurgy, university of belgrade, karnegijeva 4, belgrade, serbia and 5vinča institute of nuclear sciences, university of belgrade, p.o. box 522, belgrade, serbia (received 2 november; revised 17 december; accepted 21 december 2022) abstract: a biphasic [mn(dipya)(h2o)4](tpht)/{[zn(dipya)(tpht)]·h2o}n complex material, i, (dipya = 2,2’-dipyridylamine, tpht2– = dianion of terephthalatic acid) was synthesized by ligand exchange reaction and characterized by xrpd and ftir spectroscopy. a zno/znmn2o4 composite, ii, has been prepared via thermal decomposition of precursor i in an air atmosphere at 450 °c. xrpd, ftir and fesem analyses of ii revealed the simultaneous presence of spherical nanoparticles of wurtzite zno and elongated nanoparticles of spinel znmn2o4. the specific surface area of ii was determined by the bet method, whereas the volume and average size of the mesopores were calculated in accordance with the bjh method. the measurements of the mean size, polydispersity index and zeta potential showed colloidal instability of ii. two band gap values of 2.4 and 3.3 ev were determined using uv–vis diffuse reflectance spectroscopy, while the measurements of photoluminescence revealed that ii is active in the blue region of the visible spectrum. testing of composite ii as a pigmentary material showed that it can be used for the colouring of a ceramic glaze. keywords: zinc(ii)/manganese(ii) complex; 1,4-benzenedicarboxylate; thermoslysis; zincite; hetaerolite introduction metal oxides and mixed metal oxides belong to the largest and most useful class of solid materials which have been extensively studied from various aspects due to structural, compositional and functional diversities. 1,2 zinc oxide (zno, *corresponding author e-mail: lradovanovic@tmf.bg.ac.rs https://doi.org/10.2298/jsc221102090r ac ce pt ed m an us cri pt mailto:lradovanovic@tmf.bg.ac.rs https://doi.org/10.2298/jsc221102090r 2 radovanović et al. zincite) is a multifunctional material with excellent properties, such as high chemical, thermal and mechanical stability, low toxicity, as well as high photostability, which is why it has been used in ceramics, medicine and photocatalysis. 3 zinc manganese oxide (znmn2o4, hetaerolite) with spinel structure has been widely known for its magnetic, electronic or catalytic properties. 4 during the last decades the search for new materials that can be used as inorganic pigments is in grow, with a special emphasis on environmental suitability. 5,6 inorganic pigments are important materials for colouring glazes, ceramics, plastics and glasses, owing to their high opacity, thermal stability and chemical resistance. 7 zno is a non-toxic alternative for lead white, 8 while znmn2o4, obtained from spent alkaline batteries, is suitable as brown pigment. 9 transition metal (tm) complexes with the anion of 1,4-benzenedicarboxylic (terephthalatic, h2tpht) acid are functional materials with numerous applications in chemistry and material science. 10 the usage of the tpht 2– anion as a linker between metal centres can result in the formation of fascinating supramolecular topologies because of diversity of coordination modes ranging from monodentate to even dodecadentate. 11,12 until now, a vast number of tm–tpht compounds with different nuclearity and dimensionality, have been prepared and characterized, with many of them having tpht as a bridging ligand. 13 the possibility of using tm complexes as single-source precursors for obtaining functional oxide and mixed metal oxide nanomaterials by the direct thermal decomposition process has been assessed lately. 14,15 this approach, compared with the conventional synthetic methods, has several advantages such as the possibility of stoichiometry control and homogeneity from both aspects, in terms of metals distribution as well as in the terms of size and morphology of nanoparticles of obtained oxides. 14 as a contribution to our previous research 16 relating the design and synthesis of monoand heteronuclear tm complexes to prepare new functional materials, here we presented the synthesis, spectral and structural properties of a new biphasic mn/zn complex precursor (i) composed of coordination compounds [mn(dipya)(h2o)4](tpht) and {[zn(dipya)(tpht)]·h2o}n (dipya = 2,2’-dipyridylamine), whose crystal structures have been described previously. 17,18 following the preparation of the oxide nanomaterials by solid-state thermal decomposition of tm complexes, the biphasic complex i has been used as a single-source precursor for the synthesis of a composite powder ii containing zno and znmn2o4. the structural, spectral, morphological, optical and photoluminescence properties of ii have been investigated, as well as the possibility of using this material as a pigment. ac ce pt ed m an us cri pt properties of zno/znmn2o4 composite 3 experimental materials except for dipya, which was of purum quality, all reagents were of analytical grade and used without further purification. synthesis of biphasic mn/zn complex precursor (i) a solution of dipya (0.34 g, 2.0 mmol) in 7.5 cm3 of etoh was added into solution prepared by dissolving a mixture of 1m mn(no3)2 (1.0 cm 3, 1.0 mmol) and 1m zn(no3)2 (1.0 cm3, 1.0 mmol) in 50 cm3 of distilled water. then, 25 cm3 of an aqueous solution of na2tpht (10 cm 3, 2.0 mmol) was added drop wise at room temperature under continuous magnetic stirring. the obtained beige microcrystalline precipitate was filtered off after standing overnight, washed with small amounts of distilled water, etoh and et2o and dried at room temperature. solid-state synthesis of zno/znmn2o4 composite (ii) the zno/znmn2o4 composite (ii) has been obtained by the thermal degradation of precursor i in the air atmosphere. the mass of 0.45 g of i was heated at the constant rate up to 150 °c, isothermally calcinated at 150 °c for 30 min, then heated at the constant rate up to 450 °c, and isothermally calcinated at 450 °c during 1 h, and finally, spontaneously cooled to the room temperature. yield: 19 %. measurements the x-ray powder diffraction (xrpd) measurements for i and ii were performed on a rigaku smartlab diffractometer using cukα radiation, at 40 kv and 30 ma, in braggbrentano geometry. diffraction data were collected in the range 3° < 2θ < 120° (scan speed: 1° min–1, step width: 0.01°) for i and in the range 10° < 2θ < 70° (scan speed: 1° min–1, step width: 0.01°) for ii at room temperature. the crystal structure refinement of i and ii was obtained by the full structure matching mode of the rietveld refinement technique,19 using the fullprof software.20 the average crystallite size (<d>) for ii was calculated using the rigaku pdxl2 software and the whole powder pattern fitting (wppf) method. atr-ftir spectra of i and ii were recorded in absorbance mode using a nicolet™ is™ 10 ftir spectrometer (thermo fisher scientific) with smart itr™ atr sampling accessories, within the range of 4000–400 cm–1, at a resolution of 4 cm–1 and in 20 scan mode. field emission scanning electron microscopy (fesem) tescan mira 3 xmu was used for the morphological characterization of ii. using mira software, the micrographs were analysed and the average diameters of the particles of ii (more than 100 particles) were determined. diffuse reflectance uv-vis spectrum for ii was measured over the 200–800 nm spectral region (baso4 was used as a reference standard) by shimadzu uv-2600 spectrophotometer equipped with an integrating sphere. the specific surface area (ssa) of ii was calculated according to the brunauer, emmett and teller (bet) method from the linear part of the nitrogen adsorption isotherm at 77 k on a micrometrics asap 2020 instrument. before the measurements, the samples were out-gassed at 150 °c for 10 h under a vacuum. the total pore volume (vtot) was given at relative pressure p/p0 = 0.998. the volume of the mesopores was calculated according to the barrett, joyner and halenda (bjh) method from the desorption branch of the isotherm. the mean size, polydispersity index (pdi) and zeta potential of ii were measured by photon correlation spectroscopy and by electrophoretic light scattering using zetasizer nano ac ce pt ed m an us cri pt 4 radovanović et al. zs (malvern instruments ltd., malvern, uk). the measurements were performed at the room temperature, and each sample was measured three times. photoluminescence (pl) measurements of ii were performed at room temperature on fluorolog-3 model fl3-221 spectrofluorimeter system (horiba jobin yvon), utilizing a 450 w xenon lamp as the excitation source for the steady-state measurements and xenon– mercury pulsed lamp for the time-resolved measurements. the emission spectrum of ii was scanned in the range of wavelengths from 380 to 650 nm under 350 nm excitations. the tbx04-d pmt detector was used for both time-resolved and steady state acquisitions. the line intensities and positions of the measured spectra were calibrated with a standard mercury– argon lamp. pl measurements were performed on pellets prepared from the powders under a pressure of 10 mpa. to test the synthesized material ii as a pigment for ceramic tiles glazing, several probes were done. the specimens in the shape of discs were prepared by dry hydraulic pressing of the raw clays ground to the fraction below 0.5 mm. the methodology is explained in more detail in the literature.22 the specimens were dried to a constant mass in laboratory conditions. the blank transparent glaze was composed of ceramic glass frit in a quantity of 50 mas.%, and the rest was distilled water. the freshly prepared batch was applied to the samples by immersing them in the mixture solution. the other probe consisted of the same glazing batch with the addition of 5 mas.% of dried powder of pigment ii. both kinds of discs were dried overnight at 105 °c. the single-firing process was conducted in an oxidizing laboratory kiln using the usual regime for illitic-kaolinitic clays21 to obtain a highly vitrified product.22 the final firing temperature was set to 1200 °c based on preliminary probes with blank glazed samples. the chemical composition of the ceramic frit is obtained by energy dispersive x-ray fluorescence (xrf) by using the spectro xepos instrument that contains 50 w / 60 kv x-ray tube. the colour-space l*a*b* coordinates (l* = lightness, a* = saturation, b* = intensity) of the obtained ceramic glazes were determined by using a portable spectrophotometer colorlite (sph870) by a spectral scan in the steps of 7 recordings in 1 s. the certified white standard cl20602 is used as a reference. the obtained results showed values of l*, a* and b*, by providing information on red (a* > 0) or green a* < 0 and yellow (b* > 0) or blue (b* < 0) hues. the lightness of 0 is a standard of black, while 100 presents white colour. glazed ceramic samples were recorded using a microscope at a magnification in the range of 40– 400. the samples were illuminated by an 1800 lm led light source during shooting. results and discussion characterization of precursor i xrpd pattern for biphasic precursor i is presented in fig. 1 and it showed that the system is composed of complexes [mn(dipya)(h2o)4](tpht) and {[zn(dipya) (tpht)]·h2o}n (table i, fig. s-1 (supplementary material)). the structural characterization of each phase was performed by the rietveld method according to the known crystal structures of [mn(dipya)(h2o)4](tpht) and {[zn(dipya)(tpht)]·h2o}n determined from the single crystal data. 17,18 the quantitative analysis showed that [mn(dipya)(h2o)4](tpht):{[zn(dipya)(tpht)]·h2o}n phase-ratio was 31:69 mas.%. the rietveld refinement results (tables i and s-i) displayed a minor deviation from the final structural parameters measured in the original structures. 17,18 ac ce pt ed m an us cri pt properties of zno/znmn2o4 composite 5 fig. 1. two-phased rietveld refinement pattern of i. the bragg positions of the [mn(dipya)(h2o)4](tpht) and {[zn(dipya)(tpht)]·h2o}n phases are denoted by orange and green dashes, respectively table i. structural and fitting parameters obtained by rietveld refinement for i phase [mn(dipya)(h2o)4](tpht) {[zn(dipya)(tpht)]·h2o}n crystal system monoclinic monoclinic space group p21/c p21/n a, å 7.62706(8) 9.83335(13) b, å 23.8574(2) 14.40389(15) c, å 11.09296(19) 12.27168(15) β, ° 102.2957(10) 95.7916(9) v, å3 1972.19(4) 1729.27(4) rb, % 2.39 3.15 rf, % 1.99 3.16 number of parameters refined 314 rwp, % 5.60 rp, % 4.28 rexp, % 4.42 χ2 1.60 the existence of water molecules, dipya and tpht ligands in i were confirmed from the ftir spectrum shown in fig. s-2. a strong ν(o–h) stretching vibration at 3418 cm –1 corresponds to the lattice water molecules. characteristic vibrations of the aromatic ring, ν(c=n) and ν(c=c), as well as ν(n–h) bands, are observed at 1659, 1483 cm –1 and in the 3333–3207 cm –1 region, respectively, confirming ac ce pt ed m an us cri pt 6 radovanović et al. the coordination of the dipya ligand. the presence of coordinated tpht caused the appearance of asymmetrical (νas) and symmetrical (νs) coo – vibrations at 1599 and 1385 cm –1 , respectively, while the vibrations found at 1639 and 1232 cm –1 , respectively, confirmed the presence of non-coordinated tpht ligand. in the fingerprint region, a strong peak positioned at 750 cm –1 is due to the presence of overlapped ν(n–h) and ν(c–h) vibrations. 23 a band ascribed to the ν(m–o) stretching vibration at 413 cm –1 verified the coordination of the water molecules as well as tpht ligands to the metal atom. 24 characterization of composite ii the xrpd pattern of ii, the calculated pattern, as well as the difference profile, are shown in fig. 2. the rietveld refinement revealed the coexistence of zno, which crystallizes in a hexagonal wurtzite structure and p63mc space group, and znmn2o4, which crystallizes in a tetragonal spinel structure and i41/amd space group. the quantitative phase fraction analysis revealed 62 mas.% of zno phase and 38 mas.% of znmn2o4 phase. the refined unit cell parameters (table ii) are in good agreement with pdf cards #36-1451 and #24-1123 for zno and znmn2o4 phases, respectively. the reliability factors of less than 5 % (table ii) pointed out that the experimental and calculated data are in good agreement. the calculated values of <d> for zno phase were similar in all directions meaning that its crystallites were almost spherical (table ii). the corresponding value for znmn2o4 phase along the c-axis was almost two times smaller than along aand b-axes implying elongated crystallites of znmn2o4. fig. 2. two-phase rietveld refinement pattern of ii. the bragg positions of the zno and znmn2o4 phases are denoted by green and orange dashes, respectively ac ce pt ed m an us cri pt properties of zno/znmn2o4 composite 7 the ftir spectrum of ii is presented in fig. 3. the broad bands positioned at 611 and 483 cm –1 was ascribed to mn–o stretching vibrations of mno6 octahedron, whereas the weak peaks observed in the region 424–402 cm –1 may be due to the presence of zn–o bonds in zno4 tetrahedral group. 25,26 the fesem micrographs of ii are presented in fig. 4. the powder is composed of deformed spherical nanoparticles of zno phase and the elliptical particles of znmn2o4 phase, which is in agreement with the results found by xrpd analysis. the fesem micrograph made at higher magnification (fig. 4b) shows that the particles of both phases have smooth surfaces with an average diameter of about 67 nm for zno phase and with average width and length of 156 and 290 nm, respectively, for znmn2o4 phase. table ii. structural and fitting parameters obtained by rietveld refinement for ii phase zno znmn2o4 crystal system hexagonal tetragonal space group p63mc i41/amd a / å 3.2574(1) 5.7299(3) c / å 5.2175(2) 9.3000(8) v / å3 47.945(3) 305.34(3) <d> / nm 26.5 [0,0,1]; 30.8 [–0.356,–0.935,0] 30.8 [0.935,–0.356,0] 19.5 [0,0,1] 36.9 [–0.356,–0.935,0] 36.9 [0.935,–0.356,0] number of parameters refined 39 rwp / % 4.80 rp / % 3.82 rexp / % 3.80 s 1.2633 χ2 1.5960 fig. 3. ftir spectrum of ii ac ce pt ed m an us cri pt 8 radovanović et al. fig. 4. fesem images of ii at different magnifications zeta potential is very important for the stability of colloidal dispersions. in general, dividing line between stable and unstable dispersions is taken at ±30 mv. particles with absolute zeta potentials higher than 30 mv are mainly considered stable. 27 the mean particle size was found to be (445.6±53.1) nm. this can be explained by the formation of agglomerates which was also observed by fesem. the value of zeta potential was (–7.80±0.86) mv, indicating that particles of ii carried the negative surface charge and that colloidal dispersion is unstable. the estimated pdi was high with a value of 0.347±0.100, implying non-uniform dispersion of ii during dyeing. 28-30 the results of the bet analysis of ii are presented in table iii and fig. 5. the value of ssa is small, being equal to 16.95 m 2 g –1 , while the average pore size was estimated to be 23.3 nm (table iii). up to p/p0 = 0.8, the slope of the n2 adsorption/desorption isotherms of ii is small due to the presence of a little number of small size pores (fig. 5a). a slight separation of the adsorption and desorption isotherms of ii was observed in the region 0.8–1 at p/p0 axis, meaning that the quantity of micropores is also small, which further implied that pores were a consequence of the voids between the nanoparticles. the small ssa value of ii denoted that composite could be uneven and weakened in colouring strength. 31 table iii. the results of bet analysis of ii. ssa / m2 g–1 vtota / cm3 g–1 vmesob / cm3 g–1 vmicroc / cm3 g–1 daverd / nm dmaxe* / nm 16.95 0.0813 0.0772 0.0048 23.3 23.5 and 34.7 avtot – total pore volume; bvmeso – mesopore volume; cvmicro – micropore volume; ddaver – average pore diameter; edmax – the diameter of the pores that occupy the largest part of the volume; *two maxima exist on the curve. ac ce pt ed m an us cri pt properties of zno/znmn2o4 composite 9 fig. 5. adsorption-desorption curves (a) and pore volume and pore size distribution (b) for ii the energy band gap (eg) values for ii were calculated from the plot of the modified kubelka-munk function (f(r)hν) 2 vs the energy of the adsorbed light (hν) using the linear fits close to the absorption edge as it is shown in fig. 6a. the absorption spectra exhibited double absorption edges and two different eg values were determined: one at 2.4 ev and another at 3.3 ev. a lower eg value could be ascribed to d-d transitions and the dark colour of ii, as it is already observed for samples with a high concentration of tm in the structure, while a higher eg value could be due to an increase in the intensity of tm–o 2– charge transfer. 32 the steady-state emission spectrum of ii obtained at room temperature is presented in fig. 6b. upon excitation at 350 nm, this analysis revealed a band centred at 422 nm in the blue region of the visible part of the spectrum followed by low or negligible absorption in the red and orange region, which is associated with the brown colour of ii. 32 fig. 6. direct band gap energies (a) and the emission spectrum (b) of ii the composite ii is tested for application as a pigment for colouring the ceramic glaze. the transparent glaze is obtained from a glass frit containing a high quantity of sio2, and a high sio2/al2o3 ratio (table iv). ac ce pt ed m an us cri pt 10 radovanović et al. table iv. chemical composition of the ceramic glass used for glazing. share, % share, % loi* 3.47 so3 0.04 sio2 62.16 p2o5 0.06 al2o3 15.76 mno 0.00 fe2o3 0.19 tio2 0.06 cao 9.84 pb 0.03 mgo 0.46 cd 0.00 na2o 6.00 ba 0.89 k2o 0.92 sum 99.87 *loi – loss on ignition the low quantity of lead and absence of cadmium shows that the glaze is not toxic to living organisms. 33 the pigment material ii was of a brownish-black colour with a value of l* being 26.85 (table v, fig. 7a) and with a* and b* coordinates similar to other materials with spinel structure used as pigments. 6,32 the dark pigment decreased the lightness of the glaze by about 37 %, whereas, at the same time, redness and yellowness increased. in both transparent and pigmented glazes, a smooth surface is obtained (fig. 7b–c and fig. s-3). the unevenness of the pigment distribution may be caused by an insufficient quantity of the pigment particles that grouped and spread over the transparent glaze during the sintering process. in addition, unevenness of the pigment ii is in accordance with its small value of ssa (table iii), high pdi and zeta potential close to zero. table v. cie l*a*b* colorimetric coordinates of the pigment ii and glazes sample l* a* b* ii 26.85 2.94 4.96 transparent glaze 64.88 2.60 15.38 pigmented glaze 40.70 11.26 22.04 fig. 7. the appearance of the pigment ii (a), transparent (b) and pigmented glazed ceramics (c) the advantages of composite materials considered as not harmful to health may make these glazes promising and widely used on surfaces where glazed ceramics come into contact with food or chemicals. 34 ac ce pt ed m an us cri pt properties of zno/znmn2o4 composite 11 conclusion direct solid-state decomposition of terephthalate precursor i, composed of 31 mas.% of [mn(dipya)(h2o)4](tpht) and 69 mas.% of {[zn(dipya)(tpht)]·h2o}n, gave as result the nanocrystalline zno/znmn2o4 brown composite material, ii, with phase ratio 68:32 for zno and znmn2o4, respectively. rietveld structure refinement results revealed the presence of wurtzite zno and spinel znmn2o4. particle size of zno phase was about 67 nm, while width and length of znmn2o4 particles were about 156 and 290 nm, respectively. the pdi of 0.347, small value of ssa of 16.95 m 2 g −1 and zeta potential value of −7.80 mv resulted in unstable pigmentary dispersion of ii and uneven distribution of pigment during dyeing of transparent ceramic glaze. since the composite ii is composed of nontoxic oxides, it is expected to be the environmentally safe for application as pigmentary material in paints, polymers and ceramics. acknowledgements: this work was supported by the ministry of education, science and technological development of the republic of serbia (contract no. 451-03-68/202214/200287, contract no. 451-03-68/2022-14/200135, contract no. 451-03-68/202214/200012, contract no. 451-03-68/2022-14/200053). supplementary material additional data are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/12126, or from the corresponding author on request. извод структура и својства композита zno/znmn2o4 добијеног термичком разградњом терефталат–прекурсора лидија радовановић1, жељко радовановић1, бојана симовић2, милица в. васић3, бојана баланч1, александра дапчевић4, мирослав драмићанин5 и јелена роган4 1иновациони центар технолошко-металуршког факултета, универзитет у београду, карнегијева 4, београд, србија; 2институт за мултидисциплинарна истраживања, универзитет у београду, кнеза вишеслава 1, београд, србија; 3институт за испитивање материјала имс, универзитет у београду, булевар војводе мишића 43, београд, србија; 4технолошко-металуршки факултет, универзитет у београду, карнегијева 4, београд, србија и 5институт за нуклеарне науке „винча“, универзитет у београду, поштански преградак 522, београд, србија двофазни [mn(dipya)(h2o)4](tpht)/{[zn(dipya)(tpht)]·h2o}n комплексни материјал, i, (dipya = 2,2’-дипиридиламин, tpht2– = дианјон 1,4-бензендикарбоксилне киселине) синтетисан је реакцијом измене лиганада и окарактерисан xrpd мeтoдом и ftir спектроскопијом. композит zno/znmn2o4, ii, добијен је термичком разградњом прекурcopa i у атмосфери ваздуха на 450 °c. xrpd мeтoдом, ftir спектроскопијом и fesem микроскопијом композита ii утврђено је истовремено присуство сферних наночестица zno вирцитне структуре и издужених наночестица znmn2o4 са структуром спинела. специфична површина ii одређена је bet методом, док су запремина и просечна величина мезопора израчунати у складу са bjh методом. средња величина, индекс полидисперзије ac ce pt ed m an us cri pt https://www.shd-pub.org.rs/index.php/jscs/article/view/12126 12 radovanović et al. и цета потенцијал измерени су фотонском корелационом спектроскопијом и електрофоретским расејањем светлости и показали су нестабилност композита ii. вредности ширине забрањене зоне 2,4 и 3,3 ev одређене су uv-vis дифузно-рефлексионом спектроскопијом, док су мерења фотолуминесценције показала да је ii активан у плавој области видљивог дела спектра. испитивање композита ii као пигментног материјала показало је да се може користити за бојење керамичке глазуре. 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http://journals.iucr.org/iucr-top/comm/cpd/newsletters/ https://doi.org/10.1016/j.bsecv.2020.11.006 https://doi.org/10.1016/j.ceramint.2020.05.302 https://doi.org/10.1016/0301-0104(79)85204-0 https://doi.org/10.1016/j.physe.2018.10.027 https://doi.org/10.1080/01496395.2018.1444054 http://dx.doi.org/10.1016/s0955-2219(98)00208-8 https://doi.org/10.1081/dis-120015367 https://doi.org/10.3390/coatings10080741 https://doi.org/10.1016/j.progsolidstchem.2017.02.002 https://doi.org/10.1149/2.0811501jes ac ce pt ed m an us cri pt j. serb. chem. soc.00(0)s1-s3 (2022) supplementary material s1 supplementary material to structure and properties of zno/znmn2o4 composite obtained by thermal decomposition of terephthalate precursor lidija radovanović1*, željko radovanović1, bojana simović2, milica v. vasić3, bojana balanč1, aleksandra dapčević4, miroslav dramićanin5 and jelena rogan4 1innovation centre of the faculty of technology and metallurgy, university of belgrade, karnegijeva 4, belgrade, serbia; 2institute for multidisciplinary research, university of belgrade, kneza višeslava 1, belgrade, serbia; 3institute for testing of materials ims, university of belgrade, bulevar vojvode mišića 43, belgrade, serbia; 4faculty of technology and metallurgy, university of belgrade, karnegijeva 4, belgrade, serbia and 5vinča institute of nuclear sciences, university of belgrade, p.o. box 522, belgrade, serbia fig. s-1. the asymmetric unit of [mn(dipya)(h2o)4](tpht) phase (a) and the structural fragment of {[zn(dipya)(tpht)]·h2o}n phase (b) in i. *corresponding author e-mail: lradovanovic@tmf.bg.ac.rs ac ce pt ed m an us cri pt mailto:lradovanovic@tmf.bg.ac.rs s2 radovanović et al. table s-i. selected bond lengths (å) for [mn(dipya)(h2o)4](tpht) and {[zn(dipya)(tpht)]·h2o}n phases in i. phase bond bond length, å [mn(dipya)(h2o)4](tpht) mn1–n1 mn1–n2 mn1–o5 mn1–o6 mn1–o7 mn1–o8 2.217(14) 2.356(18) 2.23(3) 2.22(4) 2.23(4) 2.44(5) {[zn(dipya)(tpht)]·h2o}n zn1–n1 zn1–n2 zn1–o1 zn1–o3 zn1–o4 2.153(14) 2.062(8) 2.029(18) 2.408(15) 2.060(16) figurte s-2. ftir spectrum of i ac ce pt ed m an us cri pt supplementary material s3 figure s-3. transparent (a–d) and pigmented (e–h) glaze at different magnifications: 40 (a, e), 100 (b, f), 200 (c, g) and 400 (d, h) ac ce pt ed m an us cri pt effect of an industrial chemical waste on the uptake j. serb. chem. soc. 87 (0) 1–15 (2022) original scientific paper jscs–11740 published 30 september 2022 1 pictorial based learning: promoting conceptual change in chemical kinetics habiddin habiddin1*, herunata herunata1, oktavia sulistina1, aceng haetami2, maysara maysara2 and dušica rodić3# 1department of chemistry, universitas negeri malang, indonesia, 2department of chemistry education, universitas halu oleo, kendari, indonesia and 3department of chemistry, biochemistry and environmental protection, university of novi sad, novi sad, serbia (received 27 april, revised 24 june, accepted 18 july 2022) abstract: this study aimed to examine the effect of pictorial based learning (pcbl) on conceptual change in the topic of chemical kinetics. the four-tier instrument (ftdick) previously developed was deployed to map conceptual change within chemical kinetics concepts. first-year chemistry students at an indonesian university formed an experimental and a control group. the experimental group experienced the pcbl approach while the control one experienced direct instruction (di). the conceptual changes demonstrated by the two groups are classified into four categories, namely complete, partial, false and random. complete conceptual change (ccc) had the highest occurrence rate among the four categories. however, generalising that pcbl and di are influential in promoting conceptual change in the field of chemical kinetics may be too ambitious. therefore, further research is needed to reach that conclusion. the effectiveness of pcbl and di in promoting conceptual change in this study was almost equal. however, in answering the ftdick questions, the pcbl students showed a better performance reflecting more sound scientific understanding than di students did. keywords: pictorial representation; direct instruction; four-tier instrument; misconception. introduction the topic of chemical kinetics is a challenge for many students at both secondary and university levels.1 similar to other physical chemistry topics, chemical kinetics demands the integration of conceptual understanding and mathematical ability as well as a correlation with other chemistry topics such as thermodynamics, the theory of molecular kinetics, and other aspects of chemical react * corresponding author. e-mail: habiddin_wuni@um.ac.id # serbian chemical society member. https://doi.org/10.2298/jsc220403070h 2 habiddin et al. ivity. many studies have revealed that students can successfully acquire knowledge on chemical phenomena following the elaboration of the associated academic content. however, they may still harbour ingrained misconceptions.2,3 efforts to promote conceptual changes in situations where students hold misconceptions have been made over the last few decades4 and they constitute an important part of chemistry teaching. conceptual change presupposes a restructuring or modification of existing knowledge2,5–7 such that it becomes a solid piece of scientific knowledge.2 various teaching strategies relevant to the theory of conceptual change can be applied to modify students’ conceptions.2 the implementation of such material related to acids and bases resulted in significant conceptual changes as well as changes in students’ attitudes toward chemistry.4 another study8 employed the predict-discuss-explain-observe-discuss-explain (pdeode) teaching model to promote a conceptual change related to the concept of evaporation. another study revealed that the conceptual change on inorganic chemistry was successful in overcoming students' misconceptions related to the topic of the state of matter.9 presenting a visual representation of relevant chemical concepts can be a way of challenging students’ thinking processes. many studies support the theory that a pictorial representation prompts meaningful learning, which helps students master scientific concepts and encourages concept management, concepts acquisition and integration underpinning the cognitive activities.10 according to the cognitive psychology of instruction, the visualisation approach is a helpful strategy in aiding students to solve multistep tasks.11 thus, the use of pictorial representations can be helpful in teaching and learning,12 including promoting conceptual change. scientific concepts, including chemical concepts, can be presented, and communicated in many formats, including visual representations (pictures, graphs, photographs, diagrams), tables and mathematical formulae.12 depicting chemical concepts within appropriate representations can stimulate students’ cognitive development and promote their information processing abilities.13 in some studies, the advantage of pictorial representation over verbal-based learning has produced some contradictory results. one study revealed that pictorial and verbal-based instruction had an equal effect on student achievement.14 another study reported that verbal-based learning resulted in better student achievement than pictorial-based learning,15 while the other revealed that students who experienced pictorial-based learning performed better on a pictorialbased test but not on a verbal or multiple-choice one.16 arnold and dwyer17 found that the implementation of pictorial representations in teaching and learning produced higher student attainment. pictorial based learning in chemistry 3 regardless of the small number of contradictory results, the majority of research established those pictorial representations promoted a more productive science learning environment.17–19 berg et al.20 revealed that when students had been asked to draw a representational picture after observing a chemical process, they were more likely to produce a reasonable explanation of this process. in another study, macroscopic and sub-microscopic visualisations positively impacted students’ discourse.21 pictorial representations emphasising the sub-microscopic level have been a tool to improve students’ scientific understanding. students’ mastery of chemical concepts is profoundly affected by their ability to interpret abstract concepts.22 therefore, the practice in visualising and interpreting these abstract concepts should be incorporated in chemistry teaching.23 the pcbl in this study combined pictorial representations, involving the sub-microscopic level, with verbal explanations within the students’ pair work class discussions, and written explanations. experimental research design and instrument this quasi-experimental study applied the pre-test – post-test non-equivalent group design. it involved two groups of first-year chemistry students (with the age range of 19–21 years old) from an indonesian university taking a fundamental chemistry course. one group with 23 students was chosen as the experimental group, and the other with 25 students as the control group. the allocation of students in each class within the university is managed by the department. students are distributed equally in terms of their academic background to ensure homogeneity between classes. this procedure explains the equal prior knowledge between the two groups. both groups completed a pre-test and post-test with identical questions in the form of the four-tier diagnostics instrument of chemical kinetics (ftdick) developed in our previous study.24 ftdick is a four-tier assessment tool having answer reason tiers along with attached confidence rating indices (cri) on each tier. the full features of the ftdick instruments are provided in the supplementary material to this paper. the pre-test was carried out simultaneously for both groups, while the post-test was conducted separately. due to covid-19 precautions, the post-test for the control group was carried out online using the zoom platform. to ensure that students answered the questions independently, they were requested to turn on the video during the test. the post-test for the experimental group was conducted when the government regulation for delivering all the classes online had not been applied. procedure this study encompassed the following steps. pre-test within the pre-testing, the students from experimental and control groups completed the ftdick test, which provided insight into the misconception that students held before they embarked on the chemical kinetics course. several common misconceptions were uncovered and used to design the pictorial representations to be implemented in chemical kinetics teaching. the most prevalent misconceptions demonstrated by 8.70–45.60 % of students were 4 habiddin et al. found in the successive half-lives of first-, secondand zero-order reactions, the effect of concentration on the rate of chemical reaction, and the process of catalysis. intervention within this study, the experimental group was exposed to pcbl that aimed to address the misconceptions revealed by the pre-test, while the students in the control group learned through di. the teaching textbooks and duration of teaching for the two groups was the same. the pcbl process was initiated at the start of the kinetics course by presenting images or graphics representing the sub-microscopic processes occurring in various chemical reaction scenarios. the pictorial representations functioned as a cognitive trigger for students. below is an example of a pictorial trigger presented at the beginning of the session on successive halflives in chemical reactions. fig. 1 provides pictorial representations of successive half-lives for firstand secondorder reactions and was used as the trigger at the start of instruction related to the concept of half-lives. in the next step of the procedure, students worked in pairs to extract information fig. 1. the pictorial trigger for the concept of successive half-lives of first and second orders. pictorial based learning in chemistry 5 from the two figures and make comparisons. the information gained from the pictorial representation was discussed in class. this resulted in the conclusion that the time for each half-life is different for firstand second-order reactions. similar steps were applied in the teaching of other concepts. direct instruction (di) in this study was initiated by a brief review of the previous topic from the last class, followed by teaching on the current topic. subsequently, students carried out exercises individually or in small groups depending on the tasks’ complexity. the teacher then provided constructive feedback and a brief review of concepts covered within that lesson. as explained previously, the textbook for di group was the same with the textbook for pcbl class. however, the pictorial presentations within the textbook were only used for additional information to support the teacher's explanation. post-test the post-test was employed to explore conceptual changes demonstrated by the students from the experimental and control groups. eleven questions of the original ftdick were removed to leave nine questions that focussed on concepts for which students had demonstrated misconceptions. the instrument is provided in supplementary material. data analysis there are four possible combinations of students’ responses to the ftdick, namely: correct answer correct reason (cacr), correct answer wrong reason (cawr), wrong answer correct reason (wacr) and wrong answer wrong reason (wawr). cacr combinations reflect students’ scientific understanding of the concepts under investigation. wawr is the primary indicator of students’ misconceptions related to these concepts. confidence rate index (cri) for each combination is the average of the cri for the a-tier and the r-tier. it reflects the level of students’ scientific and unscientific reasoning, as outlined in table i. table i. the criteria for classification of students' misconceptions12 no. cri of a wawr combination category 1. >4.00–5.00 strong 2. >2.75–4.00 moderate 3. >2.00–2.75 weak 4. >1.00–2.00 lack of knowledge 5. >0.00–1.00 guesswork students’ responses to the pre-test and post-test were graded according to the following procedures. score 1 was attributed to a cacr combination, while score 0 was attributed to the other combinations (wacr, cawr and wawr). the number of students that demonstrated misconception on each question in both the pre-test and post-test, was calculated according to: wawr 100 n n nt = (1) n represents the number of students with misconceptions. nwawr is the number of students providing wawr combinations in each question. nt is the total number of students participating in this study. 6 habiddin et al. the criteria for classification of students’ misconceptions the strength of students’ misconceptions is reflected by students’ cri when providing wawr combinations. the cri values are classified according to the criteria applied by habiddin.12 the criteria are presented in table i. the difference in performance between pcbl and di students the mann–whitney u test using spss software was employed to reveal the statistical difference in performance of pcbl and di students on ftdick. the non-parametric procedure (mann–whitney u test) was applied because the shapiro–wilk test prerequisite procedure revealed that the collected data were not normally distributed. results and discussion pictorial based learning (pcbl): addressing students’ misconceptions the misconceptions demonstrated by students on the pre-test were used to develop teaching material for pcbl. as discussed in the method section, pcbl intervention in this study was conducted using a pictorial representation of chemical kinetics’ concepts as a trigger for students’ cognitive and thinking processes. the pcbl was initiated by giving a pictorial presentation of a specific chemical concept. following this, students worked in pairs to extract meaningful information from the representation and associated teaching. for example, where more than one image associated with a concept was provided, students were instructed to compare the similarities and differences between the pictorial representations. to overcome the misconception that “the duration of the firstand second halflife is identical (which they are not in any but first-order reactions)”, the pcbl was initiated through the pictorial representation presented in fig. 1. this misconception was uncovered from students’ responses when given a question in which they had to determine the concentration of a reactant in its firstand second half-life. in answering such a question, some students considered that the concentration within the firstand the second half-life is the same. therefore, fig. 1 provides a cognitive challenge to students. fig. 1 was employed to prompt students understand that the time needed for each half-life for a second-order reaction is not a constant as in the case of first-order reaction. this figure is intended to overcome the misconception that the duration of successive half-lives within second-order reaction is identical and draw students’ attention to the fact that the constant duration of successive half-lives is only applicable for the first-order reactions. fig. 2 is the pictorial trigger to overcome the misconception that “a catalyst does not remain chemically unchanged after the reaction is completed.” the figure shows that the catalyst, represented by the two red spheres, is not consumed and remain chemically unchanged even though it took part in the reaction, as shown in the central box. the involvement of the catalyst is confirmed by the separation of two red spheres and their recombination. pictorial based learning in chemistry 7 fig. 2. the pictorial trigger for the concept of catalyst. fig. 3 provides several pictorial clues that the presence of a catalyst increases the reaction rate. the figure is the pictorial trigger to overcome a misconception that “the presence of a catalyst decreases the rate.” fig. 3a depicts the decomposition of hcooh without the presence of a catalyst. fig. 3b illustrates the decomposition of hcooh using the catalyst h+. the figure shows that the presence of the catalyst lowers the activation energy and leads to a higher reaction rate in comparison to the uncatalysed reaction (fig. 3a). the figure also indicates another scientific information: the presence of a catalyst provides a different reaction mechanism as depicted by the different steps in the same reaction and multiple transition states in the presence of the catalyst. some concepts are difficult to display pictorially, and so verbal descriptions had to be used. for example, table ii was used to overcome the belief that “the power of the reactants in the rate law expression for a given chemical reaction is equal to the stoichiometric coefficients in the balanced equation of that reaction.” it also demonstrates that the rate law expression cannot be obtained by applying the same procedure as for the equilibrium constant expression. both of these theories are commonly held misconceptions.25 fig. 3. the pictorial trigger for the concept of catalyst. it is likely that the misconception that the rate law expression is obtained from the stoichiometric equation derives from the nature of the examples used during the teaching. if within the examples that the students were given the values of the stoichiometric coefficients and exponents in the rate law expression were identical, this could have led to the conclusion that the latter could be obtained directly from the former. table ii provides examples demonstrating that 8 habiddin et al. the rate law is determined experimentally and does not depend on the balanced stoichiometric equation. this table also demonstrates that the rate law expression and the equilibrium-constant expression are different concepts. table ii. the triggering table for the concept of rate law no. chemical equation rate law 1. co(g) + no2(g) → co2(g) + no(g) rate = kccocno2 2. 2h2o2(aq) → 2h2o(l) + o2(g) rate = kch2o2 3. 2no(g) + cl2(g) → 2nocl(g) rate = kcno2ccl2 4. 2nh3(g) → n2(g) + 3h2(g) rate = kc(nh3) 0 = k 5. pt(nh3)2cl2(aq) + h2o(l) →[pt(nh3)2o)cl] +(aq) + cl-(aq) rate = kcpt(nh3)2cl2 the figures and table were presented at the beginning of elaboration of the concept of interest. following this, students worked in pairs to extract valuable pieces of information and relate them to the targeted concept. in the next step, students shared their conclusions with the whole class, and this was followed by a class discussion led by the class teacher. the process of extracting valuable information from a picture or table, relating the information to the relevant concept and sharing and discussing ideas is expected to trigger students’ cognitive processes and finally lead to the conceptual change. these activities are appropriate to the following components of scientific practices formulated by the national research council,26 namely: asking questions, analysing and interpreting data, constructing explanations and designing solutions, engaging in discussion based on evidence, and also obtaining, evaluating and communicating information. these scientific practices were in the focus of science education research over the last decade.27–29 description of conceptual change occurrence rates students’ conceptual change as a result of the implementation of pcbl and di approach is determined based on the shift from students’ misconceptions demonstrated on the pre-test to a scientific understanding and decrease in the frequency of occurrence of these misconceptions on the post-test. table iii shows the percentages of students holding misconceptions before and after the implementation of the abovementioned teaching approaches. the table presents the complete conceptual change (ccc), partial conceptual change (pcc), false conceptual change (fcc) and random conceptual change (rcc) occurrence rates. ccc means that all the students who previously held misconceptions demonstrated a full scientific understanding given on the post test. pcc means that a only certain portion of the students demonstrated a scientific understanding of given concept on the post-test. a smaller portion of students is still retaining the misconception. fcc means that the number of students demonstrating misconception (mc) on the post-test is even higher compared to the pre-test. rcc pictorial based learning in chemistry 9 means the number of students holding the misconception decreased, but their cri increased and vice versa. the comparison of the conceptual change occurrence rates among pcbl and di students is depicted in fig. 4. table iii. conceptual change occurrence rates misconception (mc) pre-test post-test pcbl di n % cri category n % cri cat egory n % cri cat egory mc1: the terminalogy of the first half-life and the second half-life means the same 21.74 3.3 moder -ate 5.26 1.5 lack of knowledge 4.17 1.5 lack of knowledge mc2: the reaction rate always increases with a decrease in concentration of reactant 21.74 2.8 moder -ate 0 0 over come 25 4.5 strong mc3: the t1/2 value of each successive half-life for the firstand the second-order reactions is identical 26.09 3.1 moder -ate 36.84 3.9 moder ate 8.33 4.0 strong mc4: the duration of the half-lives of a second-order reaction is constant 23.91 3.6 moder -ate 36.84 3.5 moder ate 25 3.0 moder ate mc5: the power of the reactants in the rate law expression for a given chemical reaction is equal to the stoichiometric coefficients in the balanced equation of that reaction 8.70 2.0 weak 5.26 1.5 lack of knowledge 8.33 3.5 moder ate mc6: the equilibriumconstant expression can be used to derive the rate law expression 45.65 3.3 moder -ate 10.52 3.0 moder ate 16.6 7 3.3 moder ate mc7: the rate law equation can only be expressed in terms of the rate of disappearance of reactants 26.09 3.3 moder -ate 0 0 over come 0 0 over come mc8: a catalyst does not remain chemically unchanged after the reaction is completed 17.39 3.1 moder -ate 0 0 over come 0 0 over come mc9: the presence of a catalyst decreases the reaction rate 13.04 3.8 moder -ate 5.26 5.0 strong 4.17 1.0 lack of knowledge mc10: the reaction rate always decreases with time 10.87 2.5 weak 0 0 over come 0 0 over come 10 habiddin et al. fig. 4. a comparison of occurrence rates of the four types of conceptual change among students from pcbl and di groups. the ccc is the most frequent type of conceptual change demonstrated by pcbl students, whereas the occurrence rates of ccc and pcc are equal within the di students. the ccc for pcbl students is detected for mc2, mc7, mc8, while for the di students it occurred in relation to mc10 and mc7, mc8 and mc10. before the intervention, 21.74 % of pcbl students with a cri of 2.80 held the misconception that the rate always increases with a decrease in concentration (mc2). after the intervention, all pcbl students held a correct scientific understanding regarding this concept. the opposite phenomenon, i.e., fcc, is shown by di students. the number of students holding the misconception that fell in the “strong” category increased after the intervention. for mc7, mc8 and mc10, both groups demonstrated a ccc. within mc8, students assumed that “a catalyst does not remain chemically unchanged after the reaction is completed.” after the interventions encompassing fig. 2, they realised that the catalyst remains chemically unchanged even after it took part in the reaction. the absence of the two connected red spheres representing a catalyst in the central box implies that the catalyst is involved in the reaction. the two connected red spheres in the final box indicated the correct scientific idea to the pcbl students, which is that the catalyst is not consumed in the reaction and that it remains chemically unchanged after it. this study confirmed the work published by canpolat et al.30 that students taught by a conceptual change approach performed better than those taught by a more traditional approach. the successful conceptual change was also reported in relation to the teaching about acid and base.4 the improvement in students’ understanding after implementing pictorial representations has also been reported in regard to the other chemistry topics.20,21,31,32 a pcc was detected in relation to mc1, mc5, and mc6 for pcbl students. a significant decrease in a number of students holding the misconceptions to successive half-lives after experiencing pcbl is strong evidence of conceptual change regarding mc1. fig. 1 shows that the label t1/2 at every point where the concentration of the reactant is half the previous one triggered students’ aware pictorial based learning in chemistry 11 ness that half-life terminology is applicable every time the concentration is halved. the 5.26 % of students who still held this misconception after the intervention showed a very low cri of 1.5, which falls in the “lack of knowledge” category. another significant decrease in the number of students holding the misconception that “used the equilibrium-constant expression to derive the rate law” is revealed in mc6. however, the remaining 10.52 % of students who still held this misconception showed a moderate cri. meanwhile, a small decrease in the number of students holding the misconception that “the power of the reactants in the rate law expression for a given chemical reaction is equal to the stoichiometric coefficients in the balanced equation of that reaction” is revealed for mc5. providing various chemical equations accompanied by their rate law expressions (table ii) did not totally overcome students’ misconceptions in regard to this concept. pcc for di students appears for mc1, mc6 and mc9. as explained before, pcc for mc1 and mc6 also appear for pcbl students. at mc9, a small portion of di students (4.17 %) still held the misconception that “the presence of a catalyst decreases the rate” but with a very low cri falling in the lack of knowledge category. some unexpected results regarding fcc are found in relation to mc3 for pcbl students. even after experiencing pcbl and studying fig. 1, some students still firmly believed that “the t1/2 value of each successive half-life for the firstand the second-order reactions is identical.” although the cri of those misconceptions remained in the “moderate” category, the number of students holding them increased. this phenomenon confirms the strong familiarity of students with the concept of constant half-life in first-order reactions.12 greater emphasis on first-order reactions in chemistry textbooks could be the cause of this misunderstanding.12 this could also explain the unexpected in finding related to mc4, namely, “the duration of the half-lives of a second-order reaction is constant.” this finding is in line with the one published by tirr et al.15 di students demonstrated an rcc in this instance. the number of students holding the misconception decreased significantly, but the cri of those still holding it increased significantly, falling into the “strong category.” the rcc is demonstrated by pcbl students regarding mc9 related to the misconception that “the presence of a catalyst decreases the reaction rate.” this misconception is a novel finding. fig. 3 was used to explore this point with an additional question designed to establish a link between the energies of the different transition states and the relative rates of each reaction. a discussion of the different mechanisms was also instigated. after experiencing pcbl, a complete conceptual change was also demonstrated regard to this concept. the number of students demonstrating this misconception significantly decreased, but the cri of those holding it also increased, falling in the “strong” category. 12 habiddin et al. the fact that pcbl in this study did not completely remediate students’ misconception could have a variety of explanations. firstly, students may not yet be familiar with this teaching approach. students may need more practice in extracting meaningful information from visual prompts. secondly, the pcbl approach implemented in this study was provided by the lecturers (who are also the authors). the approach may be enhanced by asking students to draw their pictorial representations of the relevant concepts.33 thirdly, the ways in which students initially develop their misconceptions needs to be explored2 in order to develop better strategies to overcome them. further studies should consider these shortcomings. pcbl vs. di students’ performances the results of this study imply that it is not possible to determine which of the two approaches (pcbl and di) is better in promoting conceptual change. as displayed in fig. 4, the ccc occurrence rate for pcbl students is higher than that for the di students. the occurrence rate of pcc and fcc is equal for the two groups. the di students demonstrate higher rcc occurrence rates compared to the pcbl students. reflecting on these facts, it seems that pcbl students showed only a slightly better performance than di students did in terms of the conceptual change. this phenomenon is in line with the work of wang,34 who compared inquiry-based teaching and di. in terms of students’ performance of ftdick, the mann whitney u test revealed that the mean rank of the pcbl students (29.16) is higher than that of the di students (16.33). specifically, the u statistic and the asymptotic significance (2-tailed) p-value show that pcbl students’ performance in answering ftdick questions was significantly higher compared to the di students (u = = 92.000, p = 0.001). this result reinforces the finding that pcbl is effective in achieving ccc for most students who experience it. conclusion the implementation of pcbl and di triggers a conceptual change among students who previously harboured misconceptions in regard to chemical kinetics. complete conceptual change (ccc) had the highest occurrence rate among the pcbl students, while ccc and pcc occurred with equal frequency among the di students. fcc also occurred with the same frequency among the students for the two groups, but at the same time, this type of conceptual change was least frequently observed. the existence of pcc, rcc, and even fcc reflects that the implementation of pcbl may not instantly provide a complete conceptual change. to achieve this more practice, particularly in developing students’ ability to interpret pictures, graphics and tables, and finally extract valuable information from these representations, is needed. in dealing with pictorial representations, some students face two challenging issues, which are: i) difficulty in extracting pictorial based learning in chemistry 13 the relevant information12,35 and ii) errors in transforming the information extracted from the pictorial context into chemical behaviour.35 providing more opportunities for students to deal with pictorial representations both in teaching and learning and in the evaluation process will encourage students to become more familiar with such exercise and more confident in their ability to deal with them. a study has shown a link between students’ confidence in chemistry with their achievement in chemical kinetics.36 although the results show that the frequency of ccc among the pcbl students is slightly higher in comparison to the di students, we don’t attempt to generalize the conclusions of this study in regard to pcbl for the entire field of chemistry teaching, or even the field of chemical kinetics. however, the conclusions reached within this study may prompt further studies with larger sample sizes and in other chemistry topics and a more readily generalizable inference can be reached in the future. in terms of students' answers to ftdick questions, the pcbl students demonstrated a better performance in comparison to the di students. this may infer that the pcbl approach is more potent in improving students’ understanding of chemical kinetics than the di approach. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/11740, or from the corresponding author on request. acknowledgement. we thank universitas negeri malang for providing the grant of this research with the grant number 20.3.169/un32.14.1/lt/2019. и з в о д учење засновано на илустрацијама: промовисање концептуалне промене у оквиру теме хемијска кинетика habiddin habiddin1, herunata herunata1, oktavia sulistina1, aceng haetami2, maysara maysara и душица родић3 1department of chemistry, universitas negeri malang, indonesia, 2department of chemistry education, universitas halu oleo, kendari, indonesia и 3департман за хемију, биохемију и заштиту животне средине, природно-математички факултет, универзитет у новом саду, нови сад ова студија је имала за циљ да испита утицај учења заснованог на илустрацијама (pcbl) на концептуалне промене у оквиру теме хемијска кинетика. за мапирање концептуалних промена у оквиру концепата хемијске кинетике, коришћен је претходно развијени четворослојни инструмент (ftdick). студенти прве године хемије на једном универзитету у индонезији формирали су експерименталну и контролну групу. експериментална група је обучавана применом pcbl методе, док је контролна група обучавана применом директне инструкције (di). концептуалне промене које су уочене у оквиру ове две групе класификоване су у четири категорије: потпуне, парцијалне, лажне и насумичне. потпуна концептуална промена (ccc) је имала највећи степен учесталости од четири наведене категорије. међутим, било би исувише амбициозно генерализовати да су и pcbl и di ефикасни у поспешивању концептуалних промена у области хемијске кинетике. да би се извео такав закључак, потребна су додатна истраживања. https://www.shd-pub.org.rs/index.php/jscs/article/view/11740 14 habiddin et al. ефикасност pcbl и di методе у поспешивању концептуалних промена у овој студији била је готово једнака. међутим, у решавању задатака на ftdick, студенти pcbl групе су остварили бољи учинак од студената di групе и тиме показали боље научно разумевање. 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https://doi.org/10.1039/c9rp00018f https://doi.org/10.1007/s10763-016-9716-z https://doi.org/10.1007/s10763-019-10010-7 https://doi.org/10.1007/s10763-019-10010-7 https://doi.org/10.1007/s10763-019-10037-w https://doi.org/10.1063/5.0000502 european analytical column no. 50 j. serb. chem. soc. 87 (11) 1341–1345 (2022) euchems news division of analytical chemistry european chemical society 1341 euchems news european analytical column no. 50• slavica ražić1*#, marcela a. segundo2**, diane turner3***, manuel miró4× and antje j. baeumner5∧ 1department of analytical chemistry, faculty of pharmacy, university of belgrade, vojvode stepe 450, 11222 belgrade, serbia,2department of chemical sciences, faculty of pharmacy, university of porto, r jorge viterbo ferreira, 228, 4050-313 porto, portugal, 3anthias consulting ltd., 1 hamden way, papworth everard, cambridgeshire, cb23 3ug, uk, 4fitrace group, department of chemistry, university of the balearic islands, carretera de valldemossa km 7.5, e-07122 palma de mallorca, spain and 5institute of analytical chemistry, chemoand biosensors, university of regensburg, universitätsstr. 31, 93053 regensburg, germany the european analytical column is the voice of the division of analytical chemistry (dac) as a professional network of chemical societies and their members working in all fields of analytical sciences within the european chemical society (euchems). the strategy for 2021–2023 comprehends the promotion of analytical chemistry to a wider community, co-operation with other professional networks and to support members’ activities, particularly through study groups and task forces. this year we have invited active scientists to share their thoughts about the near future of analytical chemistry. please help us to expand this discussion among the community and feel free to share your opinion and thoughts through our social media and networks! 1. dac-euchems activities one of the main activities of dac-euchems is the promotion of organization of euroanalysis conference. every two-years, one of the participating scientific chemical societies will host euroanalysis, with active involvement of local scientists in the organization. euroanalysis xxi is scheduled to 2023 and it will * correspondence e-mails: (*)slavica.razic@pharmacy.bg.ac.rs; (**)msegundo@ff.up.pt; (***)dct@anthias.co.uk; (×)manuel.miro@uib.es; (∧)antje.baeumner@ur.de # serbian chemical society member. • reprint from analytical and bioanalytical chemistry, https://doi.org/10.1007/s00216-022 -04373-0, with permission from springer. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1342 ražić et al. take place in geneva, switzerland, under the auspices of the swiss chemical society (https://www.euroanalysis2023.ch/), organized by prof. eric bakker (university of geneva), dr. marc suter (eawag), dr. franka kalman (hes sion) and dr. bodo hattendorf (eth zurich). other ongoing activities of dac-euchems are performed within study groups. these include “bioanalytics”, “chemometrics”, “education”, “electroanalytical chemistry”, “history”, “nanoanalytics”, “quality assurance”, and “sample preparation”. please visit the dac-euchems website for updated reports (https://www.euchems.eu/divisions/analytical-chemistry/) and contact the heads of the study groups in order to have more information or to participate in their activities. collaboration with other professional networks within euchems is also sought. in particular, exchange of invited lectures and organization of special thematic sessions in is planned to take place in the 18th international conference on chemistry and the environment (https://icce2023.com/) and euro food chem xxii conferences, organized by the division of chemistry and the environment (dce-euchems) and by the division of food chemistry (dfc-euchems), respectively. lastly, one of dac-euchems objectives is to support its delegates on the organization of local events open to the international community through dissemination of the event within the professional network, including online events. the steering committee of dac-euchems will be happy to receive input for additional activities. feel free to contact one of the following persons: slavica ražić, university of belgrade, serbia (chair), marcela segundo, university of porto, portugal (secretary), martin vogel, university of münster, germany (treasurer), jiří barek, charles university, czech republic, charlotta turner, lund university, sweden, sibel a. özkan, ankara university, turkey, and franka kalman, university of applied sciences and arts of western switzerland, switzerland. 2. analytical chemistry in 2030: a glimpse of opportunities and challenges in the second part of this column, we would like to share our thoughts of the present state and future perspectives of analytical chemistry, as views of our guest authors, diane turner, manuel miró and antje baeumner. in that sense and order, and without ambition to cover all fields, let us tackle some of general and particular points and impacts in a few research areas. and, maybe, just maybe, to open the discussion with the next edition of eac. analytical chemistry is used in most industries from petrochemical to clinical and consumer products to food and environment. in academia, not only is it extensively researched and used in chemistry departments, but also in engineer________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ european analytical column no. 50 1343 ing, earth sciences, planetary sciences, archaeology, biology and beyond. the uses of analytical chemistry are many, from quality control to the identification of components in a sample mixture, and the most important questions when thinking and preparing for sample analyses is why you are performing their analysis and what questions you are seeking to answer. the results from sample analysis can be used to determine the next steps in a manufacturing environment, to decide if a product is safe for use or to consume, to present in a phd thesis or as evidence in a court of law. there has, for many years, unfortunately been a large gap between using analytical chemistry in an academic setting compared to using it in regulated industry. even though method validation and quality control are taught at universities, it has been a long-time coming in reaching research projects, with many publications not showing or even mentioning any basic quality control. there are many aspects to consider when developing a method and validating it, from sample collection, storage and transportation, to sample preparation, sample analysis, data analysis and reporting, the use of blanks, standards, internal standards, the consideration of different sample matrices and method performance parameters. then there is on-going quality control and quality assurance (qc/qa) with system suitability checks, analytical quality control (aqc) standards, replicates, checks for carryover and extraction efficiency and the monitoring of a wide range of other method performance parameters, dependent on the analytical technique employed and the variables that can affect the results of that technique. when performing quantitative sample analysis there are a lot of standard qc procedures that can be followed, but many of these can be extended into qualitative analysis too. if two samples appear to give different results, are they truly different or is the method not robust and there is poor accuracy and/or precision due to the analytical instrument or any of the steps from sample collection through to reporting? is a compound genuinely not in a sample or is it below the method detection limit? if so, what is the method detection limit, not just the instrument limit of detection? what is the uncertainty of your result? for many projects a full method validation to iso 17025 standards is not required however, a method should always be checked that it is suitable for the sample(s) and compound(s) to be analysed. generally, the more samples to be analysed, especially if the matrix varies, the more validation and on-going quality control is needed to ensure that not only is the method suitable but remains suitable and gives the correct results for all samples. at the start, a well-thought-out plan for method development, validation and quality control is needed and that needs to be referred to and evidenced in any reporting, whether that is a publication, poster or a document for court. the current drive towards proof of the quality of results is welcomed and in the not-too-far distance future could be ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1344 ražić et al. mandatory for all analytical methods in research as well as industry, especially if presenting into the public domain. considering another perspective and its multidisciplinary nature, analytical chemistry is proven superb to tackle the current societal challenges as recently demonstrated by the fast development of sensing platforms in response to the demands occasioned by the sars-cov-2 pandemic. the intimate relationship between analytical chemistry and material science ranging from paper-based (fluidic) systems to nanotechnology including silica, polymeric and metal nanoparticles trigger the development of simple point-of-need/care sensing devices for customized screening applications. nanotechnology actually is acting as a bridge to expand analytical chemistry to a plethora of companion disciplines to help answering scientific challenges. theranostic applications are clear examples on how the interplay between analytical chemistry, nanoscience and biosciences offer unrivalled opportunities against relevant biomarker identification. inspired by the industry 4.0 technological pillars (e.g., internet of things), wearable sensors and remote sensing approaches enable real-time identification of changing physiological/environmental conditions, yet further developments are still called for to improve detectability and selectivity issues. instrumental developments for confirmatory results are linked to efforts toward designing advanced mass spectrometry analysers to leverage imaging, ambient detection and collision crosssections for reliable high-throughput analysis. material science has impacted the entire analytical process right from the first step of sampling to offer new devices for in-situ sample preparation and microextraction for targeted or suspect analysis to the detection step by exploiting the unique plasmonic and electrochemical features of nanoparticles and nanoclusters. a technological breakthrough in this field, mostly linked to polymers but also to noble metal chemistry, which nurtured analytical chemistry is the so -called “additive manufacturing” (i.e., 3d-printing). advanced technologies such as laser sintering, photopolymer inkjet, low-force stereolithography and fused deposition modelling are revolutionizing several subdisciplines, such as microfluidics, electrochemical and optical sensing, and sample preparation in terms of fast prototyping of devices and straightforward printing of electrodes and optical detection components, aided by the plethora of functional materials available with distinct optical, conducting and thermal properties and chemical resistance that can be simultaneously processed. we foresee that higher resolution printers at affordable prices with improved minimum printing size features in x, y and z axes will be available in most of the analytical chemistry labs and also technological companies in the near future, because mass production and commercialization of 3d printed analytical devices are becoming a reality. going via next road of highly potential science, the field of biosensors is currently seeing a dramatic increase in societal interest and demands for imp________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ european analytical column no. 50 1345 rovement much beyond the current state of the art. wearables and point-of-care tests (poct) receive the greatest public attention due to the increasingly ubiquitous availability of simple, portable, read-out devices (cell phones, watches) and the critical need for poct during the recent pandemic. in the future, such sensors must achieve lower limits of detection than they provide today, they must allow for multiplexing and especially enable quantitative detection with high reliability and not only provide qualitative or semi-quantitative results. since the absolute requirement for simplicity of use puts strict limits on technologies used within such sensors, one can predict innovation and advancements to not be derived necessarily from new detection principles, but rather from improved signaling agents, advanced (nano)materials, and artificial-intelligence-assisted readout apps. furthermore, the improvement of sample preparation strategies for bioand chemosensors in general will be of great demand. it is obvious that good results cannot be obtained from a badly acquired sample, and some sample clean-up through centrifugation, multiple pipetting steps and the like is difficult to implement in most applications and cannot be employed at all in wearable sensors. thus, in the next 2–5 years, analytical and engineering research should focus on overcoming these ubiquitous challenges that are limiting most of the current bio and chemosensors targeting on-site applications. as mentioned earlier, another area of great development in the coming years will be the move toward digitalization, particularly as far as bioand chemosensors are concerned regarding their integration into the internet-of-things, digital diagnostics, telemedicine, and remote environmental and food monitoring. as much as physical sensors are seamlessly integrated into large systems, also bio and chemosensors will continue moving in this direction. alongside this will come a need to develop sensor technologies that allow for easy mass production, use of sustainable materials and production technologies, and result in products that do not increase the environmental burden caused by their waste. considering that a dramatic increase in the use of bioand chemosensors is predictable, these last points need to become design criteria in the development of innovative new bioand chemosensors in the coming decade. we can continue our walk from one to another area of analytical chemistry and ask ourselves is the sky the limit? it's allowed in our minds but advancements in other scientific fields, such as material science, physics and biology will certainly have their impacts and pave the road in advancements of analytical chemistry in close future. far future? we do not dare to think about it yet. we can follow the best science, that is sure… ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ @article{razic2022, author = {ra{\v{z}}i{\'{c}}, slavica and segundo, marcela a and turner, diane and 4×, manuel mir{\'{o}} and baeumner, antje j}, journal = {journal of the serbian chemical society}, title = {{european analytical column no. 50}}, year = {2022}, issn = {1820-7421}, month = {nov}, number = {11}, pages = {1341--1345}, volume = {87}, abstract = {the european analytical column is the voice of the division of analytical chemistry (dac) as a professional network of chemical societies and their members working in all fields of analytical sciences within the european chem­ical society (euchems). the strategy for 2021–2023 comprehends the pro­motion of analytical chemistry to a wider community, co-operation with other professional networks and to support members' activities, particularly through study groups and task forces. this year we have invited active scientists to share their thoughts about the near future of analytical chemistry. please help us to expand this discussion among the community and feel free to share your opinion and thoughts through our social media and networks!}, doi = {10.1007/s00216-022}, file = {:d\:/onedrive/mendeley desktop/ra{v{z}}i{'{c}} et al. 2022 european analytical column no. 50.pdf:pdf;:09_euchems news_vol87_no11.pdf:pdf}, keywords = {2023, strategy for 2021}, url = {https://www.shd-pub.org.rs/index.php/jscs/article/view/12136}, } 291 erratum (printed version only) 1. volume 87, issue 1, page 157, lines 8–11 from the top should read: 1university of novi sad, faculty of technical sciences, novi sad, serbia and 2jaroslav černi water institute, belgrade, serbia ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice accepted manuscript this is an early electronic version of an as-received manuscript that hasbeen accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as j. šćepanović, m. r. pantović pavlović2, d. vuksanović, g. m. šekularac, and m. m. pavlović, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc230505031s this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing, and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230505031s j. serb. chem. soc.00(0)1-13 (2023) original scientific paper jscs–12377 published dd mm, 2023 1 impedance response of aluminum alloys with varying mg content in almg systems during exposure to chloride corrosion environment jelena šćepanović 1 , marijana r. pantović pavlović 2,3 , darko vuksanović 1 , gavrilo m. šekularac 2 , and miroslav m. pavlović 2,3 * 1 faculty of metallurgy and technology, university of montenegro, podgorica, montenegro. 2 institute of chemistry, technology and metallurgy, national institute of the republic of serbia, department of electrochemistry, university of belgrade, belgrade, serbia, and 3 center of excellence in environmental chemistry and engineering, institute of chemistry, technology and metallurgy, belgrade, serbia (received 5 may; revised 23 june; accepted 1 july 2023) abstract: this research discusses the corrosion behavior of as-cast al alloys with different mg content by potentiostatic electrochemical impedance spectroscopy (peis). the complex plane spectra of all samples feature a high-frequency loop, followed by semi-infinite diffusion impedance characteristics at low frequencies, with the corrosion-induced formation of a defined porous structure of a layer making finite diffusion through the pores dominant upon prolonged exposure. the most compact layer causes the most pronounced and well-resolved finite diffusion features in the impedance spectra of the sample with the highest mg content, while the sample with the lowest mg content has a highly porous layer unable to slow down the corrosion rate at the layer/sample surface interface. the highest layer capacitance and diffusion admittance are found in the sample with the highest mg content, with a more adherent protective film expected to form. however, the growth rate of the layer was not adequate for the remarkable closing of the pits, indicating the weakness of this sample towards pit activity. the results show that increasing mg content improves corrosion resistance and clearly separates bulky corrosion from localized pitting corrosion, but it also increases the thickness of a more compact, poorly adhesive layer. keywords: electrochemical impedance spectroscopy; aluminum alloys; corrosion behavior; chemical composition; mechanical properties introduction aluminum and its alloys have a wide range of applications in industry and consumption due to their unique properties, such as light weight, high strength, and good corrosion resistance.1–3 due to the broad application and economic importance of aluminum and its alloys, increasing attention is being paid to researching the corrosion characteristics of these materials in cast, aged, and *corresponding author e-mail: miroslav.pavlovic@ihtm.bg.ac.rs https://doi.org/10.2298/jsc230505031s a cc ep te d m an us cr ip t mailto:miroslav.pavlovic@ihtm.bg.ac.rs https://doi.org/10.2298/jsc230505031s 2 šćepanović et al. technically processed states.1,4,5 aluminum-magnesium alloys have been the subject of numerous studies on corrosion rates in various solutions.5–9 the formation of a protective oxide layer is the main reason behind its excellent corrosion resistance. however, under saline conditions, such as those encountered in marine environments, aluminum alloys are vulnerable to localized degradation in the form of pitting and crevice corrosion. this type of corrosion occurs due to the adsorption of anions, particularly chloride ions, cl-, at the oxide-solution interface.5 from the standpoint of corrosion rate, in a chloride environment, almg alloys are susceptible to localized degradation in the form of pitting corrosion. the highest corrosion rate is observed in alloys containing around 0.8 mass % of magnesium.4 a higher magnesium content in al alloys generally indicates a higher degree of surface oxidation, where magnesium content varies from 6% to 8%.9,10 the passive stable surface film serves as a barrier for the transfer of cations from the metal to the environment and for the counter diffusion of oxygen and other anions.11 chemically homogeneous, single-phase amorphous alloys free from crystalline defects such as precipitates, segregates, grain boundaries, and dislocations often create a conducive environment for the formation of a uniform passive film without any weak points.6 dragos et al.12 shows results of elongation and structural analysis of al-mg alloy before and after heat treatment and artificial aging. on the other hand, alloys with magnesium are structural metals that are lightweight. the lightweight nature of such materials is the main reason for the interest in al-mg alloys in various industrial applications.13 aluminum forms a protective oxide film in the ph range 4.0–8.5, but this depends on temperature, form of oxide present, and the presence of substances that form soluble complexes or insoluble salts with aluminum. this implies that the oxide film is soluble at ph values below 4.0 and above 8.5. several investigations reported that the pitting potential of aluminum in chloride solutions is independent of ph in the range 4–9.14–16 however, pitting corrosion rate was found increased at slightly acidic and alkaline solutions with respect to neutral solutions. the corrosion behavior of aluminum alloys is significantly affected by the presence of particles in the matrix.3 previous works2,8,14,16,19 showed that mg2si particles tend to be anodic in relation to the matrix and can act as initiation sites for corrosion. most often, the mg2si phase dissolves, leaving behind a cavity that acts as a nucleation site for pitting.14–16,20,21 these observations were made during investigations carried out on commercial aluminum alloys with low si/mg mole ratios.7,22 crevice corrosion is a highly localized form of corrosion that occurs by infiltration of water into closely fitted surfaces. the presence of aggressive ions, such as chloride, often creates extensive localized attack.14 metals like aluminum that depend on oxide films or passive layers for corrosion resistance are particularly susceptible to crevice corrosion. attack from this phenomenon can be a cc ep te d m an us cr ip t aluminum alloys and chloride corrosion 3 aggravated when combined with the presence of crystalline defects such as mg2si precipitates. the possibility exists for reducing drastically the alloy susceptibility to corrosion if its microstructure is modified by appropriate heat treatment prior to usage. the research conducted in this study aimed to determine the corrosion rate of an al alloy in its cast and aged states, as well as to compare the corrosion rate for both states. aluminum and its alloys have been used in a wide range of industries due to their favorable characteristics such as high strength-to-weight ratio, excellent formability, and resistance to corrosion. aluminum alloys are increasingly utilized in a diverse array of products, including airplane parts, components for the aerospace industry, and the shipbuilding sector.17,18 these industries require materials with high strength-to-weight ratios and superplasticity. compared to other metals, aluminum alloys offer superior strength while being lightweight. the development of materials with superplasticity aims to enhance their mechanical and forming properties.18 various manufacturing techniques have been employed to fabricate and improve these superplastic materials, such as extrusion, rolling, forging, stir casting, as well as more recent methods like friction stir processing.17 however, the corrosion resistance of these materials is influenced by factors such as alloy composition, processing history, and exposure environment. therefore, understanding the corrosion behavior of these materials is crucial for their effective and sustainable use in various applications. the aim of the presented investigation was to analyze the influence of mg content on the corrosion behavior of al alloys in order to contribute to reliable prediction of their behavior and to the development of improved corrosion-resistant al alloy for specific applications. experimental the investigated alloys were obtained by casting and air cooling in the laboratory of foundry at the faculty of metallurgy and technology in podgorica. a 5.5 kw resistance furnace with a working temperature of 1100ºc was used to obtain the alloys. the melting of the alloys was carried out in a graphite crucible placed in the furnace. high-purity magnesium was used as an alloying element in the alloys, with the mg content of 2.42, 3.14 and 7.90 mass % in alloys 1,2 and 3 respectively. copper, zinc, chromium, and manganese were also used as alloying elements in the alloys. the chemical composition of the obtained alloys was analyzed at the aluminum combine podgorica (kap) using a nondestructive x-ray quantometer method. the selection of the alloying elements was based on previous research, with a focus on the effect of magnesium content on the corrosion characteristics while keeping the content of other alloying elements approximately constant. after casting, all three alloys were subjected to heat treatment, including heating at 515±5ºc for 6 hours and quenching in warm water. corrosion and electrochemical potentiodynamic investigations were carried out using accelerated testing equipment the par system consisting of a potentiostat-galvanostat model 273, a differential electrometer, a corrosion cell k0047, a saturated calomel electrode (sce), auxiliary electrodes cylindrical electrographite, a computer with corrosion software softcorr 352 ii, and a printer. all potentials are expressed on sce scale. a cc ep te d m an us cr ip t 4 šćepanović et al. corrosion testing was performed using the following methods: monitoring the change in corrosion potential over time, ecorr=f(τ); polarization resistance method, rp; potentiodynamic method. a synthetic nacl solution with a concentration of 0.51m was used as the corrosion medium. the electrochemical behavior was also assessed using potentiostatic electrochemical impedance spectroscopy (peis). the measurements were carried out using a three -electrode cell system in a 0.51m nacl solution, with platinum wire and sce serving as the counter and reference electrodes, respectively. prior to peis measurements, the electrolyte was purged with n2 for 30 minutes. a potentiostat/galvanostat (biologic sas, sp-240, grenoble, france) was used for peis measurements at open circuit potential. optical images were taken with leica 20 mp ultra wide angle lens, f/2.2 aperture camera. results and discussion the results of the chemical composition of the obtained alloys are presented in table 1. the table shows the percentage of each alloying element used in the production of the alloys, which is an important factor that can affect their properties, including their resistance to corrosion. table 1. results of the chemical composition of al alloys in mass % fe si ti cu zn v cr mn mg sn ni pb l1 0.29 0.12 0.14 0.34 0.59 0.012 0.21 0.33 2.42 0.001 0.002 0.007 l2 0.30 0.12 0.13 0.35 0.62 0.012 0.25 0.25 3.14 0.001 0.002 0.008 l3 0.24 0.14 0.13 0.52 0.83 0.013 0.26 0.29 7.90 0.001 0.002 0.001 table 1 shows that these are multi-component alloys with different contents of alloying elements. the table lists the mass percentage of each element in the alloys. from the table, it can be observed that the alloys have varying amounts of magnesium (mg), copper (cu), zinc (zn), chromium (cr), manganese (mn), iron (fe). the presence and the amount of each element in the alloys can significantly affect their properties, such as strength, hardness, and corrosion resistance. therefore, understanding the chemical composition of the alloys is crucial for predicting and controlling their behavior in different applications. fig. 1 shows the corrosion potential over time of as-cast al alloys in 0.51m nacl solution. before starting the polarization measurements, the system needs to be stabilized, which is judged by acceptable stability in time of the corrosion potential. corrosion potential is monitored upon sample immersion into the electrolyte. from the results shown in fig. 1, a shift in potential of the tested samples towards more positive values can be observed. the shift in potential of the alloys towards more positive values is interpreted as passivation, or the formation of an oxide film on the surface of the tested samples. the protective layer becomes thicker and more compact over time. this film prevents the passage of aggressive chloride ions from the solution, thus protecting the material from further corrosion. a cc ep te d m an us cr ip t aluminum alloys and chloride corrosion 5 fig 1. corrosion potential of as-cast al alloys over time in 0.51m nacl solution. it can also be seen that corrosion potential takes more positive values as the mg content increases. this indicates that greater protective ability of the passive films are reached with lower mg content. a) b) fig. 2. a) linear polarization and b) potentiodynamic cathodic and anodic polarization curves of al alloys in the as-cast state in a 0.51m nacl solution. linear polarization is an electrochemical technique used for monitoring corrosion. it is based on the determination of the polarization resistance, rp from the slope of the polarization line near the corrosion potential. based on the results shown in fig. 2a, and numerical values of the experimental measurements which are shown in table 2, it can be concluded that the samples are stable in the tested solution. comparing the results, based on the value of the polarization resistance a cc ep te d m an us cr ip t 6 šćepanović et al. rp (table 2), it can be observed that there is highest corrosion resistance for the l2 sample, followed by l1 and l3. fig. 2b shows the potentiodynamic cathodic and anodic polarization curves of the tested samples in a 0.51m nacl solution. in the cathodic region, a small change in current density with potential is observed, indicating a low corrosion rate of the tested samples in the nacl solution, while in the anodic region, a larger change in current density with potential is observed. the results of the measurements are presented in table 3. based on the values of jcorr and ej=0 given in table 3, it can be concluded that l1 is the lowest rate of corrosion in the tested environment, followed by l3 and l2. the potentiodynamic polarization curves provide valuable information about the corrosion behavior of the materials, as well as the values of the corrosion potential and the current density at which corrosion rate is negligible. tables 2 and 3 present the complete results of corrosion studies in 0.51m nacl on alloys 1, 2, and 3 in as-cast state. the tables provide information on corrosion potential, corrosion current density, polarization resistance, corrosion rate, and other related parameters. by analyzing these data, one can gain insight into the effectiveness of different alloy composition in preventing corrosion and choose the optimal material for specific applications. table 2. change of corrosion potential over time of as-cast al alloys in a 0.51m nacl solution. sample estart / mv efin / mv l1 -836 -725 l2 -834 -743 l3 -879 -740 table 3. corrosion potential change over time of as-cast al alloys in a 0.51m nacl solution. sample ej=0 / mv rp / kω cm -2 βa / mv jcorr / μa cm -2 l1 -727.5 1.106 19.63 19.64 l2 -743.3 2.554 25.57 8.503 l3 -736.7 0.4605 16.93 47.15 fig. 3 shows the impedance characteristics of the cast samples upon initial (24 h) and prolonged (120 h) exposure to chloride-containing corrosive medium as impedance complex plane plots. a cc ep te d m an us cr ip t aluminum alloys and chloride corrosion 7 fig. 3. impedance complex plane plots of as-cast al–mg alloy samples upon initial (24 h) and prolonged (120 h) exposure to 0.51m nacl solution. symbols: registered data; lines: the data of equivalent electrical circuit. complex plane spectra of all samples feature high-frequency loop, which is, in the cases of lower mg content (l1 and l2), followed by well-defined semiinfinite diffusion impedance characteristics at low frequencies. prolonged exposure causes the deviation of a near-45-inclined straight line to more resistance-defined behavior as the corrosion proceeds. this indicates the transition from semi-infinite to more noticeable finite diffusion conditions due to the generation and growth of a passive layer on the sample surface. while electrolyte species participating in the corrosion processes diffuse initially from the electrolyte bulk, the corrosion-induced formation of defined porous structure of a layer makes dominant the finite diffusion through the pores upon prolonged exposure. the layer over l3 sample of highest mg content seems to be formed much quickly, since finite diffusion characteristics are more resolved already in the initial stage of exposure. the photographs of corroded samples (fig. 4) show that the surface layer of l3 appears the most compact with the least frequent pitting corrosion sites. however, the radii of corrosion products (white zones) around pits are largest, which indicates that pitting corrosion is dominant mechanism in the initial stage. according to fig. 4, it appears that the increase in mg content favors the pitting corrosion and induces the formation of more compact passive layer. the most compact layer apparently causes the most pronounced and well-resolved finite diffusion features in the impedance spectra of l3. the differences between the properties of a layer on l3 and that on l1 and l2 are indicated also by the measures of the high-frequency loop. although the associated resistances are similar for all of the three samples, the imaginary impedance is lower for l3 due to larger capacitance of a thinner and more compact layer. fig. 4. optical images of the tested samples after 120 h exposure to 0.51m nacl solution. the considerations of the features of impedance spectra are proved further by the structure of the most suitable equivalent electrical circuit which describes best the physicochemical properties of the samples while they corrode (figure 5). the samples of lower mg content and of less compact passive layers behaves a cc ep te d m an us cr ip t 8 šćepanović et al. equivalently to the circuit consisted of a parallel connection of capacitor and resistor, which is in series to randles–sevcik (r–s) conformation. fig. 5. equivalent electrical circuits of as-cast al–mg alloy samples a) for l1 and l2 samples and b) l3 samples upon initial (24 h) and prolonged (120 h) exposure to 0.51m nacl solution. rc in parallel describes the properties of a layer, whereas r–s relates to diffusion-controlled corrosion processes. owing to the most distinguishable pitting corrosion and an indication of more compact layer structure, the circuit for l3 required additional rc time constant in series, which indicates the difference between the properties of a bulky layer and that formed around the pits. the values of the circuit parameters are presented in fig. 6. the highest values of layer resistance is found for l2 of mid mg content, associated to the lowest layer capacitance, which indicates the thickest layer is formed on this sample. in fig. 4 it can be seen that massive layer is formed over the sample surface except the areas close to the sample surface, where pitting corrosion appears dominant. the formation of a massive layer is followed by considerable increase in charge transfer resistance (r2, which was the lowest initially), and double layer capacitance (cpe-t3). this indicates that corrosion processes take place much faster on l2 than on l1 and l3; however, fast corrosion produces the thickest layer, whose porous structure did not change significantly while the layer grows (initial layer resistance and diffusion admittance are similar to those found upon prolonged exposure (day 1–day 5). consequently, the corrosion proceeds at the sample surface accessible through the pores of the layers, which area is considerably smaller with respect to l1 and l3. hence the r2 and cpe-t3 for l2 are considerably larger. a cc ep te d m an us cr ip t aluminum alloys and chloride corrosion 9 fig 6. the values of circuit parameters of the investigated samples; r3/r4 and c1/c2 – resistances and capacitances of the passive layer; r2 and cpe-t3 – charge transfer and double layer capacitance (the values of cpe exponent are above 0.75) associated to corrosion processes; cpe-t1 – diffusion admittance. layer resistance of l1 considerably increases during exposure, but it is still lower than the resistance found for l2. this increase is associated with the lowest charge transfer resistance and diffusion admittance among the samples upon prolonged exposure. this finding is the strong indication of the highly porous layer, unable to slow down the corrosion rate at the layer/sample surface interface. huge pores homogeneously distributed over the layer surface are also seen in fig. 4, and even appearance of a cracks around the pits in the middle of the sample is clearly visible. it can be concluded that the l1–l2 increase in mg content improves the corrosion resistance and clearly separates bulky corrosion from localized pitting corrosion. however, it increases the thickness of more compact, poorly adhesive, layer. a cc ep te d m an us cr ip t 10 šćepanović et al. further increase in mg content applied in l3 decreases additionally the layer thickness and makes it considerably more compact, which should be beneficial for the formation of a more adherent protective film. this is reflected in the highest layer capacitance and diffusion admittance that is for l3 straightforwardly defined by finite diffusion mechanism (finite warburg element was only suitable in equivalent circuit for l3, whereas classical warburg element met the requirement to describe the diffusion in the cases of l1 and l3). however, for the applied exposure the growth rate of a layer was apparently not adequate for the remarkable closing of the pits, since the increase in charge transfer resistance and double layer capacitance was not registered for l3 upon prolonged exposure. the weakness of l3 sample toward the pit activity could be connect to the presence of additional rc time constant, of quite different values of the parameters with respect to that for a layer. namely, the resistance is much lower, few tens of , and even deceases to ca. 8  upon prolonged exposure. the associated capacitance is close to 0.1 mf, which is much lower than that of a layer – thus indicating its highly localized appearance on the surface. despite this poor immunity of l3 sample toward pitting corrosion, the registered propagation of the properties of a protective layer during 5-day exposure should be expected to show protective features upon much longer exposures to corrosive medium. conclusion the article describes the results of an experiment aimed at investigating the corrosion behavior of as-cast al alloys in 0.51m nacl solution. the study employed different electrochemical techniques, including corrosion potential measurements, linear polarization, and potentiodynamic polarization curves, as well as impedance measurements. the results show that the al alloys form a protective oxide film on the surface, which prevents the passage of aggressive chloride ions from the solution, thus protecting the material from further corrosion. the study found that the impedance characteristics of the cast samples change upon initial and prolonged exposure to the chloride-containing corrosive medium. the complex plane spectra of all samples feature a high-frequency loop followed by well-defined semi-infinite diffusion impedance characteristics at low frequencies. prolonged exposure causes the deviation of a near-45°-inclined straight line to more resistance-defined behavior as the corrosion proceeds. the most compact layer apparently causes the most pronounced and well-resolved finite diffusion features in the impedance spectra of l3. the article concludes that the increase in mg content favors the pitting corrosion and induces the formation of a more compact passive layer. the study used the most suitable equivalent electrical circuit to describe the physicochemical properties of the samples while they corrode. the samples of lower mg content and less compact passive layers behave equivalently to the a cc ep te d m an us cr ip t aluminum alloys and chloride corrosion 11 circuit consisted of a parallel connection of a capacitor and resistor, which is in series to the randles–sevcik conformation. the circuit for l3 required an additional rc time constant in series, indicating the difference between the properties of a bulky layer and that formed around the pits. the highest values of the layer resistance are found for l2 of mid mg content, associated with thicker and more compact layers. these results provide insights into the corrosion behavior of as-cast al alloys and can be useful for the design of new alloys with better corrosion resistance. in conclusion, the study investigated the corrosion behavior of as-cast al alloys in 0.51m nacl solution, using various electrochemical techniques. the study found that the al alloys form a protective oxide film on the surface, which prevents the passage of aggressive chloride ions from the solution, thus protecting the material from further corrosion. the study also found that the impedance characteristics of the cast samples change upon initial and prolonged exposure to the chloride-containing corrosive medium, and that the increase in mg content favors the pitting corrosion and induces the formation of a more compact passive layer. these results provide insights into the corrosion behavior of as-cast al alloys and can be useful for the design of new alloys with better corrosion resistance. the findings of this study can have significant implications for the development of new corrosion-resistant alloys for a variety of applications. the results demonstrate that the corrosion behavior of al alloys can be controlled by the composition of the alloy and the formation of a protective oxide layer on the surface. overall, the study highlights the importance of understanding the corrosion behavior of materials and provides a valuable contribution. acknowledgements: this work was supported by the ministry of science, technological development and innovation of the republic of serbia, grant no. 451-03-47/2023-01/200026. и з в о д импедансни одзив алуминијумских легура са варијабилним садржајем магнезијума у al-mg системима током изложености хлоридној корозионој средини. јелена шћепановић1 маријана р. пантовић павловић2,3, дарко вуксаноцић1, гаврило м. шекуларац2, и мирослав м. павловић2,3.* 1металуршко-технолошки факултет, универзитет у црној гори, подгорица, црна гора, 2институт за хемију, технологију и металургију, институт од националног значаја за републику србију, центар за електрохемију, универзитет у београду, београд, и 3центар изузетних вредности за хемију и инжењеринг животне средине, институт за хемију, технологију и металургију, београд ово истраживање се бави понашањем корозије аl легура са различитим садржајем мg уз примену потенциостатске електрохемијске импедансне спектроскопије (пеис). спектри у комплексној равни свих узорака се карактеришу петљом високе фреквенције праћеном карактеристикама полу-бесконачне дифузије на ниским фреквенцијама, са формирањем a cc ep te d m an us cr ip t 12 šćepanović et al. дефинисане порозне структуре слоја услед корозије, што доводи до доминације коначне дифузије кроз поре при продуженој изложености. најкомпактнији слој узрокује најизраженије и најјасније карактеристике коначне дифузије у импедансним спектрима узорка са највећим садржајем mg, док узорак са најмањим садржајем mg има високо порозан слој који не може успорити брзину корозије на граници фаза слој/узорак. највећу капацитивност и адмитанцију дифузионог слоја има узорак са највећим садржајем mg, са очекиваном појавом адекватног заштитног филма. међутим, брзина раста слоја није била довољна за значајно затварање питова, што указује на слабост овог узорка према питинг корозији. резултати показују да повећање садржаја mg побољшава отпорност на корозију и јасно раздваја корозију у маси материјала од локализоване питинг корозије, али истовремено повећава дебљину слоја који је слабије адхезије и компактнији. 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no. 49• slavica ražić1*#, martin vogel2** and marcela a. segundo3*** 1department of analytical chemistry, faculty of pharmacy, university of belgrade, vojvode stepe 450, 11222 belgrade, serbia, 2institute for inorganic and analytical chemistry, university of münster, münster, corrensstraße, 28, 48149 münster, germany and 3department of chemical sciences, faculty of pharmacy, university of porto, r jorge viterbo ferreira, 228, 4050-313 porto, portugal the european analytical column is the voice of the division of analytical chemistry (dac) as a professional network of chemical societies and their members working in all fields of analytical sciences within the european chemical society (euchems). the strategy for 2021–2023 comprehends the promotion of analytical chemistry to a wider community, co-operation with other professional networks and to support members’ activities, particularly through study groups and task forces. this year we will focus on how our community has managed to adapt to teaching analytical chemistry during the pandemic. please feel free to share your own experience through our social media and networks! 1. dac-euchems activities one of the main activities of dac-euchems is the promotion of organization of euroanalysis conference. every two-years, one of the participating scientific chemical societies will host euroanalysis, with active involvement of local scientists in the organization. euroanalysis xxi was scheduled for august 2021, but the pandemic prevented us to meet in person as an analytical community this year. instead, we have met online last september for the webinar dedicated to our awardees prof. aldo roda (dac-euchems award) and prof. * correspondence e-mails: (*)slavica.razic@pharmacy.bg.ac.rs; (**)martin.vogel@uni-muenster.de; (***)msegundo@ff.up.pt # serbian chemical society member. • reprint from analytical and bioanalytical chemistry, https://doi.org/10.1007/s00216-02103760-3, with permission from springer. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 1128 ražić, vogel and segundo karen faulds (rober kellner lecture award), who gifted us with two wonderful lectures that can be seen at https://youtu.be/i2runlaeq5a, which were sponsored by springer. euroanalysis xxi is now rescheduled to 2023 and it will take place in geneva, switzerland, under the auspices of the swiss chemical society (https://www.euroanalysis2023.ch/), organized by prof. eric bakker (university of geneva), dr. marc suter (eawag), dr. franka kalman (hes sion) and dr. bodo hattendorf (eth zurich). the study groups are also responsible for several ongoing activities of dac-euchems. the topics addressed include “bioanalytics”, “chemometrics”, “education”, “electroanalytical chemistry”, “history”, “nanoanalytics”, “quality assurance” and “sample preparation”. please visit the dac-euchems website for updated reports (https://www.euchems.eu/divisions/analytical-chemistry/) and contact the heads of the study groups in order to have more information or to participate in their activities. lastly, one of dac-euchems objectives is to support its delegates on the organization of local events open to the international community through dissemination of the event within the professional network, including online events. the steering committee of dac-euchems will be happy to receive input for additional activities. feel free to contact one of the following persons: slavica ražić, university of belgrade, serbia (chair), marcela segundo, university of porto, portugal (secretary), jiří barek, charles university, czech republic (treasurer), charlotta turner, lund university, sweden, sibel a. özkan, ankara university, turkey, lutgarde buydens, radboud university, the netherlands, and martin vogel, university of münster, germany. 2. teaching analytical chemistry during the pandemic: an enduring endeavour – but not in vain! when the corona pandemic first hit many parts of the world in spring 2020, surely only few of us had imagined that its influence on our everyday live became that drastic, and, even worse, that enduring. it would be idle to enumerate once again what has been changing over the last 15 months or so. however, it is surely worth to have a closer look onto the dramatic changes in teaching (not only) analytical chemistry with which instructors all over the world have been faced since 2020. as social distancing had early been recognized as a key measure to interrupt the spread of covid-19, remote teaching was rapidly introduced wherever and whenever possible. this was – and still is – an endeavour for all of us teaching a subject such as analytical chemistry that is fundamentally dependent on experimental practice in the laboratory. while many instructors were thrown in at the deep end in early 2020, many of them rapidly developed innovative and smart approaches to cope with the new challenges. the many experiences made, the ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ european analytical column no. 49 1129 many lessons learned and the many conclusions drawn by instructors are worth to be shared with the analytical community as a whole. in january 2021, “abcs of education and professional development in analytical science” column started a series of articles featuring the teaching of analytical chemistry during the pandemic. the idea of this series is to support instructors all over the world in preparing their courses. several articles on different aspects of remote teaching have been published up to now and will surely be inspiring for many readers and hopefully activate some of them to share their experiences and ideas by publishing also an article for the column. for example, emily niemeyer and co-authors contributed with a paper describing some hands-on laboratory experiments that students can complete while studying off the campus.1 in their paper, easy remote laboratory experiments are described that are based on carbonate chemistry and that are used to deal with gravimetry and titrimetry, just to mention one example. supplemental material is provided, allowing readers to implement the experiments described also into their courses. another paper published in this special series by elise heiss and susan oxley describes the implementation of a flipped classroom approach for two different analytical chemistry courses.2 while presenting their experiences with different aspects of flipped classrooms, personal reflections and also recommendations of best practices, the article may serve as an inspiration for readers when thinking about the different forms of remote learning (asynchronous, synchronous, as well as assessment). all of us having taught remotely during the last months have surely been faced with the question of how to enhance student engagement in their courses. jill venton and rebecca pompano provide many good ideas and answers on this question while discussing active learning as a strategy.3 the article is a valuable source of practical tips for student engagement and provides many answers to didactic questions rising from the current zoom world. another paper dealing also with the question of student interaction and student engagement has been contributed by anna cavinato and co-authors.4 based on their experiences, four instructors describe their view on engaging students in an online environment. the article will surely inspire readers through the authors’ profound description of their experiences in different remote setting and their conclusions drawn hereof. an aspect that has turned out to be even more crucial during the pandemic under normal times is the different access to technical infrastructure and financial resources. vilmalí lópez-mejías and co-authors from the university of puerto rico therefore describe their way and their experiences of remote teaching of analytical chemistry at an institution that, prior to the pandemic, had already been hit by, e.g., hurricanes and earthquakes.5 the article is a good combination of ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 1130 ražić, vogel and segundo showing different didactic approaches for remote learning in a synchronous as well in an asynchronous mode and of presenting remote laboratory experiences to the readers. supplemental material allows having a deeper look into the functioning of the courses. all these articles presented show that teaching during the pandemic has been – and still is – a challenge, and to serve our students in the best way has even more become an endeavour. however, the experiences made and the concepts developed will surely be not in vain, as many of them can be integrated into regular courses. moreover, i am sure, we shall return to a normal life – with many lessons learned from the pandemic. i hope that this small survey on articles recently published in “abcs of education and professional development in analytical science” supports many readers while preparing and carrying out their course and stimulates them to share their experiences and concepts developed with colleagues in the field. references 1. j. f. destino, e. m. gross, e. d. niemeyer, s. c. petrovic, anal. bioanal. chem. 413 (2021) 1237 2. e. m. heiss, s. p. oxley, anal. bioanal. chem. 413 (2021)1245 3. b. j. venton, r. r. pompano. anal. bioanal. chem. 413 (2021):1507 4. a. g. cavinato, r. a. hunter, l. s. ott, j. k. robinson, anal. bioanal. chem. 413 (2021):1513 5. a. l. morales-cruz, b. m. ortiz-andrade, j. del pilar-albaladejo, l. m. díaz-vázquez, u. rivera-gonzález, v. lópez-mejías, anal. bioanal. chem. 413 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice accepted manuscript this is an early electronic version of an as-received manuscript that has been accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as d. jaćimovski, k. šućurović, m. đuriš, z. arsenijević, s. krstić and n. bošković-vragolović, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc230116016j this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230116016j j. serb. chem. soc.00(0)1-16 (2023) original scientific paper jscs–12233 published dd mm, 2023 1 mass transfer in inverse fluidized beds darko jaćimovski1, katarina šućurović1, mihal đuriš1*, zorana arsenijević1, sanja krstić2 and nevenka bošković-vragolović3 1institute of chemistry, technology and metallurgy-national institute of the republic of serbia, university of belgrade, belgrade, serbia, 2vinča institute of nuclear sciencesnational institute of the republic of serbia, university of belgrade, belgrade, serbia and 3faculty of technology and metallurgy, university of belgrade, belgrade, serbia (received 16 january; revised 27 february; accepted 22 march 2023) abstract: in this work, the coefficient of fluid-wall mass transfer in an inverse fluidized bed was determined using the adsorption method. the experiments were carried out in a column with a diameter of 45 mm with spherical and nonspherical particles of polypropylene and polyethylene with a diameter of 3.3-4.9 mm and a density of about 930 kg m-3. a diluted solution of methylene blue was used as a fluidization medium, which was adsorbed on part of the surface of the column on silica gel. the obtained results showed that the presence of particles during inverse fluidization does not contribute significantly to mass transfer compared to the influence of particles on transfer in conventional fluidized beds. therefore, the pseudofluid concept was introduced into the analysis and an empirical correlation was performed to determine the mass transfer coefficient. the obtained results were compared with literature correlations for inverse and conventional fluidized beds. keywords: inverse fluidization; fluid-wall mass transfer; pseudofluid. introduction fluidized bed contactors, because of their efficiency and transfer intensity, are often used in systems where contact between liquids and solid particles is required. when the solid phase has a lower density than the liquid phase, fluidization can be achieved by the liquid flowing into the column from the top and forming the bed in the opposite way compared to a conventional bed. such beds are inverse fluidized beds. inverse fluidization is most commonly used in wastewater treatment in bioreactors or in some adsorption processes. the beds formed in this way have been shown to be effective for formation and maintenance of biofilm. for the *corresponding author e-mail: mihal.djuris@ihtm.bg.ac.rs; tel.:+381 11 33 70 408 https://doi.org/10.2298/jsc230116016j a cc ep te d m an us cr ip t mailto:mihal.djuris@ihtm.bg.ac.rs https://doi.org/10.2298/jsc230116016j 2 jaćimovski et al. practical application of inverse fluidized beds in various contactors, it is necessary to know the fluid dynamics of these systems and the mass transfer achieved. wang et al.1 studied the removal of oil from an emulsion in water by inverse fluidization with a hydrophobic air gel. the authors used nanogel sizes: 0.5 mm-0.85 mm; 0.7 mm-1.2 mm; 1.7 mm-2.35 mm, the density was 64 kg m-3. the experiments were performed in columns with a diameter of 7.6 cm and lengths of 1.47 m and 0.77 m. the oil concentration is monitored using the cod (chemical oxygen demand). the authors have proposed a model that is consistent with the experimental data. it has been shown that the most important parameters affecting oil removal and thus mass transfer are granule size, bed height and fluid velocity. inverse fluidization is applied in bioreactors where the necessary oxygen is supplied to the microorganisms in a three-phase fluidized bed. the application of inverse fluidization in fbbr reactors (fluidized bed biofilm reactors) is shown in the work of begum and radha2 in which the aerobic biodegradation of phenol was studied using the microorganism pseudomonas fluorescens. the authors performed the experimental tests in a column with a height of 105 cm and a diameter of 10 cm. polystyrene particles with a diameter of 3.5 mm and a density of 863 kg m-3 were used as biofilm carriers. mass transfer is monitored by the parameter cod. it was shown that the value of cod removal increases with the increase of gas velocity and the ratio of the volume of settled bed to the working volume (vb/ vr). in the best case, cod reduction is 98.5%. similarly, biological treatment of wastewater in an inverse fluidized bed system was performed by sokol et al.3. these authors performed the removal of phenol, cresol, isopropylphenol, dimethylphenol, benzene and toluene from wastewater in a column with an internal diameter of 20 cm and a height of 6 m with polypropylene particles of density 910 kg m-3 as biofilm carriers in an inverse fluidized bed. mass transfer was monitored using the cod parameter. it is shown that the operating conditions that give the best results are vb/ vr = 0.55 and a gas velocity of 0.024 m s-1. the inverse fluidized bed was used for the treatment of wastewater containing starch4. the experiments were performed in a column with a diameter of 9.2 mm and a height of 1.6 m. irregularly shaped polypropylene particles with a density of 870 kg m-3 were used as carrier particles. mass transfer was monitored using the cod parameter. the reduction of the cod parameter increased with increasing air flow rate and with the duration of the experiment. wastewater treatment in an inverse fluidized system was investigated by karmanev and nikolov5. spherical polystyrene particles with an average diameter of 2.5 mm and density of 200 kg m-3 were used as carriers. it can be seen that biochemical oxygen demand (bod) decreases a cc ep te d m an us cr ip t mass transfer in inverse fluidized beds 3 with time. it has been confirmed that inverse fluidization systems are an effective system for treating wastewater with low pollutant concentrations. knowledge of mass transfer is necessary for the application of inverse fluidized beds. one of the few papers dealing with the study of mass transfer in an inverse fluidized bed is the work of nikov and karamanev6 who showed that by increasing the density difference between fluid and particles, better mass transfer is achieved. the experimental data were compared with literature correlations for mass transfer and the following correlation was proposed: 0.33 sh 0.28( sc)gamv= (1) the experiments were carried out with polystyrene and polyethylene particles with a diameter of 2.2-7.1 mm and a density of 80-930 kg m-3. the fluidization medium was water and an aqueous solution of polyethylene glycol with a concentration of 1.9% by mass. in their work, kumar et al.7 monitored mass transfer in an inverse fluidized system using electrochemical methods. mass transfer of lead ions was monitored by an electrochemical method in an inverse fluidized bed, where the fluidization medium was an aqueous solution of ferrous salts and ferricyanide in the presence of sodium hydroxide. cylindrical particles with a diameter of 2.151 mm and a height of 5 mm and a density of 877.6 kg m-3 were used. inverse fluidization was performed in a column with a diameter of 25.4 mm. as a result of these investigations, a correlation was established for the determination of the mass transfer factor: 0.30 re 0.18 1 p d j   −   =   −  (2) the aim of this work is to investigate wall-fluid mass transfer in the presence of inverse fluidized particles using the adsorption method. the experimental results are presented as a function of particle and bed parameters and compared with mass transfers in conventional fluidized beds. experimental the scheme of the experimental apparatus is shown in figure 1. a cylindrical column with an inner diameter of 45 mm was used. at the top of the column there is a ring, on the inside of which there is a thin aluminum foil coated with silica gel. the column's inner diameter was not affected by the presence of the foil. the fluidization medium was a dilute aqueous solution of methylene blue with a concentration of 2·10-3 g l-1. the experiments were performed at a temperature of 20˚c. spherical and non-spherical polypropylene and polyethylene particles were used for the experiments, and their properties are given in table i. a cc ep te d m an us cr ip t 4 jaćimovski et al. figure 1. scheme of the experimental apparatus (1cylindrical column for inverse fluidization; 2-distributor; 3-grid; 4-fluidized bed particles; 5-inlet of the solution into the column; 6-vent; 7-piezometers; 8-exit of the solution from the column; 9, 11 -valves; 10-flow meter; 12-reservoir; 13-pump,’a’-ring, a-silica gel foil, b-particles and methylene blue solution) during the fluid flow from the top of the column, an inverse fluidized bed was formed in which methylene blue adsorbed on silica gel during the experiment, i.e. fluid-wall mass transfer occurred in the presence of inert particles. one of the conditions for the application of the method is a short adsorption time to avoid saturation of the adsorption surface, therefore the experiments lasted about 5 minutes8-10. table i. properties of particles material diameter mm sphericity density kg m-3 polypropylene 3.3 1 935 polypropylene 3.7 0.86 935 polypropylene 4.4 0.85 937 polyethylene 3.9 0.87 926 a cc ep te d m an us cr ip t mass transfer in inverse fluidized beds 5 at the end of each experiment, the coloration of the surface of the silica gel foil, which is equivalent to the adsorbed amount of methylene blue, was analyzed using sigmascan pro software10-12. based on the surface concentration, the mass transfer coefficient was determined according to the following equation: 0 p c k c t = (3) results and discusion figure 2 shows the dependence of the mass transfer coefficient (equation 3) on the fluid velocity in the packed and in the inverse fluidized bed. the dependence shows a significant increase in the mass transfer coefficient at lower velocities, i.e. in a packed bed of particles. u / m s -1 0.00 0.01 0.02 0.03 0.04 0.05 0.06 k / m s -1 0 5e-6 1e-5 2e-5 2e-5 3e-5 3e-5 3.3 mm (pp) 3.7 mm (pp) 3.9 mm (pe) 4.4 mm (pp) single-phase flow figure 2. mass transfer coefficient in inverse fluidized bed and single-phase flow depending on fluid velocity after reaching the fluidized state, the mass transfer coefficient increases slightly or is approximately constant. there are no significant differences in mass transfer in the bed with different particles because the differences in diameters and sphericity are relatively small. for comparison, the mass transfer coefficient is also shown in the same diagram for a single-phase flow, i.e., when only liquid (without a cc ep te d m an us cr ip t 6 jaćimovski et al. particles) flows through the column. it can be seen that the presence of inert fluidized particles has no significant effect on mass transfer, probably due to the fact that the particle density is close to the liquid density. a somewhat clearer dependence of the mass transfer coefficient on the bed parameters can be observed from the dependence of the sherwood number sh on the reynolds number re (figure 3). from figure 3, it can be seen that the sherwood number sh in the fixed bed increases sharply with the increase of the reynolds number re (i.e., with the increase of the velocity u). in the fluidized bed, the sherwood number sh also increases with the increase in reynolds number re, but somewhat less than in the packed bed. it is also evident that there is no effect of particle size and shape on the mass transfer coefficient in the inverse fluidized bed, since all the particles studied show a very similar dependence. figure 3. dependence of sherwood on reynolds number figure 4 shows the dependence of the mass transfer coefficient on the bed porosity ε. since the bed porosity is a function of the fluid velocity u, the dependence of k=f(ε) practically follows the dependence of k=f(u) (figure 2). in a packed bed, where the porosity is constant, the mass transfer coefficient increases. during the transition to the fluidized bed state up to a bed porosity of about 0.7, the mass transfer coefficient continues to increase, while for higher values of bed porosity it becomes almost constant. figure 4 shows the appearance a cc ep te d m an us cr ip t mass transfer in inverse fluidized beds 7 of a slight maximum on the curve, which also occurs with conventional fluidization10. it is also evident that there is no visible difference in the intensity of mass transfer in the inverse fluidized bed with different particle types and sizes. 0.4 0.5 0.6 0.7 0.8 0.9 k / m s1 0.0 2.0e-6 4.0e-6 6.0e-6 8.0e-6 1.0e-5 1.2e-5 3.3 mm (pp) 3.7 mm (pp) 3.9 mm (pe) 4.4 mm (pp) figure 4. dependence of mass transfer coefficient on bed porosity for inverse fluidized bed (packed and fluidized bed) the adsorption method used in this study to determine the mass transfer coefficient is also useful for flow visualization. figure 5 shows flow visualization images for different conditions (flows) of 4.4 mm diameter polypropylene particles in the bed. the chromatograms are shown for the same experimental conditions (time duration, temperature, methylene blue concentration, etc.) but for different liquid flow rates. figures 5(a)-5(c) are characteristic of the liquid flow where the particles are stable, i.e., the packed bed. the flow patterns of the individual particles in the bed can be clearly seen. as the flow increases, an increase in the average color intensity of the silica gel foil surfaces can be seen. at the minimum fluidization velocity umf (figure 5(d)), the coloration of the silica gel foil surface is approximately uniform as the particles begin to oscillate slightly and mix the fluid. figure 5(e) and figure 5(f) are chromatograms taken in the fluidized bed and are characterized by the uniform coloration of the silica gel foil surface due to the intense mixing of the fluid by the particle fluidization. it can also be seen that the a cc ep te d m an us cr ip t 8 jaćimovski et al. coloration of the surface is approximately the same at a higher flow rate in the fluidized bed. figure 5. flow chromatogram in packed bed (a-c), at minimum fluidization (d) and in fluidized bed (e-f) a comparison of the data obtained in inverse fluidized beds with those of a conventional fluidized bed as a function of bed porosity is shown in figure 6. the results presented for the conventional fluidized bed were carried out in a bed of spherical glass particles with a diameter of 3 mm and a density of 2,500 kg m-3 with water as the fluidizing medium. these experiments were carried out under the same experimental conditions and in the same column. from the dependencies shown in figure 6, it can be seen that the influence of the particles on mass transfer in conventional fluidization is significantly greater than the influence of the particles in inverse fluidization. this can be explained by the fact that significantly higher fluid velocities were used in conventional fluidization compared to inverse fluidization. figure 6 shows the occurrence of a maximum at a porosity of about 0.7, which is consistent with our previous studies 9,13. a cc ep te d m an us cr ip t mass transfer in inverse fluidized beds 9 0.4 0.5 0.6 0.7 0.8 0.9 k / m s1 0 1e-5 2e-5 3e-5 4e-5 5e-5 6e-5 3.3 mm (pp) 3.7 mm (pp) 3.9 mm (pe) 4.4 mm( pp) conventional fluidized bed (3mm) figure 6. comparison of the mass transfer coefficient in inverse and conventional fluidized beds as a function of porosity based on the experimentally determined results for the mass transfer coefficient, the mass transfer factor jd was calculated. figure 7 shows the dependence of the mass transfer factor jd on the reynolds number re (figure 7(a)) and the porosity of the bed (figure 7(b)). both dependences show a decrease in the mass transfer factor jd with an increase in the reynolds number re and the porosity of the bed which is consistent with our previous study10. a cc ep te d m an us cr ip t 10 jaćimovski et al. a) b) figure 7. dependence of the mass transfer factor on reynolds number (a) and bed porosity (b) the diagram of the dependence of the mass transfer factor on the reynolds number also shows the data obtained for a single-phase fluid flow (figure 7(a)). it can be observed that the influence of the inverse fluidized particles on the mass transfer intensification is very small. this can be explained by the fact that in inverse fluidization the fluid velocity is low and therefore the collisions between the particles in the system are lower, so the influence on mass transfer is smaller. the drag force in conventional fluidized beds is defined as: ( ) 3 6 p d b g p f d f f f g   = − = − (4) in inverse fluidized bed is defined as: ( ) 3 , 6 p d inv g b f p d f f f g   = − = − (5) the ratio of the drag force in these two systems is: 3 3 , , p p fd d inv p inv f p df f d     − =  − (6) comparing conventional fluidization, in which particles with a diameter of 3 mm and a density of 2,500 kg m-3 were used, with inverse fluidization, in which particles with a diameter of 3.7 mm and a density of 935 kg m-3 were used (the fluid density in both cases is 1000 kg m-3), then: a cc ep te d m an us cr ip t mass transfer in inverse fluidized beds 11 , 3.63d d inv f f = (7) it is found that the friction in the inverse fluidized bed is 3.6 times lower than in the conventional fluidized bed. due to the small effect of particle-fluid friction in the inverse fluidized bed, the entire bed can be treated as a pseudofluid. all this indicates that although the conventional and inverse fluidized beds are described by the same equations, the frictional force is lower in the case of the inverse fluidized bed, so it is mainly used in biofilm reactors where the frictional forces do not disturb the formed biofilm, which is important for mass transfer in such reactors. the system behaves like a pseudofluid, and the frictional forces do not damage the biofilm. on the other hand, the particles are still present and provide gentle mixing, which is ideal for biosystems where microorganisms are involved in the reaction14,15. re p pf pf pf pf d u  = (8) where pseudofluid density is defined as: ( )1pf f p   = + − (9) and pseudofluid dynamic viscosity as: 5(1 ) exp 3 pf f     −  =     (10) superficial fluid velocity can be calculated: f p pf f p g g u a a  = + (11) since in a particulate fluidized bed the total particle motion in the column is zero (gp=0), the velocity of the pseudofluid is equal to the superficial fluid velocity: f pf f g u u a = = (12) based on the experimental results, the equation for the mass transfer factor was established: 0.48 re , 13 re 980 d pf pf j − =   (13) a cc ep te d m an us cr ip t 12 jaćimovski et al. figure 8 shows the dependence of the mass transfer factor jd on the reynolds number of a pseudofluid repf. the obtained equation agrees well with the experimental data. the mean absolute deviation of the experimental data from the data calculated by the established equation is 14.1%, while the relative deviation is -4%. figure 8. dependence of the mass transfer factor on the reynolds number of a pseudofluid table ii shows some selected correlations for the determination of mass transfer factors in conventional and inverse fluidized beds and compares them with our experimentally obtained data. the comparison of the experimental data with the data calculated according to the above correlations is shown in fig. 9. table ii. comparison of literature correlations with experimental data and the obtained model autor fluidization model σr, % σa, % yutani et al.16 (eqn. 14) conventional 0.40.4 re d p j  − = +71.5 73.5 marooka et al.17 (eqn. 15) conventional 0.5 re0.6 1 p d j   −   =   −  -6.93 14.9 a cc ep te d m an us cr ip t mass transfer in inverse fluidized beds 13 kunii & levenspil18 (eqn. 16) conventional 0.52 0.51 (1 ) re re re d p p p j  −  = + −  -0.89 14.6 nikov & karamanev6 (eqn. 17) inverse ( ) 0.330.28 re d j gamv= -82.4 81.5 our corellation (eqn. 13) inverse 0.48 re d pf j − = -4.0 14.1 figure 9. comparison of the experimental data with the obtained model and the correlations in the literature it is interesting to note that the correlation of yutani et al.16 (equation 14), was originally derived for conventional fluidized beds, and the correlation of nikov and karamanev6 (equation 17) for inverse fluidized beds, show significant deviation from the experimental data. very good agreement with the experimental data is shown by the correlations for conventional fluidization of marooka et al.17 (equation 15) and kunii and levenspiel18 (equation 16), as can be seen in тable ii. conclusion in the paper, the fluid-wall mass transfer coefficient was experimentally determined in an inverse fluidized bed. the results show a significant increase in the mass transfer coefficient with increasing flow velocity in the packed bed, while a slight increase is observed in the fluidized bed. compared to the mass transfers in a fluid flow without particles, it was found that the presence of particles does not contribute significantly to the intensification of the transfer, which is due to the low frictional force between particles and fluid. based on the obtained results, the inverse fluidized bed was classified as a pseudofluid and a new correlation was presented, which represents the dependence of the mass transfer factor on the reynolds number of a pseudofluid: 0.48 re d pf j − = 13 < repf < 980 (equation 13) the obtained experimental results deviate from the proposed correlation by less than 14.1%, with the mean relative deviation being -4%. when comparing the experimentally obtained data with the correlations from the literature, it was found that the correlations of marooka et al.17 (equation 15) and kunii and levenspiel18 (equation 16) for conventional fluidization show very good agreement with the experimental data. nomenclature bod biochemical oxygen demand cod chemical oxygen demand a cc ep te d m an us cr ip t 14 jaćimovski et al. a cross section area, m2 cp surface concentration of methylene blue on adsorbent layer, kg m-2 c0 bulk concentration of methylene blue, kg m-2 dp,inv particle diameter in inverse fluidized bed, m dp particle diameter, m dc column diametar, m fd drag force, n fb buoyancy force, n fg gravity force, n g gravitational acceleration, m s-2 gf mass flow fluids, kg s-1 gp mass flow particle, kg s-1 jd mass transfer factor k coefficient mass transfer, m s-1 t time, s u superficial fluid velocity, m s-1 upf superficial pseudofluid velocity, m s-1 vb/vr ratio of the volume of the settled bed to the working volume ε bed porosity μf viscosity fluid, pa∙s μpf viscosity pseudofluids, pa∙s ρf fluid density, kg m-3 ρp particle density, kg m-3 ρpf pseudofluid density, kg m-3 ga ( 3 2 2 p f f d g /  ) , galileo number mv ( p f f ( ) / −  ), relative density re ( f c f f u d /  ), reynolds number rep ( f p f f u d /  ), reynolds number for particle repf reynolds number for pseudofluid sh ( c ab kd / d ), sherwoods number sc ( f f ab / d  ), schmidts number σa exp exp n cal x x n x  −        1 1 , absolute deviations σr exp exp n cal x x n x  −        1 1 , relative devations acknowledgements: this work was financially supported by the ministry of science, technological development and innovation of the republic of serbia (grant no. 451-0347/2023-01/200026 and 451-03-47/202301/200135]. a cc ep te d m an us cr ip t mass transfer in inverse fluidized beds 15 извод пренос масе у инверзно флуидизованом слоју дарко јаћимовски1, катарина шућуровић1, михал ђуриш1, зорана арсенијевић1, сања крстић2 и невенка бошковић-враголовић3 институт за хемију, технологију и металургију -институт од националног значаја за републику србију, универзитет у београду, београд, србија, 2винча институт за нуклеарне наукеинститут од националног значаја за републику србију, универзитет у београду, београд, србија и 3технолошкометалуршки факултет, универзитет у београду, београд, србија у овом раду је одређиван коефицијент преноса масе флуид-зид у инверзнофлуидизованом слоју применом адсорпционе методе. експерименти су вршени у колони пречника 45 мм са сферичним и несферичним честицама полипропилена и полиетилена пречника 3.3-4.9 мм и густине око 930 кг м-3. као флуидизациони медијум коришћен је разблажени раствор метиленски плавог који је адсорбован на делу површине колоне на силикагелу. добијени резултати показали су да присуство честица при инверзној флуидизацији не доприноси значајно преносу масе у поређењу са утицајем честица на пренос масе у конвенционално флуидизованим слојевима. због тога је у анализу уведен концепт псеудофлуида и изведена је емпиријска корелација за одређивање коефицијента преноса масе. извршено је поређење добијених резултата са литературним корелацијама за инверзну и конвенционалну флуидизације. (примљено 16. јануара, ревидирано 27. фебруара, прихваћено 22. марта 2023.) references 1. d. wang, t. silbaugh, r. pfeffer y.s. lin, powder technol. 203 (2010) 298. (https://doi.org/10.1016/j.powtec.2010.05.021) 2. s.s.begum, k.v. radha, korean j. chem. eng. 31 (2014) 436 (https://doi.org/10.1007/s11814-013-0260-z) 3. w. sokol, a. ambaw, b. woldeyes, chem. eng. j. 150 (2009) 63 (https://doi.org/10.1016/j.cej.2008.12.021) 4. m. rajasimman, c.karthikeyan, int. j. environ re. 3 (2009) 569 (https://doi.org/10.22059/ijer.2010.72) 5. d.g. karamanev, l.n. nikolov, environ. prog. 15 (1996) 3. 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(2023) https://doi.org/10.2298/jsc230626052s this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230626052s j. serb. chem. soc.00(0)1-14 (2023) original scientific paper jscs–12456 published dd mm, 2023 1 kinetic investigation of reactions of a 3-arylidene-2-thiohydantoin derivative with palladium(ii) salts petar b. stanić*, darko p. ašanin, tanja v. soldatović1 and marija d. živković2,3* university of kragujevac, institute for information technologies, department of science, jovana cvijića bb, 34000 kragujevac, serbia, 1state university of novi pazar, department of natural-mathematical sciences, vuka karadžića 9, 36300 novi pazar, serbia, 2university of kragujevac, faculty of medical sciences, department of pharmacy, svetozara markovića 69, 34000 kragujevac, serbia and 3center for harm reduction of biological and chemical hazards, faculty of medical sciences, university of kragujevac, svetozara markovića 69, 34000 kragujevac, serbia (received 26 june; revised 23 july; accepted 14 august 2023) abstract: 1h nmr spectroscopy was used to monitor the reactions of an arylidene 2-thiohydantoin derivative, 3-((phenylmethylene)amino)-2-thioxo-4imidazolidinone (3), with pdcl2, cis-[pdcl2(dmso-s)2] and k2[pdcl4] in dmsod6 in order to elucidate the reaction kinetics and mechanism. the 2-thiohydantoin derivative 3 formed cis-[pd(3-n,s)(dmso-s)2] + complex (5) in reactions with pdcl2 and cis-[pdcl2(dmso-s)2], while no reaction with k2[pdcl4] was observed. a two-step mechanism for the reactions of 3 with pdcl2 and cis-[pdcl2(dmsos)2] is proposed, in which fast coordination to the side chain nitrogen occurs in the first step, while chelation and coordination to the sulfur atom in the 2thiohydantoin ring is the second, slower, rate-determining step. the reaction rate constants were calculated and reactivities of the 2-thiohydantoin derivative 3 towards the palladium(ii) salts were compared and discussed. reaction of 3 with cis-[pdcl2(dmso-s)2] was faster than with pdcl2. the investigated palladium(ii) salts also react with the solvent, dmso-d6, and the influence of these side reactions on the outcome and kinetics of the 2-thiohydantoin derivative complexation reaction is discussed in detail. the obtained results of this study can have an impact in explanation of the coordination behavior of antitumor active palladium(ii) and platinum(ii) complexes. keywords: 1h nmr spectroscopy; reaction mechanism; 2-thioxo-4imidazolidinone; coordination; pd(ii) complexes. *corresponding authors. e-mail: mzikvovic@kg.ac.rs; petar.stanic@uni.kg.ac.rs. https://doi.org/10.2298/jsc230626052s a cc ep te d m an us cr ip t https://doi.org/10.2298/jsc230626052s 2 stanić et al. introduction thiohydantoins are sulphur derivatives of hydantoin, in which one or both of the carbonyl groups in the cyclic ureide structure are replaced with a thiocarbonyl group.1 out of this class of compounds, 2-thiohydantoins are certainly the most prominent and extensively researched. 2-thiohydantoins represent a valuable molecular scaffold, exhibiting various biological and pharmacological activities and they have found applications in both medicine and industry.2,3 they display a wide range of biological activities, such as antibacterial and antifungal,4 anti-hiv,5 anticarcinogenic,6,7 anti-ulcer and anti-inflammatory,8 anticonvulsive,9 antimutagenic10 and antimelanogenic.11 2-thiohydantoins found various applications in industry, such as c-terminal protein sequencing standards,12 textile printing reagents13 and polymerization and complexation catalysts.14 coordination of active compounds with biologically relevant transition metal ions can, at times, increase their activities, especially in regards to anticarcinogenic activity.15 a newer, hybrid approach, in discovering new potential antitumor agents is coordination of active compounds with metal ions in order to improve their activity and selectivity.16,17 2-thiohydantoins have a great affinity for coordination with transition metal ions.18,19 even though it is a small molecule, 2thiohydantoin has four derivatization points, making its derivatives very versatile ligands. in addition to the heteroatoms in the ring, 2-thiohydantoin derivatives most often contain heteroatoms in the side chains of its substituents. many kinds of various 2-thiohydantoin complexes have been synthesized and reported so far.20–26 in particular, transition metal complexes of arylidene 2-thiohydantoin derivatives have been researched extensively, largely due to the biological activities they exhibit, primarily antimicrobial and anticancer.21,27,28 the aim of this study was to investigate the kinetics and mechanism of the coordination reactions of 3-((phenylmethylene)amino)-2-thioxo-4imidazolidinone, an arylidene 2-thiohydantoin derivative, with some palladium(ii) salts. as arylidene 2-thiohydantoin metal complexes, palladium(ii) in particular,28 show some promise as prospective antitumor agents, a better understanding of the mechanisms of their formation, coordination modes and kinetics might prove beneficial for the design and conceptualization of novel, more potent compounds. experimental materials and methods all chemicals and reagents used in this investigation were commercially obtained (from either sigma-aldrich or acros) and were high in purity. they were used as received, without additional purification. nmr spectra were recorded on a varian gemini-2000 spectrometer at 50 mhz and 200 mhz. dmso-d6 was used as the solvent and all chemical shifts were referenced accordingly. downfield shifts were recorded as positive numbers. tetramethylsilane was used as the internal reference and all chemical shits were rounded to the nearest 0.01 ppm. a cc ep te d m an us cr ip t reactions of arylidene 2-thiohydantoin with palladium(ii) salts 3 synthesis and characterization of 3-((phenylmethylene)amino)-2-thioxo-4-imidazolidinone (3) 3-((phenylmethylene)amino)-2-thioxo-4-imidazolidinone (3) was synthesized using a slight modification of a formerly published procedure.28 benzaldehyde (0.01 mol) and thiosemicarbazide (0.01 mol) in methanol (30 ml) were heated for 3 h under reflux and cooled thereafter. ethyl chloroacetate (0.01 mol) and anhydrous sodium acetate (0.03 mol) were added in situ and the mixture was heated for another 6 h under reflux. upon the completion of the reaction, the mixture was cooled at room temperature and then added to cold water, for the resulting product to precipitate. the product was filtered off, rinsed with hot water, dried and re-crystallized from hot methanol. structure and purity of the compound was confirmed by nmr (1h and 13c) and ir spectroscopy. the corresponding ir and nmr (1h and 13c) spectra are provided in the esi (fig. s-1 – s-3). 1h nmr kinetic experiments 1h nmr kinetic measurements of the reactions of 3-((phenylmethylene)amino)-2-thioxo4-imidazolidinone (3) (0.021 mmol) with pdcl2, cis-[pdcl2(dmso-s)2] and k2[pdcl4] (0.021 mmol) in dmso-d6 (0.6 ml) were performed in standard 5 mm nmr tubes at room temperature in an overnight experiment. dmso-d6 solutions of the reactants (0.3 ml each) were freshly prepared right before the start of the experiment. after the mixing of the reactants, 29 spectra in total were recorded overnight for each one of the experiments. the first six spectra were recorded with no delay, then the next three with a 5 min delay, then sets of three every 10, 15 and 30 min, and finally the last 11 spectra were recorded with an hour delay between them. the concentrations of the products at given experiment intervals were determined by integrating suitable proton signals in the 1h nmr spectra. the first-order rate constants were determined according to equation 1. ln(c) = -kt + ln(c0) (1) where c is concentration, c0 is starting concentration, k is the first-order rate constant and t is experiment time. results and discussion 3-((phenylmethylene)amino)-2-thioxo-4-imidazolidinone (3) was synthesized from benzaldehyde in a reaction with thiosemicarbazide (scheme 1). nucleophilic addition of thiosemicarbazide to benzaldehyde (1) yield thiosemicarbazone (2). thiosemicarbazone (2) then undergoes a cyclocondenzation reaction with ethyl chloroacetate in the presence of anhydrous sodium acetate, forming the arylidene 2-thiohydantoin derivative, 3((phenylmethylene)amino)-2-thioxo-4-imidazolidinone (3). scheme 1. synthesis of 3-((phenylmethylene)amino)-2-thioxo-4-imidazolidinone (3). a cc ep te d m an us cr ip t 4 stanić et al. for the purpose of investigating the kinetics and mechanism of 3-arylidene-2thiohydantoin coordination with pd(ii), a kinetic time-dependent experiment, monitoring the reactions of 3-((phenylmethylene)amino)-2-thioxo-4imidazolidinone (3) with pdcl2, cis-[pdcl2(dmso-s)2] and k2[pdcl4] in dmso-d6, was performed. dmso-d6 was used because it is suitable for dissolving both the 2-thiohydantoin derivative and the metal salts. coordination of 3 to pd(ii) was tracked through the changes in specific signals in the spectra. 1h nmr spectra of the reaction of 3-((phenylmethylene)amino)-2-thioxo-4imidazolidinone (3) with cis-[pdcl2(dmso-s)2] in dmso-d6 are shown in figure 1. signals of the coordinated 3-((phenylmethylene)amino)-2-thioxo-4imidazolidinone (3) can be observed, even from the first spectrum. fig. 1. time-dependent 1h nmr spectra of the reaction of 3-((phenylmethylene)amino)-2thioxo-4-imidazolidinone (3) and cis-[pdcl2(dmso-s)2] in dmso-d6. as can be seen from fig. 1, the singlet of the 2-thiohydantoin ring ch2 group (a) is shifted downfield from 3.90 to 4.15 ppm. multiplets of the aromatic benzene ring protons (b) moved downfield from 7.25-7.95 to 9.57-9.84 ppm, while the singlet of the double bond ch proton (c) is shifted downfield from 8.41 to 9.37 a cc ep te d m an us cr ip t reactions of arylidene 2-thiohydantoin with palladium(ii) salts 5 ppm. signals of the coordinated 2-thiohydantoin derivative (a, b and c) can be observed from the first spectrum and their intensities do not change throughout the experiment. this, in fact, indicates fast initial coordination, too fast for the nmr time-scale. one more thing that implies coordination is the absence of the broad singlet of the 2-thiohydantoion ring nh proton. 2-thiohydantoins are known to exist in two tautomeric forms in equilibrium (scheme 2).29 it is proposed that the thio-enol tautomeric form (4) is responsible for coordination and furthermore, that this ‘ketoenol’ equilibrium shifts to the thio-enol form during the reaction.27 the sh protons of the thio-enol tautomer can be seen in the spectra as a singlet at 1.85 ppm (fig. 1). furthermore, a newly formed singlet at 10.15 ppm (d) can be observed increasing in intensity throughout the experiment. the new singlet at 10.15 ppm belongs to hydrochloric acid that forms from the deprotonation of the 2thiohydantoin ring and the chloride anions that are substituted from cis[pdcl2(dmso-s)2]. scheme 2. reactions of 3-((phenylmethylene)amino)-2-thioxo-4-imidazolidinone (3) with pdcl2, cis-[pdcl2(dmso-s)2] and k2[pdcl4]. no reaction of 3 was observed with k2[pdcl4] during the course of the experiment (see fig. s-4). a cc ep te d m an us cr ip t 6 stanić et al. the reaction as a whole is proposed to proceed in two steps. in the first step (equation 2), initial coordination takes place through the nitrogen in the side chain. this is the faster reaction step, which is supported by the presence of the signals of the complex (a, b and c) in the first spectrum of the experiment, that then do not change in intensity during the experiment. the second, slower reaction step is deprotonation of the 2-thiohydantoin ring and coordination through the sulfur atom in the ring (equation 3). the resulting complex (5) is a five-membered chelate with palladium(ii) coordinated to the 2-thiohydantoin ring sulfur and the double bond nitrogen in the side chain. cis-[pdcl2(dmso-s)2] + 3 → cis-[pdcl(3-n)(dmso-s)2]+ + cl(2) cis-[pdcl(3-n)(dmso-s)2]+ + h2o → cis-[pd(3-n,s)(dmso-s)2]+ (5) + h3o+ + cl(3) even though the spectral data clearly shows a reaction between the 2thiohydantoin derivative 3 and cis-[pdcl2(dmso-s)2], it does not necessarily give a clear insight in the chemistry of reaction beyond modes of coordination. concentrations of the formed complex 5 were calculated by integration of the suitable proton signal at 10.15 ppm (fig. 2). as hydrochloric acid forms equimolarly with the complex 5, according to equation 3, concentrations determined from that signal can be regarded as concentrations of complex 5. the singlet at 10.15 ppm was integrated against the singlet of the uncoordinated 2thiohydantoin derivative 3 at 8.40 ppm. the relative changes of the intensity of the singlet at 10.15 ppm is directly proportional to the change in concentration of complex 5, and the concentrations were calculated from the relative integral values. a cc ep te d m an us cr ip t reactions of arylidene 2-thiohydantoin with palladium(ii) salts 7 fig. 2. changes in product concentration, cis-[pd(3-n,s)(dmso-s)2] +, during the substitution reaction of cis-[pdcl2(dmso-s)2] with 2-thiohydantoin derivative 3 in dmso-d6. it can be seen in the plot that after about an hour into the experiment, complex formation slows down drastically. if we take into consideration the stoichiometry of the system, at the end of the experiment, more than half of the initial amount of the 2-thiohydantoin derivative 3 has not undergone any sort of reaction, implying that the reaction system is a bit more complex and that cis-[pdcl2(dmso-s)2] undergoes multiple competing reactions. in order to gain a deeper insight into the details of the mechanism of the reaction, a plot of the logarithm of the product concentration vs experiment time was analyzed (fig. 3). in the plot, it is clearly visible that this is not a linear firstorder reaction, but instead, two linear slopes can be observed. this goes along with the conclusion that multiple processes are occurring, not just the reaction of cis[pdcl2(dmso-s)2] with 2-thiohydantoin derivative 3. for the first hour, the reaction direction can be described with the equation y = (1.63±0.17)∙10-4x – 5.01. after about an hour, the reaction kinetics change course and the new direction can be described with the equation y = (3.36±0.20)∙10-6x 4.46. the first phase of the reaction is significantly faster, with the slope coefficient k1 = 1.63∙10-4 s-1, than the second phase with the coefficient k2 = 3.36∙10-6 s-1. a change in the system occurred and a chemical species emerged, the concentration of which has only become significant after an hour into the experiment. a cc ep te d m an us cr ip t 8 stanić et al. fig. 3. first-order plot for the substitution reaction of cis-[pdcl2(dmso-s)2] with 2-thiohydantoin derivative 3 in dmso-d6. cis-[pdcl2(dmso-s)2] most likely engages in a parallel reaction with the solvent, as it is known that cis-[pdcl2(dmso-s)2] can react with dmso, yielding cis-[pd(dmso-o)2(dmso-s)2]2+.30,31 cis-[pdcl2(dmso-s)2] has two molecules of dmso in cis-configuration bonded through sulfur atoms. dmso molecules coordinated through the sulfur atoms exhibit a very strong trans effect on the neighbouring chlorido ligands, which in turn weakens their bonds with palladium(ii). with this in mind, cis-[pdcl2(dmso-s)2], apart from reaction with the 2-thiohydantoin derivative 3, reacts with dmso, forming tetrakis(dimethyl sulphoxide)palladium(ii), cis-[pd(dmso-o)2(dmso-s)2]2+, with the other two dmso molecules bonded through the oxygen. there is a slight deviation from the ideal square-planar structure in cis-[pd(dmso-o)2(dmso-s)2]2+, the biggest of which being the angle between the two sulfur bonded dmso molecules, due to steric repulsions between the methyl groups of one dmso and the sulfoxy group of the other. these steric repulsions prohibit the s-bonding of the other dmso molecules, which is believed to be the main reason for coordination through oxygen.30 two parallel reactions of cis-[pdcl2(dmso-s)2] take place during the experiment. the first is with the 2-thiohydantoin derivative 3 (equation 4) and the other is with the solvent, dmso, (equation 5). cis-[pdcl2(dmso-s)2] + 3 + h2o → cis-[pd(3-n,s)(dmso-s)2]+ + h3o+ + 2cl(4) a cc ep te d m an us cr ip t reactions of arylidene 2-thiohydantoin with palladium(ii) salts 9 cis-[pdcl2(dmso-s)2] + 2dmso → cis-[pd(dmso-o)2(dmso-s)2]2+ + 2cl(5) the reaction is faster in the first phase, during the first hour of the experiment (k1 = 1.63∙10-4 s-1), up until a dynamic equilibrium is achieved and a significant amount of cis-[pd(dmso-o)2(dmso-s)2]2+ is formed, then the complex formation reaction (equation 4) slows down in the second phase (k2 = 3.36∙10-6 s-1), because there is a significantly smaller amount of the reactant, cis-[pdcl2(dmso-s)2], in the system. coordination of 3-((phenylmethylene)amino)-2-thioxo-4-imidazolidinone (3) in the reaction with pdcl2 takes place in the same manner as with cis-[pdcl2(dmsos)2]. all the same signals with identical chemical shifts can be observed in the spectra of the reaction (fig. 4). pairs of signals of the coordinated and uncoordinated 3, among which are singlets of the 2-thiohydantoin ring ch2 group protons (a), multiplets of the aromatic benzene ring protons (b) and singlets of the double bond ch proton (c), can be seen at the same chemical shift in the spectra. thio-enol tautomer -sh proton is at 1.85 ppm, the broad singlet of the 2thiohydantoin ring -nh proton is missing and the hcl singlet at 10.15 ppm increases throughout the experiment. fig. 4. time-dependent 1h nmr spectra of the reaction of 3-((phenylmethylene)amino)-2-thioxo-4-imidazolidinone (3) and pdcl2 in dmso-d6. a cc ep te d m an us cr ip t 10 stanić et al. upon calculating the concentrations of the formed cis-[pd(3-n,s)(dmso-s)2]+ complex (5) (equation 3) from the spectral data, an obvious difference in reaction rates was observed and it was noticed that the reaction with pdcl2 is slower than with cis-[pdcl2(dmso-s)2]. changes in complex 5 concentrations over the course of the experiment are shown in fig. 5. the difference in the kinetics of the systems are somewhat perplexing, as spectral data confirms that the same reaction product is formed in both cases. fig. 5. changes in cis-[pd(3-n,s)(dmso-s)2] + complex (5) concentration during the substitution reaction of pdcl2 with 2-thiohydantoin derivative 3 in dmso-d6. in order to get to the bottom of this, a plot of the logarithm of the product concentration vs experiment time was analyzed (figure 6). as with cis[pdcl2(dmso-s)2], in this case there are also two phases, with two linear slopes that intercept after little over an hour. the first phase can be described with the equation y = (1.80±0.22)∙10-4x 6.59, while the second phase can be described with the equation y = (1.25±0.09)∙10-5x – 5.77. the first phase, where most of the complex is formed, has a coefficient k1 = 1.80∙10-4 s-1, which is very close to the slope coefficient of the first phase of the reaction of cis-[pdcl2(dmso-s)2] (k1 = 1.63∙104 s-1). it is known that pdcl2 has great affinity towards dmso and reacts with it to form cis-[pdcl2(dmso-s)2],32 according to equation 6. pdcl2 + 2dmso → cis-[pdcl2(dmso-s)2] (6) it can be concluded that in both cases, basically the same reaction occurs (equation 4), which is supported by the very close values of the reaction coefficients k1. the reaction is slower with pdcl2 because the salt itself does not react with the 2-thiohydantoin derivative 3. only when a sufficient amount of cisa cc ep te d m an us cr ip t reactions of arylidene 2-thiohydantoin with palladium(ii) salts 11 [pdcl2(dmso-s)2] forms does the reaction take place. the lower rate of the reaction with pdcl2 can be explained with the lower reactant concentration. fig. 6. first-order plot for the substitution reaction of pdcl2 with 2-thiohydantoin derivative 3 in dmso-d6. in the case of the third examined palladium(ii) salt, k2[pdcl4], there was no reaction with 3-((phenylmethylene)amino)-2-thioxo-4-imidazolidinone (3) during the course of the experiment. no signals of a newly formed 2-thiohydantoin complex species of any kind could be observed (figure s-4). the four chlorido ligands in k2[pdcl4] are kinetically equivalent and a strong possibility is that all of them were substituted with dmso, as tetrachloroplatinate(ii) and also tetrachrloropalladate(ii) can react with dmso in this manner.33 this would prohibit the reaction with the 2-thiohydantoin derivative 3. conclusion reactions of an arylidene 2-thiohydantoin derivative, 3((phenylmethylene)amino)-2-thioxo-4-imidazolidinone (3) with pdcl2, cis[pdcl2(dmso-s)2] and k2[pdcl4] in dmso-d6 were monitored in a time dependent kinetic 1h nmr experiment. in the cases of pdcl2 and cis-[pdcl2(dmso-s)2], the complex cis-[pd(3-n,s)(dmso-s)2]+ (5) was formed, with palladium(ii) coordinated through the nitrogen in the side chain and the 2-thiohydantoin ring sulfur atom. the mechanism of complex 5 formation consists of two steps. the first step is fast monodentate coordination of 3 via its nitrogen atom in the side chain. this step is too fast for the nmr time-scale, but it is confirmed with the corresponding signals of the complex 5 that are unchanged during the course of the experiment. the second, rate determining step of the reaction is chelation of a cc ep te d m an us cr ip t 12 stanić et al. the intermediate cis-[pdcl(3-n)(dmso-s)2]+ complex through deprotonation of the 2-thiohydantoin ring of 3 and its coordination with the sulfur atom, finally yielding to the formation of complex 5. most of the complex 5 is formed during the first hour of the experiment. it is concluded that simultaneously, a competing reaction with the solvent occurs during which cis-[pd(dmso-o)2(dmso-s)2]2+ is formed, which ultimately halts the reaction. no reaction with k2[pdcl4] was observed during the course of the experiment. ž acknowledgements: the authors are grateful for financial support from the ministry of education, science and technological development of the republic of serbia (agreements noumbers 451-03-68/2022-14/200378, 451-03-68/2022-14/200252 and 451-03-47/202301/200111) and the faculty of medical sciences, university of kragujevac (jp02/20). supplementary material supplementary materials are available electronically from https://www.shdpub.org.rs/index.php/jscs/article/view/12456, or from the corresponding authors on request. и з в о д кинетичко испитивање реакција 3-арилиденског деривата 2тиохидантоина са солима паладијума(ii) петар б. станић, дарко п. ашанин, тања в. солдатовић1 и марија д. живковић2,3 универзитет у крагујевцу, институт за информационе технологије, сектор за природноматематичке науке, јована цвијића бб, 34000 крагујевац, србија, 1државни универзитет у новом пазару, департман за природно-математичке науке, вука караџића 9, 36300 нови пазар, србија, 2универзитет у крагујевцу, факултет медицинских наука, департман за фармацију, светозара марковића 69, 34000 крагујевац, србија и 3центар за смањење штетности биолошких и хемијских хазарда, факултет медицинских наука, универзитет у крагујевцу, светозара марковића 69, 34000 крагујевац, србија протонска нмр спектроскопија је употребљена за праћење реакције арилиденског деривата 2-тиохидантоина, 3-((фенилметилен)амино)-2-тиоксо-4-имидазолидинона (3), са pdcl2, cis-[pdcl2(dmso-s)2] и k2[pdcl4] у dmso-d6, да би се испитали кинетика и механизам реакције. испитивани дериват 2-тиохидантоина 3 је наградио комплекс cis-[pd(3n,s)(dmso-s)2]+ (5) у реакцији са pdcl2 и cis-[pdcl2(dmso-s)2], док са k2[pdcl4] није уочена реакција. претпостављен је двостепени механизам за реакције 3 са pdcl2 и cis-[pdcl2(dmsos)2], у коме се у првом кораку одиграва брза координација за азот из бочног низа, а хелатизација и координовање за тиохидантоински сумпор је други, спорији корак, који одређује брзину реакције. израчунате су константе брзине реакције и реактивности деривата 2-тиохидантоина 3 према солима паладијума(ii) су упоређене и дискутоване. реакција 3 са cis-[pdcl2(dmso-s)2] је била бржа од реакције са pdcl2. испитиване соли паладијума(ii) су такође реаговале са растварачем (dmso-d6) и утицај ових реакција на исход и кинетику реакције комплексирања деривата 2-тиохидантоина је детаљно дискутован. резултати добијени у оквиру овог истраживања могу имати утицај на појашњење координационог понашања антитуморски активних комплекса паладијума(ii) и платине(ii). 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(https://www.doi.org/10.1016/s0020-1693(00)95010-2). a cc ep te d m an us cr ip t https://www.doi.org/10.1016/j.jinorgbio.2017.07.015 https://www.doi.org/10.17344/acsi.2020.6045 https://www.doi.org/10.17344/acsi.2020.6045 https://www.doi.org/10.1080/00958979908023062 https://www.doi.org/10.1007/bf00139309 https://doi.org/10.5281/zenodo.5816598 https://www.doi.org/10.1039/d2ra05233d https://www.doi.org/10.2298/jsc120725154s https://www.doi.org/10.1255/ejms.885 https://www.doi.org/10.1107/s0567740881004743 https://www.doi.org/10.1021/ic50074a050 https://www.doi.org/10.1016/0022-1902(61)80493-4 https://www.doi.org/10.1016/s0020-1693(00)95010-2 {new 4-aminoquinolines as moderate inhibitors of p. falciparum malaria} j. serb. chem. soc. 86 (2) 115–123 (2021) original scientific paper jscs–5408 115 new 4-aminoquinolines as moderate inhibitors of p. falciparum malaria igor opsenica1#, milica selaković2#, mikloš tot1, tatjana verbić1#, jelena srbljanović3, tijana štajner3, olgica djurković djaković3 and bogdan šolaja1,4*# 1university of belgrade – faculty of chemistry, studentski trg 16, p.o. box 51, 11158 belgrade, serbia, 2innovative centre, faculty of chemistry, ltd. studentski trg 12–16, 11158 belgrade, serbia, 3institute for medical research, university of belgrade, dr. subotića 4, p.o. box 39, 11129 belgrade, serbia and 4serbian academy of sciences and arts, knez mihailova 35, 11000 belgrade, serbiа (received 25 december 2020, revised 17 january, accepted 18 january 2021) abstract: synthesis of novel aminoquinoline derivatives has been accomplished and their activity against malaria strains has been examined. the compounds showed moderate in vitro antimalarial activity against two p. falciparum strains, 3d7 (cq susceptible clone) and dd2 (cq resistant clone). three aminoquinolines were further examined for antimalarial efficacy in a mouse model using a modified thompson test. in this model, mice were infected with p. berghei-infected red blood cells, and drugs were administered orally. antimalarial 3 was found toxic at a dose of 320 (mg/kg)/day in 3/6 mice, however, 2/6 mice of the same group survived through day 31, and one of them was cured. keywords: quinoline; bromobenzyl derivatives; antimalarials; β-hematin inhibitory activity. introduction climate change and global warming are closely associated with growing threat from infectious diseases. increase in global temperatures favors the development and spread of tropical diseases and the associated vector organisms.1 warming will contribute to area expansion populated with malaria-transmitting mosquitoes. with 300–500 million clinical cases and nearly one million deaths every year, malaria is a major global public health problem.2 among the five known species of the plasmodium genus that cause human malaria (plasmodium species: p. falciparum, p. ovale, p. vivax, p. malarie and p. knowlesi), p. falci * corresponding author. e-mail: bogdan.solaja@sanu.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc201225005o ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 116 opsenica et al. parum is th-e major cause of mortality. malaria parasites use hemoglobin (hb) as a source of nutrients, and after digestion of host hb in the acidic food vacuole (fv) of p. falciparum amino acids and heme are released. the parasites use hb amino acids as building blocks for their own protein synthesis. the heme moiety ferriprotoporphyrin-ix is toxic to the host, and the parasite has developed several mechanisms of detoxification: sequestration of heme into insoluble hemozoin,3 and degradation of heme with hydrogen peroxide4,5 and glutathione-mediated mechanisms6 in the fv and cytosol, respectively. the clinical symptoms of malaria appear during the asexual intraerythrocytic stage; consequently, efforts to develop an effective drug have mainly focused on this stage of infection.7 malaria has been known for centuries, and there is a lot of information regarding transmission, prevention and treatment of the disease. despite this, it is still one of the most widespread infectious diseases in the world.8 one of the main causes is development of widespread drug resistance9,10 especially to chloroquine (cq), the most widely used antimalarial (fig. 1).11,12 most of 4-amino-7-chloroquinoline based compounds (acqs) act as inhibitors of hemozoin formation,8 but it is also reported that they may act as inhibitors of oxidative13 and gluthatione -mediated14 heme degradation. resistance to acqs is closely related to reduced accumulation of drugs caused by mutations in drug transporters (pfcrt, pgh1, and pfmrp).15,16 the synthesis and development of novel acq-based derivatives has been the subject of extensive research. efforts have focused mostly on three subclasses: bisquinolines, sidechain modified 4-aminoquinolines, and hybrid 4-aminoquinolines.17,18 fig. 1. structures of cq and potent antimalarials. during our studies a variety of acq-based compounds were identified as potential antimalarial agents.19–22 recently, we synthesized a series of simplified analogues with the acq component attached via an ethylene or propylene linker to different benzyl or pyridyl-groups (fig. 1).23 the synthesized compounds showed promising antimalarial activities, and exhibited better potencies against cq susceptible (cqs) and cq resistant (cqr) strains as compared to cq and mefloquine. the structure–activity data indicated that a substitution on the benzene ring had a significant influence on the antimalarial activity. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 4-aminoquinolines as inhibitors of malaria 117 in order to better understand the possible influence of bromine substituents on the antimalarial activity, we synthesized ten new aminoquinoline derivatives and here we analyze their activity (scheme 1). scheme 1. synthesis of aminoquinoline derivatives 4–13. in addition, we have synthesized four hybrid derivatives (fig. 2) that include previously established antimalarial pharmacophores-aminoquinoline24 and tetrahydroimidazo[1,2-a]pyrazine.12 fig. 2. structures of hybrid derivatives. experimental for the sake of journal space the full experimental details for the synthesis of tested compounds are given in the supplementary material to this paper. in vitro antimalarial activity in vitro cultures of p. falciparum, chloroquine-sensitive 3d7 and chloroquine-resistant dd2 strains were maintained as described previously.25 for drug assays, parasites were synchronized with 5 % sorbitol and ring-stage parasites were seeded in 96-well plates at a 2 % parasitemia and 0.75 % hematocrit. the compounds were initially dissolved in dmso at a concentration of 50 mm and further dilutions were made in complete culture medium (final dmso concentration was ≤0.2 %). after an initial screen at 0.5 µm concentration, compounds that inhibited parasite growth for at least 50 % were further titrated to obtain ic50 and ic90 values at eight two-fold dilutions (3 independent experiments were performed for each compound, each with 3 replicates per condition). control experiments using chloroquine and/or artemisinin were performed in parallel with the tested compounds. parasite inhibition was assayed after 48 h of incubation in the presence of a drug by the colorimetric ldh assay. the test is based on the evaluation of plasmodial lactate dehydrogenase (pldh) activity and was performed according to the previously described method.26 ic50 and ic90 values were obtained using a sigmoidal dose–response model with the variable slope fitted to results using graphpad prism. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 118 opsenica et al. in vivo antimalarial activity antimalarial activity of novel compounds was tested in mice infected with plasmodium berghei anka strain using a modified version of the thompson test.27 prior to antimalarial efficacy experiments, all compounds were tested for toxicity. female c57bl/6 mice aged 12 to 14 weeks and weighing 19–21 g were used. mice were housed at 5 to 6 animals per cage at the institute for medical research animal facility under a natural photo period and were offered drinking water and standard feed ad libitum. mice were infected intraperitoneally (i.p.) with 250 μl of a pbs suspension containing 106 parasitized erythrocytes from the peripheral blood of a donor mouse. compounds were suspended in 0.5 % hydroxyethylcellulose–0.1 % tween 80 and administered orally at designated doses in a volume of 200 μl once a day (on day 3, 4 and 5 post-infection (p.i.)). mice were monitored daily and any clinical symptoms (e.g., ruffled fur, decreased locomotion, lethargy, loss of appetite, lacrimation, salivation, diarrhoea, convulsions and weight loss) were noted. parasitaemia was monitored by microscopic examination of giemsa-stained peripheral blood smears using mouse tail blood twice a week, starting immediately before the initiation of treatment. the first time point served to check the efficacy of experimental infections, whereas all later points served to monitor compound efficacy. mice were observed for 30 days from the day of infection. cure was defined as the survival of treated mice with parasite clearance at 31 days p.i. for toxicity experiments, naive mice were treated according to the described protocol of compound administration. mice were observed daily during a period of 30 days after the first day of drug administration. all animal studies were approved by a local (institute for medical research) ethics committee. in vivo experiments were approved by the veterinary directorate of the ministry of agriculture and environmental protection of serbia (decision no. 323-07-02444/2014 -05/1). β-hematin inhibitory activity assay. the inhibition of β-hematin formation is expressed as the molar equivalent of compound, relative to hemin, that inhibits β-hematin formation by 50 % and determined by slightly modified bhia assay introduced by parapini et al.28 briefly, as described previously,29 50.0 µl of 16 mm solution of hemin in dmso was distributed to 1.5 ml centrifuge tubes; compound dissolved in dmso (ccomp≈80 mm) was added to hemin in doses ranging from 0.125 to 5 mole equivalents (pure dmso was added to control samples). 100.0 µl of ultrapure water was added to each tube, and β-hematin formation initiated by the addition of 200.0 µl of 8 m acetate buffer (ph 5.2). the final concentration of dmso per tube was kept constant at 25 %. tubes were incubated at 37.0±0.1 °c for 18 h and then centrifuged. the remaining pellet was resuspended in 0.500 ml dmso to remove unreacted hematin. tubes were then centrifuged again, dmso-soluble fraction removed and the pellet, consisting of a pure precipitate of β-hematin, dissolved in 1.000 ml 0.1 m naoh. 10.0 µl aliquots were transferred to 96-well microplate and diluted with 190.0 µl 0.1 m naoh (200.0 µl of 0.1 m naoh was used as a blank). the absorbance was measured at 405 nm, with correction at 670 nm (lkb 5060-006 micro plate reader, vienna, austria). experiments were performed in duplicate. a calibration curve of hemin dissolved in 0.1 m naoh was made in the che range of 0.4×10-5–4.0×10-5 m, samples were diluted, if needed, to fit the calibration curve range. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 4-aminoquinolines as inhibitors of malaria 119 results and discussion synthesis all compounds were synthesized in high yield by coupling commercially available aryl aldehydes with aminoquinolines via reductive amination using sodium borohydride and acetic acid (scheme 1). each of the final compounds was then purified by dry-flash chromatography, and tested as > 95 % pure based on m.a. and/or hplc analysis. antimalarial activity the in vitro antimalarial activity of the synthesized compounds was evaluated against the cqs 3d7 and the cqr dd2 p. falciparum strains, using cq and artemisinin (art) as positive controls (table i). eight compounds (1–3, 5, 6, 8, 11 and 12) were quite potent, with ic50 values within 19–48 nm range and appeared more active against the cqr dd2 strain than cq. among them, derivatives 3 and 6 were 5-fold more active than cq.30 in addition, 2 and 6 were as active as cq and art against the cqs 3d7 strain (2: ic50 = 22.05 nm; 6: ic50 = 14.02 nm). the results indicate that the length of the methylene chain affects the antiplasmodial activity. the potency of our compounds against the cqs 3d7 strain was increased up to c4 spacer, and further chain lengthening resulted in a decrease of the activity (7–9 vs. 10). on the other hand, the substitution of chlorine atom with hydrogen at the 7-position of the quinoline ring did not significantly affect the in vitro antimalarial activity against the cqs strain (9: ic50 = 30.04 nm; 12: ic50 = 25.45 nm). table i. in vitro antimalarial activity, ic50 / nm (geometric mean) compound strain 3d7a dd2b 1 32.20c 40.82c 2 22.05c 37.97c 3 30.11c 18.81c 4 n.d. (>500)d n.d.d 5 43.50 34.04 6 14.02 26.07 7 41.08 n.d.e 8 45.75 35.22 9 30.04 n.d.d 10 61.66 n.d.d 11 291.10 48 12 25.45 30.72 13 76.22 216.71 cq 16.17(5)e 129.18(5)e art 13.09(3)e 15.93(2)e ap. falciparum cq susceptible clone; bp. falciparum cq resistant clone; ctaken from reference25; dn.d. – not determined; enumber of replicates ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 120 opsenica et al. in order to get an initial insight into the compound’s antimalarial mechanism of action, antimalarials 2, 3 and 8 were submitted to bhia.28 the results showed that 3 and 8 interfered with hematin polymerization with the same potency as cq (3: ic50 = 1.40; 8: ic50 = 0.81 vs. cq: ic50 = 1.23), indicating the same mechanism of action. on the other hand, compound 2 (without bromine attached to benzene ring) showed significantly lower bhia (2: ic50 = 3.46). this difference in blocking the hemozoin formation requires further research, and we will pay full attention to this issue in the near future. reported results of in vitro antimalarial activity for 2 and 3 showed that these compounds are good candidates for in vivo testing. additionally, low toxicities to human liver carcinoma cell line hepg2 and high metabolic stability in mouse and human liver microsomes of antimalarial 2 further supported antimalarial efficacy studies in a mouse model.23 the bromo derivatives 3 and 8 were examined for antimalarial efficacy. a modified thompson test model of malaria was used to determine the blood schizonticidal efficacy of the test compounds. mice were infected with p. berghei-infected red blood cells. the test compounds were administered orally at doses of 80, 160 and 320 (mg/kg)/day on days 3, 4, and 5 after parasite inoculation; infection was confirmed by positive blood smear results for all the mice on study day 3 (prior to drug administration). at a dose of 320 (mg/kg)/day, compound 3 appeared active, since it afforded survival of 2/6 mice, with parasite clearance in one and parasite recrudescence in the other mouse. however, these results should be taken with caution since 3 experimental animals died on day 7 (d7), indicating no activity. of the two mice that survived to d31, one had a positive blood smear and was therefore not cured, but the other one (1/6 tested) was cured based on a negative blood smear as well as absence of detection of p. berghei dna by pcr (performed as previously described31) of blood, liver and spleen. at concentrations of 160 and 80 (mg/kg)/ /day only suppression activity was observed. no acute toxicity of compound 3 was detected, the mice that died or were euthanized prior to day 31 showed typical gross lesions such as gray swollen liver, dark spleen, and pale emaciated carcass (like the control mice group), which indicated fatal malaria infection. antimalarial 8 was tested at doses of 160 and 80 (mg/kg)/day. at 160 mg/kg/day it was found toxic, and at the lower concentration only suppression activity was observed, table ii. the hybrid derivatives 14–17 did not meet our expectations as they exhibited extremely poor in vitro activity, of ic50 > 1000 nm, against the cqr strain. however, their bhia (ic50 ≈ 1) indicated that they may interfere with hematin polymerization to the same extent as cq (cq: ic50 = 1.23; 14: ic50 = 0.95; 15: ic50 = 1.19; 16: ic50 = 0.72; 17: ic50 = 0.96). the inconsistency between in vitro activity and bhia could indicate the inability of hybrid derivatives to accumulate in the food vacuole of the parasite. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 4-aminoquinolines as inhibitors of malaria 121 table ii. in vivo antimalarial activity; groups of four, five, or six, p. berghei (anka strain) infected mice were treated p.o. once per day on days 3–5 post infection with aminoquinolines suspended in 0.5 % hydroxyethylcellulose-0.1 % tween 80. mice alive on day 31 with no parasites as detected by pcr are considered cured compd. dose, (mg/kg)/day mice dead/day died mice alive on day 31/total mean survival time, day 2b 160c 1/13, 1/14, 1/15, 1/17 1/5 18 80 1/12, 2/13, 1/14, 1/28 0/5 16 3 320 3/7, 1/22 2/6 18 160c 1/15, 1/16, 2/17, 1/21, 1/24 0/6 18 80 1/12, 1/13, 2/14 0/4 13 8 80 2/14, 2/15, 1/19, 1/26 0/6 17 infected controls 0 all mice died on day 6–8 ataken from reference22 conclusions in this manuscript we reported our findings on the antimalarial activity of simple aminoquinoline tethered benzyl derivatives with introduced bromines in positions c3 and c4. although the synthesized compounds showed acceptably low in vitro activities, the antimalarial 3 was found toxic in vivo at a dose of 320 (mg/kg)/day in 3/6 mice, however, 2/6 mice of the same group survived through day 31, and one of them was cured. the detected toxicity of compound 3 indicates that the basic structure of presented aminoquinolines has yet to be improved in the near future. supplementary material additional data are available electronically at the pages of journal website:.https:// //www.shd-pub.org.rs/index.php/jscs/index, or from the corresponding author on request. acknowledgments. this research was financially supported by the ministry of education, science and technological development of serbia (contract no: 451-03-68/2020-14/200168 and 451-03-68/2020-14/200288), and serbian academy of sciences and arts, project f-80. и з в о д нови деривати 4-аминохинолина као умерени инхибитори паразита plasmodium falciparum игор опсеница1, милица селаковић2, миклош тот1, татјана вербић1, јелена србљановић3, тијана штајнер3, олгица ђурковић ђаковић3 и богдан шолаја1,4 1универзитет у београду-хемијски факултет, студентски трг 16, 11158 београд, 2иновациони центар хемијског факултета, студентски трг 12–16, 11158 београд, 3институт за медицинска истраживања универзитета у београду, др. суботића 4, 11129 београд и 4српска академија наука и уметности, кнез михаилова 35, 11000 београд синтетисани су нови деривати аминохинолина и испитана је антималаријска активност једињења на два соја p. falciparum, хлорокин осетљивом клону 3d7 и хлорокин резистентом клону dd2. у наставку истраживања испитана је in vivo активност деривата који су у in vitro условима испољили највећу активност. применом аминохинолина 3 у дози 320 (mg/kg)/дан преживела су два од шест мишева, при чему је један миш излечен. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 122 opsenica et al. међутим, при истој дози у 3/6 тестиране животиње исказана је токсичност аминохинолина 3, што указује да је потребно даље истраживање на побољшању структуре овог типа једињења. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice synthesis of novel fluorinated 1,5-benzothiazepine derivatives and their biological evaluation as anticancer and antibacterial agents j. serb. chem. soc. 87 (10) 1109–1116 (2022) original scientific paper jscs–5581 published 1 august 2022 1109 synthesis of novel fluorinated 1,5-benzothiazepine derivatives and their biological evaluation as anticancer and antibacterial agents sonal r. bhabal, sarfaraz f. shaikh, ishita p. yellapurkar, ganesh s. pavale and mucheli m. v. ramana* department of chemistry, university of mumbai, santacruz (e), mumbai 400 098, india (received 28 april 2021, revised 28 april, accepted 10 may 2022) abstract: a series of novel fluorinated 1,5-benzothiazepine derivatives were synthesized, characterized and evaluated for in vitro anticancer and antibacterial activity. the in vitro anticancer activity of the synthesized compounds 4a–h was evaluated against four human cancer cell lines namely lung (a549), breast (mcf-7), liver (hepg2) and prostate (pc-3). compounds 4c, 4d, 4g and 4h exhibited good activity with gi50 <10 µg ml-1 against all four human cancer cell lines which was comparable to standard drug adriamycin. additionally, antibacterial activity of synthesized compounds was estimated using resazurin microtiter assay (rema) and compared with standard drug ampicillin. among the synthesized compounds, 4c, 4d, 4g and 4h showed good antibacterial activity and all the synthesized compounds were found to be more active towards gram negative than gram positive bacteria. these promising results obtained from in vitro anticancer and antibacterial activity, inferred that the synthesized compounds are capable of being anticancer as well as antibacterial agents. keywords: in vitro; lung cancer cell line; breast cancer cell line; liver cancer cell line; prostate cancer cell line. introduction one of the major world problems is cancer; due to the increased cancer cases and deaths. in the year 2020, it was anticipated that there will be 19.3 million new cancer cases and 10.0 million deaths worldwide.1 despite the fact that chemotherapy is most commonly used to treat cancer, the failure of existing chemotherapeutics to treat cancer highlights the need for new chemical entities to be developed.2 additionally, chemotherapy in cancer treatment is usually associated with various side effects and appearance of resistance.3 moreover, despite * corresponding author. e-mail: mmvramana@yahoo.co.in https://doi.org/10.2298/jsc210428041b ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1110 bhabal et al. of progressive development in cancer chemotherapy, there are still insufficient cytotoxic agents that act selectively to cancer cells. infections are one of the reasons for compromised immunity among cancer patients. they make patient vulnerable which leads to disturbance in treatment.4 infectious diseases put public life in jeopardy and are responsible for a large number of deaths globally. the major issue is the resistance of pathogenic microorganisms to the available antimicrobial agents, which makes it difficult to treat with conventional antibiotics and force clinicians to rely on restricted options.5 therefore, the development of both new anticancer and antimicrobial entities is a necessity. in the last decades, owing to their structural properties and wide range of biological activities, benzothiazepines have piqued the interest of researchers.6,7 1,5-benzothiazepine scaffold has been used as anticancer,8–11 antimicrobial,11–14 anti-inflammatory,14 anticonvulsant15 and anti-hiv16,17agent. also, 1,5-benzothiazepine scaffold is reported as acetylcholinesterase inhibitor,18 butyrylcholinesterase inhibitor,19 vrv-pl-8a and h+/k+ atpase inhibitor.20 clentiazem, diltiazem, thiazesim and quetiapine are among the commercially available drugs which contain the 1,5-benzothiazepine skeleton. heterocycles containing fluorine atoms have a wide range of applications in pharmaceutical industry.21 the presence of a fluorine atom alters certain physicochemical properties such as basicity, lipophilicity, bioavailability as well as binding affinity of a drug molecule to the target protein.22 ciprofloxacin (antibiotic), fluconazole (antifungal), 5-fluorouracil (anticancer), paroxetine (antidepressant), linezolid (antibacterial), favipiravir (antiviral) and midazolam (sedative) are some of the marketed drugs that contain fluorine atom. prasada rao et al. reported compounds with fluorinated 1,5 benzothiazepine skeleton as anti-cancer agents.23 similarly, upreti et al. reported 8-fluoro-1,5-benzothiazepine as promising anti-aids agent.24 a series of 1,5-benzothiazepines with fluorine and 4fluorophenyl groups have also been reported for the treatment of cancer metastasis.25 based on the wide spectrum of biological activities, 1,5-benzothiazepines are good candidate and could be taken in consideration as promising anticancer and antimicrobial agents. herein we report the synthesis, anticancer and antibacterial activity of fluorinated 1,5-benzothiazepines. anticancer activity was evaluated in vitro using four different cell lines namely human lung cancer cell line (a549), human breast cancer cell line (mcf-7), human liver cancer cell line (hepg2) and human prostate cancer cell line (pc-3). similarly, antibacterial activity was performed using two gram-positive strains and two gram-negative strains. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ biological activity of fluorinated 1,5-benzothiazepine 1111 experimental materials and method all reagents and chemicals were of analytical grade, procured from sigma aldrich (india), and used without further purification. melting points are uncorrected and were recorded on centrofix syndicate mp apparatus. merck silica gel 60 f254 tlc plates were used to monitor the reaction. ftir spectra were recorded on perkin elmer, frontier equipment with atr. 1h(300 mhz) and 13c-nmr (75 mhz) were recorded on bruker avance ii using tms as the internal standard in cdcl3 and dmso-d6. esi mass spectra were recorded on ab sciex 3200 qtrap mass spectrometer. elemental analysis (chns) was carried out on model ea300, euro vector, italy. staphylococcus aureus (atcc 25923), bacillus subtilis (atcc 6633), salmonella typhi (atcc 23564) and escherichia coli (atcc 25922) cultures were procured from national chemical technology, pune, maharashtra and were used for antibacterial assay. analytical and spectral data are given in the supplementary material to this paper. chemistry synthesis of α,β-unsaturated ketone derivatives (3a–h).26 a mixture of 3,4-difluorobenzaldehyde derivative (5 mmol) and acetophenone derivative (6 mmol) were dissolved in 15 ml ethanol, then naoh 40 % solution (5 ml) was added dropwise. the temperature of reaction was maintained below 10 °c and the reaction mixture was stirred until precipitation of solid. the precipitate was then filtered, washed several times with cold water and recrystallized from aqueous ethanol. synthesis of 1,5-benzothiazepine derivatives (4a–h), scheme 1. a mixture of α,β-unsaturated ketone (5 mmol) and 2-aminothiophenol (5 mmol) were taken in methanol (10 ml) containing catalytic amount of glacial acetic acid. the reaction mixture was allowed to reflux until completion as indicated by tlc. after completion, the reaction was allowed to cool to room temperature and the solid obtained was filtered, washed several times with methanol and recrystallized using ethanol to afford yellow solid. scheme 1. synthetic pathway of fluorinated 1,5-benzothiazepine derivatives. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1112 bhabal et al. biology in vitro anticancer activity. the sulforhodamine b (srb) assay was used to test the anticancer activity of the synthesized derivatives against four human cancer cell lines namely lung (a549), breast (mcf-7), liver (hepg2) and prostate (pc-3) cancer.27 the cell lines were grown in rpmi 1640 medium for 24 h before being inoculated into 96-well plates and incubated at 37 °c. after that, the cells were fixed with 10 % trichloroacetic acid (tca) and tested with drugs at four dose levels (10, 20, 40, and 80 µg ml-1) with doxorubicin serving as a control. after adding the compounds, the plates were incubated for 48 h until the assay was terminated with the addition of cold tca. tca (30 %) was used to fix the cells, which were then incubated at 4 °c for 1 h before being stained with srb solution for 20 min. further, the excess dye was discarded by washing with 1 % acetic acid and air dried. the protein-bound dye was then eluted with a 10mm tris base, and the absorbance was measured at 540 nm using a plate reader. growth was calculated and expressed as the ratio of average absorbance of the test well (ti) to the average absorbance of the control wells (c): growth inhibition = 100(ti/c) (1) where c = control growth and ti = test growth in the presence of drug at the four concentration levels. the experiment was done in triplicate and the average values were plotted as control growth versus drug concentrations. antibacterial assay the antimicrobial activity of synthesized compounds was investigated using the resazurin microtiter assay (rema) in aseptic conditions using a 96 well microtitre plate as described previously.28 50 μl solution of test material dissolved in 2 % dmso was added after filling all the wells of the microtiter plate with 50 μl of nutrient broth. two-fold serial dilution was achieved by transferring 50 μl test material from the top well of the first row to the following wells in the next row of the same column and resulted in a graded sequence of concentrations (500, 250, 125, 62.5, 31.25, 15.63, 7.81 and 3.90 μg ml-1). after that, 50 μl of bacterial suspension were added to each well, resulting in a final concentration of 0.5 mcfarland standard cfu ml-1. later, all plates containing test materials were incubated for 24 h at 37 °c. after 24 h, each well was given 0.2 % resazurin, and a visual change in colour was observed in the wells. the transition from purple to pink/colorless was taken as a positive. the mic value for that particular sample was recorded, as the lowest concentration in the column containing the sample at which no color shift occurred, and compared to the standard drug ampicillin. results and discussion chemistry synthesis of fluorinated 1,5-benzothiazepines 4a–h followed the path shown in scheme 1. the compounds were synthesized by reaction of α,β-unsaturated ketone with 2-aminothiophenol in presence of catalytic amount of glacial acetic acid. the synthesized novel compounds 4a–h were verified by various techniques like ir, nmr (1h and 13c), ms and elemental analyses (chn). ftir spectra of the synthesized compounds 4a–h showed appearance of c–h in the range of 2844–3105 cm–1, a characteristic band in the range of 1595– –1608 cm–1 confirms presence of c=n bond in the synthesized compound, also presence of band in range of 1311–1322 cm–1 indicates presence of c–n. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ biological activity of fluorinated 1,5-benzothiazepine 1113 presence of band in the range of 681–688 cm–1 indicates formation of c–s–c bond. 1h-nmr spectra of synthesized compounds shows presence of three characteristics peaks apart from aromatic protons (δ 8.12–6.50 ppm). the three peaks in the aliphatic region are due to ch2 and ch. two protons of ch2 are diastereotopic in nature (ha and hb), ha shows triplet at δ 2.88–2.99 ppm with jab value 12.4–12.7 hz and the other hb along with ch (hx) shows doublet of doublet at δ 3.19–3.28 ppm with jbx in range12.9–13.0 hz; jab in range 4.8–5.1 hz and δ 4.88–4.99 ppm with jax in range 11.8–12.5 hz; jab in range 4.6–5.1 hz, respectively, due to abx system. the 13c-nmr showed characteristics peak at δ 37–38 ppm denoting ch and at δ = 59 ppm confirming presence of ch2. hence, the interpreted data obtained from the spectra corroborated with the structure of synthesized compound. for further verification, esi-ms was performed, m/z values obtained were in good agreement with measured mass. further elemental analysis confirmed the purity of the synthesized compounds as the experimental composition was found to be similar with the theoretical composition. biology in vitro anticancer activity. the in vitro anticancer activity of the synthesized compounds 4a–h was assessed using sulforhodamine b (srb) assay with adriamycin as standard drug. the results were described in terms of gi50 (concentration that reduces total cell growth by 50 values) and are delineated in table i. the results concluded that among the synthesized compounds, compound 4c, 4d, 4g and 4h exhibited good activity with gi50 < 10 µg ml–1 against all four cell lines, while rest of the compounds exhibited moderate to poor activity with gi50 > 10 µg ml–1. table i. in vitro anticancer activity (gi50 / µg ml-1); gi50 = concentration of drug causing 50 % inhibition of cell grow. for pure compounds, gi50 value ≤ 10 µg ml-1 is considered to demonstrate activity compound cell line a549 mcf-7 hep g2 pc-3 4a 68.2 68.6 57.6 >80 4b 27.8 48.2 32.6 28.1 4c <10 <10 <10 <10 4d <10 <10 <10 <10 4e 22.1 <10 16.9 <10 4f 32.3 35.1 33.4 11.6 4g <10 <10 <10 <10 4h <10 <10 <10 <10 adriamycin <10 <10 <10 <10 from the structure activity relationship, the activity of heterocyclic and halogenated compounds was outstanding when compared to other substituents. the ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1114 bhabal et al. compounds were found to be in the order: –f group > –cl group > –br group > > –ome group > –me group > –h denoting that the compounds having electronegative groups showed excellent activity. antibacterial assay. the antibacterial activity of the synthesized compounds 4a–h were evaluated against two gram-positive (staphylococcus aureus and bacillus subtilis) and two gram-negative (escherichia coli and salmonella typhi) bacteria using rema. ampicillin was taken as positive control. the antibacterial potential of the synthesized compounds were assessed by minimum inhibitory concentration (mic) values and are displayed in table ii. result obtained indicates that all the synthesized compounds are potent antibacterial agents. among which, compound 4c, 4d, 4g and 4h exhibited excellent activity w.r.t the standard (ampicillin) against all the bacterial strains inferring that hetero and halogenated substances show excellent results. the results outlined suggested that the synthesized compounds are more active towards gram-negative strain than gram-positive strain. table ii. in vitro bacterial activity using rema method (mic / µg ml-1) compound strain gram-positive gram-negative s. aureus b. subtilis e. coli s. typhi 4a 62.5 62.5 62.5 62.5 4b 62.5 125 62.5 62.5 4c 31.25 31.25 15.625 31.25 4d 15.625 15.625 15.625 15.625 4e 62.5 125 62.5 62.5 4f 62.5 125 62.5 62.5 4g 15.625 31.25 15.625 15.625 4h 15.625 15.625 31.25 15.625 ampicillin 31.25 31.25 15.625 31.25 conclusion to conclude, we synthesized series of fluorinated 1,5-benzothiazepine derivatives 4a–h. the synthesized compounds were evaluated for in vitro anticancer and antibacterial activity. the in vitro anticancer activity was performed using four human cancer cell lines namely a549, mcf-7, hepg2 and pc-3. compounds 4c, 4d, 4g and 4h exhibited excellent activity with gi50 <10 µg ml–1 against all four cell lines which is comparable to standard drug adriamycin. further, the synthesized compounds were subjected to antibacterial activity using resazurin microtiter assay (rema) with ampicillin as standard drug. the compounds 4c, 4d, 4g and 4h exhibited elegant antibacterial activity and the synthesized compounds were found to be more active towards gram-negative than ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ biological activity of fluorinated 1,5-benzothiazepine 1115 gram-positive bacteria. hence, the synthesized fluorinated 1,5-benzothiazepine derivatives have potential to be an anticancer as well as antibacterial agents. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/10712, or from the corresponding author on request. acknowledgements. we are thankful to microanalytical laboratory, university of mumbai, for providing characterization facilities. we are also thankful to advanced centre for treatment research and education in cancer (actrec) anti-cancer drug screening facility (acdsf), kharghar, navi mumbai, for providing anticancer activity. и з в о д синтеза нових флуорисаних деривата 1,5-бензотиазепина и њихова биолошка процена као антиканцерогених и антибактеријских средстава sonal r. bhabal, sarfaraz f. shaikh, ishita p. yellapurkar, ganesh s. pavale и mucheli m. v. ramana department of chemistry, university of mumbai, santacruz (e), mumbai 400 098, india синтетисана је серија флуорованих деривата 1,5-бензотиазепина, једињења су окарактерисана и испитана је њихова in vitro антиканцерска и антибактеријска активност. in vitro антиканцерска активност једињења 4a–h испитана је према ћелијским линијама хуманог канцера плућа (a549), груди (mcf-7), јетре (hepg2) и простате (pc-3). једињења 4c, 4d, 4g и 4h имају добру активност, која износи gi50 < 10 μg ml -1 према свим испитиваним ћелијским линијама, и блиска је активности стандардног лека aдриамицина. такође, антибактеријска активност једињења је испитана употребом ресазурин микротитар есеја (resazurin microtiter assay, rema) и добијене вредности су упоређене са активношћу стандардног лека aмпицилина. од синтетисаних једињења, једињења 4c, 4d, 4g и 4h показују добру антибактеријску активност и утврђено је да су сва синтетисана једињења активнија према грам-негативним него према грам-позитивним бактеријама. резултати добијени из in vitro антиканцерске и антибактеријске активности су охрабрујући и указују да би синтетисана једињења могла да буду добри антиканцерски и антибактеријски агенси. (примљено 28. априла 2021, ревидирано 28. априла, прихваћено 10. маја 2022) references 1. h. sung, j. ferlay, r. l. siegel, m. laversanne, i. soerjomataram, a. jemal, f. bray, ca. cancer j. clin. 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(https://doi.org/10.3390/molecules191221473) 27. v. vichai, k. kirtikara, nat. protoc. 1 (2006) 1112 (https://doi.org/10.1038/nprot.2006.179) 28. s. f. shaikh, p. p. dhavan, p. r. singh, s. p. vaidya, b. l. jadhav, m. m. v. ramana russ. j. bioorg. chem. 47 (2021) 571 (https://doi.org/10.1134/s1068162021020242). ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ @article{bhabal2022, author = {bhabal, sonal r. and shaikh, sarfaraz f. and yellapurkar, ishita p. and pavale, ganesh s. and ramana, mucheli v.}, journal = {journal of the serbian chemical society}, title = {{synthesis of novel fluorinated 1,5-benzothiazepine derivatives and their biological evaluation as anticancer and antibacterial agents}}, year = {2022}, issn = {1820-7421}, month = {may}, number = {10}, pages = {1109--1116}, volume = {87}, abstract = {a series of novel fluorinated 1,5-benzothiazepine derivatives were synthesized, characterized and evaluated for in vitro anticancer and antibac­terial activity. the in vitro anticancer activity of the synthesized compounds 4a–h was evaluated against four human cancer cell lines namely lung (a549), breast (mcf-7), liver (hepg2) and prostate (pc-3). compounds 4c, 4d, 4g and 4h exhibited good activity with gi50 <10 µg ml-1 against all four human cancer cell lines which was comparable to standard drug adriamycin. addition­ally, antibacterial activity of synthesized compounds was estimated using res­azurin microtiter assay (rema) and compared with standard drug ampicillin. among the synthesized compounds, 4c, 4d, 4g and 4h showed good antibac­terial activity and all the synthesized compounds were found to be more active towards gram negative than gram positive bacteria. these promising results obtained from in vitro anticancer and antibacterial activity, inferred that the synthesized compounds are capable of being anticancer as well as antibacterial agents.}, doi = {10.2298/jsc210428041b}, file = {:d\:/onedrive/mendeley desktop/bhabal et al. 2022 synthesis of novel fluorinated 1,5-benzothiazepine derivatives and their biological evaluation as anticancer and.pdf:pdf;:01_10712_5581.pdf:pdf}, keywords = {breast cancer cell line, liver cancer cell line, lung cancer cell line, prostate cancer cell line}, publisher = {serbian chemical society}, url = {https://www.shd-pub.org.rs/index.php/jscs/article/view/10712}, } some nitrogen-rich heterocycles derivatives as potential explosives and propellants: a theoretical study j. serb. chem. soc. 80 (6) 687–695 (2016) udc 66.017+537.872+546.17+547.7: jscs–4877 662.215.1:662.2 original scientific paper 687 some nitrogen-rich heterocycles derivatives as potential explosives and propellants: a theoretical study dany frem* frem co., beirut, lebanon (received 18 september, revised 13 december, accepted 21 december 2015) abstract: four types of nitrogen-rich heterocycles substituted with –no2, –nhno2 and –c(no2)3 explosophoric groups were explored as potential explosives and propellants materials. the calculated crystal density 0( )ρ and the condensed phase entalpy change (heat) of formation (δfh) for each of the twelve structures investigated shows that all these derivatives possess high 0ρ (1.834–1.980 g cm-3) and δfh (605–2130 kj kg -1) values. interesting properties such as detonation velocity (d), pressure (p) and specific impulse (isp) were calculated using the kamlet–jacobs method and ispbkw thermochemical code. detonation velocity and pressure in excess of 8.44 km s-1 and 32.87 gpa was obtained in all cases. furthermore, trinitromethyl substituted derivatives show performance exceeding that of hmx with an estimated d and p in the ranges of 9.32–9.72 km s-1 and 40.61–43.82 gpa, respectively. some –no2 and –nhno2 substituted derivatives were shown to be impact-insensitive while retaining good detonation performance and thus are regarded as potential replacement for current rdx-based explosives. finally, the calculated specific impulse (isp between 248 and 270 s) of all investigated derivatives indicate that these energetic materials can be considered as possible ingredient in future rocket propellant compositions. keywords: energetic materials; detonation performance; specific impulse; gurney velocity. introduction the search for new energetic materials (explosives, propellants and pyrotechnics) is an ongoing task all over the world.1–5 traditional explosive and propellant formulations relied on carbonaceous backbone (aliphatic and aromatic) based molecules bearing explosophoric groups like nitro (–no2), nitrate ester (–ono2) and nitramine (–nhno2). however, in recent years, much attention has been devoted toward the synthesis of high nitrogen content molecules.6 for example, high heat of formation (δfh) and crystal density ( 0ρ ) has been obtained * correspondence to e-mail: frem.dany@gmail.com doi: 10.2298/jsc150918002f _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 688 frem by the incorporation of nitrogen rich moiety (e.g., 1,2,4-triazole, tetrazole, 1,2,4,5-tetrazine, etc.).7 one interesting energetic material is tkx-50 which has been recently synthesized by a research team in germany.8 tkx-50 displays excellent detonation performance (d = 9.70 km s–1, p = 42.40 gpa) which is the direct result of tkx-50 high crystal density ( 0ρ = 1.88 g cm –3) and positive heat of formation ( fδ h = 447 kj mol –1). furthermore, tkx-50 possess lower impact sensitivity (20 j) compared to common nitramine based explosives (rdx, hmx and cl-20) which are in the range of 4–7.5 j. moreover, high-nitrogen molecules are also expected to be promising propellant candidates. in 2010, zhang et al.9 synthesized a series of 3,4,5-trinitropyrazole-based energetic salts and showed using cheetah 5.010 thermochemical code that 4-amino-4h-1,2,4-triazolium 3,4,5-trinitropyrazolate possess very high specific impulse ( spi = 270 s) a value in line with those found in high performance solid rocket propellant formulations. it is a common practice, however, to perform theoretical studies in order to screen out interesting energetic molecules and this before attempting any synthesis. by this strategy, both time and cost can be reduced significantly and only those structures that show good detonation/propellant performance should be considered for synthesis and further characterization. the goal of this study is to present a series of energetic molecules based on high-nitrogen heterocycles for which the physicochemical, thermodynamic and detonation/propellant properties were predicted. four types of heterocycles (see fig. 1) were investigated, namely tetrasubstituted 12h-pyrimido[4',5':4,5][1,2,3]triazolo[2,1-a]benzotriazol-5-ium (series a), tetrasubstituted 5h,10h-bis[1,2,4]triazolo[1,5-a:1',5'-d]pyrazine (series b), trisubstituted 5h-imidazo[1,2-b:3,4 fig. 1. structures of the proposed heterocycles as new energetic materials. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ nitrogen-rich heterocycles derivatives as explosives and propellants 689 -b']bis[1,2,4]triazole (series c) and disubstituted 2-amino-2h-1,2,3-triazole, 1-oxide (series d). methods of calculation crystal density ρ0( ) and heat of formation ( h)δ f crystal density is of paramount importance in designing energetic materials. it can be shown experimentally11 that for most powerful high explosive detonation velocity (d) is a linear function of 0ρ while p varies with 0 2ρ . in the present study, the simple but reliable group additivity method of ye and shreeve12 was used for the prediction of the crystal density. for 261 explosives containing the elements c-h-n-o-f-s, this approach gives a mean absolute deviation of 0.026 g cm–3 and a mean relative absolute error of 1.5 %. as for fδ h , keshavarz13 devised correlations, eqs. (1) and (2), based on stoichiometry for the calculation of condensed heat of formation for c-h-n-o containing energetic materials: 0 0 0f ec iec decδ 105.0δ 106.6δδ= + −h h h h (1) 0ecδ 32.33 39.49 92.41 63.85= − + −h a b c d (2) where fδ h is in kj mol –1, .0iecδh and .0 decδh are increasing and decreasing energy corrective terms, respectively, while .0ecδh is the predicted energy content on the basis of elemental composition, and a, b, c and d are the number of carbon, hydrogen, nitrogen and oxygen atom, respectively. equation (1) can be used to predict the heat of formation of a large set of energetic molecules including but not limited to polynitro arenes and polynitro heteroarenes. detonation velocity (d) and pressure (p) when an explosive detonates a propagating detonation wave is established inside the charge which travel at a constant velocity (d) determined by a number of factors: crystal density of the explosive, charge diameter, quantity and rate at which the chemical energy is liberated.14 another important performance parameter is the detonation pressure (p) which represents the brisance or the shattering power of an explosive material.15 the well-known kamlet–jacobs method16 was used for the prediction of d and p: 0.5 01.01 (1 1.30 )φ ρ= +d (3) 201.558ρ φ=p (4) φ = n mq (5) where d is in km s–1 and p in gpa, while n and m are the number and molecular weight of gaseous detonation products, respectively, and q is the heat of detonation in j g–1. impact sensitivities in order to be useful in real world applications, an energetic material should possess certain degree of insensitivity toward external stimuli such as electric spark, friction and shock impact. an inexpensive test called the drop weight impact test is performed routinely to measure explosives sensitivity. a 2.5 kg weight is released vertically from a predetermined height onto a sample sandwiched between a striker and an anvil. the value generally reported is a mean height for 50 % probability of causing an initiation 50( )h or “go”. the values of 50( )h for all proposed structures shown in fig. 1 are estimated here following eqs. (6) and (7):17 cnc cnnc 50 44.4167 102.2749 log δ − − − −+ += n n h mw (6) _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 690 frem 46.2923 35.6305 7.7005 7.9425δ = + − +a b c d (7) where 50h is in cm; a, b, c and d are the number of c-h-n-o in a given composition, cnc− −n and cnnc− −n are the number of cnc− − and cnnc− − moieties, mw is the molecular weight in g mol-1. specific impulse (i )sp under right conditions, high explosives can be made to burn (or deflagrate) rather than detonate. the gases generated, produce thrust, the force that propel gun projectiles and missiles. the specific impulse sp( )i is used to characterize the energy efficiency of propellant combustion.18 it has the unit of seconds. the ispbkw code19 is used for the calculation of spi . for a given propellant, the equilibrium composition product is obtained at 6.895 mpa chamber pressure c( )p then the combustion gases are permitted to expand isentropically through the rocket motor nozzle to 0.101 mpa ambient pressure a( )p . as inputs, ispbkw needs the chemical composition of the propellant and the heat of formation obtained at 0 k. results and discussion the results of calculations for the twelve structures of interest (series a, b, c and d) are shown in table i. table i. calculated physiochemical and detonation performance for series a, b, c and d cmpd. 0 ρ g cm-3 fδ h kj mol-1 fδ 'h kj kg-1 ob % d km s-1 p gpa spi a s 50h cm a1 1.950 716 1826 –32.6 8.44 33.12 256 21 a2 1.906 941 2081 –35.4 8.50 33.17 248 25 a3 1.980 788 975 0 9.32 40.68 260 9 b1 1.881 560 1637 –23.4 8.67 34.24 265 69 b2 1.845 613 1524 –27.8 8.55 32.88 255 69 b3 1.949 459 605 6.3 9.39 40.93 255 14 c1 1.888 603 2130 –25.4 8.65 34.13 265 80 c2 1.854 634 1932 –29.3 8.53 32.87 255 79 c3 1.949 540 907 4.0 9.35 40.61 258 17 d1 1.863 220 1157 0 9.17 38.08 267 7 d2 1.834 262 1190 –7.3 9.00 36.37 270 9 d3 1.942 295 741 16.1 9.72 43.82 250 5 rdxb 1.800 62 279 –21.6 8.75 34.70 266c 24d hmxb 1.900 74 250 –21.6 9.10 39.30 266c 26d petnb 1.770 –508 –1607 –10.1 8.30 33.50 265 12d aall values were calculated using the ispbkw code19, otherwise stated; bexperimental detonation velocity (d) and pressure (p) of rdx, hmx and petn were obtained from mader;19 cthe values were obtained from kuo and acharya;20 dimpact sensitivities are obtained from storm21 tetrasubstituted 12h-pyrimido[4',5':4,5][1,2,3]triazolo[2,1-a]benzotriazol-5-ium (series a) the tetraazapentalene ring system represents an interesting building block for insensitive, thermally stable explosive. one such example is the well-known _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ nitrogen-rich heterocycles derivatives as explosives and propellants 691 tacot molecule which possesses decomposition temperature of 410°c one of the highest value recorded for an explosive material.22 series a depicted in fig. 1 exploit the tetraazapentalene system in conjunction with two substituted pyrimidine rings which result in structure having both high nitrogen content (n, 35–50 %) and 0ρ values (1.906–1.980 g cm –3). the highest and lowest density was obtained with structure bearing trinitromethyl –c(no2)3 and nitramino –nhno2 groups, respectively. it is clear that structure a3 possess the lowest fδ h (975 kj kg –1) in its series, however, a3 with a zero oxygen balance )(ob and a 0ρ = 1.980 g cm –3 has the highest calculated detonation performance (d = 9.32 km s–1, p = 40.68 gpa) and also the highest specific impulse ( spi = 260 s). the impact sensitivity of a3 ( 50h = 9 cm) is higher than that of rdx ( 50h = 24 cm) and hmx ( 50h = 26 cm) but is still close to that of petn21 ( 50h = 12 cm) keeping in mind that detonation performance of a3 greatly outperform that of petn19 ( 0ρ = 1.77 g cm –3, d = 8.30 km s–1, p = 33.50 gpa). on the other hand, a1 and a2 possess detonation performance and impact sensitivity similar to rdx; however, a1 and a2 are denser ( 0ρ = 1.95 and 1.906 g cm–3 for a1 and a2, respectively, vs 1.80 g cm–3 for rdx) and, as a consequence, a1 and a2 can be advantageous in size limiting warheads. it is worth noting that 0ρ and fδ h for structure a1 have been investigated in an old study using quantum chemical method23 which gives a 0ρ = 1.940 g cm –3 and a fδ h = 719 kj mol –1 in close agreement with the result shown in table i. furthermore a comparison between the obtained 0ρ and detonation performance for a1 (1.950 g cm–3, 8.44 km s–1 and 33.12 gpa) to that of tacot24 (1.820 g cm–3, 7.06 km s–1 and 20.30 gpa) obviously shows that the presence of a pyrimidine is advantageous over a phenyl ring for obtaining powerful tetraazapentalene based energetic materials. tetrasubstituted 5h,10h-bis[1,2,4]-triazolo-[1,5-a:1',5'-d]pyrazine (series b) and trisubstituted 5h-imidazo[1,2-b:3,4-b']bis[1,2,4]triazole derivatives (series c) unlike the tetraazapentalene nucleus none of the heterocycles of series b and c had been used or investigated as precursors for energetic material preparation. to the best knowledge of the author, 5h-imidazo[1,2-b:3,4-b']bis[1,2,4]triazole heterocycle is unknown while non-energetic 5h,10h-bis[1,2,4]triazolo[1,5 -a:1',5'-d]pyrazine derivatives can be synthesized by dimerization of 3-phenyl1h-1,2,4-triazole-5-carboxaldehydes (1) giving the semiaminals (2) or the aminals (3) by the reaction of (2) with secondary amines as shown in scheme 1.25 back to the results in table i, one can notice the increased heat of formation when going from series b to series c and this amounts to 300–500 kj kg–1. this is not the case when it comes to density as little can be gained in 0ρ (less than 0.1 g cm–3) with series c being slightly denser than series b. the same trend is _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 692 frem seen when analyzing detonation performance and specific impulse. like a1 and a2 the detonation performance of –no2 and –nhno2 substituted members of series b and c is almost equal to that of rdx (d, 8.53–8.67 km s–1 and p, 32.87– –34.24 gpa); however, b1, b2, c1 and c2 are far less impact sensitive than rdx and hmx. both c1 and c2 have a calculated 50h of 80 and 79 cm, respectively, and thus nitro and nitramine substituted b and c series can be considered as potential replacement for current rdx based formulations. the trinitromethyl derivatives b3 and c3 are denser and more efficient (d > 9.30 km s–1 and p > 40 gpa) than hmx (d = 9.10 km s-1 and p = 39.30 gpa). despite, their high performance, b3 and c3 impact sensitivity is still acceptable ( 50h , 14–17 cm) for future applications. n n n nn n phph oh h h ho nh n n ph n n n nn n phph r' h h r' h o dimerization (1) (2) (3) r'= morpholino, piperidino -2h2o base 2 scheme 1. synthesis of non-energetic derivatives based on series b heterocycle.25 disubstituted 2-amino-2h-1,2,3-triazole, 1-oxide derivatives (series d) energetic materials based on the 1,2,3-triazole, 1-oxide moiety have been rarely investigated in the literature. one such example is 2-methyl-4,5-dinitro -2h-1,2,3-triazole, 1-oxide (mdnto) an explosive compound with a moderate crystal density 0ρ = 1.730 g cm –3 and a decomposition temperature of 256 °c.26 in order to gain density, the –ch3 group at position 2 in mdnto was replaced by –nh2 which results in 7.7 % increase in 0ρ (structure d1, 0ρ = 1.863 g cm –3). a further increase in density can be accomplished by replacing the two –no2 in d1 by a –c(no2)3 group (structure d3, 0ρ = 1.942 g cm –3). all structures investigated in this series have detonation velocity in excess of 9.00 km s–1 with d3 being the most energetic (d = 9.72 km s–1 and p = 43.82 gpa). furthermore, a remarkable specific impulse value is seen for d2 ( spi = 270 s) outperforming rdx and hmx by 4 s. the impact sensitivity of series d is high ranging from 5–9 cm so members of this series can be exploited as lead-free environmentally benign primary explosives or mixed with a polymeric matrix in order to decrease their sensitivity. htpb-based plastic-bonded explosives (pbxs) explosive materials are seldom used in their pure state. therefore, in most practical applications, a given explosive is made in formulations where the energetic component is dispersed in an inert/energetic binder (polymeric matrix), so _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ nitrogen-rich heterocycles derivatives as explosives and propellants 693 as to form what is called a plastic-bonded explosive (pbx). pbxs possess low levels of vulnerability and are characterized by high mechanical strength.27 here, the most impact sensitive members of series a, b, c and d are investigated as ingredient in htpb based pbxs. their detonation performance as well as their gurney velocities (metal acceleration ability, g2e ) was calculated and the results are shown in table ii. for comparison, pbxs made of hmx, rdx, and petn were included. all pbxs investigated are made with 95/5 explosive/htpb mass ratio. table ii. detonation performance as well as acceleration ability of htbp-based pbxs composition 0 ρ g cm-3 fδ h kj (100 g)-1 d km s-1 p gpa g2e a km s-1 a3/htpbb, c1.766h0.541n2.356o2.828 1.88 91 8.77 35.04 2.88 b3/htpb, c1.608h0.791n2.261o3.014 1.85 74 8.90 35.68 2.93 c3/htpb, c1.632h0.700n2.400o2.880 1.85 85 8.80 34.93 2.90 d3/htpb, c1.310h1.018n2.392o3.109 1.85 69 9.17 37.88 3.02 hmx/htpb, c1.639h3.107n2.572o2.573 1.82 22 8.63 33.24 2.86 rdx/htpb, c1.639h3.107n2.572o2.573 1.73 25 8.34 30.09 2.81 petn/htpb, c1.858h2.944n1.207o3.612 1.71 –163 8.28 29.46 2.80 agurney velocity is calculated by following formula:28 0.5 0.4g 02 0.887φ ρ=e ; bfor htpb,29 atomic composition is: c7.1102h10.813n0.1071o0.1375, 0ρ = 0.960 g cm -3, fδ h = –31.55 kj mol -1 at 298.15 k as table ii shows, formulations based on trinitromethyl substituted derivatives are all superior to current nitramine and nitrate ester compositions. moreover, interesting high gurney velocities ( g2e ≥ 2.88 km s –1) are obtained with the theoretically proposed composition which makes them candidates for high performance fragmentation warheads. it is hoped that the addition of htpb will decrease the impact sensitivity of a3 and d3 to a level of safe handling in future applications. conclusion the results shown in tables i and ii prove that the proposed energetic heterocycles are interesting energetic derivatives and several conclusions can be drawn: 1. four structures, namely b1, b2, c1 and c2 are equally performant to rdx, but on the other hand, their 50h values are far more greater (69–80 cm) than that of rdx ( 50h = 24 cm); thus, –no2 and –nhno2 substituted b and c series can be considered as potential replacement for current rdx-based explosives. 2. all investigated heterocycles substituted with –c(no2)3 group possess the highest calculated crystal density and the lowest heat of formation on a weight basis. moreover, the –c(no2)3 shows to be the best substitute offering the highest detonation performance ( d ≥ 9.10 km s–1, p ≥ 40.60 gpa) but also the highest impact sensitivity in their series. structure b3 and c3 are more efficient than _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 694 frem hmx while retaining acceptable impact sensitivity ( 50h , 14–17 cm). furthermore, trinitromethyl substituted structures of all the series studied were investigated in pbx formulations. the pbxs are of 95/5 explosive/htpb mass ratio and the calculation shows that the detonation performance of all these formulations are superior to similar pbxs based on hmx, rdx and petn. finally, these htpb-based formulations can find their way in future high performance warheads due to their high gurney velocities ( g2e , 2.88–3.02 km s –1). 3. all derivatives investigated in this study show high calculated spi values, ranging from 248 to 270 s. nomenclature cl-20 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazatetracyclo[5.5.0.05.9.03.11]dodecane hmx octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine htpb hydroxy-terminated polybutadiene petn pentaerythritol tetranitrate rdx hexahydro-1,3,5-trinitro-1,3,5-triazine tacot 1,3,7,9-tetranitro-6h-benzotriazolo[2,1-a]-benzotriazole-5-ium, inner salt tkx-50 dihydroxylammonium 5,5’-bi-1h-tetrazole-1,1’-diolate и з в о д неки азотом богати хетероциклични деривати као потенцијални експлозиви и горива: теоријска анализа dany frem frem co., beirut, lebanon испитивана су четири типа азотом богатих хетероцикличних деривата супституисаних са –no2, –nhno2 и –c(no2)3 експлозофорним групама као потенцијани експлозиви и горивни материјали. израчунавања су показала да свих 12 испитиваних структура имају високе вредности густине кристала (1,834–1,980 g cm–3) и промену енталпије (топлоту) формирања кондензоване фазе (605–2130 kj kg–1). важна својства, као што су брзина детонације (d), притисак (p) и специфични импулс су израчуната коришћењем kamlet–jacobs методе и ispbkw термохемијског кода. вредности брзине детонације су биле веће од 8,44 km s–1, а притиска од 32,87 gpа. перформансе тринитрометил супституисаних деривата превазилазе перформансе hmx, са вредностима d од 9,32 до 9,72 km s–1 и p од 40,61 до 43,82 gpа. неки –nо2 и –nhnо2 супституисани деривати су се показали као неосетљиви на удар, али су задржали добре детонаторске перформансе, па потенцијално могу заменити постојеће експлозиве на бази rdh. израчунате вредности специфичног импулса (између 248 и 270 s) за све испитиване деривате указују да ови енергетски материјали могу бити састојци будућих ракетних горива. (примљено 18. септембра, ревидирано 13. децембра, прихваћено 21. децембра 2015) references 1. d. m. badgujar, m. b. talawar, s. n. asthana, p. p. mahulikar, j. hazard. mater. 151 (2008) 289 2. u. r. nair, s. n. asthana, a. s. rao, b. r. gandhe, def. sci. j. 60 (2010) 137 3. v. ghule, r. sarangapani, p. jadhav, s. tewari, chem. pap. 65 (2011) 380 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ nitrogen-rich heterocycles derivatives as explosives and propellants 695 4. v. thottempudi, h. gao, j. m. shreeve, j. am. chem. soc. 133 (2011) 6464 5. z. yang, q. zeng, x. zhou, q. zhang, f. nie, h. huang, h. li, rsc adv. 4 (2014) 65121 6. p. he, j.-g.zhang, k. wang, x. yin, x. jin, t.-l. zhang, phys. chem. chem. phys. 17 (2015) 5840 7. h. gao, j. m. shreeve, chem. rev. 111 (2011) 7377 8. n. fischer, d. fischer, t. m. klapotke, d. g. piercey, j. 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conference & exhibit, sacramento, ca, usa, 2006, american institute of aeronautics and astronautics, 2006. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {chemical applicability of sombor indices} j. serb. chem. soc. 86 (5) 445–457 (2021) survey jscs–5433 445 survey chemical applicability of sombor indices izudin redžepović* department of chemistry, faculty of science, university of kragujevac, p. o. box 60, 34000 kragujevac, serbia (received 15 december 2020, revised 10 january, accepted 30 january 2021) abstract: recently, a novel class of degree-based topological molecular descriptors was proposed, the so-called sombor indices. within this study, the predictive and discriminative potentials of the sombor index, the reduced sombor index, and the average sombor index were examined. all three topological molecular descriptors showed good predictive potential. the statistical data indicate that the reduced sombor index preforms with a slightly better predictive potential. an external validation confirmed this finding. it was found that these degree-based indices exert modest discriminative potential, when tested on a large group of isomers. keywords: graph invariants; molecular descriptors; degree of a vertex; physicochemical properties; predictive models; discriminative potential. introduction a proper quantification of a molecule, more precisely its structure, may be accomplished by using molecular descriptors.1,2 these quantities are derived from molecular structure, and they have found various applications in chemistry.3,4 their most important use is in qspr/qsar modeling5−8 and in virtual screening.9,10 the complexity of the obtained chemical information varies with the computational algorithm of the chosen molecular descriptor. namely, the more detailed description of a molecule is obtained, the more computing resources have to be invested. on the other hand, a vague structural depiction is often computationally very efficient. thus, the selection of an adequate molecular descriptor depends on the chemical problem on which it will be applied. encoding information on molecular structure through the utilization of topological molecular descriptors represents an efficient technique. due to the low computational cost and quite simple identification with the structural details, the number of these molecular descriptors is huge. topological molecular descriptors * e-mail: izudin.redzepovic@pmf.kg.ac.rs https://doi.org/10.2298/jsc201215006r ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 446 redžepović are mathematical values calculated from a graph representation of a molecule.11−13 therefore, they are also called graph invariants and have proved to be useful in chemistry.14−19 topological molecular descriptors were also found to be valuable in drug discovery. namely, the advantages of graph invariants arises from the fact that receptor structure or mechanism of action of drugs is not necessary for qsar development, also the conformation and alignment problems related with 3d qsar (as in comfa applications) are completely avoided.20 a vertex set, and edge set of a graph g, denoted by v(g) and e(g), corresponds to the non-hydrogen atoms and bonds of a molecule, respectively. graph invariants can be computed from the molecular graph by using various algorithms. thus, depending on which parameters are used in their definitions, degree-, distance-, and eigenvalue-based graph invariants can be differentiated. the very first degree-based graph invariant is the first zagreb index (zg1), which was introduced in 1972.21 the first zagreb index was defined as follows: 21 ( ) ( ) deg( ) v v g zg g v ∈ =  (1) where deg(v) is degree, i.e., number of first neighbors, of the vertex v. this quantity was developed during the investigation of dependence of π-electron energy on molecular structure. since its introduction, this index has gained a lot of attention, especially among mathematicians.22−26 it is interesting to point out that zg1 can also be calculated in this way:27 ( ) , 1 ( ) ( ) deg( ) deg( ) i j i j e e g zg g v v ∈ = + (2) recently, the author of the paper “geometric approach to degree-based topological indices: sombor indices”28 being inspired by euclidean metric, proposed novel graph invariants based on degree of a vertex. namely, sombor index (so), reduced sombor index (sored), and average sombor index (soavg) were introduced. these quantities are defined as follows: , 2 2 ( ) ( ) deg( ) deg( ) i j i j e e g so g v v ∈ = + (3) , 2 2 red ( ) ( ) (deg( ) 1) (deg( ) 1) i j i j e e g so g v v ∈ = − + − (4) , 2 2 avg ( ) 2 2 ( ) deg( ) deg( ) i j i j e e g m m so g v v n n∈    = − + −         (5) ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ sombor indices 447 where m in eq. (5) is the number of edges, while n denotes the number of vertices. the summation in the above equations goes over all edges. this novel class of indices was developed as the geometric representation of degree-based graph invariants. as previously mentioned, the number of topological molecular descriptors is huge and is still increasing. many of them are introduced as modifications of previously well-known indices and some of them have never been applied on solving chemical problems. therefore, to filter a useful molecular descriptor from a pool of others, a list of desirable properties was compiled.29 within several listed features, sufficient predictive potential of physico-chemical properties of molecules and a low degeneracy level are among the most important ones. since sombor indices have recently been introduced and there is not much information about them, it is interesting to test their potential chemical applicability. more precisely, within this study the predictive and discriminative potentials of sombor indices are analyzed. computational methodology construction of predictive models in order to investigate predictive capability of sombor indices, models for predicting the entropy (s°) and enthalpy change of vaporization (δh°vap) of compounds were developed. the models were constructed using an octane set of isomers and they are based on multiple linear regression. with the utilization of isomers, the influence of molecular size on a predictive model is eliminated. the experimental data used for the construction of the models were collected from www.moleculardescriptors.eu30 and they are presented in table i. the table i. the experimental data used for the development of the linear models molecule s° / j mol-1 k-1 δh°vap / kj mol-1 n-octane 467.23 41.48 2-methylheptane 459.57 39.68 3-methylheptane 465.51 39.84 4-methylheptane 457.39 39.68 3-ethylhexane 457.86 39.65 2,2-dimethylhexane 432.71 37.30 2,3-dimethylhexane 451.96 38.79 2,4-dimethylhexane 447.60 37.78 2,5-dimethylhexane 442.33 37.87 3,3-dimethylhexane 438.23 37.54 3,4-dimethylhexane 445.97 38.98 2-methyl-3-ethylpentane 443.76 38.53 3-methyl-3-ethylpentane 424.59 37.99 2,2,3-trimethylpentane 423.88 36.93 2,2,4-trimethylpentane 435.51 35.15 2,3,3-trimethylpentane 427.02 37.23 2,3,4-trimethylpentane 428.40 37.71 2,2,3,3-tetramethylbutane 389.36 35.19 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 448 redžepović coefficients occurring in the regression models were calculated by means of an in-house python script with the implementation of a scikit-learn machine-learning module using least square fitting.31 degeneracy calculation the degeneracy of the sombor index, the reduced sombor index and the average sombor index was determined using a measure proposed by konstantinova.32 this quantity was named sensitivity of a topological molecular descriptor and it is defined as follows: sensitivity ti n n n − = (6) where n is the number of isomers and nti denotes the number of isomers that cannot be distinguished by topological index (ti). nowadays, this feature is commonly presented as the degeneracy of a topological index: d = 100(1 – sensitivity) (7) within this study, two molecules (a and b) are considered to have the same value of ti if the following condition is fulfilled: |ti(a) – ti(b)| < 10-13 (8) this threshold was chosen because of the precision of common python computations, which is 10-15. results and discussion predictive potential of sombor indices the initial step for developing a linear model for predicting the entropy of molecules was to correlate the calculated values of so, sored and soavg with the experimental data of octanes. with this, it was checked whether so, sored and soavg contain information that may be used to model entropy. the entropy values are plotted in fig. 1 against the so values of the octane isomers. fig. 1. the correlation between the sombor index and entropy of octane isomers. the scatter plot depicted in fig. 1 shows a reasonable correlation between the sombor index and entropy. such a correlation indicates that a linear model for predicting the entropy of octanes could be developed using the sombor index. a deeper insight into this correlation reveals that the data points are assembled into six clusters. such grouping suggests that the data points within the same group have some parameter(s) in common. namely, by direct checking, it was discovered that the points within one group have the same value of zg1. the correlations between reduced sombor index and average sombor index and entropy ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ sombor indices 449 are available in the supplementary material to this paper (figs. s-1 and s-2). the grouping of the data points is also present in these correlations, and it was revealed that zg1 also directs this clustering. since the sombor indices showed satisfactory correlation with the entropy of octanes, the correlation between these descriptors and the enthalpy of vaporization was tested (table i). the correlation between the sombor index and δh°vap is depicted in fig. 2. fig. 2. the correlation between the sombor index and enthalpy of vaporization of octane isomers. the scatter plot presented in fig. 2 shows that a linear dependence between sombor index and enthalpy of vaporization exists. in addition, fig. 2 illustrates that the data points are grouped, as in the previous case. namely, it is obvious that the so values are clustered into six different groups. again, by direct checking, it was found that the data points within one group have the same values of zg1. the correlations between reduced and average sombor indices and enthalpy of vaporization are depicted in figs. s-3 and s-4 of the supplementary material. the same groupation of the sored and soavg values is noticed. as all examined correlations show linear relationships between the sombor indices and these two physico-chemical properties, two types of linear models were constructed, (eqs. (9) and (10)). namely, in the model based on eq. (9), the entropy or the enthalpy of vaporization is predicted only by one of the sombor indices, while in the other model physicochemical property is predicted by one of the sombor indices and the first zagreb index: s°, δh°vap ≈ ati + b (9) s°, δh°vap ≈ ati + bzg1 + c (10) in these equations ti stands for the so, sored and soavg, whereas a, b and c are the fitting coefficients. the values for the fitting parameters are presented in table ii. the entropy and the enthalpy of vaporization of octanes were calculated using these models. the results of the evaluation of the models are presented in table iii. in table iii, r2, radj2 and rmse denote the coefficient of determination, adjusted coefficient of determination, and root mean square error, respectively. f denotes the value obtained by partial f-test and p stands for the p-value. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 450 redžepović table ii. the values for the fitting parameters occurring in the models parameter ti models based on eq. (9) models based on eq. (10) a b a b c s° / j mol-1 k-1 so –6.16 584.94 11.43 –17.47 713.82 sored –5.51 520.98 11.05 –18.36 847.94 soavg –5.22 491.66 3.45 –10.17 721.85 δh°vap / kj mol-1 so –0.52 50.40 –1.97 1.44 39.77 sored –0.47 45.02 –2.17 1.88 11.49 soavg –0.44 42.49 –0.94 0.59 29.20 table iii. the results of evaluation of the linear regression models ti models based on eq. (9) models based on eq. (10) r2 / % r2adj / % rmse / j mol-1 k-1 r2 / % r2adj / % rmse / j mol-1 k-1 f p s° so 89.59 88.94 5.93 92.14 91.65 5.15 4.87 0.043 sored 89.23 88.56 6.03 92.53 92.06 5.02 6.63 0.021 soavg 89.48 88.82 5.96 91.36 90.82 5.40 3.27 0.091 δvaph° so 89.67 89.03 0.50 92.09 91.59 0.44 4.57 0.049 sored 90.65 90.07 0.48 95.47 95.19 0.33 15.96 0.001 soavg 89.84 89.21 0.50 90.71 90.13 0.47 1.41 0.254 the evaluation results presented in table iii show that the entropy and the enthalpy of vaporization of alkanes could be satisfactorily predicted using simple models based on eq. (9). namely, almost 90 % of data variance of s° and δh°vap may be explained by linear model where one of the sombor indices is the only predictor. furthermore, the errors that these models produce are small, especially for the enthalpy of vaporization. among all these models, the model for predicting δh°vap that uses reduced sombor index showed the best performance. namely, its r2 value amounts 90.65 % and this model also yields smallest error. to obtain better insight into the models, the predictive potential of sombor indices were compared with predictive capability of other degree-based indices. namely, for this purpose, the randić index33 and two adriatic indices34 (misbalance lodeg index and misbalance rodeg index) were used. it was found that sombor indices demonstrate better prediction of entropy, since randić and adriatic indices explain around 80 % of data variance. considering the enthalpy of vaporization, the adriatic indices performed somewhat better. namely, these indices describe around 96 % of data variance. since the randić and adriatic indices showed similar predictive potential, it was supposed that a correlation might exist between these indices and the sombor indices. to test this assumption, the correlations between the reduced sombor index and these indices were checked. the correlations between the reduced sombor index and randić index and adriatic indices are depicted in figs. 3–5. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ sombor indices 451 fig. 3. the correlation between reduced sombor index and randić index. the correlation coefficient is –0.9815. fig. 4. the correlation between reduced sombor index and misbalance lodeg index. the correlation coefficient is 0.9790. fig. 5. the correlation between reduced sombor index and the misbalance rodeg index. the correlation coefficient is equal to 0.9812. the reduced sombor index showed good correlations with the randić and adriatic indices. these findings indicate that mathematical relations between these indices may be established. with the introduction of first zagreb index as a second predictor, the performance of the present models is improved. now, most of the models explain a huge amount of the data variance. the statistical justification of the introduction of zg1 was examined using the partial f-test with 95 % of confidence. namely, f-value from table iii represents the result of this statistical procedure. a null hypothesis was that zg1 does not contribute to the explanation of the data variation. the results show that introduction of zg1 in the sombor index and the reduced sombor index models is statistically justified (see f and p values). since f-value associated with the average sombor index is lower than the critical value, the introduction of zg1 is not supported. therefore, models based on eq. (10) with the average sombor index are omitted from further consideration. in the case of entropy, the reduced sombor index shows slightly better performance (r2 = 92.53 %) over the sombor index. considering the enthalpy of ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 452 redžepović vaporization, the reduced sombor index exerts a noticeably better predictive potential than sombor index. namely, its r2 value amounts 95.47 %, and the error is quite small (0.33 kj mol–1). in figs. 6 and 7, correlations between the experimental values and those predicted by the reduced sombor index and the first zagreb index are illustrated. as may be seen, these figures illustrate that there is very good agreement between experimental and predicted data. the correlations for the other models are presented in the supplementary material (figs. s-5–s-8). fig. 6. the correlation between experimental and predicted values for the entropy. fig. 7. the correlation between experimental and predicted values for the enthalpy of vaporization. an external validation of the model for predicting enthalpy of vaporization by sored and zg1 was realized using the data set from table iv. this data set consists of 35 values for δh°vap of nonanes. this data set was randomly split, using scikit-learn module, into a training set and a test set (80:20). the training set was used to fit the model. the values for the fitting parameters and the coefficients of determination are given in table v. with 91.98 % of explained variance in the test set, the presented model showed excellent predictive potential. the correlation between experimental and predicted enthalpy of vaporization is depicted in fig. 8. discriminative potential of sombor indices in order to test the discriminative potential of the sombor indices, a series of chemical tree isomers were constructed. namely, the number of vertices in these series ranged from 10 up to 20. the results of this investigation are tabulated in table vi. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ sombor indices 453 fig. 8. the correlation between experimental and predicted values. table iv. the data used for the external validation molecule δh°vap / kj mol-1 n-nonane 46.44 2-methyloctane 44.65 3-methyloctane 44.75 4-methyloctane 44.75 2,2-dimethylheptane 42.28 2,3-dimethylheptane 43.79 2,4-dimethylheptane 42.87 2,5-dimethylheptane 42.87 2,6-dimethylheptane 42.82 3,3-dimethylheptane 42.66 3,4-dimethylheptane 43.84 3,5-dimethylheptane 42.98 4,4-dimethylheptane 42.66 3-ethylheptane 44.81 4-ethylheptane 44.81 2,2,3-trimethylhexane 41.91 2,2,4-trimethylhexane 40.57 2,2,5-trimethylhexane 40.17 2,3,3-trimethylhexane 42.23 2,3,4-trimethylhexane 42.93 2,3,5-trimethylhexane 41.42 2,4,4-trimethylhexane 40.84 3,3,4-trimethylhexane 42.28 2-methyl-3-ethylhexane 43.84 2-methyl-4-ethylhexane 42.98 3-methyl-3-ethylhexane 43.04 3-methyl-4-ethylhexane 43.95 2,2,3,3-tetramethylpentane 41.00 2,2,3,4-tetramethylpentane 41.00 2,2,4,4-tetramethylpentane 38.10 2,3,3,4-tetramethylpentane 41.75 2,2-dimethyl-3-ethylpentane 42.02 2,3-dimethyl-3-ethylpentane 42.55 2,4-dimethyl-3-ethylpentane 42.93 3,3-diethylpentane 43.36 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 454 redžepović table v. the values for the fitting parameters and the coefficients of determination parameter value r2 / % a / kj mol-1 –2.44 training set: 95.68 test set: 91.98 b / kj mol-1 2.20 c / kj mol-1 5.16 table vi. the degeneracy of the sombor indices n # of chemical trees d (so) / % d (sored) / % d (soavg) / % 10 75 33.33 34.67 34.67 11 159 47.80 45.91 47.80 12 355 60.56 60.56 61.41 13 802 71.32 71.57 71.82 14 1858 80.36 81.00 81.05 15 4347 87.12 87.69 87.35 16 10359 91.71 92.47 92.88 17 24894 94.85 95.49 94.97 18 60523 96.92 97.37 97.06 19 148284 98.14 98.49 98.25 20 366319 98.92 99.15 99.03 the percentages presented in table vi show that the sombor index, reduced sombor index, and average sombor index exert modest discriminative potential. all three descriptors show similar sensitivity. for example, within 355 chemical trees (n = 12), more than 60 % of the molecules cannot be distinguished by these topological molecular descriptors. such degeneracy of the sombor indices is in accordance with the degeneracy of other degree-based graph invariants. namely, this class of indices show lower discriminative potential compared to eigenvaluebased descriptors. the degeneracy is plotted against the number of vertices in fig. 9, which demonstrates that the degeneracy of the sombor indices increases with the increasing number of molecules. fig. 9. percentage of the degeneracy of the sombor index, the reduced sombor index, and the average sombor index. note that all three curves almost fully overlap. conclusions within this study, the chemical applicability of the sombor indices was examined. more precisely, their predictive and discriminative potentials. the sombor index, the reduced sombor index, and the average sombor index were ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ sombor indices 455 used to model the entropy and enthalpy of vaporization of alkanes. simple linear models that use one of these indices as the only predictor showed satisfactory predictive potential. the performance of these models was improved with the introduction of the first zagreb index as a second predictor. among these three topological molecular descriptors, the reduced sombor index showed the best performance. an external validation of the derived model was applied, and the reduced sombor index showed up well. the results of the testing of the predictive potential of the sombor indices indicate that these descriptors may be successfully applied for the modeling of thermodynamic properties of compounds. the discriminative potential of the sombor indices was tested on several groups of chemical trees. all three descriptors exhibit modest sensitivity. in a large class of isomers, a great number of molecules cannot be distinguished by these indices. this result is in accordance with the degeneracy of other degreebased graph invariants. supplementary material additional data are available electronically at the pages of journal website: https:// //www.shd-pub.org.rs/index.php/jscs/index, or from the corresponding author on request. acknowledgment. this work was supported by the ministry of education, science and technological development of the republic of serbia (agreement no. 451-03-68/2020-14/ /200122). и з в о д хемијска примењивост сомбор индекса изудин реџеповић институт за хемију, природно–математички факултет, универзитет у крагујевцу, радоја домановића 12, 34000 крагујевац недавно је предложена нова класа тополошких молекулских дескриптора заснованих на степену чвора, тзв. сомбор индекси. у оквиру ове студије испитивани су предикциони и дискриминативни потенцијал сомбор индекса, редукованог сомбор индекса и просечног сомбор индекса. сва три тополошка молекулска дескриптора показала су добар предикциони потенцијал. статистички подаци указују на то да редуковани сомбор индекс има нешто бољи предикциони потенцијал у односу на друга два индекса. спољном валидацијом модела потврђен је овај резултат. такође, тестирањем на великој групи изомера утврђено је да ови индекси имају умерен дискриминативни потенцијал. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice highly efficient production of aspergillus niger amylase cocktail by solid-state fermentation using triticale grains as a well-balanced substrate j. serb. chem. soc. 80 (11) 1375–1390 (2015) udc 582.282.123.4:664.644.5+ jscs–4804 663.142/.143:66.098 original scientific paper 1375 highly efficient production of aspergillus niger amylase cocktail by solid-state fermentation using triticale grains as a well-balanced substrate biljana dojnov1*, marica grujić2 and zoran vujčić2 1department of chemistry, institute of chemistry, technology and metallurgy, university of belgrade, njegoševa 12, belgrade, serbia and 2department of biochemistry, faculty of chemistry, university of belgrade, studentski trg 12–16, belgrade, serbia (received 17 march, revised 12 may, accepted 18 may 2015) abstract: triticale (x triticosecale, wittmack), an important industrial crop, with high grain yield, containing high amounts of starch, proteins and also major and minor mineral elements, is not yet sufficiently utilized. the simultaneous production of α-amylase and glucoamylase isoforms by aspergillus niger on triticale grains, without any nutritive supplements, was developed, optimized and scaled up 10 fold for the first time. the specific combination of the examined effects led to the production of a novel glucoamylase isoform. reduction of particle size, increase in oxygen availability and substrate height lead to an increase of 30 % in the production of amylases. reduction of the relative humidity from 65 to 30 % increased glucoamylase production 2 fold and α-amylase production by 30%. the peak production of α-amylase (158 u g-1) and glucoamylase (170 u g-1) were obtained in erlenmeyer flasks and in scaled-up trays. the obtained a. niger amylase cocktail was more efficient in raw starch hydrolysis from wheat flour, 29 % more efficient in glucose formation and 10 % more efficient in total reducing sugar formation, than the commercially available amylase cocktail san super 240l, which is widely used in industry. keywords: α-amylase; glucoamylase; fungi; enzyme production; solid-state fermentation, raw starch. introduction solid-state fermentation (ssf) is widely used for fungal amylase production. it has many advantages over submerged fermentation (smf), such as lower cost, higher efficiency, reduced catabolic repression, easier maintenance, easier scale -up, etc.1–3 production of important industrial enzymes requires testing of various types of substrates and optimization of the fermentation conditions. * corresponding author. e-mail: bdojnov@chem.bg.ac.rs doi: 10.2298/jsc150317041d 1376 dojnov, grujić and vujčić natural materials, mostly wheat, are in use as substrates for the production of industrial enzymes and bioethanol, especially in europe.4,5 triticale (x triticosecale, wittmak) is an important industrial crop that is not yet sufficiently utilized. cultivation of triticale has many benefits when compared to other crops, such as high biomass and grain yield in a wide range of soils (it is particularly resistant to low ph) and climatic conditions, with yields from 3 to 6 t ha–1 depending on the geographic region.6 it is also cultivated in serbia and gives high yields,7 especially in the region near valjevo. triticale contains high amounts of starch (about 60 %) and proteins (from 12 to 15 %).8 it also contains higher amounts of the major mineral elements (k, p and mg) and nutritionally important minor elements (na, mn, fe, cu and zn) than wheat.9 it could be a good choice as a substrate for fungal amylase production for several reasons: it is cheaper than other crops, not part of the human diet, and the obtained fermentation waste could be used as well balanced animal fodder. the use of triticale as substrate for a. niger amylase production, without any additional supplements, is described for the first time herein. glucoamylase and α-amylase are likely to be produced concomitantly in fungal fermentations.10–12 this fact is often ignored and a. niger was shown to be a producer of solely glucoamylase.13–15 in fungal enzyme production, the most efficient combination of the genetic properties of the fungus and substrate composition should be applied under optimal cultivation conditions. it would be far more efficient to use a. niger for the simultaneous production of both αamylase and glucoamylase and to optimize the fermentation process in terms of enhancement of the production of both enzymes. conventional techniques for raw starch hydrolysis involve two phases: liquefaction and saccharification. the first step (gelatinization and liquefaction) requires a high temperature (over 100 °c) at ph 6.5, as well as bacterial thermostable α-amylases.16 fungal α-amylase or glucoamylase are used in the final phase (saccharification) depending on desired products – maltose or glucose.16 saccharification requires a temperature of 55 °c at ph 5.5 in order to obtain maltose syrups, or 60 °c at ph 4.5 for glucose syrups.17 implementation of different reaction conditions and several types of amylase makes starch hydrolysis difficult and expensive. an amylase cocktail (containing α-amylase and glucoamylase) could be used for starch hydrolysis in only one hydrolysis step, which would reduce the costs and/or time required for complete starch hydrolysis. significant cost reduction could be achieved by avoidance of ph changes during starch hydrolysis, which are necessary in conventional methods. cost reduction could also be achieved by saccharification using “in situ” produced enzymes, which avoids transportation expense, and which is trending in the development of bioethanol processing nowadays. the aim of this work was to develop and optimize the simultaneous production of highly efficient a. niger α-amylase and glucoamylase using triticale amylase cocktail production using triticale 1377 grains as substrate for ssf without any additional supplements; and to test the efficiency of the obtained enzyme cocktail in raw starch hydrolysis. moreover, tests were performed with wheat flour as the starting material for raw starch hydrolysis. experimental reagents all employed reagents and solvents were of the highest available purity and purchased from merck (darmstadt, germany) and sigma–aldrich (st. louis, mo, usa). triticale (x triticosecale sp.) “odisej” line was obtained from the institute of field and vegetable crops, novi sad, serbia. san super 240l was received from novozyme as a gift. raw starch from wheat and wheat flour were produced in “fidelinka”, subotica, serbia. microorganisms the a. niger atcc 10864 strain was cultivated on potato dextrose agar (pda) at 28 °c for 7 days to obtain mature spores. a spore suspension was prepared in 0.1 % tween 80 solution at a concentration of 5.9×105 spores ml-1. optimization of ssf triticale, of three different particle sizes, was used for ssf. equal quantities of triticale and water were mixed in a 250-ml erlenmeyer flask, incubated at 60 °c for 3 h,18 and autoclaved. the spore suspension was spread on substrates in 1:0.19 ratio, w/v. ssf was performed at 28 °c for 120 h. the initial external relative humidity (rh) was 65 or 30 % during fermentation. the fermentations were terminated at 24, 29, 33, 57, 81, 96, 105 and 120 h. the influence of particle size on α-amylase and glucoamylase production triticale grains were coarsely ground in a mulinex mill, whereby obtaining mixture of particles with different sizes (30 % 1mm, 28 % 2–3 mm, 18 % 4 mm, 20 % 5–6 mm, 4 % 7 mm) was obtained. three solid-state supports were each made of 16 g of coarse ground triticale (mixture of particles with different sizes) and 16 g of water. another set of three solid-state supports was prepared, each mixed with 8 g of coarse ground triticale, 8 g of whole grain triticale and 16 g of water (combined grains). a third set of three solid-state supports was prepared each with 16 g of whole triticale grains (size 8 mm) and 16 g of water. fermentations were performed in erlenmeyer flasks (250 ml) closed off with microbiological cotton wool caps at 28 °c, rh 30 % for 96 h. the influence of aeration on α-amylase and glucoamylase production during ssf each ssf in these experiments was performed on 16 g of coarse ground triticale grains mixed with 16 g of water. oxygen availability was varied using erlenmeyer flasks covered either with microbiological cotton wool caps, or with metal microbiological caps. both ssfs were realized in duplicate at 28 °c and rh 30 %. the influence of relative humidity (rh) on α-amylase and glucoamylase production during ssf each ssf in these experiments was performed on 16 g of coarse ground triticale grains mixed with 16 g of water. the first ssf was realized at an initial external rh 30 % and the second was performed at an initial external rh of 65 %. the rh was measured using a humidity meter. humidity was kept constant on rh 65 % using mist maker immersed in sterile distilled water. both ssfs were performed in duplicate at 28 °c in erlenmeyer flasks with metal microbiological caps. 1378 dojnov, grujić and vujčić the influence of substrate height on α-amylase and glucoamylase production during ssf three different quantities of coarse ground triticale grains (16, 32 and 48 g) were used in ssf to examine the influence of the substrate height on amylase production. equal quantities of triticale grains and water were mixed in erlenmeyer flasks of identical size and shape. ssf on different quantities of triticale grains was realized in duplicate at 28 °c and at an initial external rh of 65 %, using 250 ml erlenmeyer flasks with metal microbiological caps. scale up coarse ground triticale grains (up to 160 g) were placed in trays (dimensions 20.5 cm×14.0 cm× 6.0 cm) and soaked with equal volume of water (160 ml). the trays were incubated at 60 °c for 3 h,18 and autoclaved. spore suspension was spread on the substrates in 1:0.19 ratio, w/v. ssf was performed in duplicate at 28 °c for 120 h. the initial external rh was 30 % during fermentation. ssf appearance on the tray is shown in fig. 1. fig. 1.ssf appearance on the tray after 36 h. enzyme extraction after ssf fermentation was terminated by the addition of a cold buffer solution (50 mm acetate buffer ph 4.5, 0.1 % tween 80) in a 1:5 ratio (w/v) and homogenized with an ikaturrax homogenizer in 3 cycles of 15 s. extraction was performed at room temperature for 1 h. the obtained mixture was centrifuged at 5000 g for 15 min. the crude fermentation extracts were released from small sugars, which could interfere with the assays, by gel filtration on 2 ml sephadex g25 columns. acetate buffer ph 5.0 was used for the elution of proteins from the crude fermentation extracts. the eluted fractions were concentrated by ultrafiltration and then used for further analysis. amylase activity assay the amylase activity was assayed at ph 5.0 according to the dinitrosalicylic acid (dns) procedure19 using 1.0 % (w/v) soluble starch as substrate at 35 °c for 30 min. maltose was used as a standard. each data point represents the mean of three independent assays (standard errors of measurement (sem) were less than 5 % of the means). one unit of α-amylase activity, u ml-1, was defined as the amount of enzyme required to produce 1 μmol of maltose in 1 min at 35 °c. the specific amylase activity, u g-1, was calculated as the amylase units per 1 g of dry substrate in ssf (triticale). glucoamylase activity assay the glucoamylase activity was assayed at ph 5.0 using 1.0 % (w/v) soluble starch as substrate at 35°c for 30 min. glucose (the final product of the reaction) was detected in the reaction mixture by coupled reaction with glucose oxidase and horseradish peroxidase (hrpo, trinder reagent, pointe scientific, usa). samples (10 µl) were mixed with trinder reagents (1 ml),20 incubated for 30 min and the absorbance was measured at 520 nm, according to the manufacture’s instructions. each data point represents the mean of three amylase cocktail production using triticale 1379 independent assays (the sem values were less than 5 % of the means). one unit of glucoamylase activity, u ml-1, was defined as the amount of enzyme required to produce 1 μmol of glucose in 1 min at 35 °c. the specific glucoamylase activity, u g-1, was calculated as the amylase units per 1 g of dry substrate in the ssf (triticale). zymographic detection of α-amylase and glucoamylase α-amylase and glucoamylase were detected simultaneously on a zymogram.21 after electrophoresis (ef) separation of the crude fermentation extracts on native 10 % polyacrylamide (paa, 30 % t, 2.7 % c) gel, the gel was printed simultaneously on a paa gel with copolymerized β-limit dextrin (0.8 %) and on a nitrocellulose membrane (nc). α-amylase was detected in the paa gel with copolymerized β-limit dextrins after staining with iodine solution. the α-amylase activity appeared as clear bands on a purple background. in the native ef paa gel after printing, both amylases were detected as clear bands on blue background using soluble starch as the substrate, and iodine solution for staining. glucoamylases were detected on nc membrane using starch and a reaction mixture for glucose detection (trinder’s reagent and 4-cl-1-naphthol as substrate). the nc was incubated in buffered substrate solution (1.0 % (w/v) starch, 50 mm acetate ph 5.0, 2.0 mm nacl and 0.1 mm cacl2) and reaction mixture for glucose detection in a 9:1 ratio, after 30 min at 30 °c. a specific reaction leading to an insoluble purple product occurred on the nc membrane in the bands corresponding to glucoamylase. positions and relative activities (representations) of the obtained bands were quantified using a gelanalyzer 2010. hydrolysis of raw starch by the amylase cocktail raw starch from wheat and wheat flour (per 30 ml of 2 % mixtures in acetate buffer, ph 5.0) were soaked at 65 °c. thereafter, equal quantities (in enzyme units, 106.3 u ml-1 reaction mixtures) of commercial glucoamylase product (san super 240l) or a. niger amylase cocktail were added to both substrates. hydrolysis was performed at 50 °c overnight with agitation. starch mixtures were analyzed for presence of starch, dextrins, reducing sugars and glucose prior to and after hydrolysis. the concentrations of starch and dextrin were determined by the iodine dextrin color method (idc method), measuring the absorbance at 590 nm and 540 nm, respectively.22 reducing sugars were determined by the dns method, while the glucose concentration was measured using trinder’s reagents. results and discussion optimization of ssf α-amylase and glucoamylase activity is expressed in u/g of dry substrate that could be compared to each other in optimization processes. each point on the graphs is a mean of the enzyme activities obtained from two independent fermentations ± sem. use of triticale as substrate for amylase production in ssf was justified, since it resulted in peak amylase concentrations higher than amylase concentrations obtained by ssf on wheat flour, without additional supplements.23 moreover, it is in range of the amylase produced on rice waste materials without additional supplements.24 these results indicate that triticale could be taken into consideration when choosing a substrate for amylase production. triticale is an 1380 dojnov, grujić and vujčić accessible, cheap natural substrate, and the remaining biomass could be used as fodder. simultaneously, ssf is an environmental friendly method. the influence of particle size on α-amylase and glucoamylase production. production levels of α-amylase and glucoamylase by a. niger on three forms (sizes) of triticale grains, i.e., coarsely ground grains (particles of different sizes: 30 % 1 mm, 28 % 2–3 mm, 18 % 4 mm, 20 % 5–6 mm, 4 % 7 mm, combined grains – 50 % of whole grains (8 mm) and 50 % of coarsely ground grains, and whole grains (8 mm), are shown in fig. 2. the highest α-amylase and glucoamylase activities were obtained on coarsely ground grains, 133.02 and 91.71 u g–1, respectively. the activities were 30 % higher than those obtained on whole triticale grains, fig. 2. fig. 2. impact of particle size on amylase production by ssf using 16 g, in 96 h, rh 30 % in erlenmeyer flasks with cotton cups. 1 – coarse ground triticale, 2 – combined grains and 3 – whole grains. particle size is an important factor affecting enzyme production by ssf and it should optimized.25 coarse ground triticale grains were of the most suitable particle size because they ensure better accessibility of fungi to the substrate inside the particles since the height of the penetrating hyphae layer is lower and, consequently, the restrictions for the diffusion of nutrients and products are reduced. moreover, smaller particle sizes involve higher specific surface areas and, consequently, larger areas of external mycelium with better oxygenation. these were previously shown to be a critical factor affecting enzyme production.25,26 on the other hand, larger particles (whole grains) provide a limited surface for a. niger attack, which is known to be a limiting factor for enzyme production in ssf.27 smaller particles of triticale – bran, give homogenous and impervious substrate for fungal ssf because low particle size involve particle aggregations that reduce the interparticular space and thus, the volume and removal of the air phase; therefore they have not been tested here. the use of coarse ground triticale grains also increased the availability of starch and starch hydrolysis products (dextrin chains and maltose) formed during incubation of triticale at 60 °c for 3 h.18 this further increases the production of amylase cocktail. it is amylase cocktail production using triticale 1381 known that maltose and even some dextrins are major inducers of amylase production in a. niger.28–30 the influence of aeration on α-amylase and glucoamylase production during ssf. the use of cotton wool microbiological caps (higher oxygen) resulted in better fungal growth and higher production of α-amylase and glucoamylase than the use of metal microbiological caps (lower oxygen), fig. 3. sporulation of a. niger was delayed when ssf was performed in an erlenmeyer with a metal microbiological cap. fig. 3. impact of aeration on amylase production in ssf with cotton wool caps and metal caps on 16 g of coarse grounded triticale, on rh 30 %. use of cotton wool microbiological caps results in better fungal growth and higher production of α-amylase and glucoamylase than the use of metal microbiological caps. prolonged production of α-amylase to the highest level in ssf under higher oxygen availability compared with the decreased production in ssf under lower oxygen availability seems to be more coherent with the fact that more aerated conditions generally accelerate the culture of aerobic microorganisms. the use of cotton wool caps provides for better aeration, higher oxygen consumption and co2 evaporation, thereby facilitating enhanced removal of metabolic heat. all these factors are important for the process of enzyme production in ssf.3 fungal growth and amylase production under low oxygen concentrations were also reduced in a. oryzae.26 sporulation of a. niger was delayed when ssf was performed in an erlenmeyer flask with a metal microbiological cap, maybe due to the slower evaporation of water under these terms. as part of further investigations of the influence of individual factors on the production of amylases, metal microbiological caps were nevertheless used because the results showed that the sem was lower than the sem obtained when cotton wool microbiological caps were used. influence of humidity on α-amylase and glucoamylase production during ssf. a. niger production of α-amylase and glucoamylase during ssf on 16 g of coarse ground triticale grains at rh 65 % and rh 30 % was compared, fig. 4. 1382 dojnov, grujić and vujčić the production of both α-amylase and glucoamylase was higher at rh 30 % but neither was in correlation with a. niger growth. ssf under conditions of high rh is followed by extensive biomass growth but lower production of amylases. the sporulation of a. niger was accelerated under conditions of lower rh, which could be associated with higher production of both enzymes. fig. 4. impact of rh amylase production in ssf at rh 30 % and at rh 65 % on 16 g of coarse ground triticale in erlenmeyer flasks with metal cups. fungal ssf can occur in the rh range from 20 to 80 % with the optimal rh depending on the type of substrate and the microorganism.25 maintenance of the optimal rh is important because it determines the water activity, which decreases during the fermentation part of the hydrolytic process in substrate and also because of increasing temperature in the system.25 reduction of bed porosity causes slower transport of oxygen at high rh values. additionally, a low water content in ssf increases the concentration of nutritive elements in the substrate,28 which could have contributed to higher production of a. niger amylases during ssf at the low rh value. the obtained results were not in correlation with results previously published on a. niger.31 however, it is difficult to observe the impact of rh independent of other factors affecting enzyme production in ssf.32 acceleration of sporulation at the lower rh was due to a higher dryness of the substrate, which implies a higher oxygenation of the mycelium that would explain the better amylase production. the influence of substrate heights on α-amylase and glucoamylase production during ssf. the impact of the substrate height was examined by using different quantities of coarse ground triticale grains, fig. 5. fungal growth was enhanced on higher quantities of substrate. the highest α-amylase activity was detected on 32 g of triticale grains (medium substrate height) during ssf, fig. 5. glucoamylase production was in correlation with fungal growth and was the highest on 48 g of triticale grains (highest substrate height), fig. 5. a higher substrate height increases the total surface area that may affect the slower evaporation of water from the lower layers of the substrate, thus main amylase cocktail production using triticale 1383 taining a better water activity, which would enhance fungal growth and amylases production. this may explain the higher amylase production when a. niger was cultivated on higher substrate heights. fig. 5. impact of substrate height on amylase production in ssf on 16, 32 and 48 g of coarse ground triticale grains in erlenmeyer flasks with metal cups on rh 65 %. production of α-amylase and glucoamylase isoforms during ssf under different conditions great differences in the production of amylase isoforms after 57 h were observed under different fermentation conditions of ssf. therefore, the extracts were analyzed by simultaneous zymographic detection of α-amylase and glucoamylase, figs. 6–8, and the obtained results were further quantified using a gelanalyzer 2010, table i. using 16 g of coarse ground triticale grains and 16 g of water as substrate, and microbiological cotton wool caps for the erlenmeyer flasks with rh 30 % during ssf ensured the best fermentation conditions for the production of an amylase cocktail according to the zymogram and the relative activity determined fig. 6. zymographic detection of amylase cocktail during different ssf conditions in 57, 81, 96, 105 and 120 h on 16 g of triticale grains, rh 30 % and higher aeration. arrows indicate the positions of α-amylase (left) and glucoamylase (right) isoforms, labeled with a1–a6 (amylase isoform). 1384 dojnov, grujić and vujčić fig. 7. zymographic detection of amylase cocktail during different ssf conditions in 57, 81, 96, 105 and 120 h on 16 g of triticale grains, rh 65 % and lower aeration. arrows indicate the positions of α-amylase (left) and glucoamylase (right) isoforms, labeled with a1-a6 (amylase isoform). fig. 8. zymographic detection of amylase cocktail during different ssf conditions in 57, 81, 96, 105 and 120 h on 48 g of triticale grains, rh 65 % and lower oxygen availability. arrows indicate the positions of α-amylase (left) and glucoamylase (right) isoforms, labeled with a1-a6 (amylase isoform). by the gelanalyzer, figs. 6–8 and table i. the highest numbers of α-amylase (5) and glucoamylase (2) isoforms were detected when performing ssf under these conditions, as can be seen on the specific zymograms (for α-amylase and glucoamylase) and on the zymogram for both amylases. a novel glucoamylase isoform a3, detected only under the ssf conditions described above, favors the use of more aerated conditions, figs. 6–8. in addition, under these conditions a. niger produced the highest number of α-amylase isoforms (up to 4) – a1, a2, a5 and a6, while in the other fermentations, only one major isoform a5 could be found. the intensity – relative activity of the individual α-amylase isoforms differed during the fermentation, fig. 6 and table i, column a. the maximal activity of α-amylase was detected at 120 h by the a5 isoform on 16 g of triticale, rh 30 % and higher oxygen availability (table i, column a), and also at 96 h by the a5 isoform under two other fermentation conditions, i.e., with 16 g and with 48 g of triticale, rh 65 % and lower oxygen availability (table i, columns a and b). under higher oxygen availability (table i column a), there were no significant differences between the α-amylase production at 96 h (98.81 %) and at 120 h amylase cocktail production using triticale 1385 (100 %), while the production was significantly smaller after 96 h of fermentation under lower oxygen availability (table i, columns a and b). the maximal activity of glucoamylase was detected at 120 h of fermentation under all compared conditions, but the isoforms were different, table i. table i. the relative activity of individual amylases isoforms (%) in amylase cocktails during ssf under different conditions. the most intense isoform per zymogram of the individual enzyme (in each column) is labeled as 100 % activity. positions of every individual number in table are in correlation with rf values obtained in the zymogram shown in figs. 6–8. a) 16 g of triticale grains, rh 30 % and higher oxygen availability. b) 16 g of triticale grains, rh 65 % and lower oxygen availability. c) 48 g of triticale grains, rh 65 % and lower oxygen availability. a1–a6 amylase isoforms according to the zymographic detection in figs. 6–8. 1 – α-amylase, 2 – amylase cocktail, 3 – glucoamylase fermentation time, h amylase isoform a) b) c) 1 2 3 1 2 3 1 2 3 57 a1 39.75 11.15 – – – – – – – a2 2.62 1.24 – – – – – – – a3 – – – – – – – – – a4 – 28.41 – – 26.03 18.18 – 50.90 19.85 a5 42.21 99.83 – 48.14 64.39 – 49.62 96.66 – a6 – – – – – – – – – 81 a1 10.10 3.56 – – – – – – – a2 1.91 0.40 27.89 – – – – – – a3 – – – – – – – – – a4 3.66 53.49 0.55 – 38.03 94.32 – 60.23 43.13 a5 17.17 100.00 – 37.84 64.76 – 21.67 100.00 – a6 – – – – – – – – – 96 a1 – – – – – – – – – a2 – 11.58 37.79 – – – – – – a3 – – – – – – – – – a4 – 42.11 – – 44.67 60.23 – 46.51 32.06 a5 98.81 16.25 – 100.00 100.00 – 100.00 93.41 – a6 – 2.89 – – – – – – – 105 a1 – – – – – – – – – a2 – – – – – – – – – a3 – 11.11 77.27 – – – – – – a4 – 43.79 0.75 – 42.49 54.55 – 43.31 61.45 a5 87.84 7.86 – 48.65 70.52 – 48.33 89.17 – a6 – 6.98 – – – – – – – 120 a1 – 4.43 – – 1.12 – – 12.08 – a2 – 1.98 – – – – – – – a3 – 53.19 100.00 – – – – – – a4 – 5.51 0.17 – 42.42 100.00 – 67.91 100.00 a5 100.00 10.88 – 7.43 38.36 – 8.33 62.48 – a6 – 1.07 – – – – – – – 1386 dojnov, grujić and vujčić from the results of specific zymographic techniques, obtained for optimization of amylase cocktail production, it could be concluded that particle size, initial external rh and oxygen availability have a highly influential impact on α-amylase and glucoamylase production by a. niger in ssf using triticale as the substrate. the intensity of the major α-amylase isoform (a5) was contrary to the intensity of the major glucoamylase isoform (a4) on all zymograms. amylase cocktail production during ssf under selected conditions the level of production of both amylases was analyzed each hour by spectrophotometric assays and by zymography during ssf under selected conditions, i.e., on 16 g of triticale grains, at rh 30 % and with high oxygen availability, figs. 9 and 10. curves representing the production of the two amylases were parallel, while glucoamylase activity was higher, fig. 8a. peak production of glucoamylase was achieved at 96 h of ssf (170 u g–1). after 96 h, the production of glucoamylase started to decrease, while the production of αamylase continues to rise up at 120 h (158 u g–1). fig. 9. α-amylase and glucoamylase activity after ssf with 16 g of triticale grains, at rh 30 % and with high aeration. a. niger started to produce the major α-amylase isoform (a5) during the first days of fermentation (24, 29 and 33 h). the production α-amylase isoforms with lower mobility (a1 and a2) commenced at 57 h at the same time when production of the major α-amylase (a5) started to decrease, fig. 10. α-amylase production increased from 96 h to 120 h, as shown by enzymatic assay and zymography, fig. 10. a. niger started to produce glucoamylase (a3 and a4) at 81 h of fermentation, fig. 10. selected conditions enable obtaining amylase cocktail with maximal activity of both amylases in 120 h of fermentation, which could test for raw starch hydrolysis. amylase cocktail production using triticale 1387 fig. 10. zymographic detection of α-amylase and glucoamylase after ssf with 16 g of triticale grains, at rh 30 % and with high oxygen availability. arrows indicate the positions of α-amylase (right) and glucoamylase (left) isoforms labeled with a1-a6 (amylase isoform). α-amylase and glucoamylase occur together in many fermentation processes and it has been shown that they have a one-gene precursor,12,13 but here for the first time, it was demonstrated that they could be co-produced simultaneously in one ssf. this new approach to the fermentation process facilitates peak production of both enzymes or specific α-amylase or glucoamylase isoforms, and in addition enables modeling of the process to obtain a specific amylase cocktail, depending on the industry requirements. scale-up of amylase cocktail production when the selected conditions for ssf were employed in large-scale production, very similar levels of α-amylase and glucoamylase, calculated on dry substrate, were obtained. the obtained crude extract (769 ml) contained α-amylase 31.02 u ml–1, i.e., 149.1 u g–1 of dry substrate (coarse ground triticale grains) and glucoamylase 18.14 u ml–1, i.e., 87.2 u g–1 of dry substrate (coarse ground triticale grains). scaling up of this ssf enabled larger amount of amylase cocktail to be produced. the very similar levels of amylase cocktails (expressed in u g–1 of triticale) obtained in the scale-up experiments further confirmed the employment of the selected ssf conditions and justified the usage of triticale as a favorable substrate for the production of amylase cocktails. hydrolysis of raw starch by the amylase cocktails the a. niger amylase cocktail was more efficient than a commercial enzyme product in hydrolysis of wheat flour, i.e., it was 29 % more efficient in glucose formation and 10 % more efficient in the formation of total reducing sugars, table ii, a). the hydrolysis of starch isolated from wheat was similar between the a. niger amylase cocktail and the commercial cocktail, table ii, b). in terms of the formation of glucose, san super 240l was more efficient by 8 %, while 1388 dojnov, grujić and vujčić in the formation of total reducing sugars, the a. niger amylase cocktail was more efficient by 5 %. table ii. comparison of the hydrolysis of raw starch and wheat by a. niger amylase cocktail and by a commercial enzyme product. a) soaked wheat flour as substrate for hydrolysis; b) raw soaked starch from wheat as substrate for hydrolysis state starch mg ml-1 dextrin mg ml-1 reducing sugars mm glucose mm a) wheat flour prior to hydrolysis 1.64 9.22 5.37 11.04 after hydrolysis with san super 240l, novozyme 0.42 0.00 90.43 146.18 after hydrolysis with the a. niger amylase cocktail obtained in this work 0.45 0.00 100.28 206.12 b) raw soaked wheat starch prior to hydrolysis 1.59 8.51 0.00 11.04 after hydrolysis with san super 240l, novozyme 0.01 0.00 94.46 231.36 after hydrolysis with the a. niger amylase cocktail obtained in this work 0.01 0.00 99.38 213.48 yields of the hydrolysis considering the amount of starch to be hydrolyzed, when wheat flour was the substrate were 74.39 % with the commercial cocktail and 72.56 % with the a. niger amylase cocktail obtained in this work. hydrolysis of raw starch was more efficient, 99.37 % with both amylase cocktails (commercial and the present cocktail). hydrolysis of raw starch by amylase enzymes from a. niger was the subject of several papers and it showed high efficiency.12,33,34 in addition, amylase extracts obtained by ssf from aspergillus sp. were previously used for raw starch hydrolysis with wheat flour as the initial substrate.23,35 the results mentioned above are not mutually comparable or comparable with the results obtained in this study because of individual calculations of efficiency and of the degrees of hydrolysis. in addition, the efficiencies were not compared to those of commercially available enzymes. almost complete hydrolysis of raw starch (99.37 %) was attained by the a. niger amylase cocktail produced in this work when the yield of the hydrolysis was calculated based on the amount of available starch. it was the same result comparing with the commercial amylase cocktail and was much better compared with other aspergillus amylase cocktails (69 % to 86 % depending on starch origin).33 conclusions triticale grains without the addition of any nutritive components could be used as the sole substrate for fungal production of important enzyme complexes under ssf conditions that could be industrially applicable. based on triticale amylase cocktail production using triticale 1389 properties as a nutritive rich substrate, competitive and superior amylase cocktails were obtained. the demonstrated higher efficiency of the obtained a. niger amylase cocktail in wheat flour hydrolysis indicates a possibility for implementation in starch industries. the use of the obtained amylase cocktail could unify the phases in the starch hydrolysis process, which would significantly reduce the costs of starch hydrolysis. acknowledgements. this study was supported by a grant from the ministry of education, science and technological development of the republic of serbia (project grant no. 172048). we are grateful to jasna bojčevski for the correction of the english language, as well as for useful suggestions on the writing of the article. и з в о д eфикасна производња амилазног коктела из aspergillus niger методом ферментације на чврстој подлози користећи тритикале као добро избалансирани хранљиви супстрат биљана дојнов1, марица грујић2 и зоран вујчић2 1центар за хемију, институт за хемију, технологију и металургију универзитета у београду, његошева 12, београд и 2катедра за биохемију, хемијски факултет, универзитет у београду, студентски трг 12–16, београд тритикалe (x triticosecale, wittmack) је важана индустријска житарица, високог приноса, и садржи велике количине скроба, протеина и макрои микроелемената, а која још увек није довољно искоришћена. метода истовремене производње α-амилазних и глукоамилазних изоформи из aspergillus niger, користећи само зрна тритикала као подлогу без додатака других нутритивних елемената, развијена је, оптимизована и подигнута на већу скалу (10 пута) по први пут у овом раду. специфична комбинација испитиваних фактора ферментације је довела до производње потпуно нове глукоамилазне изоформе. смањење величине честица, повећање доступности кисеоника и висине стуба супстрата су довели до повећања производње амилазе до 30 %. смањење релативне влажности са 65 на 30 % је повећало производњу глукоамилазе 2 пута и α-амилазе за 30 %. максимална производња α-амилазе је била 158 u g-1, а глукоамилазе 170 u g-1, што је потврђено и на мањој и на већој скали. добијени амилазни коктел из a. niger је био ефикаснији у хидролизи сировог скроба из пшеничног брашна од комерцијално доступног амилазног коктела, san super 240l, који се најчешће користи у индустрији, и то 29 % ефикаснији у добијању глукозе и 10 % ефикаснији у добијању укупних редукујућих шећера. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice interaction of cobalt(ii), nickel(ii) and zinc(ii) with humic-like ligands studied by the esi-ms and ion-exchange methods j. serb. chem. soc. 81 (3) 255–270 (2016) udc 547.992.2+546.732’742’472: jscs–4844 544.726:543.51 original scientific paper 255 interaction of cobalt(ii), nickel(ii) and zinc(ii) with humic-like ligands studied by the esi-ms and ion-exchange methods ivana kostić1*, tatjana anđelković1, darko anđelković1, ružica nikolić1, aleksandar bojić1, tatjana cvetković2 and goran nikolić3 1university of niš, faculty of sciences and mathematics, višegradska 33, 18000 niš, serbia, 2university of niš, faculty of medicine, bul. z. đinđića 80, 18000 niš, serbia and 3university of niš, faculty of technology, bul. oslobođenja 124, 16000 leskovac, serbia (received 17 september, revised 3 november, accepted 12 november 2015) abstract: the interactions of co(ii), ni(ii) and zn(ii) with humic acid and o-donor humic-like ligands were studied by the ion-exchange and electrospray ionization mass spectrometry (esi-ms) methods. interactions were confirmed by differences between the values of chromatogram peak areas for monocomponent (ligand) and binary systems (ligand with metal ion) by a newly developed esi-ms 5 µl-loop injection technique. the schubert ion exchange method was used for the determination of the stability constants of the formed complexes at ph 4.0 and i = 0.01 mol dm-3. comparing the values of log k for complexes formed by di-positive metal ions with humic, benzoic and salicylic acids, it could be concluded that the interaction strengths of d-metals followed the irving–williams order: co(ii) < ni(ii) < cu(ii) > zn(ii). the obtained values of log k indicated that pb(ii) ions affect the strongest interactions with all the investigated ligands. complexation of humic acids macromolecules possessing o-donor binding sites as major binding sites could be predicted and modeled following the same order of interaction strength as the irving–williams order. keywords: humic acid; humic-like ligands; heavy metals; ion exchange; esi-ms. introduction environmental pollution by heavy metals is a well-known problem that threatens nature as well as human health, due to their high toxicity even in small amounts. heavy metals can be involved in a series of complex chemical and biological interactions in soil, water and sediments.1,2 an important factor in determining toxicity, bioavailability and transport of heavy metals is the binding or complexation of metal ions by humic substances (hs).3,4 * corresponding author. e-mail: ivana.kostic83@gmail.com doi: 10.2298/jsc150917094k _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 256 kostić et al. humic substances represent a major fraction of the natural organic compounds in the environment. one of characteristics of hs is their heterogeneity in terms of elemental composition, molecular size distribution and chemical functionality.4,5 bearing in mind that humic substances can derive from any organic material, including plant and animal debris, bio-wastes and meiofauna, their heterogeneity is obvious. the elemental composition of hs is 40–60 % carbon, 30–50 % oxygen, 1–3 % nitrogen and 0.1–2 % sulfur. hs contain many functional groups such as carboxylic, alcoholic, phenolic, amino, amide and sulfhydryl groups. oxygen functional groups are the most abundant groups in hs macromolecules.5,6 due to the acidity of these groups, such as carboxyl and hydroxyl groups, the acid–base behavior of humic acids, an operational fraction of hs, play an important role in the acid–base balance of natural waters.7 the study of the ph behavior of humic acid is very significant, because the complexation of metal cations and binding of different xenobiotics, such as polycyclic aromatic hydrocarbons, pesticides etc., with humic acids is ph-dependent.8 metal ions react with humic compounds and form organometal complexes with different stabilities and solubilities. the high affinity of humic substances for interactions with metal ions affects the distribution of the metal ions in terrestrial and aquatic systems. these organometal reactions facilitate the transport, mobilization or deposition of trace metals in soil and sediments, which influences their bioavailability in the natural environment. the obtained information of the complexing properties of humic substances with metal ions is important from the ecotoxicological viewpoint. for this reason, knowledge of the most reactive functionalities of humic materials is required for a full understanding of the role and function of these constituents in the environment.9 despite the application of powerful techniques for structural investigation of humic substances, including nuclear magnetic resonance spectrometry, infrared spectroscopy with fourier transform, mass spectrometry, spectroscopic analysis, etc., the structural identification of hs at the molecular level has not been completely revealed.10–13 bearing in mind that the number and type of functional groups determine the reactivity of hs, it is important to know such characteristics and not the strict/completely defined chemical structure of hs in order to predict their complexation and acidic properties in the environment. the major binding sites in the structure of hs, i.e., carboxylic and phenolic groups, are ionizable groups, the dissociation of which gives rise to a net negative charge. these two functional groups allow two types of interactions with heavy metals: carboxylate–metal cation and phenolate–metal cation interactions. metal ions could interact with humic acid through electrostatic interactions and/or formation of chelate structures with charged carboxylic and phenolic group.14 the general rules of metal interaction with hs ligands are based on their relative ionic and covalent bonding properties. based on the pearson rule, small _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. interaction of d-metals humic-like ligands 257 metal cations prefer less polarizable sites (o-containing sites) in humic macromolecule, while the larger and more polarizable cations prefer humic functional groups with nand s-donor atoms.15 as low-molecular organic substances (such as phenolic acids) were identified as structural elements of natural humic acids, they were used in this paper as humic-like models in the investigation of the correlation between humic functional composition and their acidic/complexation properties. in order to investigate heavy metal interaction with complex and structurally heterogeneous humic substances, the investigation was performed with stoichiometrically defined substances that resemble structurally the most important parts of the binding sites of humic macromolecule. benzoic (ba) and salicylic (sa) acid have phenolic hydroxyl and/or carboxylic groups bonded on aromatic rings, and they are denoted as humic-like ligands.16 electrospray ionization mass spectrometry (esi-ms) is used for the detection and characterization of ion species in solution and is often the method used for confirming the stoichiometry of newly synthesized complexes in metal–ligand systems. the soft conditions that provide a spectrum with sufficient sensitivity and low detection limit, at concentration values down to 10–6 mol l–1, represent one of the main advantages of the esi-ms method for analysis of metal– –organic complexes. this allows the analysis of solutions at concentration levels, which are more close to those usually encountered in the environmental samples. in addition, many ionic species can be determined and simultaneously analyzed without prior sample preparation. esi-ms was used to determine the stoichiometry of metal–ligand complexes in solution, as confirmation of the results obtained from other techniques.17 the stability constants of humic acid–m(ii) complexes may be determined by different analytical techniques, such as ion selective, liquid membrane electrode (islme) techniques, equilibrium dialysis, potentiometric titration and dynamic light scattering techniques.16,18–22 among other popular methods for the determination of stability constants of metal ions and humic acid, the schubert ion-exchange method, which is based on competition of metal to bind ligand and/or resin, is often used and thus applied in this study.23,24 this paper deals with an investigation of the interaction between m(ii) ions with humic acid and o-donor humic-like ligands, i.e., benzoic and salicylic acid, in order to closely define the interaction of m(ii) ions with hs in the natural environment, by comparison of interaction data with stoichiometrically defined ligands using the ion-exchange method and electrospray ionization mass spectrometry (esi-ms). the results of this study define the stoichiometry of the complexes, i.e., whether they are mononuclear or polynuclear complexes. by comparing the results of the existence and strength of the interactions of metals with stoichiometrically defined ligands, it is possible to predict the complexing cap_________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 258 kostić et al. acity of humic substances with the investigated metals, if their chemical characteristics, such as the presence of oxygen functional groups and the total acidity, are known. the obtained results have importance in the theory of coordination compounds of transition metals and the interaction with o-donor ligands, and practical importance in defining the distribution, migration and mobility of the d-metals co, ni and zn in soil, sediments and aquatic systems. experimental chemical reagents, solutions, instrumentation all the used chemicals were p.a. or hplc (high-performance liquid chromatography) grade purity. benzoic acid was purchased from fluka (switzerland); salicylic acid from merck (usa); humic acid from sigma–aldrich (usa) and methanol from baker (analyzed reagent bio). stock solutions of each metal were prepared from metal salts: zn(no3)2·6h2o, co(no3)2·6h2o, niso4·6h2o. all metal salts were purchased from merck, germany. humic acid (aldrich) was purified by the same procedure as recommended by the international humic substances society (ihss) for purification of humic acid isolated from soil, in order to remove ash content, residual fulvic acids and heavy metals.25 an estimation of the molecular weight (m̅w) of the investigated humic acid was obtained from the measurement of the absorption coefficient at 280 nm (ε280). the molecular weight was calculated using the following equation:26 m̅w = 3.99ε280 + 490 (1) the absorption coefficient, ε280, was found to be 263, and the calculated molecular weight 1539 g mol-1. according to these results, an appropriate amount of humic acid was dissolved and a working solution prepared. the spectrophotometric measurements were performed using a shimadzu uv-1650pc spectrophotometer. all standard stock solutions were prepared by dissolving the required amounts of the chemicals, measured with an accuracy of ±0.0001 g. the solutions of all chemicals were kept in a refrigerator at 4 °c. esi-ms analysis working solutions for the esi-ms analyses were made by dilution of the standard solutions in a methanol/water mixture (80/20, v/v). the esi-ms measurements were performed on a lcq advantage, thermo finnigan (usa) instrument, with auxiliary equipment, implementing the loop injection technique, rather than syringe flow injection, in order to obtain separate peaks of the analytes of interest. the calculated peak area was considered more reliable for concentration determination than the fluctuating abundance of the analyte ions usually obtained through flow injection. solutions of ligands were analyzed by esi-ms immediately after preparation. mixed solutions of m(ii) ions and benzoic and salicylic acid were stored at 25 °c and were analyzed 24 h after mixing. the concentrations of ligands and metal ions were 1, 3, 5, 7 and 9 µmol l-1. the schubert ion exchange method three different concentrations of ligands were used (5×10-3, 10×10-3 and 15×10-3 mol l-1 for benzoic and humic acids; 5×10-3, 7.5×10-3 and 10×10-3 mol l-1 for salicylic acid). all solutions were prepared using double deionized water (conductivity less than 0.1 μs cm-1). concentrations of all metal ions were 5, 10, 15 and 20 mg l-1. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. interaction of d-metals humic-like ligands 259 the ph measurements were made with a hach sension 3 ph-meter (precision of 0.1 mv or 0.001 units of ph) using a hach gel-filled combination glass electrode (51935-00). all experiments were performed at 25±1 °c. the prepared solutions were analyzed by flame atomic absorption spectroscopy (aas) using a perkin elmer aanalyst 300. for each experiment, the cation exchange resin dowex 50wx8, 100–200 mesh, na-form was used. approximately 50 g of the resin was stirred with deionized water and the fine particles were decanted off. subsequently, the resin was transferred to a glass column and the resin was rinsed with 2 l of deionized water, followed by 2 l of 2 mol l-1 hcl, 2 l of 2 mol l-1 naoh, and finally, with 2 l of deionized water. after this rinsing, the resin was passed into the na-form using 2 l of 2 mol l-1 nacl, followed by 2 l of deionized water. after converting into na-form, the resin was air-dried for 24 h, and then stored in an airtight glass container. experimental procedure for establishing the metal/ion-exchange distribution coefficients (d0 and d) time of equilibrium was defined by taking accurate amount of na-saturated cation exchange resin and 50.0 ml of each metal ion solution, and these solutions were shaken. period of attaining the equilibrium was determined by monitoring the metal concentration in supernatant by flame aas. equilibrium was achieved in 1 h. for each metal ion (co(ii), ni(ii) and zn(ii)), the ion-exchange isotherm was measured at ph 4.0 at a constant ion strength i = 0.01 mol l-1. solutions of metal ions were prepared by adding different volumes of metal stock solutions to a 50.0 ml volumetric flask. the ph was adjusted with the addition of 0.1 mol l-1 naoh and/or 0.1 mol l-1 hcl and the ion strength was adjusted with 0.01 mol l-1 nacl. in each volumetric flask, 100.0 mg of cleaned, na-saturated cation exchange resin was added, and all solutions were shaken at a constant temperature of 25 °c for 2 h. determination of distribution coefficients of metal ions in solutions with ligand (d) was performed with same procedure as for the determination of d0, except that the solution contained ligand. each solution contained metal ions in variable concentrations 5, 10, 15 and 20 mg l-1, and ligands (benzoic, salicylic or humic acids) in concentrations from 5×10-3 to 15×10-3 mol l-1. the ph value for all mixed solutions was adjusted to ph 4.0; the ion strength was adjusted at 0.01 with a solution of nacl, and finally, 100.0 mg of cleaned dowex 50wx8 (100–200 mesh) resin in the na-form was added. all solutions were shaken for 2 h at 25 °c. the total concentration of free metal ion and complexed metal ion remaining in the equilibrium solution was determined in supernatant by flame aas. results and discussion esi-ms analysis of the m(ii) ion–humic-like ligand systems humic-like ligands with o-donor atoms in their structure, as –oh and –cooh groups, due to their tendency to deprotonate, were examined in the esi negative mode. monitoring ions [m–h]– were used in all cases, and their signal intensities were good. esi-ms total ion current chromatogram of 5 µl loop injections were obtained by analyzing series of solutions of ligands and binary systems – mixed solutions of metal–ligand. the peak areas of the selected mass range ion current chromatograms of the ligands and binary systems were used for the investigation. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 260 kostić et al. the areas of each esi-ms total ion current chromatogram were calculated for the defined mass range, [m–h]– m/z±1, for all investigated ligands by using icis© modul of xcaliburtm 1.4 software. the esi-ms selected mass range ion current chromatograms of benzoic acid and of the binary system benzoic acid–co(ii) are shown in fig. 1. a) b) fig. 1. a) esi-ms selected mass range total ion current chromatogram and mass spectrum of the loop injection of benzoic acid without m(ii) ions, c = 7.00×10-6 mol l-1; b) esi-ms selected mass range total ion current chromatogram and mass spectrum of the loop injection of the binary system benzoic acid, c = 7.00×10-6 mol l-1 and cobalt(ii), c = 3.0×10-6 mol l-1; solvent: ch3oh/h2o = 80/20, negative ionization mode, flow rate = 20 µl min-1. the values of the peak areas obtained for the selected mass range ion current chromatogram were plotted versus the ligand concentrations (fig. 2). in order to describe the interaction between the investigated metal ions and ligands, comparison of the obtained integrals under the set of experimental points for monocomponent (pl) and binary systems (pml) were compared (fig. 2). the percentage difference between these areas (δp%) was calculated from the following equation: _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. interaction of d-metals humic-like ligands 261 l ml% l 100 − δ = p p p p (2) fig. 2. a) plot of the peak areas of the esi-ms selected mass range ion current chromatograms of the loop injection of benzoic acid () and mixed solutions benzoic acid– –co(ii) () for the mass range m/z 120–122 vs. ligand concentration; b) plot of the peak areas of the esi -ms ion current chromatograms of the loop injection of salicylic acid () and mixed solutions salicylic acid–co(ii) () for the mass range m/z 136–138 vs. ligand concentration; solvent: ch3oh/h2o 80/20 mixture, negative ionization mode. the areas have no physical meaning because they are presented in absolute non-dimensional units of the xcaliburtm software (count–second units). the obtained δp% values were used for comparison of the interactions between m(ii) ions and ligands. a higher value of δp% confirms stronger interaction between metal ions and ligands in the investigated solutions. the values of the peak areas of the selected mass range ion current chromatogram for different concentrations of benzoic acid and for mixed co(ii)–benzoic acid solutions are presented in table i. each measurement was performed three times, and the mean values were calculated. the same procedure was repeated for salicylic acid. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 262 kostić et al. table i. values of the peak areas of the total ion current chromatogram (abs. units) for benzoic acid and the binary systems m(ii)–benzoic acid parameter system i ii iii iv v cba / 10-6 mol l-1 1 3 5 7 9 (peak area ± sd)×106 3.36±0.27 7.41±0.32 13.45±0.49 17.22±0.73 24.26±0.19 cba / 10-6 mol l-1 + cco(ii) / 10-6 mol l-1 1+9 3+7 5+5 7+3 9+1 (peak area ± sd)×106 3.03±0.06 5.60±0.21 13.08±0.58 16.56±0.50 23.75±0.19 the confirmation of interaction was based on the area of the recorded chromatogram being in direct relation with the peak intensities, regarding the signal of ions in the required m/z mass range. in the binary systems, interaction of investigated m(ii) ion and ligand led to changes in the molecular and ionic species in the sample and thus, the intensity of the observed characteristic ion decreased. proportionally smaller areas of the chromatographic peak were obtained in the binary systems for identical ligand concentrations. relative decrease in area could be related to the strength of the interaction of the m(ii) ions and the ligand. since many properties of a solution and the recording conditions affect the obtained result, such as the equilibrium relationship between species and stoichiometry of the investigated solutions, changes in the area of chromatogram peaks can be treated as valid only in a relative comparison.27 the values of the integrals under the set of points in fig. 2 for the ligands and binary systems of ligands with metals (pml), and the δp% values are presented in table ii. in the group of studied o-donor ligands (pl), the strength of m(ii)–ligand interaction follows the decreasing order: salicylic acid > benzoic acid. the schubert ion exchange method schubert’s method was used for determination of stability constants of complexes formed between m(ii) ions and organic ligands. the principle of ion exchange equilibrium was applied to investigate the complex composition. this method is based on measuring the distribution coefficients of a metal ion between a cation exchange resin and the solution phase, in the presence and absence of a ligand (as complexing agent). the equilibrium reactions between m(ii) ions and the ion exchange resin (r), and m(ii) ions and the ligands (l) can be given by the following equations: m r m r+  rr (3) m l m l+  m nm n (4) _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. interaction of d-metals humic-like ligands 263 table ii. integrals under the set of experimental points for monocomponent and binary systems. integrals under the set of experimental points obtained for ligands (pl), binary systems of ligands with metals (pml), and the δp% values parameter ligand without metal co ni zn benzoic acid (ba) pl 105.12 – – – pml 99.25 98.51 98.88 pl–pml 5.87 6.61 6.24 δp% 5.58 6.29 5.94 salicylic acid (sa) pl 126.30 – – – pml 108.46 102.90 99.23 pl–pml 17.84 23.40 27.07 δp% 14.12 18.53 21.43 the distribution coefficient for a metal ion, m(ii), between the resin and solution phase in absence of an organic ligand is defined by the equilibrium ratio given by eq. (5): r0 f [m ] [m ] d = (5) where [mr] is the concentration of metal ions on the resin and [mf] is the concentration of free metal ions in solution. the distribution coefficient for metal ions in the presence of an organic ligand is determined by the following equation: [ ] [ ] [ ] r f c m m m d = + (6) where [mc] is the concentration of complexed metal ions. in these distribution coefficients equations, [mr] presents the amount of metal per g of resin while [mf] and [mc] present the amount of metal per volume unit of solution, and the distribution coefficients have unit of cm3 g–1. the distribution coefficient, d0, between the resin and solution phase for metal ion in the absence of ligand and the distribution coefficient, d, between resin and solution phase for metal ion in the presence of ligand was calculated using the following equation: ( ) 0 0 0 r or 100 v d d m α α = − (7) where α0 is percentage of total metal bound to the exchange resin; (100–α0) is percentage of total metal remaining in solution; v is the volume of the solution (cm3) and mr is the weight of the exchange resin (g). _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 264 kostić et al. from eq. (4), the stability constant k could be determined using the equation: [ ] [ ] [ ] m l m l m n m n k = (8) combining equations for the determination of the distribution coefficients and the equation for the calculation of the stability constant gives the following equation: [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] 1c0 f f m m l m l 1 m l m m m −− = = = = m n m nm nm mkd mk d (9) taking the logarithm of both sides, the following equation is obtained: [ ] [ ]0log 1 log log ( 1) log m log l − = + + − +    d m k m n d (10) this equation presents the full form of the schubert method, which can be applied for polynuclear complexes. for mononuclear complexes, eq. (11) is obtained since m = 1: [ ]0log 1 log log l − = +    d k n d (11) equation (11) presents the basic equation for the determination of the stability constant of mononuclear complexes. when the complex is mononuclear, the slope of the plotted data from eq. (10) has an integer value, and the intercept is equal to the value of the stability constant. the slope, n, shows the metal:ligand ratio of the formed complex, while the intercept, log k, gives the stability constant for mononuclear complexes. this method could be applied if the following conditions are met: ph and ion strength must be constant; the equilibrium temperature of the investigated system must be constant; the experiment must be performed within the linear range of the ion exchange isotherm; there is no absorption on the resin of the ligand or formed complex and the total concentration of the organic ligand must be higher than the total concentration of the metal ion. the experiment was performed at ph 4.0 to avoid hydrolysis of metal ions and the formation of carbonate or hydroxide. the percentage of the total metal bound to the exchange resin, α0, the distribution coefficients, d0 and d, the values of the slope, n, and the stability constants, log k, obtained for the investigated interaction of m(ii) ions with benzoic acid (for concentrations of m(ii) and ligand of 5 mg l–1 and 0.005 mol l–1, respectively) at ph 4.0 are summarized in table iii. the same parameters for the _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. interaction of d-metals humic-like ligands 265 investigated systems of m(ii) ions with salicylic and humic acid are presented in tables iv and v, respectively. table iii. data obtained by the schubert method for the systems m(ii)–benzoic acid. experimentally determined percentages of the total metal bound to the cation exchange resin, α0, distribution coefficients d0 and d, values of slope, n, and values of the logarithm of the stability constants, log k, for complexes of co(ii), ni(ii) and zn(ii) (5 mg l-1) with benzoic acid, at ph 4.0 and ionic strength i = 0.01 mol l-1 metal ion cba×103 / mol l -1 α0 d0 (d) log (d0/d–1) n log k log k co(ii) 0 61.14 786.67 – 1.00 – 0.55 5 60.72 772.91 –1.75 0.55 10 60.28 758.81 –1.44 0.56 20 59.90 746.88 –1.27 0.55 ni(ii) 0 12.48 71.30 – 0.53 – 0.97 5 8.00 43.48 –0.19 0.96 10 6.96 37.40 –0.04 0.96 20 6.02 32.03 0.09 1.00 zn(ii) 0 31.04 225.06 – 1.01 – 1.03 5 29.96 213.88 –1.28 1.02 10 28.88 203.04 –0.96 1.04 20 27.98 194.25 –0.80 1.02 table iv. data obtained by the schubert method for the systems m(ii)–salicylic acid. experimentally determined percentages of the total metal bound to the cation exchange resin, α0, distribution coefficients (d0 and d), values of the slope, n, and the values of logarithm of the stability constants, log k, for complexes of co(ii), ni(ii) and zn(ii) (5 mg l-1) with salicylic acid, at ph 4.0 and ionic strength i = 0.01 mol dm-3 metal ion csa×10-3 / mol l -1 α0 d0 (d) log (d0/d–1) n log k log k co(ii) 0 61.14 786.67 3.00 6.23 5 57.06 664.42 –0.74 6.17 10 44.30 397.67 –0.01 6.37 20 39.50 326.45 0.15 6.15 ni(ii) 0 12.48 71.30 0.57 5.91 5 0.50 2.51 1.44 6.07 10 0.42 2.11 1.52 5.90 20 0.34 1.71 1.61 5.77 zn(ii) 0 31.04 225.06 2.67 6.22 5 1.34 6.79 1.51 6.11 10 0.44 2.21 2.00 6.25 20 0.22 1.10 2.31 6.31 comparison of the results obtained by the analysis of the investigated systems showed that the percentage of metal bound to the ion-exchange resin decreased with increasing concentration of the ligand in solution. the value of this parameter depended on the metal type and ligand structure. the difference _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 266 kostić et al. between the amount of total metal bound to the ion exchange resins in the absence of ligand and amounts in the presence of the ligands showed that humic and salicylic acid caused larger differences than benzoic acid, which was expected due to the higher number of o-donor binding sites in their structure. smaller differences were observed with benzoic and then with salicylic acid. moreover, the type of the metal influenced the amount of resin bound metal. table v. data obtained by the schubert method for the systems m(ii)–humic acid. experimentally determined percentages of the total metal bound to the cation exchange resin, α0, distribution coefficients (d0 and d), values of the slope, n, and the values of the logarithms of the stability constants, log k, for complexes of co(ii), ni(ii) and zn(ii) (5 mg l-1) with humic acid, at ph 4.0 and ionic strength i = 0.01 mol l-1 metal ion cha×103 / mol l -1 α0 d0 (d) log (d0/d–1) n log k log k co(ii) 0 61.14 786.67 – 0.8 – 1.95 5 50.36 507.25 –0.26 2.04 10 46.46 433.88 –0.09 1.91 20 41.78 358.81 0.08 1.90 ni(ii) 0 12.48 71.30 – 0.96 – 2.24 5 6.94 37.29 –0.04 2.26 10 4.84 25.43 0.26 2.26 20 4.10 21.38 0.37 2.19 zn(ii) 0 31.04 225.06 – 0.83 – 2.25 5 18.74 115.31 –0.02 2.28 10 14.46 84.52 0.22 2.22 20 11.02 61.92 0.42 2.24 since the values of n should be an integer, values of n ≈ 1 indicate that co(ii) ions with benzoic and humic acid form 1:1 complex. in addition, it was noticed that for co(ii)–salicylate n is 3.0, and the stoichiometry 1:3 is assigned in this case. co(ii) and zn(ii) ions form mononuclear complexes, with metal:ligand ratio 1:1, with benzoic acid. ni(ii) ion forms mononuclear complex, but with 1:0.5 stoichiometry of complex. the stability constants, log k, and metal–ligand ratio of obtained m(ii) complexes with all investigated ligands are summarized in table vi. in a previous study28 of the metal ions cu(ii) and pb(ii) with humic acid and benzoic and salicylic acid as o-donor humic-like ligands, it was found that the most stable complex of the investigated m(ii) ions with benzoic acid was the pb(ii)–benzoate complex with a log k value of 1.98, which corresponded to literature data.29 in addition, the most stable complex with humic acid was the pb(ii)–humate complex. furthermore, cu(ii) ions formed more stable complexes with all the investigated ligands than the complexes formed with co(ii), ni(ii) and zn(ii) ion. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. interaction of d-metals humic-like ligands 267 table vi. data obtained by schubert's method for humic acid and humic-like ligands. metal:ligand ratio (m:n) and log kmn for humic acid and humic-like ligands with co(ii), ni(ii) and zn(ii) ions metal ion m:n log k ba sa ha ba sa ha co(ii) 1:1 1:3 1:1 0.55 5.80 1.93 ni(ii) 1:0.5 2:1 1:1 0.92 5.57 2.19 zn(ii) 1:1 1:2 1:1 1.03 6.39 2.26 comparing the values of log k in the systems m(ii) ions with humic, benzoic and salicylic acids, it could be concluded that the strength of the interaction between the metal of the transition series of elements was in the order: co(ii) < ni(ii) < cu(ii) > zn(ii) this trend correlates with the trend in the irving–williams series for the stability of complexes of d-metals with ligands having n-donor binding sites. the obtained values of log k indicate that pb(ii) ions present the strongest interactions with all the investigated ligands. considering the relation between log k and the ligand structure, the weakest interactions were observed for m(ii) ions with benzoic acid. the strongest interactions were established between m(ii) ions and salicylic acid due to the presence of both carboxylic and hydroxyl groups and thus, a higher possibility for interaction. when considering the stability constants of m(ii) ions with humic acid, it must be taken into account that the reaction between metal ions and humic acids occurs in two ways. the main or dominant mode is when the carboxylic and phenolic groups participate simultaneously in the complexation. the secondary mode is when only the carboxylic groups participate in the complexation. this was justified by the good correlation between the complexing capacity of humic acid and its carboxylic acidity.30 the concept of hard/soft acids and bases (hsab) could explain the established order of the interaction strength between the investigated m(ii) ions and humic acids, as well as the investigated humic-like ligands. o-donor groups, due to the considerable hard properties, generate strong relationships with hard metal acids. the investigated ions belong to the d-metals group, or boundary group between typical hard and typical soft metals. comparing the obtained values of log k, it was registered that all investigated m(ii) ions form the most stable complexes with salicylic acid and the lowest values of log k were obtained for the interactions with benzoic acid. in addition, it could be seen that the strongest interaction between the m(ii) ions and salicylic acid could be interpreted as this acid providing a higher possibility of interaction due to the presence of carboxylic and hydroxyl groups. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 268 kostić et al. the results of interaction strength for all investigated systems using the ion exchange method are in accordance with literature data and results obtained by the other methods. investigation of the stability constants of complexes formed between following metal ions co(ii), ni(ii), zn(ii), cu(ii) and pb(ii) with benzoic acid in other investigations showed the same order of strength as was found in the present study.29 results found using the schubert method showed that co(ii) ions and humic acid form complexes with a 1:1 stoichiometric ratio between the metal and the ligand.31 moreover, investigations of the stability constant for ni(ii), zn(ii), cu(ii) and pb(ii) ions with humic acid showed that the strongest interaction with humic acid was achieved by pb(ii), slightly weaker by cu(ii), and ni(ii) and zn(ii).9,14,32–37 conclusions phenolic acids comprise a significant part of the chemical structure of humic substances showing an important role in complexation properties of ha. benzoic and salicylic acid as humic-like ligands can be used for setting the range of stability constants of humic complexes with the investigated m(ii) ions. the established trend of the stabilities of metal–humic-like ligand complexes could be used for predicting the strength of interaction between the o-donor atoms in humics and metal ions, as well as for predicting their competition in binding. of the five divalent metal ions that were examined in the present and previous studies, it was found that pb(ii) ions, as the only non-d-metal ions, form the most stable complexes with o-donor humic-like ligands and humic acid. comparing the obtained values of log kmn, it can be concluded that the strength of interaction between the metal of transitional series of elements is in the order: co(ii) < < ni(ii) << cu(ii) > zn(ii), which correlates with the trend in the irving–williams series for the stability of complexes between d-metals and n-donor binding site ligands. competition between pb(ii) ions and other investigated m(ii) ions in the binding for o-donor binding sites of humic ligands in the environment could be expected. in the natural environment, under conditions of increased concentrations of pb(ii) ions, pb(ii) ions may displace the other investigated m(ii) ions, and would cause their increasing mobility and bioavailability. acknowledgement. authors would like to acknowledge the financial support of the ministry of education, science and technological development of the republic of serbia (grant no. iii41018). _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. interaction of d-metals humic-like ligands 269 и з в о д испитивање интеракције кобалт(ii)-, никл(ii)и цинк(ii)-јона са моделима хуминског лиганда методама esi-ms и јонске измене ивана костић1, татјана анђелковић1, дарко анђелковић1, ружица николић1, александар бојић1, татјана цветковић2 и горан николић3 1универзитет у нишу, природно–математички факултет, вишеградска 33, 18000 ниш, 2универзитет у нишу, медицински факултет, бул. з. ђинђића 80, 18000 ниш и 3универзитет у нишу, технолошки факултет, булевар oслобођења 124, 16000 лесковац у овом раду вршено је испитивање интеракције кобалт(ii)-, никл(ii)и цинк(ii) -јона са хуминском киселином и о-донорским моделима хуминског лиганда методом јонске измене и методом масене спектрометрије са електроспреј јонизацијом (esi-ms). постојање интеракције је потврђено разликом између вредности површина пика хроматограма монокомпонентних система (лиганда) и бинарних система (лиганда са металним јоном) помоћу esi-ms техникe убризгавања петљом од 5 μl. шубертова јоноизмењивачка метода је коришћена за одређивање константи стабилности формираних комплекса на ph 4,0 и при јонској јачини од 0,01 mol l-1. упоређујући вредности log k за комплексе формиранe између двовалентних металних јона и хуминске, бензоеве и салицилне киселине, може се закључити да јачина интеракције метала прелазне серије елемената прати ирвинг–вилијамсов редослед: co(ii) < ni(ii) < cu(ii) > zn(ii). добијене вредности log k указују на то да pb(ii) јон остварује најјачу интеракцију са свим испитиваним лигандима у односу на остале м(ii) јоне. комплексирање макромолекула хуминских киселина, који садрже о-донорска везивна места као главна места везивања, може се предвидети и моделирати на основу редоследа јачине интеракције у ирвинг–вилијамсовој серији. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice effect of an industrial chemical waste on the uptake j. serb. chem. soc. 87 (0) 1–15 (2022) original scientific paper jscs–11692 published 1 august 2022 1 powdered adsorbent obtained from bathurst burr biomass for methylene blue removal from aqueous solutions giannin mosoarca1, cosmin vancea1*, simona popa1**, maria elena radulescu‑grad2 and sorina boran1 1politehnica university timisoara, faculty of industrial chemistry and environmental engineering, bd. v. parvan, no. 6, 300223, timisoara, romania and 2”coriolan dragulescu” institute of chemistry, romanian academy, mihai viteazu bd. no. 24, 300223 timisoara, romania (received 16 march, revised 30 april, accepted 4 may 2022) abstract: powdered adsorbent obtained from bathurst burr biomass was tested for methylene blue removal from aqueous solutions. sem and ftir analyses were used to characterize the adsorbent before and after adsorption. the influence of contact time, adsorbent dose, ph, initial dye concentration, ionic strength and temperature on the process were investigated. kinetic, equilibrium and thermodynamic studies were conducted to analyse the process. the taguchi method was used to establish the most suitable conditions for the dye adsorption. the process is spontaneous, favourable, and exothermic and the freundlich isotherm and pseudo-second order kinetic model best describe it. the taguchi method indicate that the ionic strength is the factor with the greatest influence on the adsorption process. keywords: low-cost adsorbent; dye adsorption; kinetic; equilibrium; thermodynamic; taguchi method. introduction methylene blue is a thiazine cationic dye used in various fields and activities. it is widely used in the textile industry for dyeing cotton, wood, and silk due to its ease of application, good material resistance and economic benefits. in medicine it is used to treat methemoglobinemia, cyanide poisoning and urinary tract infections. it is also used as a colouring agent in diagnostic examination and surgery. even if not strongly hazardous, the methylene blue can have negative effects on humans causing eye irritation, breathing difficulty, nausea, heart-beat increase, vomiting, diarrhea and jaundice. to avoid harmful impacts on aquatic *,** corresponding authors. e-mail: (*)cosmin.vancea@upt.ro; (**)simona.popa@upt.ro https://doi.org/10.2298/jsc220316039m 2 mosoarca et al. life and human health, the dye should be removed from wastewater before their discharging into natural effluents.1–5 scientific literature mentions many physicochemical and biological methods designed to remove the methylene blue dye from aqueous solutions: adsorption, coagulation, flocculation, ion exchange, precipitation, oxidation, chemical precipitation, electrochemical processes, photocatalytic processes, membrane processes and biodegradation. very often, adsorption is the chosen process for dye retention due to its many advantages such as: simplicity, high efficiency, flexibility and low costs. an important number of adsorbents are known, among which an important role is played by natural materials that are cheap and available in large quantities.1–10 bathurst burr (xanthium spinosum) is a very invasive plant widespread in europe, north america, asia, australia and partly in africa. it has a very high resistance to drought, pollution and, in general, to any aggressive environmental conditions. it grows up to one meter tall and has a branched stem full of thorns. it can be found in lowland, hilly and low mountain areas, on pastures, abandoned land and roadsides. due to its anti-inflammatory, disinfectant, diuretic, antidiabetic and antitumor properties, is used in traditional medicine.11,12 the purpose of this study was to use the bathurst burr powder to remove the methylene blue dye from aqueous solutions by adsorption. the effect of contact time, adsorbent dose, ph, initial dye concentration, ionic strength and temperature on the adsorption process were monitored. kinetic, equilibrium and thermodynamic studies were used to analyse the process. in order to establish the most suitable conditions for the dye adsorption, the process was optimized by the taguchi method. experimental the adsorbent material was obtained from the aerial part of bathurst burr mature plants, which were purchased from stefmar srl, a local company that process and pack medicinal and aromatic plants. the adsorbent powder obtaining process was described elsewhere.13 the characteristics of adsorbent material, before and after adsorption, were examined by sem analysis (quanta feg 250 scanning electron microscope at 1600× magnitude) and ftir spectroscopy (shimadzu prestige-21 ftir spectrophotometer). the point of zero charge (phpzc) was determined using the solid addition method.3 in the adsorption studies the influence of ph, adsorbent dose, dye concentration, time, temperature, and ionic strength was studied. three independent replicates were performed for each test. during the experiments a constant mixing intensity was maintained. the ph was adjusted using naoh and hcl solutions (0.1 m). nacl as background electrolyte was used to study the effect of ionic strength. the methylene blue concentration was determined using a uv–vis spectrophotometer at 664 nm wavelength. the equilibrium and kinetics studies were assessed using the nonlinear equations of the langmuir, freundlich and temkin isotherms and also, the nonlinear equations of the pseudo first-order, pseudo second-order and elovich kinetic models.14-17 each of these isotherm and kinetic models were evaluated through the statistical parameters determination coefficient bathurst burr biomass adsorbent 3 (r2), sum of square error (sse), chi-square (χ2) and average relative error (are).17 the higher value for r2 and the lower value for sse, χ2 and are were used as the criterion for determining the most suitable model. the taguchi method was used to optimize the dye removal experimental conditions. the l27 orthogonal array experimental design was employed to establish the influence of six controllable factors on methylene blue removal. table i shows these factors and their levels. the taguchi method evaluates the experimental results by signal-to-noise (s/n) ratio, defining the measurement of the response deviation from the desired value.18-20 “the larger the better” option was used to maximize the s/n ratio and implicitly the highest dye removal efficiency. all calculations were performed with the minitab 19 software. table i. controllable factors and their levels factor level 1 level 2 level 3 time, min 1 10 30 adsorbent dose, mg l-1 0.5 1.5 3.0 ph 2 6 10 initial dye concentration, mg l-1 50 150 250 ionic strength, mol l-1 0 0.1 0.2 temperature, k 284 291 315 results and discussion adsorbent surface characterization fig. 1 shows the sem images of the adsorbent material surface at 3000× magnitude. before adsorption, the adsorbent surface has a porous aspect, suitable for dye adsorption (fig. 1a). after adsorption, the surface morphology has changed (fig. 1b), indicating that pores, voids, or irregularities might be filled with the dye molecules. fig. 1. sem images of adsorbent surface: a) before and b) after adsorption. the ftir spectrum of adsorbent, depicted in fig. 2, indicates that main components of bathurst burr powder are cellulose, hemicellulose, and lignin. the 4 mosoarca et al. identified peaks of the functional groups characteristic of these components are: 3448 cm–1 corresponds to o–h stretching of cellulose, senthamaraikannan et al.21 found this peak in the spectra of natural cellulosic fibre from bark of albizia amara; 2340 and 1630 cm–1 indicate o–h bending of adsorbed water, tsuboi22 identifying both peaks in ftir spectra of cellulose extracted from natural flax, ramie and cotton fibres and karimi et al.23 found the second peak in cellulose spectrum isolated from kenaf fibres; 2053 cm–1 – can be assigned to nco from isocyanate groups, presence of this functional group being reported salim et al.24 in the lignin spectra extracted from bark of leucaena leucocephala; 1368 cm–1 corresponds to c–h bending vibration in cellulose and hemicellulose, labbe et al.25 and kubovský et al.26 found this peak in spectra of aspen tree bark and oak wood respectively; 1000 cm–1 indicate c–o stretching, liang and marchessault27 identifying this peak in in ftir spectra of native cellulose; 542 cm–1 can be attributed to c–h bend, salim et al.24 found this band in the spectra of lignin extracted from native leucaena leucocephala bark. after absorption, the ftir spectrum does not show significant differences compared to the one recorded before adsorption, which indicates that physical interactions are implied in the adsorption process.8,13 fig. 2. ftir spectra of adsorbent before and after dye adsorption. the point of zero charge provides information on how an adsorbate will be adsorbed by an adsorbent material depending on the surface electrical charge. if the solution ph is higher than phpzc, the adsorbent surface is negatively charged while a solution ph lower than phpzc leads to a positively charged ads bathurst burr biomass adsorbent 5 orbent surface. therefore, the adsorption of methylene blue will be favoured when the ph of the solution is higher than phpzc. the value of this parameter, determined using solid addition method, was 6.64 (fig. 3). fig. 3. determination of point of zero charge (phpzc) based on the solid addition method. the influence of contact time on adsorption process in the first few minutes, the adsorption process is fast (fig. 4) because many active adsorption sites are available on the surface of the material.6,7,10 as time goes on, these sites gradually take over and the adsorption capacity increases more slowly. after 30 min the equilibrium is reached, indicating that all the surface of the adsorbent is covered by dye molecules.6,7 fig. 4. influence of contact time on adsorption capacity. the equilibrium times obtained for similar adsorbents used for methylene blue removal from water were: 30 min for arthrospira platensis biomass,28 50 min for haloxylon recurvum stem biomass,29 60 min for euchema spinosum alga biomass30 and 90 min for phragmites australis biomass.31 6 mosoarca et al. the influence of adsorbent dose on adsorption process the adsorbent dose influence upon the adsorption capacity together with the methylene blue removal efficiency are presented in fig. 5. the two parameters behave differently as the adsorbent dose increases: the adsorption capacity decreases while the dye removal efficiency increases. at high adsorbent amounts the aggregation of particles can occur, and a large part of the active sites remains unsaturated. these two phenomena lead to a decrease of the adsorption capacity.7,9,10 the positive effect of a larger adsorbent quantity upon the removal efficiency is based on an increase of the surface area and thus of the number of sites available for adsorption.3,7,9 similar observations have been reported in other studies on the methylene blue adsorption on other low-cost plant materials. when salix babylonica leaves were used as adsorbent, an increase in adsorbent material dose from 0.2 to 15 g l–1 led to an increase in dye removal efficiency of 36.88 % and a decrease in adsorption capacity of 98 %.7 in the case of citrus limetta peel use, it was observed that for an increase of the adsorbent dose from 0.4 to 2.0 g l–1, removal efficiency increased rapidly by about 3 % while the adsorption capacity decreased by 91 %.10 fig. 5. influence of adsorbent dose on adsorption capacity and removal efficiency. the influence of ph on adsorption process the effect of solution ph on adsorption capacity is depicted in fig. 6. increasing the value of this parameter has a positive effect on the adsorption capacity. the best results were obtained at a ph higher than 8. at these ph values, higher than the ph corresponding to the point of zero charge (phpzc 6.64), the adsorbent material surface is negatively charged, favouring the electrostatic attraction with the dye cations.3,10,32 similar results were reported in other previous articles publish in scientific literature. at ph > 8, increased adsorption capacities were recorded for adsorbent materials such as: salix babylonica leaves,7 citrus limetta peel,10 phragmites australis biomass31 and phoenix tree’s leaves.33 bathurst burr biomass adsorbent 7 the relatively constant value of adsorption capacity at high ph indicates that in addition to the electrostatic attraction, other mechanisms are also involved in the adsorption process.32 fig. 6. influence of ph on adsorption capacity. the influence of initial dye concentration on adsorption process the adsorption capacity and the dye removal efficiency values at different initial dye concentrations are shown in fig. 7. the adsorption capacity increases while the dye removal efficiency decreases as the initial dye concentration increases. the first parameter behaviour is caused by the increase of the driving force resulting from the concentration gradient.32,33 the values of the second parameter decrease due to the accumulation of methylene blue molecules on the surface of the adsorbent which leads to saturation of the adsorption sites.6,28 similar behaviours have been mentioned previously in other adsorption studies. our study shows that an increase of the initial dye concentration from 50 to 200 mg l–1 leads to an increase of the adsorption capacity from 21.7 to 83.5 mg g–1 and a decrease of dye removal efficiency from 87.18 to 83.19 % respectively. for the same initial dye concentration variation range, other researchers reported an fig. 7. influence of dye initial concentration on adsorption capacity and removal efficiency. 8 mosoarca et al. increase in adsorption capacity from about 24 to 80 mg g–1 and a decrease dye removal efficiency from 96 to 92 % when using weeping willow leaves7 as an adsorbent. another study using a. platensis biomass28 as adsorbent, an increase of the initial dye concentration from 6.25 to 100 mg l–1 generates an increase of the adsorption capacity from 8 to 90 mg g–1 and a dye removal efficiency decrease from 60 to about 45 %. the influence of ionic strength on adsorption process increasing the solution ionic strength generates a decrease of the adsorption capacity, illustrated in fig. 8, due to competition between methylene blue cations and sodium ions to occupy the available adsorption sites on the surface of the adsorbent.32,33 the unfavourable effect of ionic strength on methylene blue adsorption process was also mentioned in other studies. thus, for a. platensis biomass,28 lotus leaves32 and phoenix tree leaves33 adsorbents an increase in ionic strength from 0 to 0.2 mol l–1 leads to an adsorption capacity decrease of 72, 22 and 7 %, respectively. fig. 8. influence of ionic strength on adsorption capacity. the influence of temperature on adsorption process according to fig. 9 the adsorption capacity decreases with increasing the temperature indicating that the process is exothermic in nature. the binding forces between the adsorbent surface and the adsorbate molecules become weaker with increasing temperature.34,35 similar observations have been reported by others previous articles. when salix babylonica leaves,7 haloxylon recurvum plant stems29 and natural muscovite clay34 were used as adsorbent materials to remove methylene blue from aqueous solutions, a negative effect of temperature rise on the adsorption capacity was observed. equilibrium isotherms in order to study the interactions between the dye molecules and the adsorbent surface three isotherms were tested: langmuir, freundlich and temkin. bathurst burr biomass adsorbent 9 fig. 10 shows the plots of the tested adsorption isotherms (non-linear forms). the values of adsorption isotherms constants and also, of the corresponding error functions (r2, sse, χ2, are) are summarized in table ii. fig. 10. the tested adsorption isotherms (non-linear forms) for the methylene blue adsorption. both the langmuir isotherm and the freundlich isotherm characterize very well the adsorption process (fig. 10). considering the higher value for determination coefficient (r2) and the lower values of sse, χ2 and are it can be concluded that freundlich isotherm best describes the process. the obtained results agree with those mentioned by other researchers in methylene blue adsorption studies, this isotherm characterizing the dye retention processes on adsorbent materials obtained from elaeis guineensis leaves,4 haloxylon recurvum stems29 and euchema spinosum alga.30 table iii shows a comparison between the maximum adsorption capacities values for methylene blue adsorption using similar low-cost adsorbents. adsorption kinetics the kinetics of the methylene blue adsorption process on the studied adsorbent was investigated by means of kinetic models (non-linear forms): pseudo fig. 9. influence of temperature on adsorption capacity. 10 mosoarca et al. -first order, pseudo-second order and elovich. the plots of these kinetic models and the value of their specific constants are illustrated in fig. 11 and table iv, respectively. the values of the coefficients of determination (r2) for all models are above 0.96, the highest recorded value was for the pseudo-second order model while the lowest value corresponds to the elovich model. the calculated values of the error functions (χ2, sse and are) are the lowest for the pseudosecond order model. table ii. adsorption isotherms models constants and the corresponding error functions isotherm model parameter value langmuir non-linear kl / l mg -1 0.006±0.001 qmax / mg g -1 485.1±9.23 r2 0.9978 χ2 0.78 sse 17.37 are / % 5.08 freundlich non-linear kf / mg g -1 4.07±0.74 1/n 0.85±0.07 r2 0.9985 χ2 0.32 sse 10.90 are / % 3.39 temkin non-linear kt / l mg -1 0.220±0.043 b / kj g-1 58.77±6.94 r2 0.9584 χ2 7.81 sse 307.29 are / % 18.81 table iii. the maximum adsorption capacities (mg g-1) for methylene blue on different lowcost adsorbents adsorbent value citrullus colocynthis seeds6 18.8 phragmites australis biomass31 22.7 haloxylon recurvum stems29 22.9 salix babylonica leaves7 60.9 daucus carota leaves3 66.5 phoenix tree leaves33 80.9 elaeis guineensis leaves4 103.0 dry bean pods husks8 121.1 fava bean peel1 140.0 lotus leaf32 221.7 citrus limetta peel10 227.3 arthrospira platensis biomass28 312.5 bathurst burr biomass (this study) 485.1 euchema spinosum algae30 833.3 bathurst burr biomass adsorbent 11 fig. 11. the tested kinetic models (non -linear forms) for the methylene blue adsorption. table iv. kinetic models constants and the corresponding error functions kinetic model parameter value pseudo-first order non-linear k1 / min -1 0.648±0.051 qe,calc / mg g -1 40.63±0.4 r2 0.9899 χ2 0.61 sse 16.37 are / % 13.48 pseudo-second order non-linear k2 / min -1 0.025±0.004 qe,calc / g mg -1 min-1 42.86±0.38 r2 0.9962 χ2 0.17 sse 5.84 are / % 11.59 elovich non-linear a / g mg-1 0.218±0.048 b / mg g-1 min-1 1434±124 r2 0.9635 χ2 1.95 sse 57.41 are / % 15.84 these results indicate that the adsorption process is best described by this kinetic model. the conclusion is also supported by the calculated value of equilibrium adsorption capacity 42.86 mg g–1 which is very close to experimental value 42.05 mg g–1. the pseudo-second order kinetic model also characterized other similar methylene blue adsorption processes on other adsorbents based on salix babylonica leaves,7 corn cobs,9 euchema spinosum macro-alga,30 phragmites australis biomass31 and lotus leaves.32 thermodynamic parameters the thermodynamic parameters of dye adsorption process, calculated bases on experimental results obtained at 284, 291, 307 and 315 k, are presented in https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/phragmites-australis https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/phragmites-australis 12 mosoarca et al. table v. the negative values of standard gibbs energy change (δg0) and standard enthalpy change (δh0) indicate a spontaneous, favourable and exothermic process. the positive value of standard entropy change (δs0) shows an increase randomness at solid–liquid interface. the value of δg0 is in the range –20 to 0 kj mol–1 and the δh0 value is lower than 40 kj mol–1, therefore the main mechanism involved in absorption is physisorption.7,29,31,34,35 table v. thermodynamic parameters for the methylene blue adsorption onto bathurst burr powder δg0 / kj mol-1 δh0 / kj mol-1 δs0 / j mol-1 k-1 284 k 291 k 307 k 315 k –19.04 –19.18 –19.78 –19.91 –1.26 3.60 optimization adsorption parameters by taguchi method the l27 orthogonal array and the obtained results after each run, for dye removal efficiency and the s/n ratios, are summarized in table vi. the order of the controllable factors’ significance (table vii) was established using the rank of s/n ratio and delta values (difference between the highest and lowest average response values for each factor).18 table vi. experimental layout of l27 orthogonal array  / min adsorbent dose, mg l-1 ph initial dye concentration, mg l-1 ionic strength mol l-1 t / k removal efficiency, % s/n 1 0.5 2 50 0.0 284 18.73 25.45 1 0.5 2 50 0.1 291 10.97 20.80 1 0.5 2 50 0.2 315 7.74 17.78 1 1.5 6 250 0.1 284 40.27 32.09 1 1.5 6 250 0.2 291 23.59 27.45 1 1.5 6 250 0.0 315 16.64 24.42 1 3.0 10 150 0.2 284 44.93 33.05 1 3.0 10 150 0.0 291 26.32 28.40 1 3.0 10 150 0.1 315 18.57 25.37 10 0.5 10 250 0.2 284 27.61 28.82 10 0.5 10 250 0.0 291 19.52 25.81 10 0.5 10 250 0.1 315 40.25 32.09 10 1.5 2 150 0.0 284 56.74 35.07 10 1.5 2 150 0.1 291 40.12 32.06 10 1.5 2 150 0.2 315 82.71 38.35 10 3.0 6 50 0.1 284 42.06 32.47 10 3.0 6 50 0.2 291 29.74 29.46 10 3.0 6 50 0.0 315 61.31 35.75 30 0.5 6 150 0.1 284 23.53 27.43 30 0.5 6 150 0.2 291 48.60 33.73 30 0.5 6 150 0.0 315 28.42 29.07 30 1.5 10 50 0.2 284 30.66 29.73 bathurst burr biomass adsorbent 13 table vi. continued  / min adsorbent dose, mg l-1 ph initial dye concentration, mg l-1 ionic strength mol l-1 t / k removal efficiency, % s/n 30 1.5 10 50 0.0 291 63.32 36.03 30 1.5 10 50 0.1 315 37.02 31.37 30 3.0 2 250 0.0 284 42.43 32.55 30 3.0 2 250 0.1 291 87.64 38.85 30 3.0 2 250 0.2 315 51.25 34.19 table vii. response table for signal-to-noise ratios level time adsorbent dose ph initial dye concentration ionic strength temperature 1 26.78 28.76 26.10 30.57 33.94 30.74 2 31.85 30.70 32.21 30.21 29.74 30.29 3 32.24 31.40 32.55 30.08 27.18 29.83 delta 5.46 2.63 6.46 0.49 6.75 0.92 rank 3 4 2 6 1 5 the factor having the highest influence on the adsorption process was the ionic strength while the factor with the least influence was initial dye concentration. correlating the data from table i and table vi, the optimum adsorption conditions were: time 30 min, adsorbent dose 3 mg l–1, ph 10, initial dye concentration 50 mg l–1, no ions and temperature 284 k. conclusion powdered material obtained from bathurst burr biomass is an efficient lowcost and easily available adsorbent for methylene blue removal from aqueous solutions. the adsorption is influenced by contact time, adsorbent dose, ph, initial dye concentration, ionic strength and temperature and is best described by freundlich isotherm and pseudo-second order kinetic model. the process is spontaneous, favourable and exothermic and the main mechanism involved is physisorption. the ionic strength is the factor with the highest influence on the process and initial dye concentration least influences the adsorption. и з в о д адсорбент у праху добијен из биомасе xanthium spinosum за уклањање метиленског плавог из водених раствора giannin mosoarca1, cosmin vancea1, simona popa1, maria elena radulescu‑grad2 и sorina boran1 1politehnica university timisoara, faculty of industrial chemistry and environmental engineering, bd. v. parvan, no. 6, 300223, timisoara, romania и 2”coriolan dragulescu” institute of chemistry, romanian academy, mihai viteazu bd. no. 24, 300223 timisoara, romania адсорбент у праху добијен из xanthium spinosum биомасе је испитиван за уклањање метиленског плавог из водених раствора. sem и ftir анализе су коришћене за карактеризацију адсорбента пре и након адсорпције. испитан је утицај времена контакта, дозе 14 mosoarca et al. адсорбента, ph, почетне концентрације боје, јонске јачине и температуре на процес адсорпције. кинетичка, равнотежна и термодинамичка испитивања су вршена ради анализе процеса адсорпције. тагучи метод је коришћен да би се одредили најбољи услови за адсорпцију боје. процес је спонтан, фаворизован, егзотерман, описује га фројндлихова изотерма и кинетички модел псеудо-другог реда. тагучи метод указује да је јонска јачина фактор који има највећи утицај на процес адсорпције. 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(2022) https://doi.org/10.2298/jsc211126083s this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc211126083s j. serb. chem. soc.00(0)1-13 (2022) original scientific paper jscs–11415 published dd mm, 2022 1 do serbian high school students possess knowledge of basic chemical facts related to real life as a prerequisite for chemical literacy? filip stašević1, nasta miletić2, jelena đurđević nikolić1* and ivan gutman1 1university of kragujevac, faculty of science, radoja domanovića 12, 34000 kragujevac, serbia and 2 gymnasium kosovska mitrovica, lole ribara 29, 38220 kosovska mitrovica, serbia (received 26 november 2021; revised 21 november 2022; accepted 22 november 2022) abstract: for a long time, literacy has had a deeper meaning than just the ability of reading and writing. chemical literacy, as a part of science literacy, represents the use of chemical knowledge and skills in solving real life problems. with the increasing influence that chemistry has on society, chemical literacy becomes one of the main goals of science education. in order to examine the knowledge of chemical facts related to real life (as a prerequisite for chemical literacy) of high school students (n = 379), we designed a knowledge test and constructed a scoring scale for evaluating achievements. the obtained results are contrary to expectations, i.e. a large number of students did not achieve a satisfactory level of knowing selected chemical facts chosen by the authors. the small number of correct answers per question indicates that the examined sample of students does not show a desirable level of chemical knowledge, implying that there is a need for new, improved, strategies in chemistry teaching. keywords: chemistry teaching; students’ achievements; basic chemical knowledge introduction literacy, in its most common usage, is defined as the ability to read and write. 1 in the 21 st century, we can say that this definition is not appropriate and that the concept of literacy is much broader. literacy is a characteristic needed not just for highly educated people, but due to a different organization of society, it is everyone's necessity. 2 contemporary time shapes literacy and determines required abilities and skills. the digital age, the accelerated flow of information that quickly becomes outdated, the development of civilization and technology demand constant learning. therefore, the definition of literacy is complex and dynamic. there are several forms of literacy: nominal, functional, conceptual, scientific, media, digital, political. 3-7 many authors worldwide maintain that scientific *corresponding author e-mail: jelena.djurdjevic@pmf.kg.ac.rs; tel: +381 34 336 223 https://doi.org/10.2298/jsc211126083s ac ce pt ed m an us cri pt mailto:jelena.djurdjevic@pmf.kg.ac.rs https://doi.org/10.2298/jsc211126083s 2 stašević et al. literacy should be one of the main goals of science education. the reason for this is the increasing impact of science and technology on everyday life and social, political, educational, technological, and economic advancement. 8-10 chemical literacy, as a part of scientific literacy, should be available to the broader public not just to chemists, which would imply the use of chemistry knowledge and skills in various situations. 11 individuals who are chemical literate are: 1. able to apply knowledge and skills from the chemistry domain in daily life, 2. aware of the significance of chemistry, 3. understand the relationship between chemistry, technology, and society.12-16 as one of the scientific disciplines, chemistry has an important role to help students to understand and use basic chemical facts and concepts which is one of the prerequisites for chemical literacy. chemical literacy is needed to understand many processes and science-related issues that occur in everyday life. 17,18 chemistry topics involve studying matter and properties of matter that are important in many disciplines such as health sciences, geography, physics, environmental science. 19,20 it is well known that chemicals can play a vital role in our daily lives. therefore, it is necessary to enhance human conscience about chemistry, prepare the population for proper use of chemistry knowledge and provide them with the ability for long-life learning. also, learning chemistry should not just lean on learning the content available in textbooks. learning must be effective, by making links between chemical knowledge and real life, involving activities based on solving life problems related to chemical issues. accordingly, published standards and benchmarks regarding content enable achieving the main goal of chemistry education, and that is chemistry literacy for all students. 21,22 the role of the teacher is to adapt the given curriculum, keeping in mind the type of class composition and student characteristics, also considering textbooks and other teaching materials, as well as the technical conditions, teaching tools and media available to the school. the chemistry curriculum in high school education describes the teaching process, goals, outcomes, contents, and educational activities. standards of achievements define the result of that process, namely, required knowledge, skills, and attitudes for solving different societal challenges. these standards specify the results expected from all students (basic level), the results that are the basis for continuing education at the university level in areas not directly related to chemistry (intermediate level), and the results in chemistry required for further education in chemistry area (advanced level). hence, there are three levels of standards of achievement, which are cumulative, built into each other, so that students at the advanced level satisfy the requirements from all three levels. standards of achievements describe the qualitative and quantitative results of the teaching process and thus give the description of what students know and can do based on their overall general education in chemistry. as one of the goals of chemistry education is the functionality and applicability of knowledge, ac ce pt ed m an us cri pt chemistry knowledge in high schools in serbia 3 achieving a certain level of standards of achievement means achieving a certain level of functional chemical knowledge. the program for international student assessment (pisa) and trends in mathematics and science studies (timss) are two programs used for monitoring the progress of the skills that are essential for every child to progress through school and life. timss assessments provide an insight into students’ achievement in mathematics and science, using the scale that measures the students’ knowledge, knowledge application, and reasoning ability. 23-25 according to testing under the trends in mathematics and science studies and obtained results (timss 2011, 2015 and 2019), the average accomplishment of the serbian students (>500) was statistically higher than the previously determined average value, which placed them at 25 th (2011) 24 th (2015) and 21 st (2019) position on the list. 26-29 it should be noted that in these studies, participants were fourth-grade students from elementary school. pisa tends to focus on practical knowledge in action, namely recognizing questions as scientific, identifying relevant evidence, critically evaluating conclusions, and communicating scientific ideas. 30-33 the last results of scientific literacy for 15-years-old students assessed under the program for international student assessment (pisa 2018) showed that a mean score (440) of students from serbia in scientific literacy was statistically significantly below the organization for economic co-operation and development (oecd) average (489), so that the students from serbia ranked 45 th from 79 countries. 34 although the timss results show solid achievements of younger primary school students, pisa assessment results are warning signs that we should be concerned about the scientific literacy of the country's population. therefore, the present researchers were aimed at examining the level of basic chemical knowledge of the high school students from different school profiles using the test conceived on knowing different chemical terms and facts related to daily life. experimental research aims and research questions the purpose of the present study was to provide an insight into the manifestation of various aspects of chemical literacy, such as knowing basic chemical facts related to everyday life, among high school students, without any hidden intentions, as discrediting students, and teachers. since chemistry knowledge is important for society, research questions that guide researchers were: 1. what is the situation in high schools with students' knowledge of basic chemical facts and terms connected to real life? 2. which level of chemical knowledge possess students based on researchers' scoring scale and the standards of achievements? 3. is there a difference in the knowledge between gymnasium students and vocational school students? ac ce pt ed m an us cri pt 4 stašević et al. sample / participants simple random sampling was used to select the participants for testing. a heterogeneous sample (n = 379) encompassed the high school students from two different types of schools in serbia, gymnasium (n = 270) and vocational school (n = 109). the participants from gymnasium were all 4th grade high school students with an orientation to natural sciences and mathematics, whereas participants from three vocational schools were 1st grade (economic school, n = 28) 2nd grade (medical school, n = 52) and 4th grade (technical school, n = 29) high school students. chemistry course as a general-educational subject lasts a different number of years depending on vocational school. the medical school has chemistry for two years (pharmacy technician), the economic school has chemistry one year (economic technician) and the technical school (industrial-pharmaceutical technician) has chemistry for four years. the curriculum for all schools emphasizes knowing basic chemical concepts and facts regardless of the different duration of the chemistry education, the number of different chemistry courses, and the different number of classes per year. looking into the curriculum of these different school profiles reveals the same main topics that provide students required knowledge. the number of classes for twhole chemistry education determined by the curriculum for each school and profile is provided in table i. table i. number of the chemistry classes per school type of school and selected profile number of the theoretical classes number of the practical classes gymnasium 288 74 technical school 484 516 medical school 134 0 economic school 74 0 the total number of classes (theoretical and practical) determined by the curriculum for selected modules are 362 (gymnasium), 1000 (technical school), 134 (medical school), and 74 (economic school). a large number of the classes in technical school is due to the fact that students in this school gain chemistry knowledge throughout several separate courses (general and inorganic chemistry, organic chemistry, analytical chemistry, physical chemistry and biochemistry). other selected schools have one course through the years that covers all chemistry disciplines listed above. instrument / design the data were collected with the knowledge test administered to students in a paper form. the knowledge test contained 8 open-ended questions with a short answer. all the questions were based on the corresponding standards of achievements. the committee of experts (high school chemistry teachers and university chemistry teachers) who were not involved in its design confirmed the instrument's validity. based on the evaluations, the revised items were held in the instrument. questions from the knowledge test defined with corresponding standards of achievements are given in table ii.22 a set of questions covered by the instrument was chosen randomly from the curriculum with the intention to examine whether students know basic chemical concepts related with real life after they accomplish general chemistry education. the instrument was designed as a pilot version for providing insight into the current situation with students' chemical knowledge. the obtained results could serve as a motive for more extensive research. ac ce pt ed m an us cri pt chemistry knowledge in high schools in serbia 5 table ii. questions from the test with the respective standards of achievements question no. question standard of achievement 1. in which human organ is the lowest ph value? 2.he.1.1.5. 2. at what temperature, ice melts, and inversely, water freezes to ice? 2.he.1.1.2; 2.he.1.2.3. 3. why is not good to squeeze the lemon into hot tea? 2.he.1.3.2; 2.he.1.4.1. 2.he.1.4.2. 4. do sugar, milk, fruits, vegetables, bread contain the same carbohydrate? 2.he.1.4.1; 2.he.1.4.2. 5. the test for drivers that measures the level of alcohol in the blood is based on which chemical reaction? 2.he.1.3.3; 2.he.1.3.4. 6. which compound provokes tingling after an ant’s sting? 2.he.1.3.1; 2.he.1.3.4. 7. which gas is more commonly known as laughing gas? 2.he.1.2.3. 8. we often hear that limescale does damage to various household appliances and that it comes from hard water. which salts contribute most to water hardness? 2.he.1.2.3. the instrument covered almost all the chemistry disciplines: general chemistry (first two questions), inorganic chemistry (second and last two questions), organic chemistry (fifth, sixth and third questions) and biochemistry (fourth and third questions). students should get the necessary knowledge and competencies to solve the test throughout their chemistry education at primary and secondary school. it is worth mentioning, that all high school students, included in this research, were in the final year of their chemistry education. all participants went through all the topics set in the instrument during the chemistry courses through their schooling. it was expected, based on the standards of achievement, that the majority would access a basic level of chemical knowledge, i.e. to recognize and connect chemical facts with daily life. since chemical literacy is a multidimensional and complex term, it is difficult to assess all its aspects and components. the instrument was designed to measure acquired chemical knowledge for the end of general secondary chemical education, as well as knowing terms and facts from the chemistry domain, related to daily life. without knowing basic chemical facts there is no functional chemical knowledge, as knowledge of these facts is a prerequisite for the existence of chemical literacy. the collected data were analysed both quantitatively and qualitatively with an emphasis on quantitative analysis, whereas the data analysis was carried out with several statistical tests (mean, standard deviation, mann-whitney test also called u-test).35,36 answers were summed and scored as correct, wrong and no answer. descriptive statistics were reported in the percentages and the mean and standard deviation for the percentages were also given. the scoring scale, used to evaluate the level of students’ chemical knowledge, was constructed by the researchers and the responses to each question from the knowledge test were transcribed and scored. the scale was constructed and used to score and evaluate the students' chemical knowledge based on general standards of achievements for the end of secondary education.22 values of correct answers on the questions were scored from low (1), moderate (2) to high level (3) of chemical knowledge as indicated in table iii. the new scoring scale is designed for better insight into the students’ accomplishment of achievements on the knowledge test and better interpretation of the results. for primary school, it is specified that 80 % of students should achieve chemical knowledge at the basic level of ac ce pt ed m an us cri pt 6 stašević et al. standards of achievements.37 as the standards of achievement for secondary school represent expanded knowledge, skills, and attitudes relative to those which are determined for primary school, it is expected that all high school students should achieve a basic level of standards of achievement.38 all questions from the knowledge test were designed to be at a basic level by standards of achievements. the scale is determined by the number of correct answers, low level is less than 50 % correct answers, the moderate level implied 50-80 % of correct responses, whereas the high level is achieved with over 80 % of correct answers per question. reaching a high level on the scale can be interpreted as satisfactory chemical knowledge on the basic level. the moderate level can be explicated as worrying, while the low level represents an alarming situation related to chemical knowledge. expectations were that more than 80 % of students would reach a high level on the scoring scale. table iii. scoring scale for categorizing responses to questions level description of each category in interpretation responses low (1) students’ chemical knowledge is low. less than 50 % of correct answers per question. the situation is alarmingly worrying. moderate (2) students’ chemical knowledge is moderate. students' correct answers are between 50-80 % per question. the situation is worrying. high (3) students’ chemical knowledge is high. more than 80 % of correct answers per question. the situation is satisfactory. results and discussion the data collected with the knowledge test are presented in the table iv. results (number of wrong, correct, and no answers) are given in percentages and the number of respondents is given in parentheses. table iv. distribution of answers on the knowledge test question no. (table ii) contribution, %a correct answers wrong answers without answers 1 70.71 (268) 15.83 (60) 13.46 (51) 2 31.66 (120) 62.80 (238) 5.54 (21) 3 65.70 (249) 15.30 (58) 19.00 (72) 4 78.90 (299) 14.51 (55) 6.60 (25) 5 27.70 (105) 16.62 (63) 55.67 (211) 6 58.05 (220) 11.61 (44) 30.34 (115) 7 46.96 (178) 40.37 (153) 12.66 (48) 8 46.17 (175) 19.26 (73) 34.56 (131) anumber of respondents are given in the parentheses transcribed to a researchers’ scale, respondents are on the transition of two levels, low and moderate. students showed a moderate level of chemical knowledge (50-80 % correct answers) on four questions (question no. 1, 3, 4 and 6) and low level (<50 % correct answers) on the same number of questions (question no. 2, 5, 7 and 8) toward scale score, but no one achieved a high level of knowledge. based on these results, the students do not reach the expected level ac ce pt ed m an us cri pt chemistry knowledge in high schools in serbia 7 of basic chemical knowledge and the situation in schools can be interpreted as not satisfactory. the question with the most correct answers is the fourth question (78 %), while the question with the lowest number of correct answers is the fifth question (27.7 %). it is important to pay attention to a low level of chemical knowledge of the students related to the second question where the number of correct answers was only 31.66 %. based on these results, the students did not achieve a sufficient level of basic chemical knowledge, as expected by standards of achievement. 37,38 according to the scoring scale and students’ accomplishments on the individual questions (1, 3, 4 and 6), students know on a moderate level: acid-base properties of some body fluids and substances from real life (2.he.1.1.5.); the role and presence of biologically important compounds as well as their physical properties and structure (2.he.1.3.2.; 2.he.1.4.1. and 2.he.1.4.2.); structure, physical properties of carbohydrates (2.he.1.4.1. and 2.he.1.4.2.); trivial names of organic compounds and corresponding names according to iupac nomenclature (2.he.1.3.1.), physical and chemical properties of organic compounds, and also their importance in everyday life (2.he.1.3.4.). more precisely, students know on the moderate level which organ in the human organism is with the lowest ph value (stomach); the physical properties of vitamin c, i.e. they know what happens with vitamin c at high temperatures (decomposition); formic acid provokes tingling after an ant’s sting and that sugar, milk, fruits, vegetables, and bread do not contain the same carbohydrate. students’ responses on the 2 nd , 5 th , 7 th , and 8 th questions disclose a low level of knowledge on the researchers' scale for the selected chemical facts: the physical and chemical properties of substances that they encounter in daily life (2.he.1.1.2.; 2.he.1.2.3.); chemical properties and some of the most significant reactions of organic compounds (2.he.1.3.3. and 2.he.1.3.4.) and the most important inorganic compounds as well their applicability (2.he.1.2.3.). accurately, students were expected to know at what temperature ice melts and water freezes; oxidation reaction of alcohol and reduction reaction of dichromate is that one on which is based test that measures the level of alcohol in the blood of drivers; nitrous oxide is laughing gas and that the salts of divalent metal cations contribute the most to the water hardness (ca 2+ ; mg 2+ , etc.). the assessment of students' chemical knowledge revealed that only a small percentage of them know basic chemical facts related to real life. the problem may be that the newly acquired knowledge is not well assimilated, and thus, does not contribute to the students’ ability to meaningfully comprehend basic chemical concepts. the results also indicate many misinterpretations and misconceptions caused by mechanical learning. for example, helium was mentioned as the laughing gas instead of dinitrogen oxide or histamine, not formic acid, as a substance that causes tingling. also, the frequent wrong answer was that ice melts at 4 °c ac ce pt ed m an us cri pt 8 stašević et al. and inversely, water becomes ice, probably because students know that this temperature is characteristic for water (on 4°c water has maximum density). fig. 1 presents the students’ correct answers to questions from the knowledge per school. the present researchers expected a non-significant difference in responses between students from two types of schools, firstly, because questions were constructed on the basic level of chemical knowledge, and secondly, because the content and curriculum from both schools have the same goal, chemical literacy, and knowledge retention. fig. 1. distribution of respondents' correct answers on questions per school; light grey gymnasium, dark grey vocational school the mean value of the correct answers of gymnasium students is 50.00 with a standard deviation of 20.43, whereas this value for students from vocational school is 61.24 with a standard deviation of 12.12. based on these values, it may be concluded that students from the vocational school give more correct answers per question with smaller deviation from the mean, and barely cross the moderate level of chemical knowledge towards researchers' scale score. on the other hand, students from gymnasium show oscillations in the number of correct answers per question, and do not cross the moderate level. u-test, that was carried out shows a significant difference with a p-value of 0.44 (p > 0.05; z = ±0.77). it cannot be known what happened in the individual classes through students' education, so, the difference may be caused by the individual characteristics of both teachers and students and the quality of the teaching process. ac ce pt ed m an us cri pt chemistry knowledge in high schools in serbia 9 the obtained results clearly show disagreement with the goals defined in curriculum which is based on applicable knowledge. a large number of wrong answers suggest that the learning process in serbia’s high schools is still grounded only on the reproduction of the content. without the opportunity to understand and link content with real life, students will develop misinterpretations and misconceptions, so it is for expecting that students would not be able to apply their knowledge. 39 regarding this finding, developing new teaching forms could impact the functionality of students' knowledge and it is also a call for chemistry teachers to provide an appropriate and conducive learning environment during the learning process. the context of chemistry toward daily life situations should provide students the possibility to improve their literacy skills. 40,41 context-based learning with engaging all students during the learning process makes chemistry more relevant and relative to the students. 42 such approach leads students to make links between real life and chemistry, with context that can be an environmental issue, an everyday life problem, or an industrial process. 43 also, it is required to enhance the students’ intrinsic motivation. without this, students will continue to form knowledge with limited understanding based only on memorized facts. conclusion for a long time, literacy does not mean just being able to read and write. no matter whether your work is connected directly or indirectly with chemistry, or you compete in a quiz, read some newspaper article, or buy a cosmetic product, you should have a basic level of chemical knowledge or, in other words, be chemical literate. chemical literacy is a target in major reforms in science education today and it is conceptualized as a main goal. with that goal achieved, the school will provide people with sufficient and functional knowledge and the ability for solving real life problems. the results obtained in this paper have highlighted that knowing basic chemical facts (as a prerequisite for chemistry literacy) among students remains low. it is worrying that the students do not know at what temperature ice melts i.e. water freezes to ice, and also, other similar chemical concepts tested with the instrument. the ignorance of the basic chemical concepts brought students to an unsatisfactory level of basic chemical knowledge. the achieved level of basic chemical knowledge is not sufficient for connecting the acquired knowledge with real life situations, and thus for the application of chemical concepts in daily life. therefore, students mostly do not have the prerequisite for chemical literacy. based on the standards of achievements, eexpectations were that more than 80 % of students would reach a high level on our scoring scale. the results reveal that the situation about students’ chemical knowledge is not satisfactory, none of the question reached a high level of the scoring scale. the authors had believed that there will be no significant difference in responses between students from different ac ce pt ed m an us cri pt 10 stašević et al. school profiles. based on the results, students from the vocational school gave more correct answers per question than students from gymnasium. this result can trigger new research with an aim to confirm this difference and to analyse the reasons. the findings reflect that there still exists a need for developing learning activities throughout chemistry education to provide a chance for students to use their knowledge in real life situations. our results were collected before the sars-cov-2 pandemic, in 2018. the obtained results could serve as a motive for broader research on students' chemical literacy. a similar study is planned, after the pandemic is put under control, to examine the impact of simultaneously, non-simultaneously, and blended elearning on students’ knowledge required for reaching even a basic level of chemical literacy. acknowledgements: this work was supported by the serbian ministry of education, science and technological development (agreement nos. 451-03-68/2022-14/200122 and 451-03-01330/2020-14/2787). извод да ли ученици средњих школа у србији имају функционално хемијско знање? филип сташевић1, наста милетић2, јелена ђурђевић николић1 и иван гутман1 1универзитет у крагујевцу, природно-математички факултет, радоја домановића 12, 34000 крагујевац, србија и 2гимназија косовска митровица, лоле рибара 29, 38220 косовска митровица, србија већ дужи временски период бити писмен не подразумева само способност писања и читања. хемијска писменост, као део научне писмености, представља употребу знања, вештина и ставова у решавању различитих друштвених изазова. свест друштва о значају хемије, хемијску писменост уводи на листу главних циљева образовања и васпитања. да би испитали ниво хемијског знања и ниво познавања основних хемијских чињеница, што је предуслов за хемијску писменост, средњошколаца (n=379) осмишљен је упитник и скала за евалуацију постигнућа. приказани резултати су у супротности очекивањима, тј. нису остварени захтеви дефинисани стандардима постигнућа на основном нивоу. мали број тачних одговора указује да испитивани узорак ученика не поседује задовољавајући ниво хемијског знања, што наводи на потребу за развијањем нових, напреднијих метода у настави хемије. 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https://doi.org/10.1007/0-306-47977-x_8 https://doi.org/10.1080/09500693.2018.1470351 https://doi.org/10.1080/09500690600702470 https://doi.org/10.1021/ed078p684 {internet pages for asynchronous online and face-to-face learning about solutions and dissolution} j. serb. chem. soc. 87 (4) 531–543 (2022) original scientific paper jscs–5539 published 1 october 2021 531 internet pages for asynchronous online and face-to-face learning about solutions and dissolution lidija r. ralevic#, biljana i. tomasevic*# and dragica d. trivic# university of belgrade – faculty of chemistry, studentski trg 12–16, belgrade, serbia (received 4 august, revised and accepted 10 august 2021) abstract: in the last decades online communication has become an important part of the realization of the educational process. in the conditions caused by the covid-19 pandemic it has become particularly significant since in most cases it was necessary to switch to some forms of online teaching-learning. this paper presents the results of a research study conducted as a pedagogical experiment with parallel groups. the aim of this research study was to compare the effects of the application of internet pages for independent online asynchronous learning outside the school environment (group a) and face-to-face learning realized by a teacher at school (group b). the content of the internet pages was created in order to enable the acquisition of the concepts of solutions and dissolution. the effects of the approaches applied were studied based on the student achievement in a post-test (immediately upon learning about the concepts of solutions and dissolution) and in a delayed post-test (a year after the acquisition of these concepts). the participants in this research study were 187 primary school students, who participated in the pedagogical experiment when they were in the seventh grade, while they were in the eighth grade when they did the delayed post-test. the results showed that there was not a statistically significant difference between the overall achievements of the students who learnt about the concepts of solutions and dissolution by independent asynchronous online learning and face-to-face learning at school. this implies that the similar results can be achieved with asynchronous online learning as with face-to-face learning when the conditions do not allow school-based education. keywords: e-teaching; digital materials; submicroscopic level; educational video. * corresponding author. e-mail: bsteljic@chem.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc210804060r ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 532 ralevic, tomasevic and trivic introduction various approaches and teaching/learning methods used for achieving educational goals/outcomes are continuously developed and proposed. online learning is one of the methods used to organize teaching/learning which has shown an increasing potential for the improvement of the educational process in the last decades. online learning is distance learning based on online communication with the application (distribution) of digital teaching materials. due to the covid -19 pandemic, teaching methods within which social contact among students at school is avoided, which reduces the risk of contracting the disease, have become particularly significant.1 faced with this novel situation, over 160 countries switched to online teaching/learning.2 online learning can be divided into synchronous and asynchronous. synchronous online learning includes real-time communication between the teachers and students via various communication media. the main advantages of this method are the possibility of obtaining an instant feedback3 and increased student motivation due to their obligation to be directly involved in the teaching process.4 asynchronous online learning is time-independent communication, which supports work relation among learners and teachers when participants cannot be online at the same time.5 one of the most available options is sending questions to the teacher via e-mail.1 this has proven to be very significant in asynchronous learning since students avoid asking their teacher questions for various reasons, for example, in order to avoid drawing other students’ attention. students find asynchronous learning convenient due to the freedom of choice regarding the time and place when they access the learning material, learning dynamics and independence in doing the activities.6 for these reasons, students find asynchronous online learning more satisfactory than synchronous online learning.1 during the covid-19 pandemic, there were situations when students were not able to participate in the organized synchronous online instruction. in these situations it was really important to enable them asynchronous instruction, for example, by posting a link to an educational video which served as a learning material.7 apart from images, an educational video can also contain textual and audio information. videos with an audio component have a greater potential for achieving learning outcomes compared to the ones accompanied only by textual explanations.8,9 on the other hand, students remember better the information introduced through an educational video than the information introduced through a text or an audio, with or without images.10 this is due to the fact that students pay more attention to the information introduced through video material than to the information introduced through a text or an audio recording.11 with video materials it is possible to adjust the viewing dynamics to the tempo of information processing by stopping the video and watching it again, playing it again, slowing it down and speeding it up.12,13 the research results have shown that multiple viewing of ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. asynchronous online learning about dissolution 533 educational videos distributed to students via the youtube platform has contributed to the improvement of their achievements by approximately 13 %.14 some research studies have shown positive effects of asynchronous learning accompanied by the use of digital materials such as, for example, educational videos, while the results of some other research studies have shown that, as far as effectiveness is concerned, this approach does not differ from face-to-face approach.15 this is explained by the fact that asynchronous learning fails to engage students’ higher-order cognitive skills to a sufficient degree.16,17 with asynchronous learning students can have difficulties with using the technology and understanding the content of the learning materials, but they can also experience a lack of motivation for this kind of learning or a lack of concentration.18 the aim of this research study is to compare the effects of learning when internet pages, with digital materials about solutions and dissolution, are used in asynchronous online learning with the effects of learning within a class period held by a teacher at school. experimental based on the aim of this research study, the following null hypothesis was formulated: there is no statistically significant difference between the achievements of the seventh-grade students in solutions and dissolution after the application of internet pages for independent asynchronous online learning and the achievements of the seventh-grade students after faceto-face learning at school. the proposed hypothesis was tested in a pedagogical experiment with parallel groups. the sample the pedagogical experiment was conducted with 187 seventh-grade students (aged 13– –14) from two primary schools in the territory of belgrade. the schools were selected based on the equipment available for the application of ict (computers, projectors and a good internet connection), necessary for the realization of the pedagogical experiment. all seventhgrade students from the selected schools were included in the sample. the students were randomly divided into two groups: group a (n = 94) and group b (n = 93), formed by two classes from each school. the consent for conducting the pedagogical experiment was obtained from the management of both schools. the cooperation agreements were signed by the dean of the university of belgrade – faculty of chemistry, and the school principals. written consents for the participation of students in this research study were obtained from the students’ parents/guardians. the parents/guardians and students were informed in detail about the aim of the research study, the method of its realization and students’ roles in the research study. it was pointed out that the participation is on a voluntary basis and data confidentiality was guaranteed. it was explained to the students that they would not be rewarded for their participation in the research study and that there would be no consequences if they decided to withdraw from the research study. students could withdraw from the research study at any moment. design and procedure the research study was organized during the lesson on solutions and dissolution, the first one within the thematic unit solutions according to the chemistry curriculum for the ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 534 ralevic, tomasevic and trivic seventh grade of primary school. understanding the contents about solutions and dissolution depends on understanding the concepts from the curricular thematic unit the structure of substance. a description of the activities done with each group of students (a and b) in the pedagogical experiment is provided in table i. table i. the plan for the pedagogical experiment implementation activity group a activities group b activities 1. pre-test pre-test 2. asynchronous online learning by independently accessing the internet pages on solutions and dissolution, outside the school face-to-face learning by the application of the internet pages on solutions and dissolution, at school with teacher guidance 3. post-test post-test 4. delayed post-test delayed post-test at the beginning of the research study both groups were tested by a pre-test, which tested the groups’ previous knowledge. students from group a were given a link for accessing the internet pages for independent asynchronous online learning and instructions on how to access the internet pages before their next chemistry lesson. students from group b learned about solutions and dissolution using the same internet pages at school, with teacher guidance. the internet pages used as a teaching material were created in line with the outcomes defined by the seventh grade chemistry curriculum. the teaching material on the internet pages was prepared with the aim of enabling students to understand the process of dissolving at the submicroscopic (particles) level. the content of the internet pages was presented in the textual, visual (images, video recordings and animations) and audio format (narration). the introductory part of the content about solutions and dissolution was given in the textual form, while the other parts were given in the form of educational videos. as a key segment of the internet pages, the educational videos included animations which showed the process of dissolution of an ionic substance and a non-polar substance at submicroscopic level, accompanied by the narration by the author/researcher. video 1 shows the dissolution of an ionic substance in a polar solvent. video 2 shows the dissolution of a non-polar substance in a non-polar solvent. apart from being shown the content of the same internet pages, the students from group b learnt about solutions and dissolution by listening to their teacher talk on this topic during the chemistry class period. after the class period, the students from group b did not have an opportunity to access the internet pages. during the 35-min lesson students listened to their teacher’s lecture which included watching educational videos with the explanations which accompanied the digital material. after that, the teacher checked students’ understanding of the process of dissolving ionic and non-polar substances through a ten-minute conversation with the students. in the next chemistry class period, students from both groups did a post-test. the time available for the completion of the post-test was 45 minutes. a year after the posttest, the groups made up of the same students were tested again. the delayed testing was conducted in order to measure retained learning about solutions and dissolution in each group a year after they had learnt about these concepts. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. asynchronous online learning about dissolution 535 instruments in order to collect data in this research study two tests for knowledge assessment were designed and administered to all students. both tests were paper-pen tests. the pre-test was used to check students’ knowledge of the concepts within the thematic unit the structure of substance, key to understanding the concepts of solutions and dissolution.19 the pre-test had six questions with eight items altogether: three open-ended (short-answer) and five closedended (multiple-choice questions). the questions included the following content: the types of chemical bonds in compounds, chemical formulae of compounds, types of particles in the crystal of an ionic compound and the dipole. the same test was used for the purpose of post-testing and delayed-testing. the test contained eight questions with ten items altogether. six questions were closed-ended questions (two questions, each with two items, were alternate-choice questions; four questions were multiple-choice questions). two questions in the test were open-ended (essay-type) questions. four questions tested students’ understanding of the dissolution of an ionic substance in a polar solvent. in the test this process was presented by an illustration at the submicroscopic level. the other four questions in the test checked the students’ understanding of the process of dissolving a non-polar substance in a non-polar solvent. an illustration showing this process at the submicroscopic level was given in this part of the test as well. during post-testing, the educational videos about the dissolution of ionic and non-polar substances, which had been used as teaching material on the internet pages, were played the whole time. the students were expected to identify the type of chemical bonding in the solutes and the solvents based on the illustrations given within the questions in the tests and the educational videos played. students were also asked to describe the formation of each solution at the submicroscopic level, considering the type of chemical bond in the solute and in the solvent. video 1 contained the information necessary for responding to items 1a, 1b, 2, 3 and 4 of the test. video 2 contained the information necessary for responding to items 5a, 5b, 6, 7 and 8. the tests were reviewed by the experts in the field of chemical education, who were not involved in their design, in order to assess their validity with respect to the defined aim of the research and the hypothesis. the two chemistry teachers who enabled the realization of the research study and the members of the department of chemical education, the university of belgrade – the faculty of chemistry, examined the content of the tests, upon which the necessary revisions of the tests were made. results and discussion the results obtained in the tests are presented below. the distribution of the results in the pre-test, post-test and delayed post-test the maximum score which students could achieve in the pre-test was 8 points, while it was 10 points in the post-test and delayed post-test. the characteristics of the distribution of the scores of both student groups are presented in table ii. the following data are presented: the number of students in each group (n), the minimum (min) and maximum (max) number of correct answers in each test, the mean (mean), the standard deviation (sd), the contribution of correct answers (c), the skewness value and the kurtosis value. the mean score of group a in the pre-test (5.11) was higher than the mean score of group b (4.83). in the post-test, group b (5.54) was more successful than group a (5.20). the mean ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 536 ralevic, tomasevic and trivic score achieved by group a in the delayed post-test (4.45) was higher than the mean score achieved by group b (4.35). table ii. descriptive statistics of student achievement in the pre-, postand delayed post-test testing group n number of correct answers c / % skewness kurtosis min max mean sd pre-test a 94 1 8 5.11 1.72 63.8 0.013 −0.655 b 93 0 8 4.83 2.16 60.4 −0.338 −0.033 post-test a 94 1 8 5.20 1.61 52.0 −0.226 −0.351 b 93 2 8 5.54 1.46 55.4 −0.162 −0.477 delayed post-test a 94 2 8 4.45 1.52 44.5 0.590 −0.079 b 93 2 8 4.35 1.28 43.5 0.283 0.018 the skewness and kurtosis values were used as the criterion for assessing the normality of the distribution of the obtained results. the obtained values range from −1 to +1, which indicates that the data have a normal distribution. based on this, it was decided to apply the independent-samples t-test in order to determine whether there was a statistically significant difference between the means in groups a and b. the obtained data are presented in table iii. table iii. the value of the t-test for the results of groups a and b value pre-test post-test delayed post-test t 0.97a −1.49a 0.45a p 0.33b 0.14b 0.66b aconfidence interval less than 95 %; bp-value – more than 0.05 the value obtained by the t-test shows that there is no statistically significant difference between the mean scores achieved by groups a and b in the pre-test. based on this, it was concluded that both groups of students were at the same level regarding their previously acquired knowledge. there were no statistically significant differences between the mean scores achieved by groups a and b in the post-test and the delayed post-test. it can be concluded based on the obtained results that the application of the internet pages on solutions and dissolution for independent asynchronous online learning outside the school environment contributes to similar achievements as face-to-face learning with the same internet pages in the school environment with teacher guidance. these results are consistent with previous research studies, which have shown the same students’ scores after online and face-to-face course in inorganic chemistry20 and after introductory chemistry lectures and laboratories.21 student achievement in the pre-test questions table iv shows the number of correct answers (frequency, f) expressed as the contribution of correct answers given by the students from both groups to the ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. asynchronous online learning about dissolution 537 pre-test items. the values of the t-test, used for assessing the statistical significance of the difference and the p-values (p) are presented. table iv. the pre-test results in groups a and b item group a group b t-test p f contribution, % f contribution, % a–b 1 69 73.4 65 69.9 0.53a 0.597b 2a 30 31.9 27 29.0 0.43a 0.671b 2b 37 39.4 25 26.9 1.82a 0.070b 2c 36 38.3 30 32.3 0.86a 0.390b 3 68 72.3 63 67.7 0.68a 0.495b 4 85 90.4 82 88.2 0.50a 0.620b 5 89 94.7 81 87.1 1.81a 0.073b 6 66 70.2 76 81.7 −1.85a 0.066b aconfidence interval less than 95 %; bp-value – more than 0.05 in group a, the contribution of correct answers to individual pre-test items ranged from 31.9 to 94.7 %, while it ranged from 26.9 to 88.2 % in group b. the students from group a were less successful than the students from group b at only one item (item 6). the results obtained by the t-test did not show a statistically significant difference between the contributions of correct answers in the groups for any of the pre-test items. this confirmed that the students from group a and group b had similar previous knowledge of the concepts from the thematic unit the structure of substance. student achievement in the post-test and delayed post-test questions table v shows the number of correct answers (frequency, f) and the contribution of correct answers to the items of the post-test and delayed post-test in both groups. table v. the results of the post-test and delayed post-test in groups a and b; number of correct answers (f) and the contribution of correct answers (ca) item post-test delayed post-test group a group b t-test p group a group b t-test p f ca / % f ca / % a–b f ca / % f ca / % a–b 1a 87 92.6 89 95.7 −0.91 .363 63 67.0 66 71.0 −0.58 .562 1b 84 89.4 91 97.8 −2.40a .018 83 88.3 86 92.5 −0.96 .336 2 45 47.9 38 40.9 0.96 .337 28 29.8 26 28.0 0.28 .784 3 36 38.3 34 36.6 0.24 .807 31 33.0 20 21.5 1.77 .079 4 6 6.4 0 0.0 2.52a .013 0 0.0 1 1.1 −1.0 .316 5a 82 87.2 80 86.0 0.24 .809 61 64.9 75 80.6 −2.45a .015 5b 82 87.2 86 92.5 −1.18 .238 74 78.7 76 81.7 −0.51 .609 6 26 27.7 42 45.2 −2.52a .013 45 47.9 31 33.3 2.04a .043 7 41 43.6 55 59.1 −2.14a .034 31 33.0 24 25.8 1.07 .284 8 0 0.0 0 0.0 0 2 2.1 0 0.0 1.41 .159 athe difference in the contributions of correct answers by groups a and b statistically significant at the level p < 0.05 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 538 ralevic, tomasevic and trivic it also shows the values obtained by the t-test, which was used to assess the statistical significance of the difference between the contributions of correct answers achieved in groups a and b, as well as the p-values for the given values of the t-test. in the post-test, the contribution of correct answers to individual items ranged from 0 to 92.6 % in group a, and from 0 to 97.8 % in group b. the students in group a had the larger number of correct answers to four items, while the students in group b had the larger number of correct answers to five items. a statistically significant difference between the contributions of correct answers given by groups a and b was found for four post-test items. the students in group a were statistically significantly more successful at one post-test item (item 4). the students from group b were statistically significantly more successful at three items (1b, 6 and 7). the results of the post-test show that, after seeing videos 1 and 2 (the content of the internet pages), over 86 % of the students were able to differentiate between the models of the solute particles and the models of the solvent particles. in one of the four items which related to differentiating between the ions of the solute and the molecules of the polar solvent and between the molecules of a non-polar substance and non-polar molecules of the solvent, the students from group b achieved a statistically significantly higher contribution of correct answers. in this item, students were asked to identify the models showing polar molecules of the solvent (item 1b). during the learning process, the students from group b watched the models of the presented solvent particles (video 1) while listening to their teacher who explained the models showing the particles. in this case, watching and listening to the content turned out to be more effective than watching and reading.22 a slightly higher contribution of students from group a identified the ionic bond in the dissolved substance, but a statistically significantly higher contribution of students from group b identified the non-polar covalent bond in the solute molecules (item 7). since students from group b were more successful at recognizing the type of particles which make up the solute (item 6), they were expectedly more successful at identifying the type of chemical bond in the solute molecules. explaining the dissolution process at the submicroscopic level presented the biggest problem for both groups. the process of dissolving both ionic and non-polar covalent compounds was presented using particle models. the explanation of the dissolution of an ionic substance in a polar solvent was provided by only 6 students from group a (item 4). none of the students provided an explanation of the dissolution of a non-polar covalent substance in a non-polar solvent. it can be concluded, based on the obtained results, that the largest number of students could differentiate between the solute and solvent particles in the watched educational videos, but fewer than half of the students named the types ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. asynchronous online learning about dissolution 539 of particles and the type of chemical bond in the solute based on the models. the representations of ion models and models of polar and non-polar molecules did not contribute to a better understanding of the dissolution process at the submicroscopic level. these results are consistent with the previous research studies which had shown that students found it difficult to interpret and explain observations of phenomena and to relate them to the models of submicroscopic level of matter.23 therefore, it is always necessary to develop learning practices in chemistry which emphasise representations of the submicroscopic level. the contribution of correct answers to the individual items in the delayed post-test ranged from 0 to 88.3 % in group a and from 0 to 92.5 % in group b. the total number of correct answers was lower in both groups in the delayed post-test compared to the post-test. in the delayed post-test, students were also most successful at differentiating between the models of solute and solvent particles (the contribution of correct answers was higher than 65 %). in both groups, less than one third of students could successfully identify the type of solute and solvent particles. both groups were more successful at identifying the type of particle in the case of a non-polar solute than in the case of an ionic solute. there were more correct answers to both items in group a, but the difference is statistically significant for the identification of the particles of the non-polar covalent substance. the students from group a were more successful than the students from group b at identifying the type of chemical bonding in the solutes. it can be observed for group a that the contribution of students who correctly named the ionic bond is higher than the contribution of students who identified an ion as the type of solute particle. in the delayed post-testing, none of the students from group a explained the process of dissolution of a polar substance in a polar solvent. the situation is slightly different with the explanation of the dissolution of a non-polar substance. two students from group a explained the dissolution process and none of the students from group b. the insight into the retention of the acquired knowledge based on the delayed post-test results indicates that the watched videos enabled more than two thirds of the students to be successful at differentiating the model of solute particles from the model of solvent particles. group a was more successful at identifying the type of particles and chemical bond in the solute, while both groups were more successful at identifying the type of particle and chemical bond in the case of a non-polar substance. the presented results show that the digital material, created for the acquisition of the concepts of solutions and dissolution and posted on the internet pages, can be used both in the conditions of regular school education and for independent asynchronous online learning. online distribution of digital materials for independent learning can be an additional method within the usual realization of the teaching process, but the fact that it can be used as a particularly use________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 540 ralevic, tomasevic and trivic ful teaching method when face-to-face teaching is not possible, as was the case in the conditions caused by the covid-19 pandemic, is particularly significant. conclusion the conducted research study is a pilot study of the effects of the internet pages with contents about solutions and dissolution on the seventh-grade students’ achievements when the pages are used for independent asynchronous online learning (student group a) and when they are used in the class with explanations provided by the teacher (student group b). according to the pre-test results, groups a and b were at the same level as far as their previous knowledge was concerned. the post-test and delayed post-test results showed that there was not a statistically significant difference between the overall achievements of the students who had studied independently asynchronously online and the ones who had studied in the face-to-face instruction. these results confirmed the proposed null hypothesis that the seventh-grade students’ achievements in solutions and dissolution do not statistically significantly differ after the application of the internet pages for independent asynchronous online learning outside their school and face-to-face learning at school. in both groups, the achievements in the delayed post-test were lower than in the post-test. there was not a statistically significant difference between the mean values achieved by the groups in the delayed post-test. this indicates that the retention of knowledge acquired by asynchronous online learning is similar to the retention of knowledge acquired with teacher guidance. even though the difference between the achievements of the two groups is not statistically significant, it is important to point out that group a, which experienced asynchronous online learning, had a worse score than group b in the post-test, while it had a slightly better score in the delayed post-test. this suggests the stability of the knowledge acquired by independent asynchronous online learning. apart from the overall results, the results achieved in individual items of the post-test and the delayed post-test were analyzed for both kinds of learning immediately upon learning and a year after learning. it was established that the application of educational videos (videos 1 and 2), in which the dissolution processes are presented through the models of solute and solvent particles, had enabled the largest number of students to identify the models of solute and solvent particles. better achievements of the students from group b in these items indicate that additional teacher narration with explanations (which were given through images and texts in the educational videos) had enabled students to better identify the presented models of particles. a relevant implication of this research is that in the preparation of the material/videos for asynchronous online learning, textual explanations should also be given in the audio format. compared to identifying the models of solute and solvent particles, a smaller number of students ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. asynchronous online learning about dissolution 541 correctly identified the types of solute particles and the type of chemical bond in the solute and the solvent. in the delayed post-test both groups were more successful at identifying the type of particle and chemical bond in the case of the dissolution of a non-polar covalent substance. the results achieved within individual items in both groups show that students find it difficult to explain the process of dissolution by relating it to the models at submicroscopic level of matter. one way of dealing with these difficulties could be a wider and constant application of the representations of the submicroscopic level in the chemistry teaching/learning practice. the results obtained within this research study are important for the situations when it will be necessary to make decisions about the methods of chemistry teaching/learning due to various circumstances. since the learning outcomes were equivalent for both teaching/learning approaches we have investigated, the application of the independent asynchronous online learning outside the school environment can be recommended in the conditions when it is not possible to organize face-to-face learning with teacher guidance in school classes. in the context of the current limitations to the realization of face-to-face teaching/learning due to the coronavirus pandemic, this conclusion has an immediate and current application. furthermore, it is relevant for some future situations in which, for various reasons, it might be estimated that pedagogic teacher-student interaction at school environment is not possible. the main limitation of this research study is the size of the sample. the number of students participating in the research study is not sufficient to make generalizations. the choice of schools which participated in the research study was influenced by the it equipment available since it was essential for conducting the pedagogical experiment. in addition to this, the duration of the study is relatively short and limited to the realization of one lesson. the future research studies should focus on the comparison of the investigated approaches when some other chemistry contents are introduced and elaborated. it should be pointed out that students from group a were new to independent online asynchronous learning as a teaching/learning method. in their learning experience prior to the conducted research study, the students had never participated in any activities which included the above-mentioned approach, so a certain adjustment was probably necessary. meanwhile, students at all educational levels have gained new experience of online teaching/ /learning. this experience will help overcome some potential problems in conducting new research studies which will aim at comparing the efficiency of online and face-to-face teaching/learning. acknowledgement. ministry of education, science and technological development of republic of serbia contract number: 451-03-9/2021-14/ 200168. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 542 ralevic, tomasevic and trivic и з в о д интернет странице за асинхроно “on-line” учење и учење у учионици о растворима и растварању лидија р. ралевић, биљана и. томашевић и драгица д. тривић универзитет у београду – хемијски факултет, студентски трг 12–16, београд последњих деценија on-line комуникација је постала важан део у реализацији образовног процеса. под условима covid-19 пандемије то је било значајно јер се у највећем броју случајева морало прећи на неке од облика on-line наставе/учења. у раду су представљени резултати истраживања, спроведеног као педагошки експеримент са паралелним групама. циљ овог истраживања је био да се упореде ефекти примене интернет страница за самостално on-line асинхроно учење ван школе (група а) и наставе/учења коју реализује наставник у школи (група б). садржај интернет страница припремљен је за усвајање појмова раствори и растварање у седмом разреду основне школе. ефекти примењених приступа испитивани су на основу постигнућа ученика на пост-тесту (непосредно након учења) и на одложеном пост-тесту (годину дана након примењених приступа). у истраживању је учествовало 187 ученика који су у педагошком експерименту учествовали као ученици седмог разреда основне школе, а потом на одложеном посттесту као ученици осмог разреда. резултати су показали да не постоји статистички значајна разлика између укупних постигнућа ученика који су појмове раствор и растварање усвајали путем самосталног асинхроног on-line учења и наставе/учења коју у школи реализује наставник. то имплицира да је могуће под условима који не дозвољавају организовање наставе у школи, асинхроним онлајн учењем постићи резултате сличне резултатима наставе у школи. 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k. faulconer, j. c. griffith, b. l. wood, s. acharyya, d. l. roberts, chem. educ. res. pract. 19 (2018) 392 (https://doi.org/10.1039/c7rp00173h) 22. r. mayer, in the cambridge handbook of multimedia learning, r. mayer, ed., cambridge university press, cambridge, 2014, p. 43 (https://doi.org/10.1017/cbo9781139547369) 23. a. berg, d. orraryd, a. jahic pettersson, m. hulten, chem. educ. res. pract. 20 (2019) 710 (https://doi.org/10.1039/c8rp00288f). ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. @article{ralevic2022, author = {{ralevic #}, lidija r and tomasevic, biljana i and trivic, dragica d}, journal = {journal of the serbian chemical society}, title = {{internet pages for asynchronous online and face-to-face learning about solutions and dissolution}}, year = {2022}, issn = {1820-7421}, month = {apr}, number = {4}, pages = {531--543}, volume = {87}, abstract = {in the last decades online communication has become an important part of the realization of the educational process. in the conditions caused by the covid-19 pandemic it has become particularly significant since in most cases it was necessary to switch to some forms of online teaching-learning. this paper presents the results of a research study conducted as a pedagogical experiment with parallel groups. the aim of this research study was to compare the effects of the application of internet pages for independent online asyn­chronous learning outside the school environment (group a) and face-to-face learning realized by a teacher at school (group b). the content of the internet pages was created in order to enable the acquisition of the concepts of solutions and dissolution. the effects of the approaches applied were studied based on the student achievement in a post-test (immediately upon learning about the concepts of solutions and dissolution) and in a delayed post-test (a year after the acquisition of these con­cepts). the participants in this research study were 187 primary school students, who participated in the pedagogical experiment when they were in the seventh grade, while they were in the eighth grade when they did the delayed post-test. the results showed that there was not a statis­tically significant difference between the overall achievements of the students who learnt about the concepts of solutions and dissolution by independent asynchronous online learning and face-to-face learning at school. this implies that the similar results can be achieved with asynchronous online learning as with face-to-face learning when the conditions do not allow school-based eduion.}, doi = {10.2298/jsc210804060r}, file = {::;:09_11026_5539.pdf:pdf}, keywords = {digital materials, educational video, submicroscopic level, teaching}, publisher = {serbian chemical society}, url = {https://www.shd-pub.org.rs/index.php/jscs/article/view/11026}, } simple one-pot synthesis of thioureas from amines, carbon disulfide and oxidants in water j. serb. chem. soc. 81 (3) 219–231 (2016) udc 547.496.3+542.913:547.857.8’279.3: jscs–4841 541.124 original scientific paper 219 simple one-pot synthesis of thioureas from amines, carbon disulfide and oxidants in water milutin m. milosavljević1, ivan m. vukićević1, saša ž. drmanić2#, jasmina b. nikolić2*#, aleksandar d. marinković2#, sanja s. krstić3 and slobodan d. petrović2# 1faculty of technical science, university of priština, knjaza miloša 7, 38220 kosovska mitrovica, serbia, 2faculty of technology and metallurgy, university of belgrade, karnegijeva 4, p. o. box 3503, 11120 belgrade, serbia and 3institute of nuclear sciences vinča, laboratory for material science, university of belgrade, p. o box 522, mike alasa 14, 11001 belgrade, serbia (received 31 august, revised 27 october, accepted 30 october 2015) abstract: the present study reports a new facile methodology for the synthesis of symmetrical and asymmetrical thioureas by a one-pot reaction of the required amine, carbon disulfide and an oxidant, i.e., hydrogen peroxide, ethylenediaminetetraacetic acid (edta)/sodium percarbonate system or air. the structures of the synthesized compounds were confirmed by ir, 1hand 13c -nmr and ms methods. a reaction mechanism is proposed based on the isolation of reaction intermediate and the determination of their structure. the synthetic benefits of the presented methods are reflected in the operational simplicity, mild reaction conditions, short reaction times, solvent recycling, high purity and yield of the products, absence of dangerous by-products and technological applicability on an industrial scale. considering the commercial importance of thioureas, it should be emphasized that implementation of the optimal synthesis of thiourea determined in this study for industrial production could provide a concurrent alternative to the existing technologies in use. keywords: hydrogen peroxide; percarbonate; air; reaction mechanism. introduction thiourea is a compound that contains sulfur and nitrogen atoms, which make it susceptible to oxidation by a large number of oxidants, resulting in different products including ureas, sulfur oxides and nitrogen. such sulfur-containing compounds have a special importance for industrial applications. it has been extensively employed in various fields of science, industry and technology such * corresponding author. e-mail: jasmina@tmf.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc150831087m _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 220 milosavljević et al. as in the rubber industry,1 for agricultural applications,2,3 as a substance that improves the ripening of certain fruit species,4 in analytical chemistry,5,6 as a photocatalyst,7 in the electronic industry, including electronic modulators and electro-acoustic devices,8 electro-optic components9 and polarization filters.10 however, thiourea is a well-known environmental organic pollutant, with carcinogenic consequences11 and presents a serious allergenic component.12 moreover, thiourea induces hypothyroidism,13 causes the damage to pulmonary endothelial cells and possibly mesothelial lining cells in animal organisms.14 in addition, it was shown that thioureas inhibit nitrification in soil and water.15 thiourea is also used in the production of thiourea derivatives16 and also as an additive for plastic materials.17 in organic chemistry, it is used for the preparation of heterocycles.18 depending on the substituents, this heterocycle possesses anthelmintic, antibiotic and immunosuppressant activities.19,20 when thioureas are oxidized, various products are obtained depending on the structure of the starting compound, oxidizing agent and ph.21 oxidation using hydrogen peroxide in the presence of a ruthenium complex led to formamidine disulphide, thiourea trioxide and sulfate.22 recent research indicated that some 2aminothiazoline derivatives of thiourea are inhibitors of enzymes, such as kinurenine-3-hydroxylase,23 or possess inhibitor activity against the enzyme cyclin-dependent kinase.24 certain 2-aminothiazole derivatives of 4-hydroxychromen-2-one were obtained and the first step was the synthesis of n-alkyl(aryl) and n,n-dialkyl thioureas from ammonium isothiocyanate and an appropriate amine in water at 80−90 °c.25 moreover, n,n,n′-trialkylthioureas may be prepared from a dialkylamine and carbon disulfide in the presence of sodium hydroxide.26 as described in previous works, there are common routes in which the synthesis of substituted thioureas involve reactions of anilines with sodium or ammonium thiocyanate in the presence of strong acids, trifluoroacetic acid (tfa) or concentrated hcl, aroyl isothiocyanates with amines followed by basic hydrolysis, silicon tetraisothiocyanate with primary and secondary amines, and unsubstituted thioureas with primary alkyl amines at 170–180 °c.27 in addition, thiourea could be obtained by reaction of isothiocyanates with ammonia or amines,28 primary amines with carbon disulfide, aqueous ammonia and the presence of mercury acetate,29 and disubstituted cyanamides with hydrogen chloride and lialhsh30 or hydrogen sulfide in the presence of ammonia.31 recently, a new and efficient reagent, 1-benzotriazole-1-carbothioamide, was used for the preparation of mono and n,n-disubstituted thioureas. benzotriazole-1-carbothioamide was prepared in 84 % yield from 1-cyanobenzotriazole in 1,2-dimethoxyethane (dme) saturated with gaseous hydrogen sulfide.32 a series of n-aroyl-n′-substituted thiourea derivatives was prepared in good to excellent yields under the condition of solid–liquid phase transfer catalysis using poly(ethylene glycol)-400 (peg-400) as the catalyst.33 the promoting _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. synthesis of thioureas from amine, carbon disulfide and oxidants in water 221 effects of some compounds on wheat growth have been tested preliminarily. in addition, n-benzoyl-n′-carboxy-substituted thiourea derivatives were synthesized by the reaction of benzoyl isothiocyanate with amino acids. the reaction conditions were experimentally investigated and the preliminary biological tests showed that some of the compounds had excellent plant growth promotion activities.34 carbamoyl isothiocyanates can be used for the synthesis of n,n′-disubstituted and n,n,n′-trisubstituted thiourea derivatives in the reaction with alkyl or aryl amines. these reagents make purification trivial, without the subsequent inclusion of a protection step. the carbamate increases the reactivity of the isothiocyanate, permitting the formation of thiourea even with hindered amines. a second amine can be coupled to the carbamoyl thiourea using edc (1-ethyl-3-(3 -(dimethylamino)propyl)carbodiimide) whereby 1,3-disubstituted and 1,1,3-trisubstituted guanidines were obtained through either stepwise or one-pot synthesis.35 several thiourea and urea derivatives were prepared by the reaction of 4-aminopyrazoles with substituted isothiocyanates or isocyanates in acetone.36 the new compounds were isolated in satisfactory yields (42–70 %). however, the reported methodologies of thiourea syntheses have some drawbacks that demand the development of efficient and eco-friendly methods applicable on an industrial production level. the syntheses of symmetrical and asymmetrical thioureas by a one-pot reaction of the appropriate amine, carbon disulfide and an oxidant, i.e., hydrogen peroxide, the ethylenediaminetetraacetic acid (edta)/sodium percarbonate system or air, was investigated in this work. the mechanism of thiourea synthesis was established based on the isolation and identification of the reaction intermediates. this study represents a practical extension of on-going research into new methods for the synthesis of sulfur-related compounds in a convenient reaction medium.37–39 the proposed method is characterized by operational simplicity, high purity and yields of the obtained products, recycling of the solvents and absence of dangerous by-products in the effluent water. there are numerous operationally simple catalytic synthesis methods performed in water as the reaction medium.40,41 this fact was especially important considering that potentially dangerous carbon disulfide was used as a reagent. experimental materials all materials used for synthesis of symmetrical and asymmetrical thioureas were provided by sigma–aldrich, and used without purification. instrumental methods the mass spectra were obtained on a thermo finnigan polaris q ion trap mass spectrometer, including tracegc 2000 (thermo finnigan corp., austin, tx, usa). a polaris q ion trap gc/ms system with electron ionization (ei), and direct insertion probe (dip) tech_________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 222 milosavljević et al. niques were used. the dip mode was used to introduce the sample and the ei-ms technique to acquire the spectra. the ionization conditions were as follows: ion source temperature 200 °c, maximum energy of electron excitation 70 ev, corona current 150 μa. the obtained data were processed using xcaliburtm 1.3 software. the 1hand 13c-nmr measurements were performed on a varian gemini 2000 (200/50 mhz) instrument at 25 °c. chemical shifts (δ) are reported in part per million (ppm) relative to tetramethylsilane (δh = 0 ppm) for 1h-nmr, and to dimethyl sulfoxide (δc = 39.5 ppm) for 13c-nmr, using the residual solvent peak as a reference standard. the fourier-transform infrared (ftir) spectra were recorded in the transmission mode using a bomem mb-series spectrometer (hartmann & braun). elemental analysis was realized on the vario el iii elemental analyzer, and the results of analysis were in good agreement with the theoretical values (±0.2 %). high performance liquid chromatograph (hplc) was performed on spectra system p4000 equipped with a uv detector (240 nm) and a zorbax sb-c8 column. benzene:methanol (hplc grade, 9:1) was used as the mobile phase at a flow rate of 0.8 ml min-1 in the isocratic operational mode. the data on the characterization of the synthesized compounds are given in supplementary material to this paper. general procedure for the synthesis of symmetric (s) thioureas using hydrogen peroxide as the oxidant, exemplified by the preparation of n,n,n′,n′-tetraethylthiourea – method s-m1 in a 500 cm3 three necked round bottom flask equipped with a reflux condenser, dropping funnel, thermometer and magnetic stirrer were added 110 cm3 of water and 34.0 cm3 (0.32 mol) of 98.0 % diethylamine. subsequently, 10.2 cm3 (0.16 mol) of 98.0 % carbon disulphide (water was added as a top layer to prevent pressure development above the carbon disulfide in the dropping funnel) was added over 1 h under efficient stirring and maintaining the temperature of the reaction mixture between 35 and 40 °c. oxidation of diethylammonium salt of diethylcarbamodithioic acid was performed by addition of 46.40 cm3 of hydrogen peroxide solution (0.18 mol; a 13.2 % solution was prepared by dissolution of 178.6 cm3 (2.08 mol) of 35.0 % hydrogen peroxide in 406.5 cm3 of deionized water) keeping the reaction temperature in the range 40–45 °c for 1 h. formation of the suspended product was observed during the addition of the hydrogen peroxide solution. the reaction product was subjected to vacuum for 5 min (10 kpa) and then filtered using a vacuum filter, whereby thiourea and sulfur were obtained as the filtration cake. the obtained material was mixed with a 1:1 mixture of ethanol and dichloromethane and the suspension was filtered to give 4.8 g of sulfur and a filtrate containing the thiourea. after solvent evaporation, the pure product was obtained by recrystallization from methanol followed by column chromatography (silica gel 60, 230–400 mesh) using methanol/dimethylformamide (9:1) as the mobile phase. after solvent evaporation and product drying at 50 °c for 10 h, 27.60 g of n,n,n′,n′-tetraethylthiourea (82 %)were obtained, m.p.: 76–78 °c (lit.: 78 °c42). the purity of the product was determined by gc analysis (99.0 %). all other symmetrical thioureas were synthesized in an analogous manner to the described method s-m1, and the results of the syntheses are presented in table i. analogous methodologies were performed for symmetric thiourea synthesis in the presence of the edta/percarbonate system, i.e., in situ generated peracetic acid, method sm2, and air, method s-m3. the procedure according to method s-m3 was performed in a mildly pressurized system to provide a higher oxygen concentration in a reaction medium. results of thioureas syntheses according to methods s-m2 and s-m3 are presented in table i. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. synthesis of thioureas from amine, carbon disulfide and oxidants in water 223 table i. yields (%) and purities (hplc method, %) of the thioureas obtained by different synthesis methods cmpd. methoda yield/purity method yield/purity method yield/purity 1a as′-m1 82/99.2 as′-m2 88/99.1 as′-m3 42/99.0 1b s-m1 72/99.1 s-m2 79/99.5 s-m3 39/98.9 2a as′-m1 73/99.0 as′-m2 76/99.4 as′-m3 33/98.7 2b as′-m1 71/99.1 as′-m2 72/99.3 as′-m3 36/98.8 2c as′-m1 62/99.5 as′-m2 69/99.2 as′-m3 39/99.0 3a s-m1 63/99.3 s-m2 71/99.4 s-m3 38/99.0 3b s-m1 64/99.2 s-m2 69/98.9 s-m3 34/98.9 3c s-m1 65/99.0 s-m2 74/98.8 s-m3 41/99.1 3d s-m1 62/98.9 s-m2 70/98.7 s-m3 40/99.2 3e s-m1 68/98.8 s-m2 71/98.8 s-m3 33/99.3 4a as′-m1 57/98.9 as′-m2 62/98.8 as′-m3 36/99.0 4b as′-m1 71/99.0 as′-m2 75/98.9 as′-m3 34/99.2 4c as′-m1 70/98.8 as′-m2 73/99.0 as′-m3 42/99.3 4d as′-m1 66/98.9 as′-m2 69/99.1 as′-m3 40/99.0 as and as designate methods used for the synthesis of symmetric and asymmetric thioureas, respectively general procedure for the synthesis of asymmetric (as) thioureas using hydrogen peroxide as the oxidant, exemplified by the preparation of n,n,n′-triethylthiourea – method as-m1 to a 500 cm3 three necked round bottom flask equipped with a reflux condenser, dropping funnel, thermometer and magnetic stirrer were added 110 cm3 of water and 17.0 cm3 (0.16 mol) of 98.0 % diethylamine. subsequently, 10.2 cm3 (0.16 mol) of 98.0 % carbon disulphide (water was added as a top layer to prevent pressure development above the carbon disulfide in the dropping funnel) was added over 1 h under efficient stirring and maintaining the temperature of the reaction mixture between 35 and 40 °c. oxidation of diethylammonium salt of ethylcarbamodithioic acid was performed by addition of 23.20 cm3 (0.09 mol; 13.2 %) of hydrogen peroxide maintaining the reaction temperature in the range 40–45 °c for 1 h. the suspended product formed during the addition of the hydrogen peroxide solution. afterwards, 12.55 cm3 (0.16 mol) of 68 % ethylamine and 23.20 cm3 (0.09 mol) of 13.2 % hydrogen peroxide was added into reaction mixture and the temperature kept in the range 40–45 °c for an additional 1 h. reaction product was worked up as described above for the s-m1 method. n,n,n′-triethylthiourea (et2nc(s)nhet) was obtained in 34 % yield (9 g), m.p.: 86–88 °c (lit. 87–88 °c42). the purity of the product was determined by gc analysis (99.0 %). method as′-m1 in a 500 cm3 three-necked round bottom flask equipped with a reflux condenser, dropping funnel, thermometer and magnetic stirrer was added 110 cm3 of water and 34.0 cm3 (0.32 mol) of 98.0 % diethylamine. subsequently, 10.2 cm3 (0.16 mol) of 98.0 % carbon disulphide was added over 1 h under efficient stirring and maintaining the temperature of the reaction mixture between 35 and 40 °c. continuous addition of 15 % hydrochloric acid solution was performed until the ph reached 2, i.e., no precipitation of waxy diethylcarbamodithioic acid was observed. decantation of the supernatant solution, washing of precipitate with distilled water, and addition of 12.55 cm3 (0.16 mol) of 68 % of ethylamine and 23.20 cm3 (0.09 mol) of 13.2 % hydrogen peroxide were followed by maintaining the temperature in the range 40–45 °c for an additional 1 h. purification of reaction product, performed according to _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 224 milosavljević et al. procedure described under method as-m1 gave 23.30 g of n,n,n′-triethylthiourea (88 %; gc purity 99.2 %), m.p.: 86–88 °c (lit. 87–88 °c42). results of asymmetrical thioureas synthesis according to the as′-m1 method are presented in table i. in an analogous manner, the syntheses of the asymmetrical thioureas were performed in presence of the edta/percarbonate system, as′-m2, and air, as′-m3 methods, and the results of these thioureas syntheses are presented in table i. the ms, ftir, 1hand 13c-nmr data for all thioureas synthesized by the abovepresented methods were identical to those given in the literature. results and discussion a new method for the synthesis of symmetrical and asymmetrical thioureas, series 1–4, using monoand di-substituted alkyl and aryl amines, carbon disulfide and different oxidants is presented in scheme 1. three oxidizing agents were used: hydrogen peroxide (method m1), in situ generated peracetic acid (edta/ /percarbonate system) (method m2) and air (method m3). the synthesis of thioureas from alkyl and aryl amines and carbon disulfide without an oxidative agent did not give any appreciable quantity of the thioureas. this result indicates that an oxidant plays a crucial role in the successful synthesis of thioureas. a large number of alkyl amines were used but only dimethylamine, ethylamine and diethylamine gave satisfactory yields of the thioureas. furthermore, a variety of aryl amines: benzylamine (bnnh2) and substituted anilines (x-phnh2) where x was: 2and 4-och3, 4-cl, 4-br, 4-me, 4-et, 4-oet and 4-no2 substituents, were used for the synthesis of thioureas. the reaction occurred in one batch in water as the reaction medium, without the presence of a catalyst and without isolation of the intermediate from the reaction mixture. scheme 1. general method applied for the synthesis of symmetric and asymmetric thioureas. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. synthesis of thioureas from amine, carbon disulfide and oxidants in water 225 a study of the influences of the synthesis parameters on the reaction yield and purity of the thiourea products was performed and the optimal synthesis of the thioureas was established. according to the optimal methods, described in the experimental part, a series of thioureas were obtained and the results are given in table i. based on the results presented in table i, it could be observed that satisfactory yields and purities of the thioureas were obtained according to the optimal methods m1 and m2. the highest yields and purities were obtained using the edta/percarbonate system, somewhat lower with hydrogen peroxide and the lowest using air as the oxidant. longer reaction times, airflow change or air enriched with oxygen did not contribute to appreciable increases in the reaction yields of the thioureas obtained by method m3. moderately higher reaction yields of thioureas were obtained with pure oxygen, around 7–13 %, which indicates that oxygen diffusional transport resistance exists at the gas/liquid interface and in the bulk solution. this means that controllable generation of oxidative species in the homogeneous edta/percarbonate system had a beneficial effect on increasing the reaction yields obtained by method m2. recycling of the residual reaction solution was performed by using water without prior purification. this method offered satisfactory improvement to the overall synthesis method regardless of the heterogeneity of the residual solution and amine oxidation by-products. determination of the amine content, as well as the oxidation potential, in recycled water was used for the calculation of the ratio of the reactants for the design of the subsequent reaction. a lower yield was obtained in second cycle, 5–10 %, while difficulties encountered with purification of the obtained product (purity 85–92 %) were solved by simple purification with active carbon. the purity of the obtained product, after filtration and purification procedure, was 95–98 %, which offers an acceptable alternative for the improvement of the overall production technology. otherwise, the design of water purification was based on simple treatment with calcium hydroxide, heating at 40–50 °c for 15 min and filtration, which produced effluent water that satisfied the criteria prescribed by the epa effluent limitation guidelines for wastewater. the benefits of the use of both technologies will be the subject of detailed techno-economic analysis. according to the results presented in table i, the highest yields were obtained in the synthesis of asymmetric alkylthioureas of 88 % for 1a and 79 % for 1b obtained by using method m2. higher basicity (nucleophilicity) of aliphatic (alkyl)amines contributes to better reactivity in the first reaction step, i.e., nucleophilic addition on thicarbonyl group of carbon disulfide (scheme 2). additionally, a flexible alkyl chain could be easily adapted in the course of transition state formation, in the low energy structure with minimal interactions in transition states, and thus, lower activation energy is a consequence. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 226 milosavljević et al. reaction mechanism synthesis of symmetric thioureas. the reaction mechanism of the synthesis of thioureas in the presence of hydrogen peroxide was studied by the isolation and identification of the reaction intermediates and their characterization by 1h-and 13c-nmr, and gc/ms methods. the obtained results indicated that the synthesis reaction involved three steps. in the first step, at a mole ratio of 1:1, amine a reacts with carbon disulfide giving the corresponding alkyl(aryl) ammonium salt of carbamodithioic acid b (scheme 2; path a). further transformation of b by reaction with amine a at a 2:1 mole ratio of amine:carbon disulfide produces compound b (scheme 2; path b). thus, in the first step, if the mole ratio amine:carbon disulfide was 1:1, half quantity of the present carbon disulfide reacts with the whole amount of present amine producing alkyl(aryl) ammonium salt of carbamodithioic acid. if the mixing was interrupted and the reaction mixture allowed to equilibrate, unreacted carbon disulfide separated at the bottom of the reaction flask. the measured volume of the separated fraction corresponded to 95 % of the extent of reaction (with respect to the formation of compound b). continuation of the mixing, followed by addition of hydrogen peroxide, enabled completion of the reaction producing tetraalkylthiuram disulfide c. based on the presented results, it was necessary to use a two-fold molar excess of amine, relative to carbon disulfide, to provide synthesis of tetraalkylthiuram disulfide (compound d). scheme 2. proposed reaction mechanism for the synthesis of symmetric and asymmetric thioureas in the presence of hydrogen peroxide. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. synthesis of thioureas from amine, carbon disulfide and oxidants in water 227 the second stage of the reaction represents the oxidation of the obtained alkyl(aryl) ammonium salt of carbamodithioic acid, compound b, by hydrogen peroxide, the resulting product being tetraalkylthiuram disulfide, compound c, accompanied with the formation of amine a. in the third step, the released amine a reacts with compound c producing thiourea d, sulfur and the alkyl(aryl) ammonium salt of carbamodithioic acid b. the slow addition of hydrogen peroxide provides oxidation of the alkyl(aryl) ammonium salt of dithiocarbamoic acid to tetraalkylthiuram disulfide, compound c, by maintaining the temperature of reaction mixture in the range 35–50 °c. in such a way of repeating cycles, the reaction successively occurred until complete conversion of compound c to symmetrical thioureas was accomplished. synthesis of asymmetric thioureas. the reaction mechanism of the synthesis of asymmetric thioureas was investigated in an analogous way to that used to study the symmetric ones, considering the complex mixture of asymmetric and symmetric reaction intermediates and thiourea products that could be obtained. the synthesis was performed by reacting amine a and carbon disulfide in a mole ratio 1:1 in the first step of the reaction (scheme 2; path a), producing the alkyl(aryl)ammonium salt of carbamodithioic acid b. in the subsequent step, the addition of an equimolar quantity of amine e to the residual cs2 (scheme 2; path e) was accompanied with the formation of a mixture of symmetric and asymmetric alkyl(aryl) salts of carbamodithioic acid. main product consisted of almost equimolar quantities of salts f and c, as well as a minor fraction of the asymmetric salt (less than 4 % according to hplc analysis). addition of hydrogen peroxide to the reaction mixture (scheme 2; path f) resulted in the oxidation of compounds f and c, producing mainly symmetric thiuram disulfide, as well as a minor fraction of the asymmetric one. the liberated amines a and e exerted heterolytic cleavage of the disulfide bonds in the symmetric thiuram disulfides producing compounds e, h and i, in almost similar amounts as the used amines, both a and e are either monoalkyl or dialkyl amines. in a similar fashion as in the synthesis of symmetric thioureas, the reaction occurred until all the amine and carbon disulfide were exhausted. in the case of the thiourea synthesis in which monoalkyl amine a and dialkyl amine e were used, the obtained product contained the highest quantity of symmetric n,n′-dialkyl thiourea. the asymmetric and symmetric n,n,n′,n′-tetraalkyl thioureas were found in significantly lower quantities. such results suggested that thiourea content was mainly dictated by the amine reactivity, i.e., the nucleophilicity of the amine. in addition, the synthesis of asymmetric thioureas was realized according to an alternative method, i.e., immediate addition of two separate solutions containing alkyl(aryl) ammonium salt of carbamodithioic acids, c and f, were mixed with hydrogen peroxide. on addition of hydrogen peroxide, both salts were oxidized to symmetric and asymmetric thiuram disulfide, and the liberated amines a _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 228 milosavljević et al. and e further successively reacted in the next step with the present thiuram disulfide in a cyclic manner until termination of the reaction. the symmetric and asymmetric thioureas e, h and i were obtained in almost similar amounts as in the previous study. difficulties encountered with the separation and purification of the product mixture to obtain pure asymmetric thiourea indicated the impracticability of this method for the synthesis of asymmetric thioureas. evidence that the reaction occurred according to reaction scheme 2, were demonstrated experimentally in the following manner: the mechanism was proved by the isolation of intermediates and products of the reaction, similarly to methodology applied in previous studies.38,39 analysis of the intermediary products syntheses of symmetric thioureas. following the s-m1 procedure, immediately after addition of amine a, e.g., diethylamine, in the first initial step of the reaction, a sample of the reaction mixture was withdrawn, acidified by addition of dilute hcl (10 vol. %) and in this manner the formation of an insoluble waxy precipitate of diethylcarbamodithioic acid was evidenced (compd. j). the results of 1hand 13c-nmr and ftir characterization of the raw product, as well as hplc analysis showed that the isolated material contained 96 % of compound j. using the unpurified product in the next experiment, i.e., dispersion in appropriate volume of water followed by the addition of hydrogen peroxide and an equimolar quantity of amine a with respect to compound c, a suspension containing sulfur and n,n,n′,n′-tetraalkylthiourea was produced. after filtration and suspension of the obtained filtration cake in ethanol and repeated extraction a solid product that mostly consisted of sulfur and a combined ethanonlic solution containing a tetraalkylthiourea (example: n,n,n′,n′-tetraethylthiourea – compd. 1b) was obtained. synthesis of tetraethylthiuram disulfide (tetd; k) was performed from diethylcarbamodithioic acid and diethylamine and addition of hydrogen peroxide, which confirmed that reaction step (c) produced thiuram disulfide derivatives. the subsequent reaction step (d), i.e., reaction of tetd with diethylamine and hydrogen peroxide yielded n,n,n′,n′-tetraethylthiourea. syntheses of asymmetric thioureas. in a similar manner to that employed in the mechanistic study of symmetric thiourea synthesis, the assumed mechanism of asymmetric thiourea synthesis was studied. after isolation of the water-insoluble diethylcarbamodithioic acid, the unpurified product was used in the subsequent reaction, i.e., dispersion in an appropriate volume of water followed by the addition of hydrogen peroxide and an equimolar ratio of ethylamine, whereby sulfur and n,n,n′-triethylthiourea (1a) were produced. the amount of sulfur, as a product of decomposition of tetd in reaction mixture after filtration, was almost stoichiometrically equal to the value obtained by calculation with respect to the reaction yield. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. synthesis of thioureas from amine, carbon disulfide and oxidants in water 229 accordingly, it could be stated that the satisfactory reaction yields obtained by methods m1–m3 and the simple work-up in the synthesis of thioureas allow the optimized laboratory technology to be implemented on a semiindustrial level. it was also confirmed that the reaction by-product was not present in water, while the determined concentrations of alkyl(aryl)ammonium salt of carbamodithioic acid and tetraalkylthiuram disulfide were under maximum permissible contamination limit. the suggested innovative methods, especially method m2, could be widely used for the synthesis of thioureas on an industrial scale. conclusions the presented work describes the optimal synthesis of thioureas from alkyl and aryl amines, carbon disulfide and three oxidants: hydrogen peroxide, in situ generated peracetic acid (edta/percarbonate) and air. high conversion of the starting materials into the products was achieved using edta/percarbonate (62– –88 %), hydrogen peroxide gave lower yields (57–82 %), while the lowest yields were obtained using air (36–42 %). the synthetic method developed in the laboratory provides a good opportunity for applications at semi-industrial levels. the present innovative method provides a powerful and versatile method for the preparation of symmetrical or asymmetrical thioureas. this method has several unique advantages, such as: simple operation, mild reaction conditions, avoidance of hazardous organic solvents, the use of moderately toxic and inexpensive reagents, short reaction times and high product yields. this environmentally friendly process represents a suitable option to existing methods. supplementary material the data on characterization of the synthesized compounds are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgments. the authors are grateful to the ministry of education, science and technological development of the republic of serbia for financial support (project 172013). и з в о д једноставна једностепена синтеза тиоуреa из амина, угљен-дисулфида и различитих оксиданаса у води милутин м. милосављевић1, иван м. вукићевић1, саша ж. дрманић2, јасмина б. николић2, александар д. маринковић2, сања с. крстић3 и слободан д. петровић2 1факултет техничких наука, универзитет у приштини, књаза милоша 7, 38220 косовска митровица, 2технолошко–металуршки факултет, универзитет у београду, карнегијева 4, п. пр. 3503, 11120 београд и 3институт за нуклеарне науке винча, лабораторија за науку о материјалима, универзитет у београду, п. пр. 522, мике аласа 14, 11001 београд резултати приказани у овом раду дају једноставне методе за синтезу симетричних и асиметричних тиоуреа у једностепеној реакцији из амина, угљен-дисулфида и оксидационих средстава: водоник-пероксида, системa етилендиаминтетрасирћетна киселина/ /натријум-перкарбонат и ваздухa. структура синтетисаних једињења је потврђена на _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 230 milosavljević et al. основу резултата ir, 1hи 13c-nmr спектроскопије и масене спектрометрије. rеакциони механизам је предложен на бази изоловања интермедијера реакције и утврђивања њихове структуре. погодности наведених метода се огледају у једноставности операција, благим реакционим условима, кратким рекационим временима, могућности рециклирања растварача, високог приноса и чистоће производа, одсуства опасних споредних производа и могућности примене освојених технологија на индустријском нивоу производње. разматрајући комерцијални значај тиоуреа, може се нагласити да примена оптималних синтеза тиоуреа, базираних на приказаним методама у овом раду, на индустријском нивоу производње обезбеђује конкурентске алтернативе технологијама које се тренутно примењују у индустријској пракси. 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soleiman-beigi, monatsh. chem. 139 (2008) 927 41. z. taherinia, j. sulfur chem. 35 (2014) 470. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice accepted manuscript this is an early electronic version of an as-received manuscript that hasbeen accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as c. manojmouli, t. y pasha, k. nagaprashant, b. ramesh, noor ul eain, k. a. purushotham, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc221228041m this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc221228041m j. serb. chem. soc.00(0)1-23 (2023) review article jscs–12201 published dd mm, 2023 1 flavonoid derivatives as anticancer moiety and its effect on cancer cell lines: an updated review c. manojmouli*, t. y pasha, k. nagaprashant, b. ramesh, noor ul eain, k. a. purushotham department of pharmaceutical chemistry, sri adichunchanagiri college of pharmacy, adichunchanagiri university, b. g. nagara, karnataka, india (received 28 december 2022; revised 8 february 2023; accepted 15 july 2023) abstract: cancer is now considered the number one leading cause of premature death in industrialized countries. chemotherapy drugs are quite expensive and cause multiple side effects. natural products have been studied in depth for their potential as anticancer agents because of their remarkable chemical variability. among the various natural metabolites, flavonoids are secondary metabolites that are extensively present in nature, have potent anti-cancer properties, have few adverse effects, and also show synergistic benefits. numerous laboratories are diligently investigating the chemistry and biology of novel flavonoid derivatives due to the demand for and worth of these drugs. in this review, we summarized clinical trials of various flavonoids, molecular pathways against various cancer cell lines, and recent updates on the anticancer activity of flavonoid derivatives against various cancer cells synthesized by various methods, more studies are needed to develop the following mentioned flavonoid derivatives as an anticancer drug. keywords: flavonoids; cancer; chemotherapy; molecular targets introduction a significant global issue in terms of human health is cancer. there are many different types of cancer, and each one is associated with an increase in the number of cells in the body. one element of the mechanisms that lead to cancer is cell proliferation and it is distinct from other tumors because of its ability to invade surrounding healthy tissues. the who reports that in 2020, there were 2.30 million new instances of breast cancer, 2.20 million cases of lung cancer, 1.90 million cases of colon and rectal cancer, 1.40 million cases of prostate cancer, 1.25 million cases of skin cancer, and 1.08 million cases of stomach cancer.1–3 anticancer therapies involve several methods, such as surgery, chemotherapy, and radiation, perhaps individually or in combination. however, side effects and * corresponding author e-mail: : manojmouli07@gmail.com, tel no: 9916194182 https://doi.org/10.2298/jsc221228041m a cc ep te d m an us cr ip t mailto:manojmouli07@gmail.com https://doi.org/10.2298/jsc221228041m 2 manojmouli et al. multidrug resistance are two major obstacles to successful cancer treatment because this condition is a complicated issue, and it has been challenging to discover new medicines to combat it.4 cancer drug sales are forecast to reach $223.21 billion in 2022, up from $199.95 billion in 2021, at a compound annual growth rate (cagr) of 11.6%. multiple laboratories are extensively investigating the chemistry and biology of novel anticancer agents due to the demand for and value of these drugs. cytotoxic drugs, many of which are of natural origin, are currently the pillars of anticancer chemotherapy. natural products can be used as important sources for the development of new active molecules that might be used as leads or scaffolds to create novel, highly effective medicines with increased biological activity. drugs that are obtained from natural sources show better anticancer activities with minimal side effects.5 among the various plant metabolites, flavonoids are plants' most significant low molecular weight secondary metabolites. these metabolites have a large number of polyphenolic chemicals, including benzopyran with a substituted keto group on the pyran ring. the configuration, substitution of a hydroxy group, and the number of the hydroxyl group on a parent moiety primarily affect the pharmacokinetics and pharmacological activity. there is a need to examine the relationship between structure and function since flavonoids are directly linked to human dietary components and health.6,7 today, flavonoids are considered a significant ingredient in a wide range of nutraceutical, pharmacological, therapeutic, and cosmetic uses. this is explained by their ability to affect important cellular enzyme activity in addition to their antitumor, anti-inflammatory, antifungal, anti-aging, antiviral, antiallergic, and antioxidant activities.8 flavanols, anthocyanidins, isoflavones, flavones, flavanones, and flavanonols are the subclasses of flavonoids.9 anti-cancer drugs containing flavonoid moiety from the natural source showed minimal side effects and exhibited synergistic activity. as anticancer drugs without undesirable side effects, quercetin, wogonin, kaempferol, silibinin, and apigenin are all recognized.10 luteolin showed synergistic activity with cisplatin against ovarian cancer. quercetin with doxorubicin exhibited a synergistic effect against neuroblastoma and anaplastic osteosarcoma cell lines, quercetin with cisplatin against human mesothelioma cancer cell lines, and quercetin with temodar (temozolomide) against human astrocytoma cell line.11 for the last decade, researchers have mainly focused on synthesizing flavonoid derivatives. in this present review, we have summarized the clinical trials of flavonoids, mode of action, molecular targets for some important flavonoids against various cancer cells, and results of the anticancer activity of different flavonoid derivatives synthesized by the various synthetic method in the past 4 years i.e., between the years 2019-2022, and describe their potential against various cancer cell lines. a cc ep te d m an us cr ip t flavonoids and its anticancer properties 3 fig 1: the basic skeleton of flavonoids and their subclasses mode of action of flavonoids in chemotherapy: flavonoids are believed to be bioactive, safe, and widely available molecules and it shows a wide variety of anticancer activity through various mechanism of action like cell cycle arrest, mutagen inhibition, antiproliferation, inducing programmed cell death, inhibits the formation of new blood vessels (angiogenesis), antioxidation, modulates ros-scavenging enzyme activities, and reversal of multidrug resistance or a combination of these mechanisms. followed by one of the significant drawbacks of anti-cancer agents is the cancer cells' susceptibility to or resistance to chemotherapeutics therapies. flavonoids like kaempferol, quercetin, or morin, exert potent activity to modulate cancer cell chemoresistance and increase the efficacy of chemotherapy by increased programmed cell death or apoptosis and induced cell cycle arrest in both chemo-resistant and sensitive cancer cells.12–15 clinical trials of flavonoid derivatives: clinical trials were undertaken on 64 individuals (18-65years) by zwicker ji et al., (2015-2019) for 56 days to investigate the effectiveness of the drug isoquercetin (3-o-glucoside of quercetin) at two doses (500mg for 28 patients and a cc ep te d m an us cr ip t 4 manojmouli et al. 1000mg for 28 patients) in preventing venous thrombosis (blood clots) in individuals suffering from pancreatic and colorectal cancer by targeting protein disulfide isomerase (pdi). a thiol isomerase called protein disulfide isomerase (pdi), is released by vascular cells and is essential for thrombus development. at 500mg, constipation affected one patient, diarrhea affected four patients, hyponatremia affected one patient, epistaxis affects one patient, and nausea affected two patients. at 1000mg, only one patient was affected by gastrointestinal reflux.16 clinical trials were performed by the university of minnesota to determine the effectiveness of purple grape juice (rich in flavonoids) in improving vascular health in pediatric cancer survivors to determine the effect of purple color grape juice on endothelial function and biomarkers of vascular and systemic oxidative stress. twenty-four individuals between the ages of 10 and 30 volunteered in the clinical studies.17 the university of hohenheim, in collaboration with university hospital tuebingen and quercegen pharmaceuticals, conducted clinical trials to examine the efficiency of genistein (isoflavonoid) and quercetin (flavonoid), polyphenolic phytochemicals in comparison with placebo on the rate of increase in prostatespecific antigen (psa). analyzing malondialdehyde and protein carbonyl as markers of oxidative status as well as assessing the prevalence of prostate cancer are the secondary goals.18 the effectiveness of quercetin in preventing and treating chemotherapyinduced oral mucositis in blood cancer patients was investigated by pegah mosannen mozafari of mashhad university of medical sciences. they give 250 mg of quercetin capsules to 10 patients in the case group and give a placebo to 10 patients in the control group containing lactose. to determine the onset and severity of oral mucositis, patients underwent examinations every other day.19 fenugreek seeds contain high concentrations of saponins and flavonoids, which are known to reduce blood lipid levels and improve insulin sensitivity. with the main goal of evaluating the decrease in ovarian volume and a decrease in the number of ovarian cysts, dr. amrita sarkari jaipuriar, ms, and garg hospital, goalghar conducted a clinical trial to examine the effectiveness of fenugreek seeds extract in patients with polycystic ovary syndrome.20 clinical trials were conducted by philip diaz of ohio state university to determine whether green tea (rich in flavonoids), may reduce the chance of developing certain cancers. the primary and secondary objective of this clinical trial is to determine the free radical scavenging and measuring nf-kappabinducing kinase by giving 4 cups of green tea for 6 weeks to patients.21 brigham, joann e, manson m d, and women's hospital conducted clinical trials by giving 2 cocoa extract capsules (containing 500mg of flavanols, 80 mg of epicatechin, and 50mg of theobromine) as a dietary supplement to evaluate a cc ep te d m an us cr ip t flavonoids and its anticancer properties 5 whether cocoa extract decreases the risk of cardiovascular diseases and cancer by reviewing the various reports like pathology, surgical, operative, and diagnostic review of both inpatients and outpatients.22 the results of the above-mentioned clinical trials were summarized in table no. 1, in that some of them not disclosed their results of clinical trials. table 1: clinical trials data of flavonoids: drugs/nct no. target results isoquercetin/ nct02195232 protein disulfide isomerase (pdi) d-dimer plasma concentration (decrease, median value= -21.9%/p=0.0002). ↑no venous thromboembolism events. protein disulfide isomerase (pdi) inhibitory activity (37% at 500mg & 73.3% at 1000mg). ↓ platelet-dependent thrombin generation (median value = -31.1% at 500mg & -57.2% at 1000mg). ↓circulation of soluble platelet selectin at 1000mg. purple grape juice & apple juice/nct01043939 endothelial function and biomarkers of vascular and systemic oxidative stress (oxidized low-density lipoprotein, myeloperoxidase, high sensitivity c-reactive protein. enhances the antioxidant activity reduces the oxidation of low-density lipoprotein improves vasodilation quercetin and genistein/ nct01538316 prostate-specific antigen (psa) no results quercetin tablets/ nct01732393 chemotherapy induced oral mucositis no results furocyst (fenugreek seed extract)/ nct02789488 reduction in ovary volume no results green tea/ nct01162642 scavenging of free radicals and nf-kappab-inducing kinase no results cocoa extract/ nct02422745 cardiovascular events and invasive cancer no results a cc ep te d m an us cr ip t 6 manojmouli et al. table 2: list of various molecular targeted pathways for flavonoids against various cancer cell lines: flavonoids quercetin cancer cell lines mcf-7, cne2, hk1, hl-60, hpb-all, and scc-9 cancer cell lines targeted molecular pathway extracellular signal-regulated kinase (erk), phosphoinositide 3 kinase target of rapamycin (pi3k/akt/mtor), mitochondrial and caspase cascade pathways. ic50 values mcf-7=28 µm, hk1= 24 µm, hpb-all=18 µm references 23 kaempferol cancer cell lines panc-1 and mia paca-2 cancer cell lines. targeted molecular pathway phosphoinositide 3 kinase target of rapamycin (pi3k/akt/mtor) signaling pathway cancer cell lines panc-1 =78.75 µm and mia paca-2 =79.07 µm references 24 myricetin cancer cell lines du145, pc3, hct-15, ht-29, a549, ovcar-3, t24, paca-2, panc1, skov3, mcf-7 and hepg2 cell lines. targeted molecular pathway nuclear factor kappa b (nf-κb), nuclear factor erythroid 2– related factor 2 (nrf2), phosphoinositide 3 kinase target of rapamycin (pi3k/akt/mtor), janus kinase/signal transducers and activators of transcription (jak/stat), and canonical wnt (wnt/β-catenin) pathways. cancer cell lines ht-29=47.6µm, and mcf-7=50µm references 27 a cc ep te d m an us cr ip t flavonoids and its anticancer properties 7 rutin cancer cell lines mcf-7, mda-mb-231, a549, ht-29, sw480 cancer cell lines. targeted molecular pathway nuclear factor kappa b (nf-κb), nuclear factor erythroid 2–related factor 2 (nrf2), extracellular signal-regulated kinase (erk), c-jun nterminal kinases (jnk), and p38 mitogen-activated protein kinases (p38 mapk) pathways. cancer cell lines mda-mb-231=40 µm, ht-29=46 µm, and sw480= 54 µm. `references 25,26 luteolin cancer cell lines hct15, mv4-11, co115, and mcf-7 cancer cell lines. targeted molecular pathway nuclear factor kappa b (nf-κb), reactive oxygen species (ros), canonical wnt (wnt/β-catenin), dna topoisomerases and heat shock protein 90, e-cadherin, mammalian target of rapamycin (mtor), integrin β1 and focal adhesion kinase, phosphoinositide-3-kinase (p13k) pathways. cancer cell lines hct15=68 µm, mv4-11= 58 µm, co115=66 µm, and mcf-7=36 µm `references 28 a cc ep te d m an us cr ip t 8 manojmouli et al. isorhamnetin cancer cell lines bt474, bt-549, mda-mb-231, panc-1, mcf7, t47d, and mdamb-468 cancer cell lines. targeted molecular pathway mitochondria-dependent intrinsic, phosphoinositide 3 kinase target of rapamycin (pi3k/akt/mtor), mitogen-activated protein kinase (mapk) pathways. cancer cell lines bt474=10 µm, bt-549=10 µm, mb-231=16 µm, panc-1= 26.5µm. `references 29–31 galangin cancer cell lines mcf-7, t47d, hs578t, amn-3, cp70, ovac-3, hela, tu212, hep-2, hct-15, ht-29, a549, b16f10, eca9760 te-1 cancer cell lines. targeted molecular pathway activated protein kinase (ampk), phosphoinositide 3 kinase target of rapamycin (pi3k/akt/mtor), poly (adp-ribose) polymerase-1 pathways. cancer cell lines mcf-7=20 µm, t47d=24 µm, hs578t=11 µm, ovac-3=34.5 µm, hela=50 µm, tu212=10 µm. `references 32 wogonin cancer cell lines sw-480, a549, hct-116, bt-549, and mcf-7 cancer cell lines. targeted molecular pathway mitogen-activated protein kinase 1 (mek1), nuclear factor kappa b (nf-κb), peroxisome proliferator-activated receptor gamma (pparγ), and tumor necrosis factor-α (tnfα) pathways. cancer cell lines sw480=47.8 µm, hct-116=44.6 µm, bt-549=36 µm. `references 33 a cc ep te d m an us cr ip t flavonoids and its anticancer properties 9 silibinin cancer cell lines mcf-7, h460, hcc827, nci-h1975, a549, and nci-h1299 cancer cell lines. targeted molecular pathway epidermal growth factor receptor (egfr/lox) pathways. cancer cell lines nci-h1975=96.56 µm, mcf-7= 46µm, h460=50.5 µm, h1975=48 µm `references 34 apigenin cancer cell lines sw480, a375, a549, bt-474, mda-mb-231, dld1, a2780, du145, and c8161, cancer cell lines. targeted molecular pathway janus kinase/signal transducers and activators of transcription (jak/stat), mitogen-activated protein kinase/ extracellular-signalregulated kinase (mapk/erk), canonical wnt (wnt/β-catenin), nuclear factor kappa b (nf-κb), phosphoinositide 3 kinase target of rapamycin (pi3k/akt/mtor) pathways. cancer cell lines mda-mb-231=35.15 µm, and a549=79.8 µm, `references 35 flavonoid derivatives as anticancer moiety: fikroh ra et al., (2020) synthesized the (2e)-3-(2-bromo-4,5-dimethoxy phenyl)-1-(2-methyl phenyl) propanone by the claisen-schmidt condensation reaction of 2-bromo-4,5-dimethoxybenzaldehyde and 2hydroxy acetophenone with a good yield of 78%. the chalcone derivative, 1a (fig 1) showed moderated action on breast cancer cell lines (mcf-7) at ic50 of 42.19 µg/ml.36 fig 2: (2e)-3-(2-bromo-4,5-dimethoxyphenyl)-1-(2-methylphenyl) propanone. a cc ep te d m an us cr ip t 10 manojmouli et al. ngameni b et al., (2021) synthesized the novel series of o-substituted chalcone moieties containing various groups like allyl-, propargylor prenylsubstituent at different positions on both rings by the claisen-schmidt condensation of o-allyl, and o-propargyl vanillin and substituted aromatic ketones. compounds 2a showed antitumor action against hct116 p53 colon adenocarcinoma cells, 2b against ccrf-cem cells and mda-mb-231-bcrp breast adenocarcinoma cells, and 2c against hct116 p53 cells and hct116 p53 human colon lung cancer cells (fig 3). all these compounds showed activity at ic50 values below 1 µm.37 fig 3: o-propargylchalcone derivatives (2a & 2b) and o-propargylated chalcone (2c). pangal a et al., (2022) synthesized the chromen-2-one compounds by grinding of coumarin, trifluoro substituted anilines, and potassium carbonate under solvent-free conditions. compounds 3a and 3c showed good anticancer activity against hela cell lines at ≤10µg/ml. components 3a and 3b (fig 4) showed moderate anticancer activity at a lower concentration against hela and mcf-7 cell lines.38 fig 4: 3-(2-(substituted-(trifluoromethyl) phenylamino) acetyl)-2h-chromen-2-one derivatives mirzaei s et al., (2020) synthesized the hybrids of quinoline and chalcones as tubulin inhibitors. compound 4a (fig 5) showed good antiproliferative activity at ld50 of 22.4 µm against four human cancer cell lines like a2780 (human ovarian a cc ep te d m an us cr ip t flavonoids and its anticancer properties 11 cancer cell lines), a2780/rcis (cisplatin resistant human ovarian cancer cell lines), mcf-7 (human breast cancer cell lines), and mcf-7/mx (mitoxantrone resistant human breast cancer cell lines) and normal huvec cancer cell lines by causing cell cycle arrest at the g2/m phase.39 fig 5: benzoyl-2-chloroquinolin-3-yl (e)-3-(1,3,4,5-trimethoxyphenyl) propanone. wang et al., (2022) synthesized the chromone-2aminothiazole scaffolds as novel ck2 inhibitors. compound 5a (fig 6) showed better activity against ck2 cells at ic50 value of 0.08 µm and exhibited more potent anticancer activity against hl-60 tumor cells at ic50 value of 0.25 µm by inhibiting the downstream of casein kinase ii, including α-catenin/akt pathway and parp/survivin pathway.40 fig 6: n-(5-methoxy-1,3-benzothiazol-2-yl)-4-oxo-4h-1-benzopyran-2-carboxamide mayer et al., (2020) reported the synthesis of novel 7-aminochrysin derivatives by alkylated with n-phenylchloroacetamides at the 7th position. compound 6a (fig 7) anticancer activity against mcf7 (gi50=30nm) cell line of breast cancer and on the hct-15 cell line of colon cancer cell line (gi50=60nm) at a nanomolar concentration.41 a cc ep te d m an us cr ip t 12 manojmouli et al. fig 7: 7-(3,5-dimethoxyanilino)-5-hydroxy-2-phenyl-4h-1-benzopyranone parvinder kaur et al., (2022) synthesized a new series of cinnamic acid derivatives by reacting 2-chloro-n-hydroxy acetamide and cinnamic acid amide. compound 7a (fig 8) showed potent activity against lung cancer cell lines (a-549) at an ic50 value of 10.36 µm among all synthesized derivatives.42 fig 8: (2e)-n-methyl-n-((hydroxycarbamoyl)methyl)-3-(3-hydroxyphenyl)prop-2-enamide rahimzadeh oskuei s et al., (2021) synthesized the novel imidazole-chalcone moities as inhibitors of tubulin polymerization and as an anticancer agent. in that series of derivatives, compound 8a (fig 9) showed a better cytotoxicity effect against adenocarcinoma human alveolar basal epithelial cells (a549), human breast cancer cells (mcf-7), mitoxantrone resistant human breast cancer cells (mcf-7/mx), and human hepatocellular carcinoma cells (hepg2) at ic50 value ranging from 7.05 to 63.43 μm.43 a cc ep te d m an us cr ip t flavonoids and its anticancer properties 13 fig 9: (e)-3-(1-benzyl-2-(ethylthio)-1h-imidazol-4-yl)-1-(3,4,5-trimethoxyphenyl)propenone sarkate ap et al., (2021) reported the one pot synthesis of new series of flavonoid derivatives with different heterocyclic moieties. compound 9a and 9b showed moderate anticancer activity by inhibiting the enzyme topoisomerase ii with ic50 values of 10.28 and 12.38 μm against cancer cell lines.44 fig 10: substituted 2-amino-4,5-dihydro-5-oxo-4-phenylpyrano[3,2-c]chromene-3carbonitrile yan x et al., (2020) reported synthesizing new genistein and chrysin nitrogen mustard derivatives according to the principle of combination and hybridization. in this series, compound 10a (fig 11) showed better cytotoxic activity against hela cancer cell line (ic50=1.43 μm), pc-3 cancer cell lines (ic50=2.32μm), du145 cancer cell lines (ic50=2.91μm), mcf-7 cancer cell lines (ic50=4.90μm), which were more than 4 times higher than melphalan.45 a cc ep te d m an us cr ip t 14 manojmouli et al. fig 11: 7-[3-[bis(2-chloroethyl)amino]propoxy]-5-hydroxy-2-phenyl-4h-chromenone thorat nm et al., (2021) synthesized the n-benzyl derivatives of 6aminofavone by using the multi-step synthetic procedure like methylation, friedel–craft acylation and in situ demethylation, bekar–venkataraman rearrangement, buchwald coupling reaction, these reactions were employed in different steps for different starting materials as a potent novel anticancer moities. in this series, compound 11a (fig 12) showed high potent topoisomerase ii enzyme inhibition activity at ic50 value of 12.10 μm.46 fig 12: 6-{(4-chlorophenyl)methyl]amino}-2-phenyl-4h-1-benzopyranone liu r et al., (2020) designed and synthesized the novel 5,6,7-trimethoxy flavonoid salicylate moieties by combining 3 different moieties like trimethoxyphenyl, flavonoid, and salicylic acid based on the principle of combination. in these derivatives, compound 12a exhibits better anticancer activity against hgc-27 cells and mgc-803 cells with ic50 values of 10.20 ± 6.90 μm and 17.20 ± 3.04 μm, respectively.47 a cc ep te d m an us cr ip t flavonoids and its anticancer properties 15 fig 13: 5-bromo-2-{3-[4-(5,6,7-trimethoxy-4-oxo-4h-chromen-2-yl)-phenoxy] -propoxy benzoate of methyl acid kozłowska j et al., (2019) synthesized the novel derivatives of aminochalcones by using classical claisen-schmidt reaction of substituted aminoacetophenone with aromatic aldehydes. in this series of aminochalcones derivatives, compound 13a (fig 14) showed better anticancer activity against different human colon carcinoma cell lines at low ic50 values i.e., ht-29 (ic50=1.43µml-1), ls180 (ic50=2.06µml-1) lovo (ic50=1.56µml-1), lovo/dx (ic50=1.43µml-1), and cos7 (ic50=26.4µml-1).48 fig 14: (2e)-1-(2-aminophenyl)-3-phenylpropanone assirey et al., (2020) synthesized the 4′, 5, 7-trihydroxy-flavanone through knoevenagal condensation of an aldehyde, followed by an intramolecular michael addition reaction. compound 14a (fig 15) exhibited potent anticancer activity against hct-116, hepg-2, mcf-7, and a-549 tumor cell lines with ic50 values at 1.08, 2.42, 2.04, & 1.39 µg/ml respectively.49 a cc ep te d m an us cr ip t 16 manojmouli et al. fig 15: 8-amino-10-(thiophene)-5-hydroxy-2-(4-hydroxy-phenyl)-4-oxo-3,4-dihydro-2h10h-pyrano[2,3-f] chromene-9-carbonitrile al-oudat ba et al., (2019) designed and synthesized the derivatives of chrysin bearing the n′-alkylidene/arylideneacetohydrazide core by the reaction of hydrazide with different aldehydes. compound 15a (fig 16) with 4-benzyloxy substituent showed good antitumor activity against mda-mb-231 and mcf-7 cell lines with ic50 values of 3.3 µm, and 4.2 µm respectively.50 fig 16: n-(4-(benzyloxy)benzylidene)-2-((5hydroxy-4-oxo-2-phenyl-4h-chromen-7-yl) oxy)acetohydrazide hou et al., (2021) designed and synthesized the novel derivatives of icaritin as inhibitors of putative deptor by multi-step reaction. compound 16a (fig 17) exhibited a good antimultiple myeloma activity with an ic50 of 1.09 μm for human multiple myeloma cell lines (rpmi 8226), induced rpmi 8226 apoptosis, and acts by blocking s phase of the cell cycle.51 a cc ep te d m an us cr ip t flavonoids and its anticancer properties 17 fig 17: 2-(3,4-dichlorophenyl)-9-(3-fluorobenzyl)-3-hydroxy-9,10-dihydro-4h,8hchromeno[8,7-e]-[1,3]-oxazin-4-one kumar et al., (2021) synthesized the chalcone derivatives incorporated benzothiazole-imidazopyridine by employing various reactions like the suzukicross coupling reaction and claisen-schmidt condensation reaction. compound 17a (fig 18) showed potent cytotoxic activity against human prostate cancer cell line (pc3), human lung cancer cell line (a549), human breast cancer cell line (mcf-7), and human prostate cancer cell line (du-145) at ic50 value of 0.03, 0.01, 0.12, and 0.17 μm.52 fig 18: 3-(2-(6-(3,4,5-trimethoxyphenyl)benzo[d]thiazol-2-yl)−3h-imidazo[4,5-b]pyridin-6yl)−1-(pyridin-4-yl)propanone silva et al., (2021) designed and synthesized the acetylated derivative of quercetin by acetylation of quercetin with acetic anhydride in the presence of pyridine. compound 18a (fig 19) exhibited a better cytotoxicity activity against a cc ep te d m an us cr ip t 18 manojmouli et al. hepatocellular cells (hepg2) and promyelocytic leukemia (hl-60) cell lines with ic50 values of 53.9 and 33.6 μm respectively.53 fig 19: quercetin pentaacetate zhong g et al., (2022) synthesized the novel hesperetin derivatives by the electrophilic substitution reaction in methanol at 40oc at the c-6 position. compound 19a (fig 20) showed better antiproliferative effect on breast cancer cell lines (mcf-7), human liver cancer cell lines (hepg2), and cervical carcinoma cell lines (hela) at ic50 value of 5.3, 8.8, and 8.6 μm respectively.54 fig 20: hesperetin derivative a cc ep te d m an us cr ip t flavonoids and its anticancer properties 19 insuasty et al., (2021) synthesized a novel symmetrical and unsymmetrical quinoline-based bis-chalcone series by claisen-schmidt condensation reaction. among the synthesized derivatives, compound 20a (fig 21) showed potent anticancer action against the different carcinoma cell lines like hct-116 and ht29 with a gi50 value ranging from 0.16-5.45 µm.55 figure 21: benzyl‐7‐chloro‐3‐((1e,4e)‐5‐(4‐hydroxy‐3‐methoxyphenyl)‐3‐ oxopenta‐1,4‐ dien‐1‐yl)quinolin‐2(1h)‐one lu et al., (2020) prepared a novel amino chalcone derivatives as antiproliferative agents. among the synthesized compounds, compound 21a (fig 22) showed potent anticancer activity against mcf-7, hct-116, and mgc-803 tumor cell lines with ic50 values of 2.54 μm, 1.83 μm, and 1.52 μm respectively.56 figure 22: 4-chloro-n-(4-(3-oxo-3,4,5-trimethoxyphenyl) propenyl)phenyl butanamide conclusion world health organization (who) expressed worry about the rising cancer incidence and it has been expected to continue with the number of new cancer cases. anticancer drugs cause numerous side effects and it also affects various healthy organs and tissues. flavonoids are secondary metabolites and this is unique a cc ep te d m an us cr ip t 20 manojmouli et al. lead compound to design and for developing of potent anticancer drugs, particularly in chemotherapy. we summarized the results of clinical trials of flavonoids and highlighted the synthesized flavonoid derivatives which showed better cytotoxic activity at lower concentrations against various cancer cells, it helps the researchers in future to develop flavonoid derivatives as anticancer drugs by carrying out further clinical studies. further, need to understand key enzymes related to neoplastic cells and metastasis in-vitro and in-vivo process and it helps in providing novel potent flavonoid derivatives for fighting cancer. conflict of interest: the authors confirm that the contents of this article present no conflicts of interest. извод деривати флавоноида као антиканцерске група једињења и њихов ефекат на ћелијске линије канцера: ажурирани преглед c. manojmouli, t. y pasha, k. nagaprashant, b. ramesh, noor ul eain, k. a. purushotham department of pharmaceutical chemistry, sri adichunchanagiri college of pharmacy, adichunchanagiri university, b. g. nagara, karnataka, india данас, канцер се сматра примарним узроком преране смрти у индустријализованим земљама. хемотерапије лековима су скупе и изазивају вишеструке споредне ефекте. природни производи су детаљно испитани због њиховог великог потенцијала као антиканцерских агенаса због њихове хемијске разноликости. између многих природних метаболита, флавоноиди су секундарни метаболити широко распрострањени у природи, који имају значајна анти-канцерска својства, мало штетних ефеката, и показују синергистички користан утицај. велики број истраживачких група марљиво истражује хемијске и биолошке особине нових деривата флавоноида због потреба за овим једињењима. у овој ревији, сумирали смо резултате клиничких испитивања различитих флавоноида, ефекат на различите ћелијске линије канцера и нове резултате активности флавоноида, синтетисаних различитим поступцима, према ћелијама канцера. нова изучавања су неопходна за даљи развој нових деривата флавоноида као антиканцерских лекова. 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http://dx.doi.org/10.3390/molecules27030879 http://dx.doi.org/10.1002/ardp.202100094 http://dx.doi.org/10.3390/molecules25235530 professor nikos katsaros (1938–2022) j. serb. chem. soc. 87 (11) 1347–1348 (2022) 1347 in memory to professor nikos katsaros (1938–2022) on january 19, 2022, our dear colleague, and friend, professor nikos katsaros passed away. he worked at the “demokritos” natural sciences research center and was the former president of the association of greek chemists (agc, eex). nikos katsaros was born in gimari, corfu, in 1938, lived and grew up in athens. he studied at the university of athens, where he was admitted 2nd in the department of chemistry out of 750 candidates and received his b.s. degree in chemistry in 1961. he completed his studies at the university of massachusetts, usa, wherefrom he received an m.s. in 1967 and a ph.d. in 1968. he then served as a visiting research fellow at brookhaven national laboratory, upton, ny, usa. in 1969, he joined the university of georgia, usa, as an assistant professor of chemistry. in 1970, nikos was appointed researcher at the “demokritos” nuclear research center. in 1972, he served as visiting research fellow at imperial college london, united kingdom. from 1976 to 2004, he worked as a principal researcher at the national center for natural sciences research (ekefe) “demokritos”. also, in 1983–1998, he served as visiting professor of chemistry at the university of maryland, usa. from 1989 to 1993 he served in the hellenic ministry of industry, energy and technology, as secretary general for research and technology and in 2004–2005 as president of the hellenic food organization (efet). professor katsaros served for four terms as president of the association of greek chemists (agc) and twice as its vice-president. he was also a member of the chemistry europe fellow and was honored in 2020 for his services. he was the honorary member of the serbian chemical society. his research interests included inorganic biochemistry, food chemistry, food safety, radiochemistry, environmental chemistry, and toxic waste management. he published over 200 scientific articles and texts on these subjects. prof. katsaros was the member of editorial board of the journal of the serbian chemical society. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1348 in memory to prof. katsaros the field of education has always been at his heart and therefore he had unceasing activity either in organizing and participating in postgraduate programs of greek universities (ioannina, patras and national and kapodistrian university of athens), or in teaching at private colleges such as deree college and new york college, where he worked until recently as head of the department of human nutrition and life sciences. nikos katsaros was distinguished for his intelligence. he was an emblematic president of the eec, agc. he had distinct qualifications, which he used in his studies, in research, in his scientific activities and in his social struggles. in the context of the activities of the association of greek chemists, he had the initiative of organizing many panhellenic chemistry conferences, greece–cyprus chemistry conferences and the establishment of the institution of the panhellenic student chemistry competitions. as part of european and western balkan community, he, as president of the association of greek chemists, was founder and main driving force of series of international conferences of the chemical societies of the south-eastern european countries (seec). as a consequence of this visionary approach the conferences brought together and interconnected seec chemists into close partnership where from the scientific collaboration on many bilateral and multilateral projects emerged. while being president of agc in spring 1999 he, and complete agc leadership, made a self-organized visit to serbian chemical society bringing dioxin standards and hplc/ms spare parts to our institutions analyzing the toxic chemical effects after nato bombing of serbia. also, the agc management insisted, and visited freshly bombed hcl, ammonia and chlorine production sites at petrohemija, pančevo, and bombed oil refinery in novi sad. this friendly gesture of nikos, and agc, will be kept in serbian chemists’ hearts forever. we, your colleagues, your friends and associates say goodbye today to nikos katsaros, a worthy scientist, family man, visionary and useful social worker. have a nice trip, kiss nikos, dear colleague, and late partner. may your memory be eternal. prof. panayotis p. siskos prof. bogdan a. šolaja prof. branislav nikolić ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ @article{siskos2022, author = {siskos, panayotis p. and {\v{s}}olaja, bogdan a. and nikoli{\'{c}}, branisalav {\v{z}}.}, journal = {journal of the serbian chemical society}, title = {{professor nikos katsaros (1938–2022)}}, year = {2022}, issn = {1820-7421}, month = {nov}, number = {11}, pages = {1347--1348}, volume = {87}, abstract = {on january 19, 2022, our dear colleague, and friend, professor nikos katsaros passed away. he worked at the “demokritos” natural sciences research center and was the former president of the association of greek chemists (agc, eex).}, file = {:d\:/onedrive/mendeley desktop/siskos, {v{s}}olaja, nikoli{'{c}} 2022 professor nikos katsaros (1938–2022).pdf:pdf;:10_katsaros.pdf:pdf}, keywords = {environmental chemistry, food chemistry, inorganic biochemistry, toxic waste management}, url = {https://www.shd-pub.org.rs/index.php/jscs/article/view/12137}, } lc/dad determination of biogenic amines in serum of patients with diabetes mellitus, chronic urticaria or hashimoto's thyroiditis j. serb. chem. soc. 81 (5) 487–498 (2016) udc 543.9+547.233:575+547.415–38: jscs–4862 616.379–008.64+616.51 original scientific paper 487 lc/dad determination of biogenic amines in serum of patients with diabetes mellitus, chronic urticaria or hashimoto's thyroiditis jelena trifunović-macedoljan1, nebojša pantelić2*, ana damjanović3, sanvila rašković4, marina nikolić-đurović5, georgina pudar6, milka jadranin7, ivan juranić7# and zorica juranić3 1faculty of chemistry, innovation centre, university of belgrade, studentski trg 12–16, p. o. box 158, 11000 belgrade, serbia, 2faculty of agriculture, university of belgrade, nemanjina 6, 11080 zemun, serbia, 3department of experimental oncology, institute of oncology and radiology of serbia, pasterova 14, 11000 belgrade, serbia, 4instuitute of allergology and immunology, clinical center of serbia, school of medicine, university of belgrade, dr subotića 8, 11000 belgrade, serbia, 5institute of endocrinology, clinical center of serbia, dr subotića 13, 11000 belgrade, serbia, 6clinic for endocrinology, university clinical center “zvezdara”, dimitrija tucovića 161, 11000 belgrade, serbia and 7department of chemistry, institute of chemistry, technology and metallurgy, university of belgrade, njegoševa 12, 11000 belgrade, serbia (received 10 september 2015, revised 28 february, accepted 29 february 2016) abstract. biogenic amines are integral part of nearly every cell. in the present study, the method of acidic extraction of histamine (his), of polyamines putrescine (put), spermidine (spd) and catecholamines epinephrine (epi) and norepinephrine (ne) from human serum, precolumn derivatization with dansyl chloride, and lc/dad analysis of the biogenic amines was used with the aim of monitoring differences of their levels in patients with diabetes mellitus, chronic urticaria, and hashimoto's thyroiditis, compared to healthy subjects, and to observe them as possible markers for immune mediated diseases. the retention times were used for the determination of serum biogenic amines. statistically significant differences were found in the levels of putrescine and histamine in diabetes mellitus patients, of the levels of putrescine, histamine, spermidine and epinephrine in chronic urticaria patients and of the levels of putrescine and spermidine levels in hashimoto's thyroiditis patients, compared to those of healthy controls. norepinephrine was found only in the serum of patients with chronic urticaria. the values of recovery, evaluated in controls, varied between 85.7 and 106.7 %. the statistically significant changes in putrescine, histamine, spermidine and epinephrine levels in patients compared * corresponding author. e-mail: pantelic@agrif.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc150910020t 488 trifunović-macedoljan et al. with those in healthy people reflects the existence of biochemical disturbances in the mentioned immune-mediated diseases. keywords: biogenic amines; serum; immune-mediated diseases; markers, lc/dad. introduction polyamines and catecholamines are members of a broad group of the so-called biogenic amines.1 since the first description (by antoni van leeuwenhoek in 1678) of crystals that were much later identified as polyamine salts, these molecules have been the subject of intense research efforts, which have shed light on several biological and pathological processes, for example the repair of the extracellular matrix, cell adhesion, synthesis of nucleic acids, and involvement in cellular growth and proliferation. these molecules are also related with several types of cancer, such as breast and colon cancer, adenocarcinoma, non-hodgkin’s lymphoma etc.2,3 histamine and the polyamines putrescine and spermidine form a group of naturally occurring compounds that exert a large number of biological effects, but the mode of action of these biogenic amines at the molecular level is largely unknown.4–7 catecholamines – epinephrine and norepinephrine containing an amino function on a side chain together with the catechol function (i.e. hydroxyl groups at the 3and 4-positions on a benzene ring) play an important role in the nervous, cardiovascular and endocrine systems. catecholamines act via adrenergic receptors, and are involved in the regulation of the response to stress, psychomotor activity, emotional processes, learning, sleep, and memory.1,8–12 norepinephrine is also useful in the diagnosis of pheochromocytoma.13 polyamines as polycations are essentially involved in cell growth and differentiation.14,15 they influence the transcriptional and translational stages of protein synthesis, stabilize membranes, modulate neurophysiological functions and may act as intracellular messengers.2,4 hence, the majority of the polyamines will be sequestrated in some way and it is probable that only the “free” polyamine pool is physiologically active.16 since intracellular polyamine concentrations are much greater than extracellular, a small amount of cell lysis can markedly alter their obtained values.17 polyamines appear to have a role in immune-mediated diseases18 and have effect on dna/rna structure, chromatin condensation, translation, protein synthesis and mammalian cell growth.19 in humans, the inhibitor of polyamine biosynthesis, α-difluoromethylornithine (dfmo), with an inhibitor of cyclooxygenase 2 (cox-2) proved to be effective in prevention of the recurrence of colon cancer and adenomas, associated with the development of colon cancer.19 acetylated spermine is increased in urine in different types of human cancers.19 in colon cancer, the sensitivity of the detection of diacetylspermine (diacspm) in lc/dad determination of biogenic amines 489 urine is much higher and in breast cancer noticeably higher than the sensitivity of regularly used diagnostic serum biomarkers.20 quantification of biogenic amines in biological fluids has an important diagnostic implication in medicine,21 but direct determination of biogenic amines in biological fluids is difficult because they are small aliphatic or aromatic molecules that do not exhibit any structural features that would allow sensitive and selective detection.22 chromatographic methods are generally used for the determination of biogenic amines, but, since only few of them absorb in the uv–visible region, for their simultaneous determination, a pretreatment step of derivatization is generally performed.23 among a wide panel of analytical methods developed for the quantification of biogenic amines, hplc separation after derivatization with dansyl chloride remains the most commonly used method.24 in this study, pre-column derivatization, gradient elution, and ultraviolet detection with a dad detector were used. first, the serum levels of specific biogenic amines in three immune-mediated diseases, i.e., diabetes mellitus, chronic urticaria and hashimoto's thyroiditis, were investigated to elucidate whether there were any changes in their levels in mentioned diseases compared to healthy people. in order to determine the concentrations of serum biogenic amines in these diseases and to assess the differences among their levels,19,24,25 the method of ben-gigirey et al. modified by mao et al. was used because equipment similar to theirs was available, thereby providing reproducible results. materials and methods reagents and chemicals all chemicals were of analytical hplc grade. all biogenic amine standards were purchased as hydrochloride salts of the highest available purity. the standards of 1,4-diaminobutane dihydrochloride (putrescine), 99+ %, spermidine trihydrochloride, 99+ %, histamine dihydrochloride, 99 %, and l-(–)-epinephrine, 99 %, were purchased from acros organics (beel, belgium). (–)-norepinephrine, 99.5 %, was purchased from sigma (st louise, mo, usa). the derivatization reagent, dansyl chloride, 98%, was procured from acros organics. perchloric acid, sodium hydroxide, sodium bicarbonate, 25 % ammonium hydroxide, acetone, acetonitrile, and ammonium formate were from merck (darmstadt, germany). ultra-pure hplc grade water was prepared by passing doubly deionized water through a milli-q system. individual standard solutions of the biogenic amines were made by dissolving an exactly weighed amount of the required standard in ultra-pure water. after dissolution, each standard was diluted to 1 mg l-1 with ultra-pure water and stored as a stock solution at 4 °c in the dark. working standard dilutions were freshly prepared by diluting the stock standard solutions with ultra-pure water for each assay. a solution of dansyl chloride (the derivatization reagent) was made in acetone, diluted to 10 mg l-1 and then stored at 4 °c in the dark. 490 trifunović-macedoljan et al. serum preparation whole blood was collected after venipuncture in covered red top test tubes – vacutainers (bd – becton dickinson), without any anticoagulant. after collection of whole blood, the blood was left undisturbed to clot for 15–30 min at room temperature. the clot was removed by centrifuging at 2000g for 10 min. the resulting supernatant was designated serum. following centrifugation, the liquid component (serum) was immediately transferred into a clean polypropylene tube using a pasteur pipette. the serum samples were apportioned into 0.5 ml aliquots and stored at –20 °c until analysis. patients’ characteristics the present study included patients from the university clinical centre “zvezdara” hospital (belgrade, serbia) or the clinical centre of serbia (belgrade, serbia) and 20 healthy controls. three medical conditions/diseases – diabetes mellitus (type i and type ii), chronic urticaria and hashimoto's thyroiditis, which were newly diagnosed, were investigated in the serum. 12 of 20 diabetes mellitus patients were men, and 8 were women (age range 53–64, median age 59); 6 of 20 chronic urticaria patients were men, and 14 were women (age range 24–77, median age 48), and 8 hashimoto's thyroiditis patients were women (age range 33–75, median age 48). the chosen patients had no additional disorders beside the investigated one. healthy controls, of which 11 women and 9 men (age range 27–62, median age 36), were randomly chosen and did not have any reported diagnosis. all patients and controls provided written informed consent before entry into the study. apparatus the hplc system comprises a liquid chromatograph agilent 1200 series (agilent technologies, santa clara, ca, usa), with a degasser, a binary pump, an autosampler, a column compartment equipped with a zorbax eclipse plus c18 column (150 mm×4.6 mm; 5 μm), and a diode-array detector (dad). a personal computer system running chemstation software (agilent technologies, santa clara, ca, usa) was used for data collection and processing. chromatographic conditions a mixture of solvents a (water containing 5 mm ammonium formate) and b (acetonitrile) in a gradient mode of elution was used as the mobile phase. the gradient program was selected as follows: 55–65 % b, 0–6 min; 65–100 % b, 6–15 min; 100 % b, 15–19 min; 100– –65 % b, 19–20 min; 65 % b, 20–24 min. the mobile phase solutions were pumped at a flow rate of 1.0 ml min-1. signals were accumulated in the wavelength range 190–450 nm using a dad, and the chromatograms were recorded at 254 nm. the injection volume was 50 µl. the temperature of the column was kept at 40 °c. standard treatment the stock standard solutions of each biogenic amine were diluted with ultra pure water to the desired concentrations. each standard (500 µl) was worked up as described above for the serum samples. the qualitative analysis was realized using the retention times. the quantitative analysis was performed using the corresponding external calibration. the calibration curves were obtained by plotting the biogenic amines peak area values against the respective concentrations of biogenic amines standards. sample preparation the method proposed by mao et al. was used.25 to 500 µl of serum sample in a micro test tube, 750 µl of 0.4 m perchloric acid was added, then vortexed for 1 min, and centrifuged lc/dad determination of biogenic amines 491 at 13000 rpm for 10 min. the supernatant was aspirated into another test tube followed by the addition of 67.5 µl of 2.0 m naoh, vortexed for 1 min and then 150 µl of a saturated solution of sodium bicarbonate was added into the test tube to adjust the ph to 8.0–8.5. to a prepared sample 1000 µl of 10 g l-1 dansyl chloride was added, and the mixture was vortexed for 1 min, and left to react in a closed water bath at 40 °c for 45 min. finally, 50 µl of 25 % ammonium hydroxide was added to remove the excess derivatization reagent, and incubated for 30 min, at room temperature, in the dark. before injection, all samples were filtrated through 0.40 µm econofilters. validation of the method all quantitative analysis were performed by the external calibration procedure using standard solutions of putrescine, histamine, spermidine, epinephrine and norepinephrine. the validation of the method was accomplished by the test of linearity, precision and accuracy, limit of detection (lod), limit of quantification (loq) and recovery. the linearity of the method was tested for the biogenic amine standards by injecting 50 µl of each amine standard solution within the concentration range of 5–50×103 ng l-1 into the lc/dad system. the accuracy of this analytic method was assessed as the percentage relative error. the lod and loq values for the biogenic amines were determined by injecting progressively lower concentration of the standard solution under the chromatographic conditions. the recovery study was performed using a real serum sample from a healthy control, by spiking techniques. this control serum sample was treated in the same way as were the other samples. the recoveries were checked as three different concentration levels (low, medium and high). statistical analysis descriptive statistics and the kruskal–wallis test together with the multiple-comparison z-value test were used for statistical analysis of the experimental data employing a demo version of the number cruncher statistical systems software (kaysville, ut, usa). the linear relationship between peak areas and concentrations was calculated by linear regression. results and discussion optimal conditions of sample processing the acidic precipitation/extraction procedure is the most promising method for complete recovery of low molecular weight compounds from complex matrices.1 a solution of 0.4 m hclo4 gave slightly sharper peaks for the biogenic amines in the chromatograms, which is in agreement with the work of mao et al.25 linearity least-square regression was used for the determination of the slope, intercept, and the correlation coefficient of the calibration curves, constructed by plotting the peak area values against the respective concentrations. linearity was obtained for the range of 80–5×104 ng l–1 (fig. s-1 of the supplementary material to this paper) and the correlation coefficients were excellent (table s-i of the supplementary material). 492 trifunović-macedoljan et al. precision and accuracy the repeatability was evaluated by analyzing the quality control samples, at three different concentrations, six times per day. the intermediate precision was evaluated by analyzing the same samples once daily for two days. the relative standard deviation (rsd) of the concentrations obtained from the corresponding calibration curves was taken as the precision. the results, which are summarized in table s-ii of the supplementary material, meet the requirements of accuracy within 95.12–99.75 %, and the precision rsd values were ≤ 8.46 %. limits of detection (lod) and quantification (loq) the lowest concentrations assayed where the signal/noise ratio was at least 10:1 was regarded as the loq. the lod was defined as the concentration when the signal/noise ratio was 3:1. the determined lod values were 40 ng l–1 for putrescine, 30 ng l–1 for histamine, 250 ng l–1 for spermidine, 90 ng l–1 for epinephrine and 596 ng l–1 for norepinephrine. the determined loq values were 150 ng l–1 for putrescine, 90 ng l–1 for histamine, 820 ng l–1 for spermidine, 300 ng l–1 for epinephrine and 1980 ng l–1 for norepinephrine. recovery experiment serum samples with known concentrations of biogenic amines, were spiked with concentrations of putrescine and histamine (1000, 1500 and 2000 ng l–1), spermidine (2000, 4000 and 6000 ng l–1), epinephrine (350, 700 and 1000 ng l–1), and norepinephrine (1500, 3000 and 5000 ng l–1). the serum concentration of each biogenic amine was determined five times, and the recovery rate of each standard was calculated. the chromatogram of a spiked serum sample is presented in fig. s-2 of the supplementary material. the average recovery rate ranged from 85.7 to 106.7 %. the obtained data (table s-iii of the supplementary material) showed good recovery values for all the investigated biogenic amines. detection of the levels of the biogenic amine in the serum of the controls and patients a chromatogram of a mixture of the standard biogenic amines is shown in fig. 1. all the standard amine peaks, under the given experimental conditions, appeared within 20 min, without overlapping. the retention times were 8.9 min for putrescine, 10.1 min for histamine, 13.1 min for spermidine, 13.9 min for norepinephrine and 14.7 min for epinephrine. lc/dad determination of biogenic amines 493 fig. 1. a chromatogram of a mixture of the investigated serum biogenic amines. the peaks of the investigated biogenic amines in the serum samples could also be separated within 20 min. occasionally, the recording was spoiled by residues of the protein precipitate passing through the filter. this was resolved by additional centrifugation of the sample. representative chromatograms of biogenic amines in the sera of the control subjects and patients with the investigated diseases are shown in fig. 2. the concentration levels for the five serum biogenic amines, determined in controls, and in patients are given in table i. differences, observed by parameters of descriptive statistics, were evaluated by the kruskal–wallis test followed by the multiple-comparison z-value test. the putrescine concentrations in the sera of patients with diabetes mellitus, chronic urticaria and hashimoto's thyroidits patients were lower compared to those in the sera of the healthy controls (p < 0.0001 and z: c(d,u,h). the histamine levels were also statistically significant different between healthy controls and patients with diabetes mellitus or chronic urticaria (p < 0.0001 and z: d(c,h,u)), while patients with diabetes had higher histamine levels than healthy subjects (p < 0.0001). unexpectedly, in serum of chronic urticaria patients, the histamine levels were lower than in healthy controls (p < 0.0001). eight patients with hashimoto’s thyroiditis had detectable histamine in their serum, of which four values were the same as in the sera of the healthy controls, while the other four were lower. the histamine levels in the sera of patients with hashimoto's thyroiditis were also lower compared to those in the sera of patients with diabetes mellitus (p < 0.0001). spermidine levels in the serum 494 trifunović-macedoljan et al. fig. 2. representative chromatograms of serum biogenic amines in: a) healthy controls, b) diabetes mellitus patients, c) chronic urticaria patients and d) hashimoto's thyroiditis patients. table i. obtained values (in ng l-1 ± sd) for the five serum biogenic amines in healthy controls, diabetes mellitus patients, chronic urticaria patients and patients with hashimoto's thyroiditis; sd is the standard deviation of six replicate determinations; c = healthy controls, d = diabetes mellitus, u = chronic urticaria and h = hashimoto’s thyroiditis patients analyte controls (n = 20) diabetes mellitus (n = 20) chronic urticaria (n = 20) hashimoto's thyroiditis (n = 8) p-valuea z-valuea putrescine 1715±280 230±60 450±50 255±110 <0.0001 c(d,u,h) histamine 285±110 505±230 140±60 165±139 <0.0001 d(c,h,u) spermidine 855±180 1205±310 1825±580 1165±410 <0.0001 c(d,u,h) epinephrine 382±60 490±140 965±510 395±180 <0.0001 u(d,h,c) norepinephrine – – 700±40 – – – aresults of the statistical kruskal–wallis test with the multiple-comparison z-value test of patients with diabetes mellitus, chronic urticaria and hashimoto's thyroiditis were higher in comparison with those for the healthy controls (p < < 0.0001; z: c(d,u,h)). the epinephrine levels between chronic urticaria patients and healthy controls, diabetes mellitus and hashimoto's thyroiditis patients were also statistically significantly different (p < 0.0001; z: u(d,h,c)). the chronic urticaria patients had higher concentrations of serum epinephrine compared to those of the controls (p < 0.0001). norepinephrine (a hormone related to stress) was detected only in the serum of chronic urticaria patients. lc/dad determination of biogenic amines 495 the findings of enhanced histamine levels in diabetes mellitus patients are in agreement with the work of gill et al.26 who also found increased histamine concentrations in the plasma of patients suffering from diabetes mellitus (types i and ii). moreover, the studies of inui et al.27 and of alkan et al.28 performed on rats and mice, respectively, support the notion that endogenous histamine is involved in the pathogenesis of diabetes. the detection of norepinephrine only in the serum of chronic urticaria patients is in agreement with literature data that patients with adrenergic urticaria have enhanced levels of serum catecholamines.29 as there were no pre-analytical patients (or healthy control subjects) prepared for venipuncture, it could be concluded that enhanced levels of serum catecholamines are specific only for patients with chronic urticaria. a possible reason why the obtained values were in disagreement with those of mao et al.25 might be the influence of nutritive habits on the level of biogenic amines in body fluids, i.e., blood. patients with chronic urticaria might have a pseudo-allergic reaction (so-called exogenic allergy) caused by biogenic amines (histamine) from food such as canned fish.30,31 in clinical practice, it was recognized that patients with chronic urticaria and hashimoto’s thyroiditis diseases were alike and very often followed each other.33 this could be one of the reasons why the spectra of these two diseases were similar. in patients with hashimoto’s thyroiditis, autoimmune urticaria might appear and vice versa, and for this reason clinicians examined whether hashimoto’s thyroiditis occurs in patients with chronic urticaria.33,34 conclusions in this study, the levels of putrescine, histamine, spermidine, epinephrine, and norepinephrine in serum of healthy subjects and patients with different immune-mediated disease were investigated for the first time. it was found that the quantities of serum biogenic amines statistically differed in diabetes mellitus, chronic urticaria and hashimoto’s thyroiditis patients, compared to healthy subjects. to the best of our knowledge, there is no report on the analysis of the levels of biogenic amines in the serum of patients with chronic urticaria or hashimoto’s thyroiditis. contrary to expectations, it was found that the histamine levels were not elevated in the sera of chronic urticaria patients, compared to those in normal subjects. furthermore, it was noticed that the values of norepinephrine and epinephrine were increased in patients with chronic urticaria and hence, it is assumed that the human organism produces higher amounts of these substances as a natural defense against anaphylaxis, and simultaneously suppresses the histamine level. higher levels of spermidine were noticeable within all three investigated immune-mediated conditions compared to within healthy controls. 496 trifunović-macedoljan et al. the putrescine levels in all three immune-mediated disorders were lower than in healthy subjects. therefore, observation of the levels of spermidine and putrescine might be used as a non-specific marker that indicates that some health, possibly immune-mediated, disorder is occurring in the body. the presented results hint on biogenic amines as possible autoimmune disease markers, and some future studies could be designed for closer investigations. the possible influence of nutrition habits on the levels of biogenic amines in blood serum could also be the subject of some future studies. supplementary material the results of the calibration and validation of the developed method are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgments. this research was supported by the ministry of education, science and technological development of the republic of serbia, grant nos. 172035 and 175011. the authors thank j. trifković, faculty of chemistry, university of belgrade, for her help in the statistical discussion. и з в о д одређивање биогених амина lc/dad методом у серуму пацијената са дијагнозом diabetes mellitus, хронична urticaria или хашимотов тироидитис јелена трифуновић-мацедољан1, небојша пантелић2*, ана дамјановић3, санвила рашковић4, марина николић-ђуровић5, георгина пудар6, милка јадранин7, иван јуранић7 и зорица јуранић3 1хемијски факултет, иновациони центар, универзитет у београду, студенски трг 12–16, п .пр. 158, 11000 београд, 2пољопривредни факултет, универзитет у београду, немањина 6, 11080 земун, 3центар за експерименталну онкологију, институт за онкологију и радиологију, 11000 београд, 4институт за алергологију и имунологију, клинички центар србије, медицински факултет, универзитет у београду, др суботића 13, 11000 београд, 5институт за ендокринологију, клинички центар србије, др суботића 13, 11000 београд, 6клиника за ендокринологију, универзитетски клинички центар „звездара“, димитрија туцовића 161, 11000 београд и 7центар за хемију институт за хемију, технологију и металургију, универзитет у београду, његошева 12, 11000 београд биогени амини су саставни део готово сваке ћелије. хистамин (his), полиамини путресцин (put) и спермидин (spd), катехоламини адреналин (epi) и норадреналин (ne) изоловани су из људског серума методом киселе естракције. преколонска дериватизација је урађена помоћу данзил-хлорида, а затим је уследила lc/dad анализа биогених амина у циљу утврђивања разлика у њиховим нивоима код пацијената са дијагнозом diabetes mellitus, хронична urticaria или хашимотов тироидитис, у односу на здраве контроле, али и утврђивања њихове функције као могућих маркера за имуно-посредоване болести. за одређивање серумских биогених амина коришћена је метода ретенционих времена. утврдили смо да постоји статистички значајна разлика у нивоима путресцина и хистамина код пацијената са дијагнозом diabetes mellitus, путресцина, хистамина, спермидина и епинефрина код пацијената са дијагнозом хронична urticaria и путресцина и спермидинa код пацијената са дијагнозом хашимотов тироидитис, у поређењу са здравим контролама. норепинефрин је детектован једино код пацијената са дијагнозом хронична urticaria. вредности повратног приноса измерене у контролама lc/dad determination of biogenic amines 497 кретале су се у опсегу 85,7–106,7 %. посматрање нивоа спермидина и путресцина би могло да се користи као неспецифични индикатор који показује да се неки биохемијски, могуће имунo-посредован, здравствени поремећај дешава у телу. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {editorial} 1 editorial this issue is an in memoriam issue dedicated to professor peter pfendt, who passed away on january 14, 2021. professor pfendt is considered the founder of the scientific discipline of environmental chemistry in serbia. in the late 1980s, studying the humic substances of recent sediments, he entered the field of environment. in the years that followed, he devoted almost all his research to this field, which is today considered one of the most important in the natural sciences. for decades, he was one of the most active members of the serbian chemical society and founder of the section for environmental chemistry. it was a great honor for us to organize the collection and evaluation of scientific papers from various fields of environmental chemistry. we would like to express our gratitude to the authors, and to the editors of the journal for their understanding and help. they enabled us to prepare this special issue, which will bring back memories of the professor in the future. we would also like to take this opportunity to thank dr. gorica veselinović for her selfless help in the technical part of the preparation of this issue. belgrade, january 11, 2022 guest editors, branimir jovančićević, phd, full professor vladimir beškoski, phd, associate professor ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 2 petar pfendt (1934–2021) petar pfendt, a longtime professor at the faculty of chemistry, outstanding expert in the field of environmental chemistry and founder of the said scientific discipline in serbia, passed away in january 2021. peter pfendt was born in november 1934 in the village of jaša tomić, district of zrenjanin, to his mother marija, née prokle, housewife, and father anton, glass cutter and trader. the second world war saw him in jaša tomić where he was together with his parents. he finished elementary school in hungarian and romanian in romania, followed by a year at the teacher-training college in timisoara and the serbian gymnasium in vršac, where he graduated in 1955. in 1955, he enrolled in the chemistry department, faculty of mathematics and natural sciences, belgrade. during his studies in the period 1957–1960 he was engaged as a laboratory assistant and demonstrator at the institute of chemistry’s analytical laboratory, while simultaneously being an active member of the student union: for three years he was a member of the student union board within the chemistry division, member of the founding board of the “sima lozanić” professional club of chemistry students, vice-president of its first management board and its longtime chairman. he served in the army from september 1961 to february 1963 and graduated in march 1964 with an average grade of 9.00. he was appointed assistant at the department of applied chemistry in october 1964. in 1971 he finished his graduate studies after completing his master’s thesis entitled “determination of unsaturation of aleksinac oil shale kerogen by modified methods for iodine number”. he received his phd degree in 1975 after defending his thesis entitled “a study of the chemical nature of kerogens by bromination”. in 1975, he was appointed assistant professor for the course chemical technology at the department of applied chemistry. he was re-appointed twice, ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 3 in 1983 and 1991, for the courses industrial chemistry and environmental chemistry. in 1992 he was appointed associate professor, followed by tenureship as full professor in 1997. while initially in the capacity of an assistant and then as a full professor as well, he improved teaching of the courses on which he was engaged in terms of both holding exercises and lectures. he drafted new curriculums for courses taught in undergraduate and graduate studies and introduced several courses in the field of biochemistry and applied chemistry. during his first years as an assistant (1964–65) he expanded exercises held in chemical technology by introducing a number of latest, state-of-the-art and problem-solving exercises for students while devising tailor-made instructions for exercises. on the occasion of students’ day (april, 4), he was recognized as one of the best assistants at the university of belgrade and awarded a diploma and a badge by the university board of the student union (1972/73). in addition to teaching undergraduate studies (industrial chemistry and environmental chemistry), as of 1979 he taught the elective courses environmental chemistry and chemistry of water technology, as well as a part of the course selected processes of chemical industry. he devised pertinent curriculums and unauthorized course readers for these courses. he taught the courses basics of chemical technology and chemical technology in the period 1980–1987, followed by industrial chemistry with environmental chemistry as of 1989. in addition to working at the faculty of chemistry in belgrade, he taught in priština, kragujevac, niš, center for multidisciplinary studies in belgrade, faculty of physical sciences and faculty of geography in belgrade. in the late 1970s and early 1980s, he instituted the courses environmental chemistry (1978) and chemistry of water and wastewater (1982) at the faculty, while initiating research in the said fields. at his initiative, the environmental chemistry division was established in post-graduate studies in 1987, whereas environmental chemistry was introduced as a course for all fourth year students at the faculty (1990). in 1993, he initiated establishment of the environmental chemistry section with the department of applied chemistry in the fourth year of undergraduate studies, which included the following courses: pollutant hemodynamics, chemistry of water and wastewater, instrumental analysis 2 and environmental chemistry 2. he is the author of the textbook entitled “environmental chemistry” (2009) and co-author of “the practicum and workbook for industrial chemistry” (2021). he wrote course readers for environmental chemistry, water chemistry and technology, and selected processes of chemical industry. he is the author of instructions for student exercises for multiple courses and co-author of three secondary school textbooks in applied chemistry. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 4 he was engaged in scientific research in the field of organic geochemistry and environmental chemistry. with regards to organic geochemistry he undertook research on structures of organic substances of old sediments, primarily of bituminous shale kerogens. his master’s and doctoral theses both pertain to this scientific field. in addition to chemistry of kerogens, he studied humic acids and local peats. regarding organic and geochemistry studies, he was dedicated to researching reactivity of inorganic constituents of organic substances, primarily pyrites. prof. petar pfendt published more than 40 scientific papers. he mentored more than 65 diploma theses, eight specialist theses, 14 master’s theses and four doctoral theses. in the position of deputy chairman or chairman of the organizing committee, he organized three large symposiums (amongst which one was international) dedicated to environmental chemistry. he was a peer reviewer of papers published in organic geochemistry and the journal of the serbian chemical society. he was a contributor to the encyclopedia and worked on entries until the end of his life. as of 1973, he participated in organizing federal and international competitions for chemistry students. on several occasions he led the summer school for young researchers in the petnica research center. he went on several study visits abroad. in 1966 he attended the “borderline fields of chemistry” summer school at basf (ludwigshafen, fr germany). he went on two short visits to the department of development of wastewater treatment procedures in 1982 and 1983 at linde ag in munich (1978 and 1979). later on, in 1991, he proceeded with professional training at kf jülich (institute of applied physical chemistry – laboratory for studying pollutant hemodynamics), henkel ag (institute of ecotoxicology – biochemodynamics of surfactants) in dusseldorf and at basf’s laboratory for studying wastewater treatment procedures, ludwigshafen. he held several positions at the faculty, amongst which some were highest -ranking managerial functions: deputy head of the institute of chemistry (1976– –1978), head of the institute of chemistry (1978–1980), head of the department of applied chemistry (1988–1997), dean of the faculty of chemistry (1998– –2001). he headed the faculty in extremely difficult and sensitive times (1998– –2001). when the university was deprived of its autonomy, and freedom was taken away from professors, he acted wisely and bravely while protecting the professors and the entire staff from inadequate laws and the authorities disinclined towards the faculty and its tradition. despite the new rules of conduct and the hitherto unheard of authorizations bequest to faculty deans, pfendt respected the tradition of academic conduct in all his acts while making decisions in agreement with heads of departments and while respecting the will of the teaching-scientific council. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 5 after meeting the requirements for retirement in 1999, his employment was extended until 2003. even after he had retired, petar pfendt came to the faculty every day to pursue his scientific research until late 2020. he was fluent in german and english and had a working knowledge of french, russian, hungarian and romanian. he was married to lidija pfendt, née sumilin, full professor at the faculty of chemistry, with whom he has two sons, eduard and robert. petar pfendt, who dedicated his entire working life to teaching and science, or that is to say to the faculty of chemistry and his department, will be remembered by the professors of the faculty of chemistry and many a generation of students as an outstanding scholar, teacher and expert, while at the same time being a quiet and modest, unobtrusive, correct and tolerant person, ready to help and listen. on the other hand, he was creative and active in his endeavors to contribute to modernization and expansion of his department and the entire faculty while instituting new courses and initiating establishment of new scientific disciplines. snežana bojović, phd, full professor ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice analytical possibilities for the relative estimation of the antioxidative capacity of honey varieties harvested in different regions of serbia j. serb. chem. soc. 81 (5) 567–574 (2016) udc 638.16+543:615.279–188(497.11) jscs–4868 short communication 567 short communication analytical possibilities for the relative estimation of the antioxidative capacity of honey varieties harvested in different regions of serbia uroš m. gašić1#, dalibor m. stanković2, dragana č. dabić2#, dušanka m. milojković-opsenica1#, maja m. natić1#, živoslav lj. tešić1# and jelena j. mutić1* 1faculty of chemistry, university of belgrade, p. o. box 51, 11158 belgrade, serbia and 2innovative center, faculty of chemistry, studentski trg 12–16, 11158 belgrade, serbia (received 13 march, revised 15 december, accepted 25 december 2015) abstract: two different approaches, spectroscopic and electrochemical, were applied for the rough determination of the antioxidative capacity of honey samples. honey samples of diverse botanical origin were collected in different geographical regions of serbia. the total phenolic content (tpc) was determined by the folin–ciocalteu method. cyclic voltammograms on a glassy carbon electrode in kcl supporting electrolyte were used to check the electrode sensitivity to the presence of honey. in order to calculate the trolox equivalent antioxidant capacity (teac) of the studied honey samples, cyclic voltammograms were recorded for the trolox standard. the results were expressed as µmol of trolox equivalents per kg of sample (µmol te kg-1). good correlations were observed between the cyclic voltammetry data and the tpc determined by the folin–ciocalteu method and the radical scavenging activity (rsa) determined using the dpph·(1,1-diphenyl-2-picrylhydrazyl) radical test. cyclic voltammetry appears to be a highly attractive alternative method for a rapid estimation of the antioxidative capacity of honeys. it was found that polyfloral honey samples had the highest, whereas acacia honey showed the lowest values of tpc. keywords: antioxidant activity; folin–ciocalteu method; radical scavenging activity. introduction generally, there is a growing interest on the efficiency of natural antioxidants in food. polyphenols, i.e., flavonoids and phenolic acids, are considered as * corresponding author. e-mail: jmutic@chem.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc150313009g 568 gašić et al. one of the important groups of components for antioxidant activity identified in honey. the antioxidant activity of honey is closely related to the floral source of the honey. generally, honeys are classified as monofloral (produced from one plant species) and polyfloral (several plant sources). different honey types were subjected to antioxidant activity tests and they demonstrated significant potential, comparable to those of the other foodstuff.1 on the other hand, research on the antioxidant capacity of honey samples originating from serbia is scarce. available literature indicates that there have only been a few reports to date on the determination of both total phenolic content and antioxidant activity of serbian honeys by spectroscopic methods2–4 and an electrochemical (polarographic) method.5 cyclic voltammetry was shown to be sensitive, convenient, rapid and low-cost approach in the quality evaluation of food products beneficial for human health.6,7 studies on the evaluation of the antioxidant activity of different food products using electrochemical methods were reported.8–11 due to growing interest in the quality of serbian honey samples and continuing research on these nutritionally important products, the antioxidant potentials of honey samples of diverse botanical origin are communicated in this paper. the samples were obtained and selected directly by the beekeepers from different parts of the territory of serbia. two different approaches, spectroscopic and electrochemical, were applied to check rapidly the antioxidative capacity of the selected honey samples. the total phenolic content was determined by the well-established spectrophotometric technique using the folin–ciocalteu method. the radical scavenging activity of the honey samples was determined using the 2,2-diphenyl-1 -picrylhydrazyl radical (dpph•). cyclic voltammetry was used to check the electrochemical response of glassy carbon in the presence of the honey samples. to inspect the applicability of cyclic voltammetry for the rather rapid and preliminary determinations, the electrochemically determined results were correlated with the results obtained by spectrophotometric methods already established in the literature for these purposes. experimental chemicals and materials methanol (hplc grade), sodium carbonate, potassium chloride, hydrochloric acid, folin–ciocalteu reagent, and filter paper (whatman no. 1) were purchased from merck (darmstadt, germany). trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was purchased from sigma–aldrich (steinheim, germany). 2,2-diphenyl-1-picrylhydrazyl·(dpph•) was purchased from fluka (buch, switzerland). ultrapure water (thermofisher tka micropure water purification system, 0.055 µs cm-1) was used to prepare the standard solutions, blanks, and artificial honey analogue (30 % glucose, 40 % fructose, 10 % sucrose and 20 % water). an analogue of honey was made to check whether the main sugars in honey could interfere with the proposed electrochemical assay. sugar standards (glucose, fructose and sucrose) were purchased from tokyo chemical industry (tci, europe, belgium). determination of antioxidative capacity of honeys 569 syringe filters (13 mm, ptfe membrane, 0.45 µm) were purchased from supelco (bellefonte, pa, usa). ethanol (96 vol. %) was from j. t. baker (deventer, the netherlands). honey samples a total of 27 honey samples collected from different regions of serbia (fig. s-1 of the supplementary material to this communication) during the 2009 harvesting season were selected and provided by the association of the beekeeping organizations of serbia (spos, www.spos.info). the botanical origins of the samples were specified by the spos based on the information provided by beekeepers and sensory characteristics, and confirmed by physicochemical analyses and chemometrics.12,13 the honey samples were from: acacia (robinia pseudoacacia), sunflower (helianthus annuus), lime (tilia cordata), giant goldenrod (solidago virgaurea), basil (ocimum basilicum), oilseed rape (brassica napus), buckwheat (fagopyrum esculentum) and polyfloral meadow honey. the honeys were stored at room temperature in the dark before analysis. determination of total phenolic content and radical scavenging activity samples were prepared according to the slightly modified method proposed by meda et al.14 each honey sample (5 g) was mixed with 15 ml ultrapure water, homogenized in ultrasonic bath for 15 min at room temperature, transferred to a 50 ml volumetric flask, and filled to the mark with ultrapure water. the solution was then filtered through 0.45 µm ptfe membrane and subjected to the determination of the total phenolic content (tpc) and radical scavenging activity (rsa). the tpc was spectrophotometrically determined by the folin– –ciocalteu method reported by singleton and rossi,15 with some modifications. briefly, 0.3 ml of the sample solution and 6 ml deionized water were mixed with 0.5 ml of folin– ciocalteu reagent and solution was incubated for 6 min at room temperature. then, 3 ml of 20 % sodium carbonate solution was added and after keeping the sample at 40 °c for 30 min, the absorbance was measured at 765 nm. gallic acid was used as the standard, and the calibration curve of gallic acid was prepared in the concentration range between 50 and 250 mg l-1. a mixture of water and folin–ciocalteu reagent was used as the blank. the results are expressed as mg gallic acid equivalent (gae) per kg of honey. the rsa of the honey samples was evaluated by a modified method of li et al.16 an aliquot of 1.0 ml of sample solution was mixed with 3 ml of a methanolic solution of 1,1diphenyl-2-picrylhydrazyl (dpph, 71 mm). the mixture was left for 60 min in the dark (until stable absorption values were obtained). after that, the reduction of the dpph• absorbance was measured by monitoring continuously the decrease in absorption at 515 nm. the rsa was calculated as a percentage of dpph• discoloration using the equation: dpph sample dpph (%) 100 a a rsa a − = where adpph is the absorbance of a methanolic solution of the dpph •, asample is the absorbance in the presence of a honey extract. the assays were performed in triplicate and the results are expressed as mean values. for cyclic voltammetry, honey samples, 1 g of each, were mixed with 20 ml 0.1 m kcl, homogenized in an ultrasonic bath for 10 min at room temperature, then filtered through 0.45 µm ptfe membranes and used for cyclic voltammetry measurements. trolox was used as the standard. in order to achieve better similarity with the honey matrix, the trolox standard was prepared and recorded in a solution of artificial honey. the solution of artificial honey was prepared in the same manner as were the honey samples (1 g of artificial honey in 20 ml of 570 gašić et al. supporting electrolyte). the scan was taken in the potential range between –200 mv and 800 mv at a scan rate 100 mv s-1. cyclic voltammograms were recorded for trolox standard in the concentration range 10 to 100 µmol l-1 in this concentration range, a linear relationship between the response and concentration was obtained. parameter q was determined as the area under the oxidation voltammetric peak for the trolox solutions. the obtained calibration curve, q = f(concentration of trolox) was linear with the correlation coefficient r2 = 0.998 and was used to calculate trolox equivalent antioxidant capacity (teac) from the q parameter of the studied honeys. the results are expressed as µmol of trolox equivalents per kg of sample (µmol te kg-1). instrumentation a uv/vis spectrophotometer (gbc uv/visible cintra 6) was used for the absorbance measurements and spectra recording, using optical cuvettes of 1 cm optical path. cyclic voltammograms were recorded on a chi760b instrument (ch instruments, austin, tx, usa). the cell was equipped with a gc electrode, 3 mm in diameter (model chi104), an auxiliary platinum electrode of a larger area (model chi221, a cell top including a pt wire counter electrode) and an ag/agcl reference electrode (model chi111; all potentials in the paper are referred to ag/agcl). all measurements were performed at ambient temperature. prior to each run, the surface of the glassy carbon electrode was freshly polished with 1.0, 0.3 and 0.05 μm alumina powder, rinsed with redistilled water and degreased in ethanol in an ultrasonic bath. statistical analysis data of all measurements were obtained in triplicate and are expressed as the mean values. statistical analyses were performed by the ncss software package.17 results and discussion the results on the total phenolic content (tpc) and of the radical scavenging activiry (rsa) of the honey samples are presented in table i, together with the q parameter derived from cyclic voltammograms (cv) as trolox equivalents. different values for the cv charge correspond to oxidation of low-formal potential antioxidants and could be an indicator of the antioxidant potential of a sample.18 the highest values were registered for the polyfloral honey samples from south–west serbia, whereas the lowest cv charge values were generally obtained for the acacia honey samples, as well as for honey from basil harvested in north serbia. all honey samples were characterized with tpc values ranging between 127.76 mg (acacia – h1) to 887.18 mg (polyfloral – h22) of gallic acid per kg of honey, table i. the average content of total phenolics was in a good agreement with the values given in the literature for honeys of the surrounding regions.19–21 generally, polyfloral honey samples had the highest values of tpc, while acacia samples showed the lowest values. such findings are consistent with literature data. here, just as an example, we cite a paper published by bertoncelj et al.22 who reported higher tpc values of polyfloral honeys in comparison to monofloral honeys (lime and sunflower) is cited. as is visible from table i, the results of rsa ranged from 1.86 % (acacia honey – h4) to 23.20 % (polyfloral honey – determination of antioxidative capacity of honeys 571 h22). among all monofloral honey samples, buckwheat was found to have the highest tpc and rsa values. this was also found in a study of different monofloral honeys when buckwheat honey was reported to have the highest antioxidant activity.23 in general, the results indicated that samples from the zlatibor region (h5, h20, h22, h23, h25 and h26) were characterized with high tcp and rsa values. it was observed that polyfloral honey samples originating from this region showed different physico-chemical properties than those from the rest of serbia.24 table i. total phenolic content (tpc), radical scavenging activity (rsa), cv charge derived from cyclic voltammograms and q parameter derived from cv of monofloral and polyfloral honey samples sample botanical origin tpc mg gae kg-1 rsa % q µc q µmol te kg-1 h1 acacia 127.76 2.18 0.041 9.01 h2 279.20 2.93 0.074 16.26 h3 328.22 3.34 0.080 17.58 h4 281.35 1.86 0.084 18.46 h5 368.69 2.51 0.110 24.18 h6 sunflower 362.42 5.04 0.112 24.62 h7 465.16 9.65 0.168 36.92 h8 246.99 5.95 0.080 17.58 h9 451.19 10.64 0.124 27.25 h10 lime 320.84 4.04 0.100 21.98 h11 373.49 4.95 0.123 27.03 h12 474.19 6.43 0.090 19.78 h13 483.03 10.84 0.170 37.36 h14 giant goldenrod 467.11 6.69 0.110 24.18 h15 414.99 5.72 0.120 26.37 h16 basil 379.63 8.76 0.070 15.39 h17 395.46 4.33 0.130 28.57 h18 oilseed rape 513.64 13.51 0.180 39.56 h19 372.47 9.45 0.110 24.18 h20 buckwheat 668.58 14.44 0.210 46.15 h21 polyfloral 496.40 5.82 0.125 27.47 h22 887.18 23.20 0.300 65.93 h23 782.16 13.81 0.280 61.54 h24 540.63 7.74 0.170 37.36 h25 432.29 5.89 0.140 30.77 h26 631.85 11.86 0.230 50.55 h27 688.81 18.67 0.220 48.35 the data from table i show that tpc, rsa and q are in acceptable agreement and bring similar information about the relative antioxidant capacity of the investigated honey samples. the correlation matrix for these variables, presented in table ii, shows large positive correlations between all the values. the q para572 gašić et al. meter derived from the cv charge was strongly correlated with the tpc, with a correlation coefficient of 0.946. the level of significance for each correlation was p < 0.000001. such statistically significant correlations clearly indicate that cv is a potentially applicable fast and informative experimental tool for the relative estimation of the antioxidant capacities of honeys. table ii. correlation coefficients between tpc, rsa and q tpc rsa q tpc 1 rsa 0.879 1 q 0.946 0.859 1 conclusion serbian honey samples from different botanical origins and geographical regions were studied for their relative antioxidant capacity. two methods, spectroscopic and electrochemical, were used for this purpose. polyfloral honey samples had the highest values of tpc, while acacia samples showed the lowest values. cyclic voltammetry was shown to be a highly attractive alternative method for the rapid relative checking of the antioxidant capacity of honeys. the linear dependence between this method and the commonly used folin–ciocalteu method was high with r = 0.946. the tpc values were compared with the antioxidant activities of the honey samples and a good correlation was obtained. such a simple electrochemical technique could be considered as a valuable method for quality control, not only of honey but also for plant-derived food products in general. supplementary material a map of the geographical regions of serbia from where the 27 honey samples under study were collected is available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. this work was performed within the framework of the research projects nos. 172017 and 172030, supported by the ministry of education, science and technological development of the republic of serbia. determination of antioxidative capacity of honeys 573 и з в о д aналитичке могућности за релативну процену антиоксидативног капацитета различитих сорти меда прикупљених из различитих региона србије урош м. гашић1, далибор м. станковић2, драгана ч. дабић2, душанка м. милојковић-опсеница1, маја м. натић1, живослав љ. тешић1 и јелена ј. мутић1 1хемијски факултет универзитета у београду, студентски трг 12–16, п. пр. 158, 11000 београд и 2иновациони центар хемијског факултета универзитета у београду, студентски трг 12–16, п. пр. 158, 11000 београд помоћу два различита приступа, спектроскопског и електрохемијског, тестиране су могућности релативне процене антиоксидативног капацитета узорака меда. узорци меда различитог ботаничког порекла прикупљени су из различитих географских региона србије. садржај укупних фенола (tpc) одређен је применом folin–ciocalteu методе. циклична волтаметрија (cv) са електродом од стакластог угљеника коришћена је као индикатор антиоксидативног потенцијала кроз промену волтаметријског наелектрисања у присуству узорака меда. ради израчунавања тролокс еквивалентa антиоксидативног капацитета (teac) испитиваног меда, снимани су и циклични волтамограми са тролокс стандардом. резултати су изражени као μmol тролокс еквивалента по kg узорка (μmol te kg-1). показано је да су резултати цикличне волтаметрије у доброј корелацији са резултатима који се добијају применом folin–ciocalteu реагенса, као и са антиоксидативним потенцијалом (rsa) који је одређен употребом 1,1-дифенил-2-пикрилхидразил (dpph•) радикалa. резултати указују на то да је циклична волтаметрија ефикасна метода и да може бити алтернативна метода за брзу релативну процену антиоксидативног капацитета меда. нађено је да највеће вредности tpc имају полифлорни узорци меда, док најмање вредности показује багремов мед. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice accepted manuscript this is an early electronic version of an as-received manuscript that hasbeen accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as s. s. bekić and s. s. jovanović-šanta, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc221222019b this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc221222019b j. serb. chem. soc.00(0)1-23 (2023) review paper jscs–12188 published dd mm, 2023 1 chemically-assisted dna transfection methods-an overview sofija s. bekić* and suzana s. jovanović-šanta university of novi sad, faculty of sciences, department of chemistry, biochemistry and environmental protection, trg dositeja obradovića 3, 21000 novi sad, serbia (received 21 december 2022; revised 21 january 2023; accepted 9 april 2023) abstract: non-viral chemical-based methods for in vitro cell transfection are commonly used to incorporate foreign gene of interest into mammalian cells due to numerous benefits high efficiency, low cost and simple methodology. these powerful transfection methods generally do not possess safety risks as virusbased, and cell toxicity is significantly reduced. to obtain transfectants, host cells are usually treated with biocompatible dna carriers such as calcium phosphate, cationic lipids, deae-dextran, polyethylenimine or dendrimers, classifying these methods based on chemical reagents used. all these different approaches are based on the similar principle, formation of encapsulated amphiphilic complexes between dna and various particles, following cell uptake, most likely mediated by endocytosis. depending on the aim and design of experiment, the choice of appropriate method is made. this review article outlines strategies of the most widely used chemical transfection techniques, pointing out advantages and limitations of different dna carriers, as well as findings of researchers how to optimize and enhance efficiency of gene delivery procedure. with methodology constantly being improved, transfection methods described here find their main, biomedical application in gene therapy, a promising way to introduce functional copy of exogenous gene to genetically defective target cells. keywords: calcium phosphate; cationic polymers; gene delivery; lipofection; nonviral transfection. introduction a widely used laboratory technique called transfection underlies the introduction of foreign nucleic acids into host cells and the study of gene and protein expression in cellular environment. chemically-assisted transfection methods catalyze intracellular trafficking of nucleic acids through the use of various compounds and serve as chemical tool that enables advancement in drug *corresponding author. e-mail: sofija.bekic@dh.uns.ac.rs https://doi.org/10.2298/jsc221222019b a cc ep te d m an us cr ip t mailto:sofija.bekic@dh.uns.ac.rs https://doi.org/10.2298/jsc221222019b bekić et al.. discovery research.1 the basis of this chemical method is interaction between negatively charged nucleic acids and positively charged ions of chemical reagents.2 exogenous dna must cross different barriers prior nuclear uptake and gene expression. unprotected plasmid dna would be degraded inside the cell by nucleases in a very short period of time, so it has to be encapsulated with appropriate carriers/particles or condensed with high packing density polycationic particles.3 avoiding enzymatic degradation depends on the stability of the complex between dna and transfection agent, as well as the cell type. upon entering the cell by endocytosis, the endosome matures and vesicle fusion between matured late endosome and lysosome occurs. as a result of increased osmotic pressure and destabilization of lysosomal membrane, endosome escapes in the next step. after bursting, vesiclular content is released into the cytoplasm.4 following burst of endolysosome, complexes further enter the nucleus by not fully understood mechanism and plasmid dna is released into the nucleus, resulting in transcription of gene of interest.5,6 on the other side, destiny of complexes that did not successfully leave the endolysosome is degradation by lysosomal enzymes.4 expression of the functional eukaryotic protein in bacteria is often a problematic task. protein expression is significantly improved by using eukaryotic cell cultures due to possibility of post-translational modifications and correct protein folding in this system.7,8 introduction of foreign nucleic acids into cultured mammalian cells, enabled by a powerful transfection technique, also known as gene delivery, has revolutionized the study of gene function and expression of specific proteins.9,10 furthermore, this technique is promising in the prevention and treatment of genetic disorders and diseases such as cystic fibrosis,11 hemophilia,12 dystrophy13 and cancer,14 through an innovative approach in biomedicine, gene therapy.15 this up-to-date therapy, where genetically defective target cells are modified by the introduction of an exogenous functional gene, greatly attracts the attention of the scientific community.11–15 except repair of genetic damage, with gene therapy treatments it is also possible to treat infectious diseases16 by inhibiting life cycle stages, as well as malignancies.14 although this type of therapy promises a lot, development of new, effective and safe therapeutics in this field is very slow and challenging.17 there are two types of transfection stable and transient, with the main difference in long-term integration of foreign dna into the host genome in stable transfection, whereas in transient type transfected gene is not integrated, so expression lasts for only a limited period of time.18,19 depending on the purpose and scope of research, choice between creating stable or transient cell lines is made. when it is required to examine effects of short-term expression of a gene or protein product on a small scale transient transfection is mainly method of choice, as well as in the assays that precede creation of stable cell lines. on the other side, although stable transfection is a complex process, it plays an unavoidable role in a cc ep te d m an us cr ip t chemically-assisted dna transfection methods 3 revealing mechanism of gene regulation, large scale protein production or pharmacological studies.20,21 experimental methods used to create recombinant cell lines can be classified as direct, when the gene of interest is introduced in nucleus directly by microinjection, and indirect, where the transfer of genetic material occurs indirectly through complex formation between dna and various chemical agents, using viral vectors or by physical force-mediated cell uptake. in addition to this classification, there is another one that classifies transfection methods to biological (use of viral vectors), physical (sonoporation, electroporation, magnetofection, phototransfection) and chemical (use of calcium phosphate, cationic polymers, cationic lipids).10,19,22 the main goal of transfection process is to efficiently deliver gene of interest into cells without safety risks. non-viral carriers are developed as an alternative to viral ones, not only due to their straightforward manipulation, but also due to reduced cell toxicity and absence of immune response or potential mutagenicity.23 much effort is put into developing vectors that are not toxic for cells . motivated by the significance of chemically-assisted transfection methods in biomedicine and importance of availability of summarized information related to their methodology and efficiency improvement, we provide in this paper an overview of currently available and the most commonly used methods for dna transfection of mammalian cells based on the use of chemical reagents. short description of the chemically-based transfection methods and their advantages and disadvantages are summarized in the table 1. table 1. overview of chemically-based transfection methods. type of transfection description advantages disadvantages calcium phosphatemediated transfection widely used method for introducing foreign dna into cells that includes mixing dna with calcium chloride and phosphate ions, formation of coprecipitates, and cell uptake. -use in biomedical research -simplicity -cost-effective -applicability to large number of cell lines -biocompatibility -suitable for stable and transient transfection -safe use -low efficiency -sensitive to changes in ph and salt concentration -low reproducibility -phosphate-free medium required -serum-supplemented medium required -low efficiency in most primary cell lines lipofection this commonly used transfection method is based on the use of cationic lipids to deliver dna to -high efficiency -the most extensively studied types of dna carriers -simplicity -commercially -cytotoxicity -inactivation in the presence of serum proteins -expensive a cc ep te d m an us cr ip t bekić et al.. eukaryotic cells in the form of vesicles called liposomes. available reagents -high reproducibility -suitable for stable and transient transfection -large scale use -safe use -induction of antiinflammatory response cationic polymermediated transfection this method uses cationic polymers such as deaedextran, pei and dendrimers to form complexes with dna and introduce nucleic acids into cells of interest via electrostatic interactions. -simplicity -cost-effective -applicability to large number of cell lines -structural versatility of cationic polymers -biocompatibility -large scale use -efficient cell recovery after transfection -low efficiency (for stable transfection) -limited to short-term transient transfection inactivation in the presence of serum proteins -expensive synthetic procedures -cytotoxicity calcium phosphate-mediated transfection among chemical methods, calcium phosphate (cap) precipitation is one of the most commonly used due to its numerous advantages.24 the first method developed for mammalian cell transfection was actually cap based method initially performed in 1973 by graham and van der eb for the introduction of adenoviral dna into mammalian cells.25 adenovirus type 5 dna was successfully adsorbed by human kb cells in monolayer culture.25 widespread use of this transfection method in biomedical research was demonstrated through the generation of numerous highly productive recombinant cell lines.26 benefits of divalent calcium cations, as dna carriers in the transfection process, are related to their natural presence in many cells in the organism and physiological acceptability.27 principle of this method includes mixing dna with calcium chloride in phosphate buffer and adherence of resulting dna-cap coprecipitates on the surface of the cell membrane.9 endocytosis (fig 1.) and direct penetration through the membrane are most likely the primary mechanisms of cap-dna complex cellular entry.3,28 one of the potential mechanisms of endocytotic uptake at the intracellular level is described in the work of neuhaus et al.4 using specific inhibitors, olton et al. demonstrated that uptake of nanocaps-dna complexes into the cell and subsequent gene expression were mediated by both endocytosis types, clathrinand caveolaedependent, whereas the former one was more highlighted prior to this study.29 a cc ep te d m an us cr ip t chemically-assisted dna transfection methods 5 fig 1. proposed endocytotic mechanism of cap-mediated cell transfection. this method is characterized by high efficiency, simplicity, low cost, applicability to a large number of cell lines, biocompatibility and it is suitable for both, transient and stable transfection.27 it is also method of choice when cotransfecting multiple plasmids.27 disadvantages include significant changes in transfection efficiency due to small variations in ph and difficulty in reproducing conditions for creating coprecipitates of adequate size and quality.26 furthermore, a medium with already high amount of phosphates is undesirable for this type of transfection procedure and this gene delivery method is effective only on highly differentiated cells but not on primary cell lines or animals.30 potential difficulties related to transfection of high copy number plasmids by cap precipitation method are requirements for serum-supplemented medium, often avoided during recombinant protein production in cell lines, and low efficiency.24 the highest efficiency of exogenous dna uptake was achieved in 80-90% confluent cells that divide quickly, as well as in those growing in monolayer due to uniform precipitation.2 moreover, glycerol and dimethyl sulfoxide (dmso) are shown to increase efficiency of dna delivery into some cell lines, however, exposure time to these agents is limited since they may exhibit cytotoxicity.20,21 in order to examine optimal conditions and achieve the highest efficiency, the original cap method has undergone numerous modifications and optimizations since it was published. in the original protocol hepes buffer (n-(2hydroxyethyl)piperazine-n'-(2-ethanesulfonic acid)) was used.25 however, many variations in buffer composition were subsequently tested and conditions were individually optimized for specific cell lines. chen and coworkers significantly improved effectiveness of this method by replacing hepes with lower ph buffer, bes (n,n-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid).31 during cell growth in 3% co2 atmosphere at lower ph (6.95), dna-cap precipitates are formed gradually and uniformly on the cell surface, resulting in reduced cytotoxicity and increased transfection efficiency, most likely due to uptake of precipitates by a larger number of cells. furthermore, transfection a cc ep te d m an us cr ip t bekić et al.. efficiency was reported by chen et al. to be enhanced with the use of circular plasmids instead of linear ones, that are easily degraded by nucleases from culture medium.31 in the paper of jordan et al. it was stated that formation of cap-dna coprecipitates for successful transfection is possible only in a narrow range of conditions and is mostly dependent on the concentration of calcium and phosphate ions and other physicochemical factors dna concentration, temperature and reaction time.32 optimized procedures were applied to both, transient and stable transfection, and, using them, greater efficacy than in previous protocols was achieved.32 study of ling et al. posed the same question.30 key parameters responsible for high transfection efficiency of highly differentiated cells were estimated to be primarily characteristics of the buffer, suggesting hbs buffer with ph 7.10 as an optimal, then presence of fetal bovine serum in the medium, vortexing cells with precipitant particles and glycerol shock, whereas replacement of the consumed medium with fresh before transfecting cells had no effect on transfection potency.30 sun et al. demonstrated that, with optimization of this method, it is possible to transfect cell types such as primary neurons, a popular target in neural cell biology.33 moreover, study of sariyer provides a highefficiency protocol for transfection of primary neuronal cultures.34 due to many factors that affect efficiency including ph, salt and dna content, the period between precipitation and transfection,type of the cell line, and the researcher's skills, it is very challenging to standardize the cap transfection technique.15 these conditions must be optimized for each cell line and laboratory. transfection has to be performed shortly after precipitation, otherwise cap will lose activity when it reaches microcrystal size.27,28 in order to maintain appropriate size of calcium phopshate particles and inhibit their further growth, functionalization with various organic molecules is preferred.14 to overcome this problem, several strategies for controlled growth of cap nanoparticles have been developed, including polymer stabilization or lipid coating.35 dna may be adsorbed onto cap nanoparticles with added additives to maintain their size.27 obtaining particle size in submicrometer range facilitates penetration of dna-cap complexes through the cell membrane. additionally, it is of great importance to focus on enhancing stability of nanoparticle-dna complexes, as well as on biodegradable properties of carrier, especially useful in gene therapy. working in the field of cancer gene therapy, liu and coworkers developed modified formulations of cap nanoparticles as vectors for efficient dna delivery into cancer cells.14,36 lipofection cationic lipid mediated transfection (lipofection) is a bright spot in the field of gene therapy and significantly superior in clinical trials compared to other a cc ep te d m an us cr ip t chemically-assisted dna transfection methods 7 transfection methods 37. therapeutic potential of cationic liposomes was tested many times in clinical trials in patients with cystic fibrosis.38 in the late 1970s felgner et al. first demonstrated procedure for cell transfection using a positively charged cationic lipid, n-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (dotma), which forms spherical vesicles in aqueous solution with one (unilamellar) or more (multilamerllar) concentric phospholipid bilayers, called liposomes.39 cationic lipids are actually the largest and the most extensively studied group of non-viral dna carriers widely used today. currently, there is a growing interest in the development of new reagents for successful lipofection and some of them have already found their way to the market. numerous commercially available cationic lipid transfection reagents from different manufacturers (lipofectamine 3000 or 2000, lipofectin, cellfectin, effectene, fugene 6, and dotap) vary in price, efficiency and application in different cell types.40 cationic lipids are amphiphilic molecules generally built of three structural domainspositively charged polar heads, hydrophobic domain and linker between them and even small changes in the structure of any of them can significantly affect transfection efficiency. cationic head is usually composed of primary, secondary or tertiary amines, but can also contain imidazole and guanidino groups.40 byk et al. described synthesis of novel cationic lipids as gene delivery agents and conducted structure-activity relationship (sar) studies to explain differences in transfection potential of geometrically different, asymmetric polyamine groups in cationic heads.41 hydrophobic moiety generally represents doubly saturated or unsaturated hydrocarbon chain of varying lengths, which does not have to be symmetrical in structure, whereas linkers are usually esters, ethers, amides or carbamates.40 a wide range of different linkers, building elements of cationic lipids, is described in detail in review article of zhi et al..42 within the liposomes, hydrophobic components of cationic lipids are facing inside of the vesicle and they are protected from aqueous solution by the presence of polar heads on the surface of the molecule. in the central part of the liposome there is a cavity where is dna of interest packed for delivery and protected from degradation by various enzymes after cell uptake. review article of niculescu-duvaz et al. covers sar studies of cationic lipid domains and helps in understanding mechanism of liposome formation and action providing an excellent basis for the rational design of new improved transfection vectors.40 briefly, the mechanism of cationic lipid mediated transfection is divided into several stepslipoplex formation, membrane binding, entry into the cell, endosomal escape and finally nuclear entry and expression of gene of interest.40 electrostatic interactions are formed between liposomes, due to polar heads with overall net positive charge, and negatively charged phosphate groups of transfecting nucleic acids. penetration of lipid-associated dna through the hydrophobic cell membrane is facilitated by neutralization of anionic groups and a cc ep te d m an us cr ip t bekić et al.. entry into the target cell in the form of lipoplex is probably mediated via endocytosis or fusion (fig 2).38,39 total charge of lipoplexes is positive enabling them to bind to negatively charged surface of the cell membrane.39 following nonspecific electrostatic interaction with the cell membrane lipoplexes are introduced into intracellular compartments dominantly by endocytotic mechanism and this whole process of internalization is mostly influenced by the size of lipoplex. next phase of lipofection involves endosomal escape which is mediated by flip-flop mechanism due to lipid nature of both, lipoplex and endosomal membrane. in response to the phase behavior of these lipid bilayers, dna is released after complete neutralization of cationic lipids43,which was also investigated and described by xu and szoka.44 their conclusion was that lipoplexes destabilize endosomal membrane, reducing the intensity of electrostatic interactions between dna and cationic lipids and releasing dna to the cytoplasm.44 there are two theories about the entry of released dna into the nucleus-by passive transport during cell division, when nuclear membrane is temporarily ruptured, or by active transport through the nuclear pores.43 besides endocytosis, dominant pathway for dna delivery inside the cell fusion of cationic liposome with the cell membrane is also possible mechanism. which mechanism of these two will take place depends on the liposomal formulation itself.38 fig 2. proposed mechanism of cationic lipid-mediated transfection. key features of this cationic lipid-mediated transfection process are simplicity, high efficiency (especially in adherent cell lines), reproducibility and applicability to both, transient and stable transfection types, as well as to in vivo models, where it has shown lower efficacy indeed.39,45 genetic material owes protection from degradation to stability and structural properties of liposomes, but in order to avoid possible cell toxicity, it is necessary to find balance between optimal conditions, crucial for transfection efficiency, and potential toxic effect on cells.46 as expected, when considering the principle of this method, one of the a cc ep te d m an us cr ip t chemically-assisted dna transfection methods 9 essential factors for successful lipofection is optimal ratio between liposomal and dna component. however, if this ratio moves in favor of the first component, cytotoxicity may occur.45 liposomes are suitable for large-scale production, their use is safe 47 and, significantly, there are no limitations to the size of the dna molecules that can be transfected.48 depending on the cell type, this technique has proven to be up to 100 times more effective than transfection using cap or cationic polymers.39 routinely for performing this procedure, cells are grown in serum-free media. it is likely that negatively charged serum proteins interact with cationic liposomes and destabilize them, dramatically reducing efficiency of transfection process. yang and huang separated serum proteins by chromatography and tested efficacy of lipofection in the presence of both fractions.49 except introducing serum-free media, that is impossible in in vivo conditions, they suggested overcoming this problem by increasing the charge ratio between cationic lipid reagent and dna molecule or by adding positively charged polylysine to neutralize multiply negatively charged serum proteins.49 ross et al. attempted to overcome reduced transfection potential in the presence of serum by controlled growth of lipoplexes and concluded that key parameter for successful transfection is appropriate size of lipoplex, crucial for association with the cell membrane.50 since it was shown that inhibition of lipoplex formation by serum proteins is actually the cause of lower efficiency of lipofection-mediated gene transfer, they developed a protocol for fine regulation of lipoplex growth in polyanion-containing medium stopped at a crucial moment by serum supplementation.50 higher transfection efficiency achieved by combining cationic liposome with "helper" neutral lipid was discussed in many published studies.51–53 the most commonly used helper lipids for enhancement of cationic liposomes transfection potency are unsaturated phosphatidylethanolamines (pe), such as dioleoyl-pe (dope). this molecule is believed to facilitate fusion of cationic liposomes from lipoplexes with endosomal membrane, followed by release of dna into the cytoplasm. the efficiency of fusogenic lipid is believed to be result of its ability to form structural forms similar to membrane fusion intermediates and destabilize it.53 hui et al. attempted to explain complex role of helper lipids comparing efficiency of pe and phosphatidylcholine (pc) in transfection of cho cells.53 according to their results, former lipid led to rapid and premature aggregation of complexes in the medium resulting in formation of too large granules to enter the cell, while transfection efficiency was higher in the presence of pc as helper lipid. gradual formation of pc agregates was directed at the cell surface and after reaching appropriate size, granules were introduced into the cell. they also pointed out that endocytosis together with all other factors affecting this process is crucial for successful transfection, while fusion of cationic liposomes with the cell a cc ep te d m an us cr ip t bekić et al.. membrane is secondary and insufficient for entry of dna complexes into the cell in the absence of endocytosis.53 opposite results of efficiency of neutral helper lipids, dope and dopc, in the lipofection approach were identified by du et al. in the study about design of novel lipopolyplex formulations using combination of plasmid dna, cationic liposomes and peptide component.51 increased transfection efficiency was reported in the presence of neutral lipid dope instead of dopc, as claimed above. peptides are thought to participate in dna packaging and directing complex to membrane receptors, whereas liposomal component, stabilized by electrostatic interactions between cationic lipid and dna, presumably causes fusion with endosomal membrane, endosomal escape and release of genetic content into the cytoplasmic region.51 after cellular uptake of lipo(poly)plexes by clathrin-dependent endocytosis, release of nucleic acids from endosome to cytosol is required in order to avoid endosomolysis, degradation of genetic material by lysosomal enzymes, suggesting the need to promote destabilization of the endosomal membrane and increase lipoplex stability after entering the cell.54 it has also been noticed that, due to its nature, dope promotes formation of inverted hexagonal lipid structures that fuse with lipid bilayer of endosome leading to endosomal escape, dna release into the cytoplasm and accumulation in the nucleus, whereas non-fusogenic dopc promotes more stable laminar forms of lipid bilayer leaving lipoplexes trapped within the late endolysosomes.51,54 besides dope, effect of cholesterol, as helper lipid molecule, has also been recognized in enhancing transfection efficiency of cationic liposomes.55 biological importance of cholesterol is widely known. this molecule is essential building block of membranes, participant in many metabolic and biochemical processes, as well as highly involved in endocytosis.56 group led by safinya explained impact of cholesterol on enhancing transfection efficiency by inducing structural changes in lipoplexes.48 increase in cholesterol concentration will lead to a decrease in hydration layer of the lipoplex cationic part that, due to repulsions, acts as a barrier for endosomal fusion. in this manner not only fusion between lipoplex and endosomal membrane is facilitated, but also endosomal release of the complex.48 importance of cholesterol in enhancing efficiency of cationic polymer transfection has also been reported. replacement of poly(allylamine) primary amino group with cholesterol significantly reduced cytotoxicity of this agent and enabled hydrophobic interaction with the cell membrane.52 interestingly, cationic lipids have not only been shown to function as carriers of hydrophilic molecules (dna), but also interacting with the cell, they can modify different signaling pathways, stimulating immune and anti-inflammatory response.57 however, there is a lack of information on this topic. combination of immunostimulating and carrier properties of these molecules is highly recommended in the vaccine field.58 during their interaction with the main target, a cc ep te d m an us cr ip t chemically-assisted dna transfection methods 11 cell membrane, physiology of the cell changes, especially at the level of membrane proteins involved in numerous signaling cascades, such as mapk kinases.59 considering large number of synthesized cationic lipids of different structures and various targets of their action, it is obvious that only a small part of their possible activities has been identified to date. cationic polymer-mediated cell transfection cationic polymer-mediated transfection is a technique used in biochemistry and molecular biology to introduce nucleic acids into cells with many applications in gene therapy60, drug delivery and recombinant protein production.61 its methodology is relatively straightforward, cost-effective, and can be applicable to a wide range of cell types. cationic polymers are classified based on their chemical structure, molecular weight, morphologies and charge density to linear, branched, hybrid, and amphipathic.62 unlike cationic lipids, cationic polymers are watersoluble molecules that create polyplexes by complexation with dna. principle of cationic polymer based transfection for the most commonly used reagents is shown in fig 3. resulting complexes are smaller in the size and dna is more compactly packed than by previously described methods, implying that these advantages may be the key for efficiency.63,64cationic polymers are very flexible molecules that can be prepared as linear or branched form and due to structural versatility it is easy to manipulate with their molecular weight and geometry.64 molecular weight of cationic polymers was found to be inversely proportional to their cytotoxicity along with the distance between charged atoms in polymer.65 a cc ep te d m an us cr ip t bekić et al.. fig 3. cationic polymer based transfection with the most commonly used reagents. naturally occurring carbohydrates, such as dextrans, are characterized by biocompatibility and ability to directly bind nanoparticles from polyplex formulations to receptors on the cell membrane.66 except biocompatibility, sugarbased nanoparticles, as an alternative to non-viral vectors, exhibit low toxicity, low cost and structural modifications to improve their biological potential are easily obtained.67 in addition to dextran, chitosan and gelatin are natural polymers commonly used in biomedicine, whereas structures of synthetic gene delivery carriers are based on polyethyleneamine (pei) 68, poly-l-lysine (pll), polyprene and dendrimers.67 use of cationic polysaccharides in non-viral transfection procedures, with a focus on chitosan and its derivatives, has been recognized and extensively studied by liu et al.69 in this review more details are discussed on three the most prominent types of cationic polymers with superior efficiency compared to others, commonly used as transfection vectors due to their wide range of applications, high charge density, low cytotoxicity and immunogenicity. deae-dextran-mediated cell transfection diethylaminoethyl (deae)-dextran was among the first reagents used to deliver exogenous nucleic acids (poliovirus rna) to mammalian cells (primary a cc ep te d m an us cr ip t chemically-assisted dna transfection methods 13 rhesus monkey kidney cells) over 50 years ago.70 deae-dextran is cationic polysaccharide polymer of bacterial origin. positively charged deae moiety electrostatically binds to dna of total negative charge, resulting in formation of compact particles, polyplexes. due to the presence of three basic groups of different pka values in the structure, specific association between deae-dextran and dna is dependent on ph and solution ionic strength.71 as a result of excess positive charge, this soluble complex associates with negatively charged cell membrane and is probably introduced into the cell by a process of nonspecific endocytosis, osmotic shock and change of membrane permeability or assisted by glycerol or dmso. method for transfection of lymphocyte cell lines, described by smale, is based on an initial incubation with a mixture of deae-dextran and dna, followed by a secondary one in the presence of chloroquine.63 chloroquine participates in the neutralization of lysosomal enzymes, prevents acidification in endosomes and thereby inhibits intracellular degradation of plasmid dna.63 the increase in transfection efficiency with chloroquine treatment was investigated by luthman and magnusson.72 they assumed that, except increasing ph in lysosomes, this molecule can strongly bind to dna and protect it from degradation.72 osmotic shock probably facilitates dna uptake into the cell and prevents its breakdown by action of nucleases from lysozyme, causing pinocytotic vesicles burst due to osmotic imbalance.73 takai et al. applied combination of deaedextran and osmotic shock, by treating cells with high osmolality buffer, for transient and stable transfection of lymphocyte cell line, with less efficiency and lower gene expression observed in the latter one.73 deae-dextran-mediated dna uptake is generally limited to a short-term transient transfection, whereas in the case of stable transfection, except low number of successfully transfected cells, toxicity may represent another drawback.63 in addition to its biocompatibility, beneficial effects of dextran on the lipid status of the organism make it an attractive candidate in drug delivery and gene therapy.71,74 examining effect of polysaccharide dextran polymers of wide molecular weights range on transfection efficiency, it was concluded that efficiency positively correlates with increase in molecular weight, without impact on cell viability.74 furthermore, deae-dextran found its use in lipofection in the design of appropriate delivery carrier systems by stabilizing liposomal vesicles.75 modified cell transfection protocol with deae-dextran, published by shovel and coworkers in the early 1980s, introduced a step of "shocking" cells by exposure to dmso or glycerol, which significantly increased expression of transfected gene, up to 50 fold.76 traditional protocol was also improved by sussman et al. by treating mouse ltk cells with dmso at higher ph during initial incubation, resulting in 80% of cells successfully transfected with thymidine kinase gene from simplex virus.77 mack et al. published reproducible method for transfecting a cc ep te d m an us cr ip t bekić et al.. sensitive adherent human primary macrophages using deae-dextran.78 in this study three critical parameters for successful deae-dextran mediated transfection were identified and described: amount of added dna per transfection, concentration of cationic polymer deae-dextran and time of incubating cells in transfection medium. 78 due to its great biomedical potential, onishi et al. chose deae-dextran as a basis for the development of non-viral carriers for in vivo gene delivery.79 this polymer can be autoclaved, it exhibits low cytotoxic and high transfection activity, and plays protective role against dnases. they performed graft polymerization of methyl methacrylate on deae-dextran generating stable and efficient vector in the form of copolymer with significant therapeutic potential.79 pei-mediated cell transfection among leading and highly diverse group of non-viral carriers in the gene delivery field, pei attracts attention as cationic polymer with easily modifiable structure that allows fine regulation of its physicochemical potential.80 pei is polymer of aziridine, with amino nitrogen atoms (at every third position) available for protonation, contributing to significant cationic potential of this organic molecule and buffering properties in wide ph range.81 pei does not have defined center of symmetry.82 this molecule is easy to handle, low-cost and effective even in the treatment of cells growing in suspension.5 it can be prepared in two forms, linear and branched, with the latter proven to be more effective in dna condensation and transfection of mammalian cells and, unless otherwise stated in the literature, it refers to a branched type.6 due to branched structure and high density of positive charge, dna is certainly more easily trapped within this polymer than with linear structures such as pll and, thus, protected for safe gene transfer to target eukaryotic cell.81 pll alone does not show significant transfection efficiency in vitro and it mostly requires binding to molecules that will facilitate either cell entry or endolysosomal escape.82 pei/dna complexes enter most of the cells, but only a small number will express protein of interest.83 investigating destiny of these polyplexes inside the cell may explain why this occurs and answer a number of other important questions related to the mechanism of action. after endosome formation, polyplexes are released into the cytoplasm, destabilizing the endosomal membrane by "proton sponge" effect 81. this phenomenon is associated with buffering capacity of pei molecule and its ability to bind free protons within endosomes.63 by binding protons pei leads to an increase in endolysosomal ph that may affect folding of enzymes involved in dna degradation, whose inactivation leads to release of functional, undegraded nucleic acids. one of the speculations is that surface of these complexes, due to cationic character, adheres to negatively charged a cc ep te d m an us cr ip t chemically-assisted dna transfection methods 15 phospholipids from nuclear membrane or from membrane fragments of bursted endolysosomes and being introduced into the nucleus by fusion.5,6 optimal pei/dna ratio is essential for successful transfection. it has been documented that treating cells by complexes with low ratio showed no significant transfection efficiency, whereas too high ratio resulted in altered cellular morphology and reduced growth.81 accordingly, to optimize this method, it is crucial to find optimal ratio between these two components. on the other hand, entering large number of polyplexes by endocytosis may lead to cytotoxicity due to increased concentration of components from damaged endosomal membrane parts, upon endosomal escape.81 cytotoxicity of pei polyplexes may be related to their charge84, especially formation of non-specific interactions in the cell, so modification of their structure is popular strategy to avoid undesirable binding. circulating serum proteins can bind to pei/dna complexes and inactivate them. for these reasons, their surface is modified by non-ionic molecules such as peg85, as well as by various polysaccharides, which also play a role in targeting cells via receptors on the membrane. patnaik et al. performed complexation of pei with alginic acid and optimized appropriate ratio of these two components to achieve high mammalian cell transfection efficiency and viability.80 unmodified pei molecule showed approximately 2-16 fold lower transfection efficiency compared to pei-alginate complex against all tested cell lines. beside reducing polyplex cytotoxicity, this inserted polysaccharide molecule increases transfection efficiency by participating in endosome damage.80 godbey et al. provide an overview of physicochemical characteristics of pei, its complexes and role in transfection of cell lines.6 the protonability of this nucleic acid carrier was demonstrated to correlate with transfection efficiency. also, it has been shown that manipulation of balance of components in pei/dna complex is essential to overcome attenuation of transfection efficiency caused by off-target interactions with interfering proteins. although this undesirable binding can be bypassed by modifying complex surface with peg, polystyrene, ethoxylated glycerol, poloxamer and polylactic acid86, significant improvement of transfection efficiency was made by attaching pei to different transport proteins, such as transferrin.6 in addition to concentration of h+, formation of dna/pei polyplexes depends on the time of incubation, temperature, medium characteristics and amount of ions and various salts in solution.83 pei/dna polyplexes occur in different forms, including rods and toroids. these two structures determine the uptake efficiency and therefore overall transfection efficiency.6 taking into account the fact that all these factors affect transfection efficiency, methods that provide insight into physicochemical characteristics of these complexes are of great importance. scanning force microscopy was used to determine morphological properties and dimensions of pei/dna condensates, as well as to visualize way of dna packaging and compactness under physiological a cc ep te d m an us cr ip t bekić et al.. conditions.87 on the other hand, using particle tracking techniques (nanoparticle tracking analysis (nta), dynamic light scattering (dls)) and electron microscopy and varying incubation time of polyplex formation dominguez et al. found a strong correlation between rate of polyplex aggregation and success of transfection.5 together with previously mentioned factors, transfection efficiency is affected by concentration of dna, as well as by the type of cell being transfected and its environment.5 in comparison to cap method, where presence of serum in the medium is mandatory and complex formation time-consuming, pei transfection is more suitable for large scale use since it can be performed in serum-free medium and does not require formation of pre-complexes between pei and dna.88 comparing transfection efficiency of cho cells with pei or cap, chenuet et al. emphasized higher efficiency of the first method in terms of cell recovery and the latter one related to cell-specific productivity and copy number of integrated plasmids in stable transfection system.24 cell recovery after pei-mediated transfection is more efficient than in the case of cap, although it was believed that interaction of this polymer with genomic dna in the nucleus can lead to destabilization of chromosomes and reduced recovery.24 on the other side, when considering costs of performing on a large scale, this method requires lower budget than lipofection.88 dendrimers in cell transfection dendrimers represent another gene delivery system of unique structure and will be briefly described here. these molecules are highly branched synthetic polymers of spherical geometry composed of monomeric subunits arranged around central core.89 owing to different synthetic strategies, functional groups, monomer units, as well as structure of central dendrimer core and number of concentric layers around it differs.90 layer number around central nucleus increases molecular weight of dendritic molecules, reaching values of protein molecular weights 91, which is important for transfection process because structurally more complex vectors are able to carry and deliver more dna. more than 100 types of dendrimers have been synthesized and classified into several families peptide, polyamidoamine, pamam, polypropyleneimine, ppi, phosphorus, carbosilane and polylysine or polyornithine dendrimers.92 characteristics of listed dendrimer groups have been extensively reviewed in the article of pedziwiatr-werbicka at al.92 binding affinity and stability of peptide dendrimer-dna complex, and thus transfection efficiency, can be influenced by the type of interactions (covalent or non-covalent) between peptide and nonpeptide components in this dendritic molecule.93,94 structure of one of the most commonly used peptide dendrimers is based on polylysine.93 pamam is among the most common commercially available cores for dendrimer formation.93 core a cc ep te d m an us cr ip t chemically-assisted dna transfection methods 17 of pamam dendrimers contains ammonia or ethylenediamine, where nitrogen is trior tetravalent.91 controlled polymerization around this central molecule in pamam dendrimers leads to gradual formation of structure with spherical molecular architecture and large number of repeating amidoamine units. high density positive charge of primary amino groups on the surface, protonated at neutral ph, enables interaction with biological molecules of polyanionic character, such as dna.91 these molecules of usually radial symmetry generally exhibit low cytotoxicity, good biocompatibility and water solubility, making them vectors of choice in transfection process.95 however, due to high density charge, dendrimers may become cytotoxic, and to overcome this barrier, they undergo functionalization and coating. in terms of costs, synthetic procedures with these compounds can be expensive.93 nature of dendrimer surface affects stability of encapsulatied complexes with dna, cellular uptake and gene delivery. dendrimers showed great biomedical potential as drug delivery carriers and transfection agents in both, nucleic acid and protein transfection, as well as in gene therapy.92 functional role of these molecules in biomedicine has been described comprehensively in recent work of mirakabad et al.93 in the article of tang and szoka interaction of dendrimers and other cationic polymer structures with dna was examined, as well as morphology of resulting complexes.82 it was observed by electron microscopy that dna condensation occurs in compact toroidal form. complexes with degraded polyamidoamine dendrimers were observed as single units, while intact ones were clustered with almost 100 times larger diameter, suggesting that degree of aggregation is dependent on properties of individual polymers.82,96 unexpectedly, compared to intact, degraded dendrimers were more successful in transfection, most likely due to increased flexibility.82 wang and coworkers modified surface of dendrimer by fluorination introducing fluorine atom into aromatic ring of benzoic acid conjugated with dendrimer.95 transfection efficiency was estimated to increase with the degree of dendrimer fluorination.95 kwok et al. developed new hybrid transfection concept by studying synergistic action of peptide dendrimers and lipids as transfection reagents.97 combination of different transfection agents affects increase in their individual efficacy probably due to multiple interactions with dna and membrane on their way to the cell nucleus. this field has not yet been extensively studied because dendrimer structure modifications are very challenging.97 conclusion transfer of exogenous genes into cultured mammalian cells is a very powerful tool for manipulating dna and represents an essential step in understanding function and regulation of genes and their products, proteins. testing using this a cc ep te d m an us cr ip t bekić et al.. model system precedes in vivo studies and clinical trials converging towards the same milestone therapeutic use in humans. gene therapy, designed to introduce exogenous functional copies of genes into cells with damaged protein function represents a real revolution in medicine. mammalian cell transfection methods are developing exponentially. new generations of transfection technologies are sophisticated and easily amenable to further imrovement, such as automation with minimal operator input. in order to become commercially available products, gene delivery carriers have to meet certain criteria and overcome a lot of barriers. undesirable characteristics of these vectors are induction of immunogenicity and cytotoxicity, whereas it is preferable to be biocompatible, stable, available for structural modifications and to exhibit significant potential for cargo delivery of molecules of various types and sizes in the cell. due to simple handling procedure and reduced costs there is a great interest for developing non-viral methods, especially those based on the use of various chemical agents-inorganic amphiphilic aggregates, cationic polymers or cationic lipids. their chemical design is constantly undergoing modifications in order to increase transfection efficiency and reduce side effects. generally, transfection by chemical methods is based on electrostatic interactions between polycationic core of chemical agent and oppositely charged dna followed by entry of resulting complexes into the cell, most likely via endocytosis. chemically-assisted approaches described here have their own advantages and disadvantages and choice of the optimal one depends on the purpose of planned experiment. furthermore, remarkable transfection success is achieved by combination of individual methods and transfection agents, with promising potential in biomedicine. synergistic action leads to overcoming disadvantages of individual methods and improves their activity. transfection efficiency may also be increased by removing nonspecifically binding serum proteins from the medium, optimizing size of complexes formed between chemical reagent and dna, then modifying charge on their surface, inhibiting degradation within the lysozymes, increasing cell permeability, etc. to sum up, in vitro non-viral gene delivery to cells by chemical methods represents promising area in biomedical research with methodology constantly being improved. acknowledgements: the authors acknowledge financial support of the ministry of education, science and technological development of the republic of serbia (grant no. 45103-68/2022-14/200125).a cc ep te d m an us cr ip t chemically-assisted dna transfection methods 19 и з в о д преглед хемијских метода за днк трансфекцију софија с. бекић* и сузана с. јовановић-шанта универзитет у новом саду, природно-математички факултет, департман за хемију, биохемију и заштиту животне средине, трг доситеја обрадовића 3, 21000 нови сад, србија за in vitro уношење жељеног гена у ћелије сисара обично се користе невирусне хемијске методе за трансфекцију с обзиром на то да су веома ефикасне, јефтине и једноставне. углавном немају безбедносне ризике као оне засноване на употреби вирусних вектора, а и токсичност према ћелијама је значајно смањена. ове методе се класификују на основу хемијских реагенса који се користе за трансфекцију ћелија домаћина. углавном су то биокомпатибилни носачи днк, као што су калцијум фосфат, катјонски липиди, deae декстран, полиетиленимин или дендримери. иако различити приступи, сви су засновани на формирању инкапсулираних амфифилних комплекса између днк и различитих честица, након чега следи улазак у ћелију, највероватније посредован ендоцитозом. у зависности од циља и дизајна експеримента, врши се избор одговарајуће методе. у овом прегледном раду описане су стратегије најчешће коришћених техника хемијске трансфекције. поред тога, указано је на предности и ограничења различитих носача днк, а наведени су и резултати истраживача добијени током оптимизације протокола у циљу повећања ефикасности испоруке гена. главна биомедицинска примена овде описаних метода трансфекције је генска терапија, где се дефектни гени замењују функционалним. 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https://doi.org/10.1021/nn400343z accepted manuscript this is an early electronic version of an as-received manuscript that hasbeen accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as m. marković, m. gorgievski, n. štrbac, k. božinović, v. grekulović, a. mitovski, m. zdravković, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc221018014m this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc221018014m j. serb. chem. soc.00(0)1-19 (2023) original scientific paper jscs–12110 published dd mm, 2023 1 copper ions biosorption onto bean shells: kinetics, equilibrium, and process optimization studies miljan marković*, milan gorgievski, nada štrbac, kristina božinović, vesna grekulović, aleksandra mitovski, milica zdravković university of belgrade technical faculty in bor, vojske jugoslavije 12, bor, serbia (received 18 october 2022; revised 21 february 2023; accepted 21 march 2023) abstract: the removal of copper ions from aqueous solutions using bean shells as an adsorbent is presented in this paper. the influence of the solution solution ph on the biosorption capacity was investigated. the biosorption capacity increased with the increase in the solution ph. the pseudo-second order kinetic model showed the best agreement with the analyzed experimental data, indicating that chemisorption could be a possible way of binding the copper ions to the surface of the bean shells. the langmuir isotherm model best fitted the analyzed isotherm data. the sem-eds analysis was performed before and after the biosorption process. the change in the morphology of the sample after the biosorption process was evident, whereby k, mg, si, and ca were possibly exchanged with copper ions. response surface methodology (rsm) based on the box-behnken design (bbd) was used to optimize the biosorption process, with the selected factors: the solution ph, initial copper ions concentration, and contact time. the optimum biosorption conditions were determined to be: ph = 3-4, initial copper ions concentration 100 mg dm-3, and contact time 10-30 minutes. keywords: biosorption, copper ions, bean shells, kinetics, box-behnken design introduction concentrations of heavy metal ions in wastewaters originating from various industrial systems are often significant. these wastewaters can pose a serious threat to the surrounding ecosystems when discharged without previous treatment.1 wastewaters polluted with heavy metals are treated by well-known conventional technologies, such as: adsorption, coagulation and flocculation, ion exchange, membrane filtration, precipitation, and others.2,3,4 *corresponding author e-mail: mmarkovic@tfbor.bg.ac phone: +381628264336 https://doi.org/10.2298/jsc221018014m a cc ep te d m an us cr ip t mailto:mmarkovic@tfbor.bg.ac https://doi.org/10.2298/jsc221018014m 2 marković et al. biosorption is a possible alternative method for heavy metal ions removal. this process is considered “user-friendly” with various advantages, including specific affinity, low cost, and simple design. it is based on adsorption with agricultural or industrial by-products being used as adsorbents. these by-products are convenient for this use due to their abundant availability, favorable physical, chemical, and surface characteristics, and their low cost.4 copper is a widely used metal due to its excellent electrical and thermal conductivity, excellent corrosion resistance, and good resistance to strength and fatigue. pure copper is widely used in the production of cables and wires and many other parts in the electrical industry. due to its excellent anti-corrosion properties, copper is used for pipes, valves, and fittings in systems that carry drinking water, process water, or some other type of water. copper extraction, as well as its production, is the main source of pollution with this heavy metal. excessive copper concentrations in the environment are highly toxic for living organisms. it inhibits cell growth, impacts metabolism, and other processes. for this reason, it is very important to develop a sustainable, green remediation technique for copper removal, that is also economical, efficient, and environment-friendly.5 the aim the aim of this work is to determine whether bean shells can be used as an adsorbent for copper ions biosorption from aqueous solutions. successful usage of bean shells as an adsorbent for lead ions biosorption produced the idea of investigating the potential use of this biomass as an adsorbent for other heavy metals.6 for this purpose, kinetics, isotherm, and sem-eds analyses of the data were obtained and shown in this paper. the process was also modeled by response surface methodology using box-behnken design to analyze the influence of three variables on the biosorption process and to determine their optimal values. experimental bean shells collected on the fields in the village rudna glava (eastern serbia) were used as an adsorbent for copper ions biosorption experiments. 0.5 g of been shells were used as samples for biosorption experiments. the bean shells samples were rinsed with 200 ml of distilled water, prior to the biosorption experiments, in order to remove the physical impurities. biosorption experiments were conducted using synthetic copper ions solutions prepared with cuso4 ∙ 5h2o (p.a purity). the solutions were prepared by mixing different amounts of the copper-sulfate with distilled water, in order to obtain the concentrations needed for the specific experiment. the stock solution concentrations varied based on the specifics of the experiment. the solution phs was adjusted with 0.1 m hno3 and 0.1 m koh solutions. all experiments were performed in batch conditions. cu(ii) content was determined on a spectrophotometer (spectroquant pharo 300 merck, rahway, new jersey, usa), by forming a complex with nh4oh (p.a. purity), at 610 nm wavelength. the sem-eds analysis was performed on a sem scanning electron microscope (vega 3 lmu, tescan, brno, czech a cc ep te d m an us cr ip t biosorption of copper ions by bean shells 3 republic) with an integrated energy-dispersive x-ray detector (x act sdd 10 mm2, oxford instruments, abingdon, uk). the full characterization and preparation of the biosorbent are reported in a previous publication.6 process parameters, such as: process time, initial copper ions concentration, initial solution ph, and temperature, were adjusted depending on the performed experiment. the biosorption capacity and the % of removal were calculated using the following equations: 𝑞𝑡 = 𝐶𝑖−𝐶𝑡 𝑚 × 𝑉 (1) % 𝑟𝑒𝑚𝑜𝑣𝑎𝑙 = (1 − 𝑐𝑡 𝑐𝑖 ) × 100 (2) where: qt is the adsorbent capacity defined as mass of the adsorbed metal per unit mass of the adsorbent (mg g−1) at time t; ci is the initial metal ion concentration in the solution; ct is the metal ion concentration in the solution at time t; m is the adsorbent mass; v is the volume of the solution; % removal is the degree of the adsorbed copper ions. results and discussion the influence of the solution ph on the adsorption capacity to determine the influence of the solution ph on the biosorption capacity (figure 1), a number of experiments were performed, and the initial solution phs was adjusted in the range from 2 to 5. 50 ml of copper ions solutions (initial concentration 200 mg dm-3) was brought into contact with 0.5 g of been shells for 60 minutes. the experiments were performed in batch conditions, at room temperature, on a magnetic stirrer (with the stirring rate set at 300 rpm). as can be seen from figure 1, the solution ph had a significant effect on the biosorption capacity. an increase in the biosorption capacity with the increase of the solution ph could be noted. at ph = 2, the biosorption capacity was 1.739 mg g-1, while the maximum capacity of 12 mg g-1 was achieved at ph = 5. a lower biosorption capacity at lower solution ph could be a result of a higher concentration of h+ ions, which occupied the active sites in the structure of bean shells and suppressed the already adsorbed cu2+ ions. at higher ph, the concentration of h+ ions in the solution were lower, resulting in a higher biosorption capacity.7 a cc ep te d m an us cr ip t 4 marković et al. fig. 1. the influence of solution ph on the adsorption capacity adsorption kinetics kinetic models are often used for analyzing the experimental data to determine the biosorption rate, the step that dictates the rate of the process and its mechanism.8 in this paper, pseudo-first order kinetic model, pseudo-second order kinetic model, intraparticle diffusion (webber-morris) kinetic model, and elovich kinetic model were used to analyze obtained experimental data. in order to obtain the biosorption kinetic data, 50 ml of copper ion solutions (initial concentration 200 mg dm-3) were brought into contact with 0.5 g of bean shells, for different process time (ranging from 1 to 90 minutes). the change in the biosorption capacity with time is shown in figure 2. it can be noted that the biosorption capacity increased rapidly at the beginning of the process (first 5 minutes), as a result of a large number of available active sites in the structure of the bean shells.9 after this initial period, a slower increase in the biosorption capacity was noted (10-90 minutes), reaching a constant value after 90 minutes of the process. pseudo-first order kinetic model this model is based on the assumption that adsorption is a reversible process.10 the pseudo-first order kinetic model is given by:6 𝑑𝑞(𝑡) 𝑑𝑡 = 𝑘1(𝑞𝑒 − 𝑞(𝑡)) (3) where: q(t) – is the adsorbent capacity defined as the mass of the adsorbed metal per unit mass of the adsorbent (mg g-1) at time t; qe – is the adsorption capacity defined as mass of the adsorbed metal per unit mass of the adsorbent (mg g-1) at a cc ep te d m an us cr ip t biosorption of copper ions by bean shells 5 equilibrium; k1 – is the adsorption rate constant for the pseudo-first order kinetic model (min-1). integrating the equation (3), it follows: log(𝑞𝑒 − 𝑞(𝑡)) = log(𝑞𝑒 ) − 𝑘1 2,303 × t (4) the plot log(qe-q(t)) vs. t gives a linear dependence that serves as a base to determine the first-order kinetic model parameters. the obtained experimental data shown in figure 2 were linearized using eq. (4), and the obtained plot is shown in figure 3a. the obtained kinetic parameters corresponding to this model are given in table 1. pseudo-second order kinetic model pseudo-second order kinetic model is based on the assumption that adsorption and ion exchange takes place on the surface of the adsorbent, and that the adsorbate is bound to the adsorbent surface by chemisorption.11 the pseudo-second order kinetic model can be expressed as:12 𝑑𝑞(𝑡) 𝑑𝑡 = 𝑘1(𝑞𝑒 − 𝑞(𝑡)) 2 (5) where: q(t) is the adsorbent capacity defined as the mass of the adsorbed metal per unit mass of the adsorbent (mg g-1) at time t; qe is the adsorption capacity defined as mass of the adsorbed metal per unit mass of the adsorbent (mg g-1) at equilibrium; k2 is the adsorption rate constant for the pseudo-second order kinetic model (g mg-1 min-1).13 the linear form of eq. (5) is: 1 (𝑞𝑒−𝑞(𝑡)) = 1 𝑞𝑒 + 𝑘2𝑡 (6) plot t/q(t) vs. t, shown in figure 3b was used to determine the kinetic parameters for this model, which are given in table 1. intraparticle diffusion kinetic model (webber-morris model) this model assumes that the adsorption does not occur only on the surface of the adsorbent, but diffusion and adsorption inside the adsorbent structure are also present.13 the intraparticle diffusion kinetic model is given as:6 𝑞(𝑡) = 𝑘𝑖 𝑡 1/2 + 𝐶𝑖 (7) where: q(t) is the adsorption capacity defined as the mass of the adsorbed metal per unit mass of the adsorbent (mg g-1) at time t; ki -is the internal particle diffusion rate constant (mg g-1 min-0.5); and ci – is a constant that provides insight into the thickness of the boundary layer. if the ci value is higher, the boundary layer effect is greater, so the effect of surface adsorption in controlling the process speed is a cc ep te d m an us cr ip t 6 marković et al. greater (mg g-1). the plot t 1/2 vs q(t) shown in figure 3c is used to obtain the intraparticle diffusion kinetic model parameters, shown in table 1. elovich kinetic model this model was primarily used to analyze gas chemisorption onto solid adsorbents but was later successfully applied on the adsorption of toxic materials from aqueous solutions.14 elovich kinetic model is given in the following form:6 𝑑𝑞(𝑡) 𝑑𝑡 = αe−𝛽𝑞(𝑡) (8) where: α is the starting adsorption rate (mg g-1 min-1); β is the parameter that expresses the degree of surface coverage and activation energy for chemisorption (g mg-1); q(t) is the adsorption capacity defined as the mass of the adsorbed metal per unit mass of the adsorbent (mg g-1) at time t. from the plot q(t) = f(lnt), shown in figure 3d, elovich kinetic model parameters were determined and given in table 1. fig. 2. change in the adsorption capacity with time a cc ep te d m an us cr ip t biosorption of copper ions by bean shells 7 fig. 3. (a) pseudo-first order kinetic model; (b) pseudo-second order kinetic model; (c) intraparticle diffusion kinetic model; (d) elovich kinetic model table i kinetic model parameters for copper ions biosorption onto bean shells model parameters values pseudo-first order kinetic model k1 / min -1 0.023 qe,exp / mg g -1 19.07 qe,cal / mg g -1 4.97 r2 0.596 pseudo-second order kinetic model k2 / g mg -1 min-1 0.077 qe,exp / mg g -1 19.07 qe,cal / mg g -1 19.16 r2 0.999 intraparticle diffusion kinetic ki1 / g mg -1 min-0,5 11.722 a cc ep te d m an us cr ip t 8 marković et al. model ci1 / mg g -1 0.285 r1 2 0.989 ki2 / g mg -1 min-0,5 0.360 ci2 / mg g -1 15.995 r2 2 0.929 elovich kinetic model α / mg g-1 min-1 14.652 β / g mg-1 1.086 r2 0.837 based on the obtained correlation coefficients, it can be concluded that the adsorption kinetics could be fairly modeled with the pseudo-second order kinetic model, which led to the conclusion that chemisorption was a possible way of binding copper ions onto active sites in the adsorbent structure. this statement was also supported by the negligible difference in the values of calculated and experimentally obtained adsorption capacity (qe,cal and qe,exp). adsorption isotherms adsorption isotherms are used to gain insight into the mechanism of the adsorption process, as well as to determine the maximum adsorption capacity.15 in this paper, the linear langmuir, freundlich, and temkin isotherm models were used to describe copper ions biosorption onto bean shells. biosorption isotherm data was obtained by performing the following experiment: 0.5 g of bean shells samples were brought into contact with 50 ml of copper ions solutions, of different initial cu2+ concentrations (in the range from 50 to 500 mg dm−3). the suspension was stirred on a magnetic stirrer, at room temperature, for 90 minutes. obtained experimental adsorption isotherm data for copper ions adsorption onto bean shells is shown in figure 4a. langmuir isotherm model this model is based on the assumption that the adsorption process occurs on specific homogenous sites inside the adsorbent structure.16 the langmuir model can be expressed as:6 𝑞𝑒 = 𝑞𝑚𝐾𝐿 𝐶𝑒 1+𝐾𝑙𝐶𝑒 (9) where ce is the equilibrium concentration of metal ions (mg dm-3); qe is the equilibrium adsorption capacity (mg g-1); qm is the maximum adsorption capacity (mg g-1); and kl is the langmuir equilibrium constant (dm3 g-1). linearizing the equation (9) the following is obtained: 𝐶𝑒 𝑞𝑒 ⁄ = 1 𝐾𝐿 𝑞𝑚 + 1 𝑞𝑚 𝐶𝑒 (10) the langmuir isotherm data were calculated from the plot ce vs. ce/qe shown in figure 4b, and given in table 2. a cc ep te d m an us cr ip t biosorption of copper ions by bean shells 9 freundlich isotherm model freundlich model represents the earliest known relationship that describes the non-ideal and reversible adsorption. this model can also be used to study multilayer adsorption [18]. this model can be expressed as:6 𝑞𝑒 = 𝐾𝑓 𝐶𝑒 1/𝑛 (11) where ce is the equilibrium concentration of copper ions in the solution (mg dm-3); qe is the adsorbent capacity defined as mass of the adsorbed metal per unit mass of the adsorbent (mg g-1) at equilibrium; kf is the freundlich equilibrium constant ((mg g-1) (dm3 mg-1)1/n); and 1/n -is the coefficient of heterogeneity in the freundlich adsorption isotherm equation. linear form of equation (11) is: 𝑙𝑜𝑔𝑞𝑤 = 𝑙𝑜𝑔𝐾𝑓 + 1 𝑛 𝑙𝑜𝑔𝐶𝑒 (12) the plot log qe vs. log ce (figure 4c) provided the required data for the freundlich model isotherm data calculation, which is shown in table 2. temkin model this model assumes that the heat of sorption of all molecules linearly increases with the coverage of the adsorbent surface and that there is a uniform distribution of binding energies up to maximum binding energy.18 the temkin isotherm model is given as: 𝑞𝑒 = 𝐵𝑙𝑛(𝐾𝑇 𝐶𝑒 ) (13) where: b = rt/b is the temkin constant, which refers to the adsorption heat (j mol-1); b is the variation of adsorption energy (j mol-1); r is the universal gas constant (j mol-1 k-1); tis the temperature (k); kt is the temkin equilibrium constant (dm3 g-1); qe is the adsorption capacity (mg g-1) at equilibrium; and ce is the equilibrium concentration of metal ions in the solution (mg dm-3).6 linear form of equation (13) is: 𝑞𝑒 = 𝐵𝑙𝑛𝐾𝑇 + 𝐵𝑙𝑛𝐶𝑒 (14) temkin constants b and kt were determined from the plot ln ce vs. qe (figure 4d), and given in table 2. based on the analyzed data and the obtained results (table 2), it can be concluded that the langmuir isotherm model was the best fit for the experimental data (r2 = 0.986), which indicated that the surface of the adsorbent was homogenous, and the biosorption of copper ions onto bean shells occurred in a monolayer.19 a cc ep te d m an us cr ip t 10 marković et al. fig. 4. (a) experimental adsorption isotherm data, (b) langmuir adsorption isotherm model, (c) freundlich adsorption isotherm model, (d) temkin adsorption isotherm model table ii adsorpton isotherm model parameters for copper ions biosorption onto bean shells langmuir freundlich temkin kl / dm3 mg-1 qexp / mg g-1 qm / mg g-1 r2 kf 1/n r 2 b / j mol-1 kt / dm3 g-1 r2 24.941 17.2 18.31 0.986 1.423 0.363 0.919 0.363 3.276 0.919 a cc ep te d m an us cr ip t biosorption of copper ions by bean shells 11 the performance of the adsorbent is usually defined by the maximum biosorption capacity. based on the results in copper removal with various biosorbents reported in other works (shown in table iii), it can be concluded that bean shells could play an important role as a cost-effective biosorbent for copper ions removal. table iii cu2+ ions biosorption on bean shells in comparison with other adsorbents biosorbent maximum biosorption capacity (qm, mg g -1) work bean shells 18.31 this work wheat straw 4.3 12 sawdust of deciduous trees 9.9 20 myrica esculenta 39.37 21 activated sawdust powder 10.35 22 rosa damascena leaves 25.13 23 carbonized sunflower stem 20 24 combretum indicum 12.1 25 sem-eds analysis the sem-eds analysis was performed on samples before and after the biosorption of copper ions. the obtained results are shown in figure 5. before the biosorption of copper ions (figure 5a) a porous structure was noticed, with visible cavities and macro-pores. the presence of these pores and cavities facilitated the penetration of the aqueous phase into the adsorbent structure.26 the eds spectrum of the sample before biosorption of copper ions (figure 5b) suggested the presence of o, mg, si, k, and ca. after the biosorption process, the sem analysis (figure 5c) showed a more compact structure, with the absence of macro-pores and cavities, as a result of the incorporation of copper ions into the molecular structure of the bean shells. the obtained eds spectrum after the biosorption of copper ions (figure 5d) demonstrated the presence of o, ca, and cu. the absence of mg, si, k, and detected lower levels of ca, indicated that any of these ions could be exchanged with copper ions during the biosorption process. a cc ep te d m an us cr ip t 12 marković et al. fig. 5. sem-eds analysis before and after the biosorption of copper ions optimization of the biosorption conditions – box-behnken experimental design copper ions biosorption onto bean shells was optimized using an experimental design, in order to determine the effects of three selected independent variables on the percentage of cu2+ ions removal (dependent variable).27 the optimum biosorption conditions were determined by the means of box-behnken design (bbd) and response surface methodology (rsm). the rsm is a set of techniques useful for evaluating the relationships between a number of experimental factors and measured responses.28 the bbd was applied, comparing three factors: solution ph (x1), initial copper ions concentration (x2), and contact time (x3). the chosen experimental ranges and levels in the design are given in table 4. the experimental design matrix, as well as the response y (adsorption degree), are given in table 5. all the experiments are performed in batch conditions, at room temperature, on a magnetic stirrer (with the constant stirring rate, set at 300 rpm). table iv experimental ranges and levels in the experimental design factors range level -1 0 1 x1 – solution ph 2 3 4 x2 – initial metal ion concentration, mg/l 100 500 1000 a cc ep te d m an us cr ip t biosorption of copper ions by bean shells 13 x3 – contact time, min 10 30 60 table v box-behken design matrix for three factors along with observed response for cu2+ biosorption onto bean shells run x1: solution ph x2: initial cu 2+ ions concentration x3: contact time y: % removal 1 2 100 30 32.950 2 4 100 30 86.515 3 2 1000 30 22.576 4 4 1000 30 11.890 5 2 500 10 30.550 6 4 500 10 23.836 7 2 500 60 20.860 8 4 500 60 39.748 9 3 100 10 87.790 10 3 1000 10 6.922 11 3 100 60 69.700 12 3 1000 60 7.102 13 3 500 30 25.072 14 3 500 30 31.360 15 3 500 30 38.860 the correlation between the following independent variables: linear (β1, β2, β3), quadratic (β11, β22, β33), interaction terms (β12, β13, β23), and the response (y), was described by fitting the following polynomial equation:27 𝑌 = 𝛽0 + 𝛽1𝑋1 + 𝛽2𝑋2 + 𝛽3𝑋3 + 𝛽11𝑋1𝑋1 + 𝛽22𝑋2𝑋2 + 𝛽33𝑋3𝑋3 + 𝛽12𝑋1𝑋2 + 𝛽13𝑋1𝑋3 + 𝛽23𝑋2𝑋3 (15) the obtained results are displayed in table 6. the biosorption of copper ions onto bean shells was expressed using the following equation: 𝑌 = 31,76 + 6,88𝑋1 − 28,56𝑋2 − 1,46𝑋3 − 3,71𝑋1 ⋅ 𝑋1 + 10,42𝑋2 ⋅ 𝑋2 + +0,69𝑋3 ⋅ 𝑋3-16,06𝑋1 ⋅ 𝑋2 + 6,40𝑋1 ⋅ 𝑋3 + 4,57𝑋2 ⋅ 𝑋3 (16) the statistical significance of the model was evaluated by the analysis of variance (anova), presented in table 6. the significance of each coefficient was determined by the magnitude of the f-values and p-values, given in table 6. the larger the f-value, and the smaller p-value, the corresponding coefficient was more significant. p-values less than 0.0500 indicated high significant regression at 95 % confidence level.28 a cc ep te d m an us cr ip t 14 marković et al. table vi anova analysis for response surface model in relation to cu2+ biosorption onto bean shells source df adj ss adj ms f-value p-value model 9 8676.69 964.08 6.76 0.024 linear 3 6920.46 2306.82 16.17 0.005 x1 1 378.85 378.85 2.66 0.164 x2 1 6524.53 6524.53 45.74 0.001 x3 1 17.08 17.08 0.12 0.743 square 3 476.86 158.95 1.11 0.426 x1 2 1 50.70 50.70 0.36 0.577 x2 2 1 401.23 401.23 2.81 0.154 x3 2 1 1.76 1.76 0.01 0.916 2-way interaction 3 1279.36 426.45 2.99 0.135 x1·x2 1 1032.05 1032.05 7.23 0.043 x1·x3 1 163.87 163.87 1.15 0.333 x2·x3 1 83.45 83.45 0.58 0.479 error 5 713.24 142.65 lack-of-fit 3 617.94 205.98 4.32 0.194 pure error 2 95.30 47.65 total 14 9389.93 the suitability of the model was confirmed by the regression coefficients of the predicted and experimental responses (r 2 = 0.924 and adj-r 2 = 0.787). this suggested that 96 % of the responses were explained by the used model. the corresponding f-value (6.76) and p-value (0.024) indicated that the model was significant. p-values lower than 0.0500 in the cases of x2 (initial cu2+ ions concentration) and x1·x2 (solution ph combined with the initial cu2+ ions concentration) indicated that these were the significant model terms. the relationship between the experimental responses and the responses predicted by the model is shown in figure 6. a cc ep te d m an us cr ip t biosorption of copper ions by bean shells 15 fig. 6. plot of experimental and predicted responses as can be seen from figure 6, there was a good relationship between the experimental and predicted responses, based on the correlation coefficient (r 2 = 0.924). the contour plots showing the influence of the analyzed process parameters on the adsorption degree are presented in figure 7. figure 7a indicates that the higher solution ph and lower initial cu2+ ions concentration in the solution led to higher metal ions removal. further, the biosorption process was more favorable at lower initial cu2+ ions concentration combined with shorter contact time (figure 7b). lastly, figure 7c shows that a high solution ph combined with medium-tolonger contact time led to a higher % removal. a cc ep te d m an us cr ip t 16 marković et al. fig. 7. contour plots showing the interaction and the influence on the adsorption rate (y) of: solution ph and the initial metal ions concentration (a); initial metal ions concentration and contact time (b); and solution ph and contact time (c) response surface methodology, based on box-behnken design, was used to optimize the process of copper ions biosorption onto bean shells. the influence of three parameters (solution ph, initial metal ions concentration, and contact time) was investigated. the obtained data indicated that the used model was statistically significant. the data showed that initial metal ions concentration, as well as the combination of initial metal ions concentration, and solution ph, had a significant influence on the biosorption efficiency. using this model, the optimum biosorption conditions were determined to be: ph = 3-4; initial metal ions concentration 100 mg/l; and contact time 10-30 minutes. conclusions the study of biosorption, as a potential method for copper ions removal from aqueous solutions, is presented in this paper. bean shells were investigated as biosorption of copper ions by bean shells 17 potential adsorbent. for that purpose, kinetics, equilibrium, sem-eds, and process optimization studies were performed. the solution ph was determined to have a significant influence on the biosorption capacity. an increase in the biosorption capacity with the increase in the solution ph from 2 (1.739 mg g-1) to 5 (12 mg g-1) was noted. experimentally obtained kinetics data were analyzed using four adsorption kinetic models (the pseudo-first order kinetic model, pseudo-second order kinetic model, intraparticle diffusion kinetic model, and elovich kinetic model). the obtained kinetics parameters indicated that the pseudo-second order kinetic model best fitted the analyzed experimental data, which further suggested that chemisorption was a possible way of binding the copper ions to the surface of bean shells. experimental biosorption isotherm data were fitted using the langmuir, freundlich, and temkin adsorption isotherm models. obtained results indicated that the langmuir model served as the best fit for the analyzed data, leading to the conclusion that the surface of the adsorbent was homogenous, and the biosorption of copper ions onto bean shells occurred in a monolayer. the sem-eds analysis was performed on a bean shells sample before and after the biosorption of copper ions. the obtained sem micrographs showed that the surface morphology of the sample changed after the biosorption process, from a porous to a more compact structure, possibly as a result of the incorporation of copper ions into the structure of the bean shells. the eds spectrums of the samples before and after the biosorption process indicated that mg, si, k, and ca could potentially be involved in the biosorption process and exchanged with copper ions. process optimization studies were performed by the means of response surface methodology based on the box-behnken design. the influence of solution ph, initial metal ions concentration, and contact time was investigated and modeled. the used model was determined to be statistically significant. the data suggested that initial metal ions concentration, as well as the combination of initial metal ions concentration and solution ph, had a significant influence on the biosorption efficiency. using this model, the optimum biosorption conditions were determined to be: ph 3-4; initial metal ions concentration 100 mg dm-3; and contact time 10-30 minutes. acknowledgements: the research presented in this paper was done with the financial support of the ministry of education, science and technological development of the republic of serbia, within the funding of the scientific research work at the university of belgrade, technical faculty in bor, according to the contract with registration number 451-03-68/202214/200131. the authors express their appreciation to ms. sandra vasković, an english lecturer at the technical faculty in bor, university of belgrade, for her help in editing the manuscript. a cc ep te d m an us cr ip t 18 marković et al. и з в о д биосорпција јона бакра на љускама пасуља: испитивања кинетике, равнотеже, и оптимизација процеса миљан марковић, милан горгиевски, нада штрбац, кристина божиновић, весна грекуловић, александра митовски, милица здравковић универзитет у београду, технички факултет у бору, војске југославије 12, бор, србија у овом раду приказана је анализа уклањања јона бакра из водених раствора коришћењем љуски пасуља као адсорбенса. испитан је утицај ph вредности раствора на капацитет биосорпције. добијени резултати су показали да капацитет биосорпције расте са повећањем ph вредности раствора. кинетичка испитивања су показала да модел псеудо-другог реда најбоље описује анализиране податке, што указује да је хемисорпција могућ начин везивања јона бакра за површину љуски пасуља. испитивања равнотеже процеса су показала да ленгмиров модел адсорпционе изотерме најбоље описује анализиране податке. сем-едс анализом су испитани узорци пре и након извођења процеса биосорпције. ова анализа је показала евидентну промену у морфологији узорка након процеса биосорпције, при чему су едс спектри указали на могућу измену k, mg, si, и ca јона са јонима бакра. модел response surface methodology (rsm) базиран на boxbehnken дизајну (bbd) је коришћен за оптимизацију процеса биосорпције, са изабраним факторима: ph вредности раствора, почетна концентрација јона бакра у раствору и време контакта. помоћу модела су одређени оптимални услови за извођење процеса биосорпције, и то: ph вредности између 3 и 4, почетна концентрација јона бакра од 100 mg dm-3 и време контакта између фаза 10-30 минута. 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http://doi.org/10.31788/rjc.2022.1548035 https://doi.org/10.5004/dwt.2022.28775 https://doi.org/10.1016/j.ecoleng.2014.09.103 https://doi.org/10.1016/j.ecoenv.2017.12.008 https://doi.org/10.1186/2052-336x-12-5 synthesis and biological activity of novel zingerone–thiohydantoin hybrids j. serb. chem. soc. 87 (12) 1349–1358 (2022) original scientific paper jscs–5598 published 1 august 2022 1349 synthesis and biological activity of novel zingerone–thiohydantoin hybrids katarina d. virijević1, petar b. stanić1, jovana m. muškinja1, jelena s. katanić stanković1, nikola srećković2, marko n. živanović1 and biljana m. šmit1* 1university of kragujevac, institute for information technologies, department of science, jovana cvijića bb, 34000 kragujevac, serbia and 2university of kragujevac, faculty of science, department of chemistry, radoja domanovića 12, 34000 kragujevac, serbia (received 4 april, revised 28 may, accepted 9 june 2022) abstract: a series of zingerone–thiohydantoin hybrids were synthesized from o-alkyl zingerone derivatives by cyclocondensation with thiosemicarbazide in a two-step reaction. the obtained new potentially bioactive compounds were structurally characterized by ir and nmr spectroscopy, as well as by elemental and hrms analysis. in addition, their antimicrobial and in vitro anticancer activities were tested. the tested compounds showed low to moderate antimicrobial activity. zingerone–thiohydantoin hybrid with an o-butyl substituent exerted the significant cytotoxic activity on colon hct-116 cancer cells, without toxicity on healthy mrc-5 cells. keywords: molecular hybrids; antimicrobial activity; cytotoxic activity. introduction the development of novel synthetic molecular hybrids is one of the main challenges in the drug discovery field. hybrid drugs represent a combination of specific agents aimed to be more efficient than classic single synthesized compounds. in that way, the hybrid approach allows the connection of two distinct compounds in one molecule, increasing the biological potential of at least one of the compounds.1 many natural products play an important role in this field. for example, zingerone also called vanillylacetone, obtained from a ginger extract, is a natural compound that belongs to the methoxyphenol class, along with its derivatives. both natural and synthetic zingerone derivatives exhibit different biological and pharmacological activities such as anti-inflammatory, anti -microbial, anti-cancer and hepatoprotective.2 furthermore, zingerone appears to be a potential agent for inhibiting colon cancer progression, as the number of * corresponding author. e-mail: biljana.smit@uni.kg.ac.rs https://doi.org/10.2298/jsc220404047v ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1350 virijević et al. larger foci was found to be significantly lower after zingerone treatment, compared to dimethyl hydrazine-induced colon cancer cells.2 on the other hand, 2-thiohydantoin (2-thioxoimidazoline-4-one) is a non -aromatic five-membered heterocyclic compound with a cyclic ureide core.3 many synthesized thiohydantoin derivatives with various substituents attached to their nucleus exhibit a wide range of biological and pharmacological potentials, such as antimicrobial,4 anti-convulsive,5 anti-diabetes6 and anti-hiv.7 however, novel studies showed that thiohydantoins and their synthesized derivatives could be used as promising anti-proliferative and anti-metastatic agents.8 taking into account that colon cancer is one of the most prominent tumors in the world and less sensitive to cytostatics, the search for new effective therapeutic drugs is crucial. in this study, different zingerone derivatives were prepared as starting materials for obtaining a short series of new zingerone-thiohydantoin hybrids for the evaluation of their potential biological activity. experimental general methods all reagents and chemicals were commercially available and used without additional purification. solvents were distilled before use. anhydrous methanol was prepared by standard drying methods. zingerone, starting material for a preparation of o-alkyl zingerone derivatives, was obtained by condensation reaction of vanillin and acetone and subsequent reduction of yielded dehydrozingerone, according to well-known procedure.9 ir spectra were recorded through kbr pellets on a perkin–elmer ft-ir spectrometer model spectrum one in the 4000 to 450 cm-1 range. 1hand 13c-nmr spectra were recorded on a varian gemini 2000 nmr spectrometer using cdcl3 as the solvent and tms as the internal standard. elemental analysis was done on an elemental vario eliii chnso analyzer. hrms were measured on an agilent 6550 ifunnel q-tof lc/ms system. for biological assays microtiter plates and multiskan skyhigh microplate spectrophotometer by thermo scientific were used. general procedure for the preparation of o-alkyl zingerone derivatives 1a and b zingerone derivatives 1a and b were synthesized according to a procedure that uses dimethyl and diethyl sulfate, respectively.10 a mixture of zingerone (0.971 g, 5 mmol) and 50 ml of boiling water is heated on a steam bath. a 2 ml portion of 20 % naoh solution is heated to about 100 °c and added in one lot to the hot mixture of zingerone and water. heating is continued and 6.25 mmol of methyl/ethyl sulphate is slowly added in portions. after the addition of all methyl/ethyl sulphate, which requires about 1.5 h, the reaction mixture is heated for 45 min longer and an additional portion of 1.1 mmol of methyl/ethyl sulphate is added at the same rate as the first portion. at the end of this addition the reaction mixture should show an acid reaction. the reaction mixture is rendered slightly alkaline with naoh solution, and the addition of me/et sulfate and naoh solution is done two more times until the total amount of me/et sulfate (11.25 mmol) is added. the mixture is then made strongly alkaline by the addition of 1 ml naoh solution and is heated another 20 min. the reaction mixture is rapidly cooled at ambient temperature with continued stirring and the product is ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ zingerone–thiohydantoin hybrids 1351 extracted with diethyl ether (3×10 ml). the combined ether extracts are dried over anhydrous mgso4 and the ether is evaporated, giving yellow oil that soon solidifies. general procedure for the preparation of o-alkyl zingerone derivatives 1c–g zingerone derivatives 1c–g were synthesized according to a known procedure that uses alkyl halides with potassium carbonate in acetone.11 the mixture of zingerone (0.971 g, 5 mmol), alkyl halide (12.5 mmol) and anhydrous k2co3 (2.25 g, 16.3 mmol) in acetone (25 ml) were heated to reflux for 3 h. the mixture was cooled at ambient temperature and then poured into cold water. the products were extracted from the mixture with ethyl acetate (3×10 ml). combined extracts were rinsed with water and then dried over anhydrous na2so4. the solvent was removed by vacuum distillation and the product was separated from the residue by column chromatography with hexane/etoac (from 3:1 to 6:1). zingerone derivatives 1a, 1b and 1f are known and commercially available chemicals, while the others are novel. the structure and purity of all new products were confirmed by ir and nmr spectroscopy. synthesis of zingerone–thiohydantoin derivatives 2a–g the zingerone–thiohydantoin derivatives were synthesized according to a previously published protocol for the synthesis of arylidene thiohydantoin derivatives.12 a mixture of o-alkyl zingerone derivative 1a–g (2 mmol) and thiosemicarbazide (0.182 g, 2 mmol) in 30 ml of methanol were heated to reflux for 3 h and then cooled to ambient temperature, resulting in the corresponding intermediate thiosemicarbazone without isolation. ethyl chloroacetate (0.245 g, 2 mmol) and anhydrous sodium acetate (0.492 g, 6 mmol) were added in situ and the mixture was refluxed for another 6 h. the reaction mixture was cooled to room temperature at first and then poured into cold water. the resulting precipitate was filtered off, rinsed with hot water and re-crystallized from hot methanol, giving white amorphous powder in all cases. the structure of the synthesized compounds was confirmed by ir and nmr spectroscopy, as well as elemental analysis and hrms (supplementary material to this paper). all compounds are obtained as an inseparable mixture of z and e stereoisomers, as can be seen through the duplication of most signals in the 1h-nmr spectra. nmr spectral data are given for the major stereoisomer. antimicrobial activity determination in this preliminary testing, five microbial strains were used, four of which were bacteria, salmonella enteritidis atcc 13076, pseudomonas aeruginosa atcc 10145, staphylococcus aureus atcc 25923, escherichia coli atcc 25922, and the yeast candida albicans atcc 10259. the microorganisms were acquired from the institute of public health kragujevac, university of kragujevac, serbia, kept at 4 °c with subcultivation once a month. the broth used for bacteria cultivation was nutrient agar and yeast was cultured on sabouraud dextrose agar. for antimicrobial evaluation of synthesized compounds, a standard microdilution method by sarker et al. was used.13,14 cytotoxic activity determination all synthesized derivatives were dissolved in dimethyl sulfoxide (dmso) in order to obtain stock solutions of 5 mм concentration, followed by further dilution in dulbecco’s modified eagle medium (dmem) to obtain working concentrations (0.1, 1, 10, 50, 100 and 250 µm). at the highest applied concentration of derivatives, the concentration of dmso in the solution was lower than 0.05 %, which was previously confirmed as non-toxic to cancer cells.15 ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1352 virijević et al. the healthy human lung fibroblasts (mrc-5) and colorectal carcinoma cell lines (hct -116) were obtained from the european collection of authenticated cell cultures. cells were cultured in a complete medium in humidified conditions, at 37 °c and 5 % co2. when cells reached 70–80 % of confluence, the detachment was done using 0.25 % trypsin–edta, followed by seeding (1×104 cells/well) in 96-well flat-bottomed microtitre plates. the treatment with 100 µl of synthesized compounds solution was done 24 h after cell seeding. the effects of synthesized compounds on the viability of tested cell lines were assessed after 24 and 72 h using mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay according to the previously described protocol.16 at the end of the incubation period, 25 μl of mtt solution (from a 5 mg ml-1 stock) was added to each well, followed by incubation at 37 °c for 2 h, after which 100 μl of dmso was added. the evaluation of cytotoxic activity was done by measuring the absorbances at 550 nm wavelength. the obtained results are presented as mean ± standard error (se), expressed as a percent of cell viability (%). ic50 values (minimal inhibitory treatment concentration that induces the death of 50 % of treated cells) were calculated from dose curves obtained by the mtt test. 5-fluorouracil (5-fu) was used as positive control. the magnitude of the correlation between variables was calculated using statistical software spss (spss for windows, ver. 20, 2008, chicago, il, usa) whereat the anova test was applied, and for all comparisons p < 0.05 was considered as a statistically significant difference between the control and the tested compounds. the ic50 values were calculated by using the calcusyn software program. results and discussion synthesis of the zingerone–thiohydanoin molecular hybrids the zingerone–thiohydantoin derivatives 2a–g were obtained through a condensation reaction with thiosemicarbazide, utilizing a previously published two -step protocol (scheme 1).12 in the first step, synthesized o-alkyl zingerone derivatives 1a–g reacted with thiosemicarbazone, giving corresponding thiosemicarbazides. the thiosemicarbazides then undergo intramolecular cyclocondenzation with ethyl chloroacetate in the presence of anhydrous sodium acetate, yielding the final thiohydantoin products 2a–g. the structure and purity of the novel zingerone–thiohydantoin derivatives were confirmed by ir and nmr spectroscopy, as well as by elemental and hrms analysis. the compounds were obtained in medium to high yields, with the exception of 2d, which was obtained in a modest yield. naturally occurring zingerone itself did not react in this manner and the corresponding thiohydantoin derivative was not obtained. similar to some schiff -base derivatives,17 all newly synthesized zingerone–thiohydantoin derivatives are obtained as a mixture of z and e stereoisomers, which could be found in corresponding nmr spectra. most signals in the 1h-nmr spectra are duplicated, which indicates the presence of isomers. this is best seen through the singlets of ch2-5, ch3-6 and ch3-16, which have the most pronounced difference of the chemical shifts and thus, less overlap. the ratios of z/e isomers are obtained from relative integration of the most suitable and distinct pairs of singlets. their ratios range from 1:1.06 to 1:9.82 (scheme 1). in all cases the e isomer is favoured. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ zingerone–thiohydantoin hybrids 1353 scheme 1. synthesis of the zingerone–thiohydantoin hybrids 2a–g. furthermore, the computational data for both possible configurations of 2a were also calculated using the dft method in order to determine the more stable isomer as seen in fig. 1. when any internal interconnection between molecules for both isomers is neglected the less internal steric repulsion factor plays an important role in fixing the e isomer over z isomer, as it is with less internal repulsion. the gaseous state dft calculations also showed that the energy difference value δe = 4.0 kj mol–1 between e and z isomer was very small. fig. 1. the optimized geometries of e and z isomers of 2a, with relative free energy values indicated in kj mol-1. antimicrobial activity the results of antimicrobial effects of the zingerone–thiohydantoin hybrids are presented in table i. the compounds were tested against two gram-negative (s. enteritidis and p. aeruginosa) and two gram-positive (s. aureus and e. coli) bacterial species, as well as against the yeast c. albicans. s. aureus and c. albicans were the most resistant to the action of 2a–g, wherein the compounds had not shown any activity even at the highest applied concentration (mic > 4 mg ml–1). moreover, compound 2c was completely inactive towards all used microorganisms at the same concentration, while in the cases of other compounds mic values were quite high, mostly in range 2–4 mg ml–1. 2b was able to inhibit the ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1354 virijević et al. growth of s. enteritidis in moderate concentration (mic 0.5 mg ml–1). only e. coli was more susceptible to the action of the compounds, particularly 2a and g with mic 0.25 mg ml–1. nevertheless, the quite high concentrations at which the compounds exhibit their activity cannot be easily compared with the activity of the reference standards, erythromycin and nystatin, where mics were expressed in mg/ml. this is the first study regarding the antimicrobial potential of zingerone–thiohydantoin hybrids. the literature data about similar compounds are scarce. there are some recently published results regarding the influence of some thiohydantoin derivatives on several bacterial species.4 these compounds were the most active against gram-positive bacteria such as s. epidermidis, s. pyogenes, s. agalactiae, e. faecium and s. aureus with mics below 1 mg ml–1, but were less effective against gram-negative bacteria like k. pneumonia, p. mirabilis and e. coli. table i. antimicrobial activity (mic / mg ml-1) of the synthesized zingerone–thiohydantoin derivatives 2a–g; –: not tested compound microorganism s. enteritidis p. aeruginosa s. aureus e. coli c. albicans 2a 4 4 ˃ 4 0.25 ˃ 4 2b 0.5 4 ˃ 4 2 ˃ 4 2c ˃ 4 ˃ 4 ˃ 4 ˃ 4 ˃ 4 2d – – – – – 2e 4 4 ˃ 4 2 ˃ 4 2f ˃ 4 4 ˃ 4 4 ˃ 4 2g 4 4 ˃ 4 0.25 ˃ 4 erythromycin 20 20 1.25 2.5 – nystatin – – – – 1.25 cytotoxic activity thiohydantoin analogs have been already confirmed as potent anti-tumor agents. furthermore, apoptosis-inducing activity of some thiohydantoin derivatives has been demonstrated.18 previous research has identified zingerone as a potential anti-cancer agent, an inhibitor of colon cancer progression,2,19 with significant cytotoxicity (ic50 = 11.49 mm) when applied to mesothelioma cells. su et al. showed that the zingerone-induced cytotoxic effect on colon cancer cells (hct-116) was achieved through the mechanism of ros-mediated apoptosis.20 besides that, the cytotoxic potential of thiohydantoin derivatives on hct-116 was noticed when applied to colon cancer cells (with ic50 > 50 µm).21 in this study, the cytotoxic activity of newly synthesized zingerone–thiohydantoin derivatives was evaluated on healthy lung (mrc-5) and human colorectal carcinoma (hct-116) cell lines by mtt assay. as positive control, the commercial chemotherapeutic drug, 5-fluorouracil (5-fu) was used. 5-fu is widely used ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ zingerone–thiohydantoin hybrids 1355 in the treatment of different types of cancer, such as gastric, pancreatic, breast, and ovarian cancers.22 in regard to the influence on the viability of the healthy mrc-5 cells, a moderate cytotoxic effect was observed only for compound 2a after an extended time of exposure with ic5072 h = 184.15 µm (fig. s-44 of the supplementary material) and control with ic5072 h = 181.71 µm (fig. s-46 of the supplementary material). the tested compounds manifest their cytotoxicity potentials in a time and dose-dependent manner. the reduction of hct-116 cells viability was obtained mainly after 72 h and at the highest applied concentration of the investigated compounds (fig. s-45 of the supplementary material). the effects of the synthesized compounds were expressed by dose curves (figs. s-44 and s-45) and ic50 values (fig. 2). based on the results, 2e exhibited the most prominent antiproliferative activity on the hct-116 cell line with an ic5024 h = 209.08 µm and ic5072 h = 160.93 µm. 5-fu exerted a weaker cytotoxic effect than 2e on hct -116 after 24 and 72 h with ic5024 h > 250 µm and ic5072 h = 181.71 µm, respectively. (fig. 2). fig. 2. the cytotoxic effects of zingerone-thiohydantoin derivatives and 5-fu after 24 and 72 h exposure, represented as ic50 values. unlike other tested compounds, 2e contains a butyl group. the result of its activity is probably related to the lipophilicity of the substituents in the structure of the tested molecule. the lipophilicity of molecules plays a major role in the transport of molecules across biological membranes.23 as the length of hydrocarbon chain increases, the polarity of compounds decreases resulting in increased molecule permeability. cell membranes are relatively impermeable to hydrophilic compounds, hence the permeability of molecules depends on the hydrophobic characteristics, i.e., lipophilicity of the compound.24 in addition, a previous study reported that the lipophilicity of the substituents (ph > allyl > me) had a significant impact on the cytotoxic effect.25 compound 2f, with a benzyl group, did not exhibit a cytotoxic effect (ic50 > 250 µm). however, based on the obtained dose curves, it can be concluded that ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1356 virijević et al. the wider range of concentrations could provide promising effects. increasing the concentration range of the tested compounds would probably lead to a rise of the cytotoxic activity of some of them, and also would allow the determination of the selectivity index on tested cell lines. the presented results indicate the potential of tested compounds as anticancer agents with no significant toxicity on healthy cell lines. compound 2e exhibited the most promising bioactivity and is the leading candidate in the synthesized series. the increased cytotoxicity of 2e, compared to zingerone itself, could be attributed to the introduction of thiohydantoin moiety. conclusion this study presents the synthesis, characterization, and biological evaluation of new zingerone–thiohydantoin molecular hybrids as potential anticancer agents. in addition to the zingerone-induced cytotoxic effect, it is known that the biological activity of thiohydantoin compounds depends largely on the nature of the substitution of the thiohydantoin ring. the difference in the cytotoxic activity of the tested compounds depends on the nature of the o-alkyl substituent of the benzene ring. among the tested compounds, 2e exerted significant cytotoxic potential without toxicity to healthy mrc-5 cells. however, all tested compounds showed low to moderate antimicrobial activity. further toxicological testing is required to assess its therapeutic potential. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/11747, or from the corresponding author on request. acknowledgement. the authors are grateful for financial support from the ministry of education, science and technological development of the republic of serbia (agreements no. 451-03-68/2022-14/200378 and 451-03-68/2022-14/200122). и з в о д синтеза и биолошка активност нових зингерон–тиохидантоинских хибрида катарина д. виријевић1, петар б. станић1, јована м. мушкиња1, јелена с. катанић станковић1, никола срећковић2, марко н. живановић1 и биљана м. шмит1 1универзитет у крагујевцу, институт за информационе технологије, сектор за природно–математичке науке, јована цвијића бб, 34000 крагујевац и 2универзитет у крагујевцу, природно– –математички факултет, институт за хемију, радоја домановића 12, 34000 крагујевац серија зингерон–тиохидантоинских хибрида је синтетисана из деривата о-алкил зингерона циклокондензацијом са тиосемикарбазидом у двостепеној реакцији. добијена нова потенцијално биоактивна једињења структурно су окарактерисана ic и nmr спектроскопијом, као и елементалном анализом. поред тога, тестиране су њихове антимикробне и in vitro антиканцерогене активности. испитана једињења су показала ниску до умерену антимикробну активност. зингерон–тиохидантоински хибрид са о-бутил суп________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ zingerone–thiohydantoin hybrids 1357 ституентом је показао значајну цитотоксичну активност на ћелије рака дебелог црева hct-116 без токсичности на здраве ћелије мrc-5. 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________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ @article{virijevic2022, author = {virijevi{\'{c}}, katarina d and stani{\'{c}}, petar b and mu{\v{s}}kinja, jovana m and {katani{\'{c}} stankovi{\'{c}}}, jelena s and sre{\'{c}}kovi{\'{c}}, nikola and {\v{z}}ivanovi{\'{c}}, marko n and {\v{s}}mit, biljana m}, journal = {journal of the serbian chemical society}, title = {{synthesis and biological activity of novel zingerone–thiohydantoin hybrids}}, year = {2022}, issn = {1820-7421}, month = {aug}, number = {12}, pages = {1349--1358}, volume = {87}, abstract = {a series of zingerone–thiohydantoin hybrids were synthesized from o-alkyl zingerone derivatives by cyclocondensation with thiosemicarbazide in a two-step reaction. the obtained new potentially bioactive compounds were structurally characterized by ir and nmr spectroscopy, as well as by elemen­tal and hrms analysis. in addition, their antimicrobial and in vitro anticancer activities were tested. the tested compounds showed low to moderate antimic­robial activity. zingerone–thiohydantoin hybrid with an o-butyl substituent exerted the significant cytotoxic activity on colon hct-116 cancer cells, with­out toxicity on healthy mrc-5 cells.}, doi = {10.2298/jsc220404047v}, file = {:d\:/onedrive/mendeley desktop/virijevi{'{c}} et al. 2022 synthesis and biological activity of novel zingerone–thiohydantoin hybrids.pdf:pdf;:01_11747_5598.pdf:pdf}, keywords = {antimicrobial activity, cytotoxic activity}, publisher = {serbian chemical society}, url = {https://www.shd-pub.org.rs/index.php/jscs/article/view/11747}, } stability of bicalicene isomers – a topological study j. serb. chem. soc. 81 (1) 81–89 (2016) udc 547.534:544.131:537.872’12:66–945.3 jscs–4829 original scientific paper 81 stability of bicalicene isomers – a topological study ivan gutman*# faculty of science, university of kragujevac, p. o. box 60, 34000 kragujevac, serbia and state university of novi pazar, novi pazar, serbia (received 20 august, revised and accepted 18 september 2015) abstract: bicalicene is a conjugated hydrocarbon obtained by joining two calicene fragments. this can be realized in two different ways, thus resulting in two bicalicene isomers (tentatively referred to as cis and trans). the trans isomer is a stable compound whereas cis-bicalicene appears to be less stable and has never been prepared. the stability order of the bicalicene isomers cannot be rationalized by means of the standard topological theory of conjugated π-electron systems, and requires a special graph-theory-based analysis. keywords: calicene; bicalicene; molecular graph; cyclic conjugation; total π-electron energy. introduction calicene (compound 1 in fig. 1) is a well known non-alternant conjugated hydrocarbon.1,2 in the 1980s, yoshida et al., starting from di(t-butyl)thiocalicene (2) succeeded in synthesizing compound 3 consisting of two condensed calicene fragments. this non-alternant polycyclic conjugated hydrocarbon, named bicalicene, was found to be a reasonably stable aromatic species. it immediately attracted the attention of theoreticians, and its π-electron properties were the subject of a number of quantum chemical investigations.3,5–11 thus, from a theoretical point of view, the properties of bicalicene (3) are reasonably well understood. fig. 1. calicene (1) and the two isomeric bicalicenes (trans-bicalicene, 3, and cisbicalicene, 4). the cis-isomer 3 was synthesized from di(t-butyl)thiocalicene (2), in which reaction the trans-isomer 4 is not obtained.3,4 * e-mail: gutman@kg.ac.rs # serbian chemical society member. doi: 10.2298/jsc150820077g 82 gutman on the other hand, condensing two calicene fragments can be realized in two different ways, resulting in the isomeric compounds 3 and 4, see fig. 1. experimentally,3,4 only isomer 3 has been obtained. until today, isomer 4 has not been synthesized and the general opinion is that it is less stable than 3. curiously, however, none of the theoretical studies of bicalicene,3,5–8,10,11 with a single exception,9 considers or even mentions isomer 4. aihara9 examined the cyclic π-electron conjugation and topological resonance energy of 3 and 4 and found that the aromatic properties of both isomers should be similar. in the following, compound 3 will be named trans-bicalicene, whereas 4 will be called cis-bicalicene. in this paper, a topological (graph-theory-based) analysis of the mutual stability of cisand trans-bicalicene is undertaken. in particular, an attempt is made to discover the (topological) reasons why the cis-isomer is less stable than its trans-congener. as the considerations in the subsequent section show, the standard theory does not suffice for this, and special mathematical techniques need to be employed. standard topological approach for predicting the stability order of polycyclic conjugated hydrocarbons the relations between the structure of polycyclic conjugated π-electron systems and their chemical and thermodynamic stability can be successfully assessed by means of topological considerations, and were the subject of numerous studies; for details see the monographs,12–14 reviews15–17 and the references quoted therein. the following structural features are known to be the main factors determining the stability order of two isomeric polycyclic conjugated hydrocarbons: 1. the isomer with greater number of kekulé structures is expected to be more stable.18,19 if not all kekulé structures have the same parity, then their algebraic count needs to be taken into account.20 ionic resonance structures are not taken into account. 2. the isomer with the greater total π-electron energy and (consequently) greater resonance energy is expected to be more stable.17,21 3. the isomer with more rings of size 4 2k  and fewer rings of size 4k is expected to be more stable.22–24 4. the isomer with greater (stabilizing) cyclic conjugation in the (4 2)k  -sized rings and smaller (destabilizing) cyclic conjugation in the 4k -sized rings is expected to be more stable.15,18 5. with other structural features being equal, the species with greater steric strain is less stable.25–27 standard topological approach applied to bicalicene isomers when the above listed criteria are applied to the two bicalicene isomers, then – surprisingly – the conclusion follows that both should be equally stable aro stability of bicalicene isomers 83 matic species. in other words, the fact that cis-bicalicene has never been obtained and that it appears to be less stable than the trans-isomer cannot be accounted for. in particular, the following facts exist: 1. both isomers of bicalicene have two kekulé structures, cf. fig. 2. thus, if there is some resonance in the case of the trans-isomer, an analogous resonance should exist also in the case of the cis-isomer. it should be noted, however, that the two kekulé structures of both 3 and 4 are of opposite parity, since an even number (eight) of double bonds are cyclically rearranged. therefore, from the standard point of view, resonance should in fact destabilize both isomers 3 and 4.12,14,15,18,20 some authors would also include the ionic and doubly-ionic resonance structures 3c, 3d, 4c and 4d in the consideration but this would lead beyond the ambits of standard topological approaches. in addition, as is explained in point 4 below, such an extension of the resonance model is not necessary at all. fig. 2. the kekulé structures of trans-bicalicene (3a and b) and cis-bicalicene (4a and b), as well as of their ionic (3c and 4c) and doubly-ionic (3d and 4d) resonance forms. structure 3c is one of four ionic structures, whereas 4c is one of the two possible such structures. since, in the transformation 3a  3b, eight double bonds are cyclically moved, the kekulé structures 3a and b are of opposite parity, the algebraic structure count of 3 is zero, and no resonance stabilization is to be expected. in accordance with this, conjugated circuit theory15,18 would predict a negative resonance energy for the trans-bicalicene 3. exactly the same applies to cisbicalicene 4 and its kekulé structures. 2. the hmo total π-electron energy of cisand trans-bicalicene are, respectively, equal to 22.60 and 22.78 β-units*. the respective topological resonance energies28–30 are 0.47 and 0.55 β-units. these values are obtained under the (tacit) assumption that steric strain can be disregarded. in the case of * 1  138.9 kj mol-1 84 gutman bicalicenes, the steric strain, especially in the three-membered rings, is far from negligible. therefore, only under the (doubtful) assumption that the strain energy in both 3 and 4 is equal, would the comparison of the total π-electron energies and resonance energies of 3 and 4 be meaningful. if so, then9,13,31 the transbicalicene would be more stable by ca. 0.18β ≈ 25 kj mol–1 than its cis-isomer. if so, then according to the total-π-electron-energy/resonance-energy criterion,9,21 both bicalicene isomers would be predicted to be stable aromatic species, with isomer 4 being somewhat less aromatic than 3. 3. as seen from figs. 1 and 3, both bicalicene isomers possess equal number of rings of equal size: two three-membered (a, a′), two five-membered (b, b′) and an eight-membered ring (c). only their mutual arrangement differs. when other stability-determining structural features are equal, such a difference in the mutual arrangement of the rings should not cause a significant effect. fig. 3. labeling of the rings of the bicalicene isomers. 4. the energy effects (ef) of cyclic conjugation in the rings and pairs of rings of the bicalicene isomers are given in table i.32–34 recall that ef > 0 indicates thermodynamic stabilization, whereas rings with negative ef-values destabilize the respective π-electron system. in the case of bicalicenes, only the central 8-membered ring has such a destabilizing effect, but it is negligibly small. table i. energy effects (β-units) of cyclic conjugation in the rings and pairs of rings of trans-bicalicene (3) and cis-bicalicene (4). the labeling of the rings is indicated in fig. 2. note that individual rings in 3 and 4 have almost equal energy effects. the same is true for the pairs of rings a,a′ and b,b′. in contrast to this, the four pairs of rings a,b significantly stabilize more the cis-isomer than the trans-isomer cycle 3 4 a, a′ 0.3059 0.3207 b, b′ 0.2422 0.2430 c –0.0026 –0.0028 a+a′ 0.0338 0.0325 a+b 0.2494 0.4101 a+b′ 0.2494 0.4543 a′+b 0.2494 0.4543 a′+b′ 0.2494 0.4101 b+b′ 0.0611 0.0583 stability of bicalicene isomers 85 it should also be noted that the ef-method is insensitive to the actual distribution of the π-electron charges, which means that it properly reproduces also the cyclic-conjugation patterns pertaining to the ionic and doubly-ionic resonance structure 3c, 3d, 4c and 4d (cf. fig. 2). for individual rings of both bicalicene isomers, the ef-values were earlier calculated by aihara.9 from the data given in table i, it could be seen that the respective energy effects have very similar values. from this, aihara concluded that there is no significant difference between the aromaticity of 3 and 4. when energy effects of pairs of rings are added to the cyclic-conjugation pattern, (calculated by the method elaborated in the literature33), the picture becomes significantly different. whereas in the case of pairs of rings of equal size (a,a′ and b,b′), the ef-values are nearly the same in 3 and 4, the four pairs of rings a,b significantly stabilize more the cis-isomer 4 than the trans-isomer 3. thus, if any conclusion would be drawn based on energy effects of cyclic conjugation, then both bicalicene isomers would be predicted to be stable aromatic species, the isomer 3 somewhat less aromatic than 4. this conclusion contradicts that which would be inferred based on resonance energy (see point 2 above). 5. finally, although the presence of 3-membered rings indicates strong steric strain in the bicalicene molecules, it is difficult to envisage why this strain would be much different in the two isomers 3 and 4. summarizing the above points, it could be seen that, based on standard (usual) topological arguments, the two isomeric bicalicenes would be predicted to be nearly equally stable, and to possess nearly equal aromatic character. by such approaches, one cannot predicted with certainty whether the cisor the trans-form is the more stable isomer. the fact that trans-bicalicene is an extant, well defined, and long-known compound, whereas cis-bicalicene has never been obtained, remains obscure. in the subsequent section, it is shown how this difficulty could be overcome. a concealed graph-theoretical difference between the bicalicene isomers in view of the considerations in the previous sections, it remains to examine the effect of the different arrangements of the threeand five-membered rings in isomers 3 and 4. for this, attention is focused on the determinant of the adjacency matrix ( )a g of a molecular graph g, and its expression in terms of sachs graphs.12,13,35,36 it has been known for a long time12,17,37 that the det ( )a g is a sensitive measure of the stability of polycyclic conjugated π-electron systems. in the case of benzenoid hydrocarbons, det ( )a g is equal to the square of the number of kekulé structures. in the case of alternant non-benzenoid compounds, det ( )a g is 86 gutman equal to the square of the algebraic structure count. in any case, among pairs of conjugated isomers, the one having the greater det ( )a g is expected to have greater total π-electron and resonance energies, and a smaller homo–lumo gap,12 therefore more stable. a sachs graph of the molecular graph g is a subgraph of g consisting of isolated cycles and isolated edges.12–14 in the following, we are interested in the sachs graphs embracing all vertices of g are the focus of interest. let ( )g be the set of all such sachs graphs. as usual in the case of molecular graphs, it is assumed that the number n of vertices is even. (recall that for the molecular graphs of bicalicene, 16n  ). then: ( ) ( ) ( ) det ( ) ( 1) 2p s c s s g a g    (1) where s is a sachs graph, consisting of ( )p s components of which ( )c s are cyclic. formula (1) is now applied to the molecular graphs 3g and 4g of transand cis-bicalicene, respectively. the sets 3( )g and 4( )g have 8 elements each, depicted in fig. 4. these can be classified as follows: type 1. sachs graphs without cycles, ( ) 0 , ( ) / 2c s p s n  . both 3g and 4g have two such sachs graphs, pertaining to the two kekulé structures of 3 and 4, cf. figs. 2 and 4. type 2. sachs graphs formed by the perimeter, for which ( ) 1 , ( ) 1c s p s  . type 3. sachs graphs with two cycles. these are depicted in fig. 4. this case is discussed in more detail below. type 4. sachs graphs with four cycles, ( ) 4 , ( ) 4c s p s  . both 3g and 4g have just one such sachs graph, see fig. 4. sachs graphs of types 1, 2, and 4 contribute equally to the values of 3det ( )a g and 4det ( )a g . therefore, attention was focused on those of type 3. the sachs graphs of type 3 are 4 7 12 15 and s s s s  . by inspecting fig. 4, it could be seen that with the exception of 14s and 15s , these all have the property ( ) 6 , ( ) 2p s c s  . on the other hand, 14 14( ) 7 , ( ) 2p s c s  and 15( ) 5p s  , 15( ) 2c s  . thus, 4 7 12 13, and s s s s have an even number of components, whereas 14 15 and s s have an odd number of components. this has the consequence that all 4 7s s have an increasing contribution to 3det ( )a g , whereas the contributions of 12 15s s to 4det ( )a g cancel each other. this subtle difference may be the cause of the different stabilities of 3 and 4. indeed, by applying eq. (1), one obtains: 8 0 1 1 6 2 4 43det ( ) 2 ( 1) 2 1 ( 1) 2 4 ( 1) 2 1 ( 1) 2 32a g              and stability of bicalicene isomers 87 8 0 1 1 6 2 4 7 2 5 2 4 4 det ( ) 2 ( 1) 2 1 ( 1) 2 2 ( 1) 2 1 ( 1) 2 1 ( 1) 2 1 ( 1) 2 16 a g                     fig. 4. the 16-vertex sachs graphs of the molecular graphs of transand cis -bicalicene. sachs graphs of type 1 are 1 2 9 10, , and s s s s , and in an evident manner correspond to the kekulé structures 3a, 3b, 4a and 4b depicted in fig. 2. sachs graphs of type 2 are 3 11 and s s . the sachs graphs of type 3 are 4 7s s and 12 15s s corresponding to transand cis-bicalicene, respectively. the sachs graphs of type 4 are 8 16and s s . note that all 4 7s s and two among 12 15s s contain a threeand a five-membered cycle and for these the condition ( ) 6 ,p s  ( ) 2c s  holds. on the other hand, one among 12 15s s possesses a pair of three-membered cycles, satisfying ( ) 7 ,p s  ( ) 2c s  and one possesses a pair of five-membered cycles, satisfying ( ) 5 , ( ) 2p s c s  . this subtle difference may be the cause of the different stabilities of 3 and 4. at this point, one should recall that each sachs graph represents a particular structural detail of the underlying molecule. in view of this, the above analysis points towards those structural features that are the topological cause of the difference between the two isomeric bicalicenes. the difference may be the result of the fact that in the cis-isomer, some sachs graphs have opposite parities (equal number of cycles, but a number of components of different parity), whereas in the trans-isomer this does not happen. equivalently, in trans-bicalicene, there are no sachs graphs with two cycles of equal size, whereas in the cis-isomer such sachs graphs do exist. although in the case of non-bipartite conjugated hydrocarbons, the square root of det ( )a g is not directly related to the number of kekulé structures, it is interesting to observe that 4det ( ) 4a g  , which may be understood as if cis -bicalicene behaves like a conjugated π-electron system having four kekulé structures. then 3det ( ) 5.66a g  would imply that trans-bicalicene behaves like a π-electron systems having about six kekulé structures. as a curiosity, it is 88 gutman noticed that the number of kekulé and ionic resonance structures of cis-bicalicene is 4, and the corresponding number for those of trans-bicalicene is 6, cf. fig. 2. concluding remarks the conclusion of the presented analysis of the two isomers of bicalicene is that both satisfy all topological requirements for being stable, moderately aromatic compounds. the cis-isomer is predicted to be somewhat less stable than the trans-isomer, but this decrease in stability is not sufficient to prevent cisbicalicene from being a possibly well-defined, reasonably stable and experimentally obtainable compound. therefore, it is concluded that cis-bicalicene should be an attractive and challenging target for synthesis. и з в о д стабилност изомера бикалицена – тополошка студија иван гутман природно–математички факултет универзитета у крагујевцу и државни универзитет у новом пазару бикалицен је конјуговани угљоводоник добијен спајањем два калиценска фрагмента. то се може учинити на два начина, што доводи до два изомера бикалицена (тентативно означена као cis и trans). trans изомер је стабилно једињење док је cis-бикалицен мање стабилан и до сада није добијен. у оквиру стандардне тополошке теорије конјугованих π-електронских система, овакве разлике у стабилности изомерних дикалицена се не могу објаснити. за то је потребно да се употребе посебне граф-теоријске методе. (примљено 20. августа, ревидирано и прихваћено 18. септембра 2015) references 1. h. prinzbach, u. fischer, angew. chem. int. ed. 4 (1965) 598 2. a. s. kende, p. t. izzo, j. am. chem. soc. 87 (1965) 1609 3. z. yoshida, pure appl. chem. 54 (1982) 1059 4. s. yonedo, m. shibata, s. kida, z. yoshida, y. kai, k. miki, n. kasai, angew. chem. int. ed. 23 (1984) 63 5. a. toyota, t. nakajima, chem. lett. 11 (1982) 1049 6. t. sugimoto, m. shibata, s. yoneda, z. yoshida, y. kai, k. miki, n. kasai, t. kobayashi, j. am. chem. soc. 108 (1986) 7032 7. t. sugimoto, z. yoshida, pure appl. chem. 62 (1990) 551 8. t. sugimoto, m. sakaguchi, h. ando, y. arai, t. tanaka, z. yoshida, j. yamauchi, a. a. bothner-by, m. lisicki, j. am. chem. soc. 113 (1991) 3193 9. j. aihara, bull. chem. soc. jpn. 64 (1991) 3454 10. w. p. oziminski, m. palusiak, j. dominikowska, t. m. krygowski, r. w. a. havenith, c. m. gibson, p. w. fowler, phys. chem. chem. phys. 15 (2013) 3286 11. m. mandado, n. ramos-berdullas, phys. chem. chem. phys. 17 (2015) 16826 12. a. graovac, i. gutman, n. trinajstić, topological approach to the chemistry of conjugated molecules, springer, berlin, 1977 13. i. gutman, o. e. polansky, mathematical concepts in organic chemistry, springer, berlin, 1986 stability of bicalicene isomers 89 14. j. r. dias, molecular orbital calculations using chemical graph theory, springer, berlin, 1993. 15. m. randić, chem. rev. 103 (2003) 3449 16. i. gutman, topics curr. chem. 162 (1992) 29 17. i. gutman, j. serb. chem. soc. 70 (2005) 441 18. d. j. klein, j. chem. educ. 69 (1992) 691 19. s. radenković, i. gutman, j. serb. chem. soc. 74 (2009) 155 20. c. f. wilcox, j. am. chem. soc. 91 (1969) 2732 21. l. j. schaad, b. a. hess, chem. rev. 101 (2001) 1465 22. i. gutman, o. e. polansky, theor. chim. acta 60 (1981) 203 23. i. gutman, theor. chim. acta 66 (1984) 43 24. v. gineityte, match commun. math. comput. chem. 72 (2014) 39 25. w. c. herndon, p. c. norwalk, d. a. connor, p. lin, j. am. chem. soc. 114 (1992) 41 26. s. radenković, i. gutman, s. đorđević, chem. phys. lett. 625 (2015) 69 27. t. fujikawa, y. segawa, k. itami, j. am. chem. soc. 137 (2015) 7763 28. j. aihara, j. am. chem. soc. 98 (1976) 2750 29. i. gutman, m. milun, n. trinajstić, j. am. chem. soc. 99 (2077) 1692 30. i. gutman, int. j. chem. model. 6 (2014) 177 31. l. j. schaad, b. a. hess, j. am. chem. soc. 94 (1972) 3068 32. i. gutman, monatsh. chem. 136 (2005) 1055 33. i. gutman, s. stanković, j. đurđević, b. furtula, j. chem. inf. model. 47 (2007) 776 34. h. fatoorehchi, i. gutman, h. abolghasemi, j. math. chem. 53 (2015) 1113 35. a. graovac, i. gutman, croat. chim. acta 51 (1978) 133 36. a. graovac, i. gutman, match commun. math. comput. chem. 6 (1979) 49 37. i. gutman, k. c. das, j. serb. chem. soc. 78 (2013) 1925. accepted manuscript this is an early electronic version of an as-received manuscript that has been accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as m. stjepanović, a. janković, b. vulović, r. matović and r. saičić, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc230627046s this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230627046s j. serb. chem. soc.00(0)1-9 (2023) original scientific paper jscs–12460 published dd mm, 2023 1 synthetic study on the angular triquinanes mihailo n. stjepanović1, aleksandar v. janković2, bojan z. vulović1, radomir v. matović2 and radomir n. saičić1,3* 1university of belgrade faculty of chemistry, studentski trg 16, 11158 belgrade, serbia, 2ictm center for chemistry, njegoseva 12, 11158 belgrade, serbia, and 3serbian academy of sciences and arts, kneza mihaila 35, 11000 belgrade, serbia (received 27 june; revised 1 july; accepted 24 july 2023) abstract: the synthesis of an angular triquinane, which could serve as a suitable platform for the synthesis of several natural products (panaginsene, silphinene, senoxydene) is described. the synthesis is based on two consecutive cyclopentene annulations, where alkenes were used as latent carbonyl functionalities (via wacker reaction), and cyclopentenone annulation was effected by aldol condensation. keywords: cyclopentane; natural products; organic synthesis; wacker oxidation; aldol condensation. introduction among the thousands of structurally diverse, naturally occurring sesquiterpene compounds, the subgroup of triquinanes, although not numerous, occupies a prominent place, due to peculiar structures and a range of biological activities. these polycyclic compounds are assembled from condensed cyclopentane rings and, depending on the fusion pattern, can be classified as linear, angular, or propellane (figure 1). triquinane structural subunits are also embedded into tetraquinane skeletons. not surprisingly, they have attracted considerable interest from the synthetic community. 1 figure 1: three types of triquinane fusion *corresponding authors e-mail: rsaicic@chem.bg.ac.rs https://doi.org/10.2298/jsc230627046s a cc ep te d m an us cr ip t mailto:rsaicic@chem.bg.ac.rs https://doi.org/10.2298/jsc230627046s 2 stjepanović et al. naturally occurring angular triquinanes comprise four skeletal types, namely isocomanes, silphinanes, pentalenanes and silphiperfolanes (figure 2). some of them exhibit interesting biological activities, such as subergorgic acid (1, member of the isocomane family), which is cardiotoxic, 2 but also shows antiholinesterase activity, 3 and hence the ability to act as antidote against soman in mice. 4 oxygenated congeners of pentalenene 2, such as pentalenolactone, show antibiotic activity. 5 silphinene 3 is a biosynthetic precursor of oxygenated products such as aspergilanes, 6 or penifulvins; penifulvin a possesses significant insecticidal activity. 7 figure 2: examples of naturally occurring angular triquinanes we became interested in silphinene 3 and its congeners – panaginsene 4 8 and senoxydene 5. these compounds share the common angular triquinane skeleton, with some differences in the position of the alkene bond and one methyl substituent. one stereochemical difference is also of note: the methyl group at c11 may be of either trans-configuration (in panaginsene and senoxydene), or cisoriented (in silphinene), with respect to the cyclopentene ring. interestingly, panaginsene was initially assigned as cis-configured isomer 6, which was later revised (corrected structure: 4). 9 nine total syntheses of silphinene have been reported, the first one by ito and coworkers, who used bicyclopentadiene as the starting material. 10 paquette and leone-bay constructed the triquinane skeleton by iterative application of 3-oxo-organocuprate 1,4-addition/aldol condensation reaction tandem. 11 sternbach and coworkers relied on intramolecular diels-alder reaction, followed by oxidative fragmentation of the transient tricycle to the diquinane intermediate. 12 the synthesis by wender and ternansky hinges on arene-olefin meta-photocycloaddition, followed by reductive cleavage of cyclopropane ring in the tetracyclic intermediate. 13 crimmins and mascarella used a cc ep te d m an us cr ip t synthetic study on the angular triquinanes 3 a tactical combination of reactions: [2+2] alkene photocycloaddition/cyclobutane fragmentation to access racemic 3. 14 synthetic approach by nagarajan and rao is based on two cyclopentane annulations, the first by intramolecular hornerwadsworthemmons reaction and the second by a radical 5-exo-cyclization. 15 yamamura and coworkers created a tricyclic intermediate by an intramolecular cycloaddition of electrochemically generated phenoxy cation; this intermediate was then elaborated into a triquinane framework. 16 franck-neumannm, miesch and gross developed “the cyclobutenic way” to silphinene, which comprises cyclopropanation of bicyclo[3.2.0.]hept-6-ene cycloadduct, followed by solvolysis of a highly strained tricyclic intermediate. 17 fraser-reid and dickson used the chiron approach and synthesized (–)-3 from mannose. 18 two syntheses of panaginsene have been reported: lee and geum used as a key step an intramolecular cycloaddition via biradical intermediate, 9 whereas chakraborty and singh relied on mcmurry coupling for the cyclopentene ring closure. 19 as for senoxydene, two syntheses have been reported: paquette and coworkers used intramolecular ene-cyclization for the stereoselective formation of the key bicyclic intermediate, 20 whereas ito and coworkers exploited the common intermediate from their previous synthesis of silphinene. 21 results and discussion we set out to develop a method that would allow for efficient construction of the angular triquinane 7, which contains the skeleton of these three congeners, and where the configuration of the c-11 methyl group could be established subsequently (i.e., in late stages of synthesis), possibly giving rise to both diastereoisomers. our retrosynthetic analysis is blueprinted in scheme 1. cyclopentenone disconnection in 7 by aldol condensation transform simplifies the target to bicyclic ketone 8, which could be obtained from 9 by tactical combination of reactions: allylation/wacker oxidation. bicyclic ketone 9 should be accessible by aldolization/isomerization tandem reaction; we were aware that this step would need a study on the reaction conditions/product distribution relationship. the dicarbonyl precursor 10 of enone 9 could be prepared by wacker oxidation of 3butenylcyclopentanone derivative (not shown), on its turn obtainable from known 4,4-dimethyl-2-cyclopentene-1-one (11). 22 a cc ep te d m an us cr ip t 4 stjepanović et al. scheme 1: retrosynthetic analysis of angular triquinane 7 the synthesis commenced with 1,4-addition of a butenyl cuprate reagent to the known 4,4-dimethylcyclopent-2-ene-1-one (11), which afforded the adduct 12 (scheme 2). this transformation was reported earlier; 20b however, modifying the reaction conditions – activation by trimethylsilyl-chloride and lithium chloride – improved the yield considerably (82%, vs. 65% in the previous publication). 23 wacker oxidation of 12 under an oxygen atmosphere required 72 h at 50 °c, but provided dione 10 in 84% yield. various reaction conditions were tried to accomplish the cyclization step (i.e., 10 → 9), including potassium hydroxide in aqueous ether, potassium tert-butoxide in tert-butanol, or in thf, pyrrolidine (alone, or in the presence of acetic acid, or p-tsoh), but in all cases mixtures of conjugated (13) and deconjugated (9) product were obtained. finally, we found that the cyclization could be performed in boiling toluene, in presence of catalytic amounts of p-tsoh, to provide the desired bicyclic enone 9 in 83% yield (accompanied with 9% of the conjugated isomer 13). the next step was the installation of acetonyl moiety, to obtain dione 8. from several synthons available as synthetic equivalents of “acetonyl cation” we chose the structurally simplest one – allyl bromide – for two reasons: a) minimization of steric hindrance, and b) the allylation product 14 should be amenable also to aldehyde 15 – an intermediate whose role is explained below (in scheme 3). regioselective allylation of 9 was promoted by sodium hydride in boiling thf, where single regioand diastereoisomer 14 was obtained (85%). it is of note that the regioselectivity in reactions of this type is highly dependent on substrate structure; thus, in the allylation of the structurally related bicyclic system, the less substituted -position of the ketone required protection, in order to secure allylation at the more substituted ’-position. 20 wacker oxidation of 14 proceeded much faster than the previous one (i.e., 12 → 10), and provided bicyclic dione 8 in 50% yield (accompanied with 10% of aldehyde 15). upon treatment with potassium tertbutoxide in tert-butanol, 8 cyclized into triquinane 7 (51%), thus providing the target molecule in six steps (12% overall yield) from the starting 4,4dimethylcyclopent-2-ene-1-one (11). a cc ep te d m an us cr ip t synthetic study on the angular triquinanes 5 scheme 2: synthesis of triquinane 7 the scope of sesquiterpene natural products targeted by this methodology is not limited to angular triquinanes, but extends to bridged diquinane, i.e., cedrenes. thus, regioisomeric oxidation of intermediate 14 should provide aldehyde 15, from which the assembly of cedrene skeleton would be accomplished by an intramolecular aldol reaction (scheme 3). in this way, a single synthetic intermediate (i.e., 14) would provide access to several members of two distinct classes of quinane natural products. scheme 3: towards and -cedrene conclusion to summarize, an efficient synthesis of the advanced intermediate 7 for the synthesis of triquinane sesquiterpenes (panaginsene, senoxydene and silphinene) is described, which relies on iterative application of the wacker oxidation/aldol condensation tactical combination of reactions (i.e., 12 → 9; 14 → 7). research aiming to the conversion of this intermediate into the target natural products, as a cc ep te d m an us cr ip t 6 stjepanović et al. well as extension of this methodology to the synthesis of cedrenes (according to scheme 3), is underway in our laboratories. experimental general experimental details all chromatographic separations were performed on silica gel 60 (0.063-0.2 mm), merck. standard techniques were used for the purification of reagents and solvents. nmr spectra were recorded on varian/agilent 400 (1h nmr at 400 mhz, 13c nmr at 100 mhz in deuterated chloroform, if not otherwise stated. chemical shifts are expressed in ppm (δ) using tetramethylsilane as internal standard, coupling constants (j) are in hz. ir spectra were recorded on thermo scientific nicolet summit ft-ir instrument, and are expressed in cm-1. mass spectra were obtained on orbitrap exploris 240 spectrometer. the physical data and nmr spectra of the synthesized compounds are given in supplementary material to this paper. 4-(but-3-en-1-yl)-3,3-dimethylcyclopentan-1-one (12) to a soluton of cubr·sme2 (9.7 g, 47 mmol, 2.16 eq) and licl (2 g, 47 mmol, 2.16 eq) in thf (50 ml) a solution of grignard reagent prepared from magnesium turnings (1.42 g, 58.44 mmol) and 4-bromo-1-butene (6 g, 44.44 mmol) was added dropwise at –78 °c, and the reaction mixture was stirred for additional 30 min. a solution of ketone 11 (2.4 g, 21.8 mmol, 1 eq) and tmscl (7 ml, 5.98 g, 55 mmol, 2.52 eq) in thf (51 ml) was added dropwise over 30 min. the reaction mixture was allowed to warm to room temperature overnight, quenched with acetic acid (22 ml) and stirred for 1.5 h. the mixture was transfered into a beaker and saturated soluton of nahco3 (160 ml) was added in portions. the mixture was filtered trough celite and extracted three times with methylene chloride. the combined organic layer was washed with brine, and dried over anh. mgso4, filtered, concentrated at rotovap and purified by dry-flash chromatography (petroleum ether/ethyl acetate = 7/1) to afford 2.96 g (82%) of product 12 as a pale yellow oil. 3,3-dimethyl-4-(3-oxobutyl)cyclopentan-1-one (10) ketone 12 (610 mg, 3.67 mmol, 1 eq) was added to a solution of pdcl2 (40 mg, 0.225 mmol, 0.06 eq) and cucl (150 mg, 1.51 mmol, 0.41 eq) in dmf (2.8 ml) and h2o (0.5 ml, 27.76 mmol, 7.56 eq) under an o2 atmosphere. the reaction mixture was stirred at 50 °c for 72 h, then diluted with water and extracted with et2o. organic layer was washed with water and brine, dried over anh. mgso4, filtered, concentrated at rotovap and purified by dry-flash chromatography (petroleum ether/ethyl acetate = 7/1) to afford 561 mg (84%) of product 10 as pale yellow oil. 3,3,6-trimethyl-3,3a,4,6a-tetrahydropentalen-1(2h)-one (9) tsoh·2h2o (55 mg, 0.29 mmol, 0.22 eq) was added to a solution of diketone 10 (240 mg, 1.32 mmol) in toluene (20 ml). the flask was equiped with deak-stark apparatus and the reaction mixture was refluxed for 5 h. the mixture was transfered into a separatory funnel, diluted with dcm, washed with saturated solution of nahco3, dried over anh. mgso4, concentrated at rotovap and purified by dry-flash chromatography (petroleum ether/ethyl acetate = 95/5) to afford 182 mg (83%) of product 9 (rf = 0.32 (petroleum ether/ethyl acetate = 95/5)) followed by 15 mg (9%) of conjugated ketone 13 (rf = 0.17 (petroleum ether/ethyl acetate = 95/5)). a cc ep te d m an us cr ip t synthetic study on the angular triquinanes 7 6a-allyl-3,3,6-trimethyl-3,3a,4,6a-tetrahydropentalen-1(2h)-one (14) a solution of ketone 9 (200 mg, 1.22 mmol, 1 eq) in thf (3 ml) was added to a suspension of nah (73 mg (60%), 1.83 mmol, 1.5 eq) in thf (1 ml), mixture was heated to reflux and stirred for 30 min when allyl-bromide (0.21 ml, 2.43 mmol, 2 eq) was added. after 4 h, more allyl-bromide was added (0.21 ml, 2.43 mmol, 2 eq) and refluxing was contiued overnight. reaction was quenched with water, acidified with few drops of 1.5 m hcl, extracted with dcm, washed with brine, dried over anh. mgso4, filtered, concentrated at rotovap and purified by dry-flash chromatography (petroleum ether/ethyl acetate = 95/5) to afford 213 mg (85%) of product 14 as colorless oil. 3,3,6-trimethyl-6a-(2-oxopropyl)-3,3a,4,6a-tetrahydropentalen-1(2h)-one (8) ketone 14 (64 mg, 0.31 mmol, 1 eq) was added to a solution of pdcl2 (16.6 mg, 0.09 mmol, 0.3 eq) and cucl (40.3 mg, 0.41 mmol, 1.3 eq) in dmf (0.7 ml) and h2o (0.128 ml) under an o2 atmosphere. the reaction mixture was stirred at 50 °c for 17 h, then diluted with water and extracted with et2o. organic extract was washed with water and brine, dried over anh. mgso4, filtered, concentrated at rotovap and purified by column chromatography (hexane/ethyl-acetate = 9/1) to afford 35 mg (51%) of product 8 as colorless oil. 5,5,8-trimethyl-4,5,5a,6-tetrahydrocyclopenta[c]pentalen-2(1h)-one (7) a solution of diketone 8 (18 mg, 0.082 mmol, 1 eq), t-buok (20 mg, 0.175 mmol, 2.14 eq) and t-buoh (0.1 ml) in thf (3.5 ml) was heated to 40 °c for 1 h. after cooling to room temperature, the reaction mixture was diluted with water and few drops of 1.5 m hcl, extracted with et2o, washed with saturated solution of nahco3 and brine, dried over anh. mgso4, filtrated, concentrated at rotovap and purified by column chromatography (petroleum ether/ethyl acetate = 5/1) to afford 8.5 mg (51%) of product 7 as colorless oil. acknowledgements: this research was supported by the ministry of science, technological development and innovation of republic of serbia, contract number: 451-0347/2023-01/200168. supplementary material supplementary materials are available electronically from https://www.shdpub.org.rs/index.php/jscs/article/view/12460, or from the corresponding authors on request. и з в о д синтетичка студија ангуларних трикинана михаило н. стјепановић1, александар в. јанковић2, бојан з. вуловић1, радомир в. матовић2 и радомир н. саичић1,3 1универзитет у београду – хемијски факултет, студентски трг 16, 11158 београд, србија, 2ихтм центар за хемију, његошева 12, 11158 београд, србија, и 3српска академија наука и уметности, кнеза михаила 35, 11000 београд, србија представљена је синтеза ангуларног трикинана, који може послужити као кључни интермедијер за синтезу неколико природних производа (парагинсена, силфинена, сенокисдена). ситеза се заснива на две узастопне анулације циклопентеновог прстена, код којих су алкени искоришћени као латентне карбонилне функционалне групе (преко wacker-ове реакције), а анулације циклопентенона су остварене алдолним кондензацијама. 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23 (2017) 3178–3183 (https://doi.org/10.1002/chem.201605362). a cc ep te d m an us cr ip t https://doi.org/10.1016/s0040-4039(01)80658-4 https://doi.org/10.1016/s0040-4039(00)71256-1 https://doi.org/10.1039/c39900001440 https://doi.org/10.1002/asia.202100144 https://doi.org/10.1021/ja00361a046 https://doi.org/10.1021/jo00355a019 https://doi.org/10.1016/s0040-4039(01)80023-x https://doi.org/10.1080/00397918008062750 https://doi.org/10.15227/orgsyn.067.0121 https://doi.org/10.1021/ja021177r https://doi.org/10.1002/chem.201605362 {palladium on carbon in peg-400/cyclohexane: recoverable and recyclable catalytic system for efficient decarbonylation of aldehydes} j. serb. chem. soc. 87 (6) 669–675 (2022) original scientific paper jscs–5549 published 28 april 2022 669 palladium on carbon in peg-400/cyclohexane: recoverable and recyclable catalytic system for efficient decarbonylation of aldehydes nataša terzić-jovanović1 and vladimir ajdačić2*# 1university of belgrade – institute of chemistry, technology and metallurgy, national institute of the republic of serbia, njegoševa 12, 11000 belgrade, serbia and 2innovative centre ltd., faculty of chemistry, studentski trg 12–16, 11158 belgrade, serbia (received 28 january, revised 28 february, accepted 7 march 2022) abstract: a simple methodology for the decarbonylation of aldehydes catalysed by commercially available palladium on carbon in a green two-solvent system is reported. various aromatic, aliphatic and heteroaromatic aldehydes were transformed to the corresponding decarbonylated products in good yields. product isolation from the reaction mixture is simple in practice, and the catalyst can be reused three times. keywords: green chemistry; defunctionalization; heterogeneous catalysis. introduction the transformation of aldehydes into hydrocarbons (deformylation/decarbonylation) promoted by enzymes,1 transition-metals2 or metal-free reagents3 is an important reaction in academic research4 and industry.5 the aldehyde group is an useful promoter of certain transformations, such as the diels–alder reaction, c–h activation, and domino oxa-michael-aldol reaction, and its simple removal via decarbonylation after it has served its purpose has been extensively applied in numerous methodologies6 and in the synthesis of complex molecules and natural products.7 some metals of the first, second and third transition series, including ni,8 ru,9 rh,10 pd11 and ir12 as well as complexes thereof, efficiently perform the mentioned transformation (fig. 1). however, the toxicity and high cost of these metals is a major drawback from an economic and environmental point of view. therefore, the use of recyclable heterogeneous catalysts for decarbonylation is both a greener and more economical alternative to homogeneous catalysis.13 * corresponding author. e-mail: ajdacic@chem.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc220128024t ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 670 terzić-jovanović and ajdačić fig. 1. decarbonylation of aldehydes promoted by transition-metals. experimental the general information pd/c (10 wt.% loading) was purchased from sigma aldrich. aldehydes were mostly obtained from commercial sources and used without further purification, except for the 1-adamantanecarboxaldehyde,14 1-adamantaneacetaldehyde,14 1-prop-2-yn-1-yl-1h-indole-3-carbaldehyde15 and 1-benzyl-1h-indole-3-carbaldehyde,15 which were synthesized according to known procedures. unless stated otherwise, solvents and other reagents were obtained from commercial sources and used without further purification. dry-flash chromatography was performed on sio2 (0.018–0.032 mm). 1hand 13c-nmr spectra were recorded on a bruker ultrashield avance iii spectrometer (at 500 and 125 mhz, respectively) and varian 400/54 premium shielded spectrometer (at 400 and 101 mhz, respectively) using cdcl3 (unless stated otherwise) as the solvent and tetramethylsilane (tms) as an internal standard. the chemical shifts are expressed in ppm on the δ scale and they were calibrated relative to those of the solvent. gc–ms spectra of the synthesized compounds were acquired on an agilent technologies 7890a apparatus equipped with a db-5 ms column (30 m×0.25 mm×0.25 μm), a 5975c msd and fid detector. the selected values are as follows: carrier gas was he (1.0 ml/min), temperature linearly increased from 40–315 °c (10 °c/min), injection volume: 1 μl, temperature: 250 °c, temperature (fid detector): 300 °c, and ei mass spectra range: m/z 40–550. for determination of gc–ms yield, the internal standard (naphthalene) was added to the reaction mixture after the workup. general procedure for decarbonylation of aldehydes 2a–n decarbonylation of biphenyl-4-carbaldehyde to biphenyl (2a) (cas reg. no. 92-52-4). dry glass reaction tube purged with argon and equipped with a magnetic stir bar was charged with aldehyde (90 mg, 0.5 mmol), pd/c (26 mg, 5 mol.% pd), cyclohexane (750 μl) and peg-400 (750 μl). the sealed tube was heated at 140 °c for 24 h. the reaction medium was then cooled to room temperature. the mixture of water and cyclohexane was then added to the reaction mixture. the layers were afterwards separated and the aqueous layer was washed with cyclohexane (5×5 ml). the organic layer was dried over na2so4, and the solvent was evaporated under reduced pressure. compound 2a was obtained after dry-flash column chromatography (sio2:cyclohexane) as a white solid (69.0 mg, 90 %). decarbonylation of 2-naphthaldehyde to naphthalene (2b) (cas reg. no. 91-20-3). following the general procedure for decarbonylation, compound 2b was prepared from aldehyde (78.0 mg, 0.5 mmol) using pd/c (26.0 mg, 5 mol.% pd) in a mixture of cyclohexane (750 μl) and peg-400 (750 μl) and was obtained after dry-flash column chromatography (sio2:cyclohexane) as a white solid (45.1 mg, 70 %). ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ decarbonylation of aldehydes 671 decarbonylation of anthracene-9-carbaldehyde to anthracene (2c) (cas reg. no. 120 -12-7). following the general procedure for decarbonylation, compound 2c was prepared from aldehyde (103.2 mg, 0.5 mmol) using pd/c (26.0 mg, 5 mol % pd) in a mixture of cyclohexane (750 μl) and peg-400 (750 μl) and was obtained after dry-flash column chromatography (sio2:cyclohexane) as a white crystalline solid (73.2 mg, 82 %). decarbonylation of 4-nitrobenzaldehyde to nitrobenzene (2d) (cas reg. no. 98-95-3). following the general procedure for decarbonylation, compound 2d was prepared from aldehyde (75.6 mg, 0.5 mmol) using pd/c (26.0 mg, 5 mol % pd) in a mixture of cyclohexane (750 μl) and peg-400 (750 μl) and was obtained as a yellow oil (gc–ms yield 90 % based on naphthalene). decarbonylation of 5-fluoro-2-methoxybenzaldehyde to 4-fluoroanisol (2e) (cas reg. no. 459-60-9). following the general procedure for decarbonylation, compound 2e was prepared from aldehyde (77 mg, 0.5 mmol) using pd/c (26 mg, 5 mol % pd) in a mixture of cyclohexane (750 μl) and peg-400 (750 μl) (gc–ms yield 60 % based on methyl benzoate as standard). decarbonylation of diphenylacetaldehyde to diphenylmethyl (2h) (cas reg. no. 101-81 -5). following the general procedure for decarbonylation, compound 2h was prepared from aldehyde (89 μl, 0.5 mmol) using pd/c (26.0 mg, 5 mol % pd) in a mixture of cyclohexane (750 μl) and peg-400 (750 μl) and was obtained after dry-flash column chromatography (sio2:cyclohexane) as a colorless oil (78.1 mg, 93 %). decarbonylation of 3-(1,3-benzodioxol-5-yl)-2-methylpropanal to dihydrosafrole (2i) (cas reg. no. 94-58-6). following the general procedure for decarbonylation, compound 2i was prepared from aldehyde (83 μl, 0.5 mmol) using pd/c (26 mg, 5 mol % pd) in a mixture of cyclohexane (750 μl) and peg-400 (750 μl) and was obtained after dry-flash column chromatography (sio2:cyclohexane) as a colorless oil (59.3 mg, 72 %). decarbonylation of 1-adamantanecarboxaldehyde to adamantane (2j) (cas reg. no. 281-23-2). following the general procedure for decarbonylation, compound 2j was prepared from aldehyde (82.3 mg, 0.5 mmol) using pd/c (26.0 mg, 5 mol % pd) in a mixure of cyclohexane (750 μl) and peg-400 (750 μl) and was obtained after dry-flash column chromatography (sio2:cyclohexane) as a colorless solid (67.3 mg, 84 %). decarbonylation of 1-adamantaneacetaldehyde to 1-methyl adamantane (2k) (cas reg. no. 768-91-2). following the general procedure for decarbonylation, compound 2k was prepared from aldehyde (89.1 mg, 0.5 mmol) using pd/c (26.0 mg, 5 mol % pd) in a mixure of cyclohexane (750 μl) and peg-400 (750 μl) and was obtained after dry-flash column chromatography (sio2:cyclohexane) as a colorless solid (74.0 mg, 73 %). decarbonylation of benzo[b]thiophene-3-carboxaldehyde to benzo[b]thiophene (2l) (cas reg. no. 95-15-8). following the general procedure for decarbonylation, compound 2l was prepared from aldehyde (81.1 mg, 0.5 mmol) using pd/c (26.0 mg, 5 mol % pd) in a mixture of cyclohexane (750 μl) and peg-400 (750 μl) and was obtained after dry-flash column chromatography (sio2:cyclohexane) as a colorless solid (47.1 mg, 70 %). decarbonylation of 1-prop-2-yn-1-yl-1h-indole-3-carbaldehyde to 1-prop-2-yn-1-yl -1h-indole (2m) (cas reg. no. 19017-00-6). following the general procedure for decarbonylation, compound 2m was prepared from aldehyde (92.3 mg, 0.5 mmol) using pd/c (26.1 mg, 5 mol % pd) in a mixure of cyclohexane (750 μl) and peg-400 (750 μl) and was obtained after dry-flash column chromatography (sio2:cyclohexane) as a colorless solid (53.3 mg, 68 %). decarbonylation of 1-benzyl-1h-indole-3-carbaldehyde to 1-benzyl-1h-indole (2n) (cas reg. no. 3377-71-7). following the general procedure for decarbonylation, compound ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 672 terzić-jovanović and ajdačić 2n was prepared from aldehyde (89.2 mg, 0.5 mmol) using pd/c (118.0 mg, 5 mol % pd) in a mixture of cyclohexane (750 μl) and peg-400 (750 μl) and was obtained after dry-flash column chromatography (sio2:cyclohexane) as a colorless solid (68.0 mg, 66 %). spectral data of the compounds are given in supplementary material to this paper recycling of pd/c and peg-400 catalytic system for decarbonylation of aldehyde (1a) dry glass reaction tube purged with argon and equipped with a magnetic stir bar was charged with aldehyde (1a, 90 mg, 0.5 mmol), pd/c (26 mg, 5 mol % pd), cyclohexane (750 μl) and peg-400 (750 μl). the sealed tube was heated at 140 °c for 24 h. after the completion of the reaction, the cyclohexane layer was decanted with a pipette and peg-400 layer was washed with cyclohexane (5×2 ml). the formed residue (pd/c in peg-400) was used for next reaction cycles following the general reaction procedure. results and discussion herein we report the efficient decarbonylation of aromatic, heteroaromatic and aliphatic aldehydes mediated by palladium on carbon in ecologically acceptable solvents, cyclohexane and peg-400. to determine the optimal reaction conditions, biphenyl-4-carboxaldehyde (1а) was used as the model substrate (table i). table i. optimization of reaction conditions entry solvent t / °c time, h yielda, % 1 h2o 160 24 trace 2 peg-400 140 24 42 3 peg-400 140 44 26 4 peg-400/ cyclohexane (1:1, v,v) 140 24 90 aisolated yield the initial conditions of 5 mol % palladium on carbon in h2o at 160 °c provided only trace amounts of the corresponding decarbonylated product (entry 1). when peg-400 was used instead of h2o, the yield increased to 42 % (entry 2). increasing the reaction time from 24 to 44 h led to a significant reduction in the yield (26 %, entry 3). after a detailed analysis of the reaction mixture, it was found that the reduced yield resulted from product evaporation. finally, the addition of cyclohexane as a co-solvent increased the yield to 90 %. to our surprise, after the reaction mixture had cooled, the catalyst particles were located exclusively in the peg-400 layer (fig. 2). the product was isolated by careful decantation of cyclohexane and additional extraction of the peg-400 layer with cyclohexane. the residual catalyst in peg-400 was used successively three more times under the same reaction conditions, without a significant loss of activity (fig. 3). ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ decarbonylation of aldehydes 673 fig. 2. reaction mixture after completion of the reaction. employing the optimized decarbonylation conditions, the aldehyde substrate scope was investigated (scheme 1). the non-functional polycyclic aromatic aldehydes (1a–c) were efficiently decarbonylated and the corresponding products (2a–c) were obtained in good yields. benzaldehyde bearing an electron-withdrawing substituent (no2) 1d generated the desired product 2d in high yield. 2-fluoro-4-methoxybenzaldehyde afforded the decarbonylated product 2e in moderate yield. in the case of 4-bromobenzaldehyde (1f) and 4-formylbenzoic acid (1g) there was no reaction. fig. 3. reusability of the pd/c/peg-400 catalytic system. the substrate scope was then extended to aliphatic aldehydes. the aliphatic aldehydes with an aromatic core successfully produced the desired products (2h, and i) in good to excellent yields. furthermore, the methodology was applied to the sterically demanding aldehydes, adamantane-1-carbaldehyde (1j) and 1-adamantylacetaldehyde (1k). the decarbonylated products, adamantane (2j) and methyladamantane (2k) were isolated in good yields. the decarbonylation of several heterocyclic aldehydes was also explored under the optimal reaction conditions. the decarbonylation of benzo[b]thiophene-3-carboxaldehyde (1l) afforded the desired product benzo[b]thiophene (2l) in good yield. in addition, the n-substituted indole derivates (1m and n) gave the corresponding decarbonylated products (2m and n) in good yields. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 674 terzić-jovanović and ajdačić scheme 1. substrate scope. conclusion to conclude, the ligand-free palladium-on-carbon-catalysed decarbonylation of aldehydes in ecologically acceptable solvents as an alternative to homogeneous catalysis was reported in this study. various aldehydes were successfully decarbonylated in moderate to excellent yields. the formation of byproducts during the reaction and chromatography was not observed. also, pd/c and peg -400 system could be recycled and reused in at least four consecutive reaction cycles, without significant loss of catalytic activity. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/11599, or from the corresponding author on request. acknowledgments. this research was financially supported by the ministry of education, science and technological development of republic of serbia (contract numbers: 451-03-9/ /2021-14/200168, 451-03-9/2021-14/200288 and 451-03-9/2021-14/200026) and serbian academy of sciences and arts under strategic projects programme-grant agreement no. 01 -2019-f65. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ decarbonylation of aldehydes 675 и з в о д паладијум на угљенику у peg-400/циклохексану: каталитички систем kojи се може рециклирати и поново употребити за ефикасно декарбониловање алдехида наташа терзић-јовановић1 и владимир ајдачић2 1универзитету београду, институт за хемију, технологију и металургију (ихтм), његошева 12, 11000 београд и 2иновациони центар хемијског факултета, студентски трг 12–16, 11000 београд развијена је једноставна метода за декарбониловање алдехида користећи комерцијално доступни паладијум на угљенику уз употребу зелених растварача. различити ароматични, алифатични и хетероароматични алдехиди могу се трансформисати у декарбониловане производе у добром приносу и без настајања споредних производа. производи се једноставно изолоју из реакционе смеше, а исти катализатор се може употребити још три пута без значајног смањења приноса. (примљено 28. јануара, ревидирано 28. фебруара, прихваћено 7. марта 2022) references 1. n. li, h. nørgaard, d. m. warui, s. j. booker, c. krebs, j. m. b., jr., j. am. chem. soc. 133 (2011) 6158 (https://doi.org/10.1021/ja2013517) 2. a. modak, d. maiti, org. biomol. chem. 14 (2016) 21 (https://doi.org/10.1039/c5ob01949d) 3. v. ajdačić, s. stepanović, m. zlatovića, m. grudena, i. m. opsenica, synthesis 48 (2016) 4423 (https://doi.org/10.1055/s-0035-1562615) 4. a g. j. s. dawes, e. l. scott, j. le nôtre, j. p. m. sanders, j. h. bitter, green chem. 17 (2015) 3231(https://doi.org/10.1039/c5gc00023h) 5. u. k. bagha, j. k. satpathy, g. mukherjee, c. v. sastri, s. p. de visser, org. biomol. chem. 19 (2021) 1879 (https://doi.org/10.1039/d0ob02204g) 6. h. lu, t.-y. yu, p.-f. xu, h. wei, chem. rev. 121 (2021) 365 (https://doi.org/10.1021/acs.chemrev.0c00153) 7. ž. selaković, a. m. nikolić, v. ajdačić, i. m. opsenica, eur. j. org. chem. (2022) (https://doi.org/10.1002/ejoc.202101265) 8. k. ding, s. xu, r. alotaibi, k. paudel, e. w. reinheimer, j. weatherly, j. org. chem. 82 (2017) 4924 (https://doi.org/10.1021/acs.joc.7b00284) 9. g. domazetis, b. tarpey, d. dolphin, b. r. james, j. chem. soc. chem. commun. (1980) 939 (https://doi.org/10.1039/c39800000939) 10. m. kreis, a. palmelund, l. bunch, r. madsen, adv. synth. catal. 348 (2006) 2148 (https://doi.org/10.1002/adsc.200600228) 11. v. ajdačić, a. nikolić, m. kerner, p. wipf, i. m. opsenica, synlett 29 (2018) 1781(https://doi.org/10.1055/s-0037-1610433) 12. t. iwai, t. fujihara, y. tsuji, chem. commun. 46 (2008) 6215 (https://doi.org/10.1039/b813171f) 13. v. ajdačić, a. nikolić, s. simić, d. manojlović, z. stojanović, j. nikodinović-runić, i. m. opsenica, synthesis 50 (2018) 119 (https://doi.org/10.1055/s-0036-1590892). 14. n. terzic, j. konstantinovic, m. tot, j. burojevic, o. djurkovic-djakovic, j. srbljanovic, t. stajner, t. verbic, m. zlatovic, m. machado, i.s. albuquerque, m. prudencio, r. j. sciotti, s. pecic, s. d’alessandro, d. taramelli, b. solaja, j. med. chem. 59 (2016) 264 (https://doi.org/10.1021/acs.jmedchem.5b01374) 15. y. sawama, y. miki, h. sajiki, synlett 31 (2020) 699 (https://doi.org/10.1055/s-00401707993). ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ @article{terzic-jovanovic2022, author = {terzi{\'{c}}-jovanovi{\'{c}}, nata{\v{s}}a and ajda{\v{c}}i{\'{c}}, vladimir}, journal = {journal of the serbian chemical society}, title = {{palladium on carbon in peg-400/cyclohexane: recoverable and recyclable catalytic system for efficient decarbonylation of aldehydes}}, year = {2022}, issn = {1820-7421}, month = {apr}, number = {6}, pages = {669--675}, volume = {87}, abstract = {a simple methodology for the decarbonylation of aldehydes catal­ysed by commercially available palladium on carbon in a green two-solvent system is reported. various aromatic, aliphatic and heteroaromatic aldehydes were transformed to the corresponding decarbonylated products in good yields. pro­duct isolation from the reaction mixture is simple in practice, and the cat­alyst can be reused three times.}, doi = {10.2298/jsc220128024t}, file = {:d\:/onedrive/mendeley desktop/terzi{'{c}}-jovanovi{'{c}}, ajda{v{c}}i{'{c}} 2022 palladium on carbon in peg-400cyclohexane recoverable and recyclable catalytic system for efficient d.pdf:pdf;:01_11599_5549.pdf:pdf}, keywords = {defunctionalization, heterogeneous catalysis}, publisher = {national library of serbia}, url = {https://www.shd-pub.org.rs/index.php/jscs/article/view/11599}, } electrical conductivity of gdcl3-licl and gdcl3-licl-gd2o3 molten systems accepted manuscript this is an early electronic version of an as-received manuscript that hasbeen accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as e. v. nikolaeva, i. d. zakiryanova, a. l. bovet and i. v. korzun, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc230131051n this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230131051n j. serb. chem. soc.00(0)1-14 (2023) original scientific paper jscs–12257 published dd mm, 2023 1 electrical conductivity of gdcl3–licl and gdcl3–licl-gd2o3 molten systems elena v. nikolaeva*, irina d. zakiryanova, andrey l. bovet, iraida v. korzun institute of high temperature electrochemistry, ub ras, 620990 yekaterinburg, russia (received 31 january; revised 31 july; accepted 14 august 2023) abstract: the electrical conductivity of licl–gdcl3 molten systems with the gadolinium chloride additions ranging from 0 to 23 mol% was measured depending on both the temperature and concentration of gdcl3. the molar electrical conductivity of the molten gdcl3–licl system is calculated taking into account the assumption of additivity of the molar volume of the mixture. the obtained temperature dependencies can be approximated by arrhenius-type equation. the effective activation energy ea increased with the gdcl3 content. the liquidus temperatures of the studied systems were determined by differential scanning calorimetry. the high-temperature raman spectra of licl–gdcl3 chloride melts were recorded. in addition, the electrical conductivity of 0.77licl–0.23gdcl3 molten system with 1 mol% of gd2o3 was measured. the investigation demonstrates that the addition of gadolinium oxide results in a decrease of the electrical conductivity of the chloride molten system and growth of its liquidus temperature. keywords: ac impedance; gadolinium chloride; liquidus temperature; raman spectra. introduction gadolinium and its compounds are widely used in many areas of science and technology, such as nuclear power engineering, electronics, medicine. the unique magnetic properties of gadolinium allow it to be used for the production of laser materials. the high capability of neutron capturing makes it possible to use gadolinium compounds for controlling the operation of nuclear reactors. the study of the electric transfer processes of molten mixtures of rare earth metal (re) and alkali metal (m) chlorides is of interest in connection with the development of a number of scientific and technical problems related with the optimization of the processes of electrolytic production and refining of rare earth metals, utilization and regeneration of spent nuclear fuel (snf).1 for example, *corresponding author. e-mail: e.nikolaeva@ihte.uran.ru https://doi.org/10.2298/jsc230131051n a cc ep te d m an us cr ip t mailto:e.nikolaeva@ihte.uran.ru https://doi.org/10.2298/jsc230131051n nikolaeva et al.. pyrochemical methods of snf reprocessing are considered as promising options in the innovative nuclear fuel cycle.2,3 the development of technologies requires knowledge of the electrochemical and thermodynamic properties of molten mixtures of rare earth and alkali metal halides. in industrial production, it is desirable to have an electrolyte with high electrical conductivity and low liquidus temperatures to reduce energy consumption. lithium chloride has the highest electrical conductivity among alkali metal chlorides and a relatively low melting point. thus, the main operations of the pyrochemical processing of snf are supposed to be carried out using anhydrous technologies in molten licl and eutectic licl–kcl.4 the electrical conductivity of only certain mixtures of re chlorides with lithium chloride has been experimentally studied: licl–lacl3,5 licl–prcl3,6 licl– ndcl36 and licl–smcl3.6 in7,8 the electrical conductivity of molten mixtures of gdcl3 with nacl and kcl were investigated. earlier we measured the electrical conductivity of molten 0.515gdcl3–0.485kcl9 system and gdcl3.10 there is no available data on the electrical conductivity of molten licl–gdcl3 mixtures. wentao zhou et al.11 established the licl–gdcl3 binary phase diagram based on the earlier results of differential thermal analysis and x-ray diffraction optimized using calphad thermo-cal software. the authors11 demonstrated that the phase diagram had a simple eutectic form. the eutectic point corresponds to temperature of 678 k and a concentration of gdcl3 of 45.2 mol%. the presence of li3gdcl6 ternary compound with polymorphic transformation at 646 k and peritectoid decomposition at 660 k was identified in the solid phase. the purpose of this work was to determine the specific electrical conductivity of molten gdcl3–licl systems containing up to 23 mol% gdcl3. based on experimental data, the calculation of molar electrical conductivity was carried out. lanthanide chlorides are known to be extremely hygroscopic. when the temperature rises, they react with their own crystallization water to form very stable oxychlorides, which can dissolve in re chlorides when the latter are melted.12 the presence of oxychlorides in molten recl3 in dissolved form or in the form of solid particles leads to a decrease in the electrical conductivity of the systems.9,10 therefore, another aim of this work was to study the effect of oxygen impurities on the electrical conductivity of gdcl3–licl melts. to do this the electrical conductivity of 0.77licl–0.23gdcl3 system containing 1 mol% of gadolinium oxide was investigated. the liquidus temperatures were determined for all investigated chloride and oxide-chloride systems. in order to analyze the structural changes occurring in the chloride melt, the raman spectra of homogeneous gdcl3–licl chloride melts were obtained. a cc ep te d m an us cr ip t electrical conductivity of molten systems 3 experimental samples preparation lithium chloride (puriss. grade, “vekton,” st. petersburg, russian federation) was heated in vacuum with a gradual increase in temperature to 673 k and melted in an argon atmosphere. the resulting melt was additionally purified by directional crystallization (zone melting). according to the dsc curve (thermal analyzer sta 449c jupiter (netzsch)) of the purified licl, a single peak is observed, which corresponds to the salt melting (fig. 1, a), and its temperature position 8801 k agrees with the available data for licl, tm = 883±2 k.13 the enthalpy of melting (∆hm = 19.903 kj/mol) also agrees well with the available data 13 (∆hm = 19.83±0.2 kj/mol). fig 1. dsc curves for licl (a) and gdcl3 (b). a cc ep te d m an us cr ip t nikolaeva et al.. gadolinium chloride was prepared from gadolinium oxide (puriss. grade, “vekton,” st. petersburg, russian federation) according to the well-known technique, a detailed description of which was given in.10 the quality of anhydrous gdcl3 was investigated by a well-proven visual method for determining the transparency of a salt solution in distilled water. on the dsc curve (fig.1, b) of synthesized gdcl3 one peak corresponding to the melting of the salt is observed, and its temperature position 877 ± 1 k is consistent with the available data for gdcl3. 14-17 the gd2o3 powder was dried at 973 k for 4 h. x-ray diffraction and raman spectroscopy confirmed the monophasic nature of the obtained product.10 all the operations with the prepared reagents were carried out in a glove box in a dry nitrogen atmosphere. electrical conductivity measuring technique experiments to determine the electrical conductivity of the gdcl3–licl melt were carried out in a cell with parallel platinum electrodes. the electrical conductivity was measured using a z-1500j impedance meter, which allows measurements to be carried out in the frequency range of alternating current from 1 mhz to 1.5 mhz. the scheme of the experimental cell and the method of conducting the experiment are described in detail in.18 the measuring set was calibrated in molten licl for which the explicit data on the electrical conductivity over 917-1056 k are known.5 the electrical conductivity measurements were performed at the temperatures above the liquidus temperature of each electrolyte composition. there were at least three consequential measurements in a series of experiments. gdcl3 was gradually added to molten licl in small portions without disturbing the gaseous atmosphere of the cell. after each addion, the system was kept at a certain temperature until the values of electrical resistance became stable. only after this, measurements of the temperature dependence of the electrical conductivity started. the concentration of gdcl3 was determined after the experiment in a frozen float by emission spectral analysis with inductively coupled plasma (optima 4300 dv, "perkin elmer" usa). determination of liquidus temperature using differential scanning calorimetry liquidus temperatures were obtained by differential scanning calorimetry (dsc) using an sta 449c jupiter® netzsch thermal analyser (germany).the studies were carried out with a heating rate of 10 k/min in a high purity argon atmosphere in pt-rh crucibles. the uncertainty of the liquidus temperature values was less than 1 k. the liquidus temperature of each sample was determined during heating for the second measurement.19 high-temperature raman spectra technique raman spectra of solid samples and melts were recorded using the ava-raman fiber-optic spectrometric complex (avantes, the netherlands), which includes a 50 mw laser source of monochromatic radiation with a wavelength of λ = 532 nm. when registering the spectra, an 180o optical scattering scheme was used. the device of a high-temperature optical prefix was described earlier.19 platinum crucible was used as a container; the experiment was carried out in an argon atmosphere. results and discussion liquidus temperature of licl–gdcl3 system dsc curves were obtained for licl–gdcl3 systems containing up to 23 mol% of gdcl3. as an example, fig. 2 demonstrates the dsc and tg curves obtained a cc ep te d m an us cr ip t electrical conductivity of molten systems 5 for the 0.85licl–0.15gdcl3 system. according to wentao zhou et al.11 the temperatures of 647.7 and 664.3 k correspond to the temperatures of phase transitions in solid state. the solidus temperature is 680 k. the liquidus temperature for this composition is 819.3 k. fig 2. dsc curve for the system 0.84licl–0.16gdcl3. liquidus temperatures for all the studied systems are given in table 1. our data are in good agreement with.11 table i. liquids temperatures and coefficients in eq. (1) for licl–gdcl3 and licl–gdcl3– gd2o3 systems system tliq k a s cm–1 b s cm–1k–1  s cm–1 (923 k)  s cm–1 (1023 k)  s cm–1 (1103 k) 0.94licl–0.06gdcl3 873 0.7125 0.00461 5.07 5.54 – 0.89licl–0.11gdcl3 843 0.4292 0.00419 4.43 4.86 – 0.84licl–0.16gdcl3 819 0.0691 0.00401 3.87 4.28 – 0.77licl–0.23gdcl3 752 –0.1441 0.00356 3.16 3.51 3.79 [0.77licl–0.23gdcl3]–gd2o3 (1 mol%) 1090 0.9872 0.00243 3.67 electrical conductivity of gdcl3–licl molten systems the temperature dependences of the specific electrical conductivity of the gdcl3–licl molten systems containing up to 23 mol% of gdcl3 were investigated in the range from the temperature above the liquidus temperature of each mixture up to 1073 k (for composition 0.23gdcl3–0.77licl up to 1153 k). the obtained experimental data are shown in fig.3 together with the available data for licl.5 it can be seen that the addition of gdcl3 reduces the electrical conductivity of the systems. the temperature dependences of the specific electrical conductivity of licl–gdcl3 melts were approximated by linear equation: a cc ep te d m an us cr ip t nikolaeva et al.. κ = a + bt (1) here  is specific electrical conductivity, t – temperature (k), a and b are constants. the coefficients of eq. (1) are given in table i. fig 3. temperature dependence of the specific electrical conductivity of molten licl5 (1); molten licl, containing gdcl3 (mol %): 6 (2); 11 (3); 16 (4); 23 (5); 0.77licl–0.23gdcl3 containing 1 mol% gd2o3 (6). figure 4 shows the isotherms of the normalized conductivity of the licl – gdcl3 system containing from 0 to 23 mol% gdcl3. it can be seen that the specific electrical conductivity gradually decreases with an increase in the concentration of gadolinium chloride, deviating from additive magnitudes towards smaller values. thus, at 1023 k, the addition of 20 mol% gdcl3 reduces the specific electrical conductivity of the system by almost 40%. the largest changes in the electrical conductivity of the system occur at low temperatures. a cc ep te d m an us cr ip t electrical conductivity of molten systems 7 fig 4. isotherms of normalized electrical conductivity of licl–gdcl3 molten system at different temperatures (0 specific electrical conductivity of licl). molar electrical conductivity of molten gdcl3–licl mixtures specific electrical conductivity is the electrical conductivity of a single volume of liquid. it is the value directly measured in the experiment. for the purposes of further analysis, it has the disadvantage that a single volume of different liquids (melts) contains a different number of molecules of the studied substances and, thus, a direct comparison of specific electrical conductivity does not quite correctly reflect the properties of the compared melts. it is more correct to compare the molar electrical conductivity, that is, the electrical conductivity, of one mole of each melt. molar electrical conductivity () of licl–gdcl3 melt can be calculated according to the equation:  = .vm (2) where  is the specific electrical conductivity, vm is the molar volume. most mixtures of alkali and rare earth metal halides are formed with an increase in volume compared to its additive value.20 common to all systems is the effect of increasing deviations of the molar volume from additive values as the size of the alkali metal cation increases. a cc ep te d m an us cr ip t nikolaeva et al.. thus, according to5,20,21 for binary mixtures of rare earth metal chlorides with licl maximum the relative deviations of the additivity of the molar volume are less than 1%. while when re chlorides are mixed with cesium chlorides, this value can reach 4–5%. therefore, the molar volume of the melt xlicl–ygdcl3 can be represented by the following expression: vm = x(1d1) + y(2d2) (3) where m1 and d1 are the molecular weight and density of the melt licl; m2 and d2 are the molecular weight and density of the melt gdcl3 (extrapolated to the studied temperature range), x and y are the molar fractions of the corresponding melt components. the values of molar electrical conductivity of licl–gdcl3 melts calculated in this way are shown in figure 5 (curves 2-5) in coordinates ln() –1/t. in these coordinates, the values of molar electrical conductivity can be approximated by the linear equation ln() = a – ea/(rt) (4) where  is the molar electrical conductivity (s cm2 mol–1); a constant; t is the absolute temperature (k); r universal gas constant (j k–1 mol–1); ea is the activation energy of electrical conductivity. fig 5. temperature dependence of the molar conductivity of molten licl;5 gdcl3;10,21 licl containing 6; 11; 16 and 23 mol% of gdcl3. a cc ep te d m an us cr ip t electrical conductivity of molten systems 9 figure 5 also shows the molar electrical conductivity values for licl (curve 1) and gdcl3 (curve 6) melts calculated on the basis of the available data on specific electrical conductivity 5,10 and density.5,21 the values of the molar electrical conductivity at 923 k and 1023 k are given in table 2. the molar electrical conductivity of licl is several times higher than of gdcl3 whereas the activation energy of the gdcl3 electrical conductivity is 3 times higher than of licl. the addition of gadolinium chloride to molten licl reduces electrical conductivity of the systems. a slight increase in the activation energy can be noted with an increase in the concentration of gdcl3 up to 23 mol%. table ii. coefficients of (3) for licl–gdcl3 molten system cgdcl3 / mol% temperature range, k a ea / kj mol –1  / s cm2 mol–1   05 917–1056 6.2367 8.48 169.33 188.64 6 873–1070 6.2395 9.14 155.82 175.06 11 840–1045 6.1921 9.36 144.32 162.61 16 830–1063 6.1777 9.71 135.99 153.89 23 795–1158 6.1437 10.20 123.25 140.36 10010,21 893–1028 8.1969 37.55 27.35 43.62 all isotherms of molar electrical conductivity, both obtained by us for the gdcl3–licl system, and available data for the lacl3–licl,5 prcl3–licl,6 ndcl3– licl,6 smcl3–licl6 systems look in the similar way. as the recl3 concentration increases, the molar electrical conductivity of the licl–recl3 mixtures gradually decreases, deviating from additivity towards lower values. the maximum deviations from additivity are achieved when the recl3 content in the melt is about 25 mol% and does not exceed 10–12%. in this work, for the gdcl3–licl system containing 23 mol% gadolinium chloride, the deviation of the molar electrical conductivity from the additive values is 10%. in the gdcl3–nacl and gdcl3–kcl systems, the molar electrical conductivity isotherms show deeper minima. at gdcl3 concentrations of 30–40 mol%, the deviation of molar electrical conductivity from additive values reaches 30–50%.7 the ionic potential of alkali metal cations is substantially lower than that re3+ cation. therefore, re3+ ions act as complexing agents, coordinating chlorine anions around themselves and displacing alkali metal cations into the second coordination sphere. when a small amount (on the order of a few mole percent) of recl3 is added to the mcl melt, strong 6-coordinate gdcl63– complexes are formed in the second coordination sphere of which there are alkali metal cations. this fact has been proven by many independent research methods. 22-25 with an increase in the recl3 concentration, an increasing number of clanions are required for the formation of six-coordinated complexes. the theoretical limit is 25 mol. % recl3 when all anions are coordinated around re3+ cations. at a cc ep te d m an us cr ip t nikolaeva et al.. higher recl3 concentrations, the melt structure becomes more complicated due to the inclusion of recl63– octahedra in more complex ionic groups. analysis of the available data5-10 shows that the electrical conductivity of molten salt mixtures gradually decreases with the addition of re chloride to alkali metal chloride, while the transfer numbers of chlorine anions decrease, since a large number of cl– ions bind to complexes and do not participate in the transfer of electricity. alkali metal cations are displaced into the second coordination sphere, the mobility of na+ and k+ cations decreases slightly with increasing recl3 concentration26 whereas in licl– recl3 mixtures, in the concentration range from 0 to 80 mol % recl3, the mobility of li+ cations decreases by half.27 the ionic potential of gd3+ is only 2.4 times greater than the ionic potential of li+,28 so the counter-polarizing effect of three li+ ions can lead to strong distortions and dissociation of gdcl63– complexes. this can explain the fact that the deviations of molar electrical conductivity of molten licl–recl3 mixtures from additive values are small. raman spectra of licl–gdcl3 melts in situ raman spectroscopy has been used to obtain information about the structural features and characteristic vibrational frequencies of complex groupings in chloride melts containing lithium and gadolinium ions. figure 6 shows the raman spectra of licl–gdcl3 melts containing 0; 15; 25 mol. % gdcl3. no vibrational bands are recorded in the licl melt (fig. 6). this fact directly indicates the predominantly coulomb type of interparticle interaction in this system and the absence of stable complex structural groupings in it.29,30 a band at 252 cm–1 was recorded in gadolinium-containing melts, which is attributed to the valence symmetric oscillation of the complex grouping gdcl63–. an increase in the normalized intensity of this vibrational band was noted with an increase in the content of gadolinium chloride in the melt (fig. 6), which is associated with an increase in the concentration of such groupings. a cc ep te d m an us cr ip t electrical conductivity of molten systems 11 fig 6. the raman spectrum of the molten licl (1) and of the molten licl after the addition of gdcl3 (mol%): 15 (2); 25 (3) at 898 k. the obtained results made it possible to explain the results of the study of the conductivity of these systems. indeed, when gadolinium ions are introduced into the melt, part of the chlorine anions binds to the gdcl63– grouping, which leads to a decrease in their mobility, and, as a consequence, difficulty in electrical transfer and an increase in the activation energy of this process. electrical conductivity and liquidus temperature of the gdcl3–licl–gd2o3 molten mixture the temperature dependence of the electrical conductivity when introducing 1 mol. % gadolinium oxide into the 0.23gdcl3–0.77licl system is shown in fig. 3. two sections with different slopes can be distinguished on the curve. the temperature at which the slope changes corresponds to the liquidus temperature of the system. thus, the introduction of 1 mol% of gadolinium oxide leads to a significant increase in the liquidus temperature from 751k to 1090k, which is obviously due to the low solubility of gadolinium oxide in the melt under study. the section of the temperature dependence of the electrical conductivity above the liquidus temperature was approximated by a linear equation of the form  = a+bt, the coefficients of which are given in table 1. the decrease in the specific electrical conductivity of the system 0.77gdcl3–0.23licl after the addition of 1 mol% gd2o3 in the range of 1093–1143 k is 3–4 %. a cc ep te d m an us cr ip t nikolaeva et al.. gadolinium oxide is known10 to react with gadolinium chloride, according to the reaction: gdcl3 + gd2o3 = 3gdocl (5) the observed patterns of changes in the specific electrical conductivity of the gdcl3–licl melt when gadolinium oxide is added can be explained by the formation of complex oxychloride groups when gadolinium oxychloride is dissolved in the liquid phase. according to the results of studies of the local structure and ion dynamics in molten systems 0.5licl–0.5gdcl3 with a gd2o3 concentration up to 2 mol% obtained by ab initio molecular dynamics and in situ raman spectroscopy31 gadolinium oxide in the chloride melt dissociated to form [gd2oli] groups, which were incorporated into the network-like structure of the original chloride melt. conclusions the specific electrical conductivity of the molten gdcl3–licl system was investigated depending on the temperature and concentration of gdcl3 up to 23 mol%. a significant decrease of electrical conductivity with the concentration of gadolinium chloride was shown. so at 923 k, the additive of 20 mol% gdcl3 reduces the electrical conductivity of the system by 40%. an increase in the system temperature slightly slows down the decrease in the electrical conductivity of the system with an increase in the concentration of gdcl3. the molar electrical conductivity of the molten gdcl3–licl system is calculated taking into account the assumption of additivity of the molar volume of the mixture. the temperature dependences of the molar electrical conductivity were approximated by arrhenius type equations. a slight increase in the activation energy of molar electrical conductivity can be noted with an increase in the concentration of gdcl3. spectral studies have shown that when gadolinium ions are introduced into the licl melt, part of the chlorine anions binds to the gdcl63– grouping. this leads to a decrease in their mobility, and, as a consequence, the difficulty of electrical transfer and an increase in the activation energy of this process. introduction of 1 mol% of gadolinium oxide to gdcl3–licl melt leads to a significant increase in the liquidus temperature of the system and a decrease in electrical conductivity. this can be explained by the formation of complex oxychloride groups during the dissolution of gadolinium oxide in the liquid phase. acknowledgements: we are grateful to v. n. dokutovich for samples preparation. the emission spectral analysis and the xrd-analysis were performed using the facilities of the shared access centre "composition of compounds” (institute of high temperature electrochemistry, ural branch of ras). a cc ep te d m an us cr ip t electrical conductivity of molten systems 13 и з в о д електрична проводљивост растопа gdcl3–licl и gdcl3–licl-gd2o3 elena v. nikolaeva*, irina d. zakiryanova, andrey l. bovet и iraida v. korzun institute of high temperature electrochemistry, ub ras, 620990 yekaterinburg, russia мерена је електрична проводљивост растопа licl–gdcl3 са додатком гадолинијумхлорида у распону од 0 до 23 mol% при различитим температурама и концентрацијама gdcl3. моларна електрична проводљивост растопа gdcl3–licl је рачуната уз претпоставку адитивности моларне запремине смеше. добијене температурне зависности се могу апроксимирати аренијусовским типом једначине. ефективна енергија активације, ea, је расла са повећањем садржаја gdcl3. температуре ликвидуса испитиваних растопа су одређиване методом диференцијалне скенирајуће калориметрије. снимљени су високотемпературни раманови спектри растопа licl–gdcl3. поред тога, измерена је електрична проводљивост растопа 0,77licl–0,23gdcl3 са 1 mol% gd2o3. испитивања су показала да додатак гадолинијум-оксида смањује електричну проводљивост хлоридних растопа и повећава њихову температуру ликвидуса. 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abstract: the present work reports the synthesis of three new nitrogen-containing heteronuclear molecules (two pyrimidines and one pyrrolopyrimidine). based on the 12 principles of green chemistry, chemical methodologies are planned and executed. various phase transfer catalysts were examined in the crucial step to execute c–n bond formation (through the snar mechanism), tbai was found to be a better option than those of tbab and ctab. the one-pot synthetic methodology was optimized involving wittig olefination and bromination to achieve a key precursor for the synthesis of a new pyrrolopyrimidine. keywords: twelve principles of green chemistry; phase transfer catalysis; one -pot synthesis; wittig olefination; synthesis of new n-heterocyclic compounds; halogen solvent-free processes; catalytic reactions. introduction in the dna and rna structures, pyrimidine is a common structural motif present in the form of different nucleobases, such as cytosine, thymine and uracil. scaffolds containing tetra substituted pyrimidines occupy a prominent position based on their biological applications.1–10 recently, 2,4,6-trichloro-pyrimidine -5-carbaldehydes having cholesterol moiety are used for gelation.11 interest in the studies based on the derivatives of pyrrolo[2,3-d]pyrimidines has increased significantly after the introduction of nucleoside antibiotics, such as tubercidin, toyocamycin and sangivamycin.12 in addition these tetra substituted pyrimidines are acting as precursors in the synthesize of new biologically active pyrrolo[2,3 * corresponding author. e-mail: ramakrishnads_chem@vssut.ac.in https://doi.org/10.2298/jsc210804063u mailto:ramakrishnads_chem@vssut.ac.in https://doi.org/10.2298/jsc210804063u 2 udayasri et al. -d]pyrimidines. a few representative pyrimidines and pyrrolo[2,3-d]pyrimidine structural motifs containing examples are shown in fig. 1. fig. 1. representative examples of pyrimidine and pyrrolopyrimidine derivatives. experimental general procedures all reactions were performed under a nitrogen atmosphere using anhydrous techniques unless otherwise stated. all commercially available materials and solvents were used directly without further purification. 1h-nmr (400 mhz) and 13c-nmr (100 mhz) were recorded on a bruker avance nmr spectrometer using tms as the internal standard in cdcl3 or dmso -d6 solution. the mass spectra were determined on a thermo ltq (esi) or agilent 5973 msd (ei) spectrometer. the ir spectra were recorded on perkin–elmer model 683 or 1310 spectrometers with sodium chloride optics or kbr pellets. all reactions were monitored by thin-layer chromatography (tlc) employing 0.25 mm merck silica gel plates (60f-254). column chromatography was performed using acme silica gel (60–120 mesh). visualization of the spots on the tlc plates was achieved either by exposure to uv light, and iodine vapor or by dipping the plates in phosphomolybdic acid–ceric (iv) sulfate–sulfuric acid solution (pma solution) and heating the plates at 120 c. all the products were purified by column chromatography. the structures were characterized by analysis of spectra data, namely 1h-nmr, 13c-nmr, ir and mass spectra. analytical and spectral data are given in supplementary material to this paper. 2,4-dichloro-6-(cyclopentylamino)pyrimidine-5-carbaldehyde (7). to a stirred solution of 2,4,6-trichloro-pyrimidine-5-carbaldehyde 5 (5.0 g, 23.6 mmol) in a mixture of toluene (10 ml) and water (40 ml) was added cyclopentyl amine 6 (2.01 g, 23.6 mmol), csco3 (4.0 g, 40 mmol) and a catalytic amount of tbab (0.35 g, 1.08 mmol) at 25 to 35 c. the reaction mass was maintained for 2–3 h at 25 to 35 °c. the organic layer was separated from the aqueous layer, washed with brine solution and dried over anhydrous na2so4. the organic layer was concentrated under vacuum to give a thick oily residue that was purified by column chromatography to give 7 (5.53 g, 90 %). green synthesis of pyrimidines and pyrrolopyrimidine 3 methyl (z)-2-bromo-3-(2,4-dichloro-6-(cyclopentylamino)pyrimidin-5-yl)acrylate (9). to a stirred solution of (methoxycarbonyl methyl)triphenylphosphoniumbromide, 8 (15.8 g, 38.0 mmol) in a mixture of dioxane (9 ml) and water (45 ml) was added n-bromosuccinamide (7.5 g, 42.1 mmol) at 0–5 °c. the mixture was stirred at 0–5 °c for 45 to 60 min. the temperature of the reaction mass was raised to 25 °c and then compound 7 (9.0 g, 34.5 mmol) was added followed by csco3 (14.83 g, 46.01 mmol). after keeping the reaction mass at 25 °c for 2 to 3 h. it was dried over anhydrous na2so4 and then concentrated under reduced pressure to obtain a yellow crude oil. the crude residue was purified by column chromatography to give 9 (12.29 g, 85 %) methyl 2,4-dichloro-7-cyclopentyl-7h-pyrrolo[2,3-d]pyrimidine-6-carboxylate (10). to a stirred solution of compound 9 (4.0 g, 10.1 mmol), k2co3 (7.0 g, 50.6 mmol) and dabco (0.56 g, 5.06 mmol) in 1,4 dioxane (20 ml) was added cui (0.385 g, 2.02 mmol) under a nitrogen atmosphere at 25 c. the temperature of the reaction mass was raised to reflux and maintained for 16.0 h under a nitrogen atmosphere. then reaction mass was filtered and the filtrate washed with dioxane (4 ml). the filtrate was dried over anhydrous na2so4 and then concentrated to obtain a thick yellow syrup. using columns chromatographic technique. the product 10 was purified by column chromatography (2.92 g, 90 %) results and discussion synthesis of pyrimidine and pyrrolopyrimidine the synthesis of new and diversified pyrrolopyrimidines using pyrimidine precursors was unexplored to date. thus, it was intend to synthesize a series of n-heterocyclic compounds having a pyrimidine moiety as a common structural entity and to prepare new pyrrolo[2,3-d]pyrimidines based on the execution of green chemical approaches. in continuation of the execution of green chemical methodologies based on twelve principles of chemistry, in the current work, these advanced methodologies were extended to the synthesize n-heterocyclic compounds.13–18 the importance of one pot synthetic methodology is increasing in a fast manner.19–21 extending new synthetic methodologies to the synthesis of a series of pyrimidine analogues and their characterization were the main objectives of this work. for this purpose, the synthesis a common intermediate having functionalizable groups on each carbon of pyrimidine was envisaged. the envisaged synthetic strategy involved the usage of reliable and high yield producing reactions such as c–n bond formation (through the snar mechanism), the wittig reaction and cyclization. to generate new and different analogs having pyrimidine moiety, compound 2,4,6-trichloropyrimidine-5-carbaldehyde was chosen which is having four functionalizable sites, i.e., three chloro substituents and one aldehyde functionality. compound 5 can be synthesized using barbituric acid as the starting material since it is a cheap commercially available material. the synthesis was initiated with barbituric acid that was converted into 5 using pocl3 in dmf (vilsmeier–haack reaction conditions), the required pyrimidine derivative having four functionalizable groups was synthesized in 85 % yield (scheme 1).22 4 udayasri et al. scheme 1. c–n bond formation reaction. after obtaining the required intermediate, functionalization of the 2nd/6th position was explored using cyclopentyl amine to generate a new c–n bond. the main difficulty involved in this reaction is that there may be a chance of the formation of imine product using aldehyde and amine through condensation reaction (a parallel reaction) instead of a c–n bond formation reaction. a similar reaction with this substrate 5 using toluene solvent was reported.23 though toluene is a preferable solvent in synthetic organic chemistry in comparison with other organic solvents, such as chlorinated solvents (dcm, ccl4) or benzene, it still has some negative impacts on nature. the usage of organic solvents should be reduced to a bare minimum by employing/increasing ecofriendly solvents, such as water, to carry out the reaction. this reaction was optimized with the higher solvent contribution of water (80 %) as the major solvent in comparison with that of toluene (20 %) solvent as the minor solvent. the other advantage of using higher amounts of water is to diminish the formation of imine products that could be obtained from aldehyde and amine. water was not only a solvent but also a byproduct in this parallel imine condensation reaction. hence, the lechleiter principle was applied, i.e., by increasing the concentration of water, the formation of imine product can be reduced. the role of water in this reaction is not only to decrease the usage of the toluene solvent but also to lessen the production of imine product. the reaction was optimized based on the following parameters shown in table i. different phase transfer catalysts were examined, such as tbab, tbai and ctab. different bases, such as k2co3, khco3, na2co3, nahco3 and csco3, were employed to obtain higher yields of the required product in various solvent combinations. based on careful experimentation of the reaction under various conditions (table i), a higher yield of the product was obtained using csco3 as the base, tbab as ptc, and solvents ratio toluene:water of 1:4. the required product was obtained in good yield using tbab in comparison with other phase transfer catalysts (scheme 1). compound 7 was characterized based on its 1h-nmr spectrum, the appearance of cyclopentyl amine structure representing protons signals ranging from δ 1.5 to 4.5 ppm, the nh proton was observed at δ 9.30 ppm and this peak was absent in its d2o exchanged 1h-nmr spectrum. in addition compound 7 was confirmed by the appearance of its mass peak at m/z 260.0 (m+1) over mass green synthesis of pyrimidines and pyrrolopyrimidine 5 chromatogram with dichloro mass pattern in positive scan mode. the spectral data of 7 was in full agreement with the assigned structure. table i. optimization of the snar reaction; equivalence of base and ptc were calculated concerning the equivalence of substrates 2 and 3 s. no. ptc (4.5 mol. %, equivalence) base (1.7 equivalence) isolated yield, % (equivalence) 1 tbai k2co3 80 2 tbai khco3 65 3 tbai na2co3 75 4 tbai csco3 70 5 tbab csco3 90 6 tbab khco3 80 7 tbab na2co3 70 8 tbab nahco3 50 9 ctab k2co3 60 10 ctab khco3 50 11 ctab na2co3 60 12 ctab csco3 50 scheme 2. one pot wittig and bromination reaction. although one-pot bromination and wittig reactions were optimized previously with simple substrates such as aliphatic, and aromatic compounds most of the reactions used organic solvents such as dcm or dmso only.24 the main drawback in those reaction conditions was the non-aqueous media. to implement green chemical methodology by using an aqueous solvent along with increasing the yield of the product, the reaction conditions in the aqueous media containing a biphasic system were optimized. in this way, the usage of organic solvents was reduced by employing an eco-friendly aqueous solvent. wittig olefination of compound 7 using 8 and na2co3 at 25 c resulted in olefin 9 in 65 % yield. for improving the yield of the conversion of 7 into 8, each parameter of the reaction was systematically examined (as shown in table ii). from table ii, it is very clear that the availability of k2co3 to the reactants was greater in a bi-phasic mixture of 1,4-dioxane + water, which made the reaction faster with more complete transformation of 7 into 8 and higher yields (scheme 2). after completion of the reaction, mtbe was used in the purification step, for extraction purposes, 6 udayasri et al. tpp oxide by-products can be removed selectively from the reaction mixture and in this way the usage of a hazardous organic solvent was reduced to some extent. table ii. optimization of the wittig reaction s. no. solvent base reaction temperature, c reaction time, h isolated yield, % 1 ch2cl2 k2co3 –20 > 48 ~35 2 benzene k2co3 –20 > 48 ~ 40 3 methanol na2co3 –20 > 48 ~ 45 4 toluene + methanol na2co3 –20 > 48 ~ 40 5 1,4-dioxane na2co3 –20 7 50 6 1,4-dioxane na2co3 0 5 55 7 1,4-dioxane + water na2co3 –20 3 80 8 1,4-dioxane + water na2co3 0 2 85 9 1,4-dioxane + water csco3 25 1 85 compound 9 was analyzed using 1h-nmr by the appearance of the cyclopentyl amine structure representing protons signals ranging from δ 1.5 to 4.5 ppm, the nh proton was observed at 7.45 ppm and this peak was absent in the d2o exchanged 1h-nmr spectrum. in addition, compound 9 was confirmed by the appearance of its mass peaks at m/z 394 and 396 over mass chromatogram with bromo and dichloro mass pattern in positive scan mode. the spectral data of 9 was in full agreement with the assigned structure. compound 9 was converted to 10 using copper iodide, na2co3 and dabco in 1,4-dioxane with a 92 % yield (scheme 3).25 as shown in table iii, the best conversion of the reaction from 9 to 10 was achieved with na2co3 in 1,4-dioxane at 110 c. it was observed that inorganic bases were more effective in obtaining better cyclization conditions than the organic base diisopropylethylamine (dipea).26 after finalizing the conversion, compound 10 was purified using column chromatography. compound 10 was characterized based on 1h -nmr by the appearance of proton signal of the cyclized five-membered ring alkene at δ 7.4 ppm, in addition to the appearance of its mass peaks at m/z 314 over mass chromatogram with a dichloro mass pattern in positive scan mode. the spectral data of 10 was in full agreement with the assigned structure. scheme 3. cyclization reaction. green synthesis of pyrimidines and pyrrolopyrimidine 7 table iii. optimization of the cyclization reaction s. no. solvent base temperature, c reaction time, h isolated yield, % 1 dichloromethane k2co3 35 no reaction 2 tetrahydrofuran k2co3 65 > 36 30 3 toluene k2co3 110 > 36 60 4 toluene na2co3 110 > 36 60 5 toluene kotbu 110 > 36 25 6 1,4-dioxane et3n 110 > 36 25 7 1,4-dioxane dipea 110 > 36 20 8 1,4-dioxane na2co3 110 12 92 conclusions the outstanding features of this synthetic methodology are: i) two aqueous phase transformations such as c–n bond formation and wittig reaction, ii) one pot synthetic methodology that includes both wittig reaction and bromination, iii) one phase transfer catalytic reaction using tbab in c–n bond formation reaction, iv) all reactions are carried out by catalysts such as tbab and cui as two-room temperature reactions and v) all reactions and procedures are halogen solvent free processes. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/11028, or from the corresponding author on request. и з в о д региоселективна функционализација 2,4,6-трихлорпиримидин-5-карбалдехида нa апринципима зелене хемије: примена у синтези нових пиримидина и пиролопимидина allu udayasri1, manchikanti m. chandrasekhar1, brahmeswararao m. v. naga2, ganesh varanasi2 и duggirala s. ramakrishna1 1department of chemistry, veer surendra sai university of technology, burla-768018, odisha, india и 2prodev pharma (opc) pvt. ltd, aleap industrial estate, hyderabad-500090, india у овом раду приказана је синтеза три нова хетероциклична једињења азота (два деривата пиримидина и један дериват пиролопиримидина). план хемијске методологијe синтезе је заснован на 12 принципа зелене хемије и план је реализован. испитани су различити катализатори преноса између фаза у најважнијем реакционом кораку, формирања c–n везе (snar механизмом) и утврђено је да је најбољи резултат постигнут коришћењем tbai у поређењу са tbab и ctab. оптимизована је нова ситетичка методологија која укључује витихову (wittig) олефинацију и бромовање за добијање најважнијег прекурсора у синтези нових пиролпиримидина. 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(2022) (https://doi.org/10.1007/s11244-02201602-9). https://doi.org/10.1080/00397911.2011.614712 https://doi.org/10.1021/ol5015398 https://doi.org/10.1007/s11244-022-01602-9 https://doi.org/10.1007/s11244-022-01602-9 404 not found accepted manuscript this is an early electronic version of an as-received manuscript that has been accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as p. j. p. tjitda, f. o. nitbani, d. mbunga and t. d. wahyuningsih, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc220913045t. this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc220913045t j. serb. chem. soc.00(0)1-20 (2023) original scientific paper jscs–12064 published dd mm, 2023 1 natural flavonoids in delonix regia leaf as an antimycobacterial agent: an in silico study putra jiwamurwa pama tjitda1*, febri odel nitbani2, dominus mbunga1 and tutik dwi wahyuningsih3 1department of pharmacy, health polytechnic of kupang, indonesia, 2department of chemistry, faculty of sains and engineering, nusa cendana university, indonesia, and 3department of chemistry, faculty of mathematics and natural sciences, universitas gadjah mada, indonesia (received 13 september 2022; revised 23 december 2022; accepted 21 july 2023) abstract: multi-drug resistant (mdr) and extensively-drug resistant (xdr) as a result of continuous use of antibiotics encourage the development of new antimycobacterial drugs. in this study, 13 flavonoid compounds from the flamboyant leaf plant were studied for their inhibitory properties of mtkasa, mtdpre, and mtpank which are significant enzymes in mycobacterium tuberculosis, as well as for their molecular docking, molecular dynamics, and prediction of admet-drug likeness. the results of molecular docking studies revealed that compound f13 (apigenin) was the most potent compound because it was able to bind the most amino acids as indicated by the native ligand of each enzyme. molecular dynamics studies showed that compound f13 forms a stable complex with mtkasa. the results of the admet-drug likeness analysis concluded that compound f13 was the most promising compound. overall, compound f13 has the potential to be used as a treatment therapy against mycobacterium tuberculosis. keywords: tuberculosis, molecular docking; mtkasa; mtdpre; mtpank. introduction tuberculosis (tb) is a deadly disease caused by mycobacterium tuberculosis. many parties have and are paying special attention to the handling of tuberculosis. who, in the global tuberculosis report 2021, reported that the pandemic of covid-19 has a negative impact on efforts to treat tuberculosis. limited access to diagnosis and treatment of tuberculosis has increased the number of tuberculosis deaths by 1.2 million since 2019. 1 who also noted four countries contributed the most prominent tb cases, namely india (41%), indonesia (14%), the philippines (12%), and china (8%). another report noted that cases of drug-resistant tb against first-line drug usage for tb therapy have increased by 15%. 1 this is *corresponding authors e-mail: putrachemist_jc@yahoo.com https://doi.org/10.2298/jsc220913045t a cc ep te d m an us cr ip t mailto:putrachemist_jc@yahoo.com https://doi.org/10.2298/jsc220913045t 2 tjitda et al. primarily due to the prolonged treatment of tb, as well as the use of antibiotic combinations with side effects that result in the development of multi-drug resistant (mdr) and extensively drug-resistant (xdr). 2 as a result of all these reports, tuberculosis treatment needs urgent attention as well as appropriate and quick solutions. there are still many drug discovery studies underway to overcome the problem of drug resistance, including tuberculosis. 3 molecular docking is a computational method for new drug discovery based on structure-based design. application of molecular docking methods includes virtual screening, prediction of adverse drug reactions, drug repositioning, and drug mechanism of action against the targeted protein. 4 therefore, molecular docking is an excellent and powerful method for predicting drug activity against specific proteins. several targeted proteins to predict drug activity against the growth of mycobacterium tuberculosis are mtkasa, mtdpre1, and mtpank. 5 mtkasa and mtdpre1 are necessary enzymes used for cell wall biosynthesis. mtkasa is responsible for elongating the fatty acid carbon chain in the fas-ii pathway and provides a precursor for mycolic acid biosynthesis. 6 mtdpre1 performs the epimerization reaction of decaprenyl phosphoryl--d-ribose (dpr) to decaprenyl phosphoryl-d-arabinofuranose (dpa) in the periplasmic space of the cell membrane. dpa is used for the biogenesis of arabino galactan and lipoarabino mannan. 7,8 mtpank is also an essential drug target to overcome drug-resistant tb. mtpank enzyme plays a role in the growth of mycobacterium tuberculosis through the biosynthesis of coenzyme-a (coa). 9 various studies have been conducted to study the plant’s active compounds and their biological activity against mycobacterium tuberculosis. 10–13 the content of secondary metabolites in plants significantly contributes to certain biological activities and is always accompanied by low side effects. 14 among the secondary metabolites, flavonoids have been reported to have inhibitory activity against the growth of mycobacterium tuberculosis. 15–17 chemically, flavonoids have two benzene rings, a and b, which are connected by a pyran heterocyclic ring (c ring). flavonoid groups include flavonols, flavanones, flavones, isoflavones, flavanols, and anthocyanins. 18 flamboyant (delonix regia) is a plant that comes from the caesalpiniaceae family and is widely found in various regions of indonesia. previous studies reported that the isolation of flamboyant leaves with ethanol as a solvent produced 13 flavonoid compounds. 19 the presence of flavonoid led to the investigation of their ability to inhibit 3 key enzymes in the growth of mycobacterium tuberculosis. therefore, this study aimed to identify the potential of flavonoid compounds contained in flamboyan leaves as antimycobacterial agents, using an in silico study approach. as a result, the potential compounds obtained could be used as reference compounds for laboratory testing. a cc ep te d m an us cr ip t antimycobacterial natural flavonoids in delonix regia leaf 3 experimental materials as test ligands, natural flavonoids were obtained from previous studies investigating the hplc profiles of delonix regia leaves extract.19 the proteins analyzed in this study include mtkasa, mtdpre1, and mtpank taken from the rscb database with pdb id were 2wge, 4ff6, and 4bft, respectively. the software used for molecular docking was autodock vina; the structures of ligand compounds were drawn with chemoffice professional 16 and chem3d professional 16, and structure optimization was performed using gaussian 09. molecular docking results were visualized in discovery studio client 2021. validation of docking protocol each receptor protein was prepared by separating the native ligand from the protein using autodock tool (adt) 1.5.6. missing atoms of the receptor protein were checked and corrected through adt. furthermore, the water molecule on the receptor protein was cleaned and the protein was added with kollman charge and then stored in the pdbqt extension format. the separated native ligands were prepared by adding hydrogen atoms and gasteiger charge. prepared proteins and native ligands were then docked by following several parameters. the molecular docking for mtkasa protein used a grid box dimension of 25 x 25 x 25 å with cartesian coordinates x, y, and z respectively 37.883, 0.813, and -5.898. mtdpre1 protein used a grid box size of 12 x 12 x 12 å with cartesian coordinates x, y, and z respectively 14.697, 21.165, and 36.955 and for mtpank protein, it was 14 x 14 x 14 å with cartesian coordinates x, y, and z respectively -19.242, -10.217, and 12.648. an exhaustiveness of 16 was applied to the protocol docking validation process. in order to dock all proteins, the spacing value of 1 armstrong was used. rmsd value was determined using pymol. the docking protocol was used to dock the tested compound if it had an rmsd value below 2 å.20 preparation of natural flavonoid as tested ligand the structure of natural flavonoids from delonix regia leaves extract was taken from the pubchem database. compounds that were not found in the database were drawn with chemdraw professional 16, as shown in fig. 1. furthermore, all compounds were converted to 3-dimensional structure and geometric optimization using gaussian 09 with the dft method with a base set b3lyp 6-31g.21 furthermore, the test compound was prepared using adt, which involved the addition of hydrogen atoms, merging non-polar hydrogen atoms, and adding gasteiger charge. all compounds that had been prepared were then stored in a pdbqt format file. antimycobacterial profile prediction the antimycobacterial properties of 13 flavonoids contained in flamboyant leaf extract were predicted. each smile code of the flavonoid compound was inputted into pass online (http://www.way2drug.com/passonline/predict.php). activity predicted values (pa) and inactive predictions were recorded, tabulated, and presented in graphical form. molecular docking natural flavonoids that had been optimized were docked to the three receptor proteins. docking was conducted using autodock vina following a validated docking protocol. the docking results generated binding energy as a value that indicated the binding energy between the ligands and the receptor protein. the 2d and 3d interactions of the tested compound with a cc ep te d m an us cr ip t http://www.way2drug.com/passonline/predict.php 4 tjitda et al. key amino acid residues of the receptor protein were visualized using discovery studio client 2021. admet-drug-likeness analysis pharmacokinetic properties of natural flavonoid compounds, including adsorption, distribution, metabolism, excretion, and toxicity, were predicted using the pkcsm tool.22 the drug-likeness properties were studied using the molsoft tool (https://www.molsoft.com/publications.html). molecular dynamic simulation the lead compounds obtained from molecular docking simulations were then studied for the stability of their interactions with amino acid receptors, in this case mtkasa, mtdpre1, and mtpank. this was done through molecular dynamic simulations. simulations used cab flex 2.0 server (http://biocomp.chem.uw.edu.pl/cabsflex2/index). the simulation setting used a number of cycles and 50 trajectory frames. furthermore, the temperature of the simulation and the global weight were respectively 1.4 and 1.0. results and discussion validation of docking protocol the molecular docking method is a powerful in silico method for predicting the interaction between ligands and receptors. 23 to study the antimycobacterial properties of natural flavonoids in the ethanolic extract of delonix regia leaves, three significant proteins were used to inhibit mycobacterium tuberculosis activities. these proteins were mtkasa, mtdpre1, and mtpank. before molecular docking of the test compounds was carried out on the three proteins, docking protocol validation was performed. as shown in fig. 1, the native ligands are superimposed and include mtkasa complexes with thiolactomycin (tlm), mtdpre1 complexes with 3-(hydroxyamino)-n-[(1r)-1-phenylethyl]-5(trifluoromethyl)benzamide (0t4), and mtpank with 2-chloro-n-[1-(5-[[2-(4fluorophenoxy)sulfanyl]-4-methyl-4h-1,2,4-triazo-3-yl]benzamide (zvt). validation results in the form of rmsd values for mtkasa, mtdpre1, and mtpank were 0.828 å, 1.512 å, and 0.689 å, respectively. antimycobacterial profile prediction prediction of the antimycobacterial profile of flavonoid compounds was carried out using the pass server and gave results as shown in fig. 2. the results of the analysis from the pass server showed that all compounds had a pa value >0.3, however the value was still <0.7 except for compound f5 (0.276). this means that the compound was predicted to have moderate anti-mycobacterial potential in the laboratory. 24 furthermore, this compound could be further confirmed through molecular docking. a cc ep te d m an us cr ip t https://www.molsoft.com/publications.html http://biocomp.chem.uw.edu.pl/cabsflex2/index antimycobacterial natural flavonoids in delonix regia leaf 5 a) b) c) fig 1. superimpose re-docking ligand (green) into native ligand (blue) of protein receptor: a) complex of mtkasa (id pdb: 2wge) tlm; b) complex of mtdpre1 (id pdb: 4ff6) 0t4; c) complex of mtpank (id pdb: 4bft) zvt f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 f11 f12 f13 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 compound id pa value pi value fig 2. graph of antimycobacterial profile prediction by flavonoid compounds from flamboyant leaves molecular docking the chemical structure of flavonoid compounds contained in flamboyant leaf extract that were used as tested ligands, is shown in table i. in this study, molecular docking of flavonoids in flamboyant plants was conducted to determine potential compounds for antituberculosis drugs. the binding energy and chemical a cc ep te d m an us cr ip t 6 tjitda et al. interactions of each flavonoid with the targeted protein were studied and compared to natural inhibitors of each enzyme. table i. natural flavonoid compounds from flamboyant leaves (delonix regia). comp. id chemical structure pubchem id iupac name (pubchem generated) f1 137832345 6-[3,4-dihydroxy-5(hydroxymethyl)oxolan2-yl]-5,7-dihydroxy-2(4-hydroxyphenyl)-8[3,4,5-trihydroxy-6(hydroxymethyl)oxan-2yl]chromen-4-one f2 74977441 5,7-dihydroxy-2-(4hydroxyphenyl)-8[3,4,5-trihydroxy-6(hydroxymethyl)oxan-2yl]-6-(3,4,5-trihydroxy6-methyloxan-2yl)chromen-4-one f3 5291488 2-(3,4dihydroxyphenyl)-5hydroxy-7-[3,4,5trihydroxy-6(hydroxymethyl)oxan-2yl]oxychromen-4-one f4 3594 5-hydroxy-2-(3hydroxy-4methoxyphenyl)-7[3,4,5-trihydroxy-6[(3,4,5-trihydroxy-6methyloxan-2yl)oxymethyl]oxan-2yl]oxy-2,3dihydrochromen-4-one f5 5315615 3-(3,4dihydroxyphenyl)-2[(e)-3-(3,4dihydroxyphenyl)prop2-enoyl]oxypropanoic acid f6 5385553 5-hydroxy-2-(4hydroxyphenyl)-7[3,4,5-trihydroxy-6(hydroxymethyl)oxan-2yl]oxychromen-4-one a cc ep te d m an us cr ip t antimycobacterial natural flavonoids in delonix regia leaf 7 f7 5459217 7-[4,5-dihydroxy-6(hydroxymethyl)-3(3,4,5-trihydroxy-6methyloxan-2yl)oxyoxan-2-yl]oxy-5hydroxy-2-(4hydroxyphenyl)chromen -4-one f8 12305415 5-hydroxy-2-(4hydroxyphenyl)-3,7bis[(3,4,5-trihydroxy-6methyloxan-2yl)oxy]chromen-4-one f9 5353915 2-(3,4dihydroxyphenyl)-5,7dihydroxy-3-(3,4,5trihydroxy-6methyloxan-2yl)oxychromen-4-one f10 131752460 [2-[5,7-dihydroxy-2-(4hydroxyphenyl)-4oxochromen-3-yl]oxy4,5-dihydroxy-6(hydroxymethyl)oxan-3yl] (e)-3-(4hydroxyphenyl)prop-2enoate f11 15559329 7-[4,5-dihydroxy-6(hydroxymethyl)-3(3,4,5-trihydroxy-6methyloxan-2yl)oxyoxan-2-yl]oxy-5hydroxy-2-(4methoxyphenyl)chromen -4-one f12 5280863 3,5,7-trihydroxy-2-(4hydroxyphenyl)chromen -4-one a cc ep te d m an us cr ip t 8 tjitda et al. f13 5280443 5,7-dihydroxy-2-(4hydroxyphenyl)chromen -4-one molecular docking of the mtkasa enzyme was carried out on 13 flavonoids contained in flamboyant leaves and the results are shown in table ii. for mtkasa molecular docking, tlm which acted as a natural inhibitor, produced a binding energy value of -32.22 kj/mol, while some other test compounds also demonstrated satisfactory inhibitory activity compared to natural inhibitors. f6 compound had the highest binding energy (-38.91 kj/mol), followed by the f5 compound with a binding energy value of -36.40 kj/mol, while f3, f12, and f13 compounds showed the same binding energy value of -35.95 kj/mol. table ii. binding energy of natural flavonoid compound from delonix regia leaf. id compound name binding energy (kj/mol) mtkasa mtdpre1 mtpank tlm thiolactomycin -32.22 0t4 3-(hydroxyamino)-n-[(1r)-1-phenylethyl]5-(trifluoromethyl)benzamide -37.66 zvt 2-chloro-n-[1-(5-{[2-(4fluorophenoxy)ethyl]sulfanyl}-4-methyl4h-1,2,4-triazol-3-yl)ethyl]benzamide -39.33 flavonoid compounds f1 apigenin-6-arabinose-8-galactose -28.03 -33.47 -36.82 f2 apigenin-6-rhamnose-8-glucosa -28.87 -33.47 -34.31 f3 luteolin-7-glucose -35.98 -35.56 -36.82 f4 hesperidin -35.15 -34.31 -35.15 f5 rosmarinate -36.40 -33.47 -30.96 f6 apigenin-7-o-glucose -38.91 -37.66 -39.33 f7 apigenin-7-o-neohespiroside -31.38 -35.98 -32.64 f8 lespedin -30.96 -35.98 -31.38 f9 quercetin-3-o-rhamnoside -24.69 -33.47 -34.73 f10 2''-o-trans-p-coumaroylastragalin -30.54 -39.75 -39.33 f11 fortunellin -31.80 -34.31 -37.66 f12 kaempferol -35.98 -33.89 -34.31 f13 apigenin -35.98 -34.31 -33.05 a cc ep te d m an us cr ip t antimycobacterial natural flavonoids in delonix regia leaf 9 a) b) c) d) e) f) fig 3. chemical interaction of mtkasa enzyme with a) tlm; b) compound f6; c) compound f5; d) compound f3; e) compound f12; f) compound f13. a cc ep te d m an us cr ip t 10 tjitda et al. an investigation of the chemical interaction of tlm in the mtkasa enzyme binding pocket has been carried out (table si). the oxygen atom of the thiolactone ring is linked to cys171 by hydrogen bonds. the binding of his311 was formed by the pi-sulfur bond on the s atom of the thiolactone ring. the bond stabilization in the enzyme mtkasa binding pocket was facilitated by hydrophobic interactions with amino acids phe237, phe404, and pro280. this fact was in accordance with the results of a study conducted and reported by luckner et al. that the catalytic triad cys171-his311-his345 was the active site of the mtkasa binding pocket (fig. 3). 25 f5 and f6 compounds showed hydrogen bonds to the his311 amino acid. hydrogen bonds also supported the stabilization of f6 compounds in interacting with pro280. f5 compound has slightly lower binding energy, whereas binding of the compound to pro280 involves hydrophobic interactions. the loss of hydrogen bonds in the catalytic triad was suspected as a reason for the lower binding energy of f3, f12, and f13 compounds. additionally, f3, f12, and f13 compounds involving hydrophobic interactions also bind to essential amino acids like cys171, pro280, and phe404. this evidence was an indication that f3, f12, and f13 compounds also interact at the mtkasa enzyme active site. inhibition of the tested compounds against the mtdpre1 enzyme was also tudied. molecular docking revealed that all analyzed compounds were potent inhibitors of the mtdpre1 enzyme with binding energies ranging from -33.89 to -39.75 kj/mol. the f10 compound was the tested compound with the largest binding energy, which was -39.75 kj/mol, while the f6 compound produced the same binding energy as the native ligand (0t4), which was -37.66 kj/mol. in this study, we also found that the core structure of flavonoid compounds in apigenin and kaempferol had excellent binding ability. f7 and f13 compounds with an apigenin core structure had binding energies of -35.98 and -34.31 kj/mol, respectively. f8 and f12 compounds, which had a kaempferol core structure, gave bond affinities of -35.98 and -33.89 kj/mol, respectively. the chemical interaction visualization of the tested compound against the mtdpre1 enzyme provided data to support data binding energy. 0t4 compound as a native ligand bound several key amino acids (table sii). cys387 amino acid is found in the substrate-binding domain of mtdpre1 enzyme. 26 in addition, several amino acids, such as lys134, gly117, and gly113, which are located in the fadinteracting domain, 7 were also seen in fig. 4. f10 compound was able to bind the amino acid cys387 through hydrogen bonding. lys134 and gly117 amino acids were also bonded via hydrogen bonding with the f10 compound. for the f7 compound, adherence to cys387 and lys134 amino acids involved hydrophobic interactions, and the f13 compound only attached lys134 via hydrogen bonding. in this case, the hydroxyl group of the rhamnoside substituent is used to bond the f8 compound to cys387 essential amino acid. the weak binding affinity of the a cc ep te d m an us cr ip t antimycobacterial natural flavonoids in delonix regia leaf 11 f12 compound was due to hydrophobic interactions attached to cys387 amino acid, according to binding energy data. molecular docking studies of the tested compounds were also carried out on pantothenate kinase (mtpank). binding energy data showed that all the tested compounds had excellent affinity potential for the mtpank enzyme, ranging from -30.96 to -39.33 kj/mol. this study used the native ligand zvt as a comparison ligand. the molecular docking results showed that flavonoid core structures in apigenin and kaempferol gave the most effective binding affinity, as could be seen for f6 and f10 compounds with a binding energy value of -39.33 kj/mol. it was also revealed that compounds derived from the apigenin core structure, such as those in f1 and f2 compounds resulted in slightly lower binding energy in molecular docking results. with the mtpank, the f11 compound also produced a fairly significant binding affinity of -37.66 kj/mol compared to mtkasa and mtdpre1 enzymes. in accordance with the binding energy value, the f11 compound did not have the ability to bond to mtkasa or mtdpre1. it was interesting to discuss the chemical interactions between all tested compounds and the mtpank enzyme. zvt compounds as native ligands showed interactions in the mtpank binding pocket (table siii). triazole ring of zvt compound formed hydrogen bonds with tyr235 and asn277 with bond lengths of 1.94 å and 2.46 å, respectively (fig. 5). these two amino acids were active sites for binding to pantothenate.9,27 bond stabilization was also formed through binding the zvt compound with arg238 via hydrogen bonding. in addition, the hydrophobic interaction of zvt with met242 in the presence of a benzene ring also played a role in stabilizing the bonding of the zvt-mtpank complex. in general, all the tested compounds showed the same inhibitory mechanism. all tested compounds were in the binding pocket and bound to the active site of the mtpank enzyme. for the f10 compound, the hydroxyl group of the glucose substituent facilitated hydrogen bonding with tyr235. in contrast, the asn277 amino acid was bonded by the oxygen atom of the coumaroyl carbonyl group. f6 compound only binds tyr235 with a bond length greater than 3.87 å, which matches the binding energy value and is slightly lower than the f10 compound. a cc ep te d m an us cr ip t 12 tjitda et al. a) b) c) d) e) fig 4. chemical interaction of mtdpre1 enzyme with a) 0t4; b) compound f10; c) compound f7; d) compound f13; e) compound f8. a cc ep te d m an us cr ip t antimycobacterial natural flavonoids in delonix regia leaf 13 a) b) c) fig 5. chemical interaction of mtpank enzyme with a) zvt; b) compound f10; c) compound f6. it was strongly suspected that the presence of apigenin and kaempferol as core structures contributed to the inhibition of the tested compound against the mtpank enzyme. this was shown by the interactions of f12 and f13 compounds. visualization of the bond between these two compounds exhibited the presence of binding to two key amino acid residues of the mtpank enzyme. f12 and f13 compounds showed very effective binding to tyr235 and asn277. this means that these two compounds were in the binding pocket of mtpank and had the same inhibitory mechanism. these molecular docking results confirm that f12 and f13 compounds were potentially active as mtpank enzyme inhibitors. a cc ep te d m an us cr ip t 14 tjitda et al. admet-drug-likeness analysis the pharmacokinetic properties of the tested compounds were further analyzed using pkcsm web tools to reveal their bioavailability. the results are presented in table ii. in terms of absorption, the highest absorption ability across the intestinal membrane was shown by f12 and f13 compounds with values of >80%. furthermore, distribution character was studied through the value of volume of distribution (vd), blood-brain barrier (bbb) permeability, and central nervous system (cns) permeability. all of the tested compounds showed moderate (-0.15-0.45 log l kg -1 ) to low (vd < -0.15 log l kg -1 ) vd values. f8, f9, f10, f11, and f13 compounds with low vd indicated that these compounds tended to be distributed in plasma. furthermore, the bbb permeability value revealed that all compounds were unable to penetrate the bbb (<1.00). 22 the same case was also shown by the cns permeability value of all tested compounds. the results of the analysis showed that all compounds had poor absorption into the cns. table ii. admet properties of natural flavonoid compounds from delonix regia leaf. id comp absorption distribution metabolism excretion toxicity intestinal absorption volume of distribution (vdss) bbb permeability cns permeability substrate cyp inhibitor total clearance ames toxicity 2d6 3a4 2d6 3a4 numeric (%absorbed) numeric (log l kg-1) numeric (log bb) numeric (log ps) categorial (yes/no) numeric (log ml min-1 kg-1) categorial (yes/no) flavonoid compounds f1 21.25 0.01 -2.87 -5.82 no no no no 0.32 yes f2 30.96 0.04 -2.80 -5.79 no no no no 0.34 yes f3 56.15 0.10 -1.82 -4.51 no no no no 0.69 no f4 38.87 0.36 -2.58 -5.18 no yes no no 0.59 yes f5 48.16 0.41 -1.59 -3.62 no no no no 0.42 no f6 41.10 0.11 -1.52 -4.03 no no no no 0.60 no f7 30.44 0.03 -2.33 -5.33 no no no no 0.50 no f8 26.98 -0.40 -1.81 -5.22 no no no no 0.09 no f9 60.01 -0.24 -2.11 -4.83 no no no no 0.64 no f10 61.35 -0.98 -1.80 -4.15 no no no yes 0.14 no f11 34.94 -0.22 -2.32 -5.29 no no no no 0.60 yes f12 84.79 0.01 -1.32 -2.44 no no no yes 0.62 yes f13 91.64 -0.18 -1.05 -2.25 no no no yes 0.67 no cytochrome p450 is an enzyme in charge of drug metabolism, and in this study, the isoforms used were 2d6 and 3a4. the analysis showed that only the f4 compound could act as a cyp3a4 substrate and f10, f12, and f13 compounds were inhibitors of cyp3a4. the next analysis was total clearance, a value describing the drug’s ability to be metabolized in the liver and excreted through the kidney. 28 the results revealed that almost all compounds had a fairly acceptable a cc ep te d m an us cr ip t antimycobacterial natural flavonoids in delonix regia leaf 15 total clearance value ranging from 0.14-0.67, except for the f8 compound, which had a low total clearance value (0.09). furthermore, the toxicity properties of a drug were also a critical aspect of further drug development. the results of ames toxicity analysis found that f1, f2, f4, f11, and f12 compounds had mutagenic and carcinogenic properties, but other compounds had the opposite properties. thus, they had the potential to be further developed as drug candidates. table iii. drug-likeness of natural flavonoid compounds from delonix regia leaf. compound mw log p number of hba number of hbd psa (a2) violation of lipinski rule druglikeness score flavonoid compound f1 564.49 -0.18 14 10 198.92 3 0.26 f2 578.16 -0.00 14 10 196.75 3 0.36 f3 448.10 0.47 11 7 151.29 2 0.60 f4 610.19 -0.81 15 8 186.70 3 0.94 f5 360.08 1.54 8 5 114.28 0.37 f6 432.11 0.91 10 6 135.81 1 0.59 f7 578.16 0.30 14 8 182.25 3 0.81 f8 578.16 -0.71 14 8 179.86 3 0.73 f9 448.10 0.32 11 7 150.41 2 0.82 f10 578.14 1.71 12 7 167.32 3 0.63 f11 592.18 0.82 14 7 172.17 3 0.70 f12 286.05 1.61 6 4 87.13 0.50 f13 270.05 3.22 5 3 73.57 0.39 drug-likeness evaluation of all tested compounds was then carried out and used as a screening stage based on the lipinski rule. 29 table iii presents the data from the drug-likeness analysis of all tested compounds. f5, f12, and f13 compounds did not violate the lipinski rule. the molecular weight of the compound (mw, >500) indicated that the compound had poor absorption ability. log p value described the lipophilic character and all tested compounds had a log p value below the cut-off value (>5). referring to the lipinski rule, hydrogen bonding acceptor (hba) must be below 10, and hydrogen bonding donor (hbd) must be below 5. 30 these two values were correlated with the polar surface area (psa) value. when a compound has a violation of hba and hbd, it tends to have low bioavailability with a psa value greater than 140.00 a 2 . all the tested compounds had hba >10 and hbd > 5 except compounds f5, f12, and f13. for the drug-likeness score, all compounds gave a fairly acceptable value with a range of 0.26-0.94. compound f4 had the largest drug-likeness score, namely 0.94. unfortunately, this compound also had toxic properties based on the results of the ames toxicity analysis. compared with the results of admet analysis and drug-likeness analysis, the f12 compound had toxic properties while the f5 compound had poor adsorption a cc ep te d m an us cr ip t 16 tjitda et al. characteristics. as a result, compound 13 as the lead compound is further investigated for its stability against all 3 enzymes. molecular dynamic simulation molecular dynamics using the cabs flex 2.0 server was performed and provided a root mean square fluctuation (rmsf) graph to evaluate the stability of f13 key important enzymes of mycobacterium tuberculosis (fig. 6). as reported by jamroz et al., rmsf analysis using the cabs flex server demonstrated significant correlation with nmr spectroscopy analysis. 31 as shown in fig. 6, compound f13 formed the most stable complex against the mtkasa enzyme. it was found that the fluctuating value of all amino acid residues was 1-3 å. 32 the f13-mtdpre1 complex showed high fluctuations at residue numbers 17, 60, 166, 258, and 284. meanwhile, high fluctuation level for residue numbers 85, 248, 249, 250, 251, and 252 were found in the f13-mtpank complex. the high fluctuating value means that the flexibility of amino acid residues is very considerable. this reduces the stability of the complex. although the results of in silico studies indicated that compound f13 was the most effective compound in inhibiting mycobacterium tuberculosis by forming a stable complex with the mtkasa enzyme. there is still a need to confirm compound f13's activity in vitro and in vivo through laboratory experiments. a cc ep te d m an us cr ip t antimycobacterial natural flavonoids in delonix regia leaf 17 a) b) c) fig 6. multimodel and rmsf of compound f13 against: a) mtkasa enzyme; b) mtdpre1 enzyme; and c) mtpank enzyme. a cc ep te d m an us cr ip t 18 tjitda et al. conclusion in silico study of natural flavonoid compounds from delonix regia leaves extract showed that all tested compounds had antimycobacterial properties based on in silico analysis. admet and drug-likeness screening showed that among all the existing test compounds, only compound f13 had the potential to be studied further as an antimycobacterial candidate with binding energy values for mtkasa, mtdpre1, and mtpank respectively -35.98 kj/mol, -34.31 kj/mol, and -33.05 kj/mol. the molecular dynamics simulation results suggested that compound f13 formed the most stable complex against the mtkasa enzyme compared to mtdpre1 and mtpank. supplementary material supplementary materials are available electronically from https://www.shdpub.org.rs/index.php/jscs/article/view/12064 , or from the corresponding authors on request. и з в о д природни флавоноиди у листовима delonix regia као антимикобактеријски агенси: in silico студија putra jiwamurwa pama tjitda1, febri odel nitbani2, dominus mbunga1 и tutik dwi wahyuningsih3 1department of pharmacy, health polytechnic of kupang, indonesia, 2department of chemistry, faculty of sains and engineering, nusa cendana university, indonesia, и 3department of chemistry, faculty of mathematics and natural sciences, universitas gadjah mada, indonesia отпорност на многоструке лекове (mdr) и екстензивна отпорност на лекове (xdr) као резултат сталне употребе антибиотика охрабрује развијање нових антимикобактериских лекова. у овом раду је проучавано 13 флавоноидних једињења из листова биљке delonix regia због њихових инхибиторних особина за mtkasa, mtdpre, и mtpank, који су значајни ензими код mycobacterium tuberculosis, као и њиховог молекулског докинга, молекулске динамике, те предиђања admet сличности са лековима. резултати студије молекулског докинга откривају да је једињење f13 (apigenin) било најпотентније једињење пошто је било у стању да веже већину аминокиселина на које указује нативни лиганд сваког ензима. студије молекулске динамике су показале да једињење f13 формира стабилан комплекс са mtkasa. резултати admet-анализе сличности са лековима закључују да је једињење f13 оно које највише обећава. све у свему, једињење f13 има потенцијал да се користи као терапија лечења mycobacterium tuberculosis. 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https://doi.org/10.1016/j.ica.2022.121043 https://doi.org/10.1093/bioinformatics/btu184 https://doi.org/https:/doi.org/10.56499/jppres22.1375_10.4.660 hptlc-based metabolomics for the investigation of metabolic changes during plant development: the case study of artemisia annua j. serb. chem. soc. 87 (11) 1237–1244 (2022) original scientific paper jscs–5590 published 10 october 2022 1237 hptlc-based metabolomics for the investigation of metabolic changes during plant development: the case study of artemisia annua jovana stanković jeremić1*, dejan gođevac1, stefan ivanović1, katarina simić1, antoaneta trendafilova2, milica aćimović3 and slobodan milosavljević4,5# 1university of belgrade – institute of chemistry, technology and metallurgy – national institute of the republic of serbia, 11000 belgrade, serbia, 2institute of organic chemistry with centre of phytochemistry, bulgarian academy of sciences, 1113 sofia, bulgaria, 3institute of field and vegetable crops, 21000 novi sad, serbia and 4university of belgrade – faculty of chemistry, studentski trg 12–16, 11000 belgrade, serbia and 5serbian academy of sciences and arts, knez mihailova 35,11000 belgrade, serbia (received 7 may, revised 12 november 2021, accepted 14 february 2022) abstract: the application of high performance thin layer chromatography (hptlc)-based non-targeted metabolomics as a holistic approach to compare fingerprints of metabolite changes during artemisia annua development is described. images of hptlc chromatograms obtained after derivatization with anisaldehyde-sulphuric acid reagent were used as a dataset for multivariate analysis. principal component analysis and orthogonal partial least squares discriminant analysis confirmed the differentiation of samples belonging to vegetative phase, flowering stage, and seed formation stage of the plant development. the obtained results showed that the hptlc-based metabolomics approach can be a very reliable technique for the investigation of metabolic changes during plant development, complementary to gas chromatography–mass spectrometry and nuclear magnetic resonance-based metabolomics. keywords: plant metabolomics; pca; opls-da. introduction in this study, the cultivated artemisia annua was used as a model system for the assessment the application of high performance thin layer chromatography (hptlc) based untargeted metabolomics to probe unique metabolites during the life cycle of the plant. since the discovery of its constituent antimalarial artemisinin, a. annua, which is the only viable resource of this drug, has been the * corresponding author. e-mail: jovanas@chem.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc210507007s ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1238 stanković jeremić et al. subjects of extensive research on its chemical composition. more than 600 secondary metabolites have been identified throughout the plant including several sesquiterpenoids, triterpenoids, monoterpenoids, steroids, flavonoids, coumarins, alkaloids and benzenoids.1,2 the method of untargeted metabolomics considers simultaneous measurement of as many metabolites as possible from each sample, thus providing a holistic, general overview of a sample composition.3 hptlc is an emerging analytical tool in metabolomics research in the last decade.4,5 even conventional thin layer chromatography (tlc) offers the advantages over other analytical methods, such as short measuring time and capability of parallel analysis, and the availability of chemical reagents. many improvements in hptlc over conventional tlc such as high resolution and data robustness, also improved some metabolomics and profiling studies. furthermore, metabolites which are difficult to be identified by nuclear magnetic resonance (nmr) or mass spectrometry (ms)-based methods could be visualized on hptlc plates and isolated from the plate for further chemical elucidation.6 the main idea of this study is the application of an hptlc-based non-targeted metabolomics as a holistic approach to compare fingerprints of metabolites changing during the plant development. experimental general methods all used solvents were of analytical grade. glass hptlc silica gel 60 cn f254s plates 10 cm×20 cm were purchased from merck (darmstadt, germany); anisaldehyde-sulphuric acid used as a spray reagent was freshly made according to wagner et al.7 artemisinin standard was isolated previously in the laboratory of bulgarian academy of sciences. all nmr spectra (1h, 13c, correlated spectroscopy (cosy), heteronuclear single quantum coherence (hsqc) and heteronuclear multiple bond coherence (hmbc)) were recorded in cdcl3 (with tetramethylsilane (tms) for referencing) on a bruker avance iii 500 nmr spectrometer operating at a proton nmr frequency of 500.26 mhz, equipped with a 5 mm broadband probe (bbi) probehead. gas chromatography (gc)/ms analyses were performed according to stankovic et al. (2019)8 with some modifications of the temperature program and split ratio. the oven temperature was programmed from 60 to 240 °c at 3 °c/min, then from 240 to 310 °c at 35 °c/min, and then held isothermally for 8 min. injection volume was 1 μl, split ratio, 20:1. for lipid fraction the oven temperature was kept constant at 40 °c for 1 min, then was linearly programmed from 40 to 315 °c at 10 °c/min and then held isothermally for 6.5 min. injection volume was 1 μl, split ratio 50:1. plant material artemisia annua l. is grown at institute of field and vegetable crops, novi sad. the plants samples were collected 14 times at intervals of 7 or 14 days, successively in different phenophases – from may 27 to november 3, 2018. thus, vegetative phase (collections 1–6), flowering stage (collections 7–11), and seed formation stage (collections 12–14) were collected. the voucher specimens (determined by milica rat, m.sc., as no. 2-1514) were confirmed and deposited at the herbarium of the department of biology and ecology (buns herbarium), faculty of natural sciences, university of novi sad. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ hptlc metabolomics of a. annua 1239 extraction of plant material dry and ground plant materials of each collection (150 mg) were extracted with a mixture of hexane:ethyl acetate, 95:5 volume ratio (1 ml) for 15 min in an ultrasound water bath. the extracts were centrifuged for 15 min at 13,600 rpm. the extraction procedure was independently repeated with four biological replicates of each collection. the obtained extracts were used for hptlc analysis. hptlc analysis the 3 μl of the extracts were sprayed with a 25-μl syringe as an 7-mm band on the hptlc plate using the linomat 5 (camag, switzerland). the application position y was 8.0 mm, the first application position x was 10.0 mm, and the distance between tracks was 10.0 mm. the plates were developed in a previously saturated camag automatic developing chamber 2 with a mobile phase consisting of hexane:ethyl acetate:formic acid (19:11:0.5 volume ratio). after a drying time of 4 min developed hptlc plates were derivatized by dipping in the anisaldehyde reagent (camag chromatogram immersion device 3), followed by heating at 100 °c for 5 min. fractionation by dry-column flash chromatography dry and ground average samples of all collections (167.0 g) were extracted with hexane:ethyl acetate, 95:5 volume ratio(2×600 ml) for 15 min in an ultrasound water bath. the extracts were combined, filtered, and concentrated under reduced pressure to give 1.1 g of residue. dry-column flash chromatography fractionation was performed in a glass column (300 mm×22 mm) packed with silica gel (100 g merck, <0.08 mm) using a water vacuum pump. the column was equilibrated with the mobile phase n-hexane:ethyl acetate (95:5 volume ratio) mixture. the extract (m = 1.1 g) was diluted with the mobile phase (1:1) before applying to the column. the elution was performed with hexane:ethyl acetate mixture with increasing polarity (table s-i, supplementary material to this paper) and 34 fractions were collected. methylation of non-polar lipid fraction with meoh/h2so4 a mixture of the lipid fraction (30 mg) and meoh (6 ml+ two drops of conc. h2so4) was refluxed in a test tube fitted with condenser for 2 h. after neutralization with saturated sodium bicarbonate solution, the methyl esters were extracted with n-hexane. the organic layer was dried over anhydrous na2so4 and evaporated under reduced pressure in a rotavapor. image and multivariate data analysis the hptlc chromatograms were processed with the image j processing program (version 1.47q). the chromatograms were denoised using the median filter function with two pixels width filter, and the tracks were outlined with rectangular selection tool. the line profile plots (gray scale) were generated using plot profile option for each sample. on this way, two-dimensional graphs containing the pixel intensities toward distance along the line were obtained. the hptlc image data were then normalized to the total area, mean centred and pareto scaled. principal component analysis (pca) and orthogonal partial least squares to latent structures – discriminant analysis (opls-da) method was performed with simca software (version 15, sartorius, göttingen, germany). results and discussion the hptlc conditions regarding amount of sample applied and mobile phase composition were optimised to detect the maximum number of resolved ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1240 stanković jeremić et al. bands per sample. four biological replicates for each of the 14 collections of a. annua were then fingerprinted using the hptlc method. thus, vegetative phase (collections 1-6), flowering stage (collections 7–11), and seed formation stage (collections 12–14), were covered. the fingerprint patterns visualized after derivatization with anisaldehyde-sulphuric acid reagent are depicted in fig. 1. fig. 1. hptlc metabolic fingerprint of 14 samples (red) a. annua collected in different developmental stages. for the identification of the components found in the studied extracts, drycolumn flash chromatography of the plant extract was used, followed by nmr and/or gc/ms analyses. as a result of the purification, the isolated fractions appeared as a single band on hptlc plate. each band of the purified fraction was then connected to those of a. annua extracts by co-chromatography comprising their rf values and colours after derivatization (table i). the identity of the purified metabolites was then performed by nmr and/or gc/ms analyses (supplementary material). only artemisinin was not isolated as a pure compound, and its hptlc band was compared to that of the standard compound. table i. identified metabolites in a. annua extracts no. rf value identified metabolites colour after derivatization identification 1 0.94 triacylglycerol dark green nmr; gc/ms after transesterification 2 0.86 α-pinene, α-copaene, caryophyllene e and β-selinene purple gc/ms 3 0.78 artemisia ketone orange nmr; gc/ms 4 0.68 1,8-cineole purple gc/ms 5 0.66 caryophyllene oxide purple gc/ms 6 0.58 artemisia alcohol dark green gc/ms 7 0.46 artemisinin pink comparison to standard 8 0.20 arteannuin b pink gc/ms ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ hptlc metabolomics of a. annua 1241 the line profile plots generated from the images obtained using visible light were used as a dataset for multivariate analysis. firstly, the principal component analysis (pca), as a variable reduction technique to develop a smaller number of novel variables that will account for most of the variation in the observed variables, was performed. it has resulted in five principal components (pcs) model explaining 84.3 % of the total data of variances. based on pca score plot (fig. 2a), three groups of samples were separated to some extent, according to the plant development stages. fig. 2. a) pca score plot; b) opls-da score plot containing vegetative and flowering phase of a. annua; c) opls-da score plot containing vegetative and seed formation phase of a. annua; d) opls-da sus-plot, all obtained from hptlc fingerprints; four (4) terpenes: α-pinene, α-copaene, caryophyllene e and β-selinene. next, the orthogonal partial least squares to latent structures–discriminant analysis (opls-da) was applied, where novel variables will account for the maximum separation between predefined classes. an additional advantage of the orthogonal model is the facilitated interpretation due to separation of the systematic variation of the variables into two parts: one linearly related to class information and one orthogonal to the class information.9 two opls-da models were ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1242 stanković jeremić et al. created containing hptlc data of: a) vegetative phase versus flowering stage and b) vegetative phase versus seed formation stage of a. annua development. the quality of the obtained models was assessed by goodness of fit (r2) indicating how well the variation of variables is explained using the predictive components and predictive ability of the model (q2), indicating how well the model predicts new data, estimated by cross validation. in both created opls-da models, large r2 and q2 values over 0.5 and close to 1 indicated high goodness of fit and good predictivity (table ii). table ii. parameters of the opls-da models model number of components (predictive + orthogonal) r 2 q2 p (cv-nova) f (cv-anova) vegetative/ flowering 1 + 4 0.951 0.897 4.88×10-13 28 vegetative/ seed formation 1 + 3 0.918 0.784 6.45×10-7 12 the models were validated by permutation tests and cv-anova, the significance of the models was clearly shown with p values far less than 0.05 (table ii). in the score plots (fig. 2b and c) a clear separation between the samples was obtained along to the predictive components. the most influential variables were selected based on the variable influence on projection (vip) scores of the predictive components, and the loadings scaled as a correlation coefficient (p(corr)). vip > 1 and |p(corr)| > 0.5 were considered as important for the separation. shared and unique structure plot (sus-plot) containing p(corr) from both opls-da models was used to reveal the changes in the metabolomes in each of the three predefined group of samples (fig. 2d). the unique metabolites were found close to either the x or y axis, and the shared were located on the diagonals. also, the metabolites which are on the extreme ends of the axes contribute more significantly than those close to the centre. thus, the accumulation of triacylglycerol (1) is characteristic for the seed formation phase since it is an unique metabolite in vegetative/seed formation model. similarly, four terpenes (α-pinene (2), α-copaene (2), caryophyllene e (2) and β-selinene (2)) and artemisia ketone (3) were accumulated in the flowering stage since they are unique metabolites in the vegetative/flowering model. artemisia alcohol (6), caryophyllene oxide (5), and 1,8-cineole (4) were the main metabolites in the vegetative stage, since they are shared in both models. for artemisinin (7) and arteannuin b (8) there was no significant change in their amount during plant development. metabolomics fingerprinting of a. annua in different stages of development has also been performed previously by gc/ms.10 our objective was to highlight the advantages of hptlc: no need for derivatization prior to chromatography, ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ hptlc metabolomics of a. annua 1243 and thus detection of thermolabile and non-volatile metabolites, which is not possible using gc/ms. hptlc is also complementary technique to nmr-based metabolomics, offering solutions where crowded regions in the nmr spectra are difficult to resolve.4 conclusion this work presents an efficient way for analysing metabolic fingerprinting data of a. annua l. generated by hptlc. as a result, we demonstrated that hptlc may be applied as a simple and reliable untargeted approach to rapidly differentiate the extracts originating from various developmental stages of the plant. hptlc also demonstrated to be the method of choice for the routine study of complex matrices and could be used as an alternative and/or complementary method to techniques, such as gc/ms or nmr. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/10744, or from the corresponding author on request. acknowledgments. the authors are grateful for the support from joint research project between serbian academy of science and arts and bulgarian academy of sciences, “phytochemical investigation of secondary metabolites from plants and fungi and their biotransformed products“. и з в о д mетаболомикa базиранa на hptlc за испитивање метаболичких промена током развоја биљке: студија случаја artemisia annua јована станковић јеремић1, дејан гођевац1, стефан ивановић1, катарина симић1, antoaneta trendafilova2, mилица аћимовић3 и слободан милосављевић4,5 1универзитет у београду, институт за хемију технологију и металургију, институт од националног значаја за републику србију, студентски трг 12–16, 11000, београд, 2institute of organic chemistry with centre of phytochemistry, bulgarian academy of sciences, 1113 sofia, bulgaria, 3институт за ратарство и повртарство, 21000 нови сад, 4универзитет у београду – хемијски факултет, студентски трг 16, 11000 београд и 5српска академија наука и уметности, кнез михајлова 35, 11000, београд описана је холистичка примена нетаргетне метаболомике базиране на високоефикасној танкослојној хроматографији (hptlc) која омогућава поређење метаболичких профила artemisia annua и праћење њихових промена током развоја биљке. hptlc хроматограми након развијања анизалдехидом и сумпорном киселином су коришћени за генерисање података за мултиваријантну анализу. анализа главних компоненти и ортогонална дискриминанта анализа најмањих квадрата су потврдиле разлике између узорака који припадају различитим фенофазама – вегетативној фази, фази цветања и фази формирања семена. добијени резултати указују да метаболомички приступ заснован на hptlc методи која је комплементарна са gc/ms и nmr анализом, може бити веома поуздана техника за анализу промене током развоја биљке. (примљено 7. маја, ревидирано 12. новембра 2021, прихваћено 14. фебруара 2022) ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1244 stanković jeremić et al. references 1. v. vajs, a. jokić, s. milosavljević, nat. prod. commun. 12 (2017) 1157 (https://doi.org/10.1177/1934578x1701200802) 2. g. d. brown, molecules 15 (2010) 7603 (https://doi.org/10.3390/molecules15117603) 3. j. l. wolfender, s. rudaz, y. h. choi, h. k. kim, curr. med. chem. 20 (2013) 1056 (https://doi.org/10.2174/0929867311320080009) 4. l. f. salomé-abarca, m. mandrone, c. sanna, f. poli, c. a. m. j. j. van der hondel, p. g. l. klinkhamer, y. h. choi, phytochemistry 176 (2020) 112402 (https://doi.org/10.1016/j.phytochem.2020.112402) 5. m. maldini, g. d’urso, g. pagliuca, g. l. petretto, m. foddai, f. r. gallo, g. multari, d. caruso, p. montoro, g. pintore, foods 8 (2019) 294 (https://doi.org/10.3390/foods8080294) 6. y. ge, x. chen, d. gođevac, p. c. p. bueno, l. f. salomé abarca, y. p.jang, m. wang, y. h. choi, planta med. 85 (2019) 917 (https://doi.org/10.1055/a-0947-5797) 7. h. wagner, s. bladt, e. m. zgainski, plant drug analysis, springer-verlag, heidelberg, 1984 (https://doi.org/10.1007/978-3-662-02398-3) 8. j. stanković, m. novaković, v. tešević, a. ćirić, m. soković, g. zdunić, z. dajić-stevanović, d. gođevac, j. serb. chem. soc. 84, (2019) 1355 (https://doi.org/10.2298/jsc190513106s) 9. s. wiklund, e. johansson, l. sjöström, e. j. mellerowicz, u. edlund, j. p. shockcor, j. gottfries, t. moritz, j. trygg, anal. chem. 80 (2008) 115 (https://doi.org/10.1021/ac0713510) 10. m. chenfei, w. huahong, l. xin, x. guowang, l. benye, j. chromatogr., a 1186 (2008) 412 (https://doi.org/10.1016/j.chroma.2007.09.023). ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ @article{stankovic-jeremic2022, author = {stankovic-jeremic, jovana and godjevac, dejan and ivanovic, stefan and simic, katarina and trendafilova, antoaneta and acimovic, milica and milosavljevic, slobodan}, journal = {journal of the serbian chemical society}, title = {{hptlc-based metabolomics for the investigation of metabolic changes during plant development: the case study of artemisia annua}}, year = {2022}, issn = {1820-7421}, month = {oct}, number = {11}, pages = {1237--1244}, volume = {87}, abstract = {the application of high performance thin layer chromatography (hptlc)-based non-targeted metabolomics as a holistic approach to compare fingerprints of metabolite changes during artemisia annua development is des­cribed. images of hptlc chromatograms obtained after derivatization with anisaldehyde-sulphuric acid reagent were used as a dataset for multivariate ana­lysis. principal component analysis and orthogonal partial least squares dis­criminant analysis confirmed the differentiation of samples belonging to veget­ative phase, flowering stage, and seed formation stage of the plant develop­ment. the obtained results showed that the hptlc-based metabolomics appro­ach can be a very reliable technique for the investigation of metabolic changes during plant development, complementary to gas chromatography–mass spec­trometry and nuclear magnetic resonance-based metabolomics.}, doi = {10.2298/jsc210507007s}, file = {:d\:/onedrive/mendeley desktop/stankovic-jeremic et al. 2022 hptlc-based metabolomics for the investigation of metabolic changes during plant development the case.pdf:pdf;:01_10744_5590.pdf:pdf}, keywords = {da, opls}, publisher = {national library of serbia}, url = {https://www.shd-pub.org.rs/index.php/jscs/article/view/10744}, } accepted manuscript this is an early electronic version of an as-received manuscript that has been accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as s. a. ejaz, m. aziz, a. fayyaz, t. a. wani, and s. zargar, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc230307050e this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230307050e j. serb. chem. soc.00(0)1-19 (2023) original scientific paper jscs–12309 published dd mm, 2023 1 computer-aided approach for the identification of lead molecules as the inhibitors of cholinesterase’s and monoamine oxidases: novel target for the treatment of alzheimer’s disease syeda abida ejaz1*, mubashir aziz1, ammara fayyaz1, tanveer a. wani2, and seema zargar3 1department of pharmaceutical chemistry, faculty of pharmacy, the islamia university of bahawalpur, bahawalpur 63100, pakistan, 2department of pharmaceutical chemistry, college of pharmacy, king saud univeristy, p.o.box 2457, riyadh 11451, saudi arabia, and 3department of biochemistry, college of science, king saud univeristy, p.o. box 22452, riyadh 11451, saudi arabia (received 7 march; revised 22 june; accepted 14 august 2023) abstract: molecular docking is a promising and reliable technology for the purpose of discovering lead compounds via virtual screening. in addition to allowing for the testing of a large number of compounds, it also allows for the determination of how the selected compounds inhibit the targeted protein/receptor based on the scoring function and ranking. because selective cholinesterase and monoamine oxidase inhibitors play a critical role in the treatment of alzheimer's disease, this research focuses on elucidating the mechanism of binding interactions of a few quinolone derivatives within the active sites of cholinesterase (acetylcholinesterase (ache) and butyrylcholinesterase (bche) and monoamine oxidase (mao) (monoamine oxidase a &b). as a result of these discoveries, it is possible that the newly identified inhibitors will be used as lead compounds in the development of novel enzyme inhibitors for the treatment of specific diseases, hence enabling the development of novel therapeutic approaches. keywords: molecular docking; acetyl-cholinesterase (ache); butyrylcholineesterase (bche); active pocket; monoamine oxidases a & b, alzheimer disease. introduction the actions of brain neurons, particularly neurotransmission, are grouped into systems of individually distinct neurons, neurotransmitters, neuromodulators, receptors, and hormones. the monoaminergic and cholinergic systems are essential in these systems. a crucial component of the cholinergic system of neurotransmission is the neurotransmitter acetylcholine. 1 neuromuscular * corresponding author. e-mail: abida.ejaz@iub.edu.pk; abidaejaz2010@gmail.com https://doi.org/10.2298/jsc230307050e a cc ep te d m an us cr ip t mailto:abida.ejaz@iub.edu.pk mailto:abidaejaz2010@gmail.com 2 ejaz et al. synapses, the brain, and autonomic ganglia all contain acetylcholine (ach) which is fast acting. two enzymes; acetylcholinesterase (ache; ec 3.1.1.7) and butyrylcholinesterase are present in tissues that catalyze the breakdown of acetylcholine (bche; ec 3.1.1.8). 2 the two enzymes are both glycoproteins that can either be membrane-bound or soluble. ache is a critical enzyme in cholinergic neurotransmission on both the central and peripheral level. it is mostly membranebound and located in the neuromuscular junctions and synapses between neurons in the central nervous system, where it inhibits the activity of acetylcholine (ach) produced during nerve stimulation. 3 the enzyme is also present in soluble forms and linked with the erythrocyte membrane. bche is found in a broad variety of human tissue types. it is expressed in several different groups of neurons in the human brain that are not connected with ache, as well as glial cells. bche is significantly more abundant in the serum than ache is. bche, like ache, is a soluble enzyme that may also be membrane-bound. 4 ache is highly effective at hydrolyzing ach, but its catalytic site is buried deep inside a deep cavity, which restricts its ability to cleave esters with heavier acyl groups. 5 as compared to ache, butyrylcholinesterase has a bigger cavity and is a less selective esterase capable of cleaving a variety of esters with larger acyl groups. 6 the network of monoamine neurotransmission systems, each of which is regulated by a unique monoamine neurotransmitter, is known as the monoamine neurotransmission system. these include the dopaminergic, noradrenergic, and serotonergic systems, which are each regulated by the neurotransmitters dopamine, noradrenaline, and serotonin, respectively. 7 two isoforms of the monoamine oxidase (mao) enzyme, mao a and mao b, are involved in the oxidative deamination of biogenic amines, including neuroamines, vasoactive amines, and exogenous amines and hence regulates the concentration of amine neurotransmitters and numerous amine medications. 8 due to their location on the mitochondrial outer membrane, the mao flavoproteins metabolize amines inside the cell. each mao has a different tissue concentration, with the two versions functioning identically in the human liver. mao b is highly expressed in platelets, glial cells, and serotonergic neurons, whereas mao a is expressed primarily in the stomach, placenta, and dopaminergic and noradrenergic neurons. 9 despite having the identical catalytic center structure, with substrate-orienting tyrosinase leading to covalently bound fad, 10 the two forms exhibit distinct intrinsic activity (kcat and km values) with each substrate. serotonin is metabolized mostly by mao a in the human brain, but dopamine is metabolized by both forms.11 alzheimer's disease (ad) is the most common neurodegenerative ailment, affecting millions of individuals worldwide, including 5% of the over-65 population. the primary clinical manifestation of ad is increasing memory loss and other abnormalities in brain function, such as aberrant behavior and impairments in language, comprehension, and visual-spatial abilities. 12 a cc ep te d m an us cr ip t novel target for alzheimer’s disease 3 it has been postulated that bche collaborates with ache in the brain to regulate ach neurotransmission when ach levels become excessive, especially given that ache is inhibited by high concentrations of its substrate. 13 in the average human brain, bche activity is lower than ache activity, whereas ad patients have much higher bche/ache ratios, 14 indicating that bche inhibition can get critical as ad advances. this has generated the concept that inhibiting both ches may result in enhanced therapeutic advantages or that inhibiting bche alone may be advantageous. 15 the first therapy methods for ad were aimed at boosting cholinergic transmission in the brain, in accordance with the "cholinergic hypothesis" of memory loss. among the several strategies used to raise synaptic ach levels, inhibiting acetylcholinesterase (ache) has shown to be the most effective. inhibiting the enzyme butyrylcholinesterase (buche), which is present in trace amounts in normal brains but is elevated in the brains of ad patients with plaques and tangles, may enhance cholinergic transmission. 16 dopamine, serotonin, and noradrenaline, which are all metabolized by mao or catechol-omethyltransferase (comt), are reduced as a consequence of other neurons being gradually eliminated in ad. mao inhibitors have long been used as antidepressants, and an early study against parkinson's disease led in the acceptance of l-deprenyl (selegiline) as a disease-delaying medication for the condition. thus, mao inhibitors may promote amine neurotransmission and have beneficial biochemical effects in the therapy of alzheimer's disease. 17 increased mao b levels have been found in ad patients' brains as a result of increased astrogliosis, 18 suggesting that combined mao a/b inhibition may be an effective ad treatment. mao inhibitors' beneficial features also include a decrease in the generation of reactive oxygen species, which may contribute to greater neuronal damage. 19 current novel therapeutic approaches suggest that drugs targeting a single target may be insufficient for treating multifactorial neurodegenerative diseases such as alzheimer's disease (ad), parkinson's disease (pd), huntington's disease (hd), and amyotrophic lateral sclerosis (als), which are all characterized by the coexistence of multiple etiologies. these include, but are not limited to, the development of oxidative stress (os) and reactive oxygen species (ros), protein misfolding and aggregation, mitochondrial dysfunction, inflammation, metal dyshomeostasis, and accumulation at neurodegenerative sites. 20 thus, it is plausible to assume that ad treatment will likely need a combination of drugs to address the disease's many clinical manifestations. due to the multifactorial character of ad and the variety of brain pathways involved in its control, 21 multitargeted ligands have been widely investigated as potential therapeutic candidates with favorable benefits in ad treatment. 22 a cc ep te d m an us cr ip t 4 ejaz et al. the current study is focused on the identification of quinolone derivatives as the inhibitors of either class of enzymes i.e., cholinesterases (ache and bche) and monamine oxidases (mao-a and mao-b). quinolones have been previously identified for their anti-bacterial activity in the treatment of various infections. they are specifically designed to selectively target two bacterial topoisomerase enzymes: dna gyrase that targets gram-negative bacteria and topoisomerase iv that effects gram-positive bacteria. 23 the approved quinolone derivative drug tacrine lowers ache levels in alzhiemer’s disease. one study showed that quinolone derivatives were active against ache with an ic50 values ranging from 7.31 to 88.10 μm. 24 the long-term use, however, of quinolones is not recommended as it may be carcinogenic to adults and arthropathogenic in children. however, they can be used for the short-term treatment and symptomatic management of alzheimer’s disease. this study will lead a comprehensive knowledge of the structures, activity correlations, and roles of the discovered inhibitors/drug-like compounds which promises well enough for the future development of novel pharmaceuticals. the following compounds were selected for this study. table 1: list of the chosen derivatives code iupac name structure 3 6-fluoro-2-[4-(2-fluorophenyl) phenyl]-3methylquinoline-4-carboxylic acid 3-a 6-chloro-2-[4-(2-fluorophenyl) phenyl]-3methylquinoline-4-carboxylic acid 3-b 6-chloro-3-methyl-2-(4-phenylphenyl) quinoline-4carboxylic acid 3-c 6-fluoro-3-methyl-2-(4-phenylphenyl) quinoline-4carboxamide 4 4-chloro-1-(2-methylpropyl) imidazole[4,5-c] quinoline a cc ep te d m an us cr ip t novel target for alzheimer’s disease 5 4-a 1-(2-methylpropyl) chromeno[3,4-d] imidazol-4-one 4-b 1-(2-methylpropyl)-5h-imidazo[4,5-c] quinolin-4-one 4-c 1-(2-methylpropyl) chromeno[3,4-d] triazol-4-one experimental molecular docking: in the present work, we employed molecular docking using moe software23 to predict the inhibitory mechanism of chosen quinolone and quinoline derivatives. the crystal structure of the chosen target (ache, pdb id: 4bdt), (bche, pdb id: 4bds), (mao-a, pdb id: 2z5y), and (mao-b, pdb id: 2v5z) was obtained from the protein data bank and utilized for further research. the force field mmff94x with an rmsd gradient of >0.01 kcal/mol/å was used to minimize the energy of the selected quinoline and quinolone compounds. additionally, when the ligands and protein were created, charges were assigned and hydrogen atoms were added to all molecules, the moe site finder tool was used to locate the binding site, as mentioned before. for considering the conformational flexibility parameters, we have employed a simulation approach for docking purposes in which the ligand is allowed to interact with the groove of a targeted protein after a series of moves in its conformational space. these moves include changes in the structures of ligands either internally by rotating the torsional angles or the number of rotatable bonds, or externally by translation and rotation. each rotation of ligand produced intermolecular interactions with the targeted protein. this approach is preferred for taking into account the conformational flexibility of ligands, and it may take longer time depending upon the performance of computing system. in a current study, two series of ligands were docked. quinoline derivatives have four rotatable bonds, whereas quinolone derivatives have only two rotatable bonds. by employing a simulation approach, ligands were allowed to interact with targeted protein through torsional angles. the rigid receptor model was employed and total numbers of poses were set to 100. finally, the moe software was used to produce 3d and 2d poses for further investigation.25 the docking of all the compounds was reconfirmed by seesar analysis.26 results and discussion ache results: as shown in table 1, the derivative 4 which has the most binding score -26.9 kj/mol showed binding interactions. the amino acid his447 formed carbon hydrogen bonding with the hydrogen attached with nitrogen in the imidazole. similarly, trp86 and tyr337 formed π-π stacked interaction with imidazole and pyridine ring. the binding energy and bonding and non-bonding interactions of quinolone derivatives within the active pocket of ache is given in supplementary file. a cc ep te d m an us cr ip t 6 ejaz et al. the alkyl interaction was formed with the amino acid pro446 which formed interaction with the chlorine attached with the pyridine ring and similarly, the amino acid trp439 formed π-alkyl interaction with the chlorine attached with the pyridine ring. the amino acids which formed van der waals interactions were ile451, glu202, ser203, tyr133, gly122, gly121, asp74, tyr341, thr83, tyr77. the derivative 4-c which has the best binding score -26.5 kj/mol after the derivative 5 formed the following interactions. the amino acids trp86, tyr337 which formed π-π stacked interaction with the imidazole ring and with pyridine ring. the derivative 4-a which has the binding score -26.1 kj/mol. the amino acids glu202 and gly126 which formed carbon hydrogen bond with the oxygen attached with pyran ring and with the hydrogen of imidazole ring. the π -sigma interaction was formed with the amino acid trp86. similarly, the binding score of the derivative 4-b is -25.6 kj/mol made c-h bond with the amino acid thr83 involving oxygen attached with pyran ring. the π-π stacked interaction involved with the amino acids trp86, tyr337 with the imidazole and pyridine ring. the derivative 3-c showed binding score -22.3 kj/mol and formed interactions. the amino acid trp86 formed conventional hydrogen bonding with the amino group and the amino acid gly126, gly121 formed carbon hydrogen bonding with the oxygen attached with pyridine ring and the derivative 3 that has the binding score -21.8 kj/mol formed alkyl interaction with amino acid his447 with the substitution on pyridine ring. the amino acid trp86 formed π-sigma bond with the substituted benzene ring. similarly, the derivative 3-a has the binding score -20.8 kj/mol formed conventional hydrogen bonding with the amino acid his447. the amino acids tyr119, tyr133, leu130 formed π-alkyl interaction with chlorine attached with the benzene ring and the derivative 3-b which has a binding score of -20.7 kj/mol formed conventional hydrogen bonding involving amino acid his447 with the hydrogen attached with the oxygen in pyridine ring. the amino acids tyr119, tyr133 and leu130 which formed alkyl and π-alkyl chlorine attached with the benzene ring. a cc ep te d m an us cr ip t novel target for alzheimer’s disease 7 figure 1. 3d binding interactions of selected compounds within the active pocket of ache seesar analysis the best derivative 4 was subjected to seesar analysis in order to predict the virtual visualization of binding interactions. green colored coronas were used to indicate the structural components of potent compounds, whereas red colored coronas were used to indicate those parts that had an adverse impact on binding interactions. a colorless corona was used to shade structural elements that made little contribution. corona size predicts the contribution of a structural component. seesar visualization of potent derivative 4 is given in (figure 2). hyde energy of favorable corona (green colored) for 4 was -8.3 kj/mol while the hyde energy of unfavorable corona (red colored) was 9.0 kj/mol. a cc ep te d m an us cr ip t 8 ejaz et al. figure 2. left; seesar analysis of the active pocket of ache. right; showing docked conformations with coronas. red colored coronas represent undesirable characteristics, green colored coronas show favorable contributions, and colorless coronas represent no structural component contribution. bche results as shown in table 3, the derivative 3-c which has the most binding score 29.3648 kj/mol showed binding interactions. the amino acids thr122, tyr128 and gly121 which formed conventional hydrogen bonding and carbon hydrogen bonding with the chlorine attached with the benzene ring. similarly, the amino acids phe329 and trp231 made π-π t shaped interaction with the benzene ring. binding energy and bonding and non-bonding interactions of quinolone derivatives within the active pocket of bche is provided in supplementary file. the π-alkyl interaction was formed with the amino acid his438 and leu286 which formed interaction with the substitution attached with the pyridine ring and with the benzene ring. similarly, the amino acid trp82 formed π-sigma interaction with the substituted benzene ring. the amino acid glu197 formed unfavorable acceptor-acceptor with the oxygen attached with pyridine ring. the amino acids which formed van der waals interactions were val288, pro285, gly116, leu125, gly115, ile442, gly439, phe398. the derivative 3 which has the best binding score -28.6 kj/mol after the derivative 1 produced the following interactions. the amino a cc ep te d m an us cr ip t novel target for alzheimer’s disease 9 acids gly116 which formed π-π t shaped interaction with the pyridine ring and with substituted benzene ring. the amide-π stacked interaction was formed with the amino acid trp82 involving the benzene ring and the amino acid ala328 formed π-alkyl interaction with the substituted benzene ring. the derivative 3-b which has the binding score -26.6 kj/mol. the amino acids gly117 and ser198 which formed conventional hydrogen bond with the oxygen attached with pyridine ring. the amide-π stacked interaction was formed with the amino acid gly116. similarly, the binding score of the derivative 3-a is -26.5 kj/mol formed conventional hydrogen bond with the amino acid gly117 and ser198 involving oxygen attached with pyridine ring. the amide-π stacked interaction involved with the amino acids gly116 with the pyridine ring and substituted benzene ring. the amino acid his438 formed π-alkyl interaction with the chlorine. the derivative 4b showed binding score -25.3 kj/mol and formed interactions. the amino acid tyr128 formed conventional hydrogen bonding with the oxygen attached with the pyridine ring and the amino acid his438 formed -alkyl interaction with the dimethylpropane and the derivative 7 which has the binding score -25.2 kj/mol formed hydrogen bond and c-h bond with the amino acid trp82, tyr128 and gly121 with the oxygen attached with the pyridine ring. similarly, the derivative 8 has the binding score -24.9328 kj/mol formed hydrogen bond with the amino acid gly121 with the oxygen attached with pyridine ring. the amino acids his438 formed π-alkyl interaction with dimethylpropane and the derivative 5 which has a binding score of -24.1 kj/mol formed π-alkyl interaction involving amino acid tyr128 with chlorine attached with pyridine ring. the amino acid trp82 formed π-sigma interaction with pyridine ring. a cc ep te d m an us cr ip t 10 ejaz et al. figure 3. 3d binding interactions of selected compounds within the active pocket of bche seesar analysis seesar analysis of the best derivative 3-c was executed which anticipate virtual display of binding interactions. green colored coronas were used to positively present the structural components of potent compounds, whereas red colored coronas were used to favorably convey the structural members that had a detrimental impact on binding interactions. a colorless corona was used to represent structural elements that made little contribution. corona size determines the contribution of a structural component. seesar visualization of potent derivative 3-c is given in figure 4. hyde energies of favorable coronas a cc ep te d m an us cr ip t novel target for alzheimer’s disease 11 (green colored) for 3-c was -4.7 and -4.5 kj/mol while the hyde energy of unfavorable corona (red colored) was 13.8 kj/mol. figure 4. left; seesar analysis of the active pocket of bche. right; showing docked conformations with coronas. red colored coronas show unfavorable interactions whereas green coronas are representing favorable contributions and colorless coronas are showing no contribution of components. mao-a results: as shown in table 4, the derivative 3 which has the most binding score -37.5 kj/mol showed binding interactions. the amino acids thr52 and ile23 which formed conventional hydrogen bond and π-donor hydrogen bond with the fluorine attached with the benzene ring and with the benzene ring. similarly, the amino acid trp397 which formed π-π stacked interaction with the substituted benzene ring. binding energy and bonding and non-bonding interactions of quinolone derivatives within the active pocket of mao-a is given in supplementary file. amide-π stacked interaction was formed involving the amino acid residue tyr444 with the benzene ring. the amino acids ala448 and met445 formed πalkyl interaction with the substituted benzene ring and with the benzene ring. the amino acids which formed van der waals interactions were ser24, gly434, thr435, gly66, lys305, phe352, ala68, gly50. the derivative 3-a which has the a cc ep te d m an us cr ip t 12 ejaz et al. best binding score -37.5 kj/mol after the derivative 1 formed the following interactions. the substituted benzene ring and the pyridine ring produced conventional hydrogen bonds and carbon hydrogen bonds with the amino acids arg51, thr52, and tyr444. the benzene ring and the amino acid met444 established π -sulphur interaction. the pyridine ring was involved in the formation of the amide-stacked interaction with the amino acid tyr407. the derivative 3-b which has the binding score -37.1 kj/mol. the amino acids tyr444 which formed carbon hydrogen bond with the benzene and pyridine ring. the amino acid ala448 and cys406, met445 formed alkyl and π-alkyl interactions with the benzene and the pyridine ring. the π-cation interaction was formed with the amino acid arg51. similarly, the binding score of the derivative 3-c is -36.9 kj/mol formed amide-π stacked interaction involving the amino acid trp397 with the substituted benzene ring. the amino acid tyr407 formed π-π stacked interaction with the pyridine ring. the amino acids met445 and ala448 formed π-alkyl interaction with the benzene and pyridine ring. the derivative 4 showed binding score of -30.3 kj/mol. the amino acid tyr407 and gly443 formed carbon hydrogen bond with the hydrogen attached with the nitrogen in imidazole ring. the amino acid arg51 and phe352 formed alkyl and π-alkyl interaction with the imidazole ring and with the chlorine attached with benzene ring and similarly, the derivative 4-c which has the binding score -29.5 kj/mol formed carbon hydrogen bond with the amino acid tyr407 and gly443 with the hydrogen of dimethylpropan and the amino acid arg51 formed alkyl interaction with the imidazole ring. similarly, the derivative 4-a has the binding score -29.1 kj/mol formed carbon hydrogen bond with the amino acid tyr407 with the hydrogen of dimethylpropan. the amino acids met445 formed πalkyl interaction with benzene ring and the derivative 4-b which has a binding score of -28.4 kj/mol formed carbon hydrogen bond involving the amino acid gly67 with the hydrogen of dimethylpropan and the amino acid arg51 and met445 formed alkyl and π-alkyl interaction imidazole ring. a cc ep te d m an us cr ip t novel target for alzheimer’s disease 13 figure 5. 3d binding interactions of selected compounds within the active pocket of mao-a seesar analysis seesar analysis of the best derivative 3 was executed which anticipate virtual display of binding interactions. green colored coronas were used to favorably identify the structural elements of potent compounds, whereas red colored coronas were used to distinguish those elements that had a negative impact on binding interactions. colorless coronas were used to shade structural elements that made no contribution. the size of the corona predicts the contribution of structural components. seesar display of potent derivative 3 is given in figure 6. hyde energies of favorable coronas (green colored) for 3 was -8.7 and -4.8 kj/mol while the hyde energy of unfavorable corona (red colored) was 12.1 kj/mol. a cc ep te d m an us cr ip t 14 ejaz et al. figure 6. left; seesar analysis of the active pocket of mao-a. right; showing docked conformations with coronas. colorless coronas represent no structural component contribution, while green coronas indicate favorable contributions and red coronas indicate undesirable aspects. mao-b results: the derivative 3-b which has the most binding score -36.9 kj/mol showed binding interactions. the amino acids trp388 and tyr398 established interactions with the substituted benzene ring and the pyridine, respectively, that were ππ stacked and a t shape, respectively. the amino acids ala439 and lys296 and val294 and cys397 and phe343 developed alkyl and π-alkyl interactions with the benzene, substituted benzene ring and with chlorine attached with benzene ring. binding energy and bonding and non-bonding interactions of quinolone derivatives within the active pocket of mao-b is provided in supplementary file. the -cation interaction was also observed involving the amino acid arg42 with the benzene ring. the amino acids which formed van der waals interactions were ala263, ile264, gly13, ile14, thr43, gly57, leu56, tyr435, gly434, thr426, gly425, ser15. the derivative 3-a which has the best binding score -36.7 kj/mol after the derivative 1 formed the following interactions. the amino acids gly58 and arg42 which formed carbon hydrogen bond and conventional hydrogen bond with the oxygen attached with pyridine ring and with fluorine and benzene a cc ep te d m an us cr ip t novel target for alzheimer’s disease 15 ring. the amino acid thr43 formed π-cation interaction with the fluorine attached with benzene ring. the derivative 3-c which has the binding score -35.9 kj/mol. the amino acid ala439 formed alkyl interactions with the benzene ring. the ππ interaction was formed involving the amino acid arg42 with the benzene ring. similarly, the binding score of the derivative 3 is -34.9 kj/mol formed amide-π stacked interaction involving the amino acid gly57 with the substituted benzene ring. the amino acid tyr398 formed π-π stacked interaction with the substituted benzene ring. the amino acids met445 and ala439 formed π-alkyl interaction with the substituted benzene ring. the π-cation interaction was formed involving the amino acid arg42 with the benzene ring. the derivative 4-c showed binding score -28.7 kj/mol and formed interactions. the amino acid tyr398 formed carbon hydrogen bond with the dimethylpropan. the amino acid val294 and arg42 formed alkyl and π-alkyl interaction with the dimethylpropan and with the imidazole ring. the π-sulphur interaction was formed involving the amino acid cys397 with the imidazole ring and similarly, the derivative 4 which has the binding score -28.6 kj/mol formed alkyl and π-alkyl interaction involving the amino acid val294, tyr398 and met436 with the substituted benzene ring and with the imidazole ring. the derivative 7 has the binding score -28.5 kj/mol formed carbon hydrogen bond with the amino acid tyr398 with the hydrogen of dimethylpropan. the amino acid gly57 formed amide-π stacked interaction involving the amino acid with the imidazole ring and the derivative 4-a which has a binding score of -27.7 kj/mol formed conventional hydrogen bond involving the amino acid lys296 and the amino acid which formed carbon hydrogen bond tyr398, arg42 with the oxygen of pyridine ring and with the hydrogen of imidazole ring. a cc ep te d m an us cr ip t 16 ejaz et al. figure 7. 3d binding interactions of selected compounds within the active pocket of mao-b seesar analysis seesar analysis of the best derivative 3-b was executed which anticipate virtual display of binding interactions. green colored coronas were used to favorably present structural elements of active compounds, whereas red colored coronas are used to present elements that had a negative effect on binding interactions. a colorless corona was used to shade structural elements that made zero contribution. corona size reflects the contribution of a structural element. seesar visualizations of potent derivative 3-b is given in figure 8. hyde energies of favorable coronas (green colored) for 3-b was -5.4 and -5.3 kj/mol. a cc ep te d m an us cr ip t novel target for alzheimer’s disease 17 figure 8. left; seesar analysis of the active pocket of mao-b. right; showing docked conformations with coronas. red colored coronas show unfavorable features whereas green colored coronas represent favorable contributions and colorless coronas are showing no contribution of structural components. conclusion to identify new bche inhibitors, which may serve as a potential lead candidate for the treatment of ad, the structure-based virtual screening method was used to examine the interactions between proteins and ligands. the pubchem database was filtered, and then screened for both ache and bche protein. furthermore, their predicted inhibition constant values and binding energy values were both correlated. the results revealed that these compounds might serve as a foundation for the future development of novel bche inhibitors. supplementary material additional data are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/12309, or from the corresponding author on request. acknowledgements: the authors extend their appreciation to researchers supporting project number [rsp-2023/357], king saud university, riyadh, saudi arabia, for funding this research. a cc ep te d m an us cr ip t https://www.shd-pub.org.rs/index.php/jscs/article/view/12309 18 ejaz et al. conflict of interest: no conflicts of interest have been disclosed by the authors. competing interest: the authors declare no competing interests. и з в о д рачунарски потпомогнут приступ за идентификацију наводећих (lead) молекула за инхибиторе холинестераза и моноамин оксидаза: нове мете за третирање алцхајмерове болести syeda abida ejaz1, mubashir aziz1, ammara fayyaz1, tanveer a. wani2, and seema zargar3 1department of pharmaceutical chemistry, faculty of pharmacy, the islamia university of bahawalpur, bahawalpur 63100, pakistan, 2department of pharmaceutical chemistry, college of pharmacy, king saud univeristy, p.o.box 2457, riyadh 11451, saudi arabia, and 3department of biochemistry, college of science, king saud univeristy, p.o. box 22452, riyadh 11451, saudi arabia молекулски докинг је обећавајућа и поуздана технологија за откривање наводећих једињења путем виртуелног скрининга. поред тога што дозвољава тестирање великог броја једињења, он такође допушта одређивање како одабрана једињења инхибишу циљани протеин/рецептор на бази оцењивачке функције и рангирања. пошто селективни инхибитори холинестеразе и моноамин оксидазе играју критичну улогу у третирању алцхајмерове болести, ово истраживање се фокусира на расветљавање механизма везивних интеракција неколико хинолинских деривата у активним местима холинестеразе (ацетилхолинестеразе (ache) и бутирилхолинестеразе (bche) и моноамин оксидазе (mao) (моноамин оксидазе a &b). као резултат ових открића, могуће је да се новоидентификовани инхибитори употребе као наводећа једињења у развоју нових инхибитора ензима за третирање специфичних болести, па тако омогуће развој нових терапијских приступа. 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https://doi.org/10.3892/mmr.2014.2040 https://doi.org/10.1016/j.ejmech.2020.112787 https://doi.org/10.1016/j.bmc.2013.02.017 https://doi.org/10.2174/1568026617666170607114232 https://doi.org/10.1038/461895a https://doi.org/10.1039/c9md00337a https://doi.org/10.2174/1568026613666131113153251 https://doi.org/10.1002/jhet.3234 https://www.biosolveit.de/seesar j. serb. chem. soc. 85 (12) 1667–1673 (2020) 1667 author index abdulagatov, i., 237 abniki, m., 1223 acer, ö., 609 acharjee, n., 765 agbaba, j., 697 ahmad, d., 265 ahmadi, m., 1405 aidarova, s., 369 akbas, e., 481 akyurek, z., 967 ali akyüz, a., 967 ali, f., 493, 661 ali, j., 265 ali, s., 203, 751 alipaşa ayas, a., 559 aliyev, a., 237 allahabadi, h., 505 allahverdiyeva, s., 923 alvarez, n. m., 279 ameuru, u. s., 1253 amiri, r., 467 andjelkovic, k., 1279 annuar, m. s. m.., 353 ansari, n. f., 353 antonijevič nikolić, m., 637 antov, m. g., 1643 apostolović, t., 697 arman, i., 1137 atlagić, k., 291 avdalović, j., 421, 821, 1067 avram, s., 335 ayati, a., 1405 azizinejad, f., 1223 bajić, b., 177 bajuk-bogdanović, d., 1329 balaž, a. m. j., 25 bao, j., 111 bao, j., 857 barać, m. b., 305 barbalat, d., 89 begam, s., 53 beitollahi, h., 505 bekler, f. m., 609 bello, k. a., 1253 belošević, s. k., 453 beškoski, v., 821 beyramabadi, s. a., 37, 1033 bhavani, a. k. d., 601 bibi, i., 493, 661 bigović, d., 1553 bikić, v., 1329 bilal, m., 265 bisesti, f. m., 125, 547 blagojev, n. t., 1643 blažić, m. b., 25 bojić, a. lj., 1185 bora, a., 335 borota, a., 335 bouakkadia, a., 467 bozorgmehr, m. r., 1033 brahmchari, g., 53 branimir pavlić, b., 9 branković, s. r., 141 brčić karačonji, i., 1011 brkić, d., 979 brugnoni, l. i., 1317 bučko, s., 369 bulatović, s.s., 1067 bursić, v. p., 809 bussemer, c., 237 cabello, l. o., 279 carvajal, m. t. l., 279 chafi, n., 531 chaib, m., 531 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1668 journal of the serbian chemical society chakroun, i., 1 chaudhari, p. k., 1357 chebotarev, a., 89 chen, q., 191 ciobotă, v., 125 crevar sakač, m. a., 897 crisan, l., 335 curpan, r., 335 červenski, j., 623 čobeljić, b., 1279 čokeša, dj. m., 406 čović, j. s., 1185 dabbagh, r., 1083 dabetić, n., 439 dai, x., 671 dalila mtat, d., 1 damjanović vasilić, lj., 1329 dang, c.-h., 155 dang, v.-s., 155 danojević, d., 623 dekanski, a. b., 1237 dhandapani, m., 215 dharmadhikari, s., 1357 djokic, j. m., 163 djonlagić, j., 1197 dodić, s., 177 dramićanin, a. m., 305 dramićanin, m. d., 1475 dražić, b., 637 drinić, z., 1553 drmanić, s., 979 dubovyi, v., 89 đorđević, b. s., 1303 đorđević, i. s., 1429 đorđević, t., 885 đuriš, j., 439 džodan, đ., 1329 elkady, m. f., 381 el-shazly, a. h., 381 erdogan, a., 481 espinoza, i., 547 etinski, m. r., 1021 faraj, m. a. m., 227, 1489 farajtabar, a., 227 fethiye karsli baydere, f. k., 559 filipović, j. s., 737 filipović, v. s., 737 fischer, r., 25 fraj, j., 369 francuz, j., 1539 fuladgar, a. m., 687 fuseini, m., 381 gadžurić, s., 291 gagić, t., 97, 867 gajić, g. m., 141 gajić-kvaščev, m. d., 406 galaguz, v., 1047 gao, y., 909 garófalo-villalta, s., 125 gáspár, i. l., 1629 gašić, u., 1011 ghafourian, h., 1083 ghorbani, p., 37 giester, g., 885 gokmen, f. o., 939 golic, n. e., 163 gondudey, s., 1357 goodarzi, e., 79 gorjanović, s. ž., 517 grabež, s., 1539 grbović, f. j., 141 gruden, m., 1279, 1577 gungor, a., 967 guo, y., 191 güven, r. g., 609 guerrero, l. r., 547 gulab, h., 203 haleem, m. a., 203 halimehjani, a. z., 1253 halip, l., 335 hassel, e., 237 hatamjafari, f., 1445 he, d., 1265 heidelberg, t., 353 homayouni-tabrizi, m., 37 hou, h., 909 hussain, n., 265 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. vol. 85: author index 1669 ickovski, j. d., 575, 1565 ignjatović, n. l., 1383 iličić, m. d., 1629 ilić, m. v., 421, 1067 iqbal, m., 203, 751 irandoukht, a., 1345 isakovski, m. k., 697 ismail, n. h., 353 ivaniš, g., 237 ivanović, m. d., 711 ivanovska, a. m., 1621 jaćimović, ž. k., 453, 885 jadhav, s., 251 jadranin, m. b., 721 jafarzadeh, k., 795 jakovljević, d. m., 721 jaspal, d., 251 jentzsch, p. v., 125, 547 jeremic, s., 1507 jevtić, i. i., 711 jevtić-mučibabić, r., 177 jiao, c., 909 jin song gu, j. s., 1175 joksimović, k., 421 jovančićević, b., 1067, 1489 jovanović, m., 177 jovanović, o. p., 1001 jovanović, u. d., 406 јovanović-šanta, s., 291 jović, b., 697 ju, x., 651 jugović, d., 1383 jurič, a., 1011 jurica, k., 1011 kaluđerović, l. m., 809 kamatchi, n., 321 kamran, a. w., 751 karaman, m., 291 katona, j., 369 kemal güven, k., 609 keškić, t., 1279 keskin, e., 923 khoramnejhadian, s., 1083 kijevčanin, m. lj., 237, 1303 klaus, a. s., 721 klochkova, a., 89 knez, ž., 97, 867 knudsen, t. š., 1489 kochkodan, o., 781 kocić-tanackov, s. d., 1149 kojić, p. s., 9, 1149 kojić, v., 291, 1539 korduban, o., 1047 kosović, m., 885 kostić, a. ž., 305 kostić, d. a., 575 kostić, m. m., 1621 kostić-rajačić, s. v., 711 košutić, m. b., 737 kousar, t., 493 kozarski, m. s., 721 kukić, d. v., 1643 kumar, a. k., 53, 601 kumar, a., 53 kumar, c. n. s. s. p., 1129 latinović, n., 885 lazarevic, m. n., 163 lazović, s., 831 le, t. t.-v., 155 ledezma, o. e. v., 279 lehr, i. l., 1317 leovac, v. m., 453 li, j., 1055 li, q., 671 li, w., 1055 li, z., 111, 857 libowitzky, e., 885 lilić, j. a., 406 liu, h., 1055 liu, l., 1265 liu, w., 909 liu, y., 191 lješević, m., 821 lončarski, m., 697 loperena, a. p., 1317 lopez, a. d. f., 1317 lópez-orozco, w., 1291 loupassaki, s., 517 luo, j., 1055 ma, j., 1463 ma, x., 1463 madram, a. r., 795 maksin, v., 781 maletaškić, j., 1197 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1670 journal of the serbian chemical society malovanyi, s., 1047 manojlović, j. ž., 67 mansour, h. b., 1 marić, n., 1067 marinković, a., 979 markov, s. l., 1417 marković, m. m., 406 marković, n., 885 marković, s., 1383 marković, z. m., 1095 martìnez-casares r. m., 279 medić-pap, s., 623 medina-espinosa, t., 125 mehrez, a., 1 méndez, h. i. p., 279 mendoza-huizar, l. h., 1163, 1291 meng yao wu, m. y., 1175 menković, n., 1553 mentus, s., 1329 merdoud, a., 531 meseldžija, s. 1371 meshram, s., 953 mesli, a., 531 messadi, d., 467 miar, m., 1445 mihajlovic, s. b., 163 mihajlovski, k., 1475 mijatović, n. n., 1605 mikhailovskaya, t., 427 miletić, s., 421, 821 milić, j., 421 miličić, lj. r., 1605 milinčić, d. d., 305 milinković budinčić, j., 369 miljkovic, dj. m., 163 milojković-opsenica, d., 1011 milovanović, b. ž., 1021 milovanović, i. lj., 1149 milovanovic, j., 1507 mirković, i., 1197 mirza, b., 79 miškov-pajić, v., 453 misra, n., 53 mitić, d., 1279, 1577 mitić, m. n., 575 mitić, z. s., 1001 mitrović, t. đ., 831 moghimi, a., 1223 mohamed, s. m. d. s., 353 mohammadi, s. z., 505 mojicevic, m., 1507 morsali, a., 1033 mouffok, m., 531 muammer çalik, m., 559 mudrić, j., 1553 naz, m. y.., 967 nešić, a. 1371 nguyen, h. t.-a., 155 nguyen, t.-d., 155 ni, z., 857 nikić, j., 697 nikodinovic-runic, j., 1507 nikolić, j. b., 979 nikolić, m. s., 1197 nikolić, n. d., 347 nikolovski, z., 1489 nikšić, m. p., 721 nkeonye, p. o., 1253 novaković, s., 885 novković, n. đ., 737 nurjamal, k., 53 nytoft, h. p., 1489 oancea, s., 845 oba, a. s., 279 odović, j. v., 897 okwundu, o. s., 381 oliaey, a. r., 1445 olosutean, h., 845 onjia, a. 1371 orouj, r., 1345 ostafe, r. v., 25 ostojić, s., 1197 ozbay, n., 939 pagilla, s., 601 palaniswamy, r. v., 215 palić, i. r., 575 pan, x., 1265 panov, e., 1047 pastor, f. t., 517 pauker, c. s., 547 pavić, a. b., 1591 pavlović, j. lj., 575 pavlović, p. ž., 141 penjišević, j. z., 711 perić-grujić, a., 831 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. vol. 85: author index 1671 periyasamy, a., 321 perju, m., 845 perva-uzunalić, a., 97 pešić, m. b., 305 petković, m. m., 1021 petrović, g. m., 575, 1001 petrović, j. 1371 petrović, l., 369 petrović, p., 979 petrović, z., 1197 pevec, a., 1279 pezo, l. l., 9, 517, 1149, 1417, 1605 plavšić, d. v., 1149 popovic, d. d., 163 popović, d. m., 1429 popović, n., 25 popović, s. s., 1629 popsavin, m., 1539 popsavin, v., 1539 pouramiri, b., 1405 pouramiri, f., 1405 pourshasian, k., 1445 prodanović, j. m., 1643 prodanović, o. l., 25 prodanović, r. m., 25 prvulović, d., 623 psodorov, d. đ., 1149 psodorov, đ. p., 1149 qiao, y., 671 qin, x., 191 radaković, n. s., 1591 radanović, d., 1279 radanović, m. m., 453 radmanović, s. b., 406, 809 radojevic, d. d., 163 radovanović, l., 1475 radovanović, v. b., 737 radovanović, ž., 1475 radović, i. r., 237, 1303 rajendran, n., 321 ramanujan, v., 1129 ranđelović, m. s., 1185 ranitović, a. s., 1417 rashidzadeh, m., 1345 ražić, s., 1501 rehman, w., 493 rios-reyes, c. h., 1163, 1291 ristić, m. đ., 831 rodić, m. v., 453 rodić, m. v., 1591 rogan, j., 1475 rohani, t., 505 ruas-madiedo, p., 163 sadighi, s., 1345 sadikha, s., 1129 safarov, j., 237 saheed, r. m. m., 1489 saidman, s. b., 1317 sandoval-pauker, c., 125 savić-vujović, k. r., 711 segundo, m. a., 1501 serebryanskaya, a., 427 shafiei, f. k. t., 795 shah, n. a., 203 shah, s. w. h., 493, 661 shaikhan, b. a., 609 sharipova, a., 369 shiroudi, a., 1445 shujah, s., 265 simić, z. b., 141 simović, b., 1475 skaro bogojevic, s., 1507 snigur, d., 89 sokovic bajic, s. s., 163 soldatović, t. v., 1591 solomon, v., 321 song, l., 857 soni, a. b., 953 spasić, s., 421 spasojević, lj., 369 srebro, d. p., 711 stanić, p. b., 1591 stanisavljevic, s. m., 163 stanković, n. k., 1095 stepić, k. d., 1565 stojanović, g. s., 575, 1001, 1565 stojanović, s., 1329 subramanian, c., 215 sulaiman, s. a., 967 sun, l., 1055 sunitha, v., 601 suyambulingam, j., 215 şeyho, e., 923 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1672 journal of the serbian chemical society šantrić, lj. r., 809 šavikin, k., 1553 šćiban, m. b., 1643 škerget, m., 97, 867 škrinjar, m. m., 1149 šmit, b. m., 1591 šovljanski, o. lj., 1417 šolević knudsen, t., 1067 špirović trifunović, b. d., 305 tahir, m. n., 203, 751 tajik, s., 505 tan, f., 671 tanasković, s., 637 tančić-živanov, s., 623 tang, h., 671 temel, s., 939 terzić, a. m., 1605 tešić, o., 821 tešić, ž. lj., 305 teslić, n., 9 thach, t.-d., 155 thakur, c., 953 thakur, r. s., 1357 toauti, r., 1 todorović marković, b. m., 1095 todorović, v., 439 todorović, z. b., 1303 todosijević, m., 1553 tolinacki, m. s., 163 tomić, a. m., 1417 tomić, z. p., 809 topuzović, m. d., 141 tosti, t. b., 305 tot, a., 291 troter, d. z., 1303 troter, t. m., 1185 tubić, a., 697 turel, i., 1279 umar, m., 265 uskoković, d. p., 1383 uskoković, v., 1383 varga, d., 335 vasić, v. m., 1643 vatani, z.,687 veljković, v. b., 1303 veljović, m., 517 vidović, b., 439 villacis, w., 125 vlahović, f., 1577 vlajkov, v., 177 vojinović-ješić, lj. s., 453 volkov-husović, t. 1371 vorobyev, p., 427 vraneš, m., 291 vrvić, m. m., 821, 1067 vučković, s. m., 711 vučurović, d., 177 vučurović, v. m., 737 vujić, z. b., 897 vukelić, n., 737, 1371 vukosavljević, p., 517 vuković, n. l., 141 vunduk, j. đ., 721 wadood, a., 751 wang, b., 1463 wang, x., 111, 857 wei, z., 909 wu, j. j., 395 xiao jun tan, x. j., 1175 xing, x., 651 xu, p. y., 395 yadav, k. k., 53 yaghmaei, p., 37 yakubu, m. k., 1253 yaman, e., 939 yan, h. d., 395 yao, m., 111 yardım, y., 923 yavari, m., 1033 yi lin wang, y. l., 1175 yildiz, e., 481 yin, p., 191 yugay, o., 427 zahedi, r., 1083 zahedifar, m., 1405 zahoor, m., 751 zamanil, y., 1083 zarf, s., 1137 zarubica, a. r., 1185 zdravković, j. d., 1475 zdunić, g., 1553 zelenović, b. s., 1539 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. vol. 85: author index 1673 zhang, d., 1265 zhang, h., 111, 857 zhang, m., 909 zhang, q., 191 zhao, s., 651 zhao, y., 651 zhou, y., 909 zlatanović, s., 517 zlatar, m., 1279, 1577 zlatović, m., 1577 zrnić ćirić, m., 439 zuñiga-trejo, h., 1163 žerađanin, a. n., 421, 821 živković, m. d., 1591 živković-radovanović, v., 453 živojinović, d. z., 1605 žižak, ž., 637 subject index of vol. 85 and list of referees in 2020 are given in the electronic form at the internet address of the journal of the serbian chemical society: http://www.shd.org.rs/jscs end of volume 85. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice whole grain phenolics and antioxidant activity of triticum cultivars and wild accessions j. serb. chem. soc. 81 (5) 499–508 (2016) udc 633.11+581.144.9:547.56’914:547.972: jscs–4863 547.98:615.279 original scientific paper 499 whole grain phenolics and antioxidant activity of triticum cultivars and wild accessions djordje malenčić1, biljana kiprovski2*, vojislava bursić1, gorica vuković3, nikola hristov2 and ankica kondić-špika2 1faculty of agriculture, university of novi sad, trg dositeja obradovića 8, 21000 novi sad, serbia, 2institute of field and vegetables crops, maksima gorkog 30, 21000 novi sad, serbia and 3institute of public health of belgrade, bulevar despota stefana 54, 11000 belgrade, serbia (received 21 october 2015, revised 23 january, accepted 29 january 2016) abstract: the contents of soluble free phenolics (sp), pvpp-bound tannins (pvpp-p), flavonoids (tf) and proanthocyanidins (pa), as well as the dpph -radical scavenging capability, of the whole grain of 17 different triticum spp. accessions were analyzed. the contents of sp ranged from 976 to 2927 μg ce g-1, of pvpp-p from 335 to 1412 μg ce g-1 and of tf from 0.16 to 1.12 µg re g-1 of dry whole grain. the presence of pa in the whole grains was not established. the values for dpph-radical scavenging activity were rather moderate, under 40 % of neutralized radicals. a positive correlation between dpph-activity and analyzed phenolics was recorded in twelve of the seventeen genotypes (r from 0.73 to 0.99). trans-cinnamic acid was the major compound among acids and quercetin among flavonoids found in the wheat varieties. the data obtained should enable the selection of the accessions rich in biologically active compounds that could further be processed into functional food with possible health benefits associated with higher antioxidant properties. keywords: wheat; polyphenolics; grain; tannins; flavonoids; antioxidants. introduction wheat (triticum aestivum l.) is an important agricultural crop and a primary food ingredient worldwide. its products are essential components of the human diet, mainly because of the energy they provide, due to their high carbohydrate contents. wheat and wheat-based food ingredients rich in natural antioxidants can serve as a basis for the development of functional foods. growing evidence indicates that the intake of whole wheat foods may be associated with many health benefits, which are attributed to bioactive factors, such as non-digestible * corresponding author. e-mail: bkiprovski@gmail.com doi: 10.2298/jsc151021013m 500 malencić et al. carbohydrates and phytochemicals with antioxidant properties.1 many phenolic classes found in plant tissues (in addition to tocopherols) are potential antioxidants: flavonoids, tannins, proanthocyanidins, anthocyanins and lignin precursors may all act as scavenging compounds for reactive oxygen species (ros).2 phenolic acids, as well as their associated antioxidant activity, have been widely studied in recent years.3 along with flavonoids, phenolic acids represent the most common form of phenolic compounds found in whole grains. they are among the major and most complex groups of phytochemicals with a number of types that exist as soluble free compounds, soluble conjugates that are esterified to sugars and other low molecular mass compounds, and insoluble bound forms.4 however, the crucial obstacle to the use of these compounds is their bioavailability, which is determined by their bioaccessibility. mateo anson et al.5 reported that only the free and conjugated forms phenolic acids in breads are bioaccessible, due to their solubility. the aim of this study was to examine the content of phenolics, the antioxidant activity and the composition free phenolic acids of the whole grain of triticum spp. accessions growing in serbia. the data obtained should enable the selection of the genotypes rich in biologically active compounds that could be further processed as functional food. experimental materials thirteen samples of the whole grain of wild relatives of winter wheat from the gene-bank collection of the institute of field and vegetable crops, novi sad were analyzed and compared to four commercially grown winter wheat cultivars, i.e., pobeda, simonida, ns 40s (triticum aestivum) and nirvana (t. spelta). the study was realized at the experimental field of the institute of field and vegetable crops, novi sad, serbia (45°33′n, 19°85′e, 82 m altitude). the location is characterized by semi-arid conditions with dry, hot springs and summers, neutral autumns and moderately cold winters. the soil type was chernozem chernic.6 the seeds were sown in a randomized complete block design with three replicates. each plot consisted of 6 rows, 15 cm apart and 2.2 m long (the harvested area was 2 m2). seedling density was 350 seeds m-2. all the genotypes were sown in the middle of october (optimum sowing date) and plants reached maturity in late june 2010. weeds were controlled by hand. the grains of all seventeen genotypes were collected at the full maturity stage for the in vitro experiments. the collected plant material was dried in a shaded and well-ventilated place and kept refrigerated (–20 °c) in dark all-glass containers until extracted. extraction of soluble free phenolic compounds plant material (1 g of whole grain per sample) was ground to a fine powder in a mill and extracted for 20 min under sonication in an ultrasonic bath at ambient temperature (1 g:50 ml). soluble free phenolics, tannins and proanthocyanidins were determined in 70 % aqueous acetone extracts, and the total flavonoids in meoh:h2o:ch3cooh (volume ratio of 140:50:10) extracts. the dpph-radical scavenging activity was assayed in absolute etoh grain extracts. the extracts were rapidly vacuum-filtered through a sintered glass funnel and kept refrigerated until assayed. grain phenolics of triticum spp. 501 determination of soluble free phenolics, pvpp-bound tannins, flavonoids and proanthocyanidins content acetone-soluble phenolics, soluble free phenolics (sp) were determined by the folin– –ciocalteu procedure.7 aliquots (0.1 ml) of aqueous acetone extracts were transferred into test tubes and their volumes were made up to 0.5 ml with distilled water. after addition of folin–ciocalteu reagent (0.25 ml) and 20 % aqueous sodium carbonate solution (1.25 ml), the tubes were vortexed and after 40 min, the absorbance of the resulting blue-colored mixture was recorded at 725 nm against a blank containing only extraction solvent (0.1 ml). the amount of sp was calculated as the catechin equivalent from the calibration curve of catechin standard solutions (covering the concentration range between 0.1 and 1.0 mg ml-1) and expressed as µg catechin equivalents per g dry whole grain (µg ce g-1 d.w.g.). the content of poly(vinylpyrrolidone)-bound phenolics (pvpp-p) was determined by the folin–ciocalteu procedure as above, after removal of tannins by adsorption on an insoluble matrix (pvpp). insoluble, cross-linked pvpp (sigma, germany; 0.1 g) was weighed into test tubes and the aqueous acetone extracts (1 ml) added. after 15 min at 4 °c, the tubes were vortexed and centrifuged for 10 min at 4350g. aliquots of supernatant (0.1 ml) were transferred into test tubes and non-absorbed phenolics determined as described. the calculated values were subtracted from the sp contents and the pvpp-p content is expressed as µg ce g-1 d.w.g, for the determination of total flavonoids (tf), the extracts (2.5 ml) were transferred into 50 ml volumetric flasks and their volumes made up with water. to each 10 ml of analysis solution, water (2 ml) and alcl3 reagent (0.13 g crystalline aluminum chloride and 0.40 g crystalline sodium acetate dissolved in 100 ml of extraction solvent, 5 ml) were added and the absorbances were recorded at 430 nm against a blank (10 ml of analyzed solution plus 5 ml of water). the amount of flavonoids was calculated as the rutin equivalent (re) from the calibration curve of rutin standard solutions and are expressed as µg re g-1 d.w.g.8 proanthocyanidins (pa) were determined by a butanol–hcl assay.7 in brief, aliquots of prepared extracts (0.5 ml) were transferred into test tubes. after addition of butanol–hcl reagent (volume ratio of 95:5; 3 ml) and 2 % ferric reagent (2 % ferric ammonium sulfate in 2 m hcl, 0.1 ml), the test tubes were vortexed and placed in a boiling water-bath for 60 min. after cooling, the absorbances were recorded at 550 nm against a blank containing solvent (0.5 ml) instead of the extract. proanthocyanidins are expressed as µg leucoanthocyanidin per g d.w.g., assuming that the specific absorbance of leucoanthocyanidin was at 460 nm. antioxidant activity (dpph-test) for this investigation, the total potential antioxidant activity of the investigated whole grain extracts was assessed based on their scavenging of 1,1-diphenyl-2-picrylhydrazyl (dpph) free radicals.9 one ml of the absolute etoh solution of the sample (1:10, w/v) was mixed with an ethanolic 0.5 mmol dpph solution (0.5 ml) and 0.1 mol acetate buffer (ph 5.5, 1 ml). after standing for 30 min, the absorbance of the mixture was measured at 517 nm against a blank containing absolute etoh (0.5 ml) instead of a sample aliquot. the dpphradical scavenging activity is expressed as % of the control. separation and identification of phenolic acids and flavonoids all solvents used were of chromatographic grade and were obtained from j. t. baker (deventer, netherlands). the analytical standards used in the research were gallic acid (99.9 %), chlorogenic acid (95.0 %), trans-cinnamic acid (99.0 %), o-coumaric acid (97.0 %), p-coumaric acid (98.0 %), caffeic acid (98.0 %), ferulic acid (99.0 %), (+)-catechin hydrate (96.0 %), quercetin (98.0 %) and kaempferol (97.0 %), all manufactured by sigma–aldrich. 502 malencić et al. acetic acid was of “pure for analysis” grade from carl roth. the basic standard solutions were prepared by dissolving an analytical standard in methanol while the working solution, i.e., a mixture of the studied phenolic compounds, was obtained by mixing and diluting the basic standards with mobile phase resulting in a final mass concentration of 10 μg ml-1. the composite mixtures of all phenol acids at appropriate concentrations were used to spike samples in recovery experiments. the extraction of phenolic compounds was performed from 0.5 g of the sample with the addition of 10 ml of etoh at a constant 60 °c temperature.10,11 the chromatographic separation for phenolic compounds was achieved using an agilent 1100 (agilent technologies, usa) hplc system with a binary pump and diode array detector (dad). the phenolic acids were separated on a zorbax sb-aq column (5 μm particle size: 4.6 × 250 mm, agilent) and the other phenolic compounds on a zorbax sb-c18 column (5 μm particle size: 3.0 mm×250 mm, agilent). the extracts were filtered through 0.45-µm syringe filters and directly injected through a 20 µl fixed loop onto the column. two methods were used for the separation of the phenolic compounds. the analysis of phenolic acids was performed in the negative ionization mode. an agilent 1200 system (agilent technologies, usa) with a binary pump was used. this was equipped with a reversed-phase c18 analytical column of 150 mm×4.6 mm and 1.8 μm particle size (agilent zorbax eclipse xdbc18). the mobile phases were 0.1 % formic acid in methanol (solvent a) and 0.1 % formic acid in milli-q water (solvent b). the gradient was 0 min (80 % b), 10 min (50 % b), 20 min (5 % b), 24 min (0 % b), and 25 min (80 % b), at a flow rate 0.6 ml min-1. an agilent 6410 triple-quad lc/ms and agilent masshunter data acquisition system was applied for the mass spectrometric analysis. qualitative analysis and quantitative analysis software were used for method development and data acquisition. the linearity and precision values for all the tested phenolic compounds are presented in table i. table i. phenolic compounds analyzed by lc–ms/ms and some of their ms and analytical performance characteristics; rt – retention time, frag – fragmentor, ce – collision energy, loq – limits of quantification, r2 – linearity, rsd – relative standard deviation phenolic compound rt min precursor→product ion frag v ce v loq µg ml-1 r 2 rsd % gallic acid 1.65 169→125 110 10 0.1 0.9969 11.13 chlorogenic acid 4.01 353→190 100 10 0.1 0.9945 8.65 trans-cinamic acid 13.27 147→147 100 10 0.1 0.9816 10.79 o-coumaric acid 7.25 163→117 163→119 100 100 25 10 0.1 0.9954 12.29 p-coumaric acid 9.05 163→93 163→119 100 100 10 10 0.1 0.9941 15.6 caffeic acid 5.15 179→135 100 10 0.1 0.9918 11.62 ferulic acid 10.16 193→134 193→177.5 100 100 10 10 0.1 0.9972 12.10 (+)-catechin 3.32 289→205 289→245 135 120 10 10 0.1 0.9941 9.94 quercetin 11.62 301→151 301→179 135 100 10 10 0.1 0.9944 11.79 kaempferol 14.89 285→169 285→257 100 100 25 15 0.1 0.9920 6.79 grain phenolics of triticum spp. 503 statistical analyses values of the biochemical parameters are expressed as means ± standard error of determinations made in triplicates. the correlations between antioxidant activity and investigated phenolics contents are expressed as correlation coefficients (r) and tested for significance by the t-test (p < 0.05). to discover natural groupings of the samples, cluster analysis was performed with unweighted pair-group average analysis using the euclidean distance. all statistical analyses were performed using statistica for windows version 11.0. results and discussion the content of sp ranged from 976 to 2927 μg ce g–1 d.w.g. the highest levels of all phenolic classes investigated were recorded in the whole grain of wild triticum accessions (genotype no. 17 (triticum l. var. vulga i), followed by genotype no. 15 (t. dicoccum var. inerne d), 16 (t. dicoccum sherik var. lig) and 6 (t. macha 1)), while the sp contents in the wheat cultivars (particularly genotype no. 3) were significantly lower (table ii). in other samples, the sp levels were lower and rather uniform being close to 1300 μg ce g–1 d.w.g., on average. table ii. the content of soluble free (sp) and pvpp-bound (pvpp-p) phenolics, flavonoids (tf) and dpph scavenging activity in wheat cultivars and wild accessions ( x sem± ) genotype no. genotype spa pvpp-pa tfb dpphc 1 pobeda (t. aestivum) 1368.8±105.0 480.5±140.4 0.16±0.04 31.5±4.0 2 simonida (t. aestivum) 1150.4±38.5 334.9±14.5 0.60±0.11 31.4±2.2 3 ns 40s (t. aestivum) 975.6±38.5 364.0±14.5 0.81±0.04 29.5±4.0 4 nirvana (t. spelta) 1397.9±151.3 946.5±162.1 0.28±0.01 31.8±1.5 5 t. zhuhovky 1427.1±52.5 1033.9±145.6 0.53±0.01 37.2±2.5 6 t. macha 1 1995.0±267.3 1106.7±317.3 0.47±0.01 39.9±2.3 7 t. macha 2 1237.8±95.5 800.9±58.2 0.56±0.01 31.6±4.0 8 t. dicoccoides 1 1296.0±52.5 815.5±95.5 1.12±0.01 29.2±3.4 9 t. dicoccoides 2 1368.8±124.4 815.5±20.4 0.37±0.01 33.6±5.8 10 t. turgidum var. nigrobarbatum 1310.6±133.5 655.3±151.3 0.58±0.01 31.9±0.2 11 t. araraticum 1033.9±138.9 902.8±177.1 0.67±0.01 33.1±0.5 12 t. durum var. caerulescens 1689.2±58.2 1412.5±177.1 0.79±0.01 31.3±2.5 13 t. polonicum var. levissimum 1397.9±50.4 1092.1±50.4 0.74±0.01 31.2±1.2 14 t. dicoccum var. farum ja 1397.9±90.9 1092.1±115.5 1.00±0.02 33.8±2.4 15. t. dicoccum var. inerne d 2839.6±87.4 1135.8±87.4 0.67±0.01 32.2±1.3 16 t. dicoccum sherik var. lig 2009.6±115.5 640.7±52.5 0.60±0.01 29.0±1.6 17 triticum l. var. vulga i 2927.0±248.4 1310.6±341.2 0.33±0.01 38.5±1.5 aexpressed as µg catechin g-1 dry whole grain; bexpressed as µg rutin g-1 dry whole grain; cexpressed as % of neutralized dpph free radicals the affinity of pvpp is proportional to the number of phenolic hydroxyl groups, since the adsorption of polyphenols by pvpp is through hydrogen bonding between the proton donor from the polyphenol and the carbonyl group from 504 malencić et al. pvpp.12 the content of pvpp-p ranged from 335 to 1413 μg ce g–1 d.w.g. (table ii). the lowest amount was recorded in the wheat cultivars (genotypes no. 1–3), which also showed low sp contents. the highest pvpp-p content was detected in t. durum var. caerulescens. according to makkar et al.,7 pvpp strongly binds with tannins. contrary to phenolic acids, flavanols (epicatechin and catechin) and flavonols (quercetin and kaempferol) have higher affinities toward pvpp, especially at lower ph value and in the aglycone form.13 this implies that pvpp-bound phenolics in the present research could belong to these groups of phenolics (flavanols and flavonols). the presence of pa in the whole grain of the specimens examined in this study was not established, which is in agreement with the results of žilić et al.14 for bread and durum wheat. compared to some other cultivated plants, such as maize and soybean,15,16 the wheat whole grains were poor in flavonoids. the tf content ranged from 0.16 to 1.12 μg re g–1 d.w.g. the highest content was recorded in the genotype no. 8 (t. dicoccoides 1), followed by the genotype no. 14 (t. dicoccum var. farum ja, table ii). the structure of phenolic compounds is the key determinant of their antioxidant activity, since antioxidant activity of phenolic acids and their ester derivates depends on the number of hydroxyl groups in the molecule that are affected by steric hindrance from their carboxylate group.17 the dpph-radical scavenging activity is a measure of the non-enzymatic antioxidant activity. the dpph values for the investigated extracts varied in the range between 29.0 % (t. dicoccum sherik var. lig) and 39.9 % (t. macha 1). having a high content of all the investigated phenolic classes, the whole grain of samples t. macha 1, triticum l. var. vulga and t. dicoccum var. inerne d expressed the highest dpph-radical scavenging activity among the tested genotypes. interest in grain phenolics has increased in recent years because of their activity as antioxidants. as the total phenolic content and antioxidant activity were shown to be highly correlated,18 the correlation between the results of the antioxidant activity test and the contents of phenolics was tested in this study. positive correlations between the dpph values and sp contents were established in the genotypes no. 1, 3, 4, 6, 9, 11 and 14 (r from 0.85 to 0.99) and it was significant in the t. macha 1 genotype (r = 0.99). the genotypes no. 1, 3, 4, 6, 11 and 14 exhibited positive correlations between the dpph values and the contents of pvpp-p (r from 0.77 to 0.95), while the genotypes no. 3–6, 9, 12 and 17 showed a positive correlation between the dpph values and tf content (r from 0.73 to 0.99). significant correlations between the dpph values and the pvpp-p and tf contents were recorded only in nirvana (t. spelta, r = 0.99) and ns 40s (t. aestivum, r = 0.99). a tendency towards grouping of the genotypes in accordance with phenolic contents and antioxidant activity is graphically presented by the dendrogram in fig. 1. the first, phenolics-high group, includes the genotypes with sp contents grain phenolics of triticum spp. 505 above 2000 μg ce g–1 d.w.g. (triticum l. var. vulga i and t. dicoccum var. inerne d), which were clearly separated from the other genotypes. the second, the intermediate one, includes those with sp content between 1000 and 2000 μg ce g–1 d.w.g. finally, the phenolics-low group includes the genotypes that contain less than 1000 μg ce g–1 d.w.g. of sp (samples no. 2, 3 and 11). the grouping based on the sp content was made to contribute to a better classification. despite the difference in the sp contents, it could be clearly distinguished that the cultivars (sample nos. 1–3) were separated from the rest of the investigated genotypes and sample no. 11 with a low sp content was connected with the intermediate group. fig. 1. dendrogram of the results of a cluster analysis using the content of phenolics and antioxidant activity as variables in the estimated triticum spp. genotypes. for the genotype codes see table ii (l – low, i – intermediate, h – high (eclipse) content of phenolics). the free phenolic acids and flavonoid composition of the extracts from the wheat whole grain preparations identified by hplc are given in table iii. the major free acids in the extracts of the cultivars and wild accessions were trans -cinnamic and gallic acid (3,4,5-trihydroxybenzoic acid). żuchowski et al.,19 reported that ferulic acid (3-methoxy-4-hydroxycinnamic acid) was the predominant phenolic acid in the grain of the wheat varieties they tested. in addition, they detected, in low amounts, other phenolic acids, i.e., sinapic acid (4-hydroxy -3,5-dimethoxycinnamic acid), p-coumaric acid (4-hydroxycinnamic acid), vanil506 malencić et al. lic acid (4-hydroxy-3-methoxybenzoic acid) and p-hydroxybenzoic acid. cinnamic acid derivatives, such as p-coumaric and ferulic acids, are known to be important components of cell walls of cereals, where they dominantly occur in bound form to polymers of the wall.20–23 contrary to this, the contents of p-hydroxybenzoic and sinapic acids were comparable in the inner and outer aleurone layers, as they are mainly located in the aleurone intracellular compartment, and are mostly found in conjugated form.18,22 for the whole grain flour samples, the flavonoids catechin and apigenin (red sorghum), apigenin (white sorghum) and quercetin occurred in higher quantities compared with the other identified flavonoids. overall, most of the flavonoids identified in whole grain flour were not detected in the cell wall preparation. the present results also confirmed presence of quercetin in one of t. aestivum cultivars (ns 40s), as well as in all the investigated t. dicoccum accessions (table iii). if the results from the individual and group phenolics are compared, it could be proposed that the antioxidant capacity of the t. dicoccum accessions mostly depended on and correlates with the flavonols content, since the contents of phenolic acids were quite low compared to those of the other accessions (tables ii and iii). table iii. contents of free monomeric compounds in the whole grain of the wheat cultivars and their wild relatives ( x sem± , expressed as µg g-1 dry whole grain, nd – not detected) genotype gallic acid trans-cin-namic acid o-coumaric acid p-coumaric acid quercetin pobeda (t. aestivum) nd 1.27±0.00 nd 0.59±0.01 nd simonida (t. aestivum) 2.05±0.02 1.59±0.00 nd 0.44±0.01 nd ns 40s (t. aestivum) 2.23±0.02 0.64±0.00 nd nd 4.0±0.05 nirvana (t. spelta) 2.77±0.01 3.5±0.05 nd nd nd t. zhuhovky 2.82±0.00 3.82±0.01 1.23±0.00 nd nd t. macha 1 3.40±0.05 5.09±0.03 1.49±0.00 nd nd t. macha 2 2.57±0.01 2.86±0.01 nd 0.79±0.00 nd t. dicoccoides 1 3.52±0.01 6.36±0.01 nd nd nd t. dicoccoides 2 2.94±0.01 4.13±0.02 nd nd nd t. turgidum var. nigrobarbatum 2.67±0.01 0.32±0.02 nd nd nd t. araraticum 3.03±0.04 3.18±0.03 nd nd nd t. durum var. caerulescens 2.29±0.06 0.64±0.00 nd 0.91±0.01 nd t. polonicum var. levissimum 3.54±0.04 nd nd nd nd t. dicoccum var. farum ja nd 1.59±0.00 nd nd 2.87±0.01 t. dicoccum var. inerne d 3.07±0.04 4.77±0.01 nd nd 1.44±0.02 t. dicoccum sherik var. lig nd nd nd 0.72±0.00 0.70±0.02 triticum l. var. vulga i 3.39±0.03 1.53±0.01 nd nd nd many studies have been devoted to the evaluation of the total antioxidant capacity of foods, its correlation with the phenolic content, and the evaluation of grain phenolics of triticum spp. 507 the changes with cultivation and processing factors. however, recent evidence has indicated that the effects of antioxidants are less relevant than expected, as the antioxidant phenolics are poorly absorbed and extensively metabolized to non-antioxidant metabolites. in addition, many of these compounds have a very low bioavailability and never reach the tissues where they have to exert their antioxidant action in sufficient amounts. according to adam et al.,23 phenolic compounds bound to endosperm arabinoxylans might be more easily released with respect to phenolics linked to the pericarp or the outer aleurone layer. moreover, zaupa et al.24 explained that hydroxybenzoic acids presented the highest bioaccessibility in all wheat fractions, likely because of their predominant free form in the undigested fractions. in the previous paper, the authors suggested the isolation and use of wheat fractions in the formulation of new cereal-based products could be a strategy for improving the offer of whole grain products. according to the results, it seems that capability to accumulate all investigated classes of phenols varies within the cultivated wheat genotypes, as well as within the wild accessions. however, there is a great and significant similarity in the phenolic contents between them, which points to the fact that there is a considerable gene pool to be explored for enhancing the quality of wheat whole grain in future selection work. the whole grain of phenolics-rich genotypes (cultivars and wild accessions) may become potentially attractive, inexpensive and readily available on a large-scale raw material for food with higher antioxidant properties. acknowledgments. the research was supported by the ministry of education, science and technological development of the republic of serbia (project no. tr-31066). the authors would like to thank the principal research fellow novica mladenov, phd, for providing the plant material. и з в о д садржај фенола и антиоксидативна активност семена гајених и дивљих врста roda triticum ђорђе маленчић1, биљана кипровски2, војислава бурсић1, горица вуковић3, никола христов2 и анкица кондић-шпика2 1 пољопривредни факултет, универзитет у новом саду, трг доситеја обрадовића 8, 21000 нови сад, 2 институт за ратарство и повртарство, максима горког 30, 21000 нови сад и 3 градски завод за јавно здравље београд, булевар деспота стефана 54, 11000 београд у екстрактима семена 17 различитих гајених и дивљих triticum spp. одређен је садржај растворљивих фенола (976–2927 μg катехина g-1 суве масе), pvpp-везујућих танина (335–1412 μg катехина g-1 суве масе), флавоноида (0,16 дo 1,12 μg рутина g-1 суве масе) и проантоцијанидина, као и антиоксидативна способност датог екстракта (% неутрализације dpph радикала). присуство проантоцијанидина у екстрактима целог семена испитиваних врста није потврђено. антиоксидативна способност ектраката била је умерена (до 40 % неутралисаних dpph радикала), а позитивна корелација између датог параметра и анализираних фенолних једињења утврђена је за 12 од 17 генотипова (r, 0,73–0,99). доминантна киселина је била trans-циметна, док је кверцетин био најдо508 malencić et al. минантнији међу флавоноидима. добијени резултати представљају значајан допринос селекцији генотипова са повећаним садржајем биолошки активних компоненти као извора потенцијалне функционалне хране. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice j. serb. chem. soc. 81 (4) 369–382 (2016) udc 542.9+547.571’551+546.562’732’472’712’: jscs–4853 66.094.3:66.094.941:548.512 original scientific paper 369 schiff base ligand derived from (±)trans-1,2-cyclohexanediamine and its cu(ii), co(ii), zn(ii) and mn(ii) complexes: synthesis, characterization, styrene oxidation and a hydrolysis study of the imine bond in the cu(ii) schiff base complex marzieh sarkheil and maryam lashanizadegan* department of chemistry, faculty of physics and chemistry, al-zahra university, p. o. box 1993893973, tehran, iran (received 18 september, revised 30 november, accepted 14 december 2015) abstract: a schiff base ligand (h2l) derived from 2′-hydroxypropiophenone and (±)trans-1,2-cyclohexanediamine was synthesized. the reactions of mcl2·xh2o (m = cu(ii), co(ii), zn(ii) and mn(ii)) with the di-schiff base ligand (h2l) were studied. when stirred with 1 equivalent of cucl2.2h2o in a solution of ethanol and chloroform, this ligand undergoes partial hydrolysis of the imino bond and the resultant tridentate ligand (hl′) immediately forms the complex[cul′cl]·3/2chcl3 (1) with an n2o coordination sphere. under the same condition, the reaction of h2l with mcl2·xh2o (m = co(ii) (3), zn(ii) (4) and mn(ii) (5)) gave the complexes [ml]·1/2chcl3·3/2h2o (3–5) with an n2o2 coordination sphere and no hydrolytic cleavage occurred. in addition, the reaction of h2l with cucl2·2h2o in thf gave the complex cul (2) with an n2o2 coordination sphere. the ligand and the complexes were characterized by ftir, uv–vis, 1h-nmr spectroscopy and elemental analysis. the homogeneous catalytic activities of complexes 1, 3 and 5 were evaluated for the oxidation of styrene using tert-butyl hydroperoxide (tbhp) as oxidant. finally, the copper(ii) complex 1 was encapsulated in the nanopores of zeolite y by the flexible ligand method (cul′–y) and its encapsulation was demonstrated in different studies. the catalytic performance of heterogeneous catalyst in the styrene oxidation with tbhp was investigated. the catalytic tests showed that the homogeneous and heterogeneous catalysts were active in the oxidation of styrene. keywords: hydrolytic cleavage, solvent effect; catalyst; homogeneous; heterogeneous. introduction during the last decades, great attention has been paid by many researchers to the schiff base ligands and their metal complexes due to their crucial role in * corresponding author. e-mail: m_lashani@alzahra.ac.ir doi:10.2298/jsc150918006s _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 370 sarkheil and lashanizadegan many fields, such as catalysis,1 optoelectronic materials,2 inhibition of corrosion,3 magnetochemistry4,5 and biological models.6 they also exhibit antibacterial7,8 and anticancer9,10 activity. various reactions have been catalyzed by transition metal schiff base complexes, such as ring opening reactions of epoxides,11 aldol condensations12,13 and oxidation.14 in the catalytic oxidation reactions, these complexes represent a very useful class of compounds because as their structures are similar to the porphyrine ring, they are good at loading oxygen and mimicking enzymes.15 especially, olefin oxidation has received considerable attention as several types of chemicals that find widespread applications in the chemical and pharmaceutical industries are produced by this method, including aldehydes, ketones, alcohols, acids and epoxides,.16 therefore, the homogeneous or heterogeneous catalytic role of several schiff base transition metal complexes, such as cu(ii),17–19 co(ii),20,21 mo(vi),22–24 v(v),25,26 ni(ii)27,28 and mn(ii)29–31 complexes in the oxidation of olefins have been extensively studied. however, homogeneous catalysts have some drawbacks, such as difficulty in separation from the product for reuse and instability at high temperatures, which preclude their industrial utilization. hence, many efforts involving encapsulation in zeolites,32,33 grafting on polymers34,35 and silica36,37 have been made to heterogenize homogeneous catalysts. entrapment of metal complexes into the supercages of zeolites is an interesting technique because of reusability, chemical and thermal stability, and improved selectivity.38 although the synthesis of schiff base metal complexes is well documented,39–41 there are some reports concerning the hydrolysis of the schiff base during complex formation. the hydrolytic cleavage of a schiff base depends on different parameters, such as solvent,42,43 nature of the metal ion,44–47 the counter anion,48,49 ph of the reaction medium50 and the nature of the carbonyl compound.51 in this study, the synthesis and characterization of the di-schiff base ligand (h2l) derived from 2′-hydroxypropiophenone and (±)-trans-1,2-cyclohexanediamine and its complexes (1–5) were investigated. in addition, the influence of solvent and metal ion on the hydrolytic behavior of the azomethine linkage (c=n) of h2l were examined. the homogeneous catalytic potential of the complexes 1, 3 and 5 in the oxidation of styrene with tert-butyl hydroperoxide (tbhp) were studied. moreover, the copper(ii) schiff base complex encapsulated in the nanopores of zeolite-y by the flexible ligand method and its catalytic performance in the oxidation of styrene was tested. experimental materials all the starting materials and solvents, except (±)trans-1,2-cyclohexanediamine (alfa aesar) and 2′-hydroxypropiophenone (across), were purchased from merck and used without further purification. the synthetic reactions and work-up were performed in open air. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ schiff base ligand derived from (±)trans-1,2-cyclohexanediamine 371 physical measurements the ir spectra (kbr discs, 500–4000 cm-1) were recorded using a bruker ftir model tensor 27 spectrometer. the far ir spectrum (csi disc, 150–700 cm-1) was recorded using a perkin–elmer, model spectrum 400 imaging system. the elemental analyses were realized in a 2400 series ii chn analyzer, perkin-elmer, usa. the uv–vis absorption spectra were recorded on a perkin–elmer lambda 35 spectrophotometer. diffuse reflectance spectra (drs) were registered on an ava spec 2048 tech spectrometer, using baso4 as the reference. the 1h-nmr spectra were recorded on a bruker 500 mhz model drx spectrometer in cdcl3 or dmso-d6 with tetramethylsilane (tms) as the internal reference. the x-ray diffraction patterns were obtained using a philips x’pert diffractometer with cokα radiation (λ = 1.78897 å). the oxidation products were analyzed by gc and gc–mass spectrometry using an agilent 6890 series with a fid detector, an hp-5 phenyl methyl siloxane capillary and an agilent 5973 network, and a mass selective detector, hp-5ms 6989 network gc system, respectively. physical and spectral data of the synthesized compounds are given in supp preparation of the ligand (h2l) an ethanolic solution (10 ml) of 2′-hydroxypropiophenone (0.150 g, 2 mmol) was added to an ethanolic solution (10 ml) of (±)trans-1,2-cyclohexanediamine (0.114 g, 1 mmol). the bright yellow solution was stirred and heated to reflux for 1 h. the mixture was kept in air to allow the solvent to evaporate, whereby yellowish crystals of the ligand were obtained. yield: 97 %. preparation of complexes 1–5 [cul′cl]·3/2chcl3 (1). this copper complex was prepared by adding an ethanolic solution (10 ml) of cucl2⋅2h2o (0.170 g, 1 mmol) to a chloroform solution (10 ml) of h2l (0.378 g, 1 mmol). the resulting mixture was stirred for about 1 h. finally, the precipitate of the complex was recovered by filtration, washed several times with absolute ethanol and dichloromethane and dried at 65 °c for 2 h. yield: 30 %. cul (2). this complex was prepared by adding a thf solution (20 ml) of h2l (0.378 g, 1 mmol) to a thf solution (10 ml) of cucl2·2h2o (0.170 g, 1 mmol). after stirring the resulting mixture for about 1 h, the precipitated complex was recovered by filtration, washed with thf and et2o and finally dried at 65 °c for 1 h. yield: 40 %. [col]·1/2chcl3·3/2h2o (3).the cobalt complex was prepared in a similar manner to 1 but using cocl2·6h2o (0.237 g, 1 mmol). yield: 70 %. [znl]·1/2chcl3·3/2h2o (4). the zinc complex was prepared in a similar manner to 1 but using zncl2 (0.136 g, 1 mmol). yield: 77 %. [mnl]·1/2chcl3·3/2h2o (5). the manganese complex was prepared in a similar manner to 1 but using mncl2·4h2o (0.197 g, 1 mmol). yield: 56 %. incorporation of copper(ii) in na–y (metal exchanged zeolite y) the cu–y was prepared using the standard procedure.52 na–zeolite y (4 g) was suspended in 100 ml distilled water that contained copper(ii) nitrate (4 mmol). the mixture was then stirred for 24 h. the solid was filtered and washed with deionized water and dried at room temperature to give a light blue powder of cu–y. cu content: 6.7 % immobilization of h2l in cu–y cu–y (0.29 g, 0.3 mmol of cu) and ligand h2l (0.67 g, 1.8 mmol) were mixed in a 1.5:1 volume ratio of acetonitrile and chloroform solution (25 ml) and the reaction mixture was refluxed for 8 h in an oil bath under constant stirring. the resulting material was taken out and _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 372 sarkheil and lashanizadegan soxhlet extracted with acetonitrile to remove the unreacted ligand from the cavities of the zeolite as well as those located on the surface of the zeolite along with neat complexes, if any. the non-complexed metal ions present in the zeolite were removed by exchanging with aqueous 0.01 m nacl solution. the resulting solid was finally washed with hot distilled water until no precipitation of agcl was observed on reaction of the filtrate with agno3 solution. the product was then dried at 150 °c for several hours until constant weight was achieved. general procedure for the homogeneous oxidation of styrene catalyzed by complexes 1, 3 and 5 all oxidation reactions were performed in 50 ml round bottom flasks equipped with a water condenser. typically, to a solution of styrene (10 mmol) and catalyst (complex 1: 0.04 mmol, complexes 3 and 5: 0.045 mmol) in ch3cn (10 ml), tbhp (for complex 1: 35 mmol, for complexes 3 and 5: 30 mmol) was added. the resulting mixture was refluxed for 6 h for complex 1 and 10 h for complexes 3 and 5. the products were identified and quantified by gc and verified by gc–ms. general procedure for the heterogeneous oxidation of styrene catalyzed by cul′–y catalyst (0.04 g), styrene (10 mmol) and tbhp (35 mmol) were mixed in 10 ml of ch3cn and the reaction mixture was refluxed with continuous stirring in an oil bath for 4 h. the products were collected at different times and identified and quantified by gc, and verified by gc–ms. results and discussion synthesis and formulation the ligand h2l was synthesized by condensation of a 1:2 mole ratio of (±)trans-1,2-cyclohexanediamine with 2′-hydroxypropiophenone (scheme 1). when h2l reacted with cucl2·2h2o in a solution of ethanol and chloroform, it underwent partial hydrolytic cleavage to form the n2o coordination sphere of complex 1. notably, a subtle change in the reaction conditions, such as changing the solvent, caused a change in the type of complex produced. thus, when h2l scheme 1. synthetic routes to the ligand (h2l) and the complexes 1–5. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ schiff base ligand derived from (±)trans-1,2-cyclohexanediamine 373 reacted with cucl2·2h2o in thf, complex 2 with a n2o2 coordination sphere was formed (scheme 1). it is likely that under the conditions of the reaction, an activated nucleophile was generated at the metal, which was responsible for the ensuing cleavage reaction.43,53 additionally, no hydrolytic cleavage was observed when h2l reacted with co(ii), zn(ii) and mn(ii) chloride salts and complexes 3–5 were obtained. ftir and uv–vis studies in the infrared spectrum of the ligand (h2l), a strong and sharp band corresponding to the azomethine group ν(c=n) appeared at 1609 cm–1. the bands at 2857 and 2931 cm–1 are indicative of the presence of the 1,2-cyclohexanediyl groups. in the ir spectrum of 1, the two primary nh2 stretching modes were seen at around 3288 and 3223 cm–1 as sharp bands (doublet) for the asymmetric and symmetric vibrations, respectively. strong bands at 2857 and 2931 cm–1 corroborated the presence of 1,2-cyclohexanediyl groups in the complex. in addition, the bands due to azomethine ν(c=n) and ν(cu–cl) were observed at 1603 and 304 cm–1,54 respectively. in the ir spectrum of complex 2, strong bands appeared at 2863 and 2936 cm–1, corresponding to the presence of 1,2-cyclohexanediyl group in the complex. the sharp band due to azomethine ν(c=n) was centered at 1603 cm–1. a comparison of the ir spectra of 1 with that of 2 provided clear evidence of the hydrolytic cleavage that had occurred in one imine bond of h2l (fig. 1). the ir spectra of complexes 3–5 showed a strong band due to the azomethine group at 1604 cm–1. moreover, the spectra of these complexes showed bands in the range 2858–2936 cm–1, corresponding to the 1,2-cyclohexanediyl group (fig. 1). the c=n stretching vibration of the complexes 1–5 showed a slightly lower frequency shift in comparison to the corresponding vibration in the spectrum of the free ligand. this indicates the involvement of azomethine nitrogen in the coordination to the metal centers.55 the band corresponding to c–o of h2l appeared at 1273 cm–1. for the synthesized complexes, the c–o band was shifted to a lower frequency and was observed in the 1257–1265 cm–1 region, indicating coordination through the phenolic oxygen.56 in the ir spectra of cul′–y, an intense band appeared at 1021 cm–1, attributable to the asymmetric stretching of the al–o–si chain of the zeolite. the symmetric stretching and bending frequency bands of the al–o–si framework of the zeolite appeared at 789 and 458 cm–1, respectively.57 the band corresponding to ν(c=n) appeared at 1580 cm–1. in addition, the bands due to the two primary nh2 stretching modes were observed at 3224 and 3288 cm–1. other bands at 2856 and 2931 cm–1 were indicative of the presence of 1,2-cyclohexanediyl groups (fig. 2). these observations confirmed the partial hydrolysis of the azomethine group in the encapsulated complex. the intensities of the peaks _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 374 sarkheil and lashanizadegan in the spectrum of the encapsulated complex were weak due to their low concentration in the zeolite matrix. the electronic absorption spectra of complexes 1–3 consist of higher energy bands at 258–400 nm which are due to π–π*or n–π* transition. the diffuse reflectance spectra of complexes 1 and 2 showed a broad d–d band with a maximum at 508 and 502 nm, respectively. this is consistent with square-planar geometry around the copper ion.58 however, the electronic spectra of 1 and 2 in dmso are entirely different and show d–d bands at 675 and 887 nm, respectively. this suggests that in solution, the solvent molecules are coordinated to metal ions.59 the cobalt(ii) complex 3 exhibited d–d transition bands at 611 and 673 nm, suggesting a tetrahedral geometry.60 in the electronic spectra of 4 and 5, absorption bands appeared in the range 260–387 nm, which may be assigned to intra-ligand or charge-transfer transitions. 1h-nmr studies of the ligand (h2l) and complex 4 in the 1h-nmr spectrum of h2l, the phenolic protons were present at 16.6 ppm. the aromatic protons were found in the range 6.74–7.45 ppm as a multiplet. the spectrum showed signals at 3.91–3.93 ppm, 2.69–2.90 ppm and 1.53– –1.76 ppm with integrations corresponding to ha, hb and hc, respectively. furthermore, the signals of the methyl protons were observed at 1.22–1.25 ppm and those of the methylene protons at 1.93–1.98 ppm. fig. 1. ir spectra of the ligand (h2l) and complexes 1–5. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ schiff base ligand derived from (±)trans-1,2-cyclohexanediamine 375 a comparison of the 1h-nmr spectrum of h2l with that of complex 4 showed the disappearance of the phenolic protons present in the free ligand, which is in agreement with a bis-deprotonation of the ligand. in the 1h-nmr spectrum of 4, the aromatic protons appeared in the range 6.72–7.53 ppm. the signals at 3.66–3.98 ppm, 2.70–2.84 ppm and 1.51–1.86 ppm were related to ha, hb and hc, respectively. the signals of the methyl and methylene protons were observed at 1.08–1.21 ppm and 2.5–2.7 ppm, respectively. xrd studies the x-ray powder diffraction patterns of cu–y and cul′–y were recorded at 2θ values between 5° and 80° (fig. 3). the xrd of cu–y and cul′–y were essentially similar except the intensities were slightly changed in the encapsulated complex. this fact indicates that the framework of the zeolite had not structurally changed during encapsulation. fig. 2. ir spectra of na–y, cu–y and cul′–y. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 376 sarkheil and lashanizadegan fig. 3. xrd patterns of cu–y and cul′–y. catalytic activity studies in the oxidation of styrene the oxidation of styrene catalyzed by complexes 1, 3, 5 and cul′–y was realized using tbhp as the oxidant. a series of blank experiments (table i) showed that the presence of both catalyst and oxidant was essential for an effective catalytic reaction. different reaction parameters, such as reaction time, reaction solvent, amount of catalyst, the nature and the amount of oxidant that may affect the conversion and selectivity of the reaction were optimized. table i. blank experiments on the catalytic oxidation of styrene; reaction conditions: styrene (10 mmol), tbhp (30 mmol), acetonitrile (10 ml); the reactions were run for 6 h under reflux entry catalyst oxidant conversion, % 1 none tbhp 0 2 none h2o2 0 3 1 none 0 4 3 none 0a 5 5 none 0a athe reaction was run for 10 h under reflux catalytic activity of 1 and cul′–y in the oxidation of styrene the influence of reaction time and nature of the solvent in the oxidation of styrene catalyzed by 1 are illustrated in fig. 4. to find the best reaction solvent, the oxidation reactions were performed in various solvents, i.e., acetonitrile, _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ schiff base ligand derived from (±)trans-1,2-cyclohexanediamine 377 1-butanol, dichloromethane and chloroform, and the highest conversion was obtained in acetonitrile, which may be due to the polarity, hydrophilicity and size of the solvent molecule of acetonitrile.61 to optimize the reaction time, the oxidation of styrene was performed for different times. it was found that 6 h was the best time for maximum conversion. increasing the time to 8 h partially increased the conversion, but decreased the selectivity of the product. fig. 4. effect of time and solvent on the oxidation of styrene by tbhp in the presence of complex 1. reaction condition: styrene (10 mmol), catalyst (0.03 mmol), tbhp (30mmol), solvent (10 ml; acetonitrile , chloroform , 1-butanol or dichloromethane ) and reflux. in order to investigate the effect of the oxidizing agent in the oxidation reaction, h2o2 and tbhp were used (table ii). in the presence of h2o2, the reactions did not proceed under reflux. table ii. the influence of kind of oxidant on the oxidation of styrene; reaction condition: styrene (10 mmol), catalyst (0.03 mmol), ch3cn (10 ml), oxidant (30 mmol), reaction time, 6 h and reflux; catalyst: 1 entry oxidant conversion, % 1 tbhp 89 2 h2o2 0 the influence of amount of catalyst has been studied in the oxidation of styrene. as seen in fig. 5a, the highest conversion (92 %) was obtained with 0.04 mmol of catalyst. different amount of oxidant (tbhp) have been used in the oxidation of styrene (fig. 5b). the results indicate that the highest conversion (100 %) was obtained at 1:3.5 molar ratio of styrene to tbhp. in order to heterogenize the homogenous catalyst, the copper(ii) schiff base complex was encapsulated in the nanopores of zeolite y by flexible ligand method. the catalytic activity data of 1 and cul′–y in the oxidation of styrene are given in table iii. the results showed that when cul′–y is used, the reaction times decreased, but no important change in the selectivity of products is observed. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 378 sarkheil and lashanizadegan fig. 5. a) the effect of amount of catalyst 1 on the oxidation of styrene. reaction condition: styrene (10 mmol), ch3cn (10 ml) and tbhp (30 mmol), reaction time, 6 h and reflux; b) the effect of amount of oxidant on the oxidation of styrene. reaction condition: styrene, 10 mmol, catalyst, 0.04 mmol and ch3cn, 10 ml, oxidant, tbhp, reaction time, 6 h and reflux. table iii. oxidation of styrene using tbhp catalyzed by 1 and cul′–y; reaction conditions: catalyst, 25 mmol, cul′–y, 0.04 g, styrene, 10 mmol, tbhp, 35 mmol, acetonitrile, 10 ml; reflux entry catalyst conversion, % selectivity, % time, h 1 1 100 63a 28b 9c 6 2 cul′–y 100 56a 44b 4 astyrene epoxide; bbenzoic acid; cbenzaldehyde catalytic activity of complexes 3 and 5 in the oxidation of styrene the oxidation of styrene, catalyzed by 3 was carried in the presence of h2o2 and tbhp. the effect of the solvent nature in the catalytic activity of 3 for oxidation of styrene has been studied (fig. 6). therefore, acetonitrile, ethanol and chloroform were used and the highest conversion was obtained in acetonitrile. as indicated in fig. 6, increasing the reaction time from 2 to 10 h increases the conversion and it was found that 10 h is the best time for maximum conversion (98 %). fig. 6. effect of time and solvent on the oxidation of styrene with tbhp in the presence of complex 3. reaction condition: styrene (10 mmol), catalyst (0.03 mmol), tbhp (35 mmol), solvent (10 ml; acetonitrile , ethanol , chloroform ) and reflux. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ schiff base ligand derived from (±)trans-1,2-cyclohexanediamine 379 in order to investigate the influence of the oxidizing agent in the oxidation reaction, tbhp and h2o2 were used (table iv). in the presence of tbhp, a higher conversion was achieved. to optimize the amount of catalyst, oxidation of styrene was performed with different amounts of 3. as indicated in fig. 7a, 0.045 mmol of catalyst proved to be sufficient for the maximum conversion. different amounts of oxidant (tbhp) were studied in the oxidation of styrene (fig. 7b). the results indicated that the best mole ratio of styrene to tbhp for the oxidation of styrene was 1:3. table iv. the influence of the kind of oxidant on the oxidation of styrene; reaction conditions: styrene, 10 mmol, catalyst, 0.03 mmol, ch3cn, 10 ml, oxidant, 35 mmol, reaction time, 10 h; reflux; catalyst: 3 entry oxidant conversion, % 1 tbhp 98 2 h2o2 0 fig. 7. a) the influence of the amount of catalyst 3 on the oxidation of styrene. reaction conditions: styrene, 10 mmol, ch3cn, 10 ml and tbhp, 35 mmol; reaction time, 10 h; reflux; b) the effect of the amount of oxidant on the oxidation of styrene. reaction conditions: styrene, 10 mmol, catalyst, 0.045 mmol and ch3cn, 10 ml; oxidant, tbhp, reaction time, 10 h; reflux. the catalytic activity of 5 was investigated under the optimized condition for 3. the results are given in table v. table v. oxidation of styrene using tbhp catalyzed by 3 and 5; reaction conditions: catalyst, 0.045 mmol, styrene, 10 mmol, tbhp, 35 mmol, acetonitrile, 10 ml, reaction time, 10 h; reflux entry catalyst conversion, % selectivity, % 1 3 100 47a 53b 2 5 40 46a 54b astyrene epoxide; bbenzoic acid _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 380 sarkheil and lashanizadegan conclusions a schiff base ligand (h2l) derived from (±)trans-1,2-cyclohexanediamine and 2′-hydroxypropiophenone was prepared. it was found that the reaction between h2l and cucl2·2h2o in ethanol and chloroform led to partial hydrolysis of h2l and complex 1 was obtained, while under the same conditions, the reaction of h2l with mcl2·xh2o (m = co(ii), zn(ii) or mn(ii)) yielded complexes 3–5 without hydrolytic cleavage of the azomethine linkage. moreover, the reaction between h2l and cucl2·2h2o in thf gave complex 2. therefore, it is reasonable to conclude that the solvent and metal ion can affect the hydrolysis of the schiff base during complex formation. complexes 1, 3 and 5 were used for the oxidation of styrene with tbhp under homogenous conditions. furthermore, the copper(ii) schiff base complex 1 encapsulated in the nanopores of zeolite y by the flexible ligand method (cul′–y) and its catalytic potential in the oxidation of styrene was examined. the oxidation of styrene catalyzed by 1, 3, 5 and cul′–y gave 100 % conversion with 63, 47, 46 and 56% selectivity for styrene epoxide, respectively. the results revealed that the homogeneous and heterogeneous catalysts were efficient in the oxidation of styrene. supplementary material physical and spectral data of the synthesized compounds are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. the financial support from alzahra university is acknowledged. и з в о д лиганд типа шифове базе добијен из (±)trans-1,2-циклохександиамина и његови бакар(ii), кобалт(ii), цинк(ii) и манган(ii) комплекси: синтеза, карактеризација, оксидација стирена и хидролиза имино везе у бакар(ii) комплексима са шифовом базом као лигандом marzieh sarkheil и maryam lashanizadegan department of chemistry, faculty of physics and chemistry, al-zahra university, p. o. box 1993893973, tehran, iran полазећи из 2′-хидроксипропиофенона и (±)trans-1,2-циклохександиамина синтетизован је лиганд типа шифове базе (h2l). изучаване су реакције овог лиганда са солима опште формуле mcl2⋅xh2o (m =cu(ii), co(ii), zn(ii) или mn(ii)). када се овај лиганд помеша са еквивалентном количином cucl2⋅2h2o у смеши етанола и хлороформа као растварача долази до парцијалне хидролизе имино везе и грађење тридентатног лиганда (hl′). овај лиганд се тренутно координује са cu(ii) јоном при чему настаје [cul′cl]⋅3/2chcl3 (1) комплекс n2o хромофоре. под истим експерименталним условима лиганд h2l у реакцији са солима mcl2⋅xh2o (m = co(ii) (3), zn(ii) (4) или mn(ii) (5)) гради комплексе n2o2 хромафоре чија је општа формула [ml]⋅1/2chcl3∙3/2h2o (3–5). нађено је да у овим реакцијама не долази до хидролитичких реакција. такође, у реакцији h2l са cucl2⋅2h2o у растварачу thf долази до формирања cul комплекса 2 са n2o2 координованим атомима. лиганд и одговарајући комплекси су окарактерисани помоћу ftir, uv–vis и 1h-nmr спектроскопских метода, _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ schiff base ligand derived from (±)trans-1,2-cyclohexanediamine 381 као и на основу резултата елементалне микроанализе. испитивана је хомогена каталитичка активност комплекса 1, 3 и 5 у реакцији оксидације стирена у присуству tert -бутил-хидропероксида (tbhp) као оксидационог средства. на крају, применом различитих метода изучавано је капсулирање cu(ii) комплекса у нанопоре зеолита y помоћу методе флексибилног лиганда (cul′–y). испитивана су каталитичка својства хетерогеног катализатора на оксидацију стирена у присуству tbhp као оксидационог средства. на основу добијених резултата може се закључити да су сви испитивани хомогени и хетерогени катализатори показали активност у реакцији оксидације стирена. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {editorial} 1131 editorial this issue of the journal of the serbian chemical society is dedicated to an accomplished scientist and excellent mentor academician slobodan milosavljević, professor emeritus at the university of belgrade – faculty of chemistry, on the occasion of his 80th birthday, in honor of his many achievements in chemistry, his contribution to university education, structural instrumental analysis and natural products chemistry at the university of belgrade. slobodan was born in belgrade on 30 december, 1941. he grew up in belgrade, where his mother was philologist and the father university professor, and where he finished the high school. he received b.s. (in 1965) from faculty of technology and metallurgy, university of belgrade, m.s. (in 1970) as well as phd (in 1974) at department of chemistry, faculty of science and mathematics (later faculty of chemistry) university of belgrade. the two years of postdoctoral studies (1974– –1976) included the synthesis of natural products, at the polytechnic of north london, with dr. a. p. johnson. the first job was in inep institute for application of nuclear energy 1965, but slobodan soon moved to institute for chemistry 1966, and finally to faculty of chemistry 1970, where stayed till retirement. he became assistant, associate, and full professor at the university of belgrade in 1970, 1986 and 1992, respectively. slobodan’s lifelong interest for structural analysis started when he joined prof jeremić at the institute for chemistry in 1966, and helped develop modern laboratory for instrumental analysis. the laboratory, later center for instrumental analysis (cia), equipped with ir, uv–vis, ms and nmr gradually became a core for further teaching and ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 1132 editorial scientific work in chemistry. main task and interest was structure determination of organic compounds. slobodan dedicated most of his energy creating better conditions for scientific work in chemistry and similar, related sciences, like biology, that use the aforementioned analytical tools. he is still involved in work of this centre assisting in solving problems concerning structure elucidation of various compounds and commercial products. as a researcher, slobodan mostly deals with investigation and interpretation of nmr and mass spectra of syntetised and natural compounds. it presents a permanent scientific interest and have started with m.s. thesis. the main research topic in last two decades was isolation and identification of natural products, the constituents of wild-growing plant species from serbia and montenegro by means of spectroscopic and chromatographic methods (1d and 2d nmr, ms, glc–ms, ir, uv–vis, hplc). lately he is involved in metabolomics investigations of plants. besides intensive scientific work (about 150 original papers and reviews), slobodan was dedicated to teaching 1979-1986 “structural instrumental methods” at the same faculty. from 1979 teaching in “organic chemistry of natural products” for the third-year undergraduate students of molecular biology at the faculty of biology, belgrade; from 2000 teaching in “structural instrumental methods ii” at the faculty of chemistry. in his very good textbook “structural instrumental methods” (in serbian), ii edition published at the faculty of chemistry (2004) slobodan treats the subjects in more than 500 pages. slobodan also served as a visiting professor, teaching structural instrumental methods at the university of niš and kragujevac. he led numerous students in their diploma (b.s.) works, m.s. theses, and ph.d. dissertations. as a member of committees, he selflessly helped students and degree candidates mentored by his colleagues. students, associates and colleagues respect slobodan as a scholar and admire him as a man for his modesty and fine humor. he has trained many academics, collaborated with others in research, and influenced large number of students over the decades of his academic career. some of these students contribute to this special issue. serbian academy of sciences and arts elected slobodan as a corresponding member in 2009 and as a full member in 2018. serbian chemical society gave slobodan a medal for his lasting and outstanding contribution to science in 2006. university of belgrade appointed him professor emeritus 2012. besides chemistry, slobodan enjoys fishing, tennis and skiing. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ editorial 1133 the papers in this issue deal mostly with natural products chemistry. space limits prevented us from inviting contributions by other, equally deserving authors affiliated with faculties chemistry, biology and pharmacy. we thank all contributors for their efforts, as well as for financial support. on behalf of your colleagues, collaborators and students, we dedicate this special issue to you, slobodan, and hope that you will enjoy it for years to come. belgrade, december 2021 guest editors dr. vele tešević, full professor university of belgrade – faculty of chemistry dr. ljubodrag vujisić, associate professor university of belgrade – faculty of chemistry selected publications s. milosavljević, d. jeremić and m. lj. mihailović, 1,5-shift of hydrogen from carbon to carbon in the lead tetraacetate oxidation of 5,5-dimethyl-2heptanol, tetrahedron 29 (1973) 3547 f. w. wehrli, d. jeremić, m. lj. mihailović and s. milosavljević, long-range deuterium -induced 13c nuclear magnetic resonance isotope shifts for signal assignment and determination of deuteriation site in cyclodecanone isotopomers, chem. commun. (1978) 302 m. stefanović, i. aljančić-šolaja and s. milosavljević, a 3,4-seco-ambrosanolide from ambrosia artemisiifolia, phytochemistry 26 (1987) 850 v. vajs, d. jeremić, s. milosavljević and s. macura, two-dimensional nmr spectra of sesquiterpenes. i noesy study of caryophyllenes from inula spiraeifolia, magnetic reson. chem. 25 (1987) 889 s. milosavljević, i. aljančić, s. macura, d. milinković and m. stefanović, sesquiterpene lactones from achillea crithmifolia, phytochemistry 30 (1991) 3464 v. bulatović, v. vajs, s. macura, n. juranić and s. milosavljević, highly oxygenated guaianolides from anthemis carpatica, j. nat. prod. 60 (1997) 1222 s. trifunović, v. vajs, s. macura, n. juranić, z. djarmati, r. jankov and s. milosavljević, oxidation products of hyperforin from hypericum perforatum, phytochemistry 49 (1998) 1305-1310 m. malinar, dj. miodragović, s. milosavljević, m. ćelap and d. vučelić, mixed cobalt(iii) complexes with s-tyrosine and 1,3-diaminopropane, enantiomer 3 (1998) 349-356 s. milosavljević, v. vajs, v. bulatović, d. djoković, i. aljančić, n. juranić and s. macura, application of two-dimensional nuclear magnetic resonance methods for structure elucidation of sesquiterpene lactones (guaianolides) from anthemis carpatica and diterpenes (kauranes) from achillea clypeolata, recent res. dev. phytochem. 2 (1998) 383 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 1134 editorial s. milosavljević, v. bulatović and m. stefanović, sesquiterpene lactones from the yugoslavian wild growing plant families asteraceae and apiaceae, j. serb. chem. soc. 64 (1999) 397 i. aljančić, v. vajs. n. menković, i. karadžić, n. juranić, s. milosavljević and s. macura, flavones and sesquiterpene lactones from achillea atrata subsp. multifida: antimicrobial activity, j. nat. prod. 62 (1999) 909-911 v. vajs, n. todorović, m. ristić, v. tešević, b. todorović, p. janaćković, p. marin and s. milosavljević, guaianolides from centaurea nicolai: antifungal activity, phytochemistry 52 (1999) 383 n. menković, k. šavikin-fodulović, v. bulatović, i. aljančić, n. juranić, s. macura, v. vajs, s. milosavljević, xanthones from swertia punctata, phytochemistry 61 (2002) 415 k. šavikin-fodulović, i. aljančić, v. vajs, n. menković, s. macura, g. gojgić, s. milosavljević, hyperatomarin, an antibacterial prenylated phloroglucinol from hypericum atomarium ssp. degenii, j. nat. prod. 66 (2003) 1236 i. djordjević, v. vajs, v. bulatović, n. menković, v. tešević, s. macura, p. janaćković, s. milosavljević, guaianolides from two subspecies of amphoricarpos neumayeri from montenegro, phytochemistry 65 (2004) 2337 s. trifunović, v. vajs, z. juranić, ž. žižak, v. tešević, s. macura, s. milosavljević, cytotoxic constituents of achillea clavennae from montenegro, phytochemistry 67 (2006) 887 i. s. aljancic, v. e. vajs, v. v. tesevic, s. m. milosavljevic, some wild-growing plant species from serbia and montenegro as the potential sources of drugs, curr. pharmaceut. des. 14 (2008) 3089 k. šavikin, t. janković, d. krstić-milošević, n. menković, s. milosavljević, secondary metabolites and biological activities of some gentianaceae species from serbia and montenegro, in comprehensive bioactive natural products extraction, isolation & characterization, studium press llc, usa, 2010, pp. 323–340 i. s. aljančić, m. pešić, s. m. milosavljević, n. m. todorović, m. jadranin, g. milosavljević, d. povrenović, j. banković, n. tanić, i. d. marković, s. ruždijić, v. e. vajs and vele v. tešević, isolation and biological evaluation of jatrophane diterpenoids from euphorbia dendroides, j. nat. prod. 74 (2011) 1613 d. gođevac, v. tešević, v. vajs, s. milosavljević, g. zdunić, b. ordević and m. stanković, chemical composition of currant seed extracts and their protective effect on human lymphocytes dna, j. food. sci. 77 (2012) 779 d. godevac, v. tesevic, v. vajs, s. milosavljevic and m. stankovic, antioxidant evaluation of heterocyclic compounds by cytokinesis-block micronucleus assay, in mini-reviews in medicinal chemistry, bentham sci. 13 (2013) 431 i. s. aljančić, i. vučković, m. jadranin, m. pešić, i. đorđević, a. podolski-renić, s. stojković, n. menković, v. e. vajs, s. m. milosavljević, two structurally distinct chalcone dimers from helichrysum zivojinii and their activities in cancer cell lines, phytochemistry 98 (2014) 190 m. novaković, m. pešić, s. trifunović, i. vučković, n. todorović, a. podolski-renić, j. dinić, s. stojković, v. tešević, v. vajs, s. milosavljević, diarylheptanoids from the bark of black alder inhibit the growth of sensitive and multi-drug resistant non-small cell lung carcinoma cells, phytochemistry 97 (2014) 46 n. menković, k. šavikin, g. zdunić, s. milosavljević and j. živković, medicinal plants in northern montenegro: traditional knowledge, quality, and resources, in ethnobotany and biocultural diversities in the balkans perspectives on sustainable rural development and ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ editorial 1135 reconciliation, pieroni, andrea, quave, cassandra l. (eds.), springer new york, 2014, pp. 197–228 k. šavikin, i. s. aljančić, v. e. vajs, s. m. milosavljević, m. jadranin, i. đorđević and n. r. menković, bioactive secondary metabolites in several genera of gentianaceae species from the central regions of the balkan peninsula, in the gentianaceae vol. 2: biotechnology and applications, j. j rybczyński, m. r. davey, m. anna (eds.), , springer-verlag, heidelberg, 2015, pp. 319–348 d. gođevac, m. jadranin, i. aljančić, v. vajs, v. tešević, and s. milosavljević, application of spectroscopic methods and hyphenated techniques to the analysis of complex plant extracts, in medicinal and aromatic plants of the world, scientific, production, commercial and utilization aspects, a. máthé (ed.), springer-verlag, heidelberg, 2015, pp. 61–85 v. vajs, a. jokić, s. milosavljević, artemisinin story from the balkans, nat. prod. commun. 12 (2017) 1157 m. cvetković, b. anđelković, v. stevanović, m. jadranin, i. đorđević, v. tešević, s. milosavljević., d. gođevac nmr-based metabolomics study of amphoricarpos species from montenegro, phytochemistry lett. 25 (2018) 1 m. novakovic, j. nikodinovic-runic, j. veselinovic, t. ilic tomic, v. vidakovic, v. tesevic, s. milosavljevic, bioactive pentacyclic triterpene ester derivatives from alnus viridis ssp. viridis bark, j. nat. prod. 80 (2017) 1255 m. novakovic, d. bukvicki, v. vajs, v. tesevic, s. milosavljevic, p. marin, y. asakawa, microbial transformation of calamintha glandulosa essential oil by aspergillus niger, nat. prod. commun. 13 (2018) 479 m. cvetković, b. anđelković, v. stevanović, m. jadranin. i. đorđević, v. tešević, s. milosavljević, d. gođevac, nmr-bsed metabolomics study of amphoricarpos species from montenegro, phytochemistry lett. 25 (2018) 1 v. vajs, i. đorđević, lj. vujisić, and s. m. milosavljević, nmr spectroscopy in the analysis of illegal drugs, in chromatographic techniques in the forensic analysis of designer drugs, t. kowalska, m. sajewicz and j. sherma (eds.), ch. 10, crc press, taylor and francis group, boca raton, fl, 2018, pp. 177–198 m. novakovic, i. djordjevic, n. todorovic, s. trifunovic, b. andjelkovic, b. mandic, m. jadranin, i. vuckovic, v. vajs, s. milosavljevic, v. tesevic, new aurone epoxide and auronolignan from the heartwood of cotinus coggygria scop., j. nat. prod. res. 33 (2019) 2837 m. cvetković, i. djordjević, m. jadranin, m. stanković, b. mandić, s. milosavljević, lj. vujisić, leaf-surface guaianolides from amphoricarpos neumaeyri showing protective effect on human lymphocytes dna, nat. prod. res. 35 (2019) 3040 m. cvetković, a. damjanović, t. p. stanojković, i. đorđević, v. tešević, s. milosavljević, d. gođevac, integration of dry-column flash chromatography with nmr and ftir metabolomics to reveal cytotoxic metabolites from amphoricarpos autariatus, talanta 206 (2020) 120248. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 1136 1136 my collaboration with prof. slobodan milosavljevic it is hard for me to say when exactly i met prof. milosavljevic (sloba) for the first time, it was so long ago, further than my memory could reach. but i remember how prof. jeremic (then the chair of the center for instrumental analysis) brought us together. after i returned from two years postdoc at eth in september of 1981, prof. jeremic shown a keen interest in my postdoc experience. i was telling him what i did and about new development in the field: 2d nmr, cosy, noesy, double -quantum spectroscopy, etc. he was really impressed as he understood immediately that this was the future. then prof. jeremic asked sloba, then his young collaborator to come over and join us in the conversation, and in no time, we realized that we have complementary interests and expertise that could be aligned and that we can collaborate fruitfully. at that time, we did not have proper equipment as the only nmr instrument in the center was outdated a-60. (a year earlier i saw the same model in london’s science museum as an exhibit of the lab of 1960s.) since i was travelling around the globe for my own research, i was taking along sloba’s samples and kept recording the spectra wherever i went. first few papers went through smoothly but on one sent to magnetic resonance in chemistry we got a mixed review with major complaint that “2d nmr spectrum could be recorded by any average lab”. we were flattered by this rejection as we interpreted this that in previous papers we were above the average. then we wrote back that there is nothing wrong being an average. (in usa there was very famous show about fictitious place called lake wobegon where “… all the children are above average.”) this paper was eventually published but the lesson learned was that we must raise the papers on higher level. and sloba indeed did just that. there was nothing more i could contribute, i was just recording and occasionally interpreting the spectra. and we went on and on. later when i moved to the usa we included other colleagues from the chemistry department who had interest in nmr. many years ago, our collaboration naturally winded down as 2d nmr became quite common and generating useful spectra no longer needed specialized expertise. but our friendship has continued till today. we rarely met in belgrade outside his lab. but i am grateful for the time we got to spent together in rochester (minnesota), minneapolis, chicago, rio de janeiro…). even more interesting is that both of us had passion for skiing, tennis, and fishing but never went together to enjoy these activities. i gave up sports long time ago, but i hope there’s time to go fishing. happy anniversary, my friend. slobodan macura, ph.d., professor of biochemistry department of biochemistry and molecular biology mayo clinic graduate school of biomedical sciences mayo clinic rochester, mn, usa ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ editorial 1137 slobodan milosavljević, dear friend, distinguished scientist it is difficult to describe dedication and devotion of prof. slobodan milosavljević, member of academy of sciences and arts, tireless scientist, postdoc at london university, researcher and promoter, who selflessly transferred knowledge to students and associates of faculty for chemistry as well as biology. my dear friend slobodan is specially excelling in the field of education and formation of young people through mutual work and numerous tasks performed on the faculty of chemistry in belgrade. he led students from basic level to the highest degrees, with great knowledge and enthusiasm, so later they felt at home in world of chemistry and biology. slobodan is still in this noble mission. i shall remark on his work as much as i can see it from our longstanding collaboration and friendship. we have spent time mostly on the montenegro mountains and in the botanic garden dulovina in kolašin. academician slobodan took part, with group of botanist led by dr nebojsa menković, in long-term quest to discover medicinal and endemic plants of east balkans, namely montenegro and serbia. the stress was on the complex mountain system prokletije, and the mountain massif orjen. going through unexplored landscape, through rocky paths and inaccessible remote stones they have collected many precious medicinal as well as endemic plants. all mentioned activities were well documented, with photographs and botanical specimens for herbarium. those investigations were important for botanical garden in kolašin and later for garden velemun in plav. furthermore, the research was significant and useful for many institutions in serbia and montenegro such as: faculty for chemistry, faculty for biology, institute josif pančić in belgrade and biotechnical institute, in podgorica. one of the results of the explorations is the book on medicinal plants by nebojsa menković et al. entitled:”a guide to medicinal plants”. slobodan made a great contribution to development and growth of the botanic garden dulovina, however it was not only scientifically significant, but the garden has become more visible. in other words, papers published in domestic and international magazines made mountains of montenegro and their botanical gardens more popular and more frequently visited by general public. daniel vincek (1926–2021), founder and director of the botanic garden dulovine 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice 404 not found miroslav j. ga\vsi\'c j. serb. chem. soc. 87 (12) 1447–1449 (2022) 1447 in memoriam miroslav j. gašić (1932–2022) miroslav gašić, a longtime professor of оrganic chemistry at the faculty of chemistry, university of belgrade, member of the serbian academy of sciences and arts, and former president of the serbian chemical society passed away in march 2022. miroslav gašić was born in belgrade in december 1932. he graduated at the chemistry department, faculty of natural sciences and mathematics, university of belgrade in 1959, and was appointed as an assistant at the same department in 1960. he defended his phd thesis „acetoxylation of steroid hormones“, supervised by milutin stefanović and mihailo mihailović, at the faculty of natural sciences and mathematics, university of belgrade, in 1964. immediately after getting his phd, he did his postdoctoral research on oxidation of steroid ketones with eliahu caspi at the worcester foundation for experimental biology, shrewsbury, massachusetts, and on non-classical homoallylic carbocations with saul winstein at the university of california, los angeles. in 1970 he was appointed as an assistant professor at the chemistry department, faculty of natural sciences and mathematics, university of belgrade, in 1976 as an associate professor, and in 1983 as a full professor. he spent a year (1972/73) as a visiting professor at the university of indiana, bloomington. in 1985, miroslav gašić was elected as the corresponding member of the serbian academy of sciences and arts, and in 1994 as the full member. research activity of miroslav gašić is very varied, encompassing diverse areas of organic chemistry and biochemistry. he published 120 papers, mostly in the most outstanding journals, and several monographs. he was an invited lecturer at many universities in usa and europe, as well as at many scientific conferences. his principal research fields were: chemistry of steroids; physical organic chemistry, spectroscopy and organic electrochemistry; isolation, chemical and biological characterization of natural products. a large part of his res________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1448 in memoriam: miroslav j. gašić earch activities was dedicated to partial synthesis and synthetic transformations of steroids: synthesis and reactions of 5,10-secosteroids, oxidation of steroid 3-ketones, acetoxylation of steroid lactones using lead tetraacetate, and oxidation of estrogen-type steroids. since the beginning of his scientific career, miroslav gašić was very interested in physical organic chemistry. at the university of california he investigated a hot topic at that time, non-classical carbocations. this work is intersting both from a mechanistic and a synthetic point of view. very early he realized huge possibilities of 13c-nmr spectroscopy and published many works in this area, including very useful review articles. his studies in organic electrochemistry were mainly focused on redox behaviour of biologically active quinones and hydroquinones as well as on electrochemical synthesis of drugs. during the late seventies miroslav gašić started research on isolation and characterization of natural products, both from terrestrial plants and marine organisms. he was the pioneer of marine natural product chemistry in serbia, studying principally marine invertebrates (sponges, corals, ascidians), but also marine algae and seagrasses. his research was mostly focused on terpenes, steroids, fatty acids, halogen-containing lipids, but also on nucleosides and proteins (lectins, enzymes). especially interesting are his publications on sponge sesquiterpene hydroquinone avarol and its derivatives, which show strong and selective antineoplastic activity. his papers on avarol and related compounds have been cited in more than 600 publications. he had a very intense collaboration with scientists from all over the world (usa, germany, italy, montenegro, greece, bulgaria), and was a leader and participant at many international and national scientific projects. miroslav gašić was an outstanding teacher. he drafted a very modern curriculum in organic chemistry for students of the new group of molecular biology and physiology, adapted for students of this group. his erudition and profound knowledge of various areas of chemistry and biology contributed to the high quality of his lectures. he supervised a great number of phd theses. as a mentor, he was allowing a lot of freedom to his students, stimulating their creativity and supporting their ideas. many of his phd students pursued a successful scientific career. miroslav gašić held several important positions: president of the serbian chemical society, president of the union of chemical societies of yugoslavia, member of the chemical nomenclature division of iupac, secretary of the department of chemical and biological sciences of the serbian academy of science, head of the department of chemistry and physical chemistry of the faculty of science and mathematics, university of belgrade, head of the department of organic chemistry of the faculty of chemistry, university of belgrade, member of the crown council of the royal family of serbia, etc. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ in memoriam: miroslav j. gašić 1449 he was awarded the order of labour with the red flag (1988), the october award of the city of belgrade (1988), the medal of the serbian chemical society for lasting and outstanding contribution to science (1997) and was elected as honorary president of the serbian chemical society in 2006. miroslav gašić will be remembered and respected as an outstanding scientist and teacher, with broad interests, brilliant intelligence and problem-solving ability, but also for his erudition, tact, benevolence, sense of humour and other human qualities. prof. dušan sladić ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ @article{sladic2022, author = {sladi{\'{c}}, d}, journal = {journal of the serbian chemical society}, title = {{miroslav j. ga{\v{s}}i{\'{c}}}}, year = {2022}, issn = {1820-7421}, month = {dec}, number = {12}, pages = {1447--1449}, volume = {87}, abstract = {miroslav ga{\v{s}}i{\'{c}}, a longtime professor of оrganic chemistry at the faculty of chem­istry, university of belgrade, member of the serbian academy of sciences and arts, and former president of the serbian chemical society passed away in march 2022.}, file = {:d\:/onedrive/mendeley desktop/ga{v{s}}i{'{c}} 2022 miroslav j. ga{v{s}}i{'{c}}.pdf:pdf;:09_gasic.pdf:pdf}, keywords = {in memoriam}, url = {https://www.shd-pub.org.rs/index.php/jscs/article/view/12192}, } accepted manuscript this is an early electronic version of an as-received manuscript that has been accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as s. ceauranu, v. ostafe and a. isvoran, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc230210022c this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230210022c j. serb. chem. soc.00(0)1-18 (2023) original scientific paper jscs–12271 published dd mm, 2023 1 impaired local hydrophobicity, structural stability and conformational flexibility due to point mutations in sult1 family of enzymes silvana ceauranu, vasile ostafe and adriana isvoran* department of biology-chemistry and advanced environmental research laboratories, west university of timisoara, 4 v. pirvan, 300223 timisoara, romania (received 10 february; revised 15 march; accepted 9 april 2023) abstract: sulfotransferases (sults) are enzymes involved in phase ii of the metabolism of xenobiotics. single nucleotide polymorphisms were identified for genes encoding the sults leading to allozymes with modified sulfating activity. this study aims to analyze the effects of the most frequently identified amino acid mutations in the sequences of enzymes belonging to the sult1 family on their local properties and structural stability. the outcomes reveal that single point mutations alter the local hydrophobicity and flexibility, mainly due to destabilization of the protein structures, and consequently may lead to changes in the dynamic of the active site activity reducing the affinity for the substrate. elucidation of how the single point mutations influence the activity of enzymes contributes to understanding the molecular basis of the specificity of enzymatic activity and mitigating anomalies in the metabolism of xenobiotics. keywords: protein plasticity; protein stability; hydrophobicity profile; mutations; metabolism, bioinformatics. introduction sulfotransferases (sults) are enzymes involved in phase ii of the metabolism of a wide range of both xenobiotics and endogenous compounds (hormones, bile acids, neurotransmitters, carbohydrates, proteins). they act by transferring a sulfate group from the cofactor 3′-phosphoadenosine 5′phosphosulfate (paps) to the hydroxyl group of an acceptor substrate.1 sulfoconjugation increases the water solubility of chemical compounds and the formation of more excretable products contributing to detoxification, but it also may lead to potentially carcinogenic metabolites.2 * corresponding author e-mail: adriana.isvoran@e-uvt.ro https://doi.org/10.2298/jsc230210022c a cc ep te d m an us cr ip t mailto:adriana.isvoran@e-uvt.ro https://doi.org/10.2298/jsc230210022c 2 ceauranu et al. there were identified 13 human cytosolic sulfotransferase genes in humans conducting to proteins divided into four families differing in the tissue distribution and substrate specificity3: sult1, sult2, sult4, and sult6. the present study focuses on the sult1 family, respectively on the subfamilies sult1a1, sult1a2, sult1a3, sult1b1, sult1c2 (former sult1c1), and sult1e1 as they reveal frequently identified allozymes with modified biological functions. these enzymes are involved in sulfation of phenols, thyroid hormones, and numerous drugs.4 sult1a1 and sult1a2 are usually active against the phenolic compounds, with sult1a1 revealing a higher activity. furthermore, sult1a1 and sult1b1 have an extensive overlap in their substrate profiles, but the sulfation efficiency of sult1a1 is higher.5 sult1a3 displays selectivity for catecholamines and structurally related compounds (serotonin, dopamine).6 sult1c2 enzyme sulfonates thyroid hormones, and sult1e1 is involved in the sulfation of hormones, mainly estrogens and iodothyronines.7,8 single nucleotide polymorphisms (snps) were identified for genes encoding the sults conducting to sults allozymes with modified stability and/or sulfating activity impairing the therapeutic response of numerous drugs.2, 9-11 in the case of sult1a1, the frequent identified polymorphic variants are: sult1a1*1 (the wild type, wt), sult1a1*2 (amino acid substitution r213h), sult1a1*3 (m223v) and sult1a1*4 (r37q), with sult1a*2. sult1a*3 and sult1a*4 usually reveal lower catalytic activity than the wt enzyme.2, 10,12,13 in the case of sult1a2, the frequent allozymes are sult1a2*1 (wt), sult1a2*2 (i7t, n235t) and sult1a2*3 (p19l). sult1a2*2 displays lower thermostability and decreased catalytic activity, and sult1a2*3 exposes higher thermostability and increased activity compared to wt.14 the frequent variants of the sult1a3 are sult1a3*1 (wt), sult1a3*2 (k234n), sult1a3*3 (p101l), sult1a3*4 (p101h), and sult1a3*5 (r144c). sult1a3*2 and sult1a3*3 usually reveal decreased activity, and sult1a3*4 and sult1a3*5 reveal increased activities against numerous drugs when compared to wt.6, 15,16 only two allozymes are known for sult1b1, sult1b1 (wt) and sult1b1l145v, the last one showing significantly decreased sulfation of p-nitrophenol than the wt.17 there are registered five variants for sult1c2: sult1c2*1 (wt), sult1c2*2 (s255a), sult1c2*3 (d60a), sult1c2*4 (r73q) and sult1c2*5 (s111f). sult1c2*3 and sult1c2*4 reveal reduced activity toward p-nitrophenol when compared to the wt and sult1c2*2, whereas sult1c2*5 did not show detectable activity toward this substrate.18 the frequent variants of sult1e enzyme are sult1e1*1 (wt), sult1e1*2 (d22y), sult1e1*3 (a32v) and sult1e1*4 (p253h). the allelic variants exhibit lower sulfation activity for estradiol compared to wt.19,20 an up-to-date synthesis a cc ep te d m an us cr ip t local effects of mutations in sult1 3 regarding the catalytic activities of all these variants toward various substrates and drugs has been recently published. 21 elucidation of the structures of the sult1 enzymes in complex with different ligands highlights the dominant role of their structural flexibility/plasticity in controlling both the activity and specificity.22-28 furthermore, it is also widely presumed that structural features and biological functions of proteins are closely connected to their sequence compositions.29 a single amino acid change in the protein sequence can disturb the network of intramolecular interactions and affect how the protein folds, its structural stability, dynamics, and, consequently, its biological function.30 consequently, in order to understand the molecular effects of a single point mutation, it is also necessary to consider changes in protein structural stability and dynamics. this study aims to predict, compare and analyze the changes in the local hydrophobicity, structural stability and flexibility due to single point mutations in the sequences of the sult1 enzymes using a computational approach. experimental this study focuses on the human sult1 enzymes having frequently identified polymorphic variants (table ⅰ). the sequences of the wild type enzymes were extracted from the uniprot database31 and used for further analysis. for assessing the changes produced by the mutations present in the frequently identified allozymes of the sult1 family in the profiles of hydrophobicity and average flexibility, the protscale computational tool32 has been considered. several parameters can be chosen when using the protscale computational tool: window size, the window edge relative weight value, weight variation model, and scale normalization. the window size is defined as the number of amino acids considered for determining one point of the computed property. it means that computing the value of the investigated property for a given residue i, the amino acids in the interval of the chosen length, positioned around residue i, are considered. regarding the window edge relative weight value, the computational tools always consider that amino acid from the center of the window has a weight of 100%, and the user may choose weight values between 0 and 100% for the amino acids at the remaining positions in that window. if weight values are chosen lower than 100%, the user may select a linear or exponential decrease of the weight between the center and the edges. furthermore, the user may choose whether to use the unmodified selected scale values or to normalize these values so that they fit into the range from 0 to 1.32 the following settings were considered in this study: windows of 3, 5, and respectively 9 amino acids, the relative weight of the window edges compared to the window center was set to 100%, and the unmodified selected scales values were used. we also specify that for obtaining the hydrophobicity profiles, the kyte&doolitle scale32 has been used. a cc ep te d m an us cr ip t 4 ceauranu et al. table ⅰ. the frequently identified polymorphic variants of the enzymes belonging to sult1 family considered in this study: wtthe wild type enzyme. sult1 subfamily polymorphic variants amino acid substitutions sult1a1 sult1a1*1 wt sult1a1*2 r213h sult1a1*3 m223v sult1a1*4 r37q sult1a2 sult1a2*1 wt sult1a2*2 i7t, n235t sult1a2*3 p19l sult1a3 sult1a3*1 wt sult1a3*2 k234n sult1a3*3 p101l sult1a3*4 p101h sult1a3*5 r144c sult1b1 sult1b1 wt sult1b1-l145v l145v sult1c2 sult1c2*1 wt sult1c2*2 s255a sult1c2*3 d60a sult1c2*4 r73q sult1c2*5 s111f sult1e sult1e1*1 wt sult1e1*2 d22y sult1e1*3 a32v sult1e1*4 p253h in order to explore the local flexibility in the structures of the sult1 enzymes, the pdbflex database33 has been considered. this database offers information on the intrinsic global and local flexibilities of protein structures based on the analysis of variations appearing between the different structural files of the same protein deposited in the protein data bank (pdb). pdbflex collects information on all depositions having at least 95% sequence identity with the sequence of the query structural file, performs the analysis of the structural differences, and clusters them according to the structural similarities.33 consequently, the available elucidated structural files for the investigated enzymes and their complexes with various ligands (cofactor, substrates, drugs) have been considered. these structural files are available in pdb34, and the information regarding their active and binding sites has been also retrieved (table ⅱ). a cc ep te d m an us cr ip t local effects of mutations in sult1 5 table ⅱ. uniprot and protein data bank (pdb) identifiers (id) for the analyzed sequences and structures of the enzymes belonging to sult1 family: aa – amino acid, paps 3'phosphoadenosyl-5'-phosphosulfate, the cofactor for these enzymes. sult1 enzyme uniprot id pdb id binding site mutations corresponding to allozymes sult1a1 p50225 4graa, 36, 1ls6b, 1z28c, 2d06b, 3u3jb, 3u3kb, 3u3mb, 3u3ob, 3u3rb, 3qvub, 3qvvb, paps: 48-53, 106108, 130, 138, 193, 227-232, 255-259; substrate: 106-108 r37q, r213h, m223v sult1a2 p50226 1z29 paps: 48-53, 106108, 130, 138, 193, 227-232, 255-259; substrate: 106-108 i7t, p19l, n235t sult1a3 podmm9 2a3ra, 1cjm paps: 48-53, 130, 138, 146, 193, 227232, 255-259; substrate: 86 p101l, p101h, r144c, k234n sult1b1 o43704 2z5f, 3ckla paps: 48-53, 131, 139, 194, 228-233, 256-260; substrate: 107-109 l145v sult1c2 o00338 3bfxa paps: 49-54, 131, 139, 194, 228-233, 256-260; substrate: 107-109 d60a, r73q, s111f, s255a sult1e1 p49888 1g3ma, 1hy3, 4jvl, 4jvm, 4jvn paps: 47-52, 129, 137, 192, 226-231, 256-258 substrate: 105-107 d22y, a32v p253h awhen multiple structural files have been detected for an enzyme, these structural files have been chosen as they have a better resolution or a lower number of missing residues and/or missing atoms. b these structural files correspond to the allelic variant sult1a1*2 (r213h). 23, 26, 37, 38 c structural file 1z28 corresponds to the allelic variant sult1a1*3 (m223v).25 structures of the wt variants of the sult 1 enzymes highlighting the positions of the amino acids that suffer mutations are revealed in figure 1. ucsf chimera tool35 has been used to visualize these structures. data presented in table ⅱ and figure 1 reveal that some amino acids that support mutations corresponding to allozymes situated in the regions or their close vicinity are involved in the interactions with the cofactor and/or substrate: m223v for sult1a1*3, n235t for sult1a2*2, k234n for sult1a3*2, p253h for sult1e1*4. some residues that support mutations are missing in the structural files: i7t for sult1a2, r73 and s255 for sult1c2. a cc ep te d m an us cr ip t 6 ceauranu et al. fig. 1 illustration of the positions of the residues that support mutations in the sult1 enzymes: sult1a1 (a), sult1a2 (b), sult1a3 (c), sult1b1 (d), sult1c2 (e), sult1e1 (f). some residues that support mutations are missing in the structural files: i7t for sult1a2, r73 and s255 in sult1c2. a3p adenosine-3'-5'-diphosphate. a cc ep te d m an us cr ip t local effects of mutations in sult1 7 ucsf chimera tool has been also considered for illustrating the changes in the hydrophobicity and coulombic electrostatic potential of the regions of sult1a1 containing the point mutations r213h and m223v compared to the wt enzyme. this analysis has been made only for sult1a1, as this enzyme has solved structures of mutants deposited in pdb. dynamut2 web server (http://biosig.unimelb.edu.au/dynamut2/) has been considered for predicting changes in stability caused by single point mutations in the sequences of investigated enzymes.30 the changes in the enzymes stability are assessed by predicting the variations in folding free energy values (δδg, expressed in kj mol-1) for single point mutations: δδg<0.0 corresponds to mutations destabilizing the structure and δδg>0.0 to mutations contributing to the stabilization of the structure. the predictions are based on an experimental data set collected for 4633 mutations (2640 destabilizing and 1993 stabilizing) that were divided into 4022 entries for the training set and 611 entries for the test set. dynamut2 has a good accuracy of predictions achieving pearson's correlation of up to 0.72 for single point mutations across 10‐fold cross‐ validation and independent blind tests.30 results and discussion analysis of the influence of mutations present in the frequently identified allozymes belonging to sult1 family on their average flexibility and hydrophobicity profiles. the effect of the mutations corresponding to the main allelic variants of the sult1 family of enzymes on their local hydrophobicity are shown in figure 2 for a window of 3 amino acids and in the supplementary tables s-ⅰ – s-ⅵ for windows of 5 and 9 amino acids, respectively. figure 2 and data presented in supplementary tables s-ⅰ – s-ⅵ reveal that the punctual amino acid mutations corresponding to the frequently identified allozymes of the sult1 enzymes conduct to altered local hydrophobicity profiles. some mutations cause decreased local hydrophobicity, but others produce increased local hydrophobicity. even if only one amino acid is changed, this point mutation affects the local hydrophobic profile over a range of at least 9 residues. a molecular dynamics study revealed that in the case of sult1a1*3, the m223v mutation led to the loss of a hydrophobic contact between m223 and m60 and may be responsible for the altered sulfonation activity of the sult1a1*3.21 the flexibility profiles of the enzymes belonging to the sult1 family and of their frequently identified allozymes are shown in figure 3 for a window of 3 amino acids and in the supplementary tables s-ⅶ – s-ⅻ for windows of 5 and 9 amino acids, respectively. figure 3 and supplementary tables s-ⅶ – s-ⅻ reveal that the amino acid mutations corresponding to the frequently identified allozymes of the sult1 enzymes also alter the local flexibility profiles. a cc ep te d m an us cr ip t 8 ceauranu et al. fig. 2 hydrophobicity profiles obtained using protscale tool for a window of 3 amino acids for the enzymes belonging to the sult1 family and for their frequently identified allozymes: wt – wild type protein. a cc ep te d m an us cr ip t local effects of mutations in sult1 9 fig. 3 average flexibility profiles obtained using protscale tool for a window of 3 amino acids for the enzymes belonging to the sult1 family and for their frequently identified allozymes: wt – wild type protein. being well known that the flexibility of sults is responsible for recognizing the diverse types of substrates39, this outcome becomes important. a molecular dynamics simulation study involving the major allozymes of sult1a1 (wt, r213h, and m223v) revealed increased flexibility in the region of the binding site a cc ep te d m an us cr ip t 10 ceauranu et al. for the mutants compared to the wt enzyme and alteration of the protein dynamics.21 it should be noted that neither r213 nor m223 are in the substratebinding loops,39 but their mutations induce conformational changes affecting the flexibility of at least one of these loops.40 these changes may be responsible for the observed alternation of sulfation activities of mutants compared with wt toward numerous endogenous compounds and drugs.2, 10, 12, 13 these outcomes revealing the altered local hydrophobicity and flexibility for the allozymes are not unexpected, being known that polar or charged residues are more flexible and less hydrophobic, whereas nonpolar residues are more hydrophobic and quite inflexible.41 furthermore, the alteration of the local hydrophobicity and/or flexibility may cause the proteins dysfunctionalities, as long-range correlations concerning hydrophobicity and flexibility along the proteins chains have been observed for sequences of numerous proteins.42-45 as the protein hydrophobicity and flexibility are closely related to the primary structure, it is expected that the amino acid mutations affect not only the spatial structure of the protein but also the structural flexibility of the protein and its biological function.46 consequently, a better understanding of the relationship between the local hydrophobicity and flexibility of sult1 enzymes and their functional properties is essential for understanding the metabolism of numerous drugs. analysis of the flexibility of enzymes belonging to the sult1 family taking into account structural data. for the investigated sult1 enzymes, their local structural flexibility has been analysed using pdbflex. the identified clusters for the structural files corresponding to these enzymes are revealed in supplementary table s-ⅻⅰ. sult1a1 and sult1a2 enzymes are considered as members of the same cluster due to their high sequence similarity, about 95%.47 the regions with local flexibility identified in the structures of sult1 enzymes are presented in table 3. the nand c-terminal regions are not mentioned in this table as it is known that they can be disordered and flexible in many proteins.48 as expected, the regions with higher structural flexibility involve amino acids that interact with the cofactor and/or the substrate. several mutations corresponding to allozymes of the investigated sults correspond to the identified flexible regions revealing their possible effects on the local structural stability and flexibility of the proteins. a cc ep te d m an us cr ip t local effects of mutations in sult1 11 table ⅲ. regions with local structural flexibility in sult1 enzymes identified using pdbflex computational utility. sult1 member maximum rsmd in the cluster (å) average rsmd in the cluster (å) region (amino acid interval) average rmsd (å) ligand interacting with amino acids belonging to this region amino acid mutation corresponding to allelic variant that is present in the flexible region sult1a1, sult1a2 0.596 for 3u3k and 1z28 0.363 110-122 0.053 p-nitrophenol, 3-hydroxy-7,7dimethyl-2phenyl-4(thiophen-2-yl)2,6,7,8tetrahydro-5hpyrazolo[3,4b]quinolin-5one, 7-hydroxy2-oxo-2hchromene-3carbonitrile, estradiol 210-225 0.034 r213h, m223v sult1a3 1.167 for 2a3r and 1cjma 0.793 24 63 0.072 adenosine-3'-5'diphosphate, ldopamine 94 -104 0.182 l-dopamine p101l in sult1a3*3, p101h in sult1a3*4 141 – 156 0.036 adenosine-3'-5'diphosphate, ldopamine r144c in sult1a3*5 sult1b1 0.562 for 2z5f and 3ckl 0.395 83 89 0.282 l-dopamine sult1e1 0.625 for 1hy3 and 4jvl 0.384 79 94 0.362 adenosine-3'-5'diphosphate 141 157 0.036 l-dopamine 210 229 0.027 resveratrol 240-252 0.041 p243h în sult1e1*4 amany regions are missing in the structure of sult1a3 with the pdb id 1cjm, and these regions were not considered for the local flexibility analysis. a cc ep te d m an us cr ip t 12 ceauranu et al. analysis of the influence of mutations that are present in the frequently identified allozymes belonging to sult1 family on their local hydrophobicity and electrostatic potential for sult1a1, the output of the pdbflex tool reveals local structural flexibility for the region 210-225, taking into account the different structural files of this protein deposited in the pdb. this region incorporates two mutations corresponding to sult1a1*2 (r213h) and sult1a1*3 (m223v), respectively. the output of the protscale tool reveals that mutations corresponding to sult1a1*2 and sult1a1*3 reduce the local hydrophobicity and flexibility and may facilitate the electrostatic interactions with the solvent. this output strongly correlates with data presented in figure 4 obtained using chimera software for the regions 210-225 of the sult1a1 wt and of the enzyme containing the mutations r213h and m223v. figure 4 reveals changes in both the local hydrophobicity and coulombic electrostatic potential of this region for the allozymes compared with the wt enzyme. these changes may influence the activity of these allozymes as their sulfation activity toward both endogenous and numerous xenobiotic compounds is decreased compared with the sulfation activity of the wt.21 furthermore, literature data reveal that other two mutations in the sult1a1 sequence, d249g and y240c (both amino acids located in the vicinity of the active site), conduct to lower affinity for 3-cyano-7-hydroxycumarin and p-nitrophenol. analysis of the d249g mutant structure shows an increase in the local flexibility of this region and a significant change in the charge distribution around the active site.26 the importance of the local electrostatic interactions for determining the structure, stability, and conformational adaptabilities has been revealed for numerous proteins.49, 50 there are no structures determined for the allozymes of the others sult1 enzymes considered in this study, and consequently, the changes in the local hydrophobicity and electrostatic potential could not be analyzed for these enzymes by using structural data. taking into account the very good correlation between the information obtained using protscale and pdbflex computational tools for the sult1a1 subfamily, we may extrapolate that the punctual amino acid mutations that appear in the allozymes of the sult1 enzymes conduct to alteration of the local structural flexibility, local hydrophobicity, and consequently on the electrostatic potential. a cc ep te d m an us cr ip t local effects of mutations in sult1 13 fig. 4 changes in the local hydrophobicity (a) and coulombic electrostatic potential (b) of the 210-225 region of sult1a1 due to the point mutations r213h and m223v compared to the wild type (wt) enzyme. the following structural files have been considered when mapping the hydrophobicity and electrostatic potential: 4gra (wt), 1ls6 (r213h), and 1z28 (m223v). blue regions in figure 4a correspond to the hydrophilic surface, and orange regions correspond to the hydrophobic surface. as the color is more intense, the higher is hydrophilicity/hydrophobicity of the surface. in figure 4b, red regions illustrate the negative potential, and blue regions correspond to the positive potential. analysis of the influence of mutations that are present in the frequently identified allozymes belonging to sult1 family on their structural stability it is already known that even small reductions in protein stability can lead to dysfunctional proteins.51 the stabilization/destabilization effects of the single point mutations in the sequences of sult1 enzymes conducting to the most frequently identified allozymes have been analyzed using dynamut2 webserver, and the results are presented in table iv. a cc ep te d m an us cr ip t 14 ceauranu et al. table ⅳ. illustration of the δδg values produced by the single point mutations corresponding to the frequently identified allozymes of the sult1 enzymes. δδg<0.0 values correspond to mutations destabilizing the structure, and δδg>0.0 values to mutations contributing to the stabilization of the structure. sult1 enzyme single point mutation δδg / kj mol-1 effect observations sult1a1 r37q -2.18 destabilizing r213h -0.54 destabilizing m223v -1.92 destabilizing sult1a2 i7t t7 residue is missing in the crystallographic structure p19l -1.84 destabilizing n235t 0.09 stabilizing sult1a3 p101l -2.05 destabilizing p101h -1.04 destabilizing r144c -3.93 destabilizing k234n 0.29 stabilizing sult1b1 l145v -9.07 destabilizing sult1c2 d60a 1.04 stabilizing r73q q73 residue is missing in the crystallographic structure s111f -1.71 destabilizing s255a a225 residue is missing in the crystallographic structure sult1e1 d22y 5.68 stabilizing a32v -2.38 destabilizing p253h -5.85 destabilizing data presented in table iv reveal that the typical point mutations corresponding to allozymes usually destabilize the structure. it is in good correlation with known data revealing that the allozymes containing point mutations that destabilize the structure usually have lower sulfation activity compared to the wt enzymes. in the case of sult1a1, it was shown that the position of residue 213 precedes a flexible region, whereby mutation of this residue affects both stability and flexibility of the enzyme.38 among the frequent point mutations appearing in the sult1a1 enzyme, r213h produces the lower destabilizing effect, which is also in correlation with published data revealing that r213h induces local conformational changes affecting the substrate-binding loop and has only a low impairing effect on the overall stability of the protein structure.40 a cc ep te d m an us cr ip t local effects of mutations in sult1 15 it should be noted that several amino acids that correspond to the mutants of sult1 enzymes have ionizable sidechains. the frequently identified polymorphic variants of sult1a are r37q and r213h, with arginine and histidine being among the amino acids having ionizable sidechains. histidine is neutral, and arginine is protonated under physiological conditions. consequently, the standard protonation states have been considered for arginine and histidine residues. both histidine and arginine play essential structural and functional roles in proteins, which correlate with the ionization state of their side chains. it emphasizes that a more accurate approach should include electrostatic calculations by solving the poisson-boltzmann equation with subsequent monte carlo titration52 or employing dft/solvation electrostatic calculations.53 the mutation l145v corresponding to sult1b1 conducts to a substantial destabilization of the structure (δδg=-9.07 kj mol-1), and literature data show that this mutation results in a significantly decreased sulfation of p-nitrophenol compared to the wt.17 single amino acid mutations in the sequences of the enzymes belonging to the c2 family of the human cytochromes (cyp2c) have also been suggested to be structurally destabilizing in close connection with the observed interindividual differences in cyp2c-mediated drug metabolism.54 conclusions data obtained in the current study reveal that the point mutations present in the most frequently observed polymorphic variants of the enzymes belonging to the sult1 family result in altering the local hydrophobicity and flexibility and usually conduct to destabilize the protein structure. such changes may be responsible for the reduced affinity for the substrate due to possible effects on the dynamics and flexibility of the binding region of the protein. the outcomes of this study contribute to elucidating how sult snps may influence the metabolism of drugs and endogenous compounds and may allow for the improvement of strategies for mitigating anomalies in the metabolism of xenobiotics. furthermore, these results may contribute to understanding the molecular basis for the altered specificity of other enzymes having polymorphic variants. supplementary material supplementary tables s-ⅰ s-ⅻⅰ. hydrophobicity and average flexibility profiles of the sult1a1 (table s-ⅰ and s-ⅶ); sult1a2 (table s-ⅱ and s-ⅷ); sult1a3 (table s-ⅲ and s-ⅸ); sult1b1 (table s-ⅳ and s-ⅹ); sult1c1 (table s-ⅴ and s-ⅺ); sult1e1 (table s-ⅵ and s-ⅻ) enzyme (wt) and of its frequently identified allozymes obtained using protscale tool for a window of 5 and 9 amino acids; the clusters identified by pdbflex computational tool for assessing the structural flexibility for the sult1 enzymes that reveal frequently identified allozymes (table s-ⅻⅰ). a cc ep te d m an us cr ip t 16 ceauranu et al. additional data are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/12271, or from the corresponding author on request. извод смањена локална ходрофобност, структурна стабилност и флексибилност конформације услед тачкастих мутација sult1 фамилије ензима silvana ceauranu, vasile ostafe и adriana isvoran department of biology-chemistry and advanced environmental research laboratories, west university of timisoara, 4 v. pirvan, 300223 timisoara, romania сулфотрансферазе (sult) су ензими укључени и фазу ii метаболизма ксенобиотика. идентификовани су полиморфизми појединачних нуклеотида за гене који кодирају sult ензиме, доводећи до синтезе алоензима са измењеном сулфатационом активношћу. у овој студији су испитани ефекти најчешћих мутација амино киселина у секвенци ензима sult1 фамилије на њихове локалне особине и структурну стабилност. показано је да тачкасте мутације мењају локалну хидрофобност и флексибилност, претежно због дестабилизације структуре протеина, што може довести до промене у активности активног места и смањења афинитета за супстрат. сазнања о начину на који тачкасте мутације утичу на активност ензима доприносе разумевању молекулских основа специфичности ензимске активности и ублажавање аномалија у метаболизму ксенобиотика. 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loriot, b. o. villoutreix, m. a. miteva, drug discov today, 22 (2017) 366 (http://doi.org/10.1016/j.drudis.2016.09.015) a cc ep te d m an us cr ip t http://doi.org/10.1385/1-59259-584-7:531 http://doi.org/10.1385/1-59259-584-7:531 http://doi.org/10.1093/nar/gkv1316 http://doi.org/10.1093/nar/28.1.235 http://doi.org/10.1002/jcc.20084 https://doi.org/10.1021/bi301492j https://doi.org/10.1074/jbc.m207246200 https://doi.org/10.1016/j.jmb.2011.06.037 https://doi.org/10.1021/acs.biochem.5b00406 http://doi.org/10.3390/ijms20246256 http://doi.org/10.1002/prot.1038 https://rjp.nipne.ro/2011_56_1-2/0185_0195.pdf http://doi.org/10.1063/1.3482227 http://doi.org/10.1016/j.physa.2009.07.042 http://doi.org/10.1016/j.chaos.2011.01.008 http://doi.org/10.1016/j.foodhyd.2020.105709 http://doi.org/10.1016/s0076-6879(05)00009-1 http://doi.org/10.1371/journal.pcbi.1000958 http://doi.org/10.2174/1389203023380431 http://doi.org/10.1007/s00249-006-0123-1 http://doi.org/10.1007/s00239-015-9717-5 https://doi.org/10.2298/jsc200720047p https://doi.org/10.1021/jp0608630 http://doi.org/10.1016/j.drudis.2016.09.015 {book review: radoslav ad{\v{z}}i{\'{c}} and neboj{\v{s}}a marinkovi{\'{c}}: platinum monolayer electrocatalysis} j. serb. chem. soc. 86 (2) 209–211 (2021) book review jscs–5416 209 book review platinum monolayer electrocatalysis authors: radoslav adžić and nebojša marinković publisher: springer nature switzerland ag 2020, isbn 978-3-030-49565-7 slavko mentus1,2* 1faculty of physical chemistry, university of belgrade, studentski trg 12–14, 11158 belgrade, serbia and 2serbian academy of sciences and arts, knez mihajlova 35, 11158 belgrade, serbia (received and accepted 6 january 2021) at first glance, this book captures attention with its splendid, dominantly orange colored hardcover. within a volume of 167 pages, the reader encounters 11 chapters bearing the following titles: 1. short introduction to the science of electrocatalysis, 2. electrocatalytic reactions, 3. electrochemical energy conversion in fuel cells, 4. studies of electrocatalytic reactions, 5. important electrosorption reactions, 6. important electrocatalytic reactions, 7. platinum monolayer electrocatalysts, 8. catalytic properties of pt monolayer electrocatalysts, 9. performance stability and scale-up syntheses of pt monolayer electrocatalysts, and, 11. prospects for platinum and platinum group metal monolayer electrocatalysts. one deals here with a scientific monograph based primarily on the authors personal research achievements. after a short introduction to the electrocatalysis, covering four reactions important for energy conversion in fuel cells in the first four chapters, in the fifth one the authors describe the properties of metal monolayers on electrode surfaces and underpotential deposition of metals.the period covered by this chapter begins with the adžićs famous discovery of huge enhancement of electrocatalytic effectivenes of pt surfaces upon adsorption of metal (pb, bi) submonolayers in 1970s. the main part of the book covers the descript * e-mail: slavko@ffh.bg.ac.rs ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 210 mentus ion of very concept of pt monolayer electrocatalysts, the ways of their synthesis and application. through the description of numerous technically demanding investigations of four main reactions in energy conversion processes: oxygen reduction reaction and oxidation reactions of hydrogen, methanol and ethanol reactions on single-crystal surfaces, the book matter continues up to the recent application oriented systems. they involve stable, long-living core-shell structured electrocatalyst with durable, frugal pt monolayers covering the cores of more abundant metals (au, pd and their alloys, and non-noble metals and alloys). in the concluding chapter the authors comment the predictions in scale-up syntheses and catalysts’ stability in practical use. what one might say about the temporary circumstances in which this book appeared? the investigation of room-temperature fuel cells, the main subject of the book, experienced sudden acceleration after famous world oil crisis in 1970s. since then, the developed countries funded many projects dedicated to the replacement of liquid fossil fuels in trafic by chemical sources of electric energy. with the oil market stabilization, this tension declined, however, in the period 1990–2000 a new threat splashed the world: climate changes caused by huge consumption of fossil fuels, which actualised the activities oriented to the development of new chemical power sources. platinum seemed to be unavoidable electrocatalyst providing satisfactory effectiveness of fuel cells. in 2010s, an ambitious worlds action, supporeted by relevant un bodies, to replace gradually all oil-driven automobile engines by electrically powered ones, seemed to be unattainable due to the too high demand of platinum in comparison to available earth crust resources. this is the reason why automobile companies started to use li-ion batteries rather than fuel cells, and a majority of both research grants and research institutions reoriented themselves toward the development of batteries. however, the authors of this book persisted in their research orientation, what resulted in many valuable new contributions in saving platinum as fuel cell electrocatalyst. this proved onself as reasonable performance, since metal resources required for li-ion batteries became also critical, susceptible to rapid exhaustion in the next few decades. this new threat is the reason why the search for advanced fuel cell electrocatalysts is under permanent progress. as the springer’s editor said, “platinum monolayer electrocatalysts present a groundbreaking discovery that will likely have impact on future electrocatalysis. unlike non-noble metal monolayer, platinum monolayer can have great stability and activity that can overcome three major obstacles of conventional platinum electrocatalysts – catalysts’ cost, activity, and stability for a broad range of fuel cell applications.” this is a reason why important automobile producers persisted in the development of fuel cells-driven automobiles. on dispasal stands the platinum monolayer electrocatalyst developed by the authors of this book, produced ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ book review: platinum monolayer electrocatalysis 211 by n.e. chemcat corporation in japan. it is licensed to two other companies. an actual difficulty for broader use of fuel cell powered automobiles, compared to battery driven ones, is the technically unsolved hydrogen distribution network, however this may be considered only as temporary obstacle. this book may be highly recommended as an useful theoretical and practical guide to graduate, especially phd students, preparing themselves for studies in surface science and electrocatalysis. furthermore, reading this book, the researchers active in development of fuel cells will face the most recent research techniques available in worlds leading research institutions. thanks to a selected literature surveys placed at the end of each chapter, the reader may follow a chronological development of fundamental concepts and practical aspect of functioning of electrocatalysts for contemporary room temperature fuel cells. particularly, numerous practical examples illustrated by coloured graphs, discussed in a comprehensive language, offers a plenty part of knowledge presented in a concise and clear manner, as, otherwise, is really expected from the authors deeply personally involved in the subject matter of the book. i really enjoyed reading it. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice accepted manuscript this is an early electronic version of an as-received manuscript that hasbeen accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as l. todan, d. c. culita, m. e. soare, r. m. ion, r. c. fierascu and m. maganu, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc221028010t this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc221028010t j. serb. chem. soc.00(0)1-12 (2022) original scientific paper jscs–12113 published dd mm, 2023 1 immobilization of natural betalain pigments in inorganic hosts ligia todan1* daniela c culita1, mirabela e. soare1, rodica m. ion2, radu c. fierascu2 and maria maganu3 1ilie murgulescu institute of physical chemistry, romanian academy, 202 splaiul independentei, 060021 bucharest, romania, 2national institute for research & development in chemistry and petrochemistry-icechim, 202 splaiul independentei, 060021 bucharest, romania, and 3„costin d. nenitescu”institute of organic and supramolecular chemistry, romanian academy, 202 b splaiul independentei, 060023 bucharest, romania (received 28 october 2022; revised 21 january 2023; accepted 1 march 2023) abstract: in search of new food-grade ph sensitive formulations, red beet extract rich in betacyanin was included in different inorganic matrices based on silica and aluminosilicate to improve the stability of the dye. by the direct method of encapsulation of the pigments in silica support, stabilizing agents such as inclusion complex forming β-cyclodextrin and ascorbic acid were added. the post loading system assumes the synthesis of porous silica and aluminosilicate powders and the adsorption of the beet extract by these supports. the unloaded carriers were structurally and texturally characterized (x-ray diffraction, ftir, n2-physisorption). the presence of betanin, approved as a red food colorant, was evidenced by uv-vis spectroscopy in all the hosts. color properties were investigated as well as the ph generated color variations of the powders exposed to ammonia in the head-space of a sealed vessel. the obtained results could widen the field of applications of beet extract, the prepared ecological formulations could provide added value to edible products packaging. keywords: betanin; encapsulation; mesoporous silica; aluminosilicate; color parameters introduction betalains are the major pigments abundantly found in beetroot (beta vulgaris) which are chemically defined as the derivatives of betalamic acid. 1 betalains can be divided into yellow-orange betaxanthins and red-violet betacyanins. of the betalains, betanin is approved for use as colorant in food industry (e number e162, ci natural red 33) and is considered a better choice than chemically synthesized pigments. it has commercial applications for foodstuff, cosmetics, and pharmaceuticals. 2 betanin is almost totally extracted from beetroot crops and sold *corresponding author e-mail: l_todan@yahoo.co.uk https://doi.org/10.2298/jsc221028010t a cc ep te d m an us cr ip t mailto:l_todan@yahoo.co.uk https://doi.org/10.2298/jsc221028010t todan et al. as beetroot extract. 3 all betalains are water soluble. the main factors influencing their stability are light, oxygen concentration, temperature, the presence of water, and ph. they retain their tinctorial strength and color shade over a ph range of 3 7. 1,2 the fact that betacyanins show sensitivity to ph makes them suitable for use as colorimetric indicators for detecting changes of the environment especially in case of edible products, improving thus the protection and shelf life of foodstuffs or pharmaceuticals. 4 to preserve the chemical structure of the pigment and to facilitate its use in commercial applications, different ways of stabilization were used. metal-chelates formation, cyclodextrin complexes, addition of organic acids, inclusion in different matrices should be mentioned. 5,6 it was shown that cyclodextrins are promising stabilizing agents of betalain molecules, βcyclodextrin (β-cd) forming an inclusion complex with betanin. 5,6 copigmentation was used to preserve betalains color intensity and stability. it involves interactions between the dyes and other naturally occurring compounds such as ascorbic or citric acid which can be neutralizing agents for the electrophilic center of betalains. 5,6 immobilization of the beet pigments on a solid carrier, organic or inorganic, is a promising approach for improving the properties of the plant derived pigments, enabling thus the inclusion of certain additives that can confer better functionality to encapsulated betalain pigments. a wide range of encapsulation systems have been designed: natural products derived nanocarriers (maltodextrin, caseins), lipidbased nanocarriers, biopolymers based nanocarriers (chitosan, proteins), and inorganic carriers (carbon nanotubes, clay minerals, γ-alumina). 6-8 two approaches for the inclusion of drugs into silica based particles of matrix type have been mentioned, namely direct drug encapsulation when the drug is dissolved in the liquid precursor prior to formation of the matrix and indirect encapsulation or post loading when carrier particles are first synthesized followed by encapsulation of the drug. 9,10 the purpose of the present work was to obtain formulations betalains-sio2 and betalains-aluminosilicate, respectively, and to explore the two above mentioned loading modes of beet pigments into the silica-based carriers. our results provide novel information about the structure and properties of the final hybrid pigments, especially the color response toward ammonia, a marker of food spoilage, the obtained data contributing to the development of product formulations which can favour the properties of food packaging. experimental materials ludox®sm-30 colloidal silica (30 wt. % suspension in water) and sodium hydroxide were from sigma aldrich. β-cyclodextrine was purchased from wacker-chemie gmbh. ascorbic a cc ep te d m an us cr ip t betalain pigments in inorganic hosts 3 acid pharmaceutical grade was purchased from intra-laboratories, cetyltrimethylammonium bromide (ctab) from carl roth, aluminium oxide from merck millipore, sodium dodecyl sulfate (sds) analytical grade from applichem. the concentrated beetroot juice (100 %, biotta food grade) produced by extraction of pigments from red beet and its subsequent pasteurization, with betanin the major coloring principle was used as natural dye. characterization x-ray diffraction (xrd) analyses were performed using a 9 kw rigaku smartlab diffractometer (rigaku corp., tokyo, japan, operated at 45 kv and 200 ma, cukα radiation λ =1.54059 å), in scanning mode 2θ/θ, between 2 and 90° (2θ) for wide-angle analysis and between 0.05 and 8° (2θ) for small angle analysis, measuring scan step 0.02°, scan speed 8° min-1 (wide angle) and 4° min-1 (small angle). components were identified by comparison with icdd data. ft-ir spectra were recorded on a brucker vertex 70 spectrometer with horizontal device for attenuated reflectance and diamond crystal, on a spectral window ranging from 4000 to 400 cm-1, at a spectral resolution of 2 cm-1. spectra were recorded without any sample preparation and were processed with opus 5.5 program (brucker). nitrogen adsorptiondesorption isotherms at 77k were recorded on a micromeritics asap 2020 automated gas adsorption system (norcross, ga, usa). the samples were degassed at 250 °c for 4 h under vacuum before analysis. specific surface areas (sbet) were calculated according to the brunauer-emmett-teller (bet) equation, using adsorption data in the relative pressure range between 0.05 and 0.30. the total pore volume (vtotal) was estimated from the amount adsorbed at the relative pressure of 0.99. the pore size distribution curves were obtained from the desorption data using the barrett-joyner-halenda (bjh) model. uv-vis spectra were recorded using a jasco v-750 spectrophotometer equipped with an integrating sphere that allows measurements on solid samples. chromatic parameters were registered with a konica minolta cr-410 colorimeter with a white calibration plate (y=94.27, x= -1.06, y= 3.45), measurements were done in triplicate and means reported. in the cie l*a*b* uniform color space the color coordinates are: l* (brightness), a* (green-red) chromaticity, b* (blue-yellow) chromaticity to describe the color of the matrices.11 preparation the two approaches mentioned above were used to include beet extract into the carriers. in the direct encapsulation procedure, an aqueous mixture of 5 ml beet juice and 0.1 g β-cd is added, under constant stirring and room temperature, to 20 ml colloidal silica which was previously diluted with 12 ml water and brought to a ph of ~ 4 with an ascorbic acid solution of 15 %. then the dye containing composition was introduced in a vacuum rotary evaporator and kept at 30 °c under vacuum until the liquid was evaporated and the precipitate was separated and crushed into powder. it was denoted silica-bet 1. two mesoporous supports were prepared for beet juice adsorption. one of them, silica 2, was obtained from colloidal silica and ctab as template agent, the molar ratio sio2/ctab being equal to 0.12/0.3 10-3. the mixture was brought to an acid ph with an acetic acid solution 20 %, under stirring and kept for 5 days at room temperature. the other one, an aluminosilicate carrier (silicaal 3) is obtained by a hydrothermal process (90 °c, 48 h) in a basic medium from colloidal silica and na2o·al2o3 using sds as a structure directing surfactant. the molar ratios of the components were sio2/al2o3 = 6, sio2 /na2o = 10/7, sio2/sds = 10/1.4. the silica 2 and silicaal 3 powders were separated by repeated centrifugation and washing with distilled a cc ep te d m an us cr ip t todan et al. water and then they were thermally treated at 600 °c, with 5 h plateau and a heating rate of 1 °c min-1. 0.8 g of each host material was mixed with 1.2 ml beet juice, stirred for homogenization and followed by 5 h vacuum drying in dark conditions. a colored powder was obtained. equal quantities of the three dye loaded powders were spread on plates and were put in a pet container together with a petri dish containing ammonium hydroxide solution 25 %. the container was sealed for about 15 min so that the powders should be in contact with the ammonium vapours developed into the headspace. the color changes were determined. results and discussion structural characterization of the unloaded matrices x-ray diffraction the crystallographic structure of the as prepared samples was evaluated by xrd analysis. the diffraction patterns of the powders are presented in fig. 1. as can be seen in fig. 1 a, the diffraction patterns indicate that the silica carrier is amorphous, marked by the existence of a broad peak at 2θ in the range 20-35° attributed to amorphous silica. 12 the pattern of the aluminosilicate sample in fig. 1 b shows the formation of a crystalline phase of the zeolite y type (identification was performed using pdxl software from rigaku connected to icdd database). the inset of fig. 1 a shows the saxs diagram of the two as prepared powders. the variation of the scattering intensities with the scattering vector (q) is due to the electron density difference between the matrices and the empty pores. 13,14 the scattering curves of each of the two samples present a regular structure, confirming the long-range well-ordered mesostructured. 15,16 a cc ep te d m an us cr ip t betalain pigments in inorganic hosts 5 fig. 1. xrd patterns of silica 2 and silicaal 3 (the inset shows saxs diagram of the same unloaded carriers) (a) and details of crystal structure of silicaal 3 (b) n2 physisorption in fig.2 one can see that both samples show type iv isotherms according to iupac classification, typical for mesoporous materials. a cc ep te d m an us cr ip t todan et al. fig. 2. n2 adsorption–desorption isotherms and pore size distributions (inset of the figures) of silica 2 and silicaal 3 supports silica 2 support exhibits a h2 hysteresis loop that indicate the existence of a complex pore structure in which network effects are important. in the case of silicaal 3 support, the hysteresis loop is of h3 type suggesting an assemblage of slit-shaped pores. the pore size distribution graphs show a unimodal distribution a cc ep te d m an us cr ip t betalain pigments in inorganic hosts 7 for both types of supports with peak maxima at 12.6 nm for silica and 3.8 nm for aluminosilicate. a t-plot analysis indicated the absence of microporosity in the silica support, whereas in aluminosilicate an important fraction of micropores was detected (~70 %). bet surface area, as well as total pore volume are higher for silica type carrier than for zeolite-y type aluminosilicate (table i). table i. textural parameters of silica 2 and silicaal 3 supports sample sbet /m2g-1 vtotal /cm3g-1 silica 2 205.3 0.557 silicaal3 148.5 0.138 porosity measurements as well as the x-ray analysis prove that the supports are highly ordered materials. infrared absorption spectra fig. 3 shows the ftir spectra of the unloaded matrices. the strongest absorption bands of the two samples lie in the wavenumbers range 1200-950 cm -1 described as an asymmetric stretching mode o-(si,al)-o. 17 silica 2 sample shows a peak at 1110 cm -1 while silicaal 3 at 986 cm -1 , the presence of al atoms in the structure generating a shift to lower wavenumbers. 16 fig 3. ftir spectra of silica 2 and silicaal 3 supports a cc ep te d m an us cr ip t todan et al. in the region 420-500 cm -1 the bands are assigned to o-(si, al)-o bending mode. 17 a strong sharp band can be seen in the spectrum of silica 2 matrix at 464 cm -1 . in the aluminum containing one it is shifted to 434 cm -1 and becomes weaker. bands corresponding to symmetric stretching modes appear in the range 650-820 cm -1 . 17 in the spectrum of the silica 2 sample a single band at 810 cm -1 can be attributed to si-o-si sym stretching 18 , while the presence of al in the sample generates a widening and flattening of the bands (781 cm -1 and 700 cm -1 ). 18 the absorption bands at 1636 cm -1 show the presence of water in the matrices. 12 ftir spectra confirm the formation of an aluminosilicate type frame work in accordance with xrd results and show that the structure directing agent was removed by thermal treatment. characterization of matrices loaded with beet juice pigment uv-vis spectroscopy of the dye-carrier formulation there is an electron resonance system based on the two nitrogen atoms of the betalains molecules which is linked to the colour properties. 19 bethaxanthins with a more simple structure around nitrogen atom have a typical uv-vis absorption maximum in the 457-480 nm region and are yellow while the betacyanins with an extended resonance system generate wavelengths in the range 524-542 nm that makes them violet. 19-22 fig. 4 presents the uv-vis spectra of the three prepared samples by inclusion and adsoption. all display the betanin absorption maximum (535 -543 nm). compared to silica-bet 1 spectrum, in which case the pigments can be stabilized by inclusion complex formation with β-cd 5 , the silica 2 and silicaal 3 samples have different profiles. in case of silica 2, an overlapping of the bands is observed in the 472-535 nm range and silicaal 3 spectrum shows shifts to lower (415 nm) and higher values (543 nm). betalains being known as electron donating compounds 21 , this fact can be due to interactions with the matrices which become more significant for a high specific surface area and micro/mesoporosity. 23,24 aluminium coordinative unsaturated sites in the network of silicaal 3 enhances the interactions with adsorbed betalains. 8,24 a cc ep te d m an us cr ip t betalain pigments in inorganic hosts 9 fig. 4. uv-vis spectra of the beetroot dye formulations based on silica and aluminosilicate effect of nh3 vapors on dye-carrier formulations color change mean l* a* b* values for the dye loaded powders before and after ammonium exposure are presented in table ii. table ii. color parameters for beet juice in different matrices before and after exposure to base atmosphere sample color parameters before exposure color parameters in nh3 atmosphere l* a* b* l* a* b* e silica 2 84.78 ± 0.43 4.49 ± 0.17 -1.16 ± 0.03383.13 ± 0.29 3.60 ± 0.14 0.70 ± 0.089 1.87 silicaal 3 86.21 ± 0.35 3.69 ± 0.38 -1.78 ± 0.014 86.34 ± 0.11 1.99 ± 0.05 0.25 ± 0.080 2.65 silica-bet1 80.02 ± 0.21 7.68 ± 0.12 -0.24 ± 0.010 85.12 ± 0.47 2.80 ± 0.20 1.72 ± 0.270 7.32 *data are presented as mean ± standard deviation of triplicate analyses the other parameter in table ii, e, is the color difference between before (l0 , a0 , b0) and after exposure to nh3 (l, a, b) and was determined using the following equation. 25 δe = [(l0-l*) 2 + (a0-a*) 2 + (b0-b*) 2 ] 1/2 a cc ep te d m an us cr ip t todan et al. the values of the color parameters presented above indicate that the as prepared matrices containing beet juice are different, silica-bet 1 and silica 2 are redder while silicaal 3 is lighter and yellower. values δe between 1.5 -3 (silica 2 and silicaal 3 sample) indicate distinct and over 6 (silica-bet 1 sample) great differences between the initially prepared samples and the ones exposed to ammonia. 26 conclusion betalains pigments were included in silica and respectively aluminosilicate carriers to stabilize and improve their properties for practical applications. the direct encapsulation approach enabled the incorporation of other natural occurring compounds besides the dye, such as the complex forming β-cd and ascorbic acid, upgrading thus the prepared formulation regarding the color properties and the color variation with ph change. structure directing agents generated mesoporous silica and zeolite y-type supports with a complex, regular pore layout for the colorant adsorption. betalains color properties depend on the variation in the physico-chemical characteristics of the micro/mesoporous supports. a cc ep te d m an us cr ip t betalain pigments in inorganic hosts 11 и з в о д имобилизација природних беталаинских пигмената у неорганским материјалима ligia todan1, daniela c culita1, mirabela e. soare1, rodica m. ion2, radu c. fierascu2 и maria maganu3 1ilie murgulescu institute of physical chemistry, romanian academy, 202 splaiul independentei, 060021 bucharest, romania, 2national institute for research & development in chemistry and petrochemistryicechim, 202 splaiul independentei, 060021 bucharest, romania , и 3„costin d. nenitescu”institute of organic and supramolecular chemistry, romanian academy, 202 b splaiul independentei, 060023 bucharest, romania у циљу испитивању нових ph осетљивих формулација хране, екстракт црвене цвекле богат бетацијанином, ради побољшања стабилности његове боје, је наношен на различите неорганске подлоге које садрже силицијум-диоксид и алуминосиликате. директном методом инкапсулације пигмента на неорганску подлогу која садржи силицијум-диоксид и алуминосиликат додат је стабилизатор који представља инклузиони комплекс βциклодекстрина и аскорбинске киселине. систем накнадног пуњења укључује синтезу порозног силицијум-диоксид и алуминосиликатног праха и адсорпцију екстракта репе на овим подлогама. пре наношења екстракта извршена је структурна карактеризација неорганске подлоге (дифракција рендгенских зрака, ftir и n2-физиорпција). присуство бетанина (одобрен као црвена боја за храну) у испитиваним узорцима је одређено применом uv-vis спектроскопије. испитиване су промене боје, као и ph генерисане промене боје прахова изложених дејству амонијака при врху капсулираних посуда. добијени резултати могу допринети већој примени екстракта репе, док предложене еколошке формулације могу бити од значаја код паковања хране. 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(2023) https://doi.org/10.2298/jsc230327042p this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230327042p j. serb. chem. soc.00(0)1-5 (2023) note jscs–12332 published dd mm, 2023 1 thermal investigation of material derived from the species apatura iris marina simović pavlović1*, maja pagnacco2, dimitrije mara3, aleksandra radulović3, bojana bokić4, darko vasiljević4 and branko kolarić4,5 1faculty of mechanical engineering, university of belgrade, kraljice marije 16, belgrade, serbia; 2institute of chemistry, technology and metallurgy, university of belgrade, njegoševa 12, belgrade, serbia; 3institute of general and physical chemistry, studentski trg 12/v, belgrade, serbia; 4photonics center, institute of physics, university of belgrade, pregrevica 118, belgrade, serbia, 5microand nanophotonic materials group, university of mons, place du parc 20, 7000 mons, belgium (received 27 march; revised 20 april; accepted 21 july 2023) abstract: the material's size and shape influence its physical, chemical, and mechanical properties. this study describes an investigation of natural photonic structure of the butterfly’s wing, mainly composed of chitin. the effect of corrugations at the nanoscale on material's optical response is unambiguously revealed in presented thermal measurements. furthermore, the presented study shows the possibility of exploiting holography to monitor dynamics in situ. keywords: apatura iris butterfly; biopolymer chitin; sensing dynamics in situ. introduction apatura iris butterfly’s wing used for this study is shown in figure 13. butterfly wings are made of the biopolymer chitin4, with general formula (c8h13o5n)n. the chitin composition of different parts of the butterfly's body is described elsewhere.5 the paper revealed that chitins from different parts are chemically very similar, but with significant differences in their surface morphologies. *corresponding author. e-mail: simovicmarina99@gmail.com https://doi.org/10.2298/jsc230327042p a cc ep te d m an us cr ip t https://doi.org/10.2298/jsc230327042p simović pavlović et al.. fig 1. apatura iris butterfly: (a) an optical image of the whole butterfly; (b) sem image of a ground scale of the wing. in this study surface morphology is characterized by jeol jsm 6610 lv (japan), scanning electron microscope (sem) in conjunction with the energy dispersive spectroscopy (eds) detector model x-max large area analytical silicon drift connected with inca energy 350 microanalysis (detection of elements z ≥ 5, detection limit: ~ 0.1 mas. %, resolution 126 ev). micro-elemental (eds) analysis of apatura iris butterfly’s wing in two selected points, scale cell and the wing membrane, given in figure 2, showed presence of carbon (c), oxygen (o) and nitrogen (n) originated from chitin. as it can be seen, the content of c, n, and o slightly differs in scale cell and the wing membrane, indicating different chitin compositions in different surface structures. the presence of gold (au) originates from sample preparation for sem/eds analysis. thermal camera "flir a65" (640 x 512 pixel, thermal resolution 50 mk, focal length 13 mm, field of view angle 45° x 37°) is used to measure the temperature of the sample after the irradiation with laser. later, holographic method will be used to characterize the interaction of the photonic structure with light 6,7. a scheme of the holographic setup that is going to be used in the experiment is described elsewhere8. the setup will allow simultaneous recording of deformation and temperature. a cc ep te d m an us cr ip t thermal investigation of apatura iris 3 fig 2. (a) sem image of the scale cells and the wing membrane of apatura iris butterfly’s wing, (b) eds analysis of the wing at the scale cell (spectrum 1), (c) eds analysis of the wing at the wing membrane (spectrum 2). results and discussion six samples are individually irradiated by external lasers operating at four different wavelengths (450 nm, 532 nm, 660 nm, and 980nm) keeping the power and illuminated spot diameter constant at 1 mw and 1 mm, respectively. thermal measurement is made over the period that includes the time before the start of heating (interaction with laser), during the heating itself, and after the irradiation stopped, more precisely the cooling of the sample. the difference in temperature due to heating by laser at different wavelengths has been observed. the highest temperature is caused by the interaction with 450 nm light, while the lowest is recorded for the wavelength of 532 nm. however, the complete reversible cooling (reaching the initial state) has not been observed for the wavelengths of 450 nm and 980 nm. a complete return to the initial state is observed for the wavelengths of 532 nm and 660 nm. analyzing data in depth is vital to link thermal measurement with the reflectance spectrum9 (figure 3a) and the heating/cooling process as a function of time. figure 3b is showing the change in temperature over time, as a function of wavelengths. finally, the thermal measurements match the reflectance pattern and the heating/cooling dynamics as a function of time. it is evident that the temperature maximum in figure 3b at 450 nm corresponds to the reflectance maximum. the maximum value recorded at 450 nm is followed by 660 nm, while reflectance is at minimum around 532 nm. a cc ep te d m an us cr ip t simović pavlović et al.. fig 3. (a) apatura iris reflectance spectrum; (b) cooling dynamics as a function of time after the irradiation with four different wavelengths. the lasers have been switched on at 2nd second and switched off at 9th second in order to record heating/cooling dynamics. (reflectance spectrum is taken from the reference 9) the only observed discrepancy refers to the wavelength which does not belong to the visible part of the spectrum and for which completely different rules apply. the photon at 980 nm carries the energy that cannot cause any electronic transitions but can affect the vibrational one within the system as well as thermal management by vibrational relaxation. the observed asymmetric heating/cooling response scales perfectly with the measured reflectance response. conclusion this paper presents an investigation of apatura iris's natural photonic structures under the light irradiation at different wavelengths. the correlation between the reflectance at different wavelengths and thermal response is revealed. acknowledgements: b.k., d.v., and b.b. acknowledge funding provided by the institute of physics belgrade, through the institutional funding by the ministry of education, science, and technological development of the republic of serbia. additionally, b.k. acknowledges support from f.r.s.-fnrs. m.p. acknowledges support from the ministry of education, science and technological development of the republic of serbia (grant no. 451-03-47/202301/200026). d.m. and a.r. acknowledges support from ministry of science, technological development, and innovation of the republic of serbia contract number: 451-03-47/202301/200051. all authors acknowledge the support of the office of naval research global through the research grant n62902-22-1-2024. a cc ep te d m an us cr ip t thermal investigation of apatura iris 5 и з в о д термално испитивање материјала из лептира apatura iris марина симовић павловић1, маја пањако2, димитрије мара3, александра радуловић3, бојана бокић4, дарко васиљевић4 и бранко коларић4,5 1машински факултет – универзитет у београду, краљице марије 16, београд, србија; 2институт за хемију, технологију и металургију, универзитет у београду, његошева 12, београд, србија; 3институт за општу и физичку хемију, студентски трг 12/v, београд, србија; 4центар за фотонику, институт за физику, универзитет у београду, прегревица 118, београд, србија, 5група за микро и нанофотонске материјале, универзитет у монсу, парк трг 20, 7000 монс, белгија облик и величина материјала утичу на његове физичке, хемијске и механичке особине. ова студија описује проучавање природних фотонских структура, крила лептира која се претежно састоје од полимера хитина. ефекат нано коругације на оптички одговор материјала је презентован кроз термална мерења. такође је представљена могућност примене холографске методе за праћење динамике in situ. (примљено 27. марта; ревидирано 20. априла; прихваћено 21. јула 2023.) references 1. s.r. mouchet, p. vukusic, advances in insect physiology 54 (2018) 1 (https://doi.org/10.1016/bs.aiip.2017.11.002) 2. d. mara, b. bokic, t. verbiest, s. r. mouchet, b. kolaric, biomimetics 7(4), (2022), 153 (https://doi.org/10.3390/biomimetics7040153) 3. z. han, l. wu, z. qiu, h. guan, l. ren, j. bionic eng. 5 (2008) 14 (https://doi.org/10.1016/s1672-6529(08)60066-9) 4. h.i. leertouwer, b. d. wilts, d. g. stavenga, opt. express 19 (2011) 24061 (https://doi.org/10.1364/oe.19.024061) 5. m. kaya, b. bitim, m. mujtaba, t. koyuncu, int. j. biol. macromol. 81 (2015) 443 (https://doi.org/10.1016/j.ijbiomac.2015.08.021) 6. d. pantelić, d. grujić, d. vasiljević, j. biomed. opt. 19 (2014) 127005 (https://doi.org/10.1117/1.jbo.19.12.127005) 7. j. liu, w. kuang, j. liu, z. gao, s. rohani, j. gong, j. chem. eng. 438 (2022) 135554 (https://doi.org/10.1016/j.cej.2022.135554) 8. m. simovic-pavlovic, m.c. pagnacco, d. grujic, b. bokic, d. vasiljevic, s. mouchet, t. verbiest, b. kolaric, j. vis. exp. (2022) e63676 (https://dx.doi.org/10.3791/63676) 9. d. pantelić, s. ćurčić, s. savić-šević, a. korać, a. kovačević, b. ćurčić, b. bokić, optics express 19(7) (2011) 5817-5826 (https://opg.optica.org/oe/fulltext.cfm?uri=oe-19-75817#:~:text=https%3a//doi.org/10.1364/oe.19.005817) a cc ep te d m an us cr ip t https://doi.org/10.1016/bs.aiip.2017.11.002 https://doi.org/10.3390/biomimetics7040153 https://doi.org/10.1364/oe.19.024061 https://doi.org/10.1016/j.ijbiomac.2015.08.021 https://doi.org/10.1117/1.jbo.19.12.127005 https://doi.org/10.1016/j.cej.2022.135554 https://dx.doi.org/10.3791/63676 https://opg.optica.org/oe/fulltext.cfm?uri=oe-19-7-5817#:~:text=https%3a//doi.org/10.1364/oe.19.005817 https://opg.optica.org/oe/fulltext.cfm?uri=oe-19-7-5817#:~:text=https%3a//doi.org/10.1364/oe.19.005817 health risk assessment of potentially harmful substances from fly ashes generated by coal and coal waste combustion accepted manuscript this is an early electronic version of an as-received manuscript that has been accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as j. z. buha marković, a. d. marinković, j. z. savić, a. d. krstić, a. b. savić and m. đ. ristić, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc220130048m this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc220130048m j. serb. chem. soc.00(0)1-14 (2023) original scientific paper jscs–12208 published dd mm, 2023 1 health risk assessment of potentially harmful substances from fly ashes generated by coal and coal waste combustion jovana z. buha marković1*, ana d. marinković1, jasmina z. savić1, aleksandar d. krstić1, andrija b. savić1 and mirjana đ. ristić2 1university of belgrade, vinča institute of nuclear sciences national institute of the republic of serbia, belgrade, serbia and 2university of belgrade, faculty of technology and metallurgy, karnegijeva 4, belgrade, serbia (received 30 january 2022; revised 10 march 2023; accepted 3 august 2023) abstract: lignite and coal waste used as feed fuels in thermal power plants (tpps) and semi-industrial fluidized bed boiler (fbb), as well as their representative fly ashes (fas), were examined. fly ashes were compared employing anions and cations content in correspondent water extracts, trace elements and polycyclic aromatic hydrocarbon concentrations, as well as health risk assessments of substances known to be of concern for public health. fluoride and sulfate contents in water extracted fas are far below the legislation limits for waste, classifying all investigated fas as non-hazardous. among investigated trace elements, cd content is the lowest, while mn content is the highest. the highest enrichment ratios are noticed for as, pb, hg, cu, v and cr. the results indicate that total pahs content is elevated in fa from the combustion of coal waste (afb), with fluoranthene prevailing. the cancer risk of as and the non-cancer risk of as and ni in some fas surpass their respective permissible limits. the incremental lifetime cancer risk of an adult population indicates a potential pahs risk in afb, whereas all other fly ashes are within safe limits. keywords: coal ashes; leaching; trace elements; pahs; carcinogenic risk; total hazard impact. introduction despite limited coal supplies, its consumption in europe is expected to rise due to the uncertainty of the energy sector, so therefore many eu countries extended the life of coal-fired power plants.1,2 the choice of the appropriate coal as a feed fuel for particular combustion systems relies on coal characteristics, such as its moisture, ash content and gross calorific value.3 fluidized bed combustion is * corresponding author e-mail: jbuha@vin.bg.ac.rs https://doi.org/10.2298/jsc220130048m a cc ep te d m an us cr ip t mailto:jbuha@vin.bg.ac.rs https://doi.org/10.2298/jsc220130048m 2 buha marković et al. regarded as an environmentally friendly way of producing energy from low grade coals, due to continuous operation and low nox and so2 emissions.4,5 coal is the dominant energy source in serbia, with over 7 billion tons of estimated lignite reserves. annually, the electric power industry (epi) of serbia produces around 560, 2010 and 7878 gwh in thermal power plants (tpps) kolubara a, kostolac b and nikola tesla a, respectively, which brings to the generation of 246.60 kt, 610.82 kt and 2.08 mt fly ash, accordingly.6 since lignite with particle sizes lower than 10 mm cannot be used further in thermal power plant boilers, it is considered waste. however, coal waste might have a significant energy perspective and can be used as a feed fuel in other combustion technologies, such as fluidized bed combustion.4 in these circumstances, coal waste originating from the kolubara basin, discarded as waste in tpp kolubara a, was tested as a feed fuel in a semi-industrial fbb with a thermal power of up to 500 kw. most studies have shown that potentially harmful trace elements emitted during coal combustion are distributed in bottom ash, fly ash particles of different parameters and flue gases so that they can reach soil and water.7 content of heavy metals salts, such as chlorides or sulfates, affect leaching mechanisms of potentially harmful compounds in fas.8,9 ca and mg are the most dominant cations in fly ash water leachates, while anions primarily include sulfates, carbonates and fluorides.10 furthermore, anions and cations content were determined to complement the scarce literature data considering water extracted fas. in addition, persistent organic compounds, such as polycyclic aromatic hydrocarbons (pahs), represent significant environmental pollutants generated during coal combustion.11 the us environmental protection agency (us epa) regulated 16 priority pahs due to their harmful effects on people and the environment.12 physicochemical properties of pahs and, consequently, their environmental fate depends on their structure and number of fused aromatic rings.13 pahs are usually classified into low molecular weight (lmw), medium molecular weight (mmw) and high molecular weight (hmw). as the molecular weight of a particular pah increases, its carcinogenicity rises, while its acute toxicity decreases.14 the fate and partitioning of toxic elements and pahs in coal combustion by-products depends on the used feed fuel, combustion temperature, burner type and structure.10 therefore, a thorough analysis of the used coals and produced fas is necessary to optimize combustion processes in terms of environmental and health issues.15 intake of potentially toxic substances by humans can be through three pathways i.e. ingestion, inhalation and dermal contact. model of human exposure (adults and children) to potentially harmful substances is developed by the us epa guidelines.16 in this study, feed coals and fas from tpps kolubara a (tpkb), kostolac b (tpks) and nikola tesla a (tpnt), as well as coal waste and fa from semia cc ep te d m an us cr ip t health impact of coal ashes. 3 industrial fbb were investigated. this paper characterizes and compares different coals based on proximate and ultimate analysis, along with trace element concentrations, and analyzes corresponding fly ashes, determining their particle size diameters, trace elements and pahs content, as well as anions and cations content in fly ash water leachates. the aim of this study was to perform a human health risk assessments of potentially harmful substances in fly ashes by estimating the carcinogenic and non-cancer risk for trace elements and the incremental life cancer risk of seven carcinogenic pahs associated with different exposure routes. experimental a sampling of coals and fly ashes a sampling of coals from tpp kolubara a (ckb), tpp kostolac b (cks), tpp nikola tesla a (cnt) and coal waste from a fluidized bed boiler (cfb) was done according to the standard method.17 the same method was used for the collection of coal fly ashes from tpkb (akb), tpks (aks), tpnt (ant) and from the cyclone of fbb (afb). the samples were prepared and stored in a glass container at a dark place under a temperature below 15 °c.18,19 granulometric analysis of fly ashes the granulometric analysis of investigated fly ashes was performed using a set of sieves with round hole diameters of 90 μm, 200 μm, 500 μm and 1000 μm.20 proximate and ultimate analysis of coals the proximate analysis of investigated coals was done by leco tga 701 according to a standard test method.21 the ultimate analysis was performed by a leco chn 628 series with a sulfur add-on module.22-24 determination of anions and cations by ion chromatography 5 g of each fa was mixed with 50 ml of deionized water in an ika ks130 orbital shaker (800 rpm) for 180 min. obtained extracts were filtered and further used to determine cations and anions by ion chromatograph dionex. the details are given in the supplementary material. determination of trace elements in coals and fas extraction of 18 elements (as, be, cd, co, cr, cs, cu, ga, ge, hg, mn, mo, ni, pb, sb, sr, u, v) was done by sequential extraction.25 trace elements concentrations were determined by the inductively coupled plasma mass spectrometry (icp-ms) using an agilent 7500ce instrument equipped with octopole reaction system in fullquant mode. the details about icpms measurements are described in the supplementary material. each element's total concentration is the sum of its six representative fractions. pahs analysis the extraction of 16 priority pahs (naphthalene, nap; acenaphthylene, acy; acenaphthene, ace; fluorene, flu; phenanthrene, phe; anthracene, ant; fluoranthene, fla; pyrene, pyr; benzo[a]anthracene, baa; chrysene, chry; benzo[b]fluoranthene, bbf; benzo[k]fluoranthene, bkf; benzo[a]pyrene, bap; dibenzo[a,h]anthracene, daha; benzo[g,h,i]perylene, bghip and indeno[1,2,3-cd]pyrene, ip) from fly ashes was done according to literature.26 the prepared extracts were analyzed by hplc/dad. the details are explained in the supplementary material. a cc ep te d m an us cr ip t 4 buha marković et al. enrichment ratios (ers) of trace elements the er of a particular trace element was calculated as a quotient of its concentration in ash and correspondent coal. er higher than 1 indicates trace elements enhancement in ash compared to the corresponding feed fuel. human health assessment for trace elements and pahs from fas the human health assessment associated with trace elements and pahs found in fas was performed for adults and children. human health assessment comprises the calculation of total risk indexes (r) for carcinogenic substances (ascc, cdcc, crcc, cocc, nicc), as well as total hazard indexes (hi) for non-carcinogenic substances (asncc, pb, hg, cdncc, crncc, concc, nincc and cu). total r and hi were calculated for each element by the following equations: r = dig × sfig + dih × sfih + dd × sfd (1) hi = dig / rfig + dih / rfih + dd / rfd (2) di/ mg kg -1 day-1 is the daily intake dose, sfi/ kg day mg -1 is the corresponding carcinogenicity slope factor and rfi/ mg kg -1 day-1 is the reference dose for each exposure route i, where i stands for ingestion (ig), inhalation (ih) or dermal contact (d). parameters used to calculate di are given in supplementary material (table s-i, a and s-ii), and the toxicity values for rfi and sfi are in table s-iii. 27,28 generally, a risk less than 10−6 can be ignored; a carcinogenic risk in the range of 10−6 to 10−4 is acceptable or tolerable, while a risk exceeding 10−4 is considered unacceptable for any element. if hi is higher than 1, negative health effects are probable. the incremental lifetime cancer risk (ilcr) was estimated as the sum of 7 carcinogenic pahs (baa, chry, bbf, bkf, daha, bghip and ip) for three exposure routes. ilcr ≤ 10-6 generally denotes virtual safety, 10-4 < ilcr < 10-6 indicates potential risk, while ilcr > 10−4 represents a high risk. the health assessment calculations for pahs and their parameter values are shown in supplementary material (table s-i, b and s-ii). results and discussion proximate and ultimate analysis the fuels used in four combustion facilities were examined by proximate and ultimate analysis and results on air dried basis are shown in table i. compared to coal waste, all feed fuels from tpps have better properties as a combustion feedstock due to lower ash content, as well as higher volatile matter, carbon content and heating value.29 because high volatile matter can be associated with spontaneous combustion (particularly with low-rank coals such as lignite), knowing the volatile content of the coal simplifies transportation and handling. the total sulfur of cks is fourfold higher than other coal samples. ckb, cnt and cfb originate from the same basin (kolubara), while cks derive from the kostolac basin. the combustible sulfur proportion of cfb (36 %) is substantially lower than for other coals from the kolubara basin (62 69 %).30 a cc ep te d m an us cr ip t health impact of coal ashes. 5 table i. proximate and ultimate analysis of lignite from tpps kolubara a (ckb), kostolac b (cks) and nikola tesla a (cnt) and coal waste from fbb (cfb) ckb cks cnt cfb content, % (proximate analysis) total moisture* 42.94 40.34 48.90 36.74 inherent moisture** 6.04 8.18 7.14 7.04 ash 37.21 36.29 36.86 61.85 coke 61.55 60.56 57.31 77.43 combustible 61.79 63.71 63.14 38.15 volatile 38.45 39.44 42.69 22.57 c-fix 23.34 24.27 20.45 15.58 heating value, mj kg-1 high 16.56 16.56 16.41 10.16 low 15.75 15.62 15.60 9.75 content, % (ultimate analysis) carbon 41.26 41.64 38.80 24.81 hydrogen 3.74 3.78 3.74 1.96 total sulfur 0.64 2.76 0.55 0.66 combustible sulfur 0.43 1.91 0.34 0.26 nitrogen 0.58 0.67 0.44 0.34 oxygen 16.77 15.65 19.92 10.78 *as-received; **as determined; all other data are given on a dry basis granulometric analysis ash particle size is an important parameter since finer ashes provide a greater surface area for the sorption of harmful substances.31 the granulometric analysis results are shown in fig. 1. akb mainly comprises finer particles with diameters less than 90 μm (64.61 %), while afb has the highest yield in the f3 fraction (92.96 %). fas from tpps have mean particle diameters ranging from 126 μm to 131 μm, while for afb, it is 341 μm. the variations of fas particle size are affected by combustion system characteristics, burning temperatures, used feed fuels, as well as the system treatment of the gaseous effluents.32 a cc ep te d m an us cr ip t 6 buha marković et al. fig. 1. granulometric analysis of fly ashes from tpps kolubara a (akb), kostolac b (aks), nikola tesla a (ant) and from fluidized bed boiler (afb) anions and cations content in water extracted fly ashes fig. 2 depicts the leaching of anions and cations in water extracted fas. among determined cations, calcium prevails with concentrations ranging from 2.06 mg/g (in ant) to 5.32 mg/g (in aks). it is in line with the literature since calcium salts easily dissolve.33 potassium is the most dominant in akb with a concentration of 3.38 mg/g, which is more than tenfold higher than in other fas. sulfates are the most abundant among the other anions, ranging from 2.32 mg/g (in ant) to 10.32 mg/g (in aks), whereas chlorides, phosphates, and nitrates are undetected. fluorides vary from 0.10 mg/g (in aks) to 0.18 mg/g (in akb). most of the fluorides in fa are insoluble, while the water-soluble form of fluoride mainly originates from naf and kf.34 all water extracted fas can be regarded as non-hazardous waste since fluoride and sulfate contents are far below upper legislation limit values for waste classification given in table s-iv. a cc ep te d m an us cr ip t health impact of coal ashes. 7 fig. 2. leaching of cations (a) and anions (b) in water extracted fly ashes from tpps kolubara a (akb), kostolac b (aks), nikola tesla a (ant) and from fluidized bed boiler (afb) the concentration of trace elements in coals and representative fas; enrichment ratios (ers) fig. s-1 and table s-ishow the overall trace elements concentrations in feed coals and their corresponding fly ashes. cfb has the lowest overall trace element content among all the investigated feed coals (256.72 mg/kg). trace element concentrations in coals are the highest for mn (up to 209.63 mg/kg), while decreased content for hg and ge is observed (fig. s-1, a-b). trace element contents in fas vary from the lowest values for cd (up to 0.76 mg/kg in aks) to the highest content for mn, ranging from 210.48 mg/kg in afb to 607.29 mg/kg in akb (fig. s-1, c-d). finer tpp ashes have elevated trace element concentrations than afb due to higher concentrations in corresponding feed fuels and larger surface area of ash particles.35 furthermore, the reason for significantly lower concentrations of as, co, cs and hg in afb compared to other fas from tpps can be higher combustion temperatures in tpps. it is known that higher combustion temperatures can imply enhanced trace element concentrations in flue gases which can further easily condensate on fly ash particles.36 in contrast, cu, a cc ep te d m an us cr ip t 8 buha marković et al. ga, ge and sb contents in afb are not the lowest of all fas, which is consistent with the literature where these trace elements do not show a significant correlation with ash particle diameters.31 fig. 3. enrichment ratios for tpps kolubara a (tpkb), kostolac b (tpks), nikola tesla a (tpnt), and fluidized bed boiler (fbb) the enrichment ratios (ers) are presented in fig. 3. as (from 13.58 to 18.60), pb (from 6.55 to 8.85), hg (from 2.97 to 5.68), cu (from 4.08 to 6.13), v (from 3.14 to 5.45), and cr (from 2.60 to 5.04) have the highest er values. these elements are enhanced in fas due to their vaporization during coal combustion and subsequent condensation on the fly ash particles.37 at relatively low temperatures, arsenic easily forms volatile compounds.38 in addition, pb related to organic matters volatilizes at around 850 °c, while hg may react with flue gas components and form oxidized mercury in a wide temperature range.39 other elements, such as be, co, ni, u, sb and sr, have lower ers because they are correlated with less volatile minerals.5 pahs content in investigated fas fig. 4a shows the distributions of lmw, mmw, and hmw pahs. the total pah content and the concentration of 10 pahs defined in serbian legislation for soil are presented in fig. 4b. the total pahs content varies from 278.95 ng/g a cc ep te d m an us cr ip t health impact of coal ashes. 9 (ant) to 32548.66 ng/g (afb), which is consistent with pahs ranges for fas found in the literature.40 the mmw pahs are the most abundant in akb (68.25 %) and afb (70.03 %), while lmw pahs prevail in aks (75.23 %) and ant (67.28 %). among examined fas, afb and akb contain the highest fluoranthene contents, while aks and ant have the highest fluorene concentrations (table si,b). these findings are in accordance with literature revealing fla and flu as the most abundant pahs due to incomplete combustion of fossil fuels.26 the content of 10 pahs is in the range from 124.13 ng/g (ant) to 23075.48 ng/g (afb), which is lower than permissible limits in serbian soil guidance (table s-iv).41 fig. 4. distribution of pahs according to molecular weight (a); the overall and 10 pahs content (b); fly ashes from tpps kolubara a (akb), kostolac b (aks), nikola tesla a (ant) and fluidized bed boiler (afb) health impact for potentially toxic trace elements and pahs from fas risk indices, as well as total hazard indices for children and adults, are presented in table ii. the non-cancer risks for children demonstrate that ni values in akb, aks and ant, as well as as for all fas, exceed the acceptable limit. furthermore, his for adults are higher than safe values for as in akb, aks and ant (table ii). hazard indices for cdncc, concc, cu, hg and pb are about two orders lower than the regulatory level.42 to acquire better insight into the health impact of each fas, the overall his, as well as the overall rs, are determined as the sum of hi or r for all investigated elements, respectively. the estimated overall hi is the highest for akb (7.15 for children and 2.15 for adults). trace elements hazard quotients (hqig, hqih and hqd) and risk indices (rig, rih and rd) for three exposure routes are shown in table s-v, a-b. the dominant exposure routes for a cc ep te d m an us cr ip t 10 buha marković et al. the non-cancer risk are dermal contact for asncc, cdncc, nincc and cu, and ingestion for concc, crncc, hg and pb. table ii. trace elements cancer risk (r) and total hazard impact (hi); pahs total risk (ilcr) for fly ashes from tpps kolubara a (akb), kostolac b (aks) and nikola tesla a (ant) and from fluidized bed boiler (afb) carcinogenic elements r/ unitless akb aks ant afb children as 1.81×10-4 1.51×10-4 1.61×10-4 4.82×10-5 cd 2.87×10-17 4.92×10-17 1.70×10-17 / co 1.55×10-9 1.11×10-9 2.13×10-9 6.79×10-10 cr 4.25×10-11 3.25×10-11 4.93×10-11 2.91×10-11 ni 1.24×10-10 1.14×10-10 1.05×10-10 8.08×10-11 overall r 1.81×10-4 1.51×10-4 1.61×10-4 4.82×10-5 adults as 2.51×10-4 2.09×10-4 2.23×10-4 6.69×10-5 cd 4.49×10-17 7.69×10-17 2.66×10-17 / co 2.42×10-9 1.74×10-9 3.33×10-9 1.06×10-9 cr 6.65×10-11 5.08×10-11 7.71×10-11 4.54×10-11 ni 2.26×10-9 2.08×10-9 1.91×10-9 1.47×10-9 overall r 2.51×10-4 2.09×10-4 2.23×10-4 6.69×10-5 non-carcinogenic elements hi/ unitless akb aks ant afb children as 4.68 3.90 4.16 1.25 cd 8.71×10-4 1.49×10-3 5.16×10-4 / co 7.66×10-3 5.51×10-3 1.06×10-2 3.36×10-3 cr 9.71×10-1 7.41×10-1 1.13 6.64×10-1 cu 9.56×10-2 1.96×10-1 1.11×10-1 1.57×10-1 hg 1.02×10-1 6.61×10-2 1.23×10-1 2.80×10-2 ni 1.18 1.09 1.00 7.73×10-1 pb 1.11×10-1 1.20×10-1 1.41×10-1 7.91×10-2 overall hi 7.15 6.13 6.68 2.95 adults as 1.30 1.08 1.16 3.47×10-1 cd 3.12×10-4 5.35×10-4 1.85×10-4 / co 5.86×10-4 4.21×10-4 8.07×10-4 2.57×10-4 cr 2.32×10-1 1.77×10-1 2.69×10-1 1.59×10-1 cu 3.85×10-2 7.88×10-2 4.46×10-2 6.33×10-2 hg 2.43×10-2 1.58×10-2 2.93×10-2 6.69×10-3 ni 5.32×10-1 4.90×10-1 4.50×10-1 3.47×10-1 pb 2.41×10-2 2.60×10-2 3.06×10-2 1.71×10-2 overall hi 2.15 1.87 1.98 9.40×10-1 pahs ilcr/ unitless akb aks ant afb children 1.32×10-7 2.12×10-8 1.65×10-7 6.05×10-6 adults 4.29×10-7 6.91×10-8 5.40×10-7 1.98×10-5 a cc ep te d m an us cr ip t health impact of coal ashes. 11 afb displays the lowest total risk index (4.82×10-5 for children and 6.69×105 for adults). the calculated total risk indices decrease in the following order: ascc>cocc>nicc>crcc>cdcc (table ii). total cancer risk of as (up to 2.51×10-4 in akb) exceed the safe limits, while cocc, nicc, crcc and cdcc risk values are far below permissible limit values. the arsenic content should be thoroughly monitored and controlled. the most dominant exposure route among carcinogenic substances is the inhalation for cocc, nicc, crcc and cdcc, while for ascc, it is dermal contact (table s-v, a-b). three exposure routes were used, both for children and adults, to determine human health issues caused by pahs. table ii demonstrates that only afb for adults indicates a potential risk for pahs, while all other fas are within safe limits. the literature provides health assessments of pahs for various soil types, while there is a lack of information regarding health assessments of pahs from fas generated during coal combustion.43 conclusions potassium is the most dominant among cations (akb), while sulfates have the highest content in all fas among anions. investigated fas can be considered nonhazardous since fluorides and sulfates content are far below legislation limits for waste classification. the ers are the highest for as, pb, cu, v, hg and cr. among all investigated fas, the highest concentration of fla was noticed in akb and afb, while flu concentrations are maximal in aks and ant. health calculations associated with trace elements and pahs in fas lead to some general conclusions: • the obtained results for non-cancer risk show that ni in akb and aks and ant, as well as as for all fas, exceed the permissible limit for children, while his for adults are higher than safe values for as in akb, aks and ant. • as exceeds the safe limit for cancer risk in all fas, apart for afb. • pahs potential risks for adults (except for afb) are within safe values. due to a lack of information on anions and cations analysis in water extracted fas, as well as health risks related with exposure to pahs and trace elements, this research could contribute to the current state of knowledge for health issues associated with fly ash disposal. supplementary material supplementary materials are available electronically from https://www.shdpub.org.rs/index.php/jscs/article/view/12208, or from the corresponding authors on request. acknowledgements: this work was supported by the ministry of education, science and technological development of republic of serbia; grant number 451-03-68/2022-14/200017. a cc ep te d m an us cr ip t https://www.shd-pub.org.rs/index.php/jscs/article/view/12208 https://www.shd-pub.org.rs/index.php/jscs/article/view/12208 12 buha marković et al. и з в о д процена ризика за потенцијално опасне супстанце из летећих пепела добијених сагоревањем угља и отпадног угља joвана з. буха марковић1, ана д. маринковић1, јасмина з. савић1, александар д. крстић1, андрија б. савић1 и мирјана ђ. ристић2 1универзитет у беогрдау, инсититут ѕа нукларне науке винча институт од националог значаја за републику србију, београд, србија и 2универзитет у београду, технолошко-металуршки факултет, карнегијева 4, београд, србија у овом раду, испитивана су горива (лигнит и отпадни угаљ) која се користе у термоелектранама (те) и полуиндустријском постројењу са флуидизованим слојем (фбб), као и летећи пепели добијени њиховим сагоревањем. летећи пепели су упоређени на основу: садржаја анјона и катјона у њиховим воденим екстрактима, концентрације елемената у траговима и полицикличних ароматичних угљоводоника (пах-ова), као и процене здравственог ризика који потиче од претходно поменутих потенцијално опасних супстанци. садржај флуорида и сулфата у воденим екстрактима летећих пепела далеко је испод законски дозвољених граница за отпад, на основу чега се могу сврстати у безопасне. од испитиваних елемената у траговима, садржај cd је најнижи, док је концентрација mn највиша. највеће обогаћење пепела у односу на одговарајући угаљ, примећено је за as, pb, hg, cu, v и cr. на основу добијених резултата показано је да је укупни садржај пах-ова највећи за летећи пепео добијен сагоревањем отпадног угља. међу испитиваним паховима, највишу концентрацију има флуорантен. ризици који потичу од арсена (међу канцерогеним елементима), као и арсена и никла (међу неканцерогеним елементима), премашују дозвољене граничне вредности. вредност процењеног ризика од рака код одрасле популације у случају пах-ова, показује да за летећи пепео добијен сагоревањем отпадног угља постоји потенцијални ризик, док су вредности за остале пепеле унутар дозвољених граница. 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podgorica, montenegro, 2vinča institute of nuclear sciences, laboratory of theoretical physics and condensed matter physics, university of belgrade, p. o. box 522, 11001 belgrade, serbia, 3institut für mineralogie und kristallographie, fakultät für geowissenschaften, geographie und astronomie, university of vienna, althanstraße 14, a-1090 vienna,, austria and 4accreditation body of montenegro, jovana tomaševića 1, 81000 podgorica, montenegro (received 22 july, revised 30 december 2014, accepted 30 january 2015) abstract: in the reaction of 1,3-dimethylpyrazole-5-carboxylic acid (hl) with m(oac)2·4h2o, (m = cu or co), two novel complexes were prepared, the square-planar [cul2(h2o)2] and the octahedral [col2(meoh)4]. the crystal structures were determined by single-crystal x-ray diffraction. in both complexes, the deprotonated acid displays monodentate coordination to the metal ions. according to the results of a csd survey, this is the first structural report on the metal complexes with an n1-substituted pyrazole-5-carboxylic ligand. keywords: pyrazole-based ligand; transition metal complex; crystal structure. introduction pyrazole-based compounds and their transition metal complexes have attracted considerable research interest because of their potentially beneficial biological properties. the wide biological activity of this class of compounds (anticancer, antimicrobial, antiviral, anti-inflammatory and others) is described in several reviews.1 apart from the investigation of the biological activity of pyrazole derivatives, they have been extensively used as ligands or synthons in coordination chemistry2 and crystal engineering including the metal organic frameworks.3 the most of these valuable functions of pyrazole ligands originate in their various bonding modes to metal ions that can be further influenced and rationally designed by the attachment of substituents with additional coordination sites.3–5 another important property of pyrazole derivatives is the presence of hydrogen bonding sites, either on the pyrazole ring (pyrrolic n–h donor and pyridinic n * corresponding author. e-mail: snovak@vin.bg.ac.rs doi: 10.2298/jsc140722009j _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 868 jaćimović et al. acceptor) or its substituents, which facilitates molecular association into the hydrogen-bonded supramolecular structures. ligands derived from pyrazole-5-carboxylic acid also display various bonding modes as they can coordinate in monoor dianionic form through the pairs of nand o-donors from the heterocyclic ring and carboxyl groups. despite this coordination variety, the cambridge structural databank (csd)6 does not contain information about metal complexes with n1-substituted derivatives of pyrazole-5-carboxylic acid. herein, the syntheses, ir characterization and crystal structures of cu(ii) and co(ii) complexes with 1,3-dimethylpyrazole-5-carboxylate (scheme 1) are presented, whereby the x-ray experiments revealed the not very common monodentate coordination of this pyrazole-5-carboxylic acid based ligand. scheme 1. 1,3-dimethylpyrazole-5-carboxylic acid (hl) with atom numbering. experimental preparation of the complexes the [cul2(h2o)2] complex was synthesized in the reaction of a warm methanolic solution (10 cm3 ch3oh) of 0.25 mmol (0.049 g) cu(oac)2·4h2o with a warm methanolic solution (5 cm3 ch3oh) of 0.5 mmol (0.070 g) 1,3-dimethylpyrazole-5-carboxylic acid (hl) mixed in a 1:2 mole ratio. after two days, the blue single crystal product was filtered off and washed with methanol. the yield was 84.29 % (0.080 g). the [col2(meoh)4] complex was synthesized in the reaction of a warm methanolic solution (3 cm3 ch3oh) of 0.125 mmol (0.031 g) co(oac)2·4h2o with a warm methanolic solution (3 cm3 ch3oh) of 0.25 mmol (0.035 g) 1,3-dimethylpyrazole-5-carboxylic acid (hl) mixed in a 1:2 mole ratio. after two days, the single crystal product was filtered off and washed with methanol. yield was 32.75 % (0.019 g). the synthesis resulted in the mixture of reactants, hl and co(oac)2·4h2o, and a very small amount of the [col2(meoh)4] complex. after filtering off, the purple crystals of the complex were mechanically separated for ir and x-ray analysis. for both syntheses, the 1,3-dimethylpyrazole-5-carboxylic acid was used as purchased from sigma–aldrich. infrared spectra the infrared spectra (ir) of the synthesized complexes and corresponding uncoordinated ligand were recorded as kbr pellet on a thermo nicolet nexus 670 ft-ir instrument in the wavenumber range of 4000–400 cm-1. as expected, the ir spectra of complexes show rather similar features, especially concerning the position of the relevant absorption band ν(coo-). the spectrum of the uncoordinated ligand displayed ν(cooh) at 1712.27 cm-1, while in spectra of the complexes, the band was replaced with νas(coo-) at ca. 1600 cm-1 and νs(coo-) _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ cu(ii) and co(ii) complexes with 1,3-dimethylpyrazole-5-carboxylic acid 869 at ca. 1350 cm-1. the approximate δν of 250 cm-1 is in accordance with monodentate coordination of the carboxyl ligand.7 ir (cm-1): 2926.26, 2578.64, 2481.96, 1712.27, 1542.66, 1471.80, 1245.67; [cul2(h2o)2]: 3434.32, 2926.48, 1600.12, 1538.44, 1460.62, 1354.18, 1293.23; [col2(meoh)4]: 3380.68, 2927.31, 1605.24, 1536.72, 1459.10, 1350.08, 1287.29. x-ray crystal structure determination the diffraction data from selected single crystals of [cul2(h2o)2] and [col2(meoh)4] were collected at 200 and 150 k, respectively on a nonius kappa ccd diffractometer equipped with a monocapillary optics collimator, mokα radiation (λ = 0.71073 å). the data were corrected for absorption by evaluation of multi-scans. the crystal structure was solved by direct methods, using shelxs and refined with shelxl.8 the h atoms attached to c atoms were placed at geometrically calculated positions with the c–h distances fixed to 0.93 and 0.96 å from aromatic and methyl c atoms, respectively. the corresponding isotropic displacement parameters of the hydrogen atoms were equal to 1.2ueq and 1.5ueq of the parent c atoms. the h atoms attached to o atoms were located in the final electron density maps and refined isotropically. the geometrical calculations were performed with parst9 and platon.10 the programs ortep11 and mercury12 were used for molecular graphics. the details of the x-ray structural analysis are given in table s-i of the supplementary material to this paper. results and discussion coordination modes of pyrazole-5-carboxylic acid ligands in metal complexes extracted from csd the csd analysis6 focused only on the donor abilities of the pyrazole-5 -carboxylic acid ligands (with four potential coordination sites in total) and hence, the crystal structures of complexes with derivatives comprising additional donor sites were not considered. in the complex compounds extracted from csd, the pyrazole-5-carboxylic ligands displayed seven different ways of coordination. in over 70 % of the structures (40 of 56 extracted), the ligands derived from pyrazole-5-carboxylic acid coordinate only as n1, o bidentates forming the five membered chelate rings. the coordination of the remaining donors leads to the formation of bior polynuclear metal complexes, while the formation of a chelate ring is still preferential. there are only two examples of monodentate coordination of pyrazole-5-carboxylic ligands.13,14 in both of these complexes, the monodentate coordination can be related to the possible steric hindrance between the ligands in the coordination spheres of the corresponding metal ions. up to now, the crystal structures of complexes with n1-substituted derivatives of pyrazole-5-carboxylic acid have not been reported. in the case of the ligand present in the title complexes (scheme 1), the methyl substituent on the n1-pyrazole prevents the most frequently occurring chelating form and confines the coordination to the carboxyl oxygen donor. moreover, the steric hindrance that this substituent produces on the third donor site, n2-pyrazole, significantly reduces the coordination ability of this donor, consequently monodentate coordination could be expected. it should be men_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 870 jaćimović et al. tioned that the crystal structures of complexes comprising n2-substituted pyrazole-5-carboxylic ligands are also scarce in the literature. in several reported cases with phenyl substituents on pyrazole n2, the corresponding ligands coordinate in the n1o-chelating mode.15 description of crystal structures the [cul2(h2o)2] complex crystallizes in the space group p21/c. the cu atom is placed in a nearly ideal square planar environment formed by pairs of oxygen donors from the deprotonated carboxylic acid and h2o molecules, fig. 1a. the lengths of the two types of cu−o bonds coincide within the s.u. values, while the angle o2−cu1−o1w of 91.09(6)° actually shows the largest deviation from the ideal square-planar geometry (table s-ii of the supplementary material). the cu(ii) ion lies on an inversion center. the cu−o1w and cu−o1 bond lengths are within the ranges of previously reported square-planar cu(ii) complexes comprising monodentately-coordinated carboxyl and h2o ligands.16 in comparison to the crystal structure of the uncoordinated 1,3-dimethylpyrazole-5 -carboxylic acid (hl),17 the geometry of the coordinated ligand is slightly altered (table s-ii). apart from the expected changes in the carboxyl fragment due to the acid deprotonation, the dihedral angle between coo− and pyrazole planes increases upon the ligand coordination form 4.0 to 8.7(2)°. concerning the coordination plane of the four oxygen donors, the pyrazole ring is rotated by 69.2(1)°. fig. 1. molecular structures of: a) [cul2(h2o)2] with indicated positions of pseudo-coordinated o1w atoms (dashed lines) and b) [col2(meoh)4], independent molecule a. the displacement ellipsoids are drawn at the 40 % probability level. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ cu(ii) and co(ii) complexes with 1,3-dimethylpyrazole-5-carboxylic acid 871 two types of rather strong hydrogen bonds having h…a distances shorter than 2.0 å stabilize the crystal structure of the [cul2(h2o)2] complex (table i). the o1w−h1w…o1 interaction between the h2o molecule and the uncoordinated carboxyl oxygen atom links the complex units into a chain extending along the a axis (fig. 2a). it should be noticed that within this chain, each h2o oxygen atom is closely adjacent to the cu(ii) ions of the neighboring complex unit. the cu1…o1wi (i = x–1, y, z) distance is 2.802(1) å, which is significantly shorter than the sum of the van der waals radii of these two atoms (3.84 å).18 taking into account these short cu…o1wi contacts, the environment of each cu(ii) ion could be described as pseudo-octahedral. the positions of pseudocoordinated o1wi atoms are shown in fig. 1a. the angles formed between the atom o1wi and the o2 and o1w donor atoms from the cu(ii) coordination sphere are 87.9 and 105.8°, respectively. the separation distance between two cu(ii) in the chain is 3.826(1) å and coincides with the length of the shortest a axis. both h2o molecules coordinated to cu(ii) further engage as hydrogen bonding donors to the non-substituted pyrazole n atoms of the neighboring chain (o1w−h2w…n2), leading to cross-linkage and the formation of a three-dimensional network. a fragment of this three-dimensional crystal packing is shown in fig. 2a. table i. the geometry of hydrogen bonding (å, °) for the cu(ii) and co(ii) complexes; symmetry codes for [cul2(h2o)2]: i) x+1, y, z; ii) –x+1, y–1/2,–z+1/2; symmetry codes for [col2(meoh)4]: i) x, y, z; ii) x, y–1, z structure d–h…a d–h d…a h…a d–h…a [cul2(h2o)2] o1w−h1w …o3i 0.81(3) 2.611(2) 1.82(3) 165(3) o1w−h2w…n2ii 0.83(3) 2.716(2) 1.89(3) 170(4) [col2(meoh)4] o4a−h4a …o2ai 0.79(3) 2.587(2) 1.83(3) 160(3) o4b−h4b…o1bi 0.82(3) 2.582(2) 1.78(3) 167(3) o3a−h3a…n2aii 0.73(3) 2.707(3) 1.98(3) 175(3) o3b−h3b…n2bii 0.79(3) 2.765(2) 1.97(3) 177(3) the second complex [col2(meoh)4] crystallizes in space group p-1, with the asymmetric unit containing two halves of the corresponding crystallographically independent complex molecules (a and b). the two co(ii) ions, which are placed in the inversion centers (0,0,0 and 0.5,0.5,0.5), adopt deformed octahedral coordination geometry (table s-ii) built by two monodentately coordinated l ligands and four molecules of methanol, fig. 1b. the co−o1 coordination bonds in the two independent molecules are of similar lengths, while the dihedral angles between the carboxyl and pyrazole planes are 8.6(2) and 3.5(2)° for molecules a and b, respectively. the bonds within the l ligand show only small variations with respect to the uncoordinated molecule17 and the same ligand in the cu(ii) complex. comparison of the monodentate coordination of the l _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 872 jaćimović et al. ligand to the co(ii) and cu(ii) ions in the two complexes revealed a pronounced difference between the cu1−o1−c1 and each of the two co1−o1−c1 angles (9.3° on average) (table s-ii). in both molecules of [col2(meoh)2], the carboxyl group of l is placed in the level of vicinal meoh ligand and engages in strong intramolecular (o4−h4…o2) hydrogen bonding (table i). this is in contrast to the cu(ii) complex in which a carboxyl group points away from the vicinal ligand to engage in intermolecular o−h…o interaction. fig. 2. crystal packing of: (a) [cul2(h2o)2] and (b) [col2(meoh)4]. intermolecular interactions are indicated by dashed lines. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ cu(ii) and co(ii) complexes with 1,3-dimethylpyrazole-5-carboxylic acid 873 the meoh ligands mostly contribute to the dissimilarity of the two independent [col2(meoh)4] molecules. thus, the co−o coordination bonds involving meoh ligands (table s-ii) are both longer in molecule b by 0.04å on average. a closer comparison of the coordinated meoh also showed marked differences in the orientation of their methyl groups. considering the meoh ligands that form strong intramolecular hydrogen bonds, it could be observed that their c8 methyl atoms deviate from corresponding o1/o1′/o4/o4′ planes by 0.18(1) and 0.96(1) å in a and b, respectively. in both type of molecules another pair of meoh serves as donor in the o3−h3…n2 intermolecular hydrogen bond to the pyrazole acceptor from the neighboring molecule (table i). these interactions lead to the formation of two distinct chains composed of the same type of molecules, a or b. both chains run in the direction of the b axis; the mutual inclination between the pyrazole rings belonging to different chains is 47.4(1)°. the chains composed of a and b molecules mutually interact by van der waals interactions (fig. 2b). conclusion the report describes the synthesis, ir characterization and crystal structures of cu(ii) and co(ii) complexes with 1,3-dimethylpyrazole-5-carboxyle ligand (hl), the square-planar [cul2(h2o)2] and the octahedral [col2(meoh)4]. the presence of a methyl substituent on the n1 atom of hl prevents bidentate coordination common for pyrazole-5-carboxylic acid-based ligands; therefore, in both complexes the ligand is monodentately coordinated to the metal ion. the crystal structures of the complexes are stabilized by extensive o−h…o and o−h…n hydrogen bonds, in which h2o and methanol ligands play a significant role as hydrogen bonding donors. supplementary data crystallographic data of the structural analysis have been deposited with the cambridge crystallographic data centre ccdc no. 1015253 and 1015254. the data are available free of charge via www.ccdc.cam.ac.uk/data_request/cif (or from the ccdc, 12 union road, cambridge cb2 1ez, uk; +44 1223 336033; e-mail: deposit@ccdc.cam.ac.uk). the crystallographic data, and selected bond lengths and angles for the cu and co complexes are given in tables s-i and s-ii, respectively, of the supplementary material to this paper, which is available electronically from http://www.shd.org.rs/jscs/ or from the corresponding author on request. acknowledgement. s. b. n. thanks the ministry of education and science of the republic of serbia (project no. 172014 and 172035) for financial support. ž. j. and m. k. thank to the ministry of science of the republic of montenegro/natonal project for the financial support. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 874 jaćimović et al. и з в о д синтеза и кристална структура комплекса cu(ii) и co(ii) са 1,3-диметил-пиразол-5-карбоксилном киселином жељко к. јаћимовић 1 , милица косовић 1 , слађана новаковић 2 , gerald giester 3 и ана радовић 4 1металуршко–технолошки факултет универзитета у црној гори, подгорица , црна гора, 2институт за нуклеарне науке „винча“, лабораторија за физику кондензоване материје,универзитет у београду, п. пр. 522, 11001 београд, 3institut für mineralogie und kristallographie, fakultät für geowissenschaften, geographie und astronomie, university of vienna althanstraße 14, a-1090 vienna, austria и 4aкредитационо тело црне горе, јована томашевића 1, 81000 подгорица, црна гора у реакцији 1,3-диметилпиразол-5-карбоксилне киселине (hl) и m(oac)2·4h2o (m = cu или co) синтетисана су два нова комплекса, квадратно-планарни [cul2(h2o)2] и октаедарски [col2(meoh)4]. некоординовани лиганд и синтетисани комплекси су окарактерисани инфрацрвеним спектрима. кристалне стуктуре комплекса су одређене рендгенском стуктурном анализом. у оба комплекса депротонована киселина се координује за јон метала као монодентатни лиганд. према резултатима претраге кембричке банке података ово је први стуктурни опис комплекса метала са n1-супституисаним дериватом пиразол-5-карбоксилне киселине. 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ruschewitz, z. anorg. allg. chem. 638 (2012) 1424; b) y. kani, m. tsuchimoto, s. ohba, acta crystallogr., c 56 (2000) e79 17. e. foresti serantoni, r. mongiorgi, l. riva di sanseverino, atti accad. naz. lincei cl. sci. fis. mat. nat, rend. 54 (1973) 787 18. a. bondi, j. phys. chem. 68 (1964) 441. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice accepted manuscript this is an early electronic version of an as-received manuscript that hasbeen accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as s. a. emmanuel, a. a. sallau, o. adedirin, h. d. ibrahim, m. l. buga, a. okereke, g. n. ozonyia and f. m. alabi, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc221126040e this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc221126040e j. serb. chem. soc.00(0)1-15 (2023) original scientific paper jscs–12148 published dd mm, 2023 1 synthesis of sodium silicate crystals from rice husk ash stella adedunni emmanuel 1 , alhassan adeku sallau 1 *, oluwaseye adedirin 1 , hussain doko ibrahim 2 , mohammed lawal buga 2 , anthony okereke 2 , gertrude ngozi ozonyia 2 and fortune miebaka alabi 2 1 chemistry advanced research center, shestco, abuja, nigeria, and 2 raw materials research and development council, rmrdc, maitama, abuja (received 26 november 2022; revised 20 january 2023; accepted 10 july 2023) abstract: the rich husk is an agricultural waste of rice cultivation worldwide, which is highly rich in amorphous silica. rice husk obtained from dagiri was pyrolysed at 750 ℃ to give white ash (rha) which was further treated with acid (arha). the ash was reacted with sodium hydroxide at 90 ℃ for 2 hours 30 min to produce sodium silicate crystals. sodium silicates synthesized in the study were characterized with some physicochemical parameters. their structural and morphological properties were assessed using a fourier transform infra-red spectrophotometer (ftir), x-ray diffractometer (xrd) and scanning electrode microscope (sem). the mineralogical composition of the ash and sodium silicate was investigated with energy-dispersive x-ray fluorescence (edxrf) spectrometer. the sodium silicate produced has a melting point of 61 ℃, a ph of 12.03 and appeared as brownish-white to clear-white in colour. the rha and arha from xrd investigation showed patterns which match the mineral phase cristobalite, while that of the sodium silicate xrd patterns match the mineral heptahydrate disodiumtrioxosilicate as the most dominant phase. rietveld refinement of the xrd pattern for the sodium silicate gave rwp = 12.81, rexp = 5.55, χ 2 = 5.3274 and gof = 2.3081 against a dual phase analysis. the sodium crystal-synthesized is suitable for use in cosmetic formulations. keywords: acid treated ash, cosmetic, crystal structure, paddy, silica, water glass. introduction rice husk is an agricultural waste material produced via the cultivation of rice1. rice production worldwide was pegged at over 952 million tons in 2016 according to a 2018 data report by fao. the rice husk, which is the outer cover of the rice, accounts for 20 % of the paddy produced annually (about 190 million tons).2 *corresponding author e-mail: mailto:creamysal@yahoo.co.uk https://doi.org/10.2298/jsc221126040e a cc ep te d m an us cr ip t mailto:creamysal@yahoo.co.uk https://doi.org/10.2298/jsc221126040e 2 emmanuel et al. the world has seen a tremendous increase in annual rice production in recent years when compared with the previous report of an estimated 40 million tons in 2003. it is regarded as the second most-produced cereal in the world and geographically concentrated in asia with over 90 % of the world output. while, nigeria is putting efforts to develop this sector of agricultural produce, the bulk of rice husks generated is mostly sent to landfills, burnt as fuel to generate energy and leading to residual ashes (rha) containing over 60 % silica content.3,4 rice husk contains high ash content,5,6 varying from 13 to 29 wt.% depending on the variety, climate, and geographic location. the ash is largely composed of silica (87–97 %) with small amounts of inorganic salts.6,7 the high ash content can be recovered as amorphous silica.8 the amorphous nature of silica in rice husk makes it extractable at a lower temperature range of 90 110 °c.3 sodium silicate solutions are complex mixtures of silicate anions and polymer silicate particles with a molar ratio of sio2 to na2o > 2 and are commercially referred to as water glass.4 the solutions are generated in different grades as characterized by their sio2:na2o ratios, varying water contents and low viscosity. 9 sodium silicate is among the alkaline elements silicate which have attracted extensive studies due to their importance in mineralogy, solid-state chemistry and chemical technology. about 12 natural sodium silicates minerals are known to exist and nine of them have been studied structurally.10 anhydrous sodium silicate are considered importance raw materials in production of finished products in both inorganic chemistry and technical mineralogy.11 some of industrial applications include production of water glass solutions or manufacture of acid-resistant enamel frits, components of refractory cements, steel and alloy castings, electrodes, dyeing, printing, textile auxiliaries as inorganic binders, or builders in washing powders (soap and detergent cake).11,12,13 crystalline sodium silicates such as na2si2o5 have been shown to exhibit high ion-exchange capacity, selectivity and multifunctionality; such as buffering capacity, water softening ability and ability to degrade fats by hydrolysis as such suggested as alternative replacement of sodium tripolyphosphate builder in detergents.14,15 it has been suggested that soluble sodium silicates play other vital role of corrosion inhibition in many of its applications such as engine antifreeze and detergents.16 sodium silicate in surfactants have been applied in detergent technology for pre-flush technology, with the main role of sodium silicate targeted at removal of hardness (ca2+, mg2+) which can complex with anionic surfactants making them less reactive or unstable. the silica anions minimize these unwanted charges by sequestering the metals via a precipitation reaction. the silica anion also competes for active, positivelycharged sites on the casing and wellbore thereby reversing their surface charge and repelling surfactants.12 sodium silicate is also used as a binder in cosmetic products, such as face powders, eyeliners and lipsticks, to give them a smooth and creamy texture. it helps to bind the different ingredients together and prevent them a cc ep te d m an us cr ip t synthesis of sodium silicate crystals 3 from separating17. sodium silicate can be used as a thickener in shampoos and conditioners, to improve viscous and consistency. sodium silicate can also be used as a ph stabilizer in lotions and creams, to help maintain their ph levels. this is important because certain cosmetic ingredients can become less effective or even unstable at certain ph levels. sodium silicate can be used as a preservative in liquid soaps and facial cleansers, to help prevent the growth of bacteria and other microorganisms. it works by altering the ph level of the product, which makes it less hospitable to microorganisms. sodium silicate can also be used as a skin protectant in sunscreens and lotions, to help shield the skin from harmful uv rays and other environmental pollutants. it forms a protective film on the skin, which helps to prevent moisture loss and maintain the skin's natural barrier function18,19. the work aims to synthesize a sodium silicate crystal from rice husk ash for possible use in the cosmetic industries. experimental rice husk was obtained from rice mill at dagiri, gwagwalada local government of federal capital territory, abuja-nigeria analar grade sodium hydroxide (naoh) and hydrochloric acid (hcl) from sigma aldrich were used. all solution was prepared with distilled de-ionized water. sample pre-treatment rice husk was washed with water to remove sand and dust particles. the washed rice husk was then spread on tray and air dried for 24 hours. dried rice husk was placed in stainless steel cylinder and ashed at 750 ℃ for 3 hours in a carbolite muffle furnace available at sheda science and technology complex (shestco) abuja. the pyrolyzed sample tagged rha were weighed (200 g) separately and treated with 1600 ml of hcl (10 % and 20 % v/v) in a pyrex beaker and heated for 2 hours at 90 ℃. the reaction mixtures were filtered and washed repeatedly with distilled deionized water until the filtrate becomes neutral to litmus. the residues designated 10 % arha and 20 % arha (acid treated rha) were dried at 105 ℃ for 12 hours in a carbolite hot air oven and kept in a desiccator for subsequent steps. preparation of sodium silicate modified method 20 was adopted: 150 g of arha was reacted with 1200 ml naoh (10 % w/v) in a pyrex beaker and heated at 110 °c for 2 hours 30 minutes. the reaction mixture was filtered and the filtrate concentrated by heating to 2/3 of its original volume. the concentrate was seeded with a pinch of pure sodium silicate crystals to induce rapid crystallization. the sodium silicate crystals formed was separated by decanting the supernatant liquid. the crystal was rinsed with iced cold distilled deionized water and allowed to drain on a filter. the drained crystals were stored in polyethylene container. physicochemical characterization of crystal determination of sodium oxide content (is 6773 (2008): the sodium oxide content of crystal obtained was determined by titrating, a dissolved crystal sample with standard hydrochloric acid to ph 4.3 using methyl orange indicator. 5 g sodium silicate crystals were dissolved in 50 ml deionized water and transferred to 100 ml volumetric flask then diluted to mark with deionized water. a 25 ml portion of the solution was titrated against 0.2 m hcl to orange end point colour. a cc ep te d m an us cr ip t 4 emmanuel et al. calculations 𝐶na2 𝑂/ % = v*m*3.1 𝑤 (1) where: v is volume of hydrochloric acid used, m is the molarity of the hydrochloric acid and w is the weight of crystal sample viscosity measurement the viscosity of liquid sodium silicate solutions was determined using the brookfield rotational viscometer according to the astm d2983. by insertion of number 2 spindle, rotating at 60 rpm at 25 °c in the sodium silicate liquor. the viscosity was read from the display reading. the determination of viscosity of sodium silicate liquor was essential to determine the optimum thickness for effective crystallization of the sodium silicate crystals from its water glass liquor. determination of total solid (is 6773 (2008) the total solids of liquid silicates were determined by weighing 5 ml (5 g) into a known weight of pre-fired porcelain crucible and placed in a muffle furnace at 1050 ℃ for 30 mins. a few drops of 30 % hydrogen peroxide were added to prevent spattering. after cooling in a desiccator and the crucible was reweighed. the weight of the residue was taken and the % solids were reported to the nearest 0.01 %. determination of silicon dioxide content (is 6773 (2008) the sio2 content was calculated by subtracting the % sodium oxide from the % total solids. sodium silicates are often described by the weight ratio of the silicon dioxide to the sodium oxide, with the sodium oxide as unity. it may be calculated directly by dividing the % sio2 content by the % na2o content that has been chemically determined. determination of ph (astm d4972) a 0.1 % w/v solution of sodium silicate crystal was prepared by dissolving 0.1 g of sodium silicate crystals in 100 ml deionized water. the ph of the sodium silicate liquor and 0.1 % sodium silicate solution was measured using the ph electrode and orion versa star pro meter advanced electrochemistry meter. melting point determination (din 53736) the microscopic hot stage method melting point apparatus by bristoline was used. a single crystal sample of the sodium silicate was placed on a hot stage and the heater turned on. the temperature at the point the crystal melts was read from the inserted thermometer. characterization of crystal empyrean pan-analytical xrd (with cu kα at λ = 1.54060, å v=40 va, i=40 ma and 5 to 74, 2theta angle), edxrf (vacuum and air atmosphere geological sample analysis) and phenom prox scanning electron microscope available at the central laboratory of umar musa yar’adua university, katsina were used to study the crystal structure, mineral composition and morphology of the crystal respectively. thermo-scientific nicolet is5 ft-ir spectrometer equipped with id7 atr sampler was used to asses any functional group available in the rha, arha and sodium silicate crystal over the range 500–4000 cm-1and resolution of 0.5 cm-1. the thermal behaviour was investigated by the perkinelmer mse-tga 4000 tga (30 950 °c at 10 °c /min) available at ahmadu bello university zaria. a cc ep te d m an us cr ip t synthesis of sodium silicate crystals 5 results and discussion physicochemical properties of sodium silicate crystal the physical properties of sodium silicate crystals are shown in the pictures in fig. 1 and some other chemical properties are in table 1. from the images in fig. 1, the appearance of the crystal was originally brownish white but on further washing, it became brightly white and soapy to the touch. the ph of sodium silicate solution was found to fall within the commonly reported range of 11–12.5, as a solid with alkaline properties in solution it is readily neutralized to silica solid in the presence of acid. this forms the basis for most reported work on the synthesis of silica from rice husk ash.21,22,23,24 a b c fig. 1 image of samples obtained (a) brownish silicate (b) washed silicate (c) crystalline appearance of washed silicate table 1 some physical and chemical properties of sodium silicate crystal and silica samples sodium silicate crystals sodium silicate liquor (water glass) 750°c rha 10% arha 20% arha colour brownish to white brownish white white white ph 12.03 12.03 neutral neutral neutral solubility⁄g dm −3 highly highly insoluble insoluble insoluble density ⁄ g cm −3 -1.415 ------ viscosity ⁄ mpa•s -25 --------- stability in air slow efflorescence stable stable stable stable melting point ⁄ ℃ 61 -------- na2o ⁄ % 12.40 11.54 --- sio2 ⁄ % 25.99 25.30 --- sio2/na2o 2.00 2.19 --- total solid 38.39 36.84 - a cc ep te d m an us cr ip t 6 emmanuel et al. rha, sodium silicate crystals composition analysis the elemental composition of the rha, arha and sodium silicate crystals obtained from x-ray fluorescence analysis (by the air and vacuum atmosphere) are shown in table 2. it can be noticed that the percentage composition of sio2 increases progressively as the acid treatment concentration increase from 10 to 20 %. while other metal oxides concentration decreases. this indicates the effectiveness of acid treatment in purifying the rice husk ash samples, as a means of preventing unnecessary side reactions. the sio2 and na2o content of the sodium silicate crystals were 18.665 and 14.739 % respectively. this will indicate that the ratio of sio2 to na2o is 1, which is not in agreement with the classical method result. this deviation may be attributed to the consequential presence of traces of cao, mgo, al2o3, k2o and fe2o3 which might equally react with the acid during the titrimetric determination of na2o, thus giving a higher false value of na2o. according to the study by owoeye et al., the synthesis of a sodium disilicate from rha at different concentration of 3m, 4m and 5m of naoh was found to contains 28.64 %, 42.25 % and 36.97 % sio2 content while the na2o content was 12.32 %, 17.23 % and 18.24 % respectively 25. which were all higher than the 18.665 % of sio2 and 14.739 % of na2o contents obtained in the present study at lower concentration of naoh (10 % ≡ 2.5m). similarly, siregar et al., reported the synthesis of sodium silicate from both corn cob and rice husk with content of na been 43.29 % and 22.63%, that of o been 36.48 % and 71.98 % while the si content was 20.23 % and 5.39 % respectively26. table 2 edxrf oxides composition of rha, arha and crystal oxides, % 750 ℃ rha 10 % arha 20 % arha sodium silicate fe2o3 0.4698 0.15836 0.18990 0.1576 na2o – – – 14.739 mgo 3.30 1.46 0.35 1.379 al2o3 1.443 1.147 1.063 1.3516 sio2 72.148 90.344 91.567 18.665 p2o5 4.761 0.7510 0.8647 0.8505 so3 0.1647 0.0780 0.0777 0.3631 cl 0.0256 0.01778 0.02052 0.0824 k2o 3.3802 0.5772 0.5897 0.2346 cao 0.7572 0.1326 0.1220 0.03335 mno 0.4367 0.08555 0.08706 0.00799 sno2 1.00 1.00 1.00 others 0.17478 0.07515 0.08312 0.05111 xrd of the solid obtained the diffractogram of rha obtained is shown in fig. 2. the rha, 10 % arha and 20 % arha diffractogram pattern correspond to the mineral cristobalite (96-900-8111). the diffractogram obtained for rha treated at 750 ℃, confirms that heat treatment of rice husk at a higher temperature such as 750 ℃ a cc ep te d m an us cr ip t synthesis of sodium silicate crystals 7 results in the formation of crystalline silica. a similar report, showed that treatment of rice husk between 450-700 ℃ generally results in silica predominated with amorphous phase,27 while the higher temperature of 1000 to 1350 ℃ results in silica predominant by crystalline phase. similarly, shihab and twej reported obtaining amorphous and crystalline phases when rice husk was treated at 650 ℃ and 1000 ℃ respectively.24 fig. 2 xrd patterns of rha, arha and reference pattern it was also observed that there was no specific change in the crystalline phase nature of the rha when it was acid treated at different concentrations of 10 % and 20 % hcl. the diffractogram of the sodium silicate crystals obtained (fig. 3) match the reference phase 2106900 of the crystallography open diffractogram with a chemical name of sodium hydrogen tetraoxosilicate heptahydrate [na2(h2sio4) ∙ 7h2o]. 10 a cc ep te d m an us cr ip t 8 emmanuel et al. fig.3 diffractogram pattern of synthesized sodium silicate the suggested properties associated with this crystal are that it is monoclinic, and the nature of its structure. rietveld refinement of the xrd pattern for the sodium silicate performed by the profex software v5.0 gave rwp = 12.81, rexp = 5.55, χ 2 = 5.3274, gof = 2.3081 for na2(h2sio4). 7h2o and na3hsio4 ∙ 2h2o phase analysis 28 (fig. 4). the inability of the χ2 value, to meet up to the threshold value of 1.5 may be due to the presence of some impurities (majorly al and mg and traces of ca, fe, zn etc.) associated with the synthesis. a cc ep te d m an us cr ip t synthesis of sodium silicate crystals 9 fig. 4 sodium silicate rietveld refinement ft-ir spectra of rice husk ash and sodium silicate the infrared spectra shown in fig. 5 of the rice husk ash treated under high temperature and acid treatment. there was very week absorption peak at position of 3000 cm−1 and 3500 cm−1 which are usually attributed to o-h group for moisture in the 750 °c rha sample. while for 20 % arha and 10 % arha there was no visible absorption in the same region of 3000 to 3500 cm-1, which is a representation of −o−h group from water or si−o−h.22,29 the peaks at 1021, 1098 and 1090 in 750 °c, 10 % arha and 20 % arha samples respectively are often attributed to si−o−si asymmetric bond stretching vibration.30 while the peaks at 797, 794 and 795 are attributed to the si−o bending vibration band.31 most of the absorption peaks detected in the 750 °c rha sample, were also noticed to have disappeared in the 10 % arha and 20 % arha samples. this may be linked to effective removal of remnants structural lignin, cellulose and hemicellulose by the acid treatments. the spectra of sodium silicate show a strong broad peak at 3048.42 cm−1 which was attributed to presence of −oh group, due associated moisture. the peak at 1667.16 cm−1 is similarly a complementary −oh group caused by bending vibration of water17. while the strong stretching peak at 987.89 is due the si−o bond stretching of the silicon tetrahedron.32,33 the peak at 1443.94 cm−1 may be attributed to c−o symmetric stretching of c−o−si, due to some c sneaked or co2 absorbed into the separated solid. 34 also the peak at 2355.14 cm-1 position may be attributed to o=c=o stretching. the sodium silicate a cc ep te d m an us cr ip t 10 emmanuel et al. crystals may have been exposed to atmospheric carbon (iv) oxide during the draining process of the crystal. fig. 5 infrared spectra of sodium silicate crystals thermogravimetric analysis of sodium silicate crystals the results of tga for 750 °c rha, 10 % arha, 20 % arha and sodium silicate crystals are shown in fig. 6. it can be observed that a two-step weight loss occurs during the temperature treatments of rha and 10 % arha. the first weight loss of 0.242 % and 0.122 % which occurs before 200 °c can be attributed to physically adsorbed water.35 the second step weight loss occurs between 200 °c and 400 °c, which may be attributed to loss of other volatile, probably trapped in the sample’s matrix during initial preparation. the sample 20 % arha tga plot depicts only one step weight loss of 1.05 % between 28 °c to 450 °c. this weight loss may be attributed to release of trapped moisture as well as other volatiles. overall the tga of 750 °c rha, 10 % arha and 20 % arha samples appeared to be stable to heat treatment as about 98 % of the materials remain after 850 to 900 °c. the tga for the sodium crystal samples show that there was also two step weight loss between 30–400 ℃ and between 400–500 ℃. the first weight loss is considered a dehydration step, which is attributed to the loss of moisture in the crystal near and above its melting point as evidenced by the low weight loss of 1.777 %. the second, weight loss may be attributed to the removal of molar equivalents of crystal water molecules from the crystals sample as signified by the large weight loss of 34.475 %. the residue obtained after the attainment of 550 ℃ a cc ep te d m an us cr ip t synthesis of sodium silicate crystals 11 is attributed to the formation of anhydrous sodium silicate. beyond the 550 the anhydrous silicate was relatively stable with about 55 % of sample remaining. fig. 6 loss in weight measurement sem morphological observation the surface morphological appearance of the rha obtained at 750 ℃ shows a micro-flaky and irregular shape with agglomeration. for the 10 % arha the micro flaky agglomeration possesses a wavy opening fitted with lateral pores much like an oval hexagon and the 20 % arha showed no wavy pattern opening while also showing rod-like microstructure and some micropores. the isolated rod-like structure seen in the 20 % arha bears evidence of induced crystallinity as a result of increased acid concentration. the sodium silicate morphology at 12000× magnification figure 7d, shows an uneven surface and the appearance of tiny pores on the surface of agglomerated morphology. the surface of the crystal also appears to have slight indentation while the irregular pores appears to run deep. a cc ep te d m an us cr ip t 12 emmanuel et al. (a) (b) (c) (d) fig. 7 scanning electron microscopic image of (a) 750 ℃ rha, (b) 10 % arha, (c) 20 % arha, (d) sodium silicate (ss) crystal surface significant agglomeration of particles on the surface of the amorphous silica, used in the synthesis of sodium silicate from corncob and rice husk as well as existence of pores within the agglomerates was similarly reported.26 conclusion rice husk ash was used to synthesize crystalline sodium silicates. the acid treatment of rha showed improvement on the purification of silica obtained as well as improved crystal structure. the similarity of all the rha (750 ℃, 10 % arha and 20 % arha) to the cristobalite phase also shows that treatment type, affects the crystallinity of the silica obtained. the sodium silicate sample synthesized was found to contain two phases namely disodium orthosilicate a cc ep te d m an us cr ip t synthesis of sodium silicate crystals 13 (91.91 % na2h2sio4 ∙ 7h2o) and trisodium orthosilicate (8.10 % na3hsio4 ∙ 2h2o), as revealed by the rietveld refinement analysis. since disodium orthosilicate are often used in cosmetic formulations as an exfoliating agent or abrasive. the synthesized orthosilicate will be useful in improving skin texture when added to cosmetics. conflict of interest: the authors declare that there is no conflict of interest regarding the publication of this paper. acknowledgement: this work was supported by the raw material research and development council of nigeria. list of symbols and abbreviations arhaacid treated rice husk ash atrattenuated total reflectectance bsca-brownish silicate crystal abuja codcrystallography open database dta-differential thermal analysis edxrfenergy-dispersive x-ray fluorescence spectrometer fao-food and agriculture organization ftir-fourier transform infra-red spectrophotometer rmrdc-raw materials research and development council rha rice husk ash semscanning electron microscope shestcosheda science and technology complex tgathermogravimetry analysis xrd-x-ray diffraction/diffractometer rwp – weight profile rfactor rexp – expected r factor gof – goodness of fit и з в о д синтеза кристала натријум-силиката из пепела пиринчaне љуске stella adedunni emmanuel1, alhassan adeku sallau1*, oluwaseye adedirin1, hussain doko ibrahim2, mohammed lawal buga2, anthony okereke2, gertrude ngozi ozonyia2 и fortune miebaka alabi2 1chemistry advanced research center, shestco, abuja, nigeria, и 2raw materials research and development council, rmrdc, maitama, abuja због узгоја пиринча широм света пиринчaна љуска представља веома распрострањени пољопривредни отпад богат аморфним силицијумом. у овом раду пиролизом на 750 oc пиринчaна љуска је претворена у бели пепео (rha), који је затим третиран киселином (arha). након тога, добијена суспензија је реагована са натријумхидроксидом на 90 oc за време од 2,5 сата, при чему долази до издвајања кристала натријум-силиката. кристали натријум-силиката су окарактерисани на бази различитих физичко-хемијских параметара. за одређивање структуре силиката употребљени су a cc ep te d m an us cr ip t 14 emmanuel et al. инфрацрвени спектрофотометар са фуријевом трансформацијом (ftir), рендгенски дифрактометар (xrd) и микроскоп са скенирајућим електродама (sem). за испитивање минералошког састава пепела и натријум-силиката коришћен је енергетски дисперзивни рендгенски флуоресцентни спектрометар (edxrf). тачка топљења добијених кристала натријум-силиката је 61 oc, ph = 12,03, док је њихова боја била између светлосмеђе и светлосиве. рендгенско-структурна испитивања (xrd) белог пепела, након његове реакције са киселином (arha), су показала да испитивани узорци одговарају структури минерала кристобалита, док структура натријум-силиката одговара структури минерала натријум-триоксосиликат хептахидрат. методом утачњавања (rietveld refinement method) за кристале натријум-силиката добијене су следеће вредности: rwp = 12.81, rexp = 5.55, χ2 = 5.3274 и gof = 2.3081. нађено је да су кристали натријум-силиката погодни за припрему козметичких формулација. 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https://analyticalscience.wiley.com/do/10.1002/sepspec.10120education/full/i97dca9608c7bfa88fcf79f9b29f68226.pdf https://doi.org/10.1016/j.bsecv.2019.09.006 list of referees 2020 volume 84 (2020) 1 2020 list of referees editorial board of the journal is grateful to the following referees for reviewing the manuscripts published during 2020: mohammad abid, department of chemical engineering, university of technology, p.o. box 18310, baghdad, iraq danica agbaba, department of pharmaceutical chemistry, faculty of pharmacy, university of belgrade, serbia tanweer ahmad, college of natural and computational science, madda walabu university, balerobe, ethiopia vladimir ajdačić, faculty of chemistry, university of belgrade, serbia jovana ajduković, faculty of sciences, university of novi sad, serbia filip andrić, faculty of chemistry, university of belgrade, serbia jasmina anojčić, faculty of science, university of novi sad, serbia ofélia anjos, instituto politécnico de castelo branco, castelo branco, portugal bratislav antić, institute of nuclear sciences vinča, belgrade, serbia mališa antić, faculty of agriculture, university of belgrade, serbia ozgur arar, department of chemistry, ege university, i̇zmir, turkey jelena arsеnijević, faculty of pharmacy, department of pharmacognosy university of belgrade, serbia muhammad arshad, university of veterinary and animal sciences lahore, jhang, pakistan mahmut aslani, institue of nuclear sciences, nuclear technology department, ege university, izmir, turkey darko ašanin, faculty of science, university of kragujevac, serbia jelena avdalović, institute of chemistry, technology and metallurgy, university of belgrade, serbia marija babić, innovation center of the faculty of technology and metallurgy, university of belgrade, serbia divna bajić, faculty of technology and metallurgy, university of belgrade, serbia robert baker, school of chemistry, university of dublin, trinity college, dublin, ireland vladimir beškoski, faculty of chemistry, university of belgrade, serbia nabil bouarra, center of scientific and technical research in physicochemical analyzes (crapc), tipaza, algeria danica brzić, faculty of technology and metallurgy, university of belgrade, serbia roxana bugoi, horia hulubei national institute for nuclear physics and engineering, mǎgurele, romania xin che, shandong university, p. r. china viviana consonni, department of earth and environmental sciences, università degli studi di milano-bicocca, milano, italy 2 journal of the serbian chemical society renata vidor contri, universidade federal do rio grande, porto alegre, brazil aurelien crochet, fribourg center for nanomaterials, frimat machine pool responsible, university of fribourg, germany mirjana cvetković, institute of chemistry, technology and metallurgy, university of belgrade, serbia rene csuk, martin-luther university halle-wittenberg, organic chemistry, halle, germany božidar čobeljić, faculty of chemistry, university of belgrade, serbia ljiljana damjanović vasilić, faculty of physical chemistry, university of belgrade, serbia claude daul, department of chemistry, university of fribourg, switzerland glen deacon, monash university, clayton, australia zorana dobrijević, institute for the aplication of nuclear energy, university of belgrade, serbia snežana dragović, institute for the application of nuclear energy, university of belgrade, belgrade, serbia ivana drvenica, institut za medicinska istraživanja, univerzitet u beogradu, serbia claude daul, chemistry department, university of fribourg, switzerland jasna đonlagić, faculty of technology and metallurgy, university of belgrade, serbia ivana đorđević, institute of chemistry, technology and metallurgy, university of belgrade, serbia snežana b. đorđević, medical faculty, military medical academy, university of defense, belgrade, serbia miloš i. đuran, department of chemistry, faculty of science, university of kragujevac, serbia mihajlo etinski, faculty of physical chemistry, university of belgrade, serbia alireza foroumadi, department of medicinal chemistry, faculty of pharmacy and pharmaceutical sciences research center, tehran university of medicinal sciences, tehran, iran boris furtula, department of chemistry, faculty of science, university of kragujevac, serbia georgiy a. gamov, state university of chemistry and technology, ivanovo, russia nikola gligorijević, institute for the application of nuclear energy, university of belgrade, serbia biljana đ. glišić, department of chemistry, faculty of science, university of kragujevac, serbia snežana gojković, faculty of technology and metallurgy, university of belgrade, serbia branimir grgur, faculty of technology and metallurgy, university of belgrade, serbia nikola grozdanić, faculty of technology and metallurgy, university of belgrade, serbia sonja grubišić, faculty of chemistry, university of belgrade. serbia maja gruden, faculty of chemistry, university of belgrade. serbia snežana grujić, faculty of technology and metallurgy, university of belgrade, serbia selçuk gümüş, department of chemistry, yuzuncu yil university, turkey mehmet gumustas, institute of forensic sciences, ankara university, ankara turkey ahmed hassan, department of medicinal chemistry, faculty of pharmacy, mansoura university, mansoura, egypt darko jaćimovski, institute of chemistry, technology and metallurgy, university of belgrade, serbia milka jadranin, institute of chemistry, technology and metallurgy, university of belgrade, serbia goran janjić, institute of chemistry, technology and metallurgy, university of belgrade, serbi nenad janković, department of chemistry, faculty of science, university of kragujevac, serbia slobodan janković, faculty of medical sciences, university of kragujevac, serbia igor jordanov, faculty of technology and metallurgy, university of ss cyril and methodius, skopje, republic of north macedonia dragana jovanović, university hospital of pulmonology, clinical center of serbia, belgrade, serbia 2019 list of referees 3 branislav jović, faculty of sciences, department of chemistry, biochemistry and environmental protection, university of novi sad, serbia ivan juranić, faculty of chemistry, university of belgrade, serbia ioannis konstantinos karabagias, department of chemistry, university of ioannina, greece oxana kazakova, ufa institute of chemistry, russian academy of sciences, ufa, russian federation vesna kilibarda, posoning control centre, military medical academy, belgrade, serbia arun kodoth, mangalore university, karnataka, india juraj kóña, slovak academy of sciences, bratislava, slovak republic mirjana kostić, faculty of technology and metallurgy, university of belgrade, serbia tatjana kop, institute of chemistry, technology and metallurgy department of chemistry, university of belgrade, serbia matjaž kristl, faculty of chemistry and chemical engineering, university of maribor, maribor, slovenia uroš lačnjevac, institute for multidisciplinary research, university of belgrade, serbia ping li, school of chemistry and chemical engineering, qufu normal university, qufu, china snežana maletić, faculty of sciences, university of novi sad, serbia dragan manojlović, faculty of chemistry, university of belgrade, serbia sanja marinović, institute of chemistry, technology and metallurgy, university of belgrade, serbia smilja marković, institute of technical sciences of the serbian academy of sciences and arts, belgrade, serbia zoran marković, state university of novi pazar, serbia verka meško, international balkan university, skopje, north macedonia nevena mihailović, institute for the application of nuclear energy, university of belgrade, serbia jelena miladinović, faculty of technology and metallurgy, university of belgrade, serbia miloš milčić, faculty of chemistry, university of belgrade, serbia srđan miletić, faculty of chemistry, university of belgrade, serbia dušan milivojević, institute of molecular genetics and genetic engineering, university of belgrade, serbia slobodan milosavljević, faculty of chemistry, university of belgrade, serbia djenana miodragovic, chemistry of life processes institute, northwestern university, evanston, il, usa svetlana momchilova, institute of organic chemistry, centre of phytochemistry, bulgarian academy of sciences, sofia, bulgaria aleksandra nastasović, institute of chemistry, tehnology and metallurgy, university of belgrade, serbia olgica nedić, institute for the application of nuclear energy, university of belgrade, serbia jovan nedeljković, institute of nuclear sciences vinča, belgrade, serbia eva németh-zámboriné, faculty of horticultural science, szent istván university, budapest, hungary nikola nikačević, faculty of technology and metallurgy, university of belgrade, serbia jasmina nikolić, faculty of technology and metallurgy, university of belgrade, serbia katarina nikolić, faculty of chemistry, university of belgrade, serbia tanja nikolić, academy of professional studies south serbia, leskovac, serbia jasmina novaković, apotex inc, toronto, ontario, canada miroslav novaković, institute of chemistry, tehnology and metallurgy, university of belgrade, serbia 4 journal of the serbian chemical society rodica olar, department of inorganic chemistry, faculty of chemistry, university of bucharest, romania antonije onjia, vinča institute of nuclear sciences, university of belgrade, serbia dejan opsenica, faculty of chemistry, university of belgrade, serbia maría carolina otálora, facultad de ciencias e ingeniería, universidad de boyacá, tunja, boyacá, columbia vesna panić, institute for medical research, university of belgrade, serbia selvarengan paranthaman, department of physics & irc, kalasalingam university, india szabolcs pap, environmental research institute, thurso, caithness, scotland, uk ana penezić, institute for the application of nuclear energy, university of belgrade, serbia franc perdih, faculty of chemistry and chemical technology, ljubljana, slovenia milena petković, faculty of physical chemistry university of belgrade, serbia biljana petrović, department of chemistry, faculty of science, university of kragujevac, serbia dušan petrović, forschungszentrum jülich gmbh rada petrović, faculty of technology and metallurgy, university of belgrade, serbia rada pjanović, faculty of technology and metallurgy, university of belgrade, serbia jai prakash kushwaha, department of chemical engineering, thapar university, patiala, punjab, india radivoje prodanović, department of biochemistry, faculty of chemistry, university of belgrade, serbia dušanka radanović, institute of chemistry, technology and metallurgy, university of belgrade, serbia mirjana radanović, faculty of sciences, university of novi sad, serbia sobhan rezayati, islamic azad university, teheran, iran slavica ražić, faculty of pharmacy, university of belgrade, serbia jelena rogan, faculty of technology and metallurgy, university of belgrade, serbia anna roglans, department of chemistry, universitat de girona, spain camille rouquie, univ la reunion, univ avigon, qualisud, univ. montpellier, cirad, montpellier supagro, france luca salassa, donostia international physics center, paseo m. lardizabal 4, donostia, spain marina savić, faculty of sciences, university of novi sad, serbia snežana savić, department of pharmaceutical technology and cosmetology, faculty of pharmacy, university of belgrade, serbia milica spasojević, innovation center of the faculty of chemistry, university of belgrade, serbia pavle spasojević, innovation centre of faculty of technology and metallurgy, serbia vladimir v. srdić, faculty of technology university of novi sad, serbia svetlana stanišić, singidunum university, belgrade, serbia dragomir stanisavljev, faculty of physical chemistry, university of belgrade, serbia mirko stijepović, faculty of technology and metallurgy, university of belgrade, serbia dragoslav stoiljković, faculty of technology, university of novi sad, serbia zorica stojanović, faculty of technology, university of novi sad, serbia ana stanojević, faculty of physical chemistry, university of belgrade, serbia ivana stojković-simatović, faculty of physical chemistry, university of belgrade, serbia angela maria stortini, department of molecular sciences and nanosystems, university ca’foscari of venice, italy bogdan šolaja, faculty of chemistry, university of belgrade, serbia 2019 list of referees 5 vladimir šukalović, institute of chemistry, technology and metallurgy department of chemistry, university of belgrade, serbia maja šumar ristović, faculty of chemistry, university of belgrade, serbia gordana tasić, faculty of pharmacy, university of belgrade, serbia vele tešević, faculty of chemistry, university of belgrade, serbia jelena trifković, -faculty of chemistry, university of belgrade, serbia dragica trivić, faculty of chemistry, university of belgrade, serbia milena trmčić, inovation center of faculty of chemistry, university of belgrade, serbia aleksandra tubić, faculty of sciences, university of novi sad, serbia pablo ulloa, instituto de investigaciones agropecuarias, inia, chile đorđe veljović, faculty of technology and metallurgy, university of belgrade, serbia monu verma, department of environmental engineering, university of seoul, seoul, republic of korea tanja vidaković-koch, max planck institute for dynamics of complex technical systems, magdeburg, germany željko vitnik, department of chemistry, institute of chemistry, technology and metallurgy, university of belgrade, serbia sandra vojnović, institute of molecular genetics and genetic engineering, university of belgrade, serbia vesna vučić, institute for medical research, university of belgrade, serbia ljubodrag vujisić, faculty of chemistry, university of belgrade, serbia radojka vujasin, vinča institute of nuclear sciences, belgrade, serbia milica vujković, faculty of physical chemistry, university of belgrade, serbia malcolm watson, faculty of sciences, university of novi sad, serbia saeed yousefinejad, shiraz university of medical sciences, iran aleksandra zarubica, department of chemistry, faculty of science and mathematics, university of niš, serbia matija zlatar, institute of chemistry, technology and metallurgy, university of belgrade, serbia mario zlatović, faculty of chemistry, university of belgrade, serbia emila živković, faculty of technology and metallurgy, university of belgrade, serbia the use in grass production of clinoptilolite as an ammonia adsorbent and a nitrogen carrier j. serb. chem. soc. 80 (9) 1203–1214 (2015) udc 546.171.1:541.183+549.67:537.872: jscs–4791 504.53–035.22 original scientific paper 1203 the use in grass production of clinoptilolite as an ammonia adsorbent and a nitrogen carrier jelena milovanović1, susanne eich-greatorex2, tore krogstad2, vesna rakić3 and nevenka rajić4* 1innovation centre of the faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11120 belgrade, serbia, 2faculty of environmental science and technology, norwegian university of life sciences, 1432 aas, norway, 3faculty of agriculture, university of belgrade, nemanjina 6, 11080 zemun, serbia and 4faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11120 belgrade, serbia (received 17 march, revised 14 may, accepted 18 may 2015) abstract: clinoptilolite-rich tuff (nz) from the zlatokop deposit (vranjska banja, serbia) was studied as a nitrogen carrier for grass production. the mechanism of binding ammonium cations present in aqueous solutions by nz was examined, as well as the possibility of adsorption of ammonia released in fresh cattle manure during its fermentation. the nh4+ binding from solutions proceeded via an ion-exchange process that followed pseudo-second-order kinetics. adsorption isotherms studied at 298–318 k followed the freundlich isotherm equation. the nz readily adsorbs ammonia liberated from manure and the addition of 10 wt. % of nz to manure can preserve up to 90 % of ammonia. the potential benefit of this effect was examined in greenhouse pot experiments with italian ryegrass (lolium multiflorum, var. macho) using three different types of soil (silty, clayey and sandy). the zeta potential measurements showed that the stability of their colloidal dispersions differed mutually and that the addition of nz affected the stability and nitrogen cycling differently. all results indicated that nz could be applied in grass production. keywords: zeolites; manure; freundlich isotherm; soil; italian ryegrass. introduction nitrogen is an essential nutrient for plant growth that has to be added to the soil to ensure the best growth and yield of crops. however, mineral nitrogen fertilizers have been implicated in various environmental issues. ammonium and nitrate ions are readily lost from the soil by volatilization, leaching or surface run-off. as a result, different nitrogenous species are frequently present not only in agricultural wastewater, but also in groundwater. this may cause serious envi * corresponding author. e-mail: nena@tmf.bg.ac.rs doi: 10.2298/jsc150317042m _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1204 milovanović et al. ronmental problems such as eutrophication of water bodies and deterioration of water sources, also with possible consequences for humans, in particular for the health of small children.1 the removal of nh4+ from water through adsorption using various available sorbents was studied by many authors.2–7 natural zeolites as non-toxic, ecologically advantageous and affordable materials appear to be well suited for binding nh4+ from aqueous media due to their ion exchange and adsorption properties.8–11 moreover, the use of natural zeolites for agricultural purposes is becoming widespread because zeolites are particularly useful for controlling agricultural soil fertilization and for preventing or retarding leaching and for increasing yields.12–14 clinoptilolite is a very widespread zeolitic mineral in serbia. the clinoptilolite-rich tuff from the deposit zlatokop (vranjska banja, serbia) contains more than 70 wt. % of clinoptilolite and was found to exhibit good adsorptive and ion-exchange properties.15–17 in the present study, the tuff was investigated as a sorbent for ammonium ions and ammonia, and subsequently the ammoniumenriched zeolite was evaluated as a fertilizer. in particular, the present study examined: 1) the kinetics of binding of nh4+ from aqueous solution to nz; 2) the efficiency of nz in binding the nh3 released from fresh cattle manure; 3) whether the addition of ammonium-loaded nz (am–nz) influences the ζ-potential of the soil and accordingly the availability of nh4+ for plants; 4) the effect of the use of nz in herbage grass growth. experimental materials and methods the zeolite material (nz) was obtained from a large sedimentary zlatokop deposit in vranjska banja. the particle size of the samples used was in the range of 0.063–0.1 mm. a detailed x-ray powder diffraction analysis based on quantitative rietveld refinement showed that the nz contained 72.6 % clinoptilolite, 14.6 % feldspar plagioclase and 12.8 % quartz.18 chemical analysis of the clinoptilolite phase present in the nz obtained by scanning electron microscopy and x-ray microanalysis (jeol jsm-6610lv) gave the following composition expressed by corresponding oxides (wt. %): sio2, 65.63; al2o3, 12.97; fe2o3, 1.48; na2o, 0.95; k2o, 1.33; cao and mgo. 1.41. the loss on ignition at 1073 k, obtained by thermal analysis (ta instruments, sdt, q600), was 12.9 wt. %. furthermore, the porosity of nz measured by nitrogen adsorption at 77 k (hiden isochema htp1-v volumetric analyzer) gave for the bet specific surface area (sbet) and micropore volume (vmic) 42 m2 g-1 and 0.0032 cm3 g-1, respectively. prior to the experiments, the nz was washed with deionised water and ethanol to remove soluble amorphous impurities, and then dried to a constant mass at 105 °c. adsorption/desorption studies in solution the adsorption experiments were performed in the batch mode using nh4cl solutions of different (initial) strengths, i.e., 5, 10, 25, 50 and 100 mg nh4+ dm-3. the study was realized by mixing 1 g of nz with 100 cm3 of a solution of the chosen concentration. the suspension _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ clinoptilolite as an ammonia adsprbent and a nitrogen carrier 1205 was shaken in a thermostated water bath (memmert wnb22) for a period from 30 min to 24 h. the solid, ammonia-loaded nz (am–nz) was separated by filtration. three parameters were varied in the experiment: initial concentration of nh4+ in solution, temperature and contact time. adsorption isotherms were determined at 298, 308 and 318 k. the nh4+ desorption experiments were conducted at 298 k by treating am–nz (containing 1.1 mg nh4+ g-1) with the kcl or nacl solutions varying the salt concentration from 0.1 to 0.001 mol dm-3. adsorption study in manure the capture of ammonia released during fermentation of fresh cattle manure (some of its chemical characteristics are given in table i) was studied using a modified procedure described by sharadqah and al-dwairi.19 glass jars (volume 0.5 dm3) were filled with fresh cattle manure up to 2/3 their volume and tightly closed. into four of the jars, a well homogenized mixture of the fresh cattle manure and nz was added in different 100:n weight ratios (n = 5, 10, 15 or 20). the fifth jar without nz served as the system control. table i. chemical characteristics of the manure used in the experiments (g kg-1) ph dry matter loss on ignition total ca total na no3-nb nh4-nb total p total k 7.60 28.5 713.2 390.0 18.9 0.08 27.01 8.9 67 ameasured in dried sample; bmeasured in fresh sample in each jar, a porcelain crucible containing 10 cm3 of 0.1 m h2so4 was placed on a tripod. after 24 h, the crucibles were replaced by new ones containing fresh h2so4, and the solutions from the old ones were collected in a volumetric flask. the same process was repeated every day during 10 days, taking care that the jars were held open for the shortest possible time. the experiment was performed in triplicate. zeta potential measurements zeta (ζ) potentials of the soil samples and mixtures of the soil and am–nz suspended in water were measured. prior to the measurements, a suspension of 5 g of the soil sample (or a homogenized mixture of 5 g of soil and 100 mg of am–nz) in 25 cm3 deionised water was homogenized by a standard procedure20 for 30 min by horizontal orbital shaking (120 rpm). the suspensions were left to settle overnight, and then a part of the colloid fraction was taken for the measurement. all measurements were performed in triplicate and expressed as mean values of the ζ-potential ± standard deviation. pot experiments a greenhouse pot experiment was conducted under controlled conditions (20 °c, 18 h per day) with three different soil types and the italian ryegrass (lolium multiflorum, var. macho) as the test crop. the soil types included a loam, a silt and a sandy soil. the former two were passed through a 5-mm mesh filter prior to being filled into pots of 3 dm3 volume. some chemical parameters of the soils are given in table ii. the following treatments/fertilizers were used in the pots: a) control without any fertilizer, b) mineral fertilization with nh4no3, c) fresh cattle manure, d) fresh cattle manure with the addition of 10 wt. % nz, and in the silt soil only experiment and e) am–nz (containing 1.1 mg nh4+ g-1). all treatments were performed in triplicate. in all treatments, except in the control, the amounts of fertilizer corresponded to that typically used in grass production _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1206 milovanović et al. in southern norway (a nitrogen dose at the start of the experiment being equivalent to 120 kg n ha-1). the amount of manure was determined based on the nh4+ content in the wet sample and assuming that approximately 10 % of the organically bound n would be available over the experimental period, based on a previous mineralization study (results not shown). table ii. some properties of the soils used in the pot experiments soil sand silt clay loia ph total c total n p-alb k-alb g kg-1 mg kg-1 clay 450 380 170 61.0 5.2 23.1 2.1 58 195 sand 940 30.0 30.0 13.0 5.1 2.8 0.1 16 10 silt 20 930 50.0 37.0 6.5 14.5 1.0 49 200 aloss on ignition; bammonium acetate lactate extractable in addition, an amount phosphorus equivalent to 20 kg ha-1 and an amount of potassium equivalent to approximately 100 kg ha-1, as well as all other macroand micronutrients in appropriate amounts, were added in the case of the mineral fertilizer treatment. after the first cut, in all treatments except the one without nutrients, an additional amount of mineral n, equivalent to 60 kg n ha-1, was added. the pots were sown with 0.3 g of the seeds of italian ryegrass. the moisture content in the soil was maintained at 60 % of the field capacity by irrigation with deionised water. the grass was cut three times after 5, 9 and 13 weeks of growth. analytical methods and instrumentation the nh4+ concentration in the solutions was determined photometrically (hach dr2800) using the nessler reagent (hach method 8038). the cation concentrations of na, k, mg and ca in solution ere determined by aas using a varian spectra 55b instrument; at least five measurements were performed for each determination. fourier transformed infrared (ftir) spectra of nz and am–nz were recorded in the 4000–400 cm-1 range on a digilab-fts-80 spectrophotometer, using the kbr pellet technique. measurements of the ζ-potential were performed by electrophoresis using the laser doppler method and a sz-100 (horiba co. ltd.) instrument in which a cell containing carbon electrodes is employed as a sample holder. the ζ-potential was calculated using the peak values of the mobility distributions detected by the doppler shift in light scattering – the electrophoretic mobility of the particles was automatically calculated and converted to the ζ-potential using the smoluchowski equation.21 the particle size distribution of the soils was determined by the pipette method.22 soil and manure samples were ignited overnight at 823 k in order to determine the loss on ignition. the ph of the soil samples was measured by suspending the soil in h2o, with a soil to solution ratio of 1:2.5. the ph of the manure was measured directly in a wet sample. for both the soil and manure analyses, the total c content was determined in crushed samples by dry combustion23 at 1323 k using a leco chn-1000 instrument (st. joseph, mi, usa). the total n content was measured using the same instrument according to the dumas method.24 the ammonium and nitrate contents in the manure (nh4-n, no3-n) were measured by flow injecttion analysis (fia, tecator fiastar 5010 analyzer, hillerød, denmark) after extracting a fresh sample with 2 mol dm-3 kcl. the plant-available p and k in the soil were estimated by extraction with an ammonium acetate lactate solution (0.1 m ammonium lactate and 0.4 mol dm-3 acetic acid, ph 3.75),25 followed by inductively coupled plasma optical emission spectrometry (icp-oes, perkin elmer optima 5300 dv, waltham, ma, usa). the total p and k _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ clinoptilolite as an ammonia adsprbent and a nitrogen carrier 1207 in the manure were determined on the same instrument after autoclave digestion in concentrated hno3 (0.25 g to 0.3 g sample in 5 cm3) and subsequent dilution to 50 cm3. the effect of different treatments on the yield in the pot experiments was tested statistically by analysis of the variance (general linear model). the student–newman–keuls test was performed to identify the different means. results with p < 0.05 were considered significant. the statistical analysis was realized using sas 9.3 software (sas institute inc., cary, nc, usa). results and discussion adsorption study adsorption capacity of the nz increased with the initial nh4+ solution concentration and slightly decreased with temperature. at 298 k, it varied from 0.37 mg nh4+ g–1 (for c0 = 5 mg nh4+ dm–3) to 6.45 mg nh4+ g–1 (for c0 = 100 mg nh4+ dm–3). a slight decrease in the adsorption capacity was found at 318 k: 0.32 mg nh4+ g–1 (for c0 = 5 mg nh4+ dm–3) and 6.10 mg nh4+ g–1 (for c0 = = 100 mg nh4+ dm–3). the results showed that the adsorption of nh4+ by nz is an exothermic process, which agrees with the results obtained for a turkish and clinoptilolite-rich tuff.26 the langmuir and freundlich models were used to describe the equilibrium isotherm data.27 the langmuir model can be represented as: max l ee l e1 q b c q b c = + (1) where ce is the equilibrium concentration of the solute (mg dm–3), qe is the equilibrium concentration of the adsorbed solute (mg g–1), while qmax (mg g–1) and bl (dm3 mg–1) are langmuir constants (qmax corresponding to the maximum achievable uptake by a system, and bl is related to the affinity between the adsorbate and the adsorbent). the freundlich model can be represented as: e f enq k c= (2) where qe (mg g–1) is the equilibrium solute uptake, kf (dm3 g–1) is the isotherm constant of the freundlich model, ce (mg dm–3) is the equilibrium solution concentration, and n is the exponent of the freundlich model. kf and n are characteristics of the system and are indicators of the adsorbent capacity (or affinity for the solute) and adsorption intensity, respectively. for the langmuir isotherm analysis, the value of the separation factor (rl) defined as: l l 0 1 1 r b c = + (3) _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1208 milovanović et al. is of special importance28 and for the freundlich isotherm the value of the exponent (1/n) is significant. in all the experiments, the values of rl, which can be calculated using the bl values from table iii, and the 1/n values prove, according to the literature,16,28 that the adsorption was a favourable process (0< rl <1 and 1/n <1, table iii), in accord with the fact that nz readily adsorbs nh4+ from aqueous solutions. as can be seen from table iii, the equilibrium adsorption data gave a better fit (higher values of r2) for the freundlich than for the langmuir model. table iii. isotherm constants for the sorption of nh4+ from aqueous solution by nz temperature, k langmuir isotherm freundlich isotherm qmax a / mg g-1 bl b / dm3 mg-1 r2 kf c / dm3 mg-1 1/nd r2 298 8.8879 0.0794 0.9943 0.9507 0.5531 0.9663 308 7.6329 0.1115 0.9836 1.2017 0.4663 0.9929 318 9.7514 0.0554 0.9906 0.7724 0.6150 0.9963 amaximal monolayer adsorption capacity; blangmuir constant; cfreundlich constant; dfreundlich model exponent the nh4+ adsorption kinetics were studied at 298, 308 and 318 k for solutions with c0 = 5, 10, 25, 50 and 100 mg nh4+ dm–3. the experimental data were analyzed by the lagergren pseudo-first-order model and by the pseudo-second-order kinetics model.27 a linear dependence was obtained only for the pseudo-second-order model, indicating that the binding of nh4+ by nz occurs by the pseudo-second-order reaction mechanism described by the following equation:25 ( )22 e d d t t q k q q t = − (4) where qe (mg g–1) is the adsorption capacity at equilibrium and k2 (g mg–1 min–1) is the rate constant of the pseudo-second-order adsorption. integration between the limits t = 0 to t = t and q = 0 to q = qe, gives the following expression: 2 e2 e 1 1 t t t q qk q = + (5) the plot of t/qt vs. t is a straight line if the experimental data conform to this kinetic model, and the values of qe and k2 are obtained respectively from the slope and intercept of such a plot. as representative, the results obtained at different temperatures for the initial concentration c0 = 25 mg nh4+ dm–3 are shown in fig. 1. the adsorption capacity at equilibrium (qe) decreases with temperature whereas the rate constant changes irregularly with temperature. this was also observed for the other initial concentrations studied (data not presented). no acceptable explanation could be offered nor found in the literature for this phenomenon. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ clinoptilolite as an ammonia adsprbent and a nitrogen carrier 1209 fig. 1. kinetic curves at different temperatures obtained by fitting the experimental data to the pseudo-second-order rate model (c0 = 25 mg nh4+ dm-3). the ftir spectrum of am–nz (not shown) confirmed the presence of nh4+ in the sample (vibration band at about 1400 cm–1 attributed to nh4+, which was not present in the spectrum of nz), suggesting that the binding of nh4+ from aqueous solution by nz proceeds by an ion-exchange reaction. this was proved further by elemental aas analysis of the liquid phase after the adsorption studies, i.e., after am–nz separation from the suspensions. the concentrations of na+, k+, ca2+ and mg2+ determined in the filtrate entirely corresponded to the amount of nh4+ bound by the nz. in order to check whether the nh4+ in am–nz could be desorbed and thus become available as a nutrient in the soil, nh4+-desorption experiments were performed by treating am–nz with the nacl or kcl solutions. the obtained results are given in table iv. it is evident that the percentage of nh4+ desorption depended on the initial na+/k+ concentration, showing that the desorption was _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1210 milovanović et al. also an ion-exchange process. it is interesting to note that desorption was more efficient in the kcl than in the nacl solution. this could be explained by the fact that the radius of the nh4+ (151 pm) is very similar to that of the k+ (152 pm). desorption in 0.1 m kcl was completed in 30 min. table iv. percentage nh4+ desorbed at 298 k from am–nz into solutions of nacl or kcl in dependence on the salt concentration concentration of nacl/kcl, mol dm-3 released nh4+, % in nacl(aq) in kcl(aq) 0.100 64 100 0.010 42 50 0.005 24 42 0.001 17 18 it is well known that fresh cattle manure is rich in nitrogen, making it a good fertilizer. nitrogen is present mainly in the organic matter, the content of which in the fresh manure being about 50 wt. %. however, during fermentation, manure looses significant amounts of nitrogen. thus, the loss in four days may reach up to 90 % due to the extensive liberation of ammonia.29 in order to mitigate this loss, investigations were performed to determine: a) whether the addition of nz to fresh cattle manure could retain the liberated nh3, and b) the optimal amount of nz that has to be used for nh3 capture. addition of the nz to fresh manure conserved the nh3 released during fermentation, and the percentage of the captured nh3 ranged between 67 and 98 % depending on the applied amount of nz (5–20 wt. %). since about 90 % of nh3 was captured on addition of 10 wt. % nz in comparison to the control, this amount of nz was chosen as the optimal amount for the pot experiments (vide infra). ζ-potential values the zeta potential is an important parameter for soil/zeolite suspensions in water since it could be interpreted as an indicator of the stability of the suspended colloidal dispersions with respect to particle aggregation.30 for most soils, the ζ potential has a negative value because the ground surface is usually negatively charged. moreover, the soil stability is a qualitative indicator of biological activity, energy flow and nutrient cycling. a dispersion is regarded as stable, when the ζ-potential is < –30 mv.31 the results of the ζ-potential measurements are listed in table v. it is evident that the clayey soil showed the lowest value of ζ, which could be explained by the strong electronegativity of the clays present in the soil sample. the sandy soil also showed a very low ζ-potential, which could be attributed to the presence of organic matter in which carboxyl groups are ionized.31 the silty soil exhibited the highest ζ-potential, implying a lower sta_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ clinoptilolite as an ammonia adsprbent and a nitrogen carrier 1211 bility. the addition of am–nz influenced the ζ-potential of all three types of soil but the changes were most pronounced for the clayey and sandy soils. the stability of clayey soil decreased on addition of am–nz in contrast to sandy soil in which the colloidal fraction became more stable. table v. the values of ζ-potentials, mv soil sample system soil/water soil+am–nz/water clay –45.0±0.7 –38.0±0.8 silt –23.6±1.1 –22.3±1.6 sand –37.0±0.9 –40.6±0.7 pot experiments to obtain an insight into a possible use of nz in grass production, pot experiments were performed with italian ryegrass, lolium multiflorum, var. macho. italian ryegrass was used because it is fast-growing and responds to high n fertilization by yielding an abundance of vegetative matter. the results for the three cuts in the pot experiment are given in fig. 2, expressed as tones of dry matter (dm) per hectare. fig. 2. yields of ryegrass for different treatments and different types of soil. statistically significant differences are indicated by different letters (p < 0.001). figure 2 reveals similar overall yields for the mineral fertilizer, the manure and the manure+nz treatments, with the exception of the sandy soil where con_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1212 milovanović et al. siderably less biomass was produced in the control than in the other two treatments. in general, there was little difference between the amounts of biomass harvested at different cuts for these three treatments. a slight but significant difference (statistics for the separate cuts are not shown) was found in the silt, with a somewhat lower biomass in the first cut in the manure+nz treatment compared to mineral fertilizer and manure alone. however, this slower growth at the beginning was compensated for by a good yield in the second cut. it seems that nz added to the silt binds nh4+ strongly at least in the initial phase of the growing period. this could, at least partly, have been due to the relatively high ph value of the silty soil (6.5), at which value cations are strongly adsorbed and hence less available than in the clayey and sandy soils. this suggestion is in accordance with the zeta potential measurement, which showed that the stability of colloidal fractions of the silty soil is not influenced by the addition of zeolite. the yields in the treatments without fertilizer are, as expected, much smaller than in the other treatments, especially in the sandy soil, which contained the smallest amount of nutrients. even when the sandy soil was treated with the mineral fertilizer, the plants grew poorly – most likely due to the low original ph of the soil (i.e., 5.1, table ii). treatments including both manure and manure+nz improved the growing conditions in the sandy soil, which was reflected in the much higher yields. in the silty soil, the treatment with am–nz resulted in slightly higher overall yields than in the manure+nz and the mineral fertilizer treatment but not compared to the manure alone. the may be explained by the fact that the am– nz was obtained by the ion-exchange reaction using a nh4+-solution of well -defined strength as opposed to the nz exposed to manure. in the latter case, less nh4+ was bound by nz because the manure used possessed not only nh4+ present in the liquid phase, but also organically bound n. it should be stressed that the available n from manure cannot be determined accurately in comparison to the available n in am–nz. accordingly, the resulting differences are likely to have caused the small differences in growth between the different treatments in the silty soil. conclusions this study evaluated the adsorption ability of zeolitic tuff from the zlatokop deposit towards nh4+ present in liquid medium and towards nh3 liberated in manure, as well as its capability to be a nitrogen reservoir for plant growth. the adsorption studies show that the process proceeded via an ion-exchange mechanism, which followed the pseudo-second-order kinetic model (r2 > 0.99). the adsorption isotherms studied at 25–45 °c followed the freundlich isotherm model. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ clinoptilolite as an ammonia adsprbent and a nitrogen carrier 1213 the addition of 10 wt. % of the tuff to fresh cattle manure conserved about 90 % of ammonia and preserved its nutritive value. greenhouse pot experiments with italian ryegrass suggested that the plants utilize the nh4+ bound by the tuff in a similar manner to the nh4+ in easily soluble mineral fertilizers. further work will be directed towards exploitation of the tuff in odour control as well as towards its potential use in the reduction of the nitrogen oxide emission during manure application in agriculture. the results of such work could be expected to significantly contribute not only to a less odoriferous, but also to a healthier environment. acknowledgements. this research was supported by the norwegian programme in higher education, research and development herd (project “the use of natural zeolite (clinoptilolite) for the treatment of farm slurry and as a fertilizer carrier”) and by the ministry of education, science and technological development of the republic of serbia (project no. 172018). и з в о д примена клиноптилолита као адсорбента амонијака и носача азота за примену у узгоју травњака jeленa mиловановић1, susanne eich-greatorex2, tore krogstad2, весна ракић3 и невенка рајић4 1иновациони центар технолошко–металуршког факултета,универзитет у београду, карнегијева 4, 11000 београд, 2faculty of environmental science and technology, norwegian university of life sciences, 1432 aas, norway, 3пољопривредни факултет, универзитет у београду, немањина 6, 11080 земун и 4технолошко-металуршки факултет, универзитет у београду, карнегијева 4, 11000 београд зеолитски туф (nz) са великим садржајем клиноптиолита из рудника златокоп (врањска бања) испитиван је као носач азота за потребе гајења траве. проучен је механизам и кинетика везивања амонијум-јона из водених раствора за nz као и могућност везивања амонијака који настаје ферментацијом свежег стајњака. везивање амонијум -јона је реакција јонске измене која следи кинетику псеудо-другог реда. адсорпционе изотерме испитане на 298–318 k следе фројндлихову једначину. nz лако везује амонијак који се ослобађа у стајњаку и додатак 10 мас. % nz може да сачува 90 % амонијака. потенцијална корист овог ефекта испитивана је праћењем раста италијанског љуља (italian ryegrass, lolium multiflorum, var. macho) у саксијама у стакленој башти применом три различите врсте земљишта (прашина, глина, песак). мерењем цета потенцијала утврђено је да се стабилност колоидних дисперзија земљишта међусобно разликује и да додатак nz утиче различито на стабилност, а тиме и на кружење азота у земљишту. на основу укупних резултата закључено је да се nz може користити при узгоју травњака. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {the programme for professional development of chemistry teachers' assessment competency} j. serb. chem. soc. 86 (10) 997–1010 (2021) original scientific paper jscs–5478 published 10 september 2021 997 the programme for professional development of chemistry teachers’ assessment competency biljana i. tomasevic*#, dragica d. trivic#, vesna d. milanovic# and lidija r. ralevic# university of belgrade – faculty of chemistry, studentski trg 12–16, belgrade, serbia (received 10 july, accepted 19 july 2021) abstract: the aim of this paper is to investigate the effects of the programme for professional development of chemistry teachers on their competencies for conducting formative and summative assessment in chemistry teaching. the programme participants were 30 chemistry teachers from primary and secondary schools. data were collected using a questionnaire at the beginning and at the end of the programme implementation. the programme included four workshops with the same structure: the introduction, group work and the discussion of the results obtained through group work. the workshops focused on: i) the assessment as a support for chemistry learning; ii) the harmonization of teaching and learning activities, formative and summative assessment, feedback from formative assessment and the criteria used to evaluate students in summative assessment; iii) the evaluation of the validity of tasks used for formative and summative assessment according to the curricula aims and the educational standards; iv) designing tasks for monitoring students’ progress towards certain educational standards. teachers’ responses show the impact of the programme for the development of their competencies for assessment, particularly regarding formative and summative assessment and designing various kinds of assessment in accordance with the achievement standards. keywords: in-service teacher training; formative evaluation; summative evaluation. introduction teacher knowledge was first described as a symbiosis of subject content knowledge and pedagogical knowledge necessary for the transformation of teaching topics into specific classroom activities by shulman1 through the model of pedagogical content knowledge (pck). similar models were later developed, with the emphasis on subject content knowledge as a component which provides * corresponding author. e-mail: bsteljic@chem.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc210710052t ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 998 tomasevic et al. a necessary foundation and which should be integrated with pedagogical knowledge.2 by integrating these components specific and general pedagogical context is formed.3 subject content knowledge plays a crucial role in the meaningful integration of the other components of teacher knowledge.4,5 the component “assessment of scientific literacy” was introduced by magnusson6 et al. within their pck model (1999). the component “knowledge of science assessment” includes both teacher knowledge of the dimensions of science learning and teacher knowledge of the methods used to assess students’ learning within a specific topic. when introducing the component “subject matter specific pedagogical knowledge”, tamir7 observed that testing and evaluation should also be a part of pck. the subsequent models of pck point out the need for better interconnection of the components which constitute pck,8 as well as the nature and strength of the connection among them.9 the interconnection and mutual dependence of all pck components indicate that the teachers who have better knowledge of teaching strategies can also better assess students’ knowledge. in the literature and research studies, a component of pck referred to as “teachers’ assessment competence or teacher competency in educational assessment” comprises the teachers’ competency in the situations which require assessment of students’ knowledge. based on the understanding of competences as a measurable ability the model of teachers’ assessment competence, which enables a teacher to fulfil the requirements of assessment and quantification of knowledge in a wide range of situations, was developed.10 this model integrates cognitive judgment processes, assessment practices and the products of assessments. it is also defined as assessment literacy (al). the significance and complexity of teacher knowledge of student achievement assessment can be represented using the conceptual framework teacher assessment literacy in practice (talip), which represents the sublimation of the research studies conducted and presented in the literature in the last several decades.11 this framework connects two aspects of research studies – educational assessment and teacher education. six principal components through which assessment literacy (al) should be considered are pointed out: 1. the knowledge base of assessment, 2. teacher conception of assessment, 3. institutional and socio-cultural contexts, 4. talip the core concept of the framework, 5. teacher learning and 6. teacher identity (re)construction as assessors. since education and its requirements change during every teacher’s career, it is necessary for teachers to improve their competencies through the programmes of professional development and training.12 the need for continuous teacher development is particularly important in the situation when some significant changes in the education system are introduced due to reforms or new regulations.13,17 however, it seems that not enough attention is devoted to teachers’ competences to conduct assessment of knowledge18 within these programmes. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. chemistry teachers’ assessment competency 999 the complexity of the nature of knowledge assessment is caused by many inseparable educational and social functions which result from the structure and organization of the entire education system.19,20 in a research study which investigated teachers’ attitudes towards knowledge assessment, the items in the questionnaire related to four concepts of assessment: improvement, student accountability, school accountability and irrelevance.21 there was not a statistically significant difference between the attitudes of primary and secondary school teachers towards school accountability and irrelevance. primary school teachers agreed statistically significantly more with the statements which associated assessment activities with the potential for improvement. statistically significantly more secondary school teachers agreed with the significance of assessment for student accountability. there was a correlation of the concept of improvement with school accountability, but there was no correlation with student accountability. this means that the teachers were willing to assume the responsibility for improving school outcomes and quality and fulfilling their professional accountabilities, they were willing to include assessment in their professional responsibilities in order to improve teaching and learning process, but they were less aware of the significance of student accountability. the effects of assessment on teaching and learning, certification of learning and accountability of teaching were considered to have greater pedagogical-regulation significance by the primary school teachers, while they were considered to have greater societal-accreditation significance by the secondary school teachers.19 the conducted cluster analysis showed that teachers could have different attitudes towards assessment at the same time.22 more than half of the teachers (51 %) had moderate, average and homogenous views on different purposes of assessment which related to the attitudes that “assessment improves teaching and learning”, that “assessment is valid for accountability” and that “assessment is irrelevant”. 28 % of teachers expressed their opinion that the most important purpose of assessment is to improve teaching and learning. approximately 21 % of the teachers had a marked tendency to question the accuracy of assessment and believed that it had no impact on their teaching practice. when evaluating the questionnaire items which related to the criteria of high quality assessment on an 1–5 scale, the teachers gave high grades for all ten criteria.23 they identified “transparency” as the most important criterion (4.50), while “directness” (3.93) and “reproducibility” (3.88) were considered to be the least significant. the results show that teachers consider the existing, classic assessment criteria equally important as some new ones (authenticity, cognitive complexity, costs and efficiency, directness, educational consequences and meaningfulness) which have been introduced with the orientation being shifted towards competency-based education and assessment. this finding was contrary to researchers’ expectations since teachers are sometimes reluctant to accept such ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 1000 tomasevic et al. changes, but this research study did not investigate whether the teachers actually appreciated and applied all assessment criteria in their teaching practice. in view of the theoretical framework presented above, the aim of this paper was to establish the effects of the programme for professional development of chemistry teachers on their competencies for conducting formative and summative assessment in chemistry teaching. based on the aim, the following research question was raised: how do teachers’ attitudes towards the assessment in general and the formative and summative assessment of student achievements change under the influence of the programme for professional development of chemistry teachers’ assessment competency? experimental assessment was the main focus of the two-day programme for professional development of chemistry teachers’ assessment competency (pdchtac), which was organized by the serbian chemical society and the university of belgrade – the faculty of chemistry. four workshops were held in two days. the structure of all workshops was the same and they included an introduction, group work and a discussion of the results obtained through group work. the first workshop focused on assessment as a support for chemistry learning. the second workshop focused on the issue of how to harmonize teaching and learning activities, formative and summative assessment, feedback from formative assessment and the criteria by which students are evaluated in the summative assessment. within the third workshop participants evaluated in groups the validity of tasks used for formative and summative assessment according to the curricula aims and the educational standards. in the fourth workshop the participants designed tasks for monitoring students’ progress towards certain educational standards. bearing in mind the research question, the data about the participants’ attitudes towards assessment were collected using a questionnaire both before and after the programme realization. the completion of the questionnaires was on a voluntary and anonymous basis and in accordance with the code of conduct for scientific research of the university of belgrade (http://bg.ac.rs/files/sr/univerzitet/univ-propisi/kodeks-naucnoistrazivacki-rad29.3.2018.pdf). the aim of the questionnaire completion was explained in several introductory sentences at the beginning of the questionnaire, and respondents were guaranteed the confidentiality of the data collected. sample thirty chemistry teachers from primary and secondary schools participated in the programme. the participants’ demographic data are shown in table i. out of the 30 participants, 29 were females, while one was a male. the largest number of the teachers were in 40–49 age group. four fifths of the teachers in the sample had over 10 years of experience. university courses through which teachers could develop assessment literacy (chemistry didactics, pedagogy and psychology) had been attended by 22 (73.3 %) participants. instrument the questionnaire designed for this research study consisted of four parts: i – general information about the respondents; ii – teachers’ attitudes towards their usual practice of assessment of student achievements; iii – teachers’ general attitudes towards assessment of ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. chemistry teachers’ assessment competency 1001 student achievements; iv – teachers’ attitudes towards competencies necessary for assessment of student achievements. the questionnaire contained 59 questions altogether: 6 multiple choice questions, one open-ended question, and 52 questions in which the respondents were asked to express the level of their agreement on a likert-type scale. table i. background data of the sample (n = 30), c – contribution of teachers in each category work placea c / % age c / % length of service c / % education c / % university facultyb c / % ps 16.7 30-39 6.7 1-5 10.0 undergraduate studies 80.0 ubfc 70.0 gs 36.7 40-49 60.0 6-10 10.0 undergraduate and master studies 20.0 unsfs 6.7 vs 40.0 50-59 30.0 11-15 26.7 ubftm 3.3 ps, gs, vs 3.3 60+ 3.3 16-20 20.0 ukfs 10.0 ps, gs 3.3 20-30 30.0 upkmfsm 3.3 30+ 3.3 ubfp 3.3 usfes 3.3 aps – primary school, gs – grammar school, vs – vocational school; bubfc: university of belgrade – faculty of chemistry; unsfs: university of novi sad – faculty of sciences; ubftm: university of belgrade – faculty of technology and metallurgy, ukfs: university of kragujevac – faculty of sciences; upkmfsm: university of pristina, kosovska mitrovica – faculty of sciences and mathematics; ubfp: university of belgrade – faculty of physics; usfes: university of sarajevo – faculty of educational sciences to confirm the content validity, the questionnaire was examined by the members of the department of chemical education, the university of belgrade – faculty of chemistry, who were familiar with the construct of interest and the research subject, but who were not involved in designing and conducting the actual research study. they also reviewed readability, clarity and comprehensiveness of the questions in the questionnaire and provided a coherent estimation of the content validity of the questionnaire. the reliability of the questionnaire was examined by means of cronbach’s α coefficient.24 the value of cronbach’s α coefficient in the part with likert-type questions was 0.873 for the first application of the questionnaire, and 0.866 for the second application of the questionnaire, which indicated a satisfactory level of the internal consistency of the instrument. results and discussion teachers’ evaluations of the statements in the questionnaire provide an insight into the current practice of assessing and grading student achievements in the field of chemistry in our schools (table ii). the most frequent evaluations of chemistry teachers regarding the assessment practice in our schools (table ii) will be presented in the following part of this paper. teachers rarely change grading criteria based on the results of written assessment of students and decision on grades based on students’ suggestions and arguments. however, they more often include the effort invested by the student ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 1002 tomasevic et al. in the grade and the majority of them always or almost always provide their students with an explanation for the grades awarded. the largest number of teachers sometimes or very often grade their students in the lessons during which new material is introduced, but very often or always and almost always try to make assessment contribute to further learning. table ii. the teachers’ attitudes towards teaching practice of assessment of student achievements: 1− never or almost never; 2 – very rarely; 3 − sometimes; 4 − very often; 5 − always or almost always statement contribution, % 1 2 3 4 5 1. i change the criteria for grading written assessment of knowledge after gaining insight into students’ results. 6.7 40.0 16.7 30.0 6.7 2. i include the effort invested by a student in his grade. – 3.3 30.0 30.0 36.7 3. i change my decision about the grade after students’ suggestions or arguments. 16.7 23.3 40.0 16.7 – 4. i create questions for oral assessment of knowledge before the lesson. 3.3 3.3 20.0 40.0 30.0 5. i use the material designed by my colleagues for assessment of knowledge. 20.0 30.0 36.7 13.3 – 6. i grade my students in the lessons when new material is introduced. – 10.0 36.7 46.7 6.7 7. i provide an explanation for the grades given to my students. – – – 10.0 90.0 8. i attempt to make knowledge assessment contribute to further learning. – – – 23.3 76.7 9. upon unsatisfactory results of assessing knowledge of certain concepts, i explain the same concepts again. – 6.7 13.3 50.0 30.0 10. when assessing achievements i take into account the student achievement standards.a 13.3 6.7 10.0 40.0 30.0 11. i design tasks for written assessment of knowledge bearing in mind the student achievement standards.a 6.7 6.7 6.7 43.3 36.7 12. i design tasks for oral assessment of knowledge bearing in mind the student achievement standards.a 6.7 6.7 13.3 53.3 20.0 13. i design tasks for practical assessment of knowledge bearing in mind the student achievement standards.* 10.0 3.3 20.0 53.3 13.3 14. i provide feedback on the level of achievement and recommendations for further work to each student. – 3.3 – 33.3 63.3 15. i make my decision on the final grade based on the results of formative and summative assessment of knowledge. – – 3.3 33.3 60.0 16. i give my tests to colleagues so that they evaluate their quality and validity before their application in practice. 13.3 20.0 20.0 33.3 13.3 17. i include my students in defining the criteria by which their achievements will be assessed. 6.7 20.0 13.3 53.3 6.7 18. i include my students in the assessment of other students’ achievements. 10.0 10.0 23.3 40.0 16.7 athe student achievement standards at the end of compulsory education and at the end of general secondary education ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. chemistry teachers’ assessment competency 1003 the majority of teachers very often or always and almost always explain the concepts again after unsatisfactory results of assessment of knowledge. teachers sometimes or very rarely use materials for knowledge assessment designed by their colleagues. the majority of teachers very often or always and almost always create questions for oral assessment of knowledge before the lesson. generally, teachers very often or always and almost always take into account the student achievement standards, mostly during the process of designing the tasks for written assessment of knowledge, to a lesser extent when designing questions for oral assessment of knowledge and least for practical assessment of knowledge. the majority of teachers very often or always and almost always provide their students with feedback on the level of achievement and recommendations for further work, and they make their decision on the final grade based on formative and summative assessment of knowledge. 40 % of the teachers very rarely or sometimes give their tests to colleagues for the evaluation of quality and validity before using them in the classroom, while 33 % of them do this very often. slightly more than half of the teachers very often include their students in defining the criteria for evaluating achievements, while one fifth does this very rarely. 40 % percent of the teachers very often include students in the evaluation of other students’ achievement, while 23 % does this sometimes. teachers’ attitudes towards the function of assessment before the realization of the programme for pdchtac, and after the realization of the programme can be observed from the degree of agreement with the statements presented in table iii. since the same group evaluated these variables, the sample is not independent, so the wilcoxon test was used to determine whether there were any statistically significant differences between the evaluations provided by the group before and after the realization of the programme for pdchtac. using spss output, the data analysis shows that the evaluations provided by the group are statistically significantly different for 6 out of 18 variables. in the following part the results for the attitudes of teachers which are statistically significantly different compared to the attitudes which had been expressed immediately before the beginning of the development programme will be presented. the largest percentage of teachers mainly or strongly agreed that the assessment of student achievements is as important in the teaching process as acquiring new knowledge on both occasions when their attitudes were investigated using the same question within the research study. upon the programme completion the number of teachers who strongly agreed with the above-mentioned statement increased (z = −2.32, p < 0.05). after the development programme a larger number of teachers expressed their opinion that they mainly agreed with the statement that student achievements should be assessed in each lesson (z = −4.42, p < 0.0001). a large number of teachers changed their attitude that each assessment of achievement should result in a grade, so two thirds of the participants ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 1004 tomasevic et al. stated that they strongly or mainly disagreed with this attitude (z = −2.26, p < 0.05). after the programme the number of teachers who strongly agreed that the result of formative assessment enables a student to gain an insight into his current level of achievement increased (z = −2.30, p < 0.05), while none of the teachers expressed disagreement with this attitude anymore. table iii. teachers’ attitudes towards the assessment of student achievements; i – at the beginning of the programme realization; f – at the end of the programme realization: 1 – strongly disagree; 2 – mainly disagree; 3 – both agree and disagree; 4 – mainly agree; 5 – strongly agree statement contribution, % 1 2 3 4 5 1. assessment of student achievements and acquiring knowledge are equally important parts of the teaching process. i – – 3.3 36.7 60.0 f – – – 13.3 86.7 2. student achievements should be assessed in every lesson. i – – 23.3 30.0 46.7 f – – 6.7 46.7 46.7 3. the outcome of each assessment should be a grade. i 13.3 16.7 50.0 20.0 f 23.3 43.3 26.7 6.7 – 4. written, oral and practical assessment can provide the same kind of feedback on student achievements. i 6.7 20.0 33.3 33.3 6.7 f 13.3 30.0 26.7 23.3 6.7 5. one kind of assessment is sufficient to gain an insight into student achievements. i 50.0 40.0 6.7 – 3.3 f 76.7 20.0 3.3 – – 6. the results of formative assessment provide a student with an insight into the current level of his achievements. i 3.3 6.7 13.3 63.3 13.3 f – – 13.3 43.3 43.3 7. being familiar with the assessment criteria contributes to better student achievements. i – 3.3 3.3 53.3 40.0 f – – 3.3 40.0 56.7 8. providing an explanation for the grade has a positive impact on student’s subsequent work. i – – 6.7 30.0 63.3 f – – – 36.7 63.3 9. students learn through the process of achievement assessment. i – – 13.3 50.0 33.3 f – – 13.3 36.7 50.0 10. the results of achievement assessment depend on the type of assessment applied. i – 10.0 30.0 46.7 10.0 f – – 43.3 43.3 13.3 11. good practice in achievement assessment includes a discussion on the level of knowledge achieved by students. i – – 13.3 36.7 50.0 f – – 10.0 43.3 46.7 12. unannounced assessment of knowledge leads to worse results than announced assessment. i – 3.3 36.7 30.0 30.0 f – 13.3 36.7 26.7 23.3 13. formative assessment of knowledge requires special preparation and analysis. i – 3.3 26.7 46.7 23.3 f – 10.0 20.0 36.7 33.3 14. knowledge assessment should present learning in a new context. i – 3.3 10.0 76.7 6.7 f – 6.7 10.0 40.0 43.3 15. knowledge assessment should enable the improvement of learning strategies. i – – – 53.3 43.3 f – – 6.7 43.3 50.0 16. students can draw conclusions about the level of their achievements based on their grade. i – 3.3 43.3 40.0 13.3 f – – 20.0 56.7 23.3 17. a grade higher than the current level of achievement has a positive effect on student’s further progress. i – 10.0 43.3 36.7 10.0 f – 13.3 33.3 40.0 13.3 18. formative assessment excludes summative assessment. i 23.3 23.3 43.3 6.7 3.3 f 53.3 26.7 13.3 3.3 3.3 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. chemistry teachers’ assessment competency 1005 furthermore, the number of teachers who mainly or strongly agreed with the statement that students can draw a conclusion about the level of their achievement based on the grade increased (z = −2.83, p < 0.01). there was a statistically significant difference in the number of teachers who had previously been undecided, but who expressed their opinion that they strongly or mainly disagreed with the statement that formative assessment excludes summative assessment after the programme completion (z = −2.40, p < 0.05). there were no statistically significant differences in the teachers’ attitudes for the other 12 statements after the programme completion, but the results show that there were certain shifts and changes. a higher percentage of teachers expressed their attitude that they strongly or mainly disagreed with the statements that written, oral and practical assessment can provide the same kind of feedback on student achievements and that one kind of assessment is sufficient to gain an insight into student achievements. furthermore, a higher percentage of teachers mainly or strongly agreed that being familiar with the assessment criteria contributes to better student achievements, which providing an explanation for a grade has a positive impact on students’ subsequent work and that students learn through the process of assessment of achievement. the questionnaire was also used to investigate the teachers’ attitudes towards their competencies for assessing student achievements. the results obtained through their responses are presented in table iv. the wilcoxon test was used to determine whether there were any statistically significant differences in the evaluations obtained from the group. using spss output, the data analysis shows that the evaluations obtained from the group is statistically significantly different for 8 variables. in the following part the results for teachers’ attitudes between which there was a statistically significant difference after the realization of the programme for pdchtac are presented. in comparison with the initial evaluations, a higher percentage of teachers strongly agreed that they could monitor and evaluate the effectiveness of their work based on the results obtained through knowledge assessment (z = −2.97, p < 0.005) and that they understood the concepts of formative and summative assessment (z = −3.54, p < 0.001). almost all teachers mainly or strongly agreed that they were competent to design and realize formative assessment of knowledge (z = −2.37, p < 0.05) and summative assessment of knowledge (z = −2.99, p < 0.05). the number of teachers who considered that they were competent to design and conduct written assessment in accordance with the achievement standards also increased (z = −2.07, p < 0.05) after the development programme, and none of the teachers expressed disagreement with this statement. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 1006 tomasevic et al. table iv. the teachers’ attitudes towards the competencies for assessing student achievements; i – at the beginning of the programme realization; f – at the end of the programme realization: 1 – i strongly disagree; 2 – i mainly disagree; 3 – i both agree and disagree; 4 – i mainly agree; 5 – i strongly agree statement contribution, % 1 2 3 4 5 1. i meet the competency standards for the teaching profession. i – – – 36.7 63.3 f – – 3.3 46.7 50.0 2. i am competent to design and conduct knowledge assessment in accordance with the student achievement standards. i – – 16.7 43.3 36.7 f – – 6.7 60.0 33.3 3. i successfully adapt knowledge assessment to students’ individual abilities. i – 6.7 30.0 40.0 23.3 f – – 26.7 60.0 13.3 4. i can monitor and evaluate the effectiveness of my work based on the results obtained through knowledge assessment. i – – 23.3 66.7 10.0 f – – 10.0 53.3 36.7 5. i am competent to assess student achievements using various types of assessment. i – – 6.7 50.0 40.0 f – – 3.3 56.7 36.7 6. i understand the concepts of formative and summative assessment. i 3.3 6.7 30.0 36.7 20.0 f – – 3.3 33.3 63.3 7. i am competent to design and conduct formative assessment of knowledge. i – 3.3 30.0 40.0 26.7 f – – 3.3 56.7 40.0 8. i am competent to design and conduct summative assessment of knowledge. i – 6.7 30.0 36.7 26.7 f – – – 50.0 50.0 9. i am competent to design and conduct written assessment of knowledge in accordance with the goals of chemistry education. i – – 6.7 36.7 56.7 f – – – 43.3 56.7 10. i am competent to design and conduct oral assessment of knowledge in accordance with the goals of chemistry education. i – – – 50.0 50.0 f – – – 40.0 60.0 11. i am competent to design and conduct practical assessment of knowledge in accordance with the goals of chemistry education. i 3.3 – 6.7 63.3 26.7 f – 3.3 10.0 40.0 46.7 12. i am competent to design and conduct written assessment of knowledge in accordance with the achievement standards. i 3.3 3.3 13.3 46.7 33.3 f – – 10.0 43.3 46.7 13. i am competent to design and conduct oral assessment of knowledge in accordance with the achievement standards. i 3.3 3.3 20.0 36.7 36.7 f – – 6.7 43.3 50.0 14. i am competent to design practical assessment of knowledge in accordance with the achievement standards. i 6.7 3.3 26.7 36.7 26.7 f – 3.3 16.7 46.7 33.3 15. i have improved my competencies for monitoring and evaluating student achievements through professional development programmes. i 10.0 3.3 20.0 30.0 36.7 f – – 6.7 40.0 53.3 16. i have improved my competencies for monitoring and evaluating student achievements by reading specialized and scientific publications. i 6.7 6.7 6.7 53.3 23.3 f – – 10.0 56.7 33.3 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. chemistry teachers’ assessment competency 1007 a considerably higher percentage of teachers mainly or strongly agreed that they were competent to design and conduct oral assessment of knowledge (z = = −2.54, p < 0.05) and practical assessment of knowledge (z = −2.05, p < 0.05) in accordance with the achievement standards. responding to the statement which referred to the previous improvement of competencies for monitoring and evaluating student achievements through professional development programmes, the teachers obviously included their experience of attending the programme for pdchtac (z = −2.50, p < 0.05). as far as the other statements are concerned, there was no statistically significant difference between the responses given before and after the development programme, but a higher percentage of teachers mainly or strongly agreed with the statement that they were competent to design and conduct knowledge assessment in accordance with the student achievement standards and that they adapted knowledge assessment to individual abilities of their students. conclusion teachers’ responses regarding their assessment practice, i.e., how it is conducted in schools, indicate that teachers attempt to harmonize it with the requirements prescribed by primary and secondary school regulations (they provide their students with an explanation for their grades, they attempt to make assessment contribute to further learning, they make their decisions about final grade based on the results of formative and summative assessment, they provide their students with feedback and guidelines for subsequent work, they explain the material again in case their students failed to learn it, they take into account students’ effort when assessing them, they plan assessment in accordance with the student achievement standards). however, they more rarely consider the criteria which they use to decide upon grades and they more rarely consider grades in the light of students’ arguments. based on teachers’ responses it can be observed that they do not cooperate enough with their colleagues as far as assessment is concerned, both regarding the use of material developed by others and asking their colleagues’ opinion about the quality and validity of the instruments they use to assess student achievements. the two-day professional development programme for chemistry teachers, which focused on strengthening their competencies for assessment, influenced the teachers in such a way that there were statistically significant differences regarding their more positive attitudes towards the following statements: − knowledge assessment and learning are equally important segments of teaching; − knowledge assessment should be conducted continually (in every lesson); − formative assessment helps a student gain an insight into his current level of his achievements; ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 1008 tomasevic et al. − grades enable students to gain an insight into their level of achievements. in addition to this, the programme has caused a statistically significant increase in the number of teachers who disagree with the statements that each assessment of achievement should result in a grade and that formative assessment excludes summative assessment. as far as the statements which refer to the competencies for assessment are concerned, after the programme, a statistically significant percentage of teachers have a more positive attitude towards the following statements: − they can monitor and evaluate the effectiveness of their work based on the results of knowledge assessment; − they understand the concepts of formative and summative assessment; − they are competent to design and conduct formative and summative assessment of knowledge. upon the programme completion, there was a statistically significant increase in the number of teachers who evaluated that they were more competent to design and conduct written, oral and practical assessment based on the achievement standards. teachers’ responses show the impact of the programme for the development of their competencies for assessment, particularly regarding formative and summative assessment and designing various kinds of assessment in accordance with the achievement standards. this is a unique teacher development programme in our education system which has changes identified in the knowledge, skills and attitudes of the teachers upon its realization. acknowledgement. ministry of education, science and technological development of the republic of serbia, contract number: 451-03-9/2021-14/200168. и з в о д програм професионалног развоја компетенција наставника хемије за проверу знања биљана и. томашевић, драгица д. тривић, весна д. милановић и лидија р. ралевић универзитет у београду – хемијски факултет, студентски трг 12-16, београд циљ овог рада je испитивање ефеката програма за професионални развој наставника хемије на њихове компетенције за извођење формативног и сумативног оцењивања у настави хемије. у програму је учествовало 30 наставника хемије из основних и средњих школа. подаци су прикупљени помоћу упитника, примењеног на почетку и на крају реализације програма. програм се састојао од четири радионице са истом структуром рада: увод, групни рад и дискусија резултата групног рада. радионице су биле посвећене: 1) оцењивању као подршци учењу хемије; 2) усклађености активности наставе и учења, формативног и сумативног оцењивања, повратних информација формативног оцењивања и критеријума за сумативно оцењивање; 3) процени ваљаности задатака за формативно и сумативно оцењивање у складу с циљевима наставних програма и образовним стандардима; 4) припреми задатака за праћење напретка ученика ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. chemistry teachers’ assessment competency 1009 према одређеним образовним стандардима. одговори наставника показују допринос програма развоју њихових компетенција за праћење и проверавање ученичких постигнућа, посебно у вези с формативним и сумативним проверавањем и припремањем различитих начина проверавања усклађено са стандардима постигнућа. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice cholinesterase and tyrosinase inhibitory, and antioxidant potential of randomly selected umbelliferous plant species and the chromatographic profile of heracleum platytaenium boiss. and angelica sylvestris l. var. sylvestris j. serb. chem. soc. 81 (4) 357–368 (2016) udc *apiaceae:547.261:577.25.004.12+ jscs–4852 547.587.51:543.544.5.068.7 original scientific paper s357 cholinesterase and tyrosinase inhibitory, and antioxidant potential of randomly selected umbelliferous plant species and the chromatographic profile of heracleum platytaenium boiss. and angelica sylvestris l. var. sylvestris ilkay erdogan orhan1, fatma tosun1 and krystyna skalicka-woźniak2* 1department of pharmacognosy, faculty of pharmacy, gazi university, 06330 ankara, turkey and 2department of pharmacognosy with medicinal plant unit, medical university of lublin, 1 chodzki str, 20–093 lublin, poland (received 9 september, revised 7 december 2015, accepted 21 january 2016) abstract. the neurobiological activity of the methanol extracts of thirteen umbelliferae (apiaceae) plants was tested against acetylcholinesterase (ache), butyrylcholinesterase (bche), and tyrosinase (tyr) using a high-throughput screening technique. although the extracts displayed no to a low inhibition profile against the enzymes, the highest cholinesterase inhibition was observed with heracleum platytaenium (32.52±3.27 % for ache and 46.16±1.42 % for bche) at 100 µg ml-1. since neurodegeneration is linked to oxidative damage, the antioxidant potential of the extracts were examined through radical scavenging, metal-chelating capacity, and reducing power experiments and they exerted modest levels of activity varying according to the method. the extracts had a better ability to scavenge the nitric oxide radical (19.47±2.09 to 54.91±1.98 %). since these species are known to be rich in coumarins, quantitative high-performance liquid chromatography (hplc) analysis indicated the presence of xanthotoxin, angelicin, isopimpinellin, bergapten, and pimpinellin in heracleum platytaenium and angelicin and imperatorin in angelica sylvestris var. sylvestris. keywords: neurobiological activity; coumarins; apiaceae; hplc. introduction alzheimer’s disease (ad), the most common form of dementia, is a progressive neurodegenerative disorder affecting the elderly population, particularly over the age of 60. the cholinesterase (che) enzyme family, consisting of two sister enzymes, i.e., acetylcholinesterase (ache, ec 3.1.1.7) and butyrylcholine * corresponding author. e-mail: kskalicka@pharmacognosy.org doi: 10.2298/jsc150902017o _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 358 orhan, tosun and skalicka-woźniak sterase (bche, ec 3.1.1.8), catalyzes the hydrolysis of acetylcholine (ach), which has been proved to be in lower amounts in the brains of ad patients than usual. consequently, che inhibitors, such as tacrine, rivastigmine, donepezil, and galanthamine, have been the most frequently prescribed drug class for the modern treatment of ad.1 after ad, parkinson’s disease (pd) is another common neurodegenerative disorder worldwide with clear motor symptoms instigated by the degeneration of nigrostriatal dopaminergic neurons, often accompanied with cognitive conditions. it was stated that since enhancement of the cholinergic system by ache inhibitors may cause a reduction in apathy and falls observed during pd, they might also be helpful for therapy of pd.2 on the other hand, transcriptional induction of tyrosinase (tyr, ec 1.14.18.1) is known to initiate a neurotoxic production of cellular dopamine and its oxidative metabolites in excess amounts and, therefore, inhibition of tyr may also be a promising therapeutic approach toward pd.3 nevertheless, as the current che inhibitors are only available for the symptomatic treatment of ad and pd, new therapeutic targets for these diseases still remain to be developed. it is also worth mentioning that age-associated disorders with a neurodegenerative character, such as ad and pd, are usually linked to oxidative damage and, thus, a neuroprotective effect is correlated with prevention of the oxidative stress involved in the over production of reactive oxygen species along with metal dysregulation.4 based on all the relevant data reported hitherto, it has become quite rational to imply a multi-target approach for the treatment of ad and pd. regarding our since the year of 2000 ongoing research on finding new inhibitors of che and tyr from herbal sources, some encouraging results were obtained with coumarin-rich plants from umbelliferae (apiaceae) family, such as angelica officinalis, against ches,5 and taking this finding into account, the aim now was to investigate the neurobiological effects of methanol extracts prepared from thirteen randomly selected edible umbelliferous plants, including apium graveolens l. (ag), angelica sylvestris l. var. sylvestris (ass), artedia squamata l. (as), astrantia maxima pallas subsp. maxima (amm), coriandrum sativum l. (cs), foeniculum vulgare miller (fv), heracleum platytaenium boiss. (hp), ligusticum alatum (bieb.) sprengel (la), petroselinum crispum (miller) a. w. hill (pc), pimpinella affinis ledeb (paf), pimpinella anisum l. (pan), smyrnium olusatrum l. (so), and tordylium apulum l. (ta) through their che and tyr inhibitory activity using microtiter elisa (enzyme-linked immunosorbent assay). relevantly, antioxidant potential of the extracts was evaluated using six in vitro high-throughput screening assays based on radical scavenging, metal-chelating, and reducing power mechanisms. quantitative coumarin analysis was performed on the extracts of hp and ass using a high-performance liquid chromatography (hplc) technique. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ neurobiological activity of apiaceae plants 359 experimental plant materials samples of the studied umbelliferous plants were collected throughout turkey. the plants were identified by prof. dr. mecit vural from the department of biology, faculty of arts and sciences, gazi university (ankara, turkey) and voucher specimens were deposited in the herbarium of the faculty of pharmacy, gazi university (ankara, turkey). the collection sites and herbarium numbers of the plants are listed in table s-i of the supplementary material to this paper. extraction procedure the air-dried and powdered parts used for each plant species (the leaves for ag; the fruits for fv, cs, pan; the aerial parts for the rest) were extracted with methanol and the macerates obtained were evaporated in vacuo to dryness. the extracts were kept in a freezer until the experiments were performed. phytochemical content of the extracts. determination of total phenol and flavonoid contents in the extracts total phenol content of the extracts was determined using the folin–ciocalteu reagent (sigma, st. louis, mo, usa).6 in brief, a number of dilutions of gallic acid (50–500 µg ml-1) were obtained to prepare a calibration curve. the extracts and gallic acid dilutions diluted in ethanol (75 %) were mixed with 750 μl of folin–ciocalteu reagent and 600 μl of sodium carbonate in test tubes. the tubes were then vortexed and incubated at 40 °c for 30 min. subsequently, the absorption was measured at 760 nm on a unico 4802 uv–visible double beam spectrophotometer (dayton, nj, usa). the total flavonoid content of the extracts was established by the aluminum chloride colorimetric method.7 to sum up, a number of dilutions of quercetin (50–500 µg ml-1) were obtained to prepare a calibration curve. then, the extracts and quercetin dilutions were mixed with ethanol (75 %), aluminum chloride reagent, 100 μl of sodium acetate and distilled water. following incubation for 30 min at room temperature, the absorbance of the reaction mixtures was measured at a wavelength of 415 nm using a unico 4802 uv–visible double beam spectrophotometer (dayton, nj, usa). the total phenol and flavonoid contents of the extracts are expressed as gallic acid and quercetin equivalents (mg g-1 extract), respectively. quantification of coumarin derivatives in the hp and ass extracts hplc analysis was performed on a shimadzu system (shimadzu, japan) equipped with an automatic degasser (dgu-20a 3r), a quaternary pump (lc-20ad), an autosampler (sil20a ht) and diode-array detector (dad) (spd-m20a). the chromatographic separation was realized on a zorbax eclipse xdb c18 column (agilent) (250 mm×4.6 mm, 5 μm) at 20 °c. the flow rate of the mobile phase was maintained at 1 ml min-1 and the injection volume was 10 μl. the lc pumps, autosampler, column oven, and dad were monitored and controlled by labsolutions 5.51 software (shimadzu). a gradient of methanol (a) and water (b) was used as follows: 20 % a as starting point, then 20–50 % for 10 min; 50–60 % for 5 min; 60 % kept for 25 min; 80–100 % for 2 min. then, post run was set for 18 min. compounds were identified by comparison of their retention times and diode array detector (dad) spectra with those of appropriate standards analyzed under the same conditions. the following standards were tested: simple coumarins (scopoletin, scoparone, decursin, umbelliferone, daphnetin, harniarin, esculetin (sigma–aldrich) and osthol (chromadex, usa) as well as furanocoumarins (angelicin, xanthotoxol, isopimpinellin, isoimper_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 360 orhan, tosun and skalicka-woźniak atorin (chromadex, usa), xanthotoxin, bergapten, imperatorin (sigma aldrich), byakangelicol, heraclenin, byakangelicin, and phellopterin (phytolab, germany). quantitative determination was performed at 254 and 320 nm. quantitative analysis of pimpinellin was realized by calculation to angelicin (the lack of standard of pimpinellin). in order to confirm the identification of the coumarin derivatives, an hplc coupled with an electrospray ionization (esi) time-of-flight mass spectrometry (tof-ms) was applied. an agilent 1200 hplc system equipped with 6210 msd tof mass spectrometer and zorbax stable bond rp-18 (150 mm×2.1 mm, 3.5 µm) column was used. analyses were performed using a gradient of 60 % acetonitrile in water (+ 0.005 mol l-1 ammonium formate with 0.1 % formic acid) – solvent a, and 90 % acetonitrile in water (+0.005 mol l-1 ammonium formate with 0.1 % formic acid) – solvent b, as described previously.8 compounds were identified using the mass spectra of reference compounds, as well as ms data from the literature.9 microtiter enzyme inhibition assays cholinesterase inhibition. ache and bche inhibitory activity of the samples was measured by a slightly modified spectrophotometric method of ellman.10 electric eel ache (type-vi-s; ec 3.1.1.7, sigma, st. louis, mo, usa) and horse serum bche (ec 3.1.1.8, sigma, st. louis, mo, usa) were used, while acetylthiocholine iodide and butyrylthiocholine chloride (sigma, st. louis, mo, usa) were employed as the substrates of the reaction. 5,5′ -dithiobis(2-nitrobenzoic)acid (dtnb; sigma, st. louis, mo, usa) was used for the measurement of the anticholinesterase activity. all reagents and conditions were the same as described in a previous publication.11 briefly, in this method, 140 µl of sodium phosphate buffer (ph 8.0), 20 µl of dtnb, 20 µl of test solution and 20 µl of ache/bche solution were added via a multichannel automatic pipette (gilson pipetman, paris, france) in a 96-well microplate and incubated for 15 min at 25 °c. the reaction was then initiated with the addition of 10 µl of acetylthiocholine iodide/butyrylthiocholine chloride. hydrolysis of acetylthiocholine iodide/butyrylthiocholine chloride was monitored by the formation of the yellow 5-mercapto-2-nitrobenzoate anion resulting from the reaction of dtnb with thiocholines, catalyzed by enzymes, at 412 nm utilizing a 96-well microplate reader (versamax molecular devices, sunnyvale, ca, usa). galanthamine (sigma, st. louis, mo, usa), an anticholinesterase alkaloid-type of drug obtained from the bulbs of snowdrop (galanthus sp.), was used as the reference. tyrosinase inhibition inhibition of tyrosinase (tyr, ec 1.14.18.1; 30 u, mushroom tyrosinase, sigma) by the samples was determined using a modified dopachrome method with l-3,4-dihydroxyphenylalanine (l-dopa) as the substrate.12 the assays were conducted in a 96-well microplate using an elisa microplate reader (versamax molecular devices, usa) to measure the absorbance at 475 nm. an aliquot of the extracts dissolved in dimethyl sulfoxide (dmso) with 80 μl of phosphate buffer (ph 6.8), 40 μl of tyrosinase, and 40 μl of l-dopa were put into each well. the results were compared with the control (dmso). α-kojic acid (sigma, st. louis, mo, usa) was used as the reference. data processing for enzyme inhibition assays the measurements and calculations were evaluated using softmax pro 4.3.2.ls software. the percentage inhibition of ache/bche was determined by comparison of the rates of reaction of the test samples relative to the blank sample (ethanol in phosphate buffer, ph 8). the extent of the enzymatic reaction was calculated based on eq. (1): _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ neurobiological activity of apiaceae plants 361 100 − = c t e c (1) where e is the activity of the enzyme. the e value expresses the effect of the test sample or the positive control on the activity of ache or bche enzyme, articulated as the percentage of the remaining activity in the presence of test sample or positive control. c is the absorbance of the control solvent (blank) in the presence of enzyme and t is the absorbance of the tested sample (plant extract or positive control in the solvent) in the presence of enzyme. the data are expressed as the average inhibition ± standard error mean (sem) and the results were taken from at least three independent experiments performed in triplicate. microtiter assays for antioxidant activity by radical-formation mechanisms dpph radical scavenging activity. the stable 2,2-diphenyl-1-picrylhydrazyl (dpph) radical scavenging activity was determined by modification of the method of blois.13 the samples (30 μl) and reference dissolved in ethanol (75 %) were mixed with 2700 μl of dpph solution (1.5×10-4 mol l-1). the remaining amount of dpph· was measured at 520 nm using a unico 4802 uv–visible double beam spectrophotometer (dayton, nj, usa). gallic acid (sigma, st. louis, mo, usa) was employed as the reference. dmpd radical scavenging activity. the assay is based on reduction of the purple-colored radical dmpd+ (n,n-dimethyl-p-phenylendiamine). according to the method,14 a reagent comprising of 0.1 mol l-1 dmpd, 0.1 mol l-1 acetate buffer (ph 5.25), and 0.05 mol l-1 ferric chloride solution, which led to formation of the dmpd radical, was freshly prepared and the reagent was equilibrated to an absorbance of 0.900±0.100 at 505 nm. then, the reagent was mixed with 50 µl of the extract dilutions, as well as the reference, and the absorbance was taken at 505 nm using a unico 4802 uv–visible double beam spectrophotometer (dayton, nj, usa). quercetin was employed as the reference and the experiments were performed in triplicate. nitric oxide (no) radical scavenging activity. the scavenging activity of the extracts and reference against no was assessed by the method of marcocci et al.15 briefly, the extract dilutions were mixed with 0.005 mol l-1 sodium nitroprusside and incubated for 2 h at 29 °c. an aliquot of the solution was removed and diluted with griess reagent (1 % sulfanilamide in 5 % phosphoric acid and 0.1 % n-1-naphthylethylenediamine dihydrochloride). the absorbance of the formed chromophore was measured at 550 nm using a unico 4802 uv–visible double beam spectrophotometer (usa). microtiter assays for antioxidant activity by metal-chelating and reducing power mechanisms metal-chelating capacity. the metal-chelating capacity of the extracts and reference through ferrous ion was estimated by the method of chua et al.16 briefly, dilutions of the extracts were incubated with 0.002 mol l-1 iron(ii) chloride solution. the reaction was initiated by the addition of 0.005 mol l-1 ferrozine into the mixture and left standing at ambient temperature for 10 min. the absorbance of the reaction mixture was measured at 562 nm using a unico 4802 uv–visible double beam spectrophotometer (dayton, nj, usa). the ratio of the inhibition of the ferrozine–fe2+ complex formation was calculated. ethylenediaminetetraacetic acid (edta) was employed as the reference in this assay. ferric-reducing antioxidant power assay (frap). the frap values of the samples and the reference were determined using the assay of oyaizu.17 different concentrations of the extracts were mixed with 2500 µl of phosphate buffer (ph 6.6) and 2500 µl of potassium ferricyanide. later, the mixture was incubated at 50 °c for 20 min and, then, trichloroacetic acid (10 %, 2.500 ml) was added. after the mixture had been shaken vigorously, the solution _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 362 orhan, tosun and skalicka-woźniak was mixed with distilled water (2.500 ml) and ferric chloride (0.1 %, 0.500 ml). after incubating for 30 min, the absorbance was read at 700 nm using a unico 4802 uv-visible double beam spectrophotometer (dayton, nj, usa) and compared to that of chlorogenic acid (sigma, st. louis, mo, usa) as the reference. phosphomolibdenum-reducing antioxidant power (prap) assay in order to perform prap assays on the extracts, each dilution was mixed with 10 % phosphomolybdic acid solution + ethanol (1:1 volume ratio).18 the solution was subsequently subjected to incubation at 80 °c for 30 min and the absorbance was read at 600 nm using a unico 4802 uv–visible double beam spectrophotometer (dayton, nj, usa). the analyses were run in triplicate and compared to that of trolox as the reference. data processing for antioxidant activity assays the scavenging effect on dpph, dmpd and nitric oxide radicals, and the metal -chelation capacity of the extracts was calculated using eq. (2) and the results were expressed as inhibition level (i / %): blank sample blank 100 − = a a i a (2) where ablank is the absorbance of the control reaction (containing all reagents except the test sample), and asample is the absorbance of the extracts. the analyses were run in triplicate and the results are expressed as average values ± sem. for the frap and prap assays, the analyses were also achieved in triplicate and increased absorbance of the reaction meant increased reducing power in both assays. statistical analysis of data data obtained from in vitro enzyme inhibition and antioxidant experiments were expressed as the mean standard error (sem). statistical differences between the reference and the sample groups were evaluated by one way anova. dunnett multiple comparison tests were used as post hoc tests. p < 0.05 was considered to be significant (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001). results yield as mass percentages of the extracts are given in table i. variations from no to moderate effects were observed with the umbelliferous extracts in the che and tyr inhibitory assays performed at 100 µg ml–1 (fig. 1). the inhibitory activity of the extracts varied between 7.26±1.86 (cs) and 32.52±3.27 % (hp) against ache, none to 46.16±1.42 % (hp) against bche, and 2.56±0.96 and 16.73±2.80 % against tyr. although the extracts screened possessed either no or low to modest antioxidant activity at 100 µg ml–1, the hp extract exerted the highest scavenging activity toward dpph (33.99±2.41 %) and dmpd (10.12±1.13 %) radicals (fig. 2). on the other hand, seven of the extracts showed no radical scavenging activity slightly over 50 % as follows: paf (54.91±1.98 %) ˃ la (54.34±1.13 %) ˃ amm (53.88±0.22 %) ˃ as (53.48±0.93 %) ˃ ta (52.88±0.48 %) ˃ pc (51.09±0.16 %) ˃ pan (50.05±0.49 %), fig. 2. the frap and prap values of _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ neurobiological activity of apiaceae plants 363 the extracts were revealed to change from a low to moderate level as compared to the references, while their metal-chelating capacity was diminutive (table ii). table i. yields, total phenol and flavonoid contents of the extracts species extract yieldmass % total phenol contenta ± semb total flavonoid contentc ± sem apium graveolens (ag) 36.92 15.28±0.99 20.73±0.45 angelica sylvestris var. sylvestris (ass) 12.57 43.86±1.33 10.58±0.75 artedia squamata (as) 28.21 106.43±5.30 34.91±0.45 astrantia maxima subsp. maxima (amm) 14.98 67.30±3.31 34.06±2.54 coriandrum sativum (cs) 4.73 10.24±0.17 –d foeniculum vulgare (fs) 14.17 12.70±0.33 2.22±0.90 heracleum platytaenium (hp) 16.31 5.55±1.16 – ligusticum alatum (la) 14.67 82.06±11.27 10.79±0.45 petroselinum crispum (pc) 27.89 22.54±6.63 33.75±0.60 pimpinella affinis (paf) 20.84 61.67±9.28 22.43±3.74 pimpinella anisum (pan) 11.36 34.61±3.48 13.12±0.15 smyrnium olusatrum (so) 12.03 40.35±5.63 18.19±1.94 tordylium apulum (ta) 19.57 56.05 ± 1.66 44.11 ± 2.39 adata expressed in mg equivalent of gallic acid to 1 g of extract; bstandard error mean sem (n = 3); cdata expressed in mg equivalent of quercetin to 1 g of extract; d not able to calculate due to very low absorbance fig. 1. a) acetylcholinesterase (ache) butyrylcholinesterase (bche) and b) tyrosinase (tyr) inhibitory activity (inhibition level ± sem) of the apiaceae extracts and references (gal: galanthamine) and kojic acid at 100 µg ml-1. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 364 orhan, tosun and skalicka-woźniak fig. 2. dpph (2,2-diphenyl-1-picrylhydrazyl), dmpd (n,n-dimethyl-p-phenylendiamine), and no (nitric oxide) radical scavenging activity (scavenging activity ± sem) of the apiaceae extracts and references (quercetin for dpph and dmpd radicals and gallic acid for no radical) at 100 µg ml-1. table ii. ferric(frap) and phosphomolibdenum-reducing antioxidant power (prap) activities and metal-chelating capacity of the extracts at 1000 µg ml-1; p < 0.0001 extract frap (absorbance ± sema at 700 nmb) prap (absorbance ± sem at 600 nmb) metal-chelating capacity ± sem, % ag 0.231±0.007 0.202±0.003 13.39±2.37 as 0.332±0.005 0.191±0.003 5.70±0.99 ass 0.314±0.004 0.203±0.005 9.63±1.61 amm 0.445±0.006 0.188±0.012 6.33±2.33 cs 0.195±0.002 0.188±0.001 18.94±1.51 fv 0.241±0.003 0.211±0.027 12.33±1.61 hp 0.219±0.001 0.180±0.033 8.38±1.09 la 0.541±0.003 0.193±0.006 4.64±0.42 pc 0.175±0.001 0.246±0.017 23.92±1.92 paf 0.399±0.012 0.200±0.001 14.23±0.65 pan 0.319±0.006 0.172±0.003 7.38±0.04 so 0.242±014 0.222±0.002 25.49±1.12 ta 0.343±0.006 0.216±0.012 15.96±3.01 quercetinc 1.491±0.041 troloxd 1.871±0.012 edtae 75.08±1.16 astandard error mean (n = 3); bhigher absorbance indicated the greater antioxidant activity; creference for frap assay; dreference for prap assay; eethylenediaminetetraacetic acid, reference for metal-chelating capacity assay spectrophotometric determination of total phenol and flavonoid amounts in the umbelliferous extracts indicated that the richest extract in terms of total phenol was la (82.06±11.27 mg g–1 extract), while the pc extract had the highest amount of total flavonoids (33.75±0.60 mg g–1 extract, table i). since the most active extract against the che enzymes belonged to hp, this extract was subjected to hplc-dad analysis and the following coumarins were quantified: xanthotoxin (2.97±0.019 mg 100 g–1), angelicin (1.74±0.033 mg 100 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ neurobiological activity of apiaceae plants 365 g–1), isopimpinellin (0.31±0.003 mg 100 g–1), bergapten (2.51±0.045 mg 100 g–1), and pimpinellin (7.73±0.159 mg 100 g–1). in the hp extract, the simple coumarin, osthol, was also identified, but its quantitative analysis was not realized because the concentration of osthol was out of the range. in the ass extract that had the second highest bche inhibition after hp, angelicin (0.31±0.015 mg 100 g–1) and imperatorin (2.36±0.033 mg 100 g–1) were determined using hplc -dad in the same manner (the chromatogram are presented in fig. s-1 of the supplementary material). discussion a number of coumarin derivatives along with coumarin-rich plants were reported to possess notable inhibitory potential against ache and bche, which prompted the realization of the current study. for instance, a significant antiache activity was determined for the root methanol extract of angelica gigas (umbelliferae), which led to the isolation of twelve coumarin derivatives and, among them, decursin was identified as the most promising one due to its marked ache inhibitory as well as its in vivo memory-enhancing effect.19 later, two other coumarin derivatives (nodakenin and decursinol) isolated from a. gigas were also revealed to have ache inhibitory activity.20 additionally, the dichloromethane, ethanol, and aqueous extracts of a. graveolens were previously demonstrated to have low inhibition toward ache and bche at 200 µg ml–1, in accordance with the present data on this species. a limitation of this study was that the che or tyr inhibitory effects of the coumarin standards identified in the hp and ass extracts could not be confirmed due to scarcity in their amounts. nevertheless, in an earlier study,5 three coumarin compounds, imperatorin (83.98±0.99 %), xanthotoxin (88.04±0.83 %) and bergapten (86.69±2.56 %), identified in angelica officinalis, were shown to have strong bche inhibition, which was also supported by molecular docking experiments. since presence of xanthotoxin and bergapten in hp and imperatorin in ass was confirmed by the hplc analysis, these coumarins could be considered most likely to contribute to the moderate che inhibitory effect exhibited by hp as well as ass. furthermore, isopimpinellin, earlier obtained from angelica acutiloba with ache inhibitory effect, might be suggested to contribute to some extent to the relevant activity of hp.21 previously, the methanol and petroleum ether extracts of hp were reported to exert 49.86±1.56 % and 49.28±1.28 % against ache and 65.51±1.63 % and 56.59±1.62 % against bche, respectively, at 200 µg ml–1. the extract yielded 8 furocoumarins, elucidated as psoralen, bergapten, xanthotoxin, pimpinellin, isopimpinellin, sphondin, byakangelicin, and heraclenol.22 in the same study,22 pimpinellin was determined to cause 78.57±2.86 % inhibitions of ache and 82.17±1.66 bche inhibitions. as it was quantified as the major coumarin in the hp extract in the present study, pimpinellin might be _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 366 orhan, tosun and skalicka-woźniak speculated to be the major contributor to the che inhibitory effect of this plant. additionally, the remarkable anticholinesterase effect of xanthotoxin and bergapten was also confirmed in the study by dincel et al.,22 that supported former data on the same compounds.5 moreover, the weak dpph radical scavenging activity of hp was found in the aforementioned study, which is again consistent with the present data. cs, used for memory-enhancing purpose in iranian folk medicine, was previously deduced to have a very low ache inhibitory and dpph scavenging effect, which supports the present finding on cs.23 the root ethanol extract of fv was formerly found to display neither ache nor bche inhibitory effects pertinent to the current results on the fruit methanol extract of fv.24 consistent with the present data, the methanol extracts of both pc (root) and pan (fruit) used for memory impairment in danish folk medicine were ineffective in the ache inhibition assay.25 on the other hand, there has hitherto been no report on the che inhibitory effect of so and ta. the tyr inhibitory activity of umbelliferous plants was researched, which again led to the isolation of some coumarins as the active constituents. for instance; 9-hydroxy-4-methoxypsoralen from angelica dahurica, aloesin from aloe vera, esculetin from euphorbia lathyris, and 8′-epi-cleomiscosin a from rhododendron collettianum showed potent tyr-inhibiting effects.26 among the plant species tested herein, cs was reported to possess 47.76±2.50 and 49.2 % of inhibition of tyr in two early studies, while fv showed only 29.6 % tyr inhibition.27,28 adhikari et al. also described tyr inhibitory effects 41.7±2.2 % for cs and 22.4±6.0 % for fv, and 45.8±16.9 % for pc at 50 µg ml–1,29 in contrary to the present data on these species, which might be the result of phytochemical differences. conclusion in summary, the methanol extracts of thirteen umbelliferous plant species were screened for their che and tyr inhibitory effects and their antioxidant activity. the findings obtained in this study revealed that hp shows some notable inhibition against ache and bche and the coumarins found in this plant seem to be the active substances. to the best of our knowledge, this study is the first on che and tyr inhibition and the antioxidant activities of angelica sylvestris var. sylvestris (ass), artedia squamata (as), astrantia maxima subsp. maxima (amm), ligusticum alatum (la), smyrnium olusatrum (so) and tordylium apulum (ta). supplementary material collection sites and herbarium numbers of the plant species as well as hplc chromatograms of the hp extract and the ass extract are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ neurobiological activity of apiaceae plants 367 и з в о д антихолинестеразни, антитирозиназни и антиоксидативни потенцијал биљака фамилије umbelliferае и хроматографски профил састојака биљака heracleum platytaenium boiss. и angelica sylvestris l. var. sylvestris ilkay edrogan orhan1, fatma tosun1 и krystyna skalicka-woźniak2 1department of pharmacognosy, faculty of pharmacy, gazi university, ankara, turkey и 2department of pharmacognosy with medicinal plant unit, medical university of lublin, lublin, poland неуробиолошка активност метанолних екстраката тринаест биљака фамилије umbelliferae (apiaceae) је тестирана спрам ацетилхолинестеразе (ache), бутирилхолинестеразе (bche) и тирозиназе (tyr). екстракти нису испољили или су имали слаб инхибиторни ефекат на ензиме, а највећу антихолинестеразну активност је имао екстракт биљке heracleum platytaenium (32,52±3,27 % спрам ache и 46,16±1,42 % спрам bche) у концентрацији 100 μg ml-1. пошто су неуродегенеративне промене повезане са оксидативним оштећењима, антиоксидативни потенцијал екстраката је утврђиван у тесту хватања слободних радикала, одређивањем хелатног капацитета и мерењем редукујућег потенцијала. активност екстраката није била велика, а највећа се показала у хватању слободних радикала азот-оксида (од 19,47±2,09 до 54,91±1,98 %). ове биљне врсте садрже доста кумарина, а квантитативном hplc анализом је утврђено присуство ксантотоксина, ангелицина, изопимпинелина, бергаптена и пимпинелина у heracleum platytaenium, као и ангелицина и императорина у angelica sylvestris var. sylvestris. 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khanom, h. kayahara, k. tadasa, biosci. biotechnol. biochem. 64 (2000) 1967 28. p. k. mukherjee, s. badami, a. m. wahile, s. rajan, b. suresh, j. nat. rem. 1 (2001) 125 29. a. adhikari, h. p. devkota, a. takano, k. masuda, t. nakane, p. basnet, n. skalko -basnet, int. j. cosme. sci. 30 (2008) 353. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice chemical synthesis and characterization of highly soluble conducting polyaniline in mixtures of common solvents j. serb. chem. soc. 80 (7) 917–931 (2015) udc 678.652+542.913+547.462.3: jscs–4769 544.351.3:66.095.26 original scientific paper 917 chemical synthesis and characterization of highly soluble conducting polyaniline in mixtures of common solvents hichem zeghioud1*, saad lamouri1, zitouni safidine1 and mohammed belbachir2 1laboratoire de chimie macromoléculaire, ecole militaire polytechnique, bp 17, bordj el bahri, alger, algeria and 2laboratoire de chimie des polymères, département de chimie, faculté des sciences, université d’oran, bp 1524 el’menouer oran 31000, algeria (received 19 july, revised 29 december 2014, accepted 8 january 2015) abstract: this work presents the synthesis and characterization of soluble and conducting polyaniline–poly(itaconic acid) pani–pia according to a chemical polymerization route. this polymerization pathway leads to the formation of doped polyaniline salts, which are highly soluble in a number of mixtures between organic common polar solvents and water, the solubility reaches 4 mg ml-1. the effect of synthesis parameters, such as doping level, on the conductivity was investigated and a study of the solubility and other properties of the resulting pani salts were also undertaken. the maximum of conductivity was found equal to 2.48×10-4 s cm-1 for fully protonated pani-eb. in addition, the synthesized materials were characterized by various methods, i.e., viscosity measurements, xrd analysis and ftir and uv–vis spectroscopy. finally, tga was performed to obtain some information concerning the thermal behaviour of the materials. keywords: conducting polymer; pani; itaconic acid; polymerization; solubility properties. introduction the synthesis and improvement of new materials with special properties have attracted much attention in the last decade. polyaniline (pani) is one of the most promising polymers due to its good flexibility, low cost, oxidative stability and unique conduction mechanism.1–3 however, there are some major drawbacks, which limit its application, such as low thermal stability and poor solubility in common solvents due to the stiffness of backbone and h-bonding interactions between adjacent chains.4,5 * corresponding author. e-mail: hicheming@yahoo.fr doi: 10.2298/jsc140719003z _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 918 zeghioud et al. recently a number of studies have been devoted to the search for different synthetic methods that allow pani to be obtained with good solubility in the presence of different polymeric acids with different structures,6–9 which improve processibility, special electrical conductivity and optical and spectroscopic properties. barrios et al.10 reported the electro-synthesis of pani in the presence of poly(itaconic acid) and they evaluated the effect of the presence of dicarboxylic acids on the electrochemical behaviour of the obtained films. nevertheless, the electrochemical methods generated polyaniline with very low solubility that was difficult to process and had restricted application. travas-sejdic et al.11 studied the electrochemical properties of self-assembled multilayer films based on polyaniline and two polyanions: poly(styrene sulphonate) (pssa) and an oligonucleotide (on), and found by cyclic voltammetry experiments that pani/pssa and pani/on films had different electrochemical behaviours, with pani/on films showing lower electroactivity. gizdavic-nikolaidis et al.12 recently demonstrated that a conductive polymer nanofibrilar network of poly(lactic acid) can be electrospun with pani, and its copolymers with m-aminobenzoic acid (m-aba) from dmso/thf solutions. abdul rahman et al.13 found that the number average molecular weights of the copolymers decreased significantly with increasing m-aba fraction in the copolymers, and the solubility increased with increasing proportion of m-aba. this increase was due not only to the functional cooh groups, but also to the decrease in the average length of the polymer chains. gribkova et al.14 reported the chemical polymerization of aniline in the presence of aromatic polyamides containing sulphonic groups. they observed that the presence of the flexible-chain of polyamides with a regular distribution of sulphonic groups along the polymer chain allowed the polyaniline to exhibit a random coil conformation in the presence of strong electrolytes and the formation of water-soluble interpolymer complexes of pani with polyacids. wang et al.15 prepared polyaniline nanorods by chemical oxidative polymerization using itaconic acid as dopant. the polyaniline salt obtained, compared to the undoped form, possessed high productivity, conductivity and excellent solubility in organic solvents such as nmp, thf and dmf, which reached 19 mg ml–1. the main goal of the present work was the chemical doping of pani in the presence of poly(itaconic acid) (pia). pia is a very exciting material due to its biocompatibility with natural systems. in addition, pia is very attractive because it has two negatively charged carboxylic groups in each monomer unit. the essential advantage of the resultant pani was the possibility of producing conductive pani blends with good solubility. the spectral, thermal and electrical properties of the obtained pani molecular composites were studied. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ highly soluble conducting polyaniline 919 experimental materials aniline (ani, 99 % pure) was purchased from fluka. other provided materials were hydrochloric acid (35.37 %) from organics stinnes chemicals, methanol (99.5 %) and ammonium peroxodisulphate (aps, 99 %) from prolabo. potassium peroxodisulphate (pps, 99 %) was from riedel-de haëen, acetone (99 %) from acros organics, while ammonia (25 % solution), n-methylpyrrolidone (nmp, 99.5 %) and tetrahydrofuran (thf, 99 %) were from merck. itaconic acid (99 %) was purchased from aldrich, dimethyl sulphoxide (dmso, 100 %) from analytical reagent. deionised water was used throughout the experiments and all chemicals were used without previous purification. synthesis of pani the synthesis of the pani-eb was as follows: 4.8 g of aniline (4.75 ml) and 15 ml of hcl were dispersed in 50 ml of deionised water under vigorous stirring at room temperature for 2 h to obtain a uniform solution. then, an aqueous solution of aps (11.8 g + 50 ml deionised water) was added to the above mixture in one portion (the mole ratio ani:aps = 1). the resulting solution was stirred for 30 min to ensure complete mixing, and then the reaction was followed by continuous stirring at 2 °c for 4 h. the precipitate that formed was filtered off, washed with deionised water and methanol until the filtrate was colourless to remove excess acid and possible oligomers. finally, the powder was dried under vacuum for 48 h. then, the obtained pani as the emeraldine salt (pani-es) was stirred in 1 m solution of ammonium hydroxide at room temperature for 72 h to completely convert it to emeraldine base (pani-eb) form. upon filtering and drying under a dynamic vacuum in an oven at 60 °c for 24 h, the base form of pani was obtained as a blue powder. the yield of the polymerization was 80 %. poly(itaconic acid) synthesis the synthesis was carried out similarly to that described in work of larez et al.16 thus deionised water (10 ml) was heated until boiling and left to reach ambient temperature with continuous stirring and under nitrogen bubbling. itaconic acid (ia, 0.023 mol) and pps (2.22×10-4 mol) were added and the system was sealed and placed in a thermostatic bath at 60 °c with continuous stirring during 48 h. a heterogeneous mixture was initially obtained due to an incomplete dissolving of ia that went transparent after 5 min. after the polymerization reaction time was over, the reactor was left to cool down to room temperature under continuous stirring (about 30 min). polymer was separated by precipitation in cool acetone. the repeat unit of the resulting polymer (pia) is shown in scheme 1. scheme 1. chemical structure of the repeat unit of poly(itaconic acid) pia. doping the polymeric acid-doped pani was prepared by mixing 0.9 g of pani-eb (0.0025 mol, based on the approximate tetrametric repeat unit) in a thf dispersion with the appropriate quantity _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 920 zeghioud et al. of poly(itaconic acid) solution of different mole ratios (based on its acidic repeat unit). the suspension was sonicated in a bath-type sonicator for 2 h followed by electromagnetic stirring (12 h) to make the dispersion homogeneous. the dispersion of pani–pia was filtered using polytetrafluoroethylene membrane filters of pore size 0.45 µm. the resultant powder was washed with large quantities of distilled water until the filtrate became colourless and the powder was dried in an oven at 60 °c for 24 h. the reaction mechanism is shown in scheme 2. scheme 2. mechanism of pani-eb doping with pia. characterization the fourier transform infrared (ftir) spectra were recorded between 400 and 4000 cm-1 from kbr pellets on an infrared fourier transform spectrometer (shimadzu type 8400 s). the uv– –vis spectra of all samples dissolved in different solvents were recorded using a uv–vis spectrometer shimadzu uv-2401pc, in the wavelength range of 250–900 nm. the intrinsic viscosity measurements of solutions were made using a micro-ubbelohde schott-gerate viscosimeter. the x-ray powder diffraction patterns were recorded on a panalytical x'pert pro diffractometer fitted with cukα radiation (λ = 1.5404 nm) at 40 kv and 40 ma in the 2θ range 5– –60° region. thermogravimetric (tg) analysis was performed using a setaram mtb instrument with “10-8” sensitivity, operating at a heating rate of 10 °c min-1, from room temperature up to 450 °c under an air atmosphere. the sample mass ranged between 3 and 6 mg. results and discussion conductivity measurements the electrical conductivity measurements of compressed pellets of pia -doped pani were made by the conventional four-point probe technique at room _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ highly soluble conducting polyaniline 921 temperature; the data are plotted in fig. 1. the curve showing the evolution of the electric conductivity (σ) according to the doping level follows an exponential form. indeed, the values of the electrical conductivity order of 10–4 were found for pani–pia, whereby a high conductivity value of 2.48×10–4 s cm–1 for the pani–pia was reached when the pani-eb (conductivity value of 8.14×10–10 s cm–1) was fully protonated, showing that the resultant pani–pia was less conductive than the pani–hcl, which has a conductivity of 4.6 s cm–1. this was confirmed by the narrower band gap for pani–hcl around 3.2 ev compared with the pani–pia (table i). note that each measured value of conductivity is an average of four measurements for each face of the pellet. fig. 1. the conductivity of pani–pia at different [pia repeat unit]/[eb tetramer] mole ratio. table i. conductivity and the energy band gap of different forms of pani sample uv–vis band, nm conductivity, s cm-1 energy band gap, ev 1 2 pani–hcl 387 467 4.60 3.20 pani-eb 331 626 8.14×10-10 – pani–pia 304 347 2.48×10-4 4.08 fourier transform infrared spectroscopy (ftir) ftir spectra of ia and its polymer (pia) are shown in fig. 2. the assignments of the ftir bands of ia, pia, pani-eb and pani–pia are given in table ii. the spectra of ia and pia showed a broad band between 2775 and 3480 cm–1,17 which was attributed to o–h stretching vibrations. the spectrum of ia showed peaks at around 1700,18 143018 and 1220 cm–1,19 indicating the stretching vibrations of c=o (carboxylic acid), c–o–h in plane and c–o, respectively. the spectrum of the pia showed the peaks at around 1730,20 140019 and 1193 cm–1,21 indicating the stretching vibrations of c=o, c–o–h and c–o, _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 922 zeghioud et al. respectively. the peak at 1629 cm−1 in the curve (t = 0 h) in fig. 2,18 characterizes the presence of c=c bond coming from itaconic acid. however, this peak was absent in pia spectrum, which confirmed the polymerization of ia. fig. 2. kinetics of the polymerization reaction of itaconic acid from ia (t = 0 h) to pia (t = 48 h) determined by ftir spectroscopy. table ii. assignment of ftir bands (wavenumber, cm-1) of ia, pia, pani-eb and pani–pia sample assignment ia pia 2775–348017 2775–348017 o–h stretching vibration 170018 173020 c=o stretching vibration 162918 – c=c stretching vibration 143018 140019 c–o–h stretching vibration 122019 119321 c–o in of plane bending vibration pani-eb pani–pia assignment 343522 343522 n–h stretching vibration – 171927 c=o stretching vibration 158323 156828 n=q=n stretching rings 149424 148529 n–b–n stretching ring 129225 129225 c–n+ stretching in secondary amines 51126 51126 c–h out-of-plane bending 1,4 ring _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ highly soluble conducting polyaniline 923 the position of wavenumber of c=o stretching depends on hydrogen bonding and conjugation within the molecule. with polymerisation of ia, the c=o stretching band was shifted to higher wavenumbers, because the possibility of conjugation with a c=c band resulting in delocalization of the c=o group was eliminated on polymerisation.18 in addition, shifting of the typical bands for the c–o and c–o–h groups were detected. this effect is related to hydrogen bonding between the cooh groups of pia. the ftir spectrum of pure polyaniline (pani-eb) is shown in fig. 3. the formation of polyaniline was confirmed from the predominant peaks at the wavenumber of 1583 cm–1, corresponding to c=c stretching of the quinoid ring,23 1494 cm–1 for c=c stretching of the benzenoid ring,24 1292 cm–1 for c–n+ stretching,25 and 511 cm–1 for c–h out-of-plane bending.26 after pia doping, the quinoid and benzenoid ring bands were shifted to lower wavenumbers by 15 and 9 cm−1, respectively. this red shift phenomenon, corresponding to the transformation of quinoid rings into benzenoid rings, may result from conjugation effects after doping with the polymeric acid.28 fig. 3. ftir spectra of pani-eb, pia and pani–pia. uv–vis spectroscopy absorption spectroscopy is a valuable tool for detecting the presence of pani base and its salts. all the absorption spectra of the pani–pia samples and the pia spectrum, fig. 4, showed a peak in the range of 249–285 nm, corresponding _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 924 zeghioud et al. to π—π* or n—π* transitions in the carbonyl groups of the poly(itaconic acid),30 (table iii). fig. 4. uv–vis spectra of: a) water/nmp, b) water/thf and c) pia and pani–pia salts recorded in different solvents. table iii. absorption bands (λ / nm) of pia and pani–pia samples in different solvents sample (solvent) attribution of absorption bands π–π* and/or n–π* π–π* polaron–π* π–polaron pia (water) 282 – – – pani–pia (thf/water) 249 304 347 790 pani–pia (nmp/water) 285 347 410 816 the electronic absorption spectra of the pani–pia samples in different solvents showed bands corresponding to the following transitions: π–π* (304–347 nm), polaron–π* (347–410 nm), and π–polaron (in the region from 790 to 816 nm) of the alternating benzenoid–quinoid structures, respectively.31,32 the finding of the latter two absorption bands in the electronic spectra of all the pani–pia samples indicates that these polymers were well doped. when the solvent molecules interact with a pani–pia, its chain configuration may be changed depending on the structure and polarity of the solvents, which is reflected by peak shifts.33 solubility determination pani–pia dispersion in thf/water mixture (20 ml) was oscillated in a bath-type sonicator for 2 h at room temperature. the suspension was filtrated to remove undissolved polyaniline. the obtained solution was analyzed by uv–vis spectroscopy to confirm the protonated state of pani, then it was dried in an oven _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ highly soluble conducting polyaniline 925 at 60 °c for 12 h. the maximum solubility was the quantity of resulting polyaniline powder after drying 20 ml of solvents mixture with an optimal volume ratio, which is measured in mg ml–1. note that the optimal ratio was determined by the intrinsic viscosity method. the solutions of pani–pia in different solvents are shown in fig. 5. the picture proves that the pani synthesized in the present work was highly soluble in tetrahydrofuran/water and nmp/water mixtures with a dark green colour (fig. 5a and b, respectively). fig. 5. photographs of solutions of pani–pia in mixtures, a) water/thf = 0.428 and b) water/nmp = 0.428. the carbonyl groups of extended polyacid chain of the pia facilitate the solubility of pani in some polar solvents.34 pani–pia is not soluble in pure thf and nmp. the presence of water with each organic solvent improves the solubility of the pani–pia significantly, which could be explained by the number of carboxylic groups that is higher than the number of hydroxyl groups in the dopant which require a solvent with more groups containing hydrogen to create hydrogen bond. these groups (c=o), more negatively charged can create a stronger hydrogen bond with the water hydrogen. this idea is presented schematically in scheme 3. scheme 3. effect of the presence of water as co-solvent on pani–pia solution in thf. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 926 zeghioud et al. intrinsic viscosity the viscosity of macromolecular substances in solution is one of the most commonly used methods for characterizations. intrinsic viscosity is defined as the limiting value of the ratio of specific viscosity to concentration of the solute (ηsp/c), extrapolated to zero concentration. different solutions of pani–pia were prepared with different volume ratios of solvents (thf or nmp)/water at room temperature. this method is based on the calculation of the difference between the efflux time of the mixture of solvents (t0) and polymeric solution (t) between two points in a capillary tube. basing on the most recent work performed by yilmaz et al.35 using the values of constant k and α at room temperature (26 °c) obtained for the leucoemeraldine form in nmp solution in the presence of licl: k = 2.34 × 10–2 ml g–1 and α = 0.73 [η] = km̅vα (1) if two values are introduced into the mark–houwink equation (eq. (1)),36 the obtained value of the intrinsic viscosity [η] = 13.26 ml g–1 of the emeraldine salt form results in an apparent viscosity average molar mass of m̅v = 5915 g mol–1. the evolutions of the intrinsic viscosity of solutions having various (thf or nmp)/water volume ratios of polyaniline doped with poly(itaconic acid) are shown in fig. 6. the increase in viscosity with increasing water content indicates an increase in the hydrodynamic volume of the doped polymer chains, consistent with a progressive change in molecular conformation from “compact coil” to “expanded coil”.37 this type of behaviour indicates that water interacts more strongly with the polymer chains and/or with poly(itaconic acid) (pia) dopant fig. 6. relationship between the intrinsic viscosities of polyaniline doped with poly(itaconic acid) and volume ratio of the mixtures of: a) water/thf and b) water/nmp. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ highly soluble conducting polyaniline 927 anions than does thf. conversely, when the value 0.428 for the volume ratio was exceeded, the intrinsic viscosity began to decrease with further water addition, which could be explained as follows: the pani–pia chains take an extended coil conformation also because there are h-bonding interactions of the hydrogen of the amine groups with the oxygen present in the molecular structure of thf. when the value of the volume ratio was more than 0.428, the water excess was used to decrease the interaction chain–organic solvent that led thereafter to decreases in the hydrodynamic volume of the chains of the doped polyaniline (compact coil). the change in intrinsic viscosity values is so large that also the quality of solvent/water mixture for pani-eb and pani–pia should be considered. namely, water in this case can act as a non-solvent. after reaching an optimal proportion between solvent and water (good solvation, intermolecular interaction), its increased share might worsen the solvent quality, which could lead to more compact random coils due to stronger intramolecular chain interaction.38 thermogravimetry (tga) the results of the thermogravimetric analysis of the pure pia and pani–pia samples are presented in fig. 7. four degradation steps could be observed for pia (fig. 7, curve b) in the temperature range from 26–450 °c, with a residue of 30 wt. %. in the temperature region from 50–280 °c, two processes were detected; the first one is assigned to the elimination of free water adsorbed to the hydrophilic polymer and presence of residual solvent in the polymer,39 and the second to anhydride ring formation in the pia chain.40 in the second temperature region, from 280 to 450 °c, two degradation stages were observed, probably related to some decarboxylation and carbonization processes.16,41 fig. 7. thermogravimetric (tg) and differential tg (dtg) curves for: a) pani–pia and b) pia. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 928 zeghioud et al. in the tga thermograms of the pani–pia shown in fig. 7, curve a, there are three major stages of weight losses of pani–pia powder sample. the first weight loss occurred around 67–198 °c (10 %) resulting from the elimination of water and other volatiles.42 the second stage in the temperature range 198–337 °c was assigned to the decomposition of excess dopant pia.43 the third weight loss at the higher temperature could be attributed to the detachment of doping agent and the chemical decomposition of the short chains of pani with maximum decomposition rates at 367 and 427 °c, respectively.44 it is notable that as much of 50 % of the initial mass was preserved for temperatures up to 500 °c. this residual mass relates to the existence of reticule polymer, which is formed at high temperatures,45 and the influence of the doping agent on a real thermal stability is not clear in this temperature region. x-ray diffraction pattern (xrd) the x-ray diffraction pattern of pani-eb, pia and pani–pia are given in fig. 8. the crystalline (ic) and amorphous (ia) peaks were both integrated in 2θ space. from these integrated peaks areas (ic, ia), the ratio xc/xa can be calculated by xc/xa = 1.8×(ic/ia). a ryland factor of 1.8 is commonly used for semi-crystalline polymers.45 the percentage of crystallinity xc (%) was obtained as:46 xc (%) = 100 – 100/(1 + xc/xa) (2) the xrd pattern of pani–eb (fig. 8, curve a) contains some sharps peaks at 2θ 9.57, 15.22, 20.75 and 24.29°, representing the crystal planes of (001), (011), (100) and (110) of dedoped pani, respectively.47 the peak intensities are listed in table iv, indicating the pseudo orthorhombic space.26 the peak centred at 2θ 24.29° could be ascribed to periodicity perpendicular to the polymer chain fig. 8. xrd patterns of: a) pani-eb, b) pani–pia and c) pia. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ highly soluble conducting polyaniline 929 (π stacking),47 and the peak centred at 2θ 20.75° may be ascribed to periodicity parallel to the conjugated chain of pani (emeraldine base).48 the sharpness of the peaks represents the degree of orientation of the polymer chains in that particular crystal plane, and the intensity represents the population of crystallites in that plane.49 this result suggests a low degree of crystallinity of 9.64 %. table iv. the 2θ values, intensity and indexation (hkl) of pani-eb, pia and pani–pia sample 2θ / ° intensity, a.u. d / å (hkl) crystallinity, xc / % 09.57 167.46 09.23 001 9.64 pani–eb 15.22 176.44 05.82 011 20.75 202.06 04.28 100 24.29 203.40 03.66 110 pia 11.80 016.88 07.50 – 9.50 pani–pia 19.99 008.60 04.44 100 2.03 37.27 005.71 02.41 110 as shown in fig. 8, curve c, the poly(itaconic acid) showed a moderate degree of crystallinity (xc = 9.5 %, broad peaks in the spectrum) and a single sharp peak at 11.8°. on the other hand, the diffractogram of pani–pia powder, presented in fig. 8, curve b showed two peaks; the first at 19.99° and the other at 37.27° with a characteristics distances of 4.44 and 2.41 å, respectively. it should be noted that the chains of the pani–pia were less ordered than those of the pani-eb, which was confirmed by the value of xc = 2.03 % for pani–pia. conclusions soluble conducting polyaniline salts were successfully synthesized via chemical polymerization. undoped and poly(itaconic acid)-doped polyaniline were characterized by a number of methods. the doped polyaniline had good solubility in mixtures of nmp/water and thf/water with a maximum solubility of 4 mg ml–1 in the latter mixture at room temperature; on the other hand, this polyaniline salt was not soluble in the pure polar organic solvents (thf and nmp) except when the temperature is above 60 °c. the optimum volume ratio of 0.42 between water and thf was determined by intrinsic viscosity measurements. the electrical conductivity of the doped form of polyaniline presents a proportional relationship with the concentration of the doping agent (pia). a decrease in crystallinity was detected in the new pani salt due to the presence of poly(itaconic acid) in the composite. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 930 zeghioud et al. и з в о д синтеза и карактеризација проводног и у смеши растварача веома растворног полианилина hichem zeghioud 1 , saad lamouri 1 , zitouni safidine 1 и mohammed belbachir 2 1laboratoire de chimie macromoléculaire, ecole militaire polytechnique, bp 17, bordj el bahri, alger, algeria и 2laboratoire de chimie des polymères, département de chimie, faculté des sciences,université d’oran, bp 1524 el’menouer oran 31000, algeria у раду је приказана синтеза и карактеризација растворног и проводног полианилина pani–pia добијеног хемијским поступком тј. полимеризацијом. на овај начин је добијена со полианилина допираног поли(итаконском киселином), која је веома растворна у бројним смешама поларних органских растварача и воде, при чему је достигнута растворљивост од 4 mg ml-1. анализиран је утицај параметара синтезе, као што је степен допирања, на проводљивост, растворљивост и друга својства синтетисаних pani– –pia соли. максимална проводљивост у износу од 2,48×10-4 s cm-1 је остварена при потпуном протоновању полианилина у облику емералдинске базе (pani-eb). поред тога, синтетисани материјали су додатно карактерисани вискозиметријом разблажених раствора, uv–vis спектроскопијом, дифракцијом x-зрака (xrd), инфрацрвеном спектроскопијом и термогравиметријском анализом (tga) за анализу њихових термичких својстава. 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polym. j. 41 (2009) 432 45. d. tsocheva, t. ziatkov, l. terlemezyan, j. therm. anal. 53 (1998) 895 46. p. j. rae, d. m. dattelbaum, polymer 45 (2004) 7615t. abdiryim, x.-g. zhang, r. jamal, mater. chem. phys. 90 (2005) 367 47. m. hasik, a. drelinkiewicz, e. wenda, c. paluszkiewicz, s. quillard, j. mol. struct. 596 (2001) 89 48. b. belaabed, j. l. wojkiewicz, s. lamouri, n. el kamchi, t. lasri, j. alloys compd. 527 (2012) 137. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice the β-casein–resveratrol complex: physicochemical characteristics and implications for enhanced nutrition j. serb. chem. soc. 81 (7) 739–750 (2016) udc 547.963.2:544.2.004.12+577.122– jscs–4882 188:641.3:577.164.1:541.147 original scientific paper 739 the β-casein–resveratrol complex: physicochemical characteristics and implications for enhanced nutrition hao cheng1, yanfang li1, xundi yin2, muriel subirade3 and li liang1* 1state key lab of food science and technology, school of food science and technology, jiangnan university, wuxi, jiangsu, china, 2college of textiles and clothing, jiangnan university, wuxi, jiangsu, china and 3chaire de recherche du canada sur les protéines, les bio-systèmes et les aliments fonctionnels, institut de recherche sur les nutraceutiques et les aliments fonctionnels (inaf/stela), université laval, québec, qc, canada (received 29 october 2015, revised 28 january, accepted 26 february 2016) abstract: food proteins have been widely used as carrier materials for the encapsulation and protection of bioactive molecules. clarification of the mechanism of protein–bioactive molecule interaction is important for the development of protein-based carrier systems. the interaction of β-casein with resveratrol, a natural polyphenol, was studied using ultraviolet–visible absorption and fluorescence spectroscopy. it was found that the interaction shifted the protein fluorophores to a more hydrophilic environment and the polyphenol to a more hydrophobic environment. the formation of the complex with β-casein did not affect trans–cis isomerization of resveratrol or the total antioxidant activity of the protein–polyphenol system, as analyzed respectively using spectrophotometry and the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) assay. the protective effect of resveratrol against photodecomposition of folic acid was not affected by binding to β-casein. the data obtained should provide insight into protein–polyphenol interaction mechanisms and aid the development of β-casein-based carrier systems for the delivery of bioactive molecules. keywords: β-casein; resveratrol; folic acid; complex; photosensitivity. introduction proteins have been used widely as carrier materials for the preparation of nano/micro-particles and gels because of their nutritional value and their ability to form gels and emulsions and to interact with polysaccharides.1,2 entrapped bioactive nutrients generally interact with carrier proteins, except that they may remain dissolved in the inner liquid phase of protein-stabilized emulsions. in order to develop effective delivery systems for bioactive nutrients, the mechanisms of interaction between the carrier and the nutrient must be understood. * corresponding author. e-mail: liliang@jiangnan.edu.cn doi: 10.2298/jsc151029025c _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 740 cheng et al. however, most studies have hitherto focused on globular proteins, such as bovine serum albumin and β-lactoglobulin, which contain native sites for binding bioactive molecules.3,4 collagen, soy proteins and caseins can also form complexes with bioactive molecules,5–7 although their specific native binding sites for ligands remain uncertain. bovine milk caseins are classified as intrinsically unstructured proteins consisting of four phosphoprotein fractions, namely αs1-casein, αs2-casein, β-casein, and κ-casein. of these fractions, β-casein (≈24 kda) is the most hydrophobic. the research group of tajmir-riahi has reported interactions of caseins with vitamins and polyphenols via hydrophilic and hydrophobic contacts.8–10 the formation of complexes with β-casein could delay the photodecomposition of folic acid.11 in addition, vitamin d3 appears to bind to β-casein with dissociation constants ranging from 0.06 to 0.26 μm and with 1.16–2.05 mol of vitamin d3 bound per mol of β-casein, depending on the solution ph and ionic strength.12 it was suggested that the formation of complexes with β-casein may affect the stability and bioavailability of bioactive molecules in processed food products. resveratrol (3,4′,5-trihydroxystilbene) is a polyphenol present in grapes, peanuts and mulberries, and in food products such as red wine and peanut butter. multiple biological activities generally regarded as beneficial to health have been attributed to this polyphenol, including antioxidant, anti-inflammatory, anti-atherogenic and growth-inhibiting activities, anti-platelet aggregation and estrogen -like growth-promoting effects, immunomodulation and chemoprevention.13,14 however, the bioavailability of resveratrol is low due to its poor water solubility, sensitivity to environmental factors, and inability to reach target sites in the body.13 it was reported that resveratrol could bind to β-lactoglobulin, collagen, fibrinogen and caseins to form nano-complexes.6,9,15,16 the interaction of resveratrol with β-casein was reported with a binding constant of 2.3×104 m–1 and a free binding energy of –12.35 kcal* mol–1.9 in this study, the interaction of β-casein with resveratrol was further investigated using ultraviolet–visible (uv–vis) absorption and fluorescence spectroscopy and by the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (abts) assay. the effects of β-casein and/or resveratrol on the photosensitivity of folic acid, a synthetic form of the b group vitamin known as folates, are also discussed. the data thus gathered provide insight into the protein–polyphenol interaction mechanism and should be useful in the development of β-casein-based carrier systems for the delivery of bioactive molecules. * 1 kcal = 4184 j _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ the β-casein–resveratrol complex 741 experimental materials β-casein from bovine milk (purity ≥ 90 %), resveratrol (trans isomer, >99 %), folic acid (≈98 %) and abts (≈98 %) were purchased from sigma–aldrich (st-louis, mo, usa). potassium persulfate, absolute ethanol, sodium dihydrogen phosphate (nah2po4⋅2h2o) and disodium hydrogen phosphate (na2hpo4⋅12h2o) were obtained from sinopharm (cncm ltd., shanghai, china). sample preparation a stock solution of β-casein was prepared by dissolving the protein in phosphate buffer (10 mm, ph 7.4) to obtain a concentration of 100 μm, absorbance calculated from 280 nm using a molar extinction coefficient of 11000 m-1 cm-1,5 and was stored at 4 °c until use. a stock solution of folic acid (200 μm) was prepared for each experiment in phosphate buffer. stock solution of resveratrol was freshly prepared by dissolving in 75% ethanol at 2 mm and then diluting to 200 μm in phosphate buffer. samples were prepared by blending the stock solutions and phosphate buffer as required in duplicate for the measurements. fluorescence measurements steady-state fluorescence was measured using a fluoromax-4 fluorescence spectrophotometer (horiba jobin yvon inc., edison, nj, usa) equipped with 10-mm quartz cuvettes. the spectral resolution was 2.5 nm for both excitation and emission. the intrinsic fluorescence of the protein was measured at 10 μm in the absence and presence of resveratrol at 2, 5, 10 and 20 μm. emission spectra were obtained by scanning from 295 to 450 nm using an excitation wavelength of 275 nm. buffer and resveratrol backgrounds were subtracted from the raw spectra. fluorescence emission spectra of 20 μm resveratrol were recorded from 330 to 540 nm with an excitation wavelength of 320 nm in the absence and presence of β-casein at 5, 10, 20 and 50 μm. buffer and β-casein backgrounds were subtracted from the raw spectra. fluorescence intensities of 10 μm folic acid at the emission maximum (λmax ≈455 nm) were recorded in the absence and presence of 1 μm resveratrol and/or 1 μm β-casein using an excitation wavelength of 348 nm. buffer, resveratrol and/or β-casein backgrounds were subtracted from the raw spectra. antioxidant activity the antioxidant activities of resveratrol and β-casein were analyzed using the abts radical scavenging assay reported by re et al.17 with minor modifications. in brief, abts and potassium persulfate stock solutions at final concentrations of 7.0 and 2.3 mm, respectively, were prepared, mixed in equal amounts and then kept in a dark at room temperature for 12–16 h to produce abts radical. the abts radical solution was diluted with phosphate buffer (ph 7.4) to an absorbance of about 0.7 at 734 nm. after mixing 2 ml of diluted solution with 160 μl of buffer or sample for 120 seconds, the absorbance was determined using a mapada uv -1200 uv–vis spectrophotometer (shanghai mapada instruments, china) equipped with a 1-cm cell. the total antioxidant activity of the sample was calculated as abts radical scavenging activity using the following equation: c s c abts scavenging 100 a a a = − (1) where ac is the absorbance of the abts radical plus buffer and as is the absorbance of the abts radical plus sample. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 742 cheng et al. absorbance measurements the absorption spectra were recorded with a path length of 1 cm on a cary 50 uv–vis spectrophotometer (varian inc.). the absorption spectra of 10 μm resveratrol were recorded under uv irradiation over the wavelength range of 250–370 nm in the absence and presence of 10 μm β-casein. the buffer and β-casein backgrounds were subtracted from the raw spectra. the absorption spectra of 10 μm β-casein mixtures without and with resveratrol at 0, 2, 5, 10, 20 and 40 μm were recorded from 250 to 450 nm and absorbance spectra of 20 μm resveratrol mixtures without and with β-casein at 0, 5, 10, 20 and 50 μm were recorded from 250 to 540 nm. irradiation procedure samples in closed 10 mm quartz cuvettes were exposed to uv light using an uvl-21 ultraviolet lamp (vwr international inc.) with peak λ of ≈365 nm and with the fluence rate set at 1 mw cm-2. samples were analyzed every 20 min for up to 180 min. an inherent problem of many fluorimetric procedures is the inner filter effect, referring to the absorption of light at the excitation and/or emission wavelengths by dissolved species, including the fluorophore itself. the inner filter effect caused by absorption of both exciting and emitted radiation was corrected using the following equation:18,19 ex em(corr o/ bs) 21 0 a af f+= (2) where fcorr and fobs are, respectively, the corrected and measured fluorescence intensity, aex and aem are, respectively, the measured change in absorbance at the excitation and emission wavelengths. results and discussion absorbance of β-casein–resveratrol mixtures the difference spectra of the absorbance of resveratrol–β-casein mixtures with that of β-casein at polyphenol concentrations of up to 20 μm are shown in fig. 1a. a gradual and significant increase of the maximum concentration between 300 and 320 nm, attributed to resveratrol,15 was observed with increasing polyphenol concentration. the difference spectra of the absorbance of resveratrol–β-casein mixtures with that of resveratrol at protein concentrations of up to 50 μm are presented in fig. 1b. a gradual and significant increase in the absorbance maximum at 280 nm, attributed to β-casein, was observed. influence of β-casein–resveratrol complex formation on the protein structure β-casein contains one tryptophan residue and four tyrosine residues.20 the excitation wavelength at 275 nm was used to minimize the inner-filter effect. fluorescence spectrum of β-casein had a λmax at 335 nm with a shoulder around 300 nm in phosphate buffer at ph 7.4 (fig. 2a). the λmax shifted gradually to longer wavelengths and reached 349 nm as the concentration of resveratrol was increased to 20 μm. a significant decrease was concomitantly observed in the fluorescence intensities attributed to the tryptophan and tyrosine residues. at 20 μm resveratrol, the intensity at λmax was about 59 % of that of pure β-casein. after correction for the inner filter effect, the resveratrol-induced β-casein fluor _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ the β-casein–resveratrol complex 743 fig. 1. a) difference absorption spectra between resveratrol–β-casein and pure β-casein at various concentrations of resveratrol and b) difference absorption spectra between resveratrol–β-casein and pure resveratrol at various concentrations of β-casein. fig. 2. fluorescence emission spectra of βcasein in the absence and presence of resveratrol at various concentrations: a) fobs, the measured fluorescence intensity and b) fcorr, the corrected fluorescence intensity. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 744 cheng et al. escence changes were similar but less pronounced (fig. 2b). at 20 μm resveratrol, the λmax was at around 338 nm and its intensity was about 88 % of that of pure protein, which remained invariable after 24 h (data not shown). these results suggest that the formation of resveratrol–β-casein complexes transferred the protein fluorophores to a more hydrophilic environment.9 in fig. 2b, an isofluorescence point was seen around 364 nm, suggesting the transfer is a two-state process. influence of β-casein–resveratrol complex formation on the polyphenol environment the λmax of resveratrol fluorescence in 75 % ethanol (dashed line in fig. 3a) was about 371 nm, and the intensity was about four times greater than that at λmax of 392 nm in phosphate buffer at ph 7.4. in the buffer, λmax shifted to shorter wavelengths, with a conspicuous increase in intensity as the concentration of β-casein increased. at 50 μm β-casein, the λmax was about 379 nm and the intensity was about 66 % of the value obtained for resveratrol alone in ethanol. after correction for the inner filter effect using eq. (2), fcorr as shown in fig. 3b was basically superimposed with fobs, possibly due to negligible change in the absorbance of resveratrol–β-casein mixtures at λ > 300 nm (fig. 1b). at 10 μm, fig. 3. fluorescence emission spectra of 20 μm resveratrol in ethanol and resveratrol in the presence of 0, 5, 10, 20 and 50 μm β-casein in phosphate buffer at ph 7.4: a) fobs, the measured fluorescence intensity; dashed line represents resveratrol in ethanol; b) fcorr, the corrected fluorescence intensity. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ the β-casein–resveratrol complex 745 the β-casein-caused change in the fluorescence of resveratrol were stable after 24 h (data not shown). the change in the resveratrol fluorescence indicates that formation of a complex with β-casein transfers the polyphenol from the aqueous solution to a more hydrophobic environment. this is consistent with the interaction of resveratrol with β-lactoglobulin, a major protein in milk whey.15 influence of complex formation on the antioxidant activity of the resveratrol–β-casein system abts•+ is a radical cation with a characteristic absorbance maximum at 734 nm, which decreases as the radical is scavenged.17 radical scavenging is an important attribute of antioxidants. resveratrol contains three phenolic hydroxyl groups and thus possesses antioxidant activity. the abts+ radical scavenging capacity of resveratrol increased linearly as the polyphenol concentration increased to 40 μm, as shown in the upper inset of fig. 4. β-casein was also shown to have antioxidant activity (lower inset), which is consistent with reports by aflmajano et al.,21 possibly due to the presence of five phosphate groups, in addition to yet undetermined factors.22 the scavenging capacity of β-casein increased in parabolic fashion with concentration.23 scavenging by resveratrol or β-casein increased for 360 s after mixing with abts solution. the effect of storage time on scavenging by β-casein–resveratrol mixtures was therefore fig. 4. effect of storage time on the abts•+ scavenging activity (% of initial radical concentration) of mixtures of 10 μm β-casein with resveratrol at 10 μm (square) and 20 μm (circle), based on measurements after 120 seconds. upper inset: scavenging measured after 15 (square), 120 (circle) and 360 s (triangle) after mixing with abts, as a function of resveratrol concentration; lower inset: scavenging measured after 15 (square), 55 (circle), 120 (triangle) and 360 s (inverted triangle) as a function of β-casein concentration. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 746 cheng et al. measured after 120 s. mixtures containing 10 and 20 μm resveratrol scavenged, respectively, about 23 and 31 % of the abts•+, equal to the sum of the antioxidant capacities of the individual components, suggesting that the formation of β-casein–resveratrol complexes did not affect the antioxidant activity of either component. moreover, the scavenging capacity of β-casein–resveratrol complexes remained stable after storage for 200 h. masking effects of proteins on the antioxidant activity of polyphenols have been widely reported. the formation of complexes with β-lactoglobulin decreased the antioxidant activity of epigallocatechin 3-gallate, the most active polyphenol present in green tea.24,25 bovine serum albumin and caseins were shown to reduce the total antioxidant capacity of protein–flavonoid systems, β-casein and serum albumin more so than κ-casein and α-casein.26 however, the present β-casein–resveratrol system, in which the amount of bound resveratrol was about 20 % of the total, retained the total antioxidant capacity. this might be due to two possibilities: 1) the antioxidant portions of the molecules are not involved in the binding; 2) β-casein–resveratrol complexes dissociate to re-establish the equilibrium concentrations once they were diluted during the measurements of antioxidant activity. influence of β-casein–resveratrol on resveratrol and folic acid photosensitivity resveratrol exists naturally as the trans isomer, which is believed responsible for most of its biological activity. trans-resveratrol shows a broad absorbance maximum between 304 and 319 nm (fig. 5). under exposure to uv light, the absorbance in this band decreases, with a blue shift proportional to exposure fig. 5. absorbance spectra of resveratrol (solid line) and the difference (dashed line) between resveratrol–β-casein and pure β-casein absorption spectra, after uv irradiation for 0, 10, 20, 40 and 180 min (a–e). _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ the β-casein–resveratrol complex 747 time. after 180 min, it appears as a weaker peak around 289 nm, characteristic of cis-resveratrol,27 which is found in grape wines.28,29 trans–cis isomerization of resveratrol under daylight is slower, taking about 21–30 h,15 indicating that uv irradiation accelerates this process. among the reported biological activities of cis-resveratrol are antioxidant activity, inhibition of collagen-induced platelet aggregation, and cancer-related kinase activities,30–32 although it is unclear whether the cis isomer is as active as the trans isomer. no significant change in the absorption spectra of resveratrol was obtained by adding 20 μm β-casein (fig. 5), suggesting that the formation of a complex with this protein did not affect the chemical structure of the polyphenol. the fluorescence of folic acid (due to the pterin moiety) has an extremely low quantum yield. its intensity measured at λmax ≈ 450 nm is almost negligible, as shown in fig. 6. exposure to uv radiation caused a marked increase in the intensity, indicating degradation of folic acid.33,34 addition of β-casein reduced the photo-induced change in the intensity significantly, as observed previously.11 it is noteworthy that the photo-induced change was completely inhibited in the presence of resveratrol or of β-casein with resveratrol. these results indicate that interaction between β-casein and resveratrol did not affect the ability of either to protect folic acid against photodegradation. this is consistent with a previous study on the interaction between β-lactoglobulin and resveratrol. β-lactoglobulin could interact simultaneously with resveratrol and folic acid to form protein multi-ligand complexes.35 it was reported that β-casein could interact with folic fig. 6. fluorescence intensity at λmax of 10 μm folic acid alone (square) and of folic acid in the presence of 1 μm β-casein (circle), 1 μm resveratrol (triangle) and both β-casein and resveratrol (inverted triangle) as a function of uv irradiation time. inset: the fluorescence spectra of folic acid alone at various irradiation times. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 748 cheng et al. acid to form complexes with an affinity of about 7.0×104 m–1.8,11 therefore, the lack of an effect of β-casein–resveratrol complexation on the degradation of folic acid might be attributed to the binding capacity of both folic acid and resveratrol to β-casein. in addition, together with the photo-induced isomerization of the polyphenol in the absence and presence of β-casein (fig. 5), it is suggested that the effect of resveratrol on folic acid decomposition might be attributed to its structural transition. conclusions the interaction between resveratrol and β-casein shifted the protein fluorophore residues to a more hydrophilic environment, causing a decrease in protein fluorescence. the interaction shifted resveratrol to a more hydrophobic environment, leading to an increase in the polyphnol fluorescence. the formation of a complex with β-casein did not affect the isomerization of resveratrol or the total antioxidant activity of this protein–polyphenol system. the protective effect of resveratrol against the photodecomposition of folic acid was not affected by binding to β-casein. the data obtained in this study should provide insight into protein–polyphenol interaction mechanisms and aid in the development of β-casein-based carrier systems for the delivery of bioactive molecules. acknowledgments. this work received support from the national natural science foundation of china (nsfc project 31201291 and 31571781). the authors thank dr. van de weert (department of pharmaceutics and analytical chemistry, university of copenhagen) for help with the comments about the inner filter effect. и з в о д комплекс β-казеина и ресвератрола: физичкохемијске карактеристике и значај за унапређење исхране hao cheng1, yanfang li1, xundi yin2, muriel subirade3 и li liang1 1state key lab of food science and technology, school of food science and technology, jiangnan university, wuxi, jiangsu, china, 2college of textiles & clothing, jiangnan university, wuxi, jiangsu, china и 3chaire de recherche du canada sur les protéines, les bio-systèmes et les aliments fonctionnels, institut de recherche sur les nutraceutiques et les aliments fonctionnels (inaf/stela), université laval, québec, canada протеини хране су у широкој употреби као носачи за инкапсулацију и заштиту биоактивних молекула. за развој система који се заснивају на протеинским носачима, важно је разумети механизам интеракције протеина и биоактивних молекула. у овом раду је изучавана интеракција β-казеина са ресвератролом, природним полифенолом, користећи методе uv–vis апсорпционе и флуоресцентне спектроскопије. показано је да интеракција помера протеинске флуорофоре према хидрофилнијој средини, а полифенол ка хидрофобнијој околини. стварање комплекса са β-казеином није утицало на trans–cis изомеризацију ресвератрола или на укупну антиоксидативну активност система протеин–полифенол, што је утврђено спектрофотометријски и тестом са 2,2'-азинобис(3-етилбензотиазолин-6-сулфонском киселином). заштитни ефекат ресвератрола на фотодеградацију фолне киселине није био измењен услед његовог везивања за β-казеин. добијени резултати омогућавају сагледавање механизма интеракције про_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ the β-casein–resveratrol complex 749 теин–полифенол и могу помоћи у развоју система који се заснивају на β-казеину као носачу биоактивних молекула. 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1156 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 750 cheng et al. 33. m. k. off, a. e. steindal, a. c. porojnicu, a. juzeniene, a. vorobey, a. johnsson, j. moan, j. photochem. photobiol., b 80 (2005) 47 34. p. vorobey, a. e. steindal, m. k. off, a. vorobey, j. moan, photochem. photobiol. 82 (2006) 817 35. j. zhang, x. m. liu, m. subirade, p. zhou, l. liang, food chem. 165 (2014) 256. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice sepiolite functionalized with n-[3-(trimethoxysilyl)propyl]-ethylenediamine triacetic acid trisodium salt. part i: preparation and characterization j. serb. chem. soc. 80 (9) 1193–1202 (2015) udc 549.623.83.004.12+547.292+ jscs–4790 546.28–36:543.44–77 original scientific paper 1193 sepiolite functionalized with n-[3-(trimethoxysilyl)propyl]ethylenediamine triacetic acid trisodium salt. part i: preparation and characterization slavica s. lazarević*, ivona m. janković-častvan, bojan m. jokić, djordje t. janaćković and rada d. petrović faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11000 belgrade, serbia (received 19 february, revised 11 may, accepted 14 may 2015) abstract: natural sepiolite from andrici (serbia) was functionalized by covalent grafting of n-[3-(trimethoxysilyl)propyl]ethylenediamine triacetic acid trisodium salt onto the si–oh sepiolite groups. the functionalized material, mseas, was characterized by determination of the phase composition by xray diffraction (xrd) analysis, analysis of the morphological characteristics by scanning electron microscopy (sem), using fourier transform infrared (ftir) spectroscopy, differential thermal analyses (dta), determination of the specific surface areas and pore size distribution using the bet method and point of zero charge (phpzc) determination. the crystal structure of sepiolite did not change significantly upon surface modification. the ft-ir and dta analyses confirmed that the modified sample maintained the basic structure of sepiolite and the presence of organic groups in the functionalized sepiolite sample. the point of zero charge of mseas in kno3 solutions of different concentrations, determined by the batch technique, was at ph 7.0±0.1. keywords: sepiolite; functionalization; n-[3-(trimethoxysilyl)propyl]ethylenediamine triacetic acid, trisodium salt. introduction the variety of possible reactions of the active centres on the surface of clays allows the surface properties to be changed by introduction of new groups of atoms. grafting is a process that links inorganic and organic components via strong bonds, such as covalent or ionic–covalent linkages, to obtain functionalized clays, i.e., the formation organic–inorganic hybrid materials combining the mechanical stability of a clay framework and the reactivity of organofunctional groups. the alkoxysilane part of an organosilane molecule is capable of bonding * corresponding author. e-mail: slazarevic@tmf.bg.ac.rs doi: 10.2298/jsc150219038l _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1194 lazarević et al. with a variety of mineral or metal surfaces through complex hydrolysis/condensation reactions, whereby si–o–metal bonds are ultimately formed.1 the reactions of clay minerals with reagents containing metal chelating functionalities have recently been explored in an effort to enhance the heavy metal binding capacities of clays and their selectivity to the type of metal considered.2–9 the surface of sepiolite has a great ability for grafting reactions with organosilanes due to its high content of silanol groups that are very susceptible to the reactions.10 by hydrolysis, the alkoxy-groups of the organosilane (r–six3, where x designates hydrolysable alkoxy groups (usually methoxy, –och3, or ethoxy, –oc2h5)) are converted to silanol groups, which react with silanol groups of sepiolite forming si–o–si covalent bonds.11 the most widely used functional silanes contain thiol (–sh) or amino (–nh2) groups. the surface of sepiolite was previously functionalized using silane coupling agents: 3-mercaptopropyltrimethoxysilane (mps),2 triethoxy-3-(2-imidazolin-1-yl)propylsilane,12 [3-(2-aminoethylamino)propyl]trimethoxy-silane).13 in previous papers, natural, acid activated and thermo-acid activated sepiolites were functionalized using (3-mercaptopropyl)trimethoxy-silane and [3-(2 -aminoethylamino)propyl]trimethoxysilane and the capacities of the obtained sorbents for chromium(vi) sorption from aqueous solutions were investigated.9,14,15 it was shown that adsorption capacities of the amine-functionalized sepiolites were much higher than those of mercapto-silane functionalized sepiolites under the same conditions, indicating that adsorption capacities of functionalized sepiolites depended on the type of groups covalently grafted to the sepiolite surface. the present study is the first to investigate the modification of sepiolite with n-[3-(trimethoxysilyl)propyl]ethylenediamine triacetic acid trisodium salt in order to improve sorption capacity for metal ions from aqueous solutions. the silanation reagent n-[(3-trimethoxysilyl)propyl]ethylenediamine triacetic acid contains three methoxy groups, which could react with the oh groups on clays, and an edta group, which could chelate metals. this paper reports the preparation of the modified material and its characterization by xrd analysis, scanning electron microscopy (sem), fourier transform infrared (ftir) spectroscopy, differential thermal analyses (dta), surface-area analysis and determination of the point of zero charge (phpzc). in the second part of this paper, the adsorption properties of the functionalized sepiolite sample, the sorption of ni2+ on the mseas as a function of the initial metal concentration, the equilibration time, the ph value and temperature are discussed. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ preparation and characterization of functionalized sepiolite 1195 experimental materials the natural sepiolite (sep) used for the modification was obtained from andrici (serbia). the chemical composition, specific surface area, pore volume, pore radius, x-ray diffraction, and ftir analyses of the sample were reported previously.16 the functionalization of sepiolite was performed from aqueous solution6,17 by mixing a mechanically stirred suspension of 50 g of sepiolite in distilled water, ph of 7.1, with 22.5 ml of a 45 % aqueous solution of n-[3-(trimethoxysilyl)propyl]ethylenediamine triacetic acid trisodium salt ((ch3o)3si(ch2)3n(ch2coona)n(ch2)2n(ch2coona)2), msea, gelest). the mixture was filtered after 2 h and the modified sepiolite sample, mseas, was washed with water. characterization of the modified sepiolite the scanning electron microscopic (sem) analysis of the sepiolite powder was realized on a tescan mira 3 microscope. the powder was fused with a pd–pt alloy. x-ray diffraction (xrd) analysis of the sample was performed using an ital structures apd 2000 diffractometer with cukα radiation, in the 2θ angle range from 5 to 60°, with a 0.02° step. infrared spectroscopy analysis was made on a mb boman hartmann 100 instrument in the wave number range from 4000 to 400 cm-1. the sample was prepared by the kbr method at a ratio of sample:kbr of 1:75. differential thermal analysis in air was realized using an aminco instrument with computer-controlled temperature, at a heating rate of 10 °c min-1. the specific surface areas and pore size distribution of the modified sample was estimated using nitrogen adsorption–desorption isotherms determined using a micrometrics asap 2020 instrument. before the sorption measurement, the sample was degassed at 150 °c for 10 h under reduced pressure. the specific surface area of sample (sbet) was calculated according to the brunauer–emmett–teller (bet) method from the linear part of the nitrogen adsorption isotherm.18 the total pore volume (vtot) was given at p/p0 = 0.998. the volume of the mesopores and pore size distribution were analyzed according to the barrett, joyner and halenda method from the desorption isotherm.19 the volume of the microspores was calculated according to t-plot analysis20 using the harkins–jura thickness curve. the point of zero charge (phpzc) of the modified sepiolite was determined in kno3 solutions having concentration 0.1, 0.01 and 0.001 mol l-1, using the batch equilibration method as described previously.21 in order to determine the degree of dissolution of the modified sepiolite powder, the concentration of mg2+ in the solutions after equilibration with a 0.01 mol l-1 kno3 solution was measured by atomic absorption spectroscopy (aas, perkin elmer 730). results and discussion characterization of modified sepiolite the characteristic peak positions in the xrd spectrum of the sepiolite sample had not changed after modification with n-[3-(trimethoxysilyl)propyl]ethylenediamine triacetic acid trisodium salt (fig. 1), indicating that the structure and crystallinity of sepiolite were maintained, which could be attributed to the functionalization occurring mainly on the surface or by the partial replacement of zeolitic water. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1196 lazarević et al. fig. 1. xrd spectrum of the mseas sample (s – sepiolite). the sem micrograph of mseas is shown in fig. 2. it could be seen that the modified sepiolite had a fibrous structure, as did the natural sepiolite.22 the micrographs of natural sepiolite sample showed the fibres had needle morphology of 30–50 nm in diameter and 1 μm in length. after surface modification, the needle morphology was maintained but with reduced fibre length. the fibres of the functionalized sample formed bundle-like aggregates. fig. 2. sem micrograph of the modified sepiolite. the ftir spectrum of the modified sample (fig. 3) was generally similar to that of natural sepiolite, confirming that the modified material had maintained the basic structure of sepiolite. three regions indicative for sepiolite23,24 could be observed in fig. 3: bands in the 4000–3000 cm–1 range corresponding to the vibrations of the mg–oh group (3690 cm–1), bound water coordinated to magnesium in the octahedral sheet (3570 cm–1) and zeolitic water in the channels (at 3422 cm–1); a band at 1658 cm–1 due to the vibration of zeolitic water; bands in _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ preparation and characterization of functionalized sepiolite 1197 the 1200–400 cm–1 range characteristic of silicate: bands centred at 1016 and 467 cm–1 due to si–o–si vibration; bands at 1215 and 1078 cm–1 due to si–o bonds; a band at 437 cm–1 originating from octahedral–tetrahedral bonds (si–o–mg bonds) and bands at 690 and 637 cm–1 corresponding to vibrations of the mg–oh bond. the band at 1381 cm–1 corresponds to vibrations of c–h of the ch2 groups. the c–h stretching vibrations of the methoxy (o–ch3) and chain methylene (ch2) groups could be observed at 2850 and 2930 cm–1, respectively.9,13 a band, assigned to symmetric coo– stretching vibrations,25 could be clearly observed at 1407 cm–1. the detected ch2 and cooh groups belong to the units of the msea, clearly indicating the presence of the organic modifier in the sample. fig. 3. ft-ir spectrum of the mseas sample. the dta curve of the modified sepiolite, shown in fig. 4, revealed the decomposition of the aminocarboxylic group on the surfaces of mseas and dehydration of the sepiolite structure. the endothermic peak represents the loss fig. 4. dta curve of the mseas sample. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1198 lazarević et al. of zeolite water (at 150 °c),26 the exothermic peak at 815 °c represents the dehydratation of the octahedrically coordinated hydroxyl groups, i.e., phase transformation of sepiolite into enstatite (mgsio3). the broad exothermic peak at ≈329 °c reveals the decomposition of the grafted silane ligand and clearly proves the presence of organic silane molecules on the modified material. textural properties of modified sepiolite adsorption–desorption isotherms at −196 °c for mseas, and the pore volume and pore size distribution are presented in fig. 5a and b. the mseas showsa hysteresis pattern which is associated with the filling and emptying of the mesopores by capillary condensation, but does not show a plateau at high p/p0 values, which is characteristic of a type iv isotherm. the hysteresis loop is of type h3. according to the classification of rouquerol et al.,18 such a shape of a nitrogen fig. 5. a) nitrogen adsorption/desorption isotherms; b) pore volume (1) and pore size distribution (2) of the mseas. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ preparation and characterization of functionalized sepiolite 1199 isotherm is characteristic for type iib indicating that the material contained both mesopores, which are responsible for the hysteresis, and macropores, which results in the absence of the plateau typical for mesoporous type iv isotherms. the h3 hysteresis pattern indicates the presence of slit-like pores.27 from fig. 5 and the data summarized in table i (bet surface area, volume of mesopores, vmesopore, and micropores, vmicropore, the overall pore volume, vporetotal, and the maximum, dmax, and average, d, mesopore diameters) and data obtained for a natural sepiolite,16 it could be noticed that the modification of the sepiolite with msea caused a small change in the specific surface area, an increase in the total pore volume, the micropore volume and the mesopore volume. increases in the maximum and average mesopore diameters were observed for the modified sepiolite sample. it could be assumed that silane modification occurred almost exclusively on the external sepiolite surface, with only partial entry into the sepiolite channels, i.e., that the pores of the sepiolite were not closed during the functionalization. table i. the textural properties of the mseas sbet / m2 g-1 vporetotal / cm3 g-1 vmicropore / cm3 g-1 vmesopore / cm3 g-1 dmax / nm d / nm 309.9 0.374 0.054 0.325 4.00 6.51 determination of the point of zero charge of the modified sepiolite the point of zero charge (phpzc) is an important interfacial parameter, extensively used in characterizing the ionization behaviour of a surface. it is very important for the determination of the acid–base characteristics of the surface functional groups and their interactions with ions from aqueous solutions. the surface potential exists as a direct result of the presence of surface charge. the solution ph value at which the surface charge density of the solid phase is equal to zero (σ0 = 0, ψ0 = 0), is called the point of zero charge (phpzc). the solid phase surface is positively charged if ph < phpzc and negatively if ph > phpzc. the point of zero charge of mseas in kno3 solutions of different concentrations, determined by the batch technique from the plateaus, i.e., the curve inflexions, from the dependences phf vs. phi, (fig. 6) was at ph 7.0±0.1. the phpzc determined in kno3 solutions of different concentrations were independent of the ionic strength of the kno3 solutions, indicating that kno3 was an inert electrolyte, i.e., specific sorption of k+ and no3– did not occur at the surface. the slight change in the point of zero charge of mseas compared with the phpzc of the natural sepiolite sample (7.4±0.1)16 indicates an insignificant decrease in the basicity of the sepiolite surface after functionalization. the dependence of the quantity of mg2+ released into the solution during equilibration of 0.05 g of the mseas with 25 cm3 0.01 mol l–1 kno3 solution _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1200 lazarević et al. is also shown in fig. 6 in order to determine the degree of dissolution of the modified sepiolite powder. in the investigated ph range, the quantity of mg2+ present in the solution as a result of the dissolution of sepiolite was almost constant and equal to 0.05 mmol g–1 mseas. fig. 6. a) determination of the phpzc of mseas in kno3 solutions of different concentrations; b) dependence of the quantity of mg2+ released into the solution per unit mass of mseas on phi during equilibration with 0.01 mol l-1 kno3 solutions. conclusions the obtained results showed that natural sepiolite from the andrici deposit could be modified by treating with n-[3-(trimethoxysilyl)propyl]ethylenediamine triacetic acid trisodium salt in the presence of an aqueous solution. the surface modification of the sepiolite involved chemical reaction of the silanol groups on the sepiolite surface with the alkoxy groups of the organosilane molecule. the signs of modification of the sepiolite surface were: the presence of new exothermic peaks in the dta spectrum for modified sepiolite, the detection of carboxylic functional groups present on modified sepiolite by ftir spectroscopy. the point of zero charge of mseas in kno3 solutions of different concentrations, determined by the batch technique, was ph 7.0±0.1. acknowledgements. financial support through the ministry of education, science and technological development of the republic of serbia, project no. iii 45019, and fp7 nanotech ftm no. 245916 is gratefully acknowledged. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ preparation and characterization of functionalized sepiolite 1201 и з в о д функционализација сепиолита применом соли натријума n-[(3-триметокси)пропил]етилендиаминтриацетатне киселине. први део: припрема и карактеризација славица с. лазаревић, ивона м. јанковић-частван, бојан м. јокић, ђорђе т. јанаћковић и рада д. петровић технолошко-металуршки факултет универзитета у београду, карнегијева 4, 11000 београд природни сепиолит из налазишта андрићи је функционализован применом соли натријума n-[(3-триметокси)пропил]етилендиаминтриацетатне киселине успостављањем ковалентне везе између модификатора и si–oh површинских група сепиолита. карактеризација функционализованог узорка означеног са mseas извршена је одређивањем морфолошких карактеристика применом скенирајуће електронске микроскопије (sem), одређивањем фазног састава рендгенско-дифракционом анализом (xrd) и применом инфрацрвене спектроскопске анализе (ft-ir) и диференцијално-термијске анализе (dta), као и одређивањем специфичне површине и расподеле величина пора bet методом и тачке нултог наелектрисања (phpzc). кристална структура сепиолита није битно промењена поступком функционализације. резултати ft-ir и dta анализе потврдили су присуство основне сепиолитске структуре у узорку mseas, као и присуство карбоксилних група органског модификатора. тачка нултог наелектрисања узорка mseas, одређена у растворима kno3 различитих концентрација применом методе уравнотежавања посебних проба, износи 7,0±0,1. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice alkylating ability of carbohydrate oxetanes: practical synthesis of bolaform skeleton derivatives j. serb. chem. soc. 80 (10) 1273–1278 (2015) udc 547.451.63’454+66.095.253:547.415.1 jscs–4795 original scientific paper 1273 alkylating ability of carbohydrate oxetanes: practical synthesis of bolaform skeleton derivatives pavle a. hadžić1*#, mirjana m. popsavin2# and sunčica z. borozan3# 1goša institute, milana rakića 35, 11000 belgrade, serbia, 2faculty of sciences, department of chemistry, university of novi sad, trg d. obradovića 3, 21000 novi sad, serbia and 3faculty of veterinary medicine, university of belgrade, bulevar oslobođenja 18, 11000 belgrade, serbia (received 24 february, revised 30 march, accepted 16 april 2015) abstract: the alkylating ability of the oxetane ring in the carbohydrate structure was investigated and a flexible method for the construction of a bolaform amphiplile skeleton with xylose as the polar head is proposed. the method is based on oxetane ring opening in the easily accessible 3,5-anhydro-1,2-o-cyclohexylidenexylofuranose (1). one-step nitrogen alkylation in terminal diamines with 1 gave the basic cationic bolaform skeleton with xylose as potential polar head and a deliberately chosen length of the non-polar spacer. under similar experimental conditions, but with an appropriate mole ratio of the alkylating agent, the alkylation reaction provide for selective monoalkylation of the diamines. successful alkylation in the xanthine series (theophylline) was also achieved with 1, giving a new 5-deoxy-5-(1,2,3,6-tetrahydro-1,3-dimethyl -2,6-dioxo-7h-purin-7-yl)-α-d-xylofuranose derivative. keywords: bolaform skeleton; oxetane ring; alkylation; ring opening. introduction oxiranes and oxetanes, as highly strained heterocycles, are archetypical alkylating agents. oxetanes are widely used in organic syntheses and are considered to be less carcinogenic and lack genotoxic capacity compared to epoxides and β-lactones.1 the alkylating potential of the oxetane ring is a consequence of its ring strain, which was determined to be 106 kj mol–1, a value close to that of oxirane (112 kj mol–1).2 however, the ring-opening reactions of oxetanes usually require powerful acid catalysts, or other specific catalysts providing for particular synthetic features.3 the oxetane ring is for steric reasons rarely encountered in carbohydrates. when present, it frequently possesses interesting pharmaceutical properties and offers numerous possibilities for chemical transformations.4 in * corresponding author. e-mail: pavle.hadzic@institutgosa.rs # serbian chemical society member. doi: 10.2298/jsc150224033h _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1274 hadžić, popsavin and borozan this report, the high alkylation potential of the oxetane ring in the easily accessible 3,5-anhydro-1,2-o-cyclohexylidene-α-d-xylofuranose5 (1) toward the nitrogen in aliphatic diamines and in xanthine series (scheme 1) is presented. o o o o 1 o o o oh hnch2 ch2nh 2 4 o oo oh o o o oh rhn 5 8 9 n n n n o o ch3 ch3 o o ooh scheme 1. alkylation ability of 3,5-anhydro-1,2-o-cyclohexylidene-α-d-xylofuranose (1). its use for basic bolaform skeleton synthesis (2–4, the graphical curve represent the hydrophobic spacer), selective monoalkylation of diamines (5–8), and alkylation in the xanthine series 9. from the practical standpoint, the alkylation of aliphatic diamines leads to one-step construction of basic cationic bolaform detergent skeleton, while in the xanthine series, a new type of 7-xylose substituted theophylline derivative was obtained under mild and operationally simple reaction conditions. all alkylations were performed under solvent-free reaction conditions, additionally illustrating alkylation potential of the carbohydrate oxetane ring. experimental thermal solid-state alkylation reactions are classical examples among solvent-free reactions, and not a new concept. in particular, the n-alkylation of phthalimides and heterocyclic systems, such as carbazole with alkyl halides, are known examples.6 unexpectedly, the high inclination of the oxetane structure in 1 toward nitrogen alkylation in this investigation enabled the realization of solvent-free reactions, giving benefits in product isolation procedures. general reaction conditions. 1 and a diamine (1,2-diaminoethane, 1,4-diaminobutane, piperazine, n-methylpiperazine or theophylline from commercial suppliers and without further purification) were mixed in the appropriate mole ratio for alkylation of both nitrogen atoms, or selective alkylation of a single nitrogen in diamine compounds, with addition of water as catalyst. the reaction mixture was held in a hermetically sealed stainless steel reaction flask at temperature 110–130 °c for 12 h with occasional mixing. the products were isolated either as free bases, hydrochloride or oxalate salts (table i). detailed experimental synthetic procedures and analytical data confirming the structures of the newly synthesized compounds are given in supplementary material to this paper. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ alkylation with oxetane 1275 table i. products of alkylation of diamines and alkylation in xanthine series with a carbohydrate oxetane ring cmpd. alkylation position compound name 2 –hnch2ch2nh– 5,5′-(1,2-ethanediyldiimino)bis[1,2-o-cyclohexylidene-5-deoxy-α-d-xylofuranose] 3 –hnch2(ch2)2ch2nh– 5,5′-(1,4-butnediyldiimino)bis[1,2-o-cyclohexylidene-5-deoxy-α-d-xylofuranose] 4 –n(ch2ch2)2n– 5,5′-(1,4-piperazinediyl)bis[1,2-o-cyclohexylidene -5-deoxy-α-d-xylofuranose] 5 h2nch2ch2nh– 5-((2-aminoethyl)amino)-1,2-o-cyclohexylidene-5 -deoxy-α-d-xylofuranose dioxalate 6 h2nch2(ch2)2ch2nh– 5-((4-aminobutyl)amino)-1,2-o-cyclohexylidene -5-deoxy-α-d-xylofuranose dioxalate 7 hn(ch2ch2)2n– 1,2-o-cyclohexylidene-5-deoxy-5-(1-piperazinyl) -α-d-xylofuranose 8 ch3n(ch2ch2)2n– 1,2-o-cyclohexylidene-5-deoxy-5-(4-methylpiperazin-1-yl)-α-d-xylofuranose 9 n n no o ch3 ch3 n 1,2-o-cyclohexylidene-5-deoxy-5-(1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7h-purin-7-yl) -α-d-xylofuranose results and discussion amphiphiles with two polar heads connected by a hydrophobic spacer (bolaamphiphiles) have attracted ever-increasing attention since similar structures were found in some archaebacteria cell membranes that sustain harsh environments, high salt concentrations and elevated ambient temperatures.7 a range of bolaamphiphiles have been synthesized so far, either to imitate components of a natural cell structure, or to investigate their lipophilic–hydrophilic properties.8 in attempts to realize specific properties, a variety of bolaform surfactants with a carbohydrate as the hydrophilic moiety have also been considered. carbohydratebased detergents are recognized, and convenient syntheses of compounds with variable lipophilic–hydrophilic balance based on xylose, in particular, as the polar head, or from other carbohydrate but with preserved xylo-configuration, are known.9 specifically, syntheses of bolaamphiphiles based on xylose are also described.10 the authors affirmed earlier findings11 on the significant impact of the length of the hydrophobic linkage (spacer) on the aggregation properties of bolaamphiphiles with sugar heads. consequently, a simple synthetic method for the construction of the basic bolaform skeleton with xylose-based hydrophilic heads and deliberately preferred hydrophobic spacer length is intriguing. herein, the alkylation ability of the oxetane ring in 1 toward terminal diamino compounds as a simple, practical and flexible method for the construction of the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1276 hadžić, popsavin and borozan cationic bolaform skeleton, or selective monoalkylation of aliphatic diamines and natural products are reported. the opening of oxetane ring in 3,5-anhydro-derivatives of suitably protected12 xylofuranose derivatives with aqueous ammonia13 and amines14 was investigated earlier. the reaction leads to one-step nitrogen alkylation. under simple reaction conditions, the one-step dialkylation of α,ω-diamines of the general formula h2n–(ch2)n–nh2, or cyclic diamines (piperazine) with 1 lead smoothly to the basic bolaform skeleton 2–4. for further demonstration of akylating ability of carbohydrate oxetane 1, the alkylation of diamines accomplished under essentially the same experimental conditions but with a different mole ratio of 1 toward the diamines resulted in selective monoalkylation of diamines 5–8. frequently, prior activation of oxirane and oxetane rings is a necessary step in alkylation reactions and different catalysts are used for this purpose. protonation of oxygen in oxetane ring significantly eases nucleophillic attack of nucleophillic species present and hence, the ring opening is facilitated by acids. as demonstrated earlier, water could also play the role of the catalyst,15 and the present experiments afforded further experimental evidence on the subject. particularly, water was found to be an appropriate catalyst for the alkylation tendency of 1, owing to the expected significant strain energy release in the system of three condensed small rings in 1. our previous investigations of the reactivity of the 3,5-anhydro ring in 1 towards selected nucleophiles demonstrated pronounced regioselectivity of the oxetane ring-opening process.16 again, it was found to be the case in the reaction of 1 with diamines. the only isolable products originated from the regioselective nucleophillic attack of nitrogen on the terminal carbon atom of xylofuranose (c-5). no detectable products of inversion at the c-3 carbon of starting 1 were found because of the hypothetic nucleophilic attack at c-3, i.e., the xyloconfiguration was preserved throughout. to prove further the alkylating ability of the oxetane ring in the carbohydrate structure 1, the alkylation of some natural products was considered. many nitrogen-containing heterocycles demonstrate pronounced pharmaceutical activity. among them, xanthine and substituted xanthine derivatives are an important class of compounds with a well-known spectrum of activity.17 in particular, theophylline pentosides are the subject of intensive synthetic interest because of their structural resemblance to nucleosides. theophylline-7-riboside was evaluated as a partial agonist for ubiquitous adenosine receptors18 with the conclusion that it has characteristics between full agonists and full antagonists (xanthines). following thee findings, further attempts were made to synthesize different 7-substituted xanthine derivatives with a modified ribose rest.19 to the best of our knowledge, among other theophylline 7-pentofuranosides, arabinoside and xyloside are also known.20 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ alkylation with oxetane 1277 in further experiments, it was found that alkylating potential of the oxetane ring in 1 could be used for the facile synthesis of a 7-xylose substituted theophylline derivative, however with a new pentose substitution pattern. namely, all previously synthesized pentose derivatives were connected to theophylline through c1 xylose carbon. thus, the alkylation of theophylline with 1 smoothly gave 5-deoxy-5 -(1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7h-purin-7-yl)-α-d-xylofuranose derivative 9. however, theophylline did not react under the standard procedure (heating a mixture of diamine, oxetane 1 and water as catalyst), but further activation of the 7′-theophylline position was necessary, and hence the potassium salt of theophylline was prepared.21 conclusions the use of protected 3,5-anhydroxylofuranose as alkylating agent represent a new practical and adaptive method to reach the basic cationic bolaform skeleton with xylose-based polar heads and variable lengths of non polar spacer. the described one-step synthetic procedure give products that could be easily converted into carbohydrate polar heads with retained xylo-configuration, with deliberately chosen hydrophobic spacer length and in acceptable isolated yields. in the xanthine series, the alkylation provide for new 7-xylose substituted theophylline with the substitution pattern reversed to that previously described. supplementary material description of specific synthesis methods and the analytical and spectral data of the synthesized compounds are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. и з в о д алкилујући потенцијал угљенохидратних оксетана: практична синтеза деривата болаформног скелета павле а. хаџић1, мирјана м. попсавин2 и сунчица з. борозан3 1институт гоша, милана ракића 35, 11000 београд, 2природно–математички факултет, департман за хемију, универзитет у новом саду, трг д. обрадовића 3, 21000 нови сад и 3факултет ветеринарске медицине, универзитет у београду, булевар ослобођења 18, 11000 београд истражена је алкилујућа способност оксетанског прстена у структури угљених хидрата и предложена је флексибилна метода за синтезу скелета болаформних амфифила са ксилозом као поларном главом. метода је заснована на реакцији отварања оксетанског прстена код лако достуне 3,5-анхидро-1,2-о-циклохексилиден ксилофуранозе (1). алкиловање на атому азота код терминалних диамина са 1 даје основни болаформни скелет са заштићенoм ксилозом као потенцијалном хидрофилном главом и по вољи изабраном дужином хидрофобног тела. у сличним реакционим условима, али са другачијим одговарајућим молским односом алкилујућег агенса, остварено је селективно моноалкиловање диамина. такође, остварено је успешно алкиловање у структури ксантина, при чему је добијен раније неописани дериват ксантина 1,2-о-циклохекси_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1278 hadžić, popsavin and borozan лиден-5-деокси-5-(1,2,3,6-тетрахидро-1,3-диметил-2,6-диоксо-7h-пурин-7-ил)-α-d-ксилофураноза. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice accepted manuscript this is an early electronic version of an as-received manuscript that hasbeen accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as a. eren, f. m. bekler and k. güven, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc230707053e this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230707053e j. serb. chem. soc.00(0)1-15 (2023) original scientific paper jscs–12469 published dd mm, 2023 1 pcr-based detection of alkane monooxygenase genes in the hydrocarbon and crude oil-degrading acinetobacter strains from petroleum-contaminated soils ayşe eren, fatma matpan bekler and kemal güven * department of molecular biology and genetics, faculty of science, dicle university, 21280 diyarbakır, turkey (received 7 july; revised 8 august; accepted 18 august 2023) abstract: bacterial strains d11, e1 and e2 isolated from petroleum-contaminated soils were found to be members of acinetobacter genus revealed by 16s rrna gene sequence analysis and phenotypic characteristics. after incubation for 5 days, about 43%, 9% and 12% of total petroleum hydrocarbons of crude oil were degraded by strains d11, e1 and e2, respectively, determined by gc-ms analysis. moreover, about 70% and 76% of single hydrocarbon hexadecane was degraded by the strains d11 and e1 after 3 days of short incubation time, respectively, while the strain e2 degraded about 48% of single hydrocarbon pentadecane. by using pcr-based method, gene sequences of the strains d11 and e2 were shown similarity to alkane 1-monooxygenases from acinetobacter sp. buu8 alkm with 93.06% and 92.72%, respectively, while the sequence similarity of strain e1 was 95.84% to acinetobacter sp.826659. the present study of hydrocarbon biodegradation by acinetobacter strains may provide a good advantage in bioremediation process. keywords: petroleum-degradation; bacteria; gc-ms analysis; alkm gene introduction there is a continuous great concern on petroleum hydrocarbons causing pollution in the environment, produced from various anthropogenic activities, such as accidental petroleum spills during transport and refining of oil products, industrial discharge and leakage from underground tanks.1-2 toxic oil products are known to pose a serious threat to marine habitats, fishery and human health, leading to an imbalance in the ecosystem and thus harming the wildlife which takes years or even decades to recover.3-4 aliphatic n-alkanes, formed by the reduction of organic material during the geochemical formation of petroleum, are hydrocarbons found in crude and refined oils.5 *corresponding author. e-mail: kemalg@dicle.edu.tr https://doi.org/10.2298/jsc230707053e a cc ep te d m an us cr ip t mailto:kemalg@dicle.edu.tr https://doi.org/10.2298/jsc230707053e 2 eren et al. a greater amount of aliphatic alkanes is released into the environment and thus the bioavailability and toxicity of n-alkanes vary according to the chain length.6 since crude oil and its products are biodegradable, bioremediation for cleaning up oil-contaminated sites is considered as an efficient and cost-effective technique compared to chemical and physical treatments. recently, bioremediation of oil-contaminated sites has been an important task for scientists, carrying out many studies using pure culture or mixed bacterial consortia which degrade petroleum hydrocarbons.4,7-8 in the environmental biotechnology using biodegradation and bioremediation processes, various kinds of microorganisms including bacteria and fungi have been employed.9 the bacteria using hydrocarbons as a carbon source and energy for their growth have been studied in detail, such as alcaligenes, bacillus, corynebacterium, pseudomonas, methylomonas, micrococcus, methylobacterium, nocardia, rhodococcus, achromobacter, acinetobacter, arthrobacter, flavobacterium etc. members of these groups have been evaluated for usage in bioremediation processes.10-13 among these, biodegradation studies have been also focused on acinetobacter species such as acinetobacter baylyi adp1,14-15 acinetobacter sp. mub1,16 acinetobacter oleivorans,17 acinetobacter sp. ls-1,18 a. baumannii,19 acinetobacter johnsoni.20 as identified in acinetobacter, alkane monooxygenases encoded by the alkm gene and composed of alkane hydroxylase (alkm), rubredoxin (ruba) and rubredoxin reductase (rubb) are key enzymes that catalyze the terminal oxidation of n-alkanes to alcohols.14-16, 21-23 in this study, we aimed to study and characterise acinetobacter strains isolated from petroleum-contaminated sites in batman province of turkey and to evaluate the hydrocarbon biodegradation potential of the isolated bacterial strains. experimental collection of samples the samples were collected from petroleum-contaminated soils around the petroleum wells (southern raman1. and 237. petroleum stations). the crude oil used in the experiments was obtained from batman petroleum refinery. the basal medium (bm) contained per liter: phosphate buffer (5.0 ml), magnesium sulphate (3.0 ml), calcium chloride (1.0 ml), ferric chloride (1.0 ml), as well as 1.0 ml mineral elements solution containing znso4.h2o, mnso4 and (nh4)6moo24.4h2o. filtered crude oil (1 %) was transferred to the bm by using a 0.2 mm pore size filter as the energy source. the petroleum-contaminated soils (1 g) around the petroleum wells were transferred into bm in the presence of crude oil (1 %) and then incubated in a shaking water bath at 120 rpm at 30 °c for 5 days. after incubation, aliquots (1 ml) from each sample were transferred to freshly prepared bm plus crude oil (1 %) for preparing subculture. following two subculture cycles, the microorganisms were let to grow on solid medium (nutrient broth agar) for 5 days of incubation. the morphological appearance of the colonies growing on the plates was examined and different colonies were selected and retransferred to bm in the presence or absence of crude oil for 5 days. the isolated strains were a cc ep te d m an us cr ip t petroleum degrading by acinetobacter strains 3 characterized as colonies with different morphologies for petroleum hydrocarbon degradation and utilising hydrocarbons for the carbon and energy source. morphological, biochemical and physiological characteristics gram staining was carried out by the method of dussault. 24 the temperature range of 10 °c and 45 °c was used for growth in liquid medium, whereas the ph range tested was between 4.0-12.0. the initial ph of media was adjusted with acidic and basic solutions. urease activity testing was carried out as described by lányi.25 procedures described within bergey et al.26 and claus and berkeley, 27 were followed to determine different biochemical properties such as indole, oxidase, citritase, catalase and urease activity as well as motility of the studied isolates. preparation of genomic dna extraction and 16s rrna analyses bacterial cells (d11, e1 and e2) were cultured overnight in nb medium under optimum conditions. after incubation bacterial culture was centrifuged at 14,000 rpm for 10 min and bacterial pellet was resuspended in tris-edta (te) buffer. genomic dna was isolated from tested bacteria using protocol supplied by the manufacturer of the bacterial genomic dna extraction kit. dna concentrations were determined using a uv/vis spectrophotometer (s60 double beam, libra biochrom). all dna extracts were stored at -20 °c. 16s rrna encoding genes were amplified by pcr from isolated chromosomal dna (1 µl) using universal primers (27f and 1492r, sentebiolab). pcr amplification was performed in pcr reaction mixture (25 µl) containing; primers (20 pmol for f and r), 0.3 mm dntp´s mixture and taq dna polymerase (2.5 u, sigma) in the supplied buffer (10x, sigma). the pcr temperature program used was: initial denaturation at 95 °c 5 minutes, 40 cycles of denaturing at 94 °c for 1 min, annealing at 55 °c for 1 min, and ex tension at 72 °c for 2 min and the final extension at 72 °c for 7 min, using thermal cycler (t100, bio-rad). isolated chromosomal dna samples and pcr products were subjected to analysis with agarose gel electrophoresis using 1 % of agarose gels containing red safe gel stain and visualized using a transilluminator (wuv-m20, daihan scientific). pcr products were extracted from the gel using qiaquick gel extraction kit (qiagen). phylogenetic analysis sequence analysis of 16s rrna gene sequences of hydrocarbon-degrading isolates was performed by bm laboratory system (ankara/türkiye). sanger sequencing was performed in the macrogen netherlands laboratory using the abi 3730xl sanger sequencing instrument (applied biosystems, foster city, ca) and the bigdye terminator v3.1 cycle sequencing kit. reads obtained with primers 27f and 1492r were contiguous using the cap contig assembly algorithm in bioedit software to generate a consensus sequence. the sequences of the 16s rrna gene for all strains were determined (d11: 1407 bp, e1: 1383 bp; e2: 1393 bp). the blast search tool on ncbi [national centre of biotechnology (http://www.ncbi.nlm.nih.gov)] was used to compare and search for homology of these sequences in the genbank database. the 16s rrna gene similarities were retrieved from the database, determining most closely related strains. the kimura-2 parameter algorithm kimura28 was used to calculate distance matrices. the phylogenetic tree was built using the neighborjoining method according to saitou and nei.29 the bootstrap consensus tree was set at 100 replicates to represent the evolutionary history of taxa. evolutionary analyses were performed in mega11.30 all strains were deposited in genbank database. a cc ep te d m an us cr ip t 4 eren et al. pcr amplification of alkm gene clustalw, available from ncbi, was used to align all complete sequences of the alkm.31 consensus regions were used to design degenerate primers by identifying conserved regions in alkm nucleotide sequences. the purified dna was screened by pcr to detect alkane degradation genes (alkm). therefore, thet alkm gene was amplified using the primers alkm-f (5’-cctgtctcatttggcgctcgttcctacagg-3’) and alkm-r (5’gtgatgatctgaatgtcgttgtaactgg-3’). pcr reactions mix (25 µl) were prepared containing 10x pcr buffer with mgcl2 (sigma), 0.5 µm primer alkm-f and alkm-r, dntp´s (0.2 mm of each, sigma), taq polymerase (0.05 u ml-1, sigma) and h2o for pcr (sigma). the amplification program consisted of a denaturation at 94 °c for 4 min, denaturing at 94 °c for 30 s (35 cycles), annealing at 55-65 °c for 1 min (using gradient), followed by extension at 72 °c for 7 min using a thermal cycler. pcr products were confirmed by running on 1.0 % agarose gel containing red safe staining and visualized using a transilluminator. for sequencing, pcr products were purified using gel extraction kit. detection of alkane monooxygenase gene alkm pcr products of alkm gene from the strains were sequenced on both strands by the commercial services of sentebio lab (ankara, türkiye). analysis of pcr products was done by an automated genetic analyser and sequences were aligned and compared with other alkm sequences of acinetobacter available in the genbank database using the clustal x program and all sequences were compared using a blast search tool database32 on ncbi. phylogenetic trees of aligned sequences were constructed with mega11 software.30 nucleotide sequences were translated into amino acid sequences using the transeq tool (on the website of the european bioinformatics institute) and compared directly with the protein database. conserved areas within coding nucleotide sequences were analysed by cd-search33 while using the blastp tool on ncbi to check if sequenced alkm genes have conserved motifs. the alkm protein sequence was modelled by the swiss-model workspace (https://swissmodel.expasy.org). the ramachandran plot was evaluated with verify 3d and procheck.34 growth of bacterial strains in the presence of hydrocarbons and crude oil a fresh culture of all strains grown in bm liquid medium for 24 hours was obtained, followed by centrifugation and washing with bm. this process was repeated 3 times. bacteria were then incubated in bm (25 ml) in the presence of crude oil (1 %) for 5 days and in single hydrocarbons (1 % w/v hexane, heptane, octane, decane, pentadecane, hexadecane, toluene and squalene from sigma) for 3 days under optimum growth conditions. following incubation, the bacterial growth was determined by spectrophotometer at 600 nm. each data point represents the mean of at least three experiments. gc analyses of degradation of crude oil and hydrocarbons by the strains the determination of both crude oil and single n-alkanes degradation such as hexadecane and pentadecane by bacterial strains were performed by using gas chromatography-mass spectrometry (gc-ms). bacterial cells precultured overnight in nb medium were transferred to a 100 ml erlenmeyer flask containing 25 ml of bm in the presence of 1 % crude oil or nalkanes and incubated under optimum conditions at 30 °c. following incubation, the bacterial cells were removed, and petroleum hydrocarbons remaining in the bm were used for calculating the degradation ratio of single hydrocarbons and those within crude oil. petroleum hydrocarbon fractions remaining in the culture medium after incubation time were analysed by gc-ms with a cc ep te d m an us cr ip t petroleum degrading by acinetobacter strains 5 a flame ionization detector (fid) (hp 6850, hewlett packard). authentic standards were used to determine individual hydrocarbon fraction components by matching the retention times. results and discussion morphological, physiological, and biochemical characteristics two bacterial strains designated as strains d11 and e2 were isolated from the petroleum station southern raman 237, while the strain e1 was isolated from southern raman1 in batman province of turkey, and all strains were found to degrade and use hydrocarbons within crude oil to grow (fig. 1). fig. 1. demonstration of growth and biodegradation by the strains d11, e1 and e2, compared to abiotic control at 1 % crude oil as carbon source the strain d11, e1 was gram-negative, motile, coccobacilli, while e2 strain was gram-negative, non-motile and coccobacilli (see table s1). the comparison of phenotypic properties of the strain d11, e1 and e2 with some acinetobacter strains in table s1 reveal the similarities of both strains to each other and to other acinetobacter species, phenotypically and biochemically, based on the results obtained. as henrichsen35 stated, the name "acinetobacter" means "motile rod" and it has been noted in previous studies that a nonmotile phenotype is a common feature in this genus, but it was also noted that the motility of a few a. calcoaceticus strains was very conditional and observed. as d11 and e1 was found to be motile, there are several studies on a. baylyi and a. gerneri,36 acinetobacter sp. bt1a,37 describing motile phenotypes of acinetobacter members. as shown in table 1, all strains were gram-negative and aerobic. moreover, the characteristics of oil-degrading strains d11, e1 and e2 were interestingly very similar of which the catalase, citrate and starch test results were clearly positive, whereas, oxidase, urea, indole test results were negative. the difference was that gelatine hydrolysis test was positive for e1, but negative for d11 and e2. the biochemical test results such as starch hydrolysis, catalase and citritase activities of d11, e1 and e2 strains were similarly positive as in most acinetobacter species: positive starch hydrolysis also in a. junii strain va238 and acinetobacter sp. strain bt1a,37 positive catalase and citritase activities also in acinetobacter soli, sp. nov,39 acinetobacter strain ustb-x.40 the growth values of strain d11 and e1 a cc ep te d m an us cr ip t 6 eren et al. for the temperature, ph and naci tolerance were found to be very close and observed to be between 20-40 °c (optimum 35 °c), ph 5.0 and 10.0 (optimum ph 8.0-10.0 for d11, optimum ph 9.0-10.0 for e1) and tolerated up to 3 % nacl (w/v), respectively. moreover, the temperature and ph values for the strain e2 were also found to be between 20-40 °c (optimum 35 °c), but ph growth range was 4.010.0 (a wide optimum range of 4.0-10.0), and tolerated up to 5 % nacl (w/v). compared with previous studies on other acinetobacter species, the optimum ph values obtained were 8.0 for a. soli, sp. nov,39 and 8.5 for acinetobacter sp. strain s241. similar temperature values as in the present study were reported as 20-40 °c for acinetobacter strain ustb-x40 and a. junii strain va2.38 phylogenetic analysis the amount of genomic dna isolated from d11, e1 and e2 strains was between 25-71 g/ml and each determined to be >10 kb in size on 1% agarose gel. reads obtained with primers 27f and 1492r were contiguous to form a consensus sequence. the contig of forward and reverse sequences, contig assembly algorithm (cap) was used in bioedit software to perform this process. the analysis of 16 s rrna gene sequence revealed that the most similarity of the strain d11 (1407 bp) was to a. pittii (99.93%) and e1 (1383 bp) to a. pittii (100%), while the strain e2 (1393 bp) was similar to a. calcoaceticus (100%) (figure 2). fig. 2. the 16s gene sequence analysis of the strains d11, e1 and e2 genbank accession numbers of the sequences of the strains d11, e1, e2 are mt374264, mt374266 and mt374267, respectively. the tree was generated using blast program for sequence alignment and by the neighbor joining method a cc ep te d m an us cr ip t petroleum degrading by acinetobacter strains 7 using mega 11 software. exiguobacterium sp. (km077136) was used as the outgroup. detection of alkane monooxygenase gene alkm the nucleotide and the predicted amino acid sequences for the alkm genes were compared using several databases as described in the previous section. the results of base pairs long and nucleotides similarity with related bacterial strains are presented in table 1. table 1. alkm gene sequences and conserved domains within coding nucleotide sequences isolate related bacteria strains gene base pairs long nucleotides similarity (%) description interval e-value d11 acinetobacter sp. buu8 (uniprotkb (a6n7f9) 651 93.06 the membrane fatty acid desaturase (membrane_fad s)-like cd includes membrane fadss, alkane 66-281 1.21*10-16 744-938 2.15*10-19 e1 acinetobacter sp. 826659 (a0a013tk13) 471 95.84 the membrane fatty acid desaturase (membrane_fad s)-like cd includes membrane fadss, alkane 33-293 2.00*10-20 799-1542 1.87*10-57 e2 acinetobacter sp. buu8 (uniprotkb (a6n7f9) 448 92.72 the membrane fatty acid desaturase (membrane_fad s)-like cd includes membrane fadss, alkane 36-170 9.51*10-8 analysis of the derived amino acid sequences of alkane 1-monooxygenase from tested strains showed a high degree of similarity to that of other acinetobacter strains. conserved domains within coding nucleotide sequences were analysed with cd-search using tool on ncbi. all sequences showed similarity the membrane fatty acid desaturase (accession no. cl00615) and the regions of the conserved domains of the tested strains are also given in the table 1. it was previously reported that members of acinetobacter species possess many a cc ep te d m an us cr ip t 8 eren et al. metabolic capabilities including degradation pathways.42 for example, the biodegradation studies carried out on acinetobacter species include; a. baylyi adp1,14-15 acinetobacter sp. mub1,16 a. oleivorans.17 acinetobacter species isolated from petroleum contaminated sites have been reported to possess n-alkane degrading genes including alkane monooxgenase, alkane hydoxylase and dioxygenase genes which are the key enzymes of metabolic process in the remediation oil pollution,23,18,19,22,43 taking into account the amino acid sequences of the alkm protein from strains d11, e1 and e2, three-dimensional (3d) model was analysed with swiss-model workspace through a comparative analysis of the ramachandran prochek software for each alkm protein model generated. alignment of region linked to substrate specificity for the alkm from tested bacteria (d11, e1 and e2) and other related species (a. baylyi (o31250), acinetobacter sp. sj-2 (k4hwf9), acinetobacter sp. buu8 (a6n7f9), acinetobacter sp. (q9xbm0), acinetobacter sp. 826659 (a0a013tk13), a. calcoaceticus (f0kmz3), alkanindiges hydrocarboniclasticus (a0a1s8ct34) was performed by using the uniprotkb server. phylogenetic analysis of strains and related species by the neighbor joining method based on alkm gene sequences. in fig. s1a-e, strain d11 (651 bp) sequences showed similarity to alkane 1monooxygenase. strain d11 sequences showed similarity to alkane 1monooxygenase from acinetobacter sp. buu8 (uniprotkb (a6n7f9) with 93.06 %. the three-dimension (3d) model of alkane monooxygenase (alkm) of the a. baylyi (uniprotkb o31250) was used as reference. the data shown by the ramachandran graph (94.55 %) reveal the identity/similarity of primary, secondary and tertiary structures between the strain d11 and a. baylyi (uniprotkb o31250). alkane monooxygenase gene sequences of strain e1 (471 bp) showed similarity to alkane 1-monooxygenase of acinetobacter sp. 826659 (a0a013tk13) with 95.84% and ramachandran graph was found to be 83.33 % (figure s2a-e). in figure s3a-e, strain e2 gene sequences (448 bp) showed similarity to alkane 1-monooxygenase from acinetobacter sp. buu8 (uniprotkb a6n7f9) with 92.72 % while the ramachandran graph was 100 %. as van beilen et al.44 stated, many alkane hydroxylase homologues have been characterized in addition to alkm genes in studies conducted in a. calcoaceticus strains eb104, 69-va and ncimb 8250 and also acinetobacter sp. 2769a related to alkm genes. phylogenetic analysis of alkm amino acid sequences in these studies showed high sequence variation between them and clearly differentiated alkm genes from alkb genes. therefore, most of these genes not found in acinetobacter genus have been named alkb gene. growth of bacterial strains in the presence of crude oil and various hydrocarbons the strains were tested by incubation in the presence of various short and long chain hydrocarbons and crude oil, namely hexane, heptane, octane, decane, a cc ep te d m an us cr ip t petroleum degrading by acinetobacter strains 9 pentadecane, hexadecane and squalene, as well as toluene cultivated in bm medium at 1 % concentrations under optimum conditions. as it can be seen in figure 3, the strains d11, e1 and e2 clearly use certain hydrocarbons as carbon and energy sources for growth on both 1 % crude oil and single hydrocarbons tested. fig. 3. growth of strains d11 (red), e1 (green) and e2 (purple) in bm supplemented with crude oil (incubated for 5 days) and various hydrocarbons (incubated for 3 days) at 1% concentrations as carbon sources. each data represents the mean of at least 3 different experiments figure 3 also shows that the strains d11 and e1, significantly degrade hexadecane, among the aliphatic hydrocarbons tested. the strains also grow at low rates on decane and pentadecane, but not in short chain hydrocarbons such as hexane, heptane or octane. however, the growth for strain e2 was remarkable in the presence of long chain hydrocarbon pentadecane, while the growth was relatively higher in hexadecane and squalene, compared to control (fig. 6). sun et al.45 reported that the aerobic degradation of n-alkanes by bacteria needs alkane monooxygenase encoded by alkm gene which catalyzes the terminal oxidation of n-alkanes to alcohols, suggesting that the transcription of alkm in bacterial strains directly influences the degradation of n-hexadecane. degradation of crude oil and hydrocarbons by the strains determined by gc-ms analyses members of acinetobacter sp. are capable of utilizing a broad range of nalkanes with varying chain lengths (c10-c40) as a sole carbon source.46 furthermore, acinetobacter species are dominant bacteria in most petroleum contaminated soils,47-48 as well as in various environments including sea waters and sediments,20,49 in waste waters50 and in streams.51 a cc ep te d m an us cr ip t 10 eren et al. table 2. the degradation of petroleum hydrocarbons by members of acinetobacter isolate isolation of bacteria from substrate tested degradation rate (%) incubation time literature acinetobacter sp. strain y9 sea mud c9~c22 nalkanes from diesel oil (4 %) 53.28 7 days min et al.23 acinetobacter sp. strain s2 wastewaters diesel oil 50.62 7 days sawadogo et al.41 acinetobacter sp. strain bt1a crude-oil contaminated soil crude oil (1 %) 83 7 days acer et al.37 acinetobacter baumannii mks2 crude-oil contaminated soil crude oil (1 %) 43 13 days muthukamalam et.al.19 acinetobacter sp. crude-oil contaminated soil crude oil 75(% of c20, c21 and c22 ) 50(% of shorter chains) 7 days zheng et al.52 a. pittii h9-3 crude-oil contaminated soil crude oil (1 %) 36.8 21 days wang et al.43 a. pittii strain d11 crude-oil contaminated soil crude oil (1 %) hexzadecane(1 %) 43 (c.oil) 70 (hexadecane) 5 days 3 days present study a. pittii strain e1 crude-oil contaminated soil crude oil (1 %) hexzadecane(1 %) 9 (c.oil) 76 (hexadecane) 5 days 3 days present study a. calcoaceticus strain e2 crude-oil contaminated soil crude oil (1%) pentadecane(1 %) 12 (c.oil) 48 (pentadecane) 5 days 3 days present study different degradation rates (%) of crude oil and alkane hydrocarbons by various acinetobacter species are summarised in table 2, as well as the degradation results obtained in the present study. the remaining amount of hydrocarbons in the culture medium analysed by gc-ms were used for calculating degradation rates of crude oil and alkane hydrocarbons (1%). the comparisons with the undegraded control showed that the strains d11, e1 and e2 degraded 43 %, 9 % and 12 % of total petroleum hydrocarbons, respectively within crude oil after short incubation time of only 5 days. as the growth of the strains d11, e1 and e2 in the basal media with hexadecane or pentadecane was remarkable shown in figure 3, the gas chromatographic analysis of two single hydrocarbons were also carried out for each strain in figure s4 (a-e). figure s4b shows that as much as 70 % of single hydrocarbon hexadecane (1 %) was degraded by the strain d11, compared to abiotic control (figure s4a), while 76 % of hexadecane was degraded a cc ep te d m an us cr ip t petroleum degrading by acinetobacter strains 11 by the strain e1 (figure s4c). moreover, as can be seen in figure 4e, the strain e2 degraded about 48 % of single pentadecane (1 %) after 3 days of short incubation time, compared to abiotic control (figure s4d). there have been many studies in the literature related to biodegradation rates of hydrocarbons by various bacterial species. for example, tapilatu et al.53 reported that alcanivorax venustensis strains and some other gram-positive bacteria preferably degraded n hexadecane (40-63 %), but other genera tested seemed to prefer n-heptadecane (8-44%), after long time incubation of 30 days. many other bacterial species isolated from contaminated sites were found to degrade oil hydrocarbons at varying degradation rates as; pseudomonas sp. degraded 67.57 % of the oil (1 %) and micrococcus sp. with 52.95 % after 25 days of incubation period.54 pseudomonas sp. had shown 49.93 % of diesel oil degradation in 20 days against 0.5 % of diesel.55 six isolated bacterial strains identified as p. alcaligenes (hdb-6), bacillus thuringiensis, pseudomonas alcaligenes (hdb-4), pseudomonas mendocina, bacillus flexus and lysini bacillus sp. were found to degrade between 37.88 % to 54.01 % of crude oil after 14 days.56 there have been also several studies on hydrocarbon biodegradation rates using acenitobacter species. throne-holst et al.46 determined that acinetobacter strain can use carbon sources in the range of decane and tetracontane long-chain nalkanes. min et al.23 reported that acinetobacter strain y9 degrades n-alkanes in the range of c9-c22 as sole carbon sources degrading 53.28 % in 7 days of incubation. acinetobacter sp. bt1a was also found to degrade 83 % of 1% crude oil in 7 days37. in more recent studies, various a. baumannii strains were found to degrade an average of 43 % and % 61 of oil alkanes, respectively reported by19,57. conclusion a wide variety of microorganisms including bacteria that can degrade hydrocarbons within petroleum were isolated from oil-contaminated soils. bacteria are the most dominant microorganisms determined in microbial ecology degrading petroleum hydrocarbons. among bacteria, acinetobacter is recently found to be most common genera that is able to degrade hydrocarbons. the members of acinetobacter species studied were found to biodegrade crude oil and sole hydrocarbons. gene sequences of the strains d1, e1 and e2 showed similarity to alkane 1-monooxygenases from acinetobacter sp. alkm. the present study seems to be effective in degrading n-alkanes in shorter incubation times and further microbial consortium studies with the related strains may provide a good advantage in bioremediation of oil-contaminated soil. acknowledgements: this study, a part of ayse eren’s phd project, was supported by dicle university scientific research projects coordination unit (project number: fen.19.014). conflicts of interest: the authors declare no conflict of interest. a cc ep te d m an us cr ip t 12 eren et al. author contribution statement: designing the experiments: ae and kg; analysed the data: ae and kg, phylogenetic and bioinformatic analysis: fmb and kg; article writing: ae, kg and fmb. all authors read and approved the final article. supplementary material additional data are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/12469, or from the corresponding author on request. и з в о д детекција гена за алкан монооксигеназу у acinetobacter соју који разлаже угљоводонике и сирову нафту применом pcr методе на земљишта контаминирана нафтом ayşe eren, fatma matpan bekler и kemal güven department of molecular biology and genetics, faculty of science, dicle university, 21280 diyarbakır, turkey бактеријски сојеви d11, e1 и e2 изоловани из земљишта контаминираних нафтом су припадници acinetobacter рода, што је утврђено секвенцирањем гена за 16s rrna и фенотипском карактеризацијом. након инкубације 5 дана, gc-ms анализом је утврђено да је око 43%, 9%, односно 12% укупних угљоводоника из сирове нафте разложено сојевима d11, e1, односно e2. такође, око 70% и 76% хексадекана је разложено сојевима d11 и e1 након 3 дана инкубације, док је сој e2 разложио око 48% пентадекана. pcr методом генског секвенцирања утврђена је 93,06%, односно 92,72% сличност гена за алкан-1монооксигеназу сојева d11 и e2 са геном из acinetobacter sp. buu8 alkm, док је сличност секвенце соја e1 95,84% са геном acinetobacter sp.826659. резултати ове студије биодеградације угљоводоника сојевима acinetobacter могу наћи примену у поступку биоремедијације. (примљено 7. јула; ревидирано 8. августа; прихваћено 18. августа 2023.) references 1. r. bajagain, y. park, s. w jeong, sci. total environ. 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https://doi.org/10.6088/ijes.2013030500001 https://doi.org/10.2166/wst.2019.025 https://doi.org/10.1007/s00203-021-02469-2 https://doi.org/10.1007/s00203-021-02469-2 voltammetric and theoretical studies of the electrochemical behavior of cephalosporins at a mercury electrode j. serb. chem. soc. 80 (8) 1035–1049 (2015) udc 577.182.24+543.552+544.6.076.32– jscs–4778 034.791:519.677 original scientific paper 1035 voltammetric and theoretical studies of the electrochemical behavior of cephalosporins at a mercury electrode katarina nikolić#, mara m. aleksić*#, vera kapetanović and danica agbaba# faculty of pharmacy, university of belgrade, vojvode stepe 450, 11000 belgrade, serbia (received 29 january, revised 23 february, accepted 26 february 2015) abstract: the adsorption and electroreduction behavior of cefpodoxime proxetil, cefotaxime, desacetylcefotaxime, cefetamet, ceftriaxone, ceftazidime, and cefuroxime axetil at a mercury electrode surface were studied using cyclic (cv), differential pulse (dpv) and adsorptive stripping differential pulse (adsdpv) voltammetry. the quantitative structure property relationship (qspr) study of the seven cephalosporins adsorption at the mercury electrode was based on density functional theory dft-b3lyp/6-31g(d,p) calculations of molecular orbitals, partial charges and electron densities of the analytes. the dft-parameters and qspr model explain well the process of adsorption of the examined cephalosporins. the qspr study defined that cephalosporins with lower electron density on the nitrogen atom of the n–o bond, higher number of hydrogen bond-accepting groups, and higher principal moment of inertia should express high adsorption on the mercury electrode. keywords: cephalosporins; adsdpv; cv; dft; qspr; computational electrochemistry. introduction cephalosporins are semi-synthetic β-lactam antibiotics similar to penicillins, but with a broader spectrum of antibacterial properties and a higher resistance to β-lactamase.1 these compounds contain a β-lactam/dihydrothiazine moiety bearing different substituents at the c3 and c7 position. methoxyimino cephalosporins are reducible at a mercury electrode and therefore present electrochemically a very important class of antibiotics. this characteristic enables the application of electroanalytical techniques for the sensitive determination of methoxyimino cephalosporins. these cephalosporins were extensively studied at a mercury elec * corresponding author. e-mail: mara@pharmacy.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc150129019n _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1036 nikolić et al. trode, the mechanism of methoxyimino reduction at the mercury electrode was established and voltammetric methods based on the adsorptive accumulation of cephalosporins at the mercury electrode were developed.2–6 regardless of the high sensitivity of the determination in pharmaceutical formulations, this method still could not be used for the determination of cephalosporins in a biological matrix. apart from the reduction at mercury electrode, methoxyimino cephalosporins may be reduced or oxidized at different solid electrodes that may be specially modified and activated.7–12 these determinations appeared to be less sensitive than at a mercury electrode.7–12 since the process of the reduction of methoxyimino cephalosporins is associated with adsorption at the electrode surface, adsorptive stripping voltammetry was successfully applied for their quantitative determination as a highly selective and precise electroanalytical technique.13–15 the application of adsorptive stripping method enabled the determination of low concentrations of cephalosporins in vivo from biological samples, such as urine, serum or cerebrospinal fluid.13–15 the mechanism of reduction of the methoxyimino cephalosporins, such as cefpodoxime proxetil (cpdx-pr), cefotaxime (ctx), desacetylcefotaxime (dctx) and cefetamet (cet), at a mercury electrode was established and reported in previous papers.16–20 the methoxyimino group in cephalosporin molecules undergoes reduction at mercury electrodes yielding well-defined polarographic waves and voltammetric peaks, over a wide ph interval from 2 to 12.16–20 based on previous findings,16–20 the voltammetric study is now extended to include structurally closely related cephalosporins, i.e., ceftriaxone (cro), ceftazidime (caz) and cefuroxime axetil (cxm). in this work, adsorptive techniques were applied for a study of the electroreduction behavior and for the quantitative determination of ceftriaxone, ceftazidime, and cefuroxime axetil. the experimental results of the group of seven methoxyimino cephalosporins were further used in a quantum chemical study. the theoretical study was used to describe the adsorption mechanism at the mercury electrode surface and to predict the electrochemical adsorption at mercury electrode of related cephalosporins. to the best of our knowledge, there is no scientific report either about the study of the electrochemical behavior of cephalosporins or a quantum chemical study of their electrochemical adsorption mechanism on a mercury electrode. experimental reagents and chemicals cephalosporins (cef) were from sigma and all chemicals were of analytical grade quality. britton–robinson (br) universal buffer was prepared from stock solutions of 0.04 mol dm-3 boric, orthophosphoric and acetic acids with the appropriate volumes of 0.2 mol dm-3 naoh. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ computational electrochemistry studies of cephalosporins 1037 apparatus the voltammetric measurements were performed with an amel 433-a computerized polarographic analyzer. a three-electrode system was employed with a hanging mercury dropping electrode (hmde), an ag/agcl reference electrode and a pt-auxiliary electrode. all potentials in the paper are expressed vs. ag/agcl. adsorptive stripping differential pulse voltammetry (adsdpv) was performed under the following conditions: scan speed 200 mv s-1, pulse amplitude 100 mv, pulse width 20 ms, from 0 to –1.6 v. after recording the base-line, when a fresh mercury drop has been formed, voltammograms were recorded after a certain time of adsorptive accumulation at selected accumulation potential and selected ph, in a stirred solution (300 rpm). the accumulation period in a stirred solution was followed by a 10 s settling period to allow for quiescence of the solution and uniform distribution of the deposited substance on the surface of the mercury drop. when the differential pulse voltammetry (dpv) mode was used, the following parameters were applied: pulse repetition 100 ms, pulse amplitude 25 mv and pulse width 20 mv. the cyclic voltammograms (cv) were recorded at a scan rate ranging from 5 to 100 mv s-1. a radiometer ph meter, phm 220, with a combined ph electrode radiometer gk2401b, was used and appropriate standard buffer solutions. solutions preparations a stock solution (so) of 1×10-4 mol dm-3 of cef was prepared by dissolving an accurate mass of cef in redistilled water, and stored in freezer. more dilute solutions were prepared daily from the stock solution. procedures a 15 ml aliquot of the corresponding supporting electrolyte solution (br buffer only) or 13.5 ml aliquot of the corresponding supporting electrolyte solution (br buffer) and 1.5 ml of s0 was placed in the voltammetric cell, and deaerated for 10 min with high purity nitrogen and voltammograms were recorded. computational methods the experimentally determined slope (δip/δv) and the computed molecular parameters of the examined cephalosporins were used to build qspr models and to examine electrochemical adsorption and electroreduction mechanism of the cephalosporins. calculation of the pka values and a selection of the dominant molecules/ions species at the experimental ph 2.0–3.5 (fig. 1), were performed for all the analyzed compounds using the marvin 5.5.1.0 program.21 the minimum energy conformations of the analyzed compounds were obtained by the cs gaussian 98 program22,23 using the b3lyp/3-21g basis set.24-26 the molecular refractivity (mr), the partition coefficient (clog p), the distribution coefficient (log d at ph 1.5, 2.5, and 4.0), the radius, the principal moment of inertia (pmi), the connolly accessible area (sas), the connolly molecular area (ms), the molecular surface area (msa), the polar surface area (psa), the hydrogen bond donors (hbd) and the hydrogen bond acceptors (hba) were computed for the optimized molecular models using the marvinsketch 5.1.5.021 and the chem3d ultra 7.0.022 programs. the cs gaussian 98 program23 using the b3lyp hybrid functional that included the 6-31g basis set (b3lyp/6-31g(d,p))24-26 was applied for the computation of the molecular parameters, i.e., the energies of the highest occupied molecular orbital (homo) and the lowest unoccupied molecular orbital (lumo), the chemical potential (μ), electronegativity (χ), hardness (η), global softness (s), electrophilicity index (ω) and the dipole moment. the calculated molecular descriptors were used for the development of the qspr model using partial least square (pls) regression.27 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1038 nikolić et al. f ig . 1 . d o m in an t fo rm s o f th e ce p h al o sp o ri n s at t h e an al y ti ca l p h 2 .0 – 3 .5 . _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ computational electrochemistry studies of cephalosporins 1039 the qspr study was realized using the soft independent modeling of class analogy simca p+ 12.0 program,28 for the pls regression analysis. in qspr modeling, a summary of the importance of every variable (xk) for both y and x matrices is described by the variable importance in the projection (vipk) parameter. the x variables with a vip value larger than 1 are the most relevant for explaining the regression model, while the x variables with a vip value below 1 have a smaller influence on the regression model.27 validations of the pls regression models were performed by the leave-one-out crossvalidation (loo-cv) method. the predictive power of the model is determined by the q2 value, which is the cross-validated version of r2, and the root mean square error of prediction (rmsep). the model was fitted to the data leaving one data point out. the elaborated pls model then predicts the left-out data point. this procedure was repeated until all data points had been left out, which results in a number of parallel models. the difference between the observed and the predicted values in the left-out data point (e(i)) were calculated for each model and used for the calculation of the prediction error (rmsep). in this setting, the predicted sum of squares (press), rmsep and q2(y) were defined as: 2( )1 n ii press e = =  (1) ( / )rmsep press n= (2) 2 1 ( / )q press ssto= − (3) where ssto is the variation, sum of squares (total). models with q2 ≥ 0.5 could be considered as having good predictive capabilities.27 the quality of the plsr models was estimated using such parameters as rmsep, q2, and the correlation coefficients observed vs. predicted (r2 (obs vs. pred)).27 results and discussion the mechanism of the reduction of methoxyimino group involves two steps.29–31 first, the nitrogen of the n–och3 group is protonated (1), then the n–o bond is cleaved (2), yielding an imine that was then protonated (3) and reduced to the corresponding amine (4): + +2 3 2 3r c noch h r c nh och⎯⎯→= + =←⎯⎯ (1) + +2 3 2 3r c nh och 2e h r c nh ch oh= + + ⎯⎯→ = + (2) + +2 2 2r c nh h r c nh ⎯⎯→= + =←⎯⎯ (3) + +2 2 22r c nh 2e 2h r chnh= + + ⎯⎯→ (4) the specificity of this reduction is evidenced by the appearance of only one well-defined voltammetric peak (i) in acid and neutral media, while in slightly alkaline medium, splitting of this peak occurs, and two peaks are obtained (ii and iii). peak i represents the overall reduction of the oxime group that involves transfer of four electrons yielding an amine. as the rate of protonation of oxime decreases with increasing ph, the reduction peak decreases, and above certain ph value, the reduction occurs in two steps corresponding to oxime reduction to the imine, and imine reduction to amine, respectively. this separation into two two _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1040 nikolić et al. -electron processes is caused by differences in position and rate of establishment of acid–base equilibrium resulting in the protonation of the oxime and imino group,32 i.e. their pka values. in more alkaline solutions, at ph > 10, a new peak (iv) appeared at a more negative potential, e ≈ −1.5 v (fig. 2) due to the reduction of the unprotonated form. in addition, all the cephalosporins underwent a two-electron reduction of the unsaturated c=c bond of the dihydrothiazine ring. this reduction occurred in an acid medium at potentials very close to that of hydrogen ion reduction and therefore was not convenient for analytical investigations. fig. 2. graphically presented ph influence on the dpv peak: a) potential and b) current of 1×10-5 mol dm-3 cpdx-pr in br buffer.18 a plot of the obtained peak potentials as a function of ph (fig. 2a) shows several linear segments with varying dep/dph slopes, which indicates a varying number of protons transferred before the potential determining electron transfer. the slopes, dep/dph, obtained by curve fitting were as follows: 0.058 v ph–1 at ph 2–6, 0.189 v ph–1 at ph 6–7.5 for peak i; 0.026 v ph–1 at ph 8–10 for peak ii; 0.029 v ph–1 at ph 8–10 for peak iii and ≈0 v ph–1 at ph > 9 for peak iv. the slope of 58 mv obtained for peak i at ph < 6 suggested the same number of electrons and protons involved in the electrode process, while the slope of 26–29 mv obtained for peaks ii and iii suggested fewer protons involved, (protons:electrons = 1:2). decrease in the slope at ph > 8 indicates the ph region in which the pre-protonation becomes too slow relative to diffusion. the peak potential of peak iv is ph-independent, which is in accordance to theory in the case of the reduction of unprotonated species. according to the peak current−ph dependence (fig. 2b), it is evident that all peaks show a maxima at a certain ph value. the maximum of the ip vs. ph curve indicates pronounced adsorption at the given ph. all the investigated cephalosporins showed dpv current maximum of peak i at a ph of around 3. the mechanism of the reaction of methoxyiminocephalosporins at a mercury electrode16–20 confirmed that the reduction of cephalosporins was mostly complicated by the effects of adsorption at the electrode surface. the conjugated _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ computational electrochemistry studies of cephalosporins 1041 acids of these compounds were adsorbed at the electrode surface at potentials between 0.0 and 0.2 v, and desorbed between –1.0 and –1.2 v. previous polarographic experiments proved that the reduced form was less strongly adsorbed than the oxidized one,33 while the unprotonated form of cephalosporins, which was reduced at ph ≥ 10, showed no adsorption.5,32,33 these experiments confirmed that the adsorption prevails in an acid medium.5,32,33 the symmetrical shape of the voltammetric peaks of cefpodoxime, cefotaxime, desacetylcefotaxime and cefetamet, as well as the pronounced maxima of the curves presenting the current intensity vs. ph, indicated strong adsorption of these compounds at the mercury surface. the linear dependence of the voltammetric peak current on the scan rate, ip vs. v, is also characteristic for adsorption-controlled processes (fig. 3), and a higher slope was obtained when the adsorption was more pronounced. fig. 3. effect of the scan rate on the peak current for 1×10-5 mol dm-3 ceftriaxone (■), cefpodoxime proxetil (●), ceftazidime (▲) and cefuroxime axetil (▼) in br buffer solutions. thus, the electrochemical adsorption of cro, cpdx-pr, caz, ctx, dctx, cxm and cet on the mercury electrode (table i) was monitored by measuring the slopes (δip/δv). table i. experimentally obtained slopes (δip/δv) for the investigated cephalosporins compound ph slope (δip/δv) / μa v -1 s ceftriaxone (+/–, +) 3.0 134.07 cefpodoxime proxetil 3.5 65.8018 ceftazidime 3.0 35.56 cefotaxime 2.8 17.4016,19 desacetylcefotaxime 2.8 16.3016 cefuroxime axetil 3.0 14.43 cefetamet 2.0 13.7017 in addition, the regression equations for log ip = f(log ν) were calculated for all the investigated cephalosporins. some of them are listed below: – ceftriaxone (ph 3.0): log ip = 0.898log ν + 2.016 (r = 0.9988) – cefpodoxime proxetil (ph 3.5): log ip = 0.706log ν + 1.510 (r = 0.9970) – ceftazidime (ph 3.0): log ip = 0.682log ν + 1.232 (r = 0.9986) – cefuroxime axetil (ph 2.8): log ip = 0.561log ν + 0.742 (r = 0.9961). _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1042 nikolić et al. the corresponding slopes (higher than the theoretical value of 0.5 for a diffusion controlled process but lower than 1.0 for a process controlled only by adsorption) confirmed the influence of adsorption in acid solutions at mercury surface. according to the obtained slopes, the most pronounced adsorption was observed for ceftriaxone, and it decreased in the same order as already established with the δip/δv values. the differences observed between the investigated compounds are the consequence of specific adsorption and different orientation of these molecules at the mercury surface. it was established that the adsorption was more pronounced when the c7 substituent was more bulky. since all these compounds possess a 2-aminothiazole ring at c7, it could be assumed that this ring plays a great role in adsorption on the mercury surface. the presence of the nh3+ group of the aminothiazole ring in an acid medium creates electrostatic forces with the mercury surface in the negative potential range and thus contributes to the adsorption. finally, electron donor atoms, such as nitrogen and oxygen, in the side chain, increase the electron density of the thiazole ring and enhance the adsorption of methoxyimino cephalosporins. although the substituent in c2 and c3 position shows less contribution to the adsorption than the c7 substituent, the presence of different structures causes the differences in the adsorption characteristics. therefore, the more complex structures of cro, cpdx-pr, caz and ctx compared to those of dctx, cxm and cet could generally explain the more strongly pronounced adsorption of the molecules in the first group. further, a quantum chemical and qspr study of cro, cpdx-pr, caz, ctx, dctx, cxm and cet adsorption at the mercury electrode was performed to investigate the adsorption mechanism of the cephalosporins and to create a model for the prediction of the electrochemical adsorption for related compounds. in the qspr study, the experimentally determined slope (δip/δv), obtained using the cv technique on a mercury electrode, were used as dependent variables, while the computed molecular parameters (mr, log d, sas, ms, msa, psa, hbd, hba, homo, lumo, chemical potential (μ), electronegativity (χ), hardness (η), global softness (s), electrophilicity index (ω) and dipole moment) of the examined compounds were used as independent variables. the density functional theory (dft)-based reactivity descriptors (homo, lumo, μ, χ, η, s and ω) were successfully used in many previous studies34–43 for the interpretation of various reaction mechanisms. descriptors with the highest vip and coefficient values were selected for building the qspr model (fig. 4a). the optimal combination of the most relevant descriptors (hba, electron density on the nitrogen of the n–o bond (ed(n)), pmiy, hba–hbd, psa, mr, clog p, diameter, and ovality for building _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ computational electrochemistry studies of cephalosporins 1043 the qspr models was chosen based on the r2, q2 and rmsep values of the obtained pls models. (a) (b) fig. 4. a) vip plot and b) coefficient plot of the developed qspr model. the coefficient plot (fig. 4b) of the developed qspr model indicated a negative correlation between the ed(n), clog p and ovality parameters and adsorption on the surface of a mercury electrode (table ii). therefore, the cephalosporins with lower electron density on the nitrogen atom of the n–o bond _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1044 nikolić et al. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ computational electrochemistry studies of cephalosporins 1045 lower lipophilicity should express high adsorption on a mercury electrode. the coefficient plot (fig. 4b) also indicated a positive correlation between the mr, psa, hba, hba-hbd, pmiy and diameter parameters and adsorption on the surface of a mercury electrode and hence, cephalosporins with higher psa, mr, diameter and pmiy values and a higher number of hydrogen bond accepting groups should have a high adsorption on a mercury electrode. the qspr-model with the two significant components, r2 = 0.926 and q2 = 0.725, with the lowest rmsep (11.248) and the highest r2 value (obs vs. pred; 0.962), was selected for further study (table ii). the obtained statistical parameters of the qspr model indicated to a good prognostic capacity of the developed qspr model. prediction of the slope (δip/δv) using the developed pls-qspr-model, could be applied to the other related cephalosporins. molecular models of cephalosporins (fig. 5) confirmed essential influence of electron densities and charge of nitrogen for their adsorption on the mercury electrode surface. (a) (b) (c) fig. 5. molecular models of: a) the ceftriaxone cation, b) the ceftriaxone zwitterion and c) cefpodoxime proxetil, based on the partial atomic charge values. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1046 nikolić et al. (d) (e) (f) (g) (h) fig. 5. (continued) molecular models of: d) ceftazidime, e) cefotaxime, f) desacetylcefotaxime, g) cefuroxime-axetil and h) cefetamet, based on the partial atomic charge values. conclusions a previous voltammetric study of the electroreduction behavior and adsorption of methoxyimino cephalosporins (cefpodoxime proxetil, cefotaxime, desacetylcefotaxime and cefetamet) on a mercury electrode surface has been ext_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ computational electrochemistry studies of cephalosporins 1047 ended to structurally related cephalosporins (ceftriaxone, ceftazidime, and cefuroxime axetile). the density functional theory and qspr studies were used to explain the adsorption mechanism at a mercury electrode surface and to predict the electrochemical adsorption at a mercury electrode of the related cephalosporins. the qspr study selected the hba, the electron density on the nitrogen of the n–o bond (ed(n)), pmiy, hba–hbd, psa, mr, clog p, diameter and ovality parameters as the most significant molecular determinants for the electrochemical adsorption of cephalosporins on a mercury electrode. the developed qspr model indicated a negative correlation between the ed(n), c log p, and ovality parameters and adsorption, and a positive correlation between the mr, psa, hba, hba-hbd, pmiy and diameter parameters and adsorption on the surface of a mercury electrode. therefore, cephalosporins with lower electron density on the nitrogen atom of the n–o bond, lower lipophilicity, a higher principal moment of inertia and higher number of hydrogen bond accepting groups should express high adsorption on a mercury electrode. prediction of the electrochemical adsorption using the developed qspr model could be a very helpful tool for use in future cephalosporins studies. the presented study is first reported theoretical investigation of the electrochemical behaviors of cephalosporins. acknowledgments. this work was supported by the ministry of education, science and technological development of the republic of serbia, contract no. 172033. и з в о д волтаметријско и теоријско проучавање понашања цефалоспорина на живиној електроди катарина николић, мара м. алексић, вера капетановић и даница агбаба фамацеутски факултет, универзитет у београду, војводе степе 450, 11000 београд применом цикличне (cv), диференцијално-пулсне (dpv) и адсорптивне “stripping” диференцијално-пулсне (adsdpv) волтаметрије испитано је електрохемијско понашање и адсорпција цефподоксим-проксетила, цефотаксима, дезацетил-цефотаксима, цефетамета, цефтриаксона, цефтазидима и цефуроксим-аксетила на површини живине електроде. студија квантитативних односа структуре и особина (qspr) је коришћена за испитивање адсорпције седам цефалоспорина на живиној електроди. применом теорије функционала густине dft-b3lyp/6-31g(d,p) су израчунате енергије молекулских орбитала, парцијално наелектрисање и електронске густине испитиваних аналита, које су употребљене као молекулски параметри у qspr студији. помоћу изабраних dft-параметара и qspr модела је објашњен процес адсорпције испитиваних цефалоспорина. резултати qspr анализе показали су да већу адсорпцију на живи показују цефалоспорини са нижим наелектрисањем на сумпору тиазинског дела молекула, мањом електронском густином на атому азота n–o везе, већим бројем група које граде водоничне везе и већим главним моментом инерције. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice geochemical investigation as a tool in the determination of the potential hazard for soil contamination (kremna basin, serbia) j. serb. chem. soc. 80 (8) 1087–1099 (2015) udc 550.4.001.5:504.3.054+627.8.034.7: jscs–4782 546.47/49’56’815(497.11) original scientific paper 1087 geochemical investigation as a tool in the determination of the potential hazard for soil contamination (kremna basin, serbia) tamara perunović1, ksenija stojanović1*#, milica kašanin-grubin2, aleksandra šajnović2#, vladimir simić3, branimir jovančićević1# and ilija brčeski1 1university of belgrade, faculty of chemistry, studentski trg 12–16, 11000 belgrade, serbia, 2university of belgrade, center of chemistry, ictm, njegoševa 12, 11000 belgrade, serbia and 3university of belgrade, faculty of mining and geology, djušina 7, 11000 belgrade, serbia (received 17 september, revised 31 october, accepted 6 november 2014) abstract: the geochemical composition of the soils and underlying sediments in the kremna basin was investigated. the aim was to assess whether the observed heavy metal concentrations in the soil samples represent geogenic or anthropogenic contamination. the second objective was to show that geochemical data of underlying sediments should be used as a tool in the determination of the potential hazard for soil contamination. for this purpose, the contents of as, cr, cu, hg, ni, pb and zn of soil samples were compared with standard values, a reference soil sample and local background values of the underlying sediments. the soil samples were unpolluted regarding the contents of as, hg, pb and zn. all samples had higher contents of cr and ni, whereas three samples had higher contents of cu than the limit standard values. geochemical parameters showed that the higher concentrations of cr, cu and ni in the soils could be attributed to geogenic impact. this conclusion was supported by the chemical proxy of alteration and chemical index of weathering values, which indicated intense weathering of the sediments. the obtained results showed that the kremna area is under slight to moderate hazard if a land use change would occur, and proved the importance of the geochemical composition of underlying sediments in the interpretation of heavy metal pollution. keywords: soils; sediments; geochemistry; heavy metals; pollution; weathering. * corresponding author. e-mail: ksenija@chem.bg.ac.rs; xenasyu@yahoo.com # serbian chemical society member. doi: 10.2298/jsc170914108p _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1088 perunović et al. introduction in natural systems, enrichment and depletion balance each other out. however, anthropogenic influences tend to enrichments in different parts of the system and the distribution function is skewed towards higher values.1 heavy metals are one of the serious pollutants in the natural environment due to their toxicity, persistence and bioaccumulation problems.2,3 assessment of the contamination status is regularly based on the quality guidelines. comparison of concentrations of heavy metals measured in samples and contents given by the quality guidelines provides data about the level of sample contamination. however, assessment of whether the measured heavy metal concentrations in samples represent geogenic or anthropogenic contamination is difficult based purely on such results. in order to overcome this problem, numerous geochemical parameters have been proposed. these indices are based on comparison of the contents of heavy metals in studied samples and a reference sample. it is very important to choose the most suitable reference sample for the studied area, which can be determined using geochemical or statistical methods.1,4 the geo-accumulation index (igeo)5 is expressed as follows: ngeo 2 n log 1.5   =     c i b (1) where cn represents measured concentration of a heavy metal in the sediment or soil sample and bn is the concentration of an element in the reference sample (background value). the factor 1.5 is incorporated in the equation to account for possible variation in the background data due to lithologic effects. the geoaccumulation index (igeo) scale5 consists of seven grades ranging from unpolluted to very strongly polluted: igeo < 0, unpolluted; igeo = 0–1, unpolluted to moderately polluted; igeo = 1–2, moderately polluted; igeo = 2–3, moderately to strongly polluted; igeo = 3–4, strongly polluted; igeo = 4–5, strongly to very strongly polluted; igeo > 5, very strongly polluted. the pollution load index (pli) was introduced by tomlinson et al. (1980).6 the pli is calculated using the following equation: pli = (cf1cf2cf3…cfn)1/n (2) where cf is the contamination factor and n is the number of determined metals. the contaminant factor cf is defined as: _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ potential hazard for soil contamination (kremna basin, serbia) 1089 metal backgroud = c cf c (3) where cmetal is the concentration of a metal in the sample and cbackground represents the background value for the same metal. a pli value > 1 implies pollution, whereas pli < 1 indicates no pollution.6 the enrichment factor, r is defined as the ratio:7 s back back − = c c r c (4) where cs is the content of metal in the sample, while cback is the concentration of the same metal in the reference sample. metals with r > 1 could be considered as indicators of anthropogenic metal pollution, whereas r < 1 indicates no pollution.7 the total enrichment factor (r) for each sample averages the enrichment factor (r) values of the all (n) indicator-metals as follow: r r n =  (5) r values exceeding 1.5 indicate high pollution, r values between 1.5 and 1 imply moderate pollution, whereas samples with r values below unity are considered as unpolluted or exposed to low pollution.7 hakanson (1980)8 suggested a contamination factor ( f ic ) and the degree of contamination (cd) to describe the contamination by heavy metals. f ic is given by: nf rn =i c c c (6) where nc is the mean content of a heavy metal in the investigated samples and crn is the reference value for a heavy metal. cd represents the sum of contamination factors for all analyzed metals and is given by:8 d f ic c=  (7) f ic <1 and cd <7 indicate a low degree of contamination; f ic in the range from 1 to 3 and cd in range from 7 to 14 indicate a moderate degree of contamination; f ic in the range from 3 to 6 and cd in range from 14 to 28 imply a considerable degree of contamination, whereas f ic >6 and cd >28 reflect a very high degree of contamination.8 in this study, the geochemical composition of soils and underlying sediments in the kremna basin were investigated (fig. s-1 of the supplementary material to this paper). this location was chosen, due to its importance as a potential _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1090 perunović et al. evaporite (magnesite) deposit and boron occurrence, as well as because of its proximity to the tara national park. the aim was to assess whether the observed heavy metal concentrations in the soil samples represent geogenic or anthropogenic impact. the second objective was to show that geochemical data of underlying sediments should be used as a tool in the determination of potential hazard for soil contamination. for this purpose, comparison was performed of the contents of heavy metals (as, cr, cu, hg, ni, pb and zn) of seven soil samples with standard values, a reference soil sample and the local background values of the underlying sediments. the results of the chemical composition of sixty soil samples surrounding the kremna basin (fig. s-1) were used for the calculation of the reference soil sample (table i). the local background values of the underlying sediments were calculated based on the contents of heavy metals in fortythree sediment samples from the borehole zlt-2 (depth from 11.5 to 343 m) of the kremna basin (fig. s-1; table s-i of the supplementary material to this paper). for assessment of geogenic (natural) and anthropogenic pollution of the soils, numerous geochemical parameters, explained above, were used. table i. contents of heavy metals in the reference soil sample (mg kg-1), investigated soil samples and reference standard values sample no. as cr cu hg ni pb zn the reference soil sample 3.87 89.21 16.76 0.14 423.27 48.10 52.28 1 2.59 365.74 19.84 0.00 1261.02 4.41 53.79 2 2.18 221.15 35.86 0.00 351.81 4.12 39.57 3 2.42 186.64 50.51 0.00 299.66 4.28 92.47 4 2.07 143.91 44.22 0.00 240.50 3.88 59.54 5 2.15 61.083 19.10 0.00 108.10 4.20 49.75 6 2.10 225.59 27.26 0.00 461.76 4.07 51.06 7 2.37 296.07 81.44 0.00 579.69 4.37 54.15 mean value 2.27 214.31 39.75 0.00 471.79 4.19 57.19 standards rs 88/20109 29 100 36 0.3 35 85 140 fbih 72/0910 15 100 65 1.0 40 80 150 önorm l 107511 20 100 50 1.0 40 100 150 experimental the soils were sampled at seven locations (see supplementary material). from each location, soil samples were taken with a small shovel from a surface area of 40 cm×40 cm×10 cm. each sample originally weighed 2.5 kg. in the laboratory, after removing vegetation and root remains, the samples were air dried at room temperature. soil samples were then mixed thoroughly and the quartering procedure was used to obtain a representative sample for analyses. the obtained representative samples were gently ground in an agate mortar and finally sieved through a 63-μm sieve. sediment samples were dried at 105 °c. in the next step, the samples were successively crushed to 2.36 mm in three stages using a jaw crusher, cone crusher and roller crusher, _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ potential hazard for soil contamination (kremna basin, serbia) 1091 respectively. then, the samples were homogenized. the rough milled fragmented sample was subsequently finely pulverized and sifted through a 63-μm sieve. about 0.1 g of soil or sediment sample was precisely weighed on an analytical balance. a mixture of 4 cm3 nitric acid (hno3, 65 %), 15 cm 3 hydrochloric acid (hcl, 37 %), 3 cm3 orthophosphoric acid (h3po4, 85 %) and 1 cm 3 hydrofluoric acid (hf, 50 %) was used for digestion of the samples. digestion was performed in an advanced microwave digestion system (ethos 1, milestone, italy) using an hpr-1000/10s high pressure segmented rotor. the temperature was controlled with a predetermined power program. the temperature was typically increased to 220 °c in the first 15 min. the temperature of 220 °c was maintained for an additional 20 min. and then cooled down rapidly. the contents of major elements and heavy metals were determined by inductively coupled plasma atomic emission spectrometry (icp-aes) using a thermo scientific icap 6500 duo icp (thermo fisher scientific, cambridge, uk) spectrometer equipped with an racid86 charge injector device (cid) detector, a pneumatic cross-flow type nebulizer, a quartz torch, and an alumina injector, which enabled samples containing hf in a small amount to be detected. the optical system was purged with argon and the echelle polychromator was thermostated at 38 °c. two multi-elemental plasma standard solutions (multi-element plasma standard solution 4, specpure®, 1000 µg ml-1 and a semi-quantitative standard 1, specpure®, 10 µg ml-1) and two single plasma standard solutions (silicon, specpure®, 1000µg ml-1 and titanium, specpure®, 1000µg ml-1) certified by alfa aesar gmbh & co kg, germany, were used to prepare the calibration solutions for the icp-aes measurements. two types of blanks were required for the analysis of the prepared samples. the calibration blank was used for establishing the analytical curve and the method blank was used to identify possible contamination resulting from either the reagents (acids) or the equipment used during sample processing. for each run, the samples were prepared in duplicate, and the icp-aes measurement for each digested sample was performed in triplicate. results and discussion heavy metal content in the reference soil sample the contents of trace elements in the reference soil sample are given in table i. the concentrations of all elements in the reference soil sample, with the exception of ni, were notably lower in comparison to the serbian regulation about the program of systematic monitoring of soil quality, indicators for risk assessment of soil degradation and methodology for development of remediation programs, rs 88/2010,9 the bosnian and herzegovinian regulation for the determination of permitted quantities of harmful and hazardous substances in the soil and methods of their investigation, fbih 72/0910 and the austrian standard, önorm l 107511 values (table i). the obtained concentration of ni was higher than all the values given in the considered standard. however, the local background value of 165.69 mg kg–1 for ni (table s-i), discussed below, is also higher than the standard values10,12–15 (table s-i), indicating that a high amount of ni in the reference soil sample is of geogenic origin. therefore, the reference soil sample could be considered as native and unpolluted and be used for an estimation of the difference between geogenic and anthropogenic impacts in the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1092 perunović et al. investigated soil samples. the elevated content of ni in the reference unpolluted soil sample (table i) shows the importance of the mineral and geochemical composition of the underlying sediments, which should be determined prior to any land use change, particularly in areas exposed to intense weathering. elevated cr and ni contents may result from ophiolites (ocean floor on land, which is usually rich in some heavy metals, such as cr and ni) occurring in the neighborhood (dinaric ophiolite belt).16 the same result was obtained in a recent investigation of sediments from the eastern posavina region.17 heavy metal content in background sediments the background levels of heavy metals in the underlying sediments were relatively uniformly distributed within borehole zlt-2, with exception of cr and ni, which were generally higher in the lower sedimentary sequence (below 200 m; table s-i). the contents of almost all heavy metals were lower compared to the limit standard values,10,12–15 with exception of cr and ni which were higher than the standard values (table s-i). elevated contents of cr and ni in the sediments are of geogenic origin and originated from the ultrabasic source rocks and serpentinites. according to the relatively uniform distribution of almost all the analyzed trace elements and their relatively low content (table s-i), it could be concluded that the sediments are unpolluted, derived predominantly from natural sediment sources. therefore, they could be used as local background levels for soil in this area to estimate the difference between geogenic and anthropogenic impacts, and to assess the enrichment of soil in heavy metals, which originate from the underlying sediments. despite the relatively low contents of almost all the heavy metals (table s-i), bearing in mind that the base and the edge of the kremna basin consist of ultrabasic rocks, serpentinite and ophiolitic mélange, which are all prone to weathering, the high concentrations of cr and ni, and the presence of as, cu, hg and pb in the borehole zlt-2 could be highly negative to the soil and water quality. this was confirmed by the very high values of the chemical proxy of alteration, cpa18 and the chemical index of weathering, ciw19,20 (> 80 and > 70 %, respectively), which remain continuously high even up to the depths of 150 m (table s-i). distribution of heavy metals in the soil samples from the kremna basin relatively thin soil (≈40 cm) developed over miocene sediments in the kremna basin. seven samples at different locations (fig. s-1) were taken from the surface soil horizon. the contents of heavy metals in the soils are listed in table i. comparison of these results with the limit standard values9–11 indicates that all samples have higher contents of cr (sample 5 being the exception) and ni (table i). moreover, three samples (3, 4 and 7; table i) show a higher content of cu than the limit _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ potential hazard for soil contamination (kremna basin, serbia) 1093 standard value indicated in the serbian regulation about the program of systematic monitoring of soil quality, indicators for risk assessment of soil degradation and methodology for the development of remediation programs, rs 88/20109 (table i). the contents of the other trace elements were below the standard values9–11 (table i). the obtained results are in a concordance with the observation of alexander (2013),21 that strong weathering of ultramafic rocks and leaching of elements from serpentinites result in high to very high concentrations of cr, cu, and ni in overlying soils, compared to the average data for world soils. the elevated contents of cr, cu and ni (table i) confirmed the notable influence of the geochemical composition of the underlying sediments on the heavy metal levels in soil. determination of geogenic and anthropogenic impact in soils for more precise estimations of the geogenic and potential anthropogenic impacts on the investigated soil samples, several indices, geo-accumulation index (igeo),5 pollution load index (pli),6 enrichment factor (r),7 total enrichment factor (r),7 contamination factor ( )fic 8 and degree of contamination (cd)8 were used. the values of mentioned indices were calculated related to both, the reference soil sample (table i) and background sediment levels (borehole zlt-2; table s-i). the results are presented in tables ii–v. table ii. values of geo-accumulation index (igeo) for the investigated soils, related to the reference soil sample and local background sediment level; n.d.: not determined due to concentration of hg being below detection limit soil sample no. as cr cu hg ni pb zn igeo, related to the reference soil sample 1 –1.17 1.45 –0.34 n.d. 0.99 –4.03 –0.54 2 –1.41 0.72 0.51 n.d. –0.86 –4.13 –0.99 3 –1.26 0.48 1.01 n.d. –1.08 –4.08 0.24 4 –1.49 0.10 0.81 n.d. –1.40 –4.22 –0.40 5 –1.43 –1.13 –0.40 n.d. –2.55 –4.10 –0.66 6 –1.47 0.75 0.12 n.d. –0.46 –4.15 –0.62 7 –1.29 1.15 1.70 n.d. –0.13 –4.05 –0.53 mean value –1.36 0.50 0.49 n.d. –0.78 –4.11 –0.50 igeo, related to local background sediment level 1 –1.74 1.11 0.87 n.d. 2.34 0.21 1.53 2 –1.99 0.38 1.73 n.d. 0.50 0.11 1.09 3 –1.84 0.14 2.22 n.d. 0.27 0.17 2.32 4 –2.07 –0.24 2.03 n.d. –0.05 0.03 1.68 5 –2.01 –1.47 0.82 n.d. –1.20 0.14 1.42 6 –2.05 0.41 1.33 n.d. 0.89 0.09 1.46 7 –1.87 0.80 2.91 n.d. 1.22 0.20 1.54 mean value –1.94 0.16 1.70 n.d. 0.57 0.14 1.58 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1094 perunović et al. the igeo values of the investigated sediments, related to both the reference soil sample and background levels from borehole zlt-2, are given in table ii. comparison of the igeo values calculated using the reference soil sample with the geo-accumulation index (igeo) scale5 (table ii) showed that the investigated samples were unpolluted regarding as, hg, ni, pb and zn. slight enrichment in almost all soil samples, in relation to the reference soil sample, was observed for cr and cu (igeo in range 0.1–1.45 and 0.12–1.70, respectively; table ii). the igeo values related to background sediment levels indicated that the investigated soil samples were unpolluted regarding as and hg. for samples 4 and 5, no pollution with cr and ni was detected. generally, the low mean values of igeo for cr and ni (0.16 and 0.57; table ii) imply a geogenic origin of these elements. slight enrichment was observed for pb, whereas moderate pollution is noticed for cu and zn in all samples, as well as for cr and ni in several samples (table ii). these results show the importance of recognition of the biochemical and mineral composition of the underlying sediments for further land usage and prevention of risk for serious soil and water contamination. table iii. values of pollution load index (pli) for the investigated soils, related to the reference soil sample and local background sediment level sample as cr cu hg ni pb zn pli, related to the reference soil sample soil samples 0.58 2.13 2.10 0.00 0.87 0.09 1.06 mean value 0.98 pli, related to local background sediment level soil samples 0.39 1.68 4.89 0.00 2.23 1.65 4.48 mean value 2.18 the values of the pollution load index (pli) higher than 1 (table iii) confirmed that the investigated soil samples were enriched in cr and cu in comparison to the reference soil sample, whereas no contamination with as, hg, ni, pb and zn was observed.6 on the other hand, the values of pli calculated in relation to the background sediment values showed moderate contamination with cr and pb and significant enrichment in cu, ni and zn, whereas pollution with as and hg was not detected (table iii).6 values of the enrichment factor (r),7 calculated using the reference soil sample showed the same result as the igeo and pli values (table iv). the soil samples were unpolluted regarding as, hg, ni, pb and zn, whereas slight enrichment was observed of the contents of cr and cu in almost all samples (table iv). in comparison to background sediment levels, significant increases were noticed for cu and zn in all samples and for cr and ni in a few samples (table iv). values of r below 1 indicated no pollution with as, cr (samples 2–6), hg and pb.7 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ potential hazard for soil contamination (kremna basin, serbia) 1095 table iv. values of enrichment factor (r) and total enrichment factor (r) for the investigated soils, related to the reference soil sample and local background sediment level soil sample no. as cr cu hg ni pb zn r, related to the reference soil sample 1 –0.33 3.10a 0.18 –1.00 1.98 –0.91 0.03 2 –0.44 1.48 1.14 –1.00 –0.17 –0.91 –0.24 3 –0.38 1.09 2.01 –1.00 –0.29 –0.91 0.77 4 –0.47 0.61 1.64 –1.00 –0.43 –0.92 0.14 5 –0.44 –0.32 0.14 –1.00 –0.74 –0.91 –0.05 6 –0.46 1.53 0.63 –1.00 0.09 –0.92 –0.02 7 –0.39 2.32 3.86 –1.00 0.37 –0.91 0.04 mean value –0.41 1.40 1.37 –1.00 0.11 –0.91 0.09 r, related to local background sediment level 1 –0.55 2.23 1.75 –1.00 6.61 0.74 3.35 2 –0.62 0.95 3.97 –1.00 1.12 0.62 2.20 3 –0.58 0.65 6.00 –1.00 0.81 0.69 6.47 4 –0.64 0.27 5.13 –1.00 0.45 0.53 3.81 5 –0.63 –0.46 1.65 –1.00 –0.35 0.65 3.02 6 –0.64 0.99 2.78 –1.00 1.79 0.60 3.13 7 –0.59 1.62 10.29 –1.00 2.50 0.72 3.38 mean value –0.61 0.89 4.51 –1.00 1.85 0.65 3.62 r, related to the reference soil sample 1 0.68 2 0.14 3 0.38 4 0.10 5 –0.39 6 0.14 7 0.88 mean value 0.28 r, related to local background sediment level 1 2.35 2 1.37 3 2.34 4 1.59 5 0.65 6 1.44 7 2.98 mean value 1.82 furthermore, the r values were use for the calculation of the total enrichment factor (r).7 the values of r related to the reference soil sample were notably lower than unity, indicating no anthropogenic impact.7 however, the r values related to the background sediment level indicated moderate to high enrichment, with the exception of sample 5 (table iv). this result is primary caused by sig_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1096 perunović et al. nificantly elevated contents of cu and zn in the soil samples in comparison to the underlying sediments. the values of contamination factor ( )fic 8 in the investigated soil related to the reference soil sample indicated a moderate degree of contamination in cr and cu and a slight enhancement in ni and zn, whereas no pollution with as, hg and pb was observed (table v). the contamination degree (cd)8 that summarizes all contamination factors for all investigated soil samples reached 7.65, implying a low to moderate enrichment in heavy metals in relation to the reference soil sample (table v). table v. values of contamination factors ( f ic ) and contamination degree (cd) for the investigated soils, related to the reference soil sample and local background sediment level sample as cr cu hg ni pb zn f , ic related to the reference soil sample soil samples 0.59 2.40 2.37 0.00 1.11 0.09 1.09 mean value 1.09 f ic , related to local background sediment level soil samples 0.39 1.89 5.51 0.00 2.85 1.65 4.62 mean value 2.41 cd, related to the reference soil sample soil samples 7.65 cd, related to local background sediment level soil samples 16.91 the contamination factors ( f ic ) related to the background sediment values showed moderate pollution with cr, ni and pb, considerable degrees of pollution by cu and zn and no pollution with as and hg. this resulted in a relatively high value of cd of 16.91, which indicates a considerable degree of contamination (table v).8 combining the results of all the mentioned parameters (tables ii–v), which showed very good agreement, it could be concluded that the investigated soil samples were unpolluted in terms of as, hg, ni, pb and zn, whereas slight enrichments in cr and cu were observed in comparison to the reference soil sample. considering the geological origin and geochemical composition of underlying sediments, which had been exposed to intense weathering, this result is rather related to geogenic than to anthropogenic impacts. numerous geochemical parameters calculated in relation to the background values of the underlying sediments also showed very consistent results (tables ii–v). no pollution of the soil samples with as, hg and pb were observed. a slight enrichment of the soils in comparison to the underlying sediments was registered for the cr content. significant increases in the concentrations of cu and zn were detected in all soil samples and of ni in samples 1 and 7. the ele_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ potential hazard for soil contamination (kremna basin, serbia) 1097 vated content of ni in these two samples could be attributed to their higher amount of organic matter. namely, sample 1 was taken from a thin soil layer developed over an outcropping coal layer, while sample 7 was taken from a more developed soil, dark in color, indicating a higher presence of organic matter. therefore, it could be assumed that in the kremna basin, cu and zn are the most prone to be readily incorporated into soils as result of weathering and leaching of ultramafic rocks and serpentinites. the obtained results showed the importance of the determination of the mineral and geochemical composition of underlying sediments prior to any land use change in order to prevent potential serious contamination of soil and water. conclusions soil samples from the kremna basin could be considered as unpolluted regarding the contents of as, hg, pb and zn. all the investigated samples had higher contents of cr and ni, whereas three samples showed higher contents of cu than the limit standard values given by the serbian regulation about the program of systematic monitoring of soil quality, indicators for risk assessment of soil degradation and methodology for the development of remediation programs. values of numerous geochemical parameters calculated in relation to the reference soil sample from this area and background values of the underlying sediments in the kremna basin (borehole zlt-2) indicated that the elevated concentration of cr and ni in the analyzed soils could be attributed to geogenic rather than to anthropogenic impact. this conclusion was support by the high values of chemical proxy of alteration (cpa) and chemical index of weathering (ciw), which indicated very intense weathering of the underlying rocks. values of the geo-accumulation index (igeo), pollution load index (pli), enrichment factor (r), total enrichment factor (r), contamination factor ( )fic and degree of contamination (cd), which indicated slight enrichment in cr and cu in comparison to the reference soil sample, and significant enrichment in cu and zn in comparison to sediment background values, imply that the kremna area is under slight to moderate hazard risk if a land use change would occur. physical deterioration and runoff that could easily occur would enhance the chemical weathering of deeper samples (below a depth of 10 m). in this way, heavy metals could get into soil and water. therefore, erosion control should be mandatory in this area. this study proved that besides topographic characteristics, climatic data, and soil properties, the mineral and geochemical composition of underlying sediments play an important role in the interpretation of the level of geogenic hazard in the investigated area and should be determined prior to any land use change. acknowledgements. investigations within this study were realized in cooperation with the company rio tinto–rio sava exploration from serbia. the study was supported by the min_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1098 perunović et al. istry of education, science and technological development of the republic of serbia (projects 176006 and 176016). dr. ivan anđelković, scientific associate of innovation center of the faculty of chemistry, university of belgrade, is greatly acknowledged for the icp-aes analysis. we are also grateful to the anonymous reviewers for useful comments and suggestions. и з в о д геохемијско испитивање као основа за утврђивање потенцијалног ризика од загађивања земљишта (басен кремна) tамара перуновић 1 , ксенија стојановић 1 , милица kaшaнин-грубин 2 , александра шајновић 2 , владимир симић 3 , бранимир јованчићевић 1 и илија брчески 1 1универзитет у београду, хемијски факултет, студентски трг 12–16, 11000 београд, 2универзитет у београду, центар за хемију, ихтм, његошева 12, 11000 београд и 3универзитет у београду, рударско–геолошки факултет, ђушина 7, 11000 београд испитиван је геохемијски састав земљишта и одговарајућих подинских седимената у басену кремна. циљ рада је био да се утврди да ли су концентрације тешких метала у узорцима земљишта последица природног или антропогеног загађења. други циљ рада је био да се покаже да геохемијски подаци о подинским седиментима морају бити разматрани при утврђивању ризика од загађивања земљишта. у ту сврху изведено је поређење садржаја as, cr, cu, hg, ni, pb и zn у земљишту са граничним стандардним вредностима, концентрацијама ових елемената у референтном нативном узорку земљишта и незагађеним подинским седиментима. узорци земљишта нису контаминирани as, hg, pb и zn. сви испитивани узорци земљишта имају већи садржај cr и ni, док је у три узорка запажен повeћaн садржај cu у односу на граничне стандардне вредности. геохемијски параметри су показали да су повишене концентрације cr, cu и ni у земљишту највероватније последица природног утицаја. овом закључку у прилог иду вредности хемијског индекса промена и хемијског индекса распадања које указују на интензивнo физичко и хемијско распадање седимената. добијени резултати показују да се подручје кремне налази под благим до умереним ризиком од било какве промене начина коришћења земљишта, и потврђују значај геохемијског састава подинских седимента при интерпретацији загађивања тешким металима. (примљено 17. септембра, ревидирано 31. октобра, прихваћено 6. новембра 2014) references 1. j. matschullat, r. ottenstein, c. reimann, environ. geol. 39 (2000) 990 2. h. pekey, environ. monit. assess. 123 (2006) 219 3. j. nouri, a. h. mahvi, a. babaei, e. ahmadpour, fluoride 39 (2006) 321 4. l. fok, m. r. peart, j. chen, catena 101 (2013) 212 5. g. müller, umschan 79 (1979) 778 6. d. c. tomlinson, j. g. wilson, c. r. harris, d. w. jeffery, helgol. wiss. meeresunters. 33 (1980) 566 7. g. adami, p. barbieri, e. reisenhofer, toxicol. environ. chem. 77 (2000) 189 8. l. hakanson, water res. 14 (1980) 975 9. http://www.sepa.gov.rs/download/uredba_o_programu_pracenja_kvaliteta_zemljista.pdf (in serbian, last accessed october 30, 2014) 10. http://www.uip-zzh.com/files/zakoni/poljoprivreda/72-09.pdf (in serbian, last accessed october 30, 2014) _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ potential hazard for soil contamination (kremna basin, serbia) 1099 11. önorm l 1075: standard values for inorganic elements in soils under agricultural and horticultural use, federal environment agency – austria, 2002 12. http://www.cis.org.rs/propisi/451?1338287527 (in serbian, last accessed october 30, 2014) 13. önorm s 2088-2: austrian standards on contaminated land management: risk assessment for polluted soil concerning impacts on surface environments, federal environment agency – austria, 2000 14. https://www.elaw.org/system/files/sediment_summary_table.pdf (last accessed october 30, 2014) 15. criteria for the assessment of sediment quality in quebec and application frameworks: prevention, dredging and remediation, environment canada and ministère du développement durable, de l’environnement et des parcs du québec, 2007, p. 7, 8 16. k. ustaszewski, s. m. schmid, b. lugović, r. schuster, u. schaltegger, d. bernoulli, l. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice theoretical study on the diels–alder reaction of bromo-substituted 2h-pyran-2-ones and some substituent vinyls j. serb. chem. soc. 80 (9) 1139–1148 (2015) udc 546.14’11+547.81+519.677+ jscs–4786 541.124:66.095.252.091.7:543.637 original scientific paper 1139 theoretical study on the diels–alder reaction of bromo-substituted 2h-pyran-2-ones and some substituent vinyls mina haghdadi*, hamed amani and nasim nab department of chemistry, islamic azad university, p. o. box 755, babol branch, babol, iran (received 5 december 2014, revised 8 february, accepted 8 february 2015) abstract: a dft study of the reactivity, regioand stereoselectivity of diels– –alder reactions between 3-bromo, 5-bromo, and 3,5-dibromo-2h-pyran-2 -ones and some weakly activated and unactivated alkenes was performed using the density functional theory (dft). four possible reaction channels, which are related to the formation of metaand paraand endoand exo-cycloadducts, were explored and characterized. the energy and natural bond orbital analysis showed that the meta-regioselectivity on the exo pathway was preferred and followed an asynchronous concerted mechanism with a polar nature in all diels–alder cycloadditions. moreover, the activation free energies of the diels– alder cycloadditions of 3,5-dibromo-2h-pyran-2-one were lower than those for 3-bromo-2h-pyran-2-one and 5-bromo-2h-pyran-2-one, which is in line with experimental observations. dft-based reactivity indices clearly predicted the regiochemistry of the isolated cycloadducts. keywords: bromo-2h-pyran-2-ones; dft study; reaction mechanism; reactivity indices; regioand stereoselectivity. introduction during investigations on the role of substituents on the cycloaddition reaction of 2h-pyran-2-ones, it was found that 3-bromo and 5-bromo-2h-pyran-2 -ones are the most interesting and unique, and have useful features.1–5 these two 2h-pyran-2-ones are ambident dienes3 and react with electron-rich, electron-poor and electron-neutral dienophiles with good regioand stereoselectivity.4,6,7 the cycloadditions of 2h-pyran-2-one itself are not selective.5 moreover, in contrast to the bromo-pyrones, 4-chloro-2h-pyran-2-one, in line with 2h-pyran-2-one itself, is neither ambident diene nor undergoes regioselective cycloadditions.8 it undergoes cycloadditions only with electron-deficient dienophiles that were stereoselective, but not regioselective.3a during the course of a study of 2h * corresponding author. e-mail: mhaghdadi2@gmail.com doi: 10.2298/jsc141205014h _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1140 haghdadi, amani and nab -pyran-2-ones, cho and co-workers investigated 3,5-dibromo-2h-pyran-2-one in diels–alder (da) reactions with a series of electronically and sterically distinct dienophiles.3b their results showed that it is a highly potent ambident diene, being more reactive and stereoselective than monobromo-2h-pyran-2-ones, and thus capable of generating a variety of bicycloadducts in much higher chemical yields and endo/exo rations than monobromo-2h-pyran-2-ones.3 afarinkia and co-workers studied the diels–alder reactions of 3and 5-halo -substituted 2h-pyran-2-ones with poor electron-rich, electron-rich and deficient dienophiles.7–9 their experimental results showed that these cycloadditions proceed with excellent regioselectivity and very good stereoselectivity. in contrast, the 4-halo-substituted-2h-pyran-2-ones reactions proceed with only moderate regioand stereoselectivity.9 furthermore, their results showed that the nature of halogen substituent had only a small, sometimes negligible, influence on the cycloaddition of 2h-pyran-2-ones, and also, in both the 3and 5-substituted series, the distribution of the products did not appear to be significantly different.8 therefore, changing the halogen substituent did not significantly change the electronic demand of the 3and 5-halo-substituted 2h-pyran-2-one, although it may influence their reactivity. furthermore, they performed a range of calculations on substituted 2h-pyran-2-one cyclo-additions, at the b3lyp/6-31g level of theory, to demonstrate the advantage of 3and 5-halo-substituted 2h-pyran-2 -ones over 4-halo-substituted 2h-pyran-2-ones.8,9 although there are many reports about the alternative synthetic routs,3–9 there are no theoretical investigations about the detailed molecular mechanism and electronic parameters. as a part of a program directed toward the investigation of related da cycloadditions, herein the results of a theoretical study on the mechanism of cycloaddition reactions between 3-bromo, 5-bromo and 3,5dibromo-2h-pyran-2-ones 1a–c, with a range of vinyl derivatives: vinyl acetate (2a), vinyl benzoate (2b), 2-ethenyl-1h-isoindole-1,3(2h)-dione (n-vinylphthalimide) (2c) and 2-propenenitrile (2d), to give the bridged bicyclic lactones 3–13 are presented (scheme 1). the purpose of the present study was to provide a better understanding the mechanistic features of these processes, especially by localization and characterization of all stationary points involved in these formally [2+4]cycloadditions. a density functional theory (dft) analysis was performed to explain both the exo/endo stereocontrol and regioselectivity of these processes in order to find a possible mechanism that may explain the different reactivity observed in each case. although da cycloadditions of 5-bromo and 3,5-dibromo-2h-pyran-2-ones with poor electron-rich dienophiles 2a–c were not prepared as part of experimental studies, the calculations based on them provided a better understanding of the trends, differences, and similarities between halogen substituted 2h-pyran -2-ones. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ theoretical study on the diels-alder reaction 1141 scheme 1. the calculated possible reaction channels for the da reaction of bromo-substituted 2h-pyran-2-ones 1a–c with the vinyl derivatives 2a–d at the b3lyp/cc-pvdz level. computational details the density functional theory calculations were realized using the gaussian 09 package.10 the relative energies and free energies were computed at 298 k for the various stationary points at the b3lyp/cc-pvdz level. the electronic structures of the stationary points were analyzed by the natural bond orbital (nbo) method.11 the global reactivity indexes were estimated according to the equations recommended by parr.12 the global electrophilicity index, ω, is given by the following expression:13 2 2 μ ω η = (1) in terms of the electronic chemical potential, μ, and the chemical hardness, η. both quantities may be approached in terms of the one-electron energies of the frontier molecular orbitals homo and lumo, ɛh and ɛl,14 as: h l( ) / 2μ ε ε= + (2) l hη ε ε= − (3) recently, domingo introduced an empirical (relative) nucleophilicity index, n,15 based on the homo energies obtained within the kohn sham scheme,13 and defined as: homo homo(nu) (tce)ε ε− (4) nucleophilicity is referred to tetracyanoethylene (tce), because it presents the lowest homo energy in a large series of molecules already investigated within the context of polar cycloadditions. this choice allows the convenient handling of a nucleophilicity scale of positive values. recently, domingo proposed two new electrophilic, kp + , and nucleophilic, kp − , parr functions based on the atomic spin density distribution at the radical anion and cation of a neutral molecule.16 the electrophilic, kp + , and nucleophilic, kp − , parr functions, were obtained through the analysis of the mulliken atomic spin density of the radical anion and cation by single-point energy calculations over the optimized neutral geometries using the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1142 haghdadi, amani and nab unrestricted ub3lyp formalism for radical species. the local electrophilicity indices, ωk,17 the local nucleophilicity indices, nk,198 were calculated using the following expressions: k k pω ω += (5) k kn np −= (6) where kp + and kp − are the electrophilic and nucleophilic parr functions, 16 respectively. results and discussions in the present study, the regioand stereoselectivity of the cycloaddition reaction between bromo-substituted 2h-pyran-2-ones 1a–c and vinyl substituents 2a–d were studied, and then an analysis based on the reactivity indices of stationary points was performed. study of the da reactions of bromo-substituted 2h-pyran-2-ones 1a–c with some vinyl derivatives (2a–d) due to the asymmetry of bromo-substituted 2h-pyran-2-ones 1a–c, four regio-isomeric channels are feasible for each of the da reactions, meta and para, which are related to the endo and exo approach modes of the diene systems 1a–c relative to the r group of the vinyl compounds 2a–d (scheme 1). analysis of the stationary points associated with these da reactions indicated that they could occur via a one-step mechanism and consequently, four stereoisomeric tss, named ts1, ts2, ts3, and ts4, and the corresponding products 3–13 were located and characterized. the activation and relative energies associated with these stationary points are given in table i. analysis of the geometries at the ts structures shows that the tss of meta pathways correspond to asynchronous bond formation processes. the extent of bond formation along a reaction pathway is provided by the concept of bond order (bo).19 these values are within the range of 0.180 to 0.636. these results show that for all da reactions, ts1 and ts2 (meta pathways) are more asynchronous than ts3 and ts4 (para pathways), and that the tsca, tscb and tscc (for the n-phthalimide substituent) are the most asynchronous ones. the asynchronicity shown by the geometrical data is accounted for by the bo values. furthermore, the asynchronicity in bond formation at the tss measured by ∆r = (r2–r1) ranges from 0.72 to 1.10 at ts1 and ts2, indicating that the tss of meta process correspond to highly asynchronous bond-formation processes. natural population analysis (npa)11 allowed the evaluation of the charge transfer (ct) along these da reactions, at the tss. charge transfer (ct) plays a relevant role in most of organic reactions. in fact, in diels–alder reactions, the ct value is one of the most relevant characteristics of their transition states (tss) and, in most cases, it is responsible of the height of their energy barrier. the calculated ct values for these da reactions are given in fig. 1. in general, the ct values in the tss associated with the para pathways were lower than 0.090 e, in clear agree _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ theoretical study on the diels-alder reaction 1143 table i. activation energies, δe#, activation free energies, ∆g#, and reaction energies, ∆er, (all in kj mol-1), with the formation of da cycloadducts between bromo-substituted 2h-pyran-2-ones 1a–c and vinyl derivatives 2a–d in the meta pathways entry species ts ∆e# ∆g# ∆er 1 1a+2a→3a-exo ts1aa 108.48 166.33 –48.53 2 1a+2a→4a-endo ts2aa 112.94 169.31 –46.22 3 1a+2b→3b-exo ts1ab 112.12 168.26 –49.94 4 1a+2b→4b-endo ts2ab 116.66 170.56 –45.98 5 1a+2c→3c-exo ts1ac 106.75 163.59 –28.80 6 1a+2c→4c-endo ts2ac 112.64 168.23 –27.73 7 1a+2d→3d-exo ts1ad 117.59 171.64 –29.45 8 1a+2d→4d-endo ts2ad 107.65 161.71 –33.67 9 1b+2a→5a-exo ts1ba 92.82 150.75 –69.94 10 1b+2a→6a-endo ts2ba 102.04 157.18 –65.45 11 1b+2b→5b-exo ts1bb 98.36 154.17 –68.60 12 1b+2b→6b-endo ts2bb 100.47 155.97 –66.38 13 1b+2c→5c-exo ts1bc 64.58 120.92 –75.89 14 1b+2c→6c-endo ts2bc 76.74 131.41 –71.40 15 1b+2d→5d-exo ts1bd 108.51 162.17 –51.16 16 1b+2d→6d-endo ts2bd 100.17 153.82 –54.83 17 1c+2a→7a-exo ts1ca 94.23 151.39 –62.01 18 1c+2a→8a-endo ts2ca 100.63 156.30 –59.89 19 1c+2b→7b-exo ts1cb 98.40 153.68 –63.50 20 1c+2b→8b-endo ts2cb 100.22 155.34 –59.66 21 1c+2c→7c-exo ts1cc 91.26 148.47 –43.06 22 1c+2c→8c-endo ts2cc 101.83 157.25 –41.22 23 1c+2d→7d-exo ts1cd 106.23 160.32 –41.75 24 1c+2d→8d-endo ts2cd 98.22 152.36 –45.00 ment with the non-polar character of these pathways. on the other hand, the ct values at the tss of the da reactions of 1a–c and 2a–c in the most favorable regioisomeric pathways (meta–exo), were between 0.205 and 0.157 e, which indicate the polar nature of the meta channels in these da reactions. only the most unfavorable da reactions of 1a–c and 2d presented low ct values (lower than 0.050 e). these results with the proposal that for the da reactions of 1a–c with 2a–d, an increase in the polar character as the reaction proceeds is accompanied by an acceleration of the reaction.7–9 the energy barrier (∆e#) and activation gibbs free energy values (∆g#), related to the occurrence of transition states for the da reactions of 1a–c with 2a–d are lower for the meta approaches than those for the para ones (table i). the measured stereoselectivity indicated that the meta–exo cyclization modes are more favorable than the meta–endo ones, leading to the formation of meta–exo adducts for the da reactions of 1a–c with 2a–c, while the lowest barrier energies for the da reactions of 1a–c with 2d occur on the meta–endo pathway, which yields the meta–endo cycloadducts 4d, 6d and 8d. therefore, the presence of a cyano group _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1144 haghdadi, amani and nab ts2ab ts1ab ts2aa ts1aa ts2ad ts1ad ts2ac ts1ac ts2bb ts1bbts2ba ts1ba ts2bd ts1bd ts2bc ts1bc ts2cb ts1cbts2ca ts1ca ts2cd ts1cdts2cc ts1cc fig. 1. optimized geometries (b3lyp/cc-pvdz) of the transition structures involved in the meta pathways of the da reactions between the bromo-substituted 2h-pyran-2-ones 1a–c and the vinyl derivatives 2a–d. the bond distances are given in å, the wiberg bond indices are given in parenthesis and the natural charges (ct) of the tss are also given. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ theoretical study on the diels-alder reaction 1145 on the dienophile, neither changes the stereoselectivity (exo to endo), nor increase the energy barriers relative to the other dienophiles. on the other hand, the results of energy values in table i showed that the da reactions of 1a–c with 2a and 2b are less stereoselective than the cycloadditions to dienophiles 2c and 2d. these differences in stereoselectivity could be explained as follows. the lack of stereoselectivity in the cycloadditions of vinyl acetate 2a presumably arises from a lack of strong secondary orbital interactions, suggesting that the cycloaddition to the weakly activated dienophile may be much more susceptible to steric interaction.7 this was confirmed from the results of the cycloadditions of 2-ethenyl-1h-isoindole-1,3(2h)-dione (2c), where the reactions are highly exo selective. here, the steric congestion arises directly from an unfavorable steric interaction between the second nitrogen substituent and the bromine atom in the ts, leading to the endo cycloadduct. therefore, ts1ac, ts1bc and ts1cc leading to the exo cycloadduct are favored. this does not arise in the ts of endo cycloadduct of vinyl benzoate 2b since the benzoate group can swing away from the bromine in the transition state. moreover, the endo predomination in the cycloaddition of 2-propenenitrile (2d) is attributed to secondary orbital interactions and therefore it was not expected that cycloaddition to the bromo2h-pyran-2-ones 1a–c would give an endo to exo ratio of nearly one. as can be seen in table i and scheme 1, it is possible to correlate the calculated energy of the transition state to the final yield of the cycloadducts 3–13. the calculated values of all transition states confirmed that the ones likely to be the most abundant are the 3, 5 and 7 isomers in all cases, which occurred in the da reactions of 1a–c with 2c. the cycloadditions of 1a–c with 2d in all of reactions had the highest relative energy and it was expected to be the most disfavored cycloadduct. the results of calculated free activation energies (∆g#) for da reactions of 3,5-dibromo-2h-pyran-2-one (1c) with 2a, 2b and 2d demonstrate the lowest activation free energy, while an increasing barrier energy has been seen for 1c and 2c.with considering fmo approach (table ii), broadly speaking, 3-bromo, 5-bromo and 3,5-dibromo-2h-pyran-2-one should undergo normal and inverse electron demand cycloadditions with dienophiles bearing weakly electron-donating (1a–c) and electron-withdrawing (2d) substituents, respectively. dft-based reactivity indices the molecular dft-based parameters, electronic chemical potential (μ), chemical hardness (η), global electrophilicity (ω) and global nucleophilicity (n) of the reactants 2a–d and 1a–c are displayed in table ii. as can be seen in table ii, the bromo-2h-pyran-2-one derivatives 1a–c are more electrophilic than the dienophiles 2a–d and 3,5-dibromo-2h-pyran-2-one _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1146 haghdadi, amani and nab (1c) with the highest electrophilicity (ω = 2.43 ev) being classified as a strong electrophile on the electrophilicity scale.20 on the other hand, 1a has a high nucleophilicity index, n = 2.52 ev, and thus is classified as a strong nucleophile on the nucleophilicity scale.21 this ambiphilic behavior is the consequence of the presence of the enone and oxygen atom inside 1a–c. the electronic chemical potential (μ) of the bromo-2h-pyran-2-one derivatives 1a–c are lower than those of the dienophiles, 2a (–0.134), 2b (–0.156) and 2c (–0.130), indicating that charge transfer along the corresponding reactions will occur from the dienophiles 2a–c to the electron deficient dienes 1a–c. while as expected, a cn group (2d) decreases the chemical potential and increases the electrophilicity toward the dienophiles 2a–d, and hence, these results are in agreement with the increase in the activation energy. table ii. homo and lumo energies, electronic chemical potential, µ, chemical hardness, η, (all in a.u.), global electrophilicity, ω, and nucleophilicity, n (both in ev), for the reactants obtained at the b3lyp/cc-pvdz level of theory species ehomo elumo µ η ω n 1a –0.24366 –0.07747 –0.160 0.166 2.09 2.52 1b –0.24586 –0.07990 –0.162 0.166 2.15 2.40 1c –0.24777 –0.08881 –0.168 0.158 2.43 2.41 2a –0.25309 –0.01470 –0.134 0.238 1.02 2.27 2b –0.25205 –0.06044 –0.156 0.192 1.71 2.29 2c –0.24195 –0.01862 –0.130 0.223 1.03 2.57 2d –0.26633 –0.08384 –0.178 0.182 2.28 1.90 the polar character of a cycloaddition process can be predicted using the electrophilicity difference of the reaction pair, ∆ω.22 in this sense, the electrophilicity differences between the diene 1c and the dienophiles 2a and 2c are about 1.40, indicating a large polar character for these cycloadditions, while the small ∆ω between 1a and 2b (0.38 ev) and between 1b and 2d (0.21 ev) show a low polar character for these cycloaddition reactions. the parr indices, local electrophilicity indices and local nucleophilicity indices for the atoms c6 and c3 of the pyrones 1a–c, and c7 and c8 of the dienophiles 2a–d are given in table iii (see scheme 1 for atom numbering). the parr functions (the electrophilic, kp + , and nucleophilic, kp − ) were computed based on mulliken atomic spin density analysis. according the domingo model,15,17 along a polar cycloaddition involving asymmetric reagents, the most favorable reactive channel is that involving the initial two-center interaction between the most electrophilic center (ωk) at the electrophile and the most nucleophilic center (nk) at the nucleophile. according to this model, in the cycloaddition reactions of 1a–c with dienophiles 2a–d, the most favorable two-center interaction occurs between c6 of the dienes and c8 of _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ theoretical study on the diels-alder reaction 1147 dienophiles 2a–d, leading to the formation of the 3–13 regioisomers, which is in agreement with the experimental results.7–9 table iii. the parr functions ( k k,p p − + / au), local electrophilicity indices (ωk / ev) and local nucleophilicity (nk / ev) indices for the c6 and c3 atoms of the pyrones 1a–c and for atoms c7 and c8 of the dienophiles at the reactive sites for the reactants obtained at the b3lyp/cc -pvdz level of theory species k kp − kp + nk ωk 1a c6 0.188 0.381 0.474 0.795 c3 0.253 0.204 0.639 0.426 1b c6 0.394 0.205 0.945 0.492 c3 0.232 0.264 0.558 0.634 1c c6 0.240 0.384 0.580 0.932 c3 0.188 0.222 0.453 0.540 2a c7 0.181 0.171 0.410 0.174 c8 0.291 0.548 0.660 0.559 2b c7 0.018 0.074 0.040 0.127 c8 0.092 0.357 0.212 0.610 2c c7 0.009 0.037 0.024 0.038 c8 0.019 0.476 0.050 0.490 2d c7 0.219 0.260 0.146 0.594 c8 0.606 0.426 1.152 0.972 conclusions dft computations using the b3lyp functional in conjunction with the cc-pvdz basis set were used to analyze the outcome of the da reactions of the bromo-2h-pyran-2-ones 1a–c with some weakly activated and unactivated vinyls. the following conclusions could be inferred from the results of the energies: i. the activation energies associated with the da reaction of cyclic dienes 1a–c with dienophile 2c is more favorable than those for the reactions with 2a, b and 2d. the low reactivities of the dienophiles in these da reactions correspond with their nucleophilic character. ii. while the da reactions with 2a–c are exo selective, the reaction with 2d is endo selective. iii. 3,5-dibromo-2h-pyran-2-one is more active than 3and 5-bromo-2h -pyran-2-ones, having a lower energy barrier. iv. these da reactions proceed via a polar, regioselective and highly asynchronous process. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1148 haghdadi, amani and nab и з в о д теоријска студија дилс–алдерове реакције бромо-супституисаних 2h-пиран-2-она и неких супституисаних винила mina haghdadi, hamed amani и nasim nab department of chemistry, islamic azad university, p. o. box 755, babol branch, babol, iran извршено је dft испитивање реактивности, региои стереоселективности дилс– –алдерове реакције између 3-бромо, 5-бромо и 3,5-дибромо-2h-пиран-2-она и неких слабо активираних и неактивираних алкена. истражена су четири могућа реакциона пута, који обухватају формирање мета-, пара-, ендои егзо-циклоадукта. анализа заснована на енергији и природним орбиталама показује да је преферирана мета-региоселективност и егзо-реакциони механизам. (примљено 5. децембра 2014, ревидирано 8. фебруара, прихваћено 8. фебруара 2015) references 1. k. afarinkia, t. d. nelson, m. v. viader, g. h. posner, tetrahedron 48 (1992) 9111 2. b. t. woodward, g. h. posner, adv. cycloaddit. 5 (1999) 47 3. a) c.-g. cho, y.-w. kim, y.-k. lim, j.-s. park, h. lee, s. koo j. org. chem. 67 (2002) 290; b) c.-g. cho, j.-s. park, i.-h, jung, h. lee, tetrahedron lett. 42 (2001) 1065 4. g. h. posner, t. d. nelson, c. m. kinter, k. afarinkia, tetrahedron lett. 32 (1992) 5295 5. k. afarinkia, g. h. posner, tetrahedron lett. 33 (1992) 7839 6. g. h. posner, k. afarinkia, h. dai, org. synth. 73 (1995) 231 7. k. afarinkia, n. t. daly, s. gomez-farnos, s. joshi, tetrahedron lett. 83 (1997) 2369 8. k. afarinkia, m. j. bearpark, a. ndibwami, j. org. chem. 70 (2005) 1122 9. k. afarinkia, m. j. bearpark, a. ndibwami, j. org. chem. 68 (2003) 7158 10. gaussian 09, revision a, gaussian, inc., wallingford, ct, 2009 11. a. e. reed, r. b. weinstock, f. weinhold, j. chem. phys. 83 (1985) 735 12. r. g. parr, r. g. pearson, j. am. chem. soc. 105 (1983) 7512 13. r. g. parr, l. von szentpaly, s. liu, j. am. chem. soc. 121 (1999) 1922 14. r. g. parr, w. yang, density functional theory of atoms and molecules, oxford university press, new york, 1989, p 16 15. l. r. domingo, p. pérez, j. org. chem. 73 (2008) 4615 16. l. r. domingo, p. pérez, j. a. saez, rsc adv. 3 (2013) 1486 17. l. r. domingo, m. j. aurell, p. pérez, j. phys. chem., a 106 (2002) 6871 18. p. pérez, l. r. domingo, m. duque-noreña, e. chamorro, j. mol. struct.: theochem 895 (2009) 86 19. k. b. wiberg, tetrahedron 24 (1968) 1083 20. l. r. domingo, m. j. aurell, p. perez, r. contreras, tetrahedron 58 (2002) 4417 21. i. kim, k. a. hoff, e. t. hessen, t. haug-warberg, h. f. svendsen, chem. eng. sci. 64 (2009) 2027 22. h. chemouri, s. m. mekelleche, int. j. quantum chem. 112 (2012) 2294. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice facile synthesis and antifungal activity of dithiocarbamate derivatives bearing an amide moiety j. serb. chem. soc. 80 (11) 1367–1374 (2015) udc 547.496.2–327:542.913:615.282–188 jscs–4803 original scientific paper 1367 facile synthesis and antifungal activity of dithiocarbamate derivatives bearing an amide moiety yu-wen li* and shu-tao li school of chemistry and pharmacy, qingdao agricultural university, qingdao, p. r. china (received 14 january, accepted 24 may 2015) abstract: two series of novel dithiocarbamate derivatives bearing an amide moiety, 3a–i and 4a–i, were synthesized by a facile method, and the structures of the derivatives were confirmed by elemental analysis and 1h-nmr, 13c -nmr and high-resolution mass spectrometry (hrms). their antifungal activity against five phytopathogenic fungi were evaluated, and the results showed that most of the target compounds displayed low antifungal activity in vitro against gibberella zeae, cytospora sp., colletotrichum gloeosporioides, alternaria solani, and fusarium solani at a concentration of 100 mg l-1. however, two compounds, 4f and 4g, exhibited significant activity against a. solani and c. gloeosporioides, respectively. keywords: antifungal activity; dithiocarbamate derivatives; amide moiety; synthesis. introduction plant disease arising from phytopathogenic fungi is one of the major causes of severe losses in agriculture and horticulture crop production worldwide, and poses a threat to global food security.1 in the past years, fungicides have contributed enormously to reduce crop loss caused by phytopathogenic fungi. however, the main problem associated with the application of fungicides is the emergence of fungicide resistance. therefore, it is necessary to develop efficient fungicides with novel structures to obviate this resistance. due to their multiple biological profiles, carboxylic acid amide compounds have found application not only in medicinal chemistry but also in pesticide chemistry, i.e., as insectides,2–4 fungicides,5 herbicides6 and plant growth regulators.7 dithiocarbamate derivatives, on the other hand, have played important roles in medicinal and pesticide chemistry because of their diverse activities.8–11 in pesticide chemistry, dithiocarbamate derivatives have also served as fungicides.12 an important strategy for drug discovery has emerged that consists of * corresponding author. e-mail: ywli@qau.edu.cn doi: 10.2298/jsc150114047l 1368 li and li hybridizing two bioactive molecules or pharmacophores to generate a novel class of molecules with a potentially stronger bioactivity profile.13,14 thus, inspired by the biological importance of carboxylic acid amides and dithiocarbamates as fungicides in the pesticide field, herein, the synthesis of novel carboxylic acid amide–dithiocarbamate hybrids 3a–i and 4a–i and their antifungal activities are reported. results and discussion chemistry in this study, the starting materials, 2-chloroacetamides 2a–i, were prepared according to the literature15 with some modifications (scheme 1). the intermediates 2a–g were synthesized by treatment of 2-chloroacetyl chloride with 1a–g, respectively, in a mixture of acetic acid and a saturated solution of sodium acetate. however, this procedure was unsuitable for the synthesis of intermediates 2h and 2i due to difficulties encountered in the separation and purification of the liquid 2h and 2i from the liquid mixture of hoac and naoac. thus, intermediates 2h and 2i were synthesized by reaction of 2-chloroacetyl chloride with 1h and 1i, respectively, in the presence of triethylamine as acid-scavenger and dichloromethane as solvent. with intermediates 2a–i available, the target compounds 3a–i and 4a–i were synthesized. however, conventional synthesis of dithiocarbamates involves costly and toxic chemical reagents, such as thiophosgene.16 current strategies could partially alleviate the expense and toxicity for the synthesis by several one-pot syntheses via the reaction of carbon disulfide with amine and alkyl halides or acrylates.17 still, several drawbacks of these one -pot procedures remained inevitable, such as the use of strong bases, high reacclch2cocl + h2nr naoac hoac clch2conhr clch2cocl + yhn ch2cl2 (c2h5)3n n y o cl 1a-1g 2a-2g 1h-1i 2h-2i 2h: y = ch2 2i: y = o no2 f cl cl oh a b c d e f g r = scheme 1. synthesis of intermediates 2a–i. synthesis and antifungal activity of dithiocarbbamates 1369 tion temperatures, long reaction time, and harmful organic solvents.18 to circumvent the drawbacks associated with the previous procedures for the preparation of dithiocarbamates, the target compounds 3a–i and 4a–i were prepared according to scheme 2. scheme 2. synthesis of target compounds 3a–i and 4a–i. as illustrated in scheme 2, the synthesis method was improved such that the reactions of carbon disulfide, triethylamine and piperidine or morpholine were realized at 0 °c to obtain intermediates 3 and 4, respectively, as white solids. to the thus-obtained intermediates 3 and 4, absolute ethanol was added in situ, leading to the respective suspensions of 3 and 4 in absolute ethanol. subsequently, reaction of the suspended 3 and 4 in ethanol with the intermediates 2a–i at 50 °c led to the generation of the target compounds 3a–i and 4a–i, respectively. upon completion of the reaction, 3a–i and 4a–i were in situ precipitated by cooling the corresponding reaction mixture down to 0 °c and collected by filtration. since more or less 3a–i or 4a–i remained in the mother liquor, more sequential steps were indispensable to recover the residual 3a–i and 4a–i from their corresponding mother liquor. firstly, evaporation of the mother liquor to dryness afforded a solid mixture containing the side-product triethylamine hydrochloride and residual 3a–i and 4a–i, respectively. furthermore, given that triethylamine hydrochloride is not soluble in ether solvents while 3a–i and 4a–i are, the addition of 2-methyltetrahydrofuran to the thus-obtained solid mixtures left a white precipitate of triethylamine hydrochloride, a useful reagent in drug synthesis,19 that was removed by filtration. the filtrates were then concentrated under reduced pressure to give the respective residuals 3a–i and 4a–i. finally, recrystallization of the residuals 3a–i and 4a–i from 95 % ethanol led to the desired target compounds. the notable advantages of the present procedure over previous ones are that it is easy to conduct in terms of generation, separation, and purification of the target compounds 3a–i and 4a–i and is readily performed in one pot with only 1370 li and li one solvent by modulating the temperature in the range from 50 to 0 °c. in addition, taking the recovered 3a–i and 4a–i into account, the overall yields of the products were almost quantitative. antifungal activity: in vitro screening of compounds 3a–3i and 4a–4i all the newly synthesized target compounds 3a–i and 4a–i were evaluated in vitro for their antifungal activity against five phytopathogenic fungi, i.e., gibberella zeae, cytospora sp., colletotrichum gloeosporioides, alternaria solani and fusarium solani at concentration of 100 mg l–1. as summarized in table i, most target compounds displayed low antifungal activities against these five phytopathogenic fungi at the indicated concentration with exception of compounds 4f and 4g. compound 4f displayed 77.26 % inhibition of a. solani, while compound 4g exhibited 74.87 % inhibition of c. gloeosporioides at a concentration of 100 mg l–1. to understand further the role of different groups of the compounds in conferring the antifungal activity, it is necessary to compare their structures. structurally, compounds 3a–i and 4a–i are derived from same scaffold but with different substituents, leading to the difference in the antifungal activity. generally, compounds 4a–4i were superior to the corresponding compounds 3a–i in terms of their antifungal activity, suggesting that the presence of morpholinyl group in 4a–i conferred better antifungal activity than the corresponding pipertable i. fungicidal activities of the target compounds 3a–i and 4a–i at a concentration of 100 mg l-1; hy – hymexazol compound antifungal activity (inhibition rate, %) g. zeae a. solani c. gloeosporioides cytospora sp. f. solani 3a 3b 33.41 30.32 24.52 23.48 20.25 24.97 5 42.13 26.75 24.46 3c 34.42 21.47 14.81 36.29 34.79 3d 39.52 41.23 38.65 39.49 36.13 3e 21.78 30.14 23.41 31.73 35.47 3f 43.97 46.13 40.94 49.78 41.64 3g 40.27 43.94 39.08 46.66 39.35 3h 19.24 23.56 34.75 28.91 14.34 3i 27.88 29.76 30.51 32.14 30.09 4a 41.77 36.72 38.13 32.43 38.19 4b 46.18 39.76 30.67 51.09 32.08 4c 40.07 28.94 34.96 46.81 40.11 4d 47.14 45.97 47.08 52.13 43.93 4e 23.93 35.46 26.43 33.19 39.86 4f 58.11 77.26 59.04 57.89 53.98 4g 62.93 51.67 74.87 60.09 56.89 4h 30.08 32.77 47.69 38.06 45.77 4i 31.18 35.26 43.22 39.18 34.17 hy 90.56 82.15 53.78 46.69 79.23 synthesis and antifungal activity of dithiocarbbamates 1371 idinyl group in compounds 3a–i. more interestingly, compounds 4f and 4g, bearing two substituents on the benzene ring, displayed significantly higher antifungal activities relative to the compounds with a single substituent on the benzene ring. in addition, the compounds with fluorine substituent on the benzene ring regardless of the substituent on the carbamic moiety, such as 3d and 4d, showed higher antifungal activities (although not dramatic) compared to other corresponding compounds without fluorine substituent on the benzene ring. although most of the target compounds display low inhibition rate against mycelia growth of these five tested fungi at concentration of 100 mg/l, target compounds 4f–g could be potential lead structures for further discovery of novel antifungal agrochemicals. experimental chemistry all the employed chemicals were obtained from qingdao justness reagent company (china) and used without further purification. the melting points were measured using a wrs-1b digital melting point apparatus. the 1h-nmr spectra were recorded on a bruker drx-400 advance spectrometer at 400 mhz using tms as an internal standard. the physical, analytical and spectral data of the synthesized compounds are given in the supplementary material to this paper. general procedure for the synthesis of compounds 2a–g a substituted aniline (34.3 mmol) was added to 12.5 ml of a saturated solution of sodium acetate, followed by 12.5 ml acetic acid. the suspension was cooled to 0 °c, and 2chloroacetyl chloride (34.4 mmol, 2.75 ml) was added dropwise to the suspension at ≤5 °c. during the addition of 2-chloroacetyl chloride, the suspension dissipated and the mixture clarified. before the addition of 2-chloroacetyl chloride was complete, a white precipitate began to form. upon completion of the addition, the heterogeneous mixture was brought to 25 °c and stirred at room temperature for 2 h. the white precipitate was filtered, washed with distilled water (2×5 ml) and dried under vacuum to afford the crude product. recrystallization of crude 2a–g from absolute ethanol gave the desired 2a–g, respectively. the physical, analytic and spectral data of 2a–g are summarized in the supplementary material to this paper. general procedure for synthesis of compounds 2h and 2i to a stirred solution of triethylamine (17.2 mmol, 2.5 ml) and 17.2 mmol piperidine 1h (or morpholine 1i) in 10 ml ch2cl2, 2-chloroacetyl chloride (17.2 mmol, 1.38 ml) was added dropwise at 0 °c. upon completion of the dropwise addition, the solution was brought to room temperature and stirred for 2 h. subsequently, the resulting solution was diluted with 20 ml ch2cl2 and successively washed with 20 ml water and 20ml brine. the ch2cl2 layer was separated and concentrated under reduced pressure to give crude 1h (or 1i) which was further purified by chromatography to afford the desired 1h (or 1i) as a yellowish oil. the physical, analytical and spectral data of 2h and 2i are summarized in the supplementary material to this paper. 1372 li and li general procedure for the synthesis of compounds 3a–i and 4a–i to a solution of triethylamine (17.2 mmol, 1.5 ml) and 17.2 mmol piperidine 1h (or morpholine 1i), carbon disulfide (18.9 m mol, 1.2 ml) was added dropwise at 0 °c to form a white solid 3 (or 4). upon completion of the addition, 15 ml absolute ethanol was added to form a slurry of the white solid 3 (or 4), to which 17.2 mmol respective intermediate 2a–i was added. the mixture was heated to 50 °c to afford a clear solution. the clear solution was kept stirring at 50 °c for 3 h and then cooled to 0 °c. the cooled solution was kept for 2 h at 0 °c and the precipitate of 3a–i (or 4a–i) was collected by filtration and the corresponding filtrate was evaporated to dryness leading to a solid mixture of triethylamine hydrochloride and residual 3a–i (or 4a–i). then, the addition of 2-methyltetrahydrofuran to the thus obtained solid mixture led to triethylamine hydrochloride by filtration. the corresponding filtrate was concentrated under vacuum to give the residual 3a–i (or 4a–i). recrystallization of thus obtained residual 3a–i (or 4a–i) from 95 % ethanol led to the desired 3a–i (or 4a–i). taking the recovered 3a–i and 4a–i into account, the overall yield of 3a–i and 4a–i was almost quantitative. the physical, analytical and spectral data for 3a–i and 4a–i are summarized in the supplementary material to this paper. antifungal activity antifungal activities of target compounds 3a–i and 4a–i were evaluated in vitro against five phytopathogenic fungi (gibberella zeae, cytospora sp., colletotrichum gloeosporioides, alternaria solani and fusarium solani) using the mycelium growth rate method.20,21 all the fungi were provided by the qingdao agricultural university. the strains were retrieved from the storage tube and cultured for 2 weeks at 25 °c on potato dextrose agar (pda). the antifungal activity was assessed as follows: pda medium was prepared in flasks and sterilized. the target compounds 3a–i and 4a–i were dissolved in acetone prior to mixing with molten agar at 55 °c, and the concentration of the target compounds 3a–i and 4a–i were 100 mg l-1. the pda medium was then poured into sterilized petri dishes. the five fungi were incubated in pda at 25 °c for 7 days to obtain new mycelium for the fungicidal assays, and a mycelia disk of 4 mm in diameter cut from culture medium was picked up with a sterilized inoculation needle and inoculated in the centre of the pda petri dishes. the inoculated petri dishes were incubated at 25 °c ℃ for 3–4 days. acetone was used as the control, and the commercially available agricultural fungicide hymexazol served as the positive control. each compound was measured in three replicates, and each colony diameter of the three replicates was measured 4 times by the cross bracketing method. the inhibition rate (ir) was calculated according to the following formula: ( )% 100 4 − = − c t ir c where c is the average diameter of mycelia in the blank test and t is the average diameter of mycelia on pda treated with the target compounds. conclusions in summary, a series of dithiocarbamates bearing an amide moiety 3a–i and 4a–i were synthesized in almost quantitative yield by a facile and convenient procedure. especially, the synthesis, separation, and purification by recrystallization could be conducted in one-pot and in same medium by regulating the temperature from 50 to 0 °c. the results of the bioassay indicated that most target compounds synthesis and antifungal activity of dithiocarbbamates 1373 displayed low activities against g. zeae, cytospora sp., c. gloeosporioides, a. solani and f. solani at a concentration of 100 mg l–1. however, the compounds 4f and 4g gave significant inhibition rates against a. solani and c. gloeosporioides, respectively, at a concentration of 100 mg l–1. generally, the antifungal activities of compounds 4a–i, which have a morpholinyl substituent, are superior to the corresponding compounds 3a–i with the piperidinyl substituent. additionally, the number of substituents on benzene ring influenced the antifungal activity as evidenced by the fact that the compounds bearing two substituents on the benzene ring, such as 3f and 3g and 4f and 4g, displayed better antifungal activities than the compounds with a single substituent on the benzene ring. although most of the target compounds displayed low inhibition rates against mycelia growth of the five tested fungi at concentrations of 100 mg l–1, the target compounds 4f and 4g could be potentially leading structures for further discovery of novel antifungal agrochemicals. supplementary material physical, analytical and spectral data of the synthesized compounds are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. this work was financially supported by the natural science foundation of shandong province (zr2011bl003). и з в о д једноставна синтеза амидних деривата дитиокарбамата и њихова антифунгална активност yu-wen li и shu-tao li school of chemistry and pharmacy, qingdao agricultural university, qingdao, p. r. china синтетисана је серија нових деривата дитиокарбамата који садрже амидну групу, 3a–i и 4a–i, применом олакшаног поступка и добијеним дериватима је структура потврђена 1h-nmr, 13c-nmr спектроскопијом, елементалном анализом и масеном спектрометријом високе резолуције (hrms). испитана је антифунгална активност према пет фитопатогених гљива. резултати су показали да већина деривата показује in vitro активност према gibberella zeae, cytospora sp., colletotrichum gloeosporioides, alternaria solani и fusarium solani при концентрацији 100 mg l-1. једињења 4f и 4g показују значајну активност према a. solani и c. gloeosporioides, редом. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice evaluation of a method for phthalate extraction from milk related to the milk dilution ratio j. serb. chem. soc. 80 (8) 983–996 (2015) udc 637.12+66.061+547.584–326: jscs–4774 543.544.3:543.51 original scientific paper 983 evaluation of a method for phthalate extraction from milk related to the milk dilution ratio danica s. milojković1#, darko h. anđelković1#, gordana m. kocić2 and tatjana d. anđelković1#* 1faculty of sciences and mathematics, university of niš, višegradska 33, 18000 niš, serbia and 2faculty of medicine, university of niš, bulevar dr zorana đinđića 81, 18000 niš, serbia (received 4 december 2014, revised 26 february, accepted 19 march 2015) abstract: liquid–liquid extraction techniques coupled with gas chromatography–mass spectrometry (gc–ms) were compared for the extraction and the determination of four phthalates: dimethyl phthalate (dmp), di-n-butyl phthalate (dbp), benzyl butyl phthalate (bbp) and di-(2-ethylhexyl) phthalate (dehp) in six different kinds of milk-based samples. extraction factors: sample preparation, organic solvent type and volume, salt effect, agitation and the extraction time were optimized. the ions of the base peak (m/z 149 for dbp, bbp and dehp and m/z 163 for dmp) for the investigated phthalates were selected for the screening studies. the acquisition was performed in the selected ion-monitoring mode. the response of the mass selective detector (msd) for gc–ms phthalate calibration standards was linear between 0.25 and 2.50 µg ml-1 with calculated limit of detection (lod) values between 0.01 to 0.04 µg ml-1 and limit of quantitation (loq) values of 0.05 to 0.12 µg ml-1, while repeatability was between 1.7 to 4.9 % relative standard deviation (rsd). the study demonstrated an increase in the recovery of less polar phthalates in matrix milk standards on matrix dilution. recovery for hydrophilic phthalates, such as dmp, was not changed by matrix dilution and it was continuously low for the investigated method. two spiking levels, tested for the influence of matrix dilution on phthalate recovery, showed the same trend. keywords: extraction efficiency; phthalate esters; gas chromatography-mass spectrometry; milk samples. introduction phthalates present one of the ubiquitous chemicals in the environment. since their usage is mainly as plasticizers for polymers, such as poly(vinyl chloride) (pvc), over one million tons of phthalates are produced in western europe each year.1 the most important congeners are: di-2-ethylhexyl phthalate (dehp), * corresponding author. e-mail: tatjanaan@gmail.com # serbian chemical society member. doi: 10.2298/jsc141204028m _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 984 milojković et al. which accounts for about 50 % of the world production of phthalates,1 dimethyl phthalate (dmp), di-n-butyl phthalate (dbp) and benzyl butyl phthalate (bbp),which is prohibited by the eu in toys and childcare articles if they could be placed in the mouth by children.2 phthalates have long been considered to be potential endocrine disrupters, and several of them have shown reproductive effects in animals.3–5 the structures of the four studied phthalates as the most commonly used phthalate esters based on the 1,2-benzenedicarboxylic acid are given in fig. s-1 of the supplementary material to this paper. the permanence of phthalates in polymer materials is low because phthalates are not chemically bound to the polymer. their migration from food-packaging materials was reported as a route for food contamination with phthalates. since food is one of the major sources of human exposure to phthalates, in order to assess human exposure to these substances, monitoring of phthalates levels in various foods should be performed. fatty and oily foods are primarily contaminated with phthalates due to their lipophilic character.6 although human intake of phthalates may originate from many food sources and routes, there is a special interest in monitoring the contamination of milk and milk products because they constitute a primary food source, especially for children. tolerable daily intakes (tdi) were specified by the european food safety authority (efsa) for several phthalates, and they are 0.01, 0.05 and 0.5 and mg kg–1 body weight day–1 for dbp, dehp and bbp, respectively.7–9 the physicochemical properties of phthalates (table s-i of the supplementary material) and their amount and frequency of usage in food-packaging materials could determine their migration and leaching levels and thus the possibility of food contamination and human exposure. the majority of publications deal with phthalate determination in simple matrices, such as water10–12 or biological fluids,13 while publications about phthalates in food samples with fatty matrices14–16 are less frequent. due to usually low level of phthalates and generally high complexity of the matrices of food sample, extraction and clean up of the sample are usually considered as necessary and critical steps in phthalate determination. however, as indicated in this paper, the crucial point in phthalate extraction from complex milk matrices is considered the choice of solvent for the extraction and sample dilution step. the influence and importance of these two aspects on the recovery of phthalates, as well as analytical methodology (agitation method, extraction time, etc.) are considered herein. when sample extraction is performed with solvent mixtures of low polarity, fats are co-extracted together with the phthalates and thus, the fats must be removed and the membranes of milk fat globules should be disrupted before the chromatographic analysis. otherwise, phthalates may not be effectively extracted, yielding low recoveries. the optimization of extraction efficiency of phthalates _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ phthalate extraction from milk 985 from milk could alternatively be achieved by precipitation of milk proteins by addition of nacl for salting-out followed by the addition of acetonitrile17 or through primary addition of acetone or an alcohol.18 gel permeation chromatography (gpc) is often used for this purpose.6,19 when the sample is extracted with polar solvents, such as methanol or acetonitrile, the extract contains other interfering organic impurities in addition to fat. these may require solid-phase extraction (spe) on different sorbents.20–22 in each of these sample preparation steps, the possibility of contamination of the sample is high because even in pure solvents, solid phase extractants, laboratory water, laboratory air and laboratory glassware, phthalates could be detected.23 this can be negligible source of contamination that could be reduced by establishing whether the reduction of the accuracy of phthalate determination is higher due to phthalate contamination of the sample by using clean-up procedures or due to the complexity of co-eluting substances, which could influence the sensitivity of phthalate determination by ms analysis. since the extraction procedure could be the source of a matrix effect and is an essential step in the evaluation process of phthalate determination, different liquid solvents and solvent mixtures were investigated in this study regarding the extraction efficiency of phthalates, the possibility of phase separation, visibility of phase separation, clearness of the extracts, formation of emulsions, availability of reagents and duration of the extraction. in addition to the selection of the solvent type, different methods/procedures for extraction were also investigated in order to obtain the most optimal method for the extraction phthalates from milk and dairy products. bearing in mind that in milk with high fat content, phthalates cannot be easily extracted due to phospholipid–protein membranes that encapsulate the fat droplets containing lipophilic phthalate, dilution of milk samples prior to the analysis could enable a better extraction efficiency of phthalates and relatively clean extracts to be obtained. moreover, sample dilution is an easy and effective method to reduce interfering compounds and to diminish the matrix effect. the obtained extracts, even without a clean-up step, could be used for gc–ms analysis, as shown herein. experimental reagents and materials all solvents (hplc grade) were purchased from sigma–aldrich (st. louis, mo, usa), except n-hexane that was purchased from fisher scientific (pittsburgh, pa, usa). dimethyl phthalate (dmp), di-n-butyl phthalate (dbp), benzyl butyl phthalate (bbp) and di-(2 -ethylhexyl) phthalate (dehp) were purchased, in the highest available purity, from sigma– –aldrich (st. louis, mo, usa). dibutyl adipate (dba) was purchased from fluka (buchs, switzerland) and used as an internal standard. water from a milli-q system (millipore, bedford, ma, usa) was used. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 986 milojković et al. all reagents and water used for the analyses were checked for contamination with phthalates. to avoid phthalate contamination, all the employed laboratory dishes were made of glass, previously washed with water and soap, tap and ultrapure water, rinsed with acetone and n-hexane and dried at 200 °c in a clean oven for 4 h.23 all stock, intermediate and working solutions were prepared in n-hexane. individual stock solutions of each phthalate were prepared at a concentration of 1000 µg ml-1 and stored at 4 °c. the stock solutions were stable for up to one month. a mixed stock solution of phthalates was prepared at a concentration of 100 µg ml-1 for each phthalate. with stepwise dilutions, individual working solutions of 1 µg ml-1 were obtained for each phthalate to identify their retention times. the working solutions were stable for 10 days. furthermore, the mixed working solutions of all phthalates at 0.25, 0.50, 1.00, 1.50 and 2.50 µg ml-1 were also prepared as calibration standards with dba as the internal standard at a concentration of 1 µg ml-1. between some other commonly used internal standards, such as benzyl benzoate20 or isotope-labeled standards, dba was adopted as an internal standard being approved for this purpose according to our findings and literature data.24 the calibration curves were linear in the range from 0.25 to 2.5 µg ml-1 with correlation coefficients higher than 0.990. the linear dynamic range was broader and covered the range from 2.50 to 50 µg ml-1. samples of nine systems of commercial milk-based samples diluted with water, from 0 to 50vol. %, were all spiked phthalates at two concentration levels, 3.0 and 6.0 µg ml-1. dairy samples optimization of the phthalate liquid–liquid extraction procedure was realized using six samples of dairy products: raw bovine milk, commercial (pasteurized) milk, thawed milk, whey, human milk and yogurt. samples of raw bovine milk were collected in glass bottles from a dairy farm in south serbia avoiding any contact with plastic materials. this milk had not been pasteurized before analysis and was used as collected. samples of commercial milk from a serbian dairy were purchased at serbian market and used as received. the commercial milk was bottled in a plastic (polyethylene terephthalate, pet) bottle.6 the shelf life of milk is 10 days after packaging and the milk was used within this period. thawed commercial milk was obtained after thawing overnight the milk that had been frozen for 24 h in the original packing. whey produced in a cheese manufacturing process was purchased from the same dairy farm as the raw bovine milk. samples of human milk were collected in three successive days by a postpartum 35-year old woman (5th week postpartum). milking was carried out manually, with previously cleaned breasts and hands, directly into a glass container that was intended for this purpose. the yogurt used in this study was made from cow's milk, commercially available from a serbian market and stored in a plastic (pet) bottle. all nine systems of commercial milk-based samples diluted with water were used for the selection of optimum solvent type as extractant considering the possibility of phase separation, visibility of phase separation, clearness of the extracts, formation of emulsion, availability of the reagents and duration of extraction. in addition to selection of the solvent type, different methods/procedures for extraction were also investigated in order to obtain the most optimal method for the extraction of phthalates from milk and dairy products. for the determination of extraction efficiency of phthalates in milk, only the commercial milk was used. the milk was diluted with water from 0 to 50 vol. %. before the analysis, samples of raw bovine milk, thawed milk and human milk were stored in a glass bottle in a refrigerator at 4 °c. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ phthalate extraction from milk 987 gc–ms analysis gas chromatographic analysis was performed on a gas chromatograph 6890 (hewlett -packard) equipped with a mass selective detector (msd) 5973 (agilent) and a db-5 ms capillary column (30 m×250 mm×0.25 mm). the mass spectra were recorded under an electron impact ionization voltage of 70 ev. the gas chromatograph was operated in the split less injection mode. the oven temperatue was programmed from 60 °c (1 min) to 220 °c (1 min) at a rate of 20 °c min-1 and then to 280 °c (4 min) at a rate of 5 °c min-1. the msd was used in the single ion-monitoring mode (sim) at m/z 149 and 163. the identification and quantification of target compounds was based on the relative retention time, the presence of target ions and their relative abundance. the target ion was m/z 149 for dbp, bbp and dehp and m/z 163 for dmp. linearity was investigated in the range 0.25–2.5 µg ml-1. the linear dynamic range for the investigated phthalates by gc–ms was 0.25–50 µg ml-1. the limit of detection (lod) and limit of quantitation (loq) for each phthalate were calculated from six replicated measurements of a low concentration spiked standard solution according to the analytical detection limit guidance from the wisconsin department of natural resources.25 the laboratory contamination was monitored with blank samples obtained from milli-q water treated in the same manner as the milk samples. extraction procedures fourteen extraction procedures were examined. each entailed different conditions (type of extractant, extraction time, agitation and settling). two solvent mixtures were studied for extraction, acetone/n-hexane at a 1:1 volume ratio and methanol/n-hexane at a 1:3 volume ratio. the salting out effect was examined using acetonitrile as the extraction agent with the addition of nacl to saturation. the liquid–liquid extractions for these three systems were performed by adding a volume of the extraction agents to a volume of sample in a 2:1 ratio, followed by vigorously hand shaking for 1 h and left standing for 24 h. in extraction methods using ethyl acetate, n-hexane, acetonitrile, acetone, dichloromethane, dichloroethane, trichloroethylene and trichloroethane, the volume ratio of the extraction agent to a milk sample was 2:1, agitation was performed in an ultrasonic bath for 1 h and hand-shaking for 1 h. the extractions with extractants soluble in the feed solution, i.e., acetone and alcohols (ethanol, methanol and 2-propanol), the volume ratio of extractant to sample was also 1:2, agitation in an ultrasonic bath for 1 h and standing overnight at room temperature were applied. results and discussion gc–ms acquisition a chromatogram of the investigated phthalates is given in fig. s-2 of the supplementary material. the four phthalates were separated using the selected chromatographic conditions. the chromatogram shows that the separation of the phthalates (using the optimized conditions) occurred within a running time of 20 min and that gc–ms method is well suited for the simultaneous determination of the 4 phthalates. for the considered range of phthalate concentrations, 0.25–2.50 µg ml–1, the response of the mass-selective detector was linear. the correlation coefficients (r2) ranged from 0.990 to 0.999. the limits of detection (lod) values, calculated according to the winefordner and long criterion,26 were 0.01– –0.04 µg ml–1. the precision of the gc–ms method, expressed as the relative standard deviation (rsd, n = 3), was found to be in the range 1.7–4.9 %. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 988 milojković et al. the retention times, selected masses and the scan start times for each phthalate studied by gc–ms are listed in table i. table i. target ions, retention times and scan start times for the investigated phthalates determined by gc-ms; target ions observed in sim are shown in bold phthalate m/z retention time, min scan start time, min dmp 163, 194 8.03 7.80 dbp 149, 150, 223, 205 11.57 11.20 bbp 149, 91, 206, 238 16.02 15.50 dehp 149, 167, 279, 150 18.39 17.90 optimization of extraction procedure the choice of the optimum extraction solvent for separation was determined from a consideration of several criteria: high boiling point and a low vapor pressure in order to reduce the risk of evaporation, high selectivity that enables fewer stages to be used, insolubility of solvent for prevention of solvent losses, good chromatographic behavior, and high partitioning coefficient of the analyte. based on these considerations, several extraction solvents and mixture of solvents were investigated. as milk forms stable emulsions with the majority of solvents, all the investigated extractants were characterized by the appearance of extract and raffinate, the possibility to define the phase boundary, the possibility to perform phase separation, clearness of the extracts and formation of emulsions. among the investigated solvents and solvent systems, n-hexane presented the best extractant because it gave a homogenous and clear extract with precipitated raffinate phase, thus a well-defined interfacial boundary and the possibility to separate the phases (table ii). agitation of samples with extractants enhanced the extraction efficiency and reduced the extraction time for reaching the equilibrium. in this study, stirring, shaking and ultrasonic treatment were investigated for the extraction of the phthalates from nine milk-based samples. too vigorous agitation was avoided since it produced stable emulsions without visible phase boundary and the possibility of phase separation. in order to achieve effective phase separation, shaking of the samples and extractants was chosen as the best agitation method. the extraction time to obtain higher peak areas of phthalates relative to internal standard peak area was investigated in the range of 10 to 30 min. an extraction time of 15 min was selected for extraction since the systems reached the steady state during this period. the addition of an inorganic salt into a mixture of milk and a water-miscible organic solvent, such as acetonitrile, caused separation of the solvent from the mixture and the formation of a two-phase system. the results revealed that salt addition, although providing for a well-defined interfacial boundary, was not a satisfac _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ phthalate extraction from milk 989 table ii. extraction systems studied for phthalate extraction from milk-based samples extractant appearance of: interfacial boundary phase separation extract raffinate acetone:n-hexane 1000 ml diluted milk sample was mixed with 10 ml acetone and 10 ml n-hexane, shaken for 30 min and repeated for extract enrichment. after settling overnight, the n-hexane/acetone phase was taken. the enriched extract was evaporated to dryness and re-dissolved in n-hexane. heterogeneous, creaming yellow, opaque not defined – acetonitrile:nacl 1000 ml diluted milk sample was mixed with 20 ml acetonitrile, 20 g sodium chloride, shaken for 30 min and repeated for extract enrichment. after settling overnight, the n-hexane/acetone phase was taken. the enriched extract was evaporated to dryness and redissolved in n-hexane. homogenous, clear preci pitate well defined – methanol:n-hexane 1000 ml diluted milk sample was mixed with 5 ml methanol and 15 ml n-hexane, shaken for 30 min and repeated for extract enrichment. after settling overnight, the n-hexane/acetone phase was taken. the enriched extract was evaporated to dryness and redissolved in n-hexane. heterogeneous, opaque voluminous precipitate not defined – acetonitrile 1000 ml diluted milk sample was mixed with 20 ml acetonitrile. agitation: ultrasonic bath for 1 h and hand shaking for 1 h. extract phase submitted to new sample volume for enrichment with a final extract to solvent volume ratio of 1:50. settling overnight. the combined extract was evaporated to dryness and redissolved in n-hexane. heterogeneous, yellow voluminous precipitate not defined – ethyl acetate the same procedure as for acetonitrile homogenous, clear coalescence with creaming well defined + cyclohexane the same procedure as for acetonitrile homogenous, clear foaming, precipitate well defined + n-hexane the same procedure as for acetonitrile homogenous, clear preci pitate well defined + dichloromethane the same procedure as for acetonitrile heterogeneous, opaque preci pitate well defined + _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 990 milojković et al. table ii. continued extractant appearance of interfacial boundary phase separation extract raffinate trichloroethane the same procedure as for acetonitrile heterogeneous, opaque precipitate well defined + dichloroethane the same procedure as for acetonitrile heterogeneous, opaque precipitate well defined + extractants soluble in feed solution appearance of extract and raffinate interfacial boundary phase separation methanol heterogeneous, voluminous precipitate, flocculation not defined – ethanol – 2-propanol – acetone – tory method due to low phase separation and adhesion of milk globules on the wall of the separation funnel. based on literature data, the effect of the sample to solvent ratio on the total extracted phthalates from milk products was found to be the best at the 1:20 level. various amounts of a mixture of solvents were used in order to determine the optimum quantity of the extracting solvent, based on the appearance of extract, phase boundary and possibility of phase separation.27 this work showed that due to dilution of milk samples, the satisfactory ratio of solvent to sample volume was found to be at the 1:50 level, when the extract phase is several times submitted to a new sample volume, leading to better concentration of analyte and reduction in solvent consumption. in this way, enrichment of the extract phase with phthalates could be achieved by repeating the extraction procedure. the study showed that the influence of milk type was not a critical factor in the determination of an adequate extraction procedure. all the observed effects for optimization of the extraction procedure were more or less the same for all six investigated samples of dairy products. based on all the obtained results and observations during optimization of the extraction procedure, the parameters that provided the most efficient phthalate extraction were: ratio of sample volume to solvent volume, 1:2; the procedure of enrichment of the extract phase by submitting the extract to a new sample volume, to obtain a final extract to sample ratio 1:50, leading also to a reduction in solvent consumption; shaking as agitation method in order to avoid the formation of stable emulsions and an extraction time in the range 15 to 30 min. phthalate analysis in standard n-hexane solutions phthalates were identified by gc–ms in the full scan mode and quantified in the sim mode. linear calibration curves for the phthalates dissolved in n-hexane were obtained in the concentration range 0.25–2.50 µg ml–1. the linearity range _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ phthalate extraction from milk 991 and respective correlation coefficients (r2) calculated in the range 0.25–2.50 µg ml–1, the lod and the loq values for each phthalate investigated in this study and determined by gc–ms (in sim mode) are reported in table iii. the r2 values for all the phthalates in the linear range were above 0.990. the lod and loq values of each phthalate analyzed in this study were adequate for estimating such compounds in milk samples: the lod values ranged between 0.01 and 0.04 µg ml–1 whereas loq values ranged between 0.05 and 0.12 µg ml–1 with rsd values between 1.7–4.9 %. these values were determined according to the knoll definition,28 i.e., an analyte concentration that produced a chromatographic peak equal to three times (lod) and seven times (loq) the standard deviation of the baseline noise. in comparison with other extraction methods, this method provided comparable lod and loq values.11,19 table iii. linearity range and the respective values of correlation coefficients (r2), lod and loq values, and repeatability (rsd) of each phthalate; linearity range: 0.25–2.50 µg ml-1 phthalate r2 lod / µg ml-1 loq / µg ml-1 rsd / % dmp 0.999 0.04 0.12 4.9 dbp 0.999 0.01 0.05 1.7 bbp 0.992 0.02 0.08 2.4 dehp 0.990 0.04 0.12 3.6 phthalate analysis in matrix extracts validation of the phthalate quantification method in milk samples, considering the possible matrix effect, was performed with successive milk dilution. the high percentage of fat in milk may affect on the one hand the extraction efficiency of the phthalates and the analytical sensitivity, precision and stability of the response of the chromatographic system on the other. therefore, for an estimation of the correlation of the milk fat content with the recovery for each phthalate, milk samples with different level of dilution were spiked and examined. for an estimation of the analytical sensitivity, the slopes of the curves obtained for diluted milk samples spiked with phthalates were examined and compared to the slopes of the calibration curves of phthalate standards in n-hexane. as expected, higher phthalate recoveries were obtained by diluting the milk samples in water, whereby the recovery efficiency was improved by 60–80 %, compared to a dilution of 50 vol. % (table iv). the low recovery rate (only 10 %) for the sample with 50 % milk content was expected due to the hydrophobic nature of phthalates, which are more soluble in milk fat globules giving lower distribution ratio between the extract and the milk sample. the recovery of phthalates was the highest for water samples (without any milk), which is in accordance with previous investigations performed with commercial bottled water samples.29,30 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 992 milojković et al. table iv. phthalates recoveries (%) from spiked diluted milk samples with 3 ppb of each phthalate milk content, vol. % dmp dbp bbp dehp 0 14.00 75.00 125.00 135.00 0.5 10.82 62.30 89.51 73.61 2 7.10 60.86 92.34 69.22 3 10.12 61.78 90.18 68.00 6 13.10 54.14 72.41 65.00 10 13.10 41.72 57.24 71.03 15 13.83 34.04 46.81 60.00 30 13.33 25.45 37.27 45.00 50 11.76 4.41 8.66 10.95 the lowest recovery values were for dmp which is the phthalate with the lowest molar mass, only one carbon atom in hydrocarbon side chain and with even 103 times higher water solubility than the rest of investigated phthalates. generally, higher recovery is observed for the high-molecular weight phthalates (log kow = 7.6 for dehp), while low-molecular weight phthalates are more water-soluble relatively hydrophilic and thus the recovery decreases (log kow = = 1.5 for dmp). the same trend is observed for lower spike phthalate concentration of 3 ppb (table iv) and for higher spike phthalate concentration of 6 ppb, as shown in fig. 1. (a) (b) (c) (d) fig. 1. recoveries (average values) for the determination of phthalates in water dissolved milk samples spiked with 6 µg ml-1 of each phthalate: a) dmp, b) dbp, c) bbp and d) dehp. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ phthalate extraction from milk 993 the analytical sensitivity of diluted milk samples spiked with phthalates were examined and compared to the calibration phthalate standards in n-hexane regarding the slopes of the obtained standard curves (fig. 2). fig. 2. the slopes of the curves for each phthalate prepared in n-hexane and milk matrix: a) dmp, b) dbp, c) bbp and d) dehp. pa and pis presents the values for chromatogram peak are of analyte and internal standard. the concentrations of phthalates analyzed in the matrix extracts were in general lower than in n-hexane, showing a negative matrix effect that made for a less stable response of the chromatographic system and lower analytical sensitivity and precision. the values of the slopes of the curve, obtained by linear regression and tabulated in table v, show that the response and precision were the lowest for the milk sample with the highest fat content and for dmp as the most water soluble phthalate. this suggested a strategy for the elimination of matrix effect, i.e., _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 994 milojković et al. dilution of the milk extract, especially for more hydrophobic phthalates such as the majority of commonly used phthalates. table v. slopes of the curve obtained from solvent (n-hexane) and matrix (water and diluted milk samples) phthalate n-hexane water milk content, vol. % 2 15 50 dbp 12.8829 9.17614 7.6756 4.3494 0.28192 dehp 1.9924 1.4067 1.135 0.7638 0.099 bbp 0.905 0.75284 0.6380 0.3490 0.0371 dmp 3.1800 0.39551 0.2669 0.14242 0.02068 conclusions the method of extraction of phthalates from milk samples by n-hexane was shown to be a simple and effective procedure. the validation results of the method were satisfactory, since the recovery data and relative standard deviation values indicated good method accuracy and precision (r2 >0.990) for phthalates when evaluated in a milk matrix. an increase in the recovery of less polar phthalates in matrix milk standards by matrix dilution was observed. recovery for hydrophilic phthalates, such as dmp, was not changed by matrix dilution and was continuously low for the investigated method. the same trend for influence of matrix dilution on phthalate recovery was observed at two phthalate spiking levels. elimination of the matrix effect by dilution of the milk extract, especially for more hydrophobic phthalates such as the majority of commonly used phthalates, was shown to be a satisfactory method. supplementary material the chemical structures of the four studied phthalates, gc–ms chromatogram of a standard solution containing phthalates and physicochemical properties of the four studied phthalate esters are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgments. financial support for this investigation from ministry of education, science and technological development of the republic serbia, project tr 31060, is gratefully acknowledged. и з в о д евалуација методе екстракције фталата из млека у зависности од садржаја масти у млеку даница с. милојковић1, дарко х. анђелковић1, гордана м. коцић2 и татјана д. анђелковић1 1природно–математички факултет, универзитет у нишу, вишеградска 33, 18000 ниш и 2медицински факултет, универзитет у нишу, булевар др зорана ђинђића 81, 18000 ниш у циљу одређивања диметил-фталата (dmp), ди-n-бутил-фталата (dbp), бензил бутил-фталата (bbp) и ди-(2-етилхексил)-фталата (dehp) у шест различитих узорака млека упоређени су резултати добијени течно–течном екстракцијом куплованом са _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ phthalate extraction from milk 995 гасном хроматографијом–масеном спектрометријом. извршена је оптимизација екстракционих фактора: припрема узорака, врста и запремина органског растварача, ефекат исољавања и мешања и време екстракције. одабрани су базни максимуми испитиваних фталата (m/z 149 за dbp, bbp и dehp и m/z 163 за dmp) за даље истраживање. снимање је извршено у моду мониторинга изабраног јона. калибрациона права је линеарна у опсегу 0,25 до 2,50 μg ml-1 са израчунатим lod вредностима између 0,01 и 0,04 μg ml-1 и loq вредностима између 0,05 μg и 0,12 μg ml-1, са rsd између 1,7 и 4,9 %. истраживање је показало раст измереног аналита (“recovery”) за мање поларне фталате разблаживањем матрикса млека. “recovery” за хидрофилне фталате, као што је дмп, се не мења разблаживањем матрикса млека и константантно је низак за испитивану методу. двe концентрације унутрашњег стандарда (“spike”), тестиранe ради испитивања утицаја разблажења млека на “recovery” фталата, показују исти тренд. (примљено 4. децембра 2014, ревидирано 26. фебруара, прихваћено 19. марта 2015) references 1. d. cadogan, plast. addit. cmpd. 4 (2002) 28 2. european commission ip/99/829, ban of phthalates in childcare articles and toys, 1999 3. r. h. waring, r. m. harris, maturitas 68 (2011) 111 4. department of health and human services centers for disease control and prevention, third national report on human exposure to environmental chemicals, 2005 5. world health organization: guidelines for drinking water quality, 2008 6. x.-l. cao, compr. rev. food. sci. f. 9 (2010) 21 7. european food safety authority, opinion of the scientific panel on food additives, flavouring, processing aids and material in contact with food on a request from the commission related to dibutyl phthalate (dbp) for use in food contact materials, efsa j. 242 (2005) 1 8. european food safety authority, opinion of the scientific panel on food additives, flavouring, processing aids and material in contact with food on a request 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice modeling of density and calculations of derived volumetric properties for n-hexane, toluene and dichloromethane at pressures 0.1–60 mpa and temperatures 288.15–413.15 k j. serb. chem. soc. 80 (11) 1423–1433 (2015) udc 547.533+547.216+547.412.113: jscs–4808 541.12.034.2:532.14 original scientific paper 1423 modeling of density and calculations of derived volumetric properties for n-hexane, toluene and dichloromethane at pressures 0.1–60 mpa and temperatures 288.15–413.15 k gorica r. ivaniš#, aleksandar ž. tasić, ivona r. radović#, bojan d. djordjević#, slobodan p. šerbanović# and mirjana lj. kijevčanin*# faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11120 belgrade, serbia (received 18 march, revised 30 june, accepted 1 july 2015) abstract: densities data of n-hexane, toluene and dichloromethane at temperatures 288.15–413.15 k and at pressures 0.1–60 mpa, determined in a previous article, were fitted to the modified tait equation of state. the fitted temperature–pressure dependent density data were used to calculate the following derived properties: the isothermal compressibility, the isobaric thermal expansivity, the difference between specific heat capacity at constant pressure and at constant volume and the internal pressure, over the entire temperature and pressure intervals specified above. in order to assess the proposed modeling procedure, the obtained values for the isothermal compressibility and the isobaric thermal expansivity were compared with the corresponding literature data. the average absolute percentage deviations for the isothermal compressibility were for n-hexane 2.01–3.64 %, for toluene 0.64–2.48 % and for dichloromethane 1.81–3.20 %, and for the isobaric thermal expansivity, they were for n-hexane 1.31–4.17 %, for toluene 0.71–2.45 % and for dichloromethane 1.16–1.61 %. by comparing the obtained deviations values with those found in the literature, it could be concluded that the presented results agree well with the literature data. keywords: high pressure; isobaric thermal expansivity; isothermal compressibility; n-hexane; toluene; dichloromethane. introduction in a previous article,1 the experimental densities of compressed liquid nhexane, toluene and dichloromethane, determined using a set-up based on a density measuring cell ap dma hp coupled with a dma 5000 vibrating tube densimeter, were reported. the measurements were performed over the temperature * corresponding author. e-mail: mirjana@tmf.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc150318062i 1424 ivaniš et al. and pressure ranges: 288.15–413.15 k and 0.1–60 mpa, respectively. it was shown that the obtained data are in good and acceptable agreement with the corresponding literature values. this paper, as well as the previous one related to the density measurements for the same pure compounds-solvents, is a part of the systematic investigation of crucial thermodynamic properties of important solvents, used in both industry and the laboratory. correlation of the volumetric information mentioned above allows the evaluation of temperature and pressure effects on various thermomechanical properties,2 such as isothermal compressibility, κt, and isobaric thermal expansivity, αp; based on these properties, the internal pressure pint and the difference of the isobaric and isochoric heat capacities, cp–cv, can also be determined. by coupling the mentioned isothermal and isobaric properties κt and αp, and using the isentropic compressibility, κs, which is related to the thermodynamic speed of sound, some additional properties can be calculated; in this respect, the isobaric heat capacity, cp, and isochoric heat capacity, cv, could be mentioned. various methods, dealing with the correlation of measured densities as a function of temperature and pressure, have been established in the literature. in the present work, a tait-like equation was applied. the obtained correlation was used to compute the aforementioned parameters. calculations the experimental density data presented in a previous paper1 were fitted to the tait-like equation: ref ref ( ) ( , ) ( ) 1 ( )ln ( ) t t p b t p c t b t p ρ ρ =  + −  +  (1) where, t and p refer to temperature and pressure, respectively, and c(t) and b(t) are parameters that were assumed to be temperature dependent and can be calculated from the second order polynomials: 2 0 ( ) ii i b t b t = =  (2) 2 0 ( ) ii i c t c t = =  (3) ρref is the density at the reference pressure, pref, and it can be calculated from the second order polynomial: 2 ref 0 ( )ρ = =  ii i t a t (4) in eqs. (2)–(4), ai, bi and ci are adjustable parameters. derived volumetric properties of n-hexane, toluene and dichloromethane 1425 as stated previously, a number of derived properties exist that can be calculated from density data. the isothermal compressibility, κt, represents a measure of the sensitivity of density to a pressure change at constant temperature and it depends on the fluid structure. it can be expressed as: 1 t tp ρ κ ρ  ∂ =  ∂  (5) coupling eqs. (1) and (5) leads to the expression for the calculation of κt: ( ) ref ( ) ( ) ( ) 1 ( ) ln ( ) t c t b t p b t p c t b t p κ =  + + − +  (6) the isobaric thermal expansivity, αp, is a measure of the relative fluid density change in response to a temperature change at constant pressure: 1 1 1 ρ ρ α ρ ρ ρ −  ∂ ∂ ∂   = = −    ∂ ∂ ∂     p pt p p t t (7) when eq. (1) is included into eq. (7), an expression for the calculation of the isobaric thermal expansivity is obtained: ref ref ref ref ref ref ( )( ) ( ) ( ) ( )ln ( ) ( ( ) )( ( ) ) ( ) ( ) ( ) 1 ( )ln ( ) ρ α ρ ′ − +′+ ′− + + + = −  + −  +  p b t p p b t p c t c t t b t p b t p b t p t b t p c t b t p (8) where ρref′(t), b′(t) and c′(t) stay for the derivatives of the parameters ρref, b(t) and c(t) of eq. (1), respectively: 2 ref 1 0 ρ − = ′ =  ii i ia t (9) 2 1 0 − = ′ =  ii i b ib t (10) 2 1 0 − = ′ =  ii i c ic t (11) another thermodynamic property that can be calculated from the aforementioned equations is the ratio of the isobaric thermal expansivity to the isothermal compressibility, i.e., the thermal pressure coefficient, γ: α γ κ = p t (12) the thermal pressure coefficient is associated with the internal pressure, pint, which represents the change of the internal energy of a sample when it isothermally expands or 1426 ivaniš et al. contracts, thus describing intermolecular interactions. it is easy to calculate the internal pressure when the isobaric thermal expansivity and the isothermal compressibility are known: int ρ α γ κ ∂ ∂   = = − = − =   ∂ ∂    p tt tu p p t p t p p v t (13) where, u denotes the internal energy of a sample and v is its volume. an important thermodynamic property that can be calculated from the quantities defined above is the difference between specific heat capacity at constant pressure, cp, and specific heat capacity at constant volume, cv: 2 2 ρ ρ ρ ∂   ∂  = +  ∂  ∂  p v t p t c c t p (14) linking eqs. (5) and (7) with eq. (14) gives the expression: 2α ρκ − = pp v t t c c (15) the relation given by eq. (15) is significant because it can be used to calculate the heat capacity at constant volume instead of its experimental determination. heat capacity at constant pressure can be measured or calculated from speed of sound data and the above-defined coefficients by combining eqs. (16)–(18). isentropic compressibility, κs, is defined as: 1 ρ κ ρ  ∂ =  ∂  s sp (16) and can be calculated as follows: 2 1 κ ρ =s u (17) where, u is the speed of sound waves spreading through the sample. further, the specific heat capacity at constant pressure can be obtained from the equation: ( ) 2α ρ κ κ = − p p t s t c (18) results and discussion the criteria used to assess the quality of fitting were the absolute average percentage deviation, aad, the percentage maximum deviation, md, the average percentage deviation, bias, and standard deviation, σ: exp cal exp 1 100 ρ ρ ρ= − =  n ii i i aad n (19) derived volumetric properties of n-hexane, toluene and dichloromethane 1427 exp cal exp max 100 ρ ρ ρ  − =     ii i md , i=1,n (20) exp cal exp 1 100 ρ ρ ρ= − =  n ii i i bias n (21) ( )2exp cal 1 ρ ρ σ = − = −  n ii i n m (22) where, ρexp is the experimentally determined density, ρcal denotes the density value obtained from eq. (1), n is the number of experimental data and m represents the number of parameters included in the correlation of the tait-like equation. the reference pressure in eq. (1) was chosen to be 1 mpa. hence, the density data of a sample at 1 mpa were first used for the optimization of the ai parameters. then, all the density data, presented for the studied compounds in a previous work1 were employed for the determination of the bi and ci parameters. all the parameters were obtained applying the marquardt algorithm for the minimization of the objective function, i.e., the standard deviation. the obtained parameters of eqs. (2)–(4) and the corresponding deviations are given in table i. table i. fitting parameters of eqs. (2)–(4) parameter n-hexane toluene dichloromethane a0 / kg m-3 791.280 1082.499 1599.209 a1 / kg m-3 k-1 –0.035271 –0.55689 –0.10023 a2 / kg m-3 k-2 –0.14039×10-2 –0.60343×10-3 –0.28367×10-2 b0 / mpa 349.513 504.019 654.433 b1 / mpa k-1 –1.46894 –1.96093 –2.80478 b2 / mpa k-2 0.15855×10-2 0.20102×10-2 0.30968×10-2 c0 0.18450 0.15696 0.29779 c1 / k-1 –0.58799×10-3 –0.39307×10-3 –0.11415×10-2 c2 / k-2 0.87278×10-6 0.54981×10-6 0.15929×10-5 aad / % 0.023 0.008 0.019 md / % 0.191 0.028 0.128 bias / % 0.002 0.003 0.001 σ / kg m-3 0.209 0.090 0.332 from the presented deviation values, it could be concluded that the proposed eqs.(1)–(4) fit well the densities of the selected substances. 1428 ivaniš et al. the isothermal compressibility, the isobaric thermal expansivity, the difference between the specific heat capacities at constant pressure and constant volume and the internal pressure for all three examined compounds, at temperatures 288.15–413.15 k and pressures 0.1–60 mpa, are presented in tables s-i–s-iii of the supplementary material to this paper. in order to verify the accuracy of the density data modeling and the procedure of calculating the derived properties listed in the previous section, the obtained values of the isothermal compressibility and isobaric thermal expansivity were compared to those found in the literature. the criteria for the evaluation of the agreement were the same as in a previous paper:1 the absolute average percentage deviation (aad), the percentage maximum deviation (md) and the average percentage deviation (bias), but here, instead of density, the corresponding values of the isothermal compressibility and the isobaric thermal were used. n-hexane data on n-hexane density were modeled and the calculated values of the isothermal compressibility, given in table s-i, were compared to those published by daridon et al.3 the comparison was performed at temperatures 293.15– –373.15 k and at pressures 0.1–60 mpa; the obtained deviations were: aad = = 3.64 %, md = 5.67 % and bias = –3.64 % (fig. 1a). aiming at assessing the accuracy of the presented density modeling, the isentropic compressibility was compared. values of the speed of sound were retrieved from the literature3 and the isentropic compressibility was calculated using eq. (17); the obtained values were compared to the literature data3 at temperatures 293.15–373.15 k and pressures 5–60 mpa. the corresponding deviations were aad = 0.10 %, md = 0.29 % and bias = 0.01 %; these deviations represent one more proof that the modeling procedure was properly selected. the calculated isothermal compressibility data were compared to those given by rendzio et al.4 at several temperatures in the range 313.15–363.15 k and at pressures 0.1–60 mpa. values of the criteria were aad = 2.63 %, md = 4.73 % and bias = –2.56 %. rendzio et al.4 also reported equations for the calculation of isobaric thermal expansivity. hence, their data with those reported in table s-i were compared at temperatures 288.15–393.15 k and at pressures 0.1–60 mpa. the obtained deviations were aad = 0.99 %, md = 3.73 % and bias = –0.68 %, indicating very good agreement (shown in fig. 1b). tronkoso et al.5 gave the isothermal compressibility of n-hexane in the somewhat narrower ranges of pressure and temperature than did the aforementioned authors, and hence the comparison is realized at temperatures 293.15– derived volumetric properties of n-hexane, toluene and dichloromethane 1429 –413.15 k and at pressures 0.1–40 mpa. the deviations were aad = 2.01 %, md = 3.31 % and bias = –2.01 %. (a) (b) fig. 1. comparisons of the calculated derived properties with literature data for n-hexane: a) the isothermal compressibility with daridon et al.3 at () 293.15, () 303.15, () 313.15, () 323.15, () 323.15, () 343.15, () 353.15, () 363.15 and () 373.15 k; b) the isobaric thermal expansivity with rendzio et al.4 at () 288.15, () 303.15, () 318.15, () 333.15, () 343.15, () 353.15, () 363.15, () 373.15 and () 393.15 k. the values of the isobaric thermal expansivity presented in this work were compared to those reported by zhou et al.6 at temperatures 293.15–413.15 k and at pressures 0.1–40 mpa and the obtained deviation criteria were: aad = 4.17 %, md = 6.08 % and bias = –3.86 %. cardeirine et al.7 gave the isobaric thermal expansivities at atmospheric pressure and at temperatures 293.15–333.5 k. the results of the comparison of those data and the present ones were aad = 2.06 %, md = 3.71 % and bias = = –1.69 %. considering the values of the deviations that the cited authors reported in their papers, it could be concluded that the agreement of the present results with those found in the literature was acceptable. toluene the isothermal compressibility data of toluene, given in table s-ii, were compared to those reported by chorazewski et al.8 the comparison was performed at temperatures 303.15–373.15 k and pressures 0.1–60 mpa and the obtained results were: aad = 1.19 %, md = 1.97 % and bias = –1.18 % (fig. 2a). chorazewski et al.8 gave equations for the determination of the isobaric thermal expansivities of toluene and hence, the data presented in table s-ii were compared to these values over wide ranges of temperature and pressure, 288.15– –413.15 k and 0.1–60 mpa, respectively. the obtained deviations were aad = = 2.45 %, md = 5.18 % and bias = –2.29 % (fig. 2a). 1430 ivaniš et al. (a) (b) fig. 2. comparisons of the calculated derived properties with literature data for toluene: a) the isothermal compressibility with chorazewski et al.8 at () 303.15, () 323.15 and () 373.15 k, and b) the isobaric thermal expansivity with chorazewski et al.8 at () 288.15, () 303.15, () 318.15, () 333.15, () 343.15, () 353.15, () 363.15, () 373.15, () 393.15 and () 413.15 k. the values of the isothermal compressibility obtained in this paper were also compared to those given by zéberg-mikkelsen et al.9 at temperatures 303.15– –333.15 k and at pressures 0.1–30 mpa. the deviations were rather low: aad = = 0.64 %, md = 1.48 % and bias = –0.20 %. in addition, zéberg-mikkelsen et al.9 reported the isobaric thermal expansivities of toluene at the same temperatures and pressures as they did for the isothermal compressibility. for this property, the values of the appropriate criteria were aad = 0.71 %, md = 2.39 % and bias = –0.17 %. easteal et al.10 presented the values for the isothermal compressibility and the isobaric thermal expansivity of toluene under the same conditions. the comparison was performed at temperatures 288.15–323.15 k and pressures 0.1–50 mpa and the attained results were: for the isothermal compressibility aad = 2.48 %, md = 4.01 % and bias = –2.38 % and for the isobaric thermal expansivity aad = 1.29 %, md = 2.63 % and bias = –0.21 %. the values of the deviation criteria obtained for the isothermal compressibility and the isobaric thermal expansivity of toluene were sufficiently low for it to be concluded that the results calculated in this work agree well with the literature data. dichloromethane gonçalves et al.11 recently presented results for the isothermal compressibility and the isobaric thermal expansivity for dichloromethane at temperatures 300–400 k and pressures 0.1–100 mpa. the application of eqs. (1)–(11) enabled the calculation of the values for the mentioned properties at the same temperatures and at pressures up to 60 mpa. a comparison of the calculated data with derived volumetric properties of n-hexane, toluene and dichloromethane 1431 literature results11 resulted in the following: for the isothermal compressibility aad was 1.81 %, md was 5.04 % and bias was 0.38 % (fig. 3a) and for the isobaric thermal expansivity aad = 1.16 %, md = 3.53 % and bias = 0.19 % (fig. 3b). (a) (b) fig. 3. comparisons of the calculated derived properties with literature data for dichloromethane: a) the isothermal compressibility with ferreira et al.11 at () 300, () 320, () 340, () 360, () 380 and () 400 k, and b) the isobaric thermal expansivity with ferreira et al.11 at () 300, () 320, () 340, () 360, () 380 and () 400 k. easteal et al.10 gave the values of the isothermal compressibility and the isobaric thermal expansivity of dichloromethane at two temperatures, 288.15 and 298.15 k, and at pressures 0.1–50 mpa. the isothermal values presented in table s-iii agree with those taken from literature10 with: aad of 3.20 %, md of 3.56 % and bias of 3.20 %. when comparing the isobaric thermal expansivities from table s-iii, the following deviations were obtained: aad = 1.61 %, md = 3.33% and bias = –1.43 %. although the mentioned values of the criteria for the comparison of both properties for dichloromethane were slightly higher than those for toluene, they could still be considered as good. conclusions the density data of n-hexane, toluene and dichloromethane, reported in a previous paper,1 were fitted to a tait-like equation; the obtained parameters, and the results of assessment of the modeling, by the comparison with literature information, are given in this work. the calculated values of the isothermal compressibility, the isobaric thermal expansivity, the difference between specific heat capacity at constant pressure and at constant volume as well as the internal pressure for each of three investigated compounds, at temperatures 288.15–413.15 k and at pressures 0.1–60 mpa, are also presented herein. in order to assess the proposed modeling procedure, the obtained values of the isothermal compres1432 ivaniš et al. sibility and the isobaric thermal expansivity were compared to corresponding literature data, and the good agreement was achieved. supplementary material the calculated values of the isothermal compressibility, κt, the isobaric thermal expansivity, αp, the difference between the specific heat capacities at constant pressure and constant volume, cp–cv, and the internal pressure, pint, for n-hexane, toluene and dichloromethane, at temperatures 288.15–413.15 k and pressures 0.1–60 mpa, are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the authors gratefully acknowledge the financial support received from the research fund of ministry of education, science and technological development (project no. 172063) of the republic of serbia, and the faculty of technology and metallurgy, university of belgrade. и з в о д моделовање густине и прорачун изведених волуметријских својстава на притисцима од 0,1 до 60 mpa и на температурама од 288,15 до 413,15 k горица р. иваниш, александар ж. тасић, ивона р. радовић, бојан д. ђорђевић, слободан п. шербановић и мирјана љ. кијевчанин технолошко-металуршки факултет, универзитет у београду, карнегијева 4, 11120 београд густине n-хексана, толуена и дихлорметана на температурама 288,15–413,15 k и притисцима 0,1–60 mpa су моделоване прилагођеном тејтовом једначином. у овом раду су наведене одговарајуће једначине за рачунање изотермске стишљивости, изобарске топлотне експанзивности, разлике између специфичног топлотног капацитета при сталном притиску и оног при сталној запремини и унутрашњег притиска у читавом поменутом опсегу притисака и температура, као и вредности наведених изведених величина. у циљу процене предложене методе моделовања поређене су добијене вредности коефицијента изотермске стишљивости и коефицијента изобарског топлотног ширења са онима из литературе. просечно апсолутно процентуално одступање за изотермску стишљивост је било: за n-хексан 2,01–3,64 %, за толуен 0,64–2,48 % и за дихлорметан 1,81– –3,20 %; a за изобарско топлотну експанзивност: за n-хексан 1,31–4,17 %, за толуен 0,71–2,45 % и за дихлорметан 1,16–1,61 %. поређењем добијених одступања са оним која се могу наћи у литератури долази се до закључка да се представљени резултати добро слажу са литературним. (примљено 18. марта, ревидирано 30. јуна, прихваћено 1. јула 2015) references 1. g. r. ivaniš, a. ž. tasić, i. r. radović, b. d. djordjević, s. p. šerbanović, m. lj. kijevčanin, j. serb. chem. soc. 80 (2015) 1073 2. j. safarov, f. millero, r. feistel, a. heintz, e. hassel, ocean sci. 5 (2009) 235 3. j. l. daridon, b. lagourette, j.-p. e. grolier, int. j. thermophys. 19 (1998) 145 4. s. l. randzio, j.-p. e. grolier, j. r. quint, d. j. eatough, e. a. lewis, l. d. hansen, int. j. thermophys. 15 (1994) 415 5. j. troncoso, d. bessières, c. a. cerdeiriña, e. carballo, l. romaní, fluid phase equilib. 208 (2003) 141 6. j. zhou, r. zhu, h. xu, y. tian, j. chem. thermodyn. 42 (2010) 1429 derived volumetric properties of n-hexane, toluene and dichloromethane 1433 7. c. a. cerdeiriña, c. a. tovar, d. gonzález-salgado, e. carballo, l. romaní, phys. chem. chem. phys. 3 (2001) 5230 8. m. chorażewski, j.-p. e. grolier, s. l. randzio, j. chem. eng. data 55 (2010) 5489 9. c. k. zéberg-mikkelsen, s. i. andersen, j. chem. eng. data 50 (2005) 524 10. a. j. easteal, l. a. woolf, int. j. thermophys. 6 (1985) 331 11. f. a. m. m. gonçalves, c. s. m. f. costa, j. c. s. bernardo, i. johnson, i. m. a. fonseca, a. g. m. ferreira, j. chem. thermodyn. 43 (2011) 105. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice accepted manuscript this is an early electronic version of an as-received manuscript that has been accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as a. m. lazić, a. d. mašulović, j. m. lađarević and n. v. valentić, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc230131035l this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230131035l j. serb. chem. soc.00(0)1-16 (2023) original scientific paper jscs–12258 published dd mm, 2023 1 assessing the pharmacological potential of selected xanthene derivatives anita m. lazić1*#, aleksandra d. mašulović1#, jelena m. lađarević2# and nataša v. valentić2# 1innovation center, faculty of technology and metallurgy, belgrade, serbia, and 2university of belgrade, faculty of technology and metallurgy, belgrade, serbia (received 31 january; revised 21 february; accepted 8 july 2023) abstract: a convenient and efficient approach toward the synthesis of seven aromatically substituted xanthendiones (1‒7) and one structurally-related xanthenone (8) through condensation of dimedone and the appropriate aromatic aldehyde is reported. further, their chemical structure was confirmed by melting points, elemental analysis, ft-ir, 1h, 13c nmr and uv-vis spectroscopic methods. the relationship between the chemical structure and pharmacological activity was determined empirically using appropriate software packages and in vitro using the abts (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) method. the results of in silico prediction suggested that all investigated compounds possess good oral bioavailability. the results of the abts assay indicate that five compounds possess the ability to scavenge the abts•+ radical cation. based on the comparison of the ic50 values, the activity of the compounds was found to be as follows: 6 > 1 > 7 > 2 > 8. the effects of solvent dipolarity/polarizability and solute solvent–hydrogen-bonding interactions on the shifts of the absorption maxima were rationalized by means of the linear solvation energy relationship concepts proposed by kamlet–taft and catalán. keywords: heterocycles; chemoinformatics prediction models; antioxidant activity; lser analysis; solvatochromism. introduction the expansion of degenerative diseases induced by oxidative stress and incorporation of free radicals, including atherosclerosis, ischemic heart disease, diabetes mellitus and cancer, presents one of the main problems of today’s society.1,2 scientists are in a constant search for a novel scaffold with structural diversity and complexity to play a fundamental role in the drug discovery pipeline. herein, fused benzene or hetero benzene rings in linear, angular or clustered *corresponding authors e-mail: alazic@tmf.bg.ac.rs. https://doi.org/10.2298/jsc230131035l #serbian chemical society member a cc ep te d m an us cr ip t mailto:alazic@tmf.bg.ac.rs https://doi.org/10.2298/jsc230131035l 2 lazić et al. arrangements present interesting compounds due to their chemical structures and biological activities.3 moreover, numerous compounds containing fused rings with oxygenated phenyl moieties, are used as anticancer and anti-inflammatory agents4– 6 and have shown to inhibit the growth of cancer cells.7 on this behalf, an oxygenincorporating tricyclic compound, xanthene, arises as an interesting structural scaffold with various beneficial heterogeneous pharmacological activities.8 literature review showed that xanthene nucleus has been reported to be a versatile and suitable towards obtaining compounds for different biological targets.9 for example, xanthendiones (1,8-dioxooctahydroxanthenes) are a special class of oxygen-incorporating tricyclic compounds9 bearing as a basic feature a pyran nucleus fused on either side with cyclohex-2-enone rings.10 they are often found as a structural motif in natural products with a wide range of biological activities, such as: antioxidant11–14 antimicrobial,11,12,15 trypanocidal,16 antiinflammatory,16 antiproliferative11,12 and anticancer.17,18 besides, xanthendiones are effective inhibitors of tryparothione reductase, bone morphogenetic protein (bmp-2)targeted osteogenic agents, selective positive allosteric modulators of the δ-opioid receptors and estrogen receptors.19 as expected, the substitution pattern of the xanthene core will define the respective pharmacological response of the synthesized molecule.9 bhat et al. demonstrated that xanthendiones with electron-withdrawing halogen substituents showed a broad spectrum of antibacterial activity against both gram-positive and gram-negative bacteria. also, xanthendiones bearing the nitro group in meta position of the phenyl ring are two times more potent against c.albicans and four times more potent against a.clavatus when compared to referent drug griseofulvin.15 moreover, zukić et al.9 showed that slight variations in structure lead to different activities of xanthendiones. compounds bearing two hydroxy groups in meta and para position of the phenyl ring possess high antioxidant activity, while compounds bearing bromine atom in their structure exhibit high antimicrobial activity against e.coli and s. aureus.12 results of the antitubercular screening indicated that xanthene derivatives with the electron-withdrawing substituents possess prominent antimycobacterial activity against the mtb h37ra and m.bovis bcg.19 recent studies have shown that incorporating heterocyclic rings in molecular structure ameliorates antibacterial ability of newly synthesized compounds.20 small heterocycle-structured molecules targeting rna in living cells and providing successful therapeutic agents have been reported in the literature.21 for example, docking studies showed that these xanthendiones exhibit high binding affinity towards the amino acid residues of atp binding pocket of human pim1 kinase receptor through van der waals interactions, steric favorable interactions and hydrogen bonding, thus being promising anticancer drugs.15 ilangovan et al.8 demonstrated that 14-aryl-14h-dibenzo[a,j]xanthene derivatives act as good radical scavengers against dpph and abts•+ due to the presence of a cc ep te d m an us cr ip t pharmacological potential of xanthene derivatives 3 butterfly-like planar naphthalene rings on both sides of pyran ring and the presence of strong electron-donating substituents on the phenyl ring. besides their pharmacological activity, xanthendiones can also be used in the preparation of stable laser dyes, fluorescent sensors and protein labeling fluorophores used in laser technology, functional materials for visualization of biomolecular assemblies, photodynamic therapy and as antagonists.18 drug discovery is an inventive process of finding new potentially pharmacologically active compounds based on a combination of computational, experimental, and clinical models and the knowledge of biological targets.22 druglikeness of the compounds should present appropriate absorption, distribution, metabolism, excretion–toxicity (admet) properties leading progression from pre-clinical assessment to clinical evaluation. the rules of good bioavailability are often applied, among which the most popular is lipinski's rule. according to the "rule of five", good oral absorption can be expected for compounds whose set of physicochemical parameters is in the following ranges: partition coefficient (logp) ˂ 5, number of hydrogen bond donors ˂ 5, number of hydrogen bond acceptors ˂ 10, relative molecular mass ˂ 500.23 potentially pharmacologically active compounds often bear numerous functional groups capable of forming hydrogen bonds, making them soluble and giving them the ability to form specific interactions with their biomolecular targets.24 hydrogen bonding influences the interactions of potentially pharmacologically active organic compounds at different levels of complexity, going from those with other small molecules, up to the highest supramolecular assemblies, e.g., proteins and membranes. these interactions considerably affect the pharmacological activity, pharmacokinetics, and physicochemical properties of drugs, hence making hydrogen bonding an important subject of study in drug discovery and development.25 therefore, solvatochromic study gives an insight into possible different solute-solvent interactions mimicking the interactions of potentially pharmacologically active organic compounds with their environment. keeping in mind the above stated application and activities of xanthene derivatives, seven xanthendiones (1‒7) and one structurally-related xanthenone (8) are synthesized and thoroughly characterized. sulfur and oxygen-containing heterocycles are widely used because of their key function in fulfilling needs in medicinal chemistry,26–28 and therefore are incorporated into xanthendione scaffolds in compounds 3 and 4. in aspiration to achieve new and high potent antioxidant agents, herein, we examined radical scavenging properties of the synthetized compounds using the abts (2,2′-azinobis-(3-ethylbenzothiazoline-6sulfonic acid) method. in silico prediction was performed in order to evaluate pharmacokinetic profiles of the synthesized compounds related to absorption properties and biophysical-kinetic profiles of the synthesized compounds related to metabolism properties. the effects of specific solvent-solute interactions a cc ep te d m an us cr ip t 4 lazić et al. (hydrogen bonding) which are related to the molecular structure of a compound and nonspecific solvent-solute interactions (dipolarity/polarizability of solvents) on the absorption maxima shifts were interpreted by using the linear solvation energy relationship (lser) concept proposed by kamlet, taft and catalán.29,30 experimental the general information all chemicals, reagents and solvents were supplied by sigma-aldrich (germany) and were used without further purification. spectroscopic measurements 1h nmr spectra were recorded on a bruker ascend 400 spectrophotometer at 400 mhz, while 13c nmr spectra were recorded at 100 mhz at to the same device. 1h nmr and 13c nmr spectra of compounds 1 and 7 were recorded at room temperature in deuterated dimethyl sulfoxide (dmso-d6), while the 1h nmr and 13c nmr spectra of compounds 2–6 and 8 were recorded in deuterated chloroform (cdcl3). the chemical shifts are expressed in ppm in relation to the reference tms (δh = 0 ppm). ft-ir spectra of all synthesized compounds were recorded in the range from 400 to 4000 cm−1 using a thermo scientific nicolet is10 spectrometer, within the spectral resolution range of 400–4000 cm–1. elemental analysis of all studied compounds was performed using a microanalyzer brand elemental vario el iii. general procedure for synthesis of compounds 1 ̶8 the synthesis of compounds 1–7 is illustrated in scheme 1, while the synthesis of compound 8 is presented in scheme 2.31 3,5-dibromo-4-hydroxybenzaldehyde and 3-chloro-4hydroxybenzaldehyde, used for further synthesis of compounds 2 and 7, respectively, were synthesized according to procedures illustrated on schemes s1 and s2 (supplementary material). 5,5-dimethylcyclohaxane-1,3-dione (2 mmol) was dissolved in water prior to the addition of 1 mmol of corresponding aldehyde. the reaction mixture was stirred at room temperature for 60 min while the course of the reaction was monitored by tlc. the reaction was completed for 60 min. solid product was isolated by simple filtration and dried.32 obtained solid product (1 mmol) was dissolved in absolute ethanol (10 ml) in an erlenmeyer flask (50 ml) with gentle heating. furthermore, water (2.5 ml) and hcl (6 m, six drops) were added to the solution, and the mixture was boiled for 5 min. after cooling, water was added dropwise until the mixture became cloudy. the suspension was cooled at 0 °c, for 10 min. crystals were collected by vacuum filtration and washed with several portions (10 ml total volume) of ice cold ethanol: water (1:1 (v:v)) to yield compounds 1 ̶ 8. the solid products 1–8 were fully characterized by ft-ir, 1h and 13c nmr spectra (supplementary material, figures s1–s8) and elemental analysis. in-silico prediction determination of the relevant molecular descriptors for all synthesized compounds was assessed employing the following software packages: swissadme (swiss institute of bioinformatics, switzerland33), and preadmet (east china university of science and technology, china34). antioxidant activity the antioxidant activity of investigated compounds 1‒8 was determined using abts radical-scavenging assay.35 a stock solution of the abts•+ radical cation was prepared in the reaction of abts (4.912 ml, 7 mm in phosphate-buffered saline (pbs)) and potassium a cc ep te d m an us cr ip t pharmacological potential of xanthene derivatives 5 persulfate (0.088 ml, 140 mm in distilled water). after 16 h of incubation in the dark, the stock solution was diluted with methanol until the recorded absorbance at 734 nm was 0.700 ± 0.02. subsequently, 20 μl of the methanolic solutions of the investigated compounds (5 mm) were mixed with 2 ml of the abts radical solution, shaken and stored in the dark for 10 min. afterward the absorbance was measured at 734 nm. each test was done in triplicate. the inhibition percentage of abts•+ was calculated using the formula: inhibition = ((ac–as)/ac) 100 (1) where ac is the absorbance of the control solution (20 μl of methanol in 2 ml of abts solution) and as is the absorbance of the sample solution. ascorbic acid was used as a standard antioxidant. the antioxidant ability of the most promising derivatives 1, 2, 6, 7 and 8 was further evaluated by determination of the ic50 values. the methanolic solutions of the synthesized compounds and ascorbic acid were prepared at concentrations ranging from 5 mm to 0.5 mm, and obtained ic50 were compared. the tests were performed in triplicate. the resulting ic50 values are presented as means with standard deviation (±sd) from three experiments (n = 3). results and discussion synthesis and characterization the synthesis of compounds 1–7 is illustrated in scheme 1, while the synthesis of compound 8 is presented in scheme 2. 3,5-dibromo-4-hydroxybenzaldehyde and 3-chloro-4-hydroxybenzaldehyde, used for further synthesis of compounds 2 and 7, respectively, were synthesized according to procedures illustrated on schemes s1 and s2 (supplementary material). the synthesis of compounds 1–7 involves the formation of a knoevenagel product (a) which, through the addition of b, was further converted in the michael adduct intermediate (c) (scheme s3supplementary material). nucleophilic attack of the ‒oh group on the c=c moiety gave compounds 1 ̶7.36,37 on the other hand, when using salicylaldehyde in the reaction with dimedone, the reaction course occurs according to scheme s4 (supplementary material) and results in a formation of 9-(2-hydroxy-4,4-dimethyl6-oxocyclohex-1-enyl)-3,3-dimethyl-2,3,4,9-tetrahydro-1h-xanthen-1-one (8).38,39 a cc ep te d m an us cr ip t 6 lazić et al. scheme 1. synthesis of compounds 1 ̶7 scheme 2. synthesis of compound 8 multiparameter optimization of molecular descriptors of 1–8 first essential aspect of the potentially pharmacologically active compounds to be considered is their drug-likeness. therefore, the pharmacokinetic profile of compounds 1–8 was evaluated by predicting the appropriate admet characteristics, using various empirical rules and appropriate software packages. molecules considered to have properties similar to standard drugs must not show more than one deviation from lipinski's rule. according to veber's criterion, a cc ep te d m an us cr ip t pharmacological potential of xanthene derivatives 7 adequate oral bioavailability is achieved with molecules that have less than 10 rotatable bonds and a topological polar surface of less than 140 å2.40 according to the modified versions of these two concepts, for compounds whose physicochemical properties satisfy the following ranges: 160 ≤ relative molecular mass ≤ 500; –0.4 ≤ wlogp ˂ 5.6; 40 ≤ molar refractivity ≤ 130; 20 ≤ number of atoms ≤ 70 (goose's criterion) and wlogp ≤ 5.88, tpsa (topological polar surface area) ˂ 131.6 å2 (egan's criterion), there is a high probability of manifesting therapeutic effects.41 based on the values of the molecular descriptors described in these rules (tables s1 and s2, supplementary material), it can be concluded that the examined compounds (except compound 2 with the molecular weight > 500 g/mol and log p > 5) meet all the stated empirical criteria. tables s1 and s2 (supplementary material) show that they fulfill the theoretical condition for adequate biological availability in the body and therefore possess a pharmacological potential. based on the calculated values of the topological polar surface area of the molecule, it is expected that in in vivo conditions, the investigated compounds will show good intestinal absorption. furthermore, a small number of rotatable bonds suggests that significant conformational changes upon solvation and binding to appropriate receptors are not expected.23 hence, the complete series of compounds was further examined with swissadme and preadmet in silico tools regarding their bbb permeation, pgp inhibition, inhibition of cytochrome p450 (cyp) (1a2, 2c19, 2c9, 2d6, 3a4) and considerations were performed in accordance with the information gathered. comprehensive results are presented in supplementary material (tables s2 and s3).9 the data presented in table s2 (supplementary material) show that different partition coefficient values were obtained for the same compound, which is a consequence of different mathematical algorithms for calculating this parameter within the used software packages.19,20 the highest values of the partition coefficient were obtained for compound 2, which enables this molecule to more successfully pass through the blood-brain barrier by passive diffusion, as well as to more successfully bind to the active sites on the corresponding receptors. obtained data indicated that compounds 1, 4, 6 and 7 were found to be bbb permeant and only compound 5 was found not to be p-gp inhibitor.9 according to obtained results, it can be concluded that all investigated compounds possess high gastrointestinal absorption, inhibit isoenzymes cyp2c19 and cyp2c9 and don’t inhibit isoenzymes cyp1a2 and cyp2d6. based on the optimal values of the molecular descriptors obtained by applying appropriate software packages, it can be concluded that the compounds synthesized in this work meet all the necessary empirical criteria, which further qualify them as interesting drug candidates. antioxidant activity of the investigated compounds 1–8 evaluation of the antioxidant properties of the synthesized compounds was assessed using the abts assay and the scavenging activity was further compared a cc ep te d m an us cr ip t 8 lazić et al. to the activity of ascorbic acid (figure 1a). as antioxidants provide either hydrogen atoms or electrons to neutralize the single electron originating from free radicals, exploring effective antioxidants is of importance in medicinal chemistry.42 the obtained data indicate variable activity of the compounds, designating that substituents in position 9 of xanthendiones affect the antioxidant ability of the molecules. considering most of the compounds bear p-substituted phenyl scaffold (1, 2, 5–7) as a substituent in position 9 of xanthendione moiety, the choice of thiophene and furan rings should be further explained, in this section, from the aspect of antioxidant activity. compounds 1 (with the 4-hydroxy phenyl ring), 2 (3,5-dibromo-4-hydroxy substituted phenyl ring), 6 (with the 4-dimethylamino substituted phenyl ring), 7 (with the 3-chloro-4-hydroxy substituted phenyl ring) and 8 exhibited excellent ability to scavenge the abts•+ radical cation, comparing to the inhibition of ascorbic acid. other compounds (3–5) showed weak or no antioxidant properties. despite the expected data, wherein incorporating thiophene and furan nuclei with other heterocyclic compounds improves antioxidant activity,26–28,43,44 compounds 3 and 4 do not exhibit significant amelioration in activity when compared to analogues. incorporating these scaffolds, as pharmacophores of choice for designing antioxidant drugs, known for the excellent results through various mechanisms of action,26–28,43,44 has shown to have little to no effect on the antioxidant potential as substituents on xanthendione moiety in this case. the antioxidant activity of candidates 1, 2, 6, 7 and 8 was further evaluated by the determination of ic50 values (figure 1b). the high ic50 values generally suggest low antioxidant activity. the ic50 values of the samples, ranging from 1.53 to 3.92 mm, indicate that these compounds demonstrate good antioxidant capacity in comparison to ic50 value of ascorbic acid (1.45 mm). based on the comparison of the ic50 values, the activity of these compounds was found to be as follows: 6 > 1 > 7 > 2 > 8. based on the obtained results and literature survey, it could be concluded that the antioxidant activity is mainly associated with the presence of – oh group in the para-position of the phenyl ring which is in line with the wellestablished fact that phenolic functionality has considerable scavenging potential.43,45 notably, the most potent compound (6) bears p-dimethylamino group in the phenyl ring indicating that this group imparts significant antioxidant activity and is in accordance with the literature data.46,47 a cc ep te d m an us cr ip t pharmacological potential of xanthene derivatives 9 figure 1. the antioxidant properties of investigated compounds compared to the ascorbic acid. a) the scavenging activity at the sample concentration of 5mm; b) the ic50 values of compounds 1, 2, 6, 7, 8 and ascorbic acid used as reference. solvatochromic analysis of the investigated compounds 1–8 it is known that the physicochemical and photochemical properties of different π-conjugated structures are influenced by the extent of their π-electron conjugation and the nature of functional groups attached to the conjugated chain as well as the nature of the surrounding medium.48 bearing this in mind, we directed our solvatochromic research towards an extensive analysis of the influence of specific a cc ep te d m an us cr ip t 10 lazić et al. and non-specific solvent interactions on the absorption maxima shift of these compounds containing different chromophores and auxochromes. the solvatochromic properties of compounds 1–8 were investigated by determining the corresponding uv-vis absorption spectra in a selected solvent set of different polarity (seven protic, ten aprotic and two nonpolar solvents) in the wavelength range 200–500 nm. the uv-vis absorption spectra of the investigated compounds 1–8 showed no dependence of compound concentration suggesting that no dimmers of higher aggregates are formed. representative uv-vis spectra are presented in figures s9‒s11 (supplementary material), while the values of the absorption maxima are given in table s4 (supplementary material). the observed trends in the absorption maxima shifts are in accordance with previously published results.49–51 namely, the absorption spectra of all investigated compounds are characterized by the presence of one dominant band corresponding to the π→π* transition, which appears in the wavelength range of 220‒270 nm in the polar protic solvents and the range of 280‒300 nm in polar aprotic solvents and nonpolar solvents. in addition, based on the data presented in table s4 (supplementary material), when polar protic solvents are used, all investigated compounds show additional absorption maxima in the wavelength range of 290‒300 nm. this splitting of the absorption band could be attributed to a protonation reaction which is likely to occur at the carbonyl moieties and hydroxyl groups.30 in the case of compound 6, a shoulder at ca. 250 nm appears in diethyl ether and acetonitrile. based on the data given in table s4 (supplementary material) and the absorption spectra presented in figures s9–s11 (supplementary material), it can be concluded that increasing solvent polarity causes bathochromic shifts of absorption maximas. the influence of the solvent parameters (the effects of solvent polarity and hydrogen bonding) on the position of the absorption maxima is of very complex nature and was therefore analyzed mathematically in more detail52 using general solvatochromic equations established by kamlet-taft (2) and catalán (3): νmax = νmax0 + aα + bβ + sπ* (2) νmax = νmax0 + asa + bsb + csp + dsdp (3) where νmax is a solvent-dependent physicochemical property in a given solvent, νmax0 is a statistical quantity that corresponds to the absorption frequency in cyclohexane as a reference solvent, α is a measure of the acidity of the solvent, describing its ability to donate a proton when establishing a hydrogen bond, β is a measure of the basicity of the solvent, i.e. its ability to receive a proton when establishing a hydrogen bond, π* is a measure of dipolarity/polarizability of the solvent, (table s5, supplementary material).53 sa is a measure of the acidity of the solvent, i.e. it describes its ability to donate a proton when establishing a hydrogen bond, sb is a measure of the basicity of the solvent, i.e. its ability to receive a proton when establishing a hydrogen bond a cc ep te d m an us cr ip t pharmacological potential of xanthene derivatives 11 and spp is a measure of dipolarity/polarizability of the solvent. parameters sa, sb and spp (table s5, supplementary material) are equivalent to the kamlet-taft α, β and π* solvatochromic parameters. in 2004, catalán and hopf27 developed another important solvatochromic parameter, known as solvent polarizability-sp. a, b, c and d are regression coefficients describing the sensitivity of properties α, β and π*, that is sa, sb, spp and sp to the different solute−solvent interactions. the values of the regression coefficients with a probability of 95 %, together with the percentage contribution of solvatochromic parameters are shown in tables s6 and s7 (supplementary material). the respective correlation coefficients obtained for both solvent parameter sets are in a good agreement with each other, therefore both models are equally suitable for describing specific/nonspecific solute-solvent interactions. based on the high values of the obtained regression coefficients and the fisher parameter, the results of the correlation of absorption frequencies with solvatochromic parameters can be considered satisfactory. the data presented in table s6 (supplementary material) indicate a bathochromic shift of the absorption maxima with the increasing solvent polarity expressed by the negative coefficient s. this indicates to a better stabilization of the excited state of the molecule compared to its ground state was confirmed. the negative values of coefficient b (compounds 1‒3 and 7, table s6, supplementary material) indicate that the solvent basicity affects the molecule more in the excited than in the ground state. in the same manner, the negative values of the coefficient a (compounds 1, 3, 4, 6, 7, table s6, supplementary material) indicate that the solvent acidity affects the molecule more in the excited than in the ground state. the negative values of a term could be explained by the strong intermolecular hydrogen bonding of hbd (hydrogen bond donor) solvents with the carbonyl moieties of investigated compounds, which further reduces their electron density and thus increases the push-pull character of the chromophore. based on the values of the percentages of solvatochromic parameters (table s6, supplementary material), it can be concluded that for compounds 2, 5, 6 and 7, the dipolarity/polarizability of the solvent is more dominant in relation to its acid-base properties, while with compounds 3, 4 and 8, the acidity of the solvent is more dominant and with compound 1 the basicity of the solvent. the strongest influence of the acidity of the solvent in compounds 3 and 4 can be ascribed to furanyland thiophenylgroups used as substituents in position 9 of the xanthendione moiety.52 analogously to the previously applied model, it was observed that the negative values of the coefficients a, b, c and d in the catalán model (table s7, supplementary material) indicate that the solvent effects are more pronounced in the excited than in the ground state of the studied molecules. it is also confirmed that the dipolar investigated compounds (with the exception of compounds 3 and a cc ep te d m an us cr ip t 12 lazić et al. 5) show positive solvatochromism with regard to dipolarity/polarizability as indicated by negative c and d terms (table s7, supplementary material). considering the solvent acidity, the exception from the series are compounds 5 and 6, where positive values of the coefficient a indicate that the hydrogen bonding better stabilizes the ground than the excited state of the molecule. the disagreement between the results obtained by these two models is influenced by different solvatochromic probes used for the determination of the parameters, thus reflecting different solvent-solute interactions. the kamlet-taf empirical parameters are obtained as average experimental values of numerous solvatochromic probes, while catalán's empirical solvent scales are based on welldefined reference probe systems.54 conclusion in this study, we synthesized seven aromatically substituted xanthendiones (1‒7) and one structurally-related xanthenone (8) and screened their potential pharmacokinetic properties using convenient chemoinformatics prediction models (swissadme and preadmet). the results of chemoinformatics prediction models showed that almost all investigated compounds (with the exception of compound 2) meet lipinski’s rule of five and its extensions, such as veber’s, egan’s and goose’s criterion, indicating their good oral bioavailability. the antioxidant screening carried out according to the abts assay revealed the antiradical properties of the investigated compounds. this study also revealed that the antioxidant activities were in the following order: 6 > 1 > 7 > 2 > 8, based on the comparison of their ic50 values. compound 6 is the most active with ic50 value comparable with ic50 value of ascorbic acid. the results obtained by general solvatochromic equations established by kamlet-taft and catalán indicate that the position of the uv-vis absorption maxima depends on the nature (polarity, acidity and basicity) of the solvent. with their high antioxidant activities and good oral bioavailability, the investigated compounds have set the path for the preparation of new pharmacologically active compounds and a better understanding of the structure-activity relationship. acknowledgements: this work was supported by the ministry of science, technological development and innovation of the republic of serbia (contract no. 451-03-47/202301/200135 and 451-03-47/2023-01/200287). supplementary material supplementary materials are available electronically from https://www.shdpub.org.rs/index.php/jscs/article/view/12258, or from the corresponding authors on request. a cc ep te d m an us cr ip t https://www.shd-pub.org.rs/index.php/jscs/article/view/12258 https://www.shd-pub.org.rs/index.php/jscs/article/view/12258 pharmacological potential of xanthene derivatives 13 и з в о д одређивање фармаколошке активности деривата ксантeна анита м. лазић1*, александра д. машуловић1, јелена м. лађаревић2 и наташа в. валентић2 1иновациони центар, технолошко-металуршког факултета, београд, србија, и 2универзитет у београду, технолошко-металуршки факултет, београд, србија у овом раду, представљена је једноставна двокорачна метода за синтезу седам деривата ксантендиона и једног деривата ксантeнoна који садрже различите ароматичне супституенте, полазећи од димедона и одговарајућих ароматичних алдехида. карактеризација синтетисаних једињења извршена је одређивањем температуре топљења, као и применом елементалне анализе, ft-ir, 1h nmr и 13c nmr спектроскопских метода. веза између хемијске структуре ових једињења и њихове фармаколошке активности успостављена је емпиријски коришћењем одговарајућих софтверских пакета (за предицкцију фармаколошке активности) и in vitro одређивањем њихове антиоксидативне активности применом abts методе. резултати abts методе показују да од целокупне серије тестираних једињења, пет једињења показује значајну антиоксидативну активност. на основу међусобног поређења ic50 вредности испитиваних једињења показано је да њихова антиоксидативна активност опада у следећем низу: 6 > 1 > 7 > 2 > 8. једињење 6 је најактивније у анализираној серији и има ic50 вредност приближне вредности као аскорбинска киселина. резултати солватохромних једначина које су развили камлет, тафт и каталан, указују да положај апсорпционих максимума проучаваних једињења зависи од природе (поларности и кисело-базних својстава) употребљених растварача. са високим вредностима антиоксидативне активности и добром оралном биорасположивошћу, деривати ксантендиона и ксантенона са ароматичним супституентима, представљају добру полазну основу за синтезу нових фармаколошки активних једињења и боље разумевање утицаја структуре на фармаколошку активност. 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577.354+547.233–304.2+547.822.3+ jscs–4851 547.828:57.02:543 original scientific paper 347 synthesis, biological evaluation and docking analysis of substituted piperidines and (2-methoxyphenyl)piperazines jelena z. penjišević1#, vladimir v. šukalović1#, deana b. andrić2*#, goran m. roglić2#, irena t. novaković1#, vukić šoškić3# and slađana v. kostić-rajačić1# 1ictm – center of chemistry, university of belgrade, njegoševa 12, belgrade, serbia, 2faculty of chemistry, university of belgrade, studentski trg 12–16, belgrade, serbia and 3orgentec diagnostica gmbh, carl-zeiss-straße 49–51, mainz, germany (received 21 october, revised 25 november, accepted 26 november 2015) abstract: a series of sixteen novel substituted piperidines and (2-methoxyphenyl)piperazines were synthesized, starting from the key intermediates 1-(2 -methoxyphenyl)-4-(piperidin-4-yl)piperazine and 1-(2-methoxyphenyl)-4 -[(piperidin-4-yl)methyl]piperazine. biological evaluation of the synthesized compounds was illustrated by seven compounds, of which 1-(2-methoxyphenyl)-4-{[1-(2-nitrobenzyl)piperidin-4-yl]methyl}piperazine had the highest affinity for the dopamine d2 receptor. for all seven selected compounds, docking analysis was performed in order to establish their structure-to-activity relationship. keywords: dopamine d2 receptor; docking analysis; allosteric; orthosteric binding site. introduction g-protein-coupled receptors (gpcrs) are transmembrane receptors that mediate most of their intracellular actions through pathways involving an activation of the g-protein.1 d2 dopamine receptors (d2dar) are members of this large protein family. dysfunction of the dopaminergic system in cns can lead to a number of diseases, such as parkinson’s disease, schizophrenia, some neurohumoral disturbances, etc.2,3 therefore it is not surprising that the design and synthesis of new potential dopaminergic drugs is one of the main objectives of organic and medicinal chemistry. arylpiperazines are a common structural motif included in various compounds that interact in a specific manner with various gpcrs.4 within the scope * corresponding author. e-mail: deanad@chem.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc151021097p _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 348 penjišević et al. of the program aimed at the discovery of new dopaminergic (da-ergic) ligands and in order to explore further previously published data, a series of sixteen novel arylpiperazines were synthesized.5 for all the synthesized ligands, their in vitro binding affinities at rat d2dar were estimated and compared with the results obtained through docking analysis, using an available d2dar molecular model. experimental general melting points were measured on a boetius phmk apparatus (veb analytic, dresden, germany) and are uncorrected. the 1hand 13c-nmr (200 and 50 mhz) spectra of the compounds in deuterochloroform, unless otherwise stated, were recorded on a gemini 2000 instrument (varian, oxford). the chemical shifts (δ) are reported in ppm downfield from the internal standard tetramethylsilane. the lc-ms results were acquired on a 6210 time-of-flight lc–ms system (agilent technologies, germany); masshunter workstation software was used for data analysis. the ir spectra were taken on a thermo scientific spectrometer. a microsynth milestone and a biotage initiator 2.5 exp were used for the microwave irradiations. analytical tlc was performed on polygram sil g/uv254 plastic-backed thinlayer silica gel plates (macherey-nagel, germany). the chromatographic purifications were realized on merck-60 silica gel columns (diameter 70 mm, h = 45 mm; the same for all compounds), 230–400 mesh astm, medium pressure (dry column flash chromatography). the reagents and solvents (alfa–aesar or sigma–aldrich) were used without purification. the solutions were routinely dried over anhydrous na2so4 prior to evaporation. chemistry ethyl 4-[4-(2-methoxyphenyl)piperazin-1-yl]piperidine-1-carboxylate (3). to a stirred solution of n-carbethoxy-4-piperidone (1, 1.7 g, 0.01 mol) in methanol (25 ml; the ph value of the solution was adjusted to 7 by addition of ch3co2h), 1-(2-methoxyphenyl)piperazine (2, 3.24 g, 0.02 mol) was added, followed by the addition of nabh3cn (0.4 g, 0.0072 mol) in portions (scheme 1). stirring was continued at room temperature for 24 h. the ph value of the resulting solution was adjusted to 2 by the addition of 10 % hcl solution and the excess of the methanol was removed under vacuum. the ph value of the residue was adjusted to 9 by the addition of 10 % naoh solution and extracted with dichloromethane. the organic layer was dried over anhydrous na2so4 and evaporated in vacuo. the product was purified by dryflash chromatography using a gradient of ch3oh (0–10 %) in dichloromethane as the solvent. yield: 88 %. general procedure for the hydrolysis of the carbamates 3 and 9. carbamate 3 or 9 (0.02 mol) was suspended in cc hcl (60 ml), transferred into a sealed tube and placed into a microwave oven. irradiation at 130 °c was completed after 90 min at an initial power of 300 w (schemes 2 and 3). the reaction mixture was poured into water, the ph value adjusted to 9 by addition of 10 % naoh solution and extracted with dichloromethane. the organic layer was dried over anhydrous na2so4 and evaporated in vacuo. the product was purified by dryflash chromatography using a gradient of methanol (0–10 %) in dichloromethane as the solvent. 1-(ethoxycarbonyl)piperidine-4-carboxylic acid (6). to a solution of piperidine-4-carboxylic acid (5, 20 g, 0.155 mol) in water (200 ml), na2co3 (20 g) was added, mixture stirred at room temperature for 30 min and a solution of ethyl chloroformate (25.5 g, 0.28 mol) in toluene (240 ml) was added dropwise (scheme 2). stirring was continued at room tempe_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ substituted piperidines and (2-methoxyphenyl)piperazines 349 rature for 20 h. after separation of the layers, the aqueous layer was acidified with conc. hcl to ph ≈2, and extracted with dichloromethane. the organic layer was dried over anhydrous na2so4 and evaporated in vacuo. yield: 78 %. scheme 1. synthesis of 1-(2-methoxyphenyl)-4-(piperidin-4-yl)piperazine (4); reagents: a) nabh3cn, meoh, ph 7, r.t; b) conc. hcl, mw, 180 °c, 300 w. scheme 2. synthesis of 1-(2-methoxyphenyl)-4-[(piperidin-4-yl)methyl]piperazine (10); reagents: a) na2co3, ethyl chloroformate, toluene, r.t; b) thionyl chloride, ch2cl2, 0 °c; c) triethylamine, chloroform, 5 °c; d) nabh4, boron trifluoride diethyl etherate, diglyme, –5 °c; e) conc. hcl, mw, 180 °c, 300 w. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 350 penjišević et al. ethyl 4-(chlorocarbonyl)piperidine-1-carboxylate (7). a solution of 1-(ethoxycarbonyl) piperidine-4-carboxylic acid (6, 7.5 g, 0.0375 mol), thionyl chloride (5.35 g, 0.045 mol) and chloroform (200 ml) was stirred for 2 h at 0 °c (scheme 2). the reaction mixture was evaporated in vacuo and the product was used immediately, without further purification. yield: 67 %. ethyl 4-{[4-(2-methoxyphenyl)piperazin-1-yl]carbonyl}piperidine-1-carboxylate (8). a solution of chloride 7 in chloroform (50 ml) was added dropwise to a solution of triethylamine (3.78 g, 0.0375 mol), 1-(2-methoxyphenyl)piperazine (2, 6.07 g, 0.0375 mol) in chloroform (150 ml) at 5 °c (scheme 2). the reaction mixture was stirred at room temperature for 20 h, the resulting mixture extracted with 10 % na2co3 and the organic layer was extracted with 10 % hcl solution. the organic layer was dried over anhydrous na2so4, and evaporated in vacuo. the product was purified by dry-flash chromatography eluting with dichloromethane containing increasing amounts of methanol (0–10 %). yield: 85 %. ethyl 4-{[4-(2-methoxyphenyl)piperazin-1-yl]methyl}piperidine-1-carboxylate (9). mixture of compound 8 (0.01 mol) and nabh4 (1 g, 0.025 mol) in diglyme (1-methoxy-2-(2 -methoxyethoxy)ethane, 25 ml) was stirred for 40 min at –5 °c under argon, during which time, boron trifluoride diethyl etherate (3.9 g, 3.4 ml, 0.025 mmol) was added dropwise (scheme 2). after stirring for 1 h at 5 °c, the reaction mixture was heated to 80–90 °c, followed by stirring for additional i h. the mixture was cooled to room temperature, carefully poured into 10 ml of water and then 20 ml cc hcl was added. mixture was heated at 60–80 °c on aqueous bath for 3 h and concentrated under vacuo. to the residue, 10 % naoh solution was added until ph 9 and extracted with ch2cl2. the organic layer was dried over anhydrous na2so4 and evaporated in vacuo. the product was purified by dry-flash chromatography using the dichloromethane/methanol system as eluent. yield: 85 %. general procedure for the alkylation of compounds 4 and 10. a mixture of compound 4 or 10 (0.0018 mol), benzyl halides 11–14 (0.0018 mol), k2co3 (0.0036 mol) and acetonitrile (25 ml) was stirred at room temperature for 48 h, poured into water and extracted with dichloromethane. the organic layer was dried over anhydrous na2so4 and evaporated in vacuo. the product (15–22, scheme 3) was purified by dry-flash chromatography using a gradient of methanol (0–10 %) in dichloromethane as the solvent. general procedure for the synthesis of compounds 27–30 and 31–34. a solution of benzoyl or nitrobenzoyl chloride 23–26 (0.0017 mol) and ch2cl2 (0.34 ml) was added dropwise to a solution of 4 or 10 (0.0017 mol), ch2cl2 (1.7 ml) and et3n (0.24 ml, 0.0017 mol) at 0 °c. the reaction mixture was stirred at room temperature for 72 h. the resulting mixture was extracted with 10 % na2co3 solution, the separated organic layer was washed with 10 % k2co3 solution, dried over anhydrous na2so4 and evaporated in vacuo. the product (27–34, scheme 3) was purified by dry-flash chromatography eluting with ch2cl2 containing increasing amounts of meoh (0–10 %). analytical and spectral data for the synthesized compounds are given in the supplementary material to this paper. membrane preparation, radio-ligand binding assays and data analysis synaptosomal membranes from rat striatum were prepared for radio-ligand binding assays as previously described.6 [3h]spiperone (specific. activity: 9.25 mbq mmol-1) used to label d2dar were purchased from perkin elmer las gmbh, rodgau, germany. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ substituted piperidines and (2-methoxyphenyl)piperazines 351 hn n n h3co cl r coclr a b n n n n h3co n r n n n h3co n r o 4, n=0 10, n=1 15-18, n=0 19-22, n=1 27-30, n=0 31-34, n=1 scheme 3. synthetic route and chemical structures of the (2-methoxyphenyl)piperazine dopaminergic ligands; reagents: a) compounds 11–14 (r = h, 2-no2, 3-no2, 4-no2, respectively), k2co3, ch3cn, r.t; b) compounds 23–26 (r = h, 2-no2, 3-no2, 4-no2, respectively), triethylamine, ch2cl2, 0 °c; yields for 15–22 and 27–34: 68–89 %. [3h]spiperone–receptor binding assay. [3h]spiperone binding was assayed in 4 mm mgcl2, 1.5 mm cacl2, 5 mm kcl, 120 mm nacl, 25 mm tris–hcl solution, ph 7.4, at a membrane protein concentration of 0.7 mg ml-1 at 37 °c for 10 min in a total volume of 0.4 ml of the incubation mixture. binding of the radioligand to the 5-ht2 receptors was prevented by 50 μm ketanserin. the ki values of the tested compounds were determined by competition binding at 0.2 nm of the radio-ligand and eight to ten different concentrations of each compound (10-4 to 10-10 m). nonspecific binding was measured in the presence of 1.0 mm spiperone. the reaction was terminated by rapid filtration through whatman gf/c filters, which were further washed three times with 5.0 ml of ice-cold incubation buffer. each point was determined in triplicate. the retained radioactivity was measured by introducing dry filters into 10 ml of toluene-based scintillation liquid and counting in a 1219 rackbeta wallac scintillation counter (eg & g wallac, turku, finland) at an efficiency of 51–55 % for tritium. the results were analyzed by nonlinear curve fitting of the inhibition curves of the compounds utilizing the graph-pad prism program.7 hill slope coefficients were fixed to unity during the calculation. docking analysis docking analysis was performed with an already available d2dar model based on the d3dar crystal structure.8 the binding site of the receptor was determined by combining results from experimental data9,10 and the schrödinger maestro receptor grid generation module.11 amino acid residue charges were adjusted where needed, assuming physiological conditions. selected ligands were prepared with the ligprep maestro module and docked using the glide module from the schrödinger suite 2011.11 all ligands were docked as protonated, using the opls_2005 force field. the initial position of the ligand in the binding site was arbitrary, while the protonated nitrogen on the ligand part was kept in close proximity to asp 114 of the d2dar. after initial ligand placement, no further constraints were applied and the _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 352 penjišević et al. docking procedure was performed. the obtained structures were examined and those meeting the following criteria were selected: best docking score of the complex, shortest salt bridge formed between asp 114 of the d2dar and ligand, chair conformation of arylpiperazine ring and aryl part of the molecule positioned in the rear hydrophobic pocket of the receptor (phe 386, trp 390 and tyr 420).12 after an initial criterion was satisfied, the second step was the examination of different interactions that could be formed between the receptor and ligand (hydrogen bonds, aromatic–aromatic interactions, etc.). in that way, the best possible docking structures were selected. structures were visualized using ds visualize v2.5.113 and the obtained images were rendered using povray raytracer v3.6.14 results and discussion the general synthetic route and chemical structures of the novel substituted piperidine and (2-methoxyphenyl)piperazine are summarized in schemes 1–3. preparation of the key intermediates, 1-(2-methoxyphenyl)-4-(piperidin-4 -yl)piperazine (4) and 1-(2-methoxyphenyl)-4-[(piperidin-4-yl)methyl]piperazine (10) are described in schemes 1 and 2. ethyl 4-[4-(2-methoxyphenyl)piperazin -1-yl]piperidine-1-carboxylate (3), produced by the reductive amination of the commercially available ketone 1 was further hydrolyzed, under microwave conditions, and intermediate 4 was obtained (scheme 1). commercially available piperidine-4-carboxylic acid (5) was transformed into carbamate 6 and further into chloride 7 by reaction with thionyl chloride. acylation of (2-methoxyphenyl)piperazine with chloride 7 gave amide 8, which provided compound 9 by reduction with nabh4/boron trifluoride ethyl etherate. the carbamate 9 was converted to the secondary amine 10 by hydrolysis with cc hcl under mw conditions (scheme 2). both 4 and 10 intermediates were alkylated with benzyl or nitrobenzyl halogenide to give the final ligands 15–18 and 19–22, respectively. ligands 27–30 and 31–34 were obtained by acylation with the corresponding acyl chloride (scheme 3). the final products 15–22 and 27–34 were evaluated for their affinity to d2dar by the in vitro competitive displacement assay of [3h]spiperone (table i). as a source of d2dar, synaptosomal membranes prepared from rat striatum were used. the compound with the highest affinity for d2dar was 1-(2-methoxyphenyl)-4-{[1-(2-nitrobenzyl)piperidin-4-yl]methyl}piperazine (20, ki = 30.6 nm). compounds 19, 21, 22 and 31–34 expressed moderate binding affinity for d2dar, while 15–18 and 27–30 were completely inactive competitors of bound [3h]spiperone. compounds 20–22 and 31–34 were selected for docking analysis in order to establish their structure-to-activity relationship. d2dar model and selected compounds were prepared as described and docking analysis was performed. the obtained results showed that compound 20 binds to d2dar via a salt bridge with asp 114 on tm3. this is followed by _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ substituted piperidines and (2-methoxyphenyl)piperazines 353 multiple aromatic interactions between the aryl part of the ligand and the hydrophobic pocket (phe 386, trp 390 and tyr 420).8,15,16 in this way, the ligand establishes a favorable orientation inside the receptor binding cavity that is a prerequisite for the formation of hydrogen bonds with ser 193 on tm5. the stated interactions are formed with d2dar inside the orthosteric bind site (obs). docking analysis showed possible aromatic interactions with phe 393 and his 397, both located inside the alosteric bind site (abs, fig. 1) and the listed interactions lead to high compound activity. table i. binding constants of the synthesized compounds for the dopamine d2 receptor n n n h3co n r compound n r (ki±sem) / nm 15 0 h 736±24 16 0 2-no2 521.5±13 17 0 3-no2 937.5±35 18 0 4-no2 1512±30 19 1 h 341.5±12 20 1 2-no2 30.6±1.2 21 1 3-no2 258 ±11 22 1 4-no2 200±12 compound n r (ki±sem) / nm 27 0 h 1583.5±32 28 0 2-no2 1205±19 29 0 3-no2 755±21 30 0 4-no2 905±28 31 1 h 189.5±12.1 32 1 2-no2 219.5±14.4 33 1 3-no2 300±16.2 34 1 4-no2 334.5±17.8 in the case of compounds 21 and 22, in contrast to compound 20, the optimal hydrogen bond with the serine residues on tm5 cannot be formed. the reduced binding affinity was a clear consequence of the unfavorable orientation of ligands 21 and 22 inside the receptor binding cavity. compounds 31–34 dock, in the _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 354 penjišević et al. same manner, with aryl part oriented inside the hydrophobic pocket (phe 386, trp 390 and tyr 420), a salt bridge with asp 114 and hydrogen bonds with ser 193 (fig. 2). the only difference, compared to compound 20, is the positioning of the head part of the ligand. the reduced flexibility of the head part leads to sub-optimal positioning of the aromatic part inside the abs and the only observed aromatic interaction is with phe 394. this leads to reduced binding affinity, with respect to ligand 20. fig. 1. docking of compound 20 into the d2dar binding pocket. fig. 2. docking of compound 31 into the d2dar binding pocket. the other compounds cannot form the interactions discussed above, mostly due to their rigidity. therefore, they either cannot achieve the correct orientation inside the abs, or cannot form any hydrogen bond with serine residues on tm5. conclusions in order to achieve high binding affinity, a d2dar ligand has to fulfill several requirements. the formation of a salt bridge with asp 114 is the crucial interaction that starts the binding process, which is followed by orientation of _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ substituted piperidines and (2-methoxyphenyl)piperazines 355 arylpiperazine ligand part into the obs hydrophobic cavity. after these initial requirements are met, the ligand has to establish one or more hydrogen bonds with serine residues on tm5. failing this, the ligand could still bind to d2dar, but with reduced affinity. in order to establish hydrogen bonds, the ligand has to be of considerable length to span the entire obs between asp 114 and ser 193 and/or 197. since the obs is not linear, the ligand has to adopt a slightly curved conformation in order to bind successfully. the conformation of the arylpiperazine part is fixed at the chair conformation of the arylpiperazine ring and the rest of the ligand has to be flexible enough to fit into the obs space. in the case of compounds 15–34, only compounds 21, 22 and 31–34 can adopt the described conformation that leads to high affinity receptor binding. once the conformational requirements are fulfilled, the affinity is determined by the number and strength of particular receptor–ligand interactions. ligand 20 has the best overall fit into d2dar that, together with the formed interactions, leads to the highest affinity in the group of ligands. obtained results suggest that in future studies special attention should be paid to the synthesis of the ligands with a prolonged, flexible bridge that will provide more degrees of rotational freedom of the molecules, which allows a proper orientation of the ligands in the obs cavity, which is an essential prerequisite for high affinity d2dar ligands. supplementary material analytical and spectral data of the synthesized compounds are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgments. these results are part of project 172032, supported by the ministry of education, science and technological development of the republic of serbia. и з в о д синтеза, биолошко испитивање и докинг анализа супституисаних пиперидинских и (2-метоксифенил)пиперазинских лиганада јелена з. пењишевић1, владимир в. шукаловић1, деана б. андрић2, горан м. роглић2, ирена т. новаковић1, вукић шошкић3 и слађана в. костић-рајачић1 1ихтм –центар за хемију, универзитет у београду, његошева 12, београд, 2хемијски факултет, универзитет у београду, студентски трг 12–16, београд и 3orgentec diagnostica gmbh, carl-zeiss-straße 49–51, mainz, germany синтетисана је серија од шеснаест нових супституисаних пиперидина и (2-метоксифенил)пиперазина, полазећи од кључних интермедијера 1-(2-метоксифенил)-4-(пиперидин-4-ил)пиперазина и 1-(2-метоксифенил)-4-(пиперидин-4-илметил)пиперазина. биолошко испитивање синтетисаних једињења је истакло седам једињења, од којих 1-(2 -метоксифенил)-4-{[1-(2-нитробензил)пиперидин-4-ил]метил}пиперазин има највиши афинитет ка d2 допаминском рецептору. за свих седам једињења је урађена докинг анализа у циљу утврђивања односа структуре и активности. (примљено 21. октобра, ревидирано 25. новембра, прихваћено 26. новембра 2015) _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 356 penjišević et al. references 1. c. missale, s. r. nash, s. w. robinson, m. jaber, m. g. caron, physiol. rev. 78 (1998) 189 2. j.-m. beaulieu, r. r. gainetdinov, pharmacol. rev. 63 (2011) 182 3. n. ye, j. l. neumeyer, r. j. baldessarini, x. zhen, a. zhang, chem. rev. 113 (2013) pr123 4. a. zhang, j. l. neumeyer, r. j. baldessarini, chem. rev. 107 (2007) 274 5. j. penjišević, v. sukalović, d. andrić, s. kostić-rajačić, v. šoškić, g. roglić, arch. pharm. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice syntheses and antimicrobial activities of 1-(3-benzyl-4-oxo-3,4-dihydroquinazolin-2-yl)-4-(substituted) thiosemicarbazide derivatives j. serb. chem. soc. 80 (12) 1471–1479 (2015) udc 547.856+547.497.1:542.913: jscs–4812 615.28–188 original scientific paper 1471 syntheses and antimicrobial activities of 1-(3-benzyl-4-oxo-3,4 -dihydroquinazolin-2-yl)-4-(substituted) thiosemicarbazide derivatives veerachamy alagarsamy1*, viswas raja solomon1, g. krishnamoorthy2, m. t. sulthana1 and b. narendar1 1medicinal chemistry research laboratory, mnr college of pharmacy, sangareddy, gr. hyderabad -502 294, india and 2department of pharmaceutical chemistry, periyar college of pharmaceutical sciences for girls, trichy – 620 021, india (received 3 january, revised 11 may, accepted 10 june 2015) abstract: a series of 1-(3-benzyl-4-oxo-3,4-dihydroquinazolin-2-yl)-4(substituted) thiosemicarbazides (as1–as10) were obtained by the reaction of 3-benzyl-2-hydrazino-3h-quinazolin-4-one (6) with different dithiocarbamic acid methyl ester derivatives. the key intermediate, 3-benzyl-2-thioxo-2,3dihydro-1h-quinazolin-4-one (4), was obtained by the reaction of benzyl amine (1) with carbon disulphide and sodium hydroxide in dimethyl sulphoxide to give sodium dithiocarbamate, which was methylated with dimethyl sulphate to yield the dithiocarbamic acid methyl ester 2 and condensation with methyl anthranilate (3) in ethanol yielded the desired compound (4) via the thiourea intermediate. the sh group of compound (4) was methylated in the favourable nucleophilic displacement reaction with hydrazine hydrate, which afforded 3-benzyl-2-hydrazino-3h-quinazolin-4-one (6). the ir, and 1hand 13c-nmr spectra of these compounds showed the presence of peaks due to thiosemicarbazides, carbonyl (c=o), nh and aryl groups. the molecular ion peaks of the quinazolin-4-one moiety (m/z 144) were observed in all the mass spectra of the compounds as1–as10. elemental (c, h, n) analysis satisfactorily confirmed purity and elemental composition of the synthesized compounds. all the synthesized compounds were screened for their antimicrobial activity against selective gram positive and gram negative bacteria by agar dilution method. in the present study, compounds as8 and as9 emerged as the most active compounds of the series. keywords: quinazolinone; substituted thiosemicarbazide; anti-bacterial; antitubercular activity. * corresponding author. e-mail: drvalagarsamy@gmail.com doi: 10.2298/jsc150103053a _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1472 alagarsamy et al. introduction worldwide, tuberculosis (tb) is one of the leading causes of death. tb is an infection, primarily in the lungs (a pneumonia), caused by the bacteria mycobacterium tuberculosis. emergence of multi drug resistant tuberculosis (mdr-tb) makes the conditions most alarming.1,2 some of the mdr isolates are resistant to as many as seven of the commonly employed antimycobacterial drugs.3 quinazolines and condensed quinazolines have received the attention of medicinal chemists due to their potential biological activities. among the biological activities exhibited by quinazolines, the antimicrobial activities of 2,3-disubstituted quinazolines are promising.4 a literature survey indicated that the quinazolines nucleus substituted at the 2,3-positions (fig. 1, i and ii) showed significant antitubercular activity.5,6 pharmacophore such as thiosemicarbazides and thiosemicarbazones groups (fig 1, iii and iv) in different heterocyclic moieties were also found to exhibit antitubercular activity.7–15 the present work is an extension of ongoing efforts towards developing effective antitubercular and antimicrobial agents by a hybrid approach using the quinazoline scaffold (fig. 1). in this approach, two or more pharmacophores are merged into a single molecule. therefore, with a single molecule containing more than one pharmacophore, each pharmacophore may address the active site of targets and offer the possibility of selectivity; further it can also reduce unwanted side effects.16 in the present study, a substituted thiosemicarbazide moiety was placed at the c-2 position and a benzyl ring at the n-3 position of the quinazoline ring17,18 and the antitubercular and antibacterial activities of the resulting compounds were studied against selected gram positive and negative bacteria. fig. 1. hybrid approach design of 1-[3-benzyl-4-oxo-3,4-dihydroquinazolin-2-yl]-4 -[substituted] thiosemicarbazide analogues. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis and antimicrobial activities of quinazolinyl thiosemicarbazide derivatives 1473 experimental chemistry melting points (m.p.) were taken in open capillaries on a thomas hoover melting point apparatus (thomas hoover, usa) and are uncorrected. the ir spectra were recorded as films or in potassium bromide disks on a perkin–elmer 398 spectrometer (perkin–elmer). the 1h-spectra were recorded on a dpx-300 mhz bruker ft-nmr spectrometer (bruker, usa). the chemical shifts are reported as parts per million (δ / ppm) with tetramethylsilane (tms) as an internal standard. the mass spectra were obtained on a jeol-sx-102 instrument (jeol, japan) using fast atom bombardment (fab positive). the elemental analyses were realised on a perkin–elmer 2400 chn analyzer (perkin–elmer) and the values were within acceptable limits of the calculated values (±0.4 %). the progress of the reactions were monitored on ready-made silica gel plates (merck, norway) using chloroform–methanol (9:1) as the solvent system. iodine was used as the developing agent. all chemicals and reagents used in the synthesis were obtained from aldrich (usa), lancaster (usa) or spectrochem (india) and were used without further purification. the physical, analytical and spectral data for the compounds are given in the supplementary material to this paper. 3-benzyl-2-thioxo-2,3-dihydro-1h-quinazolin-4-one (4) a solution of benzylamine 1 (0.02 mol) in dimethyl sulphoxide (10 ml) was stirred vigorously. to this mixture was added carbon disulphide (1.6 ml) and aqueous sodium hydroxide (1.2 ml, 20 m) dropwise during 30 min under stirring. dimethyl sulphate (0.02 mol) was added gradually keeping the reaction mixture stirring in a freezing mixture for 2 h. the reaction mixture was then poured into ice water. the obtained solid 2 was filtered, washed with water, dried and recrystallised from ethanol. methyl anthranilate (3, 0.01 mol) and the above prepared methyl n-(benzyl)carbamodithioate (2, 0.01 mol), were dissolved in ethanol (20 ml). to this, anhydrous potassium carbonate (100 mg) was added and the mixture refluxed for 22 h. the reaction mixture was cooled in ice and the solid that separated was filtered and purified by dissolving in 10 % alcoholic sodium hydroxide solution and reprecipitated by treating with dilute hydrochloric acid. the thus obtained solid was filtered, washed with water, dried and recrystallised from ethanol. 3-benzyl-2-(methylsulphanyl)-3h-quinazolin-4-one (5) 3-benzyl-2-thioxo-2,3-dihydro-1h-quinazolin-4-one (4, 0.01 mol) was dissolved in 40 ml of 2 % alcoholic sodium hydroxide solution. to this, dimethyl sulphate (0.01 mol) was added dropwise with stirring. after further stirring for 1 h, the reaction mixture was poured into ice water. the obtained solid was filtered, washed with water, dried and recrystallised from ethanol–chloroform (75:25) mixture. 3-benzyl-2-hydrazino-3h-quinazolin-4-one (6) 3-benzyl-2-(methylsulphanyl)-3h-quinazolin-4-one (5, 0.01 mol) was dissolved in ethanol (25 ml). to this, hydrazine hydrate (99 %, 0.1 mol) and anhydrous potassium carbonate (100 mg) were added and refluxed for 33 h. the reaction mixture was cooled and poured into ice–water. the so obtained solid was filtered, washed with water, dried and recrystallised from chloroform–benzene (25:75) mixture. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1474 alagarsamy et al. general procedure for synthesis of 1-(3-benzyl-4-oxo-3,4-dihydroquinazolin-2-yl)-4 -(substituted) thiosemicarbazides (as1–as10) a solution of primary alkyl/aryl amine (0.02 mol) in dimethyl sulphoxide (10 ml) was stirred vigorously. to this, simultaneously, carbon disulphide (1.6 ml) and aqueous sodium hydroxide 1.2 ml (20 m) were added dropwise during 30 min with stirring. dimethyl sulphate (0.02 mol) was added gradually to the stirred reaction mixture in a freezing mixture and the stirring was continued for further 2 h. the reaction mixture was then poured into ice water and the obtained solid was filtered, washed with water, dried and recrystallised from ethanol to afford methyl n-(substituted) dithiocarbamates (7). 3-benzyl-2-hydrazino-3h-quinazolin-4-one (6, 2.32 g, 0.01 mol) and methyl n-(substituted) dithiocarbamate (7, 0.01 mol) were dissolved in ethanol and refluxed for 22–30 h (until the evolution of methanethiol ceased). after completion of the reaction, the reaction mixture was cooled to room temperature. the obtained solid was filtered, dried and recrystallised from ethanol. by adapting the above procedure, the compounds as1–as10 were prepared. it should be noted that the synthesis of compounds as1–as3, as5 and as6 were previously reported.19-21 however, none of these compounds has been examined for their antitubercular activities. pharmacology antibacterial activity. evaluation of antibacterial activity was realized using the agar dilution method.10,11 the standard strains were procured from the american type culture collection (atcc), rockville, md, usa, and the pathological strains were procured from the department of microbiology, mnr medical college, sangareddy, india. the antibacterial activity of the synthesized compounds was screened against the following bacterial strains: proteus vulgaris atcc 9484, salmonella enterica subsp. enterica sarovar typhimurium atcc 33068, klebsiella pneumoniae atcc 13883, edwardsiella tarda, pseudomonas aeruginosa atcc 27853, bacillus subtilis atcc 6051 and salmonella enterica subsp. enterica sarovar paratyphi. all bacteria were grown on muller–hinton agar (hi-media) plates (37 °c, 24 h) and the minimum inhibitory concentration (mic) was considered to be the lowest concentration that completely inhibited the growth on agar plates, disregarding a single colony or faint haze caused by the inoculums.22,23 the mic values of the test compounds were compared with those the reference drug ciprofloxacin. the data given in table i were calculated from at least three different experiments in duplicate. antitubercular activity. ten-fold serial dilutions of each test compound/drug were incurporated into middlebrook 7h11 agar slants with oadc growth supplement. inoculums of mycobacterium tuberculosis h37rv were prepared from fresh middlebrook 7h11 agar slants with oadc growth supplement adjusted to 1 mg ml-1 in tween 80 (0.05 %, w/v) saline diluted to 10-2 to give a concentrate of approximately 107 cfu ml-1. a 5 µl amount of bacterial suspension was spotted into 7h11 agar tubes containing 10-fold serial dilutions of the drugs per ml. the tubes were incubated at 37 °c, and final readings were recorded after 28 days. tubes having the compounds were compared with control tubes in which medium alone were incubated with h37rv. the concentration at which complete inhibition of colonies occurred was taken as the active concentration of test compound. the mic is defined as the minimum concentration of compound required to give complete inhibition of bacterial growth.24-26 the mic values of the test compounds were compared with that of the reference drug gatifloxacin. cytotoxicity profile of the tested compounds. for cytotoxic assay with hela, approximately 10,000 cells were seeded with 0.1 ml rpmi 1640 culture medium per well of 96-well _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis and antimicrobial activities of quinazolinyl thiosemicarbazide derivatives 1475 micro-plates. hela cells were pre-incubated for 48 h without the test substances. the solutions of the compounds of the corresponding concentrations were applied carefully on the monolayers of hela cells after the pre-incubation time. the monolayers of the adherent hela cells were fixed by glutaraldehyde and stained with a 0.05 % solution of methylene blue for 15 min. after gently washing, the stain was eluted by 0.2 ml of 0.33 m hcl in the wells. the optical densities were measured at 630 nm in a micro plate reader. in general, the compounds showed no significant cytotoxic effect at the tested concentration.27 results and discussion chemistry synthetic route depicted in scheme 1 outlines the chemistry part of the present work. the key intermediate 3-benzyl-2-thioxo-2,3-dihydro-1h-quinazolin-4 -one (4) was obtained by reacting aniline (1) with carbon disulphide and sodium hydroxide in dimethyl sulphoxide to give sodium dithiocarbamate, which was methylated with dimethyl sulphate to afford the dithiocarbamic acid methyl ester 2. compound 2 on reflux with methyl anthranilate (3) in ethanol yielded the desired 3-benzyl-2-thioxo-2,3-dihydro-1h-quinazolin-4-one (4) via the thiourea intermediate in good yield (80 %). the obtained product was cyclic and not an open chain thiourea 3a. the 3-benzyl-2-(methylsulphanyl)-3h-quinazolin-4-one (5) was obtained by dissolving 4 in 2 % alcoholic sodium hydroxide solution and methylating with dimethyl sulphate under stirring at room temperature. nucleophilic displacement of the methylthio group of 5 with hydrazine hydrate was performed using ethanol as solvent to afford 3-benzyl-2-hydrazino-3h-quinazolin-4 -one (6). the required long duration of the reaction (33 h) might be due to the presence of the bulky aromatic ring at position 3, which might have reduced the reactivity of quinazoline ring system at the c-2 position. the title compounds 1-(3-benzyl-4-oxo-3,4-dihydroquinazolin-2-yl)-4-(substituted) thiosemicarbazides (as1–as10) were obtained by the condensation of the amino group of 3-benzyl -2-hydrazino-3h-quinazolin-4-one (6) with a variety of methyl ester of dithiocarbamic esters. the formation of title products was indicated by the disappearance of peak due to nh, nh2 of the starting material in ir and 1h-nmr spectra of all the compounds as1–as10. the ir and 1h-nmr spectra of these compounds showed the presence of peaks due to thiosemicarbazides, carbonyl (c=o), nh and aryl groups. the mass spectra of the title compounds showed molecular ion peaks corresponding to their molecular formulae. in the mass spectrum of compounds as1–as10, a common peak at m/z 144 corresponding to the quinazolin-4-one moiety appeared. elemental (c, h, n) analysis satisfactorily confirmed the elemental composition and purity of the synthesized compounds. antitubercular activity the synthesized compounds were screened for their in vitro antimycobacterial activity against m. tuberculosis strain h37rv. the results are expressed in _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1476 alagarsamy et al. scheme 1. synthesis of 1-(4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-4-(substituted) thiosemicarbazides. reagents and conditions: a) cs2, naoh, dmso, 30 min; b) dimethyl sulphate, 2 h; c) methyl anthranilate, anhydrous k2co3, etoh reflux, 22 h; the product is 3a; d) 2 % alcoholic naoh, dimethyl suphate, 1 h; e) hydrazine hydrate, anhydrous k2co3, etoh reflux, 33 h; f) methyl n-(substituted) carbamodithioate, etoh reflux, 22–30 h; notation “a” in the scheme replaces notation “as” from the text. terms of minimum inhibitory concentration (mic). the results of antimycobacterial activity depicted in table i, indicate that the test compounds inhibited the growth of mycobacterium to varying degree. compounds with aliphatic substituents showed lower antitubercular activity over the aryl and heteroaryl substituents. the compounds with electron withdrawing substituent on the aryl ring showed better activity over the unsubstituted or electron donating substituent on _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis and antimicrobial activities of quinazolinyl thiosemicarbazide derivatives 1477 the aryl ring. among the test compounds, 2-(3-benzyl-4-oxo-3,4-dihydroquinazolin-2-yl)-n-(4-chlorophenyl)hydrazinecarbothioamide (as8) and 1-(3-benzyl -4-oxo-3,5-dihydroquinazolin-2-yl)-4-(4-nitrophenyl)hydrazinecarbothioamide (as9) exhibited antitubercular activity at the minimum microgram concentration (3 µg ml–1). table i. antitubercular and antibacterial activity of the synthesized compounds as1–as10; (mic in µg ml-1); na – no activity microorganism test compound standarda as1 as2 as3 as4 as5 as6 as7 as8 as9 as10 m. tuberculosis 125 63 63 6 13 13 6 3 3 6 1 s. enterica serovar typhimurium 66 63 63 63 63 125 63 8 8 16 4 p. vulgaris 63 63 125 125 63 63 63 8 16 32 1 k. pneumoniae 63 125 125 32 63 125 63 16 16 63 1 b. subtilis 63 125 63 125 63 32 32 8 8 16 1 p. aeruginosa 125 125 16 63 32 32 63 16 8 32 1 e. tarda na na na na na na na na na na na agatifloxacin was used as a reference standard against m. tuberculosis, whereas ciprofloxacin was used as a reference standard for the other bacteria antibacterial activity among the different substituents, aryl and heteroaryl substituents exhibited better activity over the aliphatic cyclic substituents. compounds with electron withdrawing substituents, such as –cl and –no2 showed better activity over the unsubstituted and electron donating substituents. compounds as8 and as9 emerged as the most active compounds of the series. compound as8 showed the most potent activity against e. coli, p. vulgaris, b. subtilis and s. enterica subsp. enterica serovar typhimurium, while compound as9 showed the most potent activity against e. coli, b. subtilis, p. aeruginosa and s. enterica subsp. enterica serovar typhimurium. conclusions in summary, the syntheses of a new series of 1-(4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)-4-(substituted) thiosemicarbazides was described. these derivatives exhibited significant antibacterial activity against various gram-positive and gram-negative bacteria, including m. tuberculosis. among the series, compound as8 showed the most potent activity against e. coli, p. vulgaris, b. subtilis and s. enterica subsp. enterica serovar typhimurium, while compound as9 showed the most potent activity against e. coli, b. subtilis, p. aeruginosa and s. enterica subsp. enterica serovar typhimurium. the test compounds as8 and as9 exhibited antitubercular activity at the minimum microgram concentration _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1478 alagarsamy et al. (3 µg ml–1) and show potential for further optimization and development to new antitubercular agents. supplementary material the physical, analytical and spectral data for the compounds are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the authors gratefully acknowledge the central instrumentation facility, iit chennai, india for the spectral analysis of the compounds synthesised in this study and dr. d. sriram, birla institute of technology & sciences, hyderabad campus for performing the antitubercular screening of the test compounds. и з в о д синтеза и антимикробна активност 1-(3-бензил-4-оксо-3,4 -дихидрокиназолин-2-ил)-4-супституисаних деривата тиосемикарбазида veerachamy alagarsamy1, viswas raja solomon1, g. krishnamoorthy2, m. t. sulthana1 и b. narendar1 1 medicinal chemistry research laboratory, mnr college of pharmacy, sangareddy, gr. hyderabad -502 294, india и 2department of pharmaceutical chemistry, periyar college of pharmaceutical sciences for girls, trichy – 620 021, india синтетисана је серија 1-(3-бензил-4-оксо-3,4-дихидрокиназолин-2-ил)-4-супституисаних деривата тиосемикарбазида (as1–as10), реакцијом 3-бензил-2-хидразино-3h -хиназолин-4-oна (6) и различитих деривата метил-естара дитиокарбаминске киселине. главни интермедијер 3-бензил-2-тиоксо-2,3-дихидро-1h-хиназолин-4-oн (4) добијен је после секвенције у којој је реакцијом бензиламина (1) са угљен-дисулфидом и натријум -хидроксидом у диметил-сулфоксиду добијен дитиокарбамат, који је метилован диметил-сулфатом при чему је добијен метил-естар дитиокарбаминске киселине 2 и који је кондензацијом са метил-антранилатом (3) у етанолу дао жељени производ 4 преко тиоуреидног интермедијера. тиол-група у једињењу 4 метилована је да би била извршена нуклеофилна замена помоћу хидразин хидрата, чиме је добијен 3-бензил-2хидразинохиназолин-4-oн (6). ir, 1hи 13c-nmr спектри једињења показују присуство сигнала тиосемикарбазидних, карбонилних (c=o), nh и арил-група. у свим масеним спектрима деривата as1–as10 присутан је сигнал хиназолин-4-он јона (m/z 144). елементална анализа (c, h, n) је показала добру чистоћу једињења. испитана је антимикробна активност свих синтетисаних једињења према одабраним грам-позитивним и грам-негативним бактеријама. деривати as8 и as9 показују најбоље активности у овој серији испитаних једињења. 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice accepted manuscript this is an early electronic version of an as-received manuscript that hasbeen accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as a.y. galashev, a.s. vorob’ev, yu. p. zaikov, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc230213038g this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230213038g j. serb. chem. soc.00(0)1-10 (2023) original scientific paper jscs–12274 published dd mm, 2023 1 quantum-mechanical study of the electronic properties of uxpuyoz compounds formed during the recovery of spent nuclear fuel alexander y. galashev*, alexey s. vorob’ev, yuri p. zaikov institute of high temperature electrochemistry, ural branch, russian academy of sciences, yekaterinburg, russia (received 13 february; revised 22 march; accepted 8 july 2023) abstract: a promising way to recover spent nuclear fuel (snf) is the method of extracting transuranium compounds from molten salt, which makes it possible to obtain a partial separation between transuranium compounds and lanthanides. this work is devoted to the quantum mechanical study of changes in the structure, energy and electronic properties of the main snf component, uranium dioxide, upon the removal of oxygen from the system. the influence on the considered properties of the system of the substitution of uranium by plutonium is also studied at a ratio of the number of plutonium atoms to uranium atoms of 1:7 and 1:3. the removal of oxygen leads to a narrowing of the band gap up to the transition to a conductive state at a ratio of uranium to oxygen of 2:3. the band gap narrows and metallization sets in even when uranium is replaced by plutonium. a two-stage uo2 metallization scheme based on lithium reduction and direct (electronic) reduction is proposed. keywords: geometric and band structures, oxides, plutonium, snf recovery, spectrum of electronic states, uranium introduction the uranium-oxygen system seems to be an extremely complex system, the literature data on which are very contradictory.1 there are reports in the literature about 14 types of oxides ranging from uo to uo3. a number of uranium oxides have several crystalline modifications. data on the physicochemical properties of these systems are of great importance, since stable compound uo2 is a reactor fuel. the basis of ceramic nuclear fuel, i.e. uo2, has high corrosion, radiation and thermal stability. the high density of uo2 provides a high concentration of fissile material. the production of electricity using a nuclear reactor does not emit greenhouse gases. nuclear reactors have a very high energy density. thus, when *corresponding author e-mail: alexander-galashev@yandex.ru https://doi.org/10.2298/jsc230213038g a cc ep te d m an us cr ip t mailto:alexander-galashev@yandex.ru https://doi.org/10.2298/jsc230213038g 2 galashev et al. one fuel pellet weighing 10 g is burned in a reactor, energy is released that is equivalent to the energy obtained by burning ~1300 kg of coal, ~1 m3 of oil, or ~150 m3 of natural gas.2 however, the low thermal conductivity of uo2 leads to high temperature gradients, which limits the size of the products. in addition, the thermal conductivity of the fuel during its lifetime in the reactor is greatly reduced (up to 70%).3 uranium oxides seem to be the most stable compounds of this element, which makes them suitable for storing uranium. uranium oxides can act as intermediates in the production of other uranium compounds, such as fluorides. the similar atomic weights and the largely similar atomic structure of uranium and plutonium create prerequisites for considering their effect on the near atomic environment to be practically identical. as a result, the same interaction potentials are used in molecular dynamics modeling of u and pu.4 electrical conductivity is the physical property that is determined by the electronic structure, has a feedback with it and affects the formation of a new phase. a change in electrical conductivity caused by any process can accelerate or slow down the development of this process. the reduction of uranium dioxide (uo2) to uranium metal is reduced with the removal of oxygen from the system. the electrolytic reduction method involves the removal of oxygen from uo2 by displacing uranium from this compound with lithium. the newly formed li2o compound enters the salt melt, where it decomposes into li+ and o2ions. during the electrolytic reduction of spent nuclear fuel (snf), as a rule, u and pu simultaneously deposited on the cathode. the u3+ reduction potential to metallic u0 is only 0.34 ev higher than that of pu3+.5 the subsequent separation of the alloy, consisting of u and pu, requires additional operations.6 in the field of pyroprocessing of snf, liquid cadmium is applied to recover transuranics from molten salt and provide some degree of separations between transuranics and lanthanides. analytical analysis using experimental data shows that in snf recovery using a liquid cd cathode, when both u and pu are dissolved in cadmium, the highest pu:u ratio is 8:1, and the lowest pu:u ratio is 1:4.7 expectedly higher is the proportion of plutonium in binary (u-pu) metallic fuel intended for fast reactors. in such a non-irradiated fuel, the pu:u ratio approximately corresponds to a ratio of 1:3, while the proportion of pu in the irradiated fuel increases significantly.8 a hubbard corrected density functional theory (dft+u) study of the electronic and thermal properties of stoichiometric and hypostoichiometric phases of uranium dioxide was carried out in kaloni et al.9 it was shown that the removal of oxygen can affect the electronic and thermal transport properties of uo2. in addition, such properties of hypostoichiometric uo2 as electronic properties, electronic thermal conductivity, phonon dispersion and lattice thermal conductivity were calculated. a change in the electronic properties during snf recovery can affect the rate of recovery and the completeness of this process. therefore, establishing the degree of electrical conductivity of the intermediate a cc ep te d m an us cr ip t quantum-mechanical study of uxpuyoz compounds 3 phases that appear is important for controlling the mode of the snf recovery process. the purpose of this work is to study changes in the structural, energy, and electronic properties of uranium dioxide systems, as well as uranium oxides containing plutonium, when the pu : u ratio is 1:7 and 1:3, when oxygen atoms are removed from the system. experimental these calculations were performed using the siesta software package. 10 in the work, a study was made of the reduction of uranium dioxide, as well as the effect of the presence of plutonium on the structures energy and electronic properties of uranium oxides. uranium dioxide was modeled by combining a 2x2x2 uranium fcc supercell (8 uranium atoms) and two cubic oxygen lattices (16 oxygen atoms). in what follows, we will represent the u8o16 system as uo2, and the u8o12 system as u2o3. the structure of uranium dioxide after geometric optimization is shown in fig. 1a. to simulate the reduction of uranium dioxide, two and four oxygen atoms were removed from the supercell; systems containing 14 and 12 oxygen atoms were considered. fig. 1b shows the structure of u2o3, containing 8 uranium atoms and 12 oxygen atoms after geometric optimization. the introduction of plutonium is represented by the replacement of one or two u atoms in 2x2x2 uranium fcc supercell by pu atoms. thus, we considered systems with the pu/u ratio in the uranium dioxide structure 1:7 (1 plutonium atom to 7 uranium atoms) and 1:3 (1 plutonium atom to 3 uranium atoms). the simulation was carried out using the lda+u approximation, 11 the values of the parameters ueff and jeff for both uranium and plutonium atoms were taken to be 4.5 and 0.5 ev, respectively. in all systems considered by us, geometric optimization was carried out using the local density approximation in the ca form. 12 the dynamic relaxation of atoms continued until the change in the total energy of the system became less than 0.001 ev. in the ab initio calculations, the born–karman periodic boundary conditions were used. the density of the three-dimensional grid used to calculate the electron density was set using a cutoff energy of 550 ry. the electron density of states (pdos) and band structures are often used to characterize the electronic properties of materials. the band structure was calculated in the l-γ-x-u-k-γ direction. the brillouin zone was set by the monkhorst-pack method 13 using 5×5×5 k-points. the binding energies of a uranium or plutonium atom with the rest of the compound (i.e. compound without uranium or plutonium atom) were calculated according to the expression: 𝐸bond 1u/1pu = − 𝐸tot−𝐸1−𝐸1a 𝑁 (1) where etot is the total energy of the system, e1 is the total energy calculated for the system in the absence of a uranium or plutonium atom, e1a is the energy calculated for a single uranium or plutonium atom, and n is the number of atoms in the system. the energy of bonds between atoms in a compound: 𝐸bond = − 𝐸tot−𝑁𝑂𝐸1𝑂−𝑁𝑈𝐸1u−𝑁pu𝐸1pu 𝑁 (2) where e1o, e1pu, e1u are the energies calculated for single oxygen, plutonium, and uranium atoms, respectively, and no, npu, nu are the number of oxygen, plutonium, and uranium atoms in the system, respectively. a cc ep te d m an us cr ip t 4 galashev et al. fig. 1. geometric structure of (a) uo2 and (b) u2o3 after geometric optimization results and discussion table i represented the following system characteristics: the average binding energy between atoms in the entire system (eb); the binding energy between the uranium atom and the rest of the system ( 𝐸b 1u ); binding energy between a plutonium atom and the rest of the system (𝐸b 1pu); bond lengths between atoms of uranium and oxygen (l(u-o)) and that of plutonium and oxygen (l(pu-o)). it can be seen that the replacement of uranium atoms in the uo2 compound by plutonium atoms up to get ratios of plutonium to uranium (npu/nu) of 1:7 and 1:3 leads to a decrease in the binding energy 𝐸𝑏 by 1.9 and 4.8%, respectively. the removal of oxygen in the absence of a plutonium in the system leads to a gradual decrease in the binding energy eb to 2.2%. however, eb behaves differently in the presence of plutonium. an increase in the number of oxygen vacancies similarly affects on the values of eb in systems with the ratio (npu/nu) of 1:7 and 1:3. in the hypothetical compound u7puo14 and u6pu2o14, the energy eb increases by 0.3 and 2.5%, respectively. however, after further withdrawal of oxygen, i.e. in the u7puo12 and u6pu2o12 compounds, the energy eb decreases. the calculation of the energies 𝐸𝑏 1u and 𝐸b 1pu was carried out for 4 different uranium atoms; the table shows the average values of the calculated bond energies. in all considered cases, the energy 𝐸b 1u decreases when oxygen removal from the system. the largest drop in energy 𝐸b 1u equal to 25.5% is observed when 4 oxygen atoms removed from the u7puo16 compound. at the same time, the bond energy 𝐸b 1pu increases when oxygen is withdrawn from plutonium containing systems. thus, when 4 oxygen atoms were removed from the u7puo16 and u6pu2o16 systems, the energy of 𝐸b 1pu increased by 34.0 and 13.3 %, respectively. a cc ep te d m an us cr ip t quantum-mechanical study of uxpuyoz compounds 5 table i. characteristics* of systems u8-xpuxoy (x and y is the number of plutonium and oxygen atoms in the system, respectively) npu no eb, ev 𝐸b 1u , ev 𝐸b 1pu , ev l(u-o), å l(pu-o), å 0 16 11.527 2.159 2.321 14 11.406 2.113 2.314 12 11.273 2.106 2.313 1 16 11.306 3.015 2.499 2.279 2.388 14 11.345 2.515 3.240 2.279 2.426 12 11.126 2.244 3.349 2.276 2.439 2 16 10.971 2.603 2.081 2.286 2.329 14 11.248 2.492 2.280 2.278 2.369 12 11.047 2.429 2.358 2.263 2.406 * eb – bond energy of atoms in the system, e1ub, e1pub – energy of bonds between 1 atom of uranium/plutonium with the rest of the system, l(u-o), l(pu-o) – average bond lengths between uranium/plutonium and oxygen atoms. the lengths of the u-u and pu-u bonds in all the considered compounds are practically unchanged and are approximately equal to 3.81 and 3.79 å, respectively. this agrees with the data of a small displacement of uranium atoms in hypostoichiometric uo2-x compounds. 9 the u-o lengths upon replacement of one or two u atoms by pu atoms in u8oy compounds decrease from 1.5 to 2.2% depending on the composition of the hypothetical compound. in all cases, a slight decrease from 0.1 to 1% in the lengths of the u-o bonds when 4 oxygen atoms were removed from the systems (uo2, u7puo16 and u6pu2o16) was found. while the pu-o bond lengths during the transition from the u7puo16 and u6pu2o16 to u7puo12 and u6pu2o12 hypothetical compounds increase by 2.1 and 3.3%, respectively. fig. 2 shows the band structures of the considered systems, and table ii presents the electronic properties of the systems containing plutonium. the band gap obtained for uranium dioxide is 2.23 ev, which is slightly larger than the value of 2.19 ev obtained earlier.14 the removal of two oxygen atoms from the uo2 system leads to a narrowing of the band gap to 0.99 ev. if we continue to remove oxygen from the system in the same amount (i.e., remove 2 more o atoms), then the system becomes conductive. the narrowing of the band gap and the transition to a conducting phase in hypostoichiometric uranium compounds are consistent with the data previously obtained.9 substitution of uranium by plutonium at ratios of npu/nu = 1:7 and 1:3 leads to metallization of the u7puo12, u6pu2o16, u6pu2o14 and u6pu2o12 systems, while the u7puo16 and u7puo14 systems have semiconductor properties. a cc ep te d m an us cr ip t 6 galashev et al. table ii. conductivity characteristics of uranium oxide systems containing plutonium compound electronic properties band gap, ev compound electronic properties band gap, ev u7puo16 semiconductor 0.07 u6pu2o16 metal u7puo14 semiconductor 0.21 u6pu2o14 metal u7puo12 metal u6pu2o12 metal fig. 2. band structures obtained for compounds u8o16, u8o14, u8o12, u7puo16, u7puo14, u7puo12, u6pu2o16, u6pu2o14 and u6pu2o12 figure 3 shows the partial density of the electronic states for the uo2, u2o3, u7puo12, and u6pu2o16 systems. it can be seen that the conductive properties in hypothetical compound u2o3 appear due to the interaction of the d and f orbitals of uranium with the p orbitals of oxygen. the fermi level turns out to be slightly shifted into the conduction band of u2o3, and the width of the a band gap is ~0.1 ev. at the same time, in the pdos spectra of u7puo12 and u6pu2o16, the valence band continuously passes into the conduction band. moreover, conductivity appears in these compounds due to the interaction of 6d uranium orbitals with 5f plutonium orbitals and 5f uranium and plutonium orbitals with 2p oxygen orbitals, respectively. in the pdos of the u7puo12 compound, a high bimodal peak related to the 5f electrons of uranium is in the valence band, while in the pdos of the a cc ep te d m an us cr ip t quantum-mechanical study of uxpuyoz compounds 7 u6pu2o16 compound, a similarly shaped peak also related to the 5f electrons of uranium appears in the conduction band. for the three connections (uo2, u2o3, u6pu2o16) shown in the figure in the pdos spectrum, there are adjacent bands are not wide enough to span the full range of electron energy levels. in the pdos spectrum of u7puo16, there are no such adjacent allowed bands. in other words, in this case the part of the spectrum that belongs to the valence band is continuous. fig 3. partial density of electronic states obtained for compounds uo2, u2o3, u7puo12 and u6pu2o16 the presence of many different phases of non-stoichiometric actinide oxides (aox), complex structures and their tendency to form solid solutions make it extremely difficult to understand not only the structure, but also chemical composition. nevertheless, the dft+u calculations reflect the correct trend of the band gap depending on the oxidation state of the actinides.15 the reduction of uo2 to uranium metal in the molten salt of licl with the addition of li2o takes place in close contact with the cathode and is an electrochemical process. we now represent the process of reduction of uo2 by chemical reactions. the presence of the electrical conductivity in the u2o3 phase allows us to understand more deeply the process of electrochemical pyrolysis that we consider. a small (~ 3%) initial addition of li2o to the licl electrolyte leads to an imbalance between li+ and clions in the salt melt and the creation of the necessary double charged layer near the surface of the uo2 pellet after creating a certain electrical voltage (3.0-3.3 v) between the electrodes. in general, the decay of li2o can be represented as: a cc ep te d m an us cr ip t 8 galashev et al. li2o → 2li + + o2– (3) the ions o2formed after the decay of li2o can quickly reach the anode, because o2ions are 2.2 times lighter than clones. in addition, their electric charge is 2 times greater than that of cl-. each o2ion gives out 2 electrons at the anode. the formed o atoms combine into o2 molecules which form bubbles that rise and remove oxygen from the system. to simplify, let us consider the snf recovery process using pure uo2 as an example. the first stage of the process mainly involves the external part (that is directly in contact with the electrolyte) of the uo2 pellet. after the formation of a double electric layer between the surface of a highly polarized semiconductor (uo2) and the near-surface part of an electrolyte (licl) strongly enriched with li + ions, a strong local electric field arises and "pulls" oxygen ions from the surface of the semiconductor: 2uo2 → (u2o3) 2+ + o2– (4) in the near-surface region of the electrolyte, the o2ion combines with two li+ ions to form an electrically neutral molecule li2o: 2li+ + o2– → li2o (5) the second stage of recovery begins with the electronically conductive metallized (u2o3) 2+ with an excess of positive charge takes the missing electrons and becomes electrically neutral, but the conducting substance: (u2o3) 2+ + 2e– → u2o3 (6) the continuation of the second stage of reduction is associated exclusively with the electrical conductivity of the intermediate u2o3 phase. the electrically conductive surface layer continues to receive electrons and u2o3 is reduced to the metallic uranium: u2o3 + 6e – → 2u + 3o2– (7) the mechanism for the reduction of uo2 to metallic u presented here is supported by experimental facts, among which the movement of the metallization front from the surface to the center of the pellet and the absence of lithium inside the pellet with a 98% reduced metal u.5–8 in addition, the reduction process actively occurs in a high-quality sintered uo2 powder granule, but quickly fades if the spent fuel used for recovery is taken in the form of a conventional powder. a significant increase in the electronic contribution for certain compositions of oxide nuclear fuel contributes to an increase in thermal conductivity.9 we have shown that this conclusion is also valid in the presence of pu in the fuel. thus, the use of hypostoichiometric phases seems to be favorable for obtaining a fuel that is resistant to accidents. a cc ep te d m an us cr ip t quantum-mechanical study of uxpuyoz compounds 9 conclusion in this work, based on quantum mechanical calculations, the partial reduction of uranium and uranium-plutonium oxides is studied. the change in the structure, energy, and electronic properties of these oxides was studied when uranium atoms were replaced by plutonium atoms up to the ratio of the number of atoms in the system npu/nu = 1:7 and 1:3. a decrease in the total bond energy in the uo2 compound is shown when uranium is replaced by plutonium in the ratios npu/nu = 1:7 and 1:3. an increase in the bond energy between plutonium and the rest of the compound was revealed upon the removal of oxygen from the u7puo16 and u6pu2o16 systems. it is shown that the band gap narrows up to complete metallization of the compound when oxygen is removed from the uo2 compound to the ratio nu/ no = 2:3. a transition to the conducting state of the uo2 compound was revealed when uranium was replaced by plutonium in the ratios npu/nu = 1:3. a two-stage process of uranium metallization during its reduction in licl-li2o melt is proposed. we hope that this study will improve understanding of the snf electrolytic reduction process and serve as a stimulus for its optimization. acknowledgements: the calculations were performed on a hybrid cluster-type computer "uran" at the institute of mathematics and mechanics, ural branch of the russian academy of sciences with a peak performance of 216 tflop/s and 1864 cpus. the work is executed within the framework of the multipurpose program "development of equipment, technologies and scientific research in the field of the use of atomic energy in the russian federation for the period up to 2024". и з в о д квантно-механичка студија електронских особина uxpuyoz једињења формираних током обнављања истрошеног нуклеарног горива александар ј. галашев*, алексеј с. воробјев, јуриј п. зајков институт за високотемпературну електрохемију, уралски огранак руске академије наука, јекатеринбург, русија обећавајући начин за обнављање истрошеног нуклеарног горива (snf) је метод екстракције трансуранских једињења из стопљене соли, што омогућава да се добије делимично раздвајање трансуранских једњења и лантанида. овај рад је посвећен квантно механичкој студији промена у структури, енергији и електронским особинама главне snf компоненте, уранијум диоксида, при уклањању кисеоника из система. такође је студиран утицај проучаваних особина на однос броја плутонијумових атома према уранијумовим атомима 1:7 и 1:3. уклањање кисеоника доводи до сужавања јаза између трака до преласка у проводно стање при односу уранијума и кисеоника од 2:3. јаз између трака се сужава и долази до метализације чак и када се уранијум замени са плутонијумом. предложена је двостепена шема метализације uo2 заснована на редукцији литијумом и директној (електронској) редукцији. 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aleksić, l. d., 969 alenezi, k. m., 345 alouaoui, r., 1327 al-zahrani, s. a., 345 ameri, a., 735 andrić, lj., 491 anđelković, b., 27 anđelković, k., 181, 1143 anđelković, t. d., 145 aničić urošević, m., 69 antić, i. s., 1425 antić, m., 109 antić, v., 109 antonijević-nikolić, m., 451 anjomshoa, k., 193 arsenijević, z. lj., 911 arsić, b., 1117 attar, t., 589 attard, e., 983 avdalović, j., 95 avdin, v. v., 57 avram, s., 321 aydaş, b., 363 aydin, g. b., 389 aziz, m., 293 babić, b. m., 205, 749, 1285 badis, a., 1327 baeumner, a. j., 1341 bag, b. p., 813 baitharu, i., 813 balaban, m., 109 balaji, s., 629 barta holló, b., 307 baskić, d., 545 behera, a. k., 813 behjatmanesh-ardakani, r., 1157 belić, d. s., 479 belošević, s. k., 307 benkhaled, a., 589 beškoski, v. p., 95 bhabal, s. r., 1109 bian, x., 603 bilal, m. s., 293 bjelajac, a. z., 749 blawert, c., 1035 blažić, m., 677 bogdanović, d. s., 145 borković, a. p., 1091 boros, b. v., 657 borović, b., 451 borsynbayev, a., 925 bosenko, o., 879 bošković-vragolović, n. m., 911 botić, t. t., 1091 boudokhane, c., 247 bousalem, s., 589 božić, k., 491 božović, n., 1035 brasil, d. s. b., 693 brčeski, i., 83 brugnoni, l. i., 1297 brzić, d. v., 911 bugeja douglas, a., 983 ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1462 journal of the serbian chemical society buljovčić, m. b., 1425 burmistrov, v. a., 355 butulija, s., 1285 bykov, v., 879 chakraborty, m., 707 chauhan, r., 629 chen, g., 505 cheng, x., 603 choukchou braham, e., 589 cvanić, t., 1359 cvetković, v. s., 1409 čobeljić, b., 181 čokeša, đ. m., 761 çamur elipek, b., 389 da costa, a. s. s., 693 da costa, r. a., 693 da rocha, e. c. m., 693 da rocha, j. a. p., 693 dalmacija, b., 133 damjanović-vasilić, lj., 1035 daneshpajouh, m., 1125 das, d., 829 das, s., 829 datcu, a. d., 657 de santana, h., 331 de souza madureira felício, a. l., 331 defrance, p., 479 dekić, r., 109 demirci, s., 263 deoghare, c., 629 devetak, i., 275 dezhampanah, h., 1273 dhandapani, s., 629 diaz-ballote, l., 845 dimitrijević, i., 1313 dimitrijević, s. b., 1409 dodevska, t., 401 dongliang, y., 293 dragić, d. m., 1091 dražić, b., 451 drljača, d. m., 1091 drmanić, s., 491 dubovina, m., 133 dugić, p. t., 1091 đogo mračević, s., 723 đokić, l., 1075 đonlagić, j., 1075 đorđević, d., 27 đorđević, j., 1245 đorđević, s., 1063 đorđević, s., 1439 đorđić crnogorac, m., 181 đorđievski, s., 775 đukić-ćosić, d., 723 đuriš, m. m., 911 đurkić, t. m., 205 ejaz, s. a., 293 ejaz, s. t., 293 ejaz, s., 293 ekka, d., 1171 el moll, h., 345 erdoğan, b., 1395 erdogan, i., 953 ergan, e., 575 eslami, h., 1125 eslaminejad, t., 1125 fan, c., 505 ferhat, s., 1327 filipović, i. p., 545 filipović, n. r., 1143 gajica g. đ., 7 ganguly, a., 629 gao, h., 1381 garić-grulović, r. v., 911 garipov, r., 891 gašić, u., 983 gazdić, d. v., 307 genesca, j., 845 ghosh, s., 1171 gođevac, d., 1237 gojgić-cvijović, g., 95 gonçalves, a. da s., 693 gonzález, m. b., 1297 gramatikov, d. d., 1049 grau, n. i., 657 grba, n., 133 gržetić, i., 69 gu, j., 857 gu, q.-s., 1381 güher, h., 389 ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ vol. 87: author index 1463 günal, a., 1395 gvero, p. m., 1091 hadnađev-kostić, m. s., 1011 hadzhiev, d., 401 hadži jordanov, s. a., 1049 hamdaoui, m., 247 hao, j., 437 haque, a., 345 harzallah, o. a., 247 hassanzadeh, a., 1125 havlíček, d., 925 heinemann, f. w., 561 hernández-nuñez, e., 845 homšek, i., 1063 hou, j.-x., 1381 ianovici, n., 657 ibrahim, s. m., 641 ibrir, a., 1327 ignjatović, l., 157 ilhan, s., 377 ilić, m., 95 ilijević, k., 69 isailović, j., 83 isvoran, a., 657 ivanović, s., 1237 jaćimovski, d. r., 911 jakimov, d., 545 janiak, c., 1259 jeremić, d., 83 jeremić, s., 1075 jevtić, i. i., 169 jevtić, s., 1075 jevtovic, v., 345 jevtović, m., 181 jiao, h., 1439 joksimović, k., 95 josino, l. p. c., 693 jovančićević, b. s., 7, 519 jovanović marić, j., 1245 jovanović, u. d., 761 jovanović-šanta s. s., 969 jović, j., 437 kadokami, k., 1425 kadyirov, a., 891 kaikenov, d., 925 kalijadis, a. m., 205, 749, 1285 kalpakli, a. o., 377 karanović, đ. m., 1011 kashif mahmoud, h. m., 293 kašanin-grubin, m. p., 7, 519 katanić stanković, j. s., 1349 kerkez, đ., 133 kocić, g. m., 145 koifman, o. i., 355 kojić, i. d., 41 kojić, v. v, 545, 969 kolarević, s., 1245 koleva, r., 775 koravović, m., 785 kostić kokić, i. s., 145 kostić-rajačić, s. v., 169 košević, m. g., 867 kovač, s., 83 kovačević, m., 785 krajinović, s., 157 krčmar, d., 133 krmar, j., 615 krstić, s. s., 867 krüger, b., 307 krunić, m. j., 169 kuleshov, s., 879 kumaraswamy, s., 997 lamovec, j. s., 899 landolsi, i., 247 lazarevska todevska, e., 1185 lazarova, y., 401 leovac, v. m., 307, 1259 li, s., 505 li, z., 603 lima, a. h. l., 693 liu, s., 857 lou, x., 603 luo, j., 505 ma, l., 603 madadi mahani, n., 193, 1157 maksimović, a., 775 maldonado-lopez, l., 845 maletaškić, j., 1285 maletić, m. m., 205, 749 maletić, s. p., 1091 mallick, s., 829 mallik, t., 1171 ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1464 journal of the serbian chemical society manojlović, d. d., 57 marinović-cincović, m., 451 marković, b., 785 marković, m. m., 761 matić, i. z., 181 matović, b., 1285 matović, z. d., 545, 561 medić, d., 775 menghiu, g., 677 miandehi, o. r., 1273 mihajlović, i. j., 121 mihoubi, n., 1327 mijin, n. d., 1143 milanović, m. m., 1011 miletić, s., 95 milosavljević, s., 1237 milošević, j., 1143 miljuš, g., 1025 mincea, m., 677 minović arsić, t. z., 749 miró, m., 1341 misran, m., 641 mišić, d., 1063 mitić, d., 181, 1143 mitić, m., 1117 mitić, v., 1313 mitrović, n., 723 mladenović, i. o., 899 mohammadi, s. z., 193 mohammad-salim, h. a., 707 mohanta, p. p., 813 mojović, lj., 437 mojsilović, k., 1035 momčilović, m., 939 moraru, c., 677 mraković, a., 939 mrkalić, e. m., 545 muratbekova, a., 925 mustafin, y., 925 muškinja, j. m., 1349 naghizadeh, m., 1125 nakano, t., 95 nanda, a., 829 nandi, s., 829 neanu, c., 321 nedić, o., 1025 nedjhioui, m., 1327 nenov, v., 775 nešić, i., 1063 nešović, m., 983 nikolić, a. r., 969 nikolić, b. ž., 867 nikolić, j., 491 nikolić, m. s., 1075 nikolić, n. d., 899 nikolovski, z., 83 novakovic, i., 1245 novaković, s. b., 1259 oalđe pavlović, m., 1245 obrovski, b. b., 121 omarov, k., 925 omel'chuk, a., 879 orlić, j., 69 oruc, i., 953 ostafe, v., 657, 677 ostojić, s., 1203 otašević, b., 615 öktem, m. f., 363 öterler, b., 389 panić, v. v., 867 pantelić, n., 109 papuga, s. v., 1091 pattanaik, s., 829 pavale, g. s., 1109 pavićević, i. d., 1025 pavlović, a., 1313 pavlović, m. m., 1409 pavlović, m., 491 pavlović, r. m., 145 pelosi, g., 545 penjišević, j. z., 169 pergal, m. v., 1203 pešević, d., 519 petrov, s. a., 1367 petrović, j., 939 petrović, m. z., 121 pezo, l., 1203 pinheiro, a. s., 693 piponski, m., 1185 pjanović, r. v., 911 polović, n. đ., 1143 ponjavić, m., 1075 popović, a., 27, 939 pourshojaei, y., 1125 ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ vol. 87: author index 1465 prekajski đorđević, m., 1285 prodanović, r., 677 protić, a., 615 pržulj, s., 519 puchta, r., 1439 pudov, a., 925 radanović, m. m., 307, 1259 radenković, m., 939 radenković, s., 1439 radišić, p., 983 radmanović, s. b., 761 radojević, v., 899 radojičić, a., 519 radonić, j., 157 radovanović, m. d., 561 raičević, v., 307 rakitin, a. r., 41 ralevic, l. r., 531 ramachandran, m., 219 ramana, m. m. v., 1109 ramos, c. a., 1297 ranitović, a., 1359 ražić, s., 723, 1341 relić, d., 27, 939 ristić, m. m., 479 ristić, m. s., 561 rjiba, n., 247 rmandić, m., 785 rodicheva, j. a., 355 rodić, m. v., 307, 1259 roglić, g. m., 57 rončević s., 133 rozsypal, t., 233 russi, l., 983 saeed, a., 293 sahin, o., 263 sahoo, r. n., 829 saidman, s. b., 1297 sakač, m. n., 969 sakan, s., 27 san-pedro, l., 845 savić, d. n., 109 savić, d., 109 savić, s. d., 57 savković, j. r., 1091 segundo, m. a., 1341 seker, n., 575 selvam, n. v., 219 serdechnova, m., 1035 setoodeh, n., 735 shaikh, s. f., 1109 shen, x., 603 sheveleva, a. e., 355 shi, m.-q.,1381 shroff, s., 813 shterev, i., 401 shukla, a., 219 shuyev, n., 925 simić, k., 1237 singaram, j., 997 sladić, d., 181, 1245 srećković, n., 1349 sremački, m. m., 121 srivastava, h., 629 stanić, p. b., 1349, 1409 stanković jeremić, j., 1237 stanković, d. m., 57 stefanović, i. s., 1203 stefova, m., 1185 steinhart, m., 1203 stevanović, j. z., 41 stevanović, n., 181 stojadinović, s., 1035 stojadinović, s., 69, 519 stojanović, g., 1117, 1313 stojanović, k. a., 41 stojanović, s. đ., 465 stojanović, s., 1035 svrkota, b., 615 swain, r., 829 šajnović, a. m., 7 šeregelj, v., 1359 šikoparija, b., 983 škrbić, b. d., 1425 šmit, b. m., 1349, 1409 šolevic knudsen, t. m., 7 šolić, m. d., 1091 šovljanski, o., 1359 španik, i., 121 špírková, m., 1203 štrbac, s. r., 7 švarc-gajić, j., 1359 tadić, v., 1063 tan, x., 857 ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ 1466 journal of the serbian chemical society tanasković, s. b., 451 tasić, g., 785 teo, y. y., 641 terzić-jovanović, n., 669 tešić, ž., 983 todorović, d., 491 todorović, t. r., 1143 tomasevic, b. i., 531 tosti, t., 983 tošić, s., 1313 trendafilova, a., 1237 trifković, j., 983 trifonova, i. p., 355 trifunović, v., 775 trišić, j., 723 trivic, d. d., 531 turk sekulić, m., 157 turner, d., 1341 urbain, x., 479 urošević, j., 1117 uysal, s., 1219 vachagina, e., 891 van eikema hommes, n. j. r., 1439 vasilić, r., 1035 vasiljević-radović, d., 899 veličković, s., 27 vergel, k., 69 veselinović, g. d., 7, 519 vidovic, d., 345 virijević, k. d., 1349 vojinović-ješić, lj. s., 307, 1259 vojinović miloradov, m. b., 121, 157 vojnović, m. m., 479 vujčić, m., 181 vujisić, lj., 27 vukčević, m. m., 205, 749 vukić, lj. m., 1091 vukićević, n. m., 1409 vuković, n. s., 41 vuković-gačić, b., 1245 vulić, j., 1359 vulić, t. j., 1011 vural, u. s., 1219 wissiak grm, k. s., 275 wu, q.-k., 1381 yeler, o., 389 yellapurkar, i. p., 1109 yemendzhiev, h., 775 yinanc, a., 1219 yosefelahi, r., 1157 yucel, t., 377 zarubica, a., 1285 zečević, m., 615 zhao, x., 857 zheludkevich, m. l., 1035 zheng, l., 1381 zherebtsov, d. a., 57 zinicovscaia, i., 69 zlatanović, i., 1313 zlatar, m., 561 zlatović, m. v., 465 zoraga, m., 377 zyryanov, v. v., 1367 žerađanin, a., 95 živanović, m. n., 1349 živković, m. d., 1409 živković, n. v., 761 živković, s., 939 živković-radovanović, v., 451 žižović, i., 1063 žugić, a., 1063 subject index of vol. 87 and list of referees in 2022 are given in the electronic form at the internet address of the journal of the serbian chemical society: http://www.shd.org.rs/jscs end of volume 87. ________________________________________________________________________________________________________________________ (cc) 2022 scs. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice accepted manuscript this is an early electronic version of an as-received manuscript that hasbeen accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as s. stajčić, l. pezo, g. ćetković, j. čanadanović-brunet, a. mandić, v. tumbas šaponjac, j. vulić, v. travičić and m. belović, j. serb. chem. soc. (2023) https://doi.org/10.2298/jsc230517043s this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. https://doi.org/10.2298/jsc230517043s j. serb. chem. soc.00(0)1-16 (2023) original scientific paper jscs–12397 published dd mm, 2023 1 antioxidant activity according to bioactive compounds content in dried pumpkin waste slađana m. stajčić1*, lato l. pezo2, gordana s. ćetković1, jasna m. čanadanović-brunet1, anamarija i. mandić3, vesna t. tumbas šaponjac1, jelena j. vulić1, vanja n. travičić1 and miona m. belović3 1university of novi sad, faculty of technology, bulevar cara lazara 1, novi sad, serbia, 2university of belgrade, institute of general and physical chemistry, studenski trg 12/v, belgrade, serbia, and 3university of novi sad, institute for food technology in novi sad, bulevar cara lazara 1, novi sad, serbia (received 23 may; revised 11 june; accepted 21 july 2023) abstract: in this study, for the preparation of dried pumpkin waste, freeze-drying and oven-drying (at 50 °c and 65 °c) were applied. effect of drying methods on physical properties (moisture content, water activity, hygroscopicity, water solubility, bulk and tapped density, flowability and colour), content of bioactive compounds (carotenoids and phenolics) and antioxidant activity was investigated. also, influence of one-year storage at –20 °c on the bioactive compounds content and antioxidant activity of dried pumpkin waste was examined. results indicated that drying method showed a significant impact on the investigated characteristics. bioactive compounds content and antioxidant activity by dpph test of freezedried were higher than of oven-dried pumpkin waste, while higher antioxidant activity in reducing power assay was determined by oven-dried pumpkin waste. an artificial neural network model was developed, for the anticipation of antioxidant activity according to bioactive compounds content (phenolics and carotenoids), in oven-dried (at 50 °c and 65 °c) and in freeze-dried pumpkin waste after one-year storage. these models showed good prediction properties (the r2 value during training cycle for output variables was 0.999). it was demonstrated that pumpkin waste is potentially an important source of bioactive compounds, which can be used after extraction in suitable forms in the development of functional food products. keywords: cucurbita moschata; carotenoids; polyphenols; antioxidants; artificial neural network.. introduction cucurbita moschata pumpkin is one of the most commonly cultivated pumpkin species.1,2 it contains numerous bioactive compounds (such as *corresponding author. e-mail: sladja@uns.ac.rs https://doi.org/10.2298/jsc230517043s a cc ep te d m an us cr ip t mailto:sladja@uns.ac.rs https://doi.org/10.2298/jsc230517043s stajčić et al.. carotenoids, phenolic compounds and vitamins) and possesses a wide range of pharmacological activities.3 during processing of pumpkin into various products, such as purées, dishes and juices, a large amount of wastes (i.e. thick rind, large seeds and pressed residues) is generated.1,4 unutilized wastes can lead to potential problems for their handling and disposal.4 also, these waste materials can be considered as potential source of valuable bioactive compounds.5,6 a limiting factor of using of wastes as raw materials for the extraction of bioactive compounds may be their deterioration. for inhibition of microbial growth and facilitating storage of plant materials different drying methods have been employed.7 the most commonly used drying method includes convection. this method is not expensive, but has the disadvantage of reducing the content of some valuable compounds which often undergo oxidation at higher temperature.8 freeze drying, also known as lyophilization, is based on sublimation process where the product is frozen first.9 in this process, drying is performed at low temperature and pressure, i.e. under conditions that allow removal of water by sublimation, which involves direct phase transition of water from solid to vapor without passing through the liquid phase.9 therefore, freeze drying is one of the most useful processes for drying thermosensitive substances.9,10 freeze-drying provides excellent quality characteristics such as colour, flavour and chemical composition of the product, but this method includes very high expenses and requires a lot of time.8 although, in most cases, freeze dried products exhibit much higher contents of bioactive compounds than hot air dried products, some studies have shown that freeze drying can led to greater loss of bioactive compounds than hot air drying.7 the effect of a particular drying method on the retention of raw quality is not predictable and depends on the bioactive compounds and the specific plant material involved.7 accordingly, the selection of drying treatment as preservation technique of waste materials can have a great influence on the retention of their bioactive constituents and raw quality. the isolation and utilization of bioactive compounds from waste are of special interest for the food, cosmetic and pharmaceutical industries.5,6 however, the effectiveness of bioactive ingredients depends on preserving their stability and bioactivity.11,12 since the content of bioactive compounds remain available can be significantly reduced under the adverse conditions during food processing and storage (temperature, oxygen, light), their potential health benefits can be restricted.11 therefore, the aim of our study was to determine and compare physical properties, content of bioactive compounds and antioxidant activity of c. moschata pumpkin waste subjected to oven and freeze drying. another objective of this study was to investigate the possibility of predicting the antioxidant activity of ovendried (50 °c and 65 °c) and freeze-dried (–40 °c) pumpkin waste after one-year storage, according to the content of phenols and carotenoids in the samples. these tasks were achieved using artificial neural network model. a cc ep te d m an us cr ip t antioxidant activity of pumpkin waste 3 experimental chemicals folin-ciocalteau reagent, 2,2-diphenyl-1-picrylhydrazyl radical (dpph•), trolox and trichloroacetic acid were purchased from sigma chemical co. (st louis, mo, usa), ferric chloride was obtained from j.t. baker (deventer, holland). all other chemicals and solvents used were of the highest analytical grade. plant material and dried pumpkin waste preparation fresh cucurbita moschata pumpkin waste material was obtained after pressing the pulp as by-product from the baby food industry (“juvitana”, inđija, serbia) and dried by different methods, in oven (model st-06, instrumentaria, zagreb, croatia) at 50 °c for 14 h and at 65 °c for 12 h without air flow and in freeze drier (model christ alpha 2-4 lsc, martin christ, osterode am harz, germany) at –40 °c for 72 h (until moisture content was reduced to 9–11 g/100 g). dried pumpkin waste was ground, packed in plastic bags, vacuumed and kept at –20 °c until further analysis. dry matter content was determined by weighing the initial and final weight, and calculated percentage of dried weight. physical characterization water activity (aw), moisture content, hygroscopicity, bulk density, tapped density and colour parameters (l*, a* and b*) were determined as reported previously.13 colour parameters (c* and h°) and browning index (bi) was calculated as described by phuon et al.14 the classification of the flowability was made as described by shishir et al.15 solubility was determined according to the method of yamashita et al.,16 with some modifications. water activity. the water activity (aw) was determined by placing approximately 3 g of dried pumpkin waste in the sample holder of a labswift-aw metre (novasina, switzerland) at 25 °c. moisture content. the moisture content of dried pumpkin waste was measured using an air oven method at 105 °c until a constant weight was achieved. hygroscopicity. for hygroscopicity 2 g of dried pumpkin waste from the petri dishes were placed at 25 °c in an airtight plastic container filled with nacl saturated solution (75.29 % rh). after 1 week, hygroscopic moisture (hygroscopicity) was weighed and expressed as g of moisture per 100 g dry solids. solubility. the dried pumpkin waste (0.1 g) was dissolved in 10 ml of distilled water, stirred (unimax 1010, heidolph instruments gmbh, kelheim, germany) at 150 rpm and room temperature for 30 min, and then centrifuged (centrifuge lace 24, colo lab experts, novo mesto, slovenia) at 4000 rpm for 5 min. the supernatant was transferred to a pre-weighed petri dish and dried at 105 °c until constant weight was achieved. after drying, the dried weight of the soluble solid was measured and used to calculate the percentage solubility. bulk and tapped density. for determination of bulk density (db), the sample (10 g) was poured into a measuring cylinder and the initial volume was noted as the bulk volume (vb). the db was calculated according to the formula: db = m/vb (1) where m is the mass of the sample and vb is the bulk volume of the powder. for determination of tapped density (dt), the sample was tapped 250 times and then the volume was measured. tapping was continued until the difference between successive volumes was less than 2 % and this value was registered as the tapped volume (vt) and dt was calculated by the formula: a cc ep te d m an us cr ip t stajčić et al.. dt = m/vt (2) bulk and tapped density of the samples were expressed in g ml-1. flowability. the values of the carr index and hausner ratio indicate the quality of the powder in terms of the flow property. carr index and hausner ratio were calculated according to the equations: carr index = ((dt–db)/dt) ·100 (3) hausner ratio = dt/db (4) colour. the colour parameters (cie l*, a* and b*) were determined with a minolta reflectance colorimeter (minolta chromameter cr-400, konica minolta inc., tokyo, japan) using d65 illumination source at observer angle of 2°. the colour of dried pumpkin waste was measured using attachment for granular materials cr-a50. the liquid extracts were measured in a 10 mm glass cell cm-a98 fixed using specimen holder cm-a96, while standard white calibration plate was fixed behind the cell because of the sample transparency. chroma (c*) and hue angle (h°) were calculated according to the formulas: c* = (a*2 + b*2)1/2 (5) h° = arctan (b*/a*) (6) browning index (bi) was calculated using the following expression: bi = ((x – 0.31)/0.17)·100 (7) where x = (a* + 1.75l*)/(5.645l* + a* − 3.012b*) (8) extraction procedure for determination of the content of bioactive compounds and antioxidant activity previously described extraction procedure with modification was used.10 briefly, dried pumpkin waste was extracted using acetone : ethanol mixture (36:64 v/v) in solid to solvent ratio 1:10 (w/v) using a laboratory shaker (unimax 1010, heidolph instruments gmbh, kelheim, germany) at 300 rpm, under light protection, at room temperature. the extraction was performed three times with the same volume of solvents. the obtained three extracts were filtered (whatman paper no.1), combined, and stored in dark bottles at -20 °c till further analysis. determination of β-carotene and total phenolic content the content of β-carotene was analyzed spectrophotometrically using a multiskan go microplate reader (thermo fisher scientific inc., waltham, ma, usa) according to the method of nagata and yamashita,17 adapted for 96 well microplate. the β-carotene content was expressed as mg of β-carotene equivalents per 100 g sample (dw). the total content of phenolics was determined spectrophotometrically by folin-ciocalteau method,18 adapted for 96 well microplate. results of total phenolics content were expressed as gallic acid equivalents (gae) per 100 g sample (dw). hplc analysis qualitative and quantitative analysis of flavonoids and phenolic acids was performed by hplc analysis.19 also, analysis of carotenoids was done according to the hplc method described previously.13 a cc ep te d m an us cr ip t antioxidant activity of pumpkin waste 5 determination of antioxidant activity antioxidant activity on dpph radicals (dpph assay) was estimated spectrophotometrically using a 96-well microplate reader, following the method described by girones-vilaplana et al.20 reducing power (rp assay) was performed with the method of oyaizu21 adapted for 96 well microplate. the calibration curves were made with trolox and results were expressed as mg trolox equivalents (te) per 100 g of sample (dw). storage stability test dried pumpkin waste samples were stored at –20 °c in high-density polyethylene bags for one year. the effect of storage on the stability of bioactive compounds and antioxidant activities were measured using the appropriate methods described above. ann modelling a multi-layer perceptron model (mlp), with three layers was applied for artificial neural network (ann) modelling, to investigate the antioxidant activity of oven-dried (50 °c and 65 °c) and freeze-dried (–40 °c) pumpkin waste samples after one-year storage, on the basis of the bioactive compounds content. the experimental database was normalized in order to improve the behaviour of the ann. broyden-fletcher-goldfarb-shanno (bfgs) algorithm was employed in solving nonlinear problems during the network modelling.22 a series of different topologies (more than 100,000) were tested during the modelling, changing the number of neurons in the hidden layer (from 5 to 20), randomly setting initial weights and biases.23 the accuracy of the model the numerical investigation of the developed ann model's accuracy was performed applying the common used statistical tests, such as coefficient of determination (r2), reduced chi-square (χ2), mean bias error (mbe), root mean square error (rmse), mean percentage error (mpe), average absolute relative deviation (aard) and sum of squared errors (sse).24 global sensitivity analysis the yoon’s interpretation method was used to determine the relative influence of bioactive compounds content on antioxidant activity of oven-dried and freeze-dried pumpkin waste samples. this method was applied using the weight coefficients of the developed ann model.25 statistical analysis all experiments were done in triplicate. the results were expressed as mean value ± standard deviation (± sd, n = 3). the calculations were performed using statsoft statistica 2010 software. results and discussion physical properties of dried pumpkin waste in recent years, the need for dried products of high quality has been increased. much attention has been focused to the quality of dried materials.26 in our study, oven-drying and freeze-drying were applied to obtain dried pumpkin waste materials. after oven-drying (at 50 °c and 65 °c) and freeze-drying, dry matter content was determined to be 15.42 %, 15.40 % and 15.37 %, respectively. the influence of drying methods and temperatures on the physical properties of dried pumpkin waste is shown in table i. a cc ep te d m an us cr ip t stajčić et al.. table i. physical properties of oven-dried (at 50 °c and 65 °c) and freeze-dried (–40 °c) pumpkin waste physical properties oven-dried (50 °c) oven-dried (65 °c) freeze-dried (–40 °c) moisture content, % 10.78±0.33b 10.18±0.44b 9.10±0.32a water activity (aw) 0.274±0.010 c 0.126±0.005b 0.035±0.001a hygroscopicity, g/100 g 13.29±0.47a 15.08±0.71b 15.49±0.48b bulk density, g ml-1 0.40±0.02c 0.36±0.02b 0.11±0.00a tapped density, g ml-1 0.58±0.04c 0.50±0.03b 0.17±0.01a carr index, % 30.05±1.50b 27.31±1.24a 33.35±0.93c hausner ratio 1.43±0.03b 1.38±0.00a 1.50±0.02c flowability poor poor very poor solubility, % 31.19±1.12a 33.30±1.47b 33.76±1.38b colour parameters lightness/darkness (l*) 49.86±0.01a 54.11±0.03b 67.29±0.02c greenness/redness (a*) 9.45±0.03c 7.34±0.03b 5.72±0.01a blueness/yellowness (b*) 23.05±0.00a 25.00±0.01b 29.91±0.01c chroma (c*) 24.91±0.01a 26.05±0.01b 30.45±0.01c hue angle (h°) 67.71±0.06a 73.64±0.06b 79.17±0.01c browning index (bi) 74.49±0.03c 70.36±0.05b 63.46±0.01a the results are presented as mean ± sd; different letters (a-c) in rows indicate that there is significant difference at p ≤ 0.05, according to tukey-s hsd test moisture content, i.e. quantity of water contained in a food system, is an important characteristic of dried powdered products, which is associated to the efficiency of drying process.27,28 also, moisture content of powders has a significant influence on storage stability.29 similarly as in our study, moisture content of oven-drying pumpkin material was higher than that in freeze-dried pumpkin material.3,30 aydin and gocmen31 reported that moisture contents in hotair oven-dried and freeze-dried pumpkin flour were 12.64 % and 12.56 %, respectively. probably, crust formed during hot air drying did not allow removing the moisture from material.30 water activity (aw) represents the availability of free water in a food system which has a crucial role in biochemical reactions.29 basically, the most of the adverse changes in food system during storage such as lipid oxidation, enzymatic reactions, non-enzymatic browning, and microbial growth are almost completely obstructed when the water activity value is below 0.4.32 therefore, water activity has been considered as one of the most important quality parameters for long term storage of dried products.30 in our study, water activity values of dried pumpkin waste samples provide their good storage stability. previously, oven-dried pumpkin materials also had higher water activity (0.408) than freeze-dried pumpkin material (0.239).30 hygroscopicity, i.e. capacity to adsorb ambient moisture, is one of the major factors which affect product stability.27,33 a prominent property of freeze-dried products is great hygroscopicity.34 hygroscopicity is related to the gradient of water concentration a cc ep te d m an us cr ip t antioxidant activity of pumpkin waste 7 between the product and the surrounding environment, which is greater for the less moist powder.33 indeed, higher hygroscopicity was determined in freeze-dried pumpkin waste in comparison with oven-dried. tapped density represents the real solid density, whereas the bulk density takes into account all the spaces between the particles of powdered products. lower bulk density implies that more occluded air exists within the powder, thus there is a greater possibility for oxidation and reduced stability of product during storage.28 the bulk and tapped densities were higher in oven-dried in relation to freeze-dried pumpkin waste, which is in agreement with the study of que et al.3 according to carr index and hausner ratio, oven-dried and freeze-dried pumpkin waste are classified,15 in terms of flow property as poor and very poor, respectively. solubility is a property referring to the product behaviour in an aqueous phase and is an indicator for its reconstitution quality. quick and complete reconstitution of powder is common requirement for its application.35 the powder structure with more cavities and pores allows easier passage of water, thus facilitates dissolution. pumpkin waste dried at 65 °c showed higher solubility compared to the waste dried at 50 °c. solubility can be associated with the amount of starch degradation, since at higher temperature during oven-drying more starch can be decomposed.3 colour is a significant factor for consumers in food choice.36 it can be used as an indicator of the chemical and quality changes as a consequence of thermal processing.36 in agreement with our study, oven-drying in comparison to freezedrying resulted in a darker colour (lower l* values).3,30 the lower l* value and higher a* value could be indicative of the browning reaction.31,37 compared to oven-dried waste samples, freeze-dried waste showed less yellowness and less redness (indicated by lower b* and a* values, respectively). browning is an important colour reaction in food processing such as drying because it affects appearance quality.36 as an indicator of browning, browning index (bi) is often used especially in conventional drying since it represents the purity of brown colour and is considered to be an important parameter associated with browning.30 in this study, the values of browning index of dried waste samples showing that oven-drying caused more brown compounds. the difference in browning may be related to the removal of water by sublimation and prevention of enzymatic browning reactions during freeze-drying.38,39 the yellowish colour of dried pumpkin waste powders could be mainly attributed to the presence of carotenoids naturally found in this vegetable. drying conditions, including high temperature, light and oxygen exposure can cause changes in food surface that lead to colour changes, and also to degradation of carotenoids.32 the lighter yellowness of freezedried in relation to oven-dried waste is shown by c* and h° values. similar colour values as in our study, lightness (l*), redness (a*) and yellowness (b*) of convective dried (55.57, 22.67, and 35.25, respectively) and freeze-dried (77.96, 18.54, and 31.22, respectively) slices of c. moschata pumpkin were found.30 in a cc ep te d m an us cr ip t stajčić et al.. addition, que et al.3 reported that the freeze-drying process can significantly decrease the brownish appearance of pumpkin flour and can produce pumpkin powders of high-quality colour. bioactive compounds and antioxidant activity of dried pumpkin waste effect of drying methods, drying temperatures and storage on the content of bioactive compounds and antioxidant activity of dried pumpkin waste samples is presented in table ii. table ii. the content of bioactive compounds (phenolics and carotenoids) and antioxidant activity (dpph and rp assays) of oven-dried (at 50 °c and 65 °c) and freeze-dried (–40 °c) pumpkin waste bioactive compounds/ antioxidant activity oven-dried (50 °c) oven-dried (65 °c) freeze-dried (–40 °c) total phenolics after drying 201.31±7.27a 213.61±7.60b 294.69±14.13c total phenolics after storage 183.47±7.03b 180.34±5.39b 175.60±6.11a β-carotene after drying 6.51±0.23b 5.56±0.00a 14.26±0.27c β-carotene after storage 2.85±0.12a 4.57±0.00b 7.89±0.22c gallic acid* 1.658±0.080a 1.757±0.078b 3.802±0.17c protocatechuic acid* 7.644±0.322b 4.555±0.221a 9.217±0.43c catechin* 0.490±0.021a 1.588±0.071b 1.672±0.067c caffeic acid* 0.128±0.005c 0.096±0.003a 0.116±0.003b syringic acid* 0.470±0.021b 0.288±0.011a 1.835±0.076c coumaric acid* 0.103±0.004b 0.097±0.003a 0.108±0.002b chlorogenic acid* 0.049±0.002b 0.038±0.001a 0.224±0.008c p-hydroxybenzoic acid* 2.642±0.131b 1.726±0.076a 12.606±0.598c vanillic acid* 0.171±0.006c 0.117±0.004a 1.385±0.060b myricetin* 0.010±0.001b 0.007±0.001a 1.385±0.056c rutin* 0.127±0.004 b 0.083±0.002a 0.313±0.011c total phenolics* 13.492 10.352 31.365 lutein* 0.033±0.001a 0.037±0.001b 0.174±0.006c zeaxanthin* 1.598±0.069b 0.403±0.017a 1.660±0.081b β-carotene* 4.406±0.187c 1.303±0.057a 3.247±0.158b β-cryptoxanthin* n.d. n.d. 2.820±0.134 total carotenoids* 6.037 1.743 7.901 dpph after drying 99.27±1.52b 78.87±0.55a 131.64±6.40c dpph after storage 50.56±1.75b 30.41±0.34a 65.66±1.55c rp after drying 66.23±1.97b 140.94±0.77c 17.39±0.28a rp after storage 60.40±2.68b 140.20±3.10c 16.41±0.46a the spectrophotometric determinations were done after drying of pumpkin waste and after storage at –20 °c (during one year) of dried pumpkin waste samples, while hplc analysis (indicated by *) was performed after drying of pumpkin waste; the results are presented as mean ± sd; different letters (a-c) in rows indicate that there is significant difference at p ≤ 0.05, according to tukey-s hsd test; total and individual contents of carotenoids and phenolics are presented a cc ep te d m an us cr ip t antioxidant activity of pumpkin waste 9 in mg/100 g dw, where total phenolics determined spectrophotometically are expressed in mg gae/100 g dw; antioxidant activity by dpph and rp assays is presented in mg te/100 g dw; n.d. means not detected higher content of total phenolics determined by spectrophotometric and hplc methods was determined in freeze-dried pumpkin waste in comparison to oven-dried samples. in the study of aydin and gocmen,31 higher level of phenols (236 mg gae/100 g dw) was found in oven-dried (at 60 °c) pumpkin (cucurbita moschata duch.) flour compared to freeze-dried (226 mg gae/100 g dw). also, que et al.3 reported that content of phenolic compounds (1.64 mg g-1) in hot airdried (at 70 °c) pumpkin (cucurbita moschata duch.) flour was 4.6 times higher than that in freeze-dried pumpkin (0.39 mg g-1) flour, indicating the formation of phenolics during drying at 70 °c. higher degree of heating could have a great impact on the increase of total phenolics of pumpkin flour.3 beside the difference in sample, extraction processes, drying conditions, cultivar, maturation stage, geography and climate may cause difference in determination of phenolics.30,40 in the study of nawirska-olszanska et al.,41 the content of phenolics in different varieties of c. moschata pumpkin was ranged from 141.16 mg/100 g dw to 390.61 mg/100 g dw. potosi-calvache et al.42 determined the content of total phenolics (from 27.7 mg gae/100 g to 79.9 mg gae/100 g) in samples of c. moschata pumpkin dried under different temperature (45-65 °c) and air flow (4-7 m/s). during processing and storage phenolics can be degraded or formed from the availability of their precursors or by non-enzymatic inter conversions between them.42 in our study, in investigated pumpkin waste powders, p-hydroxybenzoic and protocatechuic acid were determined in highest content of all identified phenolic acids, while catechin was determined in highest content of identified flavonoids. kulczyński and gramza-michałowska1 reported that profile of bioactive compounds in pumpkin (c. moschata) is considerably diversified in its cultivars. in agreement with our study, content of identified flavonoids were lower than content of phenolic acids, and high contents of p-hydroxybenzoic and protocatechuic acids were also found.1 higher β-carotene content, determined spectrophotometrically, was observed in freeze-dried than in oven-dried pumpkin waste samples. different factors (temperature, oxygen and light) during drying process (i.e. drying conditions) may cause carotenoid degradation.32,40 in agreement with in our study, higher total carotenoid content (between 5 mg/100 g and 160 mg/100 g) was obtained with freeze-drying than by convective drying of 12 pumpkin cultivars.8,40 previously, higher drying temperatures also produced greater pigment losses.32 colour values, l*, a* and b* of extracts obtained after extraction of oven-dried at 50 ° (59.43, – 5.27, and 66.53, respectively), oven-dried at 65 ° (59.27, –5.61, and 67.16, respectively) and freeze dried (56.01, 1.79, and 65.57, respectively) c. moschata pumpkin waste, indicated yellow colour that could be attributed to the presence of a cc ep te d m an us cr ip t stajčić et al.. carotenoids. it is interesting to notice that colour parameter a* had negative values in oven-dried samples, indicating degradation of reddish pigments in comparison to freeze-dried sample, which had positive a* values. in our study, higher content of total carotenoids determined by hplc method was found in freeze-dried pumpkin waste in comparison to oven-dried samples. β-criptoxanthin was not even identified in oven-dried waste. interestingly, somewhat higher β-carotene content was found in dried pumpkin waste after oven-drying at 50 °c than after freeze-drying. kulczyński and gramza-michałowska1 reported high variability of carotenoids among pumpkin (c. moschata) cultivars, and on average the content of zeaxanthin, lutein and β-carotene of different c. moschata cultivars was 2.64 mg/100 g dw, 6.87 mg/100 g dw and 2.92 mg/100 g dw, respectively. after storage, the content of phenolics and β-carotene in dried pumpkin waste samples was decreased (table ii). after storage, similar values of phenolics content were found in all dried pumpkin waste samples, while highest content of β-carotene was determined in freeze dried waste in comparison to oven-dried pumpkin waste samples. freeze-dried pumpkin waste exhibited higher antioxidant activity by dpph test, while higher antioxidant activity in reducing power assay was achieved by oven-dried pumpkin waste (table ii). this can be explained by various mechanisms by which different antioxidants exert their action.26 previously, hotair oven dried (at 60 °c and 70 °c) pumpkin flours showed higher antioxidant activities by different assays, than freeze-dried samples.3,31 phenolics formed from precursors in hot-air drying treatment or generation of maillard-type antioxidants might be responsible for higher antioxidant activity of hot-air dried pumpkin flour.3 samples of dried pumpkin waste in our study showed somewhat lower, but significant antioxidant activity by dpph test (from 3.15 μmol te g-1 to 5.26 μmol te g-1), and by reducing power test (from 0.69 μmol te g-1 to 5.63 μmol te g-1) in comparison to the antioxidant activity of dried pumpkin flours (from 5.57 μmol te g-1 to 7.21 μmol te g-1) reported by aydin and gocmen.31 after storage, dpph antioxidant activity in dried pumpkin waste powders was decreased, while reducing power was almost unchanged in comparison with levels determined before storage (table ii). after storage, highest reducing power was observed for waste dried at 65 °c, while highest antioxidant activity by dpph test was determined for freeze-dried waste. ann model the acquired optimal neural network model could be used to adequately anticipate the antioxidant activity of oven-dried (50 °c and 65 °c) and freeze-dried (–40 °c) pumpkin waste samples after one year storage, on the basis of phenolics and carotenoids content. the optimal number of neurons was: 9 (network mlp 149-4), while the highest r2 values during the training cycle were 0.999). a cc ep te d m an us cr ip t antioxidant activity of pumpkin waste 11 the obtained ann model for the anticipation of output variables was complex (175 weights-biases coefficients) due to the high nonlinearity of the observed system. the goodness of fit between experimental and ann model calculated values were shown in table iii. table iii. the "goodness of fit" tests for the developed ann model output variable χ2 rmse mbe mpe sse aard r2 dpph after drying 1.042 0.589 0.168 0.589 0.957 1.766 1.000 dpph after storage 0.456 0.390 –0.036 0.718 0.452 1.091 1.000 rp after drying 0.267 0.299 –0.193 0.277 0.156 0.601 1.000 rp after storage 0.690 0.480 –0.349 0.542 0.325 1.046 1.000 the ann predicted values were very close to the measured values in most cases, in terms of r2 values.43,44 the sse values obtained with the ann model was of the same order of magnitude as experimental errors for output variables reported in the literature.22,45 the ann model had an insignificant lack of fit tests, which means the model satisfactorily predicted output variables. global sensitivity analysisyoon’s interpretation method according to the fig. 1, gallic acid, syringic acid, chlorogenic acid, phydroxybenzoic acid, vanillic acid, myrcetin, rutin and lutein content were the most influential parameters with approximately relative importance of 9.222– 10.337 % for the prediction of dpph after drying, while the relative influence of these variables for the prediction of dpph after storage reached the relative importance of 9.583–10.680 %.the rp assay was mostly influenced by the catechin content, 27.82 % after drying, and 28.55 % after the on-year long storage. a cc ep te d m an us cr ip t stajčić et al.. fig. 1. the relative importance of the bioactive compounds content on antioxidant activity determined by dpph assay after drying (a) and storage (b), and by rp assay after drying (c) and storage (d) of pumpkin waste samples. conclusion different drying methods, including freeze-drying and oven-drying (at 50 °c and 65 °c) caused significant impact on physical properties, bioactive compounds content and antioxidant activity of dried pumpkin waste. in general, carotenoids and phenolics content of freeze-dried was higher than in oven-dried pumpkin waste. freeze-dried pumpkin waste exhibited higher antioxidant activity by dpph test, while higher antioxidant activity in reducing power assay was determined by oven-dried pumpkin waste. after storage at –20 °c during one year, higher content of β-carotene and antioxidant activity by dpph test, while lower content of phenolics and reducing power were achieved by freeze-dried than by oven-dried pumpkin waste samples. a cc ep te d m an us cr ip t antioxidant activity of pumpkin waste 13 also, findings of this study indicate that antioxidant activity of oven-dried (50 °c and 65 °c) and freeze-dried (–40 °c) pumpkin waste samples after one year storage could be anticipated, based on the phenolics and carotenoids content. the artificial neural network model showed to be adequate for the prediction of output variables (the r 2 values during training cycle for these variables were: 0.999). it was demonstrated that pumpkin waste is potentially an important source of bioactive compounds with significant antioxidant properties. in the future, to overcome a main drawback for application of natural bioactive compounds, namely their instability, different protection systems including encapsulates could be formulated. therefore, our results support the need for preparation of encapsulates of bioactive compounds extracted from dried pumpkin waste and evaluation of their physicochemical and stability characteristics, in order of development functional ingredients for value added food products. acknowledgements: this work was financially supported by the ministry of science, technological development and innovations of the republic of serbia (project no. 451-0347/2023-01/200134, project no. 451-03-47/2023-01/200051 and project no. 451-03-47/202301/200222). и з в о д антиоксидативнa активност на основу садржаја биоактивних једињења у сушеном отпаду тикве слађана м. стајчић*1, лато л. пезо2, гордана с. ћетковић1, јасна м. чанадановић-брунет1, анамарија и. мандић3, весна т. тумбас шапоњац1, јелена ј. вулић1, вања н. травичић1 и миона м. беловић3 1универзитет у новом саду, технолошки факултет, булевар цара лазара 1, нови сад, србија, 2универзитет у београду, институт за општу и физичку хемију, студентски трг 12/в, београд, србија, и 3универзитет у новом саду, научни институт за прехрамбене технологије у новом саду, булевар цара лазара 1, нови сад, србија у овој студији за припрему сушеног отпада тикве примењено је сушење лиофилизацијом и у сушници (на 50 °c и 65 °c). испитан је утицај метода сушења на физичка својства (садржај влаге, активност воде, хигроскопност, растворљивост у води, насипну и тапкану густину, проточност и боју), садржај биоактивних једињења (каротеноида и полифенолних једињења) и антиоксидативну активност. такође, испитан је утицај складиштења на –20 °c у периоду од годину дана на садржај биоактивних једињења и антиоксидативну активност сушеног отпада тикве. резултати су показали да метода сушења значајно утиче на испитане карактеристике. садржај биоактивних једињења и антиоксидативна активност dpph тестом лиофилизованог отпада били су већи у односу на отпад тикве сушен у сушници, док је већа антиоксидативна активност тестом редукционе способности одређена у отпаду тикве сушеном у сушници. развијен је модел вештачке неуронске мреже, за предвиђање антиоксидативне активности према садржају биоактивних једињења (полифенолна једињења и каротеноиди), у отпаду тикве сушеном лиофилизацијом и у сушници (на 50 °c и 65 °c) након годину дана складиштења. ови модели су показали добра својства предвиђања (вредност r2 током циклуса тренинга за излазне варијабле била је 0,999). показано је да је отпад тикве потенцијално важан извор a cc ep te d m an us cr ip t stajčić et al.. биоактивних једињења, која се након екстракције могу користити у одговарајућим облицима у развоју функционалних прехрамбених производа. 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hydrazinecarbothioamides and 1,2,4-triazoles, and an evaluation of their antimicrobial activities laura i. socea1*, gabriel saramet2, constantin draghici3, bogdan socea4, vlad d. constantin4 and manuela a. radu-popescu5 1organic chemistry department, faculty of pharmacy, “carol davila” university of medicine and pharmacy, 6 traian vuia street, 020956, bucharest, romania, 2pharmaceutical technology department, faculty of pharmacy, “carol davila” university of medicine and pharmacy, 6 traian vuia, 020956 bucharest, romania, 3“c. d. nenitzescu” institute of organic chemistry, romanian academy, 202b splaiul independentei, 060023 bucharest, romania, 4faculty of general medicine, “carol davila” university of medicine and pharmacy, st. pantelimon emergency hospital, 340–342, soseaua pantelimon street, 021623, bucharest, romania and 5general and pharmaceutical microbiology department, faculty of pharmacy, “carol davila” university of medicine and pharmacy, 6 traian vuia street, 020956, bucharest, romania (received 27 february, revised 15 may, accepted 18 may 2015) abstract: a new series of hydrazinecarbothioamides 6–9 bearing a 5h-dibenzo[a,d][7]annulene moiety were synthesized. cyclization of 6–9 in naoh solution produced the corresponding 4h-1,2,4-triazole-3-thiols 10–13, which proved to be axial isomers. the thioethers 14–17 were prepared by alkylation of 10–13 with methyl iodide. all new compounds were characterized by elemental analysis, and ir, uv, 1h-nmr and 13c-nmr spectroscopy. an evaluation for antimicrobial activity against staphylococcus aureus, pseudomonas aeruginosa, escherichia coli, bacillus subtilis, salmonella enterica subsp. enterica serovar typhimurium, shigella flexneri and candida albicans was performed. keywords: acylhydrazinecarbothioamide; 1,2,4-triazole-3-thiol; dibenzo[a,d][7]annulene; antimicrobial activity. introduction bacterial infection remains a serious threat to human lives because of their capacity to develop resistance to existing antibiotics, which is an increasing public health problem. for this reason, obtaining new types of antibacterial agents is a very important task. * corresponding author. e-mail: laurasocea@gmail.com doi: 10.2298/jsc150227039s _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1462 socea et al. the tricyclic framework of 5h-dibenzo[a,d][7]annulene constitutes an integral part of the structure of molecules that are known to be effective for the treatment of depressive disorders.1,2 analogs of 5h-dibenzo[a,d][7]annulene, such as protriptyline and demexiptiline, are well known tricyclic antidepressants, which are used in the treatment of migraines, tension headaches, anxiety, psychosis, aggression and violent behavior. the anti-allergic drug cyproheptadine (cyp) is known to have inhibitory activity for l-type calcium channels in addition to histamine and serotonin receptors.3 recently, studies that were trying to detect other possible pharmacological actions of already known tricyclic antidepressants have received increasing interest.4–13 dibenzo[a,d][7]annulene moieties are incorporated in biologically active compounds that exhibit muscarinic receptor antagonist properties and are useful in the treatment of parkinson’s disease, tardive dyskinesia and motion sickness.4 dibenzo[a,d][7]annulene derivatives exhibit antidiabetic,5 antiparasitic,6 metalloprotease inhibitory7 and antimicrobial activity.8–13 munoz-bellido et al. realized an extensive study that demonstrated the intense antibacterial activities presented by some antidepressant from the group of serotonin recapture inhibitors, such as clomipramine and sertraline.11 similarly some psychiatric agents, such as protriptyline or cyclobenzaprine, are associated with some chemotherapy agents (sulfathiazole) that enhance the antibacterial activity of the latter and reduce the mic up to 50 %.11 1,2,4-triazole derivatives are known to show biological properties including antimicrobial,14–19 anticancer,20 anti-inflammatory,21,22 anticonvulsant,23 antiviral,24,25 antitubercular,25 hypolipidemic,26 antioxidant activities27,28 and others. several compounds containing 1,2,4-triazole rings are used in therapy. for example, fluconazole, terconazole and itraconazole are used as antimicrobial drugs, while vorozole, letrozole and anastrozole are non-steroidal drugs used for the treatment of cancer.29 other examples are ribavirin (antiviral agent), rizatriptan (antimigraine agent) and alprazolam (anxiolytic agent),30 beside others. furthermore, a number of substituted hydrazinecarbothioamide were found to exhibit antifungal,12,31–36 tuberculostatic,36 cytostatic,37 anticonvulsant,38 antiviral39 and antioxidant activities.28,40 considering the above data, in continuation of ongoing research on biologically active compounds, the synthesis of new hydrazinecarbothioamides and 1,2,4 -triazoles bearing the 5h-dibenzo[a,d][7]annulene moiety and their antimicrobial activities were considered.41,42 experimental chemistry all reactants and solvents of the highest purity were obtained commercially and used without further purification. melting points were determined on a boetius apparatus and are _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ new hydrazinecarbothioamides and 1,2,4-triazoles 1463 uncorrected. the uv–vis spectra were recorded on a specord 40 analytik jena spectrometer, in methanol (2.5×10-5 m) in the wavelength range 200–600 nm. the ir spectra were recorded in kbr pellets using a vertex 70 bruker spectrometer. elemental analyses were performed on an ecs-40-10-costeh micro-dosimeter (and the values were within ±0.4 % of the theoretical ones). the nmr spectra were recorded on a varian gemini 300 bb instrument operating at 300 mhz for 1hand 75 mhz for 13c-nmr, using dmso-d6 as solvent for the hydrazinecarbothioamides and cdcl3 for the 1,2,4-triazole compounds. chemical shifts (δ in ppm) were assigned according to the internal standard signal of tetramethylsilane in dmso-d6 (δ = 0 ppm). coupling constants, j, are expressed in hz. analytical and spectral data of the synthesized compounds are given in the supplementary material to this paper. general procedure for the preparation of n-substituted 2-(5h-dibenzo[a,d][7]annulen-5-ylacetyl)-hydrazinecarbothioamides (6–9) a mixture of 2-(5h-dibenzo[a,d][7]annulen-5-yl)acetohydrazide (1, 4 mmol) and the required isothiocyanate 2–5 (4 mmol) in absolute ethanol (30–50 ml) was refluxed for 6–12 h. on cooling the reaction mixture to room temperature, a precipitate appeared. this was filtered off and recrystallized from ethanol to obtain the desired compound. general procedure for the preparation of 4-substituted 5-(5h-dibenzo[a,d][7]annulen-5-ylmethyl)-4h-1,2,4-triazole-3-thiols (10–13) a solution of the required hydrazinecarbothioamide (6–9, 1 mmol) in 8 ml of 8 % naoh solution was refluxed for 3–9 h and then filtered. after cooling, the filtrate was neutralized with acetic acid. the obtained white precipitate was filtered and recrystallized from chcl3:petroleum ether (1:2, v:v, boiling range: 60–80 °c). general procedure for the preparation of 4-substituted 3-(5h-dibenzo[a,d][7]annulen-5ylmethyl)-5-(methylsulfanyl)-4h-1,2,4-triazoles (14–17) to a solution of sodium ethoxide (1mmol of sodium in 10 ml of absolute ethanol) was added the required triazole 10–13 (1 mmol). the reaction mixture was stirred at room temperature until a solution was obtained. to this solution, methyl iodide (1 mmol) was added and stirring continued for 10 h. the reaction mixture was poured into ice water and the precipitate was filtered off, washed with water and recrystallized from ethanol. antimicrobial activity the antibacterial and antifungal activities of the compounds were investigated by the broth microdilution method, in 96 flat-bottomed wells microplates (nunc, denmark). dimethyl sulfoxide was used as the solvent for the preparation of stock solutions of the compounds, to obtain a concentration of 2048 μg ml-1. the antimicrobial actions of the newly-synthesized compounds were tested against 6 reference bacterial strains, i.e., staphylococcus aureus atcc 25923, pseudomonas aeruginosa atcc 27853, escherichia coli atcc 25922, bacillus subtilis atcc 6633, salmonella enterica subsp. enterica serovar typhimurium atcc 14028, shigella flexneri atcc 12022, and one reference yeast strain, i.e., candida albicans atcc 90028. gentamicin was used as a positive control for s. aureus, p. aeruginosa and e. coli, and fluconazole for c. albicans. bacterial susceptibility testing was performed according to the guidelines of the clinical and laboratory standards institute (clsi) m100-s16 and european committee on antimicrobial susceptibility testing (eucast).43,44 all 96 wells of the microplate were filled in with 100 μl of müller–hinton broth (cation -adjusted) when testing compounds against bacteria and sabouraud broth when testing against _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1464 socea et al. the yeast. series of two-fold dilutions of the newly-obtained compounds were performed in műller–hinton or sabouraud broth. in the case of the reference bacterial strains, the inoculum was prepared by suspending 5 distinct colonies from a 24 h culture obtained on columbia blood agar (biomérieux, france), in a tube with brain heart infusion broth (bhi broth). after vortex mixing and adjusting the density to the turbidity of the 0.5 mcfarland standard, the bacterial suspension was diluted 1:100 in bhi broth, in order to obtain the working inoculum. afterwards, each well of the microdilution plates containing 100 μl of müller– –hinton broth with compound was inoculated within 15 min with 100 μl of the bacterial inoculum, including the growth control wells, but not the sterility control wells that were filled with 200 μl of compound-free müller–hinton broth. in the case of the reference yeast strains, the inoculum was prepared by suspending 5 distinct colonies from a 24 h culture obtained on sabouraud dextrose agar, in a tube with 5 ml of sterile distilled water. after vortex mixing and adjusting the density to the turbidity of the 0.5 mcfarland standard, the fungal suspension was diluted in sterile distilled water in order to obtain a working inoculum. each well of the microdilution plates containing 100 μl of sabouraud broth with compound was inoculated with 100 μl yeast inoculum within 15 min, including the growth control wells, but not the sterility control wells, which were filled only with 200 μl compound-free sabouraud broth. after performing the inoculum controls from the growth control wells, the microplates were incubated at 37 °c for 24 h.48-50 the lowest concentration of each compound able to inhibit visible microbial growth was considered the minimum inhibitory concentration (mic) value. results and discussion chemistry the reaction sequences employed for the syntheses of the title compounds are shown in scheme 1. in the present work, n-aryl-2-(5h-dibenzo[a,d][7]annulen-5-ylacetyl)hydrazinecarbothioamides 6–9 were synthesized by nucleophilic addition of 2-(5h-dibenzo[a,d][7]annulen-5-yl)acetohydrazide (1) to the aryl isothiocyanates 2–5, in absolute ethanol under reflux. 2-(5h-dibenzo[a,d][7]annulen-5-yl)acetohydrazide (1) was prepared starting from dibenzosuberenone, according to a literature method.42,43,46 synthesis of the new 4-alkyl/aryl-5-(5h-dibenzo[a,d][7]annulen-5-ylmethyl)-4h-1,2,4-triazole-3-thiols 10–13 (that exist in equilibrium with their thione tautomer) consisted in intramolecular cyclization of acylhydrazinecarbothioamides 6–9 in 8 % sodium hydroxide solution under reflux.41,46,47 the treatment of 1,2,4-triazoles 10–13 with methyl iodide in basic media yielded the 4-substituted 3-(5h-dibenzo[a,d][7]annulen-5-ylmethyl)-5(methylsulfanyl)-4h-1,2,4-triazoles 14–17 and not the n-methylated derivatives. analytical and spectral data of the newly synthesized compounds all the synthesized compounds were analyzed by ir, uv–vis, 1h-nmr and 13c-nmr spectroscopy. the analytical and spectral data of the new compounds are given in the supplementary material to this paper. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ new hydrazinecarbothioamides and 1,2,4-triazoles 1465 infrared spectra of new hydrazinecarbothioamides 6–9 showed a new band at 1249–1258 cm–1 due to the stretching vibration of the c=s groups. this fact confirmed the addition of the 2-(5h-dibenzo[a,d][7]annulen-5-yl)acetohydrazides to different isothiocyanates. the c=o and n–h stretching bands were present at 1673–1699 and 3141–3359 cm–1, respectively. the hydrazinecarbothioamides 6–9 were present as two conformational isomers, 5′-axial and 5′-equatorial in about 3:1 ratio, interconvertible by inversion of the middle ring, which was confirmed by their 1h-nmr spectra.42 in 6–9-axial isomers, the h-5′(eq) is deshielded, manifested as a triplet at 4.62–4.65 ppm (j in range 7.0–7.3 hz), whereas the ch2-12′ protons are shielded by the double bond, showing a doublet at 2.57–2.60 ppm (scheme s-1 of the supplementary material to this paper). double bonds shield h-5′ axial, while aromatic rings deshield h-5′ equatorial, because of the current cycle. the h-5′(eq) appeared at δ = 4.62–4.65 ppm (triplet) and h-5′(ax) appeared at δ 3.74–3.76 ppm (triplet).42 the signals of nh protons appeared as singlets between 9.53–10.12 ppm and the double bond protons h-10′ and h-11′ appeared as a singlet at 7.02–7.03 ppm. the 13c-nmr spectrum of compounds 6–9 showed a narrow δ domain (123–140 ppm) with c-10′ and c-11′ easily recognizable at δ 130–131 ppm, corresponding to the dibenzo[a,d][7]annulene moiety. c=s carbon atom could be responsible for the appearance of a signal at δ ≈ 181 ppm. cyclization of 6–9 to 10–13 was proved by the ir spectra that showed the disappearance of the νc=o band. it appears that in kbr pellets, the 1,2,4-triazole -3-thiols 10–13 exist in their thionic tautomeric form.41 scheme 1. synthetic route to the title compounds. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1466 socea et al. the presence of a single conformational isomer (the axial one) of the triazoles 10–13 was confirmed by their 1h-nmr spectra. cyclization of 6–9 to 10– –13 and the subsequent reactions produced the loss of the minor equatorial isomer, probably due to an increased solubility in acidic water. h-5′(eq) appears at δ 4.35–4.45 ppm (triplet, j = 7.9 hz) and the ch2-12′ protons manifest as doublets at 2.67–2.93 ppm in 10–13. the nh signals of 6–9 totally disappeared, and were replaced by singlets at δ 11.44–11.90 ppm, attributable to the sh proton. thus, in solution, the above equilibrium was shifted towards the thiolic form. conversion of hydrazinecarbothioamides 6–9 to the triazoles 10–13 was also confirmed by the 13c-nmr spectra. a new quaternary carbon signal (for c-3) appeared at δ 166.57–167.97 ppm (scheme s-2 of the supplementary material) simultaneously with the disappearance of the c=s signal of 6–9 (δ = 181 ppm). furthermore, a new signal for c-5 of 10–13 appeared at δ ≈ 155.5 ppm, instead of the c=o signal from 6–9 at δ 169–170 ppm. a new band in 2929–2983 cm−1 region, due to presence of methyl group (νch3) in the ir spectra confirmed the structures of compounds 14–17, obtained by alkylation of the triazoles 10–13 with methyl iodide. proof of s-alkylation that led to the formation of compounds 14–17 was given by the disappearance of the c=s stretching band in the ir spectra. the presence of new signals at 14.8 ppm corresponding to ch3 group in the 13c-nmr spectra of compounds 14–17 was the most significant proof of alkylation of triazoles 10–13 with methyl iodide. heterocyclic carbons c-3 and c-5 from these methylated compounds resonated at 154.82–155.11 ppm (more shielded than the c-3 heterocyclic carbon from the 1,2,4-triazoles 10–13) and δ 151.28– –151.54 ppm, respectively. 1h-nmr spectra of the 3-(methylsulfonyl)-1,2,4-triazoles indicated the presence of a single conformational isomer, the axial one, except for triazole 15, which exists as two isomers, 5′-axial and 5′-equatorial in about 1:1 ratio. antimicrobial activity the antimicrobial activities of all products were investigated in vitro against s. aureus, p. aeruginosa, e. coli, b. subtilis, s. enterica subsp. enterica serovar typhimurium, s. flexneri and c. albicans by the dilution method. the mic values were determined using the dilution method with dimethyl sulfoxide as solvent. dimethyl sulfoxide showed no antimicrobial activity against the tested strains. the mic values (μg ml–1) for the new compounds against the strains are presented in table i. the investigation of the antimicrobial activity of the compounds was performed in duplicate. as control, s. aureus, e. coli and p. aeruginosa were tested against gentamicin, and c. albicans against fluconazole by the broth micro_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ new hydrazinecarbothioamides and 1,2,4-triazoles 1467 dilution method.44,45,49–51 the mic value of gentamicin was 2 μg ml–1 for all tested strains and the mic value of fluconazole was 2 μg ml–1 for the reference strain. table i. in vitro antimicrobial activity of compounds 6–17 as mic values (μg ml-1) compd. bacterial strains yeast s. aureus p. aeruginosa e. coli b. subtilis s. enterica subsp. enterica serovar typhimurium s. flexneri c. albicans 6 256 16 512 >1024 256 1024 >1024 7 512 256 >1024 >1024 256 256 >1024 8 >1024 1024 512 >1024 1024 512 >1024 9 512 256 512 >1024 512 512 >1024 10 512 >1024 64 128 64 512 >1024 11 256 128 512 128 >1024 1024 >1024 12 256 512 512 128 >1024 512 >1024 13 512 64 >1024 >1024 512 512 >1024 14 256 128 512 128 >1024 512 128 15 256 128 512 128 512 512 128 16 256 256 512 256 128 512 128 17 512 512 512 256 256 512 256 gentamicin 2 2 2 – – – – fluconazole – – – – – – 2 the antimicrobial screening data revealed that all the newly synthesized compounds exhibited weaker antimicrobial activities compared to those of the control drugs. for the reference bacterial strains, the mic values of the compounds ranged between: 16–1024 μg ml–1 for the hydrazinecarbothoamides 6–9, 64–1024 μg ml–1 for the 1,2,4-triazole-3-thiols 10–13 and 128–512 μg ml–1 for the methylsulfamyl-1,2,4-triazoles 14–17. hydrazinecarbothioamide 6 with a chlorine atom presented the strongest action against p. aeruginosa (mic 16 μg ml–1). 1,2,4-triazole-3-thiol 13 with a 4-(2-phenylethyl) fragment presented the strongest action against p. aeruginosa (mic 64 μg ml–1). comparing the results of this study, it was observed: a) compounds containing 4-chlorophenyl group had moderate antibacterial activity, hydrazinecarbothioamides against s. aureus, s. enterica subsp. enterica serovar typhimurium and p. aeruginosa, 1,2,4-triazole-3-thiol against e. coli and s. enterica subsp. enterica serovar typhimurium, and methylsulfanyl-1,2,4-triazole against p. aeruginosa; b) hydrazinecarbothioamides containing 4-chlorophenyl or 4-bromophenyl had better action compared to derivatives containing 4-iodophenyl or 4-(2-phenylethyl); c) the presence of methylsulfanyl-1,2,4-triazole ring in the structures 14–17 were favorable for the activity against the bacterial strains; d) _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1468 socea et al. hydrazinecarbothioamides 6–9 and 1,2,4-triazole-3-thiols 10–13 were almost inactive against c. albicans but methylsulfanyl-1,2,4triazole showed moderate activity against fungus strain. conclusions in this paper, the synthesis and characterization of four new acyl hydrazinecarbothioamides, four 4h-1,2,4-triazole-3-thiol derivatives and four methylsulfanyl-1,2,4-triazoles containing the 5h-dibenzo[a,d]annulene moiety were presented. the structures of new compounds were confirmed by spectral data (ir, uv, 1h-nmr and 13c-nmr) and elemental analysis. all the compounds were investigated for their antimicrobial activity against s. aureus, p. aeruginosa, e. coli, b. subtilis, s. enterica subsp. enterica serovar typhimurium, s. flexneri and c. albicans. the antibacterial screening data are given for all the tested compounds. the data indicated weak antibacterial activity, except for compound 6 (which presented good action against p. aeruginosa), 10 (which presented moderate action on s. enterica subsp. enterica serovar typhimurium and e. coli) and 13 (which presented a moderate action on p. aeruginosa). based on the mic values presented by the tested compounds, it could be concluded that, in general, the derivatives containing a chlorine or bromine atom had better antibacterial activity against the tested strains. supplementary material physical, analytical and spectral data for the synthesized compounds are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. this work was supported by the university of medicine and pharmacy “carol davila” bucharest, project no. 28331/04.11.2013. и з в о д синтеза нових деривата хидразинкарботиоамида и 1,2,4-триазола и њихова антимикробна активност laura i. socea1, gabriel saramet2, constantin draghici3, bogdan socea4, vlad d. constantin4 и manuela a. radu-popescu5 1 organic chemistry department, faculty of pharmacy, „carol davila“ university of medicine and pharmacy, 6 traian vuia street, 020956, bucharest, romania, 2 pharmaceutical technology department, faculty of pharmacy, „carol davila“ university of medicine and pharmacy, 6 traian vuia, 020956 bucharest, romania, 3 “c.d. nenitzescu” institute of organic chemistry, romanian academy, 202b splaiul independentei, 060023 bucharest, romania, 4 faculty of general medicine, „carol davila“ university of medicine and pharmacy, st. pantelimon emergency hospital, 340–342, soseaua pantelimon street, 021623, bucharest, romania и 5 general and pharmaceutical microbiology department, faculty of pharmacy, „carol davila“ university of medicine and pharmacy, 6 traian vuia street, 020956, bucharest, romania синтетисана је серија деривата хидразинкарботиоамида 6–9 који садрже 5h-дибензо[a,d][7]ануленски део. циклизацијa деривата 6–9 у раствору naoh даје одговарајуће аксијалне изомере 4h-1,2,4-триазол-3-тиола 10–13. тиоетри 14–17 су добијени алкиловањем деривата 10–13 јодметаном. сва нова једињења окарактерисана су елементалном _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ new hydrazinecarbothioamides and 1,2,4-triazoles 1469 анализом, ir, uv, 1h-nmr и 13c-nmr спектроскопијом. извршено је испитивање антимикробне активности према staphylococcus aureus atcc 25923, pseudomonas aeruginosa atcc 27853, escherichia coli atcc 25922, bacillus subtilis atcc 6633, salmonella enterica subsp. enterica serovar typhimurium atcc 14028, shigella flexneri atcc 12022 и candida albicans atcc 90028. 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(bucharest, rom.) 57 (2006) 1123 48. clinical and laboratory standards institute., clsi document m27-a3, approved standard, 3rd ed., clinical and laboratory standards institute, wayne, pa, 2008 49. clinical and laboratory standards institute. clsi document m27-s4, approved standard, 4th informational supplement, clinical and laboratory standards institute, wayne, pa, 2012 50. subcommittee on antifungal susceptibility testing (eucast-afst) of the escmid european committee for antimicrobial susceptibility testing (eucast-afst) 2008, eucast definitive document e def 7.1, clin. microbiol. infect. 14 (2008) 398. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice secondary-school chemistry textbooks in the 19th century j. serb. chem. soc. 80 (10) 1321–1338 (2015) udc 54(075.3)’’18’’:37(497.11) jscs–4800 original scientific paper 1321 secondary-school chemistry textbooks in the 19th century vesna d. milanovic1#, dragica d. trivic2*# and biljana i. tomasevic2# 1innovation centre of the faculty of chemistry, studentski trg 12–16, belgrade, serbia and 2university of belgrade, faculty of chemistry, studentski trg 12–16, belgrade, serbia (received 26 september 2014, revised 30 may, accepted 8 june 2015) abstract: the teaching of chemistry in serbia as a separate subject dates from 1874. the first secondary-school chemistry textbooks appeared in the second half of the 19th century. the aim of this study was to gain insight, by analysing two secondary-school chemistry textbooks, written by sima lozanić (1895) and mita petrović (1892), into the amount of scientific knowledge from the sphere of chemistry was presented to secondary school students in serbia in the second half of the 19th century, and the principles textbooks written at the time were based on. within the framework of the conducted research, we defined the criteria for assessing the quality of secondary-school chemistry textbooks were defined in the context of the time they were written. the most important difference found between the two textbooks under analysis pertained to the way in which their contents were organised. sima lozanić’s textbook is characterised by a greater degree of systematicness when it comes to the manner of presenting its contents and consistency of approach throughout the book. in both textbooks, the authors’ attempts to link chemistry-related subjects to everyday life, and to indicate the practical significance of various substances and their toxicity can be perceived. keywords: chemistry teaching; chemistry textbook; structural components of textbooks; textbook quality. introduction textbooks are representative samples of the time in which they were written, for they reflect the trends and principles that existed in a particular area of education.1 in a way, the books are built into the generations of students after which they were taught, for it is through them that a certain way of thinking, a strategy of learning, general intellectual skills and habits, ones attitude towards that which is being learnt, towards science and knowledge in general are cultivated.2 a review and analysis of various chemistry textbooks from their beginnings to the * corresponding author. e-mail: dtrivic@chem.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc140926052m _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1322 milanovic, trivic and tomasevic present day affords insight into the development of ideas about chemistry and the intellectual achievements of many generations of students in the realm of this science.3 a textbook reflects the characteristics of the context that it was created in, as well as the academic knowledge and beliefs of its author(s), reflecting their views of what science is or what it should be.4 it may be viewed as a conversation between chemistry as a scientific discipline, the context for teaching/learning, the author’s personal characteristics and the burden that each society imposes upon one.5 textbook writers are confined to the margins of scientific communities, they share various cultures and they are under strong social, economic, and political pressures.6 moreover, textbooks are read and used by a great variety of audiences with different aims, expectations and reading practices. scientific textbooks are, therefore, at the crossroad between disciplines such as the of science, the history of education and the history of books and reading.7 by analysing textbooks that were used in the past, one can form a picture of the quality of teaching at that time can be formed. insight can be gained into the relationship between science and education, into how fast scientific knowledge was built, into the process of education and the extent to which they represented a response to the needs of society at the time. it could be said that textbooks are sorts of archaeological traces of former regimes of knowledge.6 textbooks can be viewed as focal points for many of the historical contingencies that shape not only scientific practice, but also the roles of science and scientists in society. although most easily treated as part of the history of the book, they also carry historical significances that transcend that genre.8 textbooks turned into an independent and characteristic genre of scientific publication in the 19th century when science education became compulsory in a number of european universities as well as in primary and secondary education. a reason for the rise of the textbook was its instrumentality in the development of the national structures of education, in particular, the nineteenth-century implementation of secondary education.9 the teaching of chemistry as an independent subject in secondary schools in serbia dates back to 1874.10 prior to that, chemistry was studied within the framework of physics and mineralogy. it was taught in the fourth grade, four classes per week. in the guidelines sent to teachers in 1874, it is stated that inorganic and organic chemistry with experiments should be taught. of the four classes per week, one had to be dedicated to conducting experiments, which is indicative of the significance attached to experimental work in the course of teaching chemistry. by analysing two secondary-school chemistry textbooks written by sima lozanić (1895) and mita petrović (1892), the aim of this study was to gain insight into the amount of scientific knowledge from the sphere of chemistry _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ chemistry textbooks in the 19th century 1323 presented to young people in serbia in the second half of the 19th century, and into principles on which textbooks written at the time were based. sima lozanić (1847–1935) was a chemist, scientist, professor, chairman of the academy of sciences, the first rector of belgrade university, ambassador to london, minister of the economy and minister of foreign affairs, a diplomat. in the mid-1880’s, at the time of a reform and modernisation of grammar schools, lozanić worked on compiling a modern chemistry curriculum and introducing teaching through experiments in secondary schools.11 apart from chemistry, sima lozanić also studied pedagogy (1868–1870) at the well-known school of pedagogy in küsnacht near zurich. at zurich university, lozanić studied chemistry under johannes wislicenus (1835–1902), and subsequently spent one year at the august wilhelm von hofmann’s (1818–1892) laboratory for organic chemistry.12 for many years, mita petrović (1848–1891) worked at the serbian teachertraining school in sombor, where he taught mathematics and natural sciences. he had organized and equipped a chemical laboratory in sombor where he did research in the sphere of natural sciences, especially physics and chemistry. he also wrote a large number of textbooks on all subjects he taught. these were mainly based on contemporary german language textbooks which petrović translated and adjusted to the level and needs of his students. he was a correspondent member of the serbian academy of science, and member of numerous other learned societies. mita petrović received many accolades for his work.13 the rules on writing secondary-school textbooks in serbia were passed in 1895. a commission was formed the task of which was to establish whether suitable textbooks existed for all the subjects taught at school. in march 1895, the commission informed the board of education that there was no suitable chemistry textbook for the fourth grade of grammar school. within one year, lozanić wrote a textbook and submitted it for publication in march 1896 (although on the cover 1895 remained as the year of publication). in march 1896, lozanić sent the textbook to the minster of education and to the board of education for review. the reviewer marko leko submitted a negative review, containing 36 objections, of the textbook to the board.14 the board of education, which was composed of secondary school teachers who opposed lozanić’s terminology, decided not to accept lozanić’s book as an official textbook. it was in vain that lozanić tried to respond to the criticisms addressed to him.15–19 through the board of education, the polemic between him and leko continued throughout 1897, until the ed ucational gazette announced that the editorial board would accept no further correspondence pertaining to the said polemic. lozanić’s textbook was only recommended for use in secondary schools at the beginning of the 20th century. until then, mita petrović’s textbook was used.11 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1324 milanovic, trivic and tomasevic the methodology of the research the textbook sample analysed within the framework of this research was the first edition of sima lozanić’s textbook chemistry for secondary schools, dating from 1895, and the third edition of mita petrović’s textbook chemistry for s econdary schools, base d on proko p prohaszka and others, dating from 1892. sima lozanić’s textbook had a total of five editions (1895, 1897, 1903, 1910, and 1925). for the purpose of this analysis, the first edition was chosen in view of the above-mentioned criticism, which reflects the context in which the textbook was created. mita petrović’s textbook, despite the fact that it was not in accordance with the curriculum dating from 1881, was used in schools with the permission of the ministry of education until the beginning of the 20th century, even after the publication of sima lozanić’s textbook.12 the first edition of this textbook came out in 1883, but herein, the third edition, dating from 1892, was analysed as the preceding two editions were not available. in order to achieve the set goal, first a methodology for analysing and evaluating the quality of these textbooks within the context of the period when they were created required development. some characteristics of a textbook can be quantified, whereas others require a qualitative analysis. the main purpose of the qualitative approach is to understand and interpret various meanings that the textbook being analysed carries, following which the elements of the meanings are established.20 the present analysis of the selected secondary-school chemistry textbooks dating from the 19th century was supposed to provide answers to the following questions: 1. what contents were presented in the chemistry textbooks from the second half of the 19th century? 2. to what extent were the contents presented in the examined textbooks in keeping with the then current level of knowledge in chemistry? 3. what experiments are presented and described in the textbooks? 4. to what extent did the textbooks analysed establish a connection between the textbook contents and everyday life? 5. to what extent does the textbook caution the students to take care when dealing with certain substances? 6. to what extent were the contents of the textbooks in keeping with the chemistry curriculum then in effect? 7. to what extent did the textbooks deal with events from the history of chemistry? 8. how were the analysed textbooks supported in terms of graphic design and illustrations? 9. what were the structural and organisational components of the analysed textbooks? 10. how were the contents of the textbooks shaped in terms of didactics? the chemistry-related contents of the analysed textbooks were followed through the following: the themes, the index, the symbols and names of the elements mentioned in the textbook, the formulas of chemical compounds and their names, the equations depicting particular chemical reactions. with the exception of the themes reviewed, all the other parameters monitored within the framework of this part of the research were quantified. the degree to which the contents of the analysed textbooks were in keeping with the current level of knowledge in chemistry was assessed based the presence of current discoveries in the sphere of chemistry in the text (for example, the discovery of the periodical system of the elements, the arrhenius theory of acids and bases, etc.). _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ chemistry textbooks in the 19th century 1325 the extent to which the students’ understanding of chemistry was supported through experiments was also monitored. the experiments described in chemistry textbooks brings the question whether laboratory techniques can be appropriated by students when reading textbooks and provide information about the teaching practices in the context of the local resources.6 the possibilities for developing meanings on the basis of the textbook (the meaningfulness of the textbook material) and insight into the practical importance of the knowledge of chemistry were monitored on the basis of the existing links between the chemistry-related contents and their application in everyday life, and also on the basis of indicating the toxicity of certain substances, their influence on human health and precautions to be taken when dealing with such substances. the contents of the analysed textbooks were compared with the chemistry curriculum from 1881, the latter being closest in time to their publication dates. on the basis of the presence of contents related to the history of chemistry, we reviewed the extent to which the textbooks supported the development of an appropriate idea of this science in the students’ minds.21,22 furthermore, we monitored the ways in which the analysed textbooks were supported in terms of illustrations and graphic design and what kind of information was supplied through the illustrations, i.e., what was their purpose, were monitored. one of the ways of checking the extent to which textbooks achieve their aims is to evaluate the questions that they contain.23 in the textbooks analysed, the thought processes that they initiated, i.e., the form of learning they initiated, were evaluated. the structural and organisational components that were monitored within the framework of this analysis are given in table s-i of the supplementary material to this paper. the content of a textbook should be didactically shaped in such a way that it ensures the students’ minds are activated, that the basic structure of knowledge is accepted by those for whom the textbook is intended.20 the indicators of didactic organisation that were followed in the analysed textbooks were as follows: i) the existence of scientific terms, ii) the functional use of pictorial means of expression, iii) the diversity of the examples provided, iv) the meaningfulness of the organisation of the textbook contents and v) whether the textbook material has any connection with everyday life out of school. whether the language of the textbook in question was compatible with the current language of the science of chemistry at the time when the textbooks analysed were written was also monitored during the analysis of the textbooks. results and discussion from the introductory statements of both textbooks, their intentions and views on how the study of chemistry should be organised could be seen. in the preface to the first edition of his textbook, sima lozanić pointed out that students found it easier to understand the formulation of the laws of chemistry based on experiments conducted in class. he was of the opinion that it was easier for beginners to learn about general terms in the sphere of chemistry in a separate section of the book and that by doing so enabled the presentation of this part in a systematic manner, thus forming a basis for understanding inorganic and organic chemistry. the method of “placing the general part within the framework of a special part”, which was included in the curriculum, could lead to learning without real understanding. in his introductory statement, sima lozanić explained that his approach to organising the contents of organic chemistry was _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1326 milanovic, trivic and tomasevic through homologous series. he stressed that he paid greater attention to applied chemistry and to interpreting chemical phenomena in nature, for he was of the opinion that in this way “young men were not only enriched through knowledge but also developed a greater interest in science”. at the beginning of the third edition of mita petrović’s textbook chemistry for secondar y schools, b ased on pro kop prohasz ka and othe rs, dating from 1892, the preface to the first edition of this textbook, dating from 1883, is included. in this preface, mita petrović states that the presentation of the contents of chemistry, the body of knowledge of which has greatly increased, in a manner suitable for beginners to this area, while at the same time including its development as a science, is a problem. he indicated that the organisation of the textbook material was such that the theoretical explanations provided by chemistry were not given in succession, but alternated with experimental parts, and were placed so that the students should be able to understand them when they reached them. in his opening statement, the author pointed out that, when deciding on the scope of the textbook material, he was guided by the curriculum prescribed for grammar schools in the kingdom of serbia. the quantitative data on the analysed textbooks are presented in table s-ii of the supplementary material. the scope of the textbook material in the analysed textbooks differs: sima lozanić’s textbook has 163 pages, whereas mita petrović’s textbook has 110 and is smaller in format. in sima lozanić’s textbook, the table of chemical elements, containing their symbols, names and atomic mass, comprises 68 elements, of the 70 that were known at the time. the table presented in mita petrović’s textbook contains such data for 36 elements. in sima lozanić’s textbook, there are a greater number of chemical formulas. those are, for the most part, molecular formulas, whereas the number of structural formulas is small in both textbooks. both textbooks comprise contents related to general, inorganic and organic chemistry. in sima lozanić’s textbook, the contents are organised into three sections: general chemistry, inorganic chemistry and organic chemistry. following a brief opening part, explaining several fundamental terms in the sphere of chemistry, the contents of mita petrović’s textbook are organised into two sections: inorganic chemistry and organic chemistry. these two textbooks differ in how their contents are organised. sima lozanić’s textbook first explains the basic concepts, principles and laws in the sphere of chemistry, and only then proceeds to consider inorganic chemistry through the families of elements, and organic chemistry through the classes of compounds and homologous series. in mita petrović’s textbook, the material related to inorganic chemistry, within the framework of which chemical elements are considered individually, is interspersed with segments of material in which general chemical _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ chemistry textbooks in the 19th century 1327 principles and laws are reviewed (which is referred to as “placing the general part within the framework of a special part”). in the general section of sima lozanić’s textbook, the fundamental concepts and laws in the sphere of chemistry, the division of elements into metalloids and metals, and the nomenclature of chemical compounds are explained. the following fundamental chemical laws are discussed: the law of constant mass ratio, the law of multiple proportions, the law of conservation of mass, and avogadro’s law. the material is conceived in such a way that the corresponding experiments, or the results of experiments that preceded the discovery of the law in question are first presented, followed by the formulation of the law. at the end of the general section, the author explains how the remainder of the textbook is organised, announcing that the most important elements and compounds are discussed, that carbon compounds are especially emphasised because they are so numerous, and thus, that the entire sphere of chemistry is divided into two parts: inorganic chemistry, within the framework of which elements and their compounds are presented, and organic chemistry, within the framework of which carbon compounds are studied. as opposed to the general section of sima lozanić’s textbook, which contains 28 subtitles, in the opening section of mita petrović’s textbook, there are four subtitles. in the section entitled things change, various kinds of physical and chemical changes known from everyday life are presented (changes in the aggregation state of water, sliding a bow across a string, charging a resin bar with electricity, sliding a magnet across steel, the dissolution of sugar in water, the burning of wood and the fermentation of grapes). in the section entitled transient and essential changes, differences between the types of changes are presented, which is followed by their classification into two groups and finally their definition as physical and chemical changes. in the section entitled the directions of chemical changes, some experiments are presented, following which the meanings of the terms analysis and synthesis are defined. the last section in the introductory part of the textbook is entitled the difference between a mixture and a compound, in which the difference between the two is defined. in the section of sima lozanić’s textbook that deals with inorganic chemistry, the chemical elements are presented based on the family of elements to which they belong, they are considered in the following order: the hydrogen family (н), the halogen family (f, cl, br and i), the oxygen family (o, s, se and te), the nitrogen family (n, p, as, sb, bi, b, v, nb and ta), the carbon family (c, si, ge, sn, ti, zr and th), the alkali family (li, na, k, rb and cs), the earth alkali family (ca, sr and ba), the magnesium family (be, mg, zn and cd), the mercury family (hg, cu, ag and au), the lead family (pb), the aluminium family (al, ga, in, tl, ce, la, di, sm, y, er, yb and sc), the iron family (fe, co, ni, cr and mn), the platinum family (pt, pd, ir, rh, ru and os) and the molybdenum _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1328 milanovic, trivic and tomasevic family (mo, w and ur). a consistent manner of presentation was used for each of the above families of chemical elements. first the name of the family is given, followed by an enumeration of all elements belonging to it, together with their valences, and then the elements are studied individually. the organisation of the presentation of material when considering each individual element is as follows: after the section title, which contains the name of the element in serbian and, in most cases, in latin, its atomic mass, as well as molecular mass if the element occurs in nature in molecular form, the author proceeds to speak of the forms in which it occurs in nature, both in its elementary state and as part of compounds, then of the ways in which it can be obtained, its physical and chemical properties, and finally of its use, that is, its practical application. the physical properties include the state of aggregation, colour, taste, solubility in water and less polar solvents, for example, alcohol and cs2. the chemical properties include the reactivity of the given element with some other element or compound. concerning the use of elements, first their use in everyday life is presented, be it in their elementary state or in compounds. this order of presentation of the material is retained when considering the important compounds of the elements previously presented. in the case of compounds where characteristics analogous to those of previously presented compounds featuring elements of the same family can be observed, the similarity in their physical and chemical characteristics are emphasised. at the beginning of the presentation of the majority of the elements, the year when a particular element was discovered and who discovered it are briefly mentioned. in the section dealing with nitrogen, a three-atom molecule of nitrogen, n3, referred to as argon, as an allotropic modification of nitrogen, analogous with oxygen and ozone, was mentioned (which was deleted from the next edition of the textbook, published in 1897). the mention of n3 as an allotropic modification of nitrogen was criticised by marko leko, and constitutes one of his objections contributing to his negative review of the textbook. a lot of attention is paid to ores and minerals that contain certain elements and to where they can be found, and the most important mines are enumerated, both in serbia and outside its boundaries at the time. this points to the significance of mineralogy and to the importance that was attached to it in the sphere of education in the 19th century. as was already stated, the contents of the inorganic chemistry section of the textbook chemistry for secondary schools, based on prokop prohaszka and others are organised based on the principle of “placing the general part within the framework of a special part”, which was applied in the chemistry curriculum dating from 1881. this textbook contains separate sections for studying metals and non-metals. the section dealing with non-metals includes a lot of subjects from the sphere of general chemistry, alternating with segments wherein the characteristics of individual elements and their compounds are considered. the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ chemistry textbooks in the 19th century 1329 textbook section entitled metals deals with individual metals and their characteristics. the presentation of the contents related to inorganic chemistry begins with the section entitled on air . at the very beginning of this section, the following experiment is described – the heating of tin inside a closed vessel, along with measuring the mass of the solid substances before and after the reaction, identical to the one on which lavoisier (antoine laurent lavoisier, 1743–1794) established the law of conservation of mass. however, lavoisier’s work is not mentioned here, nor is the law that he discovered; the latter is subsequently explicitly stated within the framework of the section entitled the quantitative ratios of compounds. there follows a description of experiments for obtaining nitrogen and oxygen. after this, the characteristics of oxygen and nitrogen are discussed individually, primarily the physical ones, and when considering the characteristics of oxygen, the process of oxidation and the creation of oxides are explained. the next section is entitled on water , and it considers hydrogen. the author expounds on the dilemma of whether hydrogen should be classified among non-metals or metals. there follows a general part again in which the following are defined: chemical affinity, atoms and molecules. after neutralisation, there is a detailed presentation of chlorine and sulphur (the way they appear in nature, how they are obtained, their properties, use, etc.). within the framework of the section entitled the quantitative ratios of compounds , for the first time an equation of a chemical reaction is presented, namely, the one depicting the creation of water, wherein the reactants are presented in their atomic form. within the next section, entitled the i mportant co mpounds of t he elements considered so f ar, the following compounds are presented: hcl, h2s, hno3 (ho·no2), no2, no, h2so4 and (ho)2so4. described is how they occur in nature, how they are obtained, their properties and their use. the next element to be considered is carbon, its allotropic modifications, and the types of coal. particular attention is paid to the sites of coal deposits and the sources of mineral water in serbia. together with carbon, its compounds are considered, which was not the case for the preceding elements. valence, as a topic belonging to the general section, is introduced after carbon. there follows a section entitled on flame and burning , wherein the last considered non-metal, phosphorus is introduced. at the beginning of the section dealing with the study of metals, it is stated that metals are divided into light and heavy ones. the light metals are: k, na, ca, mg, ba, sr and al. the others are heavy metals. what is presented next is the division of metals based on valence follows: one-valence metals (k, na and ag), two-valence metals (ba, sr, ca, mg, zn, cd, pb, cu and hg), three-valence metals (bi and au), four-valence metals (al, mn, fe, co, ni and cr). it is pointed out that some metals, as well as some non-metals, have more than one valence, that they form two lines of compounds, but that it is still not known _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1330 milanovic, trivic and tomasevic what valence depends on and how it changes, except that it often occurs at high temperatures. subsequently, the important metals and metal compounds are studied individually, and it should be noted that the “affinity” of certain metals is stressed. the section of mita petrović’s textbook dealing with inorganic chemistry ends with an explanation of the spectral analysis method and a description of the main parts of the spectroscope. as opposed to mita petrović’s textbook, spectral analysis is not dealt with in sima lozanić’s textbook, even though in the general section of the textbook, the author did mention the importance of this method and said that he would discuss it later. at the beginning of the section dealing with organic chemistry, sima lozanić states that organic chemistry studies carbon compounds, whether those in the “kingdom of life” or those artificially produced in laboratories. then he presents a classification of organic compounds based on their composition: hydrocarbons, halogen derivatives of hydrocarbons, alcohols, aldehydes, acids, carbohydrates, nitro compounds, amines, amides, nitriles, unstudied compounds (terpenes, camphors, resins and caoutchouc, natural colours and the like, alkaloids, proteins). when speaking of a class of compounds, it is stated whether a particular compound exists in nature, whether it is extracted from certain plants or animals, and how this is realised, whether such compounds exist in the human body and what sort of function they perform. along with homologous series of carbohydrates, the text presents the boiling and melting temperatures of various elements of the series, on the basis of which conclusions may be draw about their state of aggregation under the same conditions. the importance that was attached to fuel is evident from the amount of space dedicated to oil, the way it is obtained, its refining and various fractions. when considering proteins, the molecular formulas of albumin, haematin and haemoglobin are presented (c72h112n18so22, c68h70n8fe2o10 and c600h960n154fes3o17), but it is stressed that these are their presumed formulas, and that the exact composition of these compounds is still unknown, except that they are characterised by a high molecular mass. the ensuing section contains detailed descriptions of various types of fermentation that contribute too many important processes unfolding in nature, and to processes for producing various compounds. within the framework of the section entitled the processes inside an animal organism , there is a description of the composition of blood and urine, of the process of breathing and digestion. this chapter also contains advice for healthy eating and presents information on the daily needs of the human organism for various kinds of food in order to function properly. in mita petrović’s textbook, the first section dealing with organic chemistry, entitled the ingredients of organic compounds , contains an explanation of the elements that are part of the composition of organic compounds, while organic chemistry is determined in the following way: “if we heat wood, a feather, paper, _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ chemistry textbooks in the 19th century 1331 cotton, egg-white, etc. in a glass tube which is heated at one end, after a while they will all turn black and turn into coal. all organic matter contains carbon without exception. an object that does not contain carbon is not organic. we can therefore say that organic chemistry is the chemistry of carbon compounds.” paraffins are considered first, of which methane is presented in some detail, as are its halogen derivatives, along with four equations depicting chemical reactions for its gradual chlorination. after that, cyan compounds, alcohols, ethers, aldehydes and acids are described. not much attention is dedicated to aldehydes, while acids are considered individually and in more detail within the framework of two sections. regarding carbohydrates, the following are described individually: plant fibre (cellulose), starch, dextrin, grape sugar, cane sugar (sucrose) and milk sugar. their occurrence in nature and the method of their extraction from natural products are described, and an experimental method of distinguishing between reducing and non-reducing sugars (fehling’s test) is presented. within the section entitled alcohol fermentation, the processes of producing wine, beer and various kinds of brandy are described in detail. a significant amount of attention is dedicated to tannin, in order to indicate its use for tanning leather, making ink, etc. subsequently, aromatic compounds are considered, i.e., how to obtain benzene and its nitro and amino derivatives, and their characteristics. several alkaloids are enumerated, and their physiological effect on man is indicated. the penultimate section is dedicated to natural colours, and the final one to proteins. sima lozanić’s textbook contains the descriptions of 13 experiments, while that of mita petrović contains 29 descriptions. a list of experiments featured in the textbooks is given in table s-iii of the supplementary material. some of the experiments are featured in both textbooks. the experiments are not individually marked and separated from the main text. from the manner of the presentation of the material, it could be concluded that the authors intended them for demonstration in class, and having presented them, both authors subsequently referred to the experiments already presented when considering new material. all the experiments featured in sima lozanić’s textbook pertain to the contents related to general and inorganic chemistry, whereas in mita petrović’s textbook, two of the experiments belong to the sphere of organic chemistry. in both textbooks, one can observe the connection between the textbook material and everyday life outside school, be it when it comes to the practical application of certain elements and compounds, or the occurrence and function of organic molecules in the human organism, plants or animals. there are 158 such examples in sima lozanić’s textbook and 174 examples in mita petrović’s textbook. in both textbooks, apart from pointing out the practical use of various substances, the authors stress that certain substances have a toxic effect on human _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1332 milanovic, trivic and tomasevic health. these include not only substances that can be found in a chemical laboratory, but also substances that are encountered in everyday life, or those that easily occur as a result of processes unfolding in the surroundings (such as carbon monoxide gas). in sima lozanić’s textbook, there are 14 examples testifying to the toxicity of certain substances and their harmful effects on humans, although, there is no direct reference to precautions that should be taken or firstaid measures to be applied if any accident should happen, which is a shortcoming of this textbook. in mita petrović’s textbook, there are 34 such examples, and in some cases the author points to precautions that should be taken when handling such substances, as well as first-aid measures to be applied. sima lozanić’s textbook contains 43 illustrations, whereas that of mita petrović contains 10. all the illustrations featured in both the analysed textbooks follow the main text and contribute to a better understanding of chemical concepts and processes. the illustrations in both textbooks are numerically marked, but contain no captions explaining their contents. the main text contains references pointing to a particular illustration through its number. in sima lozanić’s textbook, the illustrations are used to present apparatuses (ten illustrations), experiments (two illustrations), industrial plants or their segments (nine illustrations), various types of furnaces (eight illustrations), crystallographic structures (two illustrations), structures seen under a microscope (two illustrations), certain processes or parts of them (seven illustrations), chemical vessels and equipment (three illustrations). the first four illustrations are featured in the general section of the textbook, 36 illustrations are to be found within the framework of the inorganic chemistry section, whereas only three illustrations are included in the section of the textbook dealing with organic chemistry. in mita petrović’s textbook, the illustrations are used to present apparatuses (two illustrations), experiments (four illustrations), processes (two illustrations), laboratory vessels (one illustration) and instruments (one illustration). all the illustrations are included in the textbook section dedicated to inorganic chemistry, specifically, in the part dedicated to studying non-metals. contents pertaining to the history of chemistry, mention of essential discoveries in the sphere of chemistry and names of great chemists are to be found in sima lozanić’s textbook, 27 cases in all, whereas in mita petrović’s textbook, there are only two references to the historical development of chemistry. in his textbook, sima lozanić points to turning points in the development of the science of chemistry (disproving the phlogiston theory, disproving the vitalist theory by means of the wöhler synthesis of urea), and also to the development of certain production processes that have a broad scope of practical usefulness (the production of ceramic dishes, the production of glass), from time immemorial to the period when the textbook was written, based on which conclusions could be _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ chemistry textbooks in the 19th century 1333 draw about the development of science through history and its beneficial effect on mankind. concerning the monitored structural components in the analysed textbooks, both of them contain: the main text, illustrations and questions. apart from the three components, mita petrović’s textbook also contains instructions and references to other parts of the text. concerning the organisational components, the following were observed in both textbooks: a table of contents, an introductory explanation of the textbook structure as part of the preface, an index of terms arranged in alphabetical order, tables of chemical elements. in mita petrović’s textbook, there is a brief note on the author, consisting of a few sentences included on the title page. neither textbook contained a separate list of the literature used in compiling the books, but in their prefaces both authors refer to the authors on whose books they had relied when writing their textbooks. sima lozanić states that when writing the present textbook, he had relied on his own textbooks of inorganic and organic chemistry that he had previously written for the high schools, while mita petrović explains in the introductory section that his role model for writing the present textbook was the book written by prokop prohaszka, professor of the czech high school in prague, entitled chemie učebna kniha pro čtvrtu tridu škol realnih, založena na pokuseh , and that he also relied on sima lozanić’s inorganic and organic chemistry textbooks for the high schools. indicators of the didactic organisation of a textbook, such as explanations of scientific terms, a functional use of illustrative means of expression and variety of the examples used are for the most part similar in these two textbooks. in both of them, when a term is mentioned for the first time, it is printed in bold letters, and immediately next to it there is an explanation. in both textbooks, definitions of a certain concept, process or phenomenon are supported by diverse specific examples, before or after the formulation of the definition. in the general section of sima lozanić’s textbook, there are five questions, followed by answers, whereas in mita petrović’s textbook, in the course of studying inorganic chemistry, 12 questions are posed, but no specific answers to them are provided. the questions and instructions found in both textbooks analysed are presented in table s-iv of the supplementary material. in sima lozanić’s textbook, a uniform manner of presenting the material contained in the textbook is consistently applied almost throughout the book (the only exception being the section entitled unstudied compounds , which encompasses terpenes, camphors, resins and caoutchouc, natural colours, alkaloids and proteins), which is not the case with mita petrović’s textbook. the contents of both textbooks to a large degree cover the chemistry curriculum of 1881. both authors observed the recommendation from the curriculum on what should be taught about an element, i.e., how it is obtained, its physical _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1334 milanovic, trivic and tomasevic and chemical properties, how it occurs in nature, its use. even though sima lozanić’s textbook covers the contents relating to inorganic chemistry prescribed by the curriculum, there is a difference between the textbook and the curriculum concerning the organisation of the contents. mita petrović’s textbook follows the organisation of the contents prescribed by the curriculum more closely (“placing the general part within the framework of a special part”). the essential difference between the 1881 curriculum and the organisation of the contents in both textbooks lies in the way organic compounds are systematised. the curriculum, which was actually based on sima lozanić’s textbook for the high school, systematised organic compounds according to the number of carbon atoms they contain, whereas in the textbooks, the compounds are systematised in accordance with homologous series. in sima lozanić’s textbook, much emphasis is placed on this particular way of systematising compounds, whereas in mita petrović’s textbook, the compounds are systematised in this way without defining the notion of homologous series. one characteristic of textbooks is the translations from the language in a scientific discipline into the local or national language7. a difference was observed concerning the terminology used between the curriculum and sima lozanić’s textbook, whereas the terminology used in mita petrović’s textbook is in keeping with the curriculum. in mita petrović’s textbook, the scientific terms used are adjusted to the serbian language, that is to say, they are “serbianised”. in sima lozanić’s textbook, the scientific terminology corresponds to the terminology that we still use today, with minor changes. the names of compounds in sima lozanić’s textbook are mainly in the nominative case (for example, potassium chloride), while in mita petrović’s textbook the names of compounds are given in the genitive case (for example, chloride of potassium).24 at the time, a discussion between scientists (not exclusively chemists) was ongoing – should science terminology be “serbienisied” or not. sima lozanić’s textbook also contains topics that go beyond the curriculum contents, mainly related to organic chemistry, whereas mita petrović’s textbook does not cover several topics from the sphere of inorganic chemistry, nor does it cover the topics contained in the curriculum supplement relating to: the chemical difference between plants and animals, breathing and life-giving heat, and the feeding of plants. furthermore, it contains no topic beyond those prescribed by the curriculum. in the major part of both textbooks, the knowledge available at the time is accurately presented, with the exception of the valences of some elements, the symbol of fluorine, the formulas of certain compounds and the equations of chemical reactions in mita petrović’s textbook, and the controversial allotropic modification of nitrogen in sima lozanić’s textbook. neither textbook mentions arrhenius’s theory of electrolytic dissociation or mendeleev’s periodical system _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ chemistry textbooks in the 19th century 1335 of the elements. arrhenius’s theory of electrolytic dissociation was posited in 1884, and the textbooks analysed were published in 1892 and 1895, respectively. the omittance of this theory may be explained by the fact that, for a number of years, arrhenius’s theory was under dispute among the scientific public. neither of the analysed textbooks mentions the periodical system of the elements that mendeleev published in 1869. this was not the case with the second edition of sima lozanić’s textbook, dating from 1897. it is surprising that sima lozanić did not include the periodical system of the elements in his secondary-school textbook, considering it was included in his previously published inorganic chemistry textbook for high schools. conclusions towards the end of the 19th century, general, inorganic and organic chemistry in secondary schools in serbia were studied based on the textbooks chemistry for secondary scho ols by sima lozanić and chemistry for secondary schools, based on prokop prohaszka and others by mita petrović. in these two textbooks, a difference in the organisation of the contents of the textbooks is apparent. the discussion was ongoing for years: should general chemistry be presented under inorganic chemistry or as a special section of textbooks. in the former, general chemistry, with the basic chemical concepts, principles, theories and laws, is presented in the opening section, thus forming a basis for inorganic and organic chemistry in the remainder of the textbook. in the latter textbook, the two largest sections are taken up by inorganic and organic chemistry and contents related to general chemistry are presented within the inorganic chemistry section, in those places that the author considered to be the most convenient for the students’ understanding of the said contents. based on the authors’ introductory statements, the impression is given that both approaches are the result of their thinking and assessment of the type of organisation of the textbook contents would contribute to the students’ better understanding chemistry. the latter author’s approach followed more closely the organisation of the then chemistry curriculum, but the former author’s approach has been retained in today’s chemistry curricula. it may be assumed that the said approach was developed during the course of sima lozanić’s studies under wislicenus and hofmann. however, the approach applied by sima lozanić was criticised by marko leko, the reviewer of the chemistry for secondary scho ols textbook. the only justification for criticising sima lozanić’s textbook, based on the present analysis, lies in the fact that he mentioned a controversial allotropic modification of nitrogen. it may be assumed that this arose out of a wish to make the textbook’s contents as up-to-date as possible, but in this case, the said scientific information was insufficiently verified. in the second edition of the textbook, sima lozanić had already left out the controversial allotropic modification of nitrogen. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1336 milanovic, trivic and tomasevic the analysed textbooks differ in terms of the scope of their contents, sima lozanić’s textbook being the more voluminous of the two. as regards the accuracy of the textbooks’ contents, several segments of mita petrović’s textbook were assessed to be inaccurate. both textbooks link theoretical contents with corresponding experiments, with the experiments presented in sima lozanić’s textbook being better suited to the lesson at hand, thus providing a better grounding for understanding the theoretical contents. both textbooks contain examples that stress the practical significance of chemistry and its connection with everyday life. also, in both textbooks the authors point out the toxicity of certain substances. sima lozanić’s textbook, compared to that of mita petrović, to a greater extent includes contents related to the history of chemistry which, at the time, could acquaint young people with the nature of science and scientific/research work, the essential turning points in science and the contribution of science to the development of society. concerning the structural components, both textbooks contain: the main text, with in-built descriptions of experiments and their results, illustrations and questions. apart from these components, mita petrović’s textbook also contains instructions and references to other parts of the text. with regards to the organisational components, both textbooks contain: a table of contents, an introductory explanation of the structure of the textbook within the framework of the preface, an alphabetically arranged index of terms and tables of the elements. in the final analysis, it could be concluded that sima lozanić’s textbook is of a better quality compared to that of mita petrović, not so much in terms of the choice of contents but in the way the contents are presented and the presentation order. supplementary material structural and organisational components and quantitative data of the textbooks analysed, as well as experiments described and questions and instructions found in them, are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. this paper represents the result of working on the project “scientific theory and practice in society: multidisciplinary, educational and inter-generational perspectives”, reg. no. 179048, the realisation of which was financed by the ministry of education, science and technological development of the republic of serbia. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ chemistry textbooks in the 19th century 1337 и з в о д уџбеници хемије за средњу школу у 19. веку весна д. милановић1, драгица д. тривић2 и биљана и. томашевић2 1иновациони центар хемијског факултета у београду, студентски трг 12–16, београд и 2универзитет у београду, хемијски факултет, студентски трг 12–16, београд настава хемије, као самостални предмет у србији, датира од 1874. године. први уџбеници хемије за средњу школу појавили су се у другој половини 19. века. циљ овог рада јесте да се кроз анализу два средњошколска уџбеника из хемије, аутора симе лозанића (1895) и мите петровића (1892), сагледа шта се од научних знања из хемије презентовало ученицима у средњим школама у другој половини 19. века и према којим принципима су тада писани уџбеници. у оквиру истраживања дефинисани су критеријуми за праћење квалитета средњошколских уџбеника хемије у контексту времена у коме су настали. најбитнија нађена разлика између два анализирана уџбеника односи се на организацију садржаја. уџбеник симе лозанића карактерише већа систематичност у излагању садржаја и доследност у приступу кроз цео уџбеник. у оба уџбеника видљива су настојања да се садржаји хемије повежу са свакодневним животим, као и да се поред практичног значаја супстанци укаже на њихову токсичност. (примљено 26. септембра 2014, ревидирано 30 маја, прихваћено 8. јуна 2015) references 1. j. c. bailar, j. chem. educ. 70 (1993) 695 2. j. pešić, one view on c ritical thinking a nd i ts appli cation i n the de sign of textboo ks, university of belgrade, faculty of philosophy, belgrade, 2006 (in serbian) 3. s. d. tarbell, a. t. tarbell, j. chem. educ. 54 (1977) 266 4. r. seligardi, sci. educ. 15 (2006) 713 5. k. souza, p. porto, sci. educ. 21(2012) 705 6. b. bensaude-vincent, sci. educ. 15 (2006) 667 7. j. r. bertomeu-sanchez, a. garcia-belmar, a. lundgren, m. patiniotis, sci. educ. 15 (2006) 657 8. k. m. olesko, sci. educ. 15 (2006) 863 9. j. simon, hist. sci. 50 (2012) 339 10. the curriculu m, 28.8.1874, školstvo i prosveta u 19. veku, the ministry of education, belgrade, p. 101 (in serbian) 11. s. bojović, honourable serbs: sima lozanić, princip, belgrade, 1996 (in serbian) 12. s. bojović, chemistry in seco ndary sc hools in serbia in th e ninete enth a nd twe ntieth centuries, university of belgrade, faculty of chemistry, belgrade, 2009 (in serbian) 13. i. gutman, d. prodanović, mita (dimitrije) petrović (1848–1891), in lives and wo rk of the serbian scientist, m. r. sarić, ed., serbian academy of sciences and arts, belgrade, 2002, p. 29 14. m. leko, educ. gazette 17 (1896) (in serbian) 15. m. leko, educ. gazette 18 (1897) 177 (in serbian) 16. m. leko, educ. gazette 18 (1897) 369 (in serbian) 17. s. m. lozanić, educ. gazette 18 (1897) 55 (in serbian) 18. s. m. lozanić, educ. gazette 18 (1897) 298 (in serbian). 19. s. m. lozanić, educ. gazette 18 (1897) 439 (in serbian) 20. j. herron, j. chem. educ. 54 (1977) 15 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1338 milanovic, trivic and tomasevic 21. i. ivić, a. pešikan, s. antić, guide for a goo d textbook ove rall standards for textbooks quality, platoneum, novi sad, 2009 (in serbian) 22. l. leite, sci. educ. 11 (2002) 333 23. e. t. pappa, g. tsaparlis, chem. educ. res. pract. 12 (2011) 262. 24. s. bojović, chem. rev. 30 (1989) 88. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false 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<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> /ukr 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {application of biosynthesized metal nanoparticles in electrochemical sensors} j. serb. chem. soc. 87 (4) 401–435 (2022) review jscs–5531 published 12 november 2022 401 review application of biosynthesized metal nanoparticles in electrochemical sensors totka dodevska*, dobrin hadzhiev, ivan shterev and yanna lazarova department of organic chemistry and inorganic chemistry, university of food technology, 26 maritsa boulevard, plovdiv 4002, bulgaria (received 21 may, revised 17 august, accepted 1 october 2021) abstract: recently, the development of eco-friendly, cost-effective and reliable methods for synthesis of metal nanoparticles has drawn a considerable attention. the so-called green synthesis, using mild reaction conditions and natural resources as plant extracts and microorganisms, has established as a convenient, sustainable, cheap and environmentally safe approach for synthesis of a wide range of nanomaterials. over the past decade, biosynthesis is regarded as an important tool for reducing the harmful effects of traditional nanoparticle synthesis methods commonly used in laboratories and industry. this review emphasizes the significance of biosynthesized metal nanoparticles in the field of electrochemical sensing. there is increasing evidence that green synthesis of nanoparticles provides a new direction in designing of cost-effective, highly sensitive and selective electrode-catalysts applicable in food, clinical and environmental analysis. the article is based on 157 references and provided a detailed overview on the main approaches for green synthesis of metal nanoparticles and their applications in designing of electrochemical sensor devices. important operational characteristics including sensitivity, dynamic range, limit of detection, as well as data on stability and reproducibility of sensors have also been covered. keywords: biosynthesis; green synthesis; nanomaterials; nanotechnology; modified electrodes; review. contents 1. introduction 2. green synthesis of metal/metal oxide nanoparticles using plants 3. green synthesis of metal/metal oxide nanoparticles using microorganisms * corresponding author. e-mail: dodevska@mail.bg https://doi.org/10.2298/jsc200521077d ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 402 dodevska et al. 4. electrochemical sensors based on biosynthesized metal/metal oxide nanoparticles 4.1. electrochemical sensors based on biosynthesized agnps 4.2. electrochemical sensors based on biosynthesized aunps 4.3. electrochemical sensors based on other biosynthesized metal and metal oxide nanoparticles 5. conclusions and future perspectives 1. introduction nowadays green nanotechnology has remained at the forefront of scientific research due to its outstanding approaches and applications. green nanotechnology involves the application of green chemistry principles to the design of valuable and sustainable nanosized materials in a more environmentally benign approach.1 the unique properties of nanomaterials such as catalytic potential,2 optoelectrical properties,3 magnetic behavior4 and biological activity5 are the main factors determining their extremely wide applications in various fields of science, technology and industry. nanosized materials are widely used as catalysts6 and nanoelectronic components,7 in the composition of antibiotics, antiseptics and disinfectants,8 in drug delivery,9 food and material packaging,10 targeted delivery of pharmaceuticals,11 development of biosensors,12 etc. the main challenge in the development of catalyticaly active nano-sized materials is to prepare nanoparticles that are highly active, selective, stable, robust, and inexpensive. classical synthesis of metal nanoparticles (mnps) most commonly involves chemical reduction of metal ions from solutions of their salts in the presence of organic or inorganic reducing agent such as ethylene glycol, dimethylformamide and sodium borohydride (nabh4), followed by addition of a stabilizing agent.13 the reagents used are usually expensive and toxic substances which could generate hazardous by-products harmful to health and environment.14 therefore, there is a growing concern to develop new, alternative and sustainable methods for mnps preparation. research on the possibilities of using biological systems (plants, bacteria, fungi, algae) to obtain stable mnps and metal oxide nps has been particularly intense in recent years. the so-called green synthesis has received more attention as a cost effective and valuable alternative for environmentally safe and energy-efficient production of nanoparticles with desired properties.2,15–19 unlike chemical and physical processes, bioinspired synthetic methods restrict the use of sophisticated instruments, toxic chemicals and energy (high temperature, pressure, irradiation). green synthesis of mnps involves the use of plant extracts or microorganisms for the bioreduction of metal ions into their zero-valent elemental form. biomolecules such as proteins, sugars, flavonoids, alkaloids, polyphenols, etc. (fig. 1) act as reducing and capping agents. regarding the process of metal oxide nps synthesis in a ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. electrochemical sensors based on biosynthesized nanoparticles 403 green way, researchers suggest that specific biomolecules react with the metal ions to reduce or to form complexes. the resulting product is subjected to thermal treatment to get final metal oxide nps. therefore, different mechanisms of metal oxide nps formation were proposed considering the ability of the active compounds in reducing and chelating the metal ions. fig. 1. schematic illustration of the natural sources used to synthesize nps in a green way. biological systems differ in their capabilities to supply mnps, hence the production process highly varies depending on the choice of green material. the plant extracts are considered to be more suitable compared to microorganisms for green synthesis of mnps. plant extract mediated synthesis of mnps is preferable due to its economic and ecological effectiveness – easily available plant material, aqueous solvents and normal conditions are used for the synthesis of nanoparticles in a simple one-step procedure. extensive research shows that the plantassisted synthesis is relatively fast and suitable for large-scale production of stable mnps. at the same time, delicate, complicated and meticulous preparation steps are required for microbial synthesis of mnps. organisms such as bacteria and fungi need to be cultured or propagated in order to obtain sufficient starting materials. thus, synthesis protocols include prior procedures such as microorganism isolation and identification, growth optimization and culture preparation. other challenges are the slow reduction process (ranging from hour to days) and poor understanding of the mechanisms controlling the shape and dispersity of microbial synthesized mnps. in addition, development of new green synthesis methods based on the use of waste products from agriculture and food-processing is one of the current research areas that has attracted a great deal of attention over the past years.2 these waste derived mnps have found a variety of applications in biotechnology, however, data on their applications in sensor technologies are still limited. current review features recent trends in electrode catalysts based on metal and metal oxide nps synthesized by using plant extracts and microorganisms. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 404 dodevska et al. even though research on the applications of biosynthesized nanoparticles in electrochemical sensors is actually at a really early stage, there are already promising opportunities for development of novel sensing platforms. most relevant biosynthesis approaches, successful integration strategies, selected sensing applications and future prospects of these biosynthesized nanomaterials for the design of advanced sensor platforms are also highlighted. 2. green synthesis of metal/metal oxide nanoparticles using plants due to the abundance of biomass and the diversity of species, plants are most suitable for large-scale biosynthesis and they are preferred for green synthesis of mnps.20–22 plant leaf extracts are the most common choice for bioinspired synthesis of mnps but the use of seeds, bark, fruits, tubers and root extracts has also been reported.20,23,24 it was established that experimental conditions such as temperature,25–27 ph,27,28 concentration and quantity of extract27,29,30 and/or metal ions,26,27 and contact time27 affect the efficiency and rate of the process of metal reduction and nanoparticles with desired shape and size could be produced. researchers suggested that various compounds such as amino acids, citric acid, heterocyclic compounds, flavonoids, polyphenols, terpenoides, enzymes, peptides, polysaccharides, saponins, tannins were responsible for reduction of metal ions and subsequent stabilization of the produced nanoparticles.4, 32–41 green synthesis of mnps based on the reduction of precious and non-precious transition metals including silver, gold, palladium, platinum and copper using plant extracts has been investigated by some authors.20,21,25 silver nanoparticles (agnps) and gold nanoparticles (aunps) are the most common ones used for chemical, electrochemical, biomedical and environmental applications.42 owing to their high surface-to-volume ratio and unique physicochemical properties, agnps possess various important characteristics: high catalytic activity, electrical and thermal conductivity, as well as broad-spectrum bioactivities.43,44 in medicine agnps have received tremendous attention for their excellent antimicrobial, antibacterial and anticancer potential. antimicrobial properties of agnps caused the use of these nanomaterials in cosmetics, military, packaging, etc. a large number of research groups have been reported completely green, feasible, renewable and inexpensive approaches for synthesis of stable agnps. a new, simplified and rapid methodologies for green synthesis of agnps using azadirachta indica,45 crotolaria retusa46 and terminalia arjuna47 plant extracts as reducing and stabilizing agents have been proposed. experimental data show that the so-synthesized nanoparticles exhibit high catalytic activity as well as excellent antimicrobial properties against gram-negative and gram-positive bacteria. the extract of some grape by-products such as stalks, leaves, stems, seeds and dried fruits have been successfully used for synthesis of agnps,48 bimetallic ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. electrochemical sensors based on biosynthesized nanoparticles 405 fe/pd49 and fe3o4/ag50 nanoparticles. recent studies on the chemical composition of grapes have revealed that the main components are polar compounds soluble in hot water with a high content of tannins and polyphenolic compounds.51 this suggests that the grape extract will also contain polyphenolic compounds that could act as reducing and stabilizing agents in the formation of nanoparticles. aunps have drawn the attention of researchers because of their extensive applications in areas such as electronics, catalysis, sensing/biosensing, medicine, controlled drug delivery, etc. intensive studies have revealed that aunps possess potential to serve as building blocks for plasmonic devices, as well as being used as catalysts and antimicrobials against a wide range of microorganisms.31,52 to date, a number of methods for synthesis of aunps including physical, electrochemical, photochemical and liquid chemical reduction have been developed. krishnaswamy et al. have reported a single step green synthesis of aunps using agricultural wastes materials such as grape seed, skin and stalk.53 various methodologies for biosynthesis of aunps from plant extracts also have been reported – ginkgo biloba,54 sunflower (helianthus annuus), chilopsis linearis, medicago sativa, brassica juncea,55 leaves of sphaeranthus indicus and various parts from plants (bark, stem, root, etc.)56 have been used to synthesize aunps. palladium nanoparticles (pdnps) have broad application in heterogeneous catalysis due to their excellent catalytic/electrocatalytic ability, high surface-to -volume ratio and high surface energy. recently, it has been reported that pdnps could be synthesized by using extracts of filicium decipiens57 and hippophae rhamnoides linn.58 research teams have reported production of nanoparticles of metal oxides (cuo and zno) using centella asiatica59 and aloe leaf,60 respectively. reddy has reported on a new green method for the synthesis of cuo nanoparticles (cuonps) using calotropis procera.61 cuonps are widely used as catalysts due to their excellent photocatalytic properties.62 ghidan et al.63 also have described successful biosynthesis of cuonps using punica granatum bark extract, and ijaz et al.64 propose a method for synthesis of cuonps from fresh leaves of abutilon indicum. zinc oxide nanoparticles (znonps) have aroused great interest due to their low cost, attractive properties and important role as semiconductor materials, in development of catalysts, ceramic resistors, gas sensors and energy-saving materials.65 due to their antimicrobial and antibacterial potential znonps are widely used in medicine.66,67 matinise et al. have reported an eco-friendly method for the synthesis of znonps using moringa oleifera extract.68 mechanisms of formation of the znonps via the chemical reaction of the zinc nitrate precursor with the bioactive compounds of the moringa oleifera are proposed. the electrochemical analysis proved that znonps have high electrochemical activity ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 406 dodevska et al. without any modifications and therefore are considered as a potential candidate in electrochemical applications. nava et al. also have used zinc nitrate as a source of the zinc ions and different peels extracts, from lycopersicon esculentum (tomato), citrus sinensis (orange), citrus paradisi (grapefruit) and citrus aurantifolia (lemon), for the green synthesis of znonps.69 the proposed formation mechanism is based on the chemical characteristics of the flavonoids, limonoids and carotenoids that present in the peel extracts. these biomolecules are believed to chelate zn2+ and form metal coordinated complexes that are further thermally treated to form znonps. authors have analyzed the effect of the extract used on the surface morphology of the resulting znonps, and tested the efficiency of znonps in the photocatalytic degradation of methylene blue under uv irradiation. the presented results highlight that the chemical composition of the extract has a significant effect on the size and shape distribution of nanoparticles, which further is directly associated with their catalytic activity. 3. green synthesis of metal/metal oxide nanoparticles using microorganisms a variety of microorganisms are utilized in nanoparticle synthesis. prokaryotic bacteria, actinomycetes, yeasts, fungi and algae have been broadly employed for biosynthesis of metal/metal oxide nanoparticles. bacterial synthesis of mnps is accepted due to the relative ease of manipulation of bacteria.41 some examples of bacterial strains that have been widely used for synthesis of bioreduced agnps with different morphology are: escherichia coli, lactobacillus casei, bacillus cereus, aeromonas sp. sh10 phaeocystis antarctica, pseudomonas proteolytica, bacillus amyloliquefaciens, bacillus indus, bacillus cecembensis, enterobacter cloacae, geobacter spp., arthrobacter gangotriensis, corynebacterium sp. sh09 and shewanella oneidensis.70 from bacterial strains such as candida guilliermondii,71 pseudomonas denitrificans,72 pseudomonas fluorescens 417,73 staphylococcus epidermidis74 and bacillus stearothermophilus75 spherical aunps with a diameter between 5 and 80 nm have been successfully synthesized.76 agnps and aunps of various shapes (spherical and triangular) have also been synthesized using algal strains such as pithophora oedogonia,77 ecklonia cava,78 chondrus crispus and spyrogira insignis79 and sargassum wightii greville.80 fungi-mediated biosynthesis of metal/metal oxide nanoparticles is also a very efficient process for the production of monodispersed nanoparticles with well-defined morphologies.70 fungi act as good biological agents for the synthesis of nanoparticles of metals and metal oxides due to the presence of a variety of intracellular enzymes.81 compared to bacteria, fungi could be a source for a large amount production of nanoparticles.82 the probable mechanism for the formation of mnps is enzymatic reduction in the cell wall or inside the fungal cell. a variety of fungal species are used to synthesize metal/metal oxide nano________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. electrochemical sensors based on biosynthesized nanoparticles 407 particles such as agnps, aunps, tio2nps, znonps.70 successful synthesis of nanomaterials via yeast also has been reported by numerous research groups.70 4. electrochemical sensors based on biosynthesized metal/metal oxide nanoparticles research on electrochemical sensors attracts lots of current interest because of their promising applications in food industry, ecology, medicine, pharmacy, etc. electrochemical sensor systems offer advantages of a cost-effective, rapid, highly sensitive, selective, compact and convenient to handling method for quantitative detection of the target analyte. these devices provide the opportunity for an accurate and susceptible automation analysis, and are a promising alternative of the conventional analytical techniques where time-consuming procedures for sample pre-treatment and expensive instruments are required. variety of electroanalytical techniques including cyclic voltammetry (cv), constant potential amperometry (amp.), differential pulse voltammetry (dpv), square wave voltammetry (swv), linear sweep voltammetry (lsv) and stripping voltammetry are available and applicable for electroanalysis. advantages of these techniques over classical detection methods such as spectroscopy and chromatography are their accuracy, reliability, ease of use and low cost. the general principle of electrochemical detection is illustrated in fig. 2. fig. 2. illustrative representation of electrochemical detection. the analyte reacts at the surface of the sensing electrode (modified working electrode) involving either an oxidation or reduction mechanism. this reaction is catalyzed by the electrode material specifically developed for the analyte of interest. the current generated in the process is converted into a signal that could be amplified, processed and displayed easily by modern electrical instruments. by analyzing the magnitude of the electrical signal, we can obtain information about the concentration of the substance being analyzed. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 408 dodevska et al. the experimental setup used for electrochemical analysis consists of conventional three-electrode cell including working electrode (we), reference electrode (re) and counter electrode (ce), potentiostat and personal computer (fig. 3). the working electrode is the electrode at which the reaction of interest occurs; as a reference electrode ag/agcl or saturated calomel electrode is usually used; an inert conducting material (pt) is used as a counter electrode. during experiments, charge flow (current) occurs between the working electrode and the counter electrode while the potential of the working electrode is measured with respect to the reference electrode. fig. 3. experimental setup used for electrochemical analysis. in electrochemical sensors design, in order to enhance the effective electrode surface and to increase the sensitivity, various nanomaterials such as carbon nanoparticles, metal/metal oxide nanoparticles, nanosized alloys and binary nanocomposites are used for the functionalization of electrode surface as a direct active layer.83–85 as a result, modified electrodes have extremely high catalytic efficiency due to the reduced overpotentials and faster electron transfer kinetics. therefore, the importance of developing nanostrustured highly active and selective electrode-catalysts applicable for quality and safety assessment of foods, for environmental monitoring, pharmaceutical analysis and clinical diagnostics, has received considerable attention nowadays.86–91 physical, chemical and electrochemical methods including laser ablation, high energy ball milling, reactive sputtering, thermal salt decomposition, sol–gel method, electrodeposition, etc, have been developed to obtain nanosized metal or metal oxide particles. although each one of the methods mentioned above had its merits, there are considerable disadvantages in terms of preparation and cost – multi-step time-consuming methodologies and requirement of sophisticated equipment. in this connection, recently biosynthetic approaches has received ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. electrochemical sensors based on biosynthesized nanoparticles 409 great attention due to their capability to design alternative, environmentally friendly, safer, energy efficient and less toxic routes towards synthesis. the green synthesis of nanosized materials has found a wide range of applications in the field of electrochemical sensors/biosensors, revolutionizing this field.92–94 fig. 4 shows the basic steps in the preparation of electrode modified with biosynthesized mnps. in summary: 1) plant extract was mixed with the metal salt solution; 2) bio compounds reduce metal from positive oxidation state to zero oxidation state; 3) the working surface of electrode was modified through a dropwise of the resulting colloidal solution. fig. 4. scheme of the basic steps in the preparation of electrode modified with biosynthesized mnps. in the next part of this review article we have summarized and discussed the recent progress, current challenges and future perspectives in green synthesis of different metal/metal oxide nanoparticles and their applications in the development of new electrode-catalysts for electroanalytical purposes. the most commonly used metal nanoparticles as electron-transfer mediators are agnps and aunps due to their chemical stability, unique physicochemical properties, good conductivity and electrocatalytic activity. relatively simple procedures for synthesis of agnps and aunps, facile electrode modification with agnps and aunps as well as their key role in reducing the overpotentials of electrocatalytic reactions make them extremely attractive to research groups.95 4.1. electrochemical sensors based on biosynthesized agnps nowadays, considerable attention has been paid to the detection of hydrogen peroxide (h2o2) owing to its wide applications. h2o2 is used in industrial wastewater treatment, as a disinfectant in medicine, as an oxidant and bleaching agent in textile, paper, pharmaceutical and cosmetic industries. due to its inherent bactericidal properties h2o2 is also used as a sterilizing agent in milk and dairy production96,97 and in food aseptic packaging.98 reliable and rapid quantification of h2o2 is also important in various biological, medical and clinical studies since h2o2 is one of the by-products of enzyme-catalyzed reactions occurring in living organisms. h2o2 acts as a precursor in the formation of highly reactive and ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 410 dodevska et al. potentially harmful hydroxyl radicals and it is one of the most important markers of oxidative stress. excessive accumulation of h2o2 in the body causes various diseases such as cardiovascular disorders, alzheimer’s, dna fragmentation, tissue damage and cancer. a number of research groups have confirmed remarkable electrocatalytic activity of agnps for h2o2 reduction and successfully have employed agnps -modified electrodes as sensing interface to construct enzyme-free h2o2 electrochemical sensors.99–104 in the cited articles electrode surface modification with agnps has been performed applying chemical or electrochemical methods in order to enhance the rate of electron transfer and to decrease the required high overpotential – the major barrier for effective electrochemical detection of h2o2 at ordinary solid electrodes. salazar et al. have investigated the catalytic activity of electrode modified with biosynthesized agnps in the reaction of electroreduction of h2o2.105 they have presented a simple one-step eco-friendly strategy to obtain silver nanoparticle-modified reduced graphene oxide nanocomposites (rgo/agnps) using green tea extract for reducing both ag+ and graphene oxide sheets. tem image and the size distribution for agnps confirmed the quasi-spherical shape of the agnps with an average size of about 25 nm (a size range distribution from 5 to 60 nm). glassy carbon (gc) electrode was conveniently modified with rgo/ /agnps nanocomposite and electrochemical tests were carried out to study the electrocatalytic properties of the rgo/agnps/gc sensor towards h2o2 reduction. basic analytical parameters such as selectivity, sensitivity, limit of quantification and limit of detection, time of response and stability of modified electrode in 0.1 m pbs under optimized conditions (ph 8.0; applied potential of –0.4 v vs. ag/agcl, 3 m) were also studied. it was determined that the electrode has a sensitivity of 236 μa mm–1 cm–2 (r2 = 0.999) in the concentration range from 0.002 to 20 mm, rapid response (~2 s) and detection limit of 0.73 μм h2o2 estimated on the criterion signal-to-noise s/n = 3. after 7 months storage it was established that rgo/agnps/gc has no significant loss of sensitivity. the selectivity of the modified electrode was tested against different biological interferences including dopamine, glutamate, glucose and ascorbic acid with promising results. in addition, the applicability of this sensor for h2o2 detection in real samples was confirmed in antiseptic solutions, commercial milk and urine. tagates erecta (marigold) flowers extract has been used for production of agnps in a green route.106 characterization of biosynthesized nanoparticles was done using different methods: ultraviolet–visible spectroscopy (uv–vis), field emission scanning electron microscopy (fesem), elemental dispersive x-ray spectroscopy (edx), fourier transform infrared spectroscopy (ftir), x-ray diffraction (xrd) and x-ray photoelectron spectroscopy (xps). the uv–vis studies showed the occurrence of an absorption band at 430 nm which is specific ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. electrochemical sensors based on biosynthesized nanoparticles 411 for agnps. fesem analysis indicated that the biosynthesized agnps have a homogenous size distribution. the xrd patterns reflected that the particles are crystalline in nature, with a face-centered cubic structure. using agnps and chitosan (cs), modified pencil graphite electrode (pge) was fabricated by dropcasting method and the as-prepared hybrid material pge/agnps/cs was used for supercapacitator and electrochemical sensing applications. it has been shown that pge/agnps/cs electrode provided remarkable catalytic activity towards electrochemical reduction of h2o2. quantitative analysis of h2o2 was performed in supporting electrolyte 0.1 m hcl/kcl (ph 2.0) using cyclic voltammetry (cv) and a linear graph was obtained in the concentration range of 1.0–10.0 μm (limit of detection was found to be 0.52 μm). furthermore, the sensor was applied to real sample analysis. the results obtained suggested that the proposed electrochemical device can be used for traces analysis of h2o2 in cosmetic products. since the industrially steam distillation of essential oil crops never leads to complete extraction of the aroma substances, the essential oil industry wastes have a potential for extraction of residual volatile polar metabolites. the waste is rich in non-volatile polar metabolites (flavonoids, organic acids, carbohydrates, amino acids, etc.) and some of these compounds have also reduction properties and could influence the synthesis and stabilization of agnps. dodevska et al.107 have reported for the first time utilization of rosa damascena waste for synthesis of agnps and applicability of the nanoparticles for development of electrochemical sensors for h2o2 and vanillin detection. the process of agnps synthesis takes place in one-stage and it is based on the utilization of abundant and cheap waste materials. the authors stated that the main functional groups involved in the agnps formation were aromatic hydroxyl groups and carbonyl groups and substances such as phenolic acids, flavonoids, proteins, terpenes, carbohydrates, etc. having important role in reduction of ag+. furthermore, proteins, polysaccharides and carboxylic acids additionally participated in the process by capping the in situ generated nanoparticles. the data suggested that the ethanolic extracts have higher content of phenolic acids and flavonoids than water extracts. on the other hand, the water extracts were rich in carbohydrates (including reducing sugars), proteins and pectic substances (as suggested by the presence of uronic acids). tem micrographs showed that using water extract of rosa damascena agnps were obtained as sphere-like particles with an average size calculated to be 25.8±11.5 nm. biosynthesized agnps were deposited onto a spectroscopic graphite (gr) electrode and the electroactive layer was stabilized by applying thin film of chitosan onto the modified electrode surface. chitosan is commercially available natural polymer and preferable material in designing sensors/biosensors. it is a linear amine-rich polysaccharide, biocompatible polymer distinguished by its ability to form flexible, strength, highly adhesive membranes. in electrochemical sensors chitosan is commonly used to enhance the stability of ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 412 dodevska et al. nanoparticles. the coverage of chitosan on the biosynthesized agnps not only protected the nanoparticles against aggregation, but also stabilized surface properties of agnps while enhancing their catalytic activity. the electrochemical performance of the modified electrode agnps/cs/gr was studied by means of cv, dpv and chronoamperometry at ph 7.0 and its applicability for amperometric detection of h2o2 and vanillin was investigated. vanillin (4-hydroxy-3 -methoxybenzaldehyde) has a specific aroma, pronounced antioxidant and antimicrobial properties and it is one of the most commonly used food supplements. for adults the permissible daily intake of vanillin is less than 10 mg kg–1 (the addition of vanillin in baby formula and infant food is not permited). the overweight content of vanillin in food products, as the excessive ingestion via the dietary intake has potential toxic effect – symptoms of a vanillin overdose can include nausea, vomiting and headache; in cases of severe intoxication vanillin can cause irreversible damage to the liver and kidneys. therefore, the development of novel analytical techniques have been employed for fast and reliable quantitative detection of vanillin. electrochemical studies suggested that graphite electrode modified with agnps, biosynthesized using rosa damascena waste, possesses a stable response to vanillin up to 0.5 mm with a detection limit of 8.4 µm at an applied potential of 0.58 v (vs. ag/agcl, 3 m kcl). the developed electrode exhibited a sensitive and reproducible response for quantitative determination of h2o2 at applied potentials from –0.2 to –0.3 v. constant potential amperometry measurements at –0.3 v showed highly sensitive response to h2o2 up to 6.6 mm. electrochemical studies with agnps synthesized using flower aqueous extracts of achillea millefolium and lavandula angustifolia wastes as reducing agents, which is a novel simple approach, inexpensive and eco-friendly in nature, also were reported.94 the representative electron micrographs of agnps showed that the nanoparticles grew very tiny with spherical shape. from the presented histograms it can be seen the size distribution of agnps and their mean sizes were 2.8 nm for agnps/achillea millefolium and 3.1 nm for agnps/ /lavandula angustifolia, respectively. selected area electron diffraction (saed) pattern represents the (111), (220) and (222) crystal planes of the cubic structure of agnps in both samples. biosynthesized agnps were deposited onto a spectroscopic graphite surface, applying two different procedures, and stabilized using chitosan to build new electrocatalysts. the electrochemical performance of the modified electrodes was studied by means of cv and chronoamperometry in neutral medium and their applicability for amperometric quantitative determination of h2o2 was demonstrated. the modified electrodes showed a remarkable activity at applied potentials of –0.3 and –0.2 v vs. ag/agcl, 3 m kcl, rapid, stable and reproducible amperometric response. it was stated that amperometry at constant potential of –0.3 v is distinguished by extremely high sensitivity (533.5 μa mm–1 cm–2) up to 4.3 mm h2o2. in order to study the selectivity, the amp________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. electrochemical sensors based on biosynthesized nanoparticles 413 erometric response was examined in the presence of common interfering species such as nitrate, glucose, uric acid, ascorbic acid and citric acid. the authentic record of the electrode signal clearly shows that the tested species had no effect on the h2o2 detection – no response was observed in the presence of the above mentioned substances and the current response for h2o2, registered after adding the substances, corresponds to the one determined in the calibration study. these results demonstrate that the modified electrode has good selectivity for h2o2 and reveal the application potential of the so-biosynthesized agnps for sensing of h2o2 in real samples. potassium and sodium nitrites (kno2, nano2) are listed as permitted food additives (e249, e250). kno2 and nano2 show important bacteriostatic and bacteriocidal activity against several spoilage bacteria and foodborne pathogens in meat products and are widely used as preservatives in the preparation of cured meat products.108 nitrite ions not only have a pronounced antimicrobial activity, but also act as a color fixative and inhibits lipid oxidation, thereby slowing meat spoiling. in addition, nitrite ion concentration is one of the most important indicators determining the quality of drinking water – according to the recommendation of the world health organization109,110 the acceptable no2– content is 0.2 mg l–1; according to european community regulation111,112 the maximum permissible nitrite content of drinking water is 0.1 mg l–1. consumption of foods with excessive nitrite content poses a serious threat to human health. a number of clinical studies have shown that nitrite intake is associated with higher relative risk of breast cancer, gastric cancer, renal cell carcinoma, adult glioma, colorectal cancer, esophageal cancer and thyroid cancer.113 therefore, the importance of improved analytical methods for determination of nitrite in drinking water, cured food and environmental systems has received considerable attention. nitrite ion is an electroactive and can be quantified electrochemically. in comparison with the conventional analytical techniques, electrochemical analysis has been considered as a fast, low-cost and effective way due to its intrinsic simplicity and high sensitivity.114 a promising electrochemical sensor for accurate, sensitive and selective detection of nitrites is developed by shivakumar et al.115 the authors reported on a facile, cost effective, green synthesis method of silver nanospheres (agns) by using pre-hydrolyzed liquor (phl) from the nilgiri wood generated from pulp industry without any pre-treatment. the synthesis was performed at room temperature within 3 h. the presented xrd pattern of agns evidences face centered cubic crystalline structure of metallic silver; the average crystallite size of agns calculated from scherrer equation was found to be ~30 nm. it was suggested that hemicelluloses present in phl were responsible for the reduction of silver ions and stabilization of agns. the gc electrode modified with biosynthesized agns has been shown to exhibit excellent electrocatalytic activity in nitrite oxid________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 414 dodevska et al. ation – extremely low detection limit (0.031 μm) and high electrode sensitivity of 580 μa mm–1 cm–2 in the concentration range from 0.1 to 8.0 μm. it is noteworthy that after 30 days storage the presented electrode retains up to 98 % of its initial activity. ascorbic acid (aa), known as vitamin c, is a naturally occurring organic compound with antioxidant properties – it is one of the strongest reductants and free radical scavengers in living cells that is suggested to decrease oxidative damage and lowering the risk of certain chronic diseases. the antioxidant activity of aa is the main reason to be frequently used in food industry to prevent unwanted changes in the color and aroma of foods. the use of aa in meat products, with the addition of nitrites, is important for the activity of reduction dependent upon nitrousmetamyoglobin-fe (iii) converted into nitrousmetamyoglobin–fe (ii), which maintains the colour of the product most brilliant.116 additives based on аа are widely used in the production of food and beverages (jam, candy, fruit juices, fish and meat products, beer, etc.). aa is used as well as in cosmetics as a skin conditioning agent and in pharmaceutical industry as a diet supplement in various forms.114 the wide and effective therapeutic potential of aa in dermatology also has been proven. vitamin c plays a key role in maintaining skin health, provides protection against uv-induced photodamage, participates in the formation of skin barrier lipids and collagen in the dermis, as well as in the modulation of cell signal pathways of cell growth and differentiation. numerous clinical studies and in-vitro data support the use of topically applied aa for photoprotection, antiaging, anti-inflammatory and skin-lightening uses. a topical aa treatment of the epidermal surface suppressed uvb-induced cell death, apoptosis, dna damage, reactive oxygen species (ros) production, and the infammatory response by downregulating tumour necrosis factor-α (tnf-α) expression and release.117 under normal physiological conditions, melanin is produced by the epidermal melanocytes in response to uv-irradiation. an excessive production of melanin causes dermatological problems such as freckles, age spot and melasma. these skin pigmentation disorders can be caused by various factors such as an excessive sun exposure, hormonal imbalance during pregnancy or menopause, side effects from certain medications. emerging evidence has indicated that aa has therapeutic effects on facial hyperpigmentation, as it reduces melanin synthesis. aa suppresses the catalytic activity of tyrosinase, the rate-limiting enzyme in melanin biosynthesis.118 although the antipigmentary and skin-protective mechanisms of aa still need to be clarified, aa has been used widely as skinlightening, anti-aging, anti-oxidant and anti-inflammatory agent in commercially available cosmetics (creams, lotions, dental care products, etc.). due to the important role of aa, recently there is a significant research interest to develop electrochemical sensors for detection of aa content in various ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. electrochemical sensors based on biosynthesized nanoparticles 415 samples including foods, drugs, cosmetics and biological fluids.114,119 in this regard crystalline silver face-centered cubic phase, spherical in shape, with mean particle size about 5.3–10.2 nm was synthesized using onion extracts.120 the authors of this study believe that high phenolic content of the water extract of onion is responsible for production and stabilization of agnps. it was suggested that the formation of agnps was related to the reaction temperature, ph, duration, as well as concentrations of silver nitrate solution and onion extract. modified carbon paste electrode (agnps/cpe) was prepared using the agnps phytosynthesized at optimal conditions (5 mm agno3, 17 wt. % onion extract, temperature 35 °c, ph 10 and 18 h reaction time). the effect of synthesized agnps on aa electrooxidation was investigated by swv. voltammograms show that the peak current at 0.48 v remains linear in the concentration range 0.4 to 450 μm aa; detection limit was calculated to be 0.1 μm aa. the real sample analysis reveals the practical applicability of agnps/cpe for aa detection in fruit and vegetable juices. electrochemical sensor for dopamine (da) based on biosynthesized agnps was developed by sreenivasulu et al.121 dopamine (4-(2-aminoethyl)benzene -1,2-diol) is a neurotransmitter that affects numerous physiological processes and plays an important and diverse role in brain function. dopamine molecule is biomarker for diseases such as parkinson’s, depression, schizophrenia and some brain tumors. reliable detection of dopamine is important in research and clinical disease diagnosis and various types of electrochemical sensors have been developed due to its electroactive nature. sreenivasulu et al. have used aqueous root extract of mimosa pudica for facile and stable biosynthesis of agnps. the formation of agnps were identified using uv–vis spectrophotometer and thoroughly characterized by using xrd, ftir, sem, edax and tem. tem analysis showed that the synthesized agnps have a spherical shape and average sizes from 35.0 to 42.5 nm. amperometric studies revealed that the agnps-assembledgc electrode possesses high sensitivity, low limit of detection (0.5 μm) and an excellent dynamic range (10–60 μm) for quantitative detection of da. the authors stated that these results are comparable with those obtained by chemically modified gc electrodes previously reported in the literature. nitrobenzene (nb) is widely used as a precursor for aniline, pesticides, herbicides, insecticides, azo dyes, explosives, and drugs. acute (short-term) and hronic (long-term) inhalation, oral, and dermal exposure of humans to nb result in effects on the blood, central nervous system, liver and kidney. prolonged exposure may cause headache, nausea, fatigue, dizziness, impaired vision, cyanosis and anemia. unfortunately, a huge amount of nb was exited into water, soil and sediments from industries. therefore, the timely and accurate detection of nb is an important concern in public and environmental protection. different analytical methods have been used for detection of nb and electrochemical methods are ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 416 dodevska et al. considered simpler and more sensitive than available chromategraphic and spectrophotometric methods.122 shivakumar et al. have described eco-friendly synthesis of agnps using eucalyptus extract as a reducing and stabilizing agent.123 a gc electrode was modified with the so-synthesized nanoparticles and agnps/ /gc was tested for quantitative detection of nb. two electrochemical techniques – cv and dpv were used to study the electrochemical behaviour of agnps/gc. the modified electrode exhibited good electrocatalytic activity in the reaction of electroreduction of nb – linear current response in the concentration range 5 to 40 µm, sensitivity of 2.262 µa µm–1 cm–2, detection limit of 0.027 µm and good selectivity. stability studies were carried out by running the cv of the modified electrode in the presence of 1 mm nb on the day of preparation and every alternate day up to 20 days. the authors stated that the sensor exhibited great storage stability retaining up to 92.8 % of preliminary current at the end of tested period. the practical applicability of developed electrode material to detect selectively nb in tap water and lake water was tested and satisfactory results were obtained. in order to improve selectivity, sensitivity and detection limit for determination of nb, karthik et al. have modified gc electrode with sphere-like agnps biosynthesized using camellia japonica leaves.124 the fabricated electrocatalyst agnps/gc have been shown to have ability to detect nb with an excellent selectivity, extremely low limit of detection (0.012 µm) and a wide linear range (up to 2.593 mm). the agnps/gc showed excellent selectivity towards the nb detection – the results from selectivity test showed that the addition of potentially interfering species (common metal ions, some anions and nitroaromatic containing substances) into the system in 500-fold concentration relative to the analyte does not affect the electrode signal. this result is remarkable given the fact that other nitroaromatic compounds usually significantly affect the peak current response of nb owing to their similar structural activity. the practical applicability of this catalyst for selective quantitative analysis of nb in real samples was tested successfully in contaminated waste water. the sensor device is simple, cost effective and portable and can be applied in a number of industrial and research measurements. an electrochemical sensor for nb based on reduced graphene oxide (rgo) and agnps, biosynthesized using justicia glauca leaf extract, has been developed.125 the modified rgo/agnps/gc electrode showed good efficiency for selective quantitative determination of nb, compared to other modified electrodes – the electrode signal retained its linearity in the concentration range from 0.5 to 900 μm, the sensitivity was determined as 0.836 μa μm–1 cm–2 with a detection limit of 0.261 μm nb. in addition, the reduction peak current response to 100 μm nb was examined up to 52 days by cv and rgo/ /agnps/gc was stored in phosphate buffer solution (pbs) when not in use. the modified electrode retained about 90.15 % of its initial current response after 52 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. electrochemical sensors based on biosynthesized nanoparticles 417 days, which indicates the excellent storage stability of the sensor. the authors have reported that the fabricated sensor showed a satisfactory reproducibility with rsd of 3.8 % for determination of nb using 5 different sensors. the good recovery results and rsds of the devepoled sensor obtained in waste water samples proved the practical applicability towards the determination of nb in real samples. bastos-arrieta et al. have demonstrated that biosynthesized agnps may be useful in developing catalysts for electrochemical sensing of some heavy metal ions.126 heavy metal toxicity has proven to be a major threat and there are serious health risks associated with it.127 toxicity of heavy metals is due to accumulation in tissues, metabolic interference, mutagenesis and carcinogenesis. arsenic, cadmium, mercury, chromium, lead and nickel induce oxidative stress, dna damage, cell aging and cell death processes, resulting in increase the risk of cancer and cancer-related diseases.128 bastos-arrieta et al. reported on synthesis of agnps by using an aqueous extract of grape stalk waste as a reducing and capping agent, thus leading to a reagent-free procedure and valorisation of agrifood waste.126 various factors affecting the agnps synthesis such as temperature, contact time, extract/metal solution volume ratio and ph have been studied. agnps with an average diameter of 27.7±0.6 nm were selected to proof their suitability for sensing purposes. screen-printed carbon nanofiber electrode modified with biosynthesized agnps (agnps–spcnfe) was tested for the simultaneous stripping voltammetric determination of pb(ii) and cd(ii). the good reproducibility, high sensitivity and low limits of detection (around 2.7 μg l–1 for both metal ions) make this electrocatalyst a promising sensing element in electrochemical sensor device for a fast and reliable simultaneous detection of pb(ii) and cd(ii). table i summarizes electrochemical sensors based on biosynthesized agnps. important operational characteristics including sensitivity, dynamic range, limit of detection, as well as data on stability and reproducibility of sensors were presented. 4.2. electrochemical sensors based on biosynthesized aunps in nanotechnology aunps have attracted much attention due to their remarkable properties including high mechanical stability, unique tunable optical and distinct electronic properties, high electrical conductivity, strong binding affinity to thiols, and catalytic activity. adhering to the principles of green chemistry, mohd taib et al. have described a new method for synthesis of aunps, using water extract of hibiscus sabdariffa leaves (h. sabdariffa l.) as both reductant and stabilizer.136 the proposed procedure is reliable, environmentally friendly and cost-effective compared to other conventional synthesis methods. the authors suggested that chlorogenic acid (an ester of caffeic acid and quinic acid) in h. sabdariffa l. extract is the major compound involved in the reduction of au3+ to au. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 418 dodevska et al. table i. operational characteristics of electrochemical sensors based on biosynthesized agnps; amp. (amperommetry); cv (cyclic voltammetry); dpv (differential pulse voltammetry); dpasv (differential pulse anodic stripping voltammetry); swv (square wave voltammetry); swasv (square wave anodic stripping voltammetry); lod (limit of detection); agns (silver nanospheres); cs (chitosan); gc (glassy carbon); gr (graphite); go (graphene oxide); rgo (reduced graphene oxide); spcnfe (screen-printed carbon nanofiber electrode); cpe (carbon paste electrode); nb (nitrobenzene); aa (ascorbic acid); da (dopamine) modified electrode reducing agent method (e / v) analyte sensitivity, μa mm-1 cm-2 (linear range) (lod) stability (rsd) agnps/cs/gr94 achillea millefolium amp. (–0.3a) h2o2 533.5 (up to 4.3×10–3 m) (–) – (6.8 %) agnps/cs/gr94 lavandula angustifolia amp. (–0.3a) h2o2 374.7 (up to 3.5×10–3 m) (–) – (–) agnps/cs/pge106 tagetes erecta cv (–0.55a) h2o2 0.129 ma μm–1 (1.0–10.0 μm) (0.52 μm) 4 weeks (1.4 %) rgo/agnps/gc105 green tea amp. (–0.4a) h2o2 236 (0.002–20.0 mm) (0.73 μm) 7 weeks (3.6 %) agnps/cs/gr107 rosa damascena amp. (–0.3a) h2o2 115.2 (up to 6.6 mm) (–) – (6.7 %) agnps/gr107 rosa damascena amp. (–0.3a) h2o2 214.7 (up to 3.9 mm) (–) – (6.7 %) agau/rgo/gc129 azadirachta indica amp. (–0.4a) h2o2 – (0.1–5.0 mm) (1.0 μm) – (–) rgo/agnps/gc130 plectranthus amboinicus amp. (–0.32a) h2o2 – (1.0–800 μm) (0.312 μm) – (–) agnps/go/gc131 callicarpa maingayi amp. (–0.32a) h2o2 – (5.0–700 μm) (0.6 μm) – (–) agnps/gc132 bacillus subtilis amp. (–0.35b) h2o2 236 (0.05–120 mm) (8.0 μm) 30 days (3.1 %) agns/gc115 nilgiri wood amp. (0.86a) no2¯ 580 (0.1–8.0 μm) (0.031 μm) 30 days (3.6 %) agnps/gc133 piper betle amp. (1.0a) no2¯ 1642.27 (1.0–6000 μm) ( 0.046 μm ) 30 days (3.3 %) agnps/go/gc134 aa swasv (–0.6a) as3+ 180.5 µa µm–1 (13.33–375.19 nm) (0.24 nm) 90 days (–) agnps/spcnfe126 grape stalk waste dpasv (–0.42a) pb2+ 62 na μg–1 l (8.9–100.4 μg l–1) (2.7 μg l–1) – (–) agnps/spcnfe126 grape stalk waste dpasv (–0.55a) cd2+ 46 na μg–1 l (9.5–37.9 μg l–1) (2.8 μg l–1) – (–) agnps/cpe120 onion swv (0.45a) aa – (0.4–450 μm) (0.1 μm) – (–) agnps/gc121 mimosa pudica dpv (0.1a) da – (10.0–60 μm) (0.5 μm) – (–) agnps/gc135 ocimum tenuiflorum amp. (0.55a) glucose 895.8 (1.0–8.9 mm) (0.0048 μm) 10 days (1.15 %) rgo/agnps/gc125 justicia glauca dpv (–0.458a) nb 0.836 μa μm–1 cm–2 (0.5–900 μm) (0.261 μm) 52 days (3.8 %) ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. electrochemical sensors based on biosynthesized nanoparticles 419 table i. continued modified electrode reducing agent method (e / v) analyte sensitivity, μa mm-1 cm-2 (linear range) (lod) stability (rsd) agnps/gc124 camellia japonica amp. (–0.42a) nb – (0.05–21.0 μm) (23.0–2593 μm) (0.012 μm) – (–) agnps/gc123 eucalyptus dpv (–0.78a) nb 2.262 μa μm–1 cm–2 (5.0–40.0 μm) (0.027 μm) 20 days (–) agnps/cs/gr107 rosa damascena amp. (–0.3a) vanillin 56.8 (up to 0.5 mm) (8.4 μm) – (–) areference electrode: ag/agcl, 3 m kcl (0.200 v vs. she); breference electrode: saturated calomel electrode (sce, 0.242 v vs. she) previous reports оn aunps-synthesis have shown that the caffeic acid moiety of chlorogenic acid was essential to reduce au3+. tem analysis confirmed that the so-biosynthesized aunps were formed with a narrow distribution and an average particle size of 7±2 nm. a glassy carbon electrode modified with the aunps was tested as a catalyst in electrooxidation of nitrite. aunps/gc showed good electrocatalytic activity in the target reaction – sensitivity was calculated to be 917±30 µa mm–1 cm–2 in the concentration range from 0.37 to 10 mm and detection limit of 0.11 mm (s/n = 3). the stability, reproducibility and repeatability of aunps/gc electrode were investigated by voltammetric measurements. after 21 days storage the prepared electrode possesses around 80 % of its initial response. an analysis for ten sequential prepared electrodes showed rsd of 4.27 % which confirmed the repeatability of aunps/gc. the sensor-to-sensor reproducibility was investigated by measuring the current responses of five diverse electrodes prepared independently by the same procedure. the results showed that the response produced by different electrodes had a good reproducibility with rsd of 4.21 % and authors concluded that the sensor fabrication methodology was reliable. emmanuel et al. have presented a green procedure for synthesis of aunps using acacia nilotica twig bark extract at room temperature.137 the synthesis protocol shows that the formation of gold particles is within 10 min, which implies a higher reaction rate. the size of biosynthesized aunps was calculated using debye–scherrer equation which showed that the nanoparticles were in the average size of 30 nm. the aunps modified glassy carbon electrode exhibited excellent reduction ability towards nb compared to the unmodified electrode. the developed sensor aunps/gc displayed a wide linear response from 0.1 to 600 µm with high sensitivity (1.01 µa µm–1 cm–2) and a low detection limit of 0.016 µm in dpv mode. the modified electrode demonstrated exceptional selectivity in the presence of ions, phenolic and biologically active compounds. in ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 420 dodevska et al. addition, the aunps/gc exhibited an outstanding recovery results towards nb in various real water samples. electrochemical sensor for quantitative detection of hydrazine – another environmentally hazardous pollutant, was devepoled by karthik et al.138 their study envisages easy and innovative method for green synthesis of aunps on gc electrode; the fabricated modified electrode was used for the detection of hydrazine by using sensitive amperometric method. hydrazine is an inorganic base, which is an important reagent in the production of polymer foams, pesticides, insecticides, pharmaceuticals, etc. hydrazine is also used as rocket fuel – it is a high volumetric energy density liquid fuel (at room temperature and atmospheric pressure) that contains 12.6 wt. % of hydrogen. however, hydrazine is a highly toxic compound with mutagenic and carcinogenic effects. serious effects on the reproductive system are observed in animals after hydrazine inhalation. therefore, rapid and precise detection of hydrazine is of great importance. karthik et al. have used cerasus serrulata (c. serrulata) leaves extract for green synthesis of aunps. tem images confirmed that biosynthesized aunps were spherical in shape and approximately in the range of 5 to 25 nm. dft studies revealed that the coumarin present in the c. serrulata leaves extract demonstrated greater reducing and stabilizing properties compared to the properties of other compounds like butylhydroxytoluene and hydrocoumarin present in the extract. the electrochemical results showed remarkable electrocatalytic activity of the aunps-modified gc electrode towards oxidation of hydrazine. aunps/gc exhibited a wide linear range from 5 nm to 272 µm with a low detection limit of 0.05 µm. the fabricated electrode showed good selectivity towards the sensitive determination of hydrazine even in the presence of 1000-fold and 150-fold excess concentration of common ions and biological interferents (ascorbic acid, uric acid and dopamine), respectively. thus the proposed electrode seems to be a potential candidate for developing a simple, rapid and cost-effective electrochemical sensor for hydrazine detection. for the first time karthik et al. report in electrochemical chloramphenicol (cap) sensor using plant extract derived aunps.139 cap is an effective broad -spectrum antibiotic that has been widely used to treat mammalian, poultry, aquatic and bee diseases around the world. however, cap is associated with numerous toxic and fatal side effects in human, especially bone marrow suppression, aplastic anemia and agranulocytosis. therefore, development of fast, simple and reliable methods for cap monitoring in food samples are extremely important in food quality control. karthik et al. have presented a simple and rapid green synthesis using bischofia javanica blume leaves as reducing agent for the preparation of aunps. the biosynthesis procedure requires less than 40 s to reduce gold salts to aunps. they have used graphene oxide (go) as support to anchor and stabilize aunps which also avoids aggregation. the successful formation of ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. electrochemical sensors based on biosynthesized nanoparticles 421 the aunps/go composite was revealed by morphological, elemental, spectroscopic and electrochemical methods. the green synthesized aunps/go delivered high conductivity, surface area and porosity. aunps/go composite film modified electrode has shown excellent electrocatalytic ability to cap. the amperometric sensing platform possesses a sensitivity of 3.81 μa μm–1 cm–2 in the range of 1.5 μm–2.95 μm; lod of the sensor was calculated as 0.25 μm cap. the amperometric measurements proved that the modified electrode has a good anti-interference ability, a fast response (the current reached 95 % steady-state current within 5 s of cap injection), satisfactory repeatability (rsd for five repeatable measurements was calculated to be 3.18 %) and reproducibility of the procedure (five different aunps/go modified electrodes showed rsd 3.83 %). the long-term storage stability of the electrode was also reasonable – 92.15 % of the initial response current was retained over 15 days. the real sample analysis tested in milk, powdered milk, honey and eye drops samples validates excellent practical feasibilty of aunps/go modified electrode to determine cap content in food and pharmaceutical samples. table ii provides an overview of the electrochemical sensors based on biosynthesized aunps. table ii. operational characteristics of electrochemical sensors based on biosynthesized aunps; spe (screen-printed electrode); cap (chloramphenicol). other abbreviations are the same as table i modified electrode reducing agent method (e / v) analyte sensitivity (linear range) (lod) stability (rsd) aunps/cpe140 glycerol swv (0.65b) no2¯ 0.268 a l mol-1 (0.2–15 μm) (0.2 μm) 60 days (4.0 %) aunps/gc136 hibiscus sabdariffa amp. (0.8a) no2¯ 917 (370–10000 μm) (110 μm) 21 days (4.21 %) rgo/aunps/gc141 abelmoschus esculentus swasv (–0.791a) cd2+ 19.05 μa μm-1 cm-2 (5–10 μm) (31.81 nm) – (–) rgo/aunps/gc141 abelmoschus esculentus swasv (–0.54a) pb2+ 47.7 μa μm-1 cm-2 (5–10 μm) (12.69 nm) – (–) rgo/aunps/gc141 abelmoschus esculentus swasv (–0.064a) cu2+ 22.10 μa μm-1 cm-2 (5–10 μm) (27.42 nm) – (–) rgo/aunps/gc141 abelmoschus esculentus swasv (0.228a) hg2+ 29.28 μa μm-1 cm-2 (5–10 μm) (20.70 nm) – (–) aunps/spe77 pithophora oedogonia amp. (0.77a) carbendazim – (0.05–25 μm) (0.0029 μm) – (–) aunps/gc138 cerasus serrulata amp. (0.24a) hydrazyne – (0.005–272 μm) (0.05 μm) – (–) aunps/gc137 acacia nilotica dpv (–0.7a) nb 1.01 μa μm-1 cm-2 (0.1–600 μm) (0.016 μm) 8 days (2.1 %) ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 422 dodevska et al. table ii. continued modified electrode reducing agent method (e / v) analyte sensitivity (linear range) (lod) stability (rsd) aunps/go/gc139 bischofia javanica blume amp. (–0.45a) cap 3.81 μa μm-1 cm-2 (1.5–2.95 μm) (0.25 μm) 15 days (3.18 %) aunps/gc142 peanut seeds cv (–0.3a) sudan iv – (10–80 μm) (4.0 μm) 20 cycles (–) 4. 3. electrochemical sensors based on other biosynthesized metal and metal oxide nanoparticles copper nanoparticles (cunps) are particularly attractive because of high natural abundance of copper, low cost and the practical and straightforward multiple ways of preparing cu-based nanomaterials.143 cu-based materials can promote and undergo a variety of reactions due to accessible oxidation states of copper which enable reactivity via both oneand two-electron pathways. recently, copper oxide nanostructures have been given more attention as promising electrode materials for supercapacitores, gas sensors, electrochemical sensors and anode materials for lithium ion batteries. many researchers have found that the electrochemical performance of cuo/cu2o composites has been improved due to their stable multiple oxidation states and integration of their catalytic capabilities. a simple, low cost, stable and sensitive electrochemical sensor based on biosynthesized copper oxide nanoparticles (cuo/cu2o nps) was developed for formaldehyde detection.144 momeni et al. have successfully synthesized cuo/cu2o nanoparticles using gum arabic (highly branched complex polysaccharide, nontoxic and hydrophilic with abundant hydroxyl and carboxyl groups) as a stabilizing and capping agent. the cuo/cu2o nps modified carbon ionic liquid electrode (cuo/cu2o/cile) was designed and its catalytic activity was investigated towards formaldehyde oxidation in alkaline medium. formaldehyde is one of the most widely used chemicals – it acquires applications in different areas, such as resin, adhesive and plastic industry, fuel cells and electroless plating industry, agriculture and food manufacturing, as an industrial disinfectant and a preservative agent in medical labs, etc. however, a number of studies have suggested that formaldehyde exposure is associated with certain types of cancer, particularly myeloid leukemia.145 in the commented article the results showed that cuo/ /cu2o/cile electrode possesses good electrocatalytic activity in the target reaction with a linear current response in the range from 0.1 to 110 mm formaldehyde and a detection limit of 10 μm. the authors have proven the good reproducebility and stability of modified electrode. the long term stability of cuo/cu2o/cile electrode was tested by storing the electrode at room temperature for one month and the current response retained 94 % of its initial response. a simple and green route for synthesis of cuo/cu2o nps together with enhanced electrocatalytic ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. electrochemical sensors based on biosynthesized nanoparticles 423 activity toward formaldehyde oxidation show that the cuo/cu2o/cile is one of the most promising systems for detection of formaldehyde. copper oxide nanoparticles (cuonps) were synthesized using caesalpinia bonducella seed extract via a green synthetic pathway and were evaluated for electrochemical detection of riboflavin (vitamin b2).146 riboflavin is a water -soluble vitamin needed for the proper functioning of human organs, such as it plays an essential role in the sequence of protein, carbohydrate and fat metabolism. the lack of vitamin b2 leads to skin disorders and eye lesions. at the same time excess of riboflavin in the human body is dangerous because leads to damage to dna and tissues. riboflavin cannot be produced by the human body – it is provided through dietary supplements and pharmaceutical products. therefore, it is essential to monitor vitamin b2 in situ in real food and pharmaceutical samples. sukumar et al. have reported on the development of modified paraffinimpregnated graphite electrode cuonps/pig as a suitable sensor for the determination of nanomolar concentration of vitamin b2 with an observed linear range of 3.13−56.3 nm and a limit of detection of 1.04 nm. the electrode showed satisfactory stability over a period of 4 months – 95 % residual activity was recorded after 80 days and 80 % after 120 days, respectively. the practical applicability of cuonps/pig was checked with real samples – egg yolk, milk powder and commercially available b-complex tablets. the concentration of vitamin b2 was evaluated by the standard addition method, and the recovery values range from 99 to 99.75 %. the results showed high recovery and the authors stated that this method could be extended for further practical applications. kumar et al. have been demonstrated a facile and eco-friendly approach for the simultaneous reduction of graphene oxide as well as copper acetate to prepare cu2o decorated reduced go (rgo/cu2o).147 in this work an easily available and naturally occurring mango bark (m. indica) extract has been used as the reducing agent instead of hazardous and toxic chemicals. fourier transform infrared and x-ray photoelectron spectroscopy have been performed to confirm the removal of oxygen functional groups from the surface of go and the x-ray diffraction pattern reveals the formation of cu2onps. the electrocatalytic behaviour of the resultant rgo/cu2o composite has been carried by cv and constant potential amperometry. the utility of rgo/cu2o as an electrochemical sensor towards h2o2 detection was shown; the sensitivity and limit of detection were found to be 7.435 µa µm–1 and 42.35 nm, respectively. amanulla et al. have reported the development of a sensitive and selective amperometric sensor for h2o2, using for the first time biosynthesized iron nanoparticles (fenps).148 a simple and facile green process was used for the synthesis of fenps decorated rgo nanocomposite using ipomoea pestigridis leaf extract as a reducing and stabilizing agent. the physicochemical results confirmed the successful formation of rgo/fenps composite; tem images showed ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 424 dodevska et al. that the fenps were evenly distributed on rgo surface with an average diameter of 28±4 nm. the nanocomposite rgo/fenps was further modified on gc electrode and used for h2o2 sensing. cv data reveal that rgo/fenps nanocomposite has an excellent behavior to h2o2 electroreduction when compared to the response of fenps and rgo modified electrodes. amperometry was further used to quantify selectively h2o2 using rgo/fenps nanocomposite: the electrode response at an applied potential of –0.5 v (vs. ag/agcl) was linear over the concentration range from 0.1 µm to 2.15 mm and exceptionally fast (2 s). the limit of detection and sensitivity of the proposed sensor were estimated as 0.056 µm and 0.2085 µa µm–1 cm–2, respectively. the practical ability of the fabricated sensor was examined in commercial contact lens solution, human serum and urine samples. selenium nanoparticles (senps) synthesis has been achieved by either physical or chemical methods, which suffer from drawback like high pressure, low yields and longer growth times which cannot be regarded as an eco-friendly process. prasad et al. have developed an eco-friendly and simple method for senps synthesis using a selenium-resistant bacterium identified as bacillus pumilus sp. bab-3706 cell-free extract.149 a working electrode was modified by coating the surface of indium tin oxide (ito) with resulting colloidal senps (size 10–80 nm). the proposed sensor senps/ito exhibited good electrocatalytical activity towards the reduction of h2o2 and low detection limit. hence, microbial senps can be a promising source for the development of electrochemical sensor system for h2o2. among various metallic nanoparticles, platinum (pt) and palladium (pd) nanoparticles are the most widely studied due to their extremely high catalytic/ /electrocatalytic activity. due to their extensive applications in sensors, biosensors and catalysts, production of ptnps and pdnps through environment friendly methods has a significant role. in this regard, momeni et al. have presented green synthesis of pdnps using natural and low-cost crude extract derived from the marine alga sargassum bovinum.93 tem study confirmed the monodispersed and octahedral shape of pdnps within the size ranges 5–10 nm. electrocatalytic performance of the so-biosynthesized pdnps towards reduction of h2o2 was investigated. pdnps-modified carbon ionic liquid electrode (pdnps/cile) was developed as a nonenzymatic sensor for the determination of hydrogen peroxide. amperometric measurements at potential of –0.2 v (vs. ag/agcl) showed that pdnps/cile has an excellent stability and it is a reliable sensor for the detection of h2o2 in a wide range of 5.0 μm–15.0 mm with a sensitivity of 284.35 ma mm−1 cm−2 and a detection limit of 1.0 μm h2o2. the method is reliable – rsd of current response to 1 mm h2o2 of five separate electrodes made with different cile pastes was calculated to be 3.2 %, and a single electrode using five consecutive determinations showed average rsd of less than 2.5 %. the authors concluded that the interesting performances of the electrode coupled with its simple, ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. electrochemical sensors based on biosynthesized nanoparticles 425 effective and green preparation procedure of pdnps synthesis, without any surfactants and templates, make it a promising electrochemical sensing platform. rapid and eco-friendly synthesis of platinum nanoparticles (ptnps) using aqueous leaves extract of quercus glauca has been reported for first time for detection of hydrazine.150 the prepared ptnps were spherical in shape and size from 5–15 nm. the electrocatalytic performance of modified electrode ptnps/gc for hydrazine has been studied by cv and amperometric techniques. cyclic voltammogram of ptnps/gc showed a sharp peak at a very lower onset oxidation potential of –0.3 v. the fabricated hydrazine sensor showed excellent selectivity, low detection limit of 7 nm, wide linear range from 0.01 to 283 µm and a sensitivity of 1.704 µa µm–1 cm–2. the sensor was successfully used for the detection of hydrazine in spiked water samples. manganese oxide nanoparticles (mnonps) of different sizes were synthesized in aqueous medium using clove, i.e., syzygium aromaticum extract (ce) as reducing and stabilizing agent. mnonps with size ~4 nm were used for the electrochemical sensing of p-nitrophenol – a toxic pollutant released by textile and leather industries, iron and steel production, pharmaceutical manufacturing, rubber processing, production of electrical and electronic components.151 acute inhalation or ingestion of p-nitrophenol in humans leads to headache, drowsiness, nausea and cyanosis (result of methemoglobinemia). endocrine disrupting effect and hypothalamic pituitary gonadal toxicity of p-nitrophenol on animals have been documented recently.152 p-nitrophenol exposure disrupted steroidogenesis during the ovarian development in female rats, reduced testosterone synthesis, caused morphological changes in testes, and ultimately decreased semen quality in the roosters.153,154 the mnonps, prepared using ce-based green chemistry approach, were useful for p-nitrophenol sensing. mnonps-modified gold electrode detected p-nitrophenol with good sensitivity (0.16 µa µm–1 cm–2) and detection limit of 15.65 µm. an overview on the main operational parameters of electrodes modified with biosynthesized metal nps or metal oxide nps is presented in table iii. table iii. operational characteristics of electrochemical sensors based on other biosynthesized metal nps and metal oxide nps; ua (uric acid); mgpe (modified graphite paste electrode); egcg (epigallocatechin gallate); cile (carbon ionic liquid electrode); pig (paraffin-impregnated graphite electrode); ito (indium tin oxide); bca (butyl carbitol acetate). other abbreviations are the same as table i modified electrode reducingagent method (e / v) analyte sensitivity (linear range) (lod) stability (rsd) pdnps/cile93 sargassum bovinum amp. (–0.2a) h2o2 284.35 (5.0–15000 μm) (1.0 μm) – (2.5 %) ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 426 dodevska et al. table iii. continued modified electrode reducingagent method (e / v) analyte sensitivity (linear range) (lod) stability (rsd) rgo/fenps/gc148 ipomoea pes-tigridis amp. (–0.5a) h2o2 0.2085 μa μm-1 cm-2 (0.1–2150 μm) (0.056 μm) – (3.6 %) senps/ito149 bacillus pumilus amp. (–1.0b) h2o2 16.54 (5.0–600 mm) (3.0 μm) – (–) cu2o/rgo147 m. indica amp. (–0.2a) h2o2 7.435 μa μm-1 (0.2–3.6 μm) (42.35 nm) 15 days (–) pdag/cpe155 fungi dpv (0.3a) ua – (up to 273.0 nm) (5.543 nm) – (–) cu/cu2o/cuonps/gc15 6 pomegranate cv (–0.3a) ethanol 0.049 μa mm-1 (0.5–2.0 μm) (0.09 μm) – (–) cuo/cu2o/cile144 gum arabic cv ( 0.4a) formaldehyde 186.0 (0.1–110 mm) (10 μm) 30 days (4.6 %) coonps/cpe157 gelatin cv (–0.34a) glucose 609.04 mm-1 cm-2 (7.0–1000.0 μm) (5.3 μm) 4 weeks (2.9 %) ptnps/gc150 quercus glauca amp. (–0.18a) hydrazine 1.704 μa μm-1 cm-2 (0.01–283.0 μm) (7 nm) – (–) mnonps/bca/au151 syzygium aromaticum dpv (–0.69a) p-nitrophenol 0.16 μa μm-1 cm-2 (200.0–550.0 μm) (15.65 μm) – (–) cuonps/pig146 caesalpinia bonducella swv (0.03) riboflavin – (3.13–56.3 nm) (1.04 nm) 120 days (–) 5. conclusions and future perspectives over the past decade intensive research on the possibility of using plant extracts or microorganisms to produce stable metal nanoparticles with pronounced antibacterial and antitumor activity, as well as studies focused on biosynthesized nanoparticles as catalytically active components in the development of new electrocatalysts, have been observed. there is convincing evidence that green synthesis of metal and metal oxide nanoparticles has a potential to provide a new direction in the fabrication of cheap and highly effective electrocatalysts applicable in food, clinical, pharmaceutical and environmental analysis. in this review we have been summarized the main approaches for biosynthesis of metal nanoparticles and their use in the construction of novel electrochemical sensor platforms. it was shown that the biosynthesized metal nanoparticles produced by plants and microorganisms are successfully applied in designing of electrochemical sensors for detection of broad spectrum of analytes. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. electrochemical sensors based on biosynthesized nanoparticles 427 currently, researchers have focused their attention on detection of biomolecules involved in the synthesis of metal nanoparticles as well as on understanding the principles/pathways and mechanisms of nanoparticle biosynthesis. since the properties of nanomaterials strongly depend on their size and shape, efforts should also be focused on optimizing the experimental conditions to yield stable and reproducible size-controlled biosynthesis of nanoparticles. therefore, researchers should refine the biological mediated synthesis protocols to ensure increasing the sustainability of the processes involved in the production of nanoparticles. scalability of the production method also is a necessary – appropriate technologies need to be developed for safe and efficient production of these novel nanomaterials on a commercially sustainable scale while maintaining a satisfactory size distribution. in terms of electrochemical sensing applications selectivity and reproducibility of the electrode signal in real complex matrices, as well as long-term stability of the modified material are extremely important. the experimental data show that the modified with biosynthesized nanoparticles surfaces still remain challenging as they are not often as reproducible and stable as one would hope. further research needs to be done to address these issues and to improve the electrode performance. consequently the emphasis should be on achieving a higher operational and storage stability of the electrode-catalysts modified with biosynthesized metal/metal oxide nanoparticles. expectations in this area are also related with broadening the spectrum of target analytes, as well as development of surface modification methods capable of enhancing the electrode sensitivity and selectivity. in summary, the significant development of electrochemical sensor platforms based on biosynthesized nanomaterials is giving rise to new impetus of generating novel sustainable bio-based technologies for analysis and securing the environmental and food safety. и з в о д примена биосинтетисаних металних наночестица у електрохемијским сензорима totka dodevska, dobrin hadzhiev, ivan shterev и yanna lazarova department of organic chemistry and inorganic chemistry, university of food technology, 26 maritsa boulevard, plovdiv 4002, bulgaria у новије време развој еколошки прихватљивих, исплативих и поузданих метода синтезе металних наночестица привлачи значајну пажњу. такозване зелене синтезе, које користе благе реакционе услове и природна средства као што су биљни екстракти и микроорганизми, успостављене су као погодан, одржив, јефтин и еколошки безбедан приступ синтези разноврсних наноматеријала. током протекле деценије биосинтеза се наметнула као значајна метода којом се смањују штетни ефекти традиционалних метода синтезе наночестица које су уобичајене у лабораторијама и индустрији. овај прегледни ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2022 scs. 428 dodevska et al. рад наглашава значај биосинтетисаних металних наночестица у области електрохемијских сензора. све је више доказа да зелена ситеза наночестица пружа нови правац у дизајнирању исплативих, високо осетљивих и селективних катализатора за електроде које се примењују у анализи хране, као и у клиничким анализама и заштити животне средине. рад је базиран на 157 литературних навода и даје детаљан преглед главних приступа зеленој синтези металних наночестица и њиховој примени у електрохемијским сензорима. такође су обухваћене значајне радне карактеристике укључујући осетљивост, динамички опсег, границу детекције, као и податке о стабилности и репродуктивности сензора. 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sensors}}, year = {2022}, issn = {1820-7421}, month = {apr}, number = {4}, pages = {401--435}, volume = {87}, abstract = {recently, the development of eco-friendly, cost-effective and reliable methods for synthesis of metal nanoparticles has drawn a considerable atten­tion. the so-called green synthesis, using mild reaction conditions and natural resources as plant extracts and microorganisms, has established as a conve­nient, sustainable, cheap and environmentally safe approach for synthesis of a wide range of nanomaterials. over the past decade, biosynthesis is regarded as an important tool for reducing the harmful effects of traditional nanoparticle synthesis methods commonly used in laboratories and industry. this review emphasizes the significance of biosynthesized metal nanoparticles in the field of electrochemical sensing. there is increasing evidence that green synthesis of nanoparticles provides a new direction in designing of cost-effective, highly sensitive and selective electrode-catalysts applicable in food, clinical and envi­ronmental analysis. the article is based on 157 references and provided a det­ailed overview on the main approaches for green synthesis of metal nano­par­ticles and their applications in designing of electrochemical sensor devices. important operational characteristics including sensitivity, dynamic range, limit of detection, as well as data on stability and reproducibility of sensors have also been covered.}, doi = {10.2298/jsc200521077d}, file = {::;:01_10786_5531.pdf:pdf}, keywords = {bionanotechnology., green synthesis, modified electrodes, nanomaterials, review contents}, publisher = {serbian chemical society}, url = {https://www.shd-pub.org.rs/index.php/jscs/article/view/10786}, } electrocatalytic multicomponent assembling of aminouracils, aldehydes and malononitrile: an efficient approach to 7-aminopyrido[2,3-d]pyrimidine-6-carbonitrile derivatives j. serb. chem. soc. 81 (1) 29–34 (2016) udc 544.653.1.547:547.233’821.547.462’496.3– jscs–4824 304.2+547.465 original scientific paper 29 electrocatalytic multicomponent assembling of aminouracils, aldehydes and malononitrile: an efficient approach to 7-aminopyrido[2,3-d]pyrimidine-6-carbonitrile derivatives reyhaneh kazemi-rad*, javad azizian and hassan kefayati department of chemistry, science and research branch, islamic azad university, tehran, iran (received 10 february, accepted 1 june 2015) abstract: the electrocatalytic multicomponent transformation of 6-aminouracils, aromatic aldehydes and malononitrile in ethanol in an undivided cell in the presence of potassium bromide as an electrolyte leads to 7-aminopyrido[2,3 -d]pyrimidine-6-carbonitrile derivatives in short reaction times (8–20 min) and in good to high yields (70–86 %). keywords: aldehyde; electrocatalytic transformation; multicomponent reactions; malononitrile; pyrido[2,3-d]pyrimidine; uracil. introduction uracil and its derivatives, such as pyrido[2,3-d]pyrimidines, have received considerable attention over the past years because of their biological activities, such as dihydrofolate reductase inhibiting,1 antibacterial,2 antiallergic,3 antimicrobial,4 tyrosine kinase inhibiting,5 anti-inflammatory,6 analgesic,7 calcium channel antagonists,8 antihypertensive,9 antitubercular,10 antileishmanial11 and antifungal.12 several synthetic methodologies for the synthesis of 7-aminopyrido[2,3-d]pyrimidine-6-carbonitrile derivatives have been reported.13–17 however, in spite of their potential utility, most of the reported synthetic methods suffer from limitations, such as the use of expensive catalysts, long reaction times, difficult work -up and commercial non-availability. therefore, any new, facile and highly efficient synthetic approach to corresponding 7-aminopyrido[2,3-d]pyrimidine-6 -carbonitrile derivatives is welcome. in recent years, electrosynthetic, multicomponent reactions (emcrs) have been used extensively to prepare biologically active compounds and have become an important area of research in organic, combinatorial, and medicinal * corresponding author. e-mail: reyhanehkazemi83@gmail.com doi: 10.2298/jsc150210048k 30 kazemi-rad, azizian and kefayati chemistry.18 due to electron transfer between the electrode and the substrate molecules, highly reactive intermediates are formed under mild conditions, thereby avoiding reductant or oxidant agents as well as acids, bases and related waste by-products. thus, such methods could be one of the various fields in green chemistry.19 all these facts prompted the present design of a convenient and facile synthesis of 7-aminopyrido[2,3-d]pyrimidine-6-carbonitrile compounds based on the electrochemically induced, multicomponent reaction of aromatic aldehydes, malononitrile and 6-aminouracils in an undivided cell without a base or any additive catalyst (scheme 1). electrolysis etoh, kbr archo + cn cn + rn n r o x nh2 rn n r o x n nh2 cn ar r= h, ch3 x= o, s 1a-j 3a-j2 4a-j scheme 1. synthesis of 7-aminopyrido[2,3-d]pyrimidine-6-carbonitrile derivatives. results and discussion during the course of the study on the electrochemical transformation of organic compounds,20,21 a new strategy was suggested for the synthesis of pyrido[2,3-d]pyrimidines by the combined electrolysis of aromatic aldehydes 1, malononitrile 2 and 6-aminouracils 3 in alcohol in an undivided cell. to optimize the reaction conditions, the three-component reaction of 4-chlorobenzaldehyde 1a, malononitrile 2 and 6-aminouracil 3a was investigated as a model reaction in an alcohol using an undivided cell containing a zinc electrode as the cathode and a pt electrode as the anode in the presence of potassium bromide as the electrolyte. at first, the effects of solvent, temperature and current in the synthesis of 4a were investigated (table i). as indicated in table i, excellent conversions of starting compounds were obtained after 0.16f of electricity had been passed. a current density 5 ma cm–2 (i = 25 ma, electrode surface 5 cm2) in ethanol at 78 °c was found to be optimum for the electrochemically induced chain process and allowed for the highest yield (80 %) of 4a. an increase in the current density up to 10 ma cm–2 (i = 50 ma) resulted in a slight decrease of the reaction yield, which may be connected with the activation of undesired direct electrochemical processes possible under these conditions and leading to oligomerization of the starting material. under the optimal conditions (current density 5 ma cm–2, etoh as solvent), the electrolysis of aromatic aldehydes 1a–j, malononitrile 2 and 6-aminouracils electrosynthesis of pyrido[2,3-d]pyrimidines 31 3a–j in an undivided cell at 78 °c gave rise to the corresponding 7-aminopyrido[2,3-d]pyrimidine-6-carbonitrile in 70–86 % yield (scheme 1, table ii). table i. electrocatalytic transformation of 4-chlorobenzaldehyde (1a, 1 mmol), malononitrile (1 mmol) and 6-aminouracil (1 mmol) into 7-aminopyrido[2,3-d]pyrimidine-6 -carbonitrile (4a); kbr (0.1 mmol), alcohol (15 ml), zinc cathode (5 cm2), platinum anode (5 cm2) entry alcohol t °c i ma current density ma cm-2 time min electricity passed as parts of f yield % 1 etoh 20 25 5 30 0.46 – 2 etoh 60 25 5 30 0.46 – 3 etoh 78 5 1 30 0.09 32 4 etoh 78 10 2 15 0.09 78 5 etoh 78 25 5 10 0.16 80 6 etoh 78 50 10 5 0.16 70 7 meoh 60 25 5 15 0.23 – 8 n-proh 97 25 5 15 0.23 75 table ii. electrocatalytic multicomponent synthesis of 7-aminopyrido[2,3-d]pyrimidine-6-carbonitrile derivatives (4a–j) under the optimized conditions; aromatic aldehyde (1 mmol), malononitrile (1 mmol), 6-aminouracil (1 mmol), kbr (0.1 mmol), ethanol (15 ml), zinc cathode (5 cm2), platinum anode (5 cm2), 78 °c compd. ar r x time min electricity passed as parts of f yielda % 4a 4-clc6h4 h o 10 0.16 80 4b 4-och3c6h4 h o 15 0.23 78 4c 3-no2c6h4 h o 10 0.16 85 4d 4-no2c6h4 h o 8 0.12 84 4e 4-clc6h4 h s 12 0.19 82 4f 4-no2c6h4 h s 10 0.16 86 4g 4-clc6h4 ch3 o 20 0.31 71 4h 3-brc6h4 ch3 o 20 0.31 70 4i 3-no2c6h4 ch3 o 20 0.31 76 4j 4-no2c6h4 ch3 o 20 0.31 72 aisolated yields taking into consideration the above results, the following mechanism for the electrocatalytic chain transformation of aromatic aldehydes 1, malononitrile 2 and 6-aminouracils 3 into the corresponding 7-aminopyrido[2,3-d]pyrimidine-6 -carbonitrile 4 is proposed. as the initiation step of the catalytic cycle, the deprotonation of an ethanol molecule at the cathode leads to the formation of an ethoxide anion. the subsequent reaction in solution between the ethoxide anion and malononitrile gives rise to a malononitrile anion (scheme 2). then knoevenagel condensation of the malononitrile anion with an aromatic aldehyde occurs in the solution with the 32 kazemi-rad, azizian and kefayati elimination of a hydroxide anion and formation of arylidene malononitrile 5. the subsequent ethoxide-promoted michael addition of 6-aminouracil 3 to electron deficient knoevenagel adduct 5 followed by intramolecular cyclization and tautomerization of intermediate 6 leads to intermediate 7 with the regeneration of the ethoxide anion, which continues the catalytic chain process by interaction with the next molecule of malononitrile. the intermediate 7 is oxidized to afford the fully aromatized compound 4 (scheme 3). 2 2 2 2 cathode: 2etoh 2e 2eto h in solution: 2ch (cn) 2eto 2 ch(cn) 2etoh          scheme 2. formation of ethoxide anion at the cathode. the beginning of new catalytic cycle ar h o nc nc ar o ch(cn)2 rn n r nh o x ar cn c n rn n r n h o x ar cn nh oh 1 rn n r n h o x ar cn nh2 -2haromatization etoh rn n r n o x ar cn nh2 rn n r nh2 o x michael addition hh nc nc ar h 5 + etoh ch2(cn)2 eto oet oet 4 3 67 scheme 3. a proposed mechanism for the electrocatalytic transformation of aromatic aldehydes 1, malononitrile 2 and 6-aminouracils 3 into the corresponding pyrido[2,3-d]pyrimidine 4. experimental general all reagents were purchased from merck or fluka and used without further purification. melting points were obtained in open capillary tubes and were measured on an electrothermal ia 9100 apparatus. the ir spectra were recorded in kbr pellets on a shimadzu ft-ir 8600 spectrophotometer. the 1hand 13c-nmr spectra were determined on a bruker drx-400 avance instrument at 400 and 100 mhz, respectively. electrosynthesis of pyrido[2,3-d]pyrimidines 33 the physical, analytical and spectral data for compounds 4a–j are given in the supplementary material to this paper. general procedure for electrochemical synthesis of pyrido[2,3-d]pyrimidines 4a–j a mixture of aromatic aldehyde (1 mmol), malononitrile (0.66 g, 1 mmol), 6-aminouracil (1 mmol), and kbr (0.1 g, 1 mmol) in etoh (15 ml) was electrolyzed at 78 °c in an undivided cell equipped with a magnetic stirrer, a platinum anode and a zinc cathode under a constant current density of 5 ma cm-2. the progress of the reaction was monitored by thin layer chromatography. after the electrolysis was finished, the mixture was filtered and the filter cake was washed twice with an ethanol/water (1:1) solution to yield pure products 4a–j. conclusion in conclusion, the electrocatalytic transformation of aromatic aldehydes, malononitrile and 6-aminouracils into the corresponding pyrido[2,3-d]pyrimidines, in comparison with conventional methods, has advantages, such as: i) in situ generation of base and the avoidance of pollution or hazardous chemicals or the addition of base or pro-base, ii) a very fast one-pot reaction in good to excellent yields under milder conditions, iii) the procedure utilizes inexpensive reagents, simple equipment and convenient work-up and iv) it is easily performed and is fully beneficial from the viewpoint of being an ecological method for organic syntheses and suitable for large-scale processes. supplementary material physical, analytical and spectral data of compounds 4a–j are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. financial support for this work from the research council of the islamic azad university, tehran science and research branch is gratefully acknowledged. и з в о д електрокаталитичка вишекомпонентна реакција аминоурацила, алдехида и малонoнитрила: ефикасан приступ синтези деривата 7-амино-пиридо[2,3-d]пиримидин-6-карбонитрила reyhaneh kazemi-rad, javad azizian и hassan kefayati department of chemistry, science and research branch, islamic azad university, tehran, iran електрокаталитичком вишекомпонентном трансформацијом 6-аминоурацила, ароматичних алдехида и малонoнитрила у етанолу, у неподељеној ћелији у присуству калијум-бромида као електролита, добијени су деривати 7-амино-пиридо[2,3-d]пиримидин-6-карбонитрила у кратком реакционом времену (8–20 min) у добром до високом приносу. (примљено 10. фебруара, прихваћено 1. јуна 2015) references 1. a. gangjee, o. adair, s. f. queener, bioorg. med. chem. 9 (2001) 2929 2. l. nargund, y. reddy, r. jose, indian drugs 29 (1991) 45 34 kazemi-rad, azizian and kefayati 3. i. hermecz, a. horvath, z. meszaros, c. de vos, l. rodriguez, j. med. chem. 27 (1984) 1253 4. i. o. donkor, c. l. klein, l. liang, n. zhu, e. bradley, a. m. clark, j. pharm. sci. 84 (1995) 661 5. a. m. thompson, a. j. bridges, d. w. fry, a. j. kraker, w. a. denny, j. med. chem. 38 (1995) 3780 6. v. kolla, a. deyanov, f. nazmetdinov, z. kashina, l. drovosekova, khim. farm. zh. 27 (1993) 29 7. c. dave, p. shah, g. shah, p. pandya, k. dave, v. patel, indian j. pharm. sci. 48 (1986) 75 8. a. pastor, r. alajarin, j. j. vaquero, j. alvarez-builla, m. faude casa-juana, c. sunkel, j. g. priego, i. fonseca, j. sanz-aparicio, tetrahedron 50 (1994) 8085 9. l. r. bennett, c. j. blankley, r. w. fleming, r. d. smith, d. k. tessman, j. med. chem. 24 (1981) 382 10. i. bystryakova, i. burova, g. chelysheva, s. zhilinkova, n. smirnova, t. safonova, khim. farm. zh. 25 (1991) 31 11. a. agarwal, n. goyal, p. chauhan, s. gupta, bioorg. med. chem. 13 (2005) 6678 12. j. quiroga, c. cisneros, b. insuasty, r. abonia, s. cruz, m. nogueras, j. m. de la torre, m. sortino, s. zacchino, j. heterocycl. chem. 43 (2006) 299 13. a. b. deyanov, r. k. niyazov, f. y. nazmetdinov, b. y. syropyatov, v. e. kolla, m. e. konshin, khim. farm. zh. 25 (1991) 26 14. s. abdolmohammadi, s. balalaie, int. j. org. chem. 2 (2012) 7 15. e. m. hussein, z. naturforsch., b 67 (2012) 231 16. d. shi, l. niu, j. shi, x. wang, s. ji, j. heterocycl. chem. 44 (2007) 1083 17. r. sarma, k. j. borah, y. dommaraju, d. prajapati, mol. diversity 15 (2011) 697 18. m. n. elinson, v. m. merkulova, a. i. ilovaiskya, f. barba, b. batanero, electrochim. acta 56 (2011) 8219 19. (a) m. n. elinson, a. s. dorofeev, s. k. feducovich, r. f. nasybullin, s. v. gorbunov, g. i. nikishin, electrochem. commun. (2006) 1567; (b) m. n. elinson, a. s. dorofeev, r. f. nasybullin, g. i. nikishin, synthesis (2008) 1933 20. h. kefayati, s. h. amlashi, r. kazemi-rad, a. delafrooz, c. r. chim. 17 (2014) 894 21. r. kazemi-rad, j. azizian, h. kefayati, tetrahedron lett. 55 (2014), 6887. spectral, thermal, optical and biological studies on (e)-4-[(2-hydroxyphenyl)imino]pentan-2-one and its complexes j. serb. chem. soc. 81 (1) 57–66 (2016) udc 542.9+547.571+547.551:546.562’732’742’ jscs–4827 472:547.292:543.57:615.281/.282–188 original scientific paper 57 spectral, thermal, optical and biological studies on (e)-4-[(2-hydroxyphenyl)imino]pentan-2-one and its complexes nasser m. hosny1*, reyad ibrahim and ahmed a. el-asmy 1chemistry department, faculty of science, port said university, port said, egypt, 2chemistry department, faculty of science, mansoura university, mansoura, egypt and 3chemistry department, faculty of science, kuwait university, kuwait (received 1 february, revised 24 april, accepted 7 july 2015) abstract: metal complexes derived from the reaction of cu(ii), co(ii), ni(ii) and zn(ii) acetates and (e)-4-[(2-hydroxyphenyl)imino]pentan-2-one (h2l) were synthesized and characterized by elemental analyses, ms, ir, uv–vis and 1h-nmr spectroscopy, thermogravimetry (tg) and differential tg (dtg), and magnetic measurements. in all complexes except for the zn(ii) complex, the schiff base ligand acts as a mono-negative tridentate (noo) donor, through the azomethine nitrogen, the hydroxyl oxygen and the enolic carbonyl oxygen. the structure of the cu(ii) complex is square planar, the co(ii) is octahedral while, the ni(ii) and zn(ii) are tetrahedral. optical band gap measurements indicated the semi-conducting nature of these complexes. the biological activities were screened against two bacteria and two fungi. some of the studied complexes showed activity against bacteria and fungi. keywords: schiff bases; band gap; anti-fungal and anti-bacterial agents. introduction complexes of schiff bases represent an important class of compounds. the enol form of schiff bases of monoand diamines have attracted much attention because of their advantages, such as ease of synthesis and the presence of variety of coordination sites that make them invaluable in coordination chemistry.1,2 schiff base complexes have many applications, e.g., they are used as catalyst in oxygenation, decomposition, electro-reduction and hydrolysis reactions.3,4 schiff bases of β-diketones stabilize different metals in various oxidation states in many useful catalytic transformations.5 moreover, schiff base complexes showed optical6 and magnetic properties7 that enabled them to be used as good candidates in modern technologies. furthermore, schiff-base complexes have found biological applications as antibacterial,8–11 anti-hiv12 and anti-inflammatory * corresponding author. e-mail: nasserh56@yahoo.com doi: 10.2298/jsc150201058h 58 hosny, ibrahim and el-asmy agents.13 the biological activity of schiff bases comes from their ability to bind with transition metal ions in living systems.14,15 this work describes the synthesis and spectral, thermal, optical and biological activities of cu(ii), co(ii), ni(ii) and zn(ii) complexes with (e)-4-[(2hydroxyphenyl)imino]pentan-2-one with the aim of shedding more light on their structures and biological application. results and discussion all the isolated complexes were colored and soluble in coordinating solvents such as dimethyl sulfoxide (dmso) and dimethylformamide (dmf) but insoluble in h2o. the ligand can exist either in the keto (fig. 1, a) or in the enol form (fig. 1, b). fig. 1. the keto (a) and enol (b) tautomeric forms of the ligand. ir spectra of the complexes the most important ir bands of the ligand and its complexes are given in the supplementary material to this paper. the bands at 3435 and 3380 cm–1 in the spectrum of the ligand are attributed to phenolic and enolic (oh), respectively, while the bands due to (c=n), (ch=c), δ(oh), (c–o) and (c–n) appear at 1598, 1546, 1033, 1315 and 1238 cm–1 respectively.16 the absence of the band assigned to (c=o) of pentanedione and the appearance of a new band at 1546 cm–1 due to (hc=c) confirmed that the free ligand exists in the enol form. the ir spectra of the metal complexes showed the disappearance of the enolic oh band, indicating the participation of this group in bonding after deprotonation. the shift of (c=n) to 1586–1615 cm–1 confirmed that the azomethine group coordinated to the metal ion. the spectra of cu(ii), co(ii) and ni(ii) complexes exhibited bands in the region 3440–3469 cm–1, assigned to the phenolic oh. furthermore, these complexes showed two new bands assigned to as(coo–) and s(coo–) at 1534 and 1333 cm–1 in the co(ii) complex and at 1565 and 1375–1345 cm–1 in the cu(ii) and ni(ii) complexes, respectively. these bands did not appear in the spectrum of the zn(ii) complex. the difference between as(coo–) and s(coo–) bands indicates the bidentate nature in the co(ii) complex and monodentate in the schiff base complexes 59 cu(ii) and ni(ii) complexes.17–20 the shift of the bands at 1315 and 1284 cm–1, due to the phenolic and enolic c–o, supports the participation of the phenolic and enolic oxygens in the bonding. the spectra showed new bands in the regions 530–540 and 426–435 cm–1, assigned to (m–o) and (m–n), respectively.21 these findings suggest the involvement of c=n, c–o and the phenolic hydroxyl groups in the coordination (figs. 2–4).   n m o h3c ch3 oh nh2o xetoh oac .. fig. 2. suggested structure of the cu(ii) and ni(ii) complexes, n = 1.5 and x = 0 for the cu(ii), and n = 1, x = 1/2 for the ni(ii) complex. fig. 3. suggested structure for the zn(ii) complex.   n co o h3c ch3 1/2 etoh o o ch3 oh2 o h . fig. 4. suggested structure for the co(ii) complex. 1h-nmr spectra the 1h-nmr spectrum of the ligand in dmso-d6 showed two singlet signals at 2.34 and 2.50 ppm downfield from tms, assigned to the protons of the two ch3 groups. in addition, another singlet signal at 5.20 ppm corresponded to the proton of the hc=c group.22 the multiplet signal in the region 6.75–7.18 ppm was assigned to the phenyl protons. the two singlet signals at 9.93 and 12.15 ppm were attributed to the phenolic and enolic oh protons, respectively.23 the presence of the enolic proton together with hc=c confirmed that the ligand exists in the enol form. 60 hosny, ibrahim and el-asmy the 1h-nmr spectrum of the zn(ii) complex in dmso-d6 showed two singlet signals at 2.23 and 2.43 ppm due to the protons of the two ch3 groups. the multiplet signal in the region 6.96–7.11 ppm corresponded to the phenyl protons. the spectrum showed another singlet signal at 7.6 ppm, assigned to the proton of the hc=c group. the shift of the latter signal in comparison with its position in the free ligand indicates the participation of the enolic group in the bonding. the disappearance of the enolic and phenolic oh protons confirmed the participation of these groups in the bonding. mass spectra of the complexes the mass spectral fragmentation mode of h2l was investigated. it showed an intense molecular ion peak at m/z 191, corresponding to the formula c11h13no2. a stable peak at 176 was due to the loss of ch3. the loss of hydroxyl group gives a peak at m/z 160. the latter peak loses c2h2 with rearrangement to give the base peak at m/z 134. the base peak undergoes loss of ch3 to give a peak at m/z 120, which undergoes loss of ch, oh, oh and ch4 to give peaks at m/z 109, 93, 80 and 63, respectively, as indicated in scheme s-1 of the supplementary material to this paper. the ms of the cu(ii) complex exhibited the molecular ion peak at m/z 340, in good agreement with the formula [cu(hl)(oac)]·1.5h2o. its fragmentation pattern is shown in scheme s-2 of the supplementary material. the mass spectrum of the co(ii) complex showed the molecular ion peak at m/z 350, corresponding to the formula [co(hl)(oac)(h2o)]·0.5etoh. its fragmentation pattern is indicated in scheme s-3 of the supplementary material. the ni(ii) complex has a mass spectrum with the molecular ion peak at m/z 350, corresponding to [ni(hl)(oac)(h2o)]·0.5etoh. the fragmentation pattern of this formula is illustrated in scheme s-4 of the supplementary material. thermal gravimetric analysis thermal gravimetric analysis gives useful data about the thermal stability of the metal complexes. the tg and dtg curves were recorded within the temperature range 25–800 °c. the tg curves for the complexes are given in figs. s-1–s-4 and the results are tabulated in table s-i of the supplementary material to this paper. the results indicated that [cu(hl)(oac)]·1.5h2o decomposed in several steps. the first occurred in the range 26–133 °c, corresponding to the loss of 1.5 h2o (calcd. 7.9 %; found 8.0 %). the final step leaves cuo as a residue (calcd. residue 23.4 %; found 22.6 %). the tg curve of the co(ii) complex showed a decomposition step at 22–104 c, assigned to the loss of 0.5c2h5oh (calcd. 6.5 %; found 5.7 %). the last step could leave co2o3 as the residue (calcd. residue 47.4 %; found 46.5 %). schiff base complexes 61 the tg curve of the ni(ii) complex showed a decomposition step at 54–130 c due to the loss of 0.5c2h5oh (calcd. 6.5 ; found 6.0 %). the last step can generate nio as the residue (calcd. residue 19.8 %; found 20.0 %). the tg curve of the zn(ii) complex showed a decomposition step at 26–200 c, assigned to the loss of coordinated water (calcd. 6.5 %; found 5.6 %). the third step could leave zno as the residue (calcd. residue 29.6 %; found 28.7 %). the intermediate steps for all four complexes appear to represent the decomposition of the ligand to different organic moieties.2,10 electronic spectra and magnetic moments the electronic spectrum of the cu(ii) complex in dmf showed a broad band centered at 14005 cm–1, attributed to 2b1g  2a1g and 2b1g  2eg transitions in a square-planar geometry.24 the band recorded at 26775 cm–1 was assigned to ligand to metal charge transfer (lmct) (table s-ii of the supplementary material). the magnetic moment (2.1 b) of the complex was in the range expected for monomeric cu(ii) complexes. the high value of the square–planar complex compared with those reported for the octahedral may be taken as supporting evidence for the presence of a square-planar geometry.25 the electronic spectrum of the co(ii) complex in dmso showed three bands at 22222, 19379 and 15948 cm–1 due to lm charge-transfer, 4t1g(f)   4t1g(p) and 4t1g(f)  4a2g transitions, respectively. the ligand field parameters (b = 878 cm–1,  = 0.90 and 10dq = 8340 cm–1) correspond with those reported for co(ii) complexes with octahedral geometry.25,26 the value of the magnetic moment (5.2 b) supports the presence of octahedral geometry around the co(ii) ion (table s-ii). the spectrum of the ni(ii) complex, in dmso, exhibits three bands at 15128, 23201 and 25000 cm–1 assigned to 3t1  3t1(p), 3t1  1t2 and lmct transitions, respectively, in a tetrahedral geometry around ni(ii) ion.25 the value of magnetic moment (3.9 b) falls in the range reported for tetrahedral geometry around a ni(ii) ion.26 optimized modeled structures of the complexes were proposed based on the previous analytical and spectral data. the experimental and theoretical electronic spectra of the optimized structures are collected in table s-iii. selected bond lengths and angles are presented in table s-iv of the supplementary material. in case of co(ii) complex, the (n–m–o enolic) angle was 93.67°, which is slightly greater than is normal for an octahedral structure. the corresponding values for the ni(ii), cu(ii) and zn(ii) complexes were 102.59, 102.40 and 99.4°, respectively, deviate from the angle of normal tetrahedral structures. the optimization was performed for single molecules and the interactions between neighboring molecules were not taken into consideration; this may be the cause of the deviations. 62 hosny, ibrahim and el-asmy optical band gap (eg) the aim of measuring the conductive and semi-conductive properties of metal complexes is to reveal the feasibility of their potential use in molecular electronics. according to the conducting properties, metal complexes are applicable in optical devices, organic transistors and sensors.27,28 to clarify the conductivity of the isolated complexes, the optical band gaps (eg) of the co(ii), ni(ii), cu(ii) and zn(ii) complexes was determined from the absorption spectra of these complexes. the absorption coefficient (α) was determined from the relation: 1 ln a d   (1) where d is the path length of the sample. the optical band gap (eg) was calculated from the relation:  g mh a h e    (2) where m is equal to 1/2 and 2 for direct and indirect transition, respectively, and a is an energy independent constant.29,30 a plot of (αhν)2 vs. hν was used to determine the direct band gap (fig. 5) by extrapolating the linear portion of the curve to (αhν)2 = 0. the curves revealed that the values of the direct band gap (eg) were equal to 3.37 (calcd. 4.2), 3.38 (calcd. 3.9), 3.74 (calcd. 3.6) and 3.58 (calcd. 6.0) ev for the co(ii), ni(ii), cu(ii) and zn(ii) complexes, respectively. the band gap values suggest that these complexes may behave as semi-conductors. in addition, the values of eg are in the same range as those of highly fig. 5. optical band gap calculations for the isolated complexes. schiff base complexes 63 efficient photovoltaic materials. the present compounds could be used in harvesting solar radiation.31 biological activity the biological activities of the isolated complexes were tested against two bacteria (staphylococcus aureus ncmb 6571 and vibrio cholerae tcbs) and two fungi (aspergillus niger and a. flavus) by the disc diffusion method. the activities were measured as the diameter of inhibition zone (table i). it is clear that, the four complexes had anti-bacterial activities. the highest anti-bacterial activity was registered for the cu(ii) complex. only the co(ii) complex showed both antibacterial and antifungal activity and hence, it could be used as a wide spectrum antibiotic. the zn(ii) complex showed antifungal activity against a. niger only and hence, it could be considered as a selective antibiotic for this microorganism. the cu(ii) and ni(ii) complexes did not show any effect against the two fungi. table i. anti-microbial activity presented as the diameter of the inhibition zone, cm; –: negative metabolite complex with: co ni cu zn s. aureus 0.7 0.75 0.9 0.85 v. cholerae 1.0 1.0 1.1 0.7 a. niger 2.9 – – 3.0 a. flavus 1.75 – – – experimental reagents all the employed chemicals were of analytical grade and were used without further purification. equipment and measurements the carbon and hydrogen contents were determined using a chn analyzer (perkin– –elmer model 2400). infrared spectra were measured as kbr discs on a mattson 5000 ftir spectrometer. electronic spectra were recorded on a uv2 unicam uv–vis spectrometer using 1 cm silica cells. thermogravimetric measurements (tg) were performed on a shimadzu model 50 h instrument with a nitrogen flow rate and heating rate of 20 cm3 min-1 and 10 °c min-1, respectively. the 1h-nmr spectra were recorded on a bruker avance iii 400 mhz instrument using tms as an internal reference and dmso-d6 as the solvent. the physical, analytical and spectral data for the ligand and its complexes are listed in the supplementary material to this paper. synthesis of (e)-4-[(2-hydroxyphenyl)imino]pentan-2-one (h2l) 2,4-pentanedione (0.01 mol, 1.4 ml) was added dropwise to 0.01 mol (1.19 g) of a hot ethanolic solution of 2-hydroxyaniline. the reaction mixture was heated under reflux for 6 h. 64 hosny, ibrahim and el-asmy a brown crystalline precipitate formed on cooling. the precipitate was filtered off and washed with ethanol and diethyl ether. synthesis of metal complexes a general procedure was followed for the synthesis of all complexes. an aqueous solution of 0.01 mol of metal acetates was added to a hot ethanolic solution of 0.01 mol of (e)-4-[(2-hydroxyphenyl)imino]pentan-2-one (h2l) and refluxed for 8 h. the precipitated metal complexes were filtered off, washed with ethanol, and preserved over anhydrous calcium chloride. computational details molecular mechanics and the semi-empirical methods in the hyperchem series of programs were used to investigate the geometries of the complexes.32 am1 and pm3 methods were used to optimize the molecular geometries of complexes. the low lying conformers obtained by the molecular mechanics technique (mm+ force field) were then optimized at am1, pm3 (polak–ribiere) rms 0.01 kcal.33 antimicrobial assay the microbial strains were selected based on their clinical importance in causing disease in humans. two bacteria (s. aureus ncmb 6571 and v. cholerae) and two fungi (a. niger and a. flavus), ear pathogens isolated from a clinical culture and a laboratory culture of suez canal university center for environmental studies and consultations were used for the evaluation of the antimicrobial activity of the synthesized compounds. the bacteria were subcultured at 37 °c for 6 h on nutrient agar, whereas the fungi were subcultured on sabouraud dextrose agar (subcultured at 28 c for 4–7 days on malt agar). samples (20 μl) of the tested compound at a concentration of 50 mg 10 ml-1 in 0.7 cm holes were incubated against the strains at 37 °c for 48 h using the “disc diffusion method”.34 conclusions new biologically active metal complexes of cu(ii), co(ii), ni(ii) and zn(ii) with (e)-4-[(2-hydroxyphenyl)imino]pentan-2-one (h2l) were synthesized and characterized by different physicochemical techniques. optical band gap measurements indicated that the isolated complexes are semi-conductors. the optical band gap values showed that the compounds could be used as harvesting materials of the sun radiation in the uv and visible regions. the anti-fungal and anti -bacterial tests showed that the synthesized complexes are biologically active against both bacteria and fungi. one of the investigated compounds could be used as a broad-spectrum antibiotic. supplementary material physical, analytical and spectral data for the ligand and its complexes, ms fragmentation patterns, thermogravimetry results, magnetic moments, electronic bands and ligand field parameters, experimental and calculated electronic spectra and selected bond lengths and angles for the optimized ligand and its metal complexes are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. schiff base complexes 65 и з в о д спектрална, термална, оптичка и биолошка испитивања (e)-4-[(2-хидроксифенил)имино]пентан-2-она и његових комплекса са неким јонима прелазних метала nasser m. hosny1, reyad ibrahim и ahmed a. el-asmy 1 chemistry department, faculty of science, port said university, port said, egypt, 2 chemistry department, faculty of science, mansoura university, mansoura, egypt и 3 chemistry department, faculty of science, kuwait university, kuwait комплекси прелазних метала, синтетисани у реакцији између cu(ii)-, co(ii)-, ni(ii)и zn(ii)-ацетата и (e)-4-[(2-хидроксифенил)имино]пентан-2-она (h2l) као лиганда, окарактерисани су помоћу елементалне микроанализе, ms, ir, uv–vis и 1h-nmr спектроскопије, термалне анализе (tg и dtg) и магнетних мерења. у испитиваним комплексима, осим у случају zn(ii) јона, наведена шифова база се тридентатно (noo) координује преко азометинског атома азота, хидроксилног атома кисеоника и енолног атома кисеоника из карбонилне групе. нађено је да cu(ii) комплекс има квадратно-планарну геометрију, док је геометрија co(ii) комплекса октаедарска. за испитиване комплексе ni(ii) и zn(ii) нађено је да имају тетраедарску геометрију. испитивана је биолошка активност комплекса на две врсте бактерија и гљивица. нађено је да неки од испитиваних комплекса имају одређену активност према бактеријама и гљивицама. (примљено 1. фебруара, ревидирано 24. априла, прихваћено 7. јула 2015) references 1. x. pang, x. chen, h. du, x. wang, x. jing, j. organomet. chem. 692 (2007) 5605 2. n. m. hosny, m. a. hussien, f. m. radwan, n. nawar, spectrochim. acta, a 132 (2014) 121 3. n. sohrabi, n. rasouli, m. kamkar, bull. korean chem. soc. 35 (2014) 2523 4. s. sathiyaraj, k. sampath, c. jayabalakrishnan, synth. react. inorg., met.-org., nano -met. chem. 44 (2014) 1261 5. s. kumar, d. n. dhar, p. n. saxena, j. sci. ind. res. 68 (2009) 181 6. r. a. sheldon, j. k. kochi, metal catalyzed oxidation of organic compounds, academic press, new york, 1981, pp. 97, 102 and 105 7. n. m. hosny, j. mol. struct. 923 (2009) 98 8. a. ray, s. banerjee, r. j. butcher, c. desplanches, s. mitra, polyhedron 27 (2008) 2409 9. d. a. brahman, b. sinha, j. serb. chem. soc. 79 (2014) 1505 10. m. a. hussien, n. nawar, f. m. radwan, n. m. hosny, j. mol. struct. 1080 (2015) 162 11. s. kala, p. tharmaraj, c. d. sheela, synth. react. inorg., met.-org., nano-met. chem. 44 (2014) 1487 12. r. garg, a. kumari, s. c. joshi, n. fahmi, bull. korean chem. soc. 34 (2013) 2381 13. d. j. hadjipavlou-litina, a. a. geronikaki, drug des. discovery 15 (1998) 199 14. j. a. anten, d. nicholls, j. m. markopoulos, polyhedron 6 (1987) 1075 15. i. a. tossidis, c. a. bolos, p. n. aslanidis, g. a. katsoulos, inorg. chim. acta 133 (1987) 275 16. k. nakamoto, infrared spectra of inorganic and coordination compounds, wiley, new york, 1970 17. s. cakir, e. coskun, p. naumov, e. bicer, j. mol. struct. 608 (2001) 101 18. n. m. hosny, m. s. zoromba, j. mol. struct. 1027 (2012) 128 19. n. m. hosny, a. dahshan, j. mater. chem. phys. 137 (2012) 637 66 hosny, ibrahim and el-asmy 20. n. m. hosny, j. mater. chem. phys. 144 (2014) 247 21. j. r. ferraro, low frequency vibrations of inorganic and coordination compounds, plenum press, new york, 1971 22. r. l. lintvedt, h. f. holtzdaw jr., j. am. chem. soc. 88 (1966) 2713 23. k. krishnankutty, m. basheer ummathur, p. sayudevi, j. argent. chem. soc. 96 (2008) 13 24. b. jeragh, a. a. el-asmy, spectrochim. acta, a 130 (2014) 546 25. a. b. p. lever, inorganic electronic spectroscopy. elsevier, amsterdam, 1986 26. f. a. cotton, g. wilkinson, advanced inorganic chemistry (a comprehensive text), 4th ed., wiley, new york, 1980 27. f. karipcin, b. dede, y. caglar, d. hur, s. ilican, m. caglar, y. sahin, opt. commun. 272 (2007) 131 28. f. yakuphanoglu, a. cukurovali, i. yilmaz, opt. mater. (amsterdam, net.) 27 (2005) 1363 29. j. tauc, r. grigorovici, a. vancu, nature 15 (1966) 627. 30. r. k. pandey, s. n. sahu, s. chandra, handbook of semiconductor electrodeposition, marcel dekker, new york, 1996, p. 142 31. m. l. fu, g. c. guo, x. liu, l. z. cai,; j. s. huang, inorg. chem. commun. 8 (2005) 18 32. hyperchem 7, hypercube inc., gainesville, fl, 2002 33. j. j. p. stewart, j. comput. chem. 10 (1989) 221 34. u. hentschel, m. usher, m. w. taylor, fems microbiol. ecol. 35 (2001) 305. solid–solid synthesis, characterization and thermal decomposition of a homodinuclear cobalt(ii) complex j. serb. chem. soc. 80 (11) 1391–1397 (2015) udc 546.732+547.821+547.595: jscs–4805 544.421.032.4:548.7:543.57 original scientific paper 1391 solid–solid synthesis, characterization and thermal decomposition of a homodinuclear cobalt(ii) complex di li, guo-qing zhong* and zhi-xian wu school of material science and engineering, southwest university of science and technology, mianyang 621010, china (received 24 february, revised 25 may, accepted 6 june 2015) abstract: the homodinuclear cobalt(ii) complex [co2(dipic)2(h2o)5]·2h2o was synthesized using pyridine-2,6-dicarboxylic acid (h2dipic) and cobalt(ii) acetate as raw materials by room temperature solid–solid reaction. the complex was characterized by elemental analyses, single crystal x-ray diffraction, x-ray powder diffraction, fourier transform infrared spectroscopy, uv spectroscopy, thermogravimetry and differential scanning calorimetry. its crystal structure belongs to monoclinic system and space group p2(1)/c. there are two types of six-coordinated co(ii) ions, one co(ii) is coordinated by four carboxyl o atoms and two pyridine n atoms from two dipic2anions, and another co(ii) is coordinated by five o atoms from five h2o molecules and one bridged carboxyl o atom from the dipic2anion. the possible pyrolysis reactions in the thermal decomposition processes of the complex, the experimental and calculated percentage mass loss are also given. keywords: cobalt(ii) complex; pyridine-2,6-dicarboxylic acid; room temperature solid–solid reaction; crystal structure; thermal analysis. introduction in the past several years, coordination frameworks with transition metal and organic functional ligands have been rapidly developing. however, it still remains one of the most active areas in chemistry and materials science, not only due to their highly fascinating structures and topologically diverse architectures, but also because they have potential applications in gas storage, molecular separations, drug delivery, heterogeneous catalysis, ion exchange, nonlinear optics, and other biomedical applications.1−5 the complexes of pyridinecarboxylic acids and their derivatives were successfully used as model systems for the design of new metallo-pharmaceutical compounds.6 investigations on metalloproteins * corresponding author. e-mail: zgq316@163.com doi: 10.2298/jsc150224050l 1392 li, zhong and wu revealed that the carboxylate group plays an important role for structural folding and proton transfer via hydrogen bonding interactions in proteins.7 pyridine-2,6-dicarboxylic acid (abbreviated as h2dipic) can partly or fully deprotonate, the three coordination sites can undergo chelate or bridge coordination to form diverse structures.8−10 furthermore, h2dipic is usually found in the decomposition products of vitamins, coenzymes and alkaloids, which also have potential pharmacological activity.10,11 cobalt is an essential trace element for humans and animals, which has potential applications in medicine and bioinorganic chemistry, such as blood pressure regulation and proper thyroid function.12,13 the method of room temperature solid–solid reaction is a simple and convenient method for the preparation of complexes. this method is energy-saving and environmentally friendly. in this article, cobalt acetate and the biologically functional ligand h2dipic were chosen to synthesize a homodinuclear complex, using the room temperature solid–solid reaction method. experimental materials and physical measurements h2dipic was purchased from alfa aesar, while cobalt acetate was obtained from merck. all chemicals were of analytical reagent grade and used without further purification. elemental analysis data were obtained using a vario el cube elemental analyzer, and cobalt was determined using a thermo x-ii inductively coupled plasma mass spectrometer. the xrd pattern was recorded on a d/max-ii x-ray diffractometer in the diffraction angle range of 3–80°. the ir spectra were recorded from kbr pellets in the range of 400–4000 cm-1 on a perkin-elmer spectrum one spectrometer. the thermal analysis was realized using a sdt q600 thermogravimetric analyzer, and the measurement was recorded from 30 to 800 °c at a heating rate of 10 °c min-1 under air flow of 50 ml min-1. the uv spectra in aqueous solution (1.0×10-4 mol l-1) were recorded on a shimadzu uv-2450 spectrophotometer. synthesis of complex a mixture of h2dipic (0.84 g, 5 mmol) and cobalt acetate (1.25 g, 5 mmol) was carefully ground in an agate mortar at room temperature, whereby a strong irritant gas was released. the released gas was tested with moist ph paper, and the result indicated that the gas was slightly acid due to the release of acetic acid during the reaction process. when the evolution of the irritant gas ceased, the reaction was complete. the reaction was conducted by grinding at room temperature for 4–5 h, and the mixture turned into loose pink powder. after obtaining the xrd data, the powder was dissolved in a small amount of distilled water and the solution was filtered and concentrated. when the concentrated solution was kept at room temperature for about 24 h, red–brown block-shaped crystals were obtained. yield: 84 %; anal. calcd. for co2c14h20o15n2: c, 29.28; h, 3.51; n, 4.88; co, 20.53 %. found: c, 29.12; h, 3.65; n, 4.71; co, 20.68 %. x-ray crystal structure determination x-ray diffraction data for a single crystal of the complex were collected at 293(2) k on a bruker smart apex ii ccd diffractometer equipped with graphite monochromated mokα radiation using the ω and φ scan mode. a crystal with dimensions 0.40 mm×0.38 homodinuclear cobalt(ii) complex of pyridine-2,6-dicarboxylate 1393 mm×0.34 mm was mounted on a glass fiber. the structures were solved by direct methods and refined by full-matrix least squares on f2 using the shelxtl program package.14 all of the non-hydrogen atoms were refined with anisotropic displacement parameters and all of the hydrogen atoms were found objectively from difference fourier syntheses. the crystallographic data for the complex are given in table s-i of the supplementary material to this paper, the selected bond distances and angles are listed in table s-ii, hydrogen bond lengths and angles are given in table s-iii of the supplementary material, and the crystallographic data are deposited with the cambridge crystallographic data centre under deposition number ccdc 1028718. results and discussion x-ray crystal structure analysis a view of the key fragments of the structure and the atom numbering of the complex are shown in fig. 1. the complex crystallizes in the monoclinic system and space group p2(1)/c with cell parameters of a = 8.3680 å, b = 27.2976 å, c = 9.5826 å and β = 98.276°. the asymmetrical unit consists of the neutral complex [co2(dipic)2(h2o)5] and two lattice water molecules. the unit of the neutral complex is composed of two co(ii) ions, two dipic2– anions and five coordinated water molecules. there are two different coordination environments that form two octahedral cobalt cations, one dipic2– anion ligand utilizes the carboxyl bridged coordination mode to link co1 and co2. the co1 contains a bridged carboxyl oxygen atom (o2) from one dipic2– anion and five oxygen atoms (o9, o10, o11, o12 and o13) from water molecules. the co1 bridges the carboxyl oxygen (o2) of dipic2–, and is surrounded by water molecules, forming an octahedral geometry. the co2 exhibits a distorted octahedral coordination with two dipic2–, which has two anionic tridentate chelating modes. therefore, its molecular structure could be written as [(h2o)5co(μ-oco–dipic)co(dipic)]·2h2o. the molecular structure shown in fig. s-1 of the supplementary material to this paper may indicate weak face-to-face π−π stacking interactions, because the distances fig. 1. molecular structure of the title complex. 1394 li, zhong and wu between the pyridine rings of situated in neighboring mirror planes are 3.703 and 3.791 å.15 crystal packing diagram of the title complex is presented in fig. 2, which shows that the molecule of the complex is held together by intermolecular hydrogen bonds. there are three types of hydrogen bonds in the complex, the hydrogen bonds between coordinated water molecules and the o atoms of carbonyl groups, the coordinated water and lattice water molecules, and the o atoms of carboxylate groups with the lattice water molecules. the adjacent asymmetrical molecular units are interconnected through the hydrogen bond interactions between the coordinated water molecules (o13 and o9) and the carboxylic oxygen atoms (o5 and o6) of the dipic2– ligand forming an extended 1d supramolecular chain. the chains of the 1d supramolecular chain are further combined with each other by means of hydrogen bond interactions between the lattice water or coordinated water molecules and other carboxylic oxygen atoms of the dipic2– ligand, giving rise to the final extended 3d supramolecular network. fig. 2. crystal packing diagram of the title complex. x-ray powder diffraction the xrd pattern of the complex is shown in fig. s-2a of the supplementary material, from which it could be seen that the main diffraction peaks appeared at 2θ 14.60, 16.45 and 26.38°. the index was calculated from the xrd database on the computer program by the least squares method,16 and the results are shown in table s-iv of the supplementary material. the calculated spacing dhkl is consistent with the experimental ones, and the maximum relative deviation between the experimental and calculated spacing dhkl was less than 0.15 %. this indicates that the obtained powder was a single-phase compound. the crystal structure of the complex belongs to monoclinic system with the cell parameters a = 8.397 å, b = 27.409 å, c = 9.609 å and β = 98.22°. the results of indexes to the xrd data are consistent with the results of the single crystal structure. the experimental xrd pattern agrees well with the simulated pattern generated based on the single crystal analysis for the complex (fig. s-2b of the supplementary material). homodinuclear cobalt(ii) complex of pyridine-2,6-dicarboxylate 1395 ft-ir and uv spectra the ft-ir spectrum of the complex is shown in fig. s-3 of the supplementary material. an intense broad absorption band in the region of 3200–3500 cm–1, which can be assigned to stretching vibration of water molecules, could be seen.17 the presence of carboxyl groups was evidenced by the occurrence of strong absorption bands at 1618 cm–1, attributed to νas(coo–), and at 1395 cm–1, attributed to νs(coo–). the difference (∆ν = 223 cm–1) between νas(coo–) and νs(coo–) suggests monodentate or bridged coordination mode of the carboxylate group to co(ii).18 the bands at 1576 and 1430 cm–1 were assigned to ν(c=n) and ν(c=c), respectively. in addition, the bands at 594 and 544 cm–1 are attributed to ν(cu–n) and ν(cu–o), respectively. uv spectra of the ligand and the complex (fig. s-4 of the supplementary material) have only one absorption band, which was attributed to a π–π* transition of the ligand. compared with those of the ligand (273 nm), the maximum absorption peak of the complex was shifted to 265 nm. the coordination bonds formed stabilize the structure, which led to the blue shift of the absorption peak. thermal analysis studying the thermal decomposition process of complexes is helpful to understanding of the coordination structure and the mechanism of thermal decomposition.19,20 the tg–dsc curves of the complex are shown in fig. s-5 of the supplementary material. the possible pyrolysis reaction, and the experimental and calculated mass losses in the thermal decomposition process are summarized in table s-v of the supplementary material. the first mass loss occurs about 102 °c, corresponding to the release of two crystalline water molecules, then the composition of the complex corresponds to be [co2(dipic)2(h2o)5]. the second step in the mass loss of 14.61 % (calcd. 15.69 %) occurred at 150 °c, which shows the loss of five coordinated water molecules. the residue of [co2(dipic)2] was stable between 180 to 320 °c. thereafter, the exothermic peak at 469 °c corresponded to the decomposition of the ligand. in this step, two pyridine free radicals and two co2 molecules were lost, and the decomposition products were cobalt oxalate and co atoms.12 the cobalt oxalate decomposed at between 470 to 600 °c, and the result shows that the thermal decomposed product of cobalt oxalate is metallic cobalt21 at about 576 °c. due to the oxidation of cobalt, a mass increase was observed (found 5.28 %, calcd. 5.57 %) in the tg curve. the final residue was coo. conclusions the homodinuclear complex [co2(dipic)2(h2o)5]·2h2o was synthesized by room temperature solid–solid reaction, and characterized by ea, single crystal x-ray diffraction, xrd, ft-ir, uv and tg–dsc. the crystal structure of the 1396 li, zhong and wu complex belongs to monoclinic system and space group p2(1)/c. the two co(ii) ions are both six-coordinated, and the octahedral co(ii) cations show different coordination environments. the dipic2– ligands utilize the carboxyl bridged coordination mode to link the two co(ii) ions. the 3d supramolecular architecture of the complex is constructed by means of hydrogen bond interactions between water molecules and carboxyl oxygen atoms of the ligands. the thermal decomposition processes of the complex include dehydration and pyrolysis of the ligand, and the residue is cobalt oxide. supplementary material the crystal data, bond lengths and angles, the results of indexes to xrd data, thermal decomposition data, xrd patterns, ft-ir and uv spectra, and tg–dsc curves of the complex are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. the crystallographic data are deposited with the cambridge crystallographic data centre under deposition number ccdc 1028718. acknowledgements. this work was supported by the national natural science foundation of china (no. 21201142) and the scientific research funds of sichuan provincial education department (no. 10za016). и з в о д синтеза у чврстом стању, карактеризација и термичко разлагање хомодинуклеарног кобалт(ii)-комплекса di li, guo-qing zhong и zhi-xian wu school of material science and engineering, southwest university of science and technology, mianyang 621010, china полазећи од пиридин-2,6-дикарбоксилне киселине и кобалт(ii)-ацетата у чврстом стању на собној температури синтетисан је хомодинуклеарни [co2(dipic)2(h2o)5]·2h2o комплекс. комплекс је окарактерисан помоћу елементарне микроанализе, методом дифракције x-зрака са кристала и праха, фуријеровом трансформационом инфрацрвеном и електронском апсорпционом спектроскопском методом, као и на основу термогравиметријских и диференцијалних калориметријских метода. нађено је да комплекс представља моноклинични кристални систем са просторном групом p2(1)/c. у структури комплекса постоје два хексакоординована co(ii) јона. један co(ii) јон је координован за четири карбоксилна атома кисеоника и два атома азота из dipic2-, док је други јон овог метала координован за пет молекула воде и један dipic2преко моста. приказане су могуће реакције пиролизе и термичког разлагања комплекса, док су експериментално одређене масе продуката ових реакција. (примљено 24. фебруара, ревидирано 25. маја, прихваћено 6. јуна 2015) references 1. m. mirzaei, h. eshtiagh-hosseini, a. hassanpoor, v. barba, j. serb. chem. soc. 77 (2012) 67 2. g. h. cui, c. h. he, c. h. jiao, j. c. geng, v. a. blatov, crystengcomm 14 (2012) 4210 3. d. sun, g. g. luo, n. zhang, r. b. huang, l. s. zheng, chem. commun. 47 (2011) 1461 4. z. g. kong, s. n. guo, y. x. zhao, d. song, j. serb. chem. soc. 79 (2014) 669 homodinuclear cobalt(ii) complex of pyridine-2,6-dicarboxylate 1397 5. c. y. wang, z. m. wilseck, r. l. la duca, inorg. chem. 50 (2011) 8997 6. h. sakurai, y. koyima, y. yoshikawa, k. kawabe, h. yasui, coord. chem. rev. 226 (2002) 187 7. j. singh, p. singh, bioinorg. chem. appl. 2012 (2012), article id 104549 8. r. gupta, s. sanotra, h. n. sheikh, b. l. kalsotra, v. k. gupta, rajnikant, j. coord. chem. 65 (2012) 3917 9. k. y. choi, h. ryu, y. m. lim, n. d. sung, u. s. shin, m. suh, inorg. chem. commun. 6 (2003) 412 10. d. li, g. q. zhong, bioinorg. chem. appl. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice on atom–bond connectivity molecule structure descriptors j. serb. chem. soc. 81 (3) 271–276 (2016) udc 547.593:539.216:541.57–123:544.131 jscs–4845 original scientific paper 271 on atom–bond connectivity molecule structure descriptors boris furtula1*, ivan gutman1,2# and kinkar ch. das3 1faculty of science, university of kragujevac, p. o. box 60, 34000 kragujevac, serbia 2state university of novi pazar, novi pazar, serbia and 3department of mathematics, sungkyunkwan university, suwon 440–746, south korea (received 1 september, accepted 12 october 2015) abstract: the atom–bond connectivity index (abc) is a degree-based molecular structure descriptor with well-documented chemical applications. in 2010, a distance-based new variant of this index (abcgg) was proposed. hitherto, the relation between abc and abcgg has not been analyzed. in this paper, the basic characteristics of this relation are established. in particular, abc and abcgg are not correlated and both cases > ggabc abc and < ggabc abc may occur in the case of (structurally similar) molecules. however, in the case of benzenoid hydrocarbons, abc always exceeds abcgg. keywords: atom–bond connectivity index; abc index; molecular structure descriptor; molecular graph. introduction one of the most prolific areas of application of graph theory in chemistry is via molecular structure descriptors (topological indices), namely quantities that are calculated from the molecular graphs and that are used for modeling physicochemical, pharmacological, toxicological, and other properties of the underlying chemical compounds. several thousands such topological indices have been suggested,1 but only a dozen or so were proved to have true applicative power. one of these is the atom–bond connectivity (abc) index. it was introduced in 1998 by estrada et al.,2 but it attracted little attention. only after the publication of a paper,3 ten years later, did the abc index rapidly gain in popularity. it was shown2–4 that by means of the abc index, it is possible to predict the thermodynamic properties of acyclic and cyclic saturated hydrocarbons, including those with large steric strain. comparative studies5,6 confirmed that the abc index yields significantly better results than other mathematically similar molecular structure descriptors. * corresponding author. e-mail: furtula@kg.ac.rs # serbian chemical society member. doi: 10.2298/jsc150901093f _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 272 furtula, gutman and das the abc index is defined as follows. let g be a molecular graph with n vertices, 1 2, , , nv v v . the edge of g, connecting the vertices iv and jv will be denoted by ( , )e i j . the degree id of the vertex iv is the number of the first neighbors of iv . then: 2 i j i j i je d d abc d d + − =  (1) with the summation going over all edges of the graph g. nowadays, the theory of the abc index is well developed, and its mathematical properties have been duly examined; for details see the recent papers7–13 and the references cited therein. motivated by the success of the abc index, graovac and ghorbani14 introduced its new variant, defined as: 1 2 gg 1 2 ( ) ( ) 2 ( ) ( ) + − =  ij ij ij ij ije n e n e abc n e n e (2) in formula (2), 1( )ijn e is the number of vertices of g whose distance to the vertex iv is smaller than the distance to the vertex jv . analogously, 2 ( )ijn e is the number of vertices of g whose distance to the vertex jv is smaller than to iv . vertices equidistant from both iv and jv are ignored. more on the numbers 1( )ijn e and 2 ( )ijn e can be found elsewhere.15,16 this distance-based variant of the atom–bond connectivity index was until now studied only to a limited extent.14,17–19 interestingly, none of the articles14,17–19 considered the simplest and most obvious question, namely: what is the relation between the original atom–bond connectivity index abc, eq. (1), and its modified version abcgg, eq. (2)? the aim of the present work is to provide an answer to this question. numerical work the abc and abcgg indices were calculated for several classes of isomeric alkanes and cycloalkanes. in all cases studied, it was found that between these two structure descriptors there is no (either linear or any other) correlation. a typical example is presented in fig. 1. not only that the two atom–bond connectivity indices are not correlated, but they also imply opposite ordering for structurally similar compounds. for instance, for 2-methylnonane, gg6.58 , 6.49= =abc abc (thus, gg>abc abc ), whereas for 3-methylnonane, abc = = 6.47, gg 6.58=abc (thus, gg<abc abc ); for 2,2-dimethyloctane, abc = 6.84, gg 6.82=abc (thus, gg>abc abc but gg≈abc abc ), whereas for 3,3-dimethyloctane, gg6.68 , 6.95= =abc abc (thus, < ggabc abc ). bearing in mind the good correlation properties of the original abc index,2-5 it could be concluded that there is little hope that the abcgg index would ever be found useful in chemical applications. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. on atom-bond connectivity molecule structure descriptors 273 fig. 1. atom–bond connectivity indices of isomeric decanes plotted versus their abcgg values. the data points lying above (resp. below) the line, satisfy gg<abc abc (resp. gg>abc abc ). in the majority of cases (in this example: with only five exceptions), the abc index is less than the abcgg index. the two atom–bond connectivity indices of benzenoid hydrocarbons in this section, a few basic properties of the two abc indices of benzenoid molecules are established. for this, the facts from the well-elaborated topological theory of benzenoid hydrocarbons were used.20 an illustrative example is provided in fig. 2. consider thus a benzenoid system with n vertices, m edges, h hexagons, ni internal vertices, and b bay regions on its perimeter.20 more details on parameter b can be found elsewhere.20–22 an edge is of ( , )r s -type, if it end-vertices have degrees r and s. a benzenoid system has only vertices of degrees two and three and therefore its edges are only of (2,2)(2,3)and (3,3)-type. therefore, the term: ( 2) / ( )i j i jd d d d+ − in eq. (1) is equal to 2/2 , 2/2 and 2/3 if the edge ije is of (2,2)-, (2,3) and (3,3)-type, respectively. this implies: _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 274 furtula, gutman and das fig. 2. a benzenoid system with 11=h hexagons, 4=in internal vertices (indicated by heavy dots), three bays and one cove, and therefore with 3 1 1 2 5= × + × =b bay regions. this benzenoid system has 4 2 42= + − =in h n vertices and m = 5h + 1 – 52=in edges. according to eq. (4), the number of (3,3)-edges is 33 11 1 4 5= − + + =m 19. 22 23 33 2 2 2 2 2 3 abc m m m= + + where 22m , 23m and 33m stand for the number of edges of the (2,2)-, (2,3)and (3,3)-type, respectively. since 22 23 33m m m m+ + = : 33 33 2 2 ( ) 3 2 abc m m m= + − (3) and 33m can be calculated by means of the identity: 33 1 im h n b= − + + (4) as 2/3 2/2< , it follows from eq. (3) that the abc index is bounded as: 2 2 3 2 m abc m< ≤ (5) the equality on the right-hand side of (5) is attained only in the case of benzene ( 1h = ). in the case of benzenoid hydrocarbons, the quantities 1( )ijn e and 2 ( )ijn e , occurring in eq. (2), can be calculated by the method of elementary edge -cuts.15,23,24 an elementary edge-cut is a line segment that goes through the center of some hexagons, orthogonal to some edges, and intersects the perimeter exactly two times. the number of vertices lying on the two sides of an elementary edgecut c is denoted by 1( )n c and 2 ( )n c . since 1 2( ) ( )n c n c n+ = , only one among 1( )n c and 2 ( )n c requires evaluation, which is usually very simple. an illustrative example is provided in fig. 3. the number of edges intersected by the cut c will be denoted by ( )r c , cf. fig. 3. if ije is any of the edges intersected by c, then: 1 1( ) ( )ijn e n c= and 2 2( ) ( )ijn e n c= for benzenoid hydrocarbons, this has the direct consequence that: 1 2 1 2 ( ) ( ) 2 ( ) ( ) ( )gg c n c n c abc r c n c n c + − =  _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. on atom-bond connectivity molecule structure descriptors 275 holds, where the summation goes over all elementary edge-cuts. since, in addition, for all elementary edge-cuts, 1 2( ) ( )n c n c n+ = , one finally obtains: 1 2 ( ) 2 ( ) ( ) gg c r c abc n n c n c = −  (6) fig. 3. three elementary edge-cuts of the benzenoid system depicted in fig. 2. since this system has 42=n vertices, one has: 1 1 1 2 1( ) 2 , ( ) 3 , ( ) 42 3 39= = = − =r c n c n c , 2 1 2 2 2( ) 3 , ( ) 5 , ( ) 42 5 37= = = − =r c n c n c , and 3 1 3 2 3( ) 5 , ( ) 15 , ( ) 42 15 27= = = − =r c n c n c . on one side of any elementary edge-cut, there are at least three vertices and at most, one-half of the total number of vertices. therefore, 13 ( ) / 2n c n≤ ≤ , which substituted back into eq. (6), and bearing in mind that: ( ) c r c m= yields the bounds: gg 2 2 2 3( 3) − − ≤ ≤ − m n n abc m n n (7) equality on both sides of (7) is attained only in the case of benzene ( 1h = ). combining the lower bound in (5) and the upper bound in (7), one obtains: gg 4( 3) 3( 2) − ≥ − abc n abc n in which equality is attained only in the case of benzene ( 6n = ). thus, only in the case of benzene, do the two atom–bond connectivity indices coincide. for all other benzenoids, 2 , 10h n≥ ≥ and therefore: gg 4(10 3) 3(10 2) − > − abc abc i.e., gg7/6>abc abc where 7/6 1.0801= . if naphthalene is excluded, then 14n ≥ , and gg11/9>abc abc where 11 /9 1.1055= . thus, in contrast to alkanes and cycloalkanes, the atom–bond connectivity index of any benzenoid hydrocarbon is always greater than the abcgg index. acknowledgments. the first author was partially supported by the ministry of education, science and technological development of the republic of serbia, through grant no. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 276 furtula, gutman and das 174033. the third author was supported by the national research foundation funded by the korean government by grant no. 2013r1a1a2009341. и з в о д молекулски структурни дескриптори повезаности типа атом–веза борис фуртула1, иван гутман1,2 и kinkar ch. das3 1природно–математички факултет универзитета у крагујевцу, 2државни универзитет у новом пазару и 3sungkyunkwan university, suwon, south korea индекс повезаности типа атом–веза (abc) је молекулски структурни дескриптор заснован на степенима чворова, чије су хемијске примене добро документоване. године 2010. предложена је једна варијанта овог индекса (abcgg) заснована на растојању. до сада, релације између abc и abcgg нису биле истраживане. у овом раду, установљене су основне карактеристике ове релације: индекси abc и abcgg нису корелисани, и оба случаја, abc > abcgg и abc < abcgg, се могу догодити код структурно сличних молекула. међутим, у случају бензеноидних угљоводоника, abc је увек већи од abcgg. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice a new zinc(ii) supramolecular square: synthesis, crystal structure, thermal behavior and luminescence j. serb. chem. soc. 80 (10) 1289–1295 (2015) udc 546.472+539.216:548.7: jscs–4797 536.5.004.12:535.37 original scientific paper 1289 a new zinc(ii) supramolecular square: synthesis, crystal structure, thermal behavior and luminescence xiu-yan wang*, zhong-yu zhao, qian han, miao yu and de-yu kong key laboratory of the preparation and application of environmental friendly materials, ministry of education, college of chemistry, jilin normal university, siping 136000, china (received 4 february, revised 8 may, accepted 6 june 2015) abstract: a new square-shaped zn(ii) complex, [zn4(l)4(phen)4]·6h2o (1, l = = 2-hydroxynicotinate and phen = 1,10-phenanthroline), was synthesized under hydrothermal condition. the crystal of 1 belongs to triclinic, space group p–1 with a = 10.773(2) å, b = 12.641(3) å, c = 13.573(3) å, α = 107.44(3)°, β = = 102.66(3)°, γ = 93.89(3)°, c72h56n12o18zn4, fw = 1638.77, v = 1702.8(6) å3, z = 1, dc = 1.598 g/cm3, s = 1.045, μ(mokα) = 1.475 mm-1, f(000) = 836, r = 0.0472 and wr = 0.0919. four l ligands bridge four zn(ii) atoms to form a square-shaped structure, where four phen ligands are respectively located on the four corners of the square. the π–π stacking interactions extend adjacent squares into a 1d supramolecular chain. the thermal behavior of 1 was characterized. moreover, the solid-state luminescence property of the complex was studied at room temperature. keywords: crystal structure; supramolecular square; luminescence; 2-hydroxynicotinate. introduction over the past few decades, precise control over the shape and size of supramolecular architectures via self-assembly has been a major driving force for chemists.1–6 well-designed supramolecular architectures can show various physical properties and functionalities in molecular recognition, sensing and magnetism.7–10 to date, a number of complexes with regular arrays, such as grids, wires and rings, were synthesized using various synthetic methods.11,12 in a variety of typical cases, successful assemblies of molecular architectures with precise shapes and sizes mainly relied on the angles of the organic ligands and the coordination geometry of the metal ions.13–18 therefore, through rational ligand design and metal ion selection, a controllable arrangement of metal ions in multinuclear complexes could be achieved.19,20 in this regard, tetranuclear * corresponding author. e-mail: wangxiuyan2004@sohu.com doi: 10.2298/jsc150204049w _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1290 wang et al. complexes with square-shaped arrangements of metal ions are of intense interest to chemists owing to their potential application as molecular devices.21 by rationally combining the planar n-donor chelating ligand and the angular bridging carboxylate-containing ligand square grid-like complexes could be afforded.22 based on the above consideration, in this work a planar n-donor chelating 1,10-phenanthroline (phen) ligand and an angular bridging carboxylate-containing 2-hydroxynicotinate (l) were selected, and their complexation behavior was investigated. this paper describes the syntheses, crystal structure, and luminescent property of a new zn(ii) square-shaped complex, [zn4(l)4(phen)4]·6h2o (1). the compound was characterized by elemental analysis, ir spectrum, tg and x-ray crystallography. in addition, its luminescent property was also investigated. experimental all the materials were of analytical reagent grade and used as received without further purification. the c, h and n elemental analyses were conducted on a perkin–elmer 240c elemental analyzer. the emission spectra were measured on a renishaw invia raman microscope at room temperature. the ir spectrum was recorded in the range of 4000–400 cm-1 on an alpha centauri ft-ir spectrophotometer using the kbr pellet technique. thermal stability experiment was performed on a tg sdt2960 thermal analyzer (ta instruments) under a nitrogen atmosphere. synthesis of [zn4(l)4(phen)4]·6h2o (1) a mixture of zn(oac)2·2h2o (0.2 mmol, 0.044 g), h2l (0.15 mmol, 0.021 g), phen (0.1 mmol, 0.018 g) and koh (0.1 mmol, 0.006 g) was dissolved in distilled water (10 ml). then the mixture was transferred and sealed in a 25 ml teflon-lined stainless steel container. the container was heated at 417 k for 4 days. after the mixture had been cooled to room temperature at a rate of 10 °c·h-1, pale yellow crystals of 1 were obtained. yield: 21 %. anal. calcd. for c72h56n12o18zn4: c, 52.76; h, 3.44; n, 10.75 %. found: c, 52.31; h, 3.22; n, 10.26 %; ir (kbr, cm-1): 3435(w), 1615(w), 1564(m), 1474(w), 1408(m), 1254(w), 1139(s), 1070(w), 994(w), 952(w), 850(w), 832(w), 786(w), 726(w), 618(m), 539(w), 515(w). x-ray crystallography single-crystal x-ray diffraction data for 1 was obtained on a rigaku r-axis-rapid diffractometer with graphite-monochromatized mokα (λ = 0.71073 å) radiation using an ω–φ scan method at 293(2) k. the structure was solved by direct methods with shelxs-97 program and refined with shelxl-97 by the full-matrix least-squares techniques on f2.23,24 non-hydrogen atoms of the compound were refined with anisotropic temperature parameters. all h atoms on carbon atoms were positioned geometrically (c–h = 0.93 å) and refined as riding, with uiso(h) = 1.2ueq(carrier). the h atoms of o1w and o2w were included in the model. a summary of the crystallographic data and structure analysis are given in table s-i of the supplemrntary material to this paper, and selected bond lengths and bond angles are listed in table s-ii of the supplementary material. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ a new zn(ii) supramolecular square 1291 results and discussion crystal structure of 1 the asymmetric unit of 1 is composed of two zn(ii) atoms, two l ligands, two phen ligands, and three lattice water molecules. noticeably, the two zn(ii) atoms are both in a penta-coordinated environment, completed by two oxygen atoms from one hydroxyl and one carboxylate of the same l anion, and three nitrogen atoms of one phen ligand and one l anion (fig. 1). however, the two zn(ii) atoms exhibit different coordination spheres: zn1 is in a slightly distorted square-pyramidal geometry with a τ value of 0.27, and zn2 shows a nearly ideal trigonal–bipyramid sphere with a τ value of 0.90.25 fig. 1. view of square-shaped structure of 1 and the coordination environments of zn(ii) atoms in 1. as given in table s-ii, the zn–o distances range from 1.971(3) to 2.057(3) å, and the zn–n distances vary from 2.023(4) to 2.196(4) å, which are very similar to the ones found in other related compound [zn(bpea)(phen)] (bpea = = 1,4,4'-(1e)-1,2-ethenediyl-bisbenzoate).26 each l ligand adopts a tridentate coordination mode by using its one pyridine nitrogen, one hydroxyl oxygen and one carboxylate oxygen. in this way, four l ligands bridge four zn(ii) atoms to yield a rare square-shaped molecular arrangements with the zn(ii)…zn(ii) distance of 4.66 å. four phen ligands are respectively located on the four corners of the square. it is clear that the bis-chelating phen ligands play a key role in the formation of the square. significantly, π–π stacking interactions exist among neighboring l ligands in adjacent squares (the face-to-face distance is ca. 3.51 å). furthermore, these π–π stacking interactions extend adjacent squares into a 1d supramolecular chain (fig. 2). moreover, in complex 1, there are o–h…o hydrogen-bonding interactions among water oxygen atoms and the carboxylate oxygen atoms of the l ligands (table i). these hydrogen-bonding interactions further stabilize the 1d supramolecular architecture of 1. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1292 wang et al. fig. 2. view of the 1d supramolecular ladder structure of 1 constructed by π–π interactions. table i. hydrogen-bonding parameters for 1; symmetry transformations were used to generate equivalent atoms: b: x+1, y–1, z; c: –x+2, –y, –z+2; d: –x+1, –y, –z+2 d–h…a d(d–h) / å d(h…a) / å d(d…a) / å ∠dha / ° o(2w)–hw21…o(3w)b 0.854(10) 2.027(14) 2.864(6) 167(5) o(2w)–hw22…o(1w)c 0.854(10) 1.999(13) 2.845(6) 170(5) o(1w)–hw11…o(2)d 0.848(10) 2.019(14) 2.862(5) 172(6) o(1w)–hw12···o(1) 0.851(10) 1.926(16) 2.765(6) 168(6) notably in 1, the l is a doubly deprotonated species coordinated to zn(ii) centers via two o atoms and one n atom. it is evident that the ph value of the reaction mixture plays an important role in the double deprotonation of the h2l ligand and the formation of the final complex. in the present experiment, potassium hydroxide was used to adjust the ph value. nevertheless, only at ph around 6.5 can be the reaction mixture produce single crystals of 1 with the completely deprotonated l dianion. moreover, the hydrothermal reaction is necessary for the formation of 1. at room temperature or under milder conditions, no crystals of 1 were obtained. it could be inferred that the hydrothermal reaction improved the solubility of the reaction mixture, and favored the crystallization of 1 during the cooling-down process. moreover, the hydrothermal reaction accelerated the complete deprotonation of the h2l ligand. thermal behavior to know the stability of compound 1, its thermogravimetric curve was recorded at a heating rate of 10 °c min–1. as shown in fig. 3, there were two main steps of weight loss. the first step of 6.5 % from 25 to 159 °c can be ascribed to the release of the free water molecules (calcd. 6.6 %), and the second step of 73.3 % in the temperature range of 178–598 °c corresponds to the removal of the organic ligands (l and phen; calcd. 77.5 %). the residual weight 20.2 % (calcd. 19.9 %) could probably be attributed to the formation of zno. these thermal behaviors agree with the formula of the title compound. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ a new zn(ii) supramolecular square 1293 fig. 3. tg curve of 1 recorded at a heating rate of 10 °c min-1. luminescent property the d10 metal-based luminescent compounds are of great interest because of their potential applications as chemical sensors and in photochemistry.27,28 in this work, the emission spectra of the free organic ligand h2l and compound 1 were recorded in the solid state at room temperature (fig. 4). the spectrum of h2l shows a maximum emission peak at 393 nm (λex = 325 nm) which could be attributed to a π–π* transition, together with a broad shoulder at about 480 nm tentatively attributable to an n–π* transition.29 the spectrum of 1 exhibited a main peak at 523 nm (λex = 325 nm) that was red-shifted by 25 nm with respect to the band shown by the phen ligand (λem = 498 nm).26 the red shift may be attributed to a ligand to metal charge transfer (lmct) transition.30 fig. 4. emission spectra of h2l and 1 in the solid state at room temperature. conclusions a new square-shaped molecule was prepared from a planar n-donor chelating ligand, 1,10-phenanthroline, and the angular bridging carboxylate-con_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1294 wang et al. taining 2-hydroxynicotinate under hydrothermal conditions. in 1, the four l ligands bridge the four zn(ii) atoms to form a square-shaped structure, in which four phen ligands are respectively located on the four corners. the π–π stacking interactions extend adjacent squares into a 1d supramolecular chain. moreover, compound 1 exhibits intense luminescence in the solid state at room temperature. supplementary material the supplementary crystallographic data for this paper are deposited at the cambridge crystallographic data centre under ccdc-1046844 (1). these data can be obtained free of charge from www.ccdc.cam.ac.uk/data_request/cif. crystal data and structure refinement, and selected bond lengths and angles for 1 are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. this work was supported by the science and technology research projects of the education committee of jilin province (no. 2013206). и з в о д нови супрамолекулски квадратно-планарни цинк(ii) комплекс: синтеза, кристална структура, термичко понашање и луминисценција xiu-yan wang, zhong-yu zhao, qian han, miao yu и de-yu kong key laboratory of preparation and application of environmental friendly materials, ministry of education, college of chemistry, jilin normal university, siping 136000, china применом хидротермалних услова синтетизован је нови квадратно-планарни цинк(ii) комплекс, опште формуле [zn4(l)4(phen)4]·6h2o (1, l = 2-хидроксиникотинат и phen = 1,10-фенантролин). нађено је да комплекс 1 припада триклиничном кристалном систему са p–1 просторном групом и a = 10,773(2) å, b = 12,641(3) å, c = 13,573(3) å, α = 107,44(3)°, β = 102,66(3)°, γ = 93.89(3)°, c72h56n12o18zn4, fw = 1638.77, v = = 1702,8(6) å3, z = 1, dc = 1,598 g/cm 3, s = 1,045, μ(mokα) = 1,475 mm-1, f(000) = 836, r = 0,0472 и wr = 0,0919. у комплексу 1 четири лиганда l повезују четири zn(ii) јона мостом и граде структуру квадратно-планарне геометрије у којој се четири phen лиганда налазе на угловима квадрата. преко π–π интеракција квадратно-планарне јединице су повезане у 1d супрамолекуларни ланац. описано је термичко понашање комплекса 1. такође, испитивано је луминесцентно понашање овог комплекса на собној температури. (примљено 4. фебруара, ревидирано 8. маја, прихваћено 6. јуна 2015) references 1. f. a. cotton, c. lin, c. a. murillo, inorg. chem. 40 (2001) 478 2. s. leininger, b. olenyuk, p. j. stang, chem. rev. 100 (2000) 853 3. m. fujita, chem. soc. rev. 27 (1998) 417 4. y.-f. han, g.-x. jin, acc. chem. res. 47 (2014) 3571 5. s.-l. huang, y.-j. lin, z.-h. li, g.-x. jin, angew. chem. int. ed. engl. 53 (2014) 11218 6. z.-q. yu, m. pan, j.-j. jiang, z.-m. liu, c.-y. su, cryst. growth des. 12 (2012) 2389 7. j. malberg, m. bodensteiner, d. paul, t. wiegand, h. eckert, r. wolf, angew. chem. int. ed. engl. 53 (2014) 2771 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ a new zn(ii) supramolecular square 1295 8. d. whang, k. kim, j. am. chem. soc. 119 (1997) 451 9. a. noor, e. s. tamne, b. oelkers, t. bauer, s. demeshko, f. meyer, f. w. heinemann, r. kempe, inorg. chem. 53 (2014) 12283 10. v. vrdoljak, b. prugovečki, d. matković-čalogović, t. hrenar, r. dreos, p. siega, cryst. growth des. 13 (2013) 3773 11. b. manimaran, a. vanitha, m. karthikeyan, b. ramakrishna, s. m. mobin, organometallics 33 (2014) 465 12. f. a. cotton, c. y. liu, c. a. murillo, x. wang, inorg. chem. 45 (2006) 2619 13. a. m. najar, i. s. tidmarsh, h. adams, m. d. ward, inorg. chem. 48 (2009) 11871 14. v. yin, g.-c. huang, c.-k. kuo, m.-d. fu, h.-c. lu, j.-h. ke, k.-n. shih, y.-l. huang, g.-h. lee, c.-y. yeh, c.-h. chen, s.-m. peng, j. am. chem. soc. 130 (2008) 10090 15. y. hou, m. a. rodriguez, m. nyman, cryst. growth des. 12 (2012) 1422 16. j. park, y.-p. chen, z. perry, j.-r. li, h.-c. zhou, j. am. chem. soc. 136 (2014) 16895 17. s. khullar, s. k. mandal, cryst. growth des. 14 (2014) 6433 18. t. matsumoto, t. shiga, m. noguchi, t. onuki, g. n. newton, n. hoshino, m. nakano, h. oshio, inorg. chem. 49 (2010) 368 19. l. k. thompson, o. waldmann, z. xu, coord. chem. rev. 249 (2005) 2677 20. y. wang, x. ma, s. hu, y. wang, c. tan, y. wen, x. zhang, t. sheng, x. wu, polyhedron 83 (2014) 137 21. m. ruben, j. rojo, f. j. romero-salguero, l. h. uppadine, j.-m. lehn, angew. chem. int. ed. engl. 43 (2004) 3644 22. t. shiga, t. matsumoto, m. noguchi, t. onuki, n. hoshino, g. n. newton, m. nakano, h. oshio, chem. asian j. 4 (2009) 1660 23. shelxs 97, g. m. sheldrick, program for the solution of crystal structure, university of göttingen, göttingen, 1997 24. g. m. sheldrick, shelxl 97, program for the refinement of crystal structure, university of göttingen, göttingen, 1997 25. a. w. addison, t. n. rao, j. reedijk, j. van rijn, g. c. verschoor, j. chem. soc., dalton trans. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice one-pot green synthesis of isoxazol-5(4h)-one derivatives using dowex1-x8oh in water j. serb. chem. soc. 81 (9) 971–978 (2016) udc 547.786+542.913:544.478+ jscs–4901 546.212:57–188 original scientific paper 971 one-pot green synthesis of isoxazol-5(4h)-one derivatives using dowex1-x8oh in water davood setamdideh* department of chemistry, faculty of sciences, mahabad branch, islamic azad university, mahabad, iran (received 2 february, revised 9 may, accepted 9 may 2016) abstract: 4-(аrylmethylidene)-3-methylisoxazol-5(4h)-ones and 4-(arylmethylidene)-3-phenylisoxazol-5(4h)-ones were synthesized in a one-pot three -component procedure in the presence of dowex1-x8oh as catalyst in water. the products were obtained in high yields (90–95 %) and proper reaction times (1–5 h). the method is eco-friendly and operationally simple. keywords: dye; removal; polymeric sorbent; equilibrium; kinetic; thermodynamics; dowex1-x8oh; isoxazol-5(4h)-one; one-pot reaction; green chemistry. introduction multi-component reactions (mcrs) play an important role in combinatorial chemistry because of their ability to synthesize diverse and complex organic molecules, natural products, and biologically active compounds. an mcr allows compounds to be synthesized in a few steps and usually in a one-pot operation without the need to isolate any intermediate during the reactions. other typical benefits from these reactions are simplified work-up, reduced reaction times, saving of energy and raw materials, minimization of waste, easy purification, excellent functional group compatibility, versatility, and eco-friendliness.1–11 isoxazole derivatives possess a variety of biologically activities.12–30 in addition, isoxazolone moieties have been used for the design of liquid crystals, merocyanine dyes in optical research, and photochromic compounds.31–35 furthermore, 4-(arylmethylene)isoxazol-5-ones are used for the preparation of fused heterocyclic compounds.36–39 the synthesis of 4-aryl-3-methylisoxazol-5(4h)-one derivatives was performed using a variety reagents and catalysts in basic medium, such as via pyridine,40 sodium silicate,41 sodium benzoate,42 sodium azide,43 sodium saccha * corresponding author. e-mail: davood.setamdideh@gmail.com; d.setamdideh@iau-mahabad.ac.ir doi: 10.2298/jsc160202050s _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 972 setamdideh rin,44 sodium citrate,45 sodium sulfide,46 sodium ascorbate47 and sodium tetraborate.48 moreover, some methods were performed at high temperatures and for long reaction times,49 with minimal yields50 or under unconventional energies, such as ultrasound irradiation50 or visible light.51 recently, it was reported that dowex(r)50wx4 (ion-exchange resin, strong acid) could be used for the regioselective synthesis of oximes in the nh2oh·hcl/dowex(r)50wx4 system,52 the reduction of a variety of carbonyl compounds, such as aldehydes, ketones, α-diketones, acyloins and α,β-unsaturated carbonyl compounds, to their corresponding alcohols using the nabh4/ /dowex(r)50wx4 system,53 the synthesis of cyanohydrins using the nacn/ dowex(r)50wx4 system,54 the reductive–amination of a variety of aldehydes and anilines using the nabh4/dowex(r)50wx4 system,55 the reductive acylalation of aldehydes using the borohydrides/ac2o/dowex(r)50wx4-8 systems,56,57 and for the synthesis of isoxazol-5(4h)-one derivatives.58 furthermore, dowex1-x8 (ion-exchange resin, strong base) was used for the reduction of carbonyl compounds.59 these achievements encouraged this investigation of a convenient and eco -friendly procedure for the synthesis of 4-(arylmethylidene)-3-substituted isoxazol-5(4h)-ones using dowex1-x8oh as catalyst. thus, the synthesis of 4-(arylmethylidene)-3-methylisoxazol-5(4h)-ones 4a–i and 4-(arylmethylidene) -3-phenylisoxazol-5(4h)-ones 5a–e was attempted using equimolecular quantities of ethyl acetoacetate, hydroxylamine hydrochloride, and a variety of aromatic aldehydes in the presence of dowex1-x8oh in water, as shown in scheme 1. scheme 1. general procedure for the synthesis of 3-methyland 3-phenyl-4-(arylmethylidene)-isoxazol-5(4h)-ones 4a–i and 5a–e, respectively. for ar see table i. experimental general. all substrates and reagents of the best quality were purchased from commercial sources. the ft-ir and 1h-nmr and 13c-nmr spectra were recorded on a perkinelmer ft -ir rxi spectrometer and a 300 mhz bruker spectrometer, respectively. the products were characterized by their 1h-nmr or ir spectra and comparison with authentic samples (melting points). the organic layers were dried over anhydrous sodium sulfate. all yields refer to isolated pure products. the purities of the products were determined by 1h-nmr spectroscopy. in addition, the reactions were monitored by tlc using silica gel 60 f254 aluminum sheets. the analytical and spectral data are given in supplementary material to this paper. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ one-pot green synthesis of isoxazol-5(4h)-one derivatives 973 preparation of the dowex1-x8oh resin60 dowex1-x8 (10 g) was washed sequentially with 1 m aqueous hcl (3×40 ml), 1 m aqueous naoh (3×40 ml) and 100 ml of water, and then dried overnight prior to use. general procedure for the synthesis of the 4-(arylmethylidene)-3-methylisoxazol-5(4h)-ones 4a–i in a round-bottomed flask (25 ml) equipped with a magnetic stirrer, a mixture of ethyl acetoacetate (1.30 g, 10 mmol), hydroxylamine hydrochloride (0.7 g, 10 mmol), aromatic aldehyde (10 mmol) and dowex1-x8oh (1 g) in 10 ml of distilled water was prepared and stirred at room temperature for the required time (given in table i). after completion of the reaction (monitored by tlc), the precipitate was filtered off and washed with cold distilled water. then the products were recrystallized from ethanol or acetone (table i). the 4-(arylmethylidene)-3-methylisoxazol-5(4h)-ones were obtained in excellent yields (90–95 %), and characterized by ft-ir, and 1h-nmr and 13c-nmr spectroscopy. general procedure for the synthesis of the 4-(arylmethylidene)-3-phenylisoxazol-5(4h)-ones 5a–e in a round-bottomed flask (25 ml) equipped with a magnetic stirrer, a mixture of ethyl 3-oxo-3-phenylpropanoate (10 mmol), hydroxylamine hydrochloride (0.7 g, 10 mmol), aromatic aldehyde (10 mmol) and dowex1-x8oh (1 g) in 10 ml of distilled water was prepared and stirred at room temperature for the required time (given in table i). after completion of the reaction (monitored by tlc), the precipitate was filtered off and washed with cold distilled water. then products were recrystallized from ethanol or acetone (table i). the 4-(arylmethylidene)-3-methylisoxazol-5(4h)-ones were obtained in excellent yields (90–95 %) and characterized by ft-ir, and 1h-nmr and 13c-nmr spectroscopy. table i. the synthesis of 4-(arylmethylidene)-3-methylisoxazol-5(4h)-ones and 4-(arylmethylidene)-3-phenylisoxazol-5(4h)-ones with the dowex1-x8oh/h2o system entry product ar time, h yielda, % 1b 4a ph 2 92 2b 4b 4-meo-ph 1 95 3b 4c 2-meo-ph 1.5 93 4 4d 3-br-ph 4 90 5b 4e 4-f-ph 5 93 6b 4f 4-me-ph 2 94 7 4g 4-me2n-ph 1 95 8 4h 2-thienyl 2 92 9c 4i ph-ch=ch 2 92 10b 5a ph 3 94 11b 5b 4-me2n-ph 2 93 12 5c 4-meo-ph 2 95 13 5d 4-me-ph 2.5 90 14b 5e 2-thienyl 3 91 ayields refer to isolated pure products after recrystallization from an appropriate solvent; bthe products were recrystallized from ethanol (96 %); cthe products were recrystallized from acetone _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 974 setamdideh results and discussion the synthesis of 4-(arylmethylidene)isoxazol-5(4h)-ones is a multi-component reaction (scheme 1). first, dowex resin bearing quaternary ammonium hydroxide moieties, i.e., dowex1-x8oh, was prepared through chloride–hydroxide anion exchange according to a literature procedure.60 then, the reaction conditions were optimized. for this purpose, the reaction of benzaldehyde as model compound with hydroxylamine hydrochloride and ethyl acetoacetate in water was performed at room temperature with different amounts of dowex1 -x8oh ranging from 0 to 2 g, as shown in table ii. when the amount of dowex1-x8oh was increased from 0 to 1 g, the yield of product improved from 40 to 92 % (table ii, entries 1–4). however, when the amount of dowex1 -x8oh was further raised to 2 g, no significant decrease in the time of the reaction was observed (table i, entry 5). consequently, the amount of 1 g for dowex1 -x8oh was selected as the optimal amount of catalyst for these reactions. table ii. the optimization reaction conditions for the synthesis of 4-benzylidene-3-methylisoxazol-5(4h)-one (4a) from benzaldehyde (10 mmol), ethyl acetoacetate (10 mmol) and nh2oh·hcl (10 mmol) in h2o (10 ml) in the presence of dowex1-x8oh as shown in scheme 1 entry dowex1-x8oh, g time, h conversiona, % yieldb, % 1 0 1 100< 40 2 0.25 1 100< 60 3 0.5 3 100 94 4 1 2 100 92 5 2 1.75 100 94 aconversion refers to tlc monitoring; byield refers to isolated pure product the versatility of this protocol was examined by the reaction of a variety of aldehydes in the presence of dowex1-x8oh. all reactions were completed within 1–5 h with excellent yields of the products (90–95 %), as shown in table i. all of the products are known and were characterized by ft-ir, and 1hand 13c-nmr spectroscopy. the characterization data are given in the supplementary data to this paper. the proposed mechanism for the formation of the products is shown in scheme 2. the ion-exchange resin dowex1-x8oh is insoluble in h2o. therefore, the reactions took place under heterogeneous conditions. the influences of dowex1-x8oh are shown in scheme 2. thus, the oh groups on dowex1-x8oh (as an anion-exchange resin, strong base) deprotonate the oxime intermediate (a) that is more susceptible for reaction with aromatic aldehydes. the reaction proceeds via an intermolecular knoevenagel adduct (b) followed by dehydration and ring closure, which is also catalyzed by dowex1-x8oh. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ one-pot green synthesis of isoxazol-5(4h)-one derivatives 975 the reusability of the catalyst was checked using the dowex1-x8oh recovered from the synthesis of 4-benzylidene-3-methylisoxazol-5(4h)-one (4a) as shown in table iii. it was observed that the recovered catalyst could be satisfactorily used for a second run without regeneration, whereas, a third run with recovered catalyst led to poor yields and longer reaction times. after regeneration of the dowex1-x8oh,60 the reaction proceeded as in first run with fresh catalyst (table iii, entry 4). scheme 2. the proposed mechanism for the synthesis of 4-(arylmethylidene)-3-substituted isoxazol-5(4h)-ones. table iii. reusability of dowex1-x8oh in the synthesis of 4-benzylidene-3-methylisoxa zol-5(4h)-one (4a) from benzaldehyde under the optimized reaction conditions yieldc, % conversionb, % timea, h run no. entry 92 100 2 1 1 90 100 3 2 2 60 100< 3 3 3 90 100 2 – 4d ait is the highest time when the reaction ends or does not further progress; bconversion refers to tlc monitoring (eluent; ch2cl2); cyield refers to the isolated pure products (±3 %); dregeneration with 1m naoh60 in order to demonstrate the merit of dowex1-x8oh in comparison with other catalysts used for the same reaction, some of the obtained results are tabulated and compared in table iv. as is evident from the shown results, the yield was the highest with a short reaction time in the presence of dowex1 -x8oh. in addition, easy work-up, mild reaction conditions, reuse of catalyst and the use of water from the environment are advantages of the new protocol, while some procedures are strict and complicated. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 976 setamdideh table iv. the comparison of the synthesis of 4-(4-methoxybenzylidene)-3-methylisoxazol -5(4h)-one (4b) using the dowex1-x8oh and other reported systems entry catalyst and conditions time, h yielda, % recyclable catalyst reference 1 dowex1-x8oh/h2o/r.t. 1 95 yes this paper 2 dowex(r)wx4/h2o/r.t. 0.5 96 yes 58 3 na2s/etoh/r.t 1.5 88 no 46 4 pyridine/etoh/reflux 3 71 no 40 5b catalyst-free/grinding 0.8 61 – 49 6c catalyst-free/105–110 °c 0.25 66 – 49 7 pyridine/h2o/ultrasound 1 82 no 50 8 sodium tetraborate/h2o/r.t. 0.83 95 no 48 9 sodium benzoate/h2o/r.t. 1.5 87 no 42 10 visible light/aq. etoh/r.t. 0.16 82 – 51 aisolated yield; bthe mixture was allowed to stand 12 h after the completion of the reaction; cthe mixture was allowed to stand overnight after the completion of the reaction conclusion it has been shown that dowex1-x8oh in water is a convenient catalyst for the preparation of a variety of isoxazol-5(4h)-ones using aromatic aldehydes, ethyl acetoacetate and hydroxylamine hydrochloride precursors in a one-pot, three-component condensation reaction at room temperature with excellent yields of the products. the high efficiency, shorter reaction times, easy work-up, mild conditions, reuse of catalyst and use of water as a green and environmentally benign solvent make the new protocol attractive for the synthesis of isoxazol-5 -(4h)-ones. therefore, this new system could be a useful addition to the present methodologies. supplementary material characterization data for the synthesized compounds are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the authors gratefully appreciate the financial support of this work through grant no. 02-2905/22092-1393/12/17 from the research council of the islamic azad university branch of mahabad. и з в о д синтеза изоксазол-5(4h)-он деривата у води као растварачу, у једном реакционом кораку, у присуству dowex1-x8oh davood setamdideh department of chemistry, faculty of sciences, mahabad branch, islamic azad university, mahabad, iran извршена је синтеза деривата 4-(арилметилиден)-3-метилизоксазол-5(4h)-она и 4(арилметилиден)-3-фенилизоксазол-5(4h)-она, у једном реакционом кораку, у води као растварачу уз dowex1-x8oh као катализатор. производи су добијени у високом приносу (90–95%) у релативно кратком реакционом времену (1–5 h). описани поступак је еколошки прихватљив и једноставан за примену. 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chem. soc. 58 (2014) 230 58. d. setamdideh, j. mex. chem. soc. 59 (2015) 191 59. b. zeynizadeh, f. shirini, bull. korean chem. soc. 24 (2003) 295 60. l. yong, m.-l. yao, j. f. green, h. kelly, g. w. kabalka, chem. commun. 46 (2010) 2623. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice effects of solid poly (ethylene glycols) addition to the solutions of aniline or n,n-dimethylaniline with water: experimental measurements and modelling j. serb. chem. soc. 81 (7) 789–798 (2016) udc 547.422+66.091.7+547.552.2:536.247+ jscs–4886 54–145.2 original scientific paper 789 effects of solid poly (ethylene glycols) addition to the solutions of aniline or n,n-dimethylaniline with water: experimental measurements and modelling danijela soldatović1, nikola grozdanić2#, zoran višak3, ivona radović2# and mirjana kijevčanin2*# 1public company nuclear facilities of serbia, 12–14 mike petrovića alasa, vinča, 11351 belgrade, serbia, 2faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11120 belgrade, serbia and 3engineering and applied sciences, aston university, birmingham, b4 7et, united kingdom (received 17 march, accepted 18 april 2016) abstract: in this work, the liquid–liquid and solid–liquid phase behaviour of ten aqueous pseudo-binary and three binary systems containing polyethylene glycol (peg) 2050, polyethylene glycol 35000, aniline, n,n-dimethylaniline and water, in the temperature range 298.15–350.15 k and at ambient pressure of 0.1 mpa, was studied. the obtained temperature-composition phase diagrams showed that the only functional co-solvent was peg2050 for aniline in water, while peg35000 even showed a clear anti-solvent effect in the n,n -dimethylaniline aqueous system. the experimental solid–liquid equilibria (sle) data have been correlated by the non-random two-liquid (nrtl) model, and the correlation results are in accordance with the experimental results. keywords: solid–liquid equilibria; liquid–liquid equilibria; nrtl model; polyethylene glycol; aniline; n,n-dimethylaniline. introduction polyethylene glycols (pegs) are non-toxic, biodegradable polymers1 which form strong interand intra-molecular hydrogen bonds,2,3 since they are both good proton donors and acceptors.4 pegs, particularly in their liquid form, have wide spectrum of applications as solvents and co-solvents in organic synthesis,5 catalysis,2 food industry6 and biotechnology.7 in the previous study,8 liquid pegs as co-solvents for aniline and n,n-dimethylaniline in water were investigated, assessing possible impacts on the application of these aromatic compounds in dye industry. as a continuation, this study is investigating the possibility to use * corresponding author. e-mail: mirjana@tmf.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc160317058s _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 790 soldatović et al. solid pegs – average molecular mass 2050 (peg2050) and 35000 (peg35000) – in the same respect. thus, the related temperature–composition phase diagrams of the pseudo-binary systems aniline/or n,n-dimethylaniline+(peg2050 + water) and aniline/or n,n-dimethylaniline+(peg35000+ water) were constructed. diagrams showed that only the addition of peg2050 to aqueous solution of aniline provoked a considerable co-solvent effect for aniline in water, while peg35000 even appeared to be an anti-solvent, decreasing the solubility of water in n,n -dimethylaniline. the results were discussed in terms of the hydrogen bonds existing between pegs and aniline or n,n-dimethylaniline but, as well, taking into account distinct solubility of peg2050 and peg35000 in both studied organic solvents. binary solid–liquid equilibria (sle) data were correlated using the non-random-two-liquid (nrtl) activity coefficient models. experimental materials studied pure compounds, their suppliers and the related stated purities are listed in table i – all the compounds were used without further purification. water used for preparation of the solutions was redistilled and deionized (milipore co. equipment, bedford, ma). all the solutions were prepared gravimetrically using a mettler ag 204 analytical semi-microbalance having a precision of ±1×10-4 g. table i. table of the samples (pure components) used in this study chemical name source cas number melting point range, k mass fraction purity purification method aniline sigma–aldrich (switzerland) 62-53-3 – ≥ 0.995 none n,n-dimethylaniline merck (germany) 121-69-7 – ≥ 0.99 none polyethylene glycol 2050 sigma–aldrich (germany) 25322-68-3 325.15–327.15 ≥ 0.995 none polyethylene glycol 35000 sigma–aldrich (germany) 25322-68-3 337.15–339.15 ≥ 0.995 none apparatus and procedure solid–liquid equilibria (sle) experiments were performed in stirring-equipped, septum -closed conical pyrex vials, in the temperature range 298.15–339.15 k and at 0.1 mpa. a visual dynamic method was applied – after slow, continuous heating, accompanied by constant stirring, the temperature at which the last solid traces disappeared was taken as that of the solid–liquid equilibrium, following the procedure applied earlier.9-11 liquid–liquid equilibria (lle) experiments were performed in the same temperature range as the sle, using a visual method of cloud point determination, explained thoroughly in previous research.9,12,13 the experiments were carried out in a self-made stirrer-equipped pyrex glass cell introduced by domanska,14 which design and use was described in our earlier contribution.13 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ effects of solid peg addition to water solutions 791 in all the experiments, a glass beaker of 2 l was used as a thermostat bath, filled with ethanol (273.15–293.15 k), water (293.15–333.15 k), or silicon oil (333.15–373.15 k) as a thermostatic liquid. temperature was controlled using a pt100 temperature probe, which had an accuracy of ±0.03 k. the uncertainties in temperature determination, estimated from the repeated measurements, were never higher than ±0.5 k, except in the sle experiments where the errors were above the aforesaid, but never exceeded ±1.0 k. solutions were prepared gravimetrically using a mettler toledo ag204 analytical semimicrobalance with the precision of ±1×10-4 g. results and discussion peg 2050 and peg 35000 are solid and the first part of our study was to determine the solid–liquid phase behavior of the binary solutions (peg 2050 + n,n-dimethylaniline) and (peg35000 + aniline or n,n-dimethylaniline), since the system peg2050 + aniline was studied in the previous work.9 thus, the experimental solid–liquid equilibrium (sle) data of the three binary systems are listed in table s-i of the supplementary material to this paper. however, the temperature-composition phase diagrams at 0.1 mpa are presented only for the system peg2050 + n,n-dimethylaniline (fig. 1) since the solubility of peg35000 in the studied aromatic molecules is very low and unsuitable for graphical presentation. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 295 300 305 310 315 320 325 330 t / k x 1 fig. 1. solid–liquid equilibria for the binary system (peg 2050 + n,n-dimethylaniline): hollow circles – experimental data; line ( )− − − presents the correlations of these data using the nrtl model (eqs. (1)–(4) later on). filled circles refer to the experimental data for the binary system (peg 2050 + aniline),9 given for comparison; line ( )⋅ ⋅ ⋅ depicts the correlations of these data using the same nrtl model; x1 represents mole fractions of peg2050 in solution. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 792 soldatović et al. n,n-dimethylaniline is a dialkylamine, a derivative of aniline with two methyl groups bonded to the nitrogen in the amine group – thus, the hydrogen bond acidity is reduced to zero, while the same parameter for aniline is as high as 0.26.15 thus, being a better proton donor, aniline builds stronger hydrogen bonds with the oxygen atoms in the peg2050 chain which are proton acceptors. this could be the reason for a better solubility of peg 2050 in aniline than in n,n-dimethylaniline as it can be seen from the comparison given in fig. 1. the impact of the polymer chain length in the studied solid–liquid phase behaviour is that the longer chain led to lower solubility in the studied organic solvents (see table s-i). this was expected since the longer chain in polymer peg 35000 obstructs intermolecular hydrogen bonding and dipole-dipole interactions with aniline or n,n-dimethylaniline. liquid–liquid phase demixing temperatures (cloud points) were determined for the systems aniline/or n,n-dimethylaniline + (water + peg 2050) and for aniline or n,n-dimethylaniline + (water + peg 35000), for different compositions of (water + peg) mixed solvent. the experimental cloud points are listed in tables s-ii–s-v of the supplementary material and the related temperature– –composition phase diagrams of the pseudo-binary systems are shown in figs. 2– –5. these diagrams show that the only useful co-solvent effect is that for aniline in water, provoked by the addition of peg 2050 to the aqueous aniline solution – fig. 2. on the other hand, peg 35000 even provoked a clear anti-solvent effect 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 310 315 320 325 330 335 340 345 t / k x 1 fig. 2. temperature–composition liquid–liquid phase diagrams at 0.1 mpa of: i) binary system aniline (1) + water (2),8 hollow circles; ii) pseudobinary system aniline (1) + (water + peg2050) (2), for the different mole fractions (xpeg) of peg in the combined (peg2050 + water) solvent, filled circles, xpeg = 0.0022, hollow triangles, xpeg = = 0.0058, filled squares, xpeg = 0.0087. x1 represents mole fractions of aniline in solutions. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ effects of solid peg addition to water solutions 793 in the aqueous n,n-dimethylaniline, decreasing the solubility of water – fig. 5. the aforementioned low solubility of solid peg 35000 in the studied aromatic molecules (see table s-i) is probably the main reason for this behaviour. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 310 315 320 325 330 335 340 345 t / k x 1 fig. 3. temperature–composition liquid–liquid phase diagrams at 0.1 mpa of: i) binary system aniline (1) + water (2),8 hollow circles; ii) pseudobinary system aniline (1) + (water + peg35000) (2) for the different mole fractions (xpeg) of peg in the combined (peg35000 + water) solvent: filled circles, xpeg = 2.720×10 -4; filled triangles, xpeg = 1.285×10 -4. x1 represents mole fractions of aniline in solutions. 0.0 0.2 0.4 0.6 0.8 1.0 310 320 330 340 350 t / k x 1 fig. 4. temperature–composition liquid–liquid phase diagram at 0.1 mpa of: i) binary system n, n-dimethylaniline (1) + water (2),8 hollow circles; ii) pseudobinary system n,n-dimethylaniline (1) + (water + peg2050) (2), for the different mole fractions (xpeg) of peg in the combined (peg2050 + water) solvent: hollow triangles, xpeg–= 0.0058; filled circles, xpeg = 0.0022; filled squares, xpeg = 0.0087. x1 represents mole fractions of n,n-dimethylaniline in solutions. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 794 soldatović et al. 0.0 0.2 0.4 0.6 0.8 1.0 310 320 330 340 350 t / k x 1 fig. 5. temperature–composition liquid–liquid phase diagrams at 0.1 mpa of: i) binary system n,n-dimethylaniline (1) + water (2),8 hollow circles; ii) pseudobinary system n,n-dimethylaniline (1) + (water + peg35000) (2), for the different mole fractions (xpeg) of peg in the combined (peg2050 + water) solvent: filled triangles, xpeg = 2.720×10 -5; filled circles, xpeg = 1.285×10 -4. x1 represents mole fractions of n,n-dimethylaniline in solutions. modeling of the binary solid–liquid equilibria (sle) data the binary sle data for the studied systems were modelled using the nonrandom two-liquid (nrtl) excess free energy model16 described with the following equations: ( ) 2 2 12 12 21 1 2 212 1 21 22 12 1 ln g g x x g xx g x τ γ τ    = +    ++    (1) ( ) 2 2 21 21 12 2 1 122 2 12 11 21 2 ln g g x x g xx g x τ γ τ    = +    ++    (2) ( )12 12 12expg α τ= − , ( )12 21 21expg α τ= − (3) 1212 g rt τ δ = , 2121 g rt τ = (4) parameter α in eq. (3) is the nrtl excess free energy non-randomness parameter, taken herein as a constant (α12 = α21 = 0.3), which is a reasonable assumption suggested in literature16 but as well gave the lowest deviations in the correlations. parameters δg12 and δg21 are the binary interaction parameters of the nrtl model, obtained by the optimization of the objective function: _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ effects of solid peg addition to water solutions 795 ( )2exp calc 1 n jj j of t t = = − (5) where n is the number of experimental points, tjexp and tjcalc are experimental and calculated equilibrium temperatures corresponding to the experimental mole fraction xj. tjcalc are expressed using the equation:17 fus m, m, ln 1 lnii i i h t x rt t γ  δ = − −     (6) in eq. (6), γi is the activity coefficient of component i in the mixture at the corresponding temperature t, xi is the mole faction and r is the gas constant. tm,i is the melting temperature of the solid solute (peg) and fus m,ihδ is its molar enthalpy of fusion at the same temperature. the respective values for peg 2050 and peg 35000 are taken from literature – peg 2050:9 fus m,ihδ = 370.37 kj mol –1, tm = 330.14 k; peg 35000:18 fus m,ihδ = 6650 kj mol –1, tm = 343.15 k. equation (6) considers reasonable assumptions, already applied in literature10,11 that: i) the difference in the normal melting temperature and the triple point temperature is small – thus, the divergence of the enthalpies of fusion at these two temperatures can be neglected and ii) the impact of the difference of the heat capacities of solid and liquid solute can be neglected. a deviation of the calculated temperature values from the experimental values is expressed as absolute average deviations δt, and absolute average percent deviations pd(t), for each binary system: exp calc 1 1 n jj j t t t n = δ = − (7) exp calc exp 1 100 pd( ), % n jj j j t t t n t= − =  (8) the results for the binary interaction parameters and the corresponding values of δt and pd(t) are presented in table ii. for all four systems satisfactory results were obtained. graphical representation of the nrtl model is given in fig. 1 for the systems peg 2050 + aniline and peg 2050 + n,n-dimethylaniline. the modelling of the systems involving peg 35000 could not be graphically presented due to small solubility of peg 35000 in the studied aromatic compounds. total absolute average percent deviation pd(t) for all the systems was less than 0.97 %. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 796 soldatović et al. table ii. values of the nrtl model parameters δg12 and δg21, absolute average deviations δt and absolute average percent deviations pd(t) between experimental and calculated data for four binary systems –δg12 / kj kmol -1 δg21 / kj kmol -1 δt / k pd(t) / % peg 2050 + aniline 12596 9317.2 1.70 0.55 peg 2050 + n,n-dimethylaniline 9671.6 8725.2 1.10 0.36 peg 35000 + aniline 20339 19896 5.29 1.58 peg 35000 + n,n-dimethylaniline 26009 22103 4.88 1.49 conclusion in this study, solid–liquid equilibria data for the binary solutions of solid polyethylene glycols (average molecular mass 2050 and 35000) in aniline and n,n-dimethylaniline, organic aromatic solvents of common interest, were experimentally determined. also, experimental liquid-liquid phase demixing temperatures (cloud points) in the pseudobinary systems (aniline or n,n-dimethylaniline + ( peg 2050 or peg 35000 + water)) were obtained. peg 2050 exhibited slightly higher solubility in aniline compared to that in n,n-dimethylaniline, most probably due to higher hydrogen bond acidity of aniline which provided stronger hydrogen bonding with the polymer chain. on the other hand, longer polymer chain provoked dramatically lower solubility in both studied organic solvents. the obtained liquid–liquid phase diagrams showed that only the addition of peg 2050 to the aqueous solution of aniline provoked a considerable co-solvent effect for aniline in water. moreover, peg 35000 even appeared to be an anti -solvent, decreasing the solubility of water in n,n-dimethylaniline. solid–liquid equilibria data for four binary systems were correlated by nrtl model with two different temperature dependant parameters, δg12 and δg21, and correlation results agree well with the experimental results. supplementary material additional experimental data are available electronically from http://www.shd.org.rs/ /jscs/, or from the corresponding author on request. acknowledgements. the authors gratefully acknowledge the financial support received from the research fund of ministry of science and environmental protection, serbia and the faculty of technology and metallurgy, university of belgrade (project no. 172063). _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ effects of solid peg addition to water solutions 797 и з в о д ефекти додавања чврстих поли етиленгликола у водене растворе анилина или н,н-диметиланилина: експериментална мерења и моделовање данијела солдатовић1, никола грозданић2, зоран вишак2, ивона радовић2 и мирјана кијевчанин2 1јавно предузеће нуклеарни објекти србије, мике петровића аласа 12-14, винча, 11351 београд, 2технолошко-металуршки факултет, универзитет у београду, карнегијева 4, 11120 београд и 3 engineering and applied sciences, aston university, birmingham, b4 7et, united kingdom испитивана је фазна равнотежа течност–течност и чврстo–течност за десет псеудобинарних и три бинарна система који садрже поли етиленгликол (peg) 2050, peg 35000, анилин, n,n-диметиланилин и воду, у температурном опсегу 298,15–350,15 k и при атмосферском притиску. добијени фазни дијаграми температура–састав показали су да је peg 2050 једини функционални ко-растварач за анилин у води, док је peg 35000 показао јасне анти-растварачке ефекте у воденом раствору n,n-диметиланилина. експериментални подаци равнотеже чврсто–течност корелисани су помоћу nrtl модела, а добијени резултати су у складу са експерименталним резултатима. 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design develop. 8 (1969) 413 17. j. m. prausnitz, molecular thermodynamics of fluid-phase equilibria, prentice-hall ptr, upper saddle river, nj, 1969, pp. 390–391 18. k. pielichowski, k. flejtuch, polym. adv. technol. 13 (2002) 690. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {adsorption and removal of bisphenol a from aqueous solution by p-phenylenediamine modified magnetic graphene oxide} j. serb. chem. soc. 82 (1) 39–50 (2017) udc 547.623:544.35–145.2+547.553.1+ jscs–4945 546.25–162–31:544.723 original scientific paper 39 adsorption and removal of bisphenol a from aqueous solution by p-phenylenediamine-modified magnetic graphene oxide xiaosheng tang1, ping tang2,3, shihui si4 and liangliang liu2* 1hubei key laboratory of edible wild plants conservation and utilization & college of life sciences, hubei normal university, huangshi, hubei 435002, china, 2institute of bast fiber crops, chinese academy of agricultural sciences, changsha 410205, china, 3school of environmental science and engineering, hubei polytechnic university, hubei key laboratory of mine environmental pollution control and remediation, huangshi, hubei 435003, china and 4college of chemistry and chemical engineering, central south university, changsha, hunan 410083, china (received 30 april, revised 17 september, accepted 10 october 2016) abstract: p-phenylenediamine-functionalized magnetic graphene oxide nanocomposites (ppd–mgo) were prepared and utilized in the adsorption and removal of bisphenol a from aqueous solution. the novel nanomaterials were characterized by transmission electron microscopy (tem), fourier transform infrared spectroscopy (ft-ir) and a vibrating sample magnetometer (vsm). the factors affecting the adsorption of bisphenol a, including adsorption time, temperature and ph of solution, adsorption kinetics and isotherms were all investigated. the results showed that the ppd–mgo nanomaterial exhibited good adsorption ability for bisphenol a and good re-usability. the maximum adsorption capacity reached 155.0 mg g-1 at 45 °c and ph 7. the removal rate was 99.2 % after three times adsorption with the new nanomaterials. after five adsorption cycles, the adsorption capacity of the ppd–mgo remained at 94.0 %. the adsorption of bisphenol a was found to fit pseudo second order kinetics equations and the freundlich adsorption model. the experimental results showed the ppd–mgo nanomaterial had a good adsorption ability to remove organic compounds from aqueous solution. keywords: adsorption; bisphenol a; graphene oxide; magnetic nanoparticles. introduction with the development of modern industry in these years, water pollution has become a universal crisis.1 continuous concern for public and environmental health has led to increasing interest in treating toxic organic and inorganic pollutants in water.2 moreover, many new chemical compounds have been iden * corresponding author. e-mail: liuliangliang@caas.cn doi: 10.2298/jsc160430095t 40 tang et al. tified as harmful to the environment. endocrine disrupting compounds (edcs) are derived from various kinds of chemicals, including drugs, pesticides, consumer products, and pollutants.3 edcs are harmful to human health and the ecological environment, and could result in bio-accumulation and reproductive toxicity. it was reported that edcs disturb the human endocrine system by mimicking, blocking or disrupting the functions of hormones.4 bisphenol a (bpa), classified as a type of edc, is widely used in the production of polycarbonates, epoxy resins, plasticizers, flame retardants and other chemical products.5 since bpa at low concentrations could cause infertility and breast cancer, wastewater containing bpa has to be treated adequately before being discharged into environment.6 the development of a rapid and efficient method for removal of bpa is therefore of considerable importance. nowadays, many techniques have been applied in water treatment. among these techniques, adsorption seems to be the most extensively utilized and economically feasible method, possessing a relatively high capacity and low cost.7 various nanomaterials have been used as adsorbents for the removal of toxic pollutant from water in recent years.8–11 graphene oxide (go) is a novel two -dimensional nanomaterial prepared from graphite. it has attracted significant attention in wastewater treatment due to its high mechanical strength and ease of functionalization.12 go contains abundant hydrophilic oxygen-containing functional groups on its basal planes and edges, which provide go with a highly efficient adsorption ability.13 therefore, go could serve as an ideal adsorption material because of its large specific surface area, abundant groups and high water solubility.14,15 magnetic nanomaterials have drawn much attention in adsorption and separation because they are easily separated from the solution and recycled. the magnetic properties of magnetic nanomaterials enable separation using a magnet instead of filtration, centrifugation or decantation.16,17 magnetic nanomaterials have been used as adsorbents for the adsorption and removal of toxic compounds in aqueous solution, especially after modification.18 go-based magnetic nanomaterials were also widely used in pollution control.19–22 the combination of magnetic materials and go could significantly simplify the separation process and increase the adsorption capacity. in this study, p-phenylenediamine (ppd)-functionalized magnetic nanoparticles (ppd–mn) were successfully synthesized and bound onto go, and the ppd-functionalized magnetic go (ppd–mgo) was applied in the adsorption and removal of bpa. the existence of ppd could increase the interaction between adsorbent and target compounds because of the introduction of phenyl groups and amino groups. the effects of ph, temperature and adsorption time on the adsorption of bpa, the re-usability of ppd–mgo and removal of bpa were investigated. the kinetics and isotherm parameters of the adsorption were adsorption and removal of bpa by ppd–mgo 41 determined and calculated. the experimental results showed ppd–mgo had a high adsorption capacity and could be easily re-used. experimental chemicals and materials bisphenol a (bpa), p-phenylenediamine (ppd), glutaradehyde, n-hydroxyl succinimide (nhs), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (edc), and 3-aminopropyltriethoxysilane were purchased from sigma–aldrich. ferric chloride hexahydrate (fecl3·6h2o) and poly(ethylene glycol) (peg 6000) were purchased from sinopharm chemical reagents. ultrapure water (18.2 mω cm resistivity) was obtained from a milli-q water purification system. all other reagents used in this study were of analytical grade. preparation of ppd–mgo preparation of ppd functionalized fe3o4 nanoparticles (ppd–mn). fecl3·6h2o (1.350 g), peg 6000 (1.000 g) and anhydrous sodium acetate (3.600 g) were mixed in ethylene glycol (40 ml) under stirring for 30 min. the mixture was transferred to a stainless steel autoclave and maintained at 180 °c for 8 h. finally, the black fe3o4 nanoparticles were obtained by magnetic separation, washed with ethanol three times and dried under vacuum at 40 °c for 2 h. the fe3o4 nanoparticles (0.100 g) were then added into an ethanol solution (200 ml, 99.5 %) with mechanical agitation. after 30 min reaction, 3-aminopropyltriethoxysilane (2.0 ml) was slowly added and the solution was stirred at room temperature for 6 h. the obtained amino fe3o4 nanoparticles were washed with ethanol three times and dried under vacuum at 40 °c for 2 h. the amino fe3o4 nanoparticles (0.100 g) were added into a glutaradehyde solution (20 ml, 5 vol. %) and stirred for 30 min. then ppd solution (5 ml, 20 mg ml-1) was added to the mixture and stirred. after further 30 min reaction, the excess ppd was removed by magnetic separation and the ppd–mn was washed three times with ethanol. the ppd–mn was dried under vacuum at 40 °c for 2 h. preparation of go. go was prepared according to the improved synthetic method reported by marcano.23 concentrated h2so4 (120 ml), h3po4 (13 ml), graphite flakes (1.000 g) and kmno4 (6.000 g) were mixed and heated at 50 °c for 12 h with magnetic stirring. then the mixture was poured into ice (130 ml) containing 3 ml of 30 % h2o2 and centrifuged (8000 rpm) for 1 h. the supernatant was decanted and the solid material was washed with 30 % hcl and water five times respectively under the same centrifugation conditions. finally, the obtained solid material was dried under vacuum at 40 °c for 6 h. preparation of ppd–mgo. edc (5 ml, 5 mg ml-1) and nhs (5 ml, 5 mg ml-1) were added to a solution of go (50 ml, 2 mg ml-1) under continuous stirring to activate the carboxyl groups of go.24 ppd–mn (0.200 g) was then added and dispersed ultrasonically for 30 min. the obtained product was collected by magnetic separation and dried in vacuum at 40 °c for 4 h. characterizations of ppd–mgo ppd–mgo and go were characterized using a jeol200cx electron microscope (jeol, tokyo, japan) at 160 kv for tem images. ft-ir spectra were obtained using a nicolet avatar 360 ft-ir spectrophotometer (thermo fisher nicolet, orlando, usa). the magnetic property was investigated at room temperature using a lakeshore 7407 vibrating sample magnetometer (vsm, westerville, ohio, usa). 42 tang et al. adsorption of bpa ppd–mgo (5 mg) was added to solutions (20 ml) containing different concentrations of bpa and the mixtures were shaken for 120 min at 25, 35 and 45 °c. the mixtures were separated with a magnet and the concentration of bpa was monitored by uv–vis spectrophotometer (uv-2600, shimadzu, kyoto, japan) at 276 nm. for comparison, the same amount of fe3o4 nanoparticles, go, ppd–mn and ppd–mgo (5 mg) were added to bpa solution (20 ml, 250 mg l-1) and the mixtures were shaken for 120 min at 25 °c. the mixture was separated with a magnet or centrifugation and the concentration of bpa was monitored by uv–vis spectrophotometry at 276 nm. to study the effects of the initial ph on the adsorption of bpa, the initial ph value of the solution was adjusted to 5.0–9.0 by addition of hydrochloric acid or sodium hydroxide. all experiments were performed in triplicate. adsorption kinetics ppd–mgo (5 mg) and bpa solution (20 ml, 100 mg l-1) were mixed and shaken at 25 °c. the concentration of bpa was monitored by uv–vis spectrophotometry at certain intervals. in order to study the mechanism of the adsorption kinetics, the pseudo-first-order kinetic model and the pseudo-second-order kinetic models were used. the pseudo-first-order kinetic model could be expressed by the following equation: e e 1ln( ) lntq q q k t− = − (1) where qe (mg g-1) is the equilibrium adsorption capacity, qt (mg g-1) is the amount of adsorbed at time t, k1 (g mg-1 min-1) is the rate constant of adsorption. the pseudo-second-order kinetic model could be expressed by the following equation: 2 ad e e 1 1 t t t q k q q = + (2) where kad (g mg-1 min-1) is the rate constant of adsorption, qe (mg g-1) is the equilibrium adsorption capacity, qt (mg g-1) is the amount adsorbed at time t.25 adsorption isotherm ppd–mgo (5 mg) was added to bpa solution (20 ml) at a concentration in the range of 75–250 mg l-1. the mixtures were shaken at 25, 35 and 45 °c for 120 min and the concentration of bpa was determined. the langmuir and freundlich isotherm26 models were used to evaluate the adsorption isotherm. reusability of ppd–mgo to study the reusability, ppd–mgo (5 mg) was added to a bpa solution (20 ml, 100 mg l-1) and shaken for 120 min at 45 °c. the mixture was then separated with a magnet and the concentration of bpa was determined. the recycle of ppd–mgo was obtained by washing with ethanol and water three times. then the recycled ppd–mgo was added into another bpa solution to start a new adsorption. all the experiments were performed in triplicate. results and discussion characterization of ppd–mgo the tem images of go and ppd–mgo are presented in fig. 1. the sheet structure with smooth surface and wrinkled edges of go could be found in fig. 1a. it also could be seen that ppd–mn was bound with go and the shape of go adsorption and removal of bpa by ppd–mgo 43 changed in fig. 1b. the mean diameter of the fe3o4 nanoparticles was about 200 nm. these characteristics of go and the shape of fe3o4 nanoparticles could also be found in reported related research.27,28 it could be considered that ppd–mgo was successfully fabricated. fig. 1. tem images of (a) go and (b) ppd–mgo. the ft-ir spectra of fe3o4 nanoparticles, ppd–mn, go and ppd–mgo are presented in fig. 2. the ft-ir spectrum of the fe3o4 nanoparticles showed the major characteristic peak at 572 cm–1. the ft-ir spectrum of ppd–mn showed featured adsorptions due to the presence of ppd. the adsorptions at 3423, 1510, 1083 and 821 cm–1 could be identified as n–h stretching vibrations, the skeleton vibration of the benzenoid ring, and the in-plane and out-of-plane bending vibrations of c–h, respectively.29,30 additionally, the weak band at 2921 cm–1 was associated with c–h stretching vibrations. the bands at 1627 fig. 2. ft-ir spectra of: a) fe3o4 nanoparticles, b) ppd–mn, c) go and d) ppd–mgo. 44 tang et al. cm–1 and 1296 cm–1 were assigned to c–n stretching vibrations.31 the spectrum of ppd–mn also showed the characteristic peak at around 580 cm–1, assigned to fe–o vibrations. the ft-ir spectrum of go showed absorptions at 3432, 1731 and 1630 cm–1, assigned to hydroxyl, carbonyl and sp2-bonded carbon groups on the go, respectively.32,33 the absorptions at 1054 cm–1 and 1384 cm–1 were considered as the c–o stretching peak and o–h deformation peak.32 all the feature peaks of the spectra of go and ppd–mn could be observed in the spectrum of ppd–mgo. it could be assumed that ppd–mgo was successfully synthesized. the vsm characterization of the fe3o4 nanoparticles and ppd–mgo were conducted at room temperature to confirm the magnetic properties. as shown in fig. 3, the saturation magnetization values of the fe3o4 nanoparticles and ppd– –mgo were 71.7 and 33.6 emu g–1, respectively. compared to fe3o4 nanoparticles, the decrease in the saturation magnetization value of ppd–mgo could be attributed to the existence of non-magnetic materials.34,35 the sufficient magnetic response and almost zero remanence of ppd–mgo guaranteed the materials for magnetic separation with an ordinary magnet. fig. 3. magnetization curve of: a) fe3o4 nanoparticles and b) ppd–mgo. in order to confirm the adsorption capacity of ppd–mgo, the adsorption of bpa with fe3o4 nanoparticles, go, ppd–mn and ppd–mgo were conducted and compared under the same condition. the adsorption capacities of fe3o4 nanoparticles, go, ppd–mn and ppd–mgo at 25 °c were 40.4, 108.3, 62.8 and 151.6 mg g–1, respectively. it was found that the adsorption capacity was increased after the modification of ppd and the combination between go and ppd–mn. as adsorption is a surface phenomenon, the adsorbent with a higher surface area leads to a higher adsorption capacity. the excellent adsorption ability of ppd–mgo is the result of its large surface area (theoretical surface adsorption and removal of bpa by ppd–mgo 45 area up to 2675 m2 g–1) and its abundance of carboxyl, hydroxyl and epoxy groups.36–38 effect of ph the ph of a solution plays an important role in adsorption processes. it affects the adsorption capacity of an adsorbent and electrostatic interaction between compounds and the adsorbent surface. the effect of ph on the adsorption of bpa was investigated at different ph values, ranging from 5.0 to 9.0. as shown in fig. 4, the adsorption capacity reached a maximum when the ph was 7.0 and then decreased with increasing ph.39 the decrease in the adsorption capacity for bpa might be due to deprotonation of the hydroxy of phenol when the ph was above 7.40 the adsorptive effect might be mediated by hydrogen bonding between bpa and ppd–mgo and by hydrophobic adsorption.41,42 therefore, the optimum ph for adsorption of bpa was set at 7.0. fig. 4. the influence of the initial ph value on the adsorption of bpa. adsorption kinetics the data related to kinetics of bpa adsorption on ppd–mgo are shown in fig. 5a. the concentration of bpa decreased significantly in the first 30 min, and then the decrease trended to slow down until the adsorption equilibrium was achieved. when the incubation time exceeded 120 min, no increase in the adsorbed amount was apparent. considering the efficiency of the experiments, an adsorption time for the experiments was selected to be 120 min. the adsorption of bpa was fitted with the pseudo-first-order and pseudo -second-order kinetic equations. the corresponding results are shown in fig. 5b and c, respectively. for the pseudo-first-order equation, the constant k1 was calculated as 0.0343 g mg–1 min–1 and the correlation coefficient of fitting the model was 0.996, while the constant kad in the pseudo-second-order kinetic equation was calculated as 0.0055 g mg–1 min–1 and the correlation coefficient of fitting the model reached 0.999. the results indicated that the adsorption of bpa on ppd–mgo fitted better with the pseudo-second-order kinetic equation. it 46 tang et al. could be suggested that the adsorption behavior of bpa onto ppd–mgo was promoted by a chemical process involving valence forces through sharing or exchange of electrons between sorbent and sorbate as covalent forces.43 this means that the sorption of bpa on ppd–mgo might be due to chemical reaction between the negative charge on ppd–mgo and the positive charge on the bpa molecules.44 fig. 5. a) adsorption kinetics in aqueous solution of bpa with ppd–mgo, b) the pseudo-first-order adsorption rates and c) the pseudo-second-order adsorption rates. adsorption isotherm adsorption isotherm is important for investigating the adsorption behavior of an adsorbent. the correlation between the experimental equilibrium data and theoretical model is also essential for the interpretation of the adsorption. in this study, the isotherm data for the adsorption of bpa on ppd–mgo were fitted with the langmuir and freundlich models. the adsorption isotherms are shown in fig. 6 and the parameters of the langmuir and freundlich models were listed in table i. apparently, the coefficient of correlation of freundlich model was higher than that of langmuir model. the freundlich isotherm model can be used to describe sorption on heterogeneous surfaces and multilayer sorption formation.45 therefore, compared to the langmuir model, the adsorption of bpa on ppd–mgo was more coincident with the freundlich model. adsorption and removal of bpa by ppd–mgo 47 fig. 6. a) adsorption isotherms for bpa on ppd–mgo, b) fitting by the langmuir model and c) fitting by the freundlich model. table i. isotherm parameters for the adsorption of bpa on ppd–mgo model parameter t / °c 25 35 45 langmuir qm / mg g-1 152.0 155.0 158.0 b 0.0552 0.0542 0.0551 r2 0.703 0.7177 0.7114 freundlich 1/n 0.8328 0.8627 0.8665 k / mg g-1 12.1367 10.5974 9.9038 r2 0.9882 0.9863 0.9896 reusability of ppd–mgo and removal of bpa the reusability of an adsorbent is important in practical applications. the recycling of an adsorbent would reduce the cost of the adsorbent and the impact of the adsorbent on the environment.46 the results of the reusability test of ppd– –mgo are shown in fig. 7. during five cycles of use, a small drop in the adsorption capacity of ppd–mgo could be observed. however, the adsorption capacity of ppd–mgo remained at 141.0 mg g–1 (94 % of the initial adsorption capacity) after five cycles. these results showed that ppd–mgo exhibited satisfactory reusability in adsorption. the removal of bpa with successive cycles of ppd–mgo was conducted. after one cycle adsorption, the absorbent was separated with a magnet and new 48 tang et al. ppd–mgo was added to start the next cycle of adsorption. the removal efficiency was calculated using the following equation: e 0 removal efficiency, % 100 1 c c   = −    (5) where ce (mg l–1) is the equilibrium concentration of bpa and c0 (mg l–1) is the initial concentration of bpa. the removal efficiency of bpa with successive new ppd–mgo could reach 99.2 % after three cycles (fig. 8). these results showed that the prepared adsorbents could effectively remove bpa from the environment. fig. 7. reusability of ppd–mgo. fig. 8. removal of bpa using ppd– –mgo. conclusions in summary, ppd–mgo was successfully prepared and characterized. the adsorption kinetics and isotherms were investigated in detail. the adsorption of bpa followed pseudo-second-order kinetics and the equilibrium data were fitted well with the freundlich isotherm model. ppd–mgo also showed good reusability in the adsorption and bpa could be effectively removed by three times adsorption and removal of bpa by ppd–mgo 49 successive adsorptions. in conclusion, ppd–mgo could be a competitive candidate with great potential applications for the removal of bpa from water. acknowledgements. this work was supported by science and technology research project of hubei province education department (b2016164). и з в о д адсорпција и уклањање бисфенола а из водених раствора применом магнетног графен-оксида модификованог p-фенилендиамином xiaosheng tang1, ping tang2,3, shihui si4 и liangliang liu2 1hubei key laboratory оf edible wild plants conservation аnd utilization & college оf life sciences, hubei normal university, huangshi, hubei 435002, china, 2institute оf bast fiber crops, chinese academy оf agricultural sciences, changsha 410205, china, 3school of environmental science and engineering, hubei polytechnic university, hubei key laboratory of mine environmental pollution control and remediation, huangshi, hubei 435003, china и 4college оf chemistry аnd chemical engineering, central south university, changsha, hunan 410083, china нанокомпозити магнетног графен-оксида функционализованог p-фенилендиамином (ppd–mgo) су припремљени и коришћени за адсорпцију и уклањање бисфенола а из водених раствора. нови наноматеријали су окарактерисани трансмисионом електронском микроскопијом (тем), инфрацрвеном спектроскопијом са фуријеовом трансформацијом (ft-ir) и магнетометром са вибрирајућим узорком (vsm). адсорпција бисфенола а је испитивана праћењем времена адсорпције, температуре, ph раствора, адсорпционе кинетике и изотерми. резултати су показали да је ppd–mgo наноматеријал добар адсорбент бисфенола а и да је могућа његова вишеструка примена као адсорбента. максимални адсорпциони капацитет био је 155,0 mg g-1 на 45 °c и ph 7. степен уклањања је био 99,2 % након три пута поновљене адсорпције са новим наноматеријалима. после пет циклуса адсорпције, адсорпциони капацитет ppd–mgo је остао 94,0 %. адсорпција бисфенола а пратила је кинетику псеудо другог реда и фројндлихову адсорпциону изотерму. експериментали резултати су показали да ppd–mgo наноматеријал има добре адсорпционе карактеристике за уклањање органских једињења из водених раствора. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice analysis of alcohol dehydrogenase inhibitors from desmodium styracifolium using centrifugal ultrafiltration coupled with hplc–ms j. serb. chem. soc. 80 (8) 1051–1059 (2015) udc *desmodium styracifolium:547.42/.43+ jscs–4779 577.152.1:541.18.043+541.18.045 original scientific paper 1051 analysis of alcohol dehydrogenase inhibitors from desmodium styracifolium using centrifugal ultrafiltration coupled with hplc–ms liangliang liu1, miao chen1 and xiaoqing chen1,2* 1institute of bast fiber crops, chinese academy of agricultural sciences, changsha 410205, china and 2school of chemistry and chemical engineering, central south university, changsha 410083, china (received 19 september, revised 16 december 2014, accepted 9 march 2015) abstract: alcohol dehydrogenase (adh) inhibitors play an important role in the treatment of human methanol or ethylene glycol poisoning and the suppression of acetaldehyde accumulation in alcoholics. in this study, centrifugal ultrafiltration coupled with high performance liquid chromatography-mass spectrometry (hplc–ms) was utilized to screen and identify adh inhibitors from an ethyl acetate extract of desmodium styracifolium (osb.) merr. the experimental conditions of the centrifugal ultrafiltration were optimized. under the optimum conditions (adh concentration: 37.5 μg ml-1, incubation time: 90 min, ph 7.0 and temperature: 15 °c), formononetin and aromadendrin were successfully screened and identified from the ethyl acetate extract of d. styracifolium. the screening result was verified by adh inhibition assays. the ic50 values of formononetin and aromadendrin were 70.8 and 84.7 μg ml-1, which were in accordance with their degrees of binding. aromadendrin was for the first time reported to have inhibitory activity on adh. this method provides an effective way to screen active compounds from natural products. keywords: alcohol dehydrogenases; aromadendrin; centrifugal ultrafiltration; desmodium styracifolium; formononetin. introduction nowadays, natural products have become the primary source for chemical and pharmaceutical research because of their long history in clinical practice and reliable therapeutic efficacy.1,2 in the last thirty years, 46 % of the new chemical entities approved as drugs by the us food and drug administration were relevant to natural products.3 however, the chemical composition of natural products is complex. compared to the numerous compounds existing in natural pro * corresponding author. e-mail: xqchen@csu.edu.cn doi: 10.2298/jsc140919023l _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1052 liu, chen and chen ducts, the numbers and contents of active compounds are relatively low. the conventional procedures for screening active compounds in natural products are laborious and time-consuming.4 accordingly, numerous screening methods were developed, such as cell membrane chromatography, ultrafiltration and equilibrium dialysis.5–7 centrifugal ultrafiltration is a rapid method using centrifugation force and a semi-permeable membrane to retain high molecular weight solutes.8,9 compounds with a molecular weight higher than the nominal molecular weight cutoff of the membrane would be retained, while low molecular weight compounds could pass through the membrane.10,11 based on this principle, when an extract of natural products was incubated with some enzyme, the active compounds in the natural products would bind to the enzyme and be trapped by the membrane together with enzyme. combined with high performance liquid chromatography– –mass spectrometry (hplc–ms) analysis, active compounds could be identified by comparing the chromatograms before and after centrifugal ultrafiltration. therefore, centrifugal ultrafiltration coupled with hplc-ms could become a simple and powerful tool for discovering active compounds from natural products,12,13 and many examples have been performed by this method to screen enzyme inhibitors and ligands.14–19 these studies showed that the screening could be accomplished rapidly and the crude samples could be analyzed without further purification. alcohol dehydrogenases (adh) catalyzes the oxidation of alcohols with the reduction of nicotinamide adenine dinucleotide (nad+) in many organisms.20 in the human body, adh is also involved in the oxidation of methanol to formaldehyde and ethylene glycol to glycolic and oxalic acids.21,22. however, the existence of formaldehyde or glycolic acid is harmful to humans. adh inhibitors hinder the metabolisms of methanol and ethylene glycol, and, consequently, could be used in the therapies of human methanol and ethylene glycol poisonings.23,24 moreover, adh inhibitors could suppress acetaldehyde accumulation in alcoholics.25 desmodium styracifolium (osb.) merr. is known in china for its heat-clearing and diuretic properties. it is also an important chinese medicine for the treatments of renal stones and cardio-cerebrovascular disease. the experiments showed that the ethyl acetate extract of d. styracifolium showed inhibition on adh. however, the related detail research is still inadequate. in this study, adh inhibitors from d. styracifolium were analyzed by centrifugal ultrafiltration coupled with hplc-ms. the experimental conditions, including enzyme concentration, incubation time, ph and temperature, were investigated and optimized. two adh inhibitors were identified under the optimum conditions. experiment results proved this method could screen and analyze adh inhibitors without the purification of natural samples and the screening results were reliable. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ alcohol dehydrogenase inhibitors analyzed by centrifugal ultrafiltration/hplc–ms 1053 experimental materials dried desmodium styracifolium (osb.) merr. was purchased from beijing tongrentang co., ltd. (changsha, china). alcohol dehydrogenase (adh) was acquired from sigma– –aldrich chemicals (st. louis, mo, usa). nicotinamide adenine dinucleotide (nad+) was obtained from f. hoffmann-la roche ltd. (basel, switzerland). acetonitrile, methanol and acetic acid used for hplc were chromatographic grade and obtained from tedia company inc. (fairfield, oh, usa). ultrapure water (18.2 mω resistivity) was obtained from a milli-q water purification system (millipore, bedford, ma, usa). the other chemicals were analytical grade. formononetin and aromadendrin were isolated and characterized from d. styracifolium in the laboratory and their structures were identified by uv, ms and nmr.26. their purities were determined to be over 95 % by normalization of the peak areas detected by hplc-dad–ms/ms. preparation of desmodium styracifolium extract dried d. styracifolium (30.00 g) was suspended in 300 ml of ethanol (95 %,) and reflux extracted at 85 °c for 3 h. after cooled at room temperature, the solvent was removed with vacuum rotary evaporation. then the residue (1.25 g) was dissolved in 150 ml of water and extracted successively with equal volumes of petroleum ether, ethyl acetate and n-butanol. the evaporated ethyl acetate extract (0.19 g) was dissolved in 150 ml of water, filtered through a 0.45 μm membrane (acrodisc® syringe filter, pall, port washington, ny, usa) and stored at 4 °c for further experiments. screening of adh inhibitors by centrifugal ultrafiltration a mixture of d. styracifolium extract (100 μl, 0.5 mg ml-1) and adh solution (100 μl, 37.5 μg ml-1) was incubated at 15 °c for 90 min. after incubation, the mixture was poured into a centrifugal ultrafiltration device (nanosep mf centrifugal devices, ≤10 kda, pall, port washington, ny, usa) and centrifuged at 7000 rpm for 30 min at room temperature. the control experiments were performed under the same conditions using denatured enzyme instead of active enzyme. the filtrates were directly analyzed by hplc–ms without dilution. all the binding assays were performed in triplicate. hplc–ms analysis hplc was performed on an acquitytm uplc system (waters, milford, ma, usa) with a cooled autosampler and column oven enabling temperature control of the analytical column. a reversed phase sunfiretm c18 (250 mm×4.6 mm i.d., 5 μm, waters, milford, ma, usa) column was employed for the analysis. the chromatographic separation was realized using a mixture of solvents, acetonitrile (a) and water containing 0.4 % acetic acid (b). the gradient program for the d. styracifolium extract was as follows: 22–33 % a (0–10 min) and 33–40 % a (10–20 min). the flow rate was 1.0 ml min-1 and the column temperature was 25 °c. the detection wavelength was set at 254 nm and the injection volume was 20 μl. triple-quadrupole mass detection was performed on a micromass® quattro microtm api mass spectrometer (waters corp., milford, ma, usa) with an electrospray ionization (esi) interface. the esi source was set in the negative ionization mode. the following settings were applied to the instrument: capillary voltage, 3.00 kv; cone voltage, 40.0 v; extractor voltage, 3.00 v; source temperature, 120 °c; desolvation temperature, 400 °c; desolvation gas flow rate, 750 l h-1; cone gas flow rate, 50 l h-1 and dwell time, 0.05 s. nitrogen was used as the desolvation and cone gas. mass detection was performed in the full scan mode for m/z in the range 160–800. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1054 liu, chen and chen all collected data were acquired and processed using masslynxtm nt 4.1 software with quanlynxtm program (waters corp., milford, ma, usa). enzyme activity assay a mixture of ethanol (500 μl), nad+ (500 μl, 1 mg ml-1) and phosphate buffer solution (1000 μl, ph 7.0, 0.2 m, pbs) were used as the substrate. to initiate the reaction, 10 μl of adh (37.5 μg ml-1) was added to the substrate at 15 °c. after incubation for 5 min, the increase in absorbance at 340 nm was measured using an uv–vis spectrophotometer (uv-2450, shimadzu, kyoto, japan). one unit of adh activity was defined as the amount of enzyme that reduced 1 nmol of nad+ per minute under the assay conditions. when the inhibition of adh activity was studied, 1000 μl of d. styracifolium extract with different concentrations were used instead of pbs. the substrate was incubated for 5 min at 15 °c before the addition of 10 μl of adh (37.5 μg ml-1). the mixture was incubated for 5 min and the increase in absorbance at 340 nm was measured. the percentage inhibition, i%, of the adh activity was calculated using eq. (1). 1 0 % 1 100  δ = −   δ  a i a (1) where δa0 is the increase in absorbance at 340 nm and δa1 is the increase in absorbance at 340 nm in the presence of d. styracifolium extract. all experiments were conducted in triplicate under the same conditions and the results were obtained as the average of the corresponding experimental values. optimization of the screening conditions the effect of adh concentration on the screening was evaluated using different concentrations of adh (12.5, 25.0, 37.5 and 50.0 μg ml-1). various incubation times (10, 60, 90 and 120 min) were also studied to determine the appropriate incubation time for the assays. to acquire the optimum ph and temperature for screening, the experiments were conducted at different ph value from 5.0 to 9.0 and temperatures in the range from 5 to 45 °c. all experiments were conducted in triplicate and the results were obtained as the average of the corresponding experimental values. the binding strength of compounds to adh was defined as the binding degree, bd%, which can be calculated using eq. (2): b % 1 100 a  = −     a bd a (2) where aa and ab are the peak areas of a compound in the hplc chromatograms incubated with denatured and active adh, respectively. results and discussion optimization of hplc analysis the separation of each compound in the extracts of natural products is a challenge for hplc analysis. acid in the mobile phase could reduce peak tailing and increase the signal.27 therefore, different mobile phases with different flow rates, detection wavelengths and column temperatures were utilized to optimize the hplc conditions for the analysis of the d. styracifolium extract. the results showed that the optimum chromatographic separation was achieved using a mix_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ alcohol dehydrogenase inhibitors analyzed by centrifugal ultrafiltration/hplc–ms 1055 ture of solvents, acetonitrile (a) and water containing 0.4 % acetic acid (b). a gradient program was operated as follows: 22–33 % a (0–10 min) and 33–40 % a (10–20 min). the flow rate was 1.0 ml min–1 and the column temperature was 25 °c. the detection wavelength was set at 254 nm and the injection volume was 20 μl. all constituents could reach baseline separation and a relatively short analysis time was achieved. chromatograms obtained under these conditions are shown in fig. 1. fig. 1. the chromatograms of filtrates incubated with denatured (a) and active (b) adh. optimization of the screening conditions effect of adh concentration. different concentrations of adh (12.5, 25.0, 37.5 and 50.0 μg ml–1) were incubated with d. styracifolium extract to investigate the effect of the adh concentration on binding degree. the chromatograms of filtrates incubated with different concentrations of adh are shown in fig. 2a. the peaks of compounds 1 and 2 decreased with increasing adh concentration. when the concentration of adh was higher than 37.5 μg ml–1, the fig. 2. a) the chromatograms of the filtrates incubated with different concentrations of adh; b) the effect of the adh concentration on the binding degrees. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1056 liu, chen and chen binding degrees remained unchanged (fig. 2b). as superfluous enzyme is wasteful and would increase the cost of the experiment, an adh concentration of 37.5 μg ml–1 was considered optimal. effect of incubation time. a sufficient period was necessary to achieve ligand–enzyme equilibrium. hence, different incubation periods ranging from 10 to 120 min were studied to investigate the effect of time on the binding degree. the binding degrees of compounds 1 and 2 at different incubation times were calculated and are shown in fig. 3a. the binding degrees of compounds 1 and 2 became the highest when the incubation time reached 90 min, and did not increase on prolongation of the incubation time. the results manifested that 90 min incubation time was sufficient in screening experiments. fig. 3. a) effect of incubation time on the binding degrees; b) effect of ph on binding degrees; c) effect of temperature on binding degrees. effect of ph. the ph value affects the status of an enzyme and its activity. the effect of ph on the binding degree was studied at different ph values ranging from 5.0 to 9.0. as shown in fig. 3b, the maximum binding degrees of compounds 1 and 2 were obtained at ph 7.0. as reported in the literatures, adh showed optimum activity at ph 7.0, which was in accordance with the obtained experiment results.28 therefore, the screening ph value was set at 7.0. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ alcohol dehydrogenase inhibitors analyzed by centrifugal ultrafiltration/hplc–ms 1057 effect of temperature. the effect of temperature on binding degree was investigated and the results are shown in fig. 3c. it was found that the highest binding degrees were achieved at 15 °c. the enzyme is thermally sensitive in general and its activity would decrease in a high temperature environment.29 thus, the temperature was set at 15 °c in order to maintain the enzyme active during the experiments. screening and identification of adh inhibitors the ethyl acetate part of the d. styracifolium extract showed adh inhibition with an ic50 value of 36.2 μg ml–1, while the ic50 values of the petroleum ether and n-butanol parts were greater than 500 μg ml–1. these results suggested that there were compounds that inhibited adh in the ethyl acetate part of the d. styracifolium extract. therefore, the ethyl acetate part of the d. styracifolium extract was selected as the screening sample and was analyzed by centrifugal ultrafiltration combined with hplc–ms. screening experiments with denatured adh were performed to exclude the possibility of nonspecific adsorption between the compounds and enzyme. the chromatograms of the filtrates incubated with denatured and active adh are seen in fig. 1. two compounds marked as 1 and 2 clearly showed decreases in peak areas after centrifugal ultrafiltration with active adh. the chemical structure of these two compounds was identified according to their uv and hplc–ms spectroscopic data. according to previous reports, the data shown in table i were in agreement with literature values.30,31 therefore, these two compounds were identified as formononetin and aromadendrin. their chemical structures are shown in fig. 4. the screening results indicated formononetin and aromadendrin had potential inhibitory activities on adh. fig. 4. chemical structures of the screened compounds. table i. the identification, uv, and ms characteristics of compounds in d. styracifolium peak formula uv (λmax / nm) proposed ion structural assignment ref. 1 c16h12o4 248.3 [m+h] + 269 formononetin 30 2 c15h12o6 225.3 [m+h] + 289 aromadendrin 31 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1058 liu, chen and chen inhibition analysis of the screened compounds in order to confirm the screening results, the inhibitory activities of authentic formononetin and aromadendrin were analyzed. the authentic samples of formononetin and aromadendrin exhibited inhibitory activities on adh, and their ic50 values were 70.8 and 84.7 μg ml–1, respectively. in addition, the binding degrees of formononetin and aromadendrin under the optimum conditions were 90.0 % and 86.5 %, respectively. these results showed that the compound with high binding degree had the lower ic50 value. the binding degree could indicate not only the potential inhibitory activity of compound, but also the degree of inhibition. based on current literature, the inhibitory activity of formononetin on adh has been reported,32 while, the inhibitory activity of aromadendrin on adh is reported herein for the first time. this demonstrated that screening utilizing centrifugal ultrafiltration combined with hplc-ms was effective and conclusive. conclusions a facile screening method based on centrifugal ultrafiltration combined with hplc-ms was established for analyzing adh inhibitors from d. styracifolium. the experimental conditions were optimized. finally, formononetin and aromadendrin were screened, identified and analyzed as adh inhibitors. this method proved to be rapid and effective for the identification of active compounds in natural products. acknowledgements. this work was financially supported by the national natural science foundation of china (21175155), key project of philosophy and social sciences research, ministry of education, prc (no. 13jzd0016, research on institutional building of ecological civilization), the specialized research fund for the doctoral program of higher education of china (20130162110017) and the hunan provincial innovation foundation for postgraduates (cx2012b119). и з в о д анализа инхибитора алкохолне дехидрогеназе из desmodium styracifolium применом центрифугалне ултрафилтрације спрегнуте са hplc-ms liangliang liu1, miao chen1 и xiaoqing chen1,2 1institute of bast fiber crops, chinese academy of agricultural sciences, changsha 410205, china и 2school of chemistry and chemical engineering, central south university, changsha 410083, china инхибитори алкохол дехидрогеназе (adh) имају значајну улогу у третирању тровања метанолом и етиленгликолом и супресији акумулације ацеталдехида код алкохоличара. у овом раду, центрифугална ултрафилтрација је спрегнута са високоефикасном течном хроматографијом–масеном спектрометријом (hplc–ms), ради скрининга и идентификације adh инхибитора из етилацетатног екстракта desmodium styracifolium (osb.) merr. оптимизовани су услови центрифугалне ултрафилтрације. при оптималним условима (концентрација adh: 37,5 μg ml-1, време инкубације: 90 min, ph 7,0 и температура: 15 °c), формононетин и аромадендрин су успшно идентификовани из _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ alcohol dehydrogenase inhibitors analyzed by centrifugal ultrafiltration/hplc–ms 1059 етилацетатног екстракта d. styracifolium. резултати скрининга су верификовани тестом adh инхибиције. вредности ic50 за формононетин и аромадендрин су биле 70,8 и 84,7 μg ml-1, што је сагласно са афинитетом везивања ових једињења. инхибиторна активност на adh је први пут публикована за аромадендрин. ова метода обезбеђује ефикасан начин за скрининг активних једињења у природним производима. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice complexation of molybdenum(vi) with methyliminodiacetic acid in different water + methanol solutions j. serb. chem. soc. 81 (5) 541–551 (2016) udc 546.776+547–304.2+547.484.7: jscs–4866 54–145.2+547.261 original scientific paper 541 complexation of molybdenum(vi) with methyliminodiacetic acid in different water + methanol solutions saleh zeighaminezhad and kavosh majlesi* department of chemistry, science and research branch, islamic azad university, tehran, iran (received 23 october, revised 30 december 2015, accepted 12 january 2016) abstract: the complexation of molybdenum(vi) with methyliminodiacetic acid (mida) at ph 6.00, t = 298 k, i = 0.1 mol dm-3 of sodium chloride and different water + methanol solutions (0–45 vol. %) was studied using potentiometric and uv spectrophotometric measurements. the values of the stability constants were calculated and their trends interpreted using the kamlet– –abboud–taft (kat) model in order to investigate the role of different specific and non-specific interactions in aqueous solutions of methanol. keywords: methyliminodiacetic acid; kamlet–abboud–taft equation; solvent effect. introduction investigation of the solvent effect in complex formation reactions is the subject of ongoing research1–4 aimed at comparing the new results with previous data in aqueous solutions5–10 and understanding the contribution of the solvent in various interactions in alcoholic and ionic liquid solutions. the study of the solvent effect could be considered as a kind of simulation for the cases where the solvent polarity decreases.11 the solvent polarity at a protein–water surface or in the active site cavities of metalloenzymes are reduced in comparison to pure bulk water.11 therefore, it is well known that different types of water exist in cells.11 for the second and third transition periods, molybdenum and tungsten, respectively, are the most abundant metals in the oceans.12 this correlates with the fact that molybdenum and tungsten are the only elements of their respective periods that have known biological functions.12 to what extent this abundance is responsible for the extensive usage of mo in biology, as opposed to its unique chemistry, is not entirely clear.12 molybdenum is a trace element and most molybdenum enzymes catalyze a conversion, the net effect of which is to add an * corresponding author. e-mail: kavoshmajlesi@srbiau.ac.ir, kavoshmajlesi@gmail.com doi: 10.2298/jsc151023010z 542 zeighaminezhad and majlesi oxygen atom to, or remove an oxygen atom from the substrate.12 molybdenum enzymes have a considerable strategic significance in the biosphere and are found in all forms of life, from bacteria, through higher plants and animals to humans.12 molybdenum complexes can catalyze reactions that involve oxygen atom transfer.12 aminopolycarboxylic acids have been used with the aim of removing toxic metals and preventing metal precipitation at least for the last fifty years. they have vast applications in different fields, such as textile production, metal electroplating, waste treatment, pharamaceuticals, cosmetics, agriculture, cleaning operations, food products and photography.13 a new generation of bifunctional aminopolycarboxylic acids have been investigated for magnetic resonance imaging (mri) and photometric or radioactive imaging and therapy.13 they can be attached to biological macromolecules through a covalent bond.13 the stability constants data for the complexation of molybdenum(vi) with methyliminodiacetic acid (mida) at t = 298 k and different ionic strengths of sodium perchlorate (0.1 < i < 1.0 mol dm–3) were reported previously.6 therefore, in this research, molybdenum(vi) complexation with mida was studied in different aqueous solutions of methanol (0–45 vol. %) at i = 0.1 mol dm–3 of sodium chloride and 298 k. the solvent dependence of the dissociation and stability constants was fitted to the kamlet–abboud–taft (kat) equation in order to derive the solvatochromic regression coefficients. all of the uv measurements for the complex formation reaction in the current work were performed at ph 6.00 in order to avoid the hydrolysis of molybdenum and to ensure the existence of moo42– in the solution.6,14 experimental sodium chloride, 99.5 %; hydrochloric acid titrazole (1 mol dm-3), sodium hydroxide titrazole (1 mol dm-3), sodium molybdate, 99.5 %; sodium carbonate anhydrous, 99.5 %; methanol, 99.9 %; potassium hydrogen phthalate, minimum 99.9 % were purchased from merck and mida, 99 % (fig. 1) from aldrich and were used without further purification. the naoh solutions were prepared from titrazole solutions and their concentration was determined by several titrations with potassium hydrogen phthalate. the hcl solution was standardized with sodium carbonate solution (na2co3). sodium chloride and sodium carbonate were dried in an oven at 383 k for 2 h. all chemicals were analytical reagent grade. the specific conductance of double-distilled water was 1.3±0.1 µs cm-1. fig. 1. the chemical structure of mida. all of the spectrophotometric and potentiometric measurements were performed at 298 k and an ionic strength 0.1 mol dm-3 of sodium chloride. a metrohm ph-meter, model 827, was used for the ph measurements. the ph-meter had a sensitivity of 0.001 units. the hydrogen ion concentration was measured with a metrohm combination electrode, model 6.0228.010. a 0.01 mol dm-3 hydrochloric acid solution containing 0.09 mol dm-3 sodium complexation of mo(vi) with methyliminodiacetic acid 543 chloride (for adjusting the ionic strength to 0.1 mol dm-3) was employed as a standard solution of the hydrogen ion concentration. calibration of the glass electrode for different methanol mixtures was realized according to the literature.11,15,16 thus, the calibration was performed using buffers (ph 4.00, 7.00) from metrohm, herisau, switzerland, similar to the work of sigel11 and then correction factors15,16 were applied for the different solvent mixtures. many glass electrodes show the theoretical response to hydrogen ions, at least up to alcohol concentrations near 90 weight percent.15 definition of acidity in alcohol–water solvents in terms of the experimental quantity (ph) is possible by several units, but from the practical point of view, the pmh unit is unsatisfactory in water + alcohol media.15 the pah* was related directly to the experimental quantity using the following equation:15 pah* = ph – δ (1) where ah* is the hydrogen ion activity referred to the standard state in the mixed solvent.15 pah* is a useful quantity that plays a simple and distinct role in chemical equilibria in alcoholic media.15 in the present research, the values of the experimental quantity (ph) were obtained in different methanol mixtures containing known concentrations of hcl and nacl to give a constant ionic strength of 0.1 mol dm-3.15 the standard solutions of known pah* having the same solvent composition as the unknowns were used to calculate the values of the correction term δ.15 the value of δ is substantially constant for a solvent medium of given composition.15 the values of δ are small up to about 80 weight percent methanol and their values for different water + alcohol solutions are available in the literature.15 potentiometric titrations were performed with 0.2 mol dm-3 sodium hydroxide in thermostatted vessels with a magnetic stirrer. thus, 25 cm-3 solution in the thermostatted titration vessel contained the reagents with final concentrations: sodium chloride, 0.1 mol dm-3; hydrochloric acid, 0.016 mol dm-3; mida, 0.01 mol dm-3 and the desired amount of methanol and water for each volume fraction of methanol. spectrophotometric measurements were performed with a perkinelmer lambda 25 uv– –vis spectrophotometer between 245 and 280 nm in thermostatted 10-mm quartz cells at 298 k and an ionic strength 0.1 mol dm-3 of sodium chloride.5-8,10 the measurement cell was of the flow type.5-8,10 in order to measure the absorbance and ph of the solution simultaneously, a masterflux pump was used which allowed the circulation of the solution under study between the potentiometric and spectrophotometric cells.5-8,10 measurements were performed for different metal, ligand concentrations and ligand/metal mole ratios but the best results were obtained with cl = cm = 0.007 mol dm-3. the job method was used for different water + methanol solutions (0–45 vol. %).5-8,10 using the job method, the absorbances of the solutions of the mo(vi) + mida complex at total concentration of 0.0028 mol dm-3 in the uv range (260 to 270) nm at a constant ph of 6.00 and different volume fractions of methanol were measured and the results are gathered in tables s-i–s-x of the supplementary material. the plot for 35 % methanol is shown in fig. 2. the experimental procedure for the job method was described in detail in a previous paper.6 results and discussion complexation of molybdenum (vi) with mida in different aqueous solutions of methanol the dissociation reactions of mida were reviewed in the literature.13 the values of mida dissociation constants at 298 k, at i = 0.1 mol dm–3 of naclo4 544 zeighaminezhad and majlesi and different water + methanol solutions (0–45 vol. %) were gathered in tables i and ii of a previous work.16 in the current work, the values of the dissociation constants were calculated based on the following equilibria (l = mida): 2 1 2 [h ][hl ] h l h hl , [h l] k + − + −→ + = (2) 2 2 2 [h ][l ] hl h l , [hl ] k + − − + − − → + = , (3) the values of dissociation constants determined in this work that were obtained at different volume fractions of methanol (0–45 vol. %), i = 0.1 mol dm–3 of nacl using the potentiometric technique and the hyperquad 2013 program17 are reported in table i together with the literature data. three titrations were performed for each aqueous solution of methanol and approximately 110 points were used for the calculations at each volume fraction of methanol. table i. average experimental values of log k1 at i = 0.1 mol dm-3 of nacl and different aqueous solutions of methanol for mida, t = 298 k methanol content, vol. % log k1 log k2 0 2.29±0.10 9.21±0.04 5 2.46±0.03 9.25±0.02 10 2.56±0.02 9.30±0.01 15 2.60±0.05 9.30±0.02 20 2.66±0.04 9.40±0.02 25 2.74±0.03 9.49±0.02 30 2.87±0.04 9.56±0.02 35 2.87±0.02 9.58±0.01 40 3.11±0.04 9.68±0.02 45 3.17±0.05 9.78±0.03 0 2.12±0.09a 9.65±0.07a 0 2.28±0.02b 9.59±0.02b 0 2.32±0.03c 9.43±0.03c 0 2.4±0.1d 9.46±0.03d 0 2.54±0.04e 9.48±0.06e 5 2.58±0.02f 9.67±0.10f 10 2.68±0.03g 9.68±0.06g 15 2.64±0.01h 9.70±0.06h 20 2.65±0.02i 9.76±0.01i 25 2.72±0.02j 9.83±0.08j 30 2.76±0.01k 9.87±0.07k 35 2.79±0.02l 9.95±0.12l 40 2.85±0.01m 9.97±0.10m 45 2.95±0.01n 10.06±0.07n ai = 0.1 mol dm-3 kcl, t = 298 k; bi = 0.5 mol dm-3 naclo4, t = 298 k; ci = 0.5 mol dm-3 kno3, t = 298 k; di = 1.0 mol dm-3 naclo4, t = 298 k (a, b, c and d data were taken from the literature 13); e-ni = 0.1 mol dm-3 naclo4, t = 298 k (e, f, g h, i, j, k, l, m and n data were taken from the literature 16) complexation of mo(vi) with methyliminodiacetic acid 545 errors for ph and volume in the hyperquad 2013 program were 0.001. the speciation diagram and fitted titration curves from hyperquad 2013 program are presented in figs. 2 and 3, respectively, for 25 % methanol. fig. 2. speciation diagram for mida (l) at 298 k, i = 0.1 mol dm-3 nacl and 25 % methanol; cl = 0.01 mol dm-3; solid line h2l; long dashed line hl-; short dash line free l. 0 2 4 6 8 10 0 1 2 3 4 5 ph v(naoh) / ml fig. 3. potentiometric titration of mida (l) at 298 k, i = 0.1 mol dm-3 nacl and 25 % methanol; cl = 0.01 mol dm-3 vs. volume of naoh. the continuous line represents the calculated ph refined with the hyperquad 2013 program, considering h2l and hlspecies. circles show the experimental ph values. moo42– binds with this tridentate ligand as a 1:1 complex:5–8,10 2 2 (2 )4 3 2moo ( 2 )h l (moo ) h l h oz yx y zx y x z x− + − − −+ + + → + (4) (2 )3 2 ( 2 ) 24 [(moo ) h l ] [moo ] [h ] [l ] z yx y z xyz x y x z β − − − + + − = (5) where x = 1, y = 0 and z = 1. thus, the stability constants for the complex species, moo3l2–, was calculated at different volume fractions of methanol by combination of the following equations according to previous works:5–8,10 546 zeighaminezhad and majlesi 2c obs 0 4[moo ]a a ε −= − (6) c23 1 [moo l ] a ε − = (7) 2 2m 4 3[moo ] [moo l ]c − −= + (8) 2l 3[l] [moo l ]c −= + (9) ac, aobs, and ε0 are the corrected absorbance of the complex, the observed absorbance and the molar absorptivity of moo42–, respectively. the ε0 values are calculated at xmetal = 1.0. the molar absorptivity values of the complex, ε1, were calculated at low mole fraction of the metal, where all the metal ions were in the form of a complex. [moo3l2–], which is the concentration of the complex, was calculated at xmetal = 0.5 where the maximum absorbance was observed (fig. 4). the value of [l] was used for the calculation of [l2–] according to the definition of dissociation constants. the values of the conditional stability constants were obtained by inserting the values of [moo3l2–], [moo42–], [h+] and [l2–] into eq. (5). their values together with the literature values are shown in table ii. microsoft excel 2010 was used for the calculations in eqs. (5)–(9). 0 0.2 0.4 0.6 0.8 0 0.2 0.4 0.6 0.8 1 a c xmetal fig. 4. corrected absorbance data, ac, for moo3mida2vs. the mole fraction of mo(vi), xmetal, at 298 k, an ionic strength of 0.1 mol dm-3 nacl, 35 % methanol and different wavelengths: solid line 260 nm; long dashed line 265 nm; and short dashed line 270 nm. all of the lines were obtained based on the best fit to the corrected absorbance data. table ii. average experimental values of log β101 at ph 6.00, i = 0.1 mol dm-3 of nacl and different aqueous solutions of methanol for the complexation of molybdenum(vi) with mida, t = 298 k methanol content, vol. % log β101 0 18.30±0.03 5 18.40±0.04 10 18.44±0.04 15 18.46±0.06 20 18.53±0.10 25 18.64±0.01 30 18.76±0.03 complexation of mo(vi) with methyliminodiacetic acid 547 table ii. continued methanol content, vol. % log β101 35 18.84±0.02 40 18.86±0.06 45 18.89±0.10 0 18.2a 0 19.09±0.14b 0 18.78±0.18c 0 18.67±0.20d 0 18.85±0.15e 0 18.91±0.40f athis value was not at a fixed ionic strength and was obtained by the nmr technique. the concentrations are usually high in nmr (minimum of 0.1 mol dm-3 in metal and ligand18); bi = 0.1 mol dm-3 naclo4, t = 298 k, cl = cm = 0.01 mol dm -3; ci = 0.3 mol dm-3 naclo4, t = 298 k, cl = cm = 0.01 mol dm -3; di = 0.5 mol dm-3 naclo4, t = 298 k, cl = cm = 0.01 mol dm -3; ei = 0.7 mol dm-3 naclo4, t = 298 k, cl = cm = 0.01 mol dm -3; fi = 1.0 mol dm-3 naclo4, t = 298 k, cl = cm = 0.01 mol dm -3 (b, c, d, e and f data were taken from the literature6) comparison with literature data the original and further first works about the complexation of molybdenum(vi) with different aminopolycarboxylic acids were reported by kula and coworkers.18–21 a literature survey showed that although there are only a few reports about the complexation of molybdenum(vi) with ethylenediamine-n,n′ -diacetic acid (edda) in methanol–water22,23 and complexation of tungsten(vi)24 and molybdenum(vi)25 with edda in propanol–water mixtures, no paper was found on stability constants data in different water + methanol solutions for the interaction of molybdenum(vi) with mida. it was seen from literature data22–25 that the stability constants increased with decreasing polarity of alcohol–water mixtures, which confirms the pattern that was obtained in the current research. the concentration of mo(vi) and mida and the kind of background electrolyte (cl = cm = 0.007 mol dm–3, nacl) in this research were different from that used in a previous work (cl = cm = 0.01 mol dm–3, naclo4),6 therefore the values of stability constants at 0 % methanol (i = 0.1 mol dm–3) in the two works were not the same (table ii). application of the kat model for the study of the solvent effect although linear gibbs energy relationships such as the hammett equation are well established and are known to perform very well for substituent effects, solvent effects are more complicated processes that require a more detailed investigation. therefore, a more generally applicable treatment of multiple interacting solvent effects was designed by kamlet, abboud and taft (kat).26 in their treatment, it is necessary to consider non-specific and specific solute–solvent interactions separately. specific solute–solvent interactions were subdivided into solvent lewis-basicity interactions (hydrogen bond donor (hbd) solute/ 548 zeighaminezhad and majlesi /(hydrogen bond acceptor (hba) solvents) and solvent lewis acidity interactions (hydrogen bond acceptor (hba) solute/(hydrogen bond donor (hbd) solvents). thus, the following multiparameter equation was suggested for application in linear solvation energy relationships (lser):26–44 0log ( * )k a p d a bπ δ α β= + + + + (10) lser have been used in various fields, such as, ionic liquids, solubility, thermochemistry, nmr chemical shifts, pharmaceuticals, etc. equation (10) has been used in the correlation analysis by multiple regression of numerous reaction rates and equilibria, spectroscopic data and various other solvent dependent processes. the value of α shows the solvent hbd acidity and is a measure of the proton transfer from the solvent to the solute for hydrogen bond formation. the α values lie between zero for non-hbd solvents up to 1.0 for methanol (table iii).16 the ability of the solvent to accept a proton from a solute in order to form a hydrogen bond is indicated by the value of β, which is called the solvent hba basicity. the β values vary from zero for non-hbd solvents up to 1 for hexamethylphosphoric acid triamide (hmpt).16 the solvation parameters α and β have been used for the interpretation of intermolecular hydrogen bonding. the value of π* is an index of solvent dipolarity/polarizability. in other words, it is the capability of the solvent regarding charge, dipole and dielectric contributions. the π* value is 0.00 and 1.00 for cyclohexane and dimethylsulfoxide, respectively.16 the value of δ is a discontinuous polarizability correlation term. the value of δ is 0.0 for non-chloro substituted aliphatic solvents, 0.5 for poly-chloro -substituted aliphatics, and 1.0 for aromatic solvents.16 in the current work, δ value was zero. a0 is the value for log k in the setup when α, β, δ and π* are equal to zero. the kat equations given in table iv were obtained using microsoft excel 2010 software. table iii. solvatochromic parameters for different aqueous solutions of methanol from the literature16 methanol content, vol. % α β π* 0 1.17 0.47 1.09 5 1.16 0.48 1.05 10 1.15 0.49 1.04 15 1.14 0.50 1.02 20 1.13 0.51 0.99 25 1.12 0.52 0.97 30 1.11 0.53 0.94 35 1.10 0.54 0.92 40 1.09 0.55 0.89 45 1.07 0.56 0.85 complexation of mo(vi) with methyliminodiacetic acid 549 table iv. different kat equations with one and two solvatochromic parameters together with their standard errors and square values of the correlation coefficients (r2) for the dissociation and stability constants at t = 298 k, i = 0.1 mol dm-3 of nacl and different aqueous solutions of methanol. α, hydrogen bond donor acidity; β, hydrogen bond acceptor basicity; π*, dipolarity/polarizability. n = 10 kat equation r2 log k1 = (12.33±0.59) – (8.54±0.52)α 0.97 log k1 = –(1.92±0.29) + (9.04±0.56)β 0.97 log k1 = (6.25±0.18) – (3.60±0.19)π* 0.98 log k1 = (5.81±10.83) – (4.64±6.50)α + (4.14±6.88)β 0.97 log k1 = (4.06±4.90) + (3.06±6.84)α – (4.88±2.87)π* 0.98 log k1 = (5.89±4.66) + (0.40±5.18)β – (3.44±2.05)π* 0.98 log k2 = (16.18±0.33) – (5.98±0.29)α 0.98 log k2 = (6.21±0.19) + (6.31±0.37)β 0.97 log k2 = (11.91±0.11) – (2.52±0.11)π* 0.98 log k2 = (16.67±6.17) – (6.28±3.70)α – (0.31±3.92)β 0.98 log k2 = (12.97±3.00) – (1.48±4.19)α – (1.90±1.76)π* 0.98 log k2 = (12.09±2.84) – (0.20±3.15)β – (2.60±1.25)π* 0.98 log β101 = (25.97±0.57) – (6.54±0.51)α 0.95 log β101 = (15.01±0.22) + (7.00±0.42)β 0.97 log β101 = (21.30±0.19) – (2.76±0.19)π* 0.96 log β101 = (5.66±7.61) + (5.61±4.56)α + (12.91±4.83)β 0.98 log β101 = (19.29±5.07) + (2.81±7.08)α – (3.94±2.97)π* 0.96 log β101 = (14.76±4.13) + (7.27±4.58)β + (0.11±1.82)π* 0.97 conclusions the kat equations obtained in this research (table iv) could be used for the calculation of the dissociation constants of mida and the stability constants for the complexation of mo(vi) with mida at ph = 6.00, t = 298 k, i = 0.1 mol dm–3 of sodium chloride in the desired range of methanol (0–45 %) without any further experimental work. the values of dissociation and stability constants increased with a mild slope as the volume fraction of methanol increased, because the solvation decreased and stability increased (tables i and ii).11,45,46 the standard errors for a0, a, b and p are too high for the two parameter kat equation in comparison to the one parameter equation (table iv), which is due to the linear increase of solvatochromic parameters for methanol (table iii). the trend that was obtained in this research is in agreement with previous findings1,2,16 and therefore it is predicted that it could probably be valid for the complexation of molybdenum(vi) with other aminopolycarboxylic acids. supplementary material the absorbances of the solutions of the mo(vi) + mida complex at total concentration of 0.0028 mol dm-3 in the uv range (260 to 270) nm at a constant ph of 6.00 and different volume fractions of methanol are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. 550 zeighaminezhad and majlesi и з в о д формирање комплекса молибдена(vi) са метилиминодиацетатном киселином у различитим вода + метанол растворима saleh zeighaminezhad и kavosh majlesi department of chemistry, science and research branch, islamic azad university, tehran, iran формирање комплекса молибдена(vi) са метилиминодиацетатном киселином (mida) при ph 6,00, t = 298 k и i = 0,1 mol dm -3 натријум-хлорида и различитим вода + метанол растворима (0–45 запр. %) је испитивано потенциометријским и uv спектрофотометријским мерењима. вредности константи стабилности су израчунате и њихови трендови интерпретирани применом kamlet–abboud–taft (kat) модела да би се испитала улога специфичних и неспецифичних интеракција у воденим растворима метанола. за прорачуне су коришћени софтвери hyperquad 2013 и microsoft excel 2010. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis and characterization of novel benzimidazole embedded 1,3,5-trisubstituted pyrazolines as antimicrobial agents} j. serb. chem. soc. 82 (9) 985–993 (2017) udc 547.772+547.785.5:615.281/.282–188 jscs–5017 original scientific paper 985 synthesis and characterization of novel benzimidazole embedded 1,3,5-trisubstituted pyrazolines as antimicrobial agents gopal k. padhy1,2, jagadeesh panda3 and ajaya k. behera1* 1organic synthesis laboratory, school of chemistry, sambalpur university, jyoti vihar, burla 768019, india, 2maharajah’s college of pharmacy, phool baugh, vizianagaram 535002, india and 3raghu college of pharmacy, dakamarri, visakhapatnam 531162, india (received 4 june 2016, revised 3 april, accepted 17 july 2017) abstract: efficient syntheses of some new substituted pyrazoline derivatives linked to substituted benzimidazole scaffold were performed by multistep reaction sequences. all the synthesized compounds were characterized using elemental analysis and spectral studies (ir, 1d/2d nmr techniques and mass spectrometry). the synthesized compounds were screened for their antimicrobial activity against selected gram-positive and gram-negative bacteria, and fungi strain. the compounds with halo substituted phenyl group at c5 of the 1-phenyl pyrazoline ring (15, 16 and 17) showed significant antibacterial activity. among the screened compounds, 17 showed most potent inhibitory activity (mic = 64 µg ml-1) against a bacterial strain. the tested compounds were found to be almost inactive against the fungal strain c. albicans, apart from pyrazoline-1-carbothiomide 21, which was moderately active. keywords: chlacone; pyrazoline; diastereotopic protons; antibacterial activity; antifungal activity. introduction in the current scenario, bacterial infections bring about a serious threat to human lives due to their rapid resistance to existing antibiotics. thus, exploration of new types of antibacterial agents has become extremely vital. benzimidazole derivatives are very useful for the development of molecules of pharmaceutical interest due to their pharmacological activities, including antimicrobial,1–3 anticancer,4–6 antidiabetic7 and plasmin inhibitor.8 moreover, n-benzyl-substituted benzimidazoles have been synthesized showing promising antibacterial activities, among which amidine9 i and 2-(piperidin-4-yl)benzimidazole10 ii are examples (fig. 1). * corresponding author. e-mail: ajay.behera1962@gmail.com https://doi.org/10.2298/jsc160604089p available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs 986 padhy, panda and behera n n cl cl cl i nh hn cl n ncl cl nh h n ii n n s o o n s cn br iii n n o n n o cl iv fig. 1. structures of some benzimidazoles and pyrazolines with antibacterial activity. the pyrazoline motif is a core structure in numerous biologically active compounds. some representatives of this heterocycle exhibited antimicrobial,11,12 anticancer,13,14 anti-inflammatory,15,16 and monoamine oxidase inhibitory17,18 activities. series of novel thiazolyl-pyrazoline,19 such as iii and morpholinoquinoline clubbed pyrazoline20 iv were recently reported as potent antibacterial agents (fig. 1). the combination of n-benzylbenzimidazole and pyrazoline fragments in one molecule is expected to be a perspective approach to design promising antimicrobial agents. thus, a novel series of n-benzyl attached benzimidazolyl pyrazolines was designed, synthesized and evaluated against different bacteria and fungi. experimental chemistry the chemicals used were laboratory grade and procured from merck (india), fischer scientific (india) and finar (india). ir spectra were obtained on a bruker alpha-t ft-ir spectrometer (kbr pellets using opus software). 1hand 13c-nmr spectra were recorded on a bruker avance iii 500 mhz (av 500) spectrometer using tms as an internal standard in dmso-d6/cdcl3. the mass spectra were recorded on a varian inc 410 prostar binary lc– –ms and an agilent 6410 lc–ms spectrometer. melting points were determined by the open tube capillary method and are uncorrected. progress of the reaction and purity of the products available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs novel benzimidazole-substituted pyrazolines as antimicrobial agents 987 was checked by thin layer chromatography (tlc). the spots were located under iodine vapours/uv light. the physical, analytical and spectral data for the compounds are given in the supplementary material to this paper. general procedure for synthesis of 3-aryl-1-(1-benzyl-1h-benzo[d]imidazol-2-yl)-2-propen-1 -ones (8–12) chalcones 3–7 (10 mmol) were dissolved in 40 ml dry acetone and then anhydrous k2co3 (15 mmol) was added to the solution. later benzyl chloride (40 mmol) was added to the mixture and the contents were heated under reflux. the progress of the reaction was monitored by tlc (benzene–ethyl acetate, 4:1). after completion of the reaction (22–26 h), the reaction mixture was cooled and then poured into crushed ice. the obtained solid were filtered and recrystallized form alcohol. it should be noted that compounds 9– 11 crystallised out from the reaction mixture after cooling, and they were then collected by filtration. compounds 8 and 10 were previously reported.21 however, neither of them has been examined for their antimicrobial activities. general procedure for the synthesis of 1-benzyl-2-(5-aryl-1-phenyl-4,5-dihydro-1h-pyrazol -3-yl)-1h-benzimidazoles (13–17) to a solution of chalcones 8–12 (2 mmol) in acetic acid, phenylhydrazine (3 mmol) was added dropwise. the reaction mixture was heated under reflux. the progress of the reaction was monitored by tlc (benzene–ethyl acetate, 5:1). after completion of reaction (6–8 h), the reaction mixture was cooled and then poured into crushed ice. the obtained solid pyrazolines were washed with diethyl ether and recrystallized from acetone. general procedure for synthesis of 5-aryl-3-(1-benzyl-1h-benzimidazol-2-yl)-4,5-dihydro -1h-pyrazole-1-carbothioamides (18–22) a mixture of chalcones 8–12 (2 mmol), thiosemicarbazide (6 mmol) and naoh (4 mmol) was refluxed in ethanol (15 ml). the progress of the reaction was monitored by tlc (benzene–ethyl acetate, 5:1). after completion of reaction (4–6 h), the reaction mixture was cooled. the precipitate formed was filtered and washed with acetone. microbiology the compounds 8–22 were evaluated for their in vitro antimicrobial activity against bacterial strains, viz. bacillus subtilis mtcc 441, staphylococcus aureus mtcc 3160, pseudomonas aeruginosa mtcc 4673 and escherichia coli mtcc 739, and the fungi candida albicans mtcc 183. the reference cultures were procured from the institute of microbial technology (imtech), chandigarh, india-160036. minimum inhibitory concentrations (mic) were determined using nutrient broth (nb) for the bacteria and sabouraud dextrose broth (sdb) for fungi by the two-fold serial dilution method.22,23 the cultures were incubated for 24 h for the bacteria and 48 h for the fungi at 35 °c and the growth was monitored. the lowest concentration required to arrest the growth of microorganism was regarded as the minimum inhibitory concentration (mic). ciprofloxacin and fluconazole were used as positive control for the bacteria and fungi, respectively. dmso was used as the negative control. the determinations of the antimicrobial activities of the compounds were performed in duplicate. results and discussion chemistry the target compounds described in this study were prepared as outlined in scheme 1. condensation of o-phenylenediamine with lactic acid under phillips available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs 988 padhy, panda and behera conditions led to 2-(1-hydroxyethyl)benzimidazole (1), chromic oxidation of the latter followed by neutralization with ammonia led to 2-acetylbenzimidazole (2).24 the required synthons 3-aryl-1-(1h-benzimidazol-2-yl)-2-propen-1-ones (3–7) were prepared by claisen–schmidt condensation of 2-acetylbezimidazole with substituted aromatic aldehydes in presence of naoh.25 condensation of the 3-aryl-1-benzimidazolyl-2-propen-1-one derivatives 3–7 with benzyl chloride gave the corresponding 3-aryl-1-(1-benzyl-1h-benzimidazol-2-yl)-2-propen-1 -ones 8–12. the reaction of 8–12 with phenylhydrazine in the presence of acetic acid afforded 1-benzyl-2-(5-aryl-1-phenyl-4,5-dihydro-1h-pyrazol-3-yl)-1h-benzimidazoles 13–17, whereas when 8–12 were condensed with thiosemicarbazide in presence of naoh, 5-aryl-3-(1-benzyl-1h-benzimidazol-2-yl)-4,5-dihydro -1h-pyrazole-1-carbothioamides 18–22 were obtained in good yields. scheme 1. synthetic route to 1-benzyl-2-(1-substituted-5-aryl-4,5-dihydro-1h-pyrazol-3-yl) -1h-benzimidazoles. reagents and conditions: i) 4 m hcl, reflux, 8 h; ii) k2cr2o7, dil. h2so4, r.t., 2h; iii) ar-cho, 10 % aq naoh, ethanol, r.t., 4–8 h; iv) benzyl chloride, dry acetone, anhydrous k2co3, reflux, 22–26 h; v) phenylhydrazine, acetic acid, reflux, 6–8 h; vi) thiosemicarbazide, naoh, ethanol, reflux, 4–6 h. available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs novel benzimidazole-substituted pyrazolines as antimicrobial agents 989 the structure of the synthesized compounds 8–22 were assigned based on elemental and spectroscopic analysis, ir, 1h-nmr, 13c-nmr and mass spectrometry (supplementary material to this paper). the ir data were very informative and fully supported the proposed structures of the reported compounds. in the ir spectra of chalcones 8–12, the (c=o) stretching was found in the expected region at 1652–1662 cm–1. furthermore, the presence of (c=n) and (c–n) stretching frequencies at 1594–1597 cm–1 and 1283–1330 cm–1 in the ir spectra of 13–22 confirmed the subsequent cyclization of the chalcone to the pyrazoline derivatives. in addition, derivatives 18––22 showed typical absorption bands due to (–nh2) at 3290–3355 cm–1 and 3218–3250 cm–1. the 1h-nmr spectrum compounds 8–12 exhibited two doublets with j values between 15–20 hz, confirming the trans coupling and indicating the olefinic protons in the e form. the 13c-nmr spectra of 8–12 confirmed the presence of the α,β-unsaturated carbonyl system of chalcones by the presence of a peak at δ 182.40–182.53 ppm, corresponding to the propenone c1. although compounds 13–22 were racemates, their structures were unambiguously assigned with the help of spectral studies. the 1h-nmr spectra of compounds 13–22 displayed three sets of signals with an abx pattern for the pyrazoline ring protons. the sterochemical nature of the hydrogens ha, hb and hx was ascertained from a study of the coupling constants (j). the vicinal coupling constant between ha and hx was found to be 2.7–10.0 hz (jax), which indicates that these hydrogens are cis to each other, while the trans relationship between hb and hx was evident from the coupling constants of jbx in the 10.0– –15.0 hz range. the coupling value of jab in 17.5–20.0 hz range between ha and hb evidently indicates their geminal placement at c4. the ch2 protons of pyrazoline are diastereotopic and appeared as a pair of doublets of doublets at δ 2.90–3.10 ppm (ha) and δ 3.85–4.05 ppm (hb), due to the vicinal coupling with the hx proton and the geminal coupling with each other. the ch proton (hx) appeared as a doublet of doublets at δ 5.72–5.92 ppm due to vicinal coupling with the two magnetically non-equivalent protons of the methylene group (ha and hb) at position 4 of the pyrazoline ring. the two methylene protons of benzyl group on geminal coupling showed two separate doublets at δ 5.33–6.08 ppm (hc) and δ 5.37–6.17 ppm (hd), indicating the diastereotopic nature of the methylene hydrogen. this was further confirmed by the appearance of three sets of signals at δ 40.03–44.45 ppm (c4 of pyrazoline), δ 47.01–48.18 ppm (ch2 of benzyl) and δ 57.54–62.62 ppm (c5 of pyrazoline) in the 13c-nmr spectra of pyrazolines. it should be noted that in the spectra of compounds 15 and 17, the signal of c4 of pyrazoline was overshadowed by residual peaks of dmso-d6. compounds 18–22 exhibited two separate broad singlets at δ 7.81–7.89 ppm and δ 8.26–8.38 ppm for the –nh2 protons. all the other additional peaks observed were in agreement with the respective aromatic substituents and benzimidazole available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs 990 padhy, panda and behera ring. the mass spectra and elemental analyses were also in agreement with the proposed structures. furthermore, the carbon–proton correlation of pyrazoline 13 was confirmed by an hsqc experiment. the ha and hb protons (δ 3.06 and 3.71 ppm) showed a cross-peak with the carbon signal at 40.03 ppm, which confirmed that this signal is due to the c4 carbon. similarly, the doublet of doublets at δ 5.75 ppm is strongly correlated with the carbon resonance at δ 60.36 ppm. from this, it is inferred that the carbon signal is due to the c5 carbon. the carbon signal at δ 47.35 ppm correlated well with the two methylene protons (δ 5.34 and 5.39 ppm) of the benzyl group, which established the diastereotopic nature of the methylene hydrogens. antimicrobial activity the synthesized compounds 8–22 were screened for their in vitro antimicrobial activity. the results of antimicrobial activities of the benzimidazole derivatives are presented in table i. the antimicrobial screening data revealed that all the newly synthesized compounds exhibited weaker antimicrobial activities compared to those of the control drugs. for bacterial strains, the mic values of the compounds ranged between 128–1024 μg ml–1 for the chalcones 8–12, between 64–1024 μg ml–1 for the 1-phenylpyrazolines 13–17 and between 128– –512 μg ml–1 for the pyrazoline-1-carbothioamides 18–22. among all the tested compounds, compound 17 showed good activity (64 µg ml–1) against the tested bacterial strains. the mic value of ciprofloxacin was 6.25–12.5 μg ml–1 for all table i. in vitro antimicrobial activity of the synthesized compounds 8–22, mic / µg ml-1 compound gram-positive bacteria gram-negative bacteria fungi s. aureus b. subtilis e. coli p. aeruginosa c. albicans 8 512 512 ˃1024 ˃1024 ˃1024 9 ˃1024 512 ˃1024 ˃1024 ˃1024 10 256 256 512 512 ˃1024 11 128 256 256 512 ˃1024 12 128 128 256 256 ˃1024 13 256 512 512 256 ˃1024 14 ˃1024 512 ˃1024 ˃1024 ˃1024 15 64 128 256 128 512 16 128 64 256 128 512 17 64 128 64 256 256 18 ˃1024 512 ˃1024 ˃1024 512 19 ˃1024 ˃1024 512 ˃1024 512 20 512 256 ˃1024 512 512 21 256 128 512 512 128 22 128 128 256 512 256 ciprofloxacin 6.25 6.25 6.25 12.5 – fluconazole – – – – 6.25 available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs novel benzimidazole-substituted pyrazolines as antimicrobial agents 991 the tested bacterial strains. chalcones 8–12 and 1-phenylpyrazolines 13 and 14 were almost inactive against c. albicans but pyrazoline-1-carbothioamide 21 showed moderate activity against the fungi strain. the mic value of fluconazole was 6.25 μg ml–1 for the fungi c. albicans. subsequently, preliminary sar studies were performed to deduce how the structure variation and modification could affect the antimicrobial activity. the antimicrobial activities of the compounds are related to the presence of electron withdrawing or donating substituents on the benzene ring. compounds containing electron withdrawing –f, –cl and –br exhibited good antimicrobial activity, whereas non-substituted compounds and compounds substituted with electron donating groups did not exhibit the same potency. for example, compound 17 showed an mic of 64 µg ml–1 against s. aureus and e. coli, whereas compound 13 showed an mic of 256 and 512 µg ml–1, respectively. the reason for above antibacterial activities could be explained by electron density, which plays an important role for the optimum activity. conclusions in this paper, the synthesis and characterization of three new chalcones, five 1-phenylpyrazolines and five pyrazoline-1-carbothioamides containing the n-benzylbenzimidazole moiety are presented. the structures of the new compounds were confirmed by spectral data (ir, 1h-nmr, 13c-nmr and mass spectrometry) and elemental analysis. all the compounds were investigated for their antimicrobial activity against b. subtilis, s. aureus, p. aeruginosa, e. coli and c. albicans. the data indicated weak antibacterial activity, except for compound 17 (which presented good activity against s. aureus and e. coli), 16 (which presented moderate activity on s. aureus and p. aeruginosa and 15 (which presented moderate action on b. subtilis and p. aeruginosa). the weak antibacterial activity could be because the tested compounds were in a racemic form. based on the mic values presented by the tested compounds, it could be concluded that, in general, the derivatives containing a fluorine, chlorine and bromine atom had better antibacterial activity against the tested strains. the tested compounds were found to be either inactive, or moderately active (21), against the fungal strain c. albicans. supplementary material analytical and spectral data of the synthesized compounds are available electronically at the pages of the journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the authors gratefully acknowledge the sophisticated instrumentation facility (saif), iit madras and saif, iit bombay, india, for providing the spectral data. gopal is thankful to sri. p. ashok gajapathi raju, chairman, mansas, vizianagaram and dr. p. udaya shankar, principal maharajah’s college of pharmacy, vizianagaram for providing the necessary infrastructure and facility. available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs 992 padhy, panda and behera и з в о д синтеза и карактеризација нових бензимидазола који садрже 1,3,5-трисупституисани пиразолин као антимикробних једињења gopal k. padhy1,2, jagadeesh panda3 и ajaya k behera1 1organic synthesis laboratory, school of chemistry, sambalpur university, jyoti vihar, burla 768019, india, 2maharajah’s college of pharmacy, phool baugh, vizianagaram 535002, india и 3raghu college of pharmacy, dakamarri, visakhapatnam 531162, india извршена је ефикасна синтеза нових супституисаних деривата пиразолина везаних са бензимидазолским прстеном, применом вишефазне реакционе секвенције. сва синтетисана једињења окарактерисана су елементалном анализом, спектроскопским методама (ir, 1d/2d nmr) и масеном спектрометријом. испитана је антимикробна активност синтетисаних једињења према одабраним сојевима грам-позитивних и грам-негативних бактерија и одабраних сојева гљивица. једињења која поседују халоген-супституисану ароматичну групу на c5 1-фенилпиразолинског прстена (15–17) показују значајну антибактеријску активност. од испитиваних једињења, дериват 17 показује највећу инхибиторну активност (mic = 64 μg ml-1). испитивана једињења су показала готово потпуно одсуство активности према c. albicans, осим пиразолин-1-карботиоамида 21 који је показао умерену активност. 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for clinical laboratory (nccl), methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed., approved standard m7-a5, national committee for clinical laboratory standards, villanova, pa, 2000 23. c. balachandran, v. duraipandiyan, n. a. al-dhabi, k. balakrishna, n. p. kalia, v. s. rajput, i. a. khan, s. ignacimuthu, indian j. microbiol. 52 (2012) 676 24. v. malla reddy, k. ravinder reddy, chem. pharm. bull. 58 (2010) 1081 25. m. ouattara, d. sissouma, m. w. koné, h. e. menan, s. a. touré, l. ouattara, trop. j. pharm. res. 10 (2011) 767. available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice synthesis, structural characterization and antimicrobial evaluation of some novel piperidin-4-one oxime esters j. serb. chem. soc. 80 (9) 1101–1111 (2015) udc 547.822.3’288.4+547.58+ jscs–4783 542.913:615.281 original scientific paper 1101 synthesis, structural characterization and antimicrobial evaluation of some novel piperidin-4-one oxime esters k. gokula krishnan, r. sivakumar and v. thanikachalam* department of chemistry, annamalai university, annamalainagar 608 002, tamil nadu, india (received 13 november 2014, revised 25 january, accepted 16 april 2015) abstract: fifteen novel biologically active piperidin-4-one oxime esters 8–22 were synthesized in good yields. these compounds were prepared in reactions of carboxylic acids, in situ activated using pocl3 and pyridine, with piperidin -4-one oximes. the structures of the title compounds were elucidated based on ftir, nmr (1d and 2d) and mass spectral analyses. single crystal xrd studies of compounds 12 and 20 provided further unambiguous evidence for the proposed structure. all the synthesized compounds were tested for their in vitro antibacterial and antifungal activities. many of these derivatives exhibited good activity against bacillus subtilis, pseudomonas aeruginosa, escherichia coli, trichoderma viride and aspergillus flavus. keywords: piperidin-4-one oxime; aromatic acid; mixed anhydride; gauche interaction; conformation; single crystal xrd. introduction substituted piperidines and their analogues are basic structural units in numerous naturally occurring alkaloids and drug candidates. a search of piperidines and their derivatives revealed thousands of references to this simple ring system in medicinal and clinical research.1 in particular, chiral centers at c2 and/or c6 of the piperidine ring were found to be essential for a defined activity, such as cns,2 anti-hiv,3 anti-proliferative,4 anti-cancer,5 anti-inflammatory6 and antioxidant7 activities. furthermore, the piperidine ring serves as a building block in synthetic and medicinal chemistry as more complex alkaloids include acridone and morphine, which themselves exhibit biological activities.8 oximes9 and their derivatives represent an important class of organic molecules that attract the interest of both synthetic and medicinal chemists. oxime esters have showed great potential in biologically active molecules, such as rbpp9 inhibitors,10 anti-proliferative11 and anti-convulsant12 agents, and in * corresponding author: e-mail: profvt.chemau@gmail.com doi: 10.2298/jsc141113037k _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1102 gokula krishnan, sivakumar and thanikachala. agrochemical13 industries. in addition, oxime esters, such as oxe-1 and oxe-2, were employed as photoinitiators,14 as they meet the specific requirements desired for color filter displays in lcds. recently, oxime esters were reported to exhibit dna-cleaving15 ability in a process triggered by uv light. the formation of the ester is a simple dehydration process, which can be easily catalyzed by strong acids. previously, numerous methods were documented for the synthesis of esters via direct coupling of a carboxylic acid with an alcohol, often involving the use of stoichiometric activators and coupling reagents.16 in addition, several reports describe the synthesis of esters in good yields using coupling reagents such as dicyclohexylcarbodiimide/4-(n,n-dimethylamino)pyridine (dcc/dmap),17 diethyl azodicarboxylate/triphenylphosphine (dead/ph3p),18 n,n-dimethyl phosphoramidic dichloride19 and diethyl chlorophosphate.20 however, some drawbacks still remain due to the formation of byproducts, difficulties in handling and long reaction times. consequently, a practical and efficient synthesis of oxime esters in good yields under mild conditions is still needed. in this context, an attempt was made with phosphorus oxychloride (pocl3),21 which is a commercially available and relatively inexpensive superior reagent, and affects carboxylic group activation under mild conditions with excellent yields of the products. herein, the synthesis and structural characterization are reported of some novel 3-alkyl-2,6-diphenylpiperidin-4-one oxime esters 8–22 through carboxylic acids 7a–e, in situ-activated using pocl3 and pyridine as base as well as solvent, with 3-alkyl-2,6-diphenylpiperidin-4-one oximes 4–6 in good yields (scheme 1). the key compounds, 3-alkyl-2,6-diphenylpiperidin-4-ones22 1–3 and their oximes9,23 4–6 were prepared using literature methods. scheme 1. general synthetic route for the synthesis of piperidin-4-one oxime esters 8–22. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis and characterization of novel piperidin-4-one oxime esters 1103 experimental all the purchased reagents and solvents were of reagent grade and used without further purification. completion of reactions was monitored by tlc on silica gel-coated aluminum sheets (type 60 gf254). the melting points were measured in open capillaries and are uncorrected. the ftir spectra were recorded on avatar-300 ftir spectrometer in kbr disks. the nmr (1d and 2d) spectra were recorded on a bruker 400 mhz spectrometer. the chemical shift values are reported in ppm from tms. the analytical and spectral data of the synthesized compounds are given in the supplementary material to this paper. general procedure for the synthesis of piperidin-4-one oxime esters (8–22) a mixture of 3-methyl-2,6-diphenylpiperidin-4-one oxime (0.700 g, 2.5 mmol) and benzoic acid (0.335 g, 2.75 mmol) in dry pyridine (5 ml) was stirred at room temperature (29 °c), pocl3 (0.25 ml, 2.75 mmol) was added dropwise to the mixture and stirring was continued for 15 min. the progress of the reaction was monitored by tlc. after completion of the reaction, the crude product was neutralized with dilute nitric acid and the desired product was regenerated with sodium bicarbonate solution. the crude product was then recrystallized from ethanol to obtain the pure piperidin-4-one oxime ester 8 in good yield (0.6 g, 86 %). the above general method was adopted for the synthesis of compounds 9–22 of this series. results and discussion chemistry in the present investigation, first the reaction of 3-methyl-2,6-diphenyl piperidin-4-one oxime (4, 1 equiv.) and benzoic acid (7a, 1.1 equiv.) in pyridine as base as well as solvent was studied using various reagents (1.1 equiv.) to examine the feasibility of the reaction. as shown in table i, a trace amount of product was formed with pcl5, while moderate yields were obtained with socl2 and dcc. finally, pocl3 gave the corresponding 3-methyl-2,6-diphenyl piperidin-4 -one oxime ester 8 in good yield. table i. formation of oxime ester from oxime with benzoic acid utilizing various reagents entry substrate (equiv.) reagent (equiv.) time, min yielda, % 1 phcooh (1.1) pcl5 (1.1) 120 trace 2 phcooh (1.1) socl2 (1.1) 80 60 3 phcooh (1.1) dcc (1.1) 90 45 4 phcooh (1.1) pocl3 (1.1) 15 86 a isolated yield next, pocl3 was utilized as the reagent in varying amounts, from 0.5 to 2.0 equiv, for the synthesis of 3-methyl-2,6-diphenylpiperidin-4-one oxime ester 8. using equimolar quantities of benzoic acid 7a and pocl3, the desired product was obtained in 86 % yield (entry 2, table ii). however, 0.5 equiv. of pocl3 resulted in a poor yield of the product (entry 1, table ii) compared to when 1.1 equiv. of pocl3 was taken, while using 1.5 and 2.0 equiv. of pocl3 resulted in moderate yields (entries 3 and 4, table ii). _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1104 gokula krishnan, sivakumar and thanikachala. table ii. optimization of reaction conditions with varying amounts of pocl3 entry substrate (equiv.) pocl3 (equiv.) time, min yield a, % 1 phcooh (1.1) 0.5 30 trace 2 phcooh (1.1) 1.1 15 86 3 phcooh (1.1) 1.5 25 80 4 phcooh (1.1) 2.0 20 69 aisolated yield to study the scope and limitation of this protocol, pocl3 and pyridine were used with wide ranges of aromatic/heteroaromatic acids and piperidin-4-one oximes. these methodologies resulted in good yield of products with short reaction times for all the substrates (table iii). the purity of products was high after a single recrystallization; extraction steps and chromatographic separation are thus avoided. the final products were well characterized using ftir, nmr (1d and 2d) and mass spectrometry. the structures of compounds 12 and 20 were fully established by single crystal xrd analysis. table iii. substrate scope for piperidin-4-one oxime esters using pocl3/pyridine cmpd. r ar time, min yielda % cmpd. r ar time, min yielda, % 8 me c6h5 15 86 16 et p-no2-c6h4 20 95 9 me 3-c5h4n 20 90 17 et m-me-c6h4 25 75 10 me p-cl-c6h4 20 79 18 i-pr c6h5 20 81 11 me p-no2-c6h4 15 92 19 i-pr 3-c5h4n 25 89 12 me m-me-c6h4 25 77 20 i-pr p-cl-c6h4 25 73 13 et c6h5 20 85 21 i-pr p-no2-c6h4 20 86 14 et 3-c5h4n 20 86 22 i-pr m-me-c6h4 35 80 15 et p-cl-c6h4 25 80 aisolated yield ftir spectral analysis in general, compounds containing carbonyl group show absorption in the region of 1600–1750 cm–1. in all the compounds 8–22, a strong band appeared in the double bond region of the spectra at 1739–1752 cm–1, which confirmed the formation of oxime ester (c=o). the imino group (c=n) in the piperidine ring gave a band at 1641–1607 cm–1. a collection of bands observed in the region of 3272–3446 cm–1 and 2797–3087 cm–1 are due to the presence of a secondary amine (n–h) and aromatic and aliphatic c–h stretching. nmr spectral analysis for convenience, compound 8 was selected for the nmr spectral discussion. the two sets of signals appearing in the region 7.29–8.05 ppm for 15 protons revealed the presence of two ph groups at the piperidine ring and one benzene _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis and characterization of novel piperidin-4-one oxime esters 1105 ring of the o-benzoyl group. of these two sets of signals, one doublet that appeared in the downfield region at 8.03 ppm corresponded to the deshielded ortho h-atoms belonging to the o-benzoyl group. the remaining protons of ph groups and the o-benzoyl group collectively gave a multiplet at 7.29–7.57 ppm. a doublet appeared in the upfield region of 1.07 ppm (j = 6.4 hz) with a strong correlation in hsqc with the 11.84 ppm peak and a weak correlation with the 44.86 ppm; the signal at 1.07 ppm is due to me(c3). a broad singlet appeared at 2.00 ppm, that showed no correlation in hsqc, was unambiguously assigned as the nh proton. in the downfield region, there are three doublets at 3.95, 3.66 and 3.56 ppm. of these three signals, the two doublets at 3.95 (j = 11.6 hz) and 3.66 ppm (j = 10 hz) show strong correlation with 60.94 and 69.02 ppm signals and weak correlation with the 44.36 ppm signal. obviously, these two signals are due to h2ax and h6ax (benzylic) protons. the latter doublet at 3.56 ppm (j = 13.6 hz) shows strong correlation with 36.48 ppm signal, which is cross peak with the triplet center at 2.30 ppm, which confirmed that 3.56 and 2.30 ppm are due to h5eq and h5ax protons attached at the c5 carbon. a multiplet appearing at 2.67 ppm showed a strong correlation with the 44.36 ppm signal and a weak correlation with the 69.02 ppm signal, which confirmed the signal be assigned to the h3ax proton. akin to compound 8, the chemical shift and coupling constant of c3 methyl, ethyl, isopropyl substituted analogous compounds 9–22 were assigned accordingly. unlike compound 8, in compounds 13–22, the h2ax proton is deshielded by 0.14 ppm for the ethyland 0.38 ppm for the isopropyl-substituted compounds. thee deviations indicate that the piperidine ring is flattened or distorted about the c2–c3 bond to decrease the gauche interaction. this interaction operates through space irrespective of the equatorial ethyl/isopropyl group at the c3 carbon and the phenyl group at the c2 carbon with an h2ax proton. in the 13c-nmr spectrum of compound 8, the aromatic c-atoms appeared in the region of 126.9–129.6 ppm. the signals due to three ipso c-atoms were observed at 133.2, 142.0 and 143.0 ppm, respectively. of these three signals, the one appearing at 133.25 ppm is due to an ipso carbon of the o-benzoyl group and the rest are due to ipso carbons attached to c2 and c6 carbons of piperidine ring. the imino carbon (c4) and o-benzoyl carbonyl carbon appear in the downfield region at 164.22 and 169.51 ppm, respectively. in the upfield region, the methyl group attached to the c3 carbon was observed at 11.84 ppm and piperidine ring carbons c2, c3, c5 and c6 appeared at 69.02, 44.36, 36.48, and 60.94 ppm, respectively. conformational analysis the conformation of the piperidine ring and the orientation of the oxime ester group were studied from the values of the nmr coupling constant. com_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1106 gokula krishnan, sivakumar and thanikachala. pound 8 exhibited a large coupling constant j6ax,5ax (11.6 hz) about the c5–c6 bond and about the c2–c3 bond (10 hz) revealing the equatorial dispositions of the aryl rings at c2 and c6, and the alkyl group at c3. thus, the synthesized compound 8 existed in a normal chair conformation with equatorial orientation of the ph ring and me(c3) substituents. the coupling constant about c2–c3 bond (j2ax,3ax) were considerably lower than that about the c5–c6 bond (j6ax,5ax), due to the gauche interaction between the ph group and the alkyl group of c2 and c3. hence, the piperidine ring is flattened/distorted about the c2–c3 bond and hence the lower magnitude of j2ax,3ax relative to j6ax,5ax. the chemical shift difference between the c5 carbon (33.88 ppm) and c3 carbon (49.51 ppm) arises due to a1,3 interactions between the n–o and c5–h bonds. thus, the c5 carbon signal was more shielded compared to c3 carbon. it was concluded that o-benzoyl group was syn to the c5 carbon. single crystal xrd analysis of compounds 12 and 20 the crystal data and refinement parameters for compounds 1224 and 20 are summarized in table s-i of the supplementary material. the ortep view of compounds 12 and 20 are shown in figs. 1 and 2, respectively. in compound 12, the piperidine ring (n1/c7–c11) adopts a chair conformation (the n1 and c9 atoms deviate from the best plane of c7/c8 and c10/c11 by 54.9(2)° and –47.7(2)°, respectively) with an equatorial orientation of the ph rings and me group substituted on the heterocyclic. the c–c=n bond angles are different (c8–c9=n2 = 126.54(19)° and c10–c9=n2 = 117.50(18)°) and the dihedral angle between the atoms c10–c9=n2–o1 is –177.16(19)°, showing that the molecule exists as the e-isomer. fig. 1. the ortep view of compound 12 showing 30 % probability displacement ellipsoids. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis and characterization of novel piperidin-4-one oxime esters 1107 fig. 2. the ortep view of compound 20 showing 50 % probability displacement ellipsoids. in compound 20, the piperidine ring (n1/c1–c5) also exists in a chair conformation with the ring atoms n1 and c3 deviating from the best plane of c1/c2 and c4/c5 by 66.77 and –62.22°, respectively. the ph rings and the isopropyl group substituted on the heterocyclic ring are equatorially orientated. the bond angles of the c–c=n are different (c2–c3=n2 is 126.45° and c4–c3=n2 is 118.97°) and the molecule is found to exist in e-isomeric form as evidenced by the c2–c3=n2–o1 dihedral angle of 179.08°. antimicrobial screening all the synthesized compounds 8–22 were screened for their in vitro antimicrobial activity in nutrient broth (nb) for bacteria and sabouraud dextrose broth (sdb) for fungi by the twofold serial dilution method. a standard procedure was adopted for the preparation of all test samples.25 the minimum inhibitory concentration (mic) values of the synthesized compounds 8–22 against bacterial strains viz. bacillus subtilis, staphylococcus aureus, pseudomonas aeruginosa and escherichia coli are compared with the those of ampicillin in table iv and against fungal strains viz. penicillim chrysogenum, trichoderma viride, aspergillus niger and a. flavus are compared with those of amphotericin-b in table v. antibacterial activity all the synthesized piperidin-4-one oxime esters exhibited a wide range of in vitro antibacterial activity from the highest concentration (200 µg ml–1) to the lowest concentration (6.25 µg ml–1) against all the tested organisms, except 8 that _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1108 gokula krishnan, sivakumar and thanikachala. table iv. antibacterial activity (mic / µg ml-1) of compounds 8–22; –: no inhibition at 200 µg ml-1 cmpd. bacteria b. subtilis s. aureus p. aeruginosa e. coli 8 100 – 200 200 9 25 50 25 50 10 25 100 50 50 11 50 100 50 50 12 100 200 100 100 13 50 200 100 200 14 12.5 25 12.5 12.5 15 25 50 50 25 16 25 50 25 25 17 50 100 50 50 18 50 100 50 50 19 6.25 12.5 6.25 6.25 20 12.5 25 6.25 12.5 21 6.25 25 25 12.5 22 25 50 25 25 ampicillin 12.5 50 25 25 table v. antifungal activity (mic / µg ml-1) of compounds 8–22; – no inhibition at 200 µg ml-1 cmpd. fungi p. chrysogenum t. viride a. niger a. flavus 8 – 200 – – 9 200 100 100 200 10 200 200 200 200 11 – – 200 200 12 – 200 – 200 13 200 100 100 100 14 50 50 50 25 15 100 50 50 50 16 100 100 50 25 17 100 100 100 50 18 50 25 50 50 19 25 12.5 12.5 12.5 20 25 12.5 25 25 21 50 25 25 25 22 50 25 25 25 amphotericin b 25 25 50 50 failed to inhibit the growth of s. aureus at the highest concentration (200 µg ml–1). compounds 9–11 recorded moderate activity against s. aureus (100 µg ml–1) and improved activity against b. subtilis, p. aeruginosa and e. coli. however, compounds 8 and 12 showed weaker activity against s. aureus, p. aeruginosa _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis and characterization of novel piperidin-4-one oxime esters 1109 and e. coli at 200 µg ml–1. on introducing an ethyl and/or isopropyl group at the 3rd position of the piperidine ring instead of a methyl group and replacing of o-benzoyl group in the oxime part of all the compounds registered better activity against all the strains. several authors also documented that the 3-ethyl and/or 3-isopropyl substituted piperidin-4-one oxime derivatives exhibited outstanding antibacterial and antifungal activities.26,27 the 3-ethyl substituted oxime esters 13–17 showed better activity against all the tested organisms compared to compounds 8–12. among these compounds, 14 registered greater activity (12.5–25 µg ml–1) against all the tested organisms and compounds 15 and 16 recorded good activity against b. subtilis and e. coli at 25 µg ml–1. compound 17 showed moderate activity against b. subtilis, p. aeruginosa and e. coli at 50 µg ml–1 and compound 13 did not show any significant activity against s. aureus and e. coli even at the highest concentration (200 µg ml–1). the presence of an isopropyl group at the 3rd position in compounds 18–22 showed better activity with mic values of 6.25–12.5 µg ml–1 when compared with the other substituents (methyl or ethyl). especially compound 19 registered greater activity against all the tested strains at lower concentrations (6.25–12.5 µg ml–1) and compounds 20 and 21 also registered better activity (12.5–50.0 µg ml–1) against all the tested organisms compared with 18 and 22 (25–100 µg ml–1). from the above observations, it is obvious that the nicotinoyl-substituted oxime esters 14 and 19 recorded excellent activity among the substituted oxime esters. the chloroand nitro-substituted oximes esters 15, 16, 20 and 21 also registered good activity. antifungal activity the synthesized compounds 8–22 were also screened for their in vitro antifungal activity against the tested organisms. compounds 8 and 12 showed no significant inhibition of p. chrysogenum, a. niger and a. flavus even at the highest concentration of 200 µg ml–1, nor did compound 11 against p. chrysogenum and t. viride. compounds 9, 10 and 13 showed weak activity against a. flavus and p. chrysogenum at 200 µg ml–1. replacement of the methyl group by ethyl and/or isopropyl led to significant activity against all the tested organisms. compound 14 registered better activity against t. viride, p. chrysogenum and a. niger at 50 µg ml–1 and compound 15 also showed better activity against t. viride, a. flavus and a. niger. compounds 16 and 17 exhibited moderate activity against p. chrysogenum, t. viride with mic value of 100 µg ml–1. compound 18 showed good activity against p. chrysogenum, a. flavus and a. niger and improved activity against t. viride at 25 µg ml–1. introduction of nicotinoyl and/or an electron-withdrawing group (chloro or nitro) at the oxime part increased the activity of compounds 19 and 20 against t. viride, a. niger and a. flavus at 12.5–25 µg ml-1, of compounds 21 and 22 against p. chrysogenum, t. viride and a. niger at 25–50 µg ml-1. an isopropyl group at the 3rd position of the piperidine ring _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1110 gokula krishnan, sivakumar and thanikachala. resulted in greater activity against all the tested organisms (12.5–50 µg ml–1), when compared with the other compounds. conclusions in summary, the pocl3 and pyridine system was described as an in situ activating agent for direct piperidin-4-one oxime ester formation in the reaction between aromatic acids and piperidin-4-one oximes. the present method is practically efficient, involves shorter reaction times, simple purification procedures and is economic for the synthesis of piperidin-4-one oxime esters. from xrd crystallographic results, it was shown that the piperidine rings of compounds 12 and 20 adopt chair confirmations with equatorial orientation of the aryl groups. based on the antimicrobial studies, it was proved that piperidin-4-one oxime esters show better activities against standard strains than ampicillin and amphotericin-b. in particular, nicotinoyl oximes 14 and 19 showed excellent antibacterial as well as antifungal activities in comparison with the other oxime esters. supplementary material analytical and spectral data of the synthesized compounds and the crystal data and structural refinement of compounds 12 and 20 are available electronically from http:// //www.shd.org.rs/jscs/, or from the corresponding author on request. и з в о д синтеза, структурна карактеризација и испитивање антимикробне активности нових естара пиперидин-4-он-оксима k. gokula krishnan, r. sivakumar и v. thanikachalam department of chemistry, annamalai university, annamalainagar 608 002, tamil nadu, india синтетисано је петнаест нових, биолошки активних, естара пиперидин-4-он-оксима естара 8–22 у добром приносу. једињења су добијена реакцијом непосредно припремљених алканоил-хлорида, добијених из одговарајућих карбоксилних киселина и pocl3, са пиперидин-4-oн-оксимима. структура добијених деривата утврђена је ftir, nmr (1d и 2d) и масеном спектралном анализом. структура деривата недвосмислено је потврђена xrd анализом монокристала једињења 12 и 20. испитана је in vitro антибактеријска и антифунгална активност свих синтетисаних деривата. значајан број ових једињења показује добру активност према bacillus subtilis, pseudomonas aeruginosa, escherichia coli, trichoderma viride и aspergillus flavus. (примљено 13. новембра 2014, ревидирано 25. јануара, прихваћено 16. априла 2015) references 1. a) s. jegham, g. defoase, t. purcell, j. sehoernaker, us patent 5,280,030 (1994); b) m. j. schneider, alkaloids: chemical and biological perspectives; s. w. pelletier, ed., pergamon, oxford, 1996; c) a. p. kozikowski, g. l. araldi, j. boja, w. m. meil, k. m. johnson, j. l. flippen-anderson, c. george, e. saiah, j. med. chem. 41 (1998) 1962; d) s. s. hadida-ruah, h. m. binch, m. p. deninno, l. t. d. fanning, b. a. frieman, p. d. j. grootenhuis, n. hilgraf, p. joshi, e. a. kallel, m. t. miller, j. pontillo, a. silina, u. j. sheth, d. j. hurley, v. arumugam, us patent 2012/0264749 a1 (2012) _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis and characterization of novel piperidin-4-one oxime esters 1111 2. s. o. thorberg, s. berg, j. lundstrom, b. pettersson, a. wijkstrom, d. sanchez, p. lindberg, j. l. g. nilsson, j. med. chem. 30 (1987) 2008 3. g. xu, a. kannan, t. l. hartman, h. wargo, k. watson, j. a. turpin, r. w. buckheit jr., a. a. johnson, y. pommier, m. cushman, bioorg. med. chem. 10 (2002) 2807 4. p. lagisetty, p. vilekar, k. sahoo, s. anant, v. awasthi, bioorg. med. chem. 18 (2010) 6109 5. d. cheng, s. valente, s. castellano, g. sbardella, r. d. santo, r. costi, m. t. bedford, a. mai, j. med. chem. 54 (2011) 4928 6. a. m. katsori, m. chatzopoulou, k. dimas, c. kontogiorgis, a. patsilinakos, t. trangas, d. hadjipavlou-litina, eur. j. med. chem. 46 (2011) 2722 7. s. t. harini, h. v. kumar, j. rangaswamy, n. naik, bioorg. med. chem. lett. 22 (2012) 7588 8. j. p. michael, nat. prod. rep. 5 (2003) 476 9. a) d. j. lauffer, m. r. pavia, h. tecle, a. j. thomas, european patent 445,731, a1 (1991); b) i. damljanovic, m. vukicevic, r. d. vukicevic, monatsh. chem. 137 (2006) 301; c) p. politzer, j. s. murray, the chemistry of hydroxylamines, oximes and hydroxamic acids, wiley, chichester, 2009 10. d. a. bachovchin, m. r. wolfe, k. masuda, s. j. brown, t. p. spicer, v. fernandezvega, p. chase, p. s. hodder, h. rosen, b. f. cravatt, bioorg. med. chem. lett. 20 (2010) 2254 11. g. surkau, k. j. bohm, k. muller, h. prinz, eur. j. med. chem. 45 (2010) 3354 12. a. karakurt, m. a. alagoz, b. sayoglu, u. calis, s. dalkara, eur. j. med. chem. 57 (2012) 275 13. x. h. liu, l. pan, c. x. tan, j. q. weng, b. l. wang, z. m. li, pest. biochem. physiol. 101 (2011) 143 14. k. kunimoto, j. tanabe, h. kura, h. oka, m. ohwa, us patent 7 189 489 b2 (2007) 15. r. r. hwu, s. c. tsay, s. c. hong, m. h. hsu, c. f. liu, s. s. p. chou, bioconjugate chem. 24 (2013) 1778 16. j. otera, j. nishikido, esterification: methods, reactions and applications, 2nd ed., wiley-vch, weinheim, 2010 17. b. neises, w. steglich, angew. chem. int. ed. engl. 17 (1978) 522 18. o. mitsunobu, m. eguchi, bull. chem. soc. jpn. 44 (1971) 3427 19. a. k. adak, synlett (2004) 1651 20. j. mcnulty, r. vemula, v. krishnamoorthy, a. robertson, tetrahedron 68 (2012) 5415 21. f. effenberger, g. konig, h. klenk, angew. chem., int. ed. engl. 17 (1978) 695 22. a) c. r. noller, v. baliah, j. am. chem. soc. 70 (1948) 3853; b) k. gokula krishnan, r. sivakumar, v. thanikachalam, can. chem. trans. 2 (2014) 353 23. a) k. pandiarajan, r. t. sabapathy mohan, m. u. hasan, magn. reson. chem. 24 (1986) 312; b) e. abele, r. abele, o. dzenitis, e. lukevics, chem. heterocycl. compd. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice design, synthesis, characterization, and antimicrobial activity of the new 2-{(e)-[(4-aminophenyl)imino]methyl}-4,6-dichlo-rophenol and its complexes with co(ii), ni(ii), cu(ii) and zn(ii): an experimental and dft study j. serb. chem. soc. 81 (5) 509–520 (2016) udc 542.9+547.571’551+546.302:519.677: jscs–4864 615.281/.282–188 original scientific paper 509 design, synthesis, characterization, and antimicrobial activity of the new 2-{(e)-[(4-aminophenyl)imino]methyl}-4,6-dichlorophenol and its complexes with co(ii), ni(ii), cu(ii) and zn(ii): an experimental and dft study emine gulhan bakirdere1*, mehmet ferdi fellah2**, erdal canpolat3, mehmet kaya4 and seher gür5 1department of science education, yıldız technical university, 34220, i̇stanbul, turkey, 2department of chemical engineering, bursa technical university, 16190, bursa, turkey, 3department of science and mathematics for secondary education, fırat university, 23119, elazığ, turkey, 4department of chemistry, fırat university, 23119, elazığ, turkey and 5department of biology, fırat university, 23119, elazığ, turkey (received 30 october, revised 28 december, accepted 29 december 2015) abstract: in this study, the complexes of co(ii), ni(ii), cu(ii) and zn(ii) with 2-{(e)-[(4-aminophenyl)imino]methyl}-4,6-dichlorophenol were prepared and characterized by physical, spectral and analytical data. the metal:ligand stoichiometric ratio was 1:2 in all the complexes. the results suggested that the schiff bases were coordinated to the metal ions through the phenolic oxygens and the azomethine nitrogen to give mononuclear complexes. their structures were elucidated based on elemental analysis, ir, 1hand 13c-nmr, uv–vis, and magnetic susceptibility measurements and thermogravimetric analyses. both the antibacterial and antifungal activities and the minimum inhibitory concentration (mic) values of compounds are reported. among the tested compounds, the most effective compound providing an mic value of 64 µg ml-1 was zn(l)2 against candida tropicalis and bacillus subtilis. the theoretically optimized geometries of the complexes were tetrahedral structures. the computed stretching frequencies of the c=n, c–o and n–h bonds were in good agreement with the experimental data. all the calculated frequencies fell within about 5 % of the experimental frequency regions. keywords: schiff base; transition metal complex; antibacterial and antifungal activities; dft. *,**corresponding authors. e-mail: (*)bgulhan@yildiz.edu.tr; (**)mffellah@gmail.com, mferdi.fellah@btu.edu.tr doi: 10.2298/jsc151030008b 510 bakirdere et al. introduction several schiff base metal complexes have been studied in the past because of their wide applicability.1 schiff bases have often been used as chelating ligands in the field of coordinatiation chemistry for obtaining thermotropic liquid crystalline polymers and their metal complexes have been used as radio-pharmaceuticals for cancer targeting, dioxygen carriers and model systems for biological macromolecules.2–6 the presence of transition metals in human blood plasma indicates their importance in the mechanisms for the accumulation, storage and transport of transition metals in living organisms.7 the synthesis and characterization of various transition metal complexes with novel schiff bases were experimentally investigated.8–19 in a previous theoretical study, 2-{(e)-[(4-aminophenyl)imino]methyl}-6-bromo-4-chlorophenol as schiff base ligand and its complexes with co, ni, cu and zn metals were examined.19 in the present study, the synthesis and characterization of cobalt(ii), nickel(ii), copper(ii) and zinc(ii) complexes with 2-{(e)-[(4-aminophenyl)imino]methyl}-4,6-dichlorophenol (lh) were investigated. all compounds were characterized by elemental analyses, uv–vis, ir, and 1hand 13c-nmr spectroscopy, magnetic susceptibility measurements and thermogravimetric analyses (tga). moreover, density functional theory (dft) calculations were performed to examine the parameters of the optimized structure and to calculate the ir spectra of the ligand and its complexes with co, ni, cu and zn metals in order to compare them with the experimental data obtained in this study. additionally, all the complexes were assayed for their antibacterial activities against two grampositive bacterial strains (bacillus subtilis and staphylococcus aureus) and two gram-negative bacterial strains (escherichia coli and salmonella enterica subsp. enterica serovar typhimurium) by the minimum inhibitory concentration (mic) method. experimental methods elemental analyses (c, h and n) were realized using a leco-932 chnso analyzer by the technical and scientific research council of turkey (tubitak). the ir spectra were recorded on a mattson 1000 ft-ir spectrometer as kbr pellets. the nmr spectra were recorded on a bruker dpx-400 mhz high performance digital ft-nmr spectrometer. the electronic spectra were obtained on a shimadzu uv probe 2.1 spectrometer at the firat university. magnetic susceptibilities were determined on a sherwood scientific magnetic susceptibility balance (model mk1) at room temperature using hg[co(scn)4] as the calibrant; diamagnetic corrections were calculated from pascal’s constants. the tg curves were recorded on a shimadzu tg-50 thermo-balance. preparation of the ligand and its complexes 2-{(e)-[(4-aminophenyl)imino]methyl}-4,6-dichlorophenol (lh). a solution of 2.16 g (20 mmol) 1,4-phenylendiamine dissolved in 25 ml absolute ethanol was added dropwise to 1.81 g (10 mmol) 3,5-dichlorosalicylaldehyde and 0.01 g p-toluenesulfonic acid dissolved in dft study on and antimicrobial activity of a dichlorophenol and its complexes 511 25 ml absolute ethanol under reflux at 60 °c under continuous stirring for 1 h. after overnight stirring, the precipitate was filtered off, washed several times with hot water, cold ethanol and diethyl ether and dried under vacuum. bis(2-{(e)-[(4-aminophenyl)imino]methyl}-4,6-dichlorophenolato)cobalt(ii), co(l)2. the ligand (0.28 g, 1.00 mmol) was dissolved in 25 ml absolute ethanol. a solution of co(aco)2·4h2o (0.13 g, 0.50 mmol) in 25 ml of absolute ethanol was added dropwise over 1 h to the ligand solution under reflux at 60 °c under continuous stirring. after stirring overnight, the precipitated complex was filtered off, washed several times with hot h2o, cold etoh and cold et2o and dried under vacuum. bis(2-{(e)-[(4-aminophenyl)imino]methyl}-4,6-dichlorophenolato)nickel(ii), ni(l)2. ni(l)2 was prepared following the same procedure as described for co(l)2, starting with lh (0.28 g, 1.00 mmol) and ni(aco)2·4h2o (0.12 g, 0.50 mmol). bis(2-{(e)-[(4-aminophenyl)imino]methyl}-4,6-dichlorophenolato)copper(ii), cu(l)2. cu(l)2 was prepared following the same procedure as described for co(l)2, starting with lh (0.28 g, 1.00 mmol) and cu(aco)2·h2o (0.10 g, 0.50 mmol). bis(2-{(e)-[(4-aminophenyl)imino]methyl}-4,6-dichlorophenolato)zinc(ii) zn(l)2. zn(l)2 was prepared following the same procedure as described for co(l)2, starting with lh (0.28 g, 1,00 mmol) and zn(aco)2·2h2o (0.11 g, 0.50 mmol). microbiology the tested microorganisms were the gram-positive staphylococcus aureus atcc 6538p and bacillus subtilis atcc 6633, the gram-negative escherichia coli atcc 25922 and salmonella enterica subsp. enterica serovar typhimurium nrrl b 4420, and the yeast-like fungi, candida glabrata atcc 66032 and candida tropicalis atcc 13803. ampicillin (mustafa nevzat) and fluconazole (pfizer) were used as antibiotic references for the bacteria and yeasts, respectively (obtained from the department of biology, fırat university, turkey). antibacterial and antifungal assays. the bacterial cultures were obtained in mueller– –hinton broth (difco) for all the bacterial strains after 24 h incubation at 37±0.1 °c. the yeasts were propagated in sabouraud dextrose broth (difco) by incubation for 24 h at 25±0.1 °c. testing was performed in mueller–hinton broth and sabouraud dextrose broth at ph 7.4 for the bacteria and yeast, respectively. the final inoculum size for the bacteria and fungi was 105 cfu ml-1. the test compounds were dissolved in dmso at an initial concentration of 1024 µg ml-1 and then serially diluted in culture medium to 1 µg ml-1. a set of tubes containing only inoculated broth was kept as the control. the antibacterial activity was determined after incubation for 24 h at 37 °c for the bacteria and after incubation for 48 h at 25 °c for the yeasts. the mic was defined as the lowest concentration of the compounds that inhibited the visible growth of a microorganism. all experiments in the antibacterial and antifungal assays to define the mic values were performed in duplicate. computational methods in the present work, the theoretical calculations were based on dft20 implemented in gaussian 09 software.21 the b3lyp hybrid formalism 22-24 was used for the calculations. the hybrid method of b3lyp is a high-quality dft technique for theoretical calculations in organic chemistry.25 the theoretical approach applied in this study was presented in previous studies.18,19 the spin contamination value must be very small (less than 10 %).26 the <s2> values were negligible in this study. vibrational analysis was also performed to obtain vibrational frequencies. all vibrational frequencies were obtained for the 6-31g(d,p) method. all frequency values were scaled using a factor of 0.9613 to reproduce the experimental 512 bakirdere et al. fundamentals.27 mulliken population analysis28 was utilized to obtain spin density values (showing where unpaired electrons of the system are located) of the metal atoms. natural bond orbital (nbo)29 analysis was employed to obtain the nbo charge values and the electronic configurations of the metal atoms. the criteria of convergence in the calculations were 12×10-4, 18×10-4, 3×10-4 and 45×10-5 for gradients of the root-mean-square (rms) displacement, the max. displacement, the rms of the force and the max. force, respectively. results and discussion the ligand (lh) was prepared by reacting equimolar amounts of 1,4-phenylendiamine with 3,5-dichlorosalicylaldehyde in absolute ethanol (scheme 1). the structures of the ligand and the complexes were established by the use of their ir, 1h-nmr and electronic spectra, elemental analyses, magnetic susceptibility measurements and thermogravimetric analyses. scheme 1. preparation of the ligand. all the metal complexes with lh were prepared by the stoichiometric reaction of the corresponding metal(ii) acetate with lh in the mole ratio m:l = 1:2. the complexes are stable intensely colored solids. a summary of the elemental analysis data for ligand and the complexes are given in the supplementary material to this paper. the elemental analysis results agree with the calculated values showing that the complexes had 1:2 metal/ligand rations. the elemental analysis also confirmed the composition of the above-synthesized compounds. the characteristic ir bands of the ligand and its complexes are given in table s-i of the supplementary material. in the ir spectrum of the ligand, ν(o–h) (phenolic), ν(c=n) (azomethine), ν(c–o) (phenolic) and ν(n–h) stretching vibrations were observed at 3350–3200, 1609, 1290 and 3315 and 3190 cm–1, respectively. these values are in agreement with those observed for similar compounds.14,15 the ν(o–h) band of the phenolic group appeared at ca. 3350–3200 cm–1; its low value was due to the formation of intramolecular hydrogen bonds.4,30 this band was absent in the spectra of all the complex, indicating the deprotonation of schiff base prior to coordination through its oxygen atom.31,32 the azomethine vibration of the ligand at 1609 cm–1 was shifted to lower frequencies after complexation, appearing at 1608, 1607, 1602 and 1608 cm–1 for cobalt(ii), nickel(ii), copper(ii) and zinc(ii) complexes, respectively. this clearly indicates the coordination of the schiff base through the azomethine nitro dft study on and antimicrobial activity of a dichlorophenol and its complexes 513 gen.31,32 this is also supported by an increase in the absorption frequency of the phenolic ν(c–o) band from 1290 cm–1 in the spectrum of the free ligand to values in the range 1323–1293 cm–1 in the spectra of the complexes, indicating that the other coordination site of the schiff base is the phenolic oxygen atom.32 1hand 13c-nmr spectra of the ligand (lh) were recorded in cdcl3. the nmr assignments are given in detail in table s-ii of the supplementary material. characteristic 1h-nmr peaks of the ligand occurred at 15.30 (oh), 7.60 (ch=n) and 5.60–8.90 ppm (ar-h). the oh (phenolic) signal at 15.30 ppm disappeared upon addition of d2o to the solution, indicating that it is an acidic proton.15,16,33 the zinc(ii) complex did not dissolve in dmso-d6 and therefore, the nmr spectra of the zinc (ii) complex could not be obtained. the magnetic susceptibility data of the complexes are given in table s-iii of the supplementary material. the co(ii), ni(ii) and cu(ii) complexes were paramagnetic, while the zn(ii) complex was diamagnetic, as expected for a d10 configuration. according to the magnetic moment calculations, co(ii), ni(ii) and cu(ii) have three, two and one unpaired electrons, respectively. the magnetic moments of the co(ii), ni(ii) and cu(ii) complexes at room temperature were 4.45, 2.82 and 1.77 μb, respectively, which are characteristic for mononuclear, tetrahedral complexes.34,35 in the zn (ii) complex, a tetrahedral structure was proposed and the diamagnetic behavior was confirmed.36 the electronic spectra of the ligand and the co(ii), ni(ii), cu(ii) and zn(ii) complexes were recorded in dmso at room temperature. the aromatic band of the ligand at 260 nm was attributed to benzene π→π* transitions. the band around 400 nm is due to the n→π* transition of the non-bonding electrons present on the nitrogen of the azomethine group in the schiff base. all the complexes showed an intense band near 380–435 nm, which was assigned to n→π* transitions associated with the azomethine linkage.33 the spectra of all the complexes show intense band at ca. 386–450 nm, which can be assigned to charge transfer transition of the tetrahedral geometry.37 the thermal behaviors of the ligand and all the complexes were investigated by thermogravimetry in the temperature range from ambient to 600 °c at a heating rate of 10 °c min–1. thermogravimetric studies of all the complexes showed no weight loss up to 230 °c, indicating the absence of lattice/coordinated water molecules in the complexes.1,38 all these complexes underwent complete decomposition to the corresponding metal oxides, coo, nio, cuo and zno.39–43 based on the experimental results, the suggested structure of the complexes, where m = co(ii), ni(ii) cu(ii) and zn(ii), is shown in fig. 1. a comparative study of mic values of the schiff base lh and its complexes indicated that the metal complexes in general had a better activity than the free ligand. such increased activity of the metal chelates could be explained on the basis of the chelating theory.32 the synthesized schiff base compounds had com514 bakirdere et al. parable and similar inhibitory effects (low to moderate mic values ranging from 64 to 512 µg ml–1) on the growth of tested strains (table i). the antibacterial results evidently showed that the activity of the schiff base became more pronounced when coordinated to the metal ions. thus, all the complexes show high bactericidal activities against s. enterica, s. aureus and e. coli (mic = 128 µg ml–1) and b. subtilis (mic = 64 µg ml–1) as compared to corresponding schiff base. the structure of the tested compounds seemed to be the principal factor influencing the antimicrobial activity. the present investigations of the antimicrobial screening data revealed that all of the newly synthesized compounds exhibited poor activities compared to those of the control drugs. fig. 1. experimentally suggested structural formula of the complexes of the ligand (where m = co(ii), ni(ii) cu(ii) or zn(ii)). table i. the in vitro activities of the compounds and the reference drugs (the mic values are in µg ml-1) sample s. enterica s. aureus e. coli b. subtilis c. tropicalis c. glabrata lh 256 512 512 128 256 256 co(l)2 128 128 128 512 256 256 ni(l)2 128 256 256 128 256 256 cu(l)2 256 256 256 256 128 256 zn(l)2 256 256 128 64 64 128 ampicillin 2 2 2 2 – – fluconazole – – – – 8 8 theoretical geometries of the ligand and its complexes with metals were constructed using the structures experimentally suggested in this study. initially, the ligand was optimized by means of equilibrium geometry (eg) calculations. the optimized geometry of the ligand is presented in fig. 2. the spin multiplicity (sm) numbers determined for the complexes of the ligand with zn, cu, ni and co metals were 1, 2, 3 and 4, respectively. the optimized geometry of the 2-{(e)-[(4-aminophenyl)imino]methyl}-6-bromo-4-chloro dft study on and antimicrobial activity of a dichlorophenol and its complexes 515 phenol complexes with zn is shown as an example in fig. 3. in this optimization calculation, the computed <s2> values confirmed that the spin contamination was very small (max 0.5 % after annihilation). fig. 2. optimized geometry of the ligand. fig. 3. optimized geometry of the zn complex with 6-bromo analogue of l-. nbo charges with color representation for the optimized complexes are shown in fig. 4. the nbo charges, spin density values and electron configuration of metal atoms for the optimized complexes with zn, cu, ni and co metals are presented in table ii, from which it can be seen that the metal atoms have a high spin density, which indicates that the unpaired electrons are localized on the metal atoms. the optimized complexes with cu, ni and co atoms have distorted tetrahedral structures, while the complex with a zn atom has a normal tetrahedral structure. the reason for this might be that metal atoms in the optimized complexes with cu, ni and co atoms have some spin density whereas the zn atom has no spin density in its optimized complex. additionally, according to fig. 4, the atomic charges of the metal atoms in the complexes are higher than those of the other atoms of the ligands, which show that the metal atoms have the highest positive character. 516 bakirdere et al. a) b) c) d) e) fig. 4. nbo charges with color representation for optimized: a) zn-complex, b) ni-complex, c) cu-complex and d) co-complex; e) scale for charges (left side: lowest charge (–) and right side: highest charge (+)). table ii. nbo charges, spin density values and electron configurations of the metal atoms of the optimized zn, cu, ni and co complexes metal nbo charge spin density electron configuration zn 1.297 – [core]4s(0.37)3d(9.91)4p(0.42)5p(0.01) cu 1.123 0.683 [core]4s(0.35)3d(9.15)4p(0.37) ni 1.162 1.710 [core]4s(0.29)3d(8.16)4p(0.39) co 1.229 2.776 [core]4s(0.29)3d(7.13)4p(0.35) the vibrational frequencies obtained in a previous study for 2-{(e)-[(4 -aminophenyl)imino]methyl}-6-methoxy-4-nitrophenol and its complexes with co, ni, cu and zn metals using the 6-31g(d,p), dgdzvp and cep-121g basis sets were relatively similar.18 thus, in the present study, the vibrational frequencies were obtained by the 6-31g(d,p) method. the calculated vibrational frequency values of the c=n, c–o and n–h stretching modes for the optimized dft study on and antimicrobial activity of a dichlorophenol and its complexes 517 complexes are tabulated in table iii, from which it could be seen that the calculated values of the c=n, c–o and n–h stretching frequencies were in good agreement with experimental frequency data given in table s-i for the ligand and its zn, cu, ni and co complexes. the calculated c=n stretching frequencies for the ligand and its metal complexes (zn, cu, ni and co) were 1621, 1574 (1575), 1572 (1575), 1569 (1572) and 1568 (1571) cm–1, respectively. the experimental frequencies were 1609, 1608, 1602, 1607 and 1608 cm–1, respectively. the stretching frequencies of the c–o mode were computed as 1272, 1304 (1306), 1305 (1307), 1304 (1307) and 1303 (1304) cm–1 for the ligand and its zn, cu, ni and co complexes, respectively. the corresponding experimental frequency values were 1290, 1293, 1323, 1295 and 1294 cm–1, respectively. the n–h stretching frequencies were also similar to the corresponding experimental ir bands. consequently, all calculated frequencies fell within 5.1 % of the experimental frequency region reported in this study. this is a reasonable error region as pointed out in the literature.44 table iii. the calculated vibrational frequency values (cm-1) for the optimized ligand and its complexes frequency type ligand complex zn cu ni co c=n stretching 15c=14n 1621 1574 1572 1569 1568 40c=39n 1575 1575 1572 1571 c–o stretching 16c–27o 1272 1304 1305 1304 1303 49c–60o 1306 1307 1307 1304 n–h symmetrical stretching 11n–12h, 13h 3440 3438 3436 3437 3438 36n–37h, 38h 3440 3438 3440 3440 n–h anti-symmetrical stretching 11n–12h, 13h 3546 3541 3539 3540 3541 36n–37h, 38h 3545 3542 3545 3544 conclusions many schiff-base derivatives containing a substituted aromatic amine have been synthesized, characterized and used for complexation of transition metals. functional groups, such as amines are very far removed from the pendants participating in the complexation and have no effect on the complexes. tga and elemental analysis indicated no lattice/coordinated water molecules in the complexes. the satisfactory analytical data and all of the physicochemical studies suggested that these complexes might be formulated as m(l)2. dft calculations with the b3lyp method/6-31g(d,p) basis set were performed on the optimized structures and the vibrational ir spectra of the ligand and its co, ni, cu and zn complexes. the optimized geometries of complexes were tetrahedral structures. the calculated stretching frequency values of the c=n, c–o and n–h bonds were in good agreement with experimental frequency data reported in this study 518 bakirdere et al. for the ligand and its zn, cu, ni and co complexes. all the calculated frequencies fell within about 5 % of the experimental frequency regions. the antibacterial data given for the compounds presented in this paper indicated that the antimicrobial activity of metal complexes depended on the structure of the tested compound. the sensitivity spectrum of the microbial strains towards the ligand and the corresponding complexes was determined by qualitative and quantitative methods. the quantitative antimicrobial activity test results proved that both the ligand and complex combinations have specific antimicrobial activity, depending on the microbial species tested. although none of the presented compounds was as effective against the tested microorganisms as the used reference drugs, the determination of their antibacterial activity against other gram-negative and gram-positive strains is in progress. supplementary material detailed physical, analytical and spectral data for the ligand and its zn, cu, ni and co complexes are available electronically from http://www.sbd.org.rs/jscs/ or from the corresponding author on request. acknowledgement. this work was supported by the management unit of scientific research projects of fırat university (project: fübap 1276). и з в о д синтеза, карактеризација, антибактеријска и антифунгална активност 2-{(e)-[(4-аминофенил)имино]метил}-4,6-дихлорофенола и одговарајућих co(ii), ni(ii), cu(ii) и zn(ii) комплекса: експериментално и dft испитивање emine gulhan bakirdere1, mehmet ferdi fellah2, erdal canpolat3, mehmet kaya4 и seher gür5 1 department of science education, yıldız technical university, 34220, i̇stanbul, turkey, 2department of chemical engineering, bursa technical university, 16190, bursa, turkey, 3 department of science and mathematics for secondary education, fırat university, 23119, elazığ, turkey, 4department of chemistry, fırat university, 23119, elazığ, turkey и 5department of biology, fırat university, 23119, elazığ, turkey у овом раду су синтетисани и окарактерисани физичкохемијским методама комплекси co(ii), ni(ii), cu(ii) и zn(ii) са 2-{(e)-[(4-аминофенил)имино]метил}-4,6-дихлорофенолом. стехиометријски однос метала и лиганда у свим комплексима је 1:2. на основу добијених резултата, може се претпоставити да се шифова база координује за јоне метала преко фенолног атома кисеоника и азометинског атома азота, при чему настају мононуклеарни комплекси. структуре комплекса су одређене применом елементалне микроанализе, ir, 1hи 13c-nmr спектроскопије, uv–vis спектрофотометрије, мерењем магнетне сусцептибилности и термогравиметријском анализом. применом dft методе нађено је да комплекси имају тетраедарску структуру. теоријски израчунате вредности за фреквенције c=n, c–o и n–h веза су у сагласности са експерименталним подацима, при чему израчунате вредности одступају до 5 % од експериментално одређених фреквенција. испитивана је антибактеријска и антифунгална активност синтетисаних једињења и добијени резултати су приказани у облику mic вредности. од свих испитиваних једињења, zn(l)2 комплекс показује највећу активност dft study on and antimicrobial activity of a dichlorophenol and its complexes 519 према микроорганизмима candida tropicalis and bacillus subtilis, при чему је mic вредност 64 μg ml-1. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice occurrence and distribution of the cyclodiene-type organochlorine pesticides in soils of vojvodina province, serbia j. serb. chem. soc. 81 (6) 707–716 (2016) udc 546.13–039.7+632.95:504.53(497.113) jscs–4879 original scientific paper 707 occurrence and distribution of the cyclodiene-type organochlorine pesticides in soils of vojvodina province, serbia tijana zeremski1*, jovica vasin1, stanko milić1, petar sekulić1, štefan hansman1 and vojislava bursić2 1institute of field and vegetable crops, maksima gorkog 30, novi sad, serbia and 2faculty of agriculture, university of novi sad, trg dositeja obradovića 8, novi sad, serbia (received 12 december 2015, revised and accepted 18 january 2016) abstract: aldrin, endrin and dieldrin are highly toxic and persistent cyclodienetype organochlorine pesticides. although these compounds have been banned over the last few decades in many countries, they can still be found in the environment, especially in the agricultural soil. in order to determine the residue levels of cyclodiene-type organochlorine pesticides in soils of vojvodina province, and to obtain detailed information on their spatial distribution, 1370 soil samples under different land uses were collected from vojvodina province and analyzed for the presence of aldrin, dieldrin and endrin. the residues of those pesticides were detected in the soils of vojvodina in measurable concentrations and dieldrin was the most dominant compound in the soil. however, the concentrations of analysed cyclodiene-type organochlorine pesticides in the most soil samples from vojvodina province were lower than dutch target values for soil quality. soil organic matter and clay content were found to be positively associated with aldrin content while clay content was found to be negatively associated with endrin. key words: soil; aldrin; endrin; dieldrin; vojvodina province. introduction dieldrin, aldrin and endrin are highly toxic cyclodiene-type organochlorine insecticides that are extremely persistent in the environment. originally developed in the 1940s as an alternative to ddt, aldrin, dieldrin and endrin proved to be highly effective insecticides and were widely used during the 1950s to early 1970s. in spite of their high efficiency in insect control, their use have been prohibited in many countries since the 1970s due to the fact that their accumulation in crops poses a potential threat to human health.1 they were prohibited in serbia in 1991, but before that were extensively used on arable soils for insect control especially in vojvodina since it is an agricultural region with an intensive * corresponding author. e-mail: tijana.zeremski@nsseme.com doi: 10.2298/jsc151212015z _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 708 zeremski et al. agricultural production. however, these pesticides continue to be detected in different environments, especially agricultural fields where these pesticides were previously used.2–8 despite some differences in reports concerning the half-lives of aldrin, dieldrin and endrin in soil, most studies have shown that these pesticides are highly persistent in soil. according to the results of the experiment conducted by meijer et al.9 the half-life of dieldrin in soil is about 25 years. donoso et al.10 reported that the half-life of endrin in soil ranged up to 12 years. also, some studies have indicated contamination of the water environment around agricultural fields by cyclodiene-type organochlorine pesticides.11–13 aldrin, dieldrin and endrin residues in agricultural fields cause contamination of not only the water environment, but also of crops grown in contaminated soil. high levels of these pesticides have been detected in a variety of crops around the world.14 in serbia, a dieldrin residue level of 5–73 ng/g and aldrin residue level of 1– –49 ng/g were reported in wheat grown in central bačka and central banat regions.15 in some of the wheat samples concentration of dieldrin and aldrin was greater than the maximum permissible level of 10 ng/g according to european commission,16 which indicates possibility of high concentrations of dieldrin and aldrin in soils where the wheat was grown. the aim of this study was to determine the residue levels of cyclodiene-type organochlorine pesticides aldrin, dieldrin and endrin in soils of the vojvodina province, and to obtain detailed information on their spatial distribution. experimental details about soil sampling are given in supplementary material to this paper. after sampling, soil was air-dried, homogenised and sieved through a 2-mm stainless sieve before analysis. the water and potential soil ph, organic matter content and free caco3 content in soil were determined in accordance with iso methods for soil quality.17–19 particle size distribution was determined in the < 2 mm fraction by the internationally recognized pipette method. sample extraction and clean-up ten grams of the soil sample was weighed precisely and mixed with 5 g of anhydrous sodium sulfate. the mixture was spiked with surrogate compound 2,4,5,6-tetrachloro-m -xylene (tcmx), and then soxhlet-extracted for 24 h with 150 ml mixture of hexane/acetone (1:1 vol. ratio). the crude extracts were concentrated by rotary vacuum evaporation, and then solvent-exchanged by re-dissolving in 2 ml of hexane. a solid phase extraction tubes containing 1 g of silica sorbent were pre-eluted by 4ml of hexane. after introduction of 2 ml of the extract, the column was eluted with 5ml of n-hexane and 5ml of n-hexane/diethyl ether (1:1 vol. ratio). the two fractions were combined as a single fraction and then reduced to 1 ml under a gentle stream of nitrogen before analysis. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ occurrence and distribution of the cyclodiene-type pesticides in soils of vojvodina 709 analysis the determination of cyclodiene-type organochlorine pesticides was performed on an agilent 7890a gas chromatograph equipped with a 63ni micro-electron capture detector (gc -µecd). the separation was performed on a fused silica capillary column (hp-5ms, 30 m×0.25 mm i.d., and 0.25 µm film thickness). helium was used as carrier gas at a constant flow of 1.4 ml/min and high purity nitrogen was used as make-up gas (30 ml/min). the temperature program was as follows: initial temperature of 70 °c was held for 2 min, increased to 150 °c at a rate of 25 °c/min, then increased to 200 °c at a rate of 3 °c/min, and finally increased to 280 °c at rate of 8 °c/min and held for 10 min. the injector and detector temperatures were set at 300 °c. one microliter of each sample was injected in the splitless mode. identification of aldrin, dieldrin and endrin in the samples was based on the comparison of retention time between samples and the standard solution. quantification was performed by five-point calibration method using standard solutions in hexane (from 2.5 to 100 ng/ml, r2 > > 0.992). to confirm the results, typical samples selected were confirmed with an agilent 6890 gc-5975msd system. the gc parameters were the same as described above. the ms was operated in electron impact ionization mode with electron energy of 70 ev. the ion source, quadruple and transfer line temperatures were held at 230, 150 and 280 °c, respectively. target compounds were monitored in the selected ion monitoring (sim) mode. statistical analysis the gis (geographic information system) mapping technique was used to produce spatial distribution maps of the aldrin, endrin, and dieldrin concentrations. data were plotted with the arcview software 10.0 (esri) using a 16 km×16 km output grid. thus, one grid point represents in general one sampling point. descriptive statistical parameters, such as mean value, median, minimum, maximum values and relative standard deviation, were calculated to describe main physical and chemical characteristics of analyzed soils and concentrations of aldrin, endrin and dieldrin in all the samples. the correlation of analyzed pesticides with various soil parameters was analyzed by the karl pearson correlation coefficient. a p-value less than 0.05 (two tail) was considered to be statistically significant. all statistical analyses were carried out using the statistica 12 (statsoft inc., tulsa, ok, usa). results and discussion physical and chemical characteristics of analyzed soils the main physical and chemical characteristics of analyzed soils are given in table i. all soil properties ranged in wide intervals, indicating high heterogeneity of soils in vojvodina province. the ph value in kcl ranged from 3.42 to 9.27 indicating that all analyzed soils were acid to alkaline. the caco3 content ranged from 0.00 to 33.9 %, and organic matter content ranged from 0.31 to 9.66 %. the particle size distribution was also variable with the greatest variability in sand and silt content (0.20–50.07 % and 1.30–49.4 %, respectively). status of cyclodiene-type organochlorine pesticide residues in soil the mean, minimum, maximum concentrations, and detection frequencies of aldrin, endrin and dieldrin in the soils of vojvodina province are shown in table _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 710 zeremski et al. ii. dutch target values20 for cyclodiene-type pesticides in soils which indicate the level of sustainable soil quality are also given in table ii. table i.physical and chemical characteristics of analyzed soils; sd – standard deviation data partameter particle size distribution, % ph (in kcl) ph (in h2o) caco3 content % organic matter content, % sand (200– 2000 µm) fine sand (20–200 µm) silt (<20 µm) clay (<2 µm) minimum 3.42 4.68 0.00 0.31 0.20 14.80 5.30 1.30 maximum 9.27 10.3 33.9 9.66 50.7 70.10 56.5 49.4 mean 7.07 7.87 7.58 3.05 4.37 39.94 33.6 21.9 sd 0.79 0.67 6.89 0.95 8.29 11.48 7.77 9.75 median 7.37 8.09 6.40 3.07 1.50 40.05 34.6 21.8 variance 0.63 0.45 47.4 0.91 68.8 131.9 60.3 95.2 skewness –1.85 –1.48 0.73 0.61 3.87 0.245 –0.76 0.59 kurtosis 3.14 3.14 –0.19 2.36 16.9 –0.17 1.69 0.47 table ii. the concentration of aldrin, endrin and dieldrin in soils under different land uses collected from vojvodina province and dutch target values for soil;20 sd – standard deviation, nd – not detected component mean ng/g sd minimum ng/g maximum ng/g detection frequences, % target value ng/g agricultural fields (n = 1086) aldrin 0.12 0.37 nd 4.29 13.2 0.06 endrin 0.22 2.71 nd 85.33 8.7 0.04 dieldrin 0.33 1.22 nd 24.55 22.2 0.50 forestlands and grasslands (n = 214) aldrin 0.10 0.33 nd 3.15 11.9 0.06 endrin 0.04 0.23 nd 2.09 4.3 0.04 dieldrin 0.31 1.12 nd 11.39 17.6 0.50 orchards and vineyards (n = 70) aldrin 0.19 0.72 nd 4.94 11.4 0.06 endrin 0.25 1.10 nd 8.38 11.4 0.04 dieldrin 0.41 0.88 nd 5.74 32.8 0.50 the concentration of aldrin in all the samples varied from not detected to 4.94 ng/g with the highest detection frequency in soil samples taken at agricultural fields. aldrin concentrations higher than target value according to the dutch standard (0.06 ng/g)20 were measured in 176 soil samples which represent 12.8 % of all soil samples taken in the vojvodina. compared with the concentration of aldrin in soils from other countries and regions, the concentration of aldrin in agricultural field soils in this study was comparable with aldrin concentrations detected in agricultural soils in shanghai, china8 which varied from not detected to 6.62 ng/g with the detection frequency of 36 %, but it was much _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ occurrence and distribution of the cyclodiene-type pesticides in soils of vojvodina 711 lower than the mean aldrin concentration in agricultural soils from southern sonora, mexico21 which was reported to be1600 ng/g. concentration of aldrin detected in the forest soils of taurus mountains in turkey22 ranged from 0.002 to 0.185 ng/g, which is much lower than the concentration of aldrin in forestland and grassland soils within this study. dieldrin concentration in all the samples varied from not detected to 24.55 ng/g with the highest detection frequency in soil samples taken at orchards and vineyards. dieldrin concentrations higher than target value, according to the dutch standard (0.50 ng/g), were detected in 311 soil samples which represents 22.7 % of all soil samples in our study. in comparison with aldrin and endrin, dieldrin exhibited a significantly higher mean concentration and detection frequencies in all types of soils. this is probably due to the fact that aldrin is quickly transformed in the environment into dieldrin,23 and dieldrin is extremely persistent in soil.9,24 according to seike et al.,25 soils remain contaminated with dieldrin even when aldrin and dieldrin have not been used for the past 40 years. similar ratio between dieldrin and aldrin was reported in forest soils of taurus mountains in turkey,22 implying that aldrin residues in turkey have already been degraded. in the present study, soils under different land use exhibited mean concentrations of dieldrin which varied from 0.31. to 0.41 ng/g. these mean concentrations were higher than dieldrin concentrations detected in agricultural soils in shanghai (0.09 ng/g)8 but comparable with dieldrin concentrations found in the forest soils at taurus mountain in turkey (0.26–1.87 ng/g).22 dieldrin concentrations in our study were significantly lower than mean dieldrin concentrations in agricultural soils in alabama, usa (5.19 ng/g),26 north portugal (286 ng/),5 lower fraser valley, canada (450 ng/g)4 and agra, india (780 ng/g).11 endrin concentration in all the samples varied from not detected to 85.33 ng/g with the highest detection frequency in soil samples taken at orchards and vineyards. endrin concentrations higher than target value according to the dutch standard (0.04 ng/g) were detected in 113 soil samples which represents 8.25 % of all soil samples in our study. mean concentration of endrin in forestlands and grasslands soils in our study was 0.04 ng/g which is comparable with endrin concentrations found in soils of taurus mountain in turkey (0.03–0.44 ng/g). mean concentration of endrin in agricultural soils was 0.22 ng/g which is much lower than the one found in agricultural soils in shanghai (nd – 4.32 ng/g) and lower fraser valley, canada (70 ng/g), thus indicating that endrin was less used as insecticide on field crops in vojvodina than elsewhere in the world. distribution pattern of aldrin, dieldrin and endrin in soils under different land use was also investigated. comparison of three different types of land use showed the highest aldrin, dieldrin and endrin mean concentrations in orchard and vineyard soils, while the lowest were in the forestland and grasslands soils. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 712 zeremski et al. the highest aldrin detection frequency was in agricultural soils, while endrin and dieldrin detection frequencies were highest in orchard and vineyard soils. the occurrence of pesticide residues (retention, mobility and degradation) in soil depends on properties of compounds themselves and various environmental factors such as ph, soil moisture, soil organic carbon, clay content, etc.27,28 all these parameters are responsible for organochlorine pesticide (ocp) levels in soil system as well as for the possible adsorption on various soil components such as organic matter, clay and other minerals. soil organic matter is one of the most important factors affecting pesticide behavior in soils. organochlorine pesticides tend to bind with soil organic matter due to their hydrophobic nature which means that the increase in soil organic matter content may lead to the increase of the pesticide residue levels in soil.29,30 on the other hand, increased pesticide residue levels in soils can prohibit its further microbial degradation31. in addition, soil ph can affect the concentrations of organochlorine pesticides through modification of structure of soil organic matter and reduction of microbial activity at the lower ph values, which leads to the lower pesticide degradation in soil.32 also, it has been well established that soil types, especially clay content, affect the persistence and retention of organochlorine pesticide residues.33,34 correlations have been examined between aldrin, endrin and dieldrin concentrations in soil and various soil parameters. in this study, soil organic matter was found to be positively associated only with aldrin (r = 0.1694, p < 0.05), indicating that adsorption of this compound is enhanced by soil organic matter, while no significant correlation was observed between organic matter and endrin or dieldrin. the results did not show any definite pattern of soil ph. clay content in soil was found to be positively associated with aldrin (r = 0.3178, p < 0.05) and negatively associated with endrin (r = –0.236, p < 0.05). this is in good correlation with findings of baskaran et al.,35 who indicated that the role of clay minerals as pesticide sorbents is of higher importance where organic matter contents are relatively low. spatial distribution of cyclodiene-type organochlorine pesticide residues distribution pattern of aldrin, dieldrin and endrin in soils from different districts in vojvodina was investigated. the mean, minimum, maximum concentrations, and detection frequencies of aldrin, endrin and dieldrin in soils of vojvodina districts bačka, banat and srem, are shown in table iii. the maximum aldrin concentration (4.94 ng/g) was found in soil sample collected from the orchard in west bačka, while maximum dieldrin concentration (24.55 ng/g) was found in the soil sample collected from the agricultural field in north banat. the highest endrin level (85.33 ng/g) was found in a soil sample collected from the agricultural field in west bačka. this extremely high _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ occurrence and distribution of the cyclodiene-type pesticides in soils of vojvodina 713 level of endrin in the soil is probably due to it`s excessive use in the past, since this concentration was detected in soil used for crop production, and endrin was frequently used for insect control in field crops such as grains. table iii. the concentration of aldrin, endrin and dieldrin in soils from different districts of vojvodina province; sd – standard deviation, nd – not detected; n – number of samples component mean ng/g sd minimum ng/g maximum ng/g detection frequencies, % bačka (n = 523) aldrin 0.15 0.43 nd 4.94 16.6 endrin 0.41 3.87 nd 85.33 14.3 dieldrin 0.35 1.08 nd 14.68 22.9 banat (n = 606) aldrin 0.11 0.39 nd 4.29 11.5 endrin 0.07 0.45 nd 7.57 4.8 dieldrin 0.26 1.42 nd 24.55 15.3 srem (n = 240) aldrin 0.08 0.29 nd 2.13 8.3 endrin 0.05 0.29 nd 2.19 3.7 dieldrin 0.43 0.61 nd 3.02 41.2 spatial distribution maps of the aldrin, endrin, and dieldrin concentrations are shown in figs. s-2–s-4 of the supplemmentary material. in comparison with other two districts, the highest mean values of aldrin and endrin were detected in soil samples from bačka, together with the highest detection frequencies of 16.6 and 14.3 % respectively, which is in good correlation with the fact that more than 80 % of soil samples taken in bačka originated from agricultural fields, and that aldrin was frequently used in the past against soil insects. this is also supported with the findings of škrbić15 who detected aldrin in all wheat samples grown in central bačka and central banat in 2004. the highest mean value of dieldrin was found in srem district along with the highest detection frequency of 41 %, while maximum dieldrin concentration was detected in soils of banat district, which is in good correlation with the high concentrations of dieldrin in wheat samples grown in central banat in 2004 thus indicating excessive use of aldrin or dieldrin in the past.15 conclusions the present study examined the occurrence and spatial distribution of cyclodiene-type organochlorine pesticides in soils of vojvodina province, serbia. although the use of aldrin, endrin and dieldrin was prohibited in 1991, residues of those pesticides still exist in the soils of vojvodina in measurable concentrations. dieldrin was the most dominant compound in the soil. however, the concentrations of analysed cyclodiene-type organochlorine pesticides in the most soil samples from vojvodina province were lower than dutch target values for _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 714 zeremski et al. soil quality. soil organic matter and clay content were found to be positively associated with aldrin content while clay content was found to be negatively associated with endrin. other physical and chemical properties of the soils demonstrated no effect on loading the pollution in the soils of vojvodina. supplementary material details about soil sampling and spatial distribution maps of the aldrin, endrin and dieldrin concentrations are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgments: this study was conducted as part of the project no. tr 31072: “status, trends and possibilities to increase the fertility of agricultural land in the vojvodina province”, which is supported by the ministry of education, science and technological development of the republic of serbia. и з в о д присуство и дистрибуција органохлорних пестицида циклодиенског типа у земљишту аутономне покрајине војводине тијана зеремски1, јовица васин1, станко милић1, петар секулић1, штефан хансман1 и војислава бурсић2 1институт за ратарство и повртарство, максима горког 30, нови сад и 2пољопривредни факултет, универзитет у новом саду,трг доситеја обрадовића 8, нови сад алдрин, ендрин и диелдрин су органохлорни пестициди циклодиенског типа са израженом токсичношћу и дуготрајношћу. иако је у многим земљама у последњих неколико декада употреба ових једињења забрањена, она се још увек могу наћи у животној средини, нарочито у пољопривредном земљишту. како би се утврдио садржај остатака органохлорних пестицида циклодиенског типа у земљишту војводине и добили детаљни подаци о њиховој просторној дистрибуцији, прикупљено је 1370 узорака земљишта различите намене на територији целе војводине, а затим је у њима анализирано присуство алдрина, диелдрина и ендрина. остаци ових пестицида су у земљиштима војводине детектовани у мерљивим концентрацијама а најзаступљеније једињење је био диелдрин. међутим, у већини узорака земљишта из војводине концентрације анализираних пестидида циклодиенског типа биле су ниже од холандских граничних вредности за квалитет земљишта. утврђена је позитивна корелација између садржаја органске материје у земљишту концентрације алдрина као и позитивна корелација између алдрина и садржаја глине док је између конентрације ендрина и садржаја глине утврђена негативна корелација. (примљено 12. децембра 2015, ревидирано и прихваћено 18. јануара 2016) references 1. world health organization, environmental health criteria 91 aldrin and dieldrin, world health organization, geneva, 1989 2. p. manirakiza, o. akinbamijo, a. covaci, r. pitonzo, p. schepens, arch. environ. contam. toxicology 44 (2003) 171 3. y. hashimoto, j. pest. sci. 30 (2005) 397 4. m. t. wan, j. kuo, j. pasternak, j. environ. quality 34 (2005) 1186 5. c. gonçalves, m. f. alpendurada, talanta 65 (2005) 1179 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ occurrence and distribution of the cyclodiene-type pesticides in soils of vojvodina 715 6. i. hilber, p. mäder, r. schulin, g. s. wyss, chemosphere 73 (2008) 954 7. e. matsumoto, y. kawanaka, s. j. yun, h. oyaizu, appl. microbiol. biotechnol. 84 (2009) 205 8. y. jiang, x. wang, y. jia, f. wang, m. wu, 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_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 716 zeremski et al. 31. f. wang, y. r. bian, x. jiang, h. j. gao, g. f. yu, j. c. deng, pedosphere 16 (2006) 161 32. k. d. wenzel, m. manz, a. hubert,g. schuurmann, sci. total environ. 286 (2002) 143 33. c. a. edwards, residue rev. 13 (1966) 83 34. g. m. day, b. t. hart, i. d. mckelvie, r. beckett, environ. technol. 18 (1997) 769 35. s. baskaran, n. s. bolan, a. rahman, r. w. tillman, new zeal. j. agr. res. 39 (1996) 297. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {a dft study of the chemical reactivity of cimetidine a, c and d in the gas phase and in h2o, meoh and etoh solvents} j. serb. chem. soc. 82 (1) 25–37 (2017) udc 615.2cimetidine:543.637:519.677: jscs–4944 546.212+541.124–13 original scientific paper 25 a dft study of the chemical reactivity of cimetidine a, c and d in the gas phase and in h2o, meoh and etoh solvents luis humberto mendoza huizar1*, guillermo salgado-morán2, wilson cardona-villada2, alison geraldo pacheco3 and daniel glossman-mitnik4,5 1universidad autónoma del estado de hidalgo, academic area of chemistry, carretera pachuca-tulancingo km. 4.5 mineral de la reforma, hgo, méxico, 2universidad andrés bello. departamento de ciencias químicas, facultad de ciencias exactas, sede concepción, autopista concepción-talcahuano 7100, talcahuano, chile, 3instituto federal de educação ciência e tecnologia do sul de minas gerais, inconfidentes, minas gerais, brasil, 4laboratorio virtual nanocosmos, centro de investigación en materiales avanzados, chihuahua, miguel de cervantes 120, complejo industrial chihuahua, chih 31136, méxico and 5departament de química, universitat de les illes balears, 07122 palma de mallorca, spain (received 12 may, revised 7 july, accepted 16 september 2016) abstract: in the present work, the chemical reactivity of cimetidine a, c and d in different solvents was analyzed through the evaluation of global and local dft reactivity descriptors. in the gas, meoh and etoh phases, cimetidine a, c and d exhibit energy differences of 3–11 kcal** mol-1. however, in the aqueous phase, cimetidine a and d are approximately isoenergetic. the values of the hardness indicate that cimetidine a, c and d are more reactive in the presence of a solvent than in the gas phase. in addition, the results suggested that cimc and cimd are better nucleophiles that cima. the values of the fukui function suggest that the more reactive sites of cima are not modified in the different solvents. in the case of cimc, the more reactive sites to electrophilic and free radical attack are located on the thioether sulfur. for cimd, the number and place of the electrophilic and free radical sites are independent of the solvent. keywords: cimetidine; reactivity; hsab; fukui; dfts. introduction cimetidine, fig. 1, is a potent histamine-h2 receptor antagonist suitable to inhibit stomach acid production because it is able to bind to an h2-receptor located * corresponding author. e-mail: hhuizar@uaeh.edu.mx ** 1 kcal = 4184 j doi: 10.2298/jsc160512077m 26 mendoza huizar et al. fig. 1. general structure of n-cyano-n′-methyl-n″-{2 -[[(5-methyl-1h-imidazol-4-yl)methyl]thio]ethyl}guanidine (cimetidine). on the basolateral membrane of the astric parietal cell, blocking histamine effects.1 this drug is one of the most used proton pump inhibitors for treating peptic ulcers in the world, by which, it is considered the first blockbuster drug.2 cimetidine has four anhydrous polymorphic forms a (cima), b (cimb), c (cimc), and d (cimd) and the hydrated forms m1, m2 and m3.3 the crystal structures of cima, cimc and cimd are reported in the literature, but that of cimb has yet to be identified.2 notably, only cima and cimb have been employed in the manufacture of pharmaceutical tablets and suspensions, respectively.4,5 at the industrial level, cima is the preferred form of cimetidine because it can be obtained in a pure crystallographic state and its physical properties, such as good flux and low adherence to industrial equipment, allow its easy manipulation on a large scale.5,6 contrarily, cimb and cimc exhibit poor flux, while cimd is not compressible and thus, they cannot be employed to fabricate tablets.4 interestingly, cimc has not been employed in pharmaceutical formulations, even though it might be more effective than cima, cimb and cimd at lower dosages.7 in the solid state, polymorphs cima, cimc and cimd are isoenergetic,2 but cimc is readily transformed to cimb upon minor mechanical treatment.4,8 furthermore, it was observed that both cimb and cimc are transformed to cima, whereas cima is transformed to cimd only upon nucleation.4 on the other hand, in aqueous conditions, cima changes completely to cimb in a short time, whereas it is transformed to d in ethanol:water 1:1.4 here, it is important to mention that if crystals of a pharmaceutical compound exhibit polymorphism, then their physical properties, stability, biological activity, and processability will be dependent on their crystalline form.9–13 furthermore, the crystal structure of cimetidine is crucial for understanding its dissolution behavior and therapeutic effectiveness.4 thus, an extensive characterization of this drug is necessary to establish clearly the relevance of the crystal form on the biological activity.3–5,14,15 here, it is interesting to mention that the presence of a solvent may modify the chemical reactivity of cimetidine, allowing the dosages of this drug and its stability to be controlled. therefore, a good understanding of the chemical reactivity of cimetidine in different solvents may become useful to recognize how to increase the efficacy of this drug when it is employed in the fabrication of either suspensions or solutions. in this sense, the accepted theories of quantum chemistry provide adequate techniques to analyze the chemical reactivity of a molecular system. cimetidine has been studied in the gas phase, through theoretical quantum methods in order to analyze its conformation and tautomer stability at chemical reactivity of cimetidine 27 the am1, hf/6-31g**, mp2/6-31g**//hf6-31g**16 and b3lyp/6-31g(d) levels of theory.17 in addition, the intramolecular interactions in cimetidine were analyzed by karpinska et al., but it was not clarified which conformer was studied.16 additionally, the intramolecular interactions between cimetidine and the endothelium were analyzed employing docking studies and the obtained results suggest that cimetidine shows ability to bind to the endothelial e-selectin (e-sel) and its ligand sialyl lewis x (slex).18 olea-roman et al. studied the coordination behavior of cimetidine form a with transition metal ions at the b3lyp/def2-tzvp level of theory, in the gas phase. it was found through electrostatic potential maps that the more reactive zones of cima are in the imidazolic nitrogen and the thioether sulfur.19 additionally, singh et al. found, at the b3lyp/6-31g*(d,p) level, that cimetidine is able to donate electrons through nitrogen and oxygen atoms to an acceptor molecule with empty and low energy orbital.20 however, in spite of the interest dedicated to cimetidine from experimental and theoretical point of views, the chemical reactivity of cimetidine a, c and d at the molecular level in different solvents have not hitherto been analyzed. therefore, in the present work, their reactivity was evaluated through global and local dft reactivity descriptors to obtain a deeper understanding of the chemical behavior of this important anti-ulcer drug in the gas, h2o, meoh and etoh phases. theory the chemical reactivity of cimetidine was analyzed through conceptual reactivity parameters derived from density functional theory (dft).21 the global parameters used in the present work were the electronic chemical potential (μ), the electronegativity (χ), hardness (η)22–24 and the global electrophilicity index (ω):25 ( ) ( )1 2r e i a n ν μ ∂ = = − + ∂  (1) χ μ= − (2) ( ) ( ) ( ) 2 2 r r e i a n nν ν μ η  ∂ ∂ = = = −    ∂  ∂  (3) 2 2 μ ω η = (4) in eqs. (1)–(4), e, n, and v(r) are the energy, number of electrons, and the external potential of the system, respectively. the vertical electronic affinity (a) is calculated as a = e(n)–e(n+1), where e(n) and e(n+1) are the total groundstate energies in the neutral n and singly charged (n+1) configurations. the ion28 mendoza huizar et al. ization potential (i) is calculated as i = e(n–1)–e(n). in addition to the global reactivity parameters, it is possible to define local reactivity descriptors that can be used to study the reactivity on different sites within a molecule.21 this local reactivity can be evaluated through the fukui function (f(r)),26,27 which is defined as:26 ( ) ( ) ( ) ( )r n r f r n rν ρ μ ν   ∂  ∂ = =     ∂ ∂       (5) where ρ(r) is the electronic density. f(r) allows the identification of preferred regions where a chemical species will alter its electronic density when the number of electrons is modified. thus, this function indicates the susceptibility of the electronic density to deformation at a given position upon acceptance or donation of electrons.21–26 moreover, it is important to highlight that sites in chemical species with the largest values of the fukui function (ff) are those with higher reactivity.27 the ff can be evaluated using a finite difference approximation. however, the discontinuity of the electron density with respect to the number of electrons (n) leads to three types of ffs for a system, namely f+(r), f–(r), and f0(r) for nucleophilic, electrophilic, and free radical attacks, respectively:21 ( ) ( ) ( )1n nf r r rρ ρ+ += −   (6) ( ) ( ) ( )1n nf r r rρ ρ− −= −   (7) ( ) ( ) ( )0 1 1 1 2 n n f r r rρ ρ+ − = −    (8) methodology the initial geometries of cimetidine a, c and d were generated from the cif files cimetd03, cimetd04 and cimetd01, respectively, obtained from the cambridge crystallographic data centre.28 these geometries in the gas, water, methanol and ethanol phases were optimized using the dispersion-corrected density functional wb97xd.29,30 this functional was chosen because it has proved to be an excellent method for the study of molecules with intramolecular interactions.29,30 in addition, the def2tzvp basis set was used.31,32 the solvent was modeled through the smd solvation model reported by truhlar et al.33 all the calculations were realized through the gaussian 09 packages,34 and they were visualized with the gausview,35 gabedit36 and arguslab packages.37 results and discussion geometry optimization the optimized structures of cima, cimc and cimd in the gas phase at the wb97xd/def2tzvp level of theory are shown in fig. 2. note that cima (fig. 2a) and cimd (fig. 2c) are folded while cimc (fig. 2b) is extended. the total energy (electronic energy and zero-point vibrational energy correction), calculated for cima, cimc and cimd are –1117.18590, –1117.16810 and –1117.17586 chemical reactivity of cimetidine 29 ha, respectively. from these values, note that cima is slightly more stable than cimc and cimd. the energy differences between cima–cimc, cima–cimd and cimc–cimd are –11.2, –6.3 and 4.9 kcal mol–1, respectively. from these values, it is clear that in the gas phase cima, cimc and cimd are not isoenergetic and the interconversion among the cimetidine forms is more difficult than in the solid state. fig. 2. structures of: a) cima, b) cimc and c) cimd in the gas phase, optimized at the wb97xd/def2tzvp level of theory. a frequency analysis was applied to verify the stability criterion, and the frequency values were all positive in the optimized geometries. a summary of the main bands for cima, cimc and cimd in the gas phase is depicted in fig. s-1 of the supplementary material to this paper. it is possible to identify two bands at approximately 3700 and 3640 cm–1 that correspond to n–h stretching vibrations. here, it is important to mention that the n–h stretching vibrations in heterocyclic compounds are located in the region 3500–3000 cm–1.38 however, it is well 30 mendoza huizar et al. known that dft calculations overestimate the frequency values and therefore, it is necessary to apply a vibrational scaling factor. in the present case, the scaling correction factor was obtained via the reduced scale factor optimization model described by alecu et al. this factor is 0.950 when the wb97xd/def2tzvp level of theory is employed.39 if this correction factor is applied, the frequency values for the n–h stretching vibrations are 3515 and 3458 cm–1, which are in the range of the experimental values. adjacent to these bands, there is a peak at 3470 cm–1 (corrected 3297 cm–1) that was only present in the spectrum of cima, which corresponds to an n–h stretching vibration in the guanidine group. the cyano vibration stretching band was observed at 2300 cm–1 (corrected 2185 cm–1). a band at 1670–1620 cm–1 (corrected 1587–1539 cm–1) is related to n–h bending vibrations. the band at 1500–1350 cm–1 (corrected 1425–1283 cm–1) is associated with c–h bending vibrations, while a typical band for n–h bending vibrations was observed at 500–340 cm–1 (corrected 475–323 cm–1). in order to analyze the influence of the solvent on cima, cimc and cimd, the structures shown in fig. 2 were re-optimized at the wb97xd/def2tzvp level of theory and using the smd solvation model. the electronic energy values including the zero-point vibrational energy correction for cima, cimc and cimd in the different solvents are reported in table s-i of the supplementary material. the extended and folded structures of cima, cimc and cimd remain in h2o, meoh and etoh solvents and their corresponding xyz coordinates are reported as supplementary material (see tables s-ii–s-iv of the supplementary material). note that in all cases, cima exhibits the lowest electronic energy value, which suggests that cima is the most stable form of cimetidine. furthermore, the energy differences among the cimetidine conformers are reported in table i. note that under aqueous conditions, the energy difference between cima and cimd is only 0.8 kcal mol–1. these results suggest a possible interconversion between these conformers. however, in all the other solvents, a possible interconversion is energetically more restricted because the energy difference among the other cimetidine conformers is larger than 2.6 kcal mol–1. table i. energy differences among the cimetidine conformers at the wb97xd/def2tzvp level of theory solvent δecima-cimc / kcal mol-1 δecima-cimd / kcal mol-1 gas –11.2 –6.3 h2o –4.3 –0.8 meoh –5.8 –2.6 etoh –6.0 –2.8 the theoretical ir spectra for cima in the different solvents are reported in fig. s-2 of the supplementary material. note that there is a slight displacement of the bands in comparison to those obtained in the gas phase. in addition, obs chemical reactivity of cimetidine 31 erve that the peak at 3470 cm–1 exists even in the different solvents. the theoretical ir spectra for cimc and cimd in h2o, meoh and etoh solvents are similar to those obtained in the gas phase. additionally, the intramolecular interactions present in the folded structures of cima and cimd were studied. this kind of interactions may be conveniently analyzed and visualized employing the non-covalent interaction index (nci) defined by johnson et al.40 the visualization of these intramolecular interactions in cima and cimd was realized by plotting the isosurfaces of the reduced density gradient s (related to |∇ρ||/ρ4/3), and then the isosurfaces were colored according to the values of the electron density to identify attractive and repulsive interactions. the s-colored isosurfaces for cima, cimc and cimd are shown in fig. 3, in which the red color represents stabilizing interactions, while the white color suggests destabilization. fig. 3. nci isosurfaces ρ = 0.3 for: a) cima, b) cimc and c) cimd in the gas phase. in fig. 3a, note that the hydrogen bonds correspond to the stabilizing interactions, while the hydrogen–hydrogen interactions are destabilizing. furthermore, it is possible to observe that the folded structure in cima is caused by intramolecular interactions among imidazole nitrogen and hydrogen atoms. in the 32 mendoza huizar et al. case of cimc, the structure is extended, which is probably because there is no hydrogen bond in the center of the molecule, see fig. 3b. the intramolecular interactions in cimd are depicted in fig. 3c; note the presence of guanidine nitrogen–hydrogen interactions that are causing its folded structure. similar results were obtained in the solvents h2o, meoh and etoh, see figs. s-3–s-5, respectively, in the supplementary material. global reactivity parameters to analyze the chemical behavior of cima, cimc and cimd, their global and local reactivity descriptors. μ, η, and ω were evaluated through the eqs. (1), (3) and (4) and the obtained values are reported in table ii. note that cimd in the gas and aqueous phases has higher hardness values in comparison to those obtained for cima and cimc. these results suggest greater stability of cimd in the gas and aqueous phases. however, in meoh and etoh, the results suggest cimc to be more stable than cima and cimd. in addition, note that in all cases the hardness values of the cimetidine conformers were higher in the gas phase than in the other solvents, suggesting that cima, cimc and cimd are stabilized in h2o, meoh, and etoh solvents. table ii global reactivity parameters for cima, cimc and cimd obtained at the wb97xd/ /def2tzvp level of theory according to eqs. (1), (3) and (4) medium η / ev μ / ev ω / ev cima gas 9.7 –4.8 1.2 h2o 6.1 –3.1 0.8 meoh 6.2 –3.1 0.8 etoh 6.3 –3.1 0.8 cimc gas 9.8 –4.9 1.2 h2o 6.1 –3.1 0.8 meoh 7.0 –3.5 0.9 etoh 7.0 –3.5 0.9 cimd gas 10.1 –5.0 1.3 h2o 6.6 –3.3 0.8 meoh 6.6 –3.3 0.8 etoh 6.7 –3.3 0.8 moreover, considering that good electrophiles are characterized by high values of μ and ω, then it could be suggested that cima exhibits a greater electrophilic behavior in comparison to those shown by cimc and cimd, and consequently, cimc and cimd are better nucleophiles than cima. here, it is interesting to mention that according to the literature, the degree of nucleophilicity together with steric effects and pka values are important factors for predicting chemical reactivity of cimetidine 33 antiulcer activity.41 thus, the obtained results suggest that either suspensions or solutions based on cimc and cimd may become more nucleophilic than those based on cima, and thus, they might have better antiulcer drug behaviors than cima suspensions. local reactivity parameters the global reactivity trends of a molecular system can be characterized using μ, η, and ω, but it is also important to evaluate local reactivity descriptors, such as ff, to pinpoint the distribution of the reactivity in the molecule and to understand its chemical behavior. the ff was evaluated using eqs. (6)–(8). the results for cima in the gas phase are reported in fig. 4. a) b) c) fig. 4. local reactivities for cima: a) ( )f r+  , b) ( )f r−  and c) ( )0f r at the wb97xd/def2tzvp level of theory in the gas phase, according to eqs. (6)–(8). the isosurfaces in this figure show that nucleophilic attacks are favored on 8c, while electrophilic and free radical attacks would be on 6n. for cimc in the gas phase, see fig. s-6 in the supplementary material, 9n is the most nucleophilic atom, while the results showed 3c to be the most active site for electrophilic and free radical attacks. in the case of cimd in the gas phase, see fig. s-6, 4n is the most reactive site for nucleophilic attacks, while 10c is the most reactive site for electrophilic and free radical attacks. the ff values for cima, cimc and cimd in h2o, meoh and etoh are reported as supplementary material in figs, s-7–s-9, respectively. a summary of the most reactive sites for 34 mendoza huizar et al. cima, cimc and cimd is presented in table iii. thus, according to the obtained results, for cima, the most active sites for nucleophilic, electrophilic and free radical attacks are located on 8c, 10c and 8c, respectively, irrespective of the solvent, i.e., h2o, meoh or etoh. in the case of cimc, this conformer keeps the same chemical reactivity in meoh and etoh, since the most reactive sites for the different kinds of attacks are on 10c, 1s and 1s, respectively. however, in aqueous conditions, 13c, 1s and 1s correspond to the most reactive sites for nucleophilic, electrophilic and free radical attacks, respectively. table iii. summary of the more reactive sites of cima, cimc and cimd in the different solvents, according to eqs. (6)–(8) medium ( )rf + ( )rf − ( )rf 0 cima gas 8c 6n 6n h2o 8c 10c 8c meoh 8c 10c 8c etoh 8c 10c 8c cimc gas 9n 3c 3c h2o 13c 1s 1s meoh 10c 1s 1s etoh 10c 1s 1s cimd gas 4n 10c 10c h2o 15c 10c 10c meoh 15c 10c 10c etoh 10c 10c 10c on the other hand, cimd retains the same reactivity in h2o and meoh phases, and nucleophiles, electrophiles and free radicals might attack on the atoms labeled 15c, 10c and 10c, respectively. however, in etoh, 10c is the most susceptible site for the three kinds of attacks. these last results are coincident with those reported in the literature for cimetidine compounds.41–47 conclusions intramolecular interactions in cima and cimd result in their folded conformations. in the aqueous phase, cimetidine a and d are approximately isoenergetic, meaning interconversion between them is possible. the hardness values indicate that cima, cimc and cimd are more reactive in the presence of a solvent than in the gas phase. in the phases analyzed in the present work, cimc and cimd are better nucleophiles than cima. the values of the fukui function suggest that the most reactive sites of cima are not modified in the different solvents and they are located on 8c, 10c and 8c for nucleophilic, electrophilic and free radical attacks, respectively. in all cases, the most reactive sites for elec chemical reactivity of cimetidine 35 trophilic and free radicals attacks for cimc are located on the thioether sulfur. however, the nucleophilic attacks on cimc are located on 13c, 10c and 10c in h2o, meoh and etoh, respectively. for cimd, the most reactive sites are located on 10c atom for electrophilic and free radical attacks, independent of the solvent. the nucleophilic attacks on cimd are located on 15c, 15c and 10c in h2o, meoh and etoh, respectively. supplementary material additional data are available electronically at the pages of journal website: http:// //www.shd.org.rs/jscs/, or from the corresponding authors on request. acknowledgements. lhmh gratefully acknowledges financial support from conacyt (projects cb2015-257823 and infr-2014-227999) and the universidad autónoma del estado de hidalgo. guanajuato national laboratory (conacyt 123732) is acknowledged for the supercomputing resources. dgm is a researcher at cimav and conacyt and acknowledges both institutions for partial support. gsm and wcv thank the department of chemistry at the universidad andres bello, concepcion, chile. и з в о д dft прoучавање хемијске реактивности циметидина a, c и d у гасном стању и у h2o, meoh и etoh као растварачима luis humberto mendoza huizar1, guillermo salgado-morán2, wilson cardona-villada2, alison geraldo pacheco3 и daniel glossman-mitnik4,5 1universidad autónoma del estado de hidalgo, academic area of chemistry, carretera pachuca-tulancingo km, 4.5 mineral de la reforma, hgo, méxico, 2universidad andrés bello, departamento de ciencias químicas, facultad de ciencias exactas, sede concepción, autopista concepción-talcahuano 7100, talcahuano, chile, 3instituto federal de educação ciência e tecnologia do sul de minas gerais, inconfidentes, minas gerais, brasil, 4laboratorio virtual nanocosmos, centro de investigación en materiales avanzados, chihuahua, miguel de cervantes 120, complejo industrial chihuahua, chih 31136, méxico и 5departament de química, universitat de les illes balears, 07122 palma de mallorca, spain у овом раду је анализирана хемијска реактивност циметидина а, c и d у различитим растварачима путем процене глобалних и локалних dft дескриптора реaктивности. у гасном стању, као и у meoh и etoh фазама, циметидини a, c и d показују разлике у енергији од 3–11 kcal mol-1. међутим, у воденој фази, циметидини а и c су приближно изоенергетски. вредности хемијске тврдоће указују на то да су циметидини а, c и d реактивнији у присуству растварача него у гасној фази. такође, наши резултати сугеришу да су cimc и cimd бољи нуклеофили од cima. вредности фукуијеве функције сугеришу да реактивнија места cima нису измењена у различитим растварачима. у случају cimc, реактивнија места за напад електрофила и слободних радикала су лоцирана на тиоетарском сумпору. за cimd, број и положај места за електрофилне и слободнорадикалске нападе су независни од растварача. 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ganadu, g. lubinu, l. naldini, a. panzanelli, polyhedron 14 (1995) 1517 46. v. nurchi, f. cristiani, g. crisponi, m. l. ganadu, g. lubinu, a. panzanelli, l. naldini, polyhedron 11 (1992) 2723 47. f. t. greenaway, l. m. brown, j. c. dabrowiak, m. r. thompson, v. w. day, j. am. chem. soc. 102 (1980) 7782. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {hindered phenolic aminothiazoles synthesis, $\alpha$-glucosidase, $\alpha$-amylase inhibitory and antioxidant activities} j. serb. chem. soc. 82 (10) 1087–1095 (2017) udc 547.789.1+547.56+542.913:547.918: jscs–5025 66.097.8:615.279–188 original scientific paper 1087 hindered phenolic aminothiazoles – synthesis, α-glucosidase and α-amylase inhibitory and antioxidant activities sandhya vyjayanthy satheesh, akhila vijayan radha, krishnapriya krishnan nair girija, kallikat narayanan rajasekharan and priya rani maheswari* department of chemistry, university of kerala, kariavattom campus, trivandrum, kerala, 695581, india (received 5 october 2016, revised 1 july, accepted 5 july 2017) abstract: base-catalysed heterocyclization of either n-aryl-n'-[imino(nitroamino)methyl]thioureas or n-aryl-n'-cyanothioureas by reaction with 2-bromo1-(2,6-di-t-butyl-4-hydroxyphenyl)ethanone afforded 4-amino-2-(arylamino) -5-(3,5-di-t-butyl-4-hydroxybenzoyl)thiazoles, designed as molecular hybrids of hindered phenolic and 2-aminothiazole moieties. these compounds were screened for their inhibition activity on carbohydrate hydrolyzing enzymes. thus, [4-amino-2-(phenylamino)-5-thiazolyl](3,5-di-t-butyl-4-hydroxyphenyl)methanone exhibited α-glucosidase inhibition activity with an ic50 value of 117 µm while the standard compound acarbose showed an ic50 value of 48.3 µm and {4-amino-2-[(4-methylphenyl)amino]-5-thiazolyl}(3,5-di-t-butyl-4 -hydroxyphenyl)methanone showed good α-amylase inhibition activity with an ic50 value of 283 µm compared to acarbose (ic50 532 µm). the antioxidant activities of the hindered phenolic thiazoles were also investigated and the 2-[(4-methoxyphenyl)amino] derivative showed an antioxidant activity better than that of butylated hydroxyanisole in the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, better than that of either vitamin c or curcumin in the ferric ion-reducing antioxidant potential assay and comparable with that of butylated hydroxyanisole in the β-carotene bleaching assay. keywords: 2,4-diaminothiazolyl; 3,5-di-t-butyl-4-hydroxyphenyl; hindered phenol; enzyme inhibition. introduction diabetes mellitus is a chronic endocrine disease that affects the metabolism of carbohydrates. the goal of diabetes treatment is to maintain a nearly normal level of glycemic control subsequent to food intake so as to maintain the postprandial hyperglycaemia.1 this could be achieved by inhibiting the carbo * corresponding author. e-mail: priyajyothym@gmail.com https://doi.org/10.2298/jsc161005084s ___________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1088 satheesh et al. hydrate hydrolyzing enzymes involved in the breakdown of carbohydrates, such as α-glucosidase and α-amylase. hence, carbohydrate digestive enzyme inhibitors are widely investigated in the identification of lead compounds for the treatment of diabetes.2 in addition, the role oxidative stress and inflammation play in the development of diabetes mellitus has now been recognized and the significance of antioxidants in the control of diabetes mellitus was studied.3–5 hindered phenols in which the phenolic hydroxyl group is juxtaposed with a sterically demanding group, such as a t-butyl group, have found wide application as antioxidants and permissible food preservatives.6 typical examples of hindered phenols used in food preservation are 2-t-butyl-4-methylphenol (butylated hydroxytoluene, bht), 2-t-butyl-4-methoxyphenol (butylated hydroxyanisole, bha) and t-butylhydroquinone (tbhq). a recent report highlighted the importance of free phenolic groups in flavone, isoflavone and chalcone derivatives on their α-glucosidase inhibitory activity.7 the incorporation of a 2,6-di-t-butylphenolic unit to improve the bioactivities of flavonoids by designing hindered phenol–flavonoid hybrids and the antioxidant activity of hydrazones bearing a 2,6-di-t-butylphenolic unit have also been reported recently.8 the 2-aminothiazole moiety is isosteric with a phenolic unit and devoid of the acidity of the latter and hence, it finds much use in drug design.9 in connection with our interest in the anticancer10,11 and neuroprotective12 activities of 2,4-diaminothiazoles, it was noted that only a few reports exist on the antioxidant activity of aminothiazoles.13,14 with this background, it was hypothesized that 2,4-diaminothiazoles bearing a hindered phenol moiety could show promising antioxidant activities. accordingly, the design and synthesis of hitherto unreported 4-amino-2-(arylamino)-5-(3,5-di-t-butyl-4-hydroxybenzoyl)thiazoles as molecular hybrids incorporating di-t-butylphenol and 2,4-diaminothiazole moieties, along with their α -glucosidase and α-amylase inhibitory and antioxidant activities are reported herein. experimental chemistry melting points are uncorrected and were determined by the open capillary method. the thin layer chromatographic analyses were performed using silica gel 60 f254 tlc aluminium sheets purchased from merck, mumbai, india. the elemental analyses were performed on a vario el iii elemental analyzer. the ir spectra were recorded on jasco, bomem mb and shimadzu ftir spectrophotometers. the nmr spectra were recorded on bruker dpx-400 and 500 mhz spectrometers and fab mass spectra were recorded on jeol sx-102 fab mass spectrometer. hrms-esi spectrum was performed at a resolution of 61800 using a thermo scientific exactive mass spectrometer. all chemicals were from sigma–aldrich and merck. the required n-aryl-n'-[imino(nitroamino)methyl]thioureas 1a–e were obtained from nitroguanidine and aryl isothiocyanates 2a–e as reported earlier.15 reported procedures with slight modifications were used to prepare 1-(2,6-di-t-butyl-4-hydroxyphenyl)ethanone from 2,6-di-t -butylphenol16 and its bromination17 to obtain 2-bromo-1-(2,6-di-t-butyl-4-hydroxyphenyl)ethanone 4. ___________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. phenolic aminothiazoles 1089 analytical and spectral data of the synthesized compounds are given in supplementary material to this paper. general procedure for synthesis of 4-amino-2-(arylamino)-5-(3,5-di-t-butyl-4-hydroxybenzoyl)thiazoles 5a–e method a: from n-aryl-n'-[imino(nitroamino)methyl]thioureas 1a–e. to a stirred solution of n-aryl-n'-[imino(nitroamino)methyl]thioureas (1a–e, 1 mmol) in n,n-dimethylformamide (dmf, 3ml) at room temperature, 2-bromo-1-(2,6-di-t-butyl-4-hydroxyphenyl)ethanone (4, 1 mmol) was added and stirred. after 15 min, triethylamine (3 mmol) was added and the mixture was further stirred at room temperature for 75 min. the resulting deep brown reaction mixture was poured slowly with stirring into ice cold water. the brownish yellow precipitate of 4-amino-2-(arylamino)-5-(3,5-di-t-butyl-4-hydroxybenzoyl)thiazoles 5a–e obtained was collected by filtration, washed with water and dried. method b: from n-aryl-n'-cyanothioureas (3a–e) prepared in situ. cyanamide (1 mmol) and powdered potassium hydroxide (1.1 mmol) were stirred in dmf (2 ml) for 5 min at room temperature and to this mixture, aryl isothiocyanate (2a–e, 1 mmol) in dmf was added dropwise with stirring over 5 min. after further stirring at room temperature for 90 min, the mixture containing n-aryl-n'-cyanothioureas (3a–e) was treated with 2-bromo-1-(2,6-di-t-butyl-4 -hydroxyphenyl)ethanone (4, 1 mmol) and the stirring was continued for a further 30 min. triethylamine (1.2 mmol) was then added followed by stirring for 30 min. the so-obtained reddish brown mixture was poured into ice cold water and the crude 4-amino-2-(arylamino)-5 -(3,5-di-t-butyl-4-hydroxybenzoyl)thiazoles 5a–e were collected, washed with water and dried. the crude products obtained by methods a and b were purified either by crystallization from ethanol or by dry column flash chromatography on thin layer chromatography grade silica gel eluted with hexane–ethyl acetate. α-glucosidase inhibition activity the mode of α-glucosidase inhibition was studied according to apostolidis et al.18 briefly, about 50 µl of homogenized sample solutions of varying concentrations (5–250 µm) and 100 µl of 0.1 m phosphate buffer (ph 6.9) containing α-glucosidase solution (1.0 u ml-1) was incubated in 96 well plates at 25 °c for 10 min. after pre-incubation, 50 μl of p-nitrophenyl α-d-glucopyranoside solution (7.5 mg in 5 ml; 5 mm) in 0.1 m phosphate buffer (ph 6.9) was added to each well at timed intervals. before and after incubation at 25 °c for 5 min, the absorbance at 405 nm was measured using an enspire multimode reader (perkin elmer). acarbose was used as the positive control and the results are expressed as percent inhibition, calculated as: (1–asample/acontrol)×100 (1) where a is the absorbance. α-amylase inhibition activity the inhibitory activity of α-amylase enzymes (from aspergillus oryzae) was performed using a reported procedure with a slight modification.18 briefly, different concentrations of the stock solutions of the samples (100–600 µm) were incubated with an α-amylase solution (0.5 mg/ml) in 0.02 m phosphate buffer (ph 6.9 with 0.006 m nacl, 500 μl) at 25 °c for 10 min. after pre-incubation, 500 μl of a 1 % starch solution in 0.02 m sodium phosphate buffer (ph 6.9 with 0.006 m sodium chloride) was added to each tube at timed intervals. the reaction was stopped with 500 µl of 3,5-dinitrosalicylic acid (1 %) as a colour reagent. the tubes were then incubated in a boiling water bath for around 5 min, cooled to room temperature and ___________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1090 satheesh et al. diluted to 10 ml with distilled water. the absorbance was measured at 540 nm using acarbose as the standard. the percentage of inhibition was calculated using the formula (1). antioxidant capacity assays 2,2-diphenyl-1-picrylhydrazyl (dpph) radical scavenging activity. the dpph radical scavenging efficacy of the compounds was evaluated based on a reported procedure.19 briefly, aliquots of the test samples leading to a concentration range of 100–600 µm were mixed with a methanolic solution of dpph (1.5 ml; 25 mg l-1), kept in the dark for 30 min and the absorbance was measured at 517 nm against the control. bha and curcumin served as standards. the percentage radical scavenging activity, calculated as the scavenging effect (se) from formula (1), was plotted against concentration to obtain the concentration values resulting in 50 % inhibition (ic50). ferric ion reducing potential (frap) assay the frap activity was measured according to the method of benzie and strain.20 acetate buffer (300  mm; ph 3.6), 2,4,6-tripyridyl-s -triazine (tptz; 10 mm in 40 mm aq. hydrochloric acid) and fecl3·6h2o (20  mm) were mixed in the ratio of 10:1:1 to obtain the working frap reagent. test samples (500 µm) in methanol (10 ml) were mixed with 3 ml of working frap reagent and absorbance was measured at 593 nm after vortexing. methanolic solutions of feso4·7h2o ranging from 100 to 2000 μm were used for the preparation of the calibration curve of known fe2+ concentration. the parameter equivalent concentration (ec) was defined as the concentration of antioxidant having a ferric–tptz reducing ability equivalent to that of 1 mm feso4·7h2o. bha and curcumin were used as standards. β-carotene bleaching assay. the inhibition of the oxidative bleaching of β-carotene in a β-carotene/linoleic acid emulsion is measured in the β-carotene bleaching assay. it was realised using the method of hidalgo et al.21 by using an emulsion obtained by mixing β-carotene (0.2 mg), linoleic acid (20 mg) and tween 20 (200 mg, 0.180 ml) in chloroform (0.5 ml), evaporating off the chloroform and suspending in distilled water (50 ml). the thus obtained emulsion (4 ml) was treated with the test samples in methanol (180 µl) at a concentration 10-3 m. bha was used as the standard together with a control without sample and the absorbance was measured at 470 nm. antioxidant activity was expressed as the percentage inhibition relative to the control using the equation: aa = 100(drc – drs)/drc where drc is the degradation rate of the control (drc = ln (a/b)/60, where a is initial absorbance of control and b is the final absorbance of control after 60 min) and drs is the degradation rate of the thiazole sample (drs = ln (a/b)/60, where a is the initial absorbance of the sample and b is the final absorbance of sample after 60 min).22 results and discussion chemistry a retro-synthetic analysis indicated that a [4+1] heterocyclization of the type ((c4–n3–c2–s1)+c5), leading to the thiazole ring formation, could be adopted for the synthesis of the hitherto unreported 4-amino-2-(arylamino)-5-(3,5-di-t-butyl-4 -hydroxybenzoyl)thiazoles 5. the four ring atoms [(c4–n3–c2–s1) could be sourced from either n-aryl-n'-[imino(nitroamino)methyl]thioureas (1, method a; scheme 1), obtainable from nitroguanidine and aryl isothiocyanates (2a–e), as reported earlier,15 or from n-aryl-n'-cyanothioureas (3a–e), accessible from ___________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. phenolic aminothiazoles 1091 cyanamide and aryl isothiocyanates (2a–e, method b; scheme 2).23 the remaining c5 carbon required to assemble the thiazole core could arise from 2-bromo-1(2,6-di-t-butyl-4-hydroxyphenyl)ethanone (4). it was found that both the above two methods afforded the target diaminothiazoles in good yields (see supplementary material). ar: nh2o2nhn nh ar-n=c=s dmf; solid koh room temp; 90 min h no2nhn nh nhar s 1a-e 2a-e n o2nhn hn nhar sch n s nhar o2nhn h2n rco r-co-ch2br 4 oh bu-t bu-t r = hh rco 5a-e o ho t-bu bu-t s n nh2 nhar [ h2n-no2] dmf; et3n; 80-90 oc; 3hr 1; 2; 5 a: c6h5; b: 4-meo-c6h4; c: 4-cl-c6h4; d: 4-me-c6h4; e: 4-eto-c6h4 et3n: et3n: scheme 1. synthesis of [4-amino-2-(phenylamino)-5-thiazolyl](3,5-di-t-butyl-4-hydroxyphenyl)methanone – method a. ar: c nn nhar sch n s nhar hn rco r-co-ch2br 4 oh bu-t bu-t r = hh rco 5a-e o ho t-bu bu-t s n nh2 nhar 1; 3; 5 a: c6h5; b: 4-meo-c6h4; c: 4-cl-c6h4; d: 4-me-c6h4; e: 4-eto-c6h4 et3n: nc h n nhar s h2n-cn 3a-e ar-n=c=s dmf; solid koh room temp; 95 min 2a-e dmf; et3n room temp; 60 min scheme 2. synthesis of [4-amino-2-(phenylamino)-5-thiazolyl](3,5-di-t-butyl-4-hydroxyphenyl)methanone – method b. ___________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1092 satheesh et al. α-glucosidase and α-amylase inhibitory activity determination the α-glucosidase enzyme inhibition activity was measured using the enzyme from saccharomyces cerevisiae. the different concentrations of 4amino-2-(arylamino)-5-(3,5-di-t-butyl-4-hydroxybenzoyl)thiazoles (5a–e) showed dose dependent inhibition compared with the standard acarbose. the results showed that [4-amino-2-(phenylamino)-5-thiazolyl](3,5-di-t-butyl-4-hydroxyphenyl)methanone (5a) showed good α-glucosidase enzyme inhibition activity (ic50 = 117.02 µm), compared with the standard compound acarbose that showed an ic50 value of 48.26 µm (table i). the study of inhibition of the αamylase enzyme revealed that all the compounds showed good inhibition activity compared to that of acarbose. as a typical example, {4-amino-2-[(4-methylphenyl)amino]-5-thiazolyl}(3,5-di-t-butyl-4-hydroxyphenyl)methanone (5d) showed an ic50 value of 283.19 µm in comparison with acarbose which showed an ic50 value of 531.91 µm (table i). the results indicated that the phenolic aminothiazole unit could be a potential structural platform for the development of compounds with antidiabetic activity. table i. α-glucosidase and α-amylase enzyme inhibition activity of compounds 5a–e compound ic50 / µm α-glucosidase α-amylase 5a 117 350 5b 146 566 5c 180 293 5d 214 283 5e 239 325 acarbose 48.3 532 antioxidant activity the antioxidant activity of 4-amino-2-(arylamino)-5-(3,5-di-t-butyl-4-hydroxybenzoyl)thiazoles (5a–e) were assessed based on the dpph radical scavenging assay. these were selected as representative examples of aminothiazoles (5) with a 2-(arylamino) substituent bearing electron withdrawing, neutral or electron donating substituent on the phenyl ring. the 2-[(4-methoxyphenyl)amino] derivative (5b, ec50 = 250 µm) and 2-[(4-ethoxyphenyl)amino] derivative (5e, ec50 = 265 µm) showed better radical scavenging activities than the 2-phenylamino derivative (5a, ec50 = 340 µm), 2-[(4-methylphenyl)amino] derivative (5d, ec50 = 365 µm) and the 2-(4-chlorophenylamino) derivative (5c, ec50 590 µm), in comparison with the standards curcumin (ec50 = 200 µm) and bha (ec50 = = 280 µm). the results of the dpph assay showed that {4-amino-2-[(4-methylphenyl)amino]-5-thiazolyl}(3,5-di-t-butyl-4-hydroxyphenyl)methanone (5b) possessed an antioxidant activity that was better than that of bha, but lower than that of curcumin. ___________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. phenolic aminothiazoles 1093 in the case of frap assay, the 2-[(4-methoxyphenyl)amino]thiazole derivative (5b) showed a better activity, with a feso4 equivalence of 1700 µm, compared with those of the 2-[(4-ethoxyphenyl)amino]thiazole (5e, 1500 µm), 2 -(phenylamino)thiazole (5a, 1100 µm), 2-[(4-methylphenyl)amino]thiazole (5d, 900 µm) and the 2-[(4-chlorophenyl)amino)thiazole (5c, 800 µm) derivatives, in comparison with those of vitamin c (1400 µm) and of curcumin (1700 µm). the data of the β-carotene bleaching assay indicated that {4-amino-2-[(4methylphenyl)amino]-5-thiazolyl}(3,5-di-t-butyl-4-hydroxyphenyl)methanone (5b) exhibited 56 % antioxidant activity whereas the 2-[(4-ethoxyphenyl)amino]thiazole (5e), 2-(phenylamino)(5a), 2-[(4-chlorophenyl)amino](5c) and 2-[(4 -methylphenyl)amino]thiazole (5d) derivatives showed 52, 30, 27 and 24 % antioxidant activity, respectively, in comparison with the 58 % activity of bha (table ii). table ii. antioxidant activity studies on compounds 5a–e compound dpph radical scavenging activity, µm frap µm β-carotene bleaching method, % 5a 340 1100 30 5b 250 1700 56 5c 590 800 27 5d 365 900 24 5e 265 1500 52 bha 280 – 58 curcumin 200 1700 – vitamin c – 1400 – it appears that the presence of an electron donating substituent on the 2arylamino group of 4-amino-2-(arylamino)-5-(3,5-di-t-butyl-4-hydroxybenzoyl)thiazoles promotes antioxidant activity. the free radical scavenging activity largely depends on the hydrogen donating ability of phenolic compounds and the phenoxyl radicals thus formed are stabilized by resonance or intramolecular hydrogen bonding.24 the free radical scavenging activity was suggested to be enhanced by the presence of electron donating groups in the aromatic substituents.25 in the present case, the presence of a hindered phenolic group and the aminothiazole unit together could be responsible for the observed antioxidant potential of the studied 4-amino-(2-arylamino)-5-(3,5-di-t-butyl-4-hydroxybenzoyl)thiazoles. conclusions in conclusion, hitherto unreported 4-amino-2-(arylamino)-5-(3,5-di-t-butyl-4 -hydroxybenzoyl)thiazoles (5a–e) were prepared as molecular hybrids of hindered phenol and 2-aminothiazole moieties, leading to new antioxidants. among the compounds (5a–e), [4-amino-2-(phenylamino)-5-thiazolyl](3,5-di-t-butyl-4-hyd___________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1094 satheesh et al. roxyphenyl)methanone (5a) exhibited α-glucosidase inhibition activity (ic50 = = 117.02 µm) and {4-amino-2-[(4-methylphenyl)amino]-5-thiazolyl}(3,5-di-t -butyl-4-hydroxyphenyl)methanone (5d) showed α-amylase inhibition activity (ic50 = 283.19 µm). it was further found that {4-amino-2-[(4-methoxyphenyl)amino]-5-thiazolyl}(3,5-di-t-butyl-4-hydroxyphenyl)methanone (5b) exhibited better antioxidant activity in comparison with the activity of bha or vitamin c. the present results suggest that the antidiabetic potential of these newly synthesized hindered phenolic aminothiazoles, which exhibit a combination of α-glucosidase and α-amylase inhibition activities, as well as antioxidant properties, warrants further investigation. supplementary material analytical and spectral data of the synthesised compounds are available electronically at the pages of the journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. mpr thanks ugc-ds kothari post doctoral fellowship scheme for the postdoctoral fellowship. vra, kgkp and knr thank ugc, govt. of india for junior research and emeritus fellowships. svs thank kscste, govt. of kerala for financial assistance and prof. dr. t. s. anirudhan for mentorship. we also acknowledge sophisticated test and instrumentation centre (stic), cochin university, cochin, kerala and csir-niist, trivandrum, kerala for recording the nmr and mass spectra. и з в о д стерно заклоњени аминотиазоли – синтеза, инхибиција активности α-глукозидазе и α-амилазе и антиоксидативна активност sandhya vyjayanthy satheesh, akhila vijayan radha, krishnapriya krishnan nair girija, kallikat narayanan rajasekharan и priya rani maheswari department of chemistry, university of kerala, kariavattom campus, trivandrum, kerala, 695581, india у реакцији базно катализоване хетероциклизације n-арил-n'-[имино(нитроамино)метил]тиоуреа са 2-бром-1-(2,6-ди-t-бутил-4-хидроксифенил)етаноном као производи су добијени 4-амино-2-(ариламино)-5-(3,5-ди-t-бутил-4-хидроксибензоил)тиазоли, једињења која су дизајнирана као хибридни молекули стерно захтевних фенола и 2-аминотиазола. испитана је инхибиторна активност добијених једињења према хидролитичким ензимима угљених хидрата. утврђено је да [4-амино-2-(фениламино)-5-тиазолил](3,5-ди-t-бутил-4-хидроксифенил)метанон показује инхибицију активности α-глукозидазе са ic50 = 117,02 μm, док акарбоза као стандард показује ic50 од 48,26 μm, а дериват {4-амино-2-[(4-метилфенил)амино]-5-тиазолил}(3,5-ди-t-бутил-4-хидроксифенил)метанон показује добру инхибицију активности α-aмилазе са ic50 = 283,19 μm у поређењу са акарбозoм (ic50 = 531,91 μm). такође, испитана је антиоксидативна активност стерно заклоњених фенолних тиазола, и 2-[(4-метоксифенил)амино] дериват показује бољу антиоксидативну активност од бутилованог хидроксианизола у тесту са 2,2-дифенил-1-пикрилхидразилом као хватачем радикала, затим од витамина це или куркумина у тесту са фери-јонима, и сличну активност као бутиловани хидроксианизол у тесту са β-каротеном. (примљено 5. октобра 2016, ревидирано 1. јула, прихваћено 5. јула 2017) ___________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. phenolic aminothiazoles 1095 references 1. r. chakrabarti, r. rajagopalan, curr. sci. 83 (2002) 1533 2. r. subramanian, a. z. asmawi, a. sadikun, acta biochim. pol. 55 (2008) 391 3. j. l. evans, b. a. maddux, i. d. goldfine, antioxid. redox signaling 7 (2005) 1040 4. m. y. donath, s. e. shoelson, nat. rev. immunol. 11 (2011) 98 5. r. rahimi, s. nikfar, b. larijani, m. abdollahi, biomed. pharmacother. 59 (2005) 365 6. h. babich, environ. res. 29 (1982) 1 7. h. sun, y. li, x. zhang, y. lei, w. ding, x. zhao, h. wang, x. song, q. yao, y. zhang, y. ma, r. wang, t. zhu, p. yu, bioorg. med. chem. lett. 25 (2015) 4567 8. j. lebeau, c. furman, j. l. bernier, p. duriez, e. teissier, n. cotelle, free radical biol. med. 29 (2000) 900 9. a. zhang, w. xiong, j. e. hilbert, e. k. devita, j. m. bidlack, j. l. neumeyer, j. med. chem. 47 (2004) 1886 10. n. e. thomas, r. thamkachy, k. c. sivakumar, k. j. sreedevi, x. l. louis, s. a. thomas, r. kumar, k. n. rajasekharan, l. cassimeris, s. sengupta, mol. cancer ther. 13 (2014) 179 11. m. juneja, u. vanam, s. paranthaman, a. bharathan, v. s. keerthi, j. k. reena, r. rajaram, k. n. rajasekharan, d. karunagaran, eur. j. med. chem. 63 (2013) 474 12. m. mayadevi, d. r. sherin, v. s. keerthi, k. n. rajasekharan, r. v. omkumar, bioorg. med. chem. 20 (2012) 6040 13. o. uchikawa, k. fukatsu, m. suno, t. aono, t. doi, chem. pharm. bull. 44 (1996) 2070 14. k. b. kalpana, m. srinivasan, v. p. menon, mol. cell. biochem. 314 (2008) 95 15. r. binu, k. k. thomas, g. c. jenardanan, k. n. rajasekharan, org. prep. proced. int. 30 (1998) 93 16. n. v. portnykh, a. a. volod’kin, v. v. ershov, izv. akad. nauk sssr, ser. khim. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {immobilization of cell wall invertase in polyacrylamide hydrogel for invert sugar production} j. serb. chem. soc. 81 (12) 1359–1369 (2016) udc 663.6:577–15+66.094.941:664.1: jscs–4933 544.773.432:678.744.32+661.771.53 original scientific paper 1359 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. immobilization of cell wall invertase in a polyacrylamide hydrogel for invert sugar production aleksandra n. margetić1* and zoran m. vujčić2 1institute of chemistry, technology and metallurgy, center of chemistry, university of belgrade, studentski trg 12–16, belgrade, serbia and 2faculty of chemistry, department of biochemistry, university of belgrade, studentski trg 12–16, belgrade, serbia (received 20 july, revised 4 october, accepted 5 october 2016) abstract: cell wall invertase (cwi) from saccharomyces cerevisiae was immobilized in polyacrylamide hydrogels. the aim of the development of a new biocatalyst was to obtain an improved enzyme for invert sugar production. the monomer concentration and enzyme amount in the immobilizate were optimized, and the obtained biocatalyst had an enzyme activity of 138±6 iu g-1. the ph and temperature optima were 4.0 and 70 °c, respectively. the stability of immobilized enzyme was determined at several temperatures in the absence of substrate and the half-life obtained at 50 °c was 81 days, at 60 °c, 128 min and at 70 ° c, 1.24 min. the biocatalyst was tested at low ph values, 3.0 and 3.5 were tested, and showed great stability. the km values were 34.1±1.7 and 126.2±6.3 mm for free and immobilized cwi, respectively. the activation energies were 37.7 and 23.0 kj mol-1 for free and immobilized cwi, respectively. cell wall invertase immobilized in polyacrylamide hydrogel (cwi– –paa) was tested for the production of high concentrated invert sugar in a batch and a packed bed reactor. after five days of continuous process, the quality and characteristics of the produced invert sugar remained unchanged. keywords: immobilization; saccharomyces cerevisiae; enzyme; stability; cwi–paa. introduction the main goal of enzyme immobilization is for the industrial use of enzymes, process control and for as many as possible reaction cycles. simplicity and improvement of enzyme properties are strongly associated with the design of protocols for enzyme immobilization. an immobilized enzyme should be designed to be suitable for large-scale production. there are many protocols for immobilization of enzymes but very few are also very simple and/or very capable * corresponding author. e-mail: aleksandra@chem.bg.ac.rs doi: 10.2298/jsc160720094m 1360 margetić and vujčić available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. of improving enzyme properties. in addition, relatively few have been successfully commercialized on a large scale.1 immobilization of an enzyme in different types of hydrogels is one of the most frequently used and simplest immobilization techniques. unsatisfactory mechanical properties and susceptibility to microbial contamination of the enzymes immobilized in hydrogels are the main disadvantages of this kind of biocatalyst. natural polymers, such as gelatin, carrageenan and alginate, are some of the carriers frequently used for enzyme immobilization with these and some other weaknesses.2 synthetic organic polymers have the greatest variability concerning physical and chemical characteristics. in principle, they can be adapted to the requirements of nearly any enzymatic process. some of polymers can be easily synthesized, and their characteristics controlled by slight changes in the conditions of the polymerization reactions. the main disadvantage of hydrogels as a carrier for enzyme immobilization is unsuitable pore size. it has to be narrow enough to prevent leakage of the enzyme molecules out of the polymer network, but large enough to allow free diffusion of substrates and products. polyacrylamide (paa) is one of the immobilization carriers for entrapment that is strong enough to withstand the chemical stress and mechanical forces in a technical process. moreover, it possesses very suitable properties, such as high elasticity, low abrasion in stirred-tank reactors3,4 and good stability in almost all relevant reaction conditions.5 the great adventage of polyacrylamide is the possibility to modify the structure and pore-size of the obtained polymer by changing the ratio of monomer and cross-linking agent in the reaction mixture. yeast invertase is a glycoprotein and can be found in the internal (non-glycosylated) or external (glycosylated) form. invertase is easily extracted enzyme and can be easily obtained in the soluble form6,7 or in the form of cell wall invertase (cwi).8 this enzyme has found many applications in the food and beverage industry.9 therefore, there are many research papers dealing with different immobilization techniques or immobilization carriers for the immobilization of invertase. the main problem with invertase immobilization by entrapment, i.e., enzyme leakage,10–12 was resolved by using cell wall invertase instead of a soluble form.8 in addition, as invertase is one of the inexpensive enzymes, it is used frequently as a model system for the development of new enzyme immobilization techniques. polyacrylamide was used the first time for invertase immobilization in the early seventies, but the obtained immobilizate had low activity13,14 or lower thermal stability than the free enzyme.15 usage of paa as a carrier for invertase immobilization gave immobilized biocatalysts with better performance in combination with gelatin.12 invertase from s. cerevisiae could also be immobilized on paa by adsorption,16 but the obtained immobilized enzyme was used only in a dilute sucrose solutions. invertase was also immobilized by entrapment in paa immobilized cell wall invertase in polyacrylamide 1361 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. hydrogel by radiopolymerization.17 this method gave good immobilization results, but it is not a useful method for the production of an immobilized biocatalyst on the large scale for industrial application. from an industrial viewpoint, usage of low concentrated sucrose solution for hydrolysis followed by concentration step is not economically relevant. there is an example when whole bacterial cell immobilized in a similar polymer was developed for a commercial biotransformation process.2 experimental chemicals all chemicals were of analytical grade (or higher) and were purchased from sigma– –aldrich and merck. baker’s yeast was obtained from a local market. enzyme isolation cwi was isolated from the slurry obtained after cell autolysis as described in a previously published paper.8 the obtained slurry was diluted with an equal volume of 1 % sodium chloride and mixed for 30 min. the yeast cell wall was centrifuged at 4000 g for 15 min at 4 °c, and the pellet was resuspended in 5 volumes of distilled water. the procedure was repeated until proteins could no longer be detected in the supernatant. the resulting solid material was defatted and dried using cold acetone. the pellet was left to dry overnight at room temperature. immobilization of cwi the polymerization mixtures contained different amounts of cwi (invertase activity 9 iu mg-1; in the range of 0.25–3 g), 2.67 ml of 30 % acrylamide solution (with 0.8 % cross -linker monomer n,n′-methylenebisacrylamide), 4 µl of n,n,n′,n′-tetramethylethylenediamine (temed), 50 µl of 10 % ammonium persulfate (water solution) and water. the total volume was kept to 15 ml by the addition of 1.5 m tris buffer of ph 8.8. after 1 h polymerization at room temperature, the gels obtained between glass plates were washed with 50 mm acetate buffer ph 4.5 and cut into discs (2 mm in diameter and 0.75 mm thick). the obtained immobilized enzymes were stored at 4 °c in 70 mass % invert sugar solution in 50 mm acetate buffer ph 4.5 prior to use. enzyme activity assay the enzyme activity was assayed in a batch reactor at 25 °c. fifty μl of free cwi suspension (1 mg ml-1) was mixed with 450 μl of sucrose solution (0.3 m in 50 mm acetate buffer, ph 4.5). after 5 min, the reaction was stopped by the addition of 3,5-dinitrosalicylic acid (dns) reagent (500 µl) and heated in a boiling water bath for 5 min. for the determination of the activity of immobilized enzymes, 3 discs were taken and mixed with 5 ml of sucrose solution. after 5 min, 500 µl of the reaction mixture was mixed with 500 µl of dns reagent and heated in a boiling water bath for 5 min. after cooling to room temperature and dilution with 4 ml of water, the amount of reducing sugars was determined spectrophotometrically at 540 nm18 using a shimadzu uv-1800 spectrophotometer. one international unit (iu) of enzyme activity is defined as the amount of enzyme that hydrolyses one micromole of sucrose per minute under the assay conditions. 1362 margetić and vujčić available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. determination of optimum ph and ph stability the effect of ph on the activity of free and immobilized cwi was studied at various ph values at 25 °c. the ph of the reaction mixtures were in the range of 3.0−8.0 (0.05 m acetate buffer in the range of 3.0–5.6 and 0.05 m phosphate buffer in the range of 5.7–8.0 were used). the reaction conditions were as described in the previous section. the effect of different ph values during the 7 days incubation was studied at 25 °c at ph values 2.0, 3.0 and 4.5. in order to determine the ph stability, both free and immobilized cwi were incubated in 50 mm acetate buffer of various ph values. the enzyme activities were measured after various incubation times as described above (at ph 4.5). determination of the optimum temperature and thermal stability the effect of temperature on the activity of the enzymes was studied by performing the reaction in the temperature range 20−75 °c (with 5 °c increments). the reaction conditions were as described in the previous section. free and immobilized cwi were incubated at selected temperatures (50, 60 and 70 °c) in 50 mm acetate buffer ph 4.5. enzymes were incubated for various time intervals (for 57 d at 50 °c, for 4 h at 60 °c and for 20 min at 70 °c) during these times, the enzyme activity was monitored as described in the previous section. the inactivation rate constants, kd were calculated from the equation: ln(a/a0) = –kdt (1) where a0 is the initial activity and a is the activity after time t. the half-life values of the biocatalysts were calculated for all temperatures used for the thermal stability tests from the equation: t1/2 = ln 2/kd (2) determination of kinetic parameters the activation energy of free cwi and the immobilized cwi was calculated using the arrhenius equation after measuring enzyme activities at different temperature.19 the michaelis–menten kinetics was established by studying the effect of substrate concentration (1–200 mm for free cwi and 1–800 mm for immobilized cwi) on the reaction rate at 25 °c. parameters of michaelis–menten kinetics were obtained using non-linear regression (graph pad prism 5.0). production of invert sugar in a batch process production of invert sugar using cwi immobilized in a polyacrylamide hydrogel was tested in a batch reactor. five grams of immobilized cwi was incubated in 50 ml of 60 mass % sucrose solution at 50 °c with continuous stirring (150 rpm). the immobilized cwi used in this experiment was pre-equilibrated in 60 % sucrose solution. the packed bed reactor immobilized cwi in a polyacrylamide hydrogel was tested in packed bed reactor. the experiments were realized in a 5 ml water-jacketed glass column (6.5 cm long and 1 cm in diameter) at 50±1 °c. sucrose solutions (60 mass %) were used as the substrates. substrate solutions were brought to 50 °c before entering the column and pumped through the bed by means of a peristaltic pump. after steady state was attained, the ratio of conversion was evaluated at the end of the column by determining the reducing sugars, as described in the previous section. the concentration of the sucrose solution was checked using a sinotech model 2waj refractometer. immobilized cell wall invertase in polyacrylamide 1363 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. statistical analysis each data point represents the mean of three independent assays. statistical significance was determined by standard deviation (sd). the data are presented as the mean ± standard error of the mean (sem). results and discussion the usage of cwi instead of soluble invertase in a few natural hydrogels showed great enzyme characteristics, good stability and high productivity of invert sugar.8,20 polyacrylamide was chosen for the development of a new immobilized biocatalyst because it is hydrophilic and extensive previous experience established it as non-denaturing toward many proteins.21 the polymerization reaction could cause damage to the structure of the soluble enzyme, but as the stability of cwi in comparison to soluble enzyme is much higher, no damage was expected nor observed. immobilization cwi isolated from yeast cells has an enzyme activity of 9±1 iu mg–1. optimization of the immobilization of cwi in a polyacrylamide gel included optimization of amount of free cwi in the immobilizate and the percentage of monomer, acrylamide, in the polymerization mixture. changing of amount of monomer added in the reaction mixtures resulted in significant difference in the mechanical properties of the obtained immobilized biocatalysts. cwi was immobilized by entrapment in paa containing 8, 10 and 12 % of monomer. the biocatalyst with the best mechanical characteristics was composed of 8 % of monomer (data not shown). immobilized cwi in paa with the higher concentration of monomer was fragile, and the same characteristic was described earlier.22 the amount of cwi was in the range of 17–200 mg ml–1 of the immobilization mixture. the optimal concentration of enzyme for immobilization in paa by entrapment was chosen based on the results of invertase activity obtained for all tested biocatalysts, shown in fig. 1. fig. 1. optimization of the amount of cwi for immobilization in the polyacrylamide hydrogel. the cwi concentration is indicated in g cwi ml-1 of immobilization mixture; enzyme activity is expressed in iu g-1. 1364 margetić and vujčić available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. the immobilized cwi containing 133 mg ml–1 of free enzyme and 8 % monomer, with an activity of 138±6 iu g–1, was selected as optimal. the first immobilized cwi was entrapped in ca-alginate hydrogel and it had an activity of 71 iu g–1;8 a better activity of 91 iu g–1 was registered for cwi modified with glutaraldehyde immobilized in alginate,23 while the hitherto best published activity of 93 iu g–1 was evidenced for cwi immobilized in gelatin.20 the biocatalyst obtained in this work has 48 % higher activity than the best previously published. the immobilization of cwi within paa hydrogel was complete, without loss of enzyme in all tested biocatalysts. however, immobilization of soluble invertase in paa hydrogel without additional treatment resulted in leakage of the enzyme from the immobilizate particles.24 ph optimum and stability the changes in enzyme activity with changing ph values of the reaction mixtures of free and immobilized cwi were studied at 25 °c. based on the results presented in fig. 2, it could be concluded that optimal ph values for both free and immobilized cwi were in the range 3.0–5.0. however, maximum activity of free cwi was shown in the ph range 4.5–5.0 while immobilized cwi had a maximum activity at ph 4.0. fig. 2. effect of ph on the activity of free and immobilized cwi. similar ph optimums of free and immobilized soluble invertase from saccharomyces cerevisiae in a paa hydrogel14 and other carriers10,12,25 and also of free and immobilized cwi8,20 were published previously. high activity of immobilized cwi was observed also at lower ph value, 97 and 90 % of the highest activity at 3.5 and 3.0, respectively. the ability to hydrolyze sucrose at low ph could be a significant advantage of this biocatalyst, because microbial contamination is prevented under such conditions.26 invertase would not be used at ph values lower than 3.0, since under these conditions, a rapid acid hydrolysis of sucrose occurs.27 the slightly shifted ph optimum of immobilized cwi into the acid region could be the effect of secondary interactions, such as ionic interact immobilized cell wall invertase in polyacrylamide 1365 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. ions between the enzyme and the polymeric matrix or due to decreased hydrogen ion concentration in the microenvironment of the enzyme, which would require a more acid assay medium for the maximum activity of the enzyme.12 since both free and immobilized cwi showed high activity in the ph range of 3.0 to optimal, the ph stability was examined in terms of potential industrial application of this immobilized biocatalyst for invert sugar production at low ph values. the ph stabilities of both forms of the enzyme were tested at two ph values. the biocatalysts were incubated at ph 2.0, 3.0 and 4.5 at 25 °c for one week. no changes were observed in the enzyme activity (both free and immobilized enzymes) after 7 days incubation at ph 3 and 4.5, while at ph 2.0, both biocatalysts showed low stability. after 20 h at ph 2.0, only 5 % residual activity of the free cwi was detected and 11 % residual activity of the immobilized enzyme. optimum temperature the effects of temperature on enzyme activity of immobilized in paa and free cwi were obtained in the temperature range 20–75 °c and the results are shown in fig. 3. the results are expressed as percentage of the maximum activity. optimal temperatures for free and immobilized cwi were found to be 60 and 70 °c, respectively. the activation energies were calculated by the arrhenius method and found to be 37.7 and 23.0 kj mol–1 for free and immobilized cwi, respectively. a slightly better stability of the immobilized compared to that of the free enzyme could be used as an explanation of the obtained results. similar results, i.e., higher temperature optimum of immobilized than free enzyme were previously reported8,17,28,29 and the same result, shifted temperature optimum of invertase immobilized in the paa hydrogel to about 10 °c higher and a change in the temperature dependence curves was observed.14 activation energy is an important parameter as it may indicate diffusion limitation.30,31 fig. 3. effect of temperature on the activity of free and immobilized cwi. 1366 margetić and vujčić available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. thermal stability is one of the most important parameters of the improved biocatalysts, especially for their industrial application or application at higher temperatures. a study of thermal stability of cwi free and immobilized in paa was performed at 50, 60 and 70 °c. both biocatalysts were incubated at these temperatures and their activities were measured in determined intervals, during 57 d at 50 °c, 4 h at 60 °c or 20 min at 70 °c. the kinetic parameters of the thermal denaturation data of free and immobilized cwi were calculated and are given in table i. significant decreases in the stability of the free and immobilized cwi were observed, which is similar with the results observed for soluble invertase above 55 °c.14,32 the thermal stability of immobilized cwi was 65 % higher at 50 °c, 47 % at 60 °c and 42 % at 70 °c. the improvement of the thermal stabilities of immobilized soluble as well as cwi invertase are well known characteristics.8,9,20,23 table i. half-life of cwi and soluble invertase at different temperatures cwi t1/2 at 50 °c, days t1/2 at 60 °c, min t1/2 at 70 °c, min free 49 87 0.87 immobilized 81 128 1.24 determination of kinetic parameters the kinetic parameters michaelis constant (km) and maximum reaction rate (vmax) for free and immobilized cwi were determined by varying the concentration of sucrose in the reaction mixture. as expected, increasing values of km for the immobilized enzyme were observed. the obtained values for km were 34.1±1.7 and 126.2±6.3 mm for free and immobilized cwi, respectively, the values for vmax were 11.6±1.1 and 96.4±8.7 mm min–1, respectively. the increase in the km values for immobilized cwi could be the result of the limited accessibility of sucrose molecules to the active sites of the immobilized enzyme. similar results were published previously in many papers.8–10,12,20,33 production of invert sugar in a batch process and a packed bed reactor the obtained immobilized cwi was used in a batch reactor for hydrolysis of a high concentrated sucrose solution. the percent of invert sucrose was measured using dns reagent at various time intervals and the obtained results are presented in fig. 4. almost complete sucrose conversion (96.5 %) was achieved after 29 h. in a previous paper, cwi immobilized in alginate hydrogel was used in a similar experiment, but complete conversion was only achieved in almost double the time.8 a packed bed reactor is usually very useful for industrial application of an immobilized biocatalyst, especially if product inhibition occurs. immobilized cwi–paa was tested for invert sugar production in a packed bed reactor. for immobilized cell wall invertase in polyacrylamide 1367 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. this purpose, 3 g (3 ml) of immobilized cwi were used. during 5 days, the percent of sucrose inversion at a constant flow rate of 2 bed volume per hour (bvh) was measured. the invert sugar production was constant during these days and the percentage of sugar inversion was about 30 %. fig. 4. sucrose hydrolysis with immobilized cwi in a stirred batch reactor. conclusions in this study, cwi was successfully immobilized in a polyacrylamide hydrogel cross-linked with n,n′-methylenebisacrylamide. the obtained biocatalyst had a high invertase activity, 48 % higher than the previously published immobilized cwi in a gelatin hydrogel.20 since the aim of this study was to obtain immobilized enzyme with high microbial resistance, the high stability at low ph values is proof that the goal is attained, because the possibility of microbe contamination of sugar at low ph values and high sugar molarity is considerably less than at the ph value usually used for enzymatic invert sugar production. the significantly better thermal stability of immobilized than free enzyme shown for cwi–paa qualifies it for potential industrial use. this presumption was proved by testing the obtained biocatalyst in a packed bed reactor when it showed good characteristics under conditions similar to industrial ones. acknowledgement. this work was supported by the ministry of education, science and technological development of the republic of serbia (project grant no. 172048). и з в о д производња инвертног шећера зидном инвертазом имобилизованом у полиакриламидном хидрогелу александра н. маргетић1 и зоран м. вујчић2 1институт за хемију, технологију и металургију, центар за хемију, универзитет у београду, студентски трг 12–16, београд и 2хемијски факултет, катедра за биохемију, универзитет у београду, студнетски трг 12–16, београд зидна инвертаза изолована из ћелија квасца saccharomyces cerevisiae имобилизована је у хидрогелу полиакриламида. циљ добијања овог новог имобилизата је развитак и побољшање биокатализатора који се користи за производњу инвертог шећера. оптимизо1368 margetić and vujčić available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. вана је концентрација мономера и ензима у имобилизату, а као оптималан изабран је имобилизат са ензимском активношћу од 138±6 iu g-1. одређени су оптималани услови за рад имобилизата, ph 4,0 и температура 70 °c. испитивана је стабилност имобилизата на неколико различитих температура без присуства супстрата, добијени резултати изражени су као полуживот имобилизованог ензима и износе 81 дан на 50 °c, 128 min на 60 °c и 1,24 min на 70 °c. тестирана је и стабилност имобилизата и слободног ензима на ниским ph вредностима (3,0 и 3,5), а добијени резултати су показали бољу стабилност имобилизата. km вредност за слободну и имобилизовану зидну инвертазу износе 34,1±1,7 односно 126,2±6,3 mm, док енергија активације износи 37,7 односно 23,0 kj mol-1. продуктивност имобилизоване зидне инвертазе у полиакриламиду у производњи инвертног шећера тестирана је у високо концентрованим растворима шећера у шаржном и континуалном реактору. након пет дана континуалне производње инвертног шећера квалитет и карактеристике добијеног производа нису биле промењене. (примљено 20. јула, ревидирано 4. октобра, прихваћено 5. октобра 2016) references 1. r. dicosimo, j. mcauliffe, a. j. poulose, g. bohlmann, chem. soc. rev.42 (2013) 6437 2. s. cantone, v. ferrario, l. corici, c. ebert, d. fattor, p. spizzo, l. gardossi, chem. soc. rev. 42 (2013) 6262 3. h. nilsson, r. mosbach, k. mosbach, biochim. biophys. acta 268 (1972) 253 4. a. c. johansson, k. mosbach, biochim. biophys. acta 370 (1974) 339 5. c. pizarro, m. a. fernandez-torroba, c. benito, j. m. gonzalez-saiz, biotechnol. bioeng. 53 (1997) 497 6. m. carlson, r. taussig, s. kustu, d. botstein, mol. cell. biol. 3 (1983) 439 7. l. rodriguez, t. ruiz, j. r. villanueva, r. sentandreu, curr. microbiol. 1 (1978) 41 8. a. milovanović, n. božić, z. vujčić, food chem. 104 (2007) 81 9. s. m. kotwal, v. shankar, biotechnol. adv. 27 (2009) 311 10. a. tanriseven, s. dogan, process biochem. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {facile and rapid synthesis of divers xanthene derivatives using lanthanum(iii) chloride/chloroacetic acid as an efficient and reusable catalytic system under solvent-free conditions} j. serb. chem. soc. 82 (5) 483–493 (2017) udc 546.654’131+547.451.62–32+ jscs–4982 547.815:542.913 original scientific paper 483 facile and rapid synthesis of diverse xanthene derivatives using lanthanum(iii) chloride/chloroacetic acid as an efficient and reusable catalytic system under solvent-free conditions behjat pouramiri, maryam shirvani and esmat tavakolinejad kermani* department of chemistry, shahid bahonar university of kerman, kerman 76169, iran (received 3 august 2016, revised 5 march, accepted 10 march 2017) abstract: lacl3/clch2cooh was used as an efficient, and recyclable catalytic system for synthesis of 11h-benzo[a]xanthene-11-one, hexahydro-1h-xanthene-1,8(2h)-dione and 11-aryl-10h-diindeno[1,2-b:2′,1′-e]pyran-10,12(11h) -dione derivatives via a one-pot three-component reaction of aldehydes, 2-naphthol, and cyclic 1,3-dicarbonyl compounds. the reactions proceeded rapidly at 70 °c under solvent-free conditions and the desired products were obtained in good to excellent yields. keywords: multi-component reactions (mcrs); 11h-benzo[a]xanthene; hexahydro-1h-xanthene, 1,3-dicarbonyl compounds; solvent-free conditions; green chemistry. introduction in the past few decades, heterocyclic chemistry has become one of the most important disciplines in organic synthesis and pharmaceutical chemistry.1 a large numbers of the synthesized compounds have emerged as active pharmaceutical components in several drugs due to their potential anti-inflammatory,2,3 anti-tumor,4 anti-hyperlipidemic,5 anti-hypertensive,6 anti-hiv infections7 and several other biological properties.8,9 xanthenes are an important class of heterocyclic compounds with remarkable biological and medicinal properties, such as antiviral, antibacterial and anti -inflammatory activity.10–13 furthermore, they are used as leuco-dyes, ph sensitive fluorescent materials, and in laser technologies.14–16 among this class of molecules, xanthone is a prominent structural motif found in numerous natural products and synthetic compounds with important biological activities.17–21 multi-component reactions (mcrs) have emerged as efficient and powerful tools in organic and medicinal chemistry due to their ability to synthesize organic * corresponding author. e-mail: etavakoly@yahoo.com https://doi.org/10.2298/jsc160803034p _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 484 pouramiri, shirvani and tavakolinejad kermani molecules with higher efficiency and atom economy in a single step from three or more reactants. moreover, mcrs offer the advantage of simplicity and synthetic efficiency over conventional chemical reactions.22 therefore, developing new mcrs and improving known mcrs for the synthesis of diverse groups of compounds, especially ones that are biologically active, have gained great attention in current organic synthesis.23–25 in addition, solvent-free conditions make synthesis simpler, save energy, and prevent solvent waste, hazards, and toxicity.26–28 it therefore remains a challenge to develop multi-component reactions with suitable heterogeneous catalysts. an interesting example of mcrs is the synthesis of xanthene derivatives that can be realized by condensation of aldehydes with β-naphthol, cyclic 1,3-dicarbonyl compounds and/or a mixture of aldehyde and cyclic 1,3-dicarbonyl compounds. various catalysts, such as kal(so4)2·12h2o,29 nano-sncl4·sio2,30 nano-zno,31 iodine,32 silica sulfuric acid33 and [et3nso3h]cl34 have been used for the preparation of 14-aryl-14h-dibenzo[a,j]xanthenes. sbcl3/sio2,35 sio2– –rso3h,36 p-dodecylbenzenesulfonic acid,37 triethylbenzylammonium chloride38 and diammonium hydrogen phosphate39 have been used for the synthesis of 3,4,5,6,7,9-hexahydro-1h-xanthene-1,8(2h)-diones octahydroxanthenes. sulfamic acid,40 dodecatungstophosphoric acid,41 incl3/p2o542 and poly(2-acrylamido-2-methylpropane sulphonic acid) crosslinked with n,n′-methylenebis(acrylamide) (poly(amps-co-aa))43 have been used for the preparation of tetrahydrobenzo[a]xanthen-11-ones. however, some of these methods involved long reaction times, harsh reaction conditions, or unsatisfactory yields. therefore, improvements of these synthesis methods have been continuously sought. in continuation of ongoing research on green catalytic systems and their applications in organic synthesis and their applications as catalysts in organic synthesis,44–46 it was decided to investigate lanthanum(iii) chloride/chloroacetic acid (lacl3/clch2cooh) as a highly efficient, and reusable catalytic system for the practical, one-pot, three-component synthesis of 11h-benzo[a]xanthen-11 -ones, hexahydro-1h-xanthene-1,8(2h)-diones and 11-aryl-10h-diindeno[1,2 -b:2′,1′-e]pyran-10,12(11h)-diones under solvent-free conditions (scheme 1). experimental chemicals and apparatus chemicals were either laboratory-prepared or purchased from merck or fluka companies, and were used without any further purification. the ir spectra were recorded in kbr using a bruker ft-ir spectrophotometer. the 1h and 13c-nmr were recorded in cdcl3 and dmso-d6 on a bruker drx-500 spectrometer using tetramethylsilane as an internal reference. the melting points were determined with a hot-plate microscope apparatus. the purity determination of the substrates and reaction monitoring were accomplished by tlc (petroleum ether (b.p.: 35‒60 °c)–ethyl acetate (b.p.: 77.1 °c), 3:1) on silica-gel polygram sil g/uv254 plates (merck). _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ synthesis of xanthene derivatives under solvent-free conditions 485 scheme 1. one-pot synthesis of xanthene derivatives using lacl3/clch2cooh as an efficient and reusable catalytic system. general procedure for the synthesis of 12-aryl-8,9,10,12-tetrahydro11h-benzo[a]xanthen-11 -ones (5) a mixture of aldehyde (1, 1 mmol), β-naphthol (3, 1 mmol), dimedone or 1,3-cyclohexanedione (2, 1.2 mmol) and lacl3/clch2cooh (10 mol %) was stirred in an oil-bath at 70 °c for a reasonable time. after completion of the reaction, as monitored by tlc, the reaction mixture was cooled, diluted with et2o (5 ml) and filtered to remove catalyst. the crude product was recrystallized from ethanol to afford the pure product. general procedure for the synthesis of compounds 6 and 7 to a mixture of aromatic aldehyde (1, 1 mmol) and a cyclic 1,3-dicarbonyl compounds (2 or 4, 2 mmol), lacl3/clch2cooh (10 mol %) was added and the mixture was heated on an oil bath at 70 °c for a reasonable time. after completion of the reaction, hot ethanol was added to the mixture and then filtered. the residue was recrystallized from ethanol. the spectral data of the synthesized compounds 5 and 6 are presented in the supplementary material to this paper. results and discussion initially, to achieve suitable reaction conditions, the one-pot, three-component reaction of 2-naphthol, benzaldehyde, dimedone and lacl3/clch2cooh as a model reaction was investigated. subsequently efforts were focused on the optimization of various reaction parameters, such as amount of lacl3/ /clch2cooh and temperature, in terms of yield and time. this observation revealed that the use of 10 mol % lacl3/clch2cooh at 70 °c under solvent-free _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 486 pouramiri, shirvani and tavakolinejad kermani conditions produced 9,9-dimethyl-12-phenyl-8,9,10,12-tetrahydro-11h-benzo[a]xanthen-11-one (5a), after 10 min, in 96 % yield. notably, the desired product could not be obtained under similar reaction conditions, even after a long time (1 h) in the absence of the catalyst (table i). table i. screening of the reaction conditions for the synthesis of 9,9-dimethyl-12-phenyl8,9,10,12-tetrahydro-11h-benzo[a]xanthen-11-one (5a) yielda, % time, min temperature, °c amount lacl3/clch2cooh, mol%entry 0 60 70 no catalyst 1 20 35 50 lacl3 (10 mol %) 2 25 30 60 lacl3 (15 mol %) 3 35 50 70 clch2cooh (10 mol %) 4 40 55 80 clch2cooh (20 mol %) 5 70 25 70 lacl3/clch2cooh (5 mol %) 6 85 20 60 lacl3/clch2cooh (10 mol %) 7 96 10 70 lacl3/clch2cooh (10 mol %) 8 96 10 80 lacl3/clch2cooh (10 mol %) 9 aisolated yields in order to study the generality of the procedure, three series of various xanthene derivatives having different steric and electronic properties were synthesized using the optimized conditions. in all cases, the corresponding products were obtained in good to excellent yields. the results are presented in tables ii and iii. the obtained products were characterized by ir, 1h-nmr and 13c-nmr spectroscopy and physical data and the results were compared with the corresponding values for known xanthenes. table ii. one-pot preparation 12-aryl-8,9,10,12-tetrahydrobenzo[a]xanthen-11-ones m.p. / °c yield, % time, min product r x entry reported found 151–15342 152–150 96 10 ch3 h 5a _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ synthesis of xanthene derivatives under solvent-free conditions 487 table ii. continued m.p. / °c yield, % time, min product r x entry reported found 209–21142 211–209 90 11 ch3 4-oh 5b 189–19047 191– 189 92 10 h h 5c 235–23647 238–233 97 10 h 3-no25d 234–23547 234–236 92 10 h 4-no25e 269–27047 267–270 93 10 h 4-oh 5f aisolated yields in order to show the merit of the present work, the present results were compared with those reported by other groups in the synthesis of 9,9-dimethyl12-phenyl-8,9,10,12-tetrahydro-11h-benzo[a]xanthen-11-one (5a). it is important to note that lacl3/clch2cooh acts as an effective catalytic system with respect to reaction time and yield (table iv). in a plausible mechanism, at first, the carbonyl group of the aromatic aldehyde is activated through coordination with acidic lacl3/clch2cooh to give 8. _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 488 pouramiri, shirvani and tavakolinejad kermani next, the carbonyl carbon is attacked by the nucleophilic 1,3-dione derivative to form knoevenagel products. the subsequent addition of these fragments to 2–4 gives the acyclic adduct intermediate, which undergoes intramolecular cyclization with participation of two hydroxyl groups to afford the xanthene derivatives (scheme 2). table iii. one-pot preparation of hexahydro-1h-xanthene-1,8(2h)-dione and diindeno[1,2 -b:2′,1′-e]pyran-diones m.p. / °c yield, % time, min product r1 x entry reported found 201–20348 204–202 86 10 h h 6a 231–23348 232–230 92 10 h 4-cl 6b 245–24748 246–243 97 10 h 4-oh 6c _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ synthesis of xanthene derivatives under solvent-free conditions 489 table iii. continued m.p. / °c yield, % time, min product r1 x entry reported found 216–21848 217–219 92 10 h 4-ch36d 246–24843 245–248 93 8 h 4-no26e 277–27943 236–240 96 7 h 3-no26f 203–20449 202–204 90 8 ch3 h 6g 230–23249 230–233 93 9 ch3 4-cl 6h 290–29150 288–290 80 10 – h 7a _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 490 pouramiri, shirvani and tavakolinejad kermani table iii. continued m.p. / °c yield, % time, min product r1 x entry reported found – 310–312 85 9 – 4-cl 7b aisolated yields table iv. comparison of the results for the synthesis of xanthene 5a with different catalysts: dbsa, p-dodecylbenzenesulfonic acid; tbahs, tetrabutylammonium hydrogen sulfate; experimental conditions: benzaldehyde (1 mmol), 2-naphthol (1 mmol), dimedone (1 mmol) and lacl3/clch2cooh (10 mol %) catalyst catalyst load mol % solvent temp., °c time, min yielda % dbsa 10 h2o, ultrasound 30 60 8951 tmscl 100 mecn reflux 420 9552 hclo4–sio2 10 solvent-free 140 180 3253 sbcl3–sio2 10 solvent-free 120 50 9335 ppa–sio2 10 solvent-free 140 30 9353 tbahs 10 dioxane, h2o reflux 210 8854 lacl3/clch2cooh 10 solvent-free 70 10 96 aisolated yields scheme 2. the proposed mechanism for the synthesis of xanthene derivatives using lacl3/clch2cooh. _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ synthesis of xanthene derivatives under solvent-free conditions 491 finally, the possibility of recycling of lacl3/clch2cooh was investigated using the model reaction forming 5a in the presence of lacl3/clch2cooh. after completion of the reaction, ice-cold water was added to the reaction mixture and the product was filtered. after completely washing the solid product with water, the aqueous layer containing the catalytic system (lacl3 and clch2cooh are soluble in water) was evaporated under reduced pressure and catalyst was recovered and reused for subsequent reactions. the recovered catalyst showed the same activity as that of the fresh catalyst without any loss of activity in terms of yield and product purity. the catalyst was recycled and reused in the same reaction at least four times with remarkable retention of its activity (table v). table v. recycling yields (refer to pure isolated yields); reaction conditions: benzaldehyde (1 mmol), 2-naphthol (1 mmol), dimedone (1 mmol) and lacl3/clch2cooh (10 mol %) no. of cycles fresh run 1 run 2 run 3 run 4 yield, % 96 96 96 96 96 time, min 10 10 10 10 10 conclusions in conclusion, an efficient catalytic system was developed for the synthesis of various xanthene derivatives via one-pot three-component reactions of aldehydes, 2-naphthol, and cyclic 1,3-dicarbonyl compounds. the advantages of the presented procedure include simplicity of operation, high yields of products, short reaction time and solvent-free conditions. supplementary material analytical and spectral data of the synthesized compounds are available at the pages of the journal’s website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. we gratefully acknowledge the funding support received for this project from the research council of shahid bahonar university of kerman. и з в о д брза синтеза ксантенских деривата употребом каталитичког система лантан(iii)-хлорид/хлорсирћетна киселина у одсуству растварача behjat pouramiri, maryam shirvani и esmat tavakolinejad kermani department of chemistry, shahid bahonar university of kerman, kerman 76169, iran lacl3/clch2cooh је коришћен као ефикасан каталитички систем, који се успешно може поновно користити више пута после изоловања, за синтезу деривата 11h-бензо[a]ксантен-11-она, хексахидро-1h-ксантен1,8(2h)-диона и 11-арил-10h-дииндено[1,2 -b:2′,1′-e]пиран-10,12(11h)-диона. синтеза се врши у једном кораку, у трокомпонентној реакционој смеши алдехида, 2-нафтола, и цикличног 1,3-дикарбонилног једињења. _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 492 pouramiri, shirvani and tavakolinejad kermani реакција се одвија брзо, на 70 °c без присуства растварача а жељени производи се добијају у добром до одличном приносу. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice sorption recovery of platinum(ii,iv) in presence of copper(ii) and zinc(ii) from chloride solutions j. serb. chem. soc. 80 (9) 1149–1160 (2015) udc 546.92+66.081+546.56+ jscs–4787 546.47:544.35–131:544.726 original scientific paper 1149 sorption recovery of platinum(ii,iv) in presence of copper(ii) and zinc(ii) from chloride solutions olga n. kononova*, nataliya s. karplyakova and evgeniya v. duba institute of non-ferrous metals and material science, siberian federal university, 660041 krasnoyarsk, svobodny pr., 79 russian federation (received 17 december 2014, revised 22 january, accepted 19 february 2015) abstract: the sorption pre-concentration of platinum(ii,iv) ions was investigated in presence of accompanying copper(ii) and zinc(ii) ions from chloride solutions on new previously unexplored ion exchangers (cybber, russia). the initial concentrations of platinum and the accompanying ions were 0.25 and 2.0 mmol l-1, respectively, and the acidity of medium was 0.001–4.0 mol l-1 hcl. it was shown that the investigated resins – strong and weak basic anion exchangers as well as chelate ion exchangers – possessed good sorption and kinetic properties. the simultaneous sorption of the investigated ions results in the complete recovery of platinum, while the non-ferrous metal ions were sorbed at less than 20 %. followed by the selective elution of platinum by a thiourea (80 g l-1) solution in 0.3 m h2so4, the quantitative isolation of platinum was achieved (more than 90 %). therefore, the studied ion exchangers could be recommended for the recovery and separation of pt(ii,iv), cu(ii) and zn(ii) ions. keywords: ion exchange; platinum; copper; zinc; chloride solutions. introduction with growing global demand for the platinum group metals (pgm), these metals are being recovered not only from natural deposits, but also from non -traditional sources (e.g., metal-bearing high-carbon complexes) and secondary sources,1 including spent automobile and chemical catalysts, electronic scrap and the so-called tailings (wastes of ore-dressing plants at platinum-containing deposits).1–4 the distinctive feature of both primary and secondary pgm sources is their multicomponent composition. this means that the noble metals are contained in raw materials together with other metals, such as ni, zn, cu, co, pb, etc. moreover, the noble metals are micro components, whereas the accompanying metals * corresponding author. e-mail: cm2@bk.ru doi: 10.2298/jsc141217018k _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1150 kononova, karplyakova and duba are macro components.1,3–5 therefore, after the breakdown of such raw materials by acids (“aqua regia”), chlorination, fusion, etc., the obtained industrial solutions have low concentrations of noble metals.2,4–6 these solutions contain pgm complexes, varying in their stability and chemical inertness.2,4,7–9 moreover, it is known2,4,7–10 that pgm complexes are affected by aquation and hydrolysis. the sophisticated composition of the solutions makes it essential to use selective methods for the isolation of the noble metals. as a rule, the pgm are isolated from such solutions by precipitation or electrowinning. however, these methods basically do not provide a high degree of recovery, and the solid products formed in such cases make further processing significantly harder.4,6,11,12 these problems could be solved by means of sorption methods, known not only for their selectivity and efficiency, but also for environmental safety and compatibility with a variety of post-determination methods.6,11–16 among a variety of sorbents, ion exchangers with different functional groups are of special interest because of their high exchange capacity, osmotic and mechanical stability and good kinetic properties.2,12,13,15,17 these characteristics allow the recovery of even trace amounts of pgm through their pre-concentration, and also the removal of interfering components. as a result, the obtained noble metals can be of high purification grades.2,11,12,17,18 the selectivity of the ion exchangers is a matter of great importance, given that the ionic state of noble metals in solutions is distinguished by a variety of complex forms with different stabilities and kinetic inertness, and the solutions themselves are multicomponent systems. the selectivity could be improved through the complex-forming properties of ion exchangers.17 previously, the recovery of platinum (ii,iv) from chloride solutions on purolite ion exchangers was investigated.19 since the recovery of platinum in the presence of accompanying components is a matter of practical interest, the present work is focused on the simultaneous recovery of pt(ii,iv), cu(ii) and zn(ii) because these ions are often contained in real solutions obtained after breakdown of platinum-containing raw materials.2–6 it should also be noted that these non-ferrous metals are valuable materials and are the subject of industrial recycling. the sorption recovery of copper(ii) and zinc on purolite ion exchangers were also studied.20 for the present investigation, new, previously unexplored cybber ion exchangers made in russia were used. experimental the cybber ion exchangers, produced by synteznvk company, st. petersburg, russia, were taken for investigation. their physicochemical characteristics are summarized in table i. it could be seen from the data that anion exchangers, chelate resins and strong acidic cation exchangers were used to study copper and zinc sorption. before use, the resins were _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ sorption recovery of pt, cu and zn 1151 prepared according to standard methods and loaded by 2 m hcl, to convert them into the cl form (anion exchangers) or h+ form (cation exchanger and chelate sorbents). table i. physicochemical characteristics of the investigated macroporous ion exchangers cybber. resin matrix: styrene (st) – divinylbenzene (dvb); functionalization: qab – quaternary ammonia base, tag – tertiary amino-groups; ampa – aminomethyl-phosphonic acid; idaa – iminodiacetic acid; sg – sulfo-groups trade name exchanger type functional group exchange capacity, mmol g-1, in the form: swelling grade, % moisture % working ph range clh+ ax 400 strong base anion exchanger qab 1.20 – 19 44 0–14 alx 220 weak base anion exchanger tag 1.45 – 21 50 0–8 crx 300 chelating resin ampa – 1.80 23 40 1–14 crx 210 chelating resin idaa – 1.10 21 55 1–6 ev 023 strong acid cation exchanger sg – 1.80 17 45 0–14 the initial platinum stock solution of concentration 9.669 mmol⋅l-1 was prepared by dissolution of an accurately weighed quantity h2ptcl6 in concentrated hydrochloric acid, with the subsequent dilution of the solution to 500 ml with distilled water. the working platinum solutions with concentration 0.25 mmol l-1 and acidity 0.001–4.0 m hcl were prepared from this initial solution. in the present work, only freshly prepared platinum solutions were used. the solutions of copper(ii) and zinc(ii) of concentration 2.0 mmol l-1 were prepared from accurately weighed quantities of cucl2⋅2h2o and zncl2, respectively, which were dissolved in hydrochloric acid solutions of different concentrations (0.001–4.0 mol l-1). all the reagents were of analytical purification grade. the initial concentrations of the studied ions were selected with the aim of making the experiments similar to the real industrial conditions, i.e. to the technical solutions obtained after the processing of secondary materials.2,5,18 the acidity range of initial solutions was intentionally wide, to study the sorption properties of the investigated resins. the concentrations of platinum and non-ferrous metal ions were determined by spectrophotometrical methods, i.e., platinum with sncl2⋅2h2o,7,9 and copper(ii) and zinc(ii) with par (4-(2-pyridylazo)resorcinol)21,22 using a specol 1300 spectrophotometer (carl zeiss, germany). the sorption of the investigated ions was realized under batch experimental conditions. the equilibrium time determined by special tests was 24 h. the efficiency of sorption recovery of the ions investigated was estimated by means of the recovery degree, r / %, and the distribution coefficient, d. moreover, the separation coefficients s of the recovered metal ions were calculated as follows: _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1152 kononova, karplyakova and duba 1 2 me me k s k = (1) where 1mek is the distribution coefficient of platinum (or copper) and 2mek is the distribution coefficient of copper (or zinc). the sorption isotherms were plotted by varying the molar ratio of resins to the amount of metal ions in the contacting solution.23,24 the apparent constants of the ion exchange equilibrium were calculated based on these isotherms according to the law of mass action for the investigated equilibria.23,24 the kinetic behavior of ion exchangers investigated during sorption of the metal ions was studied by the “limited bath” method.23,25 after a certain time, the resins and solutions were quickly separated by filtration through a porous glass filter. then the concentrations of platinum, copper and zinc were determined in the solutions by the spectrophotometrical methods. using the obtained results, the degree of saturation f was calculated as follows: t q f q∞ = (2) where tq and q∞ , mmol, are the amounts of the metal ion sorbed at time t and at equilibrium, respectively. then the kinetic curves were plotted as dependences ( )f f t= and the half-exchange times, 1/ 2t / s, were determined from these curves at f = 0.5. subsequently, the diffusion coefficients of metal ions in a resin grain, sd / cm2 s-1, were calculated from the following equation: 2 2 1/ 24π s r d t = (3) where r / cm is the radius of the resin grain. moreover, the process rate, ν / mmol g-1 s-1, was calculated using the formula: i i a t ν = (4) where ai / mmol g-1 is the quantity of metal ion sorbed by the resin at time ti / s. all the results were subjected to statistical processing according to conventional procedures.26,27 the average experimental error for 3–4 parallel runs was less than 6 %. the details regarding the batch and kinetic experiments, as well as the sorption preconcentration and recovery data, are given in the supplementary material to this paper. results and discussion the ionic state of platinum in chloride solutions was investigated in detail.2,4,7–10 the electron absorption spectrum of a freshly prepared platinum (ii,iv) solution in 2 m hcl, which had two absorption maxims at 218 and 251 nm, was recorded this spectrum is in full compliance with literary data and corresponds to the presence of complexes [ptcl4]2– (218 nm) and [ptcl6]2– (251 nm).2,9,10 the ionic states of copper(ii) and zinc(ii) in chloride solutions were previously investigated in detail21,22 and are described in a previous work.20 there_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ sorption recovery of pt, cu and zn 1153 fore, the initial investigated solution contained different chloride complexes of the studied ions, and their subsequent sorption on the ion exchangers depended on the form of their complex compounds. since the cybber ion exchangers were investigated for the first time, the sorption recovery of platinum (ii,iv), copper(ii) and zinc ions were initially studied from their individual solutions of different acidities. these studies revealed that the studied anion exchangers and chelate sorbents exhibited high affinity for platinum (ii,iv) ions, with no dependence on the acidity of medium. as for copper (ii) and zinc (ii) recovery under the same conditions, the results correlated with their ionic state in the solutions. thus, the anion exchangers and chelate resins recovered copper(ii) ions only from the strong acidic solutions, indicating the sorption of copper anionic chloride complexes. copper was only recovered from the 0.001–0.1 mol l–1 hcl solutions by the strong acidic cation exchanger ev 023, meaning that cationic copper complexes are present in these solutions. it should be noted that the ion exchangers in a strong acidic medium exhibited high affinities for copper(ii) ions. moreover, only the chelate ion exchanger cybber crx 300 recovered zinc ions over the whole range of investigated hcl concentrations. the other resins did not sorb zn(ii) from strong acidic solutions, although it is known from the literary data that negatively charged complexes [zncl4]2– are present in such solutions. this was also supported by the fact that the cation exchanger ev 023 did not recover zinc ions from 2–4 m hcl solutions. this phenomenon is of academic interest and will be the subject of further investigations. however, the fact that copper(ii) and zinc(ii) ions could be recovered in different ways and from different media provides the opportunity to separate these ions by varying the hcl concentration in the contacting solution. based on this, the simultaneous recovery of copper and zinc from strong and weak acidic media was further investigated. the results are presented in table ii. it can be seen from these data that the presence of zinc did not affect copper(ii) sorption, which means that copper is not recovered from weakly acidic solutions by the investigated resins. as for the sorption of zinc in presence of cu(ii), in this case they were recovered from strong acidic solutions, and at a quite satisfactory level (86–89 %). no doubt, it could be considered the effect of synergy, i.e., the increase in sorption ability of ion exchanger to an ion that was not sorbed (or poorly sorbed) from individual solutions in the presence of another ion.28–30 therefore, the simultaneous action of both components has an effect on the sorption ability of the ion exchangers. the data in table ii also show that the studied sorbents were more selective towards copper(ii) ions than to zinc ions. the separation coefficients of non-ferrous metal ions during their recovery from 2 m hcl solution are shown in table iii. it can be seen that all the values exceed 1 and, therefore, copper and zinc could be completely separated. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1154 kononova, karplyakova and duba table ii. simultaneous recovery of copper(ii) and zinc(ii) from chloride solutions of different acidity on the investigated ion exchangers (c0(cu) = c0(zn) = 2.0 mmol l-1) trade name parameter recovery of: cu(ii) in presence of zn(ii) at c0(hcl) / mol l-1 zn(ii) in presence of cu(ii) at c0(hcl) / mol l-1 2.0 0.01 2.0 0.01 ax 400 r / % ≈100 – 86±4 89±5 log d 5.08±0.31 – 3.78±0.23 3.89±0.23 alx 220 r / % ≈100 – 86±4 89±5 log d 4.79±0.29 – 3.79±0.23 3.91±0.23 crx 300 r / % ≈100 – 88±4 88±5 log d 5.11±0.31 – 3.85±0.23 3.86±0.23 crx 210 r / % ≈100 – 87±4 87±4 log d 4.99±0.29 – 3.82±0.23 3.81±0.22 table iii. separation coefficients of cu and zn during their sorption from strong acidic chloride solutions (c0(hcl) = 2.0 mol l-1; c0(cu) = c0(zn) = 2.0 mmol l-1) trade name separation coefficient trade name separation coefficient ax 400 20 crx 300 18 alx 220 10 crx 210 13 furthermore, the sorption pre-concentration of platinum (ii,iv) in the presence of copper(ii) and zinc(ii) was studied from strong acidic chloride solutions, given that under industrial conditions, the noble metals are mostly present in strong acidic media after the breakdown. the results are summarized in table iv, from which it could be seen that the presence of copper and zinc ions in the system had no effect on the sorption pre-concentration of platinum, and hence, it could be completely recovered from the solution by the investigated sorbents. simultaneously, the copper ions and, especially, the zinc ions were sorbed at low levels (not more than 20 % for cu and 16 % for zn). these data clearly illustrate the distinct selectivity of ion exchangers towards noble metal complexes. on the one hand, this could be explained by the fact that complexes with greater stability are “preferable” for the resins during sorption from multicomponent solutions.31 as the stability of platinum chloride complexes is much higher than those of copper and zinc,8 the sorption centers of the resin are preoccupied by platinum complexes. however, on the other hand, it is known17,32,33 that complex-forming sorbents that contain nitrogen or sulfur atoms in their functional groups show especially high selectivity towards noble metal ions. from the investigated range of sorbents, the weak basic anion exchanger alx 220 and chelate resin crx 210 belong to this group. it is important that crx 210 contains iminodiacetic acid as functional groups, i.e. two carboxylic groups. the lewis theory of acids and bases states that platinum ions are “soft” acids, and possess lower affinity to oxy_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ sorption recovery of pt, cu and zn 1155 gen donor atoms,8,34 which explains the lower degree of recovery of platinum on the chelate resin crx 210 in comparison with that on alx 220. the strong basic anion exchanger ax 400 with quaternary ammonia bases as functional groups has a high selectivity to platinum ions, due to the strong electrostatic interaction between these large-sized and practically not hydrated groups and the aquatic complex ions of the noble metal.31 table iv. sorption pre-concentration of platinum(ii,iv) from strong acidic chloride solutions in the presence of copper(ii) and zinc(ii) on the investigated ion exchangers (c0(hcl) = 2.0 mol l-1; c0(pt) = 0.25 mmol l-1; c0(cu) = c0(zn) = 2.0 mmol l-1) trade name parameter recovery of: pt(ii,iv) in the presence of cu(ii) and zn(ii) cu(ii) in the presence of pt(ii,iv) and zn(ii) zn(ii) in presence of pt(ii,iv) and cu(ii) ax 400 r / % 95±5 14±1 10±1 log d 4.43±0.27 2.30±0.14 2.01±0.12 alx 220 r / % 95±5 20±2 16±2 log d 4.45±0.27 2.48±0.15 2.32±0.14 crx 210 r / % 77±4 12±1 7±1 log d 3.53±0.21 2.04±0.12 1.97±0.12 the separation coefficients of platinum and the accompanying metals were calculated and the results are given in table v. the values were much greater than 1, indicating the possibility of isolation of platinum from the non-ferrous metal ions. table v. separation coefficients of pt and non-ferrous metal ions during their sorption from strong acidic chloride solutions (c0(hcl) = 2.0 mol l-1; c0(pt) = 0.25 mmol l-1; c0(cu) = c0(zn) = 2.0 mmol l-1) trade name separation coefficients of pt(ii,iv) towards: cu (ii) zn (ii) ax 400 137 273 alx 220 91 137 crx 210 34 34 thus, the obtained data led to the conclusion that the sorption pre-concentration of platinum(ii,iv) proceeded on the strong basic anion exchanger according to the anion exchange mechanism: 2 22rcl [ptcl ] r [ptcl ] 2cln n− −+ ↔ + (5) the weak basic anion exchanger sorbed platinum ions not only through anion exchange, but also through additional complexation between the metal ions and the nitrogen atoms of the functional groups: _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1156 kononova, karplyakova and duba 2 -1rn [ptcl ] rnptcl cln n− − + ↔ +  (6) where n = 4 or 6. the chelate ion exchanger crx 210 recovered platinum(ii,iv) ions according to the complex-formation mechanism: 2 2 ( )[ptcl ] rl s (ptcl rl ) l sn m zmn n m mm m m− − − −−+ ⋅ ⋅ ⋅ ↔ + + (7) where n = 4 or 6; m is the number of functional groups o the resin rl with the charge z; s is the dissolvent. the sorption isotherms of platinum in presence of accompanying copper and zinc ions are shown in fig. 1 for the strong and weak basic anion exchangers. it can be seen that the curves are convex, i.e., the anion exchangers are selective towards pt(ii,iv) ions.23,24 based on the isotherms, the apparent constants of the ion exchange equilibrium were 3.0 and 2.4 for alx 220 and ax 400, respectively. these values correlate with the selectivity of the sorbents. fig. 1. isotherms of platinum sorption in presence of copper(ii) and zinc(ii) from strong acidic solutions onto: a) alx 220 and b) ax 400; c0(hcl) = 2.0 mol l-1. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ sorption recovery of pt, cu and zn 1157 furthermore, the kinetic properties of ion exchangers during the recovery of platinum(ii,iv) ions in presence of copper(ii) and zinc(ii) were investigated. the dependences of the process rate on time are presented in fig. 2. it could be seen that the process rate increased sharply at the beginning of ion exchange and then decreased gradually before reaching equilibrium. fig. 2. dependences of process rate on time during sorption pre-concentration of platinum(ii,iv) in presence of copper(ii) and zinc(ii) on the investigated ion exchangers; c0(hcl) = 2.0 mol l-1; c0(pt) = 0.25 mmol l-1; c0(cu) = c0(zn) = 2.0 mmol l-1;  (1) – alx 220;  (2) – ax 400;  (3) – crx 210. the calculated main kinetic parameters for sorption pre-concentration of pt(ii,iv) in the presence of the accompanying ions are summarized in table vi. according to the data, it proceeds coherently, with high process rate. the other kinetic parameters – half-exchange time and diffusion coefficients – correlate with the previously discussed data on selectivity. table vi. kinetic parameters during sorption pre-concentration of platinum(ii,iv) in the presence of copper(ii) and zinc(ii) from strong acidic chloride solutions on the investigated ion exchangers (c0(hcl) = 2.0 mol l-1; c0(pt) = 0.25 mmol l-1; c0(cu) = c0(zn) = 2.0 mmol l-1); ν is the average process rate trade name kinetic parameter t1/2 / s sd ×107 / cm2 s-1 ν ×105 / mmol g-1 s-1 ax 400 7200 1.1 1.8 alx 220 1800 4.6 1.9 crx 210 2700 2.3 0.7 finally, the desorption of platinum from the ion exchangers was investigated. as the amount of accompanying non-ferrous metal ions was no higher than 20 % (table iv), after the sorption pre-concentration of platinum, it was enough to wash the ion exchangers with 0.5 m hno3 once for their complete removal. then, the elution of platinum by thiourea solution (80 g l–1) in 0.3 m _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1158 kononova, karplyakova and duba h2so4 was performed, given that acidic thiourea solutions are conventional desorption agents for noble metals.2,9 as a result, 93 % of the platinum was eluted from the resin ax 400, and 90 % of the platinum – from alx 220. therefore, the anion exchangers cybber ax 400 and alx 220 could be recommended for the recovery of platinum(ii,iv) ions and their quantitative separation from accompanying copper(ii) and zinc ions. conclusions sorption recovery of platinum(ii,iv) in the presence of copper(ii) and zinc(ii) ions from chloride solutions on new cybber ion exchangers produced in russia was investigated. since these resins were studied for the first time, their sorption properties were investigated during recovery of each component from its individual solutions over a wide range of hcl concentrations (0.001–4.0 mol l–1). high affinities of the ion exchangers for platinum(ii,iv) complexes were revealed. however, copper(ii) ions were sorbed only from strong acidic solutions, while zinc ions, only from weak acidic media. an investigation of the simultaneous recovery of cu(ii) and zn(ii) from weak and strong acidic chloride solutions showed that zinc ions were sorbed at a high level in the presence of copper(ii) from both weak and strong acidic media. this phenomenon was the result of synergy. a study of sorption pre-concentration of platinum(ii,iv) in the presence of copper(ii) and zinc(ii) has revealed the high selectivity of the investigated ion exchangers towards the noble metal ions, whereas the non-ferrous metal ions were recovered to no more than 20 %. the investigation of kinetic properties of the resins revealed high rate of ion exchange process during pt(ii,iv) recovery in the presence of the accompanying ions. the main kinetic parameters, i.e., half-exchange time and diffusion coefficients in the resin grain, correlated with the previously found selectivity of the sorbents. the elution of the investigated components from the resins after sorption was realized using 0.5 m hno3 (for copper and zinc) and thiourea solution (80 g l–1) in 0.3 m h2so4 (for platinum). as a result, the quantitative isolation and separation of the ions was achieved. the obtained results allowed the recommendation of the anion exchangers cybber ax 400 and alx 220 for sorption pre-concentration of platinum(ii,iv) in presence of accompanying cu(ii) and zn(ii) ions from strong acidic chloride solutions and for the subsequent isolation of the noble metal by selective elution. supplementary material details of the batch and kinetic experiments, considerations about the ionic states of the metal in chloride solutions and sorption pre-concentration and recovery data, tables s-i–s-iii, _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ sorption recovery of pt, cu and zn 1159 are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the authors express deep gratitude to the synteznvk company (st. petersburg, russia) for the resin samples kindly provided for investigation. и з в о д сорпционо издвајање платине(ii,iv) у присуству бакра (ii) и цинка (ii) из хлоридних раствора olga n. kononova, nataliya s. karplyakova и evgeniya v. duba institute of non-ferrous metals and material science, siberian federal university, 660041 krasnoyarsk, svobodny pr., 79 russian federation концентровање јона платине (ii,iv) сорпцијом је испитивано у присуству пратећих јона бакра (ii) и цинка (ii) из хлоридних раствора на новим јоно-измњивачима cybber (русија), који до сада нису испитивани. почетна концентрација платине и пратећих јона је била 0,25 и 2,0 mmol l-1, редом, а киселост средине је била 0,001–4,0 mol l-1 hcl. показано је да испитиване смоле – јаки и слаби базни анјонски измењивачи као и измењивачи хелатних јона – имају добре сорпционе и кинетичке особине. истовременом сорпцијом испитиваних јона платина је потпуно издвојена, док су јони бакра и цинка сорбовани мање од 20 %. након тога, селективним издвајањем платине раствором тиоурее (80 g l-1) у 0,3 m h2so4, постигнута је квантитативна изолација платине (више од 90 %). на основу добијених резултата може се закључити да се испитивани јоноизмењивачи могу користити за одвајање јона pt(ii,iv), cu(ii) и zn(ii). (примљено 17. децембра 2014, ревидирано 22. јануара, прихваћено 19. фебруара 2015) references 1. anon, novel, non-traditional sources of platinum group metals, http://www.uralgold.ru/pt_innova.html (accessed 01.07.2010) 2. y. a. zolotov, g. m. varshal, v. m. ivanov, analytical chemistry of platinum group metals, editorial urss, moscow, 2003, p. 592 3. g. v. semenchenko, a. s. mukusheva, l. l. osipovskaya, direct leaching of noble metals from refractory ores of kazakhstan’s deposits, proceedings of the 2nd international congress “non-ferrous metals in siberia”, krasnoyarsk, 2010, p. 288 4. t. m. buslaeva, chemistry and technology of platinum group metals, nauka, moscow, 1999, p. 251 5. y. a. kotlyar, m. a. meretukov, l. s. strizhko, metallurgy of noble metals, misis, moscow, 2005, p. 392 6. o. v. spektor, a. l. ryumin, m. g. pochekutova, tsvetnye metally 7 (1998) 31 7. f. e. beamish, the analytical chemistry of the noble metals, pergamon press, oxford, 1966, p. 702 8. f. a. cotton, c. 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gesellschaft geest & portig k.-g., leipzig, gdr, 1960, p. 1074. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice determination of multi-class herbicides in soil by liquid–solid extraction coupled with headspace solid phase microextraction method j. serb. chem. soc. 81 (8) 923–934 (2016) udc 631.4+632.954:543.544.3:543.51+66.061 jscs–4897 original scientific paper 923 determination of multi-class herbicides in soil by liquid–solid extraction coupled with headspace solid phase microextraction method rada đurović-pejčev1*#, tijana đorđević1 and vojislava bursić2# 1institute of pesticides and environmental protection, banatska 31b, p. o. box 163, 11080 belgrade, serbia and 2faculty of agriculture, university of novi sad, trg dositeja obradovića 8, 21000 novi sad, serbia (received 23 november 2015, revised 21 april, accepted 13 may 2016) abstract: described is a method for simultaneous determination of five herbicides (metribuzin, acetochlor, clomazone, oxyfluorfen and dimethenamid) belonging to different pesticide groups in soil samples. developed headspace solid phase microextraction method (hs-spme) in combination with liquid– –solid sample preparation was optimized and applied for the analysis of agricultural samples. optimization of microextraction conditions, such as temperature, extraction time and sodium chloride content was performed using 100 μm polydimethyl-siloxane (pdms) fiber. the extraction efficiencies of methanol, methanol:acetone and methanol:acetone:hexane in 1:1 and 2:2:1 volume ratios, respectively, and the optimal number of extraction steps during the sample preparation, were tested as well. gas chromatography–mass spectrometry was used for detection and quantification, obtaining relative standard deviation (rsd) below 13 %, and recovery values higher than 83 % for multiple analyses of soil samples fortified at 30 μg kg-1 of each herbicide. limits of detection (lod) were less than 1.2 μg kg-1 for all the studied herbicides. keywords: pesticides; soil matrix; multiresidue method; gas chromatography mass spectrometry. introduction in modern agricultural production, the use of herbicides for weed control is necessary and essential. united states environmental protection agency (epa) indicated that in 2006 and 2007 the worldwide pesticide usage was approximately 5.2 billion pounds, of which herbicides constituted the majority at 40 %.1 usually, crops are showing larger sensitivity to weeds at the beginning of the growing period, due to their slower growth and lower density during that stage. * corresponding author. e-mails: rajcica76@gmail.com; rada.djurovic@pesting.org.rs # serbian chemical society member. doi: 10.2298/jsc151123044d _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 924 đurović-pejčev et al. therefore, application of herbicides is required. various pre-emergence weed control herbicides can be applied to different crops, including widely used: dimethenamid (used in corn, soybeans, sunflower, sugar beet and potatoes), acetochlor (in corn, sunflower, soybeans and potatoes), metribuzin (in soybeans, potatoes, tomatoes and peppers), clomazone (in soybeans, tobacco and rapeseed) and oxyfluorfen (in sunflower). soil-applied pre-emergence residual herbicides, especially those used prior to sowing, are usually incorporated into the soil. slow degradation of pesticides in the environment and extensive or inappropriate usage by farmers could lead to soil contamination. due to the outstanding concern for human health, and considering the manner and amount of herbicide application, programs monitoring soil contamination by agrochemicals have been established throughout the world, as well in serbia. since the herbicides are a very heterogeneous group of chemicals with different physicochemical properties, the current trend in residue analysis of these compounds is developing multi-residual methods that would provide for simultaneous determination of large number of compounds. in addition, these methods should overcome the drawbacks of the traditional approaches, which are laborious, time consuming, expensive, require large amounts of organic solvents and usually involve many steps, leading to loss of analyte quantity. solid phase microextraction (spme), as a technique that combines extraction, purification and concentration processes into a single step, is an example of such development. until recently, most of the spme applications for determination of herbicide residues in soil were based on preparation of soil mixtures with distilled water and subsequent immersion of the spme fiber in thus prepared slurry (dm-spme)2–6 or its exposing to a gas phase above the slurry (hs-spme)7–9. some researchers have suggested that combination of liquid–solid (l–s) soil preparation followed by dm-spme determination of herbicides in obtained extracts is the most reliable soil spme method.10–15 however, previous studies based on a combination of l–s extraction and hs-spme determination of herbicides in soil samples were done only with triazines (simazine, atrazine and prometryn)16,17 and mixture of the two pyridazinones (chloridazon and fluorochloridone) and pendimethalin as dinitroaniline herbicide.18 in those studies, methanol–acetone combination was used for the extraction of pesticides from the soil matrix. regarding determination of herbicides belonging to various pesticide groups, there are no published methods based on a combination of l–s procedure followed by simultaneous hs-spme herbicides determination. therefore, the aim of this study was to develop a rapid and simple hs-spme method combined with l–s sample preparation for simultaneous determination of five compounds (metribuzin, acetochlor, clomazone, oxyfluorfen and dimethenamid) having distinct chemical structures and belonging to different herbicide groups. microext_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ multi-class herbicides in soil 925 raction temperature, time and nacl content, main parameters affecting spme, were tested and optimized using a 100 µm long pdms fiber. the extraction efficiencies of pure methanol, methanol:acetone and methanol:acetone:hexane mixtures (1:1 and 2:2:1 volume ratios, respectively) were optimized, as well as the optimal number of extraction steps within sample preparation stage. the proposed method was applied in the analysis of selected agricultural soil samples. experimental reagents and materials the herbicides chosen for this study were: metribuzin, acetochlor, clomazone, oxyfluorfen and dimethenamid (dr ehrenstorfer, germany, table i). standard stock solutions containing 1 g dm-3 of each herbicide were prepared in acetone (j. t. baker, holland), and stored at –18 °c. standard working mixed solutions were prepared weekly by diluting the individual stock solutions with acetone and stored at 4 °c. sodium chloride of 99.5 % purity was purchased from merck (germany) and methanol from j. t. baker (holland). table i. physicochemical properties of herbicides studied;19,20 mr, molecular weight; sw, water solubility; log kow, partition coefficient between n-octanol and water; h, henry’s constant herbicide chemical class mr / g mol -1 sw / mg dm -3 log kow h / pa m3 mol-1 metribuzin triazinone 214.3 1050 1.6 1×10-5 acetochlor chloroacetamide 269.8 223 4.14 0.383 clomazone isoxayolidinone 239.7 1100 2.5 4.19×10-3 oxyfluorfen diphenyl ether 361.7 0.116 4.47 9.40×10-2 dimethenamid chloroacetamide 275.8 1200 2.15 8.32×10-3 pdms fibers (supelco, usa), 100 µm long, were used for spme measurements. extraction, along with constant mixing was performed in 4 cm3 vials (supelco, usa). an uncontaminated soil sample originating from region of the town of kikinda (serbia) was used in the study. the main physicochemical properties of the soil were: ph (in h2o) 8.39, organic matter content, 3.17 %, sand content, 73.96 %, silt content, 22.60 % and clay content, 3.44 % (all as mass %). the soil was air-dried and sieved (2 mm pores) before use. polypropylene centrifuge tubes with caps (50 cm3, sarstedt, germany), filter papers 1ps, 150 mm diameter (watman, uk) and a centrifuge (uz 4, slovenia) were used in the soil preparation procedure. instrumentation a gas chromatograph–mass spectrometer (gc–ms), model cp-3800/saturn 2200 (varian, australia) was used for separation and detection. column vf-5ms having dimensions 30 m×0.25 mm×0.25 µm by varian was used. the thermal desorption of analytes from pdms fiber was performed for 7 min at injector temperature of 270 °c. the gc was programmed as follows: initial temperature was 120 °c, followed by increase to 170 °c at 8 °c min-1 rate, kept constant for 4.5 min, increased to 280 °c at 9 °c min-1 rate and kept at the same temperature for 5.5 min. helium was used as a carrier gas and its flow rate was 1.1 ml min-1. the ion trap mass spectrometer operated in the electron impact/selected ion monitoring (ei/sim) mode. the ion trap and transferline temperatures were set to 220 °c and 250 °c, respectively. one specific herbicide ion was selected for detection and quantification, while _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 926 đurović-pejčev et al. the second one was used for confirmation. the ions inspected were as follows: 198 (215) for metribuzin, 223 (146) for acetochlor, 204 (125) for clomazone, 252 (317) for oxyfluorfen and 154 (230) for dimethenamid. optimization of hs–spme analysis hs–spme conditions, such as temperature, extraction time and nacl content were tested and optimized using 100 µm pdms fiber. optimization was done using 2.5 cm3 of aqueous solution containing 25 µg dm-3 of each herbicide. desorption parameters (temperature and time) for this study were initially selected according to previous research.13 optimization of soil sample preparation the efficiency of the hs-spme method, optimized for aqueous solutions, was tested using analysis of soil samples. for that part of the study, 10 g of sub-samples were placed in the polypropylene centrifuge tubes and fortified to concentration of 30 µg kg-1 for each herbicide using 1 mg dm-3 mixed standard solution. the spiked samples were homogenized for 15 min using a rotary stirrer and left to rest for 24 hours prior to further analysis. the extraction efficiencies of the pure methanol, methanol:acetone (1:1 volume ratio) and methanol:acetone:hexane (2:2:1 volume ratio) mixtures and the optimal number of extraction steps were determined by the following procedure: soil samples were extracted by dissolving in 15 cm3 of solvent for 30 min using a rotary stirrer and then centrifuged for 15 min at 4000 rpm. the extract was filtered and evaporated to dryness at 35 °c using a rotary evaporator. the residues were redissolved in 1 cm3 of acetone, and 0.2 cm3 of those solutions were diluted with water to 10 cm3 for hs-spme measurements. results and di̇scussi̇on hs-spme optimization selected 100 µm pdms fiber for spme measurements in this multiresidue herbicides analysis, as well as the desorption time of 7 min and 270 °c desorption temperature as optimal conditions for the used fiber, were chosen according to the results presented in detail in previous study.13 optimal microextraction temperature, nacl content and microextraction time, as experimental parameters affecting hs-spme measurements, were optimized by a well-structured step-bystep approach, using spiked water samples. microextraction temperature it is well known that the increase of microextraction temperature leads to increase of analyte vapor pressure, resulting in improvement of hs-spme efficiency.21,22 therefore, the temperature effect in the range of 23–90 °c on the hs -spme efficiency was analyzed. the obtained extraction-temperature profiles for each of the herbicide studied are shown in fig. 1. the results presented, clearly indicate that the temperature increase leads to the enhancement of the overall sorbed mass on the fiber, while other experimental conditions were kept constant. this effect could be explained by the increase of analyte vapor pressure, i.e., higher concentration in the gas phase. however, for temperatures above 75 °c for oxyfluorfen, further increase in temperature results in a reduction of herbicide _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ multi-class herbicides in soil 927 amount sorbed on the fiber (fig. 1). explanation for such a behaviour lies in the exothermic nature of the sorption process (enhanced analyte desorption from the fiber at high temperatures) and in the very low solubility of the oxyfluorfen in water (weak solvent–analyte interactions cannot be further weakened by the temperature increase). 20 40 60 80 0 7000 14000 21000 31500 pe ak a re a, c ou nt s t / 0c clomazone dimethenamid metribuzin acetochlor oxyfluorfen fig. 1. hs-spme temperature profiles for herbicides studied. since satisfactory sensitivity for each of the individual compounds studied was obtained at 75 °c, this temperature was chosen as optimal for the mixture of herbicides tested. effect of ionic strength as previously reported, an addition of salt to the sample could decrease the solubility of some analytes in the aqueous solution, stimulating their movement into the gas phase and consequently to the fiber coating.22 this is especially pronounced in the case of hydrophobic compounds that have low affinity for the pdms fibers. therefore, the ionic strength was a well studied experimental parameter influencing the hs-spme measurements. ionic strength was adjusted by adding different amounts of nacl to the standard herbicide aqueous solutions (0, 25, 50, 100, 150, 200, 250 and 300 g dm–3). as shown in fig. 2, the obtained results indicate that the ionic strength increases the spme efficiency for all the herbicides studied. also, it is evident that for the most hydrophobic herbicide studied (oxyfluorfen) the enhancement of mass sorbed on the fiber at higher nacl concentrations is significantly less pronounced. a possible explanation for this behaviour of oxyfluorfen could be the presence of a strong competition between this analyte and the more polar ones for pdms fiber sorption that finally results in a minor increase in its extraction efficacy. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 928 đurović-pejčev et al. fig. 2. effect of ionic strength on the analytical signal for studied herbicides. finally, based on the results obtained for all the herbicides studied, nacl concentration of 300 g dm–3 was chosen for further work. microextraction time some theoretical models proposed for explanation of the hs-spme process recommended shortening duration time of the analysis by indicating that quantification is possible before a sorption equilibrium could be reached.21,23 although microextraction using equilibrium time is advised, for practical reasons an efficient half-hour microextraction (enough to provide sufficient analytical sensitivity for all the compounds studied and in accordance with the chromatographic run time of 28.47 min), was compared only to 20 min procedure. the results obtained (fig. 3) indicate that the time period of 30 min was a better choice for all the herbicides studied, and therefore it was chosen for further work. overall, considering the results obtained for all the parameters optimized for hs-spme determination of studied herbicides, the following spme conditions _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ multi-class herbicides in soil 929 were found to be the most efficient: temperature of 75 °c, 300 g dm–3 nacl content and 30 min extraction time. 1 2 3 4 5 0 225000 450000 675000 900000 pe ak a re a, c ou nt s herbicide 20 min 30 min fig. 3. effect of microextraction time on hs-spme determination of clomazone (1), dimethenamid (2), metribuzin (3), acetochlor (4) and oxyfluorfen (5). soil extraction optimization optimized hs-spme method was tested by analyzing soil samples. literature reports on the analysis of herbicide residues from soil samples, using dmspme of a soil organic extract obtained by l–s extraction of previously diluted samples is a more efficient method than direct immersion of the spme fiber in the slurry of soil sample and water.10–15 previous study, based on the combination of conventional l–s procedure followed by dm-spme determination of the selected herbicides, showed that among different solvents tested (water, hexane, acetonitrile, acetone and methanol), two successive extractions with methanol (met) as the extraction solvent seemed to be the optimal sample preparation choice.13 in the same study, somewhat lower recovery obtained for oxyfluorfen (62.82 %) was explained by insufficient power of methanol as an extraction solvent in the sample preparation step and/or the strong influence of soil matrix on this herbicide. considering the obtained results and aiming for an improvement of the sample preparation step, the extraction efficiency of methanol was compared to the efficiencies obtained by combining it with solvents of different polarity (methanol:acetone (met:ac) and methanol:acetone:hexane (met:ac:hex) and employing a single extraction procedure as described in experimental – optimization of soil sample preparation section. the results (table ii) show that both solvent mixtures considerably amended the sample preparation step. the highest recoveries for majority of the tested herbicides were obtained after extraction with met:ac, therefore this solvent mixture was selected for future experiments. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 930 đurović-pejčev et al. table ii. dependence of liquid-solid (ls) extraction efficiency on type of organic solvent (met: methanol, ac: acetone, hex: hexane) and number of extraction steps (i-iv), using the most efficient solvent (met:ac) herbicide met i met:ac i met:ac:hex i met:ac ii met:ac iii met:ac iv metribuzin 98.41 99.33 99.02 102.94 101.62 102.09 acetochlor 91.39 94.68 95.24 98.61 98.86 96.02 clomazone 80.13 87.76 81.32 91.65 91.03 89.78 oxyfluorfen 59.36 79.68 65.78 84.26 81.38 82.69 dimethenamid 71.31 80.22 75.64 83.91 83.06 83.11 after selection of extraction solvent, the next step was to determine the optimal number of extraction steps. for that purpose, the extraction of spiked soil samples with methanol-acetone mixture was repeated up to four times using the same procedure. the results presented in table ii show that, for majority of the herbicides studied, the best recovery was achieved after two extraction steps. according to the results obtained in those two sets of experiments, two successive extractions with met:ac as the extraction solvent were chosen as the optimal sample preparation procedure. as clearly indicated in fig. 4, the additional spme step in our sample preparation method has real advantages as shown by comparative chromatograms obtained by direct injection (fig. 4c) of the extract (after liquid-solid extraction) and after additional hs-spme purification and concentration (fig. 4a) of the same extract. the chromatogram obtained after dm-spme of the selected herbicides in soil extract is presented in fig. 4b, too. the choice of experimental conditions for dm-spme determination was based on our previous investigation.13 evidently, compared to l–s method (fig. 4c), both additional spme steps (hs -spme (fig. 4a) and dm-spme (fig. 4b)) provide higher sensitivity in determination of all herbicides, and hs-spme approach is slightly better. since the spme fiber was not in direct contact with unwanted impurities (compounds that were co-extracted from the soil matrix during ls sample preparation) for hs-spme application, but was for dm-spme application, the obtained results were expected. validation of the proposed method the most important analytical parameters, such as linearity, limit of detection (lod), precision and confidence of the presented method were determined for the optimized ls extraction procedure followed by hs-spme measurement. concentration, ranging from 2 to 600 μg kg–1 was used for linearity testing of the developed method. the obtained arrangements and correlation coefficients (r) for all the herbicides under study are presented in table iii. the acquired correlation coefficients exceeded 0.99 for all the compounds tested, indicating good linearity. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ multi-class herbicides in soil 931 fig. 4. gc–ms chromatograms of soil sample fortified at 30 µg kg-1 level of each herbicide (clomazone (1), dimethenamid (2), acetochlor (3), metribuzin (4) and oxyfluorfen (5)), obtained by applying: a) l–s–hs-spme method, b) l–s–dm-spme method and c) l–s method (direct injection without additional spme step). _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 932 đurović-pejčev et al. the limit of detection (lod) was determined as 3.29×sb (where sb is the blank standard deviation), according to iupac recommendations.24 obtained lod for all the herbicides studied were less than 1.2 µg kg-1 (table iii). table iii. analytical characteristics of the proposed ls–hs-spme method for all the herbicides studied; r, correlation coefficient; lod, limit of detection; rsd, relative standard deviation; linearity range: 2–600 µg kg-1 herbicide r lod / µg kg-1 rsd / % recovery, % metribuzin 0.995 1.16 8.1 102.94 acetochlor 0.992 0.34 12.1 98.61 clomazone 0.996 0.10 6.3 91.65 oxyfluorfen 0.993 0.59 3.8 84.26 dimethenamid 0.998 0.87 10.2 83.91 confidence and precision of the method were determined by performing four consecutive measurements of the soil samples fortified at 30 µg kg-1 level. rsd and recovery values are presented in table iii. as shown, rsd for all herbicides were below 13 %. as rsd below 20 % are considered acceptable in trace analysis,25 the proposed method can be satisfactory in terms of precision. for all the herbicides studied, the recovery values were above 83 %, indicating that the proposed method could be used for efficient determination of the selected herbicides from complex matrix samples such as soil. application of the l–s–hs-spme method l–s–hs-spme method proposed in this study, as well as l–s–dm-spme method proposed in our previous study,13 were used for analysis of twelve soil samples from belgrade agricultural area. quantification was done using spiked soil samples that were used in the optimization procedure. the obtained results showed that with the exception of acetochlor, detected in only one sample (concentration of 13.0 µg kg-1 determined using l–s–hs-spme and 13.2 µg kg-1 by l–s–dm-spme method), all the other herbicides remained below detection limits. the effectiveness of both l–s–hs-spme and l–s–dm-spme methods for routine analysis of real samples were confirmed by excellent agreement with the obtained results. however, as soil represents extremely complex matrix, it seems that hs-spme approach is more appropriate for herbicide residue analysis, considering that in this method the fiber is not in direct contact with the sample, which enables an extension of its lifespan and reduces the matrix effects. acceptable precision and results repeatability obtained with the same spme fiber during experiments presented in this paper showed that a single pdms fiber could be utilized for more than 130 measurements. on the other hand, our previous studies showed that the same pdms fiber could be used for fewer injections (about 70–80 times) when dm-spme mode was used for determination of pesticides in the soil matrix.13 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ multi-class herbicides in soil 933 conclusion the multi-residue method based on a combination of liquid–solid sample preparation followed by hs-spme herbicide determination was used for the simultaneous determination of five herbicides belonging to different pesticide groups. investigation and optimization of microextraction conditions, such as temperature, extraction time and nacl content was performed using 100 μm polydimethyl–siloxane (pdms) fiber. during the optimization, the extraction efficiencies of several solvents and the optimal number of extraction steps for sample preparation were tested as well. results indicated that two successive extractions with mixture of methanol:acetone (1:1 volume ratio) as the extraction solvent could be used as optimal sample preparation procedure. subsequently for the hs-spme method, temperature of 75 °c, 300 g dm–3 nacl content and 30 min microextraction time could be set as microextraction conditions to yield most effective analysis. comparing the results obtained by application of the developed hs-spme method and those published for application of dm-spme method for the same set of herbicides and soils under study, we inferred that both methods are suitable for the routine determination of selected herbicides in the soil samples. however, since the proposed hs-spme mode secures an extended fiber lifetime, compared to the dm-spme mode, it could be more appropriate for analyzing complex soil matrix. acknowledgment. this study was carried out as part of the projects no tr31043 and iii43005, supported by the ministry of education, science and technological development of the republic of serbia. и з в о д метода микроекстракција у чврстој фази–узорковање из гасовите фазе у одређивању хербицида из различитих хемијских група у узорцима земљишта рада ђуровић-пејчев1, тијана ђорђевић1 и војислава бурсић2 1институт за пестициде и заштиту животне средине, банатска 31б,11080 београд, 2пољопривредни факултет, универзитет у новом саду, трг доситеја обрадовића 8, 21000 нови сад у раду је представљена метода за истовремено одређивање пет хербицида (метрибузин, ацетохлор, кломазон, оксифлуорфен и диметенамид) у земљишту, који на основу своје структуре припадају различитим хемијским групама пестицида. предложена метода микроекстракција у чврстој фази-узорковање из гасовите фазе (hs-spme) у комбинацији са течно–чврстом припремом узорака земљишта (l–s) је оптимизована и примењена за анализу реалних узорака пољопривредног земљишта. ортимизовање микроекстракционих услова, као што су температура, екстракционо време и садржај натријум-хлорида је извршена употребом 100 μm полидиметил-силоксанског (pdms) влакна. испитиване су такође екстракционе ефикасности различитих растварача (метанол, метанол:ацетон и метанол:ацетон:хексан у запреминским односима 1:1 и 2:2:1, редом), као и оптималан број екстракционих корака у току припреме узорака земљишта. дет_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 934 đurović-pejčev et al. екција и квантификација испитиваних хербицида су извршени методом гасно–масене спектрометрије (gc–ms). вредности релативних стандардних девијација и приноса одређивања хербицида у узорцима земљишта обогаћеним до концентрација од 30 μg kg-1 сваког једињења су биле испод 13 %, односно изнад 83 %, редом, док су границе детекције биле ниже од 1,2 μg kg-1. (примљено 23. новембра 2015, ревидирано 21. априла, прихваћено 13. маја 2016) references 1. united states environmental protection agency (us epa), http://www.epa.gov/pesticides/pestsales/07pestsales/usage2007.htm (accessed on 23/09/2015) 2. m. möder, p. popp, e. eisert, j. pawliszyn, fres. j. anal. chem. 363 (1999) 680 3. c.g. zambonin, f. palmisano, j. chromatogr., a 874 (2000) 247 4. s. bengtsson, t. berglöf, j. agric. food chem. 44 (1996) 2260 5. h. prosen, s. fingler, l. zupančiċ-kralj, v. drevenkar, chemosphere 66 (2007) 1580 6. a.a. boyd-boland, s. magdic, j.b. pawliszyn, analyst 121 (1996) 929 7. j. castro, r.a. perez, c. sanchez-brunete, j.l. tadeo, chromatographia 53 (2001) s-361 8. m. fernandez-alvarez, m. llompart, j. pablo lamas, m. lores, c. garsia-jares, r. cela, t. dagnac, j. chromatogr., a 1188 (2008) 154 9. a. navalón, a. prieto, l. araujo, j.l. vílchez, j. chromatogr., a 946 (2002) 239 10. a. navalón, a. prieto, l. araujo, j.l. vílchez, chromatographia 54 (2001) 377 11. f. hernandez, j. beltran, f.j. lopez, j.v. gaspar, anal. chem. 72 (2000) 2313 12. a. bouaid, l. ramos, m. gonzalez, p. fernandez, c. camara, j. chromatogr., a 939 (2001) 13 13. r. đurović, j. gajić umiljendić, s. cupać, lj. ignjatović, j. braz. chem. soc. 21 (2010) 985 14. l. hou, k. lee, j. chromatogr., a 1038 (2004) 37 15. h. prosen, l. zupančić-kralj, acta chim. slov. 45 (1998) 1 16. r. ðurović, t. ðorđević, lj. šantrić, s. gašić, lj. ignjatović, j. environ. sci. health, b 45 (2010) 626 17. r. ðurović, t. ðorđević, j. environ. sci. health, b 47 (2012) 851 18. r. ðurović, t. ðorđević, lj. šantrić, j. aoac int. 95 (2012) 1331 19. pesticidemanual, c d s tomlin, 12th ed., british crop protection council, alton, 2000– –2001 20. ppdb: pesticide properties database, university of hertfordshire, http://sitem.herts.ac.uk/aeru/ppdb/en/atoz.htm (accessed on 30/10/2015) 21. r. ðurović, m. marković, d. marković, j. serb. chem. soc. 72 (2007) 879 22. j. pawliszyn, solid phase microextraction – theory and practice, wiley-vch, new york, 1997, pp. 89–95, 133 23. j. ai, anal. chem. 70 (1998) 4822 24. l. a. currie, anal. chim. acta 391 (1999) 127 i. taverniers, m. de loose, e. van bockstaele, trends anal. chem. 23 (2004) 53. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice the adsorption behavior and mechanistic investigation of cr(vi) ions removal by poly(2-(dimethylamino)ethyl methacrylate)/poly(ethyleneimine) gels j. serb. chem. soc. 80 (7) 889–902 (2015) udc 546.766+541.183+66.095.26+ jscs–4767 544.713.432 original scientific paper 889 the adsorption behavior and mechanistic investigation of cr(vi) ions removal by poly(2-(dimethylamino)ethyl methacrylate)/poly(ethyleneimine) gels zhengbo hou, weixia zhu, hang song, pengfei chen and shun yao* school of chemical engineering, sichuan university, chengdu, 610065, china (received 14 july, revised 24 december 2014, accepted 4 march 2015) abstract: the composite hydrogels based on 2-(dimethylamino)ethyl methacrylate (dmaema) and polyethyleneimine (pei) were prepared by amino radical polymerization and confirmed by fourier transform infrared (ftir) spectroscopy and elemental analysis. then adsorption behavior and mechanism of cr(vi) ions adsorption on the obtained materials were investigated by scanning electron microscopy, zeta potential determination and thermogravimetric analysis, etc. the adsorption process was found to follow pseudo-second order kinetics and the langmuir model, and the maximum adsorption capacity of cr(vi) ions reached 122.8 mg g-1. x-ray photoelectron spectroscopy (xps) and ph analysis revealed that the cr(vi) ions were adsorbed into the gels through the electrostatic interaction mechanism, and so4 2in the solution had a great effect on the adsorption process. in addition, a high ph and ionic strength could reduce the uptakes of the adsorbate, which could be used for desorption of cr(vi) ions from the gels. keywords: 2-(dimethylamino)ethyl methacrylate; polyethyleneimine; adsorption; cr(vi); polymerization. introduction at present, many techniques, such as chemical reduction, adsorption, electrolysis, ion exchange, precipitation etc., are employed to remove cr(vi) ions from wastewater.1–3 cr(vi) ions are more toxic than cr(iii).4 among the reported traditional techniques, adsorption is an effective method due to the adventages of high selectivity, no secondary pollution, easy operation and the possibility of adsorbent recycling. various adsorbents have been used to remove cr(vi), such as macroporous 1,2-ethylenediamine-aminated polystyrene particles.5 crosslinked cassava residue xanthate,6 functionalized ordered mesoporous carbon,7 hard walnut shell,8 β-feooh-coated sand,9 nitrogen-enriched activated * corresponding author. e-mail: cusack@scu.edu.cn doi: 10.2298/jsc140701024h _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 890 hou et al. carbon from waste medium density fiberboard,10 a chitosan-coated fly ash composite,11 iron(iii)-impregnated sorbent prepared from sugarcane bagasse,12 etc. their maximum adsorption capacities were 100.06, 37.76, 68.21, 80.24, 0.060– –0.076, 89.21, 33.00 and 13.72 mg g–1, respectively. at present, the exploration for new practical, economic and efficient adsorbents for the removal of cr(vi) ions is still meaningful and necessary. in recent years, hydrogels prepared from 2-(dimethylamino)ethyl methacrylate (dmaema) and its co-polymers have attracted much interest due to their ph and thermal sensitivity.13,14 a large number of research reports were concerned with more special environment sensitivities and their applications for water treatment.15–17 poly-dmaema exhibited a high adsorption capacity (70 mg g–1) for cr(vi).18,19 polyethyleneimine (pei) is a water-soluble polyamine with many nitrogen atoms of amino groups on its macromolecular chains, which could interact effectively with some kinds of pollutants. therefore, the copolymerization of pdmaema and pei was expected to provide a material with a high adsorption capacity for the removal of cr(vi) ions from aqueous solution. in this study, pei macromolecules were grafted onto pdmaema via free radical polymerization on the amine nitrogen atoms, yielding pei/pdmaema for the first time as a novel cationic composite material for the adsorption of anions. the adsorption properties of cr(vi) ions on the pei/pdmaema were studied thoroughly. it was expected that this pei/pdmaema material would possess satisfactory adsorption capacity and rate for cr(vi) ions in water. experimental materials 2-(dimethylamino)ethyl methacrylate (dmaema) monomer, n,n′-methylenebisacrylamide (mbaa) cross-linker, and azobisisobutyronitrile (aibn) initiator were purchased from puguang industrial co., ltd. (each of purity >99 % , shanghai, china). branched polyethyleneimine (pei) (m̅w = 1800 g mol -1, 25 % aqueous solution, the content of primary, secondary and tertiary amino groups were 35, 35 and 30 %, respectively) was obtained from mengde electroplate chemistry co. (specific electro-plating class, jiangsu, china). k2cro4 was provided by jingqiu chemistry factory (ar, beijing, china). preparation of the adsorbents the polymerization procedures for dmaema monomer and mbaa cross-linker were studied in a previous study.19 for each polymerization, first, 3.144 g dmaema, 0.154 g mbaa, and 0.050 g aibn were dissolved in 3 ml of ethanol solution and the pei solution was diluted with distilled water to prepare a solution with the mass concentration (0–5 wt. %). then, the ethanolic mixture was added into 18 ml pei solution of different concentrations and stirred under a nitrogen atmosphere for 10 min. afterwards the solutions were immediately poured into tubes with 6 mm inner diameter, which were sealed and submerged in thermostatic water bath for 3 h at different temperatures. finally, the pei/pdmaema gels were rapidly cooled to room temperature and cut into 3-mm thick slices for further use. the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ cr(vi) adsorption on composite hydrogels 891 composite materials prepared with 0, 3 and 5 wt. % pei solution were marked as ed 0, ed 3 and ed 5, respectively. the amount of pei in the adsorbent (graft yield, gy) was determined by element analysis. according to the content of elements, gy could be calculated as follows: pei pdmaema (%) 100 w gy w = × (1) where wpei and wpdmaema are the pei and pdmaema weight percentage in the pei/ /pdmaema material, respectively, which were calculated from the results of the element analysis. characterization of the pdmaema and pei/pdmaema the ftir spectra of pdmaema and pei/pdmaema were recorded on spectrum one ftir spectrometer (perkin–elmer, usa) as kbr discs. the zeta potential analysis was performed using a pals zeta potential analyzer, version 3.43 (brookhaven instruments, usa). elemental analyses were accomplished with 2400 series ii chns/o elemental analyzer (perkinelmer, usa). the dtg curves were obtained using a frc/t-2 thermal analyzer (optical instruments factory, beijing, china) at a heating rate of 10 °c min-1 under a nitrogen atmosphere. the morphology of the dried composite materials after drying under vacuum at 50 °c for 24 h was observed using a jem-100cx-ii scanning electron microscope (sem). (jeol, japan). x-ray photoelectron spectroscopy (xps) data were obtained with an escalab220i-xl electron spectrometer (vg scientific, usa) using 300 w alkα radiation. to compensate for surface charge effects, the binding energies were calibrated using the c1s hydrocarbon peak at 284.8 ev. the hydrogels were immersed in water to reach a swollen equilibrium at 40 °c. afterwards, they were withdrawn and weighed after the removal of excess surface water. equilibrium swelling ratio (esr) is the moisture content to dry weight of hydrogel ratio: s d d w w esr w − = (2) where ws is the weight of the swollen gel at the desired time and wd is the weight of the dried gel. adsorption of cr(vi) ions a certain amount of k2cro4 was dissolved in redistilled water to obtain the stock solution. the concentrations of cr(vi) were determined by tu1810spc uv–vis spectrophotometer (puxitongyong instrument co., ltd., china) at 540 nm. for each adsorption experiment, the stock solution was diluted to desired concentration (c0, 25–225 mg l -1), and then the adsorbents were immersed into the conical flasks containing the diluted solution. finally, the conical flasks were placed in a shaking bed at different temperatures for desired time, and the concentration in supernatant was determined as ct. in addition, the solution ph was adjusted by 1 mol l-1 hcl or 1 mol l-1 naoh and monitored by a digital ph meter (yulong instrument co., ltd., china).the adsorption amount of cr(vi) (q, mg g-1) was calculated according to the following equation: 0 pei/pdmaema ( )tv c cq w − = (3) _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 892 hou et al. where v (ml) and wpei/pdmaema (g) stand for the solution volume and the mass of the adsorbent, respectively. desorption of cr(vi) ions firstly, the sorbents were immersed in 150 mg l-1 of cr(vi) solution at ph 2.0 for 4 h to reach equilibrium, respectively. the adsorption amount (qa, mg g -1) of cr(vi) ions, was calculated by eq. (3). then the sorbents were filtered and soaked in 10 ml of 0.1 mol l-1 naoh solution for 24 h at room temperature. finally, the concentration of cr(vi) ions in the eluent was determined, and the amount of desorption was denoted by qd (mg g -1). the desorption ratio was defined by the following equation: d a desorption ratio (%) 100 q q = × (4) results and discussion preparation process and characterization of pei/pdmaema as a very popular azo-initiator in polymerization processes, azobisisobutyronitrile (aibn) can easily produce nitrogen gas and radicals through splitting reaction. polyethyleneimine (pei) possesses many free amino groups. when they interact with the radicals of the aibn initiator, the amino group will lose a proton. as the result, a nitrogen radical (n•) together with 2-cyano-2-propyl radical (c•) could be obtained. then, the former (n•) could initiate the copolymerization of dmaema to form pei/pdmaema. the role of amine in vinyl radical polymerizations was summarized and analyzed in a previous study.20 in addition, the latter (c•) could either produce the polymerization among the monomer or abstract a hydrogen atom from the pei, resulting in the copolymerization of dmaema and pei. the related copolymerization process is shown in scheme 1. the ultrasound-assisted (at room temperature) and the soxhlet extraction methods with 95 % ethanol were first used to verify the copolymerization of pei on the polymer network. there was no or negligible residual remains in the extracted liquid after the removal of solvent under vacuum. the results indicated that pei had been successfully grafted onto the network and the copolymerization product was stable. furthermore, the formation of ed 0 and ed 5 could be further proved by ftir spectroscopy according to an analysis similar to that in previous reports.21–24 all of the ir spectra are shown in fig. 1. moreover, zeta potential analysis was used to characterize the pei/ /pdmaema products. the zeta potential of ed 0 was only +7.25 mv, but for ed 5, it became +20 mv in ph 2 hydrochloric acid buffer solution. this could be interpreted as proof of the incorporation of pei, which contains many amino functional groups. in order to analyze the content of nitrogen before and after introduction of pei, the ed 0, ed 3 and ed 5 materials were characterized by elemental analysis (table i). compared with ed 0 and ed 3, ed 5 had a higher content of nitrogen. according to elemental analysis, the amount of pei in the ed 5 was calculated to be 5.93 %. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ cr(vi) adsorption on composite hydrogels 893 scheme 1. copolymerization process of pei/pdmaema gels and adsorption of cr(vi). fig. 1. ftir spectra of pei, dmaema, pei/pdmaema (ed 5) and pdmaema (ed 0). _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 894 hou et al. table i. elemental analysis (%) of ed 0, ed 3 and ed 5 material element gypei / % c h n o ed 0 58.50 13.22 6.81 21.47 – ed 3 58.67 12.01 8.11 21.21 3.76 ed 5 58.60 10.08 9.32 22.00 5.93 the thermal stability of the pei, ed 0 and ed 5 gels were examined by thermal analysis (fig. 2). the removal of physically adsorbed and interlayer water occurred up to 150 °c, and the raw pei sample lost its 75 % weight in this temperature range. in the second stage, a significant weight loss of 44.68 % could be attributed to the decomposition of the functional amino-groups on the composite material when the temperature ascended to 300 °c. as shown in the differential thermogravimetric (dtg) curves, the rate of mass loss reached its maximum at 281 °c for ed 0, but was postponed by 20 °c after incorporation of pei. in the last stage, a smaller mass loss of 14.83 % in the 300–400 °c range was associated with degradation of the carbon skeletons, and the fastest decomposition rate appeared at 413 °c. these results implied that the ed 5 gel was successfully prepared and ed 5 could be applied in higher temperature system. fig. 2. thermogravimetric and differential thermogravimetric curves of pei, ed 0 and ed 5. equilibrium swelling ratios (esr) of pei/pdmaema the swelling behavior of the hydrogels in water at different temperatures is shown in fig. 3. as is clearly shown, the esr of ed 0 and the ed 5 gels _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ cr(vi) adsorption on composite hydrogels 895 decreased with increasing temperature and exhibited a marked inflexion when the temperature reached the special critical point, which is consistent with the lower critical solution temperature25–27 (lcst: around 40 °c and 65 °c for ed 0 and ed 5, respectively). figure 3 also shown the esr of ed 5 immersed in buffer solution as a function of ph value. the gel in the buffer solution with the low ph value presented higher swelling ratios than those of the gel in the alkaline solution. the results could be explained by the following reason: the free electron pair of the amino group conjugated with the carboxyl in the dmaema units when the gel was immersed in water. however, this coordination was severely weakened in the acidic solution because of protonation of the tertiary amino groups.27 fig. 3. equilibrium swelling ratios of ed 0 and ed 5 gels in purified water and ed 5 in acid or alkali solution at different temperatures. adsorption kinetics of cr(vi) ions adsorption duration is an important factor affecting the adsorption process and amount. the optimal duration of adsorption was investigated, and the results (fig. s-1 of the supplementary material to this paper) showed that the introduction of pei onto pdmaema gels had a significant effect on the adsorption behavior of cr(vi). when the concentration of pei was increased to 5 %, the amount of adsorbed cr(vi) ions increased to 122.8 mg g–1 and the adsorption rate was obviously improved, e.g., the adsorption by ed 5 reached complete saturation after 120 min, but for ed 0, 180 min were needed. a high adsorption rate is desirable to provide for a short contact time between the metal solution and the adsorbent in an actual process.28 furthermore, the influence of tempe_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 896 hou et al. rature in the preparation process could be ascribed to a change in the degree of crosslinking. the higher the reaction temperature was, the faster was the reaction rate, and the denser was the degree of crosslinking in the network. as the result, the inflexibility of polymer became stronger and the elasticity became weaker. it was very difficult for cr(vi) anions to be adsorbed by the internal active site with a too high degree of crosslinking. the kinetic model provides information regarding the mechanism and thus the adsorption data were correlated with the pseudo-first order kinetic model and pseudo-second order kinetic model.29 the adsorption kinetic parameters are summarized in table ii. it was found that the theoretical qe,cal values for the adsorbate were very close to the experimental qeq values in the case of the pseudo-second order kinetics. furthermore, all the linear fitting coefficients (r2) of the pseudo-second order kinetics were above 0.99, which were slightly higher than were those of the pseudo-first order kinetics. thus, the pseudo-second order equation fitted well with the experimental data and could describe the adsorbent behavior well. table ii. the adsorption kinetic parameters of cr(vi) ions onto the gels; qe,exp: the equilibrium adsorption amount of cr(vi) (experimental data); k1: the adsorption rate constant for the pseudo-first order model; qe,cal: the equilibrium adsorption amount of cr(vi) (calculated data); k2: the adsorption rate constant for the pseudo-second order model sample qe,exp mg g-1 pseudo-first order model pseudo-second order model k1×10 -3 min-1 qe,cal mg g-1 r2 k2×10 -3 g mg-1 qe,cal mg g-1 r2 ed 0 73.17 16.31 71.30 0.9508 0. 2929 81.94 0.9932 ed 3 95.34 22.98 87.91 0.9637 0.4276 102.1 0.9974 ed 5 122.8 25.42 88.12 0.9643 0.6052 128.0 0.9991 75 °c 91.86 29.75 116.3 0.8881 0.4094 99.41 0.9967 85 °c 122.8 25.37 88.12 0.9643 0.6047 128.0 0.9991 95 °c 85.03 15.59 68.44 0.9867 0.3897 92.25 0.9982 adsorption isotherm of cr(vi) ions adsorption isotherm is one of the important parameters for understanding the adsorption behavior and mechanism. the adsorption equilibrium curves are shown in fig. s-2 of the supplementary material, and the langmuir and freundlich isotherm models were used to fit the equilibrium data.30–33 as shown in table iii, the values of qe,cal derived from the langmuir model increased with increasing temperature up to 40 °c but decreased with further increasing of the temperature. this could be explained as follows: the elevated temperature enhanced the deprotonation of amino-groups and resulted in a weaker interaction between the amino groups and cr(vi) ions.16 in addition, from the comparison of r2, rl and k values, it was found that the langmuir model could fit much better with the experiment data. overall speaking, the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ cr(vi) adsorption on composite hydrogels 897 langmuir isotherm gave a better fit to the experimental data than the freundlich isotherm. the results indicated insights on the monolayer coverage of pei/ /pdmaema composite and that there were no side interactions among the adsorbates. furthermore, the site-specific interactions took place between the binding sites and anions, and the adsorbent surface was homogeneous. table iii. the adsorption isotherms parameters of cr(vi) ions onto the ed 5; kl: the constant of the langmuir isotherm; rl: the separation factor of langmuir isotherm (favorable (0 <rl <1), unfavorable (rl >1), linear relationship (rl = 1) and irreversible (rl =0) 30-32); kf: the constant of the freundlich isotherm; k: the slope of freundlich isotherm model (langmuir isotherm (k <1), freundlich isotherm (k >1)32-34) t / °c langmuir model freundlich model kl / 10 2 l mg-1 qe,cal / mg g -1 r2 rl kf / mg g -1 k r2 30 2.719 146.2 0.9915 0.4238–0.1379 11.23 0.4980 0.9802 40 5.366 172.7 0.9879 0.2715–0.0749 19.41 0.4771 0.8967 50 3.745 159.0 0.9943 0.3481–0.1040 15.36 0.4755 0.9764 60 3.063 153.1 0.9981 0.3950–0.1243 12.12 0.5035 0.9718 effect of initial ph value on the adsorption process the ph value influences the distribution of cr(vi) ions species, fig. 4 (main forms: h2cr2o7 (ph < 1), hcro4– and cr2o72– (1 < ph < 6) and cro42– (ph > > 6)). for pei/pdmaema, the ph value affects the protonation and deprotonation of amino groups, which are the active adsorption sites for anions. the maximum amount of cr(vi) ions adsorption onto ed 5 and ed 0 were both obtained at ph 2.0. with increasing ph values, the interaction between anions and amine groups weakened because of the deprotonation of the amino groups and the competition of oh–. however, when the ph value was below 2.0, the uptake of fig. 4. the effects of ph on the adsorption of cr(vi) ions onto the gels at 40 °c. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 898 hou et al. cr(vi) ions decreased because the cr(vi) ions converted into h2cr2o7, which unfavorably interacted with the protonated amine groups. furthermore, when the ph value was 10.0, the cr(vi) uptake was still up to 32 mg g–1 onto the ed 5. it could be concluded that the adsorption amount reached maximum values around a ph value of 2.0, and the working ph range of the composite adsorbent for cr(vi) ions was obviously improved due to the introduction of pei. effects of ionic strength and species on the adsorption process the results are shown in fig. s-3 of the supplementary material. the adsorption capacities of the gels remarkably decreased with increasing concentration of nacl because of the competition between cl– and the anionic adsorbents. the effect of ionic species could be divided into two groups: cationic group (nacl, kcl and nh4cl) and anionic group (nacl, nano3 and na2so4). the results showed that the effects of ions (na+, k+, nh4+, cl– and no3–) on the adsorption process were not obvious. comparatively speaking, the so42– had a great effect on the adsorption process. this could be summarized as the competition between cr(vi) and so42–. morphology and xps analysis figure 5 shows the sem images of ed 0, ed 5 and ed 5 after adsorption of cr(vi). these samples were coated with a thin layer of gold and examined by fig. 5. the sem images of (a) ed 0, (b) ed 5 and (c) ed 5 after cr(vi) adsorption. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ cr(vi) adsorption on composite hydrogels 899 sem. after the introduction of pei on the polymer, there were no significant changes in the morphology of the gels. however, clearly ragged topographies appeared after cr(vi) ions adsorption, which was relevant to the formation of bubbles during the adsorption process. the xps spectra of the ed 5 composite material before and after cr(vi) adsorption are shown in fig. 6a. before adsorption, only the peaks at the binding energies of ca. 284.8, 399.5 and 532.4 ev, corresponding to c, n and o, res (a) (b) fig. 6. a) the xps spectra of ed 5 composite material before (a) and after (b) cr(vi) adsorption; b) the results of a meticulous xps analysis of the ed 5 composite material for (a) cr2o7 2and (b) hcro4 adsorption. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 900 hou et al. pectively, could be found in the xps spectrum of ed 5. after adsorption, new significant bands corresponding to cr(vi) appeared at binding energy of ca. 577.2 and 586.8 ev. the result confirmed that cr(vi) had been adsorbed onto ed 5. furthermore, a meticulous xps analysis was performed using fitting software and the results were presented in fig. 6b. the main peak of ca. 277.5 ev was made up of 577.8 and 576.2 ev peaks, which indicated that the forms of the adsorbed cr(vi) ions were hcro4– and cr2o72–.35 this conclusion was in accord with the fact that hcro4– and cr2o72– exist mainly in the ph range of 1.0–6.0. desorption of cr(vi) ions according to repeated experiments, the ph value of the solution could obviously affect the interaction between anionic adsorbates and the amine groups in the composite materials. thus, 0.1 mol l–1 naoh solution was chosen for the desorption process. the results showed 96.3 % of cr(vi) ions were eluted from ed 5 by the alkali liquor, far higher than the 56.8 % by distilled water. then, the ed 5 was washed to neutral with distilled water and reused to adsorb cr(vi) ions. the adsorption capacity remained high at 108.5 mg g–1 after being recycled five times. there was no significant reduction in its adsorption efficiency. the results also revealed that the pei/pdmaema gels were stable under these experimental conditions. conclusions using the amino radical polymerization technology, the composite material pei/pdmaema was conveniently fabricated, and the complex grafting process was avoided. the composite material was characterized by ftir spectroscopy, elemental analysis, thermogravimetric analysis and zeta potential analysis. the equilibrium adsorption amount of cr(vi) on ed 5 was 122.8 mg g–1. the results of the influence of temperature, ph and xps analysis showed that the adsorption mechanism was electrostatic interaction, and the forms of cr(vi) ions adsorbed onto the ed 5 at ph 2 were hcro4– and cr2o72–. the desorption ratio of cr(vi) ions could be 96.3% using 0.1 mol l–1 naoh solution as the eluent and the composite material pei/pdmaema showed an excellent recyclability efficiency. supplementary material additional cr(vi) adsorption data are available electronically from http:// //www.shd.org.rs/ jscs/, or from the corresponding author on request. acknowledgments. preparation of this paper was supported by the national scientific foundation of china (no. 81373284) and the 2013 scientific research foundation of sichuan university for outstanding young scholars. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ cr(vi) adsorption on composite hydrogels 901 и з в о д испитивање мехнизма адсорпције при уклањању cr(vi) јона геловима поли(2-(диметиламино)етил-метакрилат)/поли(етиленимин) zhengbo hou, weixia zhu, hang song, pengfei chen и shun yao school of chemical engineering, sichuan university, chengdu, 610065, china композитни хидрогелови базирани на 2-(диметиламино)етил-метакрилату (dmaema) и полиетиленимину (pei) су добијени полимеризацијом амино радикала, што је потврђено инфрацрвеном спектроскопијом са фуријеовом трансформацијом (ftir) и елементалном анализом. мехнизам адсорпције cr(vi) на добијеним материјалима је испитиван скенирајућом електронском микроскопијом, одређивањем зета потенцијала и термогравиметријском анализом. нађено је да процес адсорпције прати кинетику псеудо-другог реда и лангмиров модел, као и да је максимални адсорпциони капацитет cr(vi) јона 122,8 mg g-1. анализом фотоелектронском спектроскопијом x-зрачења (xps) и ph мерењима је показано да су cr(vi) адсорбовани на геловима електростатичким интеракцијама, и да so4 2у раствору има значајан утицај на процес адсорпције. уз то, високе вредности ph и јонске јачине погодују редукцији адсорбованих cr(vi) јона, што се може искористити приликом њихове десорпције са гелова. 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babić, b. z. matović, j. serb. chem. soc. 79 (2014) 481 33. y. liu, y. zheng, a. wang, j. environ. sci. 22 (2010) 486 34. f. haghseresht, g. q. lu, energ. fuel 12 (1998) 1100 35. k. selvi, s. pattabhi, k. kadirvelu, bioresour. technol. 80 (2001) 87. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice antimicrobial and anticancer evaluation of a novel synthetic tetracyclic system obtained by dimroth rearrangement j. serb. chem. soc. 80 (10) 1251–1264 (2015) udc 547.556.9+66.095.252.094.3:541.124: jscs–4793 547.791’821:615.277 original scientific paper 1251 antimicrobial and anticancer evaluation of a novel synthetic tetracyclic system obtained by dimroth rearrangement sobhi m. gomha1*, ikhlass m. abbas1, mohamed a. a. elneairy1, mahmoud m. elaasser2 and bazada k. a. mabrouk1 1department of chemistry, faculty of science, cairo university, giza, egypt and 2regional center for mycology and biotechnology, al-azhar university, cairo, egypt (received 22 december 2014, revised 18 february, accepted 4 march 2015) abstract: a series of pyrido[3′,2′:4,5]thieno[2,3-e][1,2,4]triazolo[4,3-c]pyrimidines were prepared via oxidative cyclization of 4-(2-arylidenehydrazinyl)pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidines. dimroth rearrangement of such a series yielded pyrido[3′,2′:4,5]thieno[2,3-e][1,2,4]triazolo[1,5-c]pyrimidines. a reaction mechanism was proposed and the products were screened for their antimicrobial and anticancer activities. from the antimicrobial screening results, it could be seen that compounds 8c, 9f and 10c showed excellent activity against gram-positive bacteria while compounds 10d and 8c showed the highest activity against gram-negative bacteria. the results of the anticancer activities showed that compound 9c was the most active against hepg-2 and mcf-7 with ic50 values of 1.19 and 3.46 μg/ml, respectively. keywords: hydrazones; oxidative cyclizations; thienopyridines; reaction mechanism. introduction the synthesis of fused triazolopyrimidine moieties has been described by many investigators and it was shown that these have pronounced biological activities.1–6 previous observations revealed that [1,2,4]triazolo[4,3-c]pyrimidine derivatives can isomerize under different suitable reaction conditions to the thermodynamically more stable [1,2,4]triazolo[1,5-c]pyrimidines.7–9 this isomerization was first reported by miller and rose10,11 when they treated [1,2,4]triazolo[4,3-c]pyrimidine derivatives with an acid, base, or thermally. in continuation of an ongoing search for new bioactive heterocycles12–20 based on the above-mentioned research results, the goal of this study was to synthesize some novel pyrido[3′,2′:4,5]thieno[2,3-e][1,2,4]triazolo[4,3-c]pyramidines not only to study their isomerization to pyrido[3′,2′:4,5]thieno[2,3-e][1,2,4] * corresponding author. e-mail: s.m.gomha@hotmail.com doi: 10.2298/jsc141222022g _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1252 gomha et al. triazolo[1,5-c]pyrimidines, but also to obtain new compounds which were expected to find notable pharmacological applications. results and discussion chemistry detailed analytical and spectral data of the synthesized compounds are given in the supplementary material to this paper. synthesis of compound 4 was achieved through two synthetic pathways. the first pathway involved the conversion of 121 to 7,9-bis(4-methoxyphenyl)pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4(3h)-one (2) through its cyclization with formic acid (scheme 1). the ir spectra of 2 displayed no cyano group absorptions. scheme 1. synthesis of the starting materials 4 and 6. chlorination of 2 with phosphorus oxychloride under reflux afforded 3. nucleophilic displacement of the 4-chloro group in 3 was achieved by heating under reflux with hydrazine hydrate to give compound 4.22 the ir spectrum of the titled compound 4 showed the appearance of absorption bands at 3424–3291 cm–1 for nh and nh2), its 1h-nmr spectrum showed the presence of characteristic peaks at δ 5.01 and 9.02 ppm of nh2 and nh groups, respectively, while the mass spectrum showed a molecular ion peak at m/z 429 corresponding to its molecular formula c23h19n5o2s. detailed analytical and spectral data are given in the supplementary material to this paper. these results confirmed the chemical structure of compound 4 (scheme 1). _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ tetracyclic system through dimroth rearrangement 1253 the second pathway was the reaction of 1 with dmf–dma under reflux for 4 h to give n′-(2-cyano-4,6-bis(4-methoxyphenyl)thieno[2,3-b]pyridin-3-yl) -n,n-dimethylformimidamide (5). ir spectrum of 5 showed an absorption band at 2191 cm–1 assigned to the cyano group (cn). furthermore, its 1h-nmr spectrum showed two singlet signals at 2.64 and 2.87 ppm, which were indicative of the n(ch3)2 group in this structure. the mass spectrum of 5 showed the molecular ion peak at m/z 442 corresponding to its molecular formula c25h22n4o2s. reaction of the latter compound with hydrazine hydrate (99 %) in ethanol afforded 6. the formation of compound 6 was assumed to proceed via the loss of a dimethylamine from 5 followed by intramolecular cyclization into 6. the ir spectrum of 6 revealed the absence of the cyano group and the appearance of absorption bands at 3472, 3341 and 3220 cm–1 for nh and nh2. its 1h-nmr spectrum showed three singlets at δ 4.92, 9.02 and 8.44 ppm which indicated the presence of nh2, nh and the pyrimidine proton, respectively. isomerization of 6 to its corresponding more thermodynamically stable hydrazine compound 4 was realized by refluxing 6 in dioxane in the presence of sodium acetate through a dimroth-type rearrangement, which involves a sequence of ring opening and ring closure reactions under basic conditions, as shown in scheme 2.23,24 scheme 2. mechanism of the rearrangement of compound 6 to 4. the identity of compounds 4 and 6 was proven based on their melting points (m.p.), thin layer chromatography (tlc) and spectral data. condensation of equimolar quantities of 4-hydrazinylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine (4) with the aldehydes 7a–h gave the corresponding 4-(2 -benzylidenehydrazinyl)-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidines 8a–h (scheme 3). the structures of the products 8a–h were confirmed based on their elemental analysis and spectral data (see the supplementary material to this paper). _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1254 gomha et al. scheme 3. synthesis of fused [1,2,4]triazolo[1,5-c]pyrimidine derivatives 10a–h. the ir spectrum of 8a, taken as a typical example of the prepared series, revealed an absorption band at 3440 cm–1 due to the nh group. their 1h-nmr spectra showed the presence of hydrazone (–ch=n–nh–)protons as two singlets at δ 8.82 and 12.16 ppm, respectively (see supplementary material). next, the oxidative cyclization of the hydrazone derivatives 8a–h with iron (iii) chloride in ethanol yielded the respective triazolo[4,3-c]pyrimidine derivatives 9a–h (scheme 3). tlc analysis of the crude products isolated from the foregoing dehydrogenative cyclization method indicated that only one product was formed in each ease. the mass spectra of the isolated products showed high intensity molecular ion peaks at the expected m/z values that were less by two than those of the corresponding hydrazone 8. their ir spectra showed the disappearance of the nh group. their 1h-nmr spectra lacked the characteristic signals for the azomethine (–ch=n–) and the hydrazone (–c=n–nh–) protons present in the spectra of 8. the conversion of 8 into 9 is reminiscent of other related oxidative cyclization of aldehyde n-heteroarylhydrazones with bromine in acetic acid in the presence of sodium acetate or iron(iii) chloride, which have been reported to proceed via the generation of the respective nitrilimines, which undergo in situ 1,5-electrocyclization to give the respective fused heterocycles.25–27 when each of the triazolo[4,3-c]pyrimidine derivatives 9a–h was heated in ethanol in the presence of sodium acetate, they isomerized to the thermodynamically more stable triazolo[1,5-c]pyrimidine derivatives 10a–h through tandem ring opening and ring closure reactions, as shown in scheme 4. this rearrangement is consistent with those reported in some earlier reports.8,28 the structure _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ tetracyclic system through dimroth rearrangement 1255 elucidation of all compounds 10a–h, which have not been reported, hitherto, was based on their spectral and analytical data (see the supplementary material). in the mass spectra, all compounds gave the molecular ions at the expected m/z values, which in most cases are not the base peaks. the 1h-nmr spectra were also consistent with their proposed structures. scheme 4. mechanism of the rearrangement of compounds 9a–h to 10a–h. to provide decisive evidence for this rearrangement, the products 10a–h were compared with authentic samples prepared by an alternative synthesis. thus, treatment of 6 with the respective aromatic carboxylic acids 11a–h in presence of polyphosphoric acid gave products 10a–h, which proved identical in all respects (m.p., mixed m.p., ir and 1h-nmr spectra) with those obtained above from base-catalyzed rearrangement of 9a–h (scheme 3). this finding confirmed the base-catalyzed rearrangement of 9 into 10 (scheme 3). the driving force for the observed rearrangement is the fact that the [1,2,4]triazolo[1,5-c]pyrimidine ring system is thermodynamically more stable than its isomer, namely [1,2,4]triazolo[4,3-c]pyrimidine.29 antimicrobial evaluation the synthesized compounds were evaluated for their in vitro antimicrobial activity at 5 mg ml–1 using the agar well diffusion method against a representative panel of pathogenic strains, i.e., staphylococcus aureus and bacillis subtilis as examples of gram-positive bacteria as well as pseudomonas aeruginosa and escherichia coli as examples of gram-negative bacteria while aspergillus fumigatus and candida albicans were used as the fungal strains. dmso was used for dissolving the tested compounds and showed no inhibition zones, confirming that it has no influence on growth of the tested microorganisms. the results of the testing for antibacterial and antifungal effects summarized in table i showed that _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1256 gomha et al. the new derivatives tested displayed variable in vitro antibacterial and antifungal actions. in general, the chemical structure of the whole molecule, comprising the nature of the heterocyclic system as well as the type of the substituted function present in the heterocyclic ring structure, has a pronounced effect on the antimicrobial activity. most of the substituted analogues produced high inhibitory effects against bacteria, which were comparable to the effects of reference drugs. table i. in vitro antibacterial and antifungal activity of the compounds tested by the well -diffusion agar assay expressed as the diameter (mm) of the inhibition zone in the form of mean ± sd tested compound gram-positive bacteria gram-negative bacteria fungi b. subtilis s. aureus e. coli p. aeruginosa a. fumigatus c. albicans 8a 6.7±0.4 7.8±0.5 11.8±0.9 9.4±0.7 0 0 8b 6.1±0.3 6.4±0.4 9.2±0.3 7.3±0.6 0 0 8c 22.4±0.7 20.1±0.9 21.9±0.8 18.2±0.7 0 6.9±0.3 8f 6.5±0.4 6.8±0.6 7.1±0.4 6.9±0.5 22.1±0.8 13.3±0.7 9e 6.3±0.3 6.6±0.4 6.2±0.4 6.7±0.6 0 0 9f 20.9±0.6 21.4±0.8 16.9±0.9 12.1±0.7 19.3±0.4 7.4±0.3 9h 17.3±0.7 15.9±0.5 13.7±0.6 8.4±0.5 16.8±0.6 6.7±0.4 10c 19.8±0.7 13.8±0.8 7.2±0.5 6.5±0.4 19.7±0.8 10.6±0.6 10d 14.3±0.6 17.0±0.6 24.8±0.7 19.5±0.8 11.2±0.6 0 10f 18.9±0.5 14.1±0.6 6.7±0.4 6.6±0.5 10.4±0.7 0 10g 0 0 15.4±0.7 13.6±0.7 0 0 penicillin g 26.4±0.7 24.6±0.6 – – – – streptomycin – – 26.7±0.9 20.4±0.5 – – amphotericin b – – – – 25.9±0.8 20.1±0.7 from the screening results, it could be seen that compounds 8c, 9f and 10c showed excellent activity against the gram positive bacteria and compounds 10f, 9h and 10d showed moderate effects. the highest activity against gram negative bacteria were exerted by 10d and 8c. selective antimicrobial activity was observed under these screening conditions for compound 10g against the tested gram-negative bacteria. compounds 4, 6, 8d, 8e, 8g, 8h, 9a–d, 9g, 10a, 10b, 10e and 10h were completely inactive under these screening conditions. the rest of the compounds showed non-significant activity against the tested bacteria compared with the standard drugs. interestingly, compounds 8f, 10c and 9h showed promising inhibitory activity against a. fumigatus and c. albicans compared with amphotericin b as a reference drug. the mean values of the inhibition zone diameter obtained for these compounds suggested that some of the synthesized compounds possess significant antimicrobial activity against the tested organisms used in these assays, and therefore, the minimum inhibitory concentration (mic) of the active compounds were evaluated in vitro using the microdilution technique, while the lowest con_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ tetracyclic system through dimroth rearrangement 1257 centration showing no growth was taken as the mic. the fungicides amphotericin b and griseofulvin as well as the bactericides penicillin g and streptomycin were used as reference drugs to evaluate the potency of the tested compounds under the same conditions. the results of the mic determinations reported in table ii showed that compounds 8f, 9f, 9h, 10c, 10d and 10f exhibited broad spectrum action against both gram‐positive and gram-negative bacteria as well as fungi. compound 8c had broad-spectrum antibacterial activity. compound 9f reached the highest potency with mic values of 15.6 and 31.25 µg ml–1 against the gram‐positive bacteria b. subtilis and s. aureus, respectively. the highest activity (31.25 µg ml–1) against e. coli was detected for compound 10d. moreover, significant mic values were determined for compounds 8f and 9f against a. fumigatus compared with the reference drugs. table ii. antimicrobial activity expressed as minimum inhibitory concentration (mic / μg ml-1) of the synthesized bioactive compounds compared with standard drugs tested compound microorganism gram-positive bacteria gram-negative bacteria fungi b. subtilis s. aureus e. coli p. aeruginosa a. fumigatus c. albicans 8c 62.5 62.5 62.5 125 – – 8f 500 500 500 500 31.25 250 9f 15.63 31.25 250 125 31.25 500 9h 125 125 125 250 125 500 10c 62.5 250 250 250 62.5 500 10d 125 125 31.25 125 250 – 10f 125 250 500 500 250 – penicillin g 0.03 0.06 – – – – streptomycin – – 0.03 3.9 – – amphotericin b – – – – 0.3 2.9 griseofulvin – – – – 100 250 cytotoxic activity the in vitro growth inhibitory activity of the synthesized compounds was investigated in comparison with the well-known standard anticancer drug doxorubicin using the crystal violet colorimetric viability assay. for comparison purposes, the cytotoxicity of imatinib (2-substituted aminopyrimidine derivative; gleevec®), a standard antitumor drug used for the treatment of gastrointestinal tract tumors, was also evaluated under the same conditions. data generated were used to plot a dose response curve of which the concentration of test compounds required to kill 50 % of the cell population (ic50) was determined. the cytotoxic activity was expressed as the mean ic50 of three independent experiments (table iii) and the results revealed that all the tested compounds showed inhibitory activity to the tumor cell lines in a concentration dependent manner. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1258 gomha et al. the results presented in table iii and figs. s-1–s-3 of the supplementary material showed that compounds 9c, 8h, 9f, 8g and 10h had significant anticancer activity against the two tumor cell lines mcf-7 and hepg-2, compared with reference drug imatinib. interestingly, compound 9c was the most active against hepg-2 and mcf-7 with ic50 values of 1.19 and 3.46 μg ml–1, respectively, comparable to doxorubicin. however, compounds 8a–f, 6, 9a, 9e, 9g, 9h and 10b–g were less active than imatinib. moreover, compound 10a was almost inactive under the employed screening conditions. table iii. the in vitro inhibitory activity of tested compounds against tumor cell lines expressed as ic50 values (μg ml-1) ± standard deviation from six replicates tested compound tumor cell line mcf-7 hepg2 4 15 20 6 > 50 35.5 8a 32.6 40.5 8b 37.7 27.9 8c 34.3 31.1 8d 44.9 49.7 8e 22.9 21.7 8f 38.2 43.4 8g 19.7 10.7 8h 8.67±0.31 2.94±0.12 9a 39.7 40.6 9b 20.1 29.4 9c 3.46±0.24 1.19±0.07 9d 39.9 43.6 9e 24.6 35.1 9f 7.7 17.6 9g 22.5 27.9 9h 48.6 > 50 10a > 50 > 50 10b 45.6 36.9 10c 29.1 25.4 10d 30.4 35.2 10e 44.3 47.4 10f 36.1 39.3 10g 33.9 41.1 10h 22.4 16.1 doxorubicin 0.46 0.42 imatinib 24.6 18.9 experimental chemistry the melting points were recorded on a gallenkamp electrothermal apparatus. the structures of the synthesized compounds were confirmed by their spectral (ms, ir and 1h-nmr) _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ tetracyclic system through dimroth rearrangement 1259 data and elemental analyses. the infrared spectra (kbr) were determined on a pye unicam sp-3000 infrared spectrophotometer. the 1h-nmr spectra were obtained on a varian gemini 300 spectrometer (300 mhz) in dmso-d6 with tms as the internal standard. the mass spectra were recorded on a gcms-qp 1000 ex shimadzu spectrometer. elemental analyses were realized at the microanalytical center, university of cairo, giza, egypt. the biological evaluation of the products was performed at the regional center for mycology and biotechnology at al-azhar university, cairo, egypt. synthesis of 7,9-bis(4-methoxyphenyl)pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4(3h)-one (2). a mixture of 3-amino-4,6-bis(4-methoxyphenyl)thieno[2,3-b]pyridine-2-carbonitrile (1) (3.87 g, 10 mmol) and formic acid (20 ml) was refluxed for 2 h. the solution was cooled and then poured onto water. the resulting solid was collected and recrystallized from ethanol to give 2 as yellow crystals. synthesis of 4-chloro-7,9-bis(4-methoxyphenyl)pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine (3). a mixture of compound 2 (4.15 g, 10 mmol) and phosphorus oxychloride (20 ml) was heated at reflux for 12 h and the excess of phosphorus oxychloride was removed by distillation under reduced pressure. the residue was treated with dry benzene (10 ml), the solvent was distilled off under vacuum to remove the last traces of phosphorus oxychloride and the resultant gummy residue was triturated with ice and sodium bicarbonate solution. the thus obtained solid was collected, dried and taken for the next step without any purification. synthesis of 4-hydrazinyl-7,9-bis(4-methoxyphenyl)pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidine (4). a mixture of compound 3 (4.33 g, 10 mmol) and hydrazine hydrate (30 ml) was refluxed for 4 h. the reaction mixture was poured onto crushed ice. the thus obtained white solid was filtered, dried and recrystallized from dioxane as white crystals. synthesis of n′-(2-cyano-4,6-bis(4-methoxyphenyl)thieno[2,3-b]pyridin-3-yl)-n,n-dimethylformimidamide (5). a mixture of compound 1 (3.87 g, 10 mmol) and dimethylformamide dimethylacetal (dmf-dma) (1.33 ml,10 mmol) in dry dioxane (30 ml) was heated under reflux for 6 h, then allowed to cool and poured into cold water (40 ml). the solid product was collected and recrystallized from ethanol to give 5 as brown crystals. synthesis of 4-imino-7,9-bis(4-methoxyphenyl)pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin -3(4h)-amine (6). a mixture of compound 5 (4.42 g, 10 mmol) and hydrazine hydrate (10 ml) in ethanol (30 ml) was refluxed for 3 h, and then allowed to cool. the solid product was collected and recrystallized to give 6 as white crystals. preparation of hydrazones 8a–h general procedure. a mixture of hydrazine 4 (0.858 g, 2 mmol) and the appropriate aldehyde (2 mmol) in acetic acid (30 ml) was heated under reflux for 4 h and then cooled. the mixture was diluted with water and the solid that precipitated was filtered off, washed with water, dried and crystallized from dioxane to give the respective hydrazones 8a–h. the physical constants of the hydrazones 8a–h are listed in the supplementary material. synthesis of pyrido[3′,2′:4,5]thieno[2,3-e]-1,2,4-triazolo[4,3-c]pyrimidines 9a–h general procedure. to the appropriate hydrazone 8 (14 mmol) in ethanol (40 ml), a solution of iron(iii) chloride (2m, 5 ml) was added. the mixture was refluxed for 20 min, and then left overnight at room temperature. the excess solvent was distilled off under reduced pressure, and solid residue remaining was washed with water several times, dried and finally crystallized from the appropriate solvent to give the respective products 9. the physical constants of the products 9a–h are given in the supplementary material. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1260 gomha et al. rearrangement of 9a–h to 10a–h general procedure. to a solution of the appropriate 9a–h (1 mmol) in ethanol (50 ml) was added sodium acetate (0.164 g, 2 mmol) and the mixture was refluxed for 6 h and then cooled. the precipitated solid was filtered off, washed with water and then with ethanol and finally crystallized from dimethylformamide to give pyrido[3′,2′:4,5]thieno[2,3-e]-1,2,4-triazolo[1,5-c]pyrimidines 10a–h. alternate synthesis of 10a–g a mixture of 6 (0.429 g, 1 mmol) and polyphosphoric acid (10 ml) was heated to 50–60 °c under stirring. a substituted benzoic acid (1 mmol) was added portionwise. the mixture was then heated at 180–200 °c for 3 h under stirring. after completion of the reaction, the reaction mixture was poured into ice and neutralized with concentrated aqueous ammonia solution. the crude product was filtered, washed with water and recrystallized from dmf to afford a product that was found to be identical in all respects (m.p., mixed m.p. and ir) with product 10a–g but in higher yields. alternate synthesis of 10h to a solution of 6 (0.429 g, 1 mmol) in glacial acetic acid (10 ml), acetic anhydride (0.204 g, 2 mmol) was added and the mixture was refluxed for 2 h. after refluxing, the reaction mixture was cooled and poured into ice-cold water. the solid product that was formed was collected by filtration and crystallized from dmf to give a product that was identical in all respects with 10h prepared from the rearrangement of 9h. antimicrobial activity assay the preliminary antimicrobial activity was investigated on a dozen of the newly synthesized compounds in order to increase the selectivity of these derivatives towards the test microorganisms. all microbial strains were provided from culture collection of the regional center for mycology and biotechnology (rcmb), al-azhar university, cairo, egypt. the antimicrobial profile was tested against two gram‐positive bacterial species (bacillus subtilis and staphylococcus aureus), two gram-negative bacterial species (escherichia coli and pseudomonas aeruginosa) and two fungi (aspergillus fumigatus and candida albicans) using a modified well diffusion method.30,31 briefly, 100 μl of the test bacteria/fungi were grown in 10 ml of fresh media mueller–hinton and sabouraud agar (oxoid, uk), respectively, until they reached a count of approximately 108 cells ml-1 for the bacteria or 105 cells ml-1 for the fungi. one hundred μl of microbial suspension was spread onto agar plates corresponding to the broth in which they were maintained and tested for susceptibility by the well diffusion method. one hundred µl of each sample (at 5 mg ml-1) was added to each well (10 mm diameter holes cut in the agar gel). the plates were incubated for 24–48 h at 37 °c (for the bacteria and yeast) and for 48 h at 28 °c (for the filamentous fungi). after incubation, the growth of the microorganism was observed. the plates were performed in triplicate and the resulting inhibition zone diameters were measured in mm and used as criterion for the antimicrobial activity. the size of the clear zone is proportional to the inhibitory action of the compound under investigation. a solvent control (dmso) was included in every experiment as a negative control. penicillin g and streptomycin (sigma–aldrich, usa) were used as a positive control against the gram-positive and gram-negative bacteria, respectively. amphotericin b (sigma–aldrich, usa) was used as a positive control for the fungi. mic determination using the broth microdilution method. all the newly synthesized compounds were screened in vitro for their antibacterial and antifungal activities by the broth dilution method as described by clsi32 to determine the lowest concentration inhibiting the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ tetracyclic system through dimroth rearrangement 1261 growth of an organism, which was recorded as the mic value. dmso was used as the diluent. stock solutions at 1000 μg ml-1 were prepared, from which serial dilutions were prepared for screening the tested compounds. mueller–hinton broth was used as the nutrient medium to grow and dilute the drug suspensions for the tested bacteria, and sabouraud dextrose broth was used for fungal nutrition. the inoculum size for test strain was adjusted to 108 cfu (colony forming units) per ml by comparing the turbidity. for the broth microdilution test, 50 μl of each microbial suspension in suitable growth medium was added to the wells of a sterile 96-well microtiter plate already containing 50 μl of two-fold serially diluted tested compound. control wells were prepared with culture medium, microbial suspension only, tested compound only and dmso in amounts corresponding to the highest quantity present. the contents of each well were mixed on a microplate shaker (eppendorf, hamburg, germany) at 900 rpm for 1 min prior to incubation for 24–48 h under the above-described cultivation conditions. the mic was the lowest concentration where no viability was observed after 24– –48 h based on the metabolic activity. to indicate respiratory activity, the presence of color was determined after the addition of 10 μl well-1 of ttc (2,3,5-triphenyltetrazolium chloride, sigma) dissolved in water (20 mg ml-1) and incubation under appropriate cultivation conditions for 30 min in the dark.33,34 after incubation, the optical density was measured using a microplate reader (sunrise, tecan, inc., usa). positive controls were wells with a microbial suspension in an appropriate growth medium in amounts corresponding to the highest quantity present in the broth microdilution assay. negative controls were wells with growth medium and a tested compound. all measurements of the mic values were performed in triplicate. penicillin g and streptomycin (sigma aldrich, usa) were used as standard antibacterial while griseofulvin and amphotericin b (sigma aldrich, usa) were used as standard antifungal drugs. evaluation of the antitumor activity using a viability assay. human breast carcinoma (mcf-7) and human hepatocellular carcinoma (hepg2) cell lines were obtained from the american type culture collection (atcc, rockville, md, usa). the cells were grown in rpmi-1640 medium supplemented with 10 % inactivated fetal calf serum and 50 µg ml-1 gentamicin. the cells were maintained at 37 °c in a humidified atmosphere with 5 % co2 and were subcultured two to three times a week. the potential cytotoxicity of the compounds was evaluated on tumor cells using the method of gangadevi and muthumary.35 the cells were grown as monolayers in growth rpmi-1640. the monolayers of 104 cells adhered at the bottom of the wells in a 96-well microtiter plate incubated for 24 h at 37 °c in a humidified incubator with 5 % co2. the monolayers were then washed with sterile phosphate-buffered saline (0.01 m, ph 7.2) and simultaneously the cells were treated with 100 µl from different dilutions of the tested sample in fresh maintenance medium and incubated at 37 °c. a control of untreated cells was made in the absence of the tested sample. positive controls containing the drugs imatinib or doxorubicin were also tested as references for comparison. six wells were used for each concentration of the test sample. every 24 h, the cells were observed under an inverted microscope. the number of surviving cells was determined by staining the cells with crystal violet35,36 followed by cell lysing using 33 % glacial acetic acid and the absorbance at 590 nm was read using a microplate reader (sunrise, tecan, inc, usa) after well mixing. the absorbance values from untreated cells were considered as corresponding to 100 % proliferation. the number of viable cells was determined using microplate reader as previously mentioned before and the percentage viability was calculated as: _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1262 gomha et al. t c cell viability, % 100= od od where odt is the mean optical density of the wells treated with the tested sample and odc is the mean optical density of untreated cells. the relation between surviving cells and drug concentration was plotted to obtain the survival curve of each tumor cell line after treatment with a specified compound. the 50 % inhibitory concentration (ic50), the concentration required to cause toxic effects in 50 % of intact cells, was estimated from graphical plots. conclusions in conclusion, a general and convenient method for the synthesis of novel pyrido[3′,2′:4,5]thieno[2,3-e]-1,2,4-triazolo[1,5-c]pyrimidine derivatives in moderate to excellent yields was reported herein. the protocol features initial oxidative cyclization of the respective pyrimidinylhydrazones followed by dimroth rearrangement under basic conditions. the synthesized compounds were evaluated for their in vitro antimicrobial activity at 5 mg ml–1 against a representative panel of pathogenic strains using the agar well diffusion method and the results indicated that compounds 8c, 9f, 10c and 10d showed excellent activity against bacteria. in addition, the in vitro growth inhibitory activity of the synthesized compounds was investigated in comparison with the well-known anticancer standard drug doxorubicin using the crystal violet colorimetric viability assay and the results indicated that compounds 9c, 8h, 9f, 8g and 10h had significant anticancer activity against the two tumor cell lines mcf-7 and hepg-2. interestingly, compound 9c was the most active against hepg-2 and mcf-7 with ic50 values of 1.19 and 3.46 μg ml–1, respectively. supplementary material the analytical and spectral data of the synthesized compounds and in vitro inhibitory activities, figs. s-1–s-3, are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. и з в о д испитивање антимикробне и антиканцерске активности нових тетрацикличних система добијених димротовим премештањем sobhi m. gomha1, ikhlass m. abbas1, mohamed a. a. elneairy1, mahmoud m. elaasser2 и bazada k. a. mabrouk1 1department of chemistry, faculty of science, cairo university, giza, egypt и 2regional center for mycology and biotechnology, al-azhar university, cairo, egypt синтетисана је серија деривата пиридо[3',2':4,5]тиено[2,3-e]-1,2,4-триазоло[4,3-c]пиримидина оксидативном циклизацијом 4-(2-арилиденхидразинил)пиридо [3',2':4,5]тиен[3,2-d]пиримидина. добијени интермедијери димротовим премештањем дају пиридо[3',2':4,5]тиено[2,3-e]-1,2,4-триазоло[1,5-c]пиримидине. испитана је антимикробна и антиканцерска активност добијених деривата. на основу добијених резултата једињења 8c, 9f и 10c показују изузетну активност према грам-позитивним бактеријама, а деривати 10d и 8c показују најбоњу активност према грам-негативним бактеријама. испи_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ tetracyclic system through dimroth rearrangement 1263 тивања антиканцерске активности су показала да дериват 9с показујe најбољу активност према hepg-2 и mcf-7, са ic50 вредностима 1,19 и 3,46 �μg ml-1, редом. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis of novel pyrazoline based bis 1,2,3-triazole scaffolds via click chemistry} j. serb. chem. soc. 82 (3) 241–251 (2017) udc 547.772.2+547.791:542.913:615.281–188 jscs–4962 original scientific paper 241 synthesis of novel pyrazoline-based bis(1,2,3-triazole) scaffolds via click chemistry kothuri kiran1,2, dongamanti ashok1*, boddu ananda rao1, madderla sarasija1 and alapati srinivas rao3 1green and medicinal chemistry laboratory, department of chemistry, osmania university, hyderabad, 500007, telangana, india, 2department of chemistry, jntu-h, hyderabad, telangana, 500 085, india and 3vagdevi innoscience private ltd., 5-a/8, ida nacharam, hyderabad, 500 076, telangana, india (received 16 february, revised 4 september, accepted 16 september 2016) abstract: a series of novel bis(1,2,3-triazoles) derivatives 7a–m were synthesized by the 1,3-dipolar cycloaddition (click-reaction) of 1-methyl-3,5-bis(2 -(prop-2-yn-1-yloxy)phenyl)-4,5-dihydro-1h-pyrazole (5) with various aralkyl azides 6a–m in the presence of sodium ascorbate and copper sulphate with good yields. the required precursor 5 was synthesized by reacting (e)-1,3 -bis(2-hydroxyphenyl)prop-2-en-1-one (3) with methylhydrazine hydrate via 2,2′-(1-methyl-4,5-dihydro-1h-pyrazole-3,5-diyl)diphenol 4, followed by reaction with propargyl bromide. the homogeneity of all the newly synthesized compounds was checked by tlc. the ir, nmr, mass spectral data and elemental analysis were in accord with the assigned structure. the title compounds were evaluated for their antibacterial activity against various bacterial strains, i.e., escherichia coli, pseudomonas aeruginosa, staphylococcus aureus and bacillus subtilis; compounds 7f–7h and 7j were found to be moderately active against the bacteria, when compared with that of the standard drug. furthermore, the same library of compounds was evaluated for their antioxidant activity using the nitric oxide radical scavenging activity. the results of the study showed that compounds 7e–7h and 7k–7m showed good radical scavenging activity. keywords: click chemistry; chalcones; 1,2,3-triazoles; pyrazolines; antibacterial. introduction five-membered heterocyclic compounds occupy a distinctive place in the realm of natural and synthetic organic chemistry. 1,2,3-triazoles have received great attention due to their contribution in pharmaceutical drugs, regardless of their scarcity in nature. in this respect, various approaches for the preparation of * corresponding author. e-mail: ashokdou@gmail.com doi: 10.2298/jsc160216076a _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 242 kiran et al. these privileged structures with drug-like properties have been developed using various synthetic strategies. medicinally, 1,2,3-triazole derivatives have been shown to possess a wide range of diverse, interesting biological properties, such as, anti-hiv,1 antimalarial,2 anti-epileptic,3 anti-allergic,4 antileishmanial,5 anticancer,6,7 anti-inflammatory,8 antitubercular,9,10 antidiabetic,11 antifungal,12–14 antiviral15,16 and antibacterial.17,18 1,2,3-triazole derivatives are revealed in prominent pharmaceutical drugs, such as, carboxyamidotriazole, cefatrizine and tazobactam. pyrazolines are a significant class of heterocyclic compounds comprising two nitrogen atoms in a five-membered ring. pyrazoline derivatives are the electron rich nitrogen heterocycles that play an essential role in various biological activities. these heterocyclic compounds occur widely in the environment, in the form of alkaloids, vitamins, pigments and as constituents of plant and animal cells. considerable attention has been focused on pyrazolines and substituted pyrazolines because of their inspiring biological activities. pyrazolines constitute an interesting class of heterocycles due to their synthetic versatility and effective biological activities, such as anticancer,19 antioxidant,20 antibacterial,21 antifungal,22 antidepressant,23–25 anti-inflammatory,26 anticonvulsant,27 antitumor28 and analgesic29 properties. as far as the different pyrazoline isomers are concerned, 2-pyrazoline derivatives became the most frequently studied pyrazoline. various methods are used for the preparation of 2-pyrazolines. reaction of α,β-unsaturated ketones with substituted hydrazines seems to be the most popular procedure for the synthesis of 2-pyrazolines. as mentioned, 2-pyrazolines and 1,2,3-triazole derivatives possess valuable bioactivities, which stimulated the preparation of their various derivatives. recently, much consideration has been paid toward the synthesis and pharmacological evaluation of triazoles, and bis-triazoles, as potent hiv-1 protease inhibitors30,31 and size-specific ligands for mrna hairpin loops,32 respectively. some derivatives containing a triazole and pyrazoline moiety were synthesized and investigated for their potential antidepressant activities.33 according to these studies, a system combining two biolabile components, 2-pyrazoline and 1,2,3 -triazole, were synthesized and their potential antibacterial and antioxidant effects investigated. bearing in mind the tremendous biological potency of bis(1,2,3 -triazoles) with pyrazoline, the endeavour was the synthesis of pharmacologically active molecules, and then their evaluation for antibacterial and antioxidant activities. in this regard, our research group has focused on the design, synthesis of 1,2,3-triazoles and pyrazolines, thereby contributing to research34,35 on these biologically important heterocycles. in the current study, the aim was to obtain new compounds containing both pyrazolines and 1,2,3-triazole rings in the same structure, via the click reactions shown in schemes 1 and 2. _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ synthesis of bis(1,2,3-triazole) derivatives 243 scheme 1. synthesis route to bis(1,2,3-triazole) derivatives. experimental melting points were determined by the open capillary method using an electrical melting point apparatus and are uncorrected. the ir spectra were recorded as kbr pellets on a shimadzu ft-ir-8400s spectrophotometer. the 1h-nmr (400 mhz) and 13c-nmr (100 mhz) spectra were recorded on a bruker dpx 400 spectrophotometer using tetramethylsilane (tms) as the internal standard, with dmso-d6 and cdcl3 as solvents. the mass spectra were recorded on a gcms-qp 1000 ex mass spectrometer. thin layer chromatography (tlc) was performed to check the purity of the compounds, the spot being located under uv light and iodine vapours. analytical and spectral data for the synthesized compounds are given in supplementary material to this paper. synthesis of (e)-1,3-bis(2-hydroxyphenyl)prop-2-en-1-one (3)36,37 to a vigorously stirred solution of 2-hydroxy acetophenone (25 g, 184 mmol) and salicylaldehyde (22.4 g, 184 mmol) in ethanol (200 ml), koh (30.92 g, 551 mmol) was added in small portions over 1 h at 0 °c. the reaction mixture was stirred at room temperature for 2 h and refluxed for 1 h. after completion of the reaction and cooling to room temperature, the mixture was poured into ice-cold water, neutralized with concentrated hcl and stirred for 1 h. _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 244 kiran et al. the precipitated solid was filtered off, washed with water and dried under vacuum to afford pure chalcone 3. scheme 2. synthesis route to 1,2,3-triazole derivatives via bis-propargyl chalcone. synthesis of 2,2'-(1-methyl-4,5-dihydro-1h-pyrazole-3,5-diyl)diphenol (4) to a stirred solution of (e)-1,3-bis(2-hydroxyphenyl)prop-2-en-1-one (3, 10 g, 41.7 mmol) in ethanol (50 ml) was added methylhydrazine hydrate (2.3 g, 50.0 mmol) at 0 °c. later the temperature was raised to room conditions and the mixture refluxed for 1 h. the solvent was evaporated under reduced pressure and the obtained residual syrup was purified by column chromatography (100–200 mesh), eluted with ethyl acetate:hexane (3:2 volume ratio) to obtain compound 4. synthesis of 1-methyl-3,5-bis[2-(prop-2-ynyloxy)phenyl]-4,5-dihydro-1h-pyrazole (5) to a stirred solution of 60 % nah (2.2 g, 56.0 mmol) in dry dmf (40 ml) at 0 °c was added drop wise a solution of 2,2′-(1-methyl-4,5-dihydro-1h-pyrazole-3,5-diyl)diphenol (4, 5.0 g, 18.6 mmol) in dmf (10 ml) over a period of 30 min, and stirred for 1 h at room temperature. later, propargyl bromide (8.3 ml, 56 mmol, 80 % in toluene) was added at 0 °c and stirred for 16 h at room temperature. after completion of the reaction, the mixture was cooled to 0 °c and quenched by the addition of ice water (50 ml), extracted twice with ethyl acetate, washed with brine solution, dried over anhydrous na2so4 and evaporated under vacuum. the obtained syrup was purified by column chromatography (100–200 mesh), eluted with ethyl acetate:hexane (1:19 volume ratio) to obtain compound 5. synthesis of (2e)-1,3-bis[2-(prop-2-ynyloxy)phenyl]prop-2-en-1-one (8) to a stirred solution of 60 % nah (0.25 g, 6.25 mmol) in dry dmf (10 ml) was added a solution of (e)-1,3-bis(2-hydroxyphenyl)prop-2-en-1-one (3, 0.5 g, 2.08 mmol) in dmf (3 ml) dropwise over 15 min at 0 °c. the reaction mixture was stirred at room temperature for 1 h, cooled to 0 °c and propargyl bromide (0.93 ml, 6.25 mmol, 80 % in toluene) added. the mixture was stirred at room temperature for 16 h and later cooled to 0 °c, quenched with ice _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ synthesis of bis(1,2,3-triazole) derivatives 245 water (20 ml), extracted twice with ethyl acetate, washed with brine solution, dried over anhydrous na2so4 and evaporated. the obtained crude product was purified by column chromatography (100–200 mesh), eluted with ethyl acetate:hexane (1:19 volume ratio) to afford pure compound 8. synthesis of (2e)-1,3-bis({2-[(1-benzyl-1h-1,2,3-triazol-4-yl)methoxy]phenyl})prop-2-en-1 -one (9) a solution of (2e)-1,3-bis[2-(prop-2-ynyloxy)phenyl]prop-2-en-1-one (8, 0.2 g, 0.63 mmol) and benzyl azide 6a (0.17 g, 1.26 mmol) dissolved in t-buoh:h2o (5 ml, 1:1 volume ratio) was treated with sodium l-ascorbate (0.025 g, 0.126 mmol) and copper sulphate (0.014 g, 0.063 mmol), stirred at room temperature for 1 h. after completion of the reaction, the mixture was diluted with water, extracted with ethyl acetate, washed with brine solution, dried over anhydrous na2so4 and evaporated. the crude was purified by column chromatography (100–200 mesh), eluted with ethyl acetate:hexane (1:4 volume ratio) to afford pure product 9. general procedure for the synthesis of compounds (6a–m)36-46 a solution of aralkyl bromide (3 mmol) in dry dmf (5 ml) at 0 °c was treated with sodium azide (3.5 mmol) and stirred at room temperature for 16 h. the reaction mixture was quenched by adding ice water, extracted with diethyl ether, washed with brine, dried over anhydrous na2so4 and evaporated at below 40 °c to give 6a–m. the compounds 6a–m were not characterized and were treated immediately with compound 5 without further purification to obtain the compounds 7a–m. general procedure for the synthesis of compounds (7a–m) a solution of 1-methyl-3,5-bis[2-(prop-2-ynyloxy)phenyl]-4,5-dihydro-1h-pyrazole (5, 0.29 mmol) and aralkyl azide (6a–m, 0.58 mmol) dissolved in t-buoh:h2o (1:1 volume ratio) was treated with sodium l-ascorbate (0.058 mmol) and copper sulphate (0.029 mmol) and stirred at room temperature for 0.5–1 h. the progress of the reaction was monitored by tlc. after completion, the reaction mixture was diluted with water, extracted with ethyl acetate, washed with brine, dried over anhydrous na2so4 and evaporated. the crude was purified by column chromatography (100–200 mesh), eluted with ethyl acetate:hexane (7:3 volume ratio) to afford compounds 7a–m. biological activity antibacterial assay. the in vitro antibacterial studies against the test organisms were realised by the agar well diffusion method.38 nutrient broth (nb) plates were swabbed with 24 h old broth culture (100 ml) of the test bacteria. using a sterile cork borer, wells (6 mm) were made into each petri plates. different concentrations of the test samples dissolved in dmso were added into the wells by using sterile pipettes. gentamicin was used as the standard antibiotic for the antibacterial activity. the plates were incubated at 37 °c for 24 h. after the incubation, the diameter of zone of inhibition of each well was measured. duplicates were maintained and the average values were calculated for eventual antibacterial activity. the broth dilution test was used to determine minimum inhibitory concentration (mic) of the above-mentioned samples.39 freshly prepared nutrient broth was used as the diluent. the 24 h old culture of the test bacteria escherichia coli, pseudomonas aeruginosa, staphylococcus aureus and bacillus subtilis were diluted 100 fold in nutrient broth (100 μl bacterial cultures in 10 ml nb). increasing concentrations of the test samples were added to the test tubes containing the bacterial cultures. all the tubes were incubated at 37 °c for 24 h. the tubes were examined for visible turbidity and using nb as a control. the lowest concentration that inhibited visible growth of the tested organisms was recorded as the mic. _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 246 kiran et al. antioxidant activity nitric oxide radical scavenging activity. the scavenging effect on nitric oxide was measured according to the method of marcocci40 et al. with a little modification.41 briefly, 4 ml of a drug solution was added (in a test tube) to 1 ml of sodium nitroprusside (snp) solution (25 mm) and the tubes incubated at 29 °c for 2 h. a 2 ml aliquot of the incubation solution was diluted with 1.2 ml griess reagent (1 % sulphanilamide in 5 % h3po4 and 0.1 % n-1-naphthylethylenediamine dihydrochloride). the absorbance of the chromophore that was formed during diazotization of the nitrite with sulphanilamide and subsequent coupling with n-1-naphthylethylenediamine dihydrochloride was immediately read at 550 nm and the concentration determined from a standard curve (y = mx+c) of sodium nitrite salt treated in the same way with griess reagent. inhibition of nitrite formation by the drug or the standard plant antioxidant (vitamin c) was calculated relative to the control: inhibition, % = 100(acontrol – atest)/acontrol where atest is the absorbance of the control reaction mixture excluding the test compound/drug solution and acontrol is the absorbance of the test compounds/drug solution. results and discussion chemistry the route for the synthesis of the title compounds 7a–m is outlined in scheme 1. initially, the title compound 7a was synthesised as shown in scheme 2, when conversion of compound 9 (triazole derivative) to the n-substituted dihydro pyrazole derivative 7a was obtained in very low yield making the purification difficult. the bis-propargyl derivative 8 was obtained in 82 % yield from the corresponding chalcone derivative on treatment with nah and dmf. the bispropargyl derivative 8 was converted to the bis triazole derivatives 9 via coppercatalyzed click reaction with the aralkyl azides in 85 % yield. the bis-triazole derivatives 9 were treated with methylhydrazine hydrate in ethanol at reflux temperature. finally, the target compound 7a was obtained in very low yield. due to the low yield obtained following scheme 2, scheme 1 was approached, whereby the target compounds were isolated in good yields. the precursor 5 was synthesized by reacting (e)-1,3-bis(2-hydroxyphenyl)prop-2-en-1-one (3) with methylhydrazine hydrate via 2,2′-(1-methyl-4,5-dihydro-1h-pyrazole-3,5-diyl)diphenol (4), followed by reaction with propargyl bromide. the structures of newly synthesized compounds 7a–m were characterized by ir, 1h-nmr, 13c-nmr, mass and chns spectral analysis. except the aralkyl azides 6h, 6j, all the other aralkyl azides 6a,42 6b,43 6c,44 6d,45 6e,46 6f,47 6g,48 6i,49 6k,50 6l51 and 6m44 are known in the literature. after isolation, the aralkyl azides were treated immediately with compound 5 without further purification to obtain compounds 7a–m. the ir, 1h-nmr, 13c-nmr and mass spectra for the synthesised compounds are given in the supplementary material to this paper. the elucidation of the structure of pyrazoline ring protons is usually realised by 1h-nmr spectroscopy. in the 1h-nmr spectrum, the signals of the res_________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ synthesis of bis(1,2,3-triazole) derivatives 247 pective protons of the pyrazoline ring are verified based on their chemical shifts, multiplicities, and coupling constants. the formation of a pyrazoline ring was confirmed by the presence of an abx system in the 1h-nmr due to geminal– –vicinal coupling between protons ha, hb and hx. the ha and hb protons appeared as a doublet of doublet due to geminal and vicinal coupling as shown in scheme 1, bottom inset (compound 7a). these ha and hb differ in their coupling with hx and hence, they are anisogamous. ha, which appeared as a doublet of doublet at δ 2.67 ppm, is the proton cis to hx and geminal to hb (jab = 16.8 hz, jax = 14.8 hz). hb is the proton trans and vicinal to hx and appeared as a doublet of doublet at δ 3.52 ppm (jba = 16.8 hz, jbx = 9.6 hz). moreover, hx appeared as doublet of doublet at δ 4.24 ppm (jxa = 14.8, jxb = 9.6 hz).30,52 all the other signals from nmr spectra are in agreement with the proposed structures. antibacterial activity the newly synthesized compounds 7a–m were screened in vitro for their antibacterial activity against escherichia coli (atcc 11229) and pseudomonas aeruginosa (atcc 27853), as examples of gram-negative bacteria, and staphylococcus aureus (atcc 6538) and bacillus subtilis (atcc 6633), as examples of gram-positive bacteria. agar well-diffusion method was used to assay the antibacterial activity against test strains on mueller–hinton agar plates. gentamicin was employed as a standard antibacterial drug. the results obtained as minimum inhibitory concentration (mic) in µg ml–1 and measurements are presented in table i. table i. antibacterial activity (mic / µg ml-1) of the newly synthesized compounds 7a–m compound bacteria gram-negative gram-positive e. coli p. aeruginosa s. aureus b. subtilis 7a 25 12.5 25 12.5 7b 12.5 25 6.25 12.5 7c 25 25 25 12.5 7d 25 50 25 50 7e 25 50 50 25 7f 12.5 6.25 12.5 12.5 7g 6.25 6.25 6.25 6.25 7h 6.25 12.5 6.25 6.25 7i 25 50 12.5 12.5 7j 12.5 12.5 25 12.5 7k 12.5 50 25 12.5 7l 25 25 25 50 7m 12.5 50 25 12.5 gentamicin 3.125 3.125 3.125 3.125 _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 248 kiran et al. investigation of the antibacterial efficiency of the synthesized compounds revealed that most of the tested compounds displayed variable inhibitory effects on the growth of the tested gram-negative and gram-positive bacterial strains. it is evident from table i, that compound 7g exhibited the highest antibacterial effect, but was less potent than gentamicin in inhibiting the growth of e. coli, p. aeruginosa, s. aureus and b. subtilis (mic = 6.25 µg/ml). next to 7g, compounds 7f and 7h (6.25–12.5 µg ml–1) and 7j (6.25–25 µg ml–1) were found less potent as compared to the control drug gentamicin. it was envisaged from the analysis of the antibacterial activity results that the presence of methyl-, chloro and triflouromethyl-substituted derivatives had a moderate effect in determining the antibacterial activity and the exhibited antimicrobial potency. antioxidant activity the antioxidant activity of the synthesized compounds 7a–m were evaluated in vitro by the nitric oxide radical scavenging assay.40,41 the results were compared with that of the standard antioxidant ascorbic acid. most of the compounds tested significantly inhibited nitric oxide radical levels compared to the standard antioxidant used in the study (table ii). as could be seen in table ii, compounds 7e–h and 7k–m exhibited strong scavenging effects on the nitric oxide stable radical, with respective ic50 values of 2.50±0.65, 2.50±0.60, 2.50±0.24, 2.50±0.76, 2.50±0.37, 2.43±0.41 and 2.47±0.38 µg ml–1. these values were lower than the positive controls in the study (aa with 5.35±0.67 µg/ml), indicating that compounds with hydroxyl, nitro, chloro, methyl, fluoro, trifluoromethyl, methoxy and isopropyl substituents were found to be the most potent antioxidant agents towards nitric oxide. the remaining compounds showed moderate activity. table ii. antioxidant activity (ic50 / µg ml-1) of the synthesized compounds 7a–m against nitric oxide; values are the means of three replicates±sd. a lower ic50 value indicates better scavenging activity compound value 7a 2.53±0.25 7b 2.51±0.42 7c 2.52±0.24 7d 2.52±0.11 7e 2.50±0.65 7f 2.50±0.60 7g 2.50±0.24 7h 2.50±0.76 7i 2.51±0.29 7j 2.55±0.40 7k 2.50±0.37 7l 2.43±0.41 7m 2.47±0.38 ascorbic acid 5.35±0.67 _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ synthesis of bis(1,2,3-triazole) derivatives 249 conclusions in conclusion, a novel series of bis(1,2,3-triazole) derivatives were successfully synthesized through copper-catalyzed huisgen [3+2] cycloaddition of various aralkyl azides with bis -propargyl pyrazoline 5 in good to excellent yields. furthermore, this synthesis approach provided a structural framework that could be explored further in the development of new 1,2,3-triazole derivatives from 2-pyrazolines moieties. it is believed that the procedural simplicity, the efficiency, and the easy accessibility of the reaction partners give access to an array of heterocyclic frameworks. in this study, new hybrid molecules consisting of biologically important 1,2,3-triazole derivatives from 2-pyrazolines pharmacophores were synthesized and their antibacterial and antioxidant activities determined. amongst the synthesized compounds, 7f–h and 7j showed the moderate antibacterial activity against the tested bacterial strains. the radical scavenging activities of the synthesized compounds also showed that the compounds 7e–h and 7k–m exhibited potent ic50 values. the experimental results of this study will likely provide a new basis for the design of interesting pyrazoline-based bis(1,2,3-triazoles), and further studies, including the design of new analogues of the heterocyclic moiety, are in progress. supplementary material analytical and spectral data for the synthesized compounds are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding authors on request. acknowledgements. the authors are thankful to the head of department of chemistry, osmania university and jntu-h hyderabad for their valuable support. и з в о д синтеза „клик” реакцијом нових деривата пиразолина који садрже 1,2,3-триазолски прстен kothuri kiran1,2, dongamanti ashok1, boddu ananda rao1, madderla sarasija1 и alapati srinivas rao3 1 green and medicinal chemistry laboratory, department of chemistry, osmania university, hyderabad, 500007, telangana, india, 2 department of chemistry, jntu-h, hyderabad, telangana, 500 085, india и 3 vagdevi innoscience private ltd., 5-a/8, ida nacharam, hyderabad, 500 076, telangana, india извршена је синтеза серије бис(1,2,3-триазола) 7a–m реакцијом 1,3-диполарне циклоадиције („клик” реакција), 1-метил-3,5-бис[2-(проп-2-инил-окси)фенил]-4,5-дихидро -1h-пиразола 5 са арил-азидима 6a–m у присуству натријум-аскорбата и бакар -сулфата, у врло добром приносу. неопходан прекурсор 5 добијен је у реакцији (e)-1,3 -бис(2-хидроксифенил)проп-2-ен-1-она (3) са метил-хидразином хидратом преко 2,2′ -(1-метил-4,5-дихидро-1h-пиразол-3,5-диил)дифенола (4), после реакције са пропаргил-бромидом. чистоћа свих нових деривата потврђена је танкослојном хроматографијом. структура једињења је потврђена ir и nmr спектроскопијом, масеном спектрометријом и елементалном анализом. испитана је антибактеријска активност синтетисаних деривата према escherichia coli, pseudomonas aeruginosa, staphylococcus _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 250 kiran et al. aureus и bacillus subtilis. једињења 7f–h и 7j показала су умерену антибактеријску активност у поређењу са стандардима. осим тога, испитивањем антиоксидативне активности у присуству азот-моноксида испитивана једињења показују способност везивања слободних радикала. деривати 7e–h и 7k–m показују добру активност. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice qtaim investigation of a dipyrazol-1-ylmethane derivative and its zn(ii) complexes (znlx2, x = cl, br or i) j. serb. chem. soc. 80 (8) 997–1008 (2015) udc 544.112+541.135+532.14:547.772+ jscs–4775 547.211+547.759.32+546.47:537.872 original scientific paper 997 qtaim investigation of a dipyrazol-1-ylmethane derivative and its zn(ii) complexes (znlx2, x = cl, br or i) maryam dehestani and leila zeidabadinejad* department of chemistry, shahid bahonar university of kerman, 76169 kerman, iran (received 24 february, revised 27 march, accepted 31 march 2015) abstract: topological analyses of the electron density were performed on the bis(pyrazol-1-yl)methane derivative 9-(4-(di-1h-pyrazol-1-ylmethyl)phenyl) -9h-carbazole (l) and its zinc(ii) complexes: znlcl2 (1), znlbr2 (2) and znli2 (3) using the quantum theory of atoms in molecules (qtaim) at the b3pw91/6-31g(d) theoretical level. the topological parameters derived from the bader theory were also analyzed; these are characteristics of zn-bond critical points and of ring critical points. the calculated structural parameters were the frontier molecular orbital energies, the highest occupied molecular orbital energy (ehomo), the lowest unoccupied molecular orbital energy (elumo), hardness (η), softness (s), the absolute electronegativity (χ), the electrophilicity index (ω) and the fractions of electrons transferred (δn) from znlx2 complexes to l. numerous correlations and dependencies between the energy terms of the symmetry adapted perturbation theory approach (sapt), geometrical, topological and energetic parameters were detected and are described. keywords: znlx2; charge transfer; bond critical point; sapt. introduction the coordination chemistry of di/poly-pyrazolylmethane has seen significant development during the past few years.1,2 one of the current interesting topics is to rationally design and synthesize supramolecular structures based on di/poly -pyrazolylmethane units, which are capable of multiple binding modes and have the potential to participate in important non-covalent interactions that direct their self-assembly into remarkable architectures.3 compared with coordination bonds, non-covalent forces are weaker, but they are common and play critical roles in the formation of supramolecular structures due to their significant contribution to the self-assembly process.4–6 more studies focused on the weak non-covalent forces in the design and synthesis of supramolecular structures. herein, the * corresponding author. e-mail: lzeidabadi@yahoo.com doi: 10.2298/jsc150224027z _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 998 dehestani and zeidabadinejad dipyrazol-1-ylmethane derivative 9-(4-(di-1h-pyrazol-1-ylmethyl)phenyl)-9h -carbazole (l) and its zinc(ii) complexes: znlcl2 (1), znlbr2 (2) and znli2 (3) are reported. the ligand l, znlbr2 and znli2 were synthesized by wang et al.7 in the solid state, each compound showed extensive non-covalent interactions, including weak hydrogen bonds and c…h…π interactions that organize the molecules into 2d and 3d structures. it could be stated that such interactions are common not only in the present compounds but also in a large number of known polypyrazolylborate and polypyrazolylmethane compounds. qtaim,8 the chemical bonds in both isolated species and molecular crystals can be classified and quantified in terms of features of the bond critical points in the electron density, both theoretically and experimentally. the variety of the atomic interactions can be approximately divided into shared (or covalent) interactions, intermediate (partially covalent) interactions and closed-shell (van der waals, ionic, metal, etc.) interactions.9–11 the fundamental differences between the two limiting extremes in the interactions, i.e., closed-shell interactions and shared ones, are the electron density features at the bond critical point. these are the value of electron density, ρb, and the sign of the laplacian of the electron density, 2 b ,ρ∇ as well as the energy density he,b = gb+vb, where vb and gb are the potential and kinetic energy densities, respectively.10,11 shared interactions exhibit ρb ≥ 0.14 au, 2 bρ∇ < 0 and hb < 0, while closed-shell interactions show ρb ≤ 0.05 au, 2 bρ∇ > 0 and hb > 0. in the intermediate region, 2 b ( )rρ∇ > 0 and hb(r) < 0. the term non-covalent interaction may be ambiguous since covalency is attributed not only to typical chemical bonds. it is also connected with the hydrogen bond and with the other interactions, such as halogen or dihydrogen bonds. covalency is usually attributed to charge transfer and polarization interaction energy contributions. it seems that there are meaningful differences between the interaction mentioned above, such as ionic bond and covalent bonds. the goal of this study was to apply density functional theory (dft) calculations12 and the qtaim theory13 to analyze the properties of the n…zn–x bonds in znlcl2, znlbr2 and znli2. the latter gives direct information on the presence and type of chemical bonds in these structures. computational details all calculations, including optimizations were performed with gaussian 03 sets of code13 using the b3pw91/6-31g(d) level of theory without any symmetry restrains. quantum chemical calculations of l based on density functional theory was performed at the b3pw91/ /631g(d) level and calculations of complexes 1–3 were realized with b3pw91/6–31g(d) for the c, h, n, cl, br and i atoms and the lanl2dz basis set for the zn atoms. qtaim was also applied, and the characteristics of the bond critical points (bcps) and ring critical points (rcps) were analyzed in terms of the following properties: the electron density at the critical point, its laplacian and the total electron energy density at the critical point. for the latter, its components were also investigated: the potential electron energy _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ dipyrazol-1-ylmethane derivative and its zn(ii) complexes 999 density and the kinetic electron energy density. the following relations are well known if all terms are expressed in atomic units: 2 b b b 1 2 4 g vρ∇ = + (1) e,b b bgh v= + (2) the symmetry adapted perturbation theory (sapt) approach was applied to deepen the understanding of the nature of the interactions14 for complexes 1–3. the molpro package15 was used to perform the calculations. sapt is a well-established approach to calculate the interaction energy of two closed-shell moieties, whereby the interaction energy is obtained directly as the sum of defined contributions. thus, it is different from the commonly applied approaches in which the binding energy is calculated as the difference between the energy of the complex and the sum of the energies of the monomers. in the sapt approach, the interaction energy consists of the following terms: the first-order electrostatics (1)elst ,e the secondorder induction (2)inde and dispersion (2) dispe energies, and their exchange counterparts: firstorder exchange (1)exch ,e second-order exchange–induction ( 2) exch inde − and exchange–dispersion (2) exch disp.e − the sapt method up to the second order gives the main part of the interaction energy. the sapt2 interaction energy is calculated according to eq. (3): (1) (2) (2) (1) (2) (2)sapt2 hfint elst ind disp exch exch-ind exch-dispe e e e e e e eδ= + + + + + + (3) results and discussion structure of the ligand ligand l crystallizes in the monoclinic space group p21, selected bond distances and angles are listed in table i. the molecular structure of l and its 1d linear chain are shown in fig. 1. the dihedral angles between the carbazole ring and the central phenyl unit are 44.3° in molecule a and 46.9° in molecule b. in molecule a, the dihedral angles of the central phenyl unit and the pyrazole rings (r1, r2) are 71.4 and 80.4°, respectively. the dihedral angle of the two pyrazole rings (r1, r2) is 54.9°. in molecule b, the dihedral angles of the central phenyl unit and the pyrazole rings (r3, r4) are 71.8 and 80.0°, respectively. the dihedral angle of the two pyrazole rings (r3, r4) is 55.5°. there is no extended conjugation among the carbazole, phenyl and pyrazole rings. in the molecular structure of complexes 1-3, the zn(ii) ion is four coordinated by two nitrogen atoms of the pyrazolyl groups (l) and two halogen ions to form a distorted tetrahedral geometry. the two pyrazole groups linked to zn(ii) lead to changes in the structure of the ligand. in the structure of znlcl2, shown in fig. 2, the dihedral angles between the carbazole ring and the phenyl unit, between the phenyl unit and the pyrazole rings, and between the two pyrazole rings are 60, 78 and 77, and 52°, respectively. the dihedral angles between the carbazole ring and the phenyl unit, and between the phenyl unit and pyrazole rings are larger than those of the free ligand are. however, the dihedral angle of the two pyrazole rings is smaller than that observed for the free ligand, indicating that _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1000 dehestani and zeidabadinejad table i. calculated geometry parameters of l, znlcl2, znlbr2 and znli2 cmpd. bond length, å bond angle, ° l n1–n3 1.3411 n3–n1–c1 120.45 n2–n4 1.3442 n4–n2–c1 121.36 n1–c1 1.4546, (1.451)9 n1–c1–h1 104.77 n2–c1 1.4453, (1.455)9 n2–c1–h1 106.38 c1–c2 1.5112, (1.523)9 n1–c1–n2 110.65, (110.7)9 c1–h1 1.1064 c2–c1–h1 108.59 complex 1 n1–n3 1.3474 n3–n1–c1 110.73 n2–n4 1.3479 n4–n2–c1 110.73 n1–c1 1.4544 n1–c1–h1 104.76 n2–c1 1.4447 n2–c1–h1 106.31 c1–c2 1.5110 n1–c1–n2 110.73 c1–h1 1.1019 c2–c1–h1 108.64 zn–cl1 2.2091 n3–zn–n4 86.97 zn–cl2 2.1960 n3–zn–cl1 105.77 zn–n3 2.0848 n3–zn–cl2 112.71 zn–n4 2.0779 n4–zn–cl1 104.73 complex 2 n1–n3 1.3466 n3–n1–c1 120.95 n2–n4 1.3478 n4–n2–c1 120.87 n1–c1 1.4545 n1–c1–h1 104.75 n2–c1 1.4447 n2–c1–h1 106.31 c1–c2 1.5109 n1–c1–n2 110.72 c1–h1 1.1019 c2–c1–h1 108.65 zn–br1 2.3033, (2.364)9 n3–zn–n4 86.76, (89.29)9 zn–br2 2.2993, (2.355)9 n3–zn–br1 106.08, (109.6)9 zn–n3 2.1019, (2.071)9 n3–zn–br2 110.89, (120.4)9 zn–n4 2.1007, (2.064)9 n4–zn–br1 104.15, (108.0)9 complex 3 n1–n3 1.3913 n3–n1–c1 120.27 n2–n4 1.3895 n4–n2–c1 120.54 n1–c1 1.4545 n1–c1–h1 104.75 n2–c1 1.4447 n2–c1–h1 106.31 c1–h1 1.1019 c2–c1–h1 108.65 zn–i1 2.5109, (2.550)9 n3–zn–n4 91.57, (88.74)9 zn–i2 2.4882, (2.533)9 n3–zn–i1 106.39, (108.3)9 zn–n3 1.9468, (2.057)9 n3–zn–i2 116.78, (115.1)9 zn–n4 1.9448, (2.050)9 n4–zn–i1 108.28, (109.1)9 the planarity of ligand in complex 1 is worse than that for the free ligand. the zn–n3 and zn–n5 bond lengths are 2.0848 and 2.0779 å, respectively. the bond distances of zn–cl1 and zn–cl2 are 2.2091 and 2.1960 å, respectively. in complexes 2 and 3, the changes in the structure of ligand are similar to that of complex 1. the planarity of coordinated ligand is also worse than that of the free ligand. the corresponding angles and bond lengths for complexes 2 and 3 are listed in table i and displayed in figs. 3 and 4. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ dipyrazol-1-ylmethane derivative and its zn(ii) complexes 1001 fig. 1. a) the molecular structure of l and b) the 1d linear chain of ligand l. the bond lengths are in å. fig. 2. a) the molecular structure of complex 1 and b) the 1d structure of complex 1 showing the weak c–h…cl bond in å. fig. 3. a) the molecular structure of complex 2 and b) the 1d structure of complex 2 showing the weak c–h…br bond in å. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1002 dehestani and zeidabadinejad fig. 4. a) the molecular structure of complex 3 and b) the 1d structure of complex 3 showing the weak c—h…i bond in å. global reactivity descriptors a large part of theoretical chemistry related to reactivity is based on the concept of frontier molecular orbitals (fmo), especially the lowest unoccupied molecular orbital (lumo) and the highest occupied molecular orbital (homo). the interaction between these orbitals often allows a good description of the reactivity of reactions. the fmo theory says that the attack of an electrophilic species will occur where there is more density of the homo, whereas the attack of a nucleophilic species will occur in a region with a higher density of the lumo. parr and coworkers demonstrated that nearly all the frontier molecular theory could be rationalized from the dft.16 the µ and molecular η for an n-electron system with total energy et and external potential v(r) are defined respectively, as the first and second derivatives of the energy with respect to n:17,18 ( )v r e µ n χ ∂ = = − ∂   (4) and 2 2 ( ) 1 1 2 2 v r e µ n n η  ∂ ∂ = =   ∂ ∂    (5) where χ (ev) in eq. (4) is the electronegativity. in numerical applications, µ and η are calculated using of the difference approximation: ( )a 1 2 µ ip e= − + (6) ( )a 1 2 ip eη = − (7) _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ dipyrazol-1-ylmethane derivative and its zn(ii) complexes 1003 the vertical ionization potential (ip) and the electron affinity (ea) can be obtained from the energy of the neutral, anionic, and the cationic species at the geometry of the corresponding n electron neutral species, as follows: ( ) ( ) 1ip e n e n=  − −   (8) ( ) ( )a 1e e n e n=  − +   (9) equations (6) and (7) can be simplified using the koopmans theorem,12 which approximates the electronic affinity and the ionization potential to the negative of the lumo and homo energy, respectively. ( )lumo homo 1 2 e eμ = − + (10) ( )lumo homo 1 2 e eη = − (11) the electrophilicity index (ω) and global softness (s) are defined as follows: 2 2 μ ω η = (12) 1 /s η= (13) according to parr et al.,18 ω is a global reactivity index similar to the chemical hardness and chemical potential. this new reactivity index measures the stabilization in energy when the system reserves additional electronic charge (δn). the direction of the charge transfer is determined by the electronic chemical potential of the molecule because an electrophile is a chemical species capable of accepting electrons from the environment; its energy must decrease upon accepting an electronic charge. thus, its electronic chemical potential must be negative. the calculated values of ehomo, elumo, χ, µ, η, s and ω for l and its complexes are listed in table ii. in a reaction between two molecules, species can act as a nucleophile, which has a lower value of the electrophilicity index. the values of the electrophilicity index show that zncl2, znbr2 and zni2 are good nucleophiles; hence, these nucleophiles can attack the ligand l. electrophilic charge transfer (ect)19 is explained as the difference between the δnmax values of the interacting molecules. considering two molecules a (l) and b (zncl2, znbr2 and zni2) approaching each other, two cases exist, i) ect > 0, charge flow from b to a, ii) ect < 0, charge flow from a to b. the ect is calculated as follows: ( ) ( )amax max b–ect n n= δ δ (14) where (δnmax)a = µa / ηa and (δnmax)b = µb / ηb. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1004 dehestani and zeidabadinejad table ii. calculated ehomu, elumo, χ, µ, η, s and ω (in ev) for l, zncl2, znbr2 and zni2 molecule ehomo / ev elumo / ev χ / ev µ / ev η / ev s / ev ω / ev l –0.1999 –0.0271 0.1135 –0.1135 0.0864 0.0432 0.0746 zncl2 –0.3093 –0.1360 0.2226 –0.2226 0.0867 0.0433 0.2858 znbr2 –0.2877 –0.1224 0.2051 –0.2051 0.0826 0.0413 0.2546 zni2 –0.2699 –0.0678 0.1678 –0.1678 0.1010 0.0505 0.1394 the ect was calculated as 1.253, 1.169 and 0.347 for complexes 1–3, respectively. these results show that electrons are transferred from znx2 to l. therefore, the l was treated as an electron acceptor and, hence, znx2 was treated as an electron donor. thus, l has electrophilic behavior because the value of chemical potential is low. as shown in table ii, the high value of chemical potential and low value of electrophilicity index for these complexes 1–3 favor their nucleophilic behavior. local reactivity descriptors the fukui function (ff)16,20 or frontier function (fk+, fk–) measures changes in electron number (removing electrons from the homo or adding electrons to lumo, respectively) in chemical reactions and has been used to predict the reactivity of sites in a molecule. this function is a local density functional descriptor that is calculated using the proposed procedure based on a finite difference method.21 in this work, the two functions f+ and f – were used to determine electrophilic and nucleophilic attack, respectively. these functions can be given by: ( 1) ( )n n k k kf q q ++ = − for molecule k as an electrophile ( ) ( 1)n nk k kf q q −− = − for molecule k as a nucleophile where the parameters ( )nkq , ( 1)n kq − and ( 1)nkq + are the charges of molecule k calculated in the systems n, n–1 and n+1 electrons, respectively, at the optimized geometry of the molecule with n electrons. in the past few years, the condensed fukui functions have been used to explain the regioselectivity in chemical reactions.22 it is a tool that allows the prediction of which electron or moiety of a molecule will display more or less nucleophilic or electrophilic character. electrophilic reactivity descriptors +kf and nucleophilic reactivity descriptors − kf for all molecules are listed in table iii. the maximum values of the nucleophilic reactivity descriptors at zn indicate that these sites are more prone to electrotable iii. selected reactivity descriptors indexes of zncl2, znbr2 and zni2 molecule atom q(n) q(n–1) q(n+1) kf + kf − zncl2 zn 1.1269 1.2494 0.4060 –0.7208 –0.1226 znbr2 zn 0.9693 1.0814 0.2835 –0.6858 –0.1121 zni2 zn 0.7296 0.7651 0.0554 –0.6742 –0.0356 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ dipyrazol-1-ylmethane derivative and its zn(ii) complexes 1005 philic attack in the zncl2 molecule. thus, it can be stated that the zn atom attacked the n atom of l corresponding in all complexes. relationship between geometrical and topological parameters the calculations performed on the wide spectrum of related species enabled a deeper insight into the characteristics of intramolecular interactions to be obtained. here, mainly relationships between the bond length and the characteristics of the corresponding bond critical point are considered. for the systems analyzed herein, a pseudo-ring containing the n3…zn…n4 intramolecular bond is created and hence, an rcp also exist in complexes 1–3. the geometrical and topological parameters of these interactions are presented in table iv. briefly summarizing, the greater is the electron density at the rcp of an intramolecular bond, the stronger is the interaction and the shorter is the bond. the zn…n bond lengths are 2.2091, 2.3033 and 2.5109 å for complexes 1–3, respectively. the values of 2 bρ∇ for complexes 1–3 are –0.0201, –0.0388 and –0.0579 au, respectively. as can be seen, the bond length is shorter and the value of 2 bρ∇ higher in znlcl2 than in the other complexes. relatively, the values of the 2 bρ∇ , and table iv. topological properties (in au) of the charge density at the bond critical point of l, znlcl2, znlbr2, and znli2, ρ(r), 2 ( ),rρ∇ and gb, vb, he,b and |vb|/gb molecule bond ρb 2 ( )rρ∇ gb vb he,b |vb|/gb l n1–c1 0.2772 0.2120 0.1352 0.3472 –0.2120 2.568 n2–c1 0.2713 0.2030 0.1334 –0.4700 –0.3366 3.523 c1–h1 0.2744 0.2350 0.0331 –0.3011 –0.2680 9.0967 n1–n3 0.3748 0.1782 0.2132 –0.6046 –0.3914 2.8358 znlcl2 n1–c1 0.2741 0.2070 0.1514 –0.5097 –0.3583 3.3666 n2–c1 0.2688 0.1998 0.1413 –0.4823 –0.3410 3.4133 n1–n3 0.3703 0.1768 0.2027 –0.5822 –0.3795 2.8722 zn–cl1 0.0793 –0.0201 0.0829 –0.1157 –0.0328 1.395 zn–cl2 0.0810 –0.0207 0.0849 –0.1192 –0.0343 1.4040 zn–n3 0.0722 –0.0620 0.0878 –0.1135 –0.0257 1.2927 zn–n4 0.0712 –0.0617 0.0866 –0.1114 –0.0248 1.2863 znlbr2 n1–c1 0.2688 0.1998 0.1413 –0.4825 –0.3412 3.4147 n2–c1 0.2741 0.2073 0.3580 –0.5088 –0.1508 1.4212 n1–n3 0.3711 0.1775 0.2033 –0.5842 –0.3809 2.8735 zn–br1 0.0782 –0.0388 0.0722 –0.1056 –0.3340 1.4620 zn–br2 0.0779 –0.0393 0.0725 –0.1056 –0.3310 1.4566 zn–n3 0.0684 –0.0596 0.0822 –0.1047 –0.0225 1.2737 zn–n4 0.0683 –0.0593 0.0818 –0.1043 –0.0225 1.2751 znli2 n1–c1 0.2379 0.0848 0.1232 –0.3313 –0.2081 2.6891 n2–c1 0.2427 0.0888 0.1276 –0.3441 –0.2165 2.6967 n1–n3 0.2994 0.1065 0.1660 –0.4385 –0.2725 2.6416 zn–i1 0.0559 –0.0579 0.0587 –0.0596 –0.0009 1.0153 zn–i2 0.0538 –0.0563 0.0564 –0.0565 –0.0001 1.0018 zn–n3 0.0878 –0.1533 0.1571 –0.1608 –0.0037 1.0236 zn–n4 0.0882 –0.1538 0.1578 –0.1616 –0.0038 1.0241 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1006 dehestani and zeidabadinejad he,b, also the values of 1< |vb|/gb < 2 indicate that the zn–x (x = cl, br and i) and the zn–n interactions in all complexes have a significant partially covalent component. for the n1–c1, n2–c1, c1–h1 and n1–n3 interactions, the values of the laplacian are positive, which may mean that the ionic bond could not be classified as very strong one because all he,b values are negative, which may indicate that such interactions are rather strong. with reducing electronegativity of the x atom connected to zn(ii), the bond strength decreases. the sapt calculations the sapt applied here seems to be a proper approach to analyze the closedshell interactions. it was mentioned previously that the nucleophilic attack of zn atom of znx2 (x = cl, br and i) molecules on the ligand l leads to the formation of complexes 1–3. the sapt2 interaction energy as well as the interaction energy terms in eq. (3) are given in table v. if the attractive (negative) terms of the interaction energy are considered, the induction one, (2)ind ,e seems to be the most important, followed by the electrostatic term, (1)elst ,e and the dispersive term. however, the induction and dispersive terms are strongly damped by their exchange counterparts, exch i 2) nd (e − and (2) exch-disp ,e respectively. in the case of the induction interaction energy, it is reduced by 70–82 % while for the dispersive energy, its reduction is of 29–30 %. on the other hand, the first order electrostatic energy is outweighed by the first order exchange energy. this shows the role of the induction energy in spite of the fact that the latter one is strongly reduced by its counterpart. there are other observations; the absolute value of the dispersive energy is greater for the complex znlcl2 than for the complexes znlbr2 and znli2, even if the exchange counterpart of the dispersion energy is taken into account. however all interaction energy terms are interrelated and thus for stronger interaction and consequently shorter intermolecular distance, the absolute values of all interaction energy terms increase. table v. sapt interaction energies for the znli2, znlbr2and znlcl2 complexes energy term, au znli2 znlbr2 znlcl2 (1) elste –9595.54 –10084.66 –10205.6 (2) inde 11217.12 11786.44 11927.80 (2) dispe –18535.24 –19475.99 –19709.57 (1) exche –3216.07 –3403.57 –3444.39 ( 2) exch inde − 14696.23 15442.13 15627.33 (2) exch dispe − 959.7 1008.46 1020.56 hfeδ –1524.66 –1575.72 –1594.07 sapt2 inte –5998.46 –6302.91 –6378.50 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ dipyrazol-1-ylmethane derivative and its zn(ii) complexes 1007 conclusions the complexes of ligand l and the different non-covalent interactions were considered herein. it was found that for all interactions in which the zn atom acts as a nucleophile, a relationship between the zn…n distance and the laplacian of electron density at the corresponding bond critical point exists. this indicates a region of partially covalent bonds where the laplacian is negative and the electron density is lower of 0.15 au. electrophilic charge transfer confirms that the electrons are transferred from the znx2 to the ligand l; thus, the corresponding ligand can be treated as an electron acceptor. the results of fukui functions indicate zn atom attack on the n atom of the ligand. the sapt approach was also applied to analyze interactions in the zinc complexes. it was found that the induction energy is the most important attractive interaction energy term for the studied complexes, followed by electrostatic and dispersive terms. this means that for such strong interactions, the electron density shift is very important for the process of complexation but also that electrostatic interactions steer the arrangement of sub-units in the complexes. и з в о д qtaim испитивање деривата дипиразол-1-илметана и одговарајућих zn(ii) комплекса (znlx2, x = cl, br или i) maryam dehestani1 и leila zeidabadinejad1 department of chemistry, shahid bahonar university of kerman, 76169 kerman, iran применом квантне теорије атома у молекулима (qtaim) на b3pw91/6-31g(d) нивоу теорије урађена је тополошка анализа електронске густине 9-(4-(ди-1h-пиразол -1-илметил)фенил)-9h-карбазола (l) и одговарајућих цинк(ii) комплекса: znlcl2 (1), znlbr2 (2) и znli2 (3). анализирани су тополошки параметри добијени применом бадерове теорије који су карактеристични за критичне тачке координованог цинка и ароматичног прстена. израчунати су следећи структурни параметри: енергија највише попуњене молекулске орбитале (ehomo), енергија најниже непопуњене молекулске орбитале (elumo), тврди (η) и меки (s) карактер, апсолутна електронегативност (χ), индекс електрофилности (ω) и расподела електрона у комплексу znlx2 (δn). описане су корелације између енергије добијене применом sapt теорије (симетријски прилагођена пертурбациона теорија), геометријских, тополошких и енергетских параметара. 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1358 20. k. senthilkumar, m. ramaswamy, p. kolandaivel, j. quantum chem. 81 (2001) 4 21. w. yang, w. j. mortier, j. am. chem. soc. 108 (1986) 5708 22. g. f. lehr, r. g. lawler, j. am. chem. soc. 106 (1984) 4048. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice experimental study of the thermodynamic and transport properties of binary mixtures of poly(ethylene glycol) diacrylate and alcohols at different temperatures j. serb. chem. soc. 80 (7) 933–946 (2015) udc 547.421–036.7+547.42+ jscs–4770 544.032.4:532.13+532.14 original scientific paper 933 experimental study of the thermodynamic and transport properties of binary mixtures of poly(ethylene glycol) diacrylate and alcohols at different temperatures jelena m. vuksanović#, ivona r. radović#*, slobodan p. šerbanović# and mirjana lj. kijevčanin# faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11120 belgrade, serbia (received 9 october 2014, revised 16 january, accepted 19 january 2015) abstract: experimental density ρ, refractive index nd and viscosity η data of three binary systems of poly(ethylene glycol) diacrylate (pegda) + ethanol, + 1-propanol and + 1-butanol were measured at eight temperatures from 288.15 to 323.15 k, with temperature step of 5 k, and at atmospheric pressure. the experimental data were correlated as a function of the pegda mole fraction and temperature. the densities and refractive indices of the investigated mixtures could be fitted well with exponential function vs. composition, including the temperature dependence of the parameters, while in the case of the viscosities, a polynomial function fits well the composition of the mixtures. in the case of the temperature correlation, all three properties (ρ, ln η and nd) exhibited linear trends. the viscosity modeling was performed using four models: the unifac–visco, asog–visco, mcallister and the teja–rice models. for application of the unifac–visco model, interaction parameters of following groups were determined: ch2=ch/ch3, ch2=ch/ch2, ch2=ch/oh, ch2=ch/ch2o and ch2=ch/coo. in addition, in the same way, the binary interaction parameters used in the asog–visco model of the following groups were determined: ch2=ch/ch2, ch2=ch/oh, ch2=ch/ch2o and ch2=ch/coo. keywords: density; viscosity; refractive index; new unifac–visco parameters; new asog–visco parameters. introduction for a complete understanding of the thermodynamic and transport properties of pure organic compounds and multicomponent liquid mixtures, knowledge of their thermodynamic and transport properties over wide composition and tem * corresponding author. e-mail: ivonag@tmf.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc141009005v _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 934 vuksanović et al. perature ranges is necessary. studies of thermodynamic properties contribute to the understanding of the behavior of various organic compounds and their functional groups, and are of great importance for the understanding of the molecular interactions in multicomponent mixtures. this work is a continuation of ongoing research related to determination of thermophysical and transport properties of mixtures containing polymers.1–5 in this work, a biodegradable and biocompatible polymer, i.e., poly(ethylene glycol) diacrylate (pegda), was investigated in binary mixtures with small chain alcohols, i.e., ethanol, 1-propanol, and 1-butanol. poly(ethylene) glycol diacrylate (pegda) is a low volatility and medium viscosity clear liquid with good flexibility and elongation, good water dispersiblity, low skin irritancy and good reactivity. it is soluble in water and is used as a functional co-monomer for flexible plastics and as a cross linking agent between the molecular chains of polymers.6 furthermore, pegda is a synthetic, hydrophilic starting material for the production of hydrogels in the presence of a photo-initiator and uv light. this polymer is widely known as a biocompatible and non-immunogenic material suitable for various chemical manipulations, with application in tissue engineering and regenerative medicine.7 one of the important usages of pegda in biological and biomedical applications could be for controlled release of drugs by producing well defined microor nano-channels inside the polymer, which would make the release of drugs through the pathways more readily predictable and controlled. a procedure for producing pegda particles with specific, internal channels for drug release is described in detail in the literature.8 the investigated short-chain alcohols are completely miscible with water and used as solvents in various fields of industry. in this particular case, their application in pharmaceutical industry is of great importance because mixtures of pegda and alcohols could be potentially applied in the pharmaceutical industry for the controlled release of drugs. in addition, they find application for the removal of co2 from the air or in biochemical applications.6 alcohols are polar compounds with the following dipole moments: 5.67×10– 30 c·m for ethanol, 5.67×10–30 c·m for 1-propanol and 6.00×10–30 c·m for 1-butanol.9 pegda, as a hydrophilic polymer, contains polar or charged functional groups (carbonyl groups adjacent to an ether linkage) which make them soluble in polar compounds, such as alcohols. from the chemical structures of pegda and alcohols, it is evident that alcohols contain a hydrogen responsible for hydrogen bonding between the molecules of the same alcohol or with an oxygen from the polymer, while in the pegda molecule, ester coo groups are present. it could be conclude that these molecules might form intermolecular hydrogen bonds, i.e., hydrogen from the hydroxyl group in alcohols with the oxygen from the coo group in a polymer. there are also van der waals dispersion _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ volumetric properties and viscosities of poly(ethylene glycol)diacrylate + alcohols 935 forces and dipole–dipole interactions between alcohols and polymer, and molecules of the same compound. the hydrogen bonding and dipole–dipole interactions will be much the same for all the alcohols, but the dispersion forces will increase as the alcohol becomes larger. the intention of this work was to investigate how the thermodynamic and transport properties of pegda and alcohol mixtures change with respect to the alcohol chain length and with temperature, bearing in mind the possible interactions between the above-mentioned components. thus, in this work, the densities ρ, refractive indices nd and viscosities η of three binary systems of poly(ethylene glycol) diacrylate (pegda) + ethanol or + 1-propanol or + 1-butanol were measured at eight temperatures (288.15, 293.15, 298.15, 303.15, 308.15, 313.15, 318.15 and 323.15 k) and at atmospheric pressure. additionally, the viscosity data were modeled using the unifac–visco, asog–visco, mcallister and teja–rice models. generally, if a viscosity calculation is based on already determined parameters given in the literature, the unifac–visco and asog–visco models are actually predictive. however, since in the case of the systems investigated in the present study, some of the parameters were not known, the intention was to determine accurately their values and the unifac– –visco and asog–visco models were considered as correlative. experimental chemicals poly(ethylene glycol) diacrylate (pegda), with molecular formula c2n+6h4n+6on+3 and with number average molecular weight 700 g mol-1, was purchased from aldrich (cas no.: 26570-48-9, cat. no.: 455008, lot no.: mkbh4151v). alcohols of reagent grade: ethanol (≥ 99.9 mass %), 1-propanol (≥ 99.5 mass %), and 1-butanol (≥ 99.5 mass %) were purchased from merck. the chemicals were kept in dark bottles under an inert atmosphere and ultrasonically degassed before sample preparation. apparatus and procedures the density ρ measurements were performed using an anton paar dma 5000 digital vibrating u-tube densimeter (with automatic viscosity correction). the temperature in the cell was regulated to ±0.001 k with a built in solid-state thermostat. calibration of the apparatus was performed daily using ambient air and millipore quality water. a mettler ag 204 balance, with a precision 1×10-7 kg, was used for precise measurement of mass composition for all binary mixtures, using the cell and the procedure described previously.10 the uncertainty of the mole fraction calculation was less than ±1×10-4. the experimental uncertainty in density was about ±1×10-2 kg m-3. the refractive index nd measurements were performed using an automatic anton paar rxa 156 refractometer, which works with the wavelength of 589 nm. throughout this procedure, the temperature of the sample was kept constant with a built-in thermostat within an accuracy of ±0.03 k. the estimated experimental uncertainties in the refractive index were about ±1×10-4. the viscosity, η, measurements were performed using a digital stabinger viscometer (model svm 3000/g2). the instrument contains two measuring cells; one of which is used _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 936 vuksanović et al. for measuring the density of the sample, while the other one measures the dynamic viscosity. the kinematic viscosity was calculated from the measured density and dynamic viscosity. during this procedure, the temperature in the cells was regulated to ±0.01 k with a built in solid-state thermostat. the relative uncertainty in the dynamic viscosity measurements was estimated to be 0.35 %. the densities, dynamic viscosities and refractive indices of the pure substances at several temperatures and at atmospheric pressure are compared with literature values7,11-19 in table i. the agreement with the literature for the density measurements in most cases was within 0.55 kg m-3, while the viscosity measurements were within 0.02 mpa s. the experimental refractive indices of pure components agree with literature values within 8×10-4 for alcohols and within 0.002 for pegda. table i. densities, ρ, viscosities, η, and refractive indices, nd, of the pure components at temperature t and at atmospheric pressure; the standard uncertainties σ for each variables are σ(t) = 0.01 k; σ(p) = 5 %; σ(x1) = ±1×10 -4, and the combined uncertainties σc are σc(ρ) = = ±1×10-2 kg m-3; σc(nd) = ±1×10 -4; σc(η) = 0.35 %, at the 0.95 level of confidence (k ≈2) substance t / k ρ / 103 kg m-3 η / mpa s nd this work lit. this work lit. this work lit. pegda 293.15 1.47012 1.470 7 303.15 1.46618 1.465 7 313.15 1.46228 1.460 7 ethanol 293.15 0.789547 0.790011 1.1885 1.209711 298.15 0.785257 0.785711 1.0838 1.099011 1.35999 1.3592212 303.15 0.780942 0.780911 0.98999 0.997111 313.15 0.772202 0.773311 0.82807 0.828011 323.15 0.763276 0.763613 0.69427 0.708113 1-propanol 288.15 0.807931 0.8074914 293.15 0.803946 0.8037514 298.15 0.799932 0.7997514 1.9222 1.94315 1.38334 1.3837 16 303.15 1.7158 1.72515 1-butanol 293.15 0.810205 0.809714 2.9321 2.94116 1.39929 1.3992916 298.15 0.806384 0.806014 2.5656 2.57116 1.39725 1.3974116 303.15 0.802538 0.8019118 2.2518 2.27116 1.39519 1.395919 308.15 0.798659 0.7980718 results and discussion the experimental data of density, viscosity, and refractive index for three binary systems (pegda + ethanol, pegda + 1-propanol, and pegda + 1-butanol) at eight temperatures (288.15 to 323.15 k), over the entire composition range and at atmospheric pressure are reported in table s-i of the supplementary material to this paper. fitting of the experimental values of density and refractive index was performed as a function of pegda mole fraction with temperature dependant parameters. the quality of the fitting was estimated by the deviation between experi_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ volumetric properties and viscosities of poly(ethylene glycol)diacrylate + alcohols 937 mental value and those calculated by different equations. the best results were obtained using the following equation: ( ) ( ) 10 11 d 00 01 1 20 21 , exp a a t n a a t x a a t ρ  + = + +  + +   (1) where a00, a01, a10, a11, a20 and a21 are the fitting parameters, x1 is pegda mole fraction and t is temperature. the viscosity values, unlike density and refractive index values, varied greatly with change in temperature and hence, a single equation that combined the temperature and composition dependencies did not give good results and so fitting the viscosity values as a function of pegda mole fraction or temperature was performed separately using the following equations: 320 1 1 2 31 1b b x b x b xη = + + + (2) 10ln c c t η = + (3) where b0, b1, b2, b3, c0 and c1 are the fitting parameters, x1 is the pegda mole fraction and t is the temperature. for the three binary mixtures at different temperatures and compositions, the parameters of eq. (1) are presented in table s-ii and the parameters of eqs. (2) and (3) in table s-iii (supplementary material). the corresponding root-mean -square deviations (rmsd) σ, defined by eq. (4) are presented in tables s-ii and s-iii of the supplementary material: ( ) 1/2 2 exp cal σ    −   =          n i y y n (4) yexp and ycal are experimental and calculated values of ρ, η or nd, respectively, and n is the number of experimental data points. comparison of the densities, viscosities and refractive indices of binary systems of pegda and alcohols at 288.15 and 323.15 k and at atmospheric pressure are given in fig. 1a–c, respectively. it is obvious from fig. 1 that the experimental values of the density, viscosity, and refractive index data of pure pegda are significantly higher than those of the analyzed alcohols. in addition, it is evident that the densities and refractive index data of the mixtures increased exponentially with increasing pegda mole fraction, while the increase in the viscosities vs. pegda composition followed a polynomial trend. it can be observed that the density and refractive index increased considerably in the range of _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 938 vuksanović et al. lower pegda concentrations (from 0 to 0.3 mole fraction), while approaching higher pegda mole fractions, the curves trended to constant values. this means that the influence of pegda on the overall densities and refractive indices of the pegda + alcohol mixtures was far greater in comparison to the influence of the alcohols. in the case of viscosity, there was a constant increase in the values with increasing pegda mole fraction. in addition, it is noticeable that the densities, viscosities and refractive indices of pure compounds and their mixtures decreased as the temperature increased. this temperature influence was the most pronounced for the viscosity data. namely, the viscosity of pure pegda at 288.15 k was almost six times higher than at 323.15 k. fig. 1. comparison of experimental data for: a) density, b) viscosity and c) refractive index vs. mole fraction for the three systems at 288.15 and 323.15 k and at atmospheric pressure, where the symbols refer to: pegda (1) + ethanol at (■) 288.15 and (□) 323.15 k; pegda (1) + 1-propanol (2) at (▲) 288.15 and (δ) 323.15 k; pegda (1) + 1-butanol (2) at (●) 288.15 and (○) 323.15 k; the lines present the results calculated by eq. (1) for the ρ and nd data and by eq. (2) for the η data. experimental values of the density and refractive index against temperature at atmospheric pressure are presented in figs. 2 and 3, respectively, while ln η vs. 1/t changes at atmospheric pressure are depicted in fig. 4. one can conclude that density and refractive index data exhibit linear dependences on temperature, with a decreasing tendency of the property with increasing temperature. figs. 2 and 3 also prove that the changes of densities and _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ volumetric properties and viscosities of poly(ethylene glycol)diacrylate + alcohols 939 refractive indices are greater in the range of smaller pegda mole fractions (from 0 to 0.3). function of ln η vs. 1/t exhibited a linear trend as well, confirming that with increasing temperature the pegda + alcohols mixtures become less viscous (fig. 4). fig. 2. experimental data of density, ρ, vs. temperature, t (288.15 to 323.15 k), at atmospheric pressure, for the systems: a) pegda (1) + ethanol (2); b) pegda (1) + 1-propanol (2); c) pegda (1) + 1-butanol (2). the symbols refer to mole fractions of pegda: (◊) 0; (♦) 0.05; (○) 0.1; (●) 0.2; (δ) 0.3; (▲) 0.4; (□) 0.5; (■) 0.6; (∇) 0.7; (▼) 0.8; (+) 0.9; (◄) 1. in addition, modeling of viscosity experimental data was performed using predictive and correlative types of models. in this work unifac–visco20,21 and asog–visco22 models were used for the determination of the dynamic viscosity of the three binary mixtures. unifac–visco and asog–visco are group contribution models aimed at activity coefficient determination. in both methods, the activity coefficients in the mixtures are related to interactions between structural groups. consequently, the parameters characterizing interactions between pairs of structural groups, called group interaction parameters, are necessary (αnm for unifac–visco and mkl and nkl for asog–visco). if the parameters are given in the literature, it is not necessary to determine them again. in this work, new group interaction parameters were determined from the experi_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 940 vuksanović et al. mentally measured viscosities using the marquardt23 optimization technique for the minimization of the objective function: 2 exp cal exp1 1 min n i i of n η η η =  − = →      (5) where ηexp and ηcal denote the experimental and calculated values of the dynamic viscosity η and n is the number of experimental data points. fig. 3. experimental data of the refractive index, nd, vs. temperature, t (288.15 to 323.15 k), at atmospheric pressure, for the systems: a) pegda (1) + ethanol (2); b) pegda (1) + 1-propanol (2); c) pegda (1) + 1-butanol (2). the symbols refer to mole fractions of pegda: (◊) 0; (♦) 0.05; (○) 0.1; (●) 0.2; (δ) 0.3; (▲) 0.4; (□) 0.5; (■) 0.6; (∇) 0.7; (▼) 0.8; (+) 0.9; (◄) 1. the new unifac–visco interaction parameters, αnm, between the following groups: ch2=ch/ch3, ch2=ch/ch2, ch2=ch/oh, ch2=ch/ch2o and ch2=ch/coo are summarized in table ii. the remaining interaction parameters were taken from the original model20,21 and previous papers.5,24 in the similar way, new asog–visco group interaction parameters, mkl and nkl, of following groups ch2=ch/ch2, ch2=ch/oh, ch2=ch/ch2o, _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ volumetric properties and viscosities of poly(ethylene glycol)diacrylate + alcohols 941 ch2=ch/coo were determined using the original interaction parameters22 and parameters from previous papers.3,24 the results are summarized in table iii. fig. 4. experimental data of viscosity, ln η, vs. 1/t (288.15 to 323.15 k), at atmospheric pressure, for the systems: a) pegda (1) + ethanol (2); b) pegda (1) + 1-propanol (2); c) pegda (1) + 1-butanol (2). the symbols refer to mole fractions of pegda: (◊) 0; (♦) 0.05; (○) 0.1; (●) 0.2; (δ) 0.3; (▲) 0.4; (□) 0.5; (■) 0.6; (∇) 0.7; (▼) 0.8; (+) 0.9; (◄) 1. table ii. the unifac–visco interaction parameter, αnm n/m ch3 ch2 ch2=ch oh ch2o coo ch3 0 –709.5 a –570.861d 594.4a –50.17b –172.4a ch2 66.53 a 0 1161.742d 498.6a –319.930b 1172a ch2=ch –872.856 d 1489.036d 0 –869.851d –2243.897d –57.440d oh 1209a –634.5a –549.041d 0 –619.360b 68.35a ch2o 456.91 b –340.250b 248.157d 25.340b 0 –56.95c coo –44.25a 541.6a –445.344d 186.8a –137.945c 0 aoriginal unifac–visco parameters;20,21 bunifac–visco parameters from the literature;5 cunifac– –visco parameters from the literature;24 dnew unifac–visco parameters moreover, the experimental viscosity data were correlated with the oneparameter teja and rice,25,26 and mcallister27 two-parameter three-body and _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 942 vuksanović et al. three-parameter four-body models. these models are described in detail in a previous papers.28,29 table iii. the asog–visco interaction parameters k/l ch2 ch2=ch oh ch2o coo mkl ch2 0 0.2428 d –0.3570a –10.9924b 0.3682a ch2=ch 1.7603 d 0 1.6245d –6.3287d –2.5891d oh 14.1460a –0.3330d 0 –2.2661b –40.2000a ch2o –33.9591 b –173.2012d 1.5287b 0 –2.9720c coo 0.0952a 197.1496d 19.1310a –4.7468c 0 nkl ch2 0 356.102 d 469.650a –1.928b 112.590a ch2=ch 187.229 d 0 413.379d 299.095d 298.614d oh –6137.000a 476.584d 0 –14.965b 11583.000a ch2o –8.176 b 298.966d –127.018b 0 –835.188c coo –383.600a 300.577d –5747.000a –433.643c 0 aoriginal asog–visco parameters;22 basog–visco parameters from the literature;3 casog–visco parameters from the lierature;24 dnew asog–visco parameters the ability of these models to predict successfully the dynamic viscosities of the investigated binary mixtures and to correlate the experimental viscosity data is presented with percentage deviations, pdmax, between the experimental and calculated viscosities, using the following equation: exp calmax exp max1 100 ( ) n i i pd n η η η = − =  (6) where (ηexp)max is the maximum of the experimental η values. the results obtained by the unifac–visco, asog–visco, teja–rice and mcallister models for the mixtures of pegda + ethanol or 1-propanol or 1-butanol over the investigated temperature range are given in table iv. a graphical presentation of the experimental viscosity deviation from the values obtained by the selected models is given in fig. 5. table iv. results of the viscosity prediction and correlation for the investigated binary systems at the temperatures (288.15 to 323.15) k and at atmospheric pressure t / k predictive approach correlative approach unifac–visco asog–visco teja–rice mcallister-3 mcallister-4 pdmax / % pdmax / % pdmax / % pdmax / % pdmax / % pegda (1) + ethanol (2) 288.15 7.62 2.16 11.03 4.46 1.59 293.15 5.79 2.65 10.48 4.66 1.67 298.15 4.08 2.40 10.10 4.88 1.84 303.15 2.82 1.76 9.70 4.97 1.84 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ volumetric properties and viscosities of poly(ethylene glycol)diacrylate + alcohols 943 table iv. continued t / k predictive approach correlative approach unifac–visco asog–visco teja–rice mcallister-3 mcallister-4 pdmax / % pdmax / % pdmax / % pdmax / % pdmax / % pegda (1) + ethanol (2) 308.15 2.20 1.08 9.38 5.15 1.92 313.15 2.19 0.66 9.04 5.32 2.05 318.15 2.95 1.17 8.88 5.49 2.20 323.15 3.74 1.57 8.51 5.50 2.15 pegda (1) + 1-propanol (2) 288.15 4.66 1.54 5.96 3.01 1.11 293.15 3.14 0.77 5.68 3.13 1.15 298.15 1.76 0.50 5.52 3.30 1.15 303.15 0.87 0.84 5.29 3.40 1.20 308.15 1.39 1.34 5.07 3.49 1.27 313.15 2.78 1.95 4.90 3.59 1.31 318.15 4.17 2.61 4.72 3.66 1.36 323.15 5.32 3.07 4.65 3.73 1.42 pegda (1) + 1-butanol (2) 288.15 4.56 1.56 3.89 2.13 0.65 293.15 3.46 0.78 3.79 2.22 0.68 298.15 2.47 0.66 3.63 2.32 0.78 303.15 1.69 0.51 3.57 2.45 0.72 308.15 1.04 0.47 3.49 2.54 0.78 313.15 0.72 0.44 3.41 2.64 0.82 318.15 1.25 0.44 3.35 2.74 0.86 323.15 1.98 0.45 3.35 2.78 0.92 unifac–visco model gave very good results for the prediction of the viscosity of the three investigated binary systems. in almost all cases, the maximum percentage deviation pdmax did not exceed 5 %. the largest deviations were obtained at the lowest temperature (systems with ethanol and 1-butanol), or at the highest investigated temperature in the case of the system containing 1-propanol. inspection of figs. 5a and 5b confirms this conclusion. the asog– –visco model gave even better results, with maximum percentage deviations pdmax of 2.65 % in almost all cases. the best results were obtained for the pegda + 1-butanol binary mixture, with pdmax of less than 0.78 %, except at 288.15 k, when the deviation was 1.56 %. however, this model fits the experimental data satisfactorily (fig. 5c). from the results obtained from correlative models, it could be concluded that the best results for all three systems were obtained with the mcallister-4 model. this conclusion is supported by the graphical representation given for the systems pegda + ethanol, or + 1-butanol (fig. 5a and c) where the model correlates the experimental points very well. the teja–rice model gave the worst correlations of the experimental data, with the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 944 vuksanović et al. highest pdmax values, up to 11.03 % for the pegda + ethanol binary mixtures. this conclusion is obvious from figs. 5b and 5c, especially at lower temperatures. the best results for all three types of correlative models were obtained for the pegda + 1-butanol binary system, for which the lowest pdmax deviations were obtained for themcallister-4 model (less than 1 %). fig. 5. modeling of the viscosity data for the systems: a) pegda (1) + ethanol (2), b) pegda (1) + 1-propanol (2) and c) pegda (1) + 1-butanol (2). the symbols refer to experimental points (●) at 288.15 and (▲) at 323.15 k, while the lines present the results calculated by models given in table iv: (—) asog–visco, (---) teja–rice and (…) mcallister-4 models. conclusions in this work, the experimental data of the density ρ, viscosity η, and refractive index nd are reported for pegda + ethanol, pegda + 1-propanol and pegda + 1-butanol binary mixtures at temperatures in the range 288.15 to 323.15 k, with a temperature step of 5 k, over the whole composition range, and at atmospheric pressure. all measured physical properties increased with increasing pegda mole fraction and with decreasing temperature. the measured physical properties were correlated as a function of temperature and of pegda mole fraction. the densities and refractive indices of the mixtures showed _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ volumetric properties and viscosities of poly(ethylene glycol)diacrylate + alcohols 945 exponential dependence vs. composition, including the temperature dependence of the parameters, while the viscosities exhibited polynomial dependence over the mixture composition. in the case of the temperature correlation, all three properties (ρ, ln η and nd) exhibited linear trends. the influence of temperature on the viscosity of the mixtures was larger than its influence on the density and refractive index. moreover, the influence of the pegda mole fraction on the overall densities and refractive indices of the pegda + alcohol mixtures was far greater in comparison to the influence of the alcohol type. in addition, viscosity modeling was performed using two types of models: the predictive unifac– –visco and asog–visco, and the correlative mcallister and teja–rice models. the predictive asog–visco gave better results between the predictive models, while of the correlative models, the best results were obtained using the mcallister-4 equation, for all three investigated binary systems. furthermore, new unifac–visco and asog–visco binary interaction parameters were determined from the experimental viscosity data. supplementary material experimental data of densities ρ, viscosities η and refractive nd indices and fitting parameters and root-mean-square deviations for eqs. (1)–(3) of pegda + alcohol binary mixtures are available electronically from http://www.shd.org.rs/jscs/ or from the corresponding author on request. acknowledgement. the authors gratefully acknowledge the financial support received from the research fund of the ministry of education, science and technical development of the republic of serbia and the faculty of technology and metallurgy, university of belgrade (project no. 172063). и з в о д eкспериментално испитивање термодинамичких и транспортних својстава бинарних смеша поли(етиленгликол)-диакрилата и алкохола на различитим температурама јелена м. вуксановић, ивона р. радовић, слободан п. шербановић и мирјана љ. кијевчанин технолошко–металуршки факултет, универзитет у београду, карнегијева 4, 11120 београд експериментални подаци за густину, ρ, индекс рефракције, nd, и вискозност, η, три бинарна система поли(етиленгликол)-диакрилата (pegda) + етанол, + 1-пропанол и + 1-бутанол су мерени на осам температура (288,15 to 323,15 k), са кораком 5 k, и на атмосферском притиску. експериментални подаци су корелисани у функцији молског удела pegda и температуре. густине и индекси рефракције испитиваних смеша су фитовани експоненцијалном функцијом у зависности од састава, док се у случају вискозности полиномска функција показала као најбоља кроз цео опсег молских удела. у случају температурне зависности, све три величине (ρ, ln η и nd) показују линеаран тренд. вискозност је моделована помоћу четири модела: unifac–visco, asog–visco, mc-allister и teja–rice. помоћу unifac–visco модела одређени су интеракциони параметри следећих група: ch2=ch/ch3, ch2=ch/ch2, ch2=ch/oh, ch2=ch/ch2o и ch2=ch/coo. такође, на исти начин помоћу asog–visco модела су одређени и бинар_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 946 vuksanović et al. ни интеракциони параметри следећих група: ch2=ch/ch2, ch2=ch/oh, ch2=ch/ch2o and ch2=ch/coo. 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {ruo4-mediated oxidation of secondary amines 2. imines as main reaction intermediates} j. serb. chem. soc. 82 (6) 627–640 (2017) udc 546.96–31+542.943:547.239.2–304.2: jscs–4992 547.415.3+542.96 original scientific paper 627 ruo4-mediated oxidation of secondary amines. 2. imines as main reaction intermediates cristina a. florea*, anca hîrtopeanu, cristina stavarache and horia petride romanian academy, “costin d. nenitzescu” center of organic chemistry, spl. independenţei 202-b, ro-060023 bucharest, romania (received 14 september 2016, revised 4 april, accepted 24 april 2017) abstract: oxidation by ruo4 (generated in situ from ruo2 and naio4) of secondary amines such as bn–nh–ch2r (1; r=h, me) gave complex reaction mixtures, but mainly amides. in the presence of cyanide, the leading products were α-aminonitriles. comparison of the oxidation products of 1 with those from the corresponding imines phch=n–ch2r and bn–n=ch–r showed that formation of the indicated imines is the first main step in the oxidation of 1. a detailed mechanism is proposed. keywords: oxidation; secondary amines; ruthenium tetraoxide; imines; α-aminonitriles. introduction several mechanisms for the oxidation of alkanes,1 arenes,1 olefins,2 alcohols3 and amines3–6 using various ruthenium-containing catalysts are known, but they refer to totally different substrates and/or oxidants than those used in the present study. mechanisms for the oxidation of alkanes,7,8 alkenes,9 alcohols,10 ethers7 and tertiary amines11–15 with ruo4 as an oxidant16 were previously proposed. for instance, it was advanced that n,n-dialkylanilines are oxidized by rucl3/o2/nacn (i.e., in situ generated ruo4) via iminium cations, formed through single electron transfer (set) steps.11 independently, iminium ions were also proposed as key intermediates for the ruo4-oxidation of tertiary aliphatic amines.13–15 in all these cases,11–15 the transient existence of iminium ions was proved by cyanide-trapping to give α-aminonitriles (strecker reaction). information on the corresponding oxidation of secondary amines refers to lower-than-eight-valent ruthenium catalysts, from ru0 to ruvii.17–23 the formation of imines seems to be favoured in all these cases. imines are presumed to * corresponding author. e-mail: antonetaflorea@yahoo.com https://doi.org/10.2298/jsc160914055f 628 florea et al. result also from secondary amines and ruo4, a hypothesis16 advanced in 2005, but still not experimentally demonstrated. it must be added here that imines are versatile intermediates, widely used in well-known reactions, such as aza-baylis– –hillman,24 aza-diels–alder,25 mannich26 and strecker reactions.11,12 as presented in a preceding paper,27 the ruo4-mediated oxidation of secondary aliphatic amines, such n-methyl(1a) or n-ethylbenzylamine (1b) might start with the formation of imines 2a and b plus 3a and b (route a, scheme 1) and/or n-hydroxylamines 4a and b (route b). imines would result via a formal dehydrogenation from the n atom and the adjacent c atom (benzyl for 2, alkyl for 3), but hydroxylamines would derive from an oxidative attack on the n atom. comparison of the reaction products formed from 1 with those derived from 4 allowed the elimination route b as a main pathway.27 consequently, this paper analyzes route a as the mechanistic choice. by analogy with the previously discussed case of tertiary amines,11–15 the intermediacy of imines will be proved by cyano-trapping reactions. bn:benzyl a: r=h b: r=me bn-nh-ch2r phch=n-ch2r (benzyl) bn-n=ch-r attack at n (alkyl) 4a-b (route a) (route b) bn-n(oh)-ch2r 3a-b1a-b 2a-b attack at n and α−ν−c scheme 1. oxidative routes for 1a and b. experimental instrumentation melting points were taken with a boetius hot plate and are uncorrected. ft-ir spectra were registered on a bruker vertex 70 instrument, equipped with a diamond crystal atr accessory. nmr spectra were recorded with a varian unity inova 400 spectrometer, operating at 400 mhz (1h) and 100 mhz (13c). mass spectra were obtained with a gc 6890 agilent technologies gas chromatograph coupled with a ms 5975 b quadrupole mass spectrometer, using standard 70 ev ionization energy. materials hydrated ruo2 (aldrich), naio4 (merck), and the organic solvents (chemical co., iaşi, romania) were used as purchased, except for chcl3, which was stored over anhydrous na2co3 and filtered prior to use. the formulae of all substrates and identified reaction products (1–28) are given in scheme 2. compounds 1–25 were practically identical to those with the same numbers in part 1 of this work,27 except for the newly added compounds 8f and 14d. the origin and spectral characteristics (1h-nmr, 13c-nmr and ms) of compounds 1, 2 and 4–25 (except those of 8f and 14d) were previously presented.27 oxidation of secondary amines 629 8, 17 r r 1 a h h b me me c me h d h ph e me ph 2a-c: ph-ch=n-ch2r 3a-b: bn-n=ch-r 4a-b: bn-n(oh)-ch2r 5a-c: r-cho 6a-c: phco-nh-ch2r 7a-b: bn-nh-co-r 8a-f: bn-n(ch2r)-co-r 1 9a-c: ph-ch=n(o)-ch2r 10a-b: bn-n(o)=ch-r 12b-c: r-cn 11a-c: r-ch2-nh2 14a,d: [bn-n(ch2r)-]2ch2 13b-c: r-conh2 15a-c: ph-ch(cn)-nh-ch2r 16a-b: bn-nh-ch(cn)-r 17a-e: bn-n(ch2r)-ch(cn)-r 1 18a-b: ph-c(cn)=n-ch2r 19a-b: bn-n=c(cn)-r 20a-b: ph-ch=n-ch(cn)-r 21a-b: ph-c(cn)=n(o)-ch2r 22a-b: bn-n(o)=c(cn)-r 23a-b: bn-n(ch2r)-c(=nh)-cn 24a-b: bn-n(ch2r)-cn 25a-b: bn-n(ch2r)-conh2 1-7, 9-16, 18-25 r a h b me c ph n n n bn bn bn 26 8f: r=cn, r1=h 14d: r=cn bn: benzyl bn-nh-ch2r 1a-b ruo4/naio4 (nacn) imines (2, 3) hydroxylamines (4) aldehydes (5) amides i (6, 7), amides ii (8) nitrones (9, 10) others i (11-14) nitriles i (15, 16), nitriles ii (17) cyanoimines (18-20) cyanonitrones (21, 22) formamidines & related compounds (23-25) others ii (26-28) 27: bn-n(cho)-ch2-nh-bn 28: me-co-nh-et scheme 2. oxidation products of 1a–b. derivatives 3b,28 8f,29 2630,31 and 2732 are all known from the literature and were prepared according to the indicated procedures. compound 28 was purchased from aldrich. the nmr and ms data for 3b, 8f, 14d and 26–28 are presented as supplementary material to this paper. compound 14d was described either as a solid (m.p., 62–64 °c)33 or as an oil,34 but the reported nmr characteristics were quite similar. a new, simpler method of preparation of 14d (obtained as a solid) is proposed in the supplementary material. oxidations by ruo4/naio4 (with or without nacn) all oxidations were performed as described in part 1, including the identification and quantification of reaction products by nmr and gc–ms analyses of the various reaction mixtures.27 results and discussion the oxidations were performed either in the absence of cyanide (a-conditions) or in its presence (b-conditions); the respective results are presented in 630 florea et al. table i. to gain in simplicity, the desired entries (x and y) of table i are cited as t-x,y. the yield of benzaldehyde (5c) from table i actually refers to that of the sum 5c+benzoic acid, since benzoic acid is always derived from 5c.27 table i. oxidation of selected compounds entry no. compound (conversion, %)a reaction productsb (yieldc, %) a) oxidations in the absence of cyanided 1. 1a (55)e 2a (2.2), 2c (8.6), 4a (0.2), 5c (8.0), 6a (2.2), 6c (1.8), 7a (3.8), 8a (30.0), 8d (7.4), 9a (2.4), 12c (2.0), 13c (0.6), 14a (1.3) 2. 1b (81)e 2b (2.1), 2c (4.1), 5c (34.3), 6b (3.9), 6c (3.1), 7a (1.7), 7b (4.1), 8b (3.5), 8c (15.2), 8e (3.8), 9b (0.3), 12c (7.4), 13c (0.5) 3. 11c (100) 2c (36.1), 5c (9.8), 6c (0.2), 12c (11.7), 13c (4.7) 4. 2a (100) 5c (80.0), 6a (18.5) 5. 2b (98) 5c (88.4), 6b (9.7) 6. 26 (80) 2c (8.7), 5c (2.4), 7a (3.5), 12c (0.9), 13c (1.5), 27 (65.5) 7. 3b (100) 5c (16.6), 6c (1.2), 7a (34.1), 7b (5.9), 12c (16.1), 13c (0.3) 8. 11c+11bf (99) 2b (4.9), 2c (9.4), 3b (2.5), 5c (1.8), 6b (1.8), 6c (0.7), 7a (7.6), 7b (9.1), 12c (38.0), 13c (8.0), 28 (2.1) 9. 1b+11bg (87) 2b (2.2), 2c (3.2), 5c (23.2), 6b (3.8), 6c (2.2), 7a (1.0), 7b (2.5), 8b (6.1), 8c (32.5), 8e (2.6), 9b (0.2), 12c (5.4), 13c (0.3) b) oxidations in the presence of cyanided 10. 1a (13)e 2c (1.2), 5c (0.9), 6a (0.4), 7a (0.7), 8a (1.8), 8d (8.9), 11c (2.5), 12c (0.9), 15a (6.3), 16a (2.2), 17a (18.2), 17d (15.3), 18a (0.6), 20a (0.3), 21a (0.4), 22a (0.8), 23a (2.9), 24a (1.0), 25a (4.5) 11. 1b (37)e 2b (5.2), 2c (1.8), 5c (0.5), 6b (0.5), 7b (0.6), 8b (1.0), 8c (0.6), 8e (2.7), 9b (7.8), 12c (1.5), 15b (15.6), 16b (10.9), 17b (9.9), 17c (1.0), 17e (8.1), 18b (0.8), 20b (0.5), 21b (0.5), 22b (0.9), 23b (4.2), 24b (0.9), 25b (8.0) 12. 11c (100) 2c (6.5), 6c (0.4), 12c (11.2), 15c (36.5) 13. 2a (97) 5c (4.3), 6a (0.5), 15a (84.9), 18a (0.4), 21a (1.5) 14. 2b (98) 5c (2.3), 6b (0.6), 15b (84.5), 18b (1.8), 21b (1.1) 15. 3b (100) 7b (0.7), 12c (1.2), 15c (5.0), 16b (75.3), 20b (0.8), 22b (1.2) 16. 15b (10) 2b (14.3), 5c (21.3), 18b (34.2), 21b (18.0) 17. 16a (23) 5c (3.6), 6c (2.2), 8f (1.5), 12c (1.5), 14d (7.6), 15c (26.2), 20a (3.9), 22a (9.6) 18. 16b (25) 5c (5.2), 6c (1.9), 12c (1.3), 13c (0.5), 15c (19.0), 19b (4.2), 20b (8.5), 22b (15.3) acalculated against the reacted substrate; bformulae in scheme 2; cyields (mole ratios of product/reacted substrate) are calculated regardless of the stoichiometry (excepting those of entry 7); dreaction conditions (for 1 mmol of substrate): a – ruo2⋅xh2o (10–15 mg), co-oxidant naio4 (4 mmol), 20 ml chcl3/water = 1/1 volume ratio, room temperature, 3–5 h; b – as in a, but nacn (4 mmol) in water (10 ml) was also added; e data from ref. 1; f11c/11b = 1/1 mole ratio. conversion and yields are referred to 11c; g1b/11b = 1/0.4 mole ratio. conversion and yields are referred to 1b oxidation of amines 1a and b. reaction products the oxidation mixtures derived from 1a and b under a-conditions (t-1,2) were quite complex and contained imines (2), hydroxylamines (only 4a was det oxidation of secondary amines 631 ected), aldehydes (5c), amides (6–8), nitrones (9), benzonitrile (12c), benzamide (13c), and diamine 14a (from 1a only). when performed under b-conditions (t-10,11), besides some of the previously cited compounds, benzylamine (11c, from 1a only), α-aminonitriles (15–17), cyanoimines (18–20), cyanonitrones (21 and 22), and derivatives 23–25 were also obtained. as already presented,35 the formamidines 23 and their hydrolysis products 24 and 25 prevail in more acid conditions, but their formation does not involve the oxidation of 1. consequently, they do not belong in the present discussion. the steps in scheme 3 can be invoked to show how amides i (6 and 7) and nitriles i (15 and 16) of scheme 2 are formed. for instance, the condensation of benzaldehyde (5c) with benzylamine (11c) affords the hemi-aminal 29c. this intermediate can give benzamide 6c (by oxidation) and/or imine 2c (by dehydration). under b-conditions, most of the benzaldehyde should exist as its cyanohydrin 30c. condensation between 30c and 11c yields the cyanoderivative 15c. some of 15c could give imine 2c, by dehydrocyanation. the origins of 8a–e, 9a and b, 10a and b, as well as those of 17a–e were discussed in part 1.27 briefly, the oxidation of hydroxylamines 4a and 4b give nitrones 9a+10a and 9b+10b, respectively. compounds 10 are less stable than 9 and this could explain the absence of 10a and b in t-10,11. at the same time, amides 8 and nitriles 17 (amides ii and nitriles ii in scheme 2, respectively) come from a two-step sequence, similar to that presented in scheme 3: i) the condensation of 1a and b with aldehydes 5a–c affords hemi-aminals, which ii) are oxidized towards 8. analogously, nitriles 17 result from 1a and b and cyanohydrins 30a–c. since hemi-aminals and 17a–e do not contain a nh group, the formation of imines by dehydration/dehydrocyanation is no longer possible in these cases. 5, 11, 30 r/r1 a h b me c ph a ph h a b ph me b c ph ph c 2, 6, 15 r1 r 29 a h ph d b me ph e 3, 7, 16 r1 r 29 r1-co-nh-ch2rr 1-cho + r-ch 2-nh2 5a-c hcn ox ox: oxidation 29a-e11a-c 6a-c, 7a-b r1-ch(oh)-nh-ch2r 30a-c r1-ch=n-ch2r 2a-c, 3a-b + hcn h2o + h2o 15a-c, 16a-b hcn r1-ch(oh)(cn) + 11a-c r1-ch(cn)-nh-ch2r scheme 3. formation of amides i (6 and 7) and nitriles i (15 and 16). benzylamine (11c) was detected in one reaction mixture (t-10), but its transient formation should occur in all oxidations of 1a and b, in order to explain 632 florea et al. the formation of 2c and 6c. the existence of 12c, 13c, and 5c (partially) in the oxidation mixtures derived from 1a and b can be explained by the oxidation of 11c itself, as detailed below. oxidation of benzylamine (11c) it is well established18,36–39 that a primary amine, such as 11c, undergoes oxidation with ruthenium catalysts towards the corresponding nitrile and amide. this was verified with ruo4 and the respective results (t-3,12) were interpreted as shown in scheme 4. r-ch2-nh2 ox [r-ch=nh] r-cn ox h2o 32 5a 11b-c ox: oxidation (r=me) tautom. ch2=ch-nh2 ch2o + ohc-nh2 ox b: r=me c: r=ph 31b-c 12b-c r-cho + nh3 r-conh2 ox 13b-c5b-c [r-ch(oh)-nh2] scheme 4. oxidation of primary amines 11b and c. the first oxidation step gives the aldimine 31c, which can be dehydrogenated further to 12c, but which can also be trapped by water to yield the corresponding hemi-aminal. in turn, this last intermediate can undergo both oxidation to benzamide (13c) and splitting to a mixture of benzaldehyde (5c) and ammonia. formation of 2c, 6c and 15c was outlined in the preceding paragraph (scheme 3). oxidation of imines 2 and 3 in the absence of cyanide, imines 2a and 2b underwent clean transformation into mixtures of 5c+6a and 5c+6b, respectively (t-4,5), where benzaldehyde (5c) represented at least 80% of the reacted substrate. clearly, as depicted in scheme 3, hemi-aminals 29a and b, transiently formed from water and the respective imine, are oxidized towards the corresponding benzamides 6a and b, but largely split into equimolar mixtures of 5c and 11a and b. all these reactions are nucleophilic in nature, except for the oxidation step leading to 6a and b. in the presence of cyanide, about 85 % of reacted 2a and b were found as the corresponding nitriles 15a and b (t-13,14), formed by a non-oxidative step, the nucleophilic addition of hcn to the c=n double bond (scheme 3); the equilibria are largely shifted towards 15, despite the presumably low amount of available hcn in the reaction at ph around 9. the oxidation of 15a and 15b can be invoked instead to explain the formation of 18a+21a and 18b+21b, respectively oxidation of secondary amines 633 (scheme 5). this is supported by the oxidation of 15b alone (t-16), where 18b+21b account for about 50 % of the reacted substrate. aminonitrile 15a behaved similarly. 21a-b18a-b15a-b ph-c(cn)=n-ch2r ox a: r=h b: r=me ph-ch(cn)-nh-ch2r + ph-c(cn)=n(o)-ch 2r 20a-b19a-b 22a-b 16a-b ox bn: benzyl ox: oxidation bn-nh-ch(cn)-r bn-n=c(cn)-r + ph-ch=n-ch(cn)-r bn-n(o)=c(cn)-r bn-nh-ch2cn + ch2o bn-n(ch2cn)-ch2oh bn-n(ch2cn)-ch2-n(ch2cn)-bn bn-n(ch2cn)-cho 16a + 16a 8f 14d ox 5a scheme 5. some oxidation products of α-aminonitriles 15 and 16. unfortunately, imine 3a is only known as its cyclic “trimer”, the sym-triazacyclohexane derivative 26 (scheme 6).30,31 when submitted to a-oxidation, its major reaction product was 27 (corrected yield 65 % in t-6). although 26 and/or 27 were not observed in the oxidation mixture of 1a (t-1), the transient formation of 3a could not be excluded (see below). n h n bnbn cho n n n bn bn bn [bn-n=ch2] 3a 26 27 ox ox: oxidation 5a tautom. 33 bn-nh-cho + ch2obn-n=ch-ch3 ox 3b 7a bn-nh-ch=ch2 bn-nh-ch(oh)-ch3 + h2o ox 29e 7b 11c bn-nh2 + ohc-ch3 5b scheme 6. different behaviour of imines 3a and 3b. unlike 3a, the analogous imine 3b could be isolated.28 despite its poor stability, it was possible to study its oxidation reaction (t-7; scheme 6). with one notable exception (7a), the steps of scheme 3 (re-written in scheme 6, for clarity) are followed. for instance, the hemi-aminal 29e, the water adduct of 3b, 634 florea et al. undergoes oxidation to acetamide 7b, but also breaks into a 11c+5b mixture.14,15 subsequent reactions of benzylamine (11c) are responsible for the presence of 5c, 6c, 12c and 13c, as presented before. a possible explanation for the formation of 7a, the major oxidation product of 3b, is shown in scheme 6. imine 3b has β-n–h protons and, by analogy with some preceding papers,14–16 this enables tautomerization to enamine 33. oxidation of the c=c bond14,15,34–43 in 33 should give a 7a+5a equimolar mixture. when formed, 5a could give additional 7a through the sequence 5a + 11c → 29d → 7a (scheme 3). under b-conditions (t-15), imine 3b (and/or its tautomer 33) gave the expected hcn-adduct 16b (scheme 3; strecker reaction), accompanied by small amounts of 20b and 22b. nitrile 15c was also present, indicating that the following consecutive steps occurred: i) hydrolysis of 3b to 11c, ii) formation of 5c by oxidation of 11c, iii) transformation of 5c into cyanohydrin 30c in the presence of cyanide and iv) condensation of 30c with 11c to yield 15c (scheme 3). cyano compounds 19b, 20b and 22b are all oxidation products of 16b (t-18; scheme 5). the lack of 19b in entry 15 might be due to its relatively low amount. nitrile 16a (t-17) behaved similarly to 16b (t-18), but with a few exceptions. thus, cyanoimine 19a is absent in t-17 probably because of increased instability or of its insufficient amount, below the detectable limit, while compounds 8f and 14d, formed from 16a, have no analogues in the oxidation of 16b. their formation is elucidated in scheme 5: condensation of 16a with formaldehyde (5a; formed from the oxidation of 16a itself) gives a hemi-aminal, which can not only undergo oxidation to 8f, but can also react with 16a to give 14d. all these steps are identical to those forming 14a during the oxidation of 1a (t-1).27 oxidation of 1a and b. mechanistic considerations it is now possible to compare the oxidative results of 1 with those of 2+3. similarly to the comparison made above with the outputs derived from 4a+4b,27 no attention will be paid this time to the presence of amides 8a–e and nitriles 17a–e. their formation requires the presence of 1a and b, but these amines are absent when the reaction starts from imines 2 or 3. comparing the results of the oxidation of 1b (t-2,11) with those of 2b+3b (t-5,14 + t-7,15), it could be observed that all compounds formed in the first case exist within the reaction products of one or the other imine. unfortunately, a similar comparison cannot be made for 1a vs. 2a+3a, because of the lack of the data for 3a. however, the transient existence of 3a could be assumed. for example, the formation of 16a under b-conditions (t-10) implies reaction 3a + + hcn → 16a (scheme 3). these considerations suggest that the main route followed during the ruo4 -mediated oxidation of amines 1a and 1b implies the initial formation of the cor oxidation of secondary amines 635 responding imines 2a+3a and 2b+3b, respectively (scheme 1, route a). the alternative route b involving the hydroxylamines 4a and 4b has only a minor contribution to the final results.27 all information presented before (schemes 1–6) is summarized in scheme 7, but some reactions and/or compounds have been omitted, for the sake of clarity. this is the case of the transformations 1a→14a27 and 1+(cn)2→23→24+25,35 as well as of the subsequent oxidations of 15 (→18+21; t-16) and 16 (→15+19+20+22; t-17,18). at the same time, the sequence 1→4→9+10 is also missing, because it plays a minor role in the global picture.27 mecho ch2o 5a5b ch2=ch-net-bn ox ch2o + ohc-net-bn 35 5a 8c 1a 1b 2a 2b 3a 3b 6a 6b 7a 7b 8a 8c 8d 8e 11a 11b 15a 15b 16a 16b 17a 17c 17d 17e 29a 29b 29d 29e 34a 34c 34d 34e r: h me bn-nh-ch2r bn-n=ch-r bn-nh-ch=ch2 bn-nh-corbn-nh2 + ohc-r bn-nh-cho + ch2o 3a-b 1a-b hx ox ox r=me bn-nh-chx-r x=oh: 29d-e x=cn: 16a-b 5a-b 7a-b11c 7a 5a 33 5c + phcn + phconh2 phconhbn ph-ch(oh)-nhbn 12c 13c + 5c ox ox ox 6c ox h2o29c phch=nbn 2c hcn ph-ch(cn)-nhbn 15c bn: benzyl ox: oxidation x: oh or cn ph-ch=n-ch2r phco-nh-ch2r 2a-b x=oh: 29a-b x=cn: 15a-b 6a-b hx ox ph-chx-nh-ch2r (sch. 4) (sch. 6) (sch. 3) (sch. 3) (sch. 3) (sch. 3) (sch. 3) (sch. 3) (1) (3) (2) (4) (5) x=oh 5a + 1a-b x-ch2-n(ch2r)-bn ohc-n(ch2r)-bnox x=oh: 34a, c x=cn: 17a, c 8a, c phcho + h2n-ch2r 5c 11a-b ox r=me (sch. 4) x=oh 5c + 1a-b ph-chx-n(ch2r)-bn phco-n(ch2r)-bnox x=oh: 34d-e x=cn: 17d-e 8d-e x=oh: 34b x=cn: 17b x=oh 5b + 1b me-chx-net-bn me-co-net-bn ox 8b (6) x=oh h2o 30c + 1a-b 30a + 1a-b 30b + 1b scheme 7. mechanism of the oxidation of secondary amines 1a and b. 636 florea et al. oxidation of secondary amines 1a and 1b starts with the formation of imines 2a+3a and 2b+3b, respectively, by a formal dehydrogenation from the nitrogen atom and its adjacent c atom. every imine can suffer nucleophilic addition at the c=n double bond, as detailed in scheme 3. in the absence of cyanide, the addition of water yields the corresponding pairs of hemi-aminals 29a+29d and 29b+29e. in the presence of cyanide, both water and hydrocyanic acid act as nucleophiles. in this case, together with the cited hemi-aminals, the corresponding α-aminonitriles 15a+16a and 15b+16b are formed (strecker reaction). hemi-aminals can be either oxidized to yield amides (6a+7a or 6b+7b) or split into aldehyde + primary amine mixtures. in the later case, 29a gives benzaldehyde + methylamine (5c+11a) and 29d yields formaldehyde + benzylamine (5a+11c). analogously, 5c and ethylamine (11b) result from 29b, while acetaldehyde (5b) and 11c are obtained from 29e. imine 3b, through its tautomer 33, could be the source of the benzylformamide (7a)+5a equimolar mixture (scheme 6). under a-conditions, this is the main reaction if the oxidation starts from 3b (t-7). starting from 1b, this route to 5a plays a minor role, as indicated by the relatively small amount of 7a (1.7 % in t-2). except for amides 6 and 7 (6a+7a from 1a and 6b+7a+7b from 1b), all other compounds formed until now are involved in subsequent reactions. on one hand, the resulting aldehydes can react with the available amines (primary or secondary) and, on the other hand, the primary amines themselves can be further oxidized. for example, benzylamine (11c) is oxidized to benzonitrile (12c), benzamide (13c) and benzaldehyde (5c) (scheme 4). it reacts easily with 5c to yield benzamide 6c and imine 2c through the intermediacy of 29c. if hydrocyanic acid is present, the nitrile 15c also results. as aforementioned for 5c+11c, aldehydes 5a–c can react, at least in principle, with all available primary (11a–c) and secondary amines (especially 1a and b). actually, as indicated by the presence of compounds such as 8a–e or 17a–e in table i, the condensations with 1a and b seem to be more fruitful, probably because 1a and b are always in excess with respect to 11a–c. consequently, the sequences 1–5 could be written (see the lower part of scheme 7). more specifically, under a-conditions, the sequences 1 (5c+1a→34d→8d) and 2 (5a+1a→ →34a→8a) are active during the oxidation of 1a, but 3 (5c+1b→34e→8e), 4 (5a+1b→34c→8c) and 5 (5b+1b→34b→8b) in the case of 1b. under b-conditions, all aldehydes 5a–c are present as their cyanohydrins 30a–c and the sequences 1–5 must be re-written with nitriles 17a–e (as the final products) instead of hemi-aminals 34a–e. some contradictions arise in the case of 1b. according to the steps of scheme 7 hitherto discussed, 5a is generated from 33 and used (totally or partially) in sequence 4 to give formamide 8c; at the same time, 5b results from 29e oxidation of secondary amines 637 and it is used (totally or partially) in sequence 5 to obtain acetamide 8b. however, the relatively high amount of 8c (15.2 % in t-2) cannot originate only from the small amount of 5a (1.7 %, identical to 7a) generated from 33. moreover, when derived from 29e, the amount of 5b should be the same as that of the initially formed 11c (written as 11ci). taking into account that 11ci = 2c+6c+ +12c+13c+5c11c, where 5c11c means that 5c was generated from 11c (scheme 4) and using the values listed in t-2, it can be calculated that 5b = 11ci = 15.1+ +5c11c. unfortunately, the amount of 5c11c cannot be known because benzaldehyde (5c) results not only from 11c, but also from imine 2b. however, even the lower limit of 5b (15.1 %) seems to be higher than that required in sequence 5 to produce 8b (3.5 %). these considerations suggest that the aforementioned steps of scheme 7 are incomplete: the apparent deficit of 5a and excess of 5b deserve an explanation. to solve this problem, the sequence 5 was completed with the new sequence 6 (presented as a frame in the lower part of scheme 7). unlike 34c or 34e, hemi -aminal 34b has a (β-o–)c–h bond and this renders possible the dehydration to 35. oxidation of the c=c bond in 35 should give an equimolar mixture of formaldehyde (5a) and formamide 8c. since this extra amount of 5a will generate more 8c through sequence 4, the total amount of 8c becomes even higher. it is conceivable that 5a and 5b could also be supplied by an auxiliary source, namely the oxidation of ethylamine (11b, frame in scheme 7). it is necessary to remember that, during the oxidation of 1b, amine 11b results from 29b→5c+11b. analogously to the described behaviour of 11c, the primary amine 11b could be oxidized into acetonitrile (12b), acetamide (13b), and acetaldehyde (5b) (scheme 4). moreover, since the intermediate 31b has β-n-protons, tautomerization to 32 is possible, just as in the aforementioned sequence 3b→33. oxidation of 32 leaves 5a. therefore, both aldehydes 5a and 5b are generated from 11b. experimental proof of these suppositions came from the oxidation of an equimolar mixture of 11c and 11b (t-8). unlike the oxidation of 11c alone (t-3), compounds 2b and 6b, as well as 3b, 7a, 7b and 28 were detected in this case. derivatives 2b and 6b resulted from the sequence 5c+11b→29b→2b+6b (scheme 3), where here 5c came from the oxidation of 11c. at the same time, it is clear that formaldehyde (5a) was trapped as 7a (11c+5a→7a) and acetaldehyde (5b) as 3b+7b+28 (11c+5b→3b+7b; 11b+5b→28). the influence of the reaction 11b→5a+5b on the oxidation of 1b was tested by oxidizing a 1b+11b mixture (t-9). with respect to the values in t-2, all yields in t-9 were smaller by a factor of 0.6–0.8 (mean value of 0.7), except for: i) those of 2b and 6b, which apparently remained constant and ii) the yields of 8b and 8c, which were about double. in reality, the values in t-9 are the sum of those derived from the oxidation of 1b alone (the yields of t-2 multiplied by 0.7) and those attributable to the intervention of extra 11b and its oxidation products. 638 florea et al. accordingly, the aforementioned two exceptions are actually enhancements due to the presence and oxidation of 11b. the first cited exception is attributable to the sequence 5c+11b→29b→2b+6b, where 11b is that initially added and 5c is an oxidation product of 1b. this means that 2b and 6b came from two sources: the oxidation of 1b and the aforementioned sequence. the second exception is due to the extra amounts of 5a and 5b, generated from the added 11b, which give additional 8c and 8b, respectively, through the sequences 4–6 of scheme 7. some comments are required for the reaction under b-conditions (t-11). all aldehydes are present only as their cyanohydrins and this favours nitrile formation. the new sequences 4 and 5 will be largely 30a+1b→17c and 30b+1b→ 17b, respectively, and sequence 6 will have only a minor influence. for this reason, 8c and 8b are now present in such small amounts and 8c/8b = 0.6 in t-11. when sequence 6 is active, 8c/8b = 4.3 (t-2). in preceding papers,13–15 the oxidation regioselectivity (alkyl/benzyl) was calculated. unfortunately, this was no longer possible for 1a and b, because many reaction products originated from both types of initially formed imines (2 and 3). as outlined before, this is the case of 5a–c and of all amides or nitriles resulting from them. conclusions the ruo4-mediated oxidation of secondary amines 1 generates in the first step imines as the main intermediates, by attack at both benzylic and alkylic n-α -sites. reactions of imines with the available nucleophiles (water or hydrocyanic acid) explain the formation of n-monosubstituted amides (amides i) or n-monosubstituted α-aminonitriles (nitriles i), respectively. the oxidation output is complicated by subsequent reactions of these prime products giving, for example, n,n-disubstituted amides (amides ii), n,n-disubstituted α-aminonitriles (nitriles ii), and oxidation products of nitriles i. since many reaction products result from two or more sources, it is impossible to calculate the reaction regioselectivity. formation of all these compounds was rationalized and a complete reaction scheme was proposed and discussed. supplementary material details on the preparation of 14d, 1hand 13c-nmr characteristics of 3b, 8f, 14d, 26– –28, as well as ms data of 3b, 8f and 28 are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. oxidation of secondary amines 639 и з в о д оксидација секундарних амина помоћу ruo4. део 2. имини као главни реакциони интермедијери cristina a. florea, anca hîrtopeanu, cristina stavarache и horia petride romanian academy, “costin d. nenitzescu” center of organic chemistry, spl. independenţei 202-b, ro060023 bucharest, romania оксидацијом секундарних амина, као што су bn–nh–ch2r (1; r=h, me), помоћу ruo4 (добијеном in situ из ruo2 и naio4) настаје сложена смеша производа коју углавном чине амиди. у присуству цијанида, главни производи реакције су α-aминонитрили. поређењем структура производа оксидације 1 са производима оксидације одговарајућих имина phch=n–ch2r и bn–n=ch–r утврђено је да је формирање назначених имина први корак у оксидацији 1. предложен је детаљан механизам реакције. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<feff04120438043a043e0440043804410442043e043204430439044204350020044604560020043f043004400430043c043504420440043800200434043b044f0020044104420432043e04400435043d043d044f00200434043e043a0443043c0435043d044204560432002000410064006f006200650020005000440046002c0020044f043a04560020043d04300439043a04400430044904350020043f045604340445043e0434044f0442044c00200434043b044f0020043204380441043e043a043e044f043a04560441043d043e0433043e0020043f0435044004350434043404400443043a043e0432043e0433043e0020043404400443043a0443002e00200020042104420432043e04400435043d045600200434043e043a0443043c0435043d0442043800200050004400460020043c043e0436043d04300020043204560434043a0440043804420438002004430020004100630072006f006200610074002004420430002000410064006f00620065002000520065006100640065007200200035002e0030002004300431043e0020043f04560437043d04560448043e04570020043204350440044104560457002e> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice first and efficient synthesis of 4-[((3,4-dihydroxybenzoyl)-oxy)methyl]phenyl β-d-glucopyranoside, an antioxidant from origanum vulgare j. serb. chem. soc. 81 (1) 23–28 (2016) udc 635.71:547.565.2+547.211’81’828+ jscs–4823 547.537–31:542.913:66.095.12:615.27 short communication 23 short communication first and efficient synthesis of 4-[((3,4-dihydroxybenzoyl)oxy)methyl]phenyl β-d-glucopyranoside, an antioxidant from origanum vulgare yu-wen li1* and cui-li ma2 1school of chemistry and pharmacy, qingdao agricultural university, qingdao 266109, china and 2affiliated hospital, qingdao agricultural university, qingdao 266109, china (received 5 february, revised 14 september, accepted 15 september 2015) abstract: 4-[((3,4-dihydroxybenzoyl)oxy)methyl]phenyl β-d-glucopyranoside (dbpg, 1), a polyphenolic glycoside previously isolated from oregano (origanum vulgare l.) in 0.08 % isolated yield, was synthesized in five chemical steps with 41.4 % overall yield. first, 4-(hydroxymethyl)phenyl -d-glucopyranoside 2,3,4,6-tetraacetate (4) was obtained in 53.2 % yield by selective glycosylation of 4-hydroxybenzyl alcohol (3) with 2,3,4,6-tetra-o-acetyl-α-d -glucopyranosyl bromide (2) in a mixture of chlorobenzene and aqueous csoh using triethylbenzylammonium chloride (tebac) as a phase transfer catalyst. then, this product was esterified with 3,4-diacetoxybenzoyl chloride (7) to generate 4-[((3,4-diacetoxybenzoyl)oxy)methyl]phenyl -d-glucopyranoside 2,3,4,6-tetraacetate (8) in 95 % yield. finally, selectively global deacetylation of 8 was performed in a mixture of dibutyltin oxide and methanol under reflux to afford 1 in 94.8 % yield. keyword: 4-[((3,4-dihydroxybenzoyl)oxy)methyl]phenyl β-d-glucopyranoside; synthesis; glycosylation; antioxidant; dibutyltin oxide. introduction as an extensively used herb in china and a common herb in the western diet, oregano (origanum vulgare l.) is believed to display antithrombin, anti -helicobacter pylori, antimicrobial, antibiotic, antihyperglycemia and antioxidant effects.1,2 4-[((3,4-dihydroxybenzoyl)oxy)methyl]phenyl -d-glucopyranoside (dbpg) (fig. 1), a polyphenolic glycoside, was identified as a major constituent of oregano (origanum vulgare l.). previous and recent studies showed that dbpg exhibits free radical scavenging activity,3 antioxidant and cytoprotective effects on liver and skin cells.4 therefore, it is plausible to speculate the pos * corresponding author. e-mail: ywli@qau.edu.cn doi: 10.2298/jsc150205074l 24 li and ma sibility of employing dbpg as an additive to food and cosmetics for antioxidantmediated health. however, conventional access to dbpg via extraction from oregano is not only a time-consuming and expensive process, but also a challenging procedure due to the extremely low content (only 0.08 %) and the ever-increasing shortage of oregano. fig. 1. structure of dbpg 1. to circumvent these problems associated with the extraction of dbpg from oregano, herein, a first and efficient chemical synthesis of dbpg is presented (scheme 1), in which 2,3,4,6-tetra-o-acetyl-α-d-glucopyranosyl bromide 2 was employed as a glycosyl donor. scheme1. first and efficient synthesis of dbpg (1). results and discussion as shown in scheme 1, 4-(hydroxymethyl)phenyl -d-glucopyranoside 2,3,4,6-tetraacetate (4), a key intermediate for the synthesis of dbpg, was synthesized according to the literature5 with some modifications, such as replacement of sodium hydroxide by cesium hydroxide and substitution of chloroform for chlorobenzene. then, selective glycosylation of 4-hydroxybenzyl alcohol (3) with 2,3,4,6-tetra-o-acetyl-α-d-glucopyranosyl bromide (2) was performed in the synthesis of 4-[((3,4-dihydroxybenzoyl)oxy)methyl]phenyl β-d-glucopyranoside 25 system chlorobenzene–aqueous csoh in the presence of triethylbenzylammonium chloride (tebac) as a phase transfer catalyst. preliminary experiments were conducted to find the optimal conditions leading to the highest yield of compound 4, and it was found that the optimal molar ratio of compounds 2, 3 and tebac was 1:2:0.8 in terms of highest yield of 4. thus, compound 4 was prepared by heating the reaction mixtures at 60 °c for 4.5 h in a yield of 53.2 %, an increase of 14.2 % over that previously reported.5 with the intermediate 4 in hand, 3,4-diacetoxybenzoic acid (6) was prepared in 86.5 % yield by reaction of 3,4-dihydroxybenzoic acid (5) with acetic anhydride using conc. h2so4 as a catalyst according to literature6 with some modifications, such as replacement of ethyl acetate–hexane by ethyl ether–petroleum ether (60–90 c) as the recrystallization solvent. next, 3,4-diacetoxybenzoyl chloride (7) was prepared by reaction of 6 with thionyl chloride and stored hermetically for further employment. subsequently, coupling of the thusobtained 7 with 4 afforded 4-[((3,4-diacetoxybenzoyl)oxy)methyl]phenyl -d -glucopyranoside 2,3,4,6-tetraacetate (8) in 95 % yield. of special note is that triethylamine was employed as an acid scavenger in this coupling process because of the acid-sensitivity of the glycosidic bond of 8. finally, global deacetylation of 8 gave rise to 4-[((3,4-diacetoxybenzoyl)oxy)methyl]phenyl β-d-glucopyranoside (1). while global deacetylation of 8 using the conventional naome/meoh reagent combination (zemplen conditions) in carbohydrate chemistry afforded 1, methyl 3,4-dihydroxybenzoate as by-product was observed in this process, thus leading to poor yield of 1 and difficulty in purification of 1. to obviate the formation of the by-product in the global deacetylation of 8 under zemplen conditions, conversion of 8 into 1 by exclusive global deacetylation of 8 was performed using dibutyltin oxide (dbto) as a catalyst and methanol as the solvent.7 the 1h-nmr data for the title compound 1 is presented in the supplementary material to this paper. efforts were made to optimize the conditions for the synthesis of 1 by varying the molar ratio of compound 8 to dbto, and it was found that the appropriate mole ratio of 8 to dbto was 4:1 in terms of reaction time and facile purification of 1. under this condition, compound 1 was obtained in 94.8 % yield by refluxing the reaction mixture in methanol for 6 h. notably, down-regulating the mole ratio of 8 to dbto could shorten the reaction time, but disfavored the purification of compound 1 concurrent with dbto. therefore, striking a balance between the reaction time and easiness of purification of 1 is advisable. experimental materials and methods 2,3,4,6-tetra-o-acetyl-α-d-glucopyranosyl bromide, 4-hydroxybenzyl alcohol, 3,4 -dihydroxybenzoic acid, acetic anhydride, thionyl chloride, dibutyltin oxide and triethylbenzylammonium chloride were obtained from qingdao justness reagent company (china). all 26 li and ma solvents were obtained commercially and used without further purification unless otherwise stated. instrumentation the melting points were measured with a digital melting point apparatus (wrs-1b). the optical rotations were measured with jasco p1030 polarimeter. the 1hand 13c-nmr spectra were recorded on a bruker avance iii400 spectrometer, operating at 400 mhz for protons and 100 mhz for carbons. 2d nmr techniques (1h–1h-cosy and 1h–13c-hsqc) were used for full assignment of the spectra. all compounds were confirmed by physical and spectral methods. the physical, analytic and spectral data are given in the supplementary material to this paper. synthesis of 4-(hydroxymethyl)phenyl -d-glucopyranoside 2,3,4,6-tetraacetate (4) a solution of 2,3,4,6-tetra-o-acetyl-α-d-glucopyranosyl bromide (2, 12.30 g, 30 mmol) in 120 ml chlorobenzene was added to a solution of 4-hydroxybenzyl alcohol (3, 7.44 g, 60 mmol), csoh (8.99 g, 60 mmol) and triethylbenzylammonium chloride (5.4 g, 24 mmol) in 100 ml h2o. the mixture was then stirred at 60 °c for 4.5 h, after which it was cooled to room temperature and the chlorobenzene layer separated, washed with 80 ml saturated k2co3 solution, 80 ml h2o and dried over anhydrous na2so4. the filtrate was concentrated under vacuum to give a yellowish syrupy crude product 4, which was purified by crystallization from 95 % ethanol to afford the desired compound 4 as a white solid. yield: 53.2 %. synthesis of 3,4-diacetoxybenzoic acid (6) to a stirred suspension of 3,4-dihydroxybenzoic acid (5, 6.16 g, 40 mmol) in 19 ml acetic anhydride was added 0.2 ml of concentrated sulfuric acid. then, the suspension was stirred at 70 °c until a clear solution was obtained, indicating completion of the reaction. the resulting clear solution was poured into 200 ml of cold water and stirred until the crude 3,4 -diacetoxybenzoic acid (6) had precipitated completely. the white crude precipitate 6 was collected by filtration, washed with 50 ml of 95 % ethanol and purified by recrystallization from et2o–pe (3:2, v/v) to obtain the desired 3,4-diacetoxybenzoic acid (6) as white crystals. yield: 86.5 %. synthesis of 3,4-diacetoxybenzoyl chloride (7) a mixture of 3,4-diacetoxybenzoic acid (6, 2.38 g, 10 mmol) and thionyl chloride (25 mmol, 1.81 ml) was stirred at 40 °c for 5 h. the mixture was then distilled under vacuum to remove the excess thionyl chloride, leaving a yellowish oily residue (7). anhydrous ch2cl2 (6 ml) was added to the residue 7, which was then poured into a dried 10 ml dropping funnel for further employment. 4-[((3,4-diacetoxybenzoyl)oxy)methyl]phenyl -d-glucopyranoside 2,3,4,6-tetraacetate (8) to a stirred mixture of 4-(hydroxymethyl)phenyl -d-glucopyranoside 2,3,4,6-tetraacetate (4, 4.54 g 10 mmol) and triethylamine (11 mmol, 1.1 ml) in 30 ml dried ch2cl2 was added dropwise the already-made 3,4-diacetoxybenzoyl chloride (7) within 0.5 h. upon completion of the dropwise addition, the mixture was kept stirring for 5 h at room temperature to give a brown solution. the resulting brown solution was diluted with 50 ml ch2cl2 and washed successively with 100 ml h2o, 100 ml aq. nahco3 and 100 ml brine, and dried over anhydrous na2so4. the filtrate was concentrated under reduced pressure to afford crude 4-[((3,4-diacetoxybenzoyl)oxy)methyl]phenyl -d-glucopyranoside 2,3,4,6-tetraacetate (8) as synthesis of 4-[((3,4-dihydroxybenzoyl)oxy)methyl]phenyl β-d-glucopyranoside 27 brown solid. recrystallization of the crude 8 from absolute ethanol gave the desired 8 as white crystals. yield: 95 %. 4-[((3,4-dihydroxylbenzoyl)oxy)methyl]phenyl -d-glucopyranoside (1) a stirred mixture of 4-[((3,4-diacetoxybenzoyl)oxy)methyl]phenyl -d-glucopyranoside 2,3,4,6-tetraacetate (8, 5.39 g, 8 mmol) and dibutyltin oxide (0.5 g, 2 mmol) in 50 ml methanol was refluxed for 6 h, and then the mixture was concentrated under vacuum to provide crude product 1. recrystallization of crude 1 from absolute ethanol afforded the desired 1 as white crystals. yield: 94.8 % conclusions in summary, a chemical synthesis of dbpg in five chemical steps was developed for the first time with 41.4 % overall yield. this synthetic route to dbpg has the advantages of operational simplicity as well as facile separation and purification by recrystallization throughout the whole procedure. supplementary material the physical, analytic and spectral data of the synthesized compounds are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the author acknowledges the financial support from the natural science foundation of shandong province (no.zr2011bl003) and the start-up foundation of high talents in qingdao agricultural university (no. 630708). из в о д прва ефикасна синтеза 4-[((3,4-дихидроксибензоил)окси)метил] фенил--d-глукопиранозида, антиоксиданта из origanum vulgare yu-wen li1 и cui-li ma2 1 school of chemistry and pharmacy, qingdao agricultural university, qingdao 266109, china и 2 affiliated hospital, qingdao agricultural university, qingdao 266109, china 4-[((3,4-дихидроксибензоил)окси)метил]фенил-β-d-глукопиранозид (dbpg, 1), полифенолни глукозид, који је раније изолован из оригана (origanum vulgare l.) у приносу 0,08 %, синтетисан је у пет реакционих корака у укупном приносу од 41,4 %. прво је 4-(хидроксиметил)фенил--d-глукопиранозид-2,3,4,6-тетраацетат (4) добијен у приносу 53,2 % селективним гликозиловањем 4-хидроксибензил-алкохола (3) помоћу 2,3,4,6-тетра-o-ацетил-α-d-глукопиранозил-бромида (2), у смеши хлорбензена и воденог раствора csoh у присуству триетилбензиламонијум-хлорида (tebac) као катализатора фазног прелаза. потом је производ естерификован 3,4-диацетоксибензоилхлоридом (7) чиме је добијен 4-[((3,4-диацетоксибензоил)окси)метил]фенил--d-глукопиранозид-2,3,4,6-тетраацетат (8) у приносу од 95 %. на крају, селективно укупно деацетиловање деривата 8 извршено је у смеши дибутил-калај-оксида у метанолу, уз загревање на температури кључања, при чему је добијен дериват 1 у приносу 94,8 %. (примљено 5 фебруара, ревидирано 14 септембра, прихваћено 15. септембра 2015) references 1. m. mueller, b. lukas, j. novak, t. simoncini, a. r. genazzani, a. jungbauer, j. agric. food chem. 56 (2008) 11621 28 li and ma 2. f. m. pelissari, m. v. grossmann, f. yamashita, e. a. pineda, j. agric. food chem. 57 (2009) 7499 3. n. nakatani, h. kikuzaki, agric. biol. chem. 51 (1987) 2727 4. c. h. liang, l. p. chan, h. y. ding, e. c. so, r. j. lin, h. m. wang, y. g. chen, t. h. chou, j. agric. food chem. 60 (2012) 7690 5. a. e. pavlov, v. m. sokolov, v. i. zakharov, russ. j. gen. chem. 71 (2001) 1811 6. l. m. leblanc, a. f. pare, j. j. francois, m. j. g. hebert, m. e. surette, m. touaibia, molecules 17 (2012) 14637 7. w. li, h. m. liu, q. d. you, acta chim. sinica 9 (2003) 1516. {characterisation of lignite lithotypes from the “kovin” deposit (serbia) implications from petrographic, biomarker and isotopic analysis} j. serb. chem. soc. 82 (6) 739–754 (2017) udc 553.96.004.12:543:686.862.6.000.57: jscs–5000 550.4(497.11) original scientific paper 739 characterisation of lignite lithotypes from the “kovin” deposit (serbia) – implications from petrographic, biomarker and isotopic analysis danica mitrović1, nataša đoković1, dragana životić2, achim bechtel3, olga cvetković4 and ksenija stojanović5* 1university of belgrade, innovation center of the faculty of chemistry, studentski trg 12–16, 11000 belgrade, serbia, 2university of belgrade, faculty of mining and geology, đušina 7, 11000 belgrade, serbia, 3montanuniversität leoben, department of applied geosciences and geophysics, peter-tunner-str. 5, a-8700 leoben, austria, 4university of belgrade, institute of chemistry, technology and metallurgy, department of chemistry, njegoševa 12, 11000 belgrade, serbia and 5university of belgrade, faculty of chemistry, studentski trg 12–16, 11000 belgrade, serbia (received 22 november 2016, revised 17 february, accepted 27 february 2017) abstract: four lignite lithotypes (matrix coal, xylite-rich coal, mixture of matrix and mineral-rich coal and mixture of matrix and xylite-rich coal), originating from the kovin deposit, were investigated in detail. the paper was aimed to determine the main maceral, biomarker and isotopic (δ13c) characteristics of investigated lithotypes. based on these results the sources and depositional environment of organic matter in 4 lithotypes were established. these samples were also used as substrates for investigation of the influence of diagenetic alteration on δ13c signatures of biomarkers, as well as for assessment of the most convenient utilization for each lithotype. the investigated lithotypes differ in accordance with the composition of huminite macerals. xylite-rich coal notably distinguishes from other lithotypes beacuse of the highest content of conifer resins vs. epicuticular waxes. the mixture of matrix and mineral-rich coal is characterised by the greatest contribution of algae and fungi and the most intense methanotrophic activity at the time of deposition. in all coal lithotypes diagenetic aromatisation influenced isotopic composition of individual biomarkers. xylite-rich coal has the poorest grindability properties. however, this coal lithotype is the most suitable for fluidized bed gasification, whereas the mixture of matrix and mineral-rich coal has the lowest applicability for this process. the calorific value decreases in order: xylite-rich coal > matrix coal > mixture of matrix and xylite-rich coal > mixture of matrix and mineral-rich coal. the increase of organic carbon content and calorific value is controlled by the increase of contribution of wood vegetation vs. herbaceous peat-forming plants, as well as by stability of water table during peatification. keywords: lignite; lithotype; kovin deposit; macerals; biomarkers. * corresponding author. e-mail: ksenija@chem.bg.ac.rs https://doi.org/10.2298/jsc161122030m 740 mitrović et al. introduction the lignite lithotypes can be macroscopically determined based on the differences of the macropetrographic composition, structure, colour and texture of the sample. the lithotype classification system for lignite (soft brown coal) proposed by the international committee for coal and organic petrology (iccp)1 distinguishes: xylite-rich coal, matrix coal, charcoal-rich coal and mineral-rich coal. xylite-rich coal consists of a layered or lenticular concentration of xylites, representing at least 90 % of the volume. xylites include all fragments with wellpreserved wood structure with a diameter of at least 1 cm. smaller fragments are classified as humic detritus.2–4 matrix coal consists of fine humic particles (detritus), forming a more or less homogeneous macroscopic mass. as a lithotype, it can contain up to 10 % volume of other components. charcoal-rich coal (or fusain coal) is represented by charred organic matter (om) and is rarely found in larger amounts in lignite deposits. mineral-rich coal consists of components of different lithotypes and mineral matter. the inorganic materials are typically represented by quartz, clay, carbonates and sulphides.5 mixture of matrix and xylite -rich coal and mixture of xylite-rich and matrix coal belong to the complex lithotypes, built mainly of humic detritus and xylites. the former is dominated by humic detritus, whereas the latter is dominated by xylites, which should account for more than half of the xylites and humic detritus occurring in the specified layer.3 macropetrographic features of lithotypes and their applicability resulted from their composition and the degree of transformation during peat genesis and diagenesis.3 for establishing of sources of lignite om and its diagenetic alteration, micropetrographic (maceral) analysis and biomarker composition are the most useful. maceral analysis facilitates detection of the complex preserved structures of the parent organic material in the insoluble organic matter (kerogen) such as: plant tissues, represented by huminite group macerals (textinite, ulminite, densinite, atrinite, gelinite and corpohuminite); lipoidal plant components and protozoa residues, represented by liptinite group macerals (sporinite, cutinite, resinite, suberinite, alginite, liptodetrinite, bituminite, chlorophylinite, exudatinite); and the products of their humification and coalification. the maceral analysis is also very useful for the detection of the charred components in lignite om, represented by inertinite group (inertodetrinite, semifusinite, fusinite, macrinite, micrinite, funginite).6 however, the compositions and therefore technological properties of individual macerals differ even when the are from the same maceral group, as it was described in numerous investigations.4,6–11 unlike maceral analysis, biomarker analysis is related to extractable om (bitumen) and enables to identify numerous individual compounds with strong resemblance in structure of their parent organic molecules in living organisms. also, biomarker assemblages provide important information about depositional characteristics of lignite lithotypes 741 environment of the om. however, biomarker interpretation may be complicated due to the multiple geneses.12,13 compound-specific carbon isotopic measurements represent a powerful tool to overcome this problem, allowing us to establish the relationship between biological precursors and their diagenetic products.14–18 the paper was aimed to determine the main maceral, biomarker and isotopic (δ13c) characteristics of four lignite lithotypes (matrix coal, xylite-rich coal, mixture of matrix and mineral-rich coal and mixture of matrix and xylite-rich coal). lignite originating from the kovin deposit was used as a substrate. based on a comprehensive study, the sources of om and depositional environment of 4 lignite lithotypes have been reconstructed. alongside, the data from maceral analysis were served for assessment of the most convenient utilization of each lithotype, whereas the results of the isotopic analysis were used for the investigation of the influence of diagenetic alteration on δ13c signatures of biomarkers. experimental the details related to sampling locations and sample preparation are given in supplementary material to this paper. two internal standards, deuterated n-tetracosane for the aliphatic fraction and 1,1’-binaphthyl for the aromatic fraction were used. standards diluted in n-hexane in concentrations of 10 or 1 mg/cm3, depending on the weight of the respective fractions, were added to obtain a sample/standard mass ratio of 100:1. final concentration of the fractions in the vials, prior to gas chromatography-mass spectrometry (gc–ms) analysis, was set to 1 mg/cm3, by dilution with n-hexane. saturated and aromatic fractions were analysed by gc–ms. a gas chromategraph agilent 7890a gc (hp5-ms capillary column, 30 m×0.25 mm, 0.25 μm film thickness, helium carrier gas 1.5 cm3/min) coupled to a agilent 5975c mass selective detector (70 ev) was used. the column was heated from 80 to 310 °c, at a rate of 2 °c/min, and the final temperature of 310 °c was maintained for an additional 25 min. individual compounds were identified from the total ion current (tic) by the comparison of mass spectra and retention times with literature data. absolute concentrations of individual biomarkers were calculated using peak areas (gcms data analysis software) from the tics of aliphatic and aromatic fractions in relation to that of internal standards. the concentrations were normalized to the total organic carbon (toc) content. the same quantification method was used in numerous investigations.19–21 carbon isotope determination of individual biomarkers in selected samples, representing all lithotypes was performed using a trace gc instrument attached to a thermofisher delta-v isotope ratio mass spectrometer via a combustion interface (gc isolink, thermofisher). db -5ms fused silica column (30 m length; i.d. 0.25 mm; 0.25 μm film thickness) was used. the oven temperature gradient was programmed from 70 to 300 °c at 4 °c/min, followed by an isothermal period of 15 min. helium (flow 1.2 cm3/min) was used as carrier gas. for calibration, co2 was injected at the beginning and end of each analysis. stable isotope ratios are reported in delta notation (δ13c)22 relative to the vienna-pee dee belemnite (v-pdb) standard (δ13c = ((13c/12c)sample/(13c/12c)standard – 1)). delta notation is expressed in parts per thousand (‰). the analytical error was better than 0.2 ‰. 742 mitrović et al. since n-alkanes were not separated from polycyclic biomarkers by molecular sieves, due to the possible superimposition of n-alkanes and hopanoids23 which show notably different δ13c values, prior to isotopic analysis, abundances and mass spectra of these biomarkers, as well as of all individual compounds, whose δ13c were determined, were carefully checked. based on these results, δ13c was not measured for all individual biomarkers interpreted by isotopic signatures here, in all selected representative lithotypes. namely, the δ13c was measured only for those biomarkers present in certain selected sample (please see the supplementary material) in adequate amount, which showed at the same time sufficiently pure mass spectra. moreover the used instrument allows manual integration of the peaks, and therefore even peaks which have close retention times could be separated and measured. accordance between measured δ13c values of individual biomarkers and those reported in literature,16,18,24–28 indicated that selection of samples based on above mentioned characteristics was correct and that manual peak integration was done accurately. however, despite of the purity of the mass spectra of hopanoids, the interpretation should be taken with certain caution due to the generally low concentration of free hopanoids, since the part of these biomarkers, particularly in immature lignite om, is still bounded into macromolecular matrix and occurred as functionalized compounds (e.g., hopanoic acids and alcohols).29,30 results and discussion maceral composition huminite macerals predominate in all lithotypes (tables s-ii and s-iii of the supplementary material). this result indicates the typical humic coals. the content of total liptinites was similar in all lithotypes, whereas the average content of total inertinites was the lowest in the xylite-rich coal (xc) and the highest in the mixture of matrix and mineral-rich coal (mmic), as given in table s-ii. the pronounced differences were observed in the composition of the huminite group macerals. ulminite, followed by textinite or densinite is the dominant huminite maceral in xc (with exception of one sample, 79/04 where textinite prevailed; tables s-ii and s-iii). densinite predominates, whereas ulminite was the second most abundant huminite maceral in three other lithotypes. since ulminite and particularly densinite have better grindability properties than textinite,10,31 it can be supposed that xc has poorer susceptibility to grinding. gelinite and corpohuminite are present in all lithotypes in similar amounts (table s-ii). due to the substantial fragility, the gelified macerals represent an undesirable component. an important conclusion, which avoids non-rational utilization and consequent cost, is that all studied lithotypes from the kovin deposit are unsuitable for coal briquetting according to gelification of coal, σg higher than 20 vol. % (tables s-iv and s-v of the supplementary material).10 the composition of liptinite macerals was similar in all lithotypes, characterising by prevalence of sporinite and liptodetrinite (tables s-ii and s-iii). relatively low content of liptinite in all samples is unfavorable, since net calorific value is proportional to content of this maceral group.10 on the other hand, the characteristics of lignite lithotypes 743 relatively low content of inertinite macerals in all lignite lithotypes, with the exception of mmic is considered as favourable because inertinite generally hinders grinding, briquetting and drying of lignite.10,11 inertodetrinite prevailed among inertinite macerals in all lithotypes. however, elevated contents of fusinite and semifusinite were observed in mmic (tables s-ii and s-iii). the results of maceral analysis were applied for an assessment of usefulness for the fluidized bed gasification according to ternary diagram (fig. s-2 of the supplementary material) proposed by bielowicz.10 this diagram shows that xc is the most suitable for gasification, whereas mmic demonstrated the lowest applicability. diagrams based on maceral indices32,33 gelification index (gi) vs. tissue preservation index (tpi) and groundwater index (gwi) vs. vegetation index (vi) (table s-iv) are shown in fig. s-3 of the supplementary material. xc was formed in dry to wet forest swamp (slightly domed ombrotrophic to mesotrophic conditions), whereas the matrix coal (mc) and mmic originated from a topogenous fresh water peat mire with open water areas (limnic conditions). the mixture of matrix and xylite-rich coal (mxc) plotted in between matrix and xyliterich coal, however closer to the former, indicating bush moor. the values of vi suggest that the contribution of arboreal vegetation relative to the impact of herbaceous peat-forming plants decreases in the following order: xc > mc > mxc > mmic (tables s-iv and s-v). consistently the values of tpi showed the same trend of om preservation. based on gi and gwi, the fluctuations of water level were most pronounced during peatification of mmic and mxc. therefore, the lower average tpi for these two coal lithoptypes (table s-iv) is probably associated with an unstable water table which may have caused increased tissue degradation because of possible aeration and oxidation of om.34 bulk organic geochemical parameters total organic carbon (toc) was the highest in xc and lowest in mmic (table s-iv). the gross and net calorific value (dry basis) of the samples ranges from 15.7 to 28.3 mj kg–1 and from 14.3 mj to 27.2 mj kg–1, respectively (tables s-iv and s-v), which is in range for the rational utilization of lignite in thermal power plants (tpp) recommended by american lignite council.35 moreover, all lithotypes have higher net calorific values than recommended for ex -yugoslavia (8.89 mj kg–1).36 the significant positive correlation between calorific value and toc (table s-v) is observed (correlation coefficient, r = 0.94), as expected, and the average calorific values of coal lithotypes decrease in the same order as the average values of toc (table s-iv). the good accordance between maceral indices, toc and calorific value (table s-iv) indicates that the increase of toc content and consequently the calorific value is controlled by the increase of the impact of wood vegetation vs. herbaceous peat-forming plants, as well as 744 mitrović et al. by the stability of water table. as expected, contents of ash and mineral matter showed the trend opposite from toc and the calorific value (tables s-ii and s -iv). sulphur content has an uniform range for all coal lithotypes. generally low to moderate content of sulphur (0.21–4.40 %; tables s-iv and s-v) implies the deposition of om in fresh water environment. the content of bitumen, representing the extractable om is lower in xc than in other lithotypes probably due to the greater impact of arboreal vegetation. bulk composition of bitumen is uniform for all lithotypes and characterised by sharp prevalence of asphaltenes + nso-compounds over saturatedand aromatic hydrocarbons, as expected for the immature terrestrial organic material (tables s-iv and s-v). molecular composition of the organic matter general characteristics. diterpenoids are the most abundant hydrocarbons in all lithotypes, prevailing over n-alkanes, hopanoids, non-hopanoid triterpenoids and steroids (figs. s-4 and s-5; tables i and s-vi of the supplementary material). however, the proportion of total diterpenoids (related to sum of total quantified compounds) was the highest in xc. mc, mmic and mxc have high content of n-alkanes. xc notably differs from the other lithotypes according to the highest diterpenoids/n-alkanes ratio (table i), which indicates the contribution of conifer resins vs. epicuticular waxes. the content of total hopanoids is more uniform than the content of n-alkanes, showing slightly elevated values in mc and mxc, consistent with elevated gi values (tables i and s-iv). non-hopanoid triterpenoids are present in the low amount, being the lowest in xc, consistent with formation in the forest swamp (fig. s-3). the ratio of diterpenoids to the sum of diand triterpenoids, di/(di+tri) exhibits high and uniform ratios for all lithotypes (tables i and s-vi), indicating prevalence of gymnosperms (conifers) over angiosperms. predominance of gymnosperms over angiosperms derived om in matrix lithotypes possibly implies significant input of needle leaves,37 consistent with high content of n-alkanes (table i). diterpenoids and triterpenoids with non-hopanoid skeleton. among the individual diterpenoids no specific differences were observed, e.g., pimarane and particularly 16α(h)-phyllocladane are dominant by far in the saturated fraction, whereas simonellite and dehydroabietane are the major diterpenoid constituents of aromatic fraction of all investigated lithotypes (figs. s-4 and s-5 of the supplementary material). high amount of 16α(h)-phyllocladane indicates that the lignite forming plants belonged to the conifer families taxodiaceae, podocarpaceae, cupressaceae, araucariaceae and phyllocladaceae. the abundant pimarane suggests pinaceae, taxodiaceae and cupressaceae.12,38,39 the average δ13c values of beyerane, pimarane and 16α(h)-phyllocladane for lignite lithotypes differ up to 1 ‰ (tables ii and s-vii of the supplementary material) suggesting similar conifer sources. in all lithotypes average δ13c values characteristics of lignite lithotypes 745 746 mitrović et al. decrease in the following order: beyerane > pimarane > 16α(h)-phyllocladane (fig. s-6a of the supplementary material). considering that the isotopic differences evident at toc level are extend to the individual hydrocarbons that are produced from the resins,16 slightly lower average δ13c values of beyerane in xc and mmic can be attributed to the greater impact of cupressaceae. somewhat lower average δ13c values of pimarane in these two lignite lithotypes can be related to greater impact of both, cupressaceae and pinaceae (fig. s-6a; table ii), since it was shown that these two families have more negative δ13c values than araucariaceae and taxodiaceae.40 table ii. the δ13c values of individual diterpenoids and non-hopanoid triterpenoids of lignite lithotypes; values of parameters for individual samples are given in table s-vii of the supplementary material; a – 24,25-dinorlupa-1,3,5(10)-triene; b – 2,2,4a,9-tetramethyl -1,2,3,4,4a,5,6,14b-octahydropicene lithotype value beyer-ane pimarane 16α(h)-phyllocladane dehydroabietane simonellite retene a b matrix coal (mc) mean –25.8 –26.1 –26.8 –26.72 –27.1 –28.2 –28.4 –29.2 max. –25.1 –25.7 –25.9 –25.53 –25.9 –27.6 –27.6 –28.5 min. –27.0 –27.0 –27.4 –27.20 –28.1 –29.3 –29.2 –30.3 sd 0.7 0.5 0.5 0.63 0.8 0.8 0.7 0.8 xylite-rich coal (xc) mean –26.2 –26.7 –26.8 –26.83 –27.3 –28.8 –28.9 –29.6 max. –25.3 –25.9 –26.3 –26.35 –26.6 –27.5 –28.1 –28.7 min. –27.3 –27.5 –27.8 –27.54 –28.2 –30.8 –30.3 –30.0 sd 0.7 0.6 0.6 0.44 0.7 1.4 0.7 0.6 mixture of matrix and mineral-rich coal (mmic) mean –26.1 –26.6 –27.1 –26.18 –26.6 –28.4 –28.7 –29.2 max. –25.6 –25.6 –26.7 –25.16 –25.2 –28.3 –27.9 –27.9 min. –26.9 –27.4 –27.3 –27.21 –28.4 –28.5 –29.3 –30.5 sd 0.7 0.9 0.3 0.86 1.2 0.2 0.7 1.3 mixture of matrix and xylite-rich coal (mxc) mean –25.9 –26.0 –26.6 –26.58 –27.0 –28.6 –28.9 –29.5 max. –25.7 –25.4 –26.2 –25.81 –26.2 –28.3 –27.4 –27.8 min. –26.0 –26.5 –26.9 –27.05 –27.5 –28.9 –29.7 –30.7 sd 0.1 0.5 0.4 0.51 0.6 0.5 1.1 1.5 dehydroabietane, simonellite and retene belong to aromatic abietane type diterpenoids which differs from pimarane (pimarane skeleton) and particularly from tetracyclic diterpenoids, beyerane and 16α(h)-phyllocladane. abietanes are the most widespread class of diterpenoids, being identified in all conifer families with exception of phyllocladaceae.12 the average δ13c values of dehydroabietane, simonellite and retene are similar to average δ13c values of saturated diterpenoids, confirming the common conifer origin. the average δ13c values show the decreasing trend with the increasing of aromatisation (fig. s-6a; tables ii and s-vii of the supplementary material). characteristics of lignite lithotypes 747 this result is in agreement with the earlier investigations, that showed that the aromatised molecules are generally depleted in 13c by about 1–2 ‰ relative to their presumed precursors.27,41 very close average δ13c values of dehydroabietane and simonellite indicate the direct precursor-product connection (fig. s -6a; table ii). the average δ13c values of retene differ more (fig. s-6a; table ii), which probably results from the possibility of different formation pathways of retene from dehydroabietane,39,42–44 confirming that the diagenetic aromatisation influences δ13c. average δ13c values of dehydroabietane in mc, xc and mxc are depleted in 13c in comparison to those of saturated diterpenoids, whereas mmic showed opposite trend (fig. s-6a; table ii). on one hand, this result can be attributed to the wider range of abietane producing conifers.12 on the other hand, considering that in immature om, the aromatisation of biomarkers is mediated by microorganisms, but also favoured by the catalytic influence of clay minerals, the elevated δ13c values of dehydroabietane in mmic can result from different bacterial communities and/or clay catalytic processes. this latter assumption is in agreement with the highest content of aromatic diterpenoids in mmic (tables i and s-vi). the non-hopanoid triterpenoids are present in low amounts in all lithotypes (tables i and s-vi). in the saturated fraction they consist exclusively of des-a -degraded compounds (fig. s-4 of the supplementary material). in the aromatic fraction both, pentacyclic, i.e., non-degraded and des-a-degraded compounds were identified (fig. s-5 of the supplementary material). among the non-hopanoid triterpenoids, des-a-lupane was the most abundant in the saturated fraction, whereas 24,25-dinorlupa-1,3,5(10)-triene prevailed in the aromatic fraction of all samples (figs. s-4 and s-5). the predominance of lupane derivatives indicates that betulaceae was one of the main angiosperm sources.45,46 due to the low concentration of the aromatic non-hopanoid triterpenoids, δ13c was measured at very limited number of the samples. average δ13c of angiosperm derived aromatic non-hopanoid triterpenoids are 2–3 ‰ depleted in 13c compared to the aromatic diterpenoids (gymnosperm origin) (fig. s-6a and b; table ii), consistent with report of bechtel et al.,14 tuo et al.18 and schoell et al.27 comparison of δ13c values of 24,25-dinorlupa-1,3,5(10)-triene and its aromatised counterpart 2,2,4a,9-tetramethyl-1,2,3,4,4a,5,6,14b-octahydropicene indicated slight depletion in 13c during aromatisation. the obtained result is consistent with observation for aromatisation of diterpenoids (fig. s-6 a,b; table ii). gymnosperms are more resistant to degradation than angiosperms. however, the positive correlation between tpi (table s-v) and the content of diterpenoids (table s-vi) is observed only for mmic (r = 0.67), indicating that in conditions of topogenous fresh water peat mire with open water areas, the contribution of gymnosperms (most probably from the surroundings) is the dominant factor controlling om preservation. for all other lithotypes, the absence of correlation 748 mitrović et al. between tpi and content of diterpenoids suggest that tpi was predominantly controlled by relative height of the ground water table (humidity) rather than by input of decay-resistant conifers. although between tpi and content of nonhopanoid triterpenoids negative correlation could be expected, this is not observed here for any of lithotypes (tables s-v and s-vi). the obtained result can be attributed to low concentration of this biomarker class (tables i and s-vi) and already discussed impact of betulaceae, which is characterised by relatively high tree density, being therefore resistant to degradation. n-alkanes. the n-alkane patterns of all lithotypes were dominated by long -chain odd homologues (c27–c31) maximizing at n-c27 or n-c29 (fig. s-4) and showed expressed odd over even predominance (carbon preference index, cpi; tables s-viii and s-ix of the supplementary material), consistent with peat formation from terrigenous plants. all lithotypes are characterised by prevalence of long-chain (c26 to c35) n-alkanes. however, the proportion of short(c15 to c20) and medium(c21 to c25) chain homologues was higher in mmic indicating slightly higher contribution of n-alkanes derived from bacteria and algae (table s-viii of the supplementary material). the δ13c values of c25 to c33 odd n-alkanes range from –27.2 to –32.0 ‰ (fig. s-6c; tables s-x and s-xi of the supplementary material), indicating a source, corresponding to the lipid carbon pool of c3 higher plants.47 marked differences between δ13c values of c27–c33 n-alkanes and c25 n-alkane were not observed (fig. s-6c; table s-x), indicating that later mainly originated from higher plants. however, in all lithotypes c25 n-alkane is slightly more enriched in 13c than odd n-alkane homologues (c27–c33). this result can be attributed to the slight impact of the emergent aquatic macrophytes. for all homologues (with exception of n-c31), xylite-rich coal showed the most positive values (fig. s-6c; table s-x). this result is consistent with the highest relative content of woody vegetation from diterpenoids. depletion in 13c of c29 and c33 odd n-alkanes in other lithotytpes in comparison to xc imply greater influence of herbaceous plants,48 consistent with lower tpi and vi ratios (table s-iv). elevated average δ13c values of c29 and c31 n-alkanes for mmic (fig. s-6c; table s-x), having the lowest tpi and vi (table s-iv), can be attributed to the greater impact of fungi, since it was showed that fungi are relatively less depleted in the 13c isotope, and that fungal spores generally contain c14-c37 n-alkanes, often maximizing at c27, c29 and c31.49,50 this assumption is supported by the highest content of perylene in mmic (table i). hopanoids and steroids. the hopane composition in the saturated fraction is characterised by the presence of 17α(h)21β(h), 17β(h)21α(h) and 17β(h)21β(h) compounds with 27 and 29-32 carbon atoms. other hopanoid type constituents are unsaturated hopenes: c27 neohop-13(18)-ene, c27 hop-17(21) characteristics of lignite lithotypes 749 -ene, c28 neohop-13(18)-ene, c30 hop-17(21)-ene and c30 neohop-13(18)-ene. the aromatic hopanoids with one to four aromatic rings consist of series of orphan aromatic hopanoids bearing an ethyl group at c-21 (figs. s-5 and s-7 of the supplementary material). among the aromatic hopanoids, d-ring monoaromatic hopane prevailed in all samples, being even the most abundant compound in the aromatic fraction of several samples (fig. s-5). the δ13c values of individual hopanoids c30 hop-17(21)-ene, c2717β(h) -hopane and c2917β(h)21β(h)-hopane (in range –36.8 to –51.4; fig. s-6d; tables s-x and s-xi) indicate the contribution of chemoautotrophicand methanotrophic-bacteria.15,41,51 for mentioned hopanoids a decrease of average δ13c values is observed in order: xc > mxc > mc > mmic (fig. s-6d; table s-x), suggesting the decreasing methanotrophic activity (active methane cycle at the time of deposition) from forest swamp to topogenous fresh water peat mire. although measured at limited number of samples, due to the low concentration, the δ13c values of c3117α(h)21β(h)22(r)-hopane are relatively uniform and notably distinct from δ13c values of the other hopanoids, ranging from –24.2 to –27.5 ‰ (tables s-x and s-xi). the observed δ13c range indicates heterotrophic bacteria that consumed higher-plant-derived om.16,26 the average concentrations of individual hopanoids (table s-viii) imply the predominance of c30 hop-17(21)-ene in all lignite lithotypes. however, in mc, mmic and mxc the second most abundant hopanid was c2717β(h)-hopane, whereas xc is characterised by elevated content of c3117α(h)21β(h)22(r)-hopane. since isotopic composition (table s-x) suggested that the later mostly originated from heterotrophic bacteria, it can be concluded that this type of bacteria had greater impact on the peatification of xylite-rich coal. the typical feature of all lithotypes is prominent c28 28,30-bisnorneohop13 -(18)-ene (c28 neohop-13(18)-ene, table s-viii). considering lignites, to the best of our knowledge c28 neohop-13(18)-ene was only reported in the sediments and fossil conifer extracts from the upper eocene zeitz formation in the schleenhain open pit near borna (saxony, germany)42 and in samples from the miocene tokiguchi porcelain clay formation at the onada mine, central japan (although in that case without the defined position of double bond).52 the δ13c values of c28 neohop-13(18)-ene range from –33.6 to –36.6 ‰ and differ from δ13c values of other hopanoids (fig. s-6d; tables s-x and s-xi). the obtained values can imply that c28 neohop-13(18)-ene is sourced from a certain type of chemoautotrophic bacteria.15,41,51 the activity of chemoautotrophic bacteria during diagenesis has already been assumed based on the δ13c values of c2717β(h)and c2917β(h)21β(h) hopanes. c28 neohop-13(18)-ene showed the same δ13c trend in studied lignite lithotypes, as it was observed for other hopanes, with a single difference, that average δ13c values for xc and mxc were almost equal (fig. s -6d; table s-x). 750 mitrović et al. in some earlier investigations,17,53 it was supposed that c28 neohop-13(18) -ene could be a logical direct precursor of series of orphan aromatic hopanoids (containing an ethyl group at c-21) by the progressive aromatisation (fig. s-7). in order to check this assumption δ13c values of c28 neohop-13(18)-ene and orphan aromatic hopanoids (d-ring monoaromatic hopane and abcd-ring tetraaromatic hopane; fig. s-7) were measured, for the first time in the same series of samples, to the best of our knowledge. d-ring monoaromatic hopane has similar average δ13c values as c28 neohop-13(18)-ene, particularly for xc and mxc (fig. s-6d; table s-x) confirming that c28 neohop-13(18)-ene can be an important precursor of orphan aromatic hopanoids. however the greater range of δ13c values of d-ring monoaromatic hopane than of c28 neohop-13(18)-ene (fig. s -6d; tables s-x and s-xi) is in accordance with the well known fact that the degradation of a side chain and aromatisation of other hopanoids may also result in the formation of aromatic hopanes. it is interesting that the abcd-ring tetraaromatic hopane has higher average δ13c value in comparison to d-ring monoaromatic hopane for all lithotypes (fig. s-6d; table s-x) which is consistent with the observation that some type of aromatics can be enriched in 13c relative to their precursors.27 recently, liao et al.54 also established that aromatisation of hopanoids is followed by 13c isotopic enrichment. the contents of total steroids were low and relatively uniform (tables i and s-vi) which could be explained by the fact that in the investigated samples steroids mostly originate from higher plants, which contain very low amount of these biomarkers. therefore the determination of isotopic composition was impossible. the steroid biomarkers in all lithotypes are represented by c27–c29 (δ4-, δ2and δ5-) sterenes and a-ring monoaromatic sterane. distribution of sterenes in all lithotypes was notably dominated by c29 homologues (tables s-viii and s-ix), consistent with peat formation from terrigenous plants. however, average proportion of c27 sterenes was the highest in mmic (table s-viii) indicating the higher contribution of algae. this result is in accordance with the higher proportion of short-chain n-alkanes in mmic (table s-viii). in addition, this coal lithotype also contains slightly higher average proportion of c28 sterenes (table s-viii), indicating greater contribution from fungi, that has already been assumed, based on average δ13c values of c29 and c31 n-alkanes (fig. s-6c; table s-x), as well as by the highest content of perylene (table i). conclusion the coal lithotypes differ according to the composition of huminite macerals. ulminite, followed by textinite or densinite is the dominant huminite maceral in xc, whereas densinite followed by ulminite prevailed in three other lithotypes, indicating that xc has poorer susceptibility to grinding. on the other hand, xc is the most suitable for fluidized bed gasification, whereas mmic has the characteristics of lignite lithotypes 751 lowest applicability. all studied lithotypes from the kovin deposit are unsuitable for coal briquetting, but meet requirements for the rational utilization in thermal power plants. xc was formed in forest swamp, whereas mc and mmic originated from a topogenous fresh water peat mire with open water areas. mxc was formed in the bush moor. main sources of om in all lithotypes were gymnosperms (conifers). the predominance of gymnosperms over angiosperms derived om in the lithotypes comprising matrix coal implies significant input of needle leaves, consistent with high content of n-alkanes. lignite forming plants belonged to the gymnosperm families taxodiaceae, cupressaceae, araucariaceae, phyllocladaceae and pinaceae. mc and mxc are characterised by the slightly greater impact of taxodiaceae and araucariaceae, whereas two other lithotypes had greater impact of cupressaceae and pinacea. in all studied lithotypes betulaceae was the main angiosperm source. xc notably differs from the other lithotypes because of the highest contribution of conifer resins vs. epicuticular waxes. mmic is characterised by the greatest contribution of algae and fungi. toc content and consequently calorific value decreased in the following order: xc > mc > mxc > mmic, indicating that the increase of the impact of wood vegetation (mostly conifers) vs. herbaceous peat-forming plants and more stable water table during peatification in the forest swamp, in comparison to topogenous water peat mire, positively influences these parameters. diagenetic alteration of all lignite lithotypes was governed by chemoautotrophic-, methanotrophicand heterotrophic-bacteria. methanotrophic activity decreases in the following order: xc > mxc > mc > mmic. on the other hand, heterotrophic bacteria had the greatest influence on peatification of xc. these results show that activity of methanotrophs is greater in topogenous water peat mire, whereas heterotrophic bacteria more affect om in forest swamp. in all coal lithotypes the diagenetic aromatisation influenced isotopic composition of individual biomarkers. the aromatisation of diterpenoids and non -hopanoid triterpenoids may result in a depletion of 13c, whereas aromatisation of hopanoids is followed by 13c enrichment. direct precursor-product relationship between c28 neohop-13(18)-ene and the series of orphan aromatic hopanoids was confirmed for the first time in all studied lithotypes. supplementary material experimental data and theoretical models are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the study was financed by the ministry of education, science and technological development of the republic of serbia (projects 176006 and 451-03-01039/ /2015-09/05) and österreichischer austauschdienst (oaed) (project no. srb 18/2016) which are gratefully acknowledged. we are also grateful to the anonymous reviewers. 752 mitrović et al. и з в о д карактеризација литотипова лигнита из лежишта „ковин“ (србија) на основу петрографске, биомаркерске и изотопске анализе даница митровић1, наташа ђоковић1, драгана животић2, achim bechtel3, олга цветковић4 и ксенија стојановић5 1 универзитет у београду, иновациони центар хемијског факултета, студентски трг 12–16, 11000 београд, 2 универзитет у београду, рударско-геолошки факултет, ђушина 7, 11000 београд, 3 montanuniversität leoben, department of applied geosciences and geophysics, peter-tunner-str. 5, a-8700 leoben, austria, 4 универзитет у београду, центар за хемију, ихтм, његошева 12, 11000 београд и 5 универзитет у београду, хемијски факултет, студентски трг 12-16, 11000 београд четири литотипа лигнита (барски, ксилитни, мешавина барског и ксилитног и мешавина барског и земљастог угља) из лежишта ковин су детаљно проучавани. циљ рада био је да се одреде главна мацерална, биомаркерска и изотопска својства испитиваних литотипова. на основу тих резултата утврђени су порекло и средина таложења органске супстанце за ова 4 литотипа. ови узорци су такође послужили као супстрати за испитивање утицаја дијагенетских промена на δ13c вредности биомаркера, као и за процену најпогодније примене за сваки од литотипова. литотипови се разликују према саставу мацерала хуминитске групе. ксилитни угаљ се разликује од осталих литотипова по највећој заступљености смола голосеменица у односу на епикутикуларне воскове. највећи допринос гљива и алги запажен је у органској супстанци мешавине барског и земљастог угља. формирање овог литотипа било је праћено најинтензивнијом метанотрофном активношћу. код свих испитиваних литотипова дијагентска ароматизација утиче на изотопски састав индивидуалних биомаркера. ксилитни угаљ има најлошију мељивост. с друге стране, ксилитни угаљ је најпогоднији за гасификацију у флуидизованом слоју, док је мешавина барског и земљастог угља показала најлошију применљивост за овој процес. топлота сагоревања опада у следећем низу: ксилитни > барски > мешавина барског и ксилитног > мешавина барског и земљастог угља. пораст садржаја органског угљеника и топлоте сагоревања је резултат пораста удела дрвенасте вегетације у односу на зељасте биљке у тресету, као и стабилности нивоа воденог стуба током процеса хумификације. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice ultrasound and shacking-assisted water-leaching of anions and cations from fly ash j. serb. chem. soc. 81 (7) 813–827 (2016) udc 66.046.59+544.354–032.35:66.061.34+ jscs–4888 534–8:504.5 original scientific paper 813 ultrasound and shacking-assisted water-leaching of anions and cations from fly ash marjetka savić biserčić1, lato pezo2, ivana sredović ignjatović3#, ljubiša ignjatović4*#, andrija savić1, uroš jovanović1 and velibor andrić1 1university of belgrade, vinča institute of nuclear sciences, chemical dynamics laboratory, p.o. box 522, 11001 belgrade, serbia, 2university of belgrade, institute of general and physical chemistry, studentski trg 12–16, 11000 belgrade, serbia, 3university of belgrade, faculty of agriculture, nemanjina 6, 11080 belgrade, serbia and 4university of belgrade, faculty of physical chemistry, studentski trg 12–16, 11000 belgrade, serbia (received 22 december 2015, revised 18 february, accepted 22 february 2016) abstract: two mechanical extraction techniques were used for the extraction of environmentaly interesting components of coal fly ash: shaking, during which the extraction process lasted from 6 up to 24 h, and sonication that lasted from 15 up to 60 min, using water as extractant. the concentration of anions in fly ash extracts was determined by ion chromatography, while atomic absorption spectrometry was used for determination of: as, pb, cd, ni, cr, zn, cu, fe, mn and al. the ultrasonication yielded slightly higher amounts of extracted anions as well as pb, al, mn and fe cations, while shaking-assisted extraction was more efficient for the cr, as, zn and ni ions. the changes in ph value, particle size distribution within colloid solution, zeta potential and conductivity during ultrasound-assisted extraction were measured in order to explain changes that occur on the surface of fly ash particles contacting water and different processes (adsorption, ion exchange and flocculation) that develop under natural conditions. principal component analysis was used for assessing the effect of observed process parameters. it is essential to evaluate quantity of these elements leachable from coal fly ash into the surface waters in natural conditions in order to prevent contamination of the environment. keywords: fly ash; environmental impact; aqueous extracts. introduction being one of the major fossil fuels for generating electricity, coal is also the important factor in global warming and one of the main contributors to acid * corresponding author. e-mail: ljignjatovic@ffh.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc151222027b _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 814 savić biserčić et al. rain.1,2 during coal combustion in thermal power plants a series of physicochemical transformations of mineral matter present in coal particles occur, leading to dispersion of potentially toxic elements into the environment.3,4 the emission of combustion gases includes in part a fly ash that escapes the particulate matter control device. despite high retention efficiency of these devices in thermal power plants (e.g., electrostatic precipitators), considerable amounts of microparticles are emitted into the atmosphere because of the high overall rate of coal consumption in large power plants. these particles are considered to be highly contaminating, due to their large specific surface areas and the fact that potentially toxic trace elements condense on their surfaces during cooling of combustion gases. coal fly ash is a highly heterogeneous material usually consisting of silicon dioxide, aluminum oxide and iron oxide as major mineral components.5 during coal combustion, various trace elements such as as, pb, cd, mn, cr, ni, zn and cu could be present in fly ash, depending on coal characteristics and the combustion process.4,6,7 their possible release into the environment could cause many problems. along with as, pb, cd, mn, cr and ni, fluorine and chlorine are among the hazardous air pollutants mentioned in the 1990 clean air act amendments. heavy metals can be leached from fly ash and may become a hazard to the environment because of their contribution to the formation of toxic compounds.1–3,7 in order to analyze the elements contained in fly ash, it is necessary to extract the analytes from solid samples and employ an instrumental technique to determine their quantity. the extraction of analytes involves digestion process using strong acids, such as hno3, hcl, hf and h2so4 or their combinations.6–9 the use of mineral acids at elevated temperatures can lead to a loss of easily volatile elements, thus making impossible determination of anions in the same solution. apart from microwave digestion, which is widely used, another technique for sample preparation is ultrasound-assisted extraction (uae).8–13 uae can be performed by using ultrasonic baths or probes. the main advantages of the technique are speed of digestion, high sample treatment capacity and low reagent usage. when ultrasonic water bath is used, subsequently samples can be centrifuged instead of filtered if they were placed in plastic screw-top bottles or polypropylene centrifuge tubes.14,15 lower pressure and temperature used during the ultrasonic treatment makes this technique safer than the microwave assisted digestion. for metal detection, atomic absorption spectrometry (aas) or inductively coupled plasma/optical emission spectrometry (icp-oes) have been used.3,7–18 halogens and nonmetals are in most cases determined by ion chromatography (ic) or ion selective electrodes.19,20 in this study, ultrasound-assisted extraction (uae) for sample preparation was investigated in order to determine metals and nonmetals in coal fly ash and _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ water-leaching of fly ash 815 the results were compared with those obtained by shaking-assisted extraction (sae) and with the standard astm procedure.21 using our extraction techniques, the idea was to employ only water as extractant in order to predict the environmental impact of real leaching process associated with the disposal of fly ash into the ponds.22 the anion content (fluorides, chlorides, nitrates, nitrites, sulfates and phosphates) was analyzed by ic and the metals (arsenic, lead, cadmium, nickel, chromium, zinc, copper, iron, manganese and aluminum) were measured by aas. the main criterion for the selection of elements was their natural presence in coal, as well as their potentially negative effects on human health and the environment. particle size distribution (psd) and zeta potential (zp) measurements were performed, as well as ph value and conductivity estimations of suspension solution during ultrasonic extraction, with the aim of better understanding different processes during exposure of fly ash to the water treatment, under certain conditions. experimental results were subjected to analysis of variance (anova) to show relations between samples. pattern recognition technique (principal component analysis – pca) was applied on the experimental data (used as descriptors) to characterize and differentiate among the observed samples.23,24 experimental sampling and sample preparation fifty individual samples were collected from the kostolac thermal plant, located approximately 90 km east of belgrade, serbia. all individual samples were combined and bigger lumps milled in a grinder to obtain smaller pieces < 1 mm. subsequently, the samples were thoroughly homogenized, as confirmed by energy dispersive x-ray fluorescence spectrometry (edxrfs), dried at 50 °c for 24 h, and then micronized in a plate mill for 30 s. finally, they were sieved through a sieve with 0.212 mm openings which corresponds to us standard mesh 70 and tyler standard mesh 65. the moisture content determined by the karl -fisher coulometric titration method was 1.24 %. all the results were calculated for dried sample. homogeneity was confirmed by edxrfs (in-house developed spectrometer), suitable for non-invasive and non-destructive analysis of various materials. as an excitation source, air cooled x-ray tube (oxford instruments, rh – anode, max. 50 kv, 1 ma) with a pinhole lead -brass collimator and beam size at the exit of the collimator of 0.5 mm was used. for the detection of characteristic x-rays, an amptek x-123 complete x-ray spectrometer with a si-pin detector (6 mm2/500 μm, be window 12.5 μm thickness) was employed, and for spectra acquisition and peak calculations admca software was used. two laser pointers mounted on the measuring head enabled repeatable positioning of the samples. experimental parameters of 40 kv applied voltage, 800 μa current, and a measuring interval of 200 s with no filter were selected and kept constant during all measurements. the geometrical parameters chosen were as follows: detector–sample distance, was 21 mm; x-ray tube collimator tip– _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 816 savić biserčić et al. –sample distance, 16 mm; and the angle between the axes of the x-ray tube and the detector, 45°, while the sample surface was perpendicular to the excitation x-ray beam. the extraction suspensions were prepared by mixing the ash sample with deionized water at a ratio 1:10, i.e. 3 g of solid sample to 30 ml in 50 ml volumetric flasks. all chemicals used for the analysis were of analytical grade. deionized water from a milli q unit (sg gmbh) was used for the preparation of all samples. the resistivity of the deionized water was 18.0 mω cm at 22.5 °c. three shaking-assisted extractions (sae) were performed for each extraction time of 6, 12 and 24 h at room temperature (20 °c) using a custom made linear shaker. the ultrasound -assisted extractions (uae), employed a sonis 2 gt (iskra pio d.o.o., slovenia) ultrasonic bath operated at 40 khz ultrasonic frequency and 200 w effective ultrasonic power. three extractions were also performed for each extraction time of 15, 30, 45 and 60 min. the extraction times, for both procedures, were chosen according to earlier investigations of solid sample extractions.12,15,19 after the extraction processes, the resulting suspensions were filtered through a medium pore sized filter paper. measurement techniques the concentration of anions in solutions was determined by ion chromatography using a dionex ion chromatography instrument. the conductivity detection was performed after the suppression of eluent conductivity using an anion suppressor asrs 300. the separation column employed was a dionex ionpac as 11. as an eluent aqueous solution of nahco3 (0.5 mm) and na2co3 (2 mm), at a flow rate of 0.9 ml min -1 was used. samples were filtered and then injected. sample volume was 20 μl for each probe. three replicate injections of each sample were made and the results averaged. all chemicals were of analytical reagent grade. all standard solutions, eluent and reagents were prepared with milli-q water, degassed under vacuum, and then filtered prior to use. a primary multi-anion standard solution (spex certiprep group) was used. calibration standards were prepared by serial dilutions of the multi-component standard with milli-q water. for the determination of metals, an atomic absorption spectrometer (perkin–elmer aanalyst 700) was used. both flame and electrothermal atomization techniques were employed, depending on the element. the whole system was controlled by means of aa winlab 32 software. zn, mn, fe and cu were determined by air-acetylene, while al by nitrous oxide-acetylene flame aas. as, pb, cd, ni and cr were measured using electrothermal aas, coupled with standard pyrolytic graphite–coated tubes with integrated platforms (perkin–elmer part no. b3 001264) as an atomizer. graphite platforms significantly improve the instrument performance for the determination of cd, pb and as, according to astm d 6357.21 graphite tubes are purged with argon as an inert gas. samples were injected into the graphite furnace (gf) system by an autosampler (perkin–elmer as-800). the deuterium lamp background correction was used for certain elements. each hollow cathode lamp or electrode discharge lamp, used in these measurements as a radiation source, operated using current recommended by the manufacturer. calibration solutions for each element were prepared from serial dilutions of 1 g l-1 single element stock solution in 0.2 vol. % nitric acid (merck, kgaa, germany). the obtained results were compared to the results obtained by the standard procedure21 whereby good agreement was found. the ph value was determined using a ph meter (ph 1500, p11/bnc, eutech instruments, thermo fisher scientific, usa). _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ water-leaching of fly ash 817 additionally, the uaes were repeated in order to measure psd and zp by dynamic light scattering spectroscopy using a zeta-sizer nano red zs, with a 633 nm he–ne laser (malvern, uk) and the data were analyzed by the zetasizer software, version 6.20 (malvern, uk). the instrumental conditions were: 30 runs with run duration of 10 s; temperature, 25 °c; refractive index, 1.600 and absorption index of material, 0.09. the measurements were conducted during the extraction process every 15 min for the colloid psd and zp. statistical analysis descriptive statistical analyses for all the obtained results were expressed by means, for each treatment. collected data were subjected to anova to explore the effects of process variables. furthermore, pattern recognition technique pca was applied successfully to classify and discriminate between different samples. the evaluation of anova and pca of the obtained results was performed using statistica software version 12.23-26 results and discussion homogeneity proof homogeneity of the fly ash composite sample, after combining and long -term mixing, was confirmed by edxrf spectrometry. after the batch was well shaken, 5 subsamples were taken from the bulk and pastilles (0.500 g and 25 mm radius) made for each of them. every subsample pastille was then analyzed by edxrf spectrometry at two different measuring spots in each pastille. almost perfect overlap of the two acquired spectra confirmed the homogeneity of the composite sample (fig. 1). grain size distribution is represented in fig. 2. fig. 1. edxrf spectra of composite fly ash sample. results of anion concentration measurements the results of the ic analysis of chloride, fluoride, nitrate, nitrite, phosphate and sulfate anions in the samples treated with two different techniques of mech_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 818 savić biserčić et al. anical preparation, sae and uae are presented in table i. accuracy was determined by measuring each sample in triplicate and expressed as relative standard deviation. it should be noted that the fly ash sample is solid and as such is never perfectly homogenous. relative standard deviation (rsd) of the analytical measurements performed was determined: 1.2 (fluoride), 0.92 (chloride), 4.6 (sulfate) and 7.7 % (nitrate). fig. 2. grain size distribution of fly ash ( □ – sieved fraction, ▲ – unsieved fraction, x – overall). table i. the results of the anions determination (mg/100g) after uae/sae (n = 3, uncertanity as standard deviation); a, b, c, letters printed in superscript within the same column in the table show significantly different means of observed data (at p < 0.05 level) uae / min fluoride chloride nitrate sulfate 15 2.87±0.04a 1.99±0.07a 0.12±0.02a 673±50a 30 2.86±0.10a 2.17±0.11a 0.13±0.01a 675±55a 45 2.66±0.04c 2.08±0.07a 0.11±0.01a 678±58a 60 2.46±0.04b 2.37±0.56a 0.10±0.02a 778±4a sae / h fluoride chloride nitrate sulfate 6 2.83±0.16a 1.90±0.12a 0.12±0.02a 646±17a 12 2.77±0.07a 1.84±0.20a 0.14±0.01a 674±3a 24 2.43±0.07b 1.71±0.04a 0.11±0.01a 707±9b nitrites and phosphates were not detected. nitrites most likely oxidized to nitrates. macro and micro-analysis of coal confirmed the content of p2o5 to be below 0.01 %. the concentration of phosphorus in the ash was even lower and after the extraction it was below the detection limit.20 the results of fluorides extracted by sae and uae are shown in table i. a steady decrease in concentration with time was observed in samples obtained in both procedures (statistically significant at p < 0.05 level, according to tukey’s hsd test).27 in the case of uae, the concentration of fluoride remained the same for the first 30 min, but after 45 min values decresed and after 60 min were _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ water-leaching of fly ash 819 slightly higher than for the samples obtained by sae after 24 h. it can be noticed that the concentration of fluoride extracted using ultrasound was slightly higher compared to shaker procedure. the uae for chloride ions (table i) results demonstrated the extracted ion amount did not depend on the extraction time. during sae, however, the concentration of chlorides decreased after 12 h, and even more after 24 h of shaking. as shown in table i, the extracted amount of nitrate ions varied with the extraction time. in both cases, the nitrate content initially increased, but finally decreased below the initial values. the amount of sulfate anions extracted by uae gradually increased and continued rapid increase after 60 minutes. monitoring changes in the amount of sulfates with an elapsed time of shaking, the increase in their concentration is much more steady. after six-hour agitation, the extracted sulfate average was approximately 30 mg/100 g and remained at the same level for the next twelvehour agitation. results of cation concentration measurements the results of as, pb, cd, ni and cr cation contents in the samples mechanically treated by sae and uae, determined by graphite furnace atomic absorption spectrometry (gfaas) and the results of zn, cu, fe, mn and al content determined by flame atomic absorption spectrometry (faas), are shown in table ii. for the concentration ranges measured, the relative standard deviation (rsd) was: 2.1 (as), 2.2 (pb), 6.4 (cd), 9.7 (ni), 0.6 (cr), 9 (zn), 12 (cu), 11.8 (fe), 8.6 (mn) and 9.5 % (al). table ii. the results of the cation determination (μg/100 g) after uae/sae (n = 3, uncertanity as standard deviation); a,b,c – letters printed in superscript within the same column in the table, show significantly different means of observed data (at p < 0.05 level) element uae / min sae / h 15 30 45 60 6 12 24 as 27.44±1.19b 31.69±1.23a 33.35±0.57a 33.71±0.48a 25.30±1.24a 35.77±1.05b 42.45±1.36c zn 2.67±1.15a 3.00±1.00a 3.33±1.15a 10.67±2.08b 7.33±1.53a 10.67±2.08a 11.33±1.53a pb 1.15±0.14ab 1.02±0.03a 1.31±0.11b 1.90±0.07c 1.10±0.04a 1.19±0.04ab 1.47±0.19b cu 11.33±1.53a 11.67±2.08a 16.67±2.31ab 21.00±4.00b 11.00±1.73b 18.33±1.15a 21.33±1.53a fe 38.33±2.52b 48.00±6.56ab 58.67±9.07a 63.33±5.51a 43.00±8.19a 56.33±6.66ab 61.33±5.51b mn 14.50±0.87a 13.67±1.15a 13.33±1.15a 13.00±1.00a 13.00±1.00b 12.00±1.00ab 11.00±1.00a cd 0.05±0.01a 0.08±0.01a 0.13±0.04ab 0.23±0.09b 0.07±0.04a 0.19±0.08a 0.11±0.02a ni 0.52±0.09b 2.69±0.28a 2.49±0.39a 2.56±0.47a 1.57±0.41a 2.38±0.16ab 2.70±0.59b cr 34.33±3.53a 79.5±9.7ab 92.9±6.3bc 128.3±29.1c 78.8±3.2a 118.9±13.0b 162.6±6.2c al 10300±323c 5620±86bc 2070±66ab 730±18a 3770±90b 590±76a 459±12a the increased concentration of some ions is more or less proportional to the extraction time (statistically significant at p < 0.05 level, according to tukey’s _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 820 savić biserčić et al. hsd test). also, observed increase was more regular when sae was applied. in most cases, the amount of cations extracted by sae for 24 h and uae for 60 min appeared approximately the same. it can be noticed that the concentrations of as, ni, zn and cr increased in time, but the amounts obtained by sae were 20–25 % higher than the corresponding values obtained by uae. the concentration increase for all these elements was gradual, except for zn extracted by uae. the obtained values were very low after 45 min, followed by a sudden peak of about 70 % after 60 min. the concentrations of cu and fe ions in fly ash samples were almost the same at the end of the extraction period for both methods of mechanical treatment. the concentration increase was steady in each case, but the results of ultrasound were obtained much faster, so it is clear that uae accelerated the reactions. the larger quantity of pb ion was extracted by uae, even after an extraction time of 45 min. after 60 min, concentration was about 30 % higher than obtained using sae. in the case of cd ion, the amount extracted by sae varied with time, contrary to uae extraction that provided for steady increase. therefore, it can be concluded that the ultrasound usage secured consistent values of concentration for extracted ions. the total concentration of al ion in extracts obtained by uae contrary to that obtained by sae was significantly higher. but, the amount of al, as well as mn, during the extraction, decreased in both cases. in order to characterize and differentiate among the observed samples, pattern recognition technique (pca) was applied on the experimental data (used as descriptors). the pca method allows a considerable reduction in the number of variables and the detection of structure in the relationship between measured parameters. all samples were produced during different time intervals as shown by experimental design (tables i and ii) and predicted by pca score plot (fig. 3a and b, respectively). for visualizing the data trends and the discriminating efficiency of the used descriptors a scatter plot of samples, using the first two principal components (pcs) issued from pca of the data matrix, was obtained. the quality results for uae water-leaching of anions and cations from fly ash process (fig. 3a) show that the first two principal components, accounting for 95.30 % of the total variability can be considered sufficient for data representation. the content of cu (8.3 % of the total variability), fe (8.1 %), cd (8.6 %) and cr (8.4 %) were the most positively influential variables for the first factor coordinate calculation, while the most evident negative contribution for the first factor component was observed by fluoride (8.3 %). on the other hand, the most positively influential variables for the second factor component calculations _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ water-leaching of fly ash 821 were: as (12.0 %), ni content (26.0 %) and nitrate (18.8 %), while the negative influence was observed by pb (9.4 %). the first two principal components explained 100 % of total variability for sae process, as seen from fig. 3b. the content of as (8.7 % of the total variability), zn (8.1 %), cu (8.5 %), fe (8.5 %), ni (8.5 %), cr (8.6 %) and sulfate (8.6 %) were the most positively influential variables for the first factor coordinate calculation, while the most evident negative contribution for the first factor component was observed for mn (8.7 %). the change in cation and anion concentrations during uae and sae can be observed in fig. 3a and b, respectively, with higher al and mn concentrations fig. 3a, at the beginning of the process (for both uae and sae), while the concentration of pb, cr, as, cu, ni, fe and zn, and also the sulfate content is increased with uae and sae time (fig. 3b). the most negatively influential variables for the second factor component calculation were: cd (32.2 % of the total variability), fluoride (8.2 %) and nitrate content (36.6 %). (a) (b) fig. 3. biplot graphic of uae (a) and sae (b) water-leaching of anions and cations from fly ash. our results are in a very good agreement with the results obtained using the standard procedure for gfaas.24 in table iii the comparison of measured concentrations of as, pb, cd, ni and cr in fly ash after 60 min of uae, and those obtained by astm d 6357 are shown. the advantage of uae method is its simplicity, while standard procedure is time-consuming including usage of hazardous substances. compared to sae, uae technique proved to be more efficient in the case of al, on average 160 % increased concentration was extracted. it was more efficient in the case of pb, mn and fe ions, also (fig. 3). ultrasonic energy, when applied to solutions, causes acoustic cavitation. the collapse of bubbles formed by ultrasonic energy results in the generation of high temperatures and pressures at the interface of the collapsing bubble and another phase, leading to enhanced _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 822 savić biserčić et al. chemical reactivity.28,29 the surface area available for reactions with the extraction agent could increase because of the possible fragmentation and aggregate disintegration of sample particles. it is well known that the processes of adsorption and desorption could arise, but due to recombination of reactive groups under extreme conditions the formation of new chemical species in the suspension occur at prolonged sonication times.30 table iii. a comparison of concentration values (μg/100 g) determined by gfaas after 60 min uae with those obtained by a standard test method element uae astm d 6357 as 33.71±0.68 34.42±0.80 pb 1.90±0.07 2.01±0.09 cd 0.23±0.09 0.22±0.07 ni 2.56±0.47 2.69±0.55 cr 128.3±29.1 121.9±27.0 also, significantly larger amounts of zn and cr extracted by sae contrary to uae can be noticed. this could be explained either by considerably longer agitation time (24 h) and subsequent dissolution, or by the re-adsorption of extracted cations onto the newly exposed sorption sites due to influence of ultrasound. as observed in fig. 3, the extracted amounts of some metals varied a lot with the extension of extraction time, although some metals behaved in the similar way during the extraction. the concentrations of cu and zn were slightly elevated, until a sharp increase occured in both cases, after 30 and 45 min, respectively. concentrations of mn and al showed a steady decline. mn could be expected to exist as an oxide since mns forms only under extreme conditions of high sulfur and low oxygen availability. as such, it is very nonvolatile and its wide distribution in the ash could be expected. small amounts of ni, pb and cd suggest a prior geochemical association of these metals with mn. concentration of fe and as increased with extraction time, but very slowly after 45 min. this can be explained by the presence of as probably as an arsenical pyrite. both sulfides of as and fe are volatile and thus could be expected to condense on the surface of cooling ash particles.31–33 changes in ph value, particle size distribution (psd) in colloid, zeta potential and conductivity during the extraction the changes of the average particle size distribution, zeta potential, ph value and conductivity of the suspension during ultrasonic extraction of fly ash are presented in table iv. the results given in table iv were analyzed by applying pca and presented in fig. 4. the first two principal components explained 98.8 % of total variability for the changes in the average particle size distribution, zeta potential, ph value _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ water-leaching of fly ash 823 and conductivity of the suspension during ultrasonic extraction. zeta potential (25.2 % of the total variability), ph value (32.1 %) and conductivity (30.6 %) were the most negatively influential variables for the first factor coordinate calculation. the most positively influential variable for the second factor component calculation was the particle size (51.5 % of the total variability), while the negative influence was observed for zeta potential (23.8 %). table iv. changes of the average particle size distribution, zeta potential, ph value and conductivity of suspension during ultrasonic extraction extraction time min average particle size nm zeta potential mv ph value conductance μs cm-1 15 1418 –11.9 11.00 535.33 30 1390 –8.80 11.41 609.00 45 1416 –10.3 11.32 596.00 60 1262 –10.7 10.48 561.67 fig. 4. biplot graphic of the changes in the average particle size distribution, zeta potential, ph value and conductivity of suspension during ultrasonic extraction. once the fly ash particles contact an aqueous environment, solid phase dissolution reactions occur immediately. hydrolysis of oxide forms of ca, k, mg and na are suspected to cause high initial ph values of the solution. al, fe and si are particularly soluble at elevated ph values.34 the ph value did not change significantly; the largest decline (11.32 to 10.48), occurred from 45 to 60 min of uae when the concentration of sulfate was the highest (table i). large amounts of sulfate are typically released from fly ash, and there is a probability that soluble inorganic complexes are formed.31 the total content of acid cations decreased most from 15 to 30 min (table ii) and the corresponding increase in ph value can be observed. the general dividing line between stable and unstable suspensions is taken at ±30 mv. particles with zeta potentials more positive than 30 mv or more nega_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 824 savić biserčić et al. tive than –30 mv are normally considered stable.35 in this case, measured zeta potential of colloid particles ranged from –11.9 to –8.8 mv with the largest change observed between 15 and 30 minutes of sonication. the flocculation phenomena increased because the force of electrostatic rejection was low. it could be assumed that due to al and mn adsorption processes, the surface charge of colloidal particles slightly increased. the absolute value of zeta potential corresponds to the thickness of diffusion layer of ions. the valence of the ions could also influence double layer thickness. since the extracted concentration of al is higher than the concentration of other extracted cations (table ii) it could be assumed that one of the reasons for the low values of zeta potential lies in high concentrations of al3+ in solution that compresses the double layer to a great extent in comparison with a monovalent ion such as na+.36 after 60 min of sonication, concentration of al3+ dropped and absolute value of zeta potential increased, most likely due to al3+ adsorption. colloidal instability caused the particle size changes during sonication. from 15 to 30 min, average particle size declined and, as is expected, conductivity increased. after 60 min average particle size decreased, most likely because of the impact of ultrasound and consequent increase in the temperature of the solution. zeta potential, conductivity and ph values are changing in the same way during exposure to ultrasound (fig. 4). from 15 to 30 min they increased, which corresponded to an increase of charged particles in the solution and a thinner diffusion layer of ions. after 45 min, the diffusion layer thickness increased, primarily due to al3+ and mn2+ adsorption, as well as other ions to a lesser extent; ph value decreased because of increased concentration of sulfate in solution and decrease in conductivity occurred probably due to readsorption of alkali and alkaline earth metals on smaller diameter particles of fly ash. in aqueous media, the ph value of the sample is one of the most important factors that affect zeta potential. a zeta potential versus ph value curve will be positive at low ph values and negative at high ph values. since the range of ph values in this case is alkaline, low negative zeta potentials are expected, which is in accordance with the results presented in table iv and fig. 4. various cations released into the solution caused the change in ph value, which could further influence the adsorption and desorption processes to some extent. these processes are mainly related to competitive cation interactions for the sorbing phase and the exchange of sorbed cations with those from solution influenced by ultrasound. further investigations are required in order to explore in what forms are analyzed ions present during the processes of extraction, what are the relations between them, as well as the compounds they may form in the given range of ph values, which would be substantial for enhancements of extraction procedures. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ water-leaching of fly ash 825 conclusions from the obtained results it could be concluded that the techniques employed for the mechanical assisted extraction (as a preparation step) of fly ash samples are significant for estimating the concentration of trace elements and pollutants that could be released into the environment. shaking and ultrasound assisted extractions of aqueous fly ash suspensions were performed. the ultrasonic assisted extraction is less time consuming and our results showed that the prolonged extraction of 15 min did not lead to significant change in the amount of the extracted anions, except in the case of sulfate where concentration readily peaked. contrary to this, the concentration of the most cations increased over the uae extraction time and 60 min extraction was found to be optimal. only amounts of al and mn showed decrease. the larger amounts of as, ni, zn and cr, were extracted by sae, probably due to considerably longer agitation time. the interaction of ultrasonic energy with the aqueous fly ash suspensions, alternately, influenced the processes of cation adsorption and desorption, leading to a change in the extracted amounts of cations as a function of sonication time. uae was more efficient for al, pb and mn. the concentrations of cu and fe ions were almost the same after both extraction procedures, so it was clear that ultrasound accelerated the reactions, while in the case of cd ions uae promoted release as well as extracted quantity. it could be concluded that there is no significant difference in the extracted quantities for most of analyzed ions between sae and uae, but the 15 min uae is as efficient as six-hour sae. the ph value, conductivity, average particle size and zeta potential changed in accordance with extraction times and measured concentrations of cations and anions. acknowledgements. this study was supported by ministry of education, science and technologic development of the republic of serbia, project numbers: 172030, iii45012 and iii43009. и з в о д ултразвучна и механичка екстракција у води анјона и катјона из лебдећег пепела марјетка савић бисерчић1, лато пезо2, ивана средовић игњатовић3, љубиша m. игњатовић4, андрија савић1, урош јовановић1 и велибор андрић1 1универзитет у београду, институт за нуклеарне науке винча – лабораторија за хемијску динамику, п.пр. 522, 11001 београд, 2институт за општу и физичку хемију, студентски трг 12–16, 11 000 београд, 3универзитет у београду, пољопривредни факултет, немањина 6, 11080 београд и 4универзитет у београду, факултет за физичку хемију студентски трг 12–16, 11 000 београд представљене су технике екстракција анјона и катјона из лебдећег пепела помоћу дејонизоване воде. извршено је испитивање расподеле величина честица, као и потврда хомогености узорака помоћу рендгенске флуоресцентне спектрометрије. прва екстракциона процедура је изведена помоћу линеарне мућкалице у току 6, 12 и 24 h, а друга помоћу ултразвука у току 15, 30, 45 и 60 min. концентрације анјона (сулфати, хлориди, _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 826 savić biserčić et al. флуориди, фосфати, нитрати и нитрити) из водених екстраката одређиване су јонском хроматографијом, док су метали који су битни за животну средину (al, fe, mn, cd, cr, as, ni, zn, cu и pb) у раствору одређивани применом атомске апсорпционе спектрометрије. поређење класичне и ултразвучне екстракције показује већу ефикасност примене ултразвука за екстракцију анјона као и pb, al, mn и fe, док је за одређивање cr, as, ni и zn класична екстракција ефикаснија. промена ph вредности, величине колоидних честица, зета потенцијала и проводљивости су током ултразвучне екстракције мерене у циљу објашњења интеракције површине честица лебдећег пепела са водом и различитих процеса (адсорпције, јонске измене, флокулације) који се дешавају у природним условима. анализа главних компоненти је коришћена за процену утицаја испитиваних параметара. са аспекта утицаја на животну средину, од посебног значаја је квантитативна анализа испитиваних елемената који процесом испирања доспевају у природне воде. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice effect of an industrial chemical waste on the uptake accepted manuscript this is an early electronic version of an as-received manuscript that has been accepted for publication in the journal of the serbian chemical society but has not yet been subjected to the editing process and publishing procedure applied by the jscs editorial office. please cite this article as: p. vorobyev, t. mikhailovskaya, o. yugay, l. saurambaeva, a. serebryanskaya, n. chukhno, r. kurmakyzy, j. serb. chem. soc. (2017), doi: 10.2298/jsc161220023v this “raw” version of the manuscript is being provided to the authors and readers for their technical service. it must be stressed that the manuscript still has to be subjected to copyediting, typesetting, english grammar and syntax corrections, professional editing and authors’ review of the galley proof before it is published in its final form. please note that during these publishing processes, many errors may emerge which could affect the final content of the manuscript and all legal disclaimers applied according to the policies of the journal. on lin e f irs t on lin e f irs t j. serb. chem. soc. 82 (0) 1–11 (2017) udc jscs–4381 original scientific paper 1 optimization of vanadium-oxide catalyst for the oxidation of 3-methylpyridine into nicotinic acid pavel vorobyev, tatyana mikhailovskaya, olga yugay*, lyudmila saurambaeva, anna serebryanskaya, nikolay chukhno and raya kurmakyzy a.b. bekturov institute of chemical sciences jsc, almaty 050010, kazakhstan (received 20 december 2016, revised 9 february, accepted 13 february 2017) abstract: upon modification of v2o5 with sno2 or zro2, increase in the activity and selectivity of the vanadium-oxide catalyst in the vapor-phase oxidation of 3-methylpyridine into nicotinic acid were observed. it was shown that the promoting effects of sno2 and zro2 were the result of increases under their influence of the proton affinity of the vanadyl oxygen and decreases in the enthalpy of deprotonation of the methyl group of the substrate, connected by a nitrogen atom with the lewis acidic center (vanadium ion). the given characteristics were calculated by the density functional theory quantumchemical method. modification of binary v2o5–sno2 and v2o5–zro2– catalysts by tio2 addition resulted in a further increase in the nucleophility of the vanadyl oxygen and, as a consequence, an increase in the catalytic activity and selectivity for nicotinic acid formation. keywords: quantum-chemical method; oxidation; promoting effect; v2o5; sno2; zro2. introduction nicotinic acid holds an important place among pyridine carboxylic acids,1 based on which a number of medicinal preparations are obtained. nicotinic acid is also used in the production of premixes in cattle breeding. at present nicotinic acid is obtained industrially by a liquid-phase oxidation of 3-methylpyridine by inorganic oxidizers or by hydrolysis of nicotinic acid nitrile, formed upon ammoxidation of 3-methylor 2-methyl-5-ethylpyridines. earlier, a catalyst for ammoxidation of 3-methylpyridine into nicotinonitrile,2 which has been patented in 38 countries, was developed. this catalyst operates at the “lonza” plant in guangzhou (china). the disadvantages of this method are a two-stage process and the formation of harmful liquid wastes and gas emissions, containing ammonia. * corresponding author. e-mail: yu.ok@mail.ru doi: 10.2298/jsc161220023z on lin e f irs t 2 vorobyev et al. a vapor-phase oxidation of methylpyridines by air oxygen on oxide catalysts is the most useful method for obtaining pyridine carboxylic acids. increasing demands for nicotinic acid and its derivatives have promoted the interest of researchers to study the direct vapor-phase oxidation of 3-methylpyridine into nicotinic acid and to search for efficient catalysts for this process.3–6 thus, on a v2o5–tio2–na2o catalyst with a v:ti:na atomic ratio of 100:20:1.15 at 400 °с, the maximum conversion of 3-methylpyridine into nicotinic acid was up to 94 % with a selectivity of 51 %.7 the vapor-phase oxidation of 3-methylpyridine was studied on vanadates of y, bi, co, fe, mn,8 and also cr and al.8,9 it was shown that the largest total yield of nicotinic acid and pyridine-3-carbaldehyde attained 69 % at 350 °с on the cr0.5al0.5vo4 vanadate. this paper considers an experimental study of a number of catalysts, containing v2o5 and sn, zr and ti dioxides in the vapor-phase oxidation of 3methylpyridine. with the purpose of interpretation of the experimental results, a quantum-chemical study of the effect of oxide-modifiers upon the nucleophilic properties of the active oxygen connected with vanadium and the mobility of hydrogen of the methyl substituent of the initial substance chemisorbed on the catalyst surface was performed. the development of a one-stage heterogeneous catalytic process for the oxidation of 3-methylpyridine into nicotinic acid would enable the creation of new, more efficient, ecologically friendly and safe methods for the production of vitally important medicinal preparations. experimental materials the initial 3-methylpyridine after drying and distillation had the characteristics, corresponding to the pure substance: boiling t / 140 °c (692 mm), d4 20 = 0.9566, nd 20 = 1.5050. the catalysts with different contents of v2o5 and oxide-modifiers sno2, tio2 and zro2 were prepared by mixing the initial oxides in the required molar ratio. further, the obtained charge was pressed into tablets and calcined at 650–800 °с in an airflow for 2 h. the cooled catalysts were ground into grains with a size of 3–5 mm and 0.1 l was transferred to the reactor. synthesis experiments the study of vapor-phase oxidation of 3-methylpyridine was performed in a flow-type unit with a metallic reactor of length 1000 mm and diameter 20 mm, simulating an element of an industrial contact machine. the feeding rate of 3-methylpyridine was 36 g per 1 l of catalyst per hour. the molar ratio of the reagents 3-methylpyridine:о2:н2о was 1:14:90. the reaction products were captured in air-lift type scrubbers. analysis of 3-methylpyridine oxidation products the 3-methylpyridine was determined by a chromatographic method with a flameionization detector. the column was filled with sorbent 10 % lucopren g 1000 on chromatone n-aw-hmds (0.20–0.25 mm). the column temperature was 140 °с, the on lin e f irs t optimization of 3-methylpyridine oxidation catalyst 3 vaporator temperature was 230 °с) and helium was the gas carrier. a 0.1 % benzonitrile solution was used as the internal reference. nicotinic acid was determined by koh potentiometric titration using a universal ionomer ph-150mi. the products of deep oxidation were analyzed by a chromatographic method with a thermal conductivity detector. activated carbon «ag-5» (0.250.50 mm) was the adsorbent for the determination of со, polysorb-1 (0.16−0.20 mm) for the determination of со2. the temperature of the column-heating oven was 40 °с. in all experiments, the balance of the determined substances contributed 95−100 % of the total. computing details for quantum-chemical modeling the active sites on the surface of the vanadium-oxide catalysts were simulated by clusters that contain tetrahedral coordinated ions of vanadium. the sizes of the v5+ vanadium cation, as is know from the literature,10 allow both a tetrahedral and octahedral configurations. in this research on the influence of oxide-promoters, an initial v4o10 cluster was used and one or two fragments of oxide-promoter molecules were sequentially added to this v4o10 cluster. while studying the influence of the oxide-promoters on the deprotonation of ch3– groups, clusters with two atoms of vanadium were used to alleviate the calculations. it is considered that minimum surface clusters could be used for chemosorption research due to the strongly localized nature of the v=o and v–o bonds.11,12 optimization of clusters geometry was realized by the dft method (density functional theory)13 using the two-exponential lanl2dz basis set with an effective core potential. for non-empirical calculations of energy and optimization of the geometry of the molecules, the born–oppenheimer approximation was used. this approximation does not consider the movement of the nucleus. for elimination of possible errors in the definition of energy and taking into account of the effects of molecular translation, rotation and vibration, frequency calculations for the clusters with optimized geometry were performed. by default, these calculations were realized at 298.15 k and 1 atmosphere pressure. to obtain a precise value of the total energy of a system at zero degrees kelvin, the zero-point energy (zpe) must be added to the received etot. 14 for comparison to the results of the experiment on the oxidation of 3-methylpyridine, the frequency calculations were performed at a reaction temperature of 573.15 k. the values of the thermal energy correction (etherm.) received from the calculations was added to the total energy to take account of the effect of molecular translation, rotation and vibration motion at the specified temperature. in accordance with the above, the proton affinity of vanadyl oxygen (pav=o) of the investigated clusters (table i) was calculated by formula (1): pav=o = [(eneutral cluster + etherm.neutral cluster) – (echarged cluster + etherm.charged cluster)]∙2625.5(1) and enthalpy of deprotonation (de) of the methyl group of adsorbed substrate (table ii) by formula (2): de=[(eanion/clus.+ etherm.anion/clus.)–(emolecule/clus.+ etherm.molecule/clus.)]∙2625.5 (2) the calculations were realized by the gaussian 09w program, version c.01. results and discussion a comparison of the behaviors of the individual vanadium pentoxide, the binaries v2o5–sno2,v2o5–zro2 and the three-component v2o5–sno2–tio2 and v2o5–zro2–tio2 catalysts in oxidation of 3-methylpyridine is shown in fig. 1. the experimental data testify to the fact that modification of v2o5 by the on lin e f irs t 4 vorobyev et al. above oxides resulted in a considerable increase in the catalyst activity, as could be judged by the significant increase in the conversion of 3-methylpyridine (a) and selectivity to nicotinic acid formation (b), as well as a decrease in the optimal reaction temperature. fig.1. the effect of temperature and composition of the catalyst on the conversion of 3-methylpyridine (a) and yield of nicotinic acid (b) (1 v2o5, 2 v2o5–sno2, 3 v2o5–zro2, 4 v2o5– sno2–tio2, 5 v2o5–zro2–tio2). it is known15 that sno2 may increase the mobility of the v=o bond in the v2o5 lattice, thus contributing to intensification of its dissociation and reduction to vo2. it could be considered that in the vanadium–titanium catalysts with sno2 addition, this function of tin dioxide did not manifest itself because the extent of v2o5 reduction in the three-oxide vanadium–titanium–tin systems was approximately the same, or even less, than that in the two-component vanadium– titanium catalysts without sno2 additions. probably, this is connected with the fact that sno2 may play the role of an oxidizer in relation to low vanadium oxides.16 due to this function of sno2 in the vo2–sno2 system, an oxygen rearrangement is observed: tin dioxide is reduced to sno, giving its oxygen for oxidation of vo2 into v2o5, and the formed sno is easily and rapidly oxidized by air oxygen again to sno2. hence, it follows that sno2 may stabilize the structure of the oxide three-component system v2o5–sno2–tio2, giving its on lin e f irs t optimization of 3-methylpyridine oxidation catalyst 5 oxygen to vanadium and facilitating re-oxidation of the catalyst by gas phase oxygen. the results of the experiments for 3-methylpyridine oxidation on the vanadium–zirconium catalyst are also shown in fig. 1, from which it could be seen that this catalyst was more efficient than v2o5. the promoting effect of zro2 on the oxide-vanadium catalyst manifested itself in a significant increase in the conversion of the initial substance and in the yield of nicotinic acid. on the v2o5–zro2 catalyst at 330 °с, the conversion of 3-methylpyridine and yield of nicotinic acid were 30–35 % higher than that on v2o5 without zro2 addition. this showed that the binary v2o5–zro2 catalyst was more active and selective than the v2o5–sno2 catalyst. the yield of nicotinic acid on the v2o5–zro2 catalyst was 62 % (selectivity of 85 %). an earlier study of the catalytic activity of v2o5–tio2 catalysts in the oxidation of 3-methylpyridines showed that v2о5 was markedly inferior in terms of catalytic activity and selectivity to nicotinic acid formation compared to those of oxide-vanadium catalysts modified by titanium dioxide. it was established that the addition of any quantity of tio2 to v2о5 sharply increased the efficiency of the catalyst activity. thus, on the catalysts containing 18.1 and 77.7 % of tio2, the conversion of the initial pyridine base and selectivity to nicotinic acid formation increased 2–2.5-fold, and the yield of nicotinic acid increased almost five-fold as compared with the v2o5 catalyst. the obtained results testify to the fact that an increase in activity and selectivity of the oxide-vanadium catalyst under the effect of tio2 additions is connected, in our opinion, with a change in the chemical and phase composition of the catalyst during its preparation and a change thereby of reactivity of the v2о5 oxygen. in the process of calcination of the catalysts in the system v2о5– tio2, a continuous series of solid solutions for vо2–tio2 substitution are formed. irrespective of the initial mixture composition, the vо2–tio2 solid solution becomes a second main component of the catalyst alongside with v2о5. tin dioxide seems to weaken the v=o bond in the lattice of vanadium pentoxide and contributes both to an acceleration of the transformation of v2о5 into vо2, and the formation of new active centers on the catalyst surface, strengthening its oxidation ability and selectivity. judging by the increase in activity and selectivity, the concentration of such centers on the surface of the vanadium oxide catalysts modified by tio2 is much higher than that on the surface of the vanadium oxide catalyst in the absence of tio2. non-stoichiometric vanadium oxides v3o7 and v6o13, formed on the surface of the vanadium–titanium catalysts in the process of oxidation reaction under the effect of the reaction medium may serve as active centers of v2o5– tio2 catalysts. their formation was proved earlier by x-ray diffraction and ir spectroscopic measurements in a study of vanadium–titanium catalysts engaged on lin e f irs t 6 vorobyev et al. in the oxidation of 3-methylpyridine.17 hence, it follows that reactivity of vanadium pentoxide, modified by tio2 addition depends on the rate of mutual transfer in the system v2o5  v3o7  v6o13. the solid solution vо2–tio2, which may be considered as the active carrier that changes the reactivity of v2о5, limits the rate in vanadium–titanium catalysts. the change of the mutual transfer rate in this system under the effect of the vо2–tio2 solid solution determines the activity and selectivity of vanadium–titanium catalysts in dependence of the quantitative composition. the above-stated was the basis for the selection of titanium dioxide as an additive to the binary v2o5–sno2 and v2o5–zro2 catalysts. the important value, characterizing the reactivity of oxygen bonded to vanadium, is its nucleophility. the calculated analog of nucleophility is the affinity of the vanadyl oxygen proton (pav=o). using the dft method, pav=o values were obtained for clusters that model the active sites of v2o5 and of the modified v2o5–sno2, v2o5–zro2, v2o5–sno2–tio2 and v2o5–zro2–tio2. optimization of geometry of the clusters (fig. 2) and calculation of the total energy values of the protonated еtot.(bh +) and initial еtot.(b) structures were performed. the pentoxide vanadium model corresponds to the formula v4o10 and is described by the d2h symmetry group. the models containing mo2 = sno2, tio2 and zro2 promoters correspond to the formula mv2o7. the models with the given promoters possess c2v symmetry. upon protonation, the symmetry of all models decreases down to с1. the models of the initial and protonated three-component catalysts possess c1 symmetry. according to quantum-chemical calculations, the simultaneous introduction in the v4o10 cluster of two fragments of oxide-modifiers (sno2 and tio2 or zro2 and tio2) (fig. 2.) results in a considerable increase in the proton affinity of the vanadyl oxygen (table i). it was shown that indeed the proton affinity of the doubly-connected oxygen (pav=o / kj∙ mol –1) increases in the same order as the catalytic activity, i.e., v2o5 < v2o5–sno2 < v2o5–zro2 < v2o5–sno2–tio2 < v2o5–zro2–tio2. results of the calculations are given in table i. based on the regularities of the effect of the oxide-modifiers, revealed by experimental result and quantum-chemical calculations, a catalyst for direct vapor-phase oxidation of 3-methylpyridine into nicotinic acid and a waste-free technology of its preparation were developed.18 the present approach to the interpretation of the experimental results by the relative activity of the studied catalysts in the oxidation of 3-methylpyridine was based on the concepts of organic сн-acids and the theories of adsorption and catalysis on the surface of transient metal oxides. on lin e f irs t optimization of 3-methylpyridine oxidation catalyst 7 fig. 2. clusters modeling active centers of v2o5 (a), v2o5–sno2 (b), v2o5–zro2 (c), v2o5–sno2–tio2 (d) and v2o5–zro2–tio2 (e). on lin e f irs t 8 vorobyev et al. table i. total energy (еtot.) and thermal energy (etherm.) of vanadium-containing clusters and their charged forms (fig. 3), proton affinity of oxygen, bonded to vanadium-ion (pav=o). temperature 573.15 k clusters −еtot. / a.u. etherm. / a.u. pav=o / kj∙mol -1 a: neutral cluster charged cluster 1038.17019 1038.45849 0.08145 0.09380 724.5 b: neutral cluster charged cluster 672.90766 673.20234 0.05559 0.06791 741.3 c: neutral cluster charged cluster 716.26601 716.58135 0.05583 0.06826 795.3 d: neutral cluster charged cluster 1400.87024 1401.18833 0.11778 0.13014 802.7 e: neutral cluster charged cluster 1444.17841 1444.56402 0.11795 0.13034 979.9 as it is known,19 3-methylpyridine is a weak сн-acid (ан) and under the effect of a proton acceptor (в) may release the proton with the formation of the corresponding carbanion (а–). the heat of the reaction of proton transfer from сн-acid to the nucleophilic center of the catalyst surface is determined from the equation: ah + b → a– + bh+ (3) δe = еtot.(a –) + еtot.(bh +) – еtot.(b) where еtot. is the total energy of the corresponding compound. for the same сн-acid (3-methylpyridine), the heat of the proton transfer reaction depends on proton affinity of the base (в), which is determined from the equation: b + h+ → bh+ (4) pa = еtot.(bh +) – еtot. (b) – е(h +) where: pa is the proton affinity and е(h+) is the energy of proton formation. therefore, the еtot.(bh +) – еtot.(b) difference, or proton binding energy, differs from proton affinity to base в by a constant value е(h+), and may serve as a characteristic of the proton acceptor activity (in the present case, the vanadyl oxygen). modeling of the «vertical» adsorption of 3-methylpyridine by a nitrogen atom at a lewis acidic center (vanadium ion) with methyl proton transfer to the vanadyl oxygen was performed (fig. 3). it was established that the enthalpy of on lin e f irs t optimization of 3-methylpyridine oxidation catalyst 9 deprotonation of the chemisorbed substrate (de / kj·mol–1) decreased in the order v2o5 > v2o5–sno2 > v2o5–tio2 (table ii). according to the experimental data, an activity of the catalysts in the oxidation of 3methylpyridine increased in the same order. fig. 3. deprotonation of the methyl group of 3-methylpyridine connected with the lewis acidic center (vanadium ion) of v2o5 (a), v2o5–sno2 (b) and v2o5–tio2 (c). table ii. total energies (еtot.) and thermal energy (etherm.) of 3-methylpyrine and its carbanion, connected with the vanadium pentoxide and binary catalysts and deprotonation enthalpy (de) of the substrate. temperature 573.15 k structure −еtot. / a.u. etherm. / a.u. de / kj∙mol -1 a: molecule/cluster anion/cluster 883.49487 883.40403 0.22557 0.22342 232.9 b: molecule/cluster anion/cluster 960.89283 960.80488 0.21200 0.20965 224.7 c: molecule/cluster 1015.75760 0.21260 on lin e f irs t 10 vorobyev et al. anion/cluster 1015.67325 0.21036 215.6 conclusions the results of the quantum-chemical calculations complied with the experimental data on the promoting effect of additions of sno2, tio2 and zro2 to v2o5 in the oxidation of 3-methylpyridine. according to the calculations, the mechanism of the promoting effect of the studied oxide-modifiers is determined by their ability to increase the nucleophility of the vanadyl oxygen and to decrease the deprotonation enthalpy of the methyl group of the chemisorbed 3methylpyridine. catalyst for direct vapor-phase oxidation of 3-methylpyridine into nicotinic acid and a waste-free technology of its preparation was developed. the catalytic oxidation of 3-methylpyridine on the developed catalyst was tested on a pilot unit. the yield of nicotinic acid attained was 75–77 % (selectivity 90 %). и з в о д оптимизација оксидације 3-метилпиридина у никотинску киселину катализовану ванадијум-оксидом pavel vorobyev, tatyana mikhailovskaya, olga yugay, lyudmila saurambaeva, anna serebryanskaya, nikolay chukhno и raya kurmakyzy a.b. bekturov institute of chemical sciences jsc, almaty 050010, kazakhstan после модификација v2o5 помоћу sno2 и zro2 уочено је повећање селективности у оксидацији 3-метилпиридина у никотинску киселину, у гасовитој фази, помоћу ванадијум-оксида. показано је да услед присуства sno2 и zro2 долази до повећања афинитета према протону атома кисеоника у ванадијум-оксиду, и да је смањивање енталпије депротоновања метил-групе супстрата повезано са везиванјем атома азота супстрата за луисову киселину (ванадијум јон). ове карактеристике израчунате су квантно-хемијским поступцима теорије функционала густине (density functional theory). модификацијама бинарних катализатора v2o5–sno2 и v2o5–zro2 додавањем tio2 повећава се нуклеофилност ванадил кисониковог атомашто има за последицу повећавање каталитичке активности и селективности у синтези никотинск екиселине. (примљено 20. децембра 2016, ревидирано 9. фебруара, прихваћено 13. фебруара 2017) references 1. r. chuck, appl. catal., a 280 (2005) 75 2. d. kh. sembaev, f. а. ivanovskaya, е. м. guseinov, r. d. chuck, lonza ltd., ch; institute of chemical sciences of the national academy of the republic kazakhstan, kz pat. usa 5,698,701 (1997) 3. p. vorobyev, l. saurambaeva, t. mikhailovskaya, o. yugay, a. serebryanskaya, i. shlygina, russ. j. appl. chem. 87 (2014) 887 4. d. f. heinz, w. hoelderich, s. krill, w. boeck, k. huthmachery, j. catal. 192 (2000) 1 5. g. ya. popova, t. v. andrushkevich, i. i. zakharov, yu. a. chesalov, kinet. catal. 46 (2005) 217 6. h. f. huang, b. c. zhu, h. f. lu, h. y. liu, y. f. chen, j. chem. eng. chin. univ. 3 (2004) 334 on lin e f irs t optimization of 3-methylpyridine oxidation catalyst 11 7. s. järås, s. lundin, j. appl. chem. biotechnol. 27 (1977) 499 8. t. shishido, z. song, e. kadowaki, y. wang, k. takehira, appl. catal., a 239 (2003) 287 9. t. shishido, j. jpn. pet. inst. 54 (2011) 225 10. l. e. orgel, discuss. faraday soc. 26 (1958) 138 11. m. witko, r. tokarz, j. haber, j. mol. catal. 66 (1991) 205 12. m. witko, r. tokarz, j. haber, j. mol. catal. 66 (1991) 357 13. w. koсh, m. c. holthausen, chemist’s guide to density functional theory, 2nd ed., wiley–vch, weinheim, germany, 2001 14. j. b. foresman, a. frish, exploring chemistry with electronic structures methods, 2nd ed., gaussian inc., pittsburgh, pa, usa, 1996 15. w. m. h. sachtler, g. j. h. dorgelo, j. fahrenfort, r. j. h. voorhoeve, rec. trav. chim. 89 (1970) 460 16. s. yoshida, t. murakami, k. tarama, bull. inst. chem. res., kyoto univ. 51 (1973) 195 17. d. kh. sembaev, b. v. suvorov, l. i. saurambaeva, kh. t. suleimanov, kinet. katal. 20 750 (1979) (in russian) 18. d. kh. sembaev, t. p. mikhailovskaya, r. kurmakizy, p. b. vorobyev, (a.b. bekturov institute of chemical sciences jsc), kz innov. pat. 30237 (2015) 19. d. j. cram, fundamentals of carbanion chemistry, academic press, new york & london, 1965. on lin e f irs t http://link.springer.com/journal/10975 {the possibility to increase antioxidant activity of celery root during osmotic treatment} j. serb. chem. soc. 82 (3) 253–265 (2017) udc 633.42+532.71:664.151.2:615.279–188 jscs–4963 original scientific paper 253 the possibility of increasing the antioxidant activity of celery root during osmotic treatment milica r. nićetin1*, lato l. pezo2, biljana lj. lončar1, vladimir s. filipović1, danijela z. šuput1, violeta m. knežević1 and jelena s. filipović3 1faculty of technology, university of novi sad, bulevar cara lazara 1, 21000 novi sad, serbia, 2institute of general and physical chemistry, university of beograd, studentski trg 12/v, 11000 beograd, serbia and 3institute for food technology in novi sad, bul. cara lazara 1, 21000 novi sad, serbia (received 20 october, revised 22 december, accepted 30 december 2016) abstract: osmotic treatment of celery root was studied in two osmotic solutions (sugar beet molasses and a ternary solution of water, sucrose and salt), at three temperatures (20, 35 and 50 °c), and three different immersion periods (1, 3 and 5 h), at atmospheric pressure. the aim was to examine the influence of the type of the used hypertonic agent, the temperature and the immersion time on the water loss, solid gain, water activity, dry matter content, antioxidant activity (expressed by dpph) and colour attributes (described by cielab coordinates l*, a* and b*). during the experiments, the antioxidant activity of celery root was increased in sugar beet molasses, while the dpph value tended to decrease in the ternary solution. the experimental data of osmotic dehydration was used for pca modelling. the standard scores analysis revealed that the optimum process parameters were gained with an immersion time of 5 h and a temperature of 35 °c. keywords: antioxidant capacity; celery root; osmotic treatment; sugar beet molasses; optimization. introduction from old times, celery (apium graveolens l.) has been known as a special medicinal herb or spice, due to the presence of many healthful and aromatic substances.1,2 celery is a source of digestible carbohydrates, proteins, and high amount of dietary fibres, and proven rich in bioactive compounds, such as vitamins, free amino acids and minerals.3,4 the main bioactive components in celery responsible for its healing properties are flavonoids (mostly apiin and apigenin), essential oils (α-limonene and selinene, butylphthalide, celerin, apiol and * corresponding author. e-mail: milican@uns.ac.rs doi: 10.2298/jsc161020015n _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 254 nićetin et al. myristicin), organic acids (chlorogenic acid and caffeic acid), bergapten, niacin, inositol, etc.5 presently, there is an increasing interest in phenolic compounds derived from celery, mainly phenolic acids and flavonoids, because of their strong antioxidant properties.6 phenolic substances are known to be the most responsible for the antioxidant activity of celery plants, thereby for their physiological functionalities, such as lowering cholesterol levels, and anti-inflammatory, antimicrobial and anticancer activities.7 current studies have confirmed that celery can lower blood pressure, regulate heart function, as well as the blood glucose level by stimulating the pancreas to insulin secretion, and hence, it could be used to reduce complications caused by diabetes.8 antioxidants in celery also possess the potential to retard lipid oxidation, which is one of the major causes of chemical spoilage of foods, and inhibit various types of oxidizing enzymes. therefore, celery can be added to foods as a preservative, to improve nutritional quality and food safety, and to reduce the need for synthetic food antioxidants.9,10 since celery is highly perishable, with a high moisture content, several drying treatments could be used to extend its shelf life. for certain fruit and vegetable products, traditional drying processes may not be satisfactory, due to texture degradation, colour alteration and nutritional loss.11,12 the application of osmotic treatment (ot) at a mild temperature in food preservation has been widely applied as it presents many advantages as compared to traditional drying treatments.13 the foodstuff is not exposed to high temperatures, the changes in the initial sensory characteristic are minimal, while the nutritional value and the functional properties of the product are kept on the same level, or even improved. furthermore, ot is an environmentally acceptable and energy efficient process due to the low temperature and energy requirements and less waste material.14,15 ot involves soaking a food, mostly fruit or vegetable, in a hypertonic solution to reduce the moisture content of the food, with minimal processing under ambient or modified environmental conditions. the driving force for water removal is the concentration gradient between the surrounding hypertonic solution and the immersed plant material.16,17 the complex cellular structure of plant tissue acts as a semi-permeable membrane, which allows two main counter-current flows: water outflow from the plant tissue into the osmotic solution and the simultaneous migration of solids from the solution to the tissue.18 the leaching out of the own solutes from the tissue can affect the quality of the final product to a lesser extent.19 the choice of the hypertonic solution depends on the expected water loss (wl) and solid gain (sg), and the sensory properties of the final food product.20 a concentrated sucrose solution, sodium chloride solutions and their combinations are usually used as hypertonic solutions.21,22 _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ increasing the antioxidant activity of celery during osmotic treatment 255 recent research showed that sugar beet molasses is a highly effective osmotic medium for the treatment of fruits, vegetables and meat. high dry matter content and high water loss, a specific nutrient composition and nutritive quality of osmo-dehydrated product, and low costs and energy requirements are a few of the main reasons why sugar beet molasses is a useful osmotic solution.17,23 molasses, the thick, dark syrup obtained as a by-product from the processing of sugar beet into sucrose, consists of fermentable carbohydrates (sucrose, glucose and fructose) and several non-sugar organic materials (betaine and other amino acids; minerals, mainly potassium; vitamins, especially of the b-group, etc.).24,25 various studies evidenced that molasses is a rich source of phenolic compounds playing possible roles in the prevention of several chronic diseases involving oxidative stress. maillard browning, carbohydrate−amino acid condensation products, formed during sugar processing are also present in very high concentrations in molasses in the range from low organic compounds to complex aromatic polymers, and they were reported to have antioxidant activities. therefore, molasses has health benefits in the human diet, beyond its special taste and flavour, due to it being rich in minerals and antioxidants.26,27 the colour of any food product, depending upon the nature and content of pigment and coloured substances present in food material, may be represented in terms of the cielab coordinates l*, a* and b* system. the l*, a* and b* values explain a three-dimensional colour space. the l* value is the vertical axis and defines the lightness, and the a* and b* values are perpendicular horizontal axes and define red-to-green and blue-to-yellow, respectively.18,28 the objective of the presented work was to investigate the effects of the type of osmotic solution, the processing time and temperature on the mass transfer phenomena during osmotic treatment of celery root in sugar beet molasses and an aqueous ternary solution. the aim was to determine the water loss (wl), solid gain (sg), water activity (aw), dry matter (dm), antioxidant activity (expressed by dpph) and the colour attributes (described by cielab coordinates l*, a* and b*) as a function of the process variables and to determine the optimum conditions for the osmotic treatment. experimental osmotic treatment sugar beet molasses, obtained from the sugar factory crvenka, serbia, with an initial dry matter content of 85.04 % was diluted to a concentration of 80 % (this solution was marked as s1). the aqueous ternary osmotic solution was made from 1.200 g sucrose and 350 g nacl per kg distilled water. this solution (s2) was diluted with distilled water to a concentration of 60 %. celery root (apium graveolens l. var. rapaceum, alabaster variety) was purchased on a local market in novi sad, serbia, shortly prior to the experiment. prior to acquisition, the samples had been stored in a sales gondola at ambient temperature. the celery root samples were cut into cubes (1×1×1 cm3) using a kitchen knife. after preparation, the samples were measured and immersed in the hypertonic solutions. the sample to solution ratio was 1:5, _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 256 nićetin et al. which can be considered high enough to neglect the influence of solution concentration changes during the process. after each sampling time (1, 3 and 5 h), samples of celery root were removed from the solutions (s1 and s2), lightly washed with distilled water, gently blotted with paper to remove excessive water from the surface and weighed. the dry matter content of the fresh and treated samples was determined by drying the material at 105 °c for 24 h in a heat chamber (instrumentaria sutjeska, croatia). the aw values of the osmotically-treated samples were measured using a testo 650 water activity measurement instrument (testo, lenzkirch, germany) with an accuracy of ±0.001 at 25 °c. preparation of celery root extracts to prepare the extracts for antioxidant analysis, fresh and osmotically dehydrated celery root samples were dried at 50 °c in a heat chamber (instrumentaria sutjeska, croatia) until constant weight. the dried samples were ground to a powder using a universal laboratory mill type wz-1 (spolem, zbpp, bydgoszcz, poland). the powder (2 g) for each sample was extracted with 200 ml of boiled water. after extraction at room temperature for 10 min, obtained aqueous extracts were filtered using whatman no. 1 filter paper. the extracts were stored in a refrigerator (4 °c) until further use. determination of free radical scavenging ability by the dpph radical scavenging assay the antioxidant capacity of celery root during osmotic treatment was determined using the dpph radical scavenging assay,29 with some modifications. briefly, 100 μl of the extract was added to 1.9 ml of 0.094 mm 2,2-diphenyl-1-picrylhydrazyl (dpph) in methanol and made up to 2 ml, followed by vigorous vortexing. the free radical scavenging capacity of the sample was evaluated by measuring the absorbance at 517 nm after 30 min using an evolution 300 uv/vis spectrophotometer (thermo fisher scientific, waltham, ma, usa). the antioxidant capacity is expressed as mmol l-1 trolox equivalents, calculated using a calibration curve of trolox (0–1000 μm), a water-soluble vitamin e analogue. all determinations were performed in triplicate. investigation of the colour attributes colour images of the experimental results were captured by a canon powershot a550 (canon europe ltd., uxbridge, uk), which is a common digital camera for home use. all the acquired images were 24 bit rgb (16.8 million colours) with a 1024×768 spatial resolution. the macro-function of the digital camera was used to cover a scenic area of approximately ø60 mm. samples were placed on a white paper napkin set on a flat white painted surface, inside a closed chamber, 15 cm below the digital camera. paper napkins were used in order to avoid undesired reflection effects from chamber walls. with this setup, it was possible to capture images with negligible shadows and without specula reflections. the acquired images were transferred to a personal computer in the form of jpeg compressed image files. the images were imported to an originally developed computer program used for this investigation, and the colour data for each image were transformed to a three-dimensional array of r (red), g (green) and b (blue) values, ranging from 0–255. the frequency of colour indexes was recorded, and the maximum values were observed. subsequently, these data were transformed to cielab colour coordinates, using a java algorithm for rgb to xyz and xyz to lab colour coordinates.30 _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ increasing the antioxidant activity of celery during osmotic treatment 257 statistical analysis the experimental results are expressed as means and standard deviations (sd) for each treatment. collected data were subjected to anova to explore the effects of the process variables. furthermore, pattern recognition techniques, including pca and ca, were successfully applied to classify and discriminate the different samples. an evaluation of rsm, anova, pca and ca of the obtained results was performed using statistica® software, version 12 (statsoft inc. 2012, usa).31 the experimental data used for the analysis were derived using a box and behnken fractional factorial (3 level–2 parameter) design, 2 blocks, according to the rsm. independent experimental factors for each of the five mixtures are given in table i. table i. independent experimental factors and their levels experimental factor symbol coded factor’s level –1 (low) 0 (centre) +1 (high) time, h x1 1 3 5 temperature, °c x2 20 35 50 the rsm equations describe the effects of the test variables on the observed responses, determine the inter-relationships between the test variable and represent the combined effect of all test variables in the observed responses. the following second order polynomial (sop) model was fitted to the experimental data. eight models of the following form were developed to relate the eight responses (y) and two process variables (x), for each of the different osmotic treatments: 2 2 2 1 20 12 1 1 l l l l l i ik k ki kii k i i y x x x xβ β β β = = = + + +  , k,l ∈ n: k ∈ [1,8], l ∈ [1,2] (1) where: 0 l kβ , lkiβ , lkiiβ , 12lkβ are constant regression coefficients; lky is either wl, sg, aw, dm, dpph, l*, a* or b*, while x1 is time, and x2 is temperature. a model describing osmotic treatment in s1 solution is marked with l = 1, while treatment in s2 is marked with l = 2. determination of normalized standard scores in order to obtain a more complex observation of the ranking of the quality of osmotic -treated celery root, standard scores (ss) are evaluated using a chemometric approach by integrating the measured values generated from different measuring methods. min–max normalization is one of the most widely used technique to compare various characteristics of complex samples determined using multiple measurements, where samples are ranked based on the ratio of raw data and extreme values of the measurement used.27 the evaluation is performed, according to following equations: max 1 , max min i i i i i i ii x x x i x x − = − ∀ − in the case of “the higher, the better” criterion (2) or max , max min i i i i i i ii x x x i x x − = ∀ − in the case of the “the lower, the better” criterion (3) _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 258 nićetin et al. where xi represents the raw data. normalized scores of most of the properties were evaluated using these equations, except for the l*, a* and b* parameters, which were evaluated according to the initial values, as follows: ( )0min ,i i i x x x i= − ∀ (4) for a sample, the sum of the normalized scores of different measurements when averaged give a single unitless value, termed as ssi, which is a specific combination of data from different measuring methods with no unit limitation. this approach also enables the ease of employing some other sets of osmotically treated celery root samples to this elaboration in future comparisons. standard scores were calculated and the results are given in table ii. table ii. experimental design for kinetics investigation, antioxidant activity and colour attributes during osmotic treatment of celery root and standard score analysis solution sugar beet molasses solution ternary solution case 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 time 1 1 1 3 3 3 5 5 5 1 1 1 3 3 3 5 5 5 temp. 20 35 50 20 35 50 20 35 50 20 35 50 20 35 50 20 35 50 ss 0.61 0.65 0.60 0.53 0.57 0.56 0.64 0.77 0.74 0.42 0.51 0.49 0.42 0.41 0.40 0.45 0.47 0.45 results and discussion all analytical measurements were performed in accordance with aoac32 and repeated three times, according to experimental plan described in table ii. the obtained results are presented in fig. 1, which shows that the sugar beet molasses was the better solution for ot, with respect to obtaining higher values of wl and dm, and lower values of sg and aw. the maximum values of wl (0.78 g g–1 i.s.) and dm (63.02 %) were achieved after an immersion time of 5 h and a temperature of 50 °c. in addition, the superiority of sugar beet molasses as an osmotic solution was confirmed from the aspect of retaining and even increasing the antioxidant activity of the treated celery. ot in molasses provoked an increase in the dpph values in all samples of celery root from initially 0.41 to 0.45 mm te l–1. the dpph values for all samples treated in the ternary solution decreased proportionally to the increase in temperature and immersion time (from the initial 0.41 to 0.26 mm te l–1). in comparison with molasses, the use of the ternary solution in the ot of celery root could be considered less convenient, if the comparison is based on the antioxidant activity, as a general indicator of potential health effects. enhancement in dpph values in celery root treated in molasses confirmed that molasses is a rich source of antioxidants. probably, some phenolic compounds from the molasses penetrated into the tissue of the celery during ot. moreover, the results showed a reduction in the l* values and an increase in the a* values for samples treated in molasses, which indicate a darkening because of the diffusion of coloured substances from the molasses into the dehydrated samples during the process. the _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ increasing the antioxidant activity of celery during osmotic treatment 259 penetration of coloured substances from molasses was proportional to the increasing temperature. it was found that the colour cielab parameters significantly correlated with the dpph values. it seems that there is a relationship between the increase in the colour parameters and the antioxidant activity in the samples treated in molasses, and this is probably because some of the pigments in molasses are known for their antioxidant properties. 0 0.2 0.4 0.6 0.8 0 1 2 3 4 5 6 7 8 9 101112131415161718 0.00 0.04 0.08 0.12 0.16 0 1 2 3 4 5 6 7 8 9 101112131415161718 0.74 0.78 0.82 0.86 0.90 0.94 0 1 2 3 4 5 6 7 8 9 101112131415161718 0 10 20 30 40 50 60 70 0 1 2 3 4 5 6 7 8 9 1011 12131415161718 0.0 0.1 0.2 0.3 0.4 0.5 0 1 2 3 4 5 6 7 8 9 101112131415161718 0 10 20 30 40 50 60 70 0 1 2 3 4 5 6 7 8 9 1011 12131415161718 -15 -10 -5 0 5 10 15 20 25 0 1 2 3 4 5 6 7 8 9 101112131415161718 0 5 10 15 20 25 30 35 40 45 0 1 2 3 4 5 6 7 8 9 1011 12131415161718 w l sg a w d m d pp h l* a* b* a b e f d h j gh i k c fg gh i j i j d gh a cd c d e f i cd b g h i j i k m h j l a fgh def h efg def fgh abc ab defg abc cdef bcde ab cde a abcd abc def a b c e c e ij g j m d f h e i k gh k l f g g fg g fg fgg fgfg e d c de c b c b a l f d a e c b e a b hi k k j j l gh i g f p q n n j k m o l c g a d e c b i h e o h a i d c g a b m l m jk f gh ij n k untreated celery root celery root treated in sugar beet molasses celery root treated in ternary solution fig. 1. experimental results of kinetics parameters, antioxidant activity and colour attributes during osmotic treatment of celery root; wl – water loss, sg – solid gain, aw – water activity, dm – dry matter content, l*, a*, b* – colour coordinates. a-q different letters written in superscript within the same column in the table show significantly different means of observed data (at the p < 0.05 level); n = 3. the immersion time in h is given at x-axes. _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 260 nićetin et al. principal component analysis (pca) principal component analysis (pca) is a mathematical procedure used as a central tool in exploratory data analysis.33 pca is a multivariate technique in which the data are transformed into orthogonal components that are linear combinations of the original variables. pca is realized by eigenvalue decomposition of a data correlation matrix.34 this transformation is defined in such a way that the first component has the largest possible variance. this analysis is used to achieve maximum separation among clusters of parameters.27 this approach, evidencing spatial relationship between processing parameters, enabled a differentiation between the different samples in both solutions (s1 and s2). the pca, applied to the given data set, fig. 1, showed a differentiation between the samples according to the observed process parameters and was used as a tool in exploratory data analysis to characterize and differentiate neural network input parameters. as could be seen, there is a neat separation of the observed samples according to used assays. the quality results show that the first two principal components, accounting for 83.14 % of the total variability for solution s1 and s2, could be considered sufficient for data representation. considering the map of the pca performed on the data, sg (which contributed 20.4 % of total variance, based on correlations), dm (12.1 %) and l* (8.8 %) exhibited positive scores according to first principal component, whereas aw (16.6 %) and a* (11.8 %) showed negative score values according to the first principal component (fig. 2). wl (which contributed 23.5 % of total variance, based on correlations) and dm (16.7 %) showed the positive influence towards the second principal component, while a negative impact was observed by the colour coordinates l* (23.1 %) and b* (18.7 %). complete linkage, city-block (manhattan) distances 9 6 5 8 3 7 4 2 1 18 17 15 16 12 14 13 11 10 0 0 20 40 60 80 100 120 cases l in ka ge d is ta nc e fig. 2. tree diagram for the osmotic treatment of celery root in the solution of sugar beet molasses and the ternary solution. _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ increasing the antioxidant activity of celery during osmotic treatment 261 the pca graphics show quite good discrimination between solutions s1 and s2. the influence of processing parameters could be observed in fig. 3, with the samples processed for shorter immersion times and lower temperatures located at the bottom left side of the graphic. samples treated in the sugar beet molasses solution are located at the upper left side of the graphic, showing increased dpph and a* values, while samples treated with the ternary solution showed increased colour attributes l* and b*. furthermore, it is evident that sg is augmented for samples treated in the ternary solution, especially for samples with increased immersion time and temperature. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 -4 -3 -2 -1 0 1 2 3 4 factor 1: 50.51% -4 -3 -2 -1 0 1 2 3 4 wl sg aw dm dpph l* a* b* sugar beet molasses solution ternary solution fresh sample fa ct or 2 : 3 2. 63 % fig. 3. biplot graphic of the osmotic treatment of celery root in the solution of sugar beet molasses and the ternary solution. cluster analysis (ca) the ca dendrogram of for the osmotic treatment of celery root in sugar beet molasses solution and ternary solution is shown in fig. 2. the complete linkage algorithm and city block (manhattan) distances were used as the measure of proximity among the samples. city block distances (shown on the ordinate axis) are measured as the average difference across the dimensions of the observed samples. this distance measure yields results similar to the euclidean distance, but in this measuring technique, the effect of single large differences (outliers) is dampened (since they are not squared). the dendrogram presented in fig. 2 is based on experimental data. the resulting dendrogram showed three main clusters; the first cluster contained samples 3, 5, 6, 8 and 9 (samples treated with the s1 solution, with increased dm), the second cluster included 1, 2, 4 and 7, while the third cluster contained samples treated with the s2 solution. the linkage distance (shown on the ordinate axis) between the main clusters was nearly 120. _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 262 nićetin et al. response surface methodology (rsm) the anova calculation showed the effects of the independent variables on the responses (table iii). the sop models for all variables were found to be statistically significant and the response surfaces were fitted to these models. table iii. the anova calculation for the osmotic treatment of celery root in the solution of sugar beet molasses and the ternary solution; +significant at the p < 0.01 level, ++significant at the p < 0.05 level; **significant at the p < 0.10 level; unmarked terms were not statistically significant; df degrees of freedom; i.s. initial sample parameter df wl g g-1 i.s. sg g g-1 i.s. aw dm % dpph mm te l-1 l* a* b* sol. 1 0.0004 0.0094+ 0.0054+ 100.3+ 0.0679+ 5061.7+ 1183.9+ 317.2+ t 1 0.0894+ 0.0001 0.0008 985.8+ 0.0035+ 91.7** 3.7 74.9** t2 1 0.0004 0.0012+ 0.0008 15.8 0.0000 20.4 69.2** 50.6 temp 1 0.1069+ 0.0021+ 0.0086+ 800.4+ 0.0038+ 67.0 11.2 247.1+ temp2 1 0.0048++ 0.0001 0.0005 1.1 0.0000 15.7 20.9 12.2 sol.×t 1 0.0097+ 0.0000 0.0012** 77.0++ 0.0022+ 4.5 60.4** 56.7 sol.×temp 1 0.0020** 0.0001 0.0000 7.7 0.0022++ 249.4+ 32.8 211.1++ t×temp 1 0.0012 0.0001 0.0000 50.3++ 0.0000 13.5 12.4 46.6 error 9 0.0042 0.0007 0.0025 73.7 0.0002 189.8 150.2 181.5 r2 – 0.981 0.953 0.843 0.965 0.998 0.967 0.903 0.848 linear terms of immersion time and process temperature were the most influential variables for the wl and sg calculations (statistically significant, at p < 0.01 level). the linear term of solution type in the sop model was the most influential factor for sg calculation, as well as for the colour properties (l*, a* and b*). the linear term of solution type was very important for the calculation of aw and dpph, while the linear term of process temperature in the sop model for aw exerted the highest impact. the residual variance is shown in table iii, where the lack of fit represents other contributions of higher order terms. a significant lack of fit generally shows that the model failed to represent the data in the experimental domain at which points were not included in the regression.35 all sop models had an insignificant lack of fit tests, which means that all the models represented the data satisfactorily. the coefficient of determination, r2, is defined as the ratio of the explained variation to the total variation. it is also the proportion of the variability in the response variable that is accounted for by the regression analysis. a high r2 is indicative that the variation was accounted for and that the data fitted satisfactorily to the proposed model. the r2 values for wl (0.981), sg (0.953), aw (0.843), dm (0.965), dpph (0.998), l* (0.967), a* (0.903) and b* (0.848) were very good and show the good fit of experimental results to the model. _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ increasing the antioxidant activity of celery during osmotic treatment 263 standard score analysis ss as the mean value of standard score transformed from the initial data generated with different methods (assays) for each item was calculated according to the following equation: w *+ *+ * 0.2 3 l a b ss wl sg a dpph   = ⋅ + + + +     (2) the maximum value of ss represents the optimal parameters for the processing, and the optimum for the response variables. the graphs of the dependent variables with significant parameters were obtained using an objective function to determine the optimum production conditions, plotted on an optimization graph. if the value of the membership trapezoidal function is close to 1, it shows the tendency of tested processing parameters to be optimal. in this article, standard scores were calculated for various properties and the obtained data are presented in table ii. an ss above 0.60 stands for a high standard. samples with an ss value below 0.60 are attributed with poorer characteristics. using standard score analysis and the determination of the ss of different samples and different processing parameters can be referenced for developing strategies for improving the characteristics of the final product. analysis of the standard scores showed that the optimum characteristics of osmotically dehydrated celery root were realised at a temperature of 35 °c, during 5 h of treatment, with sugar beet molasses as osmotic solution (0.77), while the ss for osmotic treated celery root in ternary solution was quite lower (0.47). generally, sugar beet molasses was a much better solution for osmotic treatment of celery root, according to the ss results and dpph values. conclusions based on the presented results, it could be concluded that both solutions are adequate for effective dehydration, considering the satisfactory losses of water and decrease of the aw values during all experiments. since the quality of osmotically treated celery root is influenced by many parameters that are altered as the technological treatments change, standard score analysis was applied for evaluating the quality, in conjunction with pca and ca. these analyses compiled various properties of the products. similar results were obtained with these analyses, indicating that the osmo-dehydrated celery root samples treated in sugar beet molasses, processed at the optimal processing parameters (temperature 35 °c for 5 h), gained the best score (0.77 of 1.00). despite the fact that molasses proved to be superior as an osmotic solution, the most important finding in this study presents its effect on increasing initial antioxidant activity of celery root. this finding increases the possibility of embedding the molasses as a natural ing_________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 264 nićetin et al. redient in various food formulations, in spite of its unpleasant sensory characteristics. in addition, the use of sugar beet as the osmotic agent is economy and environmentally reasonable, because the molasses is a side product of the sugar industry. it could be concluded that celery root osmotically treated in molasses, with extended shelf life and improved antioxidant properties, is suitable as a food additive or functional food ingredient. as an additive to soups, yogurt, mayonnaise, sauces and other complex systems of food, it has the potential to contribute to an overall improvement of their oxidative stability, nutritional value and taste. likewise, its use as a natural preservative could reduce the need for applying artificial preservatives, additives and antioxidants in food. acknowledgement. these results are part of a project supported by the ministry of education, science and technological development of the republic of serbia, tr-31055, 2011–2015. и з в о д могућност да се осмотским третманом повећа антиоксидативна активност корена целера милица р. нићетин1, лато л. пезо2, биљана љ. лончар1, владимир с. филиповић1, данијела з. шупут1, виолета м. кнежевић1 и јелена с. филиповић3 1технолошки факултет, универзитет у новом саду, булевар цара лазара 1, 21000 нови сад, 2институт за општу и физичку хемију, универзитет у београду, студентски трг 12/v, 11000 београд и 3научни институт за прехрамбене технологије у новом саду, бул. цара лазара 1, 21000 нови сад у овом раду је испитиван осмотски третман корена целера у два осмотска раствора (раствору меласе шећерне репе – s1 и тројном воденом раствору – s2), на три температуре (20, 35 и 50 °c) и три различита периода потапања (1, 3 и 5 h), при атмосферском притиску. циљ рада је био да се покаже утицај врсте хипертоничног раствора, температуре и периода потапања на губитак воде, прираштај суве материје, активност воде (аw), садржај суве материје, антиоксидативну активност (изражену преко dpph) и боју корена целера (описану колорним координатама cielab, l*, а* и b*). током експеримента, антиоксидативна активност корена целера се повећавала у раствору s1, али је вредност dpph имала тенденцију смањивања у раствору s2. анализом стандардне оцене показано је да су оптимални параметри процеса постигнути при времену потапања од 5 h и температури од 35 °c. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice a novel compound isolated from sclerochloa dura has anti-inflammatory effects j. serb. chem. soc. 81 (1) 1–12 (2016) udc *sclerochloa dura:547.295.96:547.426.1+ jscs–4821 547.918:615.276–188 original scientific paper 1 a novel compound isolated from sclerochloa dura has anti-inflammatory effects syed majid bukhari1,2*, astrid jullumstrø feuerherm3**, hanna maja tunset3, stian moe isaksen1, mari sæther3, thor håkon krane thvedt1, susana villa gonzalez1, rudolf schmid1, anders brunsvik4, erik fuglseth5, bojan zlatković6, berit johansen3 and nebojša simić1 1department of chemistry, norwegian university of sciences and technology, 7491 trondheim, norway, 2department of chemistry, comsats institute of information technology, abbottabad-22060, kpk, pakistan, 3department of biology, norwegian university of sciences and technology, 7491 trondheim, norway, 4sintef materials and chemistry, department of mass spectrometry, sem sælands vei 2a, 7465 trondheim, norway, 5technology transfer office, norwegian university of sciences and technology, 7491 trondheim, norway and 6department of biology, university of niš, 18000 niš, serbia (received 19 january, revised 26 june, accepted 28 june 2015) abstract: the activation of phospholipase a2 (pla2) by means of pro-inflammatory cytokines results in the subsequent release of arachidonic acid (aa) and generates eicosanoids, which further propagate inflammation. through cyclooxygenases (cox1/2) responsible for the enzymatic conversion of aa to eicosanoids, non-steroidal anti-inflammatory drugs are effective in relieving the pain and discomfort of inflammation. using the aa release assay as a guide for biological and anti-inflammatory activity, the novel compound 1-o -(3-o-linolenoyl-6-deoxy-6-sulfo-α-d-glucopyranosyl)glycerol (1) together with five known compounds, i.e., isovitexin, byzantionoside b, tricin 4′-o -(erythro-β-guaiacylglyceryl) ether 7-o-β-glucopyranoside, 1-o-feruloylglycerol and tricin 7-glucoside, isolated from the methanol extract of the aerial parts of sclerochloa dura using lc techniques (sephadex lh-20 column chromatography, preparative hplc and semi-preparative hplc), were evaluated. all the isolated compounds were identified using spectroscopic methods, i.e., nmr spectroscopy and ms spectrometry. the novel compound 1 was found to be an effective inhibitor of aa release with an ic50 value of 0.09±0.03 mg ml -1. keywords: arachidonic acid; anti-inflammatory activity; phospholipase a2 inhibitor; sephadex lh-20; hplc; cell line sw982. *,** corresponding authors. e-mail: (*)majid_bukhari@hotmail.com; (**)astrid.j.feuerherm@bio.ntnu.no doi: 10.2298/jsc150119055b 2 bukhari et al. introduction it is generally known that plants produce many chemical compounds, such as alkaloids, flavonoids, proteins, amino acids, bioactive peptides, sugars, biopesticides and others. plants have always been the centre of attention in the history of traditional medicine, because they produce vital and complex molecules, which can be used either directly or in modified form to cure or alleviate a number of diseases. for example, berberine (alkaloid) is well known for its antiinflammatory activity.1 it is present in a number of plants, such as hydrastis canadenis, berberis vulgaris, b. aristata2 and rhizoma coptidis.3 similarly, quercetin is an anti-inflammatory compound that belongs to the class of flavonoids.4 it can be isolated from many plants, for instance helminthostachys zeylanica.5 bromelain (a mixture of sulfur-containing proteases) obtained from the stem of ananas comosus also exhibits anti-inflammatory activity.6 some other classes of compounds showing anti-inflammatory activity are terpenoids, phenols and polyphenols.7 there are many species of plants used as traditional medicines that contain potential candidate compounds for the development of anti-inflammatory drugs.6,8 sclerochloa dura (l.) p. beauv. may be an example of a plant with beneficial medicinal properties; however, its traditional use is not well documented. this plant, known also as common hardgrass or fairground grass, belongs to family poaceae barnhart, which involves more than 700 genera and almost 50,000 species. it is an annual plant, with flat leaves and a procumbent or erect steam up to 16 cm in length.9 the inflorescence is a crowded, one-sided series of flattened spikelets. the plant is a common inhabitant of areas with heavy traffic, e.g. along dirty roads, on play yards and pathways. it is widely spread in the moderate climate zone of almost all continents on the north hemisphere and was also introduced in australia.10 this plant has been used traditionally in small communities in south–east serbia for the treatment of menstrual disorders characterized by excessive bleeding and pain. professor aleksandar igić (medical faculty, university of niš, serbia) in a personal correspondence reported that in this region tea or decocts of s. dura have been used by women having menstrual disorders; according to the statements of subjects, the oral intake of a boiled water extract of the plant results in pain relief and significantly reduces bleeding.9 in a quest to identify the chemical composition and biological properties of s. dura, its crude extract and subsequent fractions were tested on human fibroblastlike synoviocyte cell line sw982 (stimulated with interleukin -1β) to determine its effects on arachidonic acid (aa) release. it was recently shown that the s. dura extract contains one or several compounds with the ability to modulate the activity of phospholipase a2 (pla2) enzymes.11 the pla2 enzymes hydrolyze fatty acids, such as aa, from the sn-2 ester bond of membrane glycerophospholipids. the -6 fatty acid (aa) is a substrate s. dura has anti-inflammatory effects 3 for eicosanoids such as prostaglandins and leukotrienes, which are potent lipid mediators of inflammation.12 the pla2 enzymes are grouped into five main categories, namely, cytosolic pla2s (cpla2), secretory pla2s (spla2), ca2+-independent pla2s (ipla2), platelet-activating factor acetylhydrolases (paf-ah), and lysosomal pla2s.13 apart from their role in normal physiological functions, the different isotypes are implicated in a variety of diseases14 and ever since the cpla2 inhibitor arachidonyl trifluoromethyl ketone (atk, aacocf3) was introduced in 1993,15 there has been great interest in the development of inhibitors that target specific pla2 subtypes.16,17 most chronic non-communicable diseases, such as atherosclerosis, rheumatoid arthritis, diabetes and chronic respiratory diseases, are characterized by a considerable inflammatory component. these are the leading cause of global death. inflammation is an acute response to harmful stimuli that normally abates after the challenge is cleared. in chronic inflammatory diseases, the response is not attenuated and the inflammation in the tissue is not completely resolved. instead, prolonged signaling ensures a persistent inflammatory environment. proinflammatory cytokines, such as tumor necrosis factor (tnf) and interleukin-1β (il-1β), are abundant and activate myriad of intracellular effector enzymes, including pla2 enzymes. the activation of pla2 enzymes results in the subsequent release of aa and the generation of eicosanoids, creating a self-perpetuating process.18–20 several studies using knockout mice or cpla2-iva inhibitors have highlighted the potential for targeting this enzyme in order to quench inflammation and ameliorate disease.21 the presence of compounds in the extracts of s. dura that inhibit aa release and may, hence, exert overall anti-inflammatory properties11 is in accordance to its traditional use to relieve discomfort associated with menstrual disorders. indeed, increased availability of aa was associated with heavy menstrual bleeding.22 an elevation of the levels of prostaglandins, such as pge2 and pgf2, in the endometrium of women with heavy menstrual bleeding compared to women with normal menses was documented.23,24 prostaglandins play an important role in uterus contractions25 and are considered as major factors in primary dysmenorrhea. non-steroidal anti-inflammatory drugs target the cyclooxygenases (cox1/2) responsible for the enzymatic conversion of aa to eicosanoids, and can therefore effectively relieve pain associated with dysmenorrheal.24 the present study covers the process of subfractionating the methanolic extract of s. dura, which lead to the isolation, as well as identification, of the novel compound 1-o-(3-o-linolenoyl-6-deoxy-6-sulfo-α-d-glucopyranosyl)glycerol (1) along with five known compounds, which have not hitherto been reported to show anti-inflammatory effects by affecting the pla2 enzyme activity (fg. 1). all of the identified compounds expressed anti-inflammatory activity by showing modulation of the pla2 activity in an in vitro aa release assay. 4 bukhari et al. experimental general for extraction, a vwr ultrasound cleaner (usa), a büchi rotavapor r-200 (uk), a büchi rotavapor rii (uk) and an isomantle from isopadisomantle (uk) were used. the medium pressure liquid chromatography (mplc) system used was from kronlab gmbh with a prepcon 4.47 data system. the stationary phase used for the mplc was polygoprep 60–50 rp-18 (macherey & nagel). water from a millipore elix 5 system, methanol from fisher scientific (uk) and 2-propanol from vwr (usa) were used for the mobile phase. a sepbox system (fa. sepiatec) was used for the preparative and semi-preparative hplc. the mobile phase components were water obtained from a millipore elix 5 system, methanol from fisher scientific (uk) and acetonitrile from vwr (usa). detection was realized using elsd (sedex 75) and uv (merck, λ = 250 nm) detectors. for preparative hplc, a kromasil c18 column (250 mm×50 mm, 10 µm) was used. for the semi-preparative hplc, a kromasil c18 column (250 mm×16 mm, 10 µm) and a nucleodur c18 isis column (100 mm10 mm, 5 µm) were used. the human fibroblastlike synoviocyte cell line sw982 was purchased from atcc (uk). recombinant human il-1β was purchased from roche (uk); arachidonyl trifluoromethyl ketone (atk, aacocf3) was from enzo life sciences, phosphate-buffered saline solution (pbs) was from oxoid (uk); dulbecco's modified eagle medium (dmem), fetal bovine serum (fbs), fatty acid-free bovine serum albumin (fbsa), gentamicin and l-glutamine were from sigma–aldrich (usa). labeled (5,6,8,9,11,12,14,15-3h)-aa (specific activity, 180–240 ci mmol-1 (6.661012–8.881012 bq)) and the liquid scintillation cocktail optiphase supermix were from nen perkin elmer (usa). deuterated methanol and dimethyl sulfoxide were used to prepare the nmr samples. 1d 1h-nmr and 13c-nmr, as well as standard 2d hmbc, cosy, noesy, hsqc and hsqc-tocsy experiments for structure elucidation of the isolated compounds were run on a bruker avance ii instrument, operating at a proton frequency of 600.18 mhz and with a triple-resonance cryo probe, 5 mm, equipped with a z-gradient. plant material sclerochloa dura (whole plant) was collected in 2009 (june–july) near the gabrovačka reka, niš, serbia. the identification of the plant was performed by bojan zlatković from the department of biology and ecology, university of niš, serbia. a specimen of the identified plant was deposited in the herbarium of the faculty of science and mathematics (hmn) of the university of niš, serbia (voucher number 6922). the plant material was dried for 10 days in the dark, with proper ventilation and at room temperature. the dried plant was kept in a closed plastic bag in a dark at room temperature until extraction. extraction aerial plant parts (1.4 kg) were crushed into smaller pieces and extracted two times with methanol (5.0 l), each time for 24 h at room temperature. the extraction was aided by 90 min of sonication. the obtained extracts were filtered (blue ribbon filter paper from schleicher and schuell) and combined. the volume was reduced to 500 ml on a rotary evaporator and the concentrated solution transferred into separation funnels. extraction with n-hexane (three times, 400 ml) was used for defatting and the separated methanolic phase was dried on rotary evaporator. the weight of the dry methanolic extract was 170 g, giving a yield of 12 %. s. dura has anti-inflammatory effects 5 isolation and identification of bioactive fractions of s. dura isolation was realized using a multistage fractionation approach by liquid chromatography. details of the isolation method are given in the supplementary material to this paper. cell culture and treatment of sw982 cells the human fibroblastlike synoviocyte cell line sw982 is used as a model system to determine pla2 activity by measuring aa release 19,26 sw982 cells are widely used as a model for rheumatoid synovial inflammation and express pla2 and cytokines after inflammatory stimulation by, e.g., il-1β. cells were sub-cultured bi-weekly by routine trypsin detachment and kept in a sub-confluent state. the cells were cultured in dmem supplemented with 10 % fetal bovine serum, 0.1 mg ml-1 gentamicin and 0.3 mg ml-1 l-glutamine, in a humidified carbon dioxide atmosphere at 37 °c. for the aa release experiments, 5×105cells per well were seeded in a 48-well per plate form. at two days post-confluency, the cells were labeled with 3h-aa in serum-free dmem overnight and processed at day three post-confluency to ensure differentiation and synchronization of the cells. the experiments were performed in serum-free dmem in triplicate. untreated cells without inducing agents or plant extract were included for non-stimulated controls; distilled water was added to these for vehicle control. the cpla2α inhibitor atk was included as a positive control of inhibition. 15 following treatments, the cells were routinely observed by microscopy to ensure unaltered cell morphology, integrity and viability. aa release assay the aa release assay determines the amount of aa released from cells under inflammatory conditions. in the presence of inhibiting compound(s), the aa release is reduced, which is taken as evidence that the compound(s) target some level in the arachidonyl cascade. at two days post-confluency, the sw982 cells were serum-starved and labeled overnight with 3h-aa at a concentration of 0.4 µci ml-1 (148108 bq). prior to the addition of s. dura fractions or atk, the cells were washed with pbs containing fatty acid-free bsa at a concentration of 2.0 mg ml-1 to remove unincorporated radioactivity. cells were pre-treated for 1 h or 2 h with various dilutions of fractions of the plant extract and atk, respectively, prior to the addition of 10 ng ml-1 il-1β to induce an inflammatory state (fig. 2a). after 24 h of il-1β stimulation, the supernatants were cleared of detached cells by centrifugation at 13000 rpm for 10 min. the adherent cells were dissolved in 1.0 m naoh in order to measure the amount of labeled aa incorporated into the cells. the activities in the supernatants and cellular fractions were assessed by liquid scintillation counting in ls 6500 multi-purpose scintillation counter (beckman coulter, inc., usa). the results are given as released 3h-aa in the supernatants relative to total 3h-aa incorporated into the cells. the inhibition was calculated as aa release % in inhibited relative to stimulated cells, normalized to the control and subtracted from 100. results and discussion several prefractions of the crude s. dura extract inhibit aa release in a cellular bioactivity test model by means of mplc, the crude extract was separated into 9 prefractions (a-i), which were tested for inhibitory effects in aa-release assay. out of these, e, f and g were found to exert the highest inhibitory effect, as shown in table i. 6 bukhari et al. table i. the inhibition activity of prefractions e, f and g. experiments were performed in triplicate. the inhibition and ic50 values were based on two independent experiments. the ic50 could not be calculated for prefraction g, as a dose-dependent inhibition was not obtained prefraction concentration, mg ml-1 inhibition (mean±sd), % ic50 / mg ml -1 e 0.10 63.1±18.4 0.09 f 0.08 63.5±25.5 0.06 g 0.10 68.3±4.8 – prefraction e was temporarily given low priority for further investigation, because its screening by hplc-dad indicated the presence of mostly kaempferol and its derivatives (data not shown). kaempferol compounds are already known by their anti-inflammatory activity27 and therefore, attention was turned to prefractions f and g, as potential sources of new anti-inflammatory compounds. by the bioactivity-guided study, from prefractions f and g, six compounds with pronounced anti-inflammatory activity were identified (fig. 1). elucidation of the structure of the inhibitory compounds of s. dura the isolated novel compound was identified as glycerol derivative (1-o-(3o-linolenoyl-6-deoxy-6-sulfo-α-d-glucopyranosyl)glycerol). further, five additional isolated compounds from the methanolic extract of s. dura with strong anti-inflammatory activity in the aa release assay included one more glycerol derivative (1-o-feruloylglycerol), three flavonoids (isovitexin, tricin 4′-o-(erythro-β-guaiacylglyceryl) ether 7-o-β-glucopyranoside and tricin 7-glucoside) and one glucoside (byzantionoside b). compound 1. compound 1 (fig. 1, 38.8 mg) was isolated from the combined fractions g50 and g51 using semi-preparative hplc (table s-i of the supplementary material). the analysis of spectral data revealed that compound 1 was a novel compound. hrms indicated to a molecular formula c27h46o11s (esi–) with a molecular mass of 578.2751 (theoretical value 578.2761). the analysis of the 1d and 2d nmr spectra with homoand hetero-nuclear direct or long range correlations allowed structure elucidation and a complete 1h and 13c assignment, as given in the supplementary material to this paper. the carbons 1–6 belonging to glucose moiety had shift values for 13c ranging from  54.4 to 100.3 ppm and for protons from  2.92 to 4.77 ppm. the shift values of both 13c and protons at c6 indicated a direct attachment to an so3h group, which was also confirmed by ms analysis. the 18 c-atoms with shift values ranging from  14.8 to 176.2 ppm with attached protons from  0.98 to 5.37 ppm belong to the linolenoyl chain of the molecule. an hmbc correlation peak of h3 (at  3.65 ppm) of glucose and carbon c1″ ( 176.2 ppm) of the linolenoyl chain revealed the attachment position. the glycerol moiety exhibited shift values for carbons ranging from  64.4 s. dura has anti-inflammatory effects 7 to 72.7 ppm and protons from  3.36 to 4.05 ppm. the correlation peaks between carbon c1 of glucose and protons ha,b1′ of glycerol in hmbc unambiguously revealed the connectivity between glucose and glycerol. the coupling constant of the h1 doublet of glucose referred to an axial position of the glycerol moiety. both the molecular formula and nmr data indicated that 1 was 1-o-(3-o-linolenoyl-6-deoxy-6-sulfo-α-d-glucopyranosyl)glycerol (fig. 1). fig. 1. the structures of the identified compounds from methanolic extract of s. dura. compound 1: 1-o-(3-o-linolenoyl-6-deoxy-6-sulfo-α-d-glucopyranosyl)glycerol; compound 2: isovitexin; compound 3: tricin 4′-o-(erythro-β-guaiacylglyceryl) ether 7-o-β-glucopyranoside; compound 4: byzantionoside b; compound 5: 1-o-feruloylglycerol; compound 6: tricin 7-glucoside. 8 bukhari et al. compound 2. compound 2 (14.3 mg) was identified as isovitexin (fig. 1). it was isolated as the main compound in fraction f07 obtained in the preparative hplc separation of prefraction f (table s-ii of the supplementary material). hrms (esi+) was 432.1055, which corresponded to molecular formula c21h20o10 (theoretical value 432.1056). the signal assignments for carbons and protons can be seen in supplementary material. the nmr data were in agreement with those already published.28 compound 3. compound 3 (17.0 mg), fig. 1, the main compound in fraction f51, was obtained by preparative hplc separation of the prefraction f (table s-ii). it was identified as tricin 4′-o-(erythro-β-guaiacylglyceryl) ether 7-o-β -glucopyranoside. the found value of hrms (esi+) was 688.2003 (theoretical value 688.2003), which corresponded to the molecular formula c33h36o16. the nmr data of the compound (supplementary material) fit the published ones.29 compound 4. semi-preparative hplc separation of groups f40–f44 (table s-iii of the supplementary material to this paper) yielded 34.3 mg of compound 4 (fig. 1). the hrms (esi+) value for 4 was 372.2147 compared to the theoretical value 372.2148, which matches the molecular formula c19h32o7. compound 4 was identified as byzantionoside b. the nmr data (supplementary material) were in agreement with those published previously.30 compound 5. the semi-preparative hplc separation of groups f70–f74 (table s-iii) gave 39.6 mg of compound 5 (fig. 1). it was identified as 1-o-feruloylglycerol. the hrms (esi+) value was found to be 268.0952 (corresponding to the molecular formula c13h16o6), with the theoretical value 268.0947. the nmr resonances of 1h and 13c for 5 (supplementary material) were in agreement with those reported previously.31 compound 6. a total of 15.5 mg of compound 6 (fig. 1) was obtained as the main compound of fraction f-32, by preparative hplc separation of prefraction f (table s-ii of the supplementary material). it was identified as tricin 7-glucoside (fig. 1). the theoretical ms (esi+) value for c23h24o12 is 492.1268 and 492.1263 was found. the nmr data of 6 (table s-iv of the supplementary material) corresponded to those reported previously.32 comparison of bioactivity of the identified compounds from s. dura the respective fractions containing each of these compounds were again tested for their bioactivity to confirm inhibitory effects on the release of arachidonic acid, thus implying that the pla2 enzyme activity is affected. the identified compounds from s. dura inhibited the il-1β-induced release of aa in sw982 cells in the following order of decreasing efficiency: 5>4>1>3>6>2. based on the observed unchanged morphology (data not shown), all the compounds were well tolerated by the sw982 cells, including the novel compound 1. to characterize more closely the inhibitor potency, a series of repeated s. dura has anti-inflammatory effects 9 dose-response experiments were performed, which led to an estimated ic50 of 0.09 mg ml-1 (table ii and fig. 2b). table ii. comparison of the activity of compounds 1–6 based on experiments performed in triplicate compound concentration, mg ml-1 inhibition, % (mean ± s.d.) 1 0.15 92.8±46.52 2 0.30 26.8±35.60 3 0.15 67.3±7.10 4 0.12 96.9a 5 0.09 99.5a 6 0.30 54.9±41.40 abased on one experiment due to limited amounts of compound 0 1 2 3 4 5 6 7 f ol d ch an ge a a re le as e ctrl il-1 il-1 +atk/ m il-1+ cpd 1/ mg ml-1 105 0.3 0.15 0.08 0.04 (a) (b) fig. 2. a) compound 1 inhibits cellular aa release in a dose-dependent manner following 24 h stimulation with il-1β (10 ng ml-1). the cpla2α specific inhibitor atk is included for reference. b) estimated ic50 for compound 1 is 0.090.03 mg ml-1 (dotted lines). the results shown are means  sd of experiments 2–5 performed in triplicate. * p<0.05, ** p<0.001 vs. il-1β alone (no inhibition). as the molecular weight of compound 1 is 578.2751 g mol–1, the mole percentage of 1 in the active fraction was estimated to be 60 %, based on the integral ratio in the 1h-nmr spectrum. hence, the estimated ic50 of 0.090.03 mg ml–1 corresponds to 114.1±31.1 µm. as expected, the cpla2α specific inhibitor atk is far more potent, with reported ic50 values of 2 µm and 8 µm in inhibiting aa release from platelets and monocytes, respectively. taken together, these results indicate that compound 1 may have potential as a novel anti-inflammatory compound, interfering with and normalizing the available levels of pro-inflammatory arachidonic acid. 10 bukhari et al. conclusions in this study, one novel compound together with five known compounds was isolated and identified from the methanolic extract of the aerial parts of sclerochloa dura. all isolated compounds had the ability to inhibit the pla2 activity as assessed by their ability to inhibit cellular aa release and, therefore, could be considered as lead compounds for the development of potent anti-inflammatory drugs. to the best of our knowledge, this is the first report that these compounds may inhibit of pla2 activity at some level, a finding that requires further investigation. it is still unclear if the compounds affect kinases or other effectors upstream of pla2, if they inhibit pla2 enzymes, or if the compounds affect the receptors at the cell surface. to help clarify such issues, work is now in progress to establish the synthesis of the novel compound and its derivatives. supplementary material details concerning the isolation and identification of the bioactive fractions of s. dura and the assignments of the 1hand 13c-nmr resonances of compounds 1–6 are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. funding of this research was provided by hec pakistan via siu norway, discovery-ntnu/tto and medtech trondheim as foundation. the experimental work was realized at the institute of chemistry and department of biology, nt faculty, norwegian university of science and technology, trondheim, norway. technical assistance was provided by dr asma zaidi. и з в о д нова једињења изолована из sclerochloa dura имају антиинфламаторно дејство syed majid bukhari1,2, astrid jullumstrø feuerherm3, hanna maja tunset3, stian moe isaksen1, mari sæther3, thor håkon krane thvedt1, susana villa gonzalez1, rudolf schmid1, anders brunsvik4, erik fuglseth5, бојан златковић6, berit johansen3 и небојша симић1 1 department of chemistry, norwegian university of sciences and technology, 7491 trondheim, norway, 2 department of chemistry, comsats institute of information technology, abbottabad-22060, kpk, pakistan, 3 department of biology, norwegian university of sciences and technology, 7491 trondheim, norway, 4 sintef materials and chemistry, department of mass spectrometry, sem sælands vei 2a, 7465 trondheim, norway, 5 technology transfer office, norwegian university of sciences and technology, 7491 trondheim, norway и 6 департман за биологију и екологију, природно–математички факултет, универзитет у нишу, ниш активирање pla2 посредством проинфламаторних цитокина узрокује ослобађање арахидонске киселине (аа) и стварање еикозаноида који изазивају упалу. нестероидни лекови против упала, који блокирају циклооксигеназу (cox1/2) одговорну за ензимску трансформацију аа у еикозаноиде, успешно делују против болова и тескоба узрокованих упалама. коришћењем теста којим се ослобађа аа као путоказа за утврђивањем биолошке и антиинфламаторне активности, изоловано је, из метанолног екстракта надземних делова sclerochloa dura, ново једињење 1-o-(3-o-линоленоил-6-деокси6-сулфо-α-d-глукопиранозил)-глицерол (1), заједно са још пет познатих једињења: изовитексин, бизантинозид б, трицин-4-o-(еритро-β-вајацилглицерил)-етар-7-o-β -глукопиранозид, 1-o-ферулоил-глицероли трицин-7-глукозид, која су окарактерисана s. dura has anti-inflammatory effects 11 на исти начин. једињења су изолована хроматографским техникама (sephadex lh-20 хроматографија на колони, hplc, препаративни и семипрепаративни hplc) и окарактерисан спектроскопским техникама (нмр спектроскопија и масена спектрометрија). инхибиторна активност ослобађања аа новог једињења 1 има ic50 = 0,090,03 mg ml -1. (примљено 19. јануара, ревидирано 26. јуна, прихваћено 28. јуна 2015) references 1. c.-l. kuo, c.-w. chi, t.-y. liu, cancer lett. 203 (2004) 127 2. w.-h. chueh, j.-y. lin, j. agric. food chem. 59 (2011) 8021 3. n. li, l. gu, l. qu, j. gong, q. li, w. zhu, j. li, eur. j. pharm. sci. 40 (2010) 1 4. g. f. anhê, m. m. okamoto, a. kinote, c. sollon, c. lellis-santos, f. f. anhê, g. a. lima, s. m. hirabara, l, a. velloso, s. bordin, u. f. machado, eur. j. pharmacol. 689 (2012) 285 5. y.-c. huang, t.-l. hwang, c.-s. chang, y.-l. yang, c.-n. shen, w.-y. liao, s.-c. chen, c.-c. liaw, j. nat. prod. 72 (2009) 1273 6. j. m. wallace, integr. cancer ther. 1 (2002) 7 7. w.-c. lin, j.-y. lin, j. agric. food chem. 59 (2011) 184 8. g. r. d. m. lima, c. d. a. montenegro, c. l. f. d. almeida, f. d. athayde-filho, j. m. barbosa-filho, l. m. batista, int. j. mol. sci. 12 (2011) 2692 9. c. a. stace, sclerochloa beauv, in flora europaea, 2nd ed., t. g. tutin, v. h. heywood, n. a. burges, d. a. moore, d. h. 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food. chem. 119 (2010) 630 29. m. bouaziz, n. c. veitch, r. j. grayer, m. s. j. simmonds, m. damak, phytochemistry 60 (2002) 515 30. k. matsunami, h. otsuka, y. takeda, chem. pharm. bull. 58 (2010) 438 31. j. luo, l. li, l. kong, food. chem. 131 (2012) 1056 32. a. s. awaad, n. h. mohamed, d. j. maitland, g. a. soliman, rec. nat. prod. 2 (2008) 76. {densities and viscosities for binary mixtures of n-heptane with alcohols at different temperatures} j. serb. chem. soc. 82 (7–8) 891–903 (2017) udc 547.217.1+547.42/.43:532.13/.14 jscs–5010 :544.032.4 original scientific paper 891 densities and viscosities for binary mixtures of n-heptane with alcohols at different temperatures maria magdalena budeanu* and vasile dumitrescu chemistry department, petroleum and gas university of ploiesti, ploiesti, romania (received 24 november 2016, revised 23 march, accepted 28 april 2017) abstract: densities (ρ) and viscosities (η) of the binary systems n-heptane with alcohols (ethanol, propan-1-ol and propan-2-ol) were measured at temperatures between 288.15 and 308.15 k and at atmospheric pressure, over the whole composition range. the excess values of molar volume (ve) and viscosity (ηe) were calculated from experimental measurements. the excess functions of the binary systems were fitted to redlich–kister equation. comparison between experimental excess molar volume and the one calculated from flory and prigogine–flory–patterson theories, has also been done. the viscosity results were fitted to the equations of grunberg–nissan, heric–brewer, jouyban– –acree and mcallister. also, the activation energies of viscous flow have been obtained and their variations with compositions have been discussed. keywords: n-heptane; ethanol; propan-1-ol; propan-2-ol; flory theory; prigogine–flory–patterson theory. introduction experimental liquid densities and viscosities of pure hydrocarbons and their mixtures are useful in the design and the simulation of processes. the physicochemical properties play an important role in the understanding of several industrial processes.1 therefore, experimental measurements are needed to understand the fundamental behaviour of these properties and then to develop new models.2 the obtained properties (excess molar volumes and excess viscosities) in combinations with other mixing properties provide valuable information for the qualitative analysis of the interactions between molecules.3 in this work, the densities and viscosities of three binary systems of n-heptane + ethanol or + propan-1-ol or propan-2-ol were measured at temperatures between 288.15 and 308.15 k, over the entire composition range and at atmospheric pressure. the investigations into the literature have shown that these systems have been studied, but not quite in the same conditions. * corresponding author. e-mail: m19magda@yahoo.com https://doi.org/10.2298/jsc151210051b (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 892 budeanu and dumitrescu jimenez et al.4 reported the values of viscosities and densities for propan-1 -ol + n-heptane at 293.15, 298.15, 303.15 and 308.15 k. keller et al.5 investigated the excess molar volumes of propan-1-ol + n-heptane at 298.15 k and tanaka et al.6 studied excess molar volumes of propan-2-ol + n-heptane at the temperature 298.15 k. pereiro et al.7 reported densities for ethanol + n-heptane at 293.15, 298.15, 303.15 k and orge et al.8 studied densities and viscosities for ethanol + n-heptane and propan-1-ol + n-heptane at 298.15 k. papaioannou et al.9 presented densities for n-heptane + ethanol at 298.15 k and zeberg-mikkelsen et al.10 investigated dynamic viscosities for ethanol + n-heptane at 293.15, 313.15, 333.15 and 353.15 k. zielkiewicz11 reported excess volumes of heptane + propan-2-ol at 313.15 k and rajendran12 studied density and viscosity of binary liquid mixtures of n-heptane with propan-2-ol at 298.15 k. from our experimental data, the excess molar volumes and the excess viscosities were calculated and correlated by the redlich–kister equation. the experimental data of excess volumes of the binary mixtures were used to test to applicability of flory13–16 and prigogine–flory–patterson17,18 theories, which has not previously been employed for the study of excess molar volume of binary mixtures of ethanol, propan-1-ol and propan-2-ol with n-heptane. the thermodynamic functions of activation of viscous flow have been estimated from the experimental data. the purpose of this paper is also to test five semiempirical equations with one to four parameters to correlate viscosity of binary mixtures. experimental materials n-heptane (mole fraction purity > 0.990) was supplied by sigma–aldrich, propan-1-ol (mole fraction purity > 0.999) was obtained from merck, ethanol (mole fraction purity > 0.993) and propan-2-ol (mole fraction purity > 0.997) were supplied by chemical company. the chemicals were dried over molecular sieves (fluka type 4 å). the purity was checked through chromatographic analysis. samples were determined by weighing at atmospheric pressure and ambient temperature using a adventurer pro av 264cm balance with an uncertainty of and precision of ±10-4 g. the error in the final mole fraction is estimated to be less than ±0.0001. conversion to molar quantities was based on the relative atomic mass table of 2006 issued by iupac.19 measurements the densities were determined by the hydrostatic weighing method of kohlrausch20 with the precision of ±0.05 kg m-3. the estimated uncertainty for density was ±0.15 kg m-3. the experimental technique has been previously described,21 an ultra-thermostat type u10 (freital) has been used to maintain a constant temperature (±0.05 k). the uncertainty in the excess molar volume was estimated to be ±5.5×10-8 m3.mol-1. viscosities of the pure compounds and of the binary mixtures were determined with an ubbelohde kinematic viscometer,22 viscosity measuring unit viscoclock (schott-gerate gmbh), that was kept in a vertical position in a water thermostat. a thermostatically controlled bath (u10 constant to ±0.05 k) was used. the kinematic viscosity was calculated using the relation (1): (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ densities and viscosities of n-heptane + alcohols binary mixtures 893 ν = at – b/t (1) where t, in s, is the flowing time of a constant volume liquid through the viscometer capillary. accuracy of time measurement for the viscosity measuring unit viscoclock is ±0.01 s. a and b are characteristic constants of the used viscometer, which were determined by taking bidistilled water and benzene (merck, mole fraction purity > 0.995) as the calibrating liquids for correction of kinetic energy deviations. the dynamic viscosity was determined from the eq. (2): η = νρ (2) where ρ is the density of the liquid. the precision of the viscosity was estimated to be ±0.001 mpa.s. in all determinations, triplicate experiments were performed at each composition and temperature, and the arithmetic mean was taken for the calculations of the viscosity. the uncertainty in the excess viscosity is estimated to be ±4×10-3 mpa.s. results and discussion the measured densities and viscosities of the pure component liquids present good agreement with the literature values, as seen in table s-i of the supplementary material to this paper. densities and viscosities of the binary mixtures of n-heptane + ethanol, n-heptane + propan-1-ol and n-heptane + propan-2-ol are reported in table s-ii of the supplementary material. the results of this study are in close agreement with the values reported in the literature. n-heptane and ethanol densities values reported in the literature differ from our experimental data with a maximum of 0.09 %. for propan-1-ol, densities values published in the literature differ by less than 0.08 % and for propan-2-ol differ by less than 0.2 %. viscosity values reported in the literature differ than our results with a maximum 0.8 % for n-heptane, with maximum 2 % for ethanol, with maximum 1.5 % for propan-1-ol and with 0.6 % for propan-2-ol. the excess molar volumes were calculated from the densities of the pure liquids and their mixtures using the following eq. (3): ve = (xm1+(1–x)m2)/ρ – (xm1/ρ1+(1–x)m2/ρ2) (3) where x is the mole fraction of a component 1, m1 and m2 are the molar masses of the components 1 and 2, and ρ, ρ1 and ρ2 are the respective densities of the solution and of the pure components. the experimental values of the dynamic viscosity are used to calculate the excess viscosity (ηe) defined by the eq. (4): ηe = η – (xη1+(1–x)η2) (4) where η1 and η2 are the viscosities of pure components. the results of ve and ηe are reported in tables s-iii and s-iv of the supplementary material. the values of the excess molar volumes and the excess viscosities for the investigated binary mixtures in this work at 298.15 k show good agreement with the literature data.4,8 (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 894 budeanu and dumitrescu for the solution n-heptane + ethanol with x ≈ 0.4, the deviation between calculated excess molar volume value and literature value8 is 1.8 %, while for excess viscosity the corresponding deviation is 0.4 %. for the solution n-heptane + propan-1-olwith x ≈ 0.5, the excess molar volume value reported in the literature4 differs from our experimental data with 2.2 % and the excess viscosity value differs with 0.7 %. the deviation between our excess molar volume value and the literature12 value for the solution n-heptane + propan-2-ol with x ≈ 0.5 is 10 % and for the excess viscosity is 5.0 %. these differences may be due to different purity of the components used and the differences in concentrations of solutions. figures s-1–s-6 of the supplementary material present comparisons of our experimental excess molar volume and excess viscosity of n-heptane + ethanol, n-heptane + propan-1-ol and n-heptane + propan-2-ol at 298.15 k with data available in the literature. the excess functions of the binary systems can be represented by a redlich– –kister type equation: ( )3e k0 2 1 k i j ikx x x a x== − (5) where xe represents any of the following properties: ve, ηe and xi and xj are the mole fractions of the components i and j, respectively, and k is the number of the polynomial coefficient, ak. the values of these coefficients are indicated in tables s-v and s-vi along with the standard deviation, σ, defined by relation: ( ) ( ) 1/22 exp calc /x x m kσ  = − −    (6) where xexp is the experimental excess function, xcalc is the calculated excess function with eq. (5), m is the number of data points and k is the number of the polynomial coefficient. several effects may contribute to the values of excess molar volumes, such as breaking of liquid order on mixing, unfavourable interactions between groups, differences in molecular volumes and differences in free volumes between liquids components.23 fig. 1 shows experimental (eq. (3)) and calculated values (eq. (5)) of ve at 298.15 k constant temperature. the experimental excess molar volumes for all the binary mixtures studied in this work are positive in the whole composition range and at all temperatures. the positive values are due to the expansion of solution volume due to the mixing caused by the hydrogen bond rupture and the dispersive interactions between unlike molecules. in this sense, the addition of non-polar solvent causes the disruption of alcohol aggregates through the breaking of hydrogen bonds, making (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ densities and viscosities of n-heptane + alcohols binary mixtures 895 ve positive, since aggregates have higher volumes than the sum of their components.24 0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.1 0.2 0.3 0.4 10 6 v e / m 3 m ol -1 x fig. 1. experimental excess molar volumes versus n-heptane mole fraction, x, at 298.15 k for the mixtures: ■) n-heptane + ethanol; ▲) n-heptane + propan-1-ol; •) n-heptane + propan-2-ol. continuous curve was calculated from eq. (5) for experimental data. the curves for n-heptane + propan-1-ol and n-heptane + propan-2-ol systems are asymmetric, with its maximum shift toward a high mole fraction of n-heptane; this behaviour agrees with the literature.25–27 the excess molar volumes decreases with the increasing alkyl chain length of the alcohol. the excess molar volumes of n-heptane + propan-2-ol solutions is greater than the excess molar volumes of n-heptane + propan-1-ol mixtures. these behaviour is in accordance with the results of ortega et al.28 that showed that ve values are greater when alkanes are mixed with secondary or tertiary alcohols than when they are mixed with primary alcohols. breaking the hydrogen bonds by adding a non-polar solvent is more pronounced for propan-2-ol compared with propan-1-ol. this may explain the higher values of the excess molar volumes in propan-2-ol solutions than in propan-1-ol solutions. the effect of temperature on the ve shows a systematic decrease with temperature for all the mixtures. the excess viscosities values may be generally explained by considering the difference in size and shape of the component molecules and specific interactions between unlike molecules, such as h-bond formation and charge transfer complexes. these interactions may cause an increase in viscosity in mixtures, rather than in a pure component.29 positive values of excess viscosities indicate strong interactions, while negative values indicate weaker interactions.30 fig. 2 shows experimental (eq. (4)) and calculated values (eq. (5)) of ηe at 303.15 k constant temperature. (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 896 budeanu and dumitrescu 0.0 0.2 0.4 0.6 0.8 1.0 -0.60 -0.45 -0.30 -0.15 0.00 ηe / m pa s x fig. 2. experimental excess viscosities versus n-heptane mole fraction, x, at 303.15 k for the mixtures: ■) n-heptane + ethanol; ▲) n-heptane + propan-1-ol; •) n-heptane + propan-2-ol. continuous curve was calculated from eq. (5) for experimental data. the excess viscosities are negative over the entire range of mole fractions at all the temperatures. the negative excess viscosity was explained by many authors through different forms.31–34 the negative values support the main factor of breaking of the self-associated alcohols and weak interactions between unlike molecules.35 the minimum of ηe–x curves occurs at low mole fractions of n-heptane. this behaviour is characteristic of binary mixtures formed by a component that presents a molecular association and the other component does not have this characteristic.4 if the temperature increases the negative values of excess viscosity decrease. this can be explained by breaking the hydrogen bonds and increasing mobility of molecules due to the increase of thermal energy.36 flory and prigogine–flory–patterson (pfp) theories experimental results of the molar excess volume have been used to test the applicability of the flory13–16 and pfp theories.17,18 in the flory theory, the excess molar volume is related to the reduced volume, v~ , the reduced temperature, t ~ , and the adjustable interactional parameter χ12. the pfp theory includes three contributions in order to explain the thermodynamic behaviour of the liquid mixtures: an interactional contribution ( eintv ), which is proportional to the interactional parameter χ12, the free volume contribution ( efvv ), which arises from the dependence of the reduced volume, upon the reduced temperature, as a result of the difference between the degree of expansion of the two components and the internal pressure contribution ( * e p v ), (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ densities and viscosities of n-heptane + alcohols binary mixtures 897 which depends on both the difference of characteristic pressures and the reduced volumes of the components. the excess molar volume given by the flory theory is: ( ) ( ) ( ) ( ) ( ) 7/3* * 0 1 1 2 2 1 1 2 2e 1/3 1 1 2 24 / 3 x v x v t t v φ ν φ ν φ ν φ ν + + − = − +      (7) the pfp equation is: ( ) ( ) ( ) ( ) ( ) ( ) ( ) * 1/3 2/3e 12 1 2* * *1/3 1 1 2 2 1 2 1/3 1 2 1 21/3 * * e1 2 1 2 int 1 2* * * * 1 2 2 1 1 1 2 2 e e fv * * * * 1 1 2 2 1 1 2 2 1 x 4 / 3 1 14 / 9 1 4 / 3 1 x x x p v v x v p p p v p p v x v vv v x v v x v ν ν χ ψ θ ν ν ν ν ψ ψ ν ν ν ν ψ ψ ψ ψ − − − − = −  + −    − −  − +  −   − − = + + + + + + +           (8) where ~v is the reduced volume: ( ) 3 1 3 1 t t α ν α   = +  +   (9) v* – characteristic volume; p * – characteristic pressure; t – reduced temperature: * * 1 1 1 2 2 2 * * 1 1 2 2 1 2 12 p t p t t p p φ φ φ φ φ θ χ + = + −    (10) 0t – “ideal” reduced temperature for the mixture; ψi – contact energy fraction: ( )* * *1 2 1 1 1 1 2 21 p p pψ ψ φ φ φ= − = + (11) φi – hard core volume fraction: ( )* * *2 1 2 2 1 1 2 21 x v x v x vφ φ= − = + (12) θi – surface site fraction: ( )*1/3 *1/3 *1/32 1 2 2 1 1 2 21 v v vθ θ φ φ φ= − = + (13) (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 898 budeanu and dumitrescu the coefficient of thermal expansion, α, was obtained from the following equation:37 α = (1/v)(∂v/∂t)p (14) where v is the molar volume of the solution; t – temperature. the various parameters involved in eqs. (7) and (8) for the pure components and the mixture are obtained from flory’s theory13,14 and are shown in tables s-vii and s-viii of the supplementary material. the values of the interaction parameter χ12, were derived by fitting the theory to experimental values of the excess molar volume for equimolar solutions. the results of ve calculated from eqs. (7) and (8) are reported in table s-ix of the supplementary material. these results were compared by means of the percentage absolute average deviation (add), with the following equation: exp calc exp1 100 n i q q add n q= − =  (15) where n is the number of experimental data and q represents ve. the results in table s-ix reveal that add values in the predictions of the excess molar volume for n-heptane + ethanol mixtures are less than 0.9 % using the flory theory and less than 1.2 % using pfp theory. for n-heptane + propan-1 -ol system the add value is 0.7 % using the flory theory and less than 2.2 % using pfp theory. for n-heptane + propan-2-ol system the add values are less than 0.7 % for flory theory and less than 1.4 % using pfp theory. these low values of percentage absolute average deviation obtained, suggest the validity of these theories. an analysis of each of the three contributions to excess molar volume shows that the interaction contribution is positive, being the most important contribution. the volume contribution is positive, while the internal pressure contribution is negative. the viscosity data correlation several empirical and semiempirical relations have been used to represent the dependence of viscosity on the concentration of components in binary systems. these relations are classified according to the number of the adjustable parameters used to account for the deviation from some average.38,39 grunberg-nissan40 proposed the following equation based on one parameter: lnη = x1lnη1+x2lnη2+x1x2d (16) heric-brewer41 expression with two parameters is: lnη = x1lnη1+x2lnη2+x1lnm1+x2lnm2-ln(x1m1+x2m2)+x1x2[α12+α21(x1-x2)] (17) the three-body mcallister42 equation with two parameters is: (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ densities and viscosities of n-heptane + alcohols binary mixtures 899 [ ] ( ) ( ) [ ] 3 2 2 3 1 1 1 2 12 1 2 21 2 2 2 1 2 2 1 1 2 2 1 2 3 1 2 2 1 2 2 1 ln ln 3 ln 3 ln ln ln / 3 ln 2 / / 3 3 ln 1 2 / / 3 ln / x x x x x x x x m m x x m m x x m m x m m η η η η η= + + + −  − + + + +   + + +  (18) we tested also the four-body mcallister equation with three parameters: [ ] ( ) ( ) ( ) [ ] 4 3 2 2 3 1 1 1 2 1112 1 2 1122 1 2 2221 4 3 2 2 1 2 2 1 1 2 2 1 2 2 3 1 2 2 1 1 2 2 1 4 2 2 1 ln ln 4 ln 6 ln 4 ln ln ln / 4 ln 3 / / 4 6 ln 1 / / 2 4 ln 1 3 / / 4 ln / x x x x x x x x x x m m x x m m x x m m x x m m x m m η η η η η η = + + + +  − + + + +     + + + +    + (19) and jouyban acree43,44 equation with four parameters: ( )( )1 1 2 2 1 2 1 20ln ln ln /n jjjx x x x a x x tη η η == + + − , n = 3 (20) in the eqs. (16)–(20) the dynamic viscosity of the liquid mixture is designated by η and η1 and η2 are the dynamic viscosities, x1 and x2 are the mole fractions of the pure components 1 and 2 constituting the liquid mixture, m1 and m2 are the molecular masses, t is the temperature; d, α12, α21 and aj are interaction parameters (viscosity coefficients) and reflect the non-ideality of the system and the mcallister model adjustable parameters are given by η12, η21, η1112, η1122 and η2221. the parameters that appear in eqs. (16)–(20) were estimated using the experimental viscosity data and a non-linear regression analysis employing the levenberg–marquardt algorithm.45 table s-x of the supplementary material shows the parameters calculated, the standard deviations (σ) and the add values between experimental values and those obtained using the semiempirical relations. the standard deviation was calculated using the eq. (6), were xexp is the experimental viscosity, xcalc is the calculated viscosity with eqs. (16)–(20) and k is the number of adjustable parameters. the add values were determined for all equations with the eq. (15), were q represents the values of viscosity. the interactional parameter, d, is negative for binary systems. nigam and mahl46 concluded from the study of binary mixtures that if ηe < 0, d < 0 and magnitude of both are large then the dispersion force would be dominant. applying the model with one parameter (grunberg–nissan model) gives the highest values of standard deviation. models with two parameters (heric–brewer and three body mcallister model) lead to the identical result of standard deviation and their application leads to a poor estimate of viscosities solutions. these models are not suitable to estimate viscosities of the systems studied in this work. (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 900 budeanu and dumitrescu use of three and four parameter equations reduces the σ values. the data show that the jouyban–acree model is suitable for 288.15, 298.15 and 303.15 k, while the four body mcallister model is adequate at 293.15 and 308.15 k for n-heptane and ethanol system. the jouyban–acree model shows the best agreement with experimental data for n-heptane + propan-1-ol system at all temperatures. a comparison of the calculated and the experimental viscosities for the n-heptane + propan-2-ol mixtures shows that the jouyban–acree model produces accurate results for 288.15, 293.15, 303.15 and 308.15 k, while for 298.15 k the four -body mcallister model is able to describe viscosities. it can be concluded from this study that the correlating ability significantly improves for these non-ideal systems, as the number of adjustable parameters is increased. thermodynamic functions of activation the energies of activation of viscous flow for the binaries studied were calculated using the following eq. (21):47 exp hn g v rt η ≠ δ =       (21) where η is viscosity of a liquid mixtures, h is planck’s constant, n is avogadro’s number, v is the molar volume of the solution, r is universal gas constant, t is temperature and δg≠ is gibbs energy change of activation for viscous flow process. the combination yields the equation: ln v h s hn rt r η ≠ ≠δ δ  = −    (22) where δh≠ and δs≠ are enthalpy and entropy changes of activation of viscous flow. the plots of ln(ηv/hn) vs. 1/t were found to be linear in the temperature range 288.15 to 308.15 k, as such the values of δh≠ and δs≠ were obtained by the corresponding slopes and the intercepts. with δh≠ and δs≠ values as input in eq. (22), the corresponding values of δg≠ were also calculated. the values of thermodynamic functions of activation of viscous flow and of correlation coefficient (r) are listed in table s-xi of the supplementary material as a function of composition. the experimental values of δg≠ against mole fraction are presented in figs. s-7–s-9 of the supplementary material. the values of δh≠ are more than two times higher for ethanol and propan-1 -ol than for n-heptane, while for propan-2-ol the values are more than three times higher than that for n-heptane, indicating that the association and the dipole– –dipole interactions increase the value of δh≠. the value of δs≠ is negative for n-heptane and positive for ethanol, propan -1-ol and propan-2-ol and shows that the overall molecular order increases for n-heptane (non-associating component) due to activated complex formation, but (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ densities and viscosities of n-heptane + alcohols binary mixtures 901 decreases in the case of ethanol, propan-1-ol and propan-2-ol. the positive δs≠ for all alcohols indicated that probably the rupturing of hydrogen bonds formed through oh groups of alcohols in the activation process for viscous flow occurs, resulting in the structural disorder.48 the values of δh≠ and δg≠ are positive for all the binary mixtures and at all temperatures used. these values of gibbs energy and enthalpy change of activation of viscous flow increase with the alcohol concentration of solution at constant temperature. the values of δg ≠ at constant concentration decrease if the temperature increases, in accord with the viscosity variation, except for the mixtures concentrated in alcohols. the values of δs ≠ are negative for all binary mixtures except for the mixtures concentrated in alcohols. these values show that overall molecular order due to activated complex formation increases for all binary mixtures except for the mixtures concentrated in alcohols.49 the excess gibbs energy change of activation of viscous flow, δg≠e, were obtained from following equation: δg≠e = rt[lnηv – (xlnη1v1+(1–x)lnη2v2)) (23) where v is the molar volume of the mixture and v1 and v2 are the respective molar volumes of the pure components. the δg≠e parameter may be considered as a valid measure to detect the presence of an interaction between molecules.50,51 negative values were observed for the excess gibbs energy change of activation of viscous flow of the all the binary mixtures and at all temperatures studied. these values lead us to conclude that dispersion forces are the predominant in these systems. the experimental values of δg≠e against mole fraction are presented in figs. s-10–s-12 of the supplementary material. conclusions the densities and viscosities of binary mixtures of n-heptane with alcohols (ethanol, propan-1-ol and propan-2-ol) were measured experimentally at temperatures between 288.15 and 308.15 k over the entire composition range. from these results, the excess values of molar volume and viscosity have been determined and fitted into the redlich–kister equation. positive deviations were observed for the excess molar volumes and negative deviations for the excess dynamic viscosity, at all of the temperatures and in the whole concentration range. the experimental results of the molar excess volume have been used to test the applicability of the flory and prigogine–flory–patterson theories. the values of the percentage absolute average deviation obtained suggest the validity of these theories. grunberg–nissan, heric–brewer, jouyban–acree, three-body and four -body mcalister models have been used to calculate viscosity coefficients and these were compared with the experimental data for the mixtures. the results of these correlations showed that jouyban–acree and four-body mcallister models are suitable to describe viscosities of the binary mixtures studied in this work. (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 902 budeanu and dumitrescu the energies of activation of viscous flow and the excess molar gibbs energies of activation of viscous for these binary mixtures were also calculated. the values of δh≠ and δg≠ are positive for all the binary mixtures at all of the temperatures and in the whole concentration range. the values of δs≠ are negative for all binary mixtures, except for the mixtures concentrated in alcohols. the negative deviations were observed for δg≠e of the all systems and at all of the temperatures. и з в о д густине и вискозности бинарних смеша n-хептана са алкохолима на различитим температурама maria magdalena budeanu и vasile dumitrescu chemistry department, petroleum and gas university of ploiesti, ploiesti, romania густине (ρ) и вискозности (η) бинарних система n-хептана са алкохолима (етанол, пропан-1-ол и пропан-2-ол) су измерене на температурама између 288,15 и 308,15 k и на атмосферском притиску. при мерењу је покривен цео концентрациони опсег испитиваних смеша. допунске вредности моларне запремине (ve) и вискозности (ηe) израчунате су из експерименталних података. допунске функције бинарних система корелисане су redlich–kister једначином. урађено је поређење између експерименталних допунских функција бинарних система и израчунатих помоћу flory и prigogine–flory– –patterson теорија. експериментални резултати вискозности корелисани су помоћу једначина grunberg–nissan, heric–brewer, jouyban–acree и mcallister. такође, израчунате су активационе енергије вискозног тока бинарних система и дискутован је утицај састава смеше на њих. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice formic acid oxidation at platinum–bismuth catalysts j. serb. chem. soc. 80 (10) 1217–1249 (2015) udc 547.291+542.943:546.92’87+544.478 jscs–4792 review 1217 review formic acid oxidation at platinum–bismuth catalysts• ksenija đ. popović*# and jelena d. lović# ictm – institute of electrochemistry, university of belgrade, njegoševa 12, p. o. box 473, 11000 belgrade, serbia (received 18 march, revised 24 april, accepted 5 may 2015) abstract: the field of heterogeneous catalysis, specifically catalysis on bimetallic surfaces, has seen many advances over the past few decades. bimetallic catalysts, which often show electronic and chemical properties that are distinct from those of their parent metals, offer the opportunity to obtain new catalysts with enhanced selectivity, activity, and stability. the oxidation of formic acid is of permanent interest as a model reaction for the mechanistic understanding of the electro-oxidation of small organic molecules and because of its technical relevance for fuel cell applications. platinum is one of the most commonly used catalysts for this reaction, despite the fact that it shows a few significant disadvantages, such as high cost and extreme susceptibility to poisoning by co. to solve these problems, several approaches have been used, but generally, they all consist in the modification of platinum with a second element. especially, bismuth has received significant attention as a pt modifier. according to the results presented in this review dealing with the effects influencing formic acid oxidation, it was found that two types of pt–bi bimetallic catalysts (bulk and low loading deposits on gc) showed superior catalytic activity in terms of lower onset potentials and oxidation current densities, as well as exceptional stability compared to pt. the findings in this report are important for an understanding of the mechanism of formic acid electro-oxidation on the bulk alloy and decorated surface, for the development of advanced anode catalysts for direct formic acid fuel cells, as well as for the synthesis of novel low-loading bimetallic catalysts. the use of bimetallic compounds as anode catalysts is an effective solution to overcoming the problems of current stability in the oxidation of formic acid during long-term applications. in the future, the tolerance of both co poisoning and electrochemical leaching should be considered as the key factors in the development of electrocatalysts for anodic reactions. keywords: formic acid oxidation; pt–bi catalysts; alloy; metal clusters; fuel cell anode catalysts. • in memory to dr rade m. stevanović, our friend and colleague. * corresponding author. e-mail: ksenija@tmf.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc150318044p _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1218 popović and lović contents 1. introduction 2. bulk catalysts for electro-oxidation of formic acid 2.1. activity of bulk catalyst 2.1.1. platinum electrodes 2.1.2. effect of bi adatoms 2.1.3. ptbi ordered intermetallic compounds 2.1.4. ptbi alloys 2.2. stability of bulk catalysts 3. low-loading pt–bi catalysts 3.1. activity of low-loading catalysts 3.1.1. pt/gc catalyst 3.1.2. pt@bi/gc clusters 3.2. stability of low-loading catalysts 3.3. pt(bi)/gc shell–core catalyst 4. conclusions 1. introduction for many years, much attention has been focused on low-temperature fuel cells with energy conversion based on the electrocatalytic oxidation of small organic molecules, such as methanol, ethanol and formic acid. the fuel cell industry has focused the majority of its research on the development of cost-effective, reactive, and durable catalysts. electrochemical oxidation of formic acid is under comprehensive investigation for two main reasons: formic acid can be used as a fuel in direct formic acid fuel cell (dfafc) and can serve as a model reaction that provides a simplified example of the oxidation of more complex organic molecules that can also be used for this purpose.1 a direct formic acid–oxygen fuel cell with a polymer electrolyte membrane (pem) has some advantages over a direct methanol fuel cell. oxidation of formic acid commences at a lower positive potential than methanol oxidation and the crossover of formic acid through the polymer membrane is lower than that of methanol.2,3 moreover, formic acid is a relatively benign and non-explosive fuel, which makes it facile in handling and distribution, as compared to hydrogen. on the other hand, it has a lower energy content with respect to hydrogen or methanol. recent data showed, however, that formic acid fuel cells are attractive alternatives for small portable fuel cell applications.4,5 electrochemical oxidation of formic acid has been widely studied on different metal electrodes. among them, platinum was shown to exhibit the highest catalytic activity of all the pure metals. this reaction has been investigated at platinum since the early work of breiter6 and the results were reviewed by par_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ formic acid oxidation at pt–bi catalysts 1219 sons and van der noot1, and jarvi and stuve.7 many results have been reported concerning the electrocatalytic oxidation of formic acid from the fundamental viewpoint8–12 in contrast to the limited information on the properties of formic acid as a fuel. however, in the last few years, this reaction has attracted more attention.13,14 as already mentioned above, formic acid oxidation on platinum is considered as a model reaction in electrocatalysis, because the oxidation of formic acid is a simple structure-sensitive process. in spite of the apparent simplicity of the process, that is, the oxidation only requires the elimination of two hydrogen atoms in the form of protons and the transfer of the two corresponding electrons, the oxidation mechanism has a double pathway. thus, the oxidation of formic acid on pt electrodes follows the dual path mechanism introduced by capon and parsons,15,16 involving a direct path (dehydrogenation) and an indirect path (dehydratation), both generating co2 as the final reaction product. this finding was later confirmed by differential electrochemical mass spectrometry (dems) measurements.3 both routes are structure sensitive and there is a clear dependence of the reactivity on the surface structure, as experiments with single crystal electrodes revealed:17–21 in the direct path, formic acid is oxidized through a reactive intermediate to co2, while in the indirect path, formic acid is first dehydrated to the adsorbed co intermediate, as a poisoning species that hinders the direct reaction path, followed by oxidation of the adsorbed co by oh formed at higher potentials. the direct path that proceeds through an active intermediate is the simplest one. formic acid adsorbs on the surface, probably transferring one electron, to form an active intermediate, and then this intermediate is oxidized to co2. regarding the nature of the active intermediate, the question is not yet fully resolved.22–24 adsorbed formate (hcoo), rather than the formic acid fragment (cooh), was proposed as the reactive intermediate23,25–27 and this assumption was confirmed by direct surface-enhanced infrared absorption spectroscopy (seiras).28 in the indirect pathway, the first step is a dehydration step with the loss of an oxygen atom to form adsorbed co, which was detected by ir spectroscopy.10,29 co adsorbs strongly on the surface, and for this reason, the route is also known as the poisoning route. thus, adsorbed co blocks the active sites on the surface and prevents the reaction from proceeding. accordingly, the catalytic performance of pt is significantly reduced at low potentials due to co poisoning. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1220 popović and lović however, besides being a poisoning species, co may act as a reactive intermediate, whereby some fraction of coads can be oxidized with ohads to produce co2.12 nevertheless, platinum is an unavoidable material and considered as one of the most efficient catalysts for the oxidation of small organic molecules but, on the other hand, has several significant disadvantages: high cost and extreme susceptibility to poisoning due to strongly adsorbed intermediates, which are formed during the oxidation processes.30 taking this into account, the ideal electrocatalysts would be one that accelerates the direct route and prevents the formation of co. it is now well known that these requirements are fulfilled by bimetallic catalysts, which often show electronic and chemical properties that are distinct from those of their constituent metals and offer the chance to obtain new catalysts with enhanced selectivity, activity and stability. several approaches have been taken to achieve these goals, but in general, they consist of the modification of platinum with a second element. this modification is usually realized by alloying or by modification of the pt surface with adsorbed foreign metals in an amount less than a full monolayer.31,32 the presence of a foreign metal alters the properties of pt in the bimetallic surfaces.12,33 the effects of these atoms can be classified in three main categories: electronic, bifunctional and third body effects. the first one, which involves ligand and strain effects, relates to a change in the electronic properties of the catalytically active material. the bifunctional effect is present when the second metal becomes the source of the oxygen required for the oxidation of the fuel. the third body effect implies a change in the distribution of the active adsorption sites due to dilution of the catalytically active material. generally, more than one of these factors controls the enhanced properties of bimetallic surfaces making separation of the individual contributions difficult. in order to improve pt electrocatalytic activity towards hcooh oxidation and tolerance to co, addition of metals such as ru, pb, os, li, pd, fe, bi etc.12,34–39 were applied. especially, bismuth has received significant attention as a pt-modifier,18,31,32,40,41 and different systems, such as ptbi intermetallics,42–46 ptbi alloys,39,47,48 electrochemically co-deposited carbon supported ptbi (ptbi/c),49 or pt modified by bi either by underpotential deposition (upd) or irreversible adsorption50,51 were proposed as good catalysts for formic acid oxidation. besides the many methods for the synthesis of bimetallic catalysts, a new method for the preparation of noble metal coatings was recently proposed. this procedure includes the replacement of the surface layer of a less precious metal (ru, cu, pb and ti) with a more noble metal (pt and pd) by spontaneous electroless exchange upon immersion into a complex solution of pt or pd ions.52,53 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ formic acid oxidation at pt–bi catalysts 1221 an additional approach for the formation of low dimensional systems is electrodeposition of mono or multilayer metals on different substrates.54–56 this concept of bimetallic mono and multilayer catalysts has received much attention regarding its possibility to reduce the noble metal quantity and maintain the activity by replacing the under-layer (bulk of the catalyst) with a less noble metal. moreover, unlike other bimetallic catalysts where the second metal is either in the form of an adatom or as a component of a surface alloy, this type of catalyst allows the study of the electronic effect of the second metal under-layer on the noble catalyst over-layer, as the only operating factor. in addition, from a practical point of view, long-term stability of the investigated catalysts for formic acid oxidation is very important. therefore, it is necessary to determine which of the factors mostly affected the improvement of the formic acid oxidation rate and the stability of the catalyst. in this paper, recent advances in hcooh oxidation research are presented with focus on the progresses that have been made on pt–bi catalysts for the possible use in dfafcs. different preparation methods were employed to adapt the properties of pt. each method will be emphasized for its advantage and discussed in terms of its limitations, based on the physicochemical and electrochemical characterizations of the catalysts in order to explain the mechanism of action of bismuth added to platinum, the importance of surface composition and surface morphology for the reaction of formic acid oxidation. 2. bulk catalysts for electro-oxidation of formic acid 2.1. activity of bulk catalysts 2.1.1. platinum electrodes platinum, the most studied catalyst for formic acid oxidation, is very susceptible to poisoning species, which significantly reduces its catalytic performance at low potentials, as is well known from the literature.12 traditional single crystals represent an ideal model surfaces for the oxidation reaction of small organic molecule and are suitable for surface characterization methods both in situ and ex situ. studies on single crystal pt samples showed that formic acid oxidation is a strongly structure sensitive reaction. the most complete study on formic acid structure sensitivity was realized by the motoo group,57 using a complete series of stepped surfaces around the stereographic triangle. the studies of this reaction on stepped pt surfaces were performed in order to clarify how exactly the step density influences this reaction, i.e. higher index or stepped surfaces were used to verify the active site assumption, whether low coordination sites are particularly active. the less poisoned surface was pt(111), as indicated by the low observed hysteresis, but the activity toward formic acid oxidation was low. it should be noted that the presence of defects, especially on (100) steps, considerably increased the activity of a pt(111) electrode. for example, in order _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1222 popović and lović to achieve higher rates at moderate poisoning, electrodes having 5–6 atoms wide (111) terraces were the best under the experimental conditions used. the oxidation of formic acid is a structure sensitive reaction, which implies the existence of adsorption steps in the process. in an electrochemical environment, the adsorption processes have to be considered as complex steps since they always involve the competitive adsorption of anions, water and/or hydrogen, which can have different dependences on concentration, and affects the relative rates of dissociative adsorption in a complex way.21 these kind of studies are relevant not only from a fundamental point of view but also from a practical one, because in practical applications, the stepped surfaces may be considered as models for surface defects always present on polycrystalline electrodes. remembering that the cyclic voltammogram for an as-prepared polycrystalline pt electrode (fig. 1a) is described by a region of hydrogen adsorption/ /desorption (e < 0.05 v vs. sce), separated by a double layer from the region of surface oxide formation (e > 0.45 v vs. sce). the absence of well-developed peaks at an as-prepared pt polycrystalline electrode in the hydrogen adsorption/ /desorption region is caused by the employed preparation procedure. the activity of pt electrode towards formic acid oxidation is given in fig. 2a. the cyclic voltammogram shows a well-established feature for formic acid oxidation.7 in the forward scan, the current slowly increases reaching a plateau at ≈0.25 v vs. sce followed by an ascending current starting at 0.5 v vs. sce, which attains a maximum at ≈0.62 v vs. sce. such behavior could be explained considering the dual path mechanism, i.e., dehydrogenation assigned as the direct path, based on the oxidation of formate,28 and dehydration, indirect path, assumes the formation of coads, both generate co2 as the final reaction product. at low potentials, hcooh oxidizes through the direct path with the simultaneous formation of coads. increasing coverage with coads reduces the pt sites available for the direct path and current slowly increases reaching a plateau. subsequent formation of oxygen-containing species on pt enables the oxidative removal of coads, more pt sites become available for hcooh oxidation and current increases until pt oxide, inactive for hcooh oxidation, is formed, which results in a current peak at ≈0.62 v vs. sce. in the backward scan, the sharp increase in the hcooh oxidation current coincides with the reduction of pt oxide. the currents are much higher than in the forward sweep, because the pt surface is freed of coads. 2.1.2. effect of bi adatoms the addition of foreign metals to pt surfaces in amounts less than a full monolayer results in modified surface catalytic properties. therefore, surfaces of bimetallic electrodes often show improved electrocatalytic behavior. it was reported in earlier studies that bi modification of platinum electrodes could exceptionally increase their reactivity toward hcooh oxidation, depending on the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ formic acid oxidation at pt–bi catalysts 1223 fig. 1. basic voltammograms for pt and pt/biirr (a), ptbi alloy (b) and pt2bi (c) bulk electrodes in 0.1 m h2so4. scan rate: 50 mv s-1. ω = 1500 rpm. t = 295 k. bi surface coverage.40,47 most current studies exploring structure/property/activity relationships based on experimental approaches either use well-defined single crystals as a model or are based on modified noble single crystal surfaces. feliu and co-workers58,59 as well as abruna and co-workers60,61 reported that bismuth-modified platinum low and high-index single crystal surfaces, which were prepared via the under-potential deposition (upd) process, exhibited extraordinary enhancement in reactivity towards formic acid oxidation. irreversibly adsorbed bi40,47,51,62 inhibits poison formation simultaneously enhancing dehydrogenation,51 i.e., this modification is an efficient way to hinder the dehydration path (co-intermediate pathway) in favor of the direct path.63 this increased selectivity for dehydrogenation was proposed to be an “ensemble effect”64,65 in which the adsorbed bi divides the pt surface into small domains _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1224 popović and lović where only dehydrogenation can occur. a correlation between ensemble size and formic acid oxidation activity was also established.66 according to literature data, the activity of pt catalysts modified with bi depends on the shape of the pt nanocrystals,67 and varies with the size of the particles68 and the loading of the pt catalyst.63 fig. 2. cyclic voltammograms for the oxidation of 0.125 m hcooh in 0.1 m h2so4 solution on: a) pt, b) pt/biirr, c) ptbi and d) pt2bi catalysts. inset: magnification of the onset potential region. scan rate: 50 mv s-1. ω = 1500 rpm. t = 295 k. the beneficial effect of bi on pt for this reaction could be due to changes in the pt–pt distance that favor the direct route in formic acid oxidation,69 or to the formation of surface bi oxides that participate in the oxidation of intermediates,39,70 or to electronic effects by lowering the electron density of the 5d orbitals, resulting in a considerable decrease of the co binding strength to pt,33,71 or to the ensemble effect creating an appropriate size of pt domains and thereby providing direct oxidation of hcooh to co2.48,64 depending on the preparation of the catalysts and their resulting surface composition, the contribution of the above effects may vary. in the research performed by our group,72 the oxidation of formic acid was studied on polycrystalline pt modified by irreversibly adsorbed bi (pt/biirr) (fig. 2b). the experiments were realized without bi cations in solution, as _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ formic acid oxidation at pt–bi catalysts 1225 opposed to modification by an upd metal, thereby avoiding competition between adsorption/reaction steps of the reactant and modifier. the results were contrasted to pure pt (fig. 2a). modification of the pt electrode was performed at the open circuit potential, as described elsewhere.65 after modification, the electrode was rinsed with water and transferred into a cell containing supporting electrolyte. the fraction of the sites covered by bi (denoted pt/biirr;θ) was estimated from the decrease in the charge for desorption of hydrogen, due to the fact that hydrogen does not adsorb on bi,73 i.e., only the pt sites not blocked by bi were available for hydrogen adsorption. in order to avoid bi dissolution, the anodic potential limit was set at 0.5 v vs. sce, since it was established that the initial amount of bi was almost completely retained when the upper potential limit was fixed below 0.75 v vs. rhe.74 this finding is in agreement with that reported using electrochemical quartz crystal microbalance (eqcm) analysis of the bi oxidation mechanism on smooth pt electrodes.75 the activities of pt/biirr and pt electrodes towards formic acid oxidation are compared in fig. 2a and b and the results showed that the onset potential for the reaction on the pt/biirr (θbi ≈ 0.3) was about 0.1 v less positive than on the pt electrode. the current reached a peak that corresponded to the oxidation of hcooh to co2 via the direct path, occurring on pt sites that were not blocked by the poisoning coads species. on the descending part of the curve, a shoulder appeared at almost the same potential as the peak on the curve for the pure pt electrode, which arises from the hcooh oxidation on the sites being freed by coads oxidation. thus, hcooh oxidation on pt/biirr;θ ≈0.3 proceeded predominantly by the dehydrogenation path with some minor degree of dehydratation also occurring. hcooh oxidation was also tested on a pt/biirr electrode with larger coverage by bi (θbi ≈ 0.5). bell-shaped voltammogram clearly suggests that oxidation of hcooh on this electrode proceeds through dehydrogenation path. it is noticeable that the dehydration path is completely suppressed. the increased selectivity toward the dehydrogenation path on pt/biirr compared to pt was mainly the result of an ensemble effect caused by bi reducing the continuous pt sites necessary for dehydration. nevertheless, the ensemble effect on the pt/biirr catalyst was enabled by adsorbed bi, which practically had no influence on the neighboring free pt atoms. 2.1.3. ptbi ordered intermetallic compounds recently, abruna and co-workers42,43,45,69 studied the electro-oxidation of formic acid on the surface of ordered intermetallic compounds ptbi and ptbi2. the choice of ptbi was based on extensive earlier studies in which the enhanced activity of pt surfaces modified with irreversibly adsorbed bi adlayers toward the oxidation of formic acid was established.40,60–62 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1226 popović and lović intermetallics are binary or multi-elemental metallic compounds that, since they have well-defined crystalline structures, offer predictable control over structural, geometric, and electronic effects in a manner that is not available when disordered alloys are used. in principle, the electronic and atomic structures, both of which are well known to be important parameters for electrocatalytic activity, can be significantly controlled. as the order in intermetallic phases arises from the high enthalpy of mixing, a high chemical and structural stability could be expected. therefore, in contrast to disordered alloys, all pt (and bi) atoms on the surface of an ordered intermetallic phase have the same local geometry and thus, the same activity. the results obtained in these studies relating to formic acid oxidation indicated that the ptbi ordered intermetallic phase has properties and reactivity that are dramatically different from those of bare platinum surfaces. especially, the onset potential for the electrocatalytic oxidation of formic acid is significantly shifted (by over 300 mv) to more negative values and the current density (at a given potential) is significantly enhanced when compared to pure pt. moreover, ptbi displayed virtual immunity to co poisoning.69 oana et al.76 found for the intermetallic structures that the susceptibility for co adsorption on pt was drastically reduced on ptbi2 and ptbi surfaces, with respect to pt, due to an increase in the fermi level of the system induced by bi. according to abruna and co-workers,45,69 the origin of the catalytic activity was related to electronic effects enhancing the affinity of ptbi for formic acid adsorption and producing surface oxides at low potentials, as well as to geometric effects that reduces the affinity for co poisoning. the shift in the onset potential and the increase in the current density are due to electronic effects. in essence, the formation of the ptbi ordered intermetallic results in a charge redistribution (as a first approximation arising from work function differences), which enhances the affinity of ptbi towards formic acid and further gives rise to the formation of surface oxides at much lower potentials. regarding the geometric effect, contrary to any pt-based alloy where the pt–pt distance for nearest-neighbor pt atoms is essentially the same as in pt metal (2.78 å), in ordered intermetallic compounds, the pt–pt distances can be modulated over a range of a factor of 2. for example, in ptbi the pt–pt distance in the (001) plane is 4.32 å. such distances were expected to diminish significantly co poisoning, by reducing bridge sites and eliminating 3-fold hollow adsorption sites. however, some electronic effects could also be involved. therefore, ordered intermetallic compounds, which were proposed as powerful catalysts for formic acid oxidation,42–46 not only exhibited greatly enhanced electrocatalytic activity (especially relative to pt), but they could also serve as model systems to explore structure/composition/property/activity relationships. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ formic acid oxidation at pt–bi catalysts 1227 2.1.4. ptbi alloys the fact that formic acid is a good candidate for dfafcs initiated the study of its oxidation on so-called real catalysts, such as bulk ptbi alloy and two-phase pt2bi catalysts.39,48,72 in one of our previous works, formic acid oxidation was investigated on ptbi alloy samples obtained according to the bi–pt phase diagram39 by melting the pure elements under an inert atmosphere in the proportion of bi to pt of 1:1, and the alloy was characterized by x-ray photoelectron spectroscopy (xps) and by x-ray diffraction (xrd) analysis.39,48 xps analysis revealed three chemical states of bi, i.e., ptbi or bi(0), bi2o3 and bio(oh) on the catalyst surface (table i).39 these results suggested a model in which the ptbi alloy was covered by a layer of bi2o3 and the very top of this layer contained bio(oh) species. table i. xps analysis of the surface composition of differently treated ptbi 1:1 alloy; be – the binding energies of the bi 4f7/2 excitation; fwhm – the full width at half maximum; takeoff angle (90 or 15°) is next to the sample name in brackets39 sample be bi 4f7/2, ev fwhm, ev approximate composition, % species polished electrode 157.3 0.9 34.3 bi–pt 158.8 1.42 65.7 bi3+ equilibrated at ocp (90°) 157.4 0.9 63.2 bi–pt 158.3 1.6 31.8 bi3+ 159.6 1.9 4.9 bio(oh) equilibrated at ocp (15°) 157.5 1.0 64.7 bi–pt 158.5 1.6 22.0 bi3+ 159.5 1.7 13.3 bio(oh) oxidized at 0.8 v vs. sce (90°) 157.5 0.9 43.2 bi–pt 158.5 1.3 44.1 bi3+ 159.3 1.8 12.6 bio(oh) oxidized at 0.8 v vs. sce (15°) 157.8 1.1 34.4 bi–pt 158.8 1.2 51.8 bi3+ 159.6 1.3 13.8 bio(oh) xrd characterization of the ptbi alloy was performed to determine its phase composition. the diffraction pattern reveals peaks characteristic for hexagonal structure of the ptbi alloy and very small additional maxima that were assigned to traces of platinum cubic phase (≈0.7 wt. %).48 electrochemical characterization showed that ptbi followed the behavior of its constituents.39 in the potential range up to 0.05 v vs. sce, the electrode activity and the processes involved were determined by the behavior of pure bi. it was established that the activity of ptbi was highly dependent on the reduction/oxidation of bi species. dissolution of bi, i.e., leaching from the alloy matrix, proceeded all along the anodic potential scan.39,42 comparison of the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1228 popović and lović basic voltammograms for ptbi and pt (fig. 1a and b) showed that the deposition/dissolution of bi completely suppressed hydrogen adsorption/desorption on pt, as well as that the surface oxidation on ptbi was initiated at significantly lower positive potentials. the fact that adsorption/desorption of hydrogen was completely suppressed on the ptbi surface, as well as absolute inactivity of both the bi and ptbi surfaces for the adsorption of co, made determination of the real surface area impossible. therefore, comparison of activity was given by the geometric surface area. the voltammograms for the oxidation of formic acid on pt and ptbi alloy electrodes, given in fig. 2a and c, respectively, clearly indicated a dependence of the activity on the reduction/oxidation processes of bi. oxidation of formic acid does not occur on pure bi39,77 and, consequently, does not occur on ptbi covered by bi. hence, the beginning of the reaction must be linked to free pt sites on the ptbi. relative to pt, the onset potential on the ptbi electrode was shifted towards negative potentials by more than 0.25 v and the current densities at 0.05 v vs. sce were higher by about two and half orders of magnitude. the exceptional activity of ptbi is caused by upd phenomena of bi on pt, which was electrochemically detected. namely, after recording the voltammogram for ptbi, this electrode was replaced with pt and voltammograms were taken in the supporting solution with and without hcooh. the voltammetric profiles obtained in the supporting solution indicated an underpotential deposition of bi on pt, evidencing that dissolved bi could be adsorbed on pt sites as an upd layer. in the presence of formic acid, voltammogram displayed typical features for formic acid oxidation on a bi-modified pt surface,12 clearly suggesting that this reaction on the ptbi alloy occurs on the bi modified pt sites on the ptbi surface and the huge increase in catalytic activity relative to polycrystalline pt was attributed to upd phenomena of bi leached from the alloy matrix and readsorbed on pt. in addition, based on xps analysis, it is proposed that some contribution of a bifunctional action, enabled by the presence of hydroxylated bi species, should be taken into consideration. our studies of pt2bi electrode,48,72 a two-phase material consisting of ptbi alloy and pure pt, revealed that this is a powerful catalyst for formic acid oxidation. characterization of the catalyst was realized by xrd spectroscopy (phase composition), stm (surface morphology) and coads stripping voltammetry (surface composition). comparison of the basic voltammograms of pt2bi electrode and pt in the potential range up to 0.05 v vs. sce revealed that the presence of bi suppressed hydrogen adsorption/desorption to a large extent (fig. 1c). the cathodic peak at approximately –0.1 v vs. sce could be correlated to the reduction of bi oxide species and adsorbed bi3+, species formed in the positive going scan.39,43 com_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ formic acid oxidation at pt–bi catalysts 1229 prehensive oxide formation/reduction was presented by the anodic and cathodic peaks that are superimposed over those of pt oxide formation and reduction, although the peak position that corresponds to oxide reduction on pt2bi was slightly shifted towards negative potentials with respect to pt, indicating some electronic interaction between pt and bi. a similar behavior was reported for smooth polycrystalline pt electrodes in the presence of bi(iii) ions78 and for ptbi (1:1) alloy.39 the cyclic voltammogram for formic acid oxidation on pt2bi is presented in fig. 2d. on pt2bi catalyst, formic acid oxidation commenced more than 0.2 v earlier than on pt. the currents increased reaching a peak with a current ≈30 times higher than the plateau on pt and than diminished up to the positive potential limit. as bi does not adsorb formic acid,39,77 the oxidation of formic acid occurred on pure pt domains and on pt atoms on ptbi domains. the bell-shaped voltammogram for formic acid oxidation suggests that the reaction on pt2bi proceeded through the dehydrogenation path with the dehydration path being completely suppressed. compared to pt, the high activity of the pt2bi catalyst could be explained by increased selectivity toward the dehydrogenation path caused by an ensemble effect originating from the interruption of continuous pt sites by bi atoms. coads stripping voltammetry recorded at pt2bi and pt48 demonstrated that onset potential and the peak position at pt2bi were slightly shifted to more negative potentials relative to pt, indicating the presence of some electronic modification of the pt surface atoms, capable for co adsorption, by bi. since bi74 and ptbi69 are inactive for co adsorption, the oxidation of co occurs only on the pt domains. therefore, the charge under the coads peak at pt2bi reflecting a process at the surface of the pt phase was used for determining the contribution of pure pt in the surface composition of the pt2bi catalyst. the estimated contribution of pure pt on the pt2bi surface corresponded closely to bulk composition, thus indicating that adsorbed co also prevents leaching of bi. in order to test whether the surface morphology of pt2bi changes during formic acid oxidation, stm measurements were performed before and after the reaction and insignificant changes in the surface morphology and roughness were found. consequently, it appears that the pt2bi surface became kinetically stabilized due to the competition between the oxidation of formic acid at the electrode/ /solution interface and bi leaching, i.e., corrosion/oxidation processes of the electrode surface itself.79 accordingly, the main reasons for high stability of pt2bi catalyst is the suppression of bi leaching, as well as inhibition of dehydration path in the reaction of formic acid oxidation. on the other hand, the lower onset potential for hcooh oxidation and higher reaction currents on pt2bi alloy compared to both pt/biirr electrodes were the result of ensembles that were created by alloyed bi atoms incorporated _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1230 popović and lović into the pt lattice, causing a shift in the d-band center of the adjacent pt atoms. therefore, bi in the alloy also exhibited an electronic effect, which could enhance the affinity towards hcooh adsorption and thus increasing the interaction of hcooh molecules with the catalyst surface. it was found that all the investigated bimetallic catalysts were more active than pt with the onset potentials shifted to more negative values and the currents at 0.0 v vs. sce (under steady state conditions) improved by up to two order of magnitude (table ii). table ii. activity of the respective catalysts at e = 0.0 v (sce) determined under steady -state conditions activity parameter catalyst pt poly pt/biirr,θ ≈0.3 pt/biirr,θ ≈0.5 pt2bi j / ma cm-2 0.0038 0.058 0.36 0.48 jpt2bi/jrespective catalyst 127 8.3 1.33 1 comparison of the results obtained for these different bulk pt–bi catalysts indicated that bi in the alloy and irreversibly adsorbed bi exhibited different effects on the catalytic activity. this enables distinguishing between the role of the ensemble and electronic effects in the oxidation of formic acid on pt–bi electrodes. the electronic effect, existing only on the alloy, contributes to an earlier start of the reaction, while the maximum current originates from an ensemble effect. during potential cycling of the pt/biirr electrode, bi was leached from the electrode surface and the ensemble effect was reduced over time, or lost. 2.2. stability of bulk catalysts from a practical point of view, long-term stability of the investigated catalysts for the oxidation of formic acid is very important. the stabilities of the pt– bi catalysts were tested by chronoamperometric measurements and by prolonged potential cycling in the supporting solution as well as in the supporting solution containing formic acid. the aim of the study was to establish which factors mostly affected the improvement of the oxidation rate of formic acid and the stability of the catalyst. additionally, a parallel study on a pt electrode was performed to verify the promotional role of bi. cyclic voltammograms recorded on pt2bi catalyst in the formic acid containing electrolyte are shown in fig. 3. over the potential cycling up to 0.8 v vs. sce, the activity of pt2bi electrode slowly decreased during the first 5–7 cycles reaching values of ≈85 % of the initial currents. after these first few sweeps, the currents remain unchanged with further cycling. on the contrary, the cycling of pt2bi in supporting electrolyte led to enhancement of the currents related to the oxidation of bi species, indicating some surface decomposition caused by bi leaching/dissolution process (fig. 3, inset a). it appears, consequently, that sta_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ formic acid oxidation at pt–bi catalysts 1231 bility of pt2bi during oxidation of formic acid could be induced by the presence of formic acid in the electrolyte. fig. 3. cyclic voltammograms (1st and 20th sweep) for the oxidation of 0.125 m hcooh in 0.1 m h2so4 solution at a pt2bi catalyst. insets: a) basic voltammograms (1st and 20th sweep) for pt2bi electrode in 0.1 m h2so4 solution and b) cyclic voltammograms (1st and 20th sweep) for the oxidation of 0.125 m hcooh in 0.1 m h2so4 solution at pt/biirr;θ≈0.3 electrode. scan rate: 50 mv s-1. ω = 1500 rpm. t = 295 k. contrary to the pt2bi catalyst, the pt/biirr electrode shows significant changes with continuous cycling in the solution containing formic acid (fig. 3, inset b).72 repetitive cycling up to 0.8 v vs. sce shifted the onset potential for formic acid oxidation to more positive values, decreased the reaction currents, while anodic peak diminished and a new peak starts to emerge and grow at ≈0.6 v vs. sce. this transformation of the cyclic voltammograms indicated modification of the surface composition due to continuous bi dissolution. apparently, re -adsorption of bi species from the solution was rather low, so the initial voltammogram was never restored, which is in accordance with results obtained for formic acid oxidation on bismuth-coated mesoporous pt microelectrodes.80 to test the assumption that the presence of formic acid stabilizes the catalyst, a pt2bi electrode was subjected to potential cycling in the supporting electrolyte and after 20 cycles the electrode was replaced by a pt electrode (fig. 4a). the results of this experiment showed a slightly reduced charge of hydrogen adsorption/desorption indicating underpotential deposition of the previously leached/ _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1232 popović and lović /dissolved bi. the same procedure was repeated in the electrolyte containing formic acid. the voltammogram of formic acid oxidation recorded on pt in this experiment almost retraced the characteristic profile of pure pt, suggesting that leaching of bi was suppressed in the presence of formic acid. this was confirmed by stm measurements performed before and after the oxidation of formic acid, indicating a small difference in roughness and an insignificant change in the surface morphology.48 fig. 4. comparison of cyclic voltammograms recorded after replacement of: a) pt2bi, b) ptbi and c) pt/biirr with polycrystalline pt in 0.1 m h2so4 solution and in 0.125 m hcooh solution. scan rate: 50 mv s-1. ω = 1500 rpm. t = 295 k. it should be noted that the experiment performed after replacing the ptbi alloy with pt revealed significant bi leaching under the same experimental conditions, meaning that pt2bi was more resistant to bi leaching than ptbi (fig. 4b). the same experiment conducted with the pt/biirr electrode showed considerable changes in surface composition due to bi dissolution. upon prolonged cycling, the electrode surface became enriched in platinum and exhibited a pt-like electrochemical behavior in acid electrolyte containing formic acid (fig 4c). although pt/biirr shows remarkable initial activity compared to pure pt, this electrode was not stabilized by formic acid oxidation, since the desorption of bi was not suppressed in the presence of formic acid. in addition, the poisoning _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ formic acid oxidation at pt–bi catalysts 1233 effect induced by the dehydration path was not avoided and biirr did not provoke any significant modification of the electronic environment. therefore, pt/biirr catalysts are less active than the corresponding alloy. chronoamperometric experiments were performed to prove the activity and stability of the investigated catalysts (fig. 5). insight into the chronoamperometric curves confirmed the advantage of alloys, i.e., the necessity of alloying pt with bi to obtain a catalyst with stable activity. fig. 5. chronoamperometric curves for the oxidation of 0.125 m hcooh at 0.2 v in 0.1 m h2so4 solution on ptbi, pt2bi, pt/biirr and pt catalysts. ω = 1500 rpm. t = 295 k. in summary, the main reason for the high stability of the pt2bi catalyst is the inhibition of the dehydration path in the reaction, as well as suppression of bi leaching in the presence of formic acid, which is specified by a minor change in the surface morphology and roughness.48,72 comparing the results obtained for the two types of pt–bi catalysts, polycrystalline pt modified by irreversible adsorbed bi and for pt2bi catalyst, the role of the ensemble effect and electronic effect in the oxidation of formic acid was distinguished.48 the electronic effect contributes to a lower onset potential of the reaction, while the maximum current comes from the ensemble effect. during the potential cycling treatment of the pt/biirr electrode, bi is dissolved from the electrode surface and the ensemble effect is reduced or lost over time. on the other hand, the high stability of the pt2bi catalyst, confirmed by chronoamperometric experiments, proves an adventage of alloys, i.e., the necessity of alloying pt with bi to obtain a corrosion resistant catalyst. according to liu et al.,79 the high stability of the ptbi intermetallic is due to suppressed leaching of bi in the presence of formic acid because of the effective competition between oxidation of the organic molecule at the electrode surface and the corrosion/oxidation of the electrode surface itself. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1234 popović and lović 3. low-loading pt–bi catalysts 3.1. activity of low-loading catalysts the concept of bimetallic mono and multilayer catalysts has received much attention regarding its possibility to reduce the noble metal loading and maintain the activity by replacing the under-layer (or bulk of the catalyst) with a less noble metal. in addition, unlike other bimetallic catalysts where the second metal is either in the form of an adatom or as a surface alloy component, this type of catalyst allows the study of the electronic effect of the second metal under-layer to the noble catalyst over-layer, as the only operating factor. 3.1.1. pt/gc catalyst pt was deposited onto a glassy carbon substrate (pt/gc) using chronocoulometry at the potential corresponding to pt limiting current plateau.70 for the sake of comparison, a pt/gc electrode was prepared using the same electrochemical procedure and the quantity analogous to one for a bimetallic electrode. afm imaging of the pt deposit on the gc substrate showed randomly distributed clusters (agglomerates), which consisted of spherical nanoparticles with a regular size distribution of 5.7±1.5 nm, as revealed by stm measurements.70 catalytic activity of pt/gc electrode for formic acid oxidation was examined by potentiodynamic and quasi steady-state measurements. the reaction proceeds through both paths featured by higher first and lower second anodic, indicating lower poisoning of this pt surface compared to the pt bulk electrode and a shift in the reaction towards the direct path. stm analysis of this electrode revealed rather small, loosely packed particles with a diameter of ≈5 nm, which should have a lower number of defects and smaller pt ensembles exposed to the reaction. such morphology of the particles should lead to a more pronounced direct path in formic acid oxidation. these results are in accordance with a previously reported conclusion81 that the particle structure, i.e., morphology, rather than the particle size plays a predominant role in the activity of pt catalysts for formic acid oxidation. the particle structure is directed by particle growth, which is influenced by the support morphology and saturation of the active centers of the support by a metal precursor. 3.1.2. pt@bi/gc clusters formic acid oxidation was studied on a pt–bi catalyst obtained using an unusual approach for the preparation, i.e., modification of the bi deposit with a pt overlayer.70 briefly, platinum–bismuth deposits on a glassy carbon (gc) rotating disk electrode were prepared by a two-step process. electrochemical deposition of a controlled amount of bi was performed at –0.1 v vs. sce onto a mirror-like polished gc substrate. subsequently, the electrodes were rinsed and transferred to the solution for deposition of pt. it should be stressed that pt deposition was realized at a potential of –0.1 v vs. sce, which corresponded to the pt limiting current plateau, in order to avoid any displacement reaction between pt _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ formic acid oxidation at pt–bi catalysts 1235 and the less noble bi and/or gc substrate. for the same reason, the gc and gc/bi electrodes were immersed and pulled out from the solution for pt deposition at –0.1 v vs. sce. the electrodes prepared in such manner, denoted as pt@bi/gc, were characterized by afm spectroscopy, which indicated that pt crystallized preferentially onto the previously formed bi particles. analysis of the current vs. time transient responses demonstrated compliance with theoretical curves for progressive 3d nucleation82 in all three cases, i.e., for bi deposition on a gc substrate, for pt deposition on bi/gc surface and pt deposited alone on a gc support. the density of nuclei at saturation imply not only higher coverage of gc surface by bi in comparison to pt, but also that pt could be better spread over bi than over gc. the issue of bi leaching (dissolution) from ptbi catalysts, and their catalytic effect alongside the hcooh oxidation is rather unresolved. in order to control bi dissolution, as prepared electrode were subjected to electrochemical oxidation by slow sweep in the supporting electrolyte within the relevant potential range. such oxidized electrodes are denoted as “treated pt@bi/gc”. this procedure led to quantitative oxidation of bi partially occluded by pt, but simultaneously to the formation of bi oxide, accordingly creating a surface composed of pt and bi oxide. the sem micrographs of the as-prepared and treated pt@bi/gc electrodes are shown in fig. 6a and b, respectively. the sem image of the as-prepared sample shows well separated, randomly distributed clusters (agglomerates), with size of about 700 nm, which were formed from smaller particles. after electrochemical treatment of pt@bi agglomerates, the sem image showed a decrease in the number and size of the isolated clusters, indicating some dissolution of uncovered/unprotected bi deposit. anodic striping charges indicated that along oxidation procedure about 70% of the quantity of deposited bi was oxidized (fig. 6c). the shape of the stripping peak implies the possibility of its deconvolution into two peaks, a sharp one that corresponds to bi dissolution and a broader one at more positive potential that suits bi oxide formation.83 icp mass spectroscopic analysis of the electrolyte after this electrochemical treatment revealed that bi was only partially dissolved, which confirmed the possibility of the formation of some bi oxide species. these assumptions were confirmed by edx spectra, which evidenced the presence of pt, bi and o, showing a decrease in bi quantity in the pt@bi/gc electrode after the slow sweep and significant increase in the oxygen content, which may be attributed to bi oxide formation (fig. 6d). thus, the composition of the treated pt@bi/gc obtained by edx analysis corresponded qualitatively well to the results obtained by the deconvolution of the stripping peak. in this way, the prepared electrode exhibited significantly high activity and exceptional stability in comparison to the pt/gc electrode. formic acid oxidation _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1236 popović and lović proceeded predominantly through the dehydrogenation path on the treated pt@bi/gc electrode, resulting in its high activity. the onset potential was shifted by 150 mv to more negative values and the currents were about 10 times higher than those at the same potential on pt/gc, as revealed both by potentiodynamic as well as by quasi-steady state measurements (fig. 7). fig. 6. sem images for pt@bi/gc electrode a) before and b) after electrochemical treatment; c) deconvoluted anodic stripping voltammogram for bi oxidation in 0.1 m h2so4 solution and d) element content in the catalyst before and after electrochemical treatment, as analyzed by edx. this high activity and increased selectivity toward dehydrogenation is the result of well-balanced ensemble effect originating from the interruption of continuous pt sites by bi-oxide domains. the possibility of some electronic effect of non-oxidized bi under the pt on the activity of the pt@bi/gc electrode could not be excluded. prolonged cycling and chronoamperometry tests revealed exceptional stability of the catalyst during formic acid oxidation.70 this low loading pt-based electrode exhibited activity for the oxidation of formic acid similar to that of bulk pt2bi alloy, which has been shown to be one of the best pt–bi bimetallic catalysts for the oxidation of formic acid.48 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ formic acid oxidation at pt–bi catalysts 1237 fig. 7. initial cyclic voltammograms: a) for treated pt/gc and treated pt@bi/gc electrodes in 0.1 m h2so4 solution and b) for the oxidation of 0.125 m hcooh in 0.1 m h2so4 solution (scan rate 50 mv s-1); c) corresponding tafel plots (scan rate 1 mv s-1). ω = 1500 rpm. t = 295 k. 3.2. stability of low-loading catalysts as already stated, upon treatment of as prepared pt@bi/gc clusters by a slow anodic sweep, a unique bimetallic structure consisting of a bi core occluded by pt and a bi-oxide was obtained. consideration of the stability of the pt@bi/ /gc catalyst was realized by applying prolonged potential cycling up to 0.8 v vs. sce (hereinafter referred to as a cycling protocol) in supporting electrolyte or in supporting electrolyte containing formic acid and the results were compared with data obtained at a pt/gc electrode treated in the same manner. an attempt was made to correlate the electrochemical response with the structural features of these two catalysts and to signify the effects that determine the electrode stability in formic acid oxidation.84 the difference in activity between pt@bi/gc and pt/gc catalyst after the application of cycling protocol in appropriate electrolyte revealed a complex phenomenology, suggesting that the interplay of several factors, such as nanoparticle size, surface morphology, influence of formic acid and its intermediate coads, determines the performance of the catalysts in formic acid oxidation. oxidation of hcooh at the pt@bi/gc electrode occurred directly through dehydrogenation to co2 enabled by the well-balanced bi-oxide domains and small pt ensembles, which resulted in remarkable stability of this catalyst. seri_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1238 popović and lović ous formic acid oxidation effectively competes with oxidation of the electrode surface, i.e., dissolution of bi.79 leaching of bi from pt@bi/gc in the presence of formic acid was not prevented but highly suppressed due to the source of bi, i.e., bi core. since this electrode is composed of a bi core occluded by pt and bi oxide, the morphology of the surface slightly changes and facilitates high activity and stability of this catalyst. pt@bi/gc catalyst exhibited excellent stability during prolonged oxidation of formic acid even above the potentials of bi dissolution, as revealed by the negligible decrease in activity (fig. 8a). such stability of the catalyst was confirmed by quasi-steady state measurements performed on the surface previously treated by potential cycling in supporting electrolyte containing formic acid. the data obtained under slow sweep conditions corroborated the difference in the activities of investigated catalysts, as was found by the potentiodynamic measurements. these measurements also showed high selectivity of this surface toward the dehydrogenation path. tafel slops of 120 mv obtained at pt@bi/gc electrode after 1, 20 and 100 cycles in the supporting electrolyte containing formic acid indicate that the dehydrogenation path in formic acid oxidation proceeded on a surface free of adsorbed co species. contrary to the pt@bi/gc electrode, which possessed high stability and unusual cycling performance in formic acid oxidation, the activity of the pt/gc electrode during the cycling protocol in the presence of formic acid in the supporting electrolyte continuously decreased (fig. 8b). even more, while at pt@bi/gc electrode, the reaction proceeded continuously through the direct path, in the case of pt/gc, the reaction mechanism changes during cycling. as can be seen from fig. 8b, in the first cycle, the current raises and reaches a peak that indicates predominant direct oxidation of hcooh, while the appearance of a well-defined shoulder on the descending part of the curve signifies the indirect path in the reaction as well. as the number of cycles increases, the currents related to dehydrogenation diminish and simultaneously, the well-defined shoulder transforms into a peak, indicating the increased role of the indirect path in the reaction. this decrease in the activity of pt/gc electrode could not only be due to a gradual accumulation of reaction residues, i.e., poisoning, on the electrode surface, but also to structural adjustments of the platinum nanoparticles as a result of the changes in the potentials during the scanning in acidic solutions, especially in the presence of the organic compound. the tafel slope obtained for pt/gc electrode of 140 mv (fig. 8b) indicates that the reaction occurred on the surface partially covered by coads, not only through the dehydrogenation path, but also via the dehydration path occurring in parallel. significant decreases in the currents and increases in the tafel slope after 20 and 100 cycles indicate retardation of the reaction due to adsorption and accumulation of co on the surface. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ formic acid oxidation at pt–bi catalysts 1239 fig. 8. cyclic voltammograms for the oxidation of 0.125 m hcooh in 0.1 m h2so4 (1st, 20th and 100th sweep at a rate of 50 mv s-1); effect of cycling – plots of current density vs. number of cycles and corresponding tafel plots (scan rate 1 mv s-1) obtained a) on a pt@bi/gc electrode and b) on a pt/gc electrode. ω = 1500 rpm. t = 295 k. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1240 popović and lović the oxidation of adsorbed co, usually used for surface characterization, was also employed to determine the electrochemically active surface area (ecsa) of a catalyst. the values calculated for pt@bi/gc and pt/gc electrodes revealed that, regardless of the presence or absence of hcooh in the supporting electrolyte, the ecsa of the pt@bi/gc electrode increased during the cycling protocol, while the ecsa decreased for the pt/gc electrode (fig. 9). however, the degree of ecsa change upon cycling for both electrodes significantly depended on whether the supporting electrolyte contained hcooh. the ecsa of the pt@bi/ /gc electrode slightly increased during this treatment in presence of formic acid due to some bi dissolution and was confirmed in the experiments with polycrystalline pt (fig. 10b). fig. 9. normalzed ecsa values calculated with cvs upon potential cycling in supporting solution and in supporting solution containing 0.125 m hcooh on: a) pt@bi/gc and b) pt/gc electrodes. the normalized ecsa values were calculated by dividing the ecsa value after a certain number of potential cycles by that of the first cycle. however, when this electrode was cycled in pure supporting electrolyte, the ecsa value increased significantly during the treatment (fig. 9) because of intense leaching of bi from the electrode, which was confirmed by the experiment with polycrystalline pt (figs. 10a and b). on the other hand, the pt/gc electrode exhibited completely opposite properties upon similar treatment in the supporting electrolyte with or without formic acid. when this electrode was subjected to potential cycling in the absence of hcooh, the ecsa value decreased slightly (fig. 9), which was primarily due to the coalescence and agglomeration of the particles. some negli_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ formic acid oxidation at pt–bi catalysts 1241 gible dissolution of pt could also be possible.85 the result of these phenomena was the formation of defects on the surface that led to some negative shift of the onset and the peak potential of the oxidation of coads, which indicates a slight change in the surface morphology had occurred. the consequence of these minor surface alternations was a small decrease in the activity for the oxidation of formic acid. fig. 10. cyclic voltammograms recorded a) after replacement of pt@bi/gc that has been exposed to cycling protocol with a polycrystalline pt electrode in 0.1 m h2so4 solution and b) for the oxidation of 0.125 m hcooh in 0.1 m h2so4. scan rate: 50 mv s-1. ω = 1500 rpm. t = 295 k. furthermore, when the pt/gc electrode was similarly treated in the presence of hcooh, the ecsa decreased much more (fig. 9) and a larger shift of the coads stripping peaks in the negative direction was observed, indicating to a considerable perturbation in the surface morphology. the consequence of these changes was a significantly lower activity of these surfaces. considering that formic acid oxidation proceeds on a pt/gc electrode through both the direct dehydrogenation and the indirect dehydration path, and that during cycling the reaction turns more to the latter one in which co adsorbs on the surface, it seems that adsorption of co and its oxidation contribute not only to particle agglomeration but even more to pt dissolution. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1242 popović and lović high stability of pt@bi/gc electrode is confirmed in chronoamperometric experiment (fig. 11). current density recorded on treated pt@bi/gc during 1800 s at a constant potential of 0.2 v was again significantly higher than on the pt/gc electrode. at pt/gc electrode, the current decayed rapidly reaching a low steady state value within a few minutes. contrarily, the current decreased slowly at the pt@bi/gc electrode and stabilized at a value that was more than 10 times higher than for the other electrode. this experiment also demonstrated the higher stability of the pt@bi/gc electrode in comparison to the pt2bi catalyst48 since under the same conditions, the decrease in the currents for hcooh oxidation at the pt@bi/gc electrode was much lower compared to the decrease registered for the pt2bi catalyst. fig. 11. chronoamperometric curves for the oxidation of 0.125 m hcooh in 0.1 m h2so4 solution at 0.2 v on pt@bi/gc and pt/gc electrodes. ω = 1500 rpm. t = = 295 k. 3.3. pt(bi)/gc shell–core catalyst the bimetallic ptbi electrodes, as catalysts for formic acid oxidation, were examined in terms of obtaining a steady state electrode surface. the catalyst, denoted as pt(bi)/gc was produced using a similar methodology of preparation as previously described for the pt@bi/gc catalyst, i.e., by sequential deposition of bi followed by deposition of pt. in contrast to the experiments to test the stability of the pt@bi/gc catalyst, when its activation was performed by two slow sweeps up to 0.8 v vs. sce, the pt(bi)/gc electrodes after metal deposition were activated by cycling the potential at a scan rate of 50 mv s–1 between hydrogen and oxygen evolution (–0.27 up to 1.2 v vs. sce) in 0.1 m h2so4 solution, prior to use as catalysts for the oxidation of formic acid. generally, potential cycling is an electrochemical treatment that determines the degree of surface reconstruction and the size of the electrochemically active area. in the case of the pt(bi)/gc electrodes, this treatment was applied to quantify the amount of remaining bi and bi oxide in order to explain the importance of the composition and morphology of the surface for the reaction of formic acid oxidation.86 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ formic acid oxidation at pt–bi catalysts 1243 edx and icp-ms analysis, and afm and electrochemical characterization, revealed that initially unfinished core–shell structures were formed. afm characterization of the electrode surface indicated that pt was preferentially deposited on the previously formed bi particles, but cyclic voltammetry revealed leaching of bi, meaning that bi was not completely occluded by pt. the pt(bi)/gc catalysts were not stable at potentials beyond 0.4 v vs. sce due to bi leaching/dissolution from the surface, which occurred through the oxidation of the less-noble metal. electrochemical treatment by potential cycling of the as-prepared electrode (fig. 12a) led to quantitative oxidation of bi from the fig. 12. cyclic voltammograms recorded on the pt(bi)/gc electrode: a) in 0.1 m h2so4 and b) for the oxidation of 0.125 m hcooh in 0.1 m h2so4 after 8, 50 and 250 cycles. scan rate: 50 mv s-1. ω = 1500 rpm. t = 295 k. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1244 popović and lović unprotected core and the simultaneous formation of bi oxide, consequently creating a shell composed of pt and bi-oxide. by prolonged cycling, the amount of surface oxides diminished creating finally a pt@bi shell–core structure. these electrodes exhibit enhanced electrocatalytic activity in formic acid oxidation in comparison to pt/gc electrode treated in the same manner (fig. 12b). this behavior could be explained primarily by an ensemble effect induced by surface bi oxides interrupting the pt domains, but to some extent, could also be attributed to the influence of the under-lying bi onto the pt surface layer, affecting the extent of poison adsorption on the pt. electronic modification of pt both by surface and sub-surface bi can play some role as well. significantly prolonged potential cycling in supporting electrolyte of pt(bi)/gc electrodes previously stabilized by bi oxide led to considerably lower bi leaching and was accompanied by dissolution and redeposition of pt, resulting in a pt shell over a bi core. the pt@bi/gc (shell–core) catalysts, with only pt in the surface layer exhibit somewhat enhanced activity due to the electronic effect of the remaining under-lying bi, which depended on the thickness of the pt layer determined by the quantity of bi in the core. this observation was confirmed by results obtained at three catalysts with different ratio of pt and bi (different underlying thickness of bi).86 in this way, by controlling the thickness of the bi and pt layers using electrochemical techniques, it was possible to improve the electrocatalytical properties of pt(bi)/gc in hcooh oxidation and to create a low-loaded pt-based catalyst with comparable activity to that of the bulk metal alloy. 4. conclusions according to the results presented in this work dealing with the effects influencing the overall formic acid oxidation, it was found that both types of pt–bi bimetallic catalysts (bulk and low-loaded deposits on gc) showed superior catalytic activity compared to pt, in terms of the lower onset potential and higher oxidation current density. both types of pt–bi catalysts were investigated in order to establish how bi atoms affect the adsorption characteristics of pt towards formic acid. it was found that among all the tested pt–bi bimetallic bulk catalysts, pt2bi is the most powerful for formic acid oxidation, exhibiting high activity and stability. high activities of the bulk bimetallic catalysts result from the fact that formic acid oxidation proceeds completely through the dehydrogenation path. increased selectivity toward dehydrogenation is caused by an ensemble effect. the high stability of pt2bi surfaces are induced by the suppression of bi leaching, as was evidenced by the insignificant changes in the morphology and roughness of the surfaces before and after electrochemical treatment in formic acid containing solution. the results presented indicate that bi in alloy and irreversibly adsorbed _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ formic acid oxidation at pt–bi catalysts 1245 bi exhibit different effects on the catalytic activity. bi in the alloy not only induces an ensemble effect, but also has an electronic effect, which could be the reason for better performance of this catalyst resulting in higher currents and a lower onset potential. the activity of ptbi alloy is caused by upd phenomena of bi on pt, which was electrochemically detected. in addition, based on xps analysis, it is proposed that the activity of ptbi could also be caused by the bifunctional action of hydroxylated bi species. comparing the results obtained for these bulk pt–bi catalysts, the role of the ensemble effect and electronic effect in the oxidation of formic acid could be distinguished. the electronic effect, existing only on the alloy, contributed to the earlier start of the reaction, while the enhanced maximum current originates from the ensemble effect. thus, the stability of the catalytic activity of pt–bi bimetallic electrodes is strongly related to the leaching tolerance of the electrode surface during formic acid oxidation. the leaching tolerance of anodic catalysts was greatly enhanced by the formation of alloy between pt and bi, i.e., it is necessary to alloying pt with bi to obtain a corrosion stable bulk catalyst. electrochemical deposition of low loading pt layer over bi deposits on a gc electrode resulted in the formation of approximately spherical clusters of bi covered by pt. treatment of the as-prepared electrode in the relevant potential range and supporting electrolyte leads to quantitative oxidation of bi partially occluded by pt, and simultaneously to the formation of bi oxide, thus creating a surface composed of pt and bi-oxide. the so-prepared electrode exhibits higher activity and exceptional stability in comparison to a pure pt/gc electrode. formic acid oxidation proceeds predominantly through dehydrogenation path on treated pt@bi/gc electrode resulting in its high activity. the increased selectivity toward dehydrogenation is caused by an ensemble effect originating from the interruption of continuous pt sites by bi-oxide domains. the possibility of some electronic effect of non-oxidized bi under the pt on the activity of pt@bi/ /gc cannot be excluded. such stability is induced by the stability of the bi-oxide formed during electrode pre-treatment. this low loading pt-based electrode exhibits activity for the oxidation of formic acid similar to the activity of bulk pt2bi alloy, which has been shown to be one of the best pt–bi bimetallic catalysts for the oxidation of formic acid. the use of bimetallic compounds as anode catalysts is an effective solution to overcome the problems with stability of the formic acid oxidation current for long-term applications. in the future, the tolerance of both co poisoning and electrochemical leaching should be considered as the key factors in the development of electrocatalysts for anodic reactions. acknowledgement. this work was financially supported by the ministry of education, science and technological development of the republic of serbia, contract no. h-172060. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1246 popović and lović и з в о д оксидација мравље киселине на катализаторима платина–бизмут ксенија ђ. поповић и јелена д. ловић ихтм – центар за електрохемију, универзитет у београду, његошева 12, п. пр. 473, 11000 београд електрохемијска оксидација мравље киселине је предмет истраживања последњих деценија као модел реакција за разумевање механизма оксидације малих органских молекула, као и због њене могуће примене у горивним спреговима. платина је један од најчешће коришћених катализатора за ову реакцију упркос томе што показује неколико значајних недостатака који спречавају њену широку практичну примену: има високу цену и показује врло изражен пад ефикасности услед тровања површине адсорбованим интермедијерима (coads). да би се превазишли ови проблеми и побољшале каталитичка својства катализатора, платина се модификује другим металима, па се стога све више користе биметални платински катализатори. посебна пажња је усмерена на bi као модификатор платине. оксидација мравље киселине испитивана је на два типа pt–bi електрода: на масивним електродама и на електродама добијеним таложењем танких филмова на носаче од стакластог угљеника. оба типа биметалних катализатора показују знатно већу активност и изузетну стабилност у поређењу са чистом pt. резултати приказани у овом прегледу значајни су за разумевање механизма електрооксидације мравље киселине на легурама pt–bi и pt модификованој бизмутом, за развој нових анода побољшаних карактеристика за примену у горивим спреговима са мрављом киселином као горивом (dfafc), као и за синтезу биметалних катализатора са малим садржајем pt на бази танких филмова. коришћењем ових биметалних катализатора превазишао би се и проблем стабилности анодног материјала за дугорочну примену у горивним спреговима. 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice errata (printed version only) j. serb. chem. soc. 80 (9) 1215 (2015) 1215 errata (printed version only) 1. vol. 75, issues no. 3 and 4 (2010): – first seven characters of the doi numbers of all articles should read: 10.2298 2. vol. 80, issue no. 8 (2015), article no. jscs-4782, page 1097: – the following paragraph is to be added before the paragraph “acknowledgements.”: supplementary material description of area and sampling location, as well as contents of heavy metals and several major components in sediment samples from zlt-2 borehole, reference standard values and weathering indices are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice coordination studies of 1,2-bis(diphenylphosphino)ethane with di-μ-hydroxo dinuclear complexes of tungsten(iv) and molybdenum(iv) j. serb. chem. soc. 81 (1) 47–55 (2016) udc 546.774+546.784+547.636’416:544.433.21 jscs–4826 original scientific paper 47 coordination studies of 1,2-bis(diphenylphosphino)ethane with di-μ-hydroxo dinuclear complexes of tungsten(iv) and molybdenum(iv) makoto minato1*, takashi ito1 and jian-guo ren2 1department of materials chemistry, graduate school of engineering, yokohama national university, 79-5 tokiwadai, hodogaya-ku, yokohama, 240-8501, japan and 2department of chemistry, shanxi university, taiyuan, 030006, china (received 1 may, revised 28 july, accepted 29 july 2015) abstract: new trifluoroethoxido phosphine complexes [cp2m(η 1-dppe)(cf3ch2o)] + and [cp2(cf3ch2o)m(μ-dppe)mcp2(cf3ch2o)] 2+ (m = mo or w, cp = η-c5h5 and dppe = ph2pch2ch2pph2) were prepared by reaction of the cationic di-μ-hydroxido dinuclear complex of molybdenocene or tungstenocene [cp2m(μ-oh)2mcp2] 2+ with dppe. from the 1hand 31p-nmr data, the configurations of the products could be assigned. furthermore, x-ray crystallography was used to definitively identify one of the products [cp2(cf3ch2o)mo(μ-dppe)mocp2(cf3ch2o)] 2+, which crystallizes in the space group p21/c(#14) with a = 12.230(5) å, b = 11.149(5) å, c = 28.966(7) å, β = 101.07(3)°, v = 3876(2) å3 and z = 2. it was ascertained that the amount of dppe added to the reaction mixture could influence the product distribution. a mechanism involving initial replacement of the hydroxido ligand by the alkoxido group followed by nucleophilic attack of the phosphine is proposed based on the reaction profile. keywords: molybdenocene; tungstenocene; dinuclear complexes; dppe. introduction previously, it was shown that cationic di-μ-hydroxo dinuclear complexes of molybdenocene and tungstenocene [cp2m(μ-oh)2mcp2]2+ (cp = η-c5h5; m = = mo (1a) or w (1b)) could be conveniently prepared by reactions of cp2mh2 and cp2m(ots)2 (ots = p-ch3c6h4so3) in aqueous acetone.1 these novel dinuclear complexes have attracted much attention due to their ability to catalyze intraand inter-molecular h/d exchange reactions,2 and reduction of ketones3 and nitriles.4 * corresponding author. e-mail: minato@ynu.ac.jp  the authors are pleased to dedicate this paper to professor rastko d. vukicevic in the year of his 65th birthday with continued best wishes for his retirement. doi: 10.2298/jsc150501066m 48 minato, ito and ren the hydroxo groups in complexes 1 were sufficiently labile to undergo displacement by a wide variety of substrates, affording molybdenocene and tungstenocene derivatives and hence, they are useful as precursors of these types of compounds.1,5,6 it was found that reactions between complexes 1 and monodentate tertiary phosphines always proceeded with the concomitant incorporation of co-existing alcohols to yield novel alkoxido phosphine complexes [cp2m(pr′3)(ro)]+(ots–) (2, r′ = et, bun, and ph; r = me, et, pri, cf3ch2 and ph) (scheme 1).1c it was proven that these reactions occurred spontaneously under mild conditions (20–50 °c). furthermore, on dissolving in benzene containing a small quantity of water, the resulting complexes 2 readily and quantitatively reverted to the original complexes 1 with liberation of the phosphine ligands, which suggests the reversibility of the reactions. scheme 1. the reactions between complexes 1 and monodentate tertiary phosphines. the syntheses of complexes 2 showed the following intriguing trends. in methanol, ethanol, or 2-propanol, only basic phosphines, such as triethylphosphine or tributylphosphine, reacted with 1 and no substitution of the hydroxo bridging groups by triphenylphosphine were observed in these solvents. however, in moderately acidic alcohols, such as trifluoroethanol, or in the presence of phenol, the less reactive triphenylphosphine reacted smoothly to afford 2 in good yields. therefore, the outcome of the reactions appears to be dependent on the nucleophilicity of the tertiary phosphines and the acidity of the co-existing alcohols. in addition, of particular interest was the fact that no compounds resulting from the incorporation of two phosphine ligands were formed; the labile alkoxido ligand bound to the central metals of 2 was not displaced by a second phosphine ligand even in the presence of excess tertiary phosphine. these results are somewhat puzzling and questions remain regarding a reasonable reaction mechanism. as a natural extension of this study, the study of the reactions of the dinuclear complexes 1 with a chelating ligand was of interest. the focus of the present paper was the reactions between 1 and dppe (where dppe represents 1,2-bis(diphenylphosphino)ethane), since green et al. reported that the reactions of analogous halogenido complexes [cp2mi2] (m = mo or w) with dppe resulted in formation of complexes [cp2m(dppe)]2+ in which the dppe ligand coordinates to the metals in a bidentate mode.7 thus, considering the chelate effect of the dppe ligand, the formation of a similar bidentate-type complex could be anticipated in the new alkoxido dppe complexes 49 the present case. the results of such experiments are reported herein. in addition, another purpose of this study was to propose a reasonable reaction mechanism pertaining to all reactions between 1 and tertiary phosphines. results and discussion the reactions of 1 with dppe were run in cf3ch2oh/c6h6 at room temperature. the preliminary results demonstrated that the reaction afforded two organometallic species that were assigned as mononuclear complexes 3 containing a monodentate dppe ligand and bridged dimetallic complexes 4 (scheme 2). the reaction of dppe did not proceed at all in methanol or in ethanol, which is similar to the reaction of triphenylphosphine. it was further ascertained that the amount of dppe added to the reaction mixtures could influence the product distribution. it was then decided to investigate the reaction conditions in order to gain a better understanding of the factors controlling the selectivity of the products. the results are summarized in table i. scheme 2. the reactions between complexes 1 and dppe to produce mononuclear complexes 3 and bridged dimetallic complexes 4. table i. products obtained from the reactions of 1 with dppe in the presence of cf3ch2oh under several conditions compound complex / mmol dppe, mmol cf3ch2oh/c6h6, ml/ml yield, % 3a 1a / 0.294 1.300 1.5/5.0 81 3b 1b / 0.135 0.880 1.5/5.0 82 4a 1a / 0.341 0.339 1.5/1.5 50 4b 1b / 0.159 0.158 2.0/2.0 43 as shown in table i, good yields of complexes 3 could be obtained if a large excess of dppe was added to 1 in solution at room temperature. on the other hand, the reactions of 1 with dppe in a mole ratio of 1:1 gave 4 as the major products. it is worth emphasizing that complexes containing a chelating dppe ligand were not observed in the reactions of complexes 1. thus, dppe does not yield the expected chelated complexes. complexes 3 are soluble in methanol, ethanol, trifluoroethanol and acetone, while 4 are soluble in the foregoing alcohols and essentially insoluble in acetone. it was found that complexes 3 and 4 were stable to air in the solid state. com50 minato, ito and ren plexes 3 and 4 were characterized by standard methods, in particular 31p{1h}and 1h-nmr spectroscopies, as well as by x-ray structural determination of 4a. in addition, the combustion analyses for 3b and 4a were consistent with their spectroscopic properties (see experimental).8 selected nmr data are collected in table ii. table ii. selected 1h-nmr (270 mhz, cd3od, 293 k) and 31p-nmr data (202 mhz, cd3od, 293 k, j in hz) for complexes 3 and 4 (δ / ppm) compound 1h-nmr 31p-nmr 3a 7.2–7.6 (20h, m, ar-h), 5.46 (10h, d, jph = 1.83, cp), 3.20 (2h, q, jfh = 9.56, cf3ch2o), 2.6–2.9 (2h, br, mopch2ch2), 1.6–1.9 (2h, br, mopch2ch2) 29.9 (s), –12.0 (br) 3b 7.2–7.6 (20h, m, ar-h), 5.42 (10h, d, jph = 1.22, cp), 3.45 (2h, q, jfh = 9.56, cf3ch2o), 2.6–2.8 (2h, br, wpch2ch2), 1.7–1.9 (2h, br, wpch2ch2) not measured 4a 7.2–7.6 (20h, m, ar-h), 5.38 (20h, d, jph = 1.83, cp), 3.20 (4h, q, jfh = 9.56, cf3ch2o), 2.2–2.4 (4h, br, mopch2) 28.0 (s) 4b 7.2–7.6 (20h, m, ar-h), 5.35 (20h, d, jph = 1.22, cp), 3.40 (4h, q, jfh = 9.56, cf3ch2o), 2.1–2.3 (4h, br, wpch2) not measured the 1h-nmr spectra of complexes 3 are similar, showing five different resonances besides the resonances due to the tso protons, and resemble those of 2 in a previous paper.1c as shown in the table ii, resonances for the cyclopentadienyl ring protons of 3a and 3b appear at around δ 5.5 ppm as a doublet coupled to phosphorus; this represents significant shielding of these protons compared with the chemical shifts in the parent dinuclear complexes 1 (δ = 6.0 ppm) but are compatible with the observed chemical shifts of 2. as expected, the spectra of 3 show two separate signals for the ch2ch2 fragment of the dppe ligand at δ around 2.7 and 1.8 ppm. the cf3ch2o units in 3a and 3b show a quartet at δ 3.20 (3a, jfh = 9.56 hz) and 3.45 ppm (3b, jfh = 9.56 hz), respectively. assignment of the monodentate coordination mode of the dppe ligand was based on the observation of two discrete equal intensity resonances at δ around 29 and –12 ppm in the 31p{1h}-nmr spectrum of 3a. the downfield resonance is assignable to a metal-bonded phosphorus atom, while the high-field resonance can readily be assigned to an uncoordinated phosphorus atom since this chemical shift is very similar to that of free dppe. in the 1h-nmr spectrum of complex 4a, the cp protons occur at δ = 5.4 ppm as a doublet with a p–h coupling constant of 1.83 hz, while the cf3ch2 protons appear as a quartet with an f–h coupling constant of 9.56 hz at δ = 3.2 ppm. unlike complex 3a, the spectrum of 4a shows only one multiplet for the the new alkoxido dppe complexes 51 ch2ch2 fragment at around δ = 2.3 ppm. furthermore, the 31p{1h}-nmr spectrum of 4a contains only one resonance at around δ 28 ppm, which was assigned to the phosphorus atom on molybdenum. evidently, these results indicate that 4a contains the mo(μ-dppe)mo group. the spectrum of complex 4b is almost identical to that of 4a, supporting a structure analogous to that of 4a. complex 4a was fully characterized by x-ray crystal structure determination. dark red crystals suitable for the x-ray analysis were obtained by recrystallization from cf3ch2oh/et2o. the more important bond lengths and bond angles are given in table iii. a summary of the crystallographic data is given in table iv (see experimental). as was anticipated from the nmr consideration, the analysis indicated that the molecule has a symmetric structure in the solid state with the formulation [cp2(cf3ch2o)mo(μ-dppe)mo(och2cf3)cp2](ots)2(cf3ch2oh)2 in which two molecules of trifluoroethanol are included as a crystallization solvent. an ortep drawing of the cation of 4a is shown in fig. 1. the tso molecules were found to be disordered and were omitted for clarity. from this drawing, it is clear that the two molybdenum centers are held together by a dppe ligand. the coordination sphere around the metal center is completed by bridging dppe ligand, cf3ch2o group, and two cyclopentadienyl rings, which are arranged in the form of a distorted tetrahedron. the cyclopentadienyl rings are bound to molybdenum in an η5 fashion and each of the ring carbon atoms are coplanar. thus, complex 4a has geometry typical of bent metallocene. table iii. selected bond distances and angles for 4a bond distance, å bond distance, å mo1–p1 2.541(4) mo1–o1 2.070(10) mo1–c1 2.29(2) mo1–c2 2.33(2) mo1–c3 2.37(3) mo1–c4 2.33(2) mo1–c5 2.31(3) mo1–c6 2.31(2) mo1–c7 2.27(2) mo1–c8 2.35(2) mo1–c9 2.37(2) mo1–c10 2.34(2) o1–c11 1.45(2) c11–c12 1.37(4) bond angle,  bond angle,  p1–mo1–o1 75.5(3) mo1–p1–c13 113.0(5) mo1–p1–c14 111.1(5) mo1–p1–c20 116.9(5) mo1–o1–c11 119.4(10) p1–mo1–c2 132.2(6) the structure can be compared with that of the related alkoxido phosphine complex [cp2mo(pbun3)(cf3ch2o)]+ (2a).1c the mo–p bond distance (2.541(4) å) is remarkably similar to that exhibited by 2a (2.540(4) å). on the other hand, the mo–o bond distance of 2.070(10) å is significantly longer than that found in 2a (2.019(8) å). furthermore, the o–mo–p angle of 75.5(3)° is slightly greater than the corresponding angle of 74.4(3)° found in 2a. the most 52 minato, ito and ren interesting feature of the structure is the observation of the long o–c bond distance of 1.45(2) å, which is considerably longer than the ca. 1.39 å found in 2a. table iv. summary of the crystal structure data for 4a•2cf3ch2oh formula c68h68f12p2o10mo2s2 m / g mol-1 1591.21 crystal system monoclinic space group p21/c (#14) a / å 12.230(5) b / å 11.149(5) c / å 28.966(7) β / ° 101.07(3) v / å3 3876(2) z 2 ρc / g cm -3 1.363 μ / cm-1 4.97 r / wr 0.102/0.143 gof 2.28 t / °c 25 fig. 1. ortep drawing of the cation of complex 4a with thermal ellipsoid plots (40 % probability). the hydrogen atoms have been omitted for clarity. a mechanism accounting for the reaction pathways is proposed in scheme 3 based on literature precedents and the reaction profile. in addition, certain general observations pertained to all reactions between 1 and the tertiary phosphines are included in this study. as mentioned in the introduction, each step in scheme 3 is likely to be reversible. it is well known that most 18-electron complexes undergo ligand substitution reactions via dissociative pathways.9 hence, it is conceivable that the first step in the sequence leading to formation of 2–4 (d) is the new alkoxido dppe complexes 53 dissociation of 1 into the monomeric 16-electron complexes a. in this case, it is possible that it is the resonance limiting form a′, which may be formally viewed as a protonated oxo-complex.10 taking into account the fact that the reaction between 1 and a tertiary phosphine is very susceptible to the co-existing alcohol as noted in the introduction, it seems likely that the next step consists of nucleophilic attack of an alcohol on the metal center. then proton transfer to the hydroxy group occurs to give alkoxido complex b; this process is quite similar to the reported hydrolysis of 1.2 subsequent elimination of water from b produces an unsaturated species c. this dehydration step seems to be a facile process since π-donation by an alkoxido ligand is well established toward early transition metals11 and so the canonical form c′ would make a greater contribution to the hybrid. inevitably, the final step in the mechanism is nucleophilic attack of a tertiary phosphine on the central metal to afford the product d. scheme 3. possible mechanism for the formation of the complexes 2–4. experimental general procedures all manipulations were performed under an inert nitrogen or argon atmosphere using standard schlenk techniques. commercially available reagent grade chemicals (wako chemical) were used as such without any further purification. solvents were purified according to standard procedures. all nmr spectra were recorded on a jeol jnmex-270 spectrometer or a jeol jnmgx-500 spectrometer. 31p{1h}-nmr peak positions were referenced to external h3po4. the di-μ-hydroxido dinuclear complexes [cp2m(μ-oh)2mcp2] 2+ (cp = η-c5h5; m = mo (1a) or w (1b)) were prepared by literature procedures. 1c reaction of 1 with excess dppe a solution containing 1a (0.244 g, 0.294 mmol) and dppe (0.500 g, 1.260 mmol) in cf3ch2oh/c6h6 (1.5 ml/5 ml) was stirred at room temperature for 140 h. during this time, the solution changed from green to red. from the resulting solution, the solvent was evaporated to dryness under reduced pressure. the residue was washed successively with hexane and diethyl ether and then extracted with acetone. the extract was reduced to dryness and the residue was washed with hexane and diethyl ether to yield 3a (0.430 g, 81 %) as an orange– 54 minato, ito and ren –red powder. this procedure was also applicable to the synthesis of the tungsten analogue 3b (yield = 82 %). 3b: anal. calcd. for c45h43f3o4sp2w: c, 55.00; h, 4.41 %. found: c, 54.28; h, 4.53 %. reaction of 1 with 1 equiv. of dppe a solution containing 1a (0.289 g, 0.341 mmol) and dppe (0.135 g, 0.339 mmol) in cf3ch2oh/c6h6 (1.5 ml/1.5 ml) was stirred at room temperature for 15 h. during this time, the solution changed from green to red. from the resulting solution, the solvent was evaporated to dryness under reduced pressure. the residue was washed successively with hexane, diethyl ether, and acetone, and then extracted with ethanol. the extract was reduced to dryness and the residue was washed with hexane and diethyl ether to yield 4a (0.237 g, 50%) as an orange–red powder. purification of the product by recrystallization from cf3ch2oh/ /et2o afforded dark red crystals in the form of flat plates. this procedure was also applicable to the synthesis of the tungsten analogue 4b (yield = 43 %). 4a: anal. calcd. for c64h62f6o8s2p2mo2: c, 55.26; h, 4.49 %. found: c, 55.03; h, 4.53 %. x-ray crystallographic study of 4a a crystal suitable for x-ray crystallography was grown in cf3ch2oh–et2o. the thus obtained dark red crystal was mounted on a glass fiber. measurement was made on a rigaku afc5r diffractometer using mo kα radiation (λ = 0.71068 å) for data collection. the unit -cell parameter was determined by least squares fitting of 25 reflections with a range 21.39 < < 2θ < 25.95°. the parameters used during the collection of diffraction data are given in table iv. the structure was solved and refined using fourier techniques. the non-hydrogen atoms were refined anisotropically. hydrogen atoms were included but not refined. conclusions new trifluoroethoxido phosphine complexes [cp2m(η1-dppe)(cf3ch2o)]+ (3) and [cp2(cf3ch2o)m(μ-dppe)mcp2(cf3ch2o)]2+ (4) were prepared by reactions of the cationic di-μ-hydroxido dinuclear complex of molybdenocene and tungstenocene [cp2m(μ-oh)2mcp2]2+ (1) with dppe. the products of the reactions were identified using 1hand 31p-nmr spectroscopy. in addition, x-ray structural data on 4a clearly established a bridged dimeric structure. it was ascertained that the amount of dppe added to the reaction mixtures could influence the product distribution. thus, the reaction of 1 with dppe in a molar ratio of 1:1 gives complexes 4 as the major products, while good yields of complexes 3 were obtained if a large excess of dppe was added to 1. supplementary material crystallographic data for the structural analysis has been deposited with the cambridge crystallographic data centre, as ccdc reference number 605332. a copy of this information may be obtained free of charge from the director, ccdc, 12 union road, cambridge cb2 1ez, u.k. (fax, +44-1223-336033; e-mail, deposit@ccdc.cam.ac.uk; web, http:// //www.ccdc.cam.ac.uk). acknowledgments. we are grateful to dr. mikio yamasaki of the rigaku corporation for the x-ray structure analysis. we also thank dr. masako tanaka of the tokyo institute of technology for the elemental analysis. the new alkoxido dppe complexes 55 и з в о д испитивање координације 1,2-бис(дифенилфосфино)етана са ди--хидроксидо динуклеарним комплексима волфрама(iv) и молибдена(iv) makoto minato1, takashi ito1 и jian-guo ren2 1 department of materials chemistry, graduate school of engineering, yokohama national university,79-5 tokiwadai, hodogaya-ku, yokohama, 240-8501, japan и 2 department of chemistry, shanxi university, taiyuan, 030006, china у реакцији катјонског ди--хидроксидо комплекса молибденоцена или волфрамоцена [cp2m(μ-oh)2mcp2] 2+ са dppe лигандом синтетисани су нови трифлуороетоксидо-фосфински комплекси, [cp2m(η 1-dppe)(cf3ch2o)] + и [cp2(cf3ch2o)m(μ -dppe)mcp2(cf3ch2o)] 2+ (m = mo или w, cp = η-c5h5 и dppe = ph2pch2ch2pph2). нађено је да дистрибуција реакционих производа зависи од количине додатог dppe лиганда. дискутован је механизам испитиване реакције који у првој фази укључује супституцију хидроксидо лиганда са алкоксидо групом, а затим у другој фази долази до нуклеофилног напада фосфинског лиганда. (примљено 1. маја, ревидирано 28. јула, прихваћено 29. јула 2015) references 1. a) j.-g. ren, h. tomita, m. minato, k. osakada, t. ito, chem. lett. (1994) 637; b) m. minato, j. ren, h. tomita, t. tokunaga, f. suzuki, t. igarashi, t. ito, j. organomet. chem. 473 (1994) 149; c) j.-g. ren, h. tomita, m. minato, k. osakada, m. yamasaki, t. ito, organometallics 15 (1996) 852 2. a) c. balzarek, d. r. tyler, angew. chem. int. ed. 38 (1999) 2406; b) c. balzarek, t. j. r. weakley, l. y. kuo, d. r. tyler, organometallics 19 (2000) 2927; c) k. l. breno, d. r. tyler, organometallics 20 (2001) 3864 3. a) l. y. kuo, t. j. r. weakley, k. awana, c. hsia, organometallics 20 (2001) 4969; b) l. y. kuo, d. m. finigan, n. n. tadros, organometallics 22 (2003) 2422 4. k. l. breno, m. d. pluth, d. r. tyler, organometallics 22 (2003) 1203 5. j.-g. ren, z.-w. wang, z.-m. zhang, d.-s. liu, m. minato, t. ito, chin. j. appl. chem. 17 (2000) 81 6. m. minato, j.-g. ren, m. kasai, k. munakata, t. ito, j. organomet. chem. 691 (2006) 282 7. t. aviles, m. l. h. green, a. r. dias, c. romao, j. chem. soc., dalton trans. (1979) 1367 8. for complexes 3a and 4b, no samples suitable for combustion analyses are available at present. 9. j. p. collman, l. s. hegedus, j. r. norton, r. g. finke, principles and applications of organotransition metal chemistry, university science books, mill valley, ca, 1987, p. 247 10. a) m. l. h. green, a. h. lynch, m. g. swanwick, j. chem. soc., dalton trans. (1972) 1445; b) n. d. silavwe, m. y. chiang, d. r. tyler, inorg. chem. 24 (1985) 4219 11. j. c. huffman, k. g. moloy, j. a. marsella, k. g. caulton, j. am. chem. soc. 102 (1980) 3009. a novel approach toward the synthesis of some new tridentate schiff bases from anil-like compounds j. serb. chem. soc. 81 (10) 1111–1119 (2016) udc 542.9+547.571’551:547.553.1+ jscs–4911 547.564.4+547.822.1:547.781’821 original scientific paper 1111 a novel approach toward the synthesis of some new tridentate schiff bases from anil-like compounds fatemeh bagheri and abolfazl olyaei* department of chemistry, payame noor university, p. o. box 19395-3697, tehran, iran (received 2 december 2015, revised 19 june, accepted 20 june 2016) abstract: a novel method was developed for synthesizing a series of new tridentate schiff base ligands starting from hydroxynaphthyl pyrimidinyl amines with o-phenylenediamines or o-aminophenol or 2-amino-3-hydroxy-pyridine in the presence of formic acid catalyst under solvent-free conditions. in these reactions [1+1], the condensation product as half-unit ligand was obtained. moreover, the reaction of hydroxynaphthylmethylene pyrimidinyl amines with 3,4-diaminopyridine and 1,8-naphthalenediamine leads to the formation of c2-naphthylated imidazopyridine and dihydropyrimidine, respectively. the attractive features of this protocol are use of an inexpensive catalyst, operational simplicity, short reaction times, easy handling, and good yields. keywords: schiff base; anil; o-phenylenediamine; o-aminophenol; dihydropyrimidine; imidazopyridine. introduction condensation of carbonyl compounds with primary amines was discovered in 1864 by hugo schiff.1 this acid-catalyzed reaction is universal and enables a broad variety of azomethines to be obtained. schiff bases are known to show biological activities, such as antimicrobial,2–5 antifungal6 and antitumor,7 and are used in the production of dyes and pigments.8 moreover, schiff bases are also employed as chelating agents9 capable of coordinating metal ions to give complexes that serve as models for biological systems.10,11 2-hydroxy schiff base ligands, derived from the reaction of salicylaldehyde and 2-hydroxy-1-naphthaldehyde with amines, and their complexes have been extensively studied.12–14 2-hydroxy schiff base ligands are of interest mainly due to the existence of (o–h…n and n–h…o) type hydrogen bonds and tautomerism between the enol–imine and keto–enamine forms. tautomerism in orthohydroxy naphthylmethylene anilines (anils) in solution and in the solid state has been investigated by different spectroscopic techniques.15–17 * corresponding author. e-mail: olyaei_a@pnu.ac.ir doi: 10.2298/jsc151202064b _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1112 bagheri and olyaei in recent years, environmentally safe synthetic methods have received considerable attention, and some solvent-free protocols have been developed. the catalyzed synthesis of imines under solvent-free conditions may be performed by microwave irradiation, ionic liquids, montmorillonite clays,18 zeolites, silica, alumina or other matrices.19,20 however, ortho-hydroxy naphthylmethyleneanilines were synthesized by refluxing in organic solvents such as ethanol and microwave irradiation of naphthaldehyde and amines. in continuation of ongoing research for the development of simple and efficient methods for the synthesis of various heterocyclic compounds under solventfree conditions21–26 and interest in anils derived from naphthols, triethyl orthoformate and heteroaryl amines,25 it was decided to prepare some new schiff bases from hydroxynaphthylmethylene pyrimidinyl amines with o-phenylenediamines or o-aminophenol or 2-amino-3-hydroxypyridine under solvent-free conditions. results and discussion previously, the synthesis of 1-((pyrimidin-2-ylimino)methyl)naphthalene -2,7-diol (1a) from 2,7-dihydroxynaphthalene, triethyl orthoformate and 2-aminopyrimidine in the presence of formic acid catalyst under solvent-free conditions was reported.25 in continuation of this work, the scope of this approach was explored for the synthesis of 1-((pyrimidin-2-ylimino)methyl)naphthalen-2 -ol (1b) and 2-((pyrimidin-2-ylimino)methyl)naphthalen-1-ol (1c), scheme 1. scheme 1. synthesis of hydroxynaphthylmethylene pyrimidinyl amines 1b and 1c. initially, to synthesize compound 1b, the reaction of 2-aminopyrimidine (1.0 mmol), triethyl orthoformate (1.0 mmol) and β-naphthol (1.0 mmol), in the presence of formic acid catalyst at 120 °c under solvent-free condition was performed and compound 1b was afforded in 85 % yield. the same reaction with α-naphthol instead of β-naphthol afforded compound 1c in 88 % yield. the 1h-nmr spectra of compounds 1b and 1c showed two doublet signals for the hydroxyl and ch-imine protons corresponding to strong intramolecular hydrogen bond (o–h…n), as expected. in the 13c-nmr spectra of compounds 1b and 1c, the signals at δ 184.19 and 183.55 ppm, corresponding to the carbonyl _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. synthesis of tridentate schiff bases 1113 group, indicated that these anil-like compounds favor the keto–enamine form over the enol–imine form. next, the reactions of hydroxynaphthylmethylene pyrimidinyl amines 1a–c with o-phenylenediamines in the presence of formic acid catalyst under solventfree conditions were studied. for initial feasibility studies, the reaction of o-phenylenediamine with compound 1b was selected as a model reaction. to optimize further the reaction temperature, the two reagents were allowed to react at temperatures ranging from 80 to 150 °c under solvent-free conditions. the results are given in table i. the yield of product 2b was increased, and the reaction time was shortened as the temperature increased from 80 to 110 °c (table i, entries 1–3). however, further increase of the temperature from 110 to 150 °c failed to improve the yield of product 2b (table i, entries 4 and 5). therefore, 110 °c was chosen as the reaction temperature for all further reactions under solvent-free conditions. in this reaction, the [1+1]-condensation product was obtained instead of c2-naphthylated benzimidazole in 80 % yield. in order to gauge the scope of these conditions, o-phenylenediamines and hydroxynaphthylmethylene pyrimidinyl amines were examined under the optimized conditions. the results indicate that the reaction proceeded smoothly via [1+1]-condensation to give the corresponding half-unit ligands 2 in good yields (scheme 2). table i. temperature optimization for the synthesis of 2b entry temperature, °c time, min yield, % 1 80 60 65 2 100 50 72 3 110 35 80 4 120 35 70 5 150 35 54 identification of 2a–d was realized based on the 1h-nmr, 13c-nmr, ir, and mass spectra, and elemental analysis. the corresponding data are given in supplementary material to this paper. it is very important to mention here that the 1h-nmr spectra of compounds 2a and 2d showed two doublet signals for the hydroxyl and ch-imine protons at about δ 15.0 and 9.0 ppm corresponding to a strong intramolecular hydrogen bond (o–h…n), as expected. in the spectrum of compound 2d, the nh proton was observed at about δ 7.06 ppm. upon addition of d2o into the nmr tube containing 2d, the signals of nh2, nh and oh disappeared and the signals of the ch-imine moieties collapsed into a single sharp singlet. therefore, the schiff base 2a favors the enol–imine form over the keto– –enamine form whereas both forms, the enol–imine and keto–enamine form, were observed in the schiff base 2d (scheme 3). the 1h-nmr spectra of compounds 2b and 2c showed two singlet signals for the hydroxyl and ch-imine protons at about δ 15.0 and 9.6 ppm, respectively. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1114 bagheri and olyaei scheme 2. synthesis of tridentate schiff base ligands 2a–d. it was suggested that a weak intramolecular hydrogen bond (o–h…n) formed and resonance caused only deshielding of chemical shift of the oh proton. in addition, in the spectrum of compound 2b, an nh proton at about δ 7.0 ppm was observed, but it is not observed in the spectrum of compound 2c. therefore, as in compound 2d, both the enol–imine and keto–enamine form were observed in the schiff-base 2b but in compound 2c the enol–imine form was favored over the keto–enamine form. the results of the reaction of hydroxynaphthylmethylene pyrimidinyl amines with o-phenylenediamines are summarized in table ii. scheme 3. tautomeric equilibrium in compound 2d. table ii. synthesis of compounds 2 entry product time, min yield, % 1 2a 25 84 2 2b 35 80 3 2c 30 78 4 2d 20 88 _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. synthesis of tridentate schiff bases 1115 based on the experimental results, a possible mechanism for the formation of compound 2b is presented in scheme 4. initially, the reaction proceeds via the formation of intermediate 3, which is formed in situ by michael addition of o-phenylenediamine to the activated keto–enamine form of the compound 1b. this intermediate undergoes de-amination to afford the corresponding product 2b. scheme 4. proposed mechanism for the synthesis of compound 2b. during the course of this work, the reaction of hydroxylnaphthylmethylene pyrimidinyl amines 1b and 1c with o-aminophenol and 2-amino-3-hydroxypyridine in the presence of formic acid catalyst under solvent-free conditions was studied. by evaluation of the temperature, it was observed that 110 °c is an effective temperature in terms of reaction time and obtained yield (scheme 5). the results are summarized in table iii. scheme 5. synthesis of schiff base ligands 4a–c. table iii. synthesis of schiff base derivatives 4 entry product time, min yield, % 1 4a 25 88 2 4b 20 85 3 4c 25 81 in these reactions, the [1+1]-condensation product was obtained instead of c2-naphthylated benzoxazoles/oxazolopyridine in 81–88 % yield. identification _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1116 bagheri and olyaei of 4a–c was realized based on 1h-nmr, 13c-nmr, ir and mass spectra and elemental analysis (supplementary material). the 1h-nmr spectra of compounds 4a–c showed two doublet signals for the hydroxyl and ch-imine protons at about δ 14.5 and 9.0 ppm, respectively. moreover, in compound 4b, an nh signal was observed at about δ 7.0 ppm. it is suggested that both the enol–imine and keto– –enamine form are observed in the schiff base 4b. on the other hand, the enol– –imine form is favored over the keto–enamine form in compounds 4a and 4c. in addition, a strong intramolecular hydrogen bond (o–h…n) was observed in these compounds. when the reaction of 3,4-diaminopyridine with anil-like 1b was conducted under the same reaction conditions, the c2-naphthylated imidazopyridine 6 was obtained in good yield (80 %) after 35 min. a mechanistic rationalization for this reaction is provided in scheme 6. similar to condensation of o-phenylenediamines with compound 1b, the [1+1]-condensation product 2e is formed in the initial step. in the second step, the reaction proceeds via dihydroimidazopyridine 5, which was formed by the intramolecular nucleophilic addition of the amino group to the activated imine group. finally, the intermediate 5 was oxidized to the corresponding imidazopyridine 6 by air oxygen (scheme 6). in addition, the 1h-nmr spectrum of compound 6 exhibited both the enol–imine form and the keto–enamine form. scheme 6. proposed mechanism for the synthesis of compound 6. next, the scope of this domino reaction of compound 1c with 1,8-naphthalenediamine was explored under the optimized conditions. the results indicated that compound 2-(2,3-dihydro-1h-perimidin-2yl)naphthalen-1-ol (7) was synthesized in 81 % yield (scheme 7). scheme 7. synthesis of compound 7. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. synthesis of tridentate schiff bases 1117 experimental all commercially available chemicals and reagents were used without further purification. the melting points were determined with an electrothermal model 9100 apparatus and are uncorrected. the ir spectra were recorded on a shimadzu 4300 spectrophotometer. the 1hand 13c-nmr spectra were recorded in dmso-d6 on a bruker drx-300 avance spectrometer. the chemical shifts (δ) are reported in parts per million and are referenced to the nmr solvent. the mass spectra of the products were obtained with a hp 5973 (agilent technologies) mass selective detector. elemental analyses were realized using a chn–o–rapid heraeus elemental analyzer (wellesley, ma). general procedure for the synthesis of compounds 1b and 1c. formic acid (98 % aqueous solution, 0.1 mmol) was added to a mixture of 2-aminopyrimidine (1 mmol), triethyl orthoformate (1 mmol), and 1-naphthol or 2-naphthol (1 mmol). the reaction mixture was magnetically stirred on a preheated oil bath at 120 °c for 15 min. the progress of the reaction was monitored by thin-layer chromatography (tlc). after completion of the reaction, the reaction mixture was cooled to room temperature, and ch3cn (5 ml) added. the precipitate was filtered, washed with cold ch3cn, dried, and purified by recrystallization from ch3cn to give the hydroxynaphthylmethylene pyrimidinyl amines 1b and 1c as colored crystals. the characterization data for 1b and 1c are given in the supplementary material to this paper. general procedure for the synthesis of compounds 2a–d, 4a–c, 6 and 7. formic acid (98 % aqueous solution, 0.1 mmol) was added to a mixture of compounds 1a or 1b or 1c and o-phenylenediamines (1 mmol) or o-aminophenol (1 mmol) or 2-amino-3-hydroxypyridine (1 mmol). the reaction mixture was magnetically stirred on a preheated oil bath at 110 °c for the appropriate time. the progress of the reaction was monitored by thin-layer chromatography (tlc). after completion of the reaction, the crude product was purified by column chromatography using ethyl acetate/hexane to give the corresponding schiff bases 2a–d, 4a–c and products 6 and 7. the characterization data for 2a–d, 4a–c, 6 and 7 are given in the supplementary material to this paper. conclusions in conclusion, a simple and efficient procedure for the synthesis of new anils using hydroxynaphthylmethylene pyrimidinyl amines with o-phenylenediamine or o-aminophenol or 2-amino-3-hydroxypyridine under solvent-free conditions was demonstrated. the reaction proceeds by [1+1]-condensation and the products were obtained in good yields, showing that the synthetic route allows blocks of half-unit ligand of the new schiff-base derivatives to be built. moreover, the simple experimental procedure combined with the easy workup and shorter reaction time, are salient features of the presented method. supplementary material analytical and spectral data of the synthesized compounds are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1118 bagheri and olyaei и з в о д нов приступ синтези нових тридентатних шифових база из једињења типа анила fatemeh bagheri и abolfazl olyaei department of chemistry, payame noor university, po box 19395-3697, tehran, iran развијен је нов поступак за синтезу тридентатних шифових база као лиганада, полазећи од хидроксинафтилметиленпиримидинилимина и o-фенилендиамина или o-аминофенола или 2-амино-3-хидроксипиридина у присуству мравље киселине као катализатора, у одсуству растварача. овим реакцијама добијен је производ [1+1] кондензације. осим тога, реакцијом хидроксинафтилметиленпиримидинилимина са 3,4-диаминопиридином и 1,8-нафталендиамином настају c2-нафтил-имидазопиридини и дихидропиримидини, редом. предности оваквог приступа синтези су: приступачан и јефтин катализатор, једноставност поступка, кратко реакционо време и добар принос. 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(2009) 656 21. a. olyaei, m. karbalaei karimi, r. razeghi, tetrahedron lett. 54 (2013) 5730 22. a. olyaei, b. shams, m. sadeghpour, f. gesmati, z. razaziane, tetrahedron lett. 51 (2010) 6086 23. a. olyaei, m. vaziri, r. razeghi, tetrahedron lett. 54 (2013) 1963 24. a. olyaei, m. ghodrat alidoust, synth. commun. 45 (2015) 94 _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. synthesis of tridentate schiff bases 1119 25. a. olyaei, f. gesmati, m. sadeghpour, b. shams, m. alizadeh, synth. commun. 42 (2012) 1650 26. a. olyaei, m. vaziri, r. razeghi, b. shams, h. bagheri, j. serb. chem. soc. 78 (2013) 4. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. << /ascii85encodepages false 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {experimental measurements and modeling of solvent activity and surface tension of binary mixtures of polyvinylpyrrolidone in water and ethanol} j. serb. chem. soc. 82 (4) 427–435 (2017) udc 544.275–188:541.123.2:54–145.2+ jscs–4978 547.262:547.745–036.7:544–971 original scientific paper 427 experimental measurements and modelling of the solvent activity and surface tension of binary mixtures of poly(vinyl pyrrolidone) in water and ethanol majid taghizadeh* and saber sheikhvand amiri chemical engineering department, babol noshirvani university of technology, p. o. box 484, babol 4714871167, iran (received 5 may 2016, revised 30 january, accepted 7 march 2017) abstract: in this paper, the density (ρ), viscosity (η) and surface tension (σ) of solutions of poly(vinyl pyrrolidone) (pvp) with molecular weights of 25000 (k25) and 40000 g mol-1 (k40) in water and ethanol were measured in the temperature range 20–65 °c and at various mass fractions of polymer (0.1, 0.2, 0.3 and 0.45). the solvent activity measurements were performed at 45 and 55 °c. thereafter, two thermodynamic models for predicting the solvent activity and surface tension of binary polymer mixtures (pvp in water and ethanol) were proposed. the flory–huggins theory and eyring model were employed to calculate the surface tension of the solution and the solvent activity, respectively. additionally, the proposed activity model was dependent on the density and viscosity of the solution. afterwards, the ability of these models at various temperatures and mass fractions were investigated by comparing the results with the experimental data. the results confirmed that, in the investigated temperature range, these models have good accuracy. keywords: poly(vinyl pyrrolidone); solvent activity; surface tension; thermosdynamic model. introduction considering the importance of density, viscosity and the activity of pure liquids and mixtures in various fields of chemical engineering, such as, environmental engineering, pharmaceuticals, paint and detergents, lubricants, catalytic reactions, liquid extraction, petroleum refining and other processes involving heat and mass transfer, measuring these parameters and providing a model in a temperature range with an acceptable response have been discussed in the literature.1–9 in recent years, aqueous polymer solutions have exhibited a wide range of applications in different industries. poly(vinyl pyrrolidone) (pvp) is one of the * corresponding author. e-mail: m_taghizadehfr@yahoo.com doi: 10.2298/jsc160505028t 428 taghizadeh and amiri most important polymers, which is used as a blood plasma expander due to its low toxicity.2,3 surface tension is one of the important properties of binary polymeric mixtures in various fields of applied science. surface tension is a physical property that is closely related to the molecular composition and structure.10–12 surface tension of pure fluids and mixtures can usually be measured directly, but the lack of resources and facilities makes the experimental measurements of the surface tension of a liquid difficult and impractical. therefore, an estimation method for the surface tension of polymeric mixtures is required if it is not possible to determine reliable experimental values.13,14 the surface tension of liquids can be measured by the drop-weight method,15 the wilhelmy plate or ring method,16,17 and the maximum bubble pressure method.18 the ring method is one of the most common methods used for the measurement of surface tension. the advantages of the ring method are that it is fast and simple and does not require calibration using solutions with known surface tension. many models have been developed to predict the thermodynamic properties of binary mixtures. some researchers proposed theoretical methods to calculate the surface tension based on thermodynamics.19 egemen et al.20 proposed a group contribution method to predict the surface tension of liquid organic solvents. polymer solution thermodynamics usually has its origin in the thermodynamics of mixtures of quasi-spherical molecules. this field has been proliferating along with polymer activity models.21–24 the activity of a pure substance, or of a specified component in a solution, is commonly defined in terms of fugacity, the activity of the material or component being the ratio of its fugacity in the given state to that in some “standard state” at the same temperature.25 solvent activity is an effective parameter in various fields and processes, such as the prediction of the crystallization of solids that have water of hydration. there are several methods to measure the solvent activity of mixtures, such as freezing point depression, boiling point elevation, dynamic and static vapour pressure measurements, osmotic pressure measurements, hygrometric, vapour sorption, isopiestic method and vapour pressure osmometry (vpo). among the methods mentioned, vapour pressure osmometry is a precise technique to determine the solvent activity of solutions.26 in this study, the density, viscosity and surface tension of solutions of poly(vinyl pyrrolidone) (pvp) with molecular weights of 25,000 (k25) and 40,000 g mol–1 (k40) in water and ethanol were measured in the temperature range 20–65 °c. solvent activity measurements were performed at 45 and 55 °c using the vapour pressure osmometry (vpo) technique. all measurements were performed in binary systems containing of 0.1, 0.2, 0.3 and 0.45 mass fractions of polymer. for the systems studied, two thermodynamic models were also developed for calculating the solvent activity, and the surface tension of pvp solutions based on physicochemical properties of polyvinylpyrrolidone solutions 429 eyring model27 and flory–huggins28 theory, respectively. the activity model was dependent on the density and viscosity of solution. next, the surface tensions of solutions and solvent activities for polymer/solvent mixtures were calculated and compared with the experimental values. experimental materials poly(vinyl pyrrolidone) (pvp) with molecular weights of 25000 and 40000 g mol-1 and absolute ethanol gr (>99 %), manufactured by merck, were used in this study. doubledistilled water was used to produce the solutions. apparatus and procedures the solutions were prepared by mass, using sartorius balance with an accuracy of ±0.1 mg. the density, viscosity, surface tension and activity measurements were realised using a pycnometer, a dv-ii + pro digital viscometer manufactured by brookfield (usa), a digital tensiometer kruss k9 (hamburg, germany), and a vapour pressure osmometer (vop) (knauer, germany), respectively. the tensiometer, which was equipped with a du noüy platinum–iridium ring (6 mm circumference), was calibrated with distilled water (σ = 72.8 mn m-1). a constant temperature water bath was used to control the temperature of the solutions with an accuracy of ±0.1 °c. measurements for each solution were repeated five times. the density, viscosity, activity and surface tension of the pvp solutions in water and ethanol were measured at different temperatures and concentrations, and the obtained results were correlated with quadratic equations relating the density and viscosity of the solutions to the mass fraction of the polymer. several types of errors, i.e., the relative error (re), average relative error (are), the average absolute relative error (aare) and root mean squared error (rmse), were calculated for evaluating the performance of the proposed model (supplementary material to this paper). density density measurements were performed using a pycnometer, the bubble volume of which was 25 cm3, at temperatures of 20, 25, 30, 35, 40, 45, 50 and 55 °c. each measurement was repeated three times and the average results of the density and their corresponding standard deviation values are reported. the following equation was used to determine the density of solution using the experimental data: 2a bw cwρ = + + (1) where w is the mass fraction of polymer, ρ is density (g cm-3), and a, b and c are adjustable parameters that were obtained by linear multiple regression using statica 7 software. the measured densities of the solutions, the coefficients of eq. (1) along with its average relative error (are), coefficient of determination (r2), and root mean squared error (rmse) are reported in table s-i of the supplementary material. as can be seen in this table, the standard deviation values of the results were relatively low and in the range of 0.001–0.003 (g cm-3), which show the good reproducibility and accuracy of the experiments. viscosity the viscosity measurements were performed three times using the dv-ii + pro digital viscometer manufactured by brookfield (usa) and the average results and their corresponding standard deviation values are reported. the following equation was used to determine the viscosity of the solutions using the experimental data: 430 taghizadeh and amiri 2 3a b w c w d wη ′ ′ ′= ′ + + + (2) where w is the mass fraction of polymer, η is viscosity (mpa s), and a′, b′, c′ and d′ are adjustable parameters that were obtained by linear multiple regression using statica7 software. the measured viscosities of the solutions, the coefficients of eq. (2) along with its average relative error, coefficient of determination, and root mean squared error are reported in table s-ii of the supplementary material. as can be seen in this table, the standard deviation values of the results were relatively low and in the range of 0.1–37 (mpa s), which show the good reproducibility and accuracy of the experiments. solvent activity the activity of the solvent in the polymer solution was measured using the vapour pressure osmometry (vop, knauer, germany) method at 45 and 55 °c and at different mass fractions of polymer (0.1, 0.2, 0.3 and 0.45). before beginning each experiment, the calibration constant of the instrument was determined using a urea–water solution and a phenylmethanol–ethanol solution. the calibration constants had different values at different temperatures and had to be determined for each temperature. two thermistors covered with platinum screens pieces were placed in the measuring chamber. in the chamber, a container of solvent provided a saturated solvent atmosphere around thermistors by using two wicks. usually, if a droplet of solvent is dripped on one of the thermistors and a droplet of solution i dripped on the other thermistor, δv begins to rise. after reaching a state of quasi-equilibrium, it remains almost unchanged. temperature differences between the two thermistors were reported as voltage differences. the activity was calculated using the following equation:29 1 1 δ ln 1000 vm a k = − (3) where a1 is the activity of the solvent, δv is the voltage difference, m1 is the molecular weight of the solvent and k is the calibration constant. each measurement was repeated 3 to 5 times, and the mean of the voltage difference is reported. the accuracy of the measured voltage difference was 0.1 mv. surface tension the surface tension measurements were conducted using a kruss k9 digital tensiometer (hamburg, germany). this instrument comprises a precise microbalance and an accurate mechanism to move vertically the liquid sample in a glass beaker. here, the surface tension of the polymer solutions was measured by the ring method (du nouy method). in the ring method, a mechanical force is required to lift a ring from the liquid surface. the ring is usually made of an alloy of platinum and iridium. measurements were performed at different temperatures using a warm-water bath; the temperature of the sample was kept at equilibrium. each measurement was performed three times and the average results of the surface tension and their corresponding standard deviation values are reported. thermodynamic models for solvent activity and surface tension are presented in the supplementary material to this paper. results and discussion solvent activity the proposed thermodynamic model for calculating the solvent activity is a function of mole fraction, mass fraction, density (or specific volume) and visco physicochemical properties of polyvinylpyrrolidone solutions 431 sity of the solution. the density and viscosity of the solutions are expressed as a function of mass fraction of polymer (in the form of a mathematical model). to calculate the solvent activity of the polymer mixtures at the mentioned temperatures and mass fractions, the density and viscosity of the solutions have to be accurately determined because they are crucial for obtaining good results with the proposed model. the experimental values have been used to validate the model. first, the solvent activities in the polymer–solvent systems were measured by the osmometry method. then the solvent activities were calculated by the model and the results were compared with the experimental data. comparisons between the experimental and calculated solvent activities for various mass fractions at temperatures of 45 and 55 °c are presented in figs. 1 and 2. solvent activity values measured for pvp solutions at different tempera0.95 0.97 0.99 1.01 0 0.1 0.2 0.3 0.4 a 1 w exp. t = 45 °c exp. t = 55 °c model t = 45 °c model t = 55 °c (a) 0.95 0.97 0.99 1.01 0 0.1 0.2 0.3 0.4 a 1 w exp. t = 45 °c exp. t = 55 °c model t = 45 °c model t = 55 °c (b) fig. 1. solvent activity in (a) k25–water; and (b) k25–ethanol solutions at 45 and 55 °c. 0.95 0.97 0.99 1.01 0 0.1 0.2 0.3 0.4 a 1 w exp. t = 45 °c exp. t = 55 °c model t = 45 °c model t = 55 °c (a) 0.95 0.97 0.99 1.01 0 0.1 0.2 0.3 0.4 a 1 w exp. t = 45 °c exp. t = 55 °c model t = 45 c model t = 55 c (b) fig. 2. solvent activity in (a) k40–water, (b) k40–ethanol solutions at 45 and 55 °c. 432 taghizadeh and amiri tures and along with their relative errors and average absolute relative errors are reported in table s-iii of the supplementary material. as seen in figs. 1 and 2, the solvent activities decreased significantly with increasing weight percentage of the polymer in the range 0.3–0.45. these results showed that the proposed model could predict with good accuracy the solvent activity in the mass fraction range of 0.1 to 0.3. surface tension the surface tensions of polymer solutions were measured using a digital tensiometer at different temperatures and mass fractions of polymer. the surface tension values and relative error percentages along with their average absolute relative errors at various temperatures are given in table s-iv of the supplementary material. a unifac model and the flory–huggins solution theory were applied to develop a thermodynamic model to predict the surface tensions of the pvp solutions. comparisons of the experimental and theoretical results for the pvp–solvent systems are shown in figs. 3 and 4. in addition, the trends in the evolution of the surface tension with temperature at various mass fractions are illustrated. 45 47 49 51 53 55 57 59 61 63 65 15 20 25 30 35 40 45 50 55 σ / m n m −1 t / °c exp. w = 0.1 exp. w = 0.2 exp. w = 0.3 model w = 0.1 model w = 0.2 model w = 0.3 (a) 15 17 19 21 23 25 27 29 15 20 25 30 35 40 45 50 55 σ / m n m −1 t / °c exp. w = 0.1 eep. w = 0.2 exp. w = 0.3 model w = 0.1 model w = 0.2 model w = 0.3 (b) fig. 3. surface tension of (a) k25–water; and (b) k25–ethanol solutions at various temperatures. on increasing the temperature, the force between the molecules is reduced and less force is required to pull the molecules into the liquid. thus, the surface tension of the liquid is reduced. moreover, the surface tension can vary due to chemical substances in contact. as could be seen in figs. 3 and 4, the surface tension decreased with increasing the mass fraction of polymer. the experimental results and the results obtained from the proposed model confirmed the inverse relationship between temperature and mass fraction with the surface tension of the solution. physicochemical properties of polyvinylpyrrolidone solutions 433 15 17 19 21 23 25 27 29 31 33 15 20 25 30 35 40 45 50 55 6 σ / m n m −1 t / °c exp. w = 0.1 exp. w = 0.2 model w = 0.1 model w = 0.2 (b) fig. 4. surface tension of (a) k40–water; and (b) k40–ethanol solutions at various temperatures. conclusions in this study, thermodynamic modelling and experimental procedures were used to study the activity and surface tension of binary polymer mixtures. first, the density, viscosity and surface tension of solutions, and solvent activities of poly(vinyl pyrrolidone) with molecular weights of 25000 and 40000 g mol–1 in water and ethanol were measured at different temperatures and mass fractions. then, two quadratic equations were presented to determine the density and viscosity of solutions as a function of mass fraction. these equations were used to present a thermodynamic model for the prediction of solvent activities. this model was developed based on the eyring viscosity model. next, a thermodynamic model based on the flory–huggins theory was presented for the prediction of the surface tension of the solutions. the results obtained from the surface tension and activity models for polymer/solvent mixtures were compared with the experimental results. the overall average absolute relative errors of the models for solvent activities and surface tensions of the solutions were estimated to be 0.35 and 5.69 %, respectively. these results show that the proposed thermodynamic models were able to predict the surface tension and activity of the solvent with good accuracy. nomenclature a molar surface area (cm2 mol-1) a activity of solvent g*e excess gibbs free energy (j mol-1) m1 molecular weight of solvent (g mol-1) q unifac parameter r gas constant (j mol-1 k-1) t absolute temperature (k) w mass fraction of polymer x mole fraction of component γ activity coefficient of solvent 434 taghizadeh and amiri δv voltage difference (mv) δ solubility parameter (j1/2 cm-3/2) η viscosity of solution (mpa s) μυ standard chemical potential (j mol-1) υ molar volume of solution (cm³ mol-1) υk number of group k ρ density of solution (g cm-3) σ surface tension of solution (mn m-1) ϕ volume fraction χ interaction parameter between polymer and solvent supplementary material experimental data and theoretical models are available electronically at the pages of the journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. и з в о д експериментално одређивање и моделовање активности растварача и површинских напона бинарних смеша поли(винилпиролидона) у води и етанолу majid taghizadeh и saber sheikhvand amiri chemical engineering department, babol noshirvani university of technology, p. o. box 484, babol 4714871167, iran у овом раду су приказани резултати експерименталног мерења густина (ρ), вискозности (η) и површинских напона (σ) смеша поли(винилпиролидона) (pvp), моларних маса 25000 (к25) и 40000 g mol-1 (к40), у води и етанолу у температурном опсегу 20–65 °c и при различитим масеним уделима полимера (0,1, 0,2, 0,3 и 0,45). мерења површинског напона вршена су на 45 и 55 °c. такође, предложена су два термодинамичка модела за предвиђање активности растварача и површинског напона бинарних смеша полимера (pvp у води и етанолу). flory–huggins и eyring модели коришћени су за израчунавање површинског напона раствора и активности растварача. предложени модели зависе од вредности густине и вискозности раствора. поређењем са експерименталним подацима испитивана је применљивост ових модела на већем броју температура и више масених удела. потврђено је да у испитиваном температурном опсегу наведени модели дају добре резултате. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {the ph measurement with glass electrode in electromagnetic field} j. serb. chem. soc. 81 (12) 1407–1414 (2016) udc 544.354.3.08+544.6.076.32–033.5: jscs–4937 537.8 original scientific paper 1407 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. the ph measurement with glass electrode in an electromagnetic field dragan veselinović1 and zoran velikić2* 1faculty of physical chemistry, university of belgrade, p. o. box 137, 11000 belgrade, studentski trg 12–16, serbia and 2institute of physics, university of belgrade, p. o. box 68, pregrevica 118, 11080 zemun, serbia (received 7 april, revised 13 september, accepted 20 september 2016) abstract: measurements of ph values of buffer solutions (ph 4.0, 7.0 and 10.0) and distilled water were performed with a glass electrode in an electromagnetic field in the frequency interval of 10–200 mhz and an output power of dispersed and reflected electromagnetic radiation of 0.01–3 w. in all the cases, a reduction of ph values occurred, i.e., a “recorded ph value” was obtained. the reduction appeared within the applied frequency interval, reaching extreme values at specific frequencies. the reduction of the ph values increased with the radiation power and depended of the solution buffer capacity. the effect of electromagnetic field on ph value change was exerted dominantly through the influence on glass electrodes. keywords: recorded ph value; frequency; power. introduction there are data in the literature on the influence of high frequency electromagnetic fields on various systems, causing changes of certain parameters of the systems. in stripping voltammetry, the anode peak currents of cd(ii), zn(ii), pb(ii) and mn(ii) are changed in the frequency range of 0–200 mhz.1 in solutions for reverse voltammetry that contain n-butanol, the peak currents of cd(ii), pb(ii) and zn(ii) were increased due to n-butanol desorption from the electrode2 as well as to the influence of the electromagnetic field. a radiofrequency electromagnetic field caused the disassembly of water molecules in water vapor bubbles.3 a high frequency electromagnetic field (2450 mhz) is phytotoxic4 and enabled the suppression of weeds.5 electromagnetic fields changed the critical micelle content and the values of thermodynamic functions in a solution of sodium dodecyl sulfate6 at frequencies of 60–100 mhz. investigations of the * corresponding author. e-mail: velikic@ipb.ac.rs doi: 10.2298/jsc160407082v 1408 veselinović and velikić available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. influence of strong electromagnetic fields formed around long-distance power lines with voltage interval 66,000–220,000 v on plants, significantly changed the fertility and production of pollen grain,7 along with other changes. it was found that high frequency magnetic fields of 30–200 mhz affected the adsorption characteristics of inorganic surface-active substances.8 water dissociation was also investigated in a radiofrequency electromagnetic field (13.56 mhz).9 previous investigations indicated that variations of both the frequency10 and the field power11 led to changes in the recorded ph values when a glass electrode was used. since ph measurement with this electrode currently prevails, better knowledge of the influence a high frequency electromagnetic field on these measurements is necessary. experimental the solution ph was measured with an iskra ma 5730 ph-meter with electronics without radio lamps. three combined glass electrodes (consisting of both glass and reference electrodes) as well as two glass and two reference electrodes used separately were applied in these measurements. the glass electrodes (five) were produced by five different manufacturers and differed in previous usage duration. three standard buffer solutions (entech instruments), with ph 4.0, 7.0 and 10.0 (at 25 °c) were used. for diluting the buffer solutions, when necessary, distilled water was used, always from the same batch. the ph of the water was also measured. for the measurements of solution ph values, with the both electrode types, a glass cell (fig. 1) was used. the part of the cell (1) is in an electromagnetic field, spreading between two copper plaques(2a and 2b), dimension 38 mm×20 mm, connected to an electromagnetic field source. the second part of the cell (1a) was not in the electromagnetic field. the diameters of the cylindrical cell parts (1 and 1a) were 20 mm and the heights were 80 mm. fig. 1. the cell for the measurement of the solution ph in an electromagnetic field; 1) the cell part in the electromagnetic field; 1a) the cell part out of the electromagnetic field; 2a) and 2b) copper plaques; 3) the electrode in the electromagnetic field; 4) the electrode out of the electromagnetic field; 5) connecting cables to the electromagnetic field source; 6) cables for the connection of the electrodes to theph-meter. ph measurement in electromagnetic field 1409 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. the electromagnetic field production to obtain electromagnetic field in the radiofrequency (rf) region, with variable frequency and variable output power, an appropriate device was designed. it consisted of a signal generator and an amplifier. the signal generator instek geg450b yields a sinusoidal voltage. the output power is up to 300 μw. the frequency and amplitude of the output voltage may be adjusted within the range of 0.1–150 mhz. to amplify the signal power from the signal generator, a signal amplifier was constructed. its basic purpose was to amplify by a constant factor (10,000 times) the signal coming from signal generator to the amplifier input and to conduct it to the amplifier output where from it is transferred to the cell metal plaques producing thus an electromagnetic field between them. details related to measuring system and procedure are described in supplementary material to this paper. results and discussion the “recorded ph values” of the buffer solution of ph 7.0 in dependence on the frequency of the electromagnetic field from three subsequent measurements with separate electrodes (the glass and the reference one) are presented in fig. 2. a change of the “recorded ph values” with the change of frequency only occurred when the glass electrode was in the electromagnetic field, with the largest shift, which is the curve minimum, at 117 mhz (curve 2) and when both electrodes were in the electromagnetic field with the largest shift at 122 mhz (curve 1). the shift was more significant when only the glass electrode was in the electromagnetic field. the “recorded ph values” of the solution did not change when only the reference electrode was in the field (curve 3). using the combined electrode in the same solution, the characteristic difference between the “recorded ph values” occurred (fig. 2, curve 4), with the largest shift at a field frequency of 95 mhz. in all cases when a glass electrode was in the electromagnetic field, some of the observed “recorded ph values” of the solution were less than the actual ph value. the results undoubtedly indicate that an electromagnetic field exerts an influence on a glass electrode. the characteristics of the curves showing the dependence of the “recorded ph values” of the solution on the frequency of the electromagnetic field include a decrease in the “recorded ph values” within a certain frequency range, which leads to the formation of a decrease band with a minimum at certain frequency, i.e., a spectrum of the “recorded ph values” is formed. analogue measurements using ph 7.0 buffer solutions were performed with four electrodes with different duration of prior usage, produced by different manufacturers, three of which were combined and two were only glass electrodes. in all these cases, the same curves types as in the fig. 2 were obtained, with minimums at frequencies 95, 117, 122 and 135 mhz. the “recorded ph 1410 veselinović and velikić available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. values” of the solution within the range of variation were always lower than 7.0 with the extreme decrease to ph 4.0. the same measurements were performed with the ph 4.0 buffer solution as well as and distilled water using the combined electrode. the obtained curves were of the same type as with the ph 7.0 buffer, i.e., in all cases, spectra of the “recorded ph values” were formed. fig. 2. changes of the “recorded ph values” of the buffer solution with electromagnetic field frequency, when 1) both electrodes, 2) glass electrode, 3) reference electrode and 4) combined electrode were in the solution (buffer solution, ph 7.0). using the same combined glass electrode, “recorded ph values” spectra of the three buffer solutions with ph value 4.0, 7.0 and 10.0 were measured. in all cases, the same type of spectra was obtained (fig. s-3 of the supplementary material). these results indicate that the electromagnetic field has a basic influence on the “recorded ph values”, i.e., the influence of this field on the glass electrode surface causes an electrode potential change and the formation of a “recorded ph values”. considering that electromagnetic fields may have different intensities, the curves are presented in fig. 3 showing the changes of the “recorded ph values” of the solution in dependence of the output power (w) of the dispersed (curve 1) and reflected (curve 2) radiation, at a constant electromagnetic field frequency of 135 mhz, for the ph 7.0 buffer solution with a glass electrode in the electromagnetic field. the obtained results show that the intensity of the radiation had a significant influence on the changes of the recorded solution ph values. similar results were obtained with the other glass electrodes, showing, however that the extent of the changes of the “recorded ph values” of solution depended also on ph measurement in electromagnetic field 1411 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. the type and the duration of previous usage of the glass electrode, which has already been mentioned. the same types of curves were obtained when distilled water was used at a constant frequency of 120 mhz. fig. 3. changes of the “recorded ph values” of a buffer solution (ph 7.0) with signal output power, 1) dispersed ray and 2) reflected ray, the field frequency was constant at 135 mhz. an electromagnetic field may affect the “recorded ph values” of the solution, through the cables that connect the electrodes with the ph-meter, if they are not protected against this influence. an investigation was performed by fixing 1, 2 or 3 ferrite rings on the connecting cables of the combined, glass and a reference electrode, positioned at the middle and the ends of the cable. the curves of the changes of the “recorded ph values” in buffer ph 7.0 solution with variation of the electromagnetic field frequency both with and without ferrite rings on the cables are presented in fig. 4. these curves are the confirmation that the achievement of the “recorded ph values” could be partly the consequence electromagnetic field activity on the connecting cable. the shape of the spectrum of the “recorded ph values” was not changed, which means that this type of electromagnetic field influence does not act on the electrodes. moreover, this shows that the connecting cables should be protected against electromagnetic fields before the measurement of the ph of a solution. similar curves were obtained with the ph 4.0 buffer solution. the determination of “recorded ph values” of bi-distilled water was performed at varying the electromagnetic field frequency, using the combined glass electrode. the curves obtained in two independent measurements of two water samples are presented in fig. 5. the obtained shape of the spectra of the water “recorded ph values” was the same as with the buffer solutions, with a minimum at 130 mhz. these results confirm that the occurrence of such “recorded ph 1412 veselinović and velikić available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. values” is the consequence of the influence of an electromagnetic field on the glass electrode. in this case, the lowest recorded ph value of bi-distilled water was lower than with buffer solutions, which shows that the solution components also affect the achievement of the solution recorded ph value and its final value. fig. 4. change of the “recorded ph values” of a ph 7.0 buffer solution with electromagnetic field frequency; 1) without a ferrite ring on the connecting cables and with 3 ferrite rings on the connecting cable of: 2) a glass electrode, 3) both electrodes, 4) the reference electrode. fig. 5. change of the “recorded ph values” of distilled water (ph 6.0) with electromagnetic field frequency. 1) first water sample and 2) second water sample. ph measurement in electromagnetic field 1413 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. conclusions based on the obtained results, it may be concluded that an electromagnetic field did influence the system glass electrode–buffer solution, inducing a “recorded ph values”. this ph value change did not arise if only the reference electrode was in the electromagnetic field, i.e., the electromagnetic field either did not influence the reference electrode or that the influence was very weak and lower than the measurement error. this conclusion applies to the frequency interval and the output power interval of the direct and reflected field used in this study. the presented results enabled the conclusion that when a combined glass electrode was used, the electromagnetic field induced a “recorded ph value” exclusively by its effect on the glass electrode. this conclusion is supported by the fact that the spectrum of the “recorded ph value” was variable, i.e., it was dependent on the age and previous use of the glass electrode. the formation of the “recorded ph value” of a solution during measurement in an electromagnetic field may be not only the consequence of the influence of the electromagnetic field on the glass electrode but also of the ph value of the solution being measured. however, when the same buffer solution was used for a series of measurements with different glass electrodes, the obtained “recorded ph values” were different, as well as the corresponding spectra, which indicates that it is the effect of electromagnetic field on the glass electrode that is dominant, and not the effect on the solution. furthermore, when using the same glass electrode was used in three different buffer solutions with ph values 4.0, 7.0 and 10.0, the same shape and position of maximal “recorded ph values” of the spectra were obtained. this leads to the conclusion that the appearance of “recorded ph values” is a consequence of a change of the adsorption characteristics of h+ ions on the surface of the glass electrode, which is in accordance with the finding9 that an electromagnetic field changes the adsorption characteristics of surface-active substances. if the influence of the electromagnetic field on the solution, i.e., on the equilibrium processes in the solution, were the dominant one, the same “recorded ph value” would be obtained when using different glass electrodes, which was not found in the present study. the influence of the electromagnetic field on the solution and the solvent cannot be excluded. this was confirmed by the appearance of “recorded ph values” for distilled water, which was within the range obtained for the buffer solutions. this is because the buffer capacity of water is negligible in comparison with the capacity of buffer solutions, i.e., buffer solution capacity also affects the height of the “recorded ph value” peak. the obtained results, i.e., the influence of electromagnetic field on glass electrode and appearance of a “recorded ph value” indicate that during ph 1414 veselinović and velikić available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. measurements, an electromagnetic field should be excluded, i.e., electromagnetic sources should be removed from the vicinity of the measuring space. supplementary material details related to measuring system and procedure, as well as the data at different ph values, are available electronically at the pages of journal website: http:// //www.shd.org.rs/jscs/, or from the corresponding author on request. и з в о д утицај електромагнетног поља на мерење ph стакленом електродом драган с. веселиновић1 и зоран великић2 1факултет за физичку хемију, универзитет у београду, 11000 београд, студентски трг 12–16 и 2институт за физику, универзитет у београду, 11080 београд, прегревица 118, земун мерена је ph вредност пуферских раствора (4,0; 7,0 и 10,0) и дестиловане воде са стакленом електродом у електромагнетном пољу у опсегу фреквенција од 10 до 200 mhz и излазној снази диспергованог и рефлектованог електромагнетног зрачења од 0,01 до 3 w, коришћењем конструисаног уређаја. у свим случајевима долази до снижења вредности ph, пуферских раствора и дестиловане воде, т.ј. до настајања ’’очитане вредности ph” зависно од фреквенције и снаге електромагнетног поља. до снижења вредности ph долази у одређеном опсегу фреквенција, са највећим снижењем при одређеној фреквенцији, тј. настаје спектар „очитаних вредности ph“. снижење „очитане вредности ph“ повећава се са излазном снагом зрачења и зависи од пуферског капацитета раствора. деловање електромагнетног поља на измену вредности ph је последица деловања овог поља превасходно на стаклену електроду. (примљено 7. априла, ревидирано 13. септембра, прихваћено 20. септембра 2016) references 1. i. e. stas, b. p. shipunov, t. s. ivonina, electroanalysis 17 (2005) 294 2. i. e. stas, t. s. ivonina, bull. tomsk polytech. univ. 310 (2007) 110 3. j. schneider, f. holzer, m. kraus, p. u. kopinke, u. roland, plasma sources sci. technol. 22 (2013) 01501 4. p. s. davis, j. r. wayland, m. g. merkle, science 173 (1971) 535 5. m. f. diprose, f. a. benson, a. j. willis, bot. rev. 50 (1984) 172 6. i. e. stas, o. p. mikhailova, zhur. fiz. khim. 83 (2009) 397 7. s. zaidi, s. khatoon, pak. j. bot. 35 (2003) 743 8. o. p. mikhailova, i. e. stas, b. p. shipunov, zhur. fiz. khim. 84 ( 2010) 2325 9. j. schneider, f. holzer, m. kraus, f. dieter kopinke, u. roland, plasma sour. sci. tech. 22 (2013) 015010 10. z. velikić, d. veselinović, in proceedings of 6th symposium chemistry and environmental protection, vršac, 21–24.05. 2013, serbian chemical society, book of abstracts, p. 130 11. d. veselinović, z. velikić, in proceedings of sixth international congress "ecology, health, work, sport″, banja luka, 2013, association “health for all”, book of abstracts, p. 211. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis, characterization and antimicrobial activity of benzofuran and thiophene substituted novel diketoxime derivatives} j. serb. chem. soc. 82 (4) 367–377 (2017) udc 547.728.1+547.73+542.913: jscs–4972 547.442.2:615.28–188 original scientific paper 367 synthesis, characterization and antimicrobial activity of novel benzofuranand thiophene-containing diketoxime derivatives demet coskun1*, seher gur2 and mehmet fati̇h coskun1 1university of fırat, faculty of science, department of chemistry , 23119 elazığ-turkey and 2university of fırat, faculty of science, department of biology , 23119 elazığ-turkey (received 17 may, revised 24 november, accepted 14 december 2016) abstract: the aim of this study was the preparation of 1,1′-(2,5-thiophenediyl)bis[1-(2-benzofuranyl)methanone] (2), the corresponding diketoxime (3), and the ether and ester derivatives (4a–e) of the diketoxime. these compounds were prepared in good yields. minimum inhibitory concentrations (mic) of the synthesized compounds 1–4 were determined against salmonella enterica subsp. enterica serovar typhimurium, escherichia coli and staphylococcus aureus. among the synthesized compounds, 1 and 4e showed good activity against e. coli, s. enterica and s. aureus. keywords: bisbenzofuran; thiophene; diketoxime; antimicrobial activity. introduction benzofuran derivatives are an interesting class of heterocyclic compounds. many benzofuran derivatives find application in several fields, including as antioxidants, brightening agents, a fluorescent sensor1 and other fields of chemistry.2 benzofuran derivatives form a number of natural products, such as bergapten, nodakenetin, xanthotoxin and khellin (fig. 1). fig. 1. several benzofuran derivatives: a) khellin; b) bergapten; c) nodakenetin; d) xanthotoxin. thiophene and its derivatives exhibit interesting biological activities and considerable electroluminescent properties.3,4 these compounds have been used * corresponding author. e-mail: dcoskun@firat.edu.tr doi: 10.2298/jsc160517003c 368 coskun, gur and coskun as fluorescent markers and sensors in energy transfer and light-harvesting systems.5 the diketoxime derivatives are very important model systems in applied chemistry. in general, they are used in medicine, photography, agriculture, textiles, dye chemistry and the manufacture of semiconductors. furthermore, they are also used as biological model compounds (i.e., vitamin b12).6–12 accordingly, the antibacterial and antifungal activities of oxime derivatives have been well explored.13–20 various heterocyclic-substituted oxime ethers were extensively studied due to their diverse biological properties.21 oxime ethers are used as anti-inflammatory drugs,22,23 β-adrenergics,24 mild inhibitory active compounds in poultry science25 and as various materials with steroidal effects.26 the biological activities of the oxime ether pharmacophore –c=n–o–r is also well documented.27 however, studies in the literature concerning bisbenzofuran-containing oxime compounds are limited. due to the applications of benzofuran, thiophene and diketoxime derivatives mentioned above, it was decided to design new benzofuranand thiophene-based systems consisting of functionalized diketoxime. the aim of this study was the synthesis of 1,1′-(2,5-thiophenediyl)bis[1-(2 -benzofuranyl)methanone], its dioxime derivative, and new reaction products of the dioxime derivative with various acyl and alkyl halides. in addition, the antimicrobial behavior of all the synthesized compounds was investigated. experimental all the chemicals were supplied from sigma–aldrich and used without further purification. melting points were measured using a differential scanning calorimeter (shimadzu dsc-50) and are uncorrected. the nmr spectra were determined on a bruker ac 400 (400 mhz) spectrometer, with tetramethylsilane (tms) as the internal standard in dmso-d6 or cdcl3 as solvents. the ft-ir spectra were recorded as kbr pellets on a perkin–elmer spectrum one ftir spectrometer. the synthesis scheme to compounds 1–4 is presented in fig. 2, whereas analytical and spectral data are given in supplementary material to this paper. synthesis of compounds 1–3 the required starting material 2-acetylbenzofuran was obtained by cyclocondensation reaction. currently, several general methods are known for the preparation of 2-acetylbenzofuran.28 1-(benzofuran-2-yl)-2-bromoethanone was obtained by reaction of 2-acetylbenzofuran with bromine in acetic acid, according to the general procedure described in the literature,29 with slight modifications. the new diketo sulfide (1) and its 2,5-diacylthiophene derivative (2) were prepared according to the procedure described by miyahara.30 synthesis of 2,2'-thiobis[1-(1-benzofuranyl)ethanone] (1). a solution of sodium sulfide nonahydrate (4.80 g, 20 mmol) in 12 ml of water was added dropwise to a stirred solution of 1-(benzofuran-2-yl)-2-bromo ethanone (9.56 g, 40 mmol) in 100 ml of acetone cooled in an ice bath. reaction mixture was stirred at room temperature for 2 h. the yellow residue was synthesis and antimicrobial activities of diketoxime derivatives 369 filtered and washed with water and methanol. the compound 1 was then recrystallized from benzene–methanol (1:3 volume ratio). fig. 2. synthesis to the benzofuranand thiophene-substituted diketoxime derivatives. synthesis of 1,1′-(2,5-thiophenediyl)bis[1-(2-benzofuranyl)methanone] (2). a solution of glyoxal was prepared by refluxing a mixture of 0.34 g (1.60 mmol) glyoxal trimer dihydrate and 10 ml of methanol and 30 ml of dioxane for 1 h under magnetic stirring. the keto sulfide (1.40 g, 4 mmol) was dissolved in the solution. a solution of sodium methoxide (0.10 g of sodium dissolved in 10 ml of methanol) was added dropwise to this solution at 40 °c. a precipitate then began to form. the mixture was stirred for 30 min. the formed solid was filtered, washed with methanol and dried. synthesis of 1,1′-(2,5-thiophenediyl)bis[1-(2-benzofuranyl)methanone], 1,1′-dioxime (3). compound 2 (1.00 g, 2.69 mmol), hydroxylamine hydrochloride (0.37 g, 5.38 mmol) and pyridine (50 ml) were mixed and refluxed for 24 h. after cooling to room temperature, the mixture was poured into water and the crude 3 that separated was filtered, washed with water several times and crystallized from ethanol. general procedure for the synthesis of 4a–d triethylamine (0.07 ml, 0.5 mmol) was added to a stirred solution of 3 (0.10 g, 0.25 mmol) in 5 ml of tetrahydrofuran. the mixture was stirred at room temperature for 10 min. the appropriate acyl chloride (0.5 mmol) was added dropwise at 0 °c and the reaction mixture was stirred at room temperature for 24 h. the mixture was poured into water and the formed precipitate was filtered, dried in air and recrystallized from ethanol. synthesis of 1,1′-(2,5-thiophenediyl)bis[1-(2-benzofuranyl)methanone], 1,1′-bis[o-(4ethenylphenyl)methyloxime] (4e). compound 3 (0.10 g, 0.25 mmol), k2co3 (0.076 g, 0.55 370 coskun, gur and coskun mmol) and the appropriate alkyl chloride (0.5 mmol) were mixed in absolute acetone and the mixture was refluxed for 5 h. after cooling to room temperature, the mixture was poured into water. the formed solid was filtered, washed with water and recrystallized from ethanol. antibacterial assays bacterial cultures were obtained in mueller–hinton broth (difco) for escherichia coli (atcc 25922) salmonella enterica subsp. enterica serovar typhimurium (nrrl 4420) and staphylococcus aureus (atcc 6538) after 24 h of incubation at 37±0.1 °c. testing for the bacteria was realized in mueller–hinton broth. the final inoculum size for all bacteria was 105 cfu ml-1. the test compounds were dissolved in dmso at an initial concentration of 1024 μg ml-1 and then serially diluted to 1 μg ml-1 in culture medium. a set of tubes containing only inoculated broth served as the control. antibacterial activity was determined after incubation for 24 h at 37 °c. the minimum inhibitory concentration (mic) and defined as the lowest concentration of the compounds that inhibited the visible growth of a microorganism. all experiments were replicated two times to define the mic values. results and discussion first, 2-acetylbenzofuran was obtained in the reaction of 2-hydroxybenzaldehyde and 1-chloroacetone. 1-(benzofuran-2-yl)-2-bromoethanone, which was the starting material, was synthesized by bromination of 2-acetylbenzofuran with bromine in acetic acid. treatment of this compound with hydroxylamine hydrochloride in pyridine gave 3. reaction of 3 with appropriate acyl chlorides in thf in the presence of triethylamine gave the oxime ester compounds 4a–d. furthermore, reaction of compound 3 with the appropriate alkyl chloride in acetone in the presence of k2co3, gave the oxime ether derivative 4e. all new compounds were characterized by 1h-nmr, 13c-nmr and ft-ir spectroscopy and elemental analysis. these data are given in the supplementary material to this paper. in ft-ir spectrum, the most important evidence of the formation 1 was the observation of two stretching vibrations at about 1663 and 1680 cm–1, belonging to the two c=o groups. additionally, in the 1h-nmr spectrum, a signal at 4.1 ppm as a singlet belonging to –sch2– protons and in the 13c-nmr spectrum, a signal at 185.48 ppm belonging to the c=o group proved the formation of compound 1. in the ftir spectrum of compound 2, the c=o stretching vibration was observed at 1618 cm–1. due to conjugation, this stretching vibration shifted to a lower wave number. in the 1h-nmr spectrum of this compound, the disappearance of the singlet signal at 4.1 ppm, belonging to –sch2 protons, was evidence for the formation of compound 2. in the ir spectrum of 3, while oh band of the oxime was displayed between 3000–3250 cm–1, the c=n stretching band and the n–o stretching band were seen at 1583 and at 1030 cm–1, respectively. as is known, there are three geometrical isomers of the dioximes due to the two oxime groups including the anti -(e,e), amphi-(e,z) and syn-(z,z) forms, which are shown in fig. 3.31 synthesis and antimicrobial activities of diketoxime derivatives 371 fig. 3. geometric isomers of the symmetrically substituted dioxime 3: a) anti-(e,e), b) amphi-(e,z) and c) syn-(z,z). it was expected that 3 would show two different isomeric structures. when the 1h-nmr spectrum for compound 3 was investigated, the n–oh resonances appeared as two singlets at 13.1 ppm (for the e,e isomer) and at 12.8 ppm (for the z,z isomer). the e isomer appeared at a higher frequency than the z isomer.32–36 in the 13c-nmr spectrum of dioxime 3, twenty-four carbon signals were seen, supporting a synand anti-isomer mixture of compound 3. in the 13c-nmr spectrum of compound 3, the c=n signal was displayed at 153 ppm while the c=o signal had disappeared. dioxime 3 was not expected to be in the amphi form due to different integral values belonging to the n–oh protons at 13.1 and 12.8 ppm. the different integral values belonging to the two n–oh protons confirmed that compound 3 consisted of a synand anti-isomer mixture. moreover, if the amphi-isomer were present in the mixture, more carbon peaks could be expected in the 13c-nmr spectrum. due to molecular balancing, the molecules in the amphi-isomer structure break down. additionally, a tlc run of a sample of the crystalline dioxime 3 was performed and doubled spots on the plate were observed. therefore, it was concluded that the dioxime 3 consisted of a mixture of ee/zz isomers. reactions of dioxime 3 with appropriate alkyl and acyl chlorides gave isomer mixtures that consisted of dioxime ether and dioxime ester type compounds, 4a–e. the disappearance of strong broad –oh band in the 3250–3000 cm–1 372 coskun, gur and coskun region in the ft-ir spectrum of 4a–d and the appearance of strong bands at around 1755–1740 and 1250–1050 cm–1, which were due to c=o and c–o stretching vibrations, respectively, indicated the formation of ester products. the ester derivatives of the dioxime 4b (fig. 4) and 4c (fig. 5) were obtained as a mixture of antiand amphi-forms. in the 1h-nmr spectrum of 4b, the signals of thiophene ring protons (ha1 and ha) in the ester unit of the amphi -form of 4b appeared as two doublets at 7.32 (j = 3.5 and 5.1 hz) and 7.14 (j = = 3.6 hz and 5.8 hz) ppm. in the 1h-nmr spectrum of 4b, the signals of the thiophene ring protons of the anti-form of 4b are probably among multiple signals at 7.33–8.15 ppm. in the 1h-nmr spectrum of compound 4c, both resonances of the furan ring proton (hb) belonging to the anti-isomer of 4c appeared as a singlet signal at 6.87 ppm. signals of the ha1 and ha protons belonging to amphi-isomer of 4c appeared as two singlets at 6.82 and 6.63 ppm. for a symmetrical dioxime in the anti-form, only a single peak was expected.37,38 these three values might correspond to a mixture of antiand amphi-forms.39,40 fig. 4. a) amphi-(e,z)and b) anti-(e,e)-isomers of 4b. fig. 5. a) amphi-(e,z)and b) anti-(e,e)-isomers of 4c. the amounts of the syn-, antiand amphi-isomers of 3 and 4c–e were calculated from the integrated heights in the 1h-nmr spectra and these values are given in table i. synthesis and antimicrobial activities of diketoxime derivatives 373 table i. e,e, z,z and e,z amounts, %, of 1,1′-(2,5-thiophenediyl)bis[1-(2-benzofuranyl)methanone], 1,1′-dioxime compound isomer e,e z,z e,z 3 55 45 – 4c 41 – 59 4d 61 39 – 4e 31 69 – the ester derivatives of the dioxime 4a, 4d and 4e (fig. 6) were obtained as a mixture of antiand syn-forms. in the 1h-nmr spectrum of 4d, signal of the och3 protons belonging to the anti-isomer of 4d was observed as a singlet signal at 3.87 ppm. in addition, the signal of the och3 protons belonging to the syn -isomer of 4d appeared as a singlet at 3.75 ppm. in the 1h-nmr spectrum of 4e, signals of the och2 protons belonging to the antiand syn-isomer were observed as two singlets at 5.47 and 5.40 ppm, respectively. fig. 6. a) anti-(e,e)and b) syn-(z,z)-isomers of 4e. in the 13c-nmr spectra of 4b and 4c, the 48 carbon signals prove that these dioxime derivatives consisted of antiand amphi-isomer mixtures. moreover, in the 13c-nmr spectra of 4a, 4d and 4e, the 34, 38 and 40 carbon signals, respectively, proved that these dioxime derivatives consisted of antiand syn-isomer mixtures. the och3 proton signals at 3.87 ppm (anti-isomer) and 3.75 ppm (syn-isomer) also have good correlation with the quaternary carbon signal at 162.72 ppm in hmbc spectrum (fig. s-7c of the supplementary material). the 13c-signals at 56.17 ppm (anti-isomer) and 56.02 ppm (syn-isomer) give cross peaks with two och3 protons belonging to the antiand syn-isomers at 3.87 and 3.75 ppm, res374 coskun, gur and coskun pectively, in the hsqc spectrum (fig. s-7d of the supplementary material). it is clear that the signals at 56.17 and 56.02 ppm belong to the aliphatic och3 carbons of antiand syn-isomers of 4d. all these data obtained from hsqc and hmbc spectra showed that the signal at 162.72 ppm is the quaternary carbon (c-17) on the disubstituted phenyl ring. the 13c-signals at 132.59 and 132.35 ppm have a cross peak with the proton resonances around 8.14–8.04 ppm (anti -isomer) and 7.94–7.92 ppm (syn-isomer) in the hsqc spectrum. these proton signals have good β-correlation with the carbon signals at 164.35 and 164.17 ppm in the hmbc spectrum. it is clear that the signals at 164.35 and 164.17 ppm belong to the carbonyl carbon (c-13) of antiand syn-isomers of 4d. in the hsqc spectrum, the signals around 7.83–7.77 ppm have a cross peak with the 13c-signals at 112.38 and 112.31 ppm. in the hmbc spectrum, the same proton signals are in correlation with the quaternary carbon (c-4) signals at 127.54 ppm (anti-isomer) and 127.46 ppm (syn-isomer). these data show that the signals around 7.83–7.77 ppm belong to the olefinic protons attached to the olefinic carbons (c-3) on benzofuran, which give signals at 112.38 and 112.31 ppm. the correlations in the hmbc spectrum show that the carbons giving the signals at 155.49 and 154.81 ppm, 148.81 and 148.51 ppm and 127.54 and 127.46 ppm belong to c-9, c-2 and c-4 of the antiand syn-isomers, respectively. the signals around 7.83–7.94 ppm have cross peaks with the 13c-signals at 123.64 ppm (anti-isomer) and 122.99 ppm (syn-isomer) in the hsqc spectrum and have α-correlation with the quaternary carbon signals at 155.49 and 154.81 ppm and 127.54 and 127.46 ppm in the hmbc spectrum. the protons giving the signals around 7.83–7.94 ppm are attached to c-5, which resonate at 123.64 ppm (anti -isomer) and 122.99 ppm (syn-isomer). the quaternary carbon signals at 155.49 and 154.81 ppm and 148.81 and 148.51 ppm have α-correlation with the signals at 7.65–7.59 ppm in the hmbc spectrum, and these proton signals have cross peak with the 13c-signal at 113.48 ppm (anti-isomer) and 113.36 ppm (syn-isomer) in the hsqc spectrum. these data show that the signals around 7.65–7.59 ppm are due to the protons attached to c-8. antibacterial activity the minimal inhibitory concentration (mic) of the synthesized compounds was determined against s. aureus, e. coli and s. enterica subsp. enterica serovar typhimurium using a standard broth dilution technique. all the mic results are presented in table ii. the obtained data showed that the compounds were able to inhibit the growth of the selected microorganisms in vitro with mic values between 32 and 128 µg ml–1. among the synthesized compounds, 4e exhibited good activity against s. aureus and e. coli and 1 against s. enterica subsp. enterica serovar typhimurium synthesis and antimicrobial activities of diketoxime derivatives 375 with an mic value of 32 µg ml–1. the other compounds showed moderate activity against the tested microorganisms. table ii. minimum inhibitory concentration (mic / µg ml-1) of compounds 1–4; positive control: chloramphenicol compound e. coli atcc 25922 s. enterica nrrl 4420 s. aureus atcc 6538 1 64 32 64 2 64 128 64 3 64 64 128 4a 64 128 128 4b 64 128 64 4c 128 128 128 4d 128 128 64 4e 32 64 32 positive control 32 32 32 conclusions in the present study, 1,1′-(2,5-thiophenediyl)bis[1-(2-benzofuranyl)methanone] (2), the corresponding diketoxime (3) and the ether and ester derivatives (4a–e) of the diketoxime were designed and synthesized. the synthesized compounds 3 and 4a–e were mixtures of different isomers, such as syn-, antiand amphi-isomers. synthesized compounds were tested for antimicrobial activity against s. aureus, e. coli and s. enterica subsp. enterica serovar typhimurium. among the synthesized compounds, 1 and 4e showed good activity against e. coli (atcc 25922), s. typhimurium (nrrl 4413) and s. aureus (atcc 6538). the biological effects of 1–4 could be helpful in the design of more potent antibacterial agents for therapeutic use. supplementary material analytical and spectral data, as well as the nmr spectra, of the synthesized compounds are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgment. the authors thank firat university for financial support of this work. и з в о д синтеза, карактеризација и антимикробна активност нових деривата дикетоксима који садрже бензофуран и тиофен demet coskun1, seher gur2 и mehmet fatih coskun1 1 university of fırat, faculty of science, department of chemistry, 23119 elazığ-turkey и 2university of fırat, faculty of science, department of biology, 23119 elazığ-turkey описана је синтеза 1,1′-(2,5-тиофендиyл)бис[1-(2-бензофуранил)метанона] (2), одговарајућих дикетоксима (3) и етарских и естарских деривата дикетоксима (4а–е). једињења су синтетисана у добром приносу. одређена је минимална инхибиторна 376 coskun, gur and coskun активност (mic) синтетисаних једињења 1–4 према salmonella enterica subsp. enterica serovar тyphimurium, escherichia coli и staphylococcus aureus. од синтетисаних једињења, деривати 1 и 4е показују добру активност према e.coli, s. enterica и s.aureus. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice reductive heck reactions of n-arylamino-substituted tricyclic imides j. serb. chem. soc. 81 (10) 1121–1125 (2016) udc 547.415.5+547.556.5:546.98–039.7+ jscs–4912 544.478:542.96:615 original scientific paper 1121 reductive heck reactions of n-arylamino-substituted tricyclic imides muge atbakar, onur topbastekin and nuket ocal* yildiz technical university, faculty of science and arts, department of chemistry, davutpasa campus, 34220 esenler-istanbul, turkey (received 19 february, revised 10 may, accepted 13 june 2016) abstract: the c–c coupling of (3ar,4s,7r,7as)-rel-2-[(3-chloro-4-fluorophenyl)amino]-3a,4,7,7a-tetrahydro-4,7-methano-1h-isoindole-1,3(2h)-dione (3) which was prepared as a new starting material and (3ar,4s,7r,7as)-rel-2-[(2,4-dinitrophenyl)amino]-3a,4,7,7a-tetrahydro-4,7-methano-1h-isoindole-1,3(2h)-dione (6) with aryland heteroaryl iodides gave the aryl(hetaryl), n-arylamino tricyclic imides 4a–d and 7a–d under reductive heck conditions. keywords: hydroarylation reactions; cyclic hydrazines; imides; c–c coupling with pd(oac)2. introduction hydrazine derivatives are widely applied as pharmaceuticals, e.g., as tuberculoci--dal, anti-tumor and psychotherapeutic agents.1 they are also used in agriculture as herbicides and protection agents for plants.2 kas’yan and co-workers studied the reactions of an endic anhydride with alkyland arylhydrazines and reported the products as biologically active compounds.3 these kinds of products also have imide forms. cyclic imides are also important intermediates of many pharmaceutical drugs, such as human therapeutic agents, and are reported to exhibit cytostatic, antibacterial, herbicide and antimicrobial activities. in addition, they can cross biological membranes in vivo due to the imide bonds.4–8 organopalladium-catalyzed c–c bond formation has become one of the most efficient approaches in the synthesis of organic molecules. due to its broad synthetic potential as a stereoselective c–c coupling method, the heck reaction has been the subject of several synthetic and mechanistic studies over the last 30 years. the heck reaction is widely used as an important method to build biologically active compounds in synthetic chemistry and the pharmaceutical indus * corresponding author. e-mail: nocal@yildiz.edu.tr doi: 10.2298/jsc160218062a _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1122 atbakar et al. try.9–11 reductive heck reactions of unsaturated n-substituted tricyclic imides, which have possible biological activities, were studied previously.12–18 the palladium-catalyzed hydroarylations of several n-substituted tricyclic imides were studied in order to find a new stereoselective access to a series of new exo-aryl(hetaryl)-substituted tricyclic imides in the presence of triphenylarsine using the kaufmann reductive heck procedure.19–21 in this study, the endic anhydride (1)22 was combined with 3-chloro-4-fluorophenylhydrazine (2), which was chosen for the active groups on the aromatic ring, and 2,4-dinitrophenylhydrazine (5), which was chosen for many applications of this group in medicinal chemistry, using the kas’yan method.3 then, their hydroarylation reactions with aryl and heteroaryl iodides were studied to obtain new possibly bioactive molecules. experimental general all the reactions were performed under a nitrogen atmosphere unless otherwise indicated. reactions were monitored using thin-layer chromatography (tlc). visualization of the developed chromatogram was performed under uv light or using kmno4 stain. the ir spectra were obtained with a perkin elmer ft-ir system and are reported in terms of frequency of absorption (cm-1). the melting points were determined using a gallenkamp digital thermometer. all melting points are uncorrected. the nmr spectra were determined with a bruker ac-500 (500 mhz) nmr spectrometer. tms (tetramethylsilane) was used as the internal standard and cdcl3 (or dmso-d6, cd3ocd3 and cd3od) were used as the solvents. signal multiplicities in the nmr spectra are reported as follows: s, singlet, brs, broad singlet, d, doublet, dd, doublet of doublets and m, multiplet. mass spectra were measured with either an agilent lc/msd trap sl or gc–ms (agilent 6890n gc-system-5973 imsd) or an agilent 6460 triple quad lc/ms instruments. the synthesis of (3ar,4s,7r,7as)-rel-2-[(3-chloro-4-fluorophenyl)amino]-3a,4,7,7a-tetrahydro-4,7-methano-1h-isoindole-1,3(2h)-dione (3) a mixture of 1 (164 mg, 1 mmol) and 2 (160.5 mg, 1 mmol) in ch3cn (5 ml) was stirred at r.t. for 4 h. the solid was filtered and recrystallized from 2-propanol to afford 3 in 80 % yield. the characterization data for 3 are given in the supplementary material to this paper. general procedure for the hydroarylation reactions a solution of pd(oac)2 (5.6 mg, 0.025 mmol) and asph3 (33.7 mg, 0.11 mmol) in dry dmf (3 ml) was stirred in a schlenk flask under nitrogen at 65 °c for 15 min in order to form the catalyst complex. then 3 (306 mg, 1.5 mmol) or 6 (1.00 mmol), triethylamine (354 mg, 3.5 mmol) and formic acid (138 mg, 3.0 mmol) were added. the mixture was heated to 65 °c for 28 h. after cooling to r.t., brine (50 ml) was added, the reaction mixture was extracted with ethyl acetate and dried over mgso4. the solvent was evaporated, and the residue purified by chromatography to afford 4a–d or 7a–d. the characterization data for the synthesized compounds are given in the supplementary material together with their ir and nmr spectra. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. hydroarylations of tricyclic imides 1123 results and discussion first, (3ar,4s,7r,7as)-rel-2-[(3-chloro-4-fluorophenyl)amino]-3a,4,7,7a-tetrahydro-4,7-methano-1h-isoindole-1,3(2h)-dione (3) was synthesized as a new compound in the reaction of the endic anhydride 1 and 3-chloro-4-fluorophenylhydrazine (2) as a new compound. reactions of 3 with iodobenzene, 2-iodothiophene, 4-chloro-1-iodobenzene and 2-chloro-5-iodopyridine under reductive heck conditions gave the pure products 4a–d after chromatographic separation on silica gel as single diastereomers in isolated yields (scheme 1). o o o f cl hn h2n n o o n h cl f 1 2 3 ch3cn 3 n o o n h cl f 4a-d tpas, dmf et3n, hcooh 65 oc + ari ar ar = s n cl cl a b c d pd(oac)2 scheme 1. synthesis of 3 and 4a–d. furthermore, (3ar,4s,7r,7as)-rel-2-[(2,4-dinitrophenyl)amino]-3a,4,7,7a -tetrahydro-4,7-methano-1h-isoindole-1,3(2h)-dione (6) was prepared in a known procedure3 from the reaction of the same endic anhydride (1) and 2,4-dinitrophenylhydrazine (5) as the second starting material. the same reductive heck arylation conditions were successfully applied to the reactions of 6 with iodobenzene, 2-iodothiophene, 4-chloro-1-iodobenzene and 2-chloro-5-iodopyridine to give the new exo-arylated heterocycles 7a–d in moderate yields after chromatographic separation (scheme 2). the stereochemistry of the compounds was investigated from their nmr spectra including diagnostic spin–spin interactions. the exo-position of the c-5 substituent was confirmed by the fact that endo-h5 showed no significant interaction with h7 but did show a cross-peak because of w-coupling to h8-syn. the geminal protons on c8 were identified by vicinal coupling to h7 and w-coupling to h3a-exo, respectively. in addition, the 13c-nmr and hsqc spectral data _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1124 atbakar et al. were in agreement with the proposed structures and the mass spectra of all new compounds showed the expected molecular ion peaks. ir spectra of the starting materials and hydroarylation products exhibited strong absorption bands due to the symmetric and asymmetric stretching vibrations of the c=o and nh bonds in the expected areas. scheme 2. synthesis of 7a–d. conclusions in summary, the palladium(ii) acetate catalyzed hydroarylation of readily accessible tricyclic hydrazino imides in the presence of triphenylarsine as ligand was shown to be a stereoselective, versatile and high yield approach to the synthesis of the aryl and heteroaryl derivatives of tricyclic hydrazino imides 4a–d and 7a–d. from the results, it was possible to observe that these compounds may be suitable for effective description of their biological activity. supplementary material characterization data and selected spectra of the synthesized compounds are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. we gratefully acknowledge the financial support for this study from the yildiz technical university scientific research projects coordination department (project no. 2014-01-02-kap03). we also thank prof. dr. dieter kaufmann for the hydroarylation reactions studies. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. hydroarylations of tricyclic imides 1125 и з в о д редуктивна хекова реакција n-ариламино-супституисаних трицикличних имида muge atbakar, onur topbastekin и nuket ocal yildiz technical university, faculty of science and arts, department of chemistry, davutpasa campus, 34220 esenler-istanbul, turkey (3ar,4s,7r,7as)-rel-2-[(4-флуор-3-хлорфенил)амино]-3a,4,7,7a-тетрахидро-4,7-метано-1h-изоиндол-1,3(2h)-дион (3), који је припремљен као ново полазно једињење, и (3ar,4s,7r,7as)-rel-2-[(2,4-динитрофенил)амино]-3a,4,7,7aтетрахидро-4,7-метано-1h -изоиндол-1,3(2h)-дион (6) у редуктивној хековој реакцији са арили хетероарилјодидима, кao производе дају n-ариламинотрицикличне имиде 4a–d и 7a–d. (примљено 19. фебруара, ревидирано 10 маја, прихваћено 13. јуна 2016) references 1. s. tsupova, u. maeorg, heterocycles 88 (2014) 129 2. z. cui, x. yang, y. shi, h. uzawa, j. cui, bioorg. med. chem. lett. 21 (2011) 7193 3. o. v. krishchik, i. n. tarabara, a. o. kas’yan, s. v. shishkina, o. v. shishkin, a. k. isaev, l. i. kas’yan, russ. j. org. chem. 40 (2004) 1140 4. m. f. brana, a. gradillas, a. gomez, n. acero, f. llinares, d. munoz-mingarrro, c. abradelo, f. rey-stolle, m. yuste, j. campos, m. a. gallo, a. espinosa, j. med. chem. 47 (2004) 2236 5. s. m. sondhi, r. rani, p. roy, s. k. agrawal, a. k. saxena, bioorg. med. chem. lett. 19 (2009) 1534 6. j. kossakowski, m. jarocka, farmaco 56 (2001) 785 7. m. m. patil, s. s. rajput, int. j. pharm. pharm. sci. 6 (2014) 8 8. m. k. hargreaves, j. g. pritchard, h. r. dave, chem. rev. 70 (1970) 439 9. e. negishi, a. meijere, handbook of organopalladium chemistry for organic synthesis, wiley, new york, 2002, p. 1113 10. i. p. beletskaya, a. v. cheprakov, chem. rev. 100 (2000) 3009 11. z. l. wei, c. george, a. p. kozikowski, tetrahedron lett. 44 (2003) 3847 12. j. storsberg, m.-l. yao, n. ocal, a. de meijere, a. e. w. adam, d. e. kaufmann, chem. commun. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice one-pot preparation of carbamoyl benzotriazoles and their applications in the preparation of ureas, hydrazinecarboxamides and carbamic esters j. serb. chem. soc. 81 (1) 13–22 (2016) udc 547.495.2.05:547.53–304.9+ jscs–4822 547.334’467.1+547.26’11 original scientific paper 13 one-pot preparation of carbamoyl benzotriazoles and their applications in the preparation of ureas, hydrazinecarboxamides and carbamic esters hui mao, huili liu, yawei tu, zhiyun zhong, xin lv* and xiaoxia wang** zhejiang key laboratory for reactive chemistry on solid surfaces, college of chemistry and life sciences, zhejiang normal university, jinhua 321004, china (received 26 january, revised 10 september, accepted 11 september 2015) abstract: carbamoyl benzotriazoles were conveniently synthesized in one-pot from carboxylic acids, diphenyl phosphorazidate (dppa) and 1h-benzotriazole (bth). the reactivity and applications of carbamoyl benzotriazoles were also explored. carbamoyl benzotriazoles react smoothly with amino acids, hydrazines and alcohols, thus providing facile access to the corresponding ureas, hydrazinecarboxamides and carbamic esters, respectively, in good to excellent yields. keywords: carbamoyl benzotriazoles; amino acid-derived ureas; hydrazinecarboxamides; carbamic esters. introduction isocyanates represent a valuable class of compounds by virtue of their widespread applications in organic synthesis and medicinal chemistry.1–3 nevertheless, isocyanates are usually toxic and unstable, and should be stored carefully due to the ease of exothermic polymerization and sensitivity to moisture. although they could be used by in situ generation, their direct availability is required in certain circumstances. capture of isocyanates formed in situ by wang resin provides a good solution and was successfully used in the synthesis of amines.4 on the other hand, carbamoyl azides5a,b and carbamoyl imidazolium salts5c have been developed as isocyanate substituents. it is noteworthy that carbamoyl benzotriazoles6–10 may be a more promising alternative since they are crystalline, moderately reactive and can be stored with reasonably long shelf life. the currently available methods for the synthesis of carbamoyl benzotriazole include: 1) condensation between phosgene, amine and 1h-benzotriazole (bth), or direct carbamoylation6 of bth with carbamoyl chloride or isocyanates; 2) *,** corresponding authors. e-mail: (*) lvxin@zjnu.cn; (**) wangxiaoxia@zjnu.cn doi: 10.2298/jsc150126069m 14 mao et al. diazotization of o-aminophenylureas7; 3) the reaction between acyl azides and bth;8,9 and 4) the reaction of carbamate salts with 1-chlorobenzotriazole in the presence of a base and pph3.10 despite the available methods, the more straightforward preparation of carbamoyl benzotriazoles from carboxylic acid remained unexplored. besides, the application of carbamoyl benzotriazoles as an isocyanate substituent in the formation of a variety of compounds has not yet been sufficiently addressed. herein, a facile one-pot synthesis of carbamoyl benzotriazoles from carboxylic acids, dppa and bth is reported. the use of carbamoyl benzotriazoles for the preparation of amino acid-derived ureas, hydrazinecarboxamides and carbamic esters is demonstrated. results and discussion the condensations between carboxylic acids, dppa and an amine to afford urea are well established.11 however, the condensation involving bth as the nucleophile to afford carbamoyl benzotriazoles, a promising carbamoyl reagent, remained unknown. in this study, 4-methylbenzoic acid, dppa and bth were initially used as the model substrates to optimize the reaction conditions (scheme 1 and table i). scheme 1. one-pot reaction of 4-methylbenzoic acid, bth and dppa to form carbamoyl benzotriazoles. table i. optimization of the conditions for the one-pot synthesis of carbamoyl benzotriazole 2a; all reactions were performed under reflux entry base base (equiv.) solvent time, h yielda, % 1 et3n 3.0 thf 8 trace 2 et3n 2.0 thf 8 trace 3 et3n 1.0 thf 10 41 4 k2co3 1.0 thf 8 trace 5 na2co3 1.0 thf 8 trace 6 et3n 1.0 toluene 6 80 7 et3n 1.0 1,4-dioxane 6 37 aisolated yields based on 1a several bases were examined. et3n (2.0 and 3.0 equiv.)11,12 afforded only a trace amount of the desired product (table i, entries 1 and 2). the use of inorg preparation of carbamoyl benzotriazoles and their applications 15 anic bases, such as k2co3 and na2co3, did not result in any improvement (entries 4 and 5). considering that isocyanates may be sensitive to acids13a and bases under elevated temperatures,13b the reaction under neutral conditions was attempted. to our delight, reducing the amount of et3n to 1 equiv. afforded the desired product 2a in 41 % yield (table i, entry 3). different solvents were also screened (entries 3, 6 and 7) and the use of toluene afforded the product in 80 % yield. with the optimal conditions in hand, various carboxylic acids were used for the preparation of carbamoyl benzotriazoles. the results are summarized in scheme 2 and table ii. scheme 2. one-pot preparation of carbamoyl benzotriazoles. table ii. one-pot preparation of carbamoyl benzotriazoles from carboxylic acids, bth and dppa; all reactions were performed under reflux entry r1 product 2 time, h yielda, % 1 2a 6 80a, 85b 2 2b 6 76 3 2c 6 71 4 2d 6 70 5 2e 6 83 6 2f 6 55 7 2g 6 53 8 2h 6 55 9 2i 6 –c 10 2j 6 49 11 2k 6 45 12 ch3– 2l 8 50 13 n-c3h7– 2m 8 57 14 i-c3h7– 2n 8 57 16 mao et al. table ii. continued entry r1 products 2 time, h yielda, % 15 n-c6h13– 2o 8 58 16 2p 8 46 17 2q 8 42 aisolated yields on 1 mmol scale; bisolated yield on 10 mmol scale; cno desired product was obtained a variety of carboxylic acids could be converted directly into the respective carbamoyl benzotriazole in moderate to good yields. in general, aromatic carboxylic acids bearing an electron-donating aryl (table ii, entries 1–5) gave better results than those with an electron-withdrawing aryl (entries 6–8). heterocyclic nicotinic acid afforded 2k in 45 % yield, while no desired product was obtained using p-nitrobenzoic acid (entry 9). 2-(naphthalen-1-yl)acetic acid and other aliphatic carboxylic acids (entries 10 and 12–15) generally afforded relatively lower yields (entries 12–17). the potential for large-scale preparation was also investigated. running the reaction of 4-methylbenzoic acid on the 10 mmol scale gave a slightly better yield than that obtained on the 1 mmol scale (entry 1). in order that carbamoyl benzotriazoles be an alternative to isocyanate, it is necessary to demonstrate their usage in the preparation of ureas and carbamates14 since isocyanates have played a very important role in the preparation of these compounds. herein, an investigation was undertaken to examine the reactivity of carbamoyl benzotriazoles towards amino acids, hydrazines and alcohols to determine their potential in the preparation of various ureas, hydrazinecarboxamides and carbamic esters. previously, it was reported that carbamoyl benzotriazoles reacted with amines smoothly under mild conditions.8 here amino acids were used to explore the application of carbamoyl benzotriazoles in the synthesis of chiral ureas (scheme 3 and table iii), which may possess important biological activities. for example, azadipeptide nitriles are used as novel cysteine protease inhibitors.15 geldern also reported the structure–activity relationship of other urea derivatives of amino acids in an attempt to improve the potency of a novel series of azole -based endothelin-a (eta) selective antagonists.16 scheme 3. the aminoacylation of amino acids with carbamoyl benzotriazoles. the reaction of carbamoyl benzotriazole 2b with l-phenylalanine was realized using the procedure reported for the acylation of amino acids using n-acyl preparation of carbamoyl benzotriazoles and their applications 17 benzotriazoles.17 to our delight, the reaction gave the desired product 4a in 91 % yield at r.t. in the presence of et3n (1.5 equiv.) in ch3cn–h2o (2:1, v/v, table iii, entry 1). table iii. preparation of ureas via aminoacylation of amino acids with carbamoyl benzotriazoles entry r1 compounds 3 products 4 time, h yielda, % 1 2b 3a 4a 0.5 91 2 3b 4b 0.5 94 3 3c 4c 0.5 90 4 3d 4d 0.5 92 5 3e 4e 0.5 80 6 3f 4f 0.5 94 7 3g 4g 0.5 72 8 3h 4h 10 85 9 2a 3a 4i 0.5 90 10 3c 4j 0.5 95 11 2h 3a 4k 0.5 91 12 n-c3h7– 2m 3a 4l 24 96 aisolated yields based on carbamoyl benzotriazoles as shown in table iii, the aminoacylation of various natural amino acids was investigated. generally, the reactions between amino acids and carbamoyl benzotriazoles afforded the corresponding ureas in excellent yields within 0.5 h (entries 1–6). for l-proline, as long as 10 h was required to obtain 85 % yield (entry 8). it is worth noting that good chemoselectivity for the acylation of lserine was observed, where the amino group reacted selectively, but not the hydroxyl group (entry 7). generally, the substituent on the aromatic ring did not affect the efficiency of the reaction (entries 9–11). the method was also applicable for the reactions of aliphatic carbamoyl benzotriazole (entry 12). 18 mao et al. maintaining the chirality of the amino acid was proved by polarimetric analysis of (r)-3-phenyl-2-(3-phenylureido)propanoic acid 4a, the [α] value of which was found to be +51.51°, in good accordance with the literature value.18 the katritzky group also demonstrated that no racemization of the amino acid derivative occurred under the same conditions.19 bis-nucleophilic hydrazines were also reacted with carbamoyl benzotriazoles (scheme 4 and table iv). for methylhydrazine, the more electrophilic nitrogen atom (with methyl attached) was aminoacylated without any promoter and compounds 6a and 6b were obtained in excellent yields (table iv, entries 1 and 2). phenylhydrazine, however, was aminoacylated on the terminal nitrogen and the yields were relatively lower (table iv, entries 3 and 4) even in the presence of nah. without nah, no aminoacylation of the phenylhydrazine occurred. scheme 4. the aminoacylation of hydrazines with carbamoyl benzotriazoles. table iv. preparation of hydrazinecarboxamides via aminoacylation of hydrazines with carbamoyl benzotriazoles; the reaction was promoted by 1.5 equiv. of nah (based on 2) in dry toluene at r.t. or at 40 c entry r1 r2 base products 6 time, h yielda, % 1 2b ch3– – 6a 10 90 2 2h ch3– – 6b 10 89 3 2b nah 6c 10 45 4 c2h5– 2r nah 6d 60 41 aisolated yields based on carbamoyl benzotriazoles finally, the use of carbamoyl benzotriazoles for the synthesis of carbamic esters was explored. thus the reaction of 2b with ethanol was performed. with toluene as the solvent at 70–78 °c, the reaction afforded the desired compound 8b in 80 % yield. as shown in scheme 5 and table v, all the carbamoyl benzotriazoles examined smoothly afforded the desired carbamic esters in good yields. in combination with the convenience in the purification of the products, the aminoacylation preparation of carbamoyl benzotriazoles and their applications 19 of alcohols with carbamoyl benzotriazoles provides a facile route to prepare carbamic esters. scheme 5. the aminoacylation of alcohols with carbamoyl benzotriazoles. table v. preparation of carbamic esters via aminoacylation of alcohols with carbamoyl benzotriazoles entry r1 r2 products 8 time, min yield, %a 1 2b c2h5 8a 30 83 2 2b n-c4h9 8b 30 80 3 2a c2h5 8c 30 77 4 2a n-c4h9 8d 30 78 5 2h c2h5 8e 45 85 6 2h n-c4h9 8f 45 85 aisolated yields based on carbamoyl benzotriazoles experimental thf and toluene were dried over na. other commercial reagents were used without further purification, and all solvents were of reagent grade. all reaction mixtures were stirred magnetically and were followed by tlc analysis at 254 nm. flash column chromatography was performed using 100–200 mesh silica. melting points are uncorrected. the ir spectra were recorded using kbr disks or as films using a nicolet-670 ftir spectrometer with absorption in cm-1. the 1h-nmr and 13c-nmr spectra were determined on a bruker ac-400 spectrometer as cdcl3 or dmso-d6 solutions. the j values are in hertz and the chemical shifts are expressed in ppm downfield from internal tms. elemental analysis was realized on an ea 1110 instrument. polarimetry was performed according to the literature18 on an anton paar mcp200 modular circular polarimeter. among the compounds, carbamoyl benzotriazoles 2a, 2b, 2e, 2f, 2h and 2o, urea 4l, hydrazinecarboxamide 6c and carbamic esters 8a–f are known. other carbamoyl benzotriazoles and products including 2c, 2d, 2g, 2j–2n, 2p, 2q, 4a–k, 6a, 6b and 6d are new compounds. the analytical and spectral data of compounds are given in the supplementary material to this paper. general procedure for the one-pot synthesis of carbamoyl benzotriazoles 2 to a solution of carboxylic acid (1.0 mmol) in dry toluene (20 ml) was added et3n (1.0 mmol, 101 mg), diphenyl phosphorazidate (1.0 mmol, 275 mg) and 1h-benzotrizole (1.0 mmol, 119 mg). the mixture was stirred under reflux until completion of the reaction 20 mao et al. (monitored by tlc). the reaction mixture was cooled to r.t., and washed successively with water (10 ml), saturated na2co3 (10 ml) and brine (10 ml). the combined organic phase was dried over anhydrous na2so4, and concentrated under reduced pressure to obtain a residue, which was purified by recrystallization from toluene or by column chromatography on silica gel using ea–pe (1:20, boiling range 60–90 c) as the eluent to give the pure products 2a–2q. general procedure for the synthesis of the amino acid-derived ureas 4 to a solution of compound 2 (1 mmol) in ch3cn (16 ml) and h2o (8 ml) was added an amino acid (1.1 mmol). et3n (1.5 mmol, 152 mg) was then added dropwise. the mixture was stirred at r.t. until completion of the reaction (monitored by tlc). the acetonitrile was removed, and the residue was extracted by ethyl acetate (10 ml). the organic phase was washed successively with 10 % hcl (5 ml), saturated na2co3 solution (5 ml) and brine (5 ml), dried over anhydrous na2so4, and concentrated under reduced pressure to give the pure products 4a–4l. general procedure for the synthesis of hydrazinecarboxamides 6a and 6b to a solution of compound 2 (1 mol) in dry toluene (5 ml) was added methylhydrazine (1.1 mmol, 51 mg). the mixture was stirred at r.t. until completion of the reaction (monitored by tlc). then thf (10 ml) was added, and the mixture was washed with saturated na2co3 (35 ml) and brine (5 ml). the combined organic phase was dried over anhydrous na2so4, concentrated under reduced pressure, and purified by column chromatography on silica gel using ea–pe (1:1) as the eluent to give the pure products 6a and 6b. general procedure for the synthesis of hydrazinecarboxamides 6c and 6d to a solution of compound 2 (1 mol) in dry toluene (5 ml) was added aromatic hydrazine (1.1 mmol, 119 mg) and nah (1.5 mmol, 36 mg). the mixture was stirred at r.t. or 40°c until the completion of the reaction (monitored by tlc). then thf (10 ml) was added, and the mixture was washed with na2co3 (35 ml) and brine (5 ml). the combined organic phase was dried over anhydrous na2so4, concentrated under reduced pressure, and purified by column chromatography on silica gel using ea/pe (ea/pe = 1:1) as the eluent to give the pure product 6c and 6d. general procedure for the synthesis of carbamic esters 8a–f a round bottom flask was charged with compound 2 (1.0 mmol), alcohol (1.1 mmol) and toluene (5 ml). the mixture was stirred at 80 °c until completion of the reaction (monitored by tlc). the reaction mixture was cooled to r.t. whereby a white solid precipitated that was filtered off. the filter cake was washed by ethanol (5 ml) to give the pure products 8a–f. conclusions in summary, a convenient one-pot synthesis of carbamoyl benzotriazoles from carboxylic acid, dppa and bth was developed. the carbamoyl benzotriazoles showed good reactivity and chemoselectivity towards amino acids, hydrazines and alcohols, and provided facile access to amino acid-derived ureas, hydrazinecarboxamides and carbamic esters. together with the properties of being stable and easily to handle, carbamoyl benzotriazoles show promise for use as a practical substitute for isocyanates. preparation of carbamoyl benzotriazoles and their applications 21 supplementary material the physical, analytical and spectral data for the synthesized compounds and their spectra are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding authors on request. acknowledgement. this work was financially supported by the national natural science foundation of china (no. 20802070). и з в о д синтеза карбамоил-бензотриазола и њихова примена у синтези урее, хидразинкарбоксамида и естара карбаминске киселине hui mao, huili liu, yawei tu, zhiyun zhong, xin lv и xiaoxia wang zhejiang key laboratory for reactive chemistry on solid surfaces, college of chemistry and life sciences, zhejiang normal university, jinhua 321004, china карбамоил-бензотриазоли су синтетисани у једном реакционом кораку, полазећи од карбоксилних киселина, дифенил фосфоразидата (dppa) и 1h-бензотриазола (bth). испитана је реактивност и примена карбамоил-триазола. они лако реагују са амино -киселинама, хидразинима и алкохолима, и на тај начин се добијају одговарајући деривати урее, хидразинкарбоксамида и естри карбаминске киселине, редом, у добром до одличном приносу. 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tamamura, bioorg. med. chem. 19 (2011) 6735; c) p. koschker, a. lumbroso, b. breit, j. am. chem. soc. 133 (2011) 20746 15. r. löser, j. gut, p. j. rosenthal, m. frizler, m. gütschow, k. t. andrews, bioorg. med. chem. lett. 20 (2010) 252 16. t. w. v. geldern, j. a. kester, r. bal, j. r. wu-wong, w. chiou, d. b. dixon, t. j. opgenorth, j. med. chem. 39 (1996) 968 17. a. r. katritzky, q. y. chen, s. r. tala, chem. biol. drug. des. 73 (2009) 611 18. w. h. schuller, c. iemann, j. am. chem. soc. 73 (1951) 1644 19. a. r. katritzky, p. p. mohapatra, d. fedoseyenko, m. duncton, p. j. steel, j. org. chem. 72 (2007) 4268. {microwave-assisted synthesis of new pyrazole derivatives bearing 1,2,3-triazole scaffold as potential antimicrobial agents} j. serb. chem. soc. 82 (4) 357–366 (2017) udc 547.772.2+547.791:542.913+ jscs–4971 537.5–962:615.28–188 original scientific paper 357 microwave-assisted synthesis of new pyrazole derivatives bearing 1,2,3-triazole scaffold as potential antimicrobial agents dongamanti ashok*, rangu kavitha, srinivas gundu and velagapuri hanumantha rao green and medicinal chemistry laboratory, department of chemistry, osmania university, hyderabad500 007, india (received 5 february, revised 7 december 2016, accepted 17 january 2017) abstract: a new series of (e)-3-(3-(4-substituted phenyl)-1-phenyl-1h-pyrazol-4-yl)-1-(2-hydroxy-4-((1-aryl-1h-1-2,3-triazol-4-yl)methoxy)phenyl)prop-2-en-1-one derivatives was synthesized. the synthesis of the title compounds involved the 1,3-dipolar cu(i)-catalyzed alkyne–azide cycloaddition (cuaac) reaction of (e)-3-(3-(4-substituted phenyl)-1-phenyl-1h-pyrazol-4 -yl)-1-(2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-ones with aromatic azides. the structures were confirmed by nmr, ft-ir, mass and elemental analysis. all the synthesized compounds (6a–j) were evaluated for their antimicrobial activity. compounds 6a, 6d and 6e demonstrated promising inhibitory effects on both bacterial and fungal strains. keywords: pyrazole; chalcone; 1,2,3-triazole; microwave irradiation; antimicrobial activity. introduction infectious diseases caused by microbes, such as bacteria and fungi, are one of the leading causes of morbidity and mortality. the major reason for the increase in microbial infections is the resistance developed by these microbial organisms.1 thus, the development of new antimicrobial or antipathogenic agents that act upon new microbial targets is a necessity.2 pyrazole scaffolds possess a wide range of bioactivities, including antiviral,3 anti-inflammatory,4 anticonvulsant,5 anticancer,6 insecticidal,7 and antifungal.8,9 in recent years, several drugs developed from pyrazole derivatives, such as celecoxib that demonstrates anti-inflammation effects and inhibits cox-2, rimonabant that functions as a cannabinoid receptor inverse agonist and is utilized in obesity treatment, fomepizole that inhibits alcohol dehydrogenase and sildenafil that inhibits phosphodiesterase (fig. 1). * corresponding author. e-mail: ashokdou@gmail.com doi: 10.2298/jsc160205016a 358 ashok et al. fig. 1. commercially available bioactive pyrazole and triazole drugs. on the other hand, 1,2,3-triazoles have received attention due to their ease of synthesis by click chemistry bearing attractive features as well as their numerous biological activities.10–14 1,2,3-triazole derivatives have gained interest for their various biological activities, such as chemotherapeutic agents,15,16 and potent antimicrobial,17 anti-inflammatory,18,19 local anaesthetic,20 anticonvulsant,21 antineoplastic,22 antimalarial23 and antiviral activity.24 among the best known examples of triazole-containing drugs, tazobactam, a commercially available β-lactamase inhibitor, plays an eminent role in combination with broad-spectrum antibiotics (fig. 1). considering the above facts, it seemed worthwhile to integrate both pyrazole and 1,2,3-triazole pharmacophore units in one molecular platform to generate a newer scaffold for biological evaluation. pharmacophore hybridization is believed to be analogous to conventional combination therapy wherein the two drugs are covalently linked and available as a single entity.4 in continuation to ongoing research activities25–27 to discover and develop potential new antimicrobial agents, an efficient method for the synthesis of (e)-3-(3-(4-substitutedphenyl)-1-phenyl-1h-pyrazol-4-yl)-1-(2-hydroxy-4-((1-aryl-1h-1,2,3-triazol-4 -yl)methoxy) phenyl)prop-2-en-1-one derivatives in excellent yields is reported herein. results and discussion the synthetic approach adapted to obtain the target compounds is depicted in schemes 1 and 2. the selective preparation of the starting material 1-(2-hydroxy -4-(prop-2-yn-1-yloxy)phenyl)ethanone28 (3) was realized by the nucleophilic substitution reaction of 1-(2,4-dihydroxyphenyl)ethanone (1) with propargyl bromide (2) in the presence of k2co3 (scheme 1). the selective mono-propargylation of the hydroxy group without altering the o-hydroxy acetyl functionality was performed in high yields when starting from compound 1 rather than the other positional isomers. the synthesis of the title compounds 6a–j was accomplished by two synthetic strategies as shown in scheme 2. in order to develop a high yield protocol for the synthesis of the title compounds by click chemistry, the yields of compound 6g were investigated using cuso4·5h2o/sodium ascorbate and cui in different solvents, such as thf/h2o, t-buoh/h2o and dmf/h2o, under both conventional conditions and mwi. in synthesis and antimicrobial effects of pyrazole–triazole hybrids 359 the above optimization study, higher yields using cui in dmf/h2o (1:3) under mwi were attained (table i). scheme 1. synthesis of 1-(2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl)ethanone (3). scheme 2. synthetic route for the preparation of pyrazole-based 1,2,3-triazole hybrids (6a–j). in route-1 (scheme 2), the (e)-3-(3-(4-substituted phenyl)-1-phenyl-1h-pyrazol-4-yl)-1-(2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one derivatives (5a and b) were synthesized by claisen–schmidt condensation of compound 3 with substituted 1h-pyrazole-4-carboxaldehydes (4a and b) in presence of koh under conventional conditions and microwave irradiation. these compounds (5a and b) via a huisgen 1,3-dipolar cycloaddition reaction with aromatic 360 ashok et al. azides using cui in dmf under mwi gave compounds 6a–j. in route-2 (scheme 2), compound 3 in a click reaction with aromatic azides using cui in dmf under mwi followed by reaction with substituted 1h-pyrazole-4-carboxaldehydes (4a and b) in the presence of koh under conventional conditions and microwave irradiation gave compounds 6a–j. table i. optimization study for the synthesis of compound 6g under different catalyst and solvent conditions entry catalyst solvent conventional microwave irradiation time, h yield, %a time, min yield, %a 1 cuso4/sod. ascorbate thf/h2o (1:2) 72 21 20 25 2 cuso4/sod. ascorbate thf/h2o (1:3) 72 27 20 32 3 cuso4/sod. ascorbate dmf/h2o (1:2) 24 35 15 40 4 cuso4/sod. ascorbate dmf/h2o (1:3) 24 54 14 61 5 cuso4/sod. ascorbate t-buoh/h2o (1:2) 24 40 16 46 6 cuso4/sod. ascorbate t-buoh/h2o (1:3) 24 44 16 49 7 cui thf/h2o (1:2) 72 30 20 42 8 cui thf/h2o (1:3) 72 34 20 44 9 cui dmf/h2o (1:2) 14 47 10 69 10 cui dmf/h2o (1:3) 12.5 67 8.5 91 11 cui t-buoh/h2o (1:2) 24 41 16 52 12 cui t-buoh/h2o (1:3) 24 50 16 63 aisolated yields by comparing the above routes, the target compounds were synthesized in excellent yields in route-1 (table ii) to give overall yields of 81–92 % in shorter reaction time, while in route-2, the overall yields were much lower in the range of 34–48 % and a longer time (24–48 h)was necessary to complete the reaction. table ii. comparison of the yields of compounds 5a,b and 6a–j under different synthetic conditions compound m.p., °c conventional mwi time, h yield, %a time, min yield, %a 5 a 150–152 12 49 8 89 5 b 128–130 10 51 7 90 6 a 142–144 14 54 10 87 6 b 101–103 12.5 48 8 81 6 c 171–173 12 50 9 84 6 d 162–164 13 49 9.5 87 6 e 184–186 12 53 8 90 6 f 133–135 13 52 9 89 6 g 147–149 12.5 54 8.5 91 6 h 197–199 13 57 10 92 6 i 142–144 12.5 46 10 91 6 j 134–136 12 52 10 92 aisolated yields synthesis and antimicrobial effects of pyrazole–triazole hybrids 361 the formation of pyrazole derivatives containing 1,2,3-triazoles 6a–j was confirmed by ir, nmr and mass analysis (the spectral data are given in supplementary material to this paper). the ir spectrum of compound 6g showed absorption bands at 3111 and 1631 cm–1 due to oh and c=o groups, respectively. the 1h-nmr spectrum of 6g showed three singlets at δ 2.40, 3.86 and 5.35 ppm corresponding to ch3, och3 and och2 protons. the appearance of a doublet at δ 7.78 ppm (j = 15.29 hz) was due to the β-proton of the α,β-unsaturated carbonyl group. two singlets at δ 8.65 and 9.46 ppm correspond to triazole and pyrazole protons, respectively. a broad singlet due to the oh protons was observed at δ 13.52 ppm.29 in the 13c-nmr spectrum of 6g, the ch3, och3 and och2 carbons resonated at δ 20.2, 55.4 and 60.7 ppm, respectively. the carbonyl carbon appeared at δ 190.8 ppm. the mass spectra of 6g showed the molecular ion peak at m/z = 584 [m+h]+. antibacterial activity the synthesized compounds 5a and b and 6a–j were screened in vitro for their antibacterial activity against bacillus subtilis (atcc 6633) and staphylococcus aureus (atcc 6538), as examples of gram-positive bacteria, and escherichia coli (atcc 11229) and proteus vulgaris (atcc 13315), as examples of gram-negative bacteria. the agar well-diffusion method was used to assay the antibacterial activity against the test strains on mueller–hinton agar plates. gentamicin was employed as the standard antibacterial drug. the results obtained as zone of inhibition in mm and minimum inhibitory concentration (mic) in µg ml–1 are presented in table iii. investigation of the antibacterial efficiency of the synthesized compounds involved varying the substitution with electronegative chloroand electron-donating methyl group on phenyl ring of the pyrazole nucleus and variable substitutions on phenyl ring of triazole nucleus. it is evident from table iii that compound 6a with chloro substitutions on both the pyrazole and triazole nucleus exhibited the highest antibacterial inhibitory efficacy with inhibition zones of 28, 27 and 31 mm and an mic of 3.125 µg ml–1 against b. subtilis, s. aureus and e. coli, respectively, and 26 mm with an mic 6.25 µg ml–1 against p. vulgaris compared to standard drug gentamicin. after 6a, came compound 6d, with zones of inhibition in range of 24–28 mm (mic 6.25–12.5 µg ml–1), with chloro substitution on the pyrazole scaffold and trifluoromethyl group on the triazole nucleus, and 6e, with inhibition zones in range of 22–24 mm (mic 12.25 µg ml–1) with chloro substitution on the pyrazole scaffold and a benzyl group on triazole nucleus. this indicates that electron withdrawing groups and an electronegative chlorine atom on the phenyl ring strongly affect the antibacterial activity. the remaining compounds displayed moderate antibacterial potency with inhibition zones in the range 15–21 mm and with mic values of 25–50 µg ml–1. 362 ashok et al. table iii. antimicrobial activity of the synthesized compounds compound gram positive bacteria gram negative bacteria fungal strains b. subtilis s. aureus e. coli p. vulgaris a. niger c. albicans 5a 19a (25)b 15 (50) 21 (25) 17 (25) 15(25) 16(50) 5b 23 (12.5) 18 (25) 19 (25) 16 (50) 12(50) 18(50) 6a 28 (3.125) 27 (3.125) 31 (3.125) 26 (6.25) 24(6.25) 25(6.25) 6b 20 (25) 17 (25) 17 (50) 21 (25) 16(50) 18(25) 6c 19 (12.5) 19 (25) 21 (25) 24 (12.5) 21(25) 19(12.5) 6d 26 (6.25) 24 (12.5) 27 (12.5) 28 (6.25) 21(12.5) 18(12.5) 6e 24 (12.5) 22 (12.5) 23 (25) 23 (12.5) 19(12.5) 21(12.5) 6f 21 (12.5) 21 (25) 23 (12.5) 19 (25) 13(50) 19(25) 6g 16 (50) 16 (50) 21 (25) 18 (50) 15(50) 14(50) 6h 18 (25) 21 (12.5) 23 (25) 23 (50) 18(25) 13(50) 6i 15 (50) 18 (25) 16 (50) 20 (50) 18(50) 16(25) 6j 21 (25) 15 (50) 18 (25) 15 (50) 12(50) 16(50) gentamicin 32 (1.56) 29 (1.56) 34 (1.56) 30 (3.125) – – fluconazole – – – – 34(3.125) 31(1.56) azone of inhibition in mm; bmic in µg ml-1 antifungal activity the compounds were evaluated for their in vitro antifungal activity against aspergillus niger (atcc 9029) and candida albicans (atcc 10231) fungal strains. the agar well diffusion method was used to evaluate the antifungal activity against test strains on pda plates. fluconazole was used as the standard antifungal drug. the results are given in table iii, from which it is evident that compound 6a displayed the best antifungal activity with zones of inhibition of 24 and 25 mm, and an mic of 6.25 µg ml–1 against a. niger and c. albicans, respectively. compounds 6d (21 and 18 mm) and 6e (19 and 21 mm) were able to induce appreciable promising growth inhibitory activity with an mic 12.5 µg ml–1 against a. niger and c. albicans. thus, it was hypothesized that compounds with a chlorine atom and electron withdrawing groups on the phenyl ring of pyrazole and the triazole moieties would exhibit the highest antimicrobial inhibitory potency. experimental materials all the used materials were obtained commercially, mostly from sigma–aldrich, and used without further purification. equipment melting points were determined in open capillaries and are uncorrected. the purity of the compounds was checked by tlc on silica gel 60 f254 (merck). the 1h-nmr and 13c-nmr spectra were recorded on bruker avance ii 400 spectrometer using tms as an internal standard. the ir spectra were recorded in kbr on a shimadzu ftir 8400s spectrophotometer. the mass spectra were recorded on shimadzu lcms-2020 mass spectrometer. ele synthesis and antimicrobial effects of pyrazole–triazole hybrids 363 mental microanalysis was performed on a perkin elmer chn-2400 analyzer. all the microwave irradiation experiments were realized in a cem discover microwave system and reaction temperatures were monitored by an equipped ir sensor. analytical and spectral data are presented in supplementary material to this paper. general procedure for the synthesis of (e)-3-(3-(4-substituted phenyl)-1-phenyl-1h-pyrazol -4-yl)-1-(2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one derivatives (5a and b) conventional heating method. to a mixture of 1-(2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl)ethanone (3, 1 mmol) and powdered koh (2 mmol), substituted 1h-pyrazole-4-carboxaldehydes (4a and b, 1 mmol) was added and the reaction mixture was heated at 80 °c for 10– 12 h. after completion of the reaction (as indicated by tlc), the reaction mixture was poured into ice-cold water and neutralized with 10 % hcl solution. the thus-obtained solid was filtered and purified by column chromatography on silica gel using hexane/ethyl acetate (9:1 volume ratio) as eluent to afford compound 5. microwave irradiation method. to a mixture of 1-(2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl)ethanone (3, 1 mmol) and powdered koh (2 mmol) in a glass vial, a substituted 1hpyrazole-4-carboxaldehydes (4a and b, 1 mmol) was added and the vial was tightly sealed. the mixture was then irradiated for 7–8 min at 90 °c, at an irradiation power of 180 w. after completion of the reaction (as indicated by tlc), the vial was cooled, the reaction mixture was poured into ice-cold water and neutralized with 10 % hcl solution. the thus-obtained solid was filtered and purified by column chromatography on silica gel using hexane/ethyl acetate (9:1) as eluent to afford compound 5. general procedure for the synthesis of (e)-3-(3-(4-substituted phenyl)-1-phenyl-1h-pyrazol -4-yl)-1-(2-hydroxy-4-((1-aryl-1h-1,2,3-triazol-4-yl)methoxy)phenyl)prop-2-en-1-one derivatives (6a–j) using cui catalyst conventional method. to a well stirred mixture of (e)-3-(3-(4-substituted phenyl)-1 -phenyl-1h-pyrazol-4-yl)-1-(2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one derivatives (5a and b, 1 mmol) and cui (0.05 equiv.) in h2o/dmf (3:1) (6 ml), an aromatic azide was added and the reaction mixture was stirred at room temperature for 12–14 h. after completion of the reaction (as indicated by tlc), the reaction mixture was poured into icecold water. the thus-obtained solid was filtered and purified by column chromatography on silica gel using hexane/ethyl acetate (7:3, v/v) as eluent to afford compound 6. microwave irradiation method. a mixture of a (e)-3-(3-(4-substituted phenyl)-1-phenyl -1h-pyrazol-4-yl)-1-(2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one derivative (5a and b, 1 mmol) and cui (0.05 equiv.) was suspended in h2o/dmf (3:1 volume ratio, 2 ml) in a glass vial equipped with a small magnetic stirring bar. to this, an aromatic azide was added and the vial was tightly sealed. the mixture was then irradiated for 8–10 min at 60 °c, at an irradiation power of 100 w. after completion of the reaction (as indicated by tlc), the vial was cooled, and the reaction mixture poured into ice-cold water. the thus-obtained solid was filtered and purified by column chromatography on silica gel using hexane/ethyl acetate (7:3, v/v) as eluent to afford compound 6. general procedure for the synthesis of (e)-3-(3-(4-substituted phenyl)-1-phenyl-1h-pyrazol -4-yl)-1-(2-hydroxy-4-((1-aryl-1h-1,2,3-triazol-4-yl)methoxy)phenyl)prop-2-en-1-one derivatives (6a–j) using cuso4/sodium ascorbate catalyst conventional method. to a well stirred mixture of (e)-3-(3-(4-substituted phenyl)-1 -phenyl-1h-pyrazol-4-yl)-1-(2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one derivative (5a and b, 1 mmol), cuso4·5h2o (0.05 equiv.) and sodium ascorbate (0.05 equiv.) in 364 ashok et al. h2o/dmf (2:1 volume ratio, 10 ml), an aromatic azide was added and the reaction mixture was stirred at room temperature for 24 h. after completion of the reaction (as indicated by tlc) the reaction mixture was poured into ice-cold water. the thus-obtained solid was filtered and purified by column chromatography on silica gel using hexane/ethyl acetate (7:3) as eluent to afford compound 6. microwave irradiation method. a mixture of a (e)-3-(3-(4-substituted phenyl)-1-phenyl -1h-pyrazol-4-yl)-1-(2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl)prop-2-en-1-one derivative (5a and b, mmol) cuso4·5h2o (0.05 equiv.) and sodium ascorbate (0.05 equiv.) were suspended in h2o/dmf (2:1 volume ratio, 2 ml) in a glass vial equipped with a small magnetic stirring bar. to this, an aromatic azide was added and the vial was tightly sealed. the mixture was then irradiated for 15 min at 60 °c, at an irradiation power of 100 w. after completion of the reaction (as indicated by tlc), the vial was cooled and the reaction mixture poured into icecold water. the thus-obtained solid was filtered and purified by column chromatography on silica gel using hexane/ethyl acetate (7:3) as eluent to afford compound 6. biological assay antimicrobial activity. the in vitro antimicrobial studies were performed by the agar well diffusion method against test organisms.30,31 nutrient broth (nb) plates were swabbed with 24 h-old broth culture (100 ml) of the test bacteria. using a sterile cork borer, wells (6 mm) were made into each petri plate. different concentrations of test samples dissolved in dmso were added into the wells using sterile pipettes. simultaneously, the standard antibiotics, gentamicin for antibacterial activity, fluconazole for antifungal activity were tested against the pathogens. the plates were incubated at 37 °c for 24 h for the bacteria and at 28 °c for 48 h for the fungi. after appropriate incubation, the diameter of zone of inhibition of each well was measured. the broth dilution test was used to determine the minimum inhibitory concentration (mic) of the samples.32,33 freshly prepared nutrient broth was used as the diluent. the 24 h-old culture of the test bacteria b. subtilis, s. aureus, e. coli and p. vulgaris and the test fungi a. niger and c. albicans were diluted 100-fold in nutrient broth (100 μl bacterial cultures in 10 ml nb). increasing concentrations of the test samples were added to the test tubes containing the bacterial and fungal cultures. all the tubes were incubated at 37 °c for 24 h for the bacteria and at 28 °c for 48 h for the fungi. the tubes were examined for visible turbidity using nb as the control. the lowest concentration that inhibited visible growth of the tested organisms was recorded as the mic. conclusion in summary, a new series of compounds 6a–j was synthesized by conventional and microwave irradiation methods. in the microwave irradiation method, reactions were completed in a short reaction time under mild reaction conditions and convenient operation in high yields. all the titled compounds were screened for their in vitro antimicrobial activity. compound 6a was found to be the most potent and compounds 6d and 6e were found to be moderately potent compared to the standard drug gentamicin against the pathogenic bacteria, while compounds 6a, 6d and 6e exhibited potent activity against the pathogenic fungi compared to the standard drug fluconazole with their respective concentrations. antimicrobial screening results revealed that, compound 6a of the synthetic library could be considered as a promising antimicrobial drug candidate. synthesis and antimicrobial effects of pyrazole–triazole hybrids 365 acknowledgements. the authors are thankful to the head of department of chemistry, osmania university, for providing the laboratory facilities and the director, central facilities for research and development (cfrd), osmania university, for providing the ir and nmr spectral analysis. financial support for rk from ugc, new delhi, india, is gratefully acknowledged. supplementary material spectral and analytical data of the synthesized compounds are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. и з в о д синтеза нових 1,2,3-триазолских деривата пиразола, под условима микроталасног зрачења, као потенцијалних антимикробних једињења dongamanti ashok, rangu kavitha, srinivas gundu и velagapuri hanumantha rao green and medicinal chemistry laboratory, department of chemistry, osmania university, hyderabad-500 007, india синтетисана је серија нових деривата (e)-3-(3-(4-супституисани фенил)-1-фенил -1h-пиразол-4-ил)-1-(2-хидрокси-4-((1-арил-1h-1,2,3-триазол-4-ил)метокси)фенил)проп-2-ен-1-она. синтеза деривата укључује cu(i)-катализовану 1,3-диполарну алкин– –азид циклоадицију (cuaac) реакцијом (e)-3-(3-(4-супституисани фенил)-1-фенил-1h -пиразол-4-ил)-1-(2-хидрокси-4-(проп-2-ин-1-илокси)фенил)проп-2-ен-1-она и ароматичних азида. структуре производа су потврђене nmr и ftir спектроскопијом, масеном спектрометријом и микроанализом. испитана је антимикробна активност свих синтетисаних деривата 6a–j. једињења 6a, 6d и 6e показују интересантне инхибиторне активности према бактеријама и гљивама. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {transport properties of binary liquid mixtures candidate solvents for optimized flue gas cleaning processes} j. serb. chem. soc. 81 (12) 1427–1439 (2016) udc 547.422+547.262’416+547.431.4: jscs–4939 532.13:536.2:66.021.2.063.8 original scientific paper 1427 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. transport properties of binary liquid mixtures – candidate solvents for optimized flue gas cleaning processes andrej m. stanimirović1*, emila m. živković2#, divna m. majstorović2# and mirjana lj. kijevčanin2# 1electric power industry of serbia, carice milice 2, 11000 belgrade, serbia and 2faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11120 belgrade, serbia (received 23 june, revised 9 september, accepted 20 september 2016) abstract: thermal conductivities and viscosities of three pure chemicals, monoethanolamine (mea), tetraethylene glycol dimethyl ether (tegdme) and polyethylene glycol 200 (peg 200) and two binary mixtures (mea+ +tegdme and mea+peg 200) were measured at six temperatures: 298.15, 303.15, 308.15, 313.15, 318.15 and 323.15 k and atmospheric pressure. measurement of the thermal conductivities was based on a transient hot wire measurement setup, while the viscosities were measured with a digital stabinger svm 3000/g2 viscometer. from these data, deviations in the thermal conductivity and viscosity were calculated and fitted to the redlich–kister equation. thermal conductivities of mixtures were correlated using the filippov, jamieson, baroncini and rowley models, while the viscosity data were correlated with the eyring-uniquac, eyring-nrtl and mcalister models. keywords: thermal conductivity; viscosity; modelling; mea; tegdme; peg 200. introduction modelling and simulation of industrial processes in thermal power plants, as well as the design of appropriate facilities for treating flue gas, require the thermophysical properties of the applied solvent as input data. among other properties, it is necessary to know the thermal conductivity and viscosity of the used solvent in the range of working temperature of the plant. multitudes of studies have been conducted in the past years in the quest for the most suitable solvent for the removal of carbon dioxide.1–6 numerous industrial plants, some of which have been in operation for over forty years, emerged from these studies. * corresponding author. e-mail: andrej_stanimirovic@yahoo.com # serbian chemical society member. doi: 10.2298/jsc160623083s 1428 stanimirović et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. primary amines exhibit the highest level of carbon dioxide binding. for many years, primary alkanolamines, monoethanolamine (mea) in the first place, were successfully used as chemical solvents in industrial plants. however, corrosion processes, which intensify with increasing solvent concentration, must also be taken into account in thermal power plants. secondary amines show slightly weaker level of binding of carbon dioxide, and this degree is further reduced with tertiary amines. however, secondary and tertiary amines exhibit better affinity for the binding of sulphur dioxide, another toxic greenhouse gas (general considerations related to the environmental impact of the industrial gases are presented in supplementary material to this paper). choice of the most suitable solvent will be affected by associated energy costs of regeneration: since the recovery of the solvent is mainly done through thermal regeneration, a compromise is made between binding affinity and ability to regenerate the solvent. tetraethylene glycol dimethyl ether (tegdme) is a polar aprotic solvent that has already found commercial application in a regenerative process for so2 removal.7 polyethylene glycols (peg) are important industrial solvents of low toxicity, low vapour pressure, high chemical stability and low melting points. the high solubility of so2 and relatively easy desorption are the main adventages of polyethylene glycol 200 (peg 200), which reduces power consumption in the solvent regeneration process.8 thanks to these characteristics, it could be used in the purification of flue gases from power plants. unlike mea, both tegdme and peg 200 are physical solvents which means that absorption process occurs without chemical reaction. investigations9,10 have shown that mixtures of physical and chemical solvents often show better characteristics regarding binding capacity and selectivity than aqueous solutions of alkanolamines, traditionally used in gas purification processes. in this work, experimental thermal conductivities and viscosities are reported for three pure substances, mea, tegdme and peg 200, and two binary mixtures, mea+tegdme and mea+peg 200, at six temperatures, 298.15, 303.15, 308.15, 313.15, 318.15, 323.15 k and atmospheric pressure. from these experimental data, deviations in thermal conductivity (δλ) and viscosity (δη) were calculated and correlated by the redlich–kister equation.11 for correlating the thermal conductivities of the mixtures, the filippov,12 jamieson,13 baroncini14 and rowley15 models were used. the viscosity data were correlated by the eyring-nrtl,16 eyring-uniquac17 and the twoand three-parameter mcalister18 models. experimental chemicals mea (99.5 %) was purchased from merck, tegdme (99.0 %) from sigma–aldrich, while peg 200 (99.0 %) was supplied from acros organics. chemicals were used without further purification. the densities, thermal conductivities and dynamic viscosities of the pure transport properties of binary liquids 1429 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. substances have been compared in table i with literature values at 298.15 k.19-27 the agreement between literature and the experimental values is satisfactory with differences of less than 0.6 kg m-3 for the density measurements, within 5×10-3 w m-1 k-1 for the thermal conductivities, and within 7×10-2 mpa s for the viscosity measurements of the less viscous fluids. table i. densities ρ, thermal conductivities λ and dynamic viscosities η of the pure components at 298.15 k; standard uncertainties, u, for each variables are u(t) = ±0.01 k; u(p) = = ±5 %; u(x1) = ±0.0001, and the combined expanded uncertainties, uc, are uc(ρ) = ±1.2 kg m-3; uc(λ) = ±4.0 % and uc(η) = ±1.0 %, at the 0.95 level of confidence (k ≈ 2) component ρ / 103 kg m-3 λ / w m-1 k-1 η / mpa s exp. lit. exp. lit. exp. lit. mea 1.0117 1.012319 0.2349 0.2399a,20 18.883 18.9521 tegdme 1.0064 1.005922,23 1.006324 0.1649 0.161525 3.3316 3.31322,23 3.39424 peg 200 1.1208 1.1209826 0.2017b 0.199b,27 51.872 48.15726 ainterpolated value; bvalues at 303.15 k apparatus and procedures thermal conductivities reported in this study were measured using transient hot wire experimental setup, which is a reliable and precise technique for measuring the thermal conductivity of liquids.28 the details are presented in the supplementary material. an analytical model for a long thin conductor heated by a constant current and cooled by the surrounding heat-conducting body of infinite dimensions can be found in the literature.29,30 the detailed considerations are given in supplementary material to this paper. uncertainty in the measurement of the thermal conductivity was evaluated in accordance with the guide to the expression of uncertainty in measurement (gum).31 the obtained uncertainty components (for the calculations see supplementary material) and the total expanded uncertainty of the performed thermal conductivity measurement are given in table ii. table ii. thermal conductivity measurement uncertainty components uncertainty component uncertainty level, % uq 2.5 uσ 2.5 urw0 0.2 uzr 1.9 uk 4.2 viscosities η of the binary mixtures and corresponding pure substances were measured with a digital stabinger svm 3000/g2 viscometer. the instrument consists of two measuring cells; one of them is used for measuring the density of the sample and the other for the viscosity measurements. the principles of the measurements are presented in the supplementary material. the temperature in the cell was regulated to ±0.01 k with a built in solid-state thermostat. the reproducibility of the dynamic viscosity and density measurements, stated by the manufacturer, were 0.35 % and 0.5 kg m-3, respectively. the relative uncertainty in dynamic viscosity measurements was evaluated as less than ±1.0 %, at the 0.95 level of confidence (k ≈ 2). densities of pure components were also measured with the stab1430 stanimirović et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. inger svm 3000/g2 model. uncertainty in density measurements, at the 0.95 level of confidence (k ≈ 2), was estimated as ±1.2 kg m-3. mixtures were prepared gravimetrically on a mettler ag 204 balance. the balance precision was 1×10-7 kg and the standard uncertainty in the mole fraction calculation was estimated as ±1×10-4. results and discussion the experimental data of thermal conductivity λ and viscosity η, as well as the calculated values of deviation in thermal conductivity δλ and viscosity δη for investigated binary systems (mea + tegdme and mea + peg 200) at six temperatures, 298.15, 303.15, 308.15, 313.15, 318.15 and 323.15 k, and atmospheric pressure are reported in table s-i of the supplementary material. the deviation functions were calculated from the equation: 2 1 i i i y y x y = δ = −  (1) in which δy refers to the deviation in the thermal conductivity, δλ, or in the viscosity, δη, y is a mixture property, thermal conductivity or viscosity, while yi is thermal conductivity, λi, or viscosity, ηi of the pure component. the deviation functions δλ and δη were further correlated with the redlich– –kister (rk) equation:11 p 0 k i j p z x x a = =  (2) in which z represents δλ or δη, while ap are the fitting parameters, optimized by the f-test, of which there are (k+1). the values of fitting parameters are presented in table s-ii of the supplementary material. the quality of the correlation was evaluated by the root-mean-square deviations (rmsd), σ. literature values of the thermal conductivities for the pure components are also included in fig. 2. for monoethanolamine, the literature values show a similar temperature dependency as the obtained experimental data with a maximum deviation of around 2 %. the same is the case for tegdme, except that the maximum deviation from the literature values for this substance was around 3.5 %.32 for peg 200, no literature data are available, except for the values measured by one of the producers.27 the experimental data decreased with increasing temperature, while literature values increased. in the investigated temperature range, the maximum deviation was 2.8 % at 323.15 k, while the minimum deviation was 0.5 % at 313.15 k. comparison of deviations in thermal conductivity, δλ, is shown in fig. 3 for both systems at 308.15 k. the deviations in thermal conductivity for investigated binary systems were negative at all temperatures and for all mixture compositions, transport properties of binary liquids 1431 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. as presented in table s-ii and displayed in fig. 3. curves for both systems are asymmetrical and shifted towards higher mea mole fractions. for both mixtures, the δλ values are more negative at higher temperatures, although the temperature influence was more significant in the mea+tegdme binary system. mixture with peg 200 showed higher deviation from ideality, as can be noticed in fig. 3. a) 300 310 320 330 0.14 0.16 0.18 0.20 0.22 0.24 0.0000 0.2000 0.4000 0.6000 0.8000 1.0000 teg dme25 teg dme32 mea20 λ / w m -1 k -1 t / k b) 300 305 310 315 320 325 330 0.180 0.195 0.210 0.225 0.240 λ / w m -1 k -1 t / k 0.0000 0.2001 0.4001 0.6000 0.8000 1.0000 mea20 peg 20027 fig. 2. experimental values of the thermal conductivity λ, as a function of temperature for the systems: a) mea+tegdme and b) mea+peg 200, at differnt molar fractions, x1 .lines are fittings of the data points. 1432 stanimirović et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. 0.0 0.2 0.4 0.6 0.8 1.0 -0.035 -0.030 -0.025 -0.020 -0.015 -0.010 -0.005 0.000 mea + tegdme mea + peg200 rk equation δλ / w m -1 k -1 x 1 fig. 3. experimental values of the deviations in the thermal conductivity δλ, as a function of mea mole fraction x1 for the mea+tegdme and mea+peg 200 systems at 308.15 k. the lines were calculated from the redlich–kister equation.11 the experimental data for viscosity, η, and the calculated viscosity deviation, δη, are presented in figs. 4–6. similar to the thermal conductivity, for system with tegdme, the viscosities of mixtures were between the values for the pure components, while for the system with peg 200, viscosities of the mixtures were higher than the viscosities of the pure components, as can be seen in fig. 4b. the viscosities of both the mixtures and the pure components decreased with increasing temperature. literature values of the viscosity for pure mea and peg 200 were incorporated in fig. 4. the agreement between the experimental data and literature values for mea was very good, while for peg 200, larger deviations were noticed. inspection of the limited number of available data showed that one literature source reported lower26 and the other higher27 viscosity values than the ones measured in this study. viscosity deviations, δη, were negative for the system with tegdme and positive for the system with peg 200, at all temperatures and for all mixture compositions, as can be seen in fig. 6. the curves for both systems are asymmetrical and shifted towards the higher mea mole fractions. for both mixtures, the δη values decreased by an absolute value with increasing temperature. transport properties of binary liquids 1433 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. a) 0.0 0.2 0.4 0.6 0.8 1.0 0 5 10 15 20 298.15 k 303.15 k 308.15 k 313.15 k 318.15 k 323.15 k η / m p a s x 1 b) 0 .0 0.2 0.4 0.6 0.8 1.0 0 10 20 30 40 50 60 η / m p a s x 1 298.15 k 303.15 k 308.15 k 313.15 k 318.15 k 323.15 k fig. 4. experimental values of viscosity η as a function of mea mole fraction x1 for the systems: a) mea+tegdme and b) mea+peg 200, at different temperatures. the lines were fitted to the data points. 1434 stanimirović et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. a) 0.00310 0.00315 0.00320 0.00325 0.00330 0.00335 0.5 1.0 1.5 2.0 2.5 3.0 ln (η / m p a s) t -1 / k-1 0.0000 0.2000 0.4000 0.6000 0.8000 1.0000 mea21 mea33 mea34 tegdme23 tegdme24 tegdme35 tegdme36 b) 0.00310 0.00315 0.00320 0.00325 0.00330 0.00335 1.8 2.4 3.0 3.6 4.2 ln (η / m p a s) t -1 / k -1 0.0000 0.2001 0.4001 0.6000 0.8000 1.0000 mea21 mea33 mea34 peg 20026 peg 20027 fig. 5. experimental values of viscosity η as a function of temperature for the systems: a) mea+tegdme and b) mea+peg200, at different mea mole fractions x1. the lines were fitted to the data points. transport properties of binary liquids 1435 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. a) 0.0 0.2 0.4 0.6 0.8 1.0 -7.5 -5.0 -2.5 0.0 δ η / m p a s x 1 29 8.15 k 30 3.15 k 30 8.15 k 31 3.15 k 31 8.15 k 32 3.15 k r k equatio n b) 0.0 0.2 0.4 0.6 0.8 1.0 0 6 12 18 24 δ η / m p a s x 1 298.15 k 303.15 k 308.15 k 313.15 k 318.15 k 323.15 k rk eq uation fig. 6. experimental values of the viscosity deviation δη as a function of the mea mole fraction x1 for the systems: a) mea+tegdme and b) mea+peg 200, at different temperatures. the lines were calculated from the redlich–kister equation.11 1436 stanimirović et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. modelling thermal conductivities of mixtures were correlated using the filippov,12 jamieson,13 baroncini14 and rowley15 models, while the viscosity data were correlated with the eyring-nrtl16 eyring-uniquac17 and the mcalister 2-and 3-parameter18 models. details of these models are given in the supplementary material to this paper. the ability of these models to correlate thermal conductivity and viscosity was estimated by calculating the percentage deviations (pd), from the following equation: exp cal exp1 100 ( ) m i i y y pd y m y= − =  (7) in which yexp and ycal represent the experimental and calculated values, respectively, of the thermal conductivity or viscosity and m is the number of experimental data points. the results of the modelling for the investigated binary mixtures are reported in table iii for the thermal conductivities and in table iv for the viscosities. table iii. results of thermal conductivity correlations (pd / %) t / k correlation filippov jamieson baroncini rowley mea (1)+tegdme (2) 298.15–323.15 2.05 1.97 5.19 2.43 mea (1)+peg 200 (2) 298.15–323.15 2.56 1.88 5.47 7.11 table iv. results of the viscosity correlations (pd / %) t / k correlation eyring-nrtl eyring-uniquac mcallister-3 mcallister-4 mea (1)+tegdme (2) 298.15 0.29 1.40 0.27 0.16 303.15 0.14 1.35 0.11 0.10 308.15 0.21 1.34 0.21 0.14 313.15 0.21 1.00 0.22 0.12 318.15 0.18 1.05 0.14 0.09 323.15 0.24 0.92 0.23 0.07 mea (1)+peg 200 (2) 298.15 4.59 0.79 2.15 0.21 303.15 3.70 0.68 2.09 0.23 308.15 2.78 0.60 1.97 0.21 313.15 2.03 0.55 1.88 0.22 318.15 1.36 0.45 1.80 0.22 323.15 1.07 0.38 1.79 0.24 transport properties of binary liquids 1437 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. the thermal conductivity correlations were realised with the same set of parameters over the complete temperature range investigated. satisfactory results were obtained with the filippov12 and jamieson13 models. the baroncini14 model gave poorer results for both systems, while the rowley model,15 both correlatively and predicatively, was satisfactory only for the system with tegdme. viscosity modelling was performed at each of the investigated temperatures. the best results in correlating viscosity were obtained with the three-parameter mcalister18 model, probably due to the larger number of adjustable parameters. the two-parameter eyring-nrtl,16 eyring-uniquac17 and mcalister18 models mostly gave satisfactory results, with the exception of the eyringnrtl16 model for system with peg 200 at lower temperatures. conclusions transport properties of two binary liquid mixtures were investigated both by measurement and by correlation to multiple models in order to gain better understanding of the conditions in liquid mixtures and to improve the models that describe their behaviour. the values of both properties decrease with an increase in temperature. the values of properties vary monotonically with composition for one system (mea+tegdme), but not for the other (mea+peg 200). in the latter case, the thermal conductivities were lower and the viscosities higher than the respective properties of the pure components. the thermal conductivity and viscosity data were successfully correlated with filippov, jamieson, eyringuniquac and mcalister models. the rowley and eyring-nrtl models were satisfactory only for the mea+tegdme system, while the baroncini model gave poorer results for both the investigated binary mixtures. supplementary material considerations related to industrial gasses emission, experimental details and comments concerning correlation models are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the authors gratefully acknowledge the financial support received from the research fund of the ministry of education, science and technological development of the republic of serbia (project no. 172063) and the faculty of technology and metallurgy, university of belgrade. 1438 stanimirović et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. и з в о д транспортна својства бинарних течних смеша – потенцијалних растварача за оптимизовани процес пречишћавања димних гасова андреј м. станимировић1, емила м. живковић2, дивна м. мајсторовић2 и мирјана љ. кијевчанин2 1електропривреда србије, царице милице 2, 11000 београд и 2технолошко–металуршки факултет, универзитет у београду, карнегијева 4, 11120 београд топлотне проводљивости и вискозности три чисте супстанце, моноетаноламина (меа), тетраетиленгликол-диметил етра (tegdme) и полиетилен-гликола (peg 200) и две бинарне смеше (меа+tegdme и меа+peg 200) су измерене на 6 температура, 298,15; 303,15; 308,15; 313,15; 318,15 и 323,15 k, и на атмосферском притиску. мерење топлотне проводљивости је засновано на нестационарној методи топле жице, док су вискозности одређене на дигиталном stabinger svm 3000/g2 вискозиметру. из ових података, израчунате су промене топлотне проводљивости и вискозности при мешању и корелисане redlich–kister једначином. топлотне проводљивости смеша корелисане су и моделима filippov, jamieson, baroncini и rowley док су за корелисање вискозности коришћени модели eyring-uniquac, eyring-nrtl и mcalister.ра за испитивање великог броја променљивих са малим бројем експеримената. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice multicenter wiener indices and their applications j. serb. chem. soc. 80 (8) 1009–1017 (2015) udc 539.6+544.131 jscs–4776 original scientific paper 1009 multicenter wiener indices and their applications ivan gutman1,2*#, boris furtula1# and xueliang li3 1faculty of science, university of kragujevac, p. o. box 60, 34000 kragujevac, serbia, 2state university of novi pazar, novi pazar, serbia and 3center for combinatorics, nankai university, tianjin, 300071, china (received 26 january, revised 12 february, accepted 13 february 2015) abstract: the wiener index w could be viewed as a molecular structure descriptor composed of increments representing interactions between pairs of atoms. a generalization of the w are the steiner–wiener indices kw , k = 3, 4,… in the quantity kw , interactions between k-tuples of atoms play a role, based on the concept of the steiner distance. it is shown that the term kw wλ+ provides an approximation for the boiling points of alkanes better than w itself. the best such approximation is obtained for 7k = . keywords: wiener index; multicenter wiener index; steiner distance; steiner– –wiener index; molecular graph. introduction the wiener index (w) is one of the oldest and most examined graph-based molecular structure descriptors. details on its mathematical properties and chemical applications are given in reviews,1–5 recent research papers,6–9 and the references cited therein. on the occasion of the fiftieth anniversary of the wiener index, three special journal issues were published.10–12 additional historical data on w can be found in a survey.13 the wiener index is defined in the following manner. let g be a molecular graph and 1 2, , , nv v v be its vertices. the distance between the vertices iv and jv , denoted by ( , )i jd v v , is the number of edges in (= the length of) the shortest path that connects iv and jv . then: ( ) ( , )i j i j w w g d v v < = =  (1) with the summation embracing all pairs of vertices (vi,vj) of the molecular graph g. * corresponding author. e-mail: gutman@kg.ac.rs # serbian chemical society member. doi: 10.2298/jsc150126015g _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1010 gutman, furtula and li bearing in mind that each vertex of the molecular graph represents an atom of the underlying molecule,14 the quantity w, defined by means of eq. (1), may be viewed as a sum of structural increments representing pairs of atoms, i.e., two -center interatomic interactions. from this point of view, one could think of three-center, four-center, etc. interactions that would lead to the following evident multicenter extension of the wiener-index concept: 3 3 ( ) ( , , )i j k i j k w w g d v v v < < = =  (2) 4 4 ( ) ( , , , )i j k l i j k l w w g d v v v v < < < = =  (3) 5 5 ( ) ( , , , , )i j k l m i j k l m w w g d v v v v v < < < < = =  (4) etc. in formulas (2)–(4), the meaning of the three-, four-, five-vertex distances requires clarification. in fact, a long time ago, chartrand et al. introduced such a multi-vertex distance into graph theory,15 which was eventually much studied under the name “steiner distance”.16 its definition is given in the subsequent section. the multicenter wiener-type indices based on the steiner distance will be referred to as “steiner–wiener indices” and are defined in the subsequent section. the present work is aimed at establishing their chemical applicability. steiner distance and steiner–wiener index let g be a connected graph with n vertices. let 1 2 { , , , } ki i i s v v v=  be a set of k distinct vertices of g. then the steiner tree, ( )t s , is a tree (= connected acyclic graph) that is a subgraph of g, containing all vertices of s, and possessing a minimal number of edges. the number of edges of ( )t s is the steiner distance of the vertices 1 2 , , , ki i i v v v . for details on the steiner-distance concept, see elsewhere.17,18 for 2,3, ,k n=  , the k-th steiner–wiener index of the (molecular) graph g is defined as: 1 2 ( ) ( , , , ) kk k i i i s w w g d v v v= =   (5) where the summation goes over all k-element subsets 1 2 { , , , } ki i i s v v v=  of the vertex set of g. steiner–wiener indices, kw , were recently considered, 19 and their basic mathematical properties determined. some of these are the following: 1. 2 ( )w g coincides with the ordinary wiener index ( )w g , eq. (1). 2. eqs. (2), (3), and (4) are special cases of eq. (5), for 3k = , 4k = and 5k = , respectively. 3. for a graph g with n vertices, if k n= , then ( ) 1kw g n= − . 4. for a graph g with n vertices, if k n> , then ( ) 0kw g = . for a tree t, and for all 2,3, ,k n=  : _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ multicenter wiener indices 1011 1 1 2 1 ( ) ( ) ( ) k k e i n e n e w t i k i − =    =   −    (6) where 1( )n e and 2 ( )n e are the number of vertices lying on the two sides of the edge e, and where the first summation goes over all edges of t. for all edges e of the tree t, 1 2( ) ( )n e n e n+ = . note that for 2k = , formula (6) reduces to the expression (7), discovered by wiener himself as early as in 1947:7,14,20 1 2( ) ( ) ( ) e w t n e n e=  (7) steiner–wiener indices and boiling points of alkanes the first chemical application of the wiener index was its usage for the prediction of the normal boiling points of alkanes.20 eventually, correlations with boiling points became a standard test for the quality of topological indices.21–24 in view of this, this physico-chemical parameter is also used in these studies of the steiner–wiener indices. the well known23 plot of the normal boiling points vs. the wiener index is reproduced in fig. 1. the curve passing through the data-points is of the form: fig. 1. correlation between normal boiling points (bp / k) and wiener index (w) for the set of all isomeric alkanes with 2 to 9 carbon atoms (74 compounds).25 the curve passing through the data-points is specified by eq. (8). statistical data pertaining to this correlation are found in tables i and ii, for k = 2. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1012 gutman, furtula and li * * calc * ( ) 1 a bw bp bp w cw + ≈ = + (8) where *w w= and where , and a b c are fitting parameters. the correlation between the experimental and calculated boiling points (i.e., between bp and calcbp , cf. eq. (8)), is shown in fig. 2. table i. fitting parameters in formulas (8) and (9), for k = 2,3,..., 9. the (a,b,c)-values were obtained by means of the scaled levenberg–marquardt algorithm.26 the λ-values are those for which the respective correlation coefficients are maximal, cf. fig. 3. the parameters for the best approximation are indicated by boldface k a b c λ 2 191.328 15.104 0.031 – 3 192.480 14.547 0.031 0.023 4 193.704 14.820 0.032 0.044 5 191.287 16.476 0.037 0.063 6 186.764 18.773 0.043 0.127 7 181.255 21.421 0.049 0.392 8 180.547 20.834 0.047 0.802 9 187.788 16.790 0.036 1.400 table ii. statistical data for the correlations between boiling points and the topological indices * kw w wλ= + , k =2,3,..., 9; r = correlation coefficient, are = average relative error (in %), mre = maximal observed relative error (in %). the data for the best approximation are indicated by boldface. for details, see eqs. (8) and (9) and the text k r are mre 2 0.98954 1.45 8.42 3 0.98957 1.45 8.83 4 0.99018 1.41 9.46 5 0.99135 1.33 8.58 6 0.99256 1.23 6.80 7 0.99323 1.18 4.63 8 0.99273 1.23 4.19 9 0.99149 1.33 6.98 the most obvious idea for testing the steiner–wiener indices would be to set * kw w= into eq. (8). this, however, did not yield any improvement, and thus had to be abandoned. a better option was to modify the wiener index as: * kw w wλ= + (9) and use the variable *w in combination with eq. (8) . for each fixed choice of k, k = 3,4,...8, the parameter λ was varied, and its value determined to maximize the correlation coefficient for the linear correlation between bp and *calc ( )bp w . in all the studied cases, an optimal value for λ exists at which the correlation coefficients attain a maximum; a characteristic example is shown in fig. 3. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ multicenter wiener indices 1013 fig. 2. correlation between the calculated boiling points ( calcbp , according to eq. (8), * =w w ) and the experimental boiling points (bp) for the same compounds as in fig. 1. statistical data pertaining to this correlation are found in tables i and ii, for k = 2. fig. 3. the λ-dependence of the correlation coefficient r for the correlation between bp and calcbp for the case 5k = . the maximum is attained at 0.063λ = , cf. table i. the results thus obtained are presented in tables i and ii, and in figs. 4 and 5. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1014 gutman, furtula and li fig. 4. normal boiling points (bp / k) vs. 7w wλ+ for the same alkanes as in fig. 1. as the data in tables i and ii show, the choice 7k = provides the best agreement between bp and bpcalc, cf. eqs. (8) and (9). fig. 5. the best correlation between bp and bpcalc, was obtained by eqs. (8) and (9) for k = 7. statistical data pertaining to this correlation are to be found in tables i and ii. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ multicenter wiener indices 1015 discussion and concluding remarks viewing at the wiener index as a structure descriptor based on two-center interatomic interactions, it could be expected that the next-important structural feature would be three-center interactions. in the case of steiner–wiener index applied to alkanes, this certainly cannot be the case, since for trees the following identity holds: 3 2 ( ) ( ) 2 n w t w t − = (10) therefore, 3w contains the exactly same structural information as the ordinary wiener index, w. relation (10) is deduced from eq. (6) as follows. for 3k = , eq. (6) has the form: 1 2 1 2 3 ( ) ( ) ( ) ( ) ( ) 1 2 2 1 e n e n e n e n e w t        = +               which, bearing in mind that 1 2( ) ( )n e n e n+ = , is transformed into: 2 2 1 1 3 1 2 1 2 1 2 1 2 ( )[ ( ) 1] ( )[ ( ) 1] ( ) ( ) ( ) 2 2 ( ) ( ) 2 2 ( ) ( ) ( ) ( ) 2 2 e e e n e n e n e n e w t n e n e n e n e n n e n e n e n e − − = +   + − − = =    formula (10) is now obtained from eq. (7). the calculations fully agree with the above argument: the accuracies of the models for 2k = and 3k = were the same, see table i. if 3k > , because of the very large number of k-tuples of vertices, the calculation of the steiner–wiener index kw based on its definition (5) becomes extremely cumbersome. in the case of acyclic systems (such as the molecular graphs of alkanes), instead of eq. (5), the calculations could be realized using eq. (6), which is significantly easier. in fact, by means of eq. (6) any steiner–wiener index kw could be calculated just as easily as the ordinary wiener index w. the fact that the accuracy of the approximations based on the indices * k w w wλ= + increases with k, and reaches its maximum at 7k = , is somewhat unexpected. it may be that this is a statistics-based artifact of the considered models. nevertheless, this phenomenon deserves further examination. the results of the present study may be considered from a pessimistic and from an optimistic point of view. a pessimist would say that there is very little difference between the figs. 1 and 4, as well as between figs. 2 and 5. an optimist would point to the fact that the average and maximal errors of the best _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1016 gutman, furtula and li model (based on 7w wλ+ ) are, respectively, by 20 and 50 % smaller than those of the starting model (based solely on w). in view of this, it may be concluded that by adding multicenter distance-contributions to the wiener index, its applicability to model physicochemical properties of alkanes is improved, but only to a limited extent. и з в о д вишецентрични винерови индекси и њихове примене иван гутман1,2, борис фуртула1 и xueliang li3 1природно–математички факултет универзитета у крагујевцу, 2државни универзитет у новом пазару и 3center for combinatorics, nankai university, tianjin, china винеров индекс w се може посматрати као молекулски структурни дескриптор састављен од сабирака који репрезентују интеракције између парова атома. једна генерализација винеровог индекса су штајнер–винерови индекси wk, k = 3,4,... у индексу wk се води рачуна о интеракцијама k атома, заснованих на појму штајнеровог растојања. показано је да формула w+λwk омогућава апроксимативно израчунавање тачке кључања алкана боље него сам винеров индекс. најбоља таква апроксимације је за k = 7. (примљено 26. јануара, ревидирано 12. фебруара, прихваћено 13. фебруара 2015) references 1. i. gutman, y. n. yeh, s. l. lee, y. l. luo, indian j. chem., a 32 (1993) 651 2. s. nikolić, n. trinajstić, z. mihalić, croat. chim. acta 68 (1995) 105 3. d. h. rouvray, in topology in chemistry – discrete mathematics of molecules, d. h. rouvray, r. b. king, eds., horwood, chichester, 2002, p. 16 4. k. xu, m. liu, k. c. das, i. gutman, b. furtula, match commun. math. comput. chem. 71 (2014) 461 5. m. liu, b. liu, match commun. math. comput. chem. 69 (2013) 491 6. a. hamzeh, s. hossein-zadeh, a. r. ashrafi, match commun. math. comput. chem. 69 (2013) 47 7. r. škrekovski, i. gutman, match commun. math. comput. chem. 72 (2014) 295 8. h. lin, match commun. math. comput. chem. 72 (2014) 783 9. k. hrinakova, m. knor, r. škrekovski, a. tepeh, match commun. math. comput. chem. 72 (2014) 791 10. 50th anniversary of the wiener index, i. gutman, s. klavžar, b. mohar, eds., discr. appl. math. 80 (1997), pp. 1–113 11. fifty years of the wiener index, i. gutman, s. klavžar, b. mohar, eds., match commun. math. comput. chem. 35 (1997), pp. 1–259 12. fifty years of the wiener index, i. gutman, ed., j. serb. chem. soc. 62 (1997), pp. 185– –294 13. d. h. rouvray, in topology in chemistry – discrete mathematics of molecules, d. h. rouvray, r. b. king, eds., horwood, chichester, 2002, p. 1 14. i. gutman, o. e. polansky, mathematical concepts in organic chemistry, springer, berlin, germany, 1986 15. g. chartrand, o. r. oellermann, s. tian, h. b. zou, časopis pest. mat. 114 (1989) 399 16. jakob steiner (1795–1863), swiss mathematician _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ multicenter wiener indices 1017 17. f. k. hwang, d. s. richards, p. winter, the steiner tree problem, north-holland, amsterdam, 1992 18. w. goddard, o. r. oellermann, in: structural analysis of complex networks, m. dehmer, ed., birkhäuser, dordrecht, 2011, pp. 49–72 19. x. li, y. mao, i. gutman, discuss. math. graph theory, in press 20. h. wiener, j. am. chem. soc. 69 (1947) 17 21. d. h. rouvray, j. comput. chem. 8 (1987) 470 22. p. g. seybold, m. may, u. a. bagal, j. chem. educ. 64 (1987) 575 23. z. mihalić, n. trinajstić, j. chem. educ. 69 (1992) 701 24. g. rücker, c. rücker, j. chem. inf. comput. sci. 39 (1999) 788 25. r. l. brown, s. e. stein, in: nist standard reference database no. 69, p. j. linstrom, w. g. mallard, eds., gaithersburg, md, 2014, http://webbook.nist.gov 26. j. nocedal, s. j. wright, numerical optimization, springer, new york, 2006. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice (n(but)4)5h4pv6mo6o40 as an efficient catalyst for the oxidative desulphurisation of gasoline j. serb. chem. soc. 81 (1) 91–101 (2016) udc 665.73+66.094.522.8:546.215:547.269.1 jscs–4830 original scientific paper 91 (n(but)4)5h4pv6mo6o40 as an efficient catalyst for the oxidative desulphurisation of gasoline mohammad ali rezvani*, mokhtar ali nia asli, liela abdollahi and mina oveisi department of chemistry, faculty of science, university of zanjan, 45371-38791 zanjan, iran (received 2 february, revised 11 may, accepted 19 may 2015) abstract: the oxidative desulphurization (ods) of gasoline and model compounds that exist in gasoline with hydrogen peroxide using (n(but)4)5h4pv6mo6o40 as a scavenger was studied. this keggin-type polyoxometalate was shown to be able to scavenge hydrogen sulphide and mercaptans in high yields. this system provides an efficient, convenient and practical method for scavenging sulphur compounds. this quaternary ammonium keggin-type polyoxometalate ((n(but)4)5h4pv6mo6o40) that has a lipophilic cation can act as a phase transfer agent and better transfer the peroxometal anion into the organic phase. the oxidation reactivity of the catalysts depends on the type of the counter-cation: ((c4h9)4n) + > nh4 + > k+. keywords: keggin-type polyoxometalate; counter-cation; mercaptan; gasoline; demercaptanization. introduction in the past decade, clean fuels research, including demercaptanization and desulphurization, has become an important subject of environmental catalysis studies worldwide.1–5 mercaptans are a kind of organic sulphide that widely exist in petroleum products. they cause foul odours and deteriorate the finished products. due to their acidity, mercaptans are corrosive to metals, which is harmful to storage and usage of oil products.5–7 therefore, it is necessary to remove them, either by transforming them to innocuous disulphides or by their extraction.7–11 in continuation of on-going research on the syntheses and application of polyoxometalate in organic reactions,12–21 herein, the applicability of quaternary ammonium keggin-type molybdovanadophosphate ((n(but)4)5h4pv6mo6o40) for the efficient desulphurization of gasoline in the preparation of ultra-clean fuels is reported. this quaternary ammonium keggin-type molybdovanadophos * corresponding author. e-mail: marezvani2010@gmail.com doi: 10.2298/jsc150202040a 92 ali rezvani et al. phate, ((n(but)4)5h4pv6mo6o40), that has a lipophilic cation can act as a phase transfer agent and transfer the peroxometal anion into the organic phase. the catalytic function of heteropolyacids (hpas) has attracted much attention because of their uncommon ability to accept an electron without deformation of their structure or reversible reduction.12–14 keggin-type polyoxoanions have been widely studied as homogeneous and heterogeneous catalyst for the oxidation of organic compounds, whereas the application of wells–dawson type polyoxoanions is mostly limited to homogeneous or gas phase applications and only a few investigations have demonstrated catalytic activity in the heterogeneous form.14–17 generally, keggin structures show more acidity and catalytic activity among the heteropolyacids.14 these solid acids are usually insoluble in non-polar solvents but highly soluble in polar ones. they can be used in bulk or supported forms in both homogeneous and heterogeneous systems.19 further catalytically important subclasses of the keggin compounds are the mixed-addenda vanadium-substituted hpas with the general formula h3+npm12–nvno40 (m = = mo or w; n = 1 to 6). in fact, vn+ is the most strongly oxidizing element and can be readily reduced to v(n–1)+ with the concomitant oxidation of an organic substrate. the introduction of vn+ into the keggin framework shifts its catalytic activity from acid-dominated to redox-dominated. various oxidants have been used in oxidative desulfurization (ods), such as no2, o3, h2o2 and solid oxidizing agents.11 among these oxidants, h2o2 is mostly chosen as an oxidant, as only water is produced as a by-product. peracids produced in situ from organic acid catalysts and h2o2 were reported to be very effective for the rapid oxidation of sulphur compounds in fuel oils under mild conditions. the catalytic activity of ((n(but)4)5h4pv6mo6o40) was tested on the oxidative desulphurization of gasoline and model sulphur compounds that exist in gasoline, i.e., benzothiophene (bt) and thiophene (t), with formic acid/hydrogen peroxide as the oxidizing reagent. the catalyst could be easily separated and reused at the end of the reaction without significant loss in its catalytic activity, which suggests that the catalyst is stable under different conditions. experimental all reagents and solvents used in this work were available commercially and used as received, unless otherwise indicated. the model compounds and chemicals, including thiophene (t) and benzothiophene (bt), solvent (n-heptane) for the experiments and analysis and hydrogen peroxide (30 vol. %) were obtained from aldrich. typical actual gasoline (density 0.7918 g ml-1 at 15 °c, total sulphur content 0.391 wt. %) was used and details of its properties are given in table i. preparation of the catalysts several heteropolyoxometalate catalysts: (nh4)5h4pv6w6o40, (nh4)6hpv4w8o40 and (nh4)5h4pv6mo6o40, etc., used for comparison, were prepared according to literature (n(but)4)5h4pv6mo6o40 as catalyst for desulfurization of gasoline 93 procedures.17,22 details of the preparation of the catalysts are given in the supplementary material to this paper. table i. properties of the examined gasoline before and after oxidative desulphurization, ods, by (n(but)4)5h4pv6mo6o40; ibp – initial boiling point; fbp – final boiling point after odsa before odsmethod unit properties of gasoline entry 0.7912 0.7918 astm d 1298g ml-1 density by hydrometer at 15 °c1 0.014 0.391 astm d 4294wt.% total sulphur by x-ray analysis2 3 39 astm d 3227ppm mercaptans 3 14 16 astm d 3230ptb salt 4 nil. nil. astm d 4006vol. % water content by distillation 5 43.6 43.9 astm d 86 c ibp* distillation 6 208.1 208.5 fbp# 67.1 68.3 vol. % 10 112.2 114.2 50 183.6 184.3 90 205.8 206.1 95 acondition for desulphurization: 20 ml of gasoline, 0.1 g catalyst, 2 ml oxidant, 10 ml of extraction solvent, time = 1 h, and temperature = 40 °c catalyst characterization the chemical characterization of the prepared catalysts was accomplished by means of elemental analysis and ir spectroscopy. elemental analysis results were obtained by integra xl inductively coupled plasma spectrometer. the fourier transform infrared (ftir) spectra of the solid samples were recorded in kbr pellets on a thermo-nicolet-is 10 instrument in the wavenumber range 400–4000 cm-1. catalytic tests oxidative desulphurization of simulated gasoline using the formic acid/h2o2 system. some typical thiophenes and benzothiophenes, which represent easy and hard to remove sulphur species in gasoline, were selected to evaluate the catalysts and the reactivity of the thiophenes and benzothiophenes in the oxidation reaction. stock solutions of the model sulphur compound were made by dissolving t or bt in n-heptane to give a final sulphur concentration of 500 ppm. then, performic acid (1:1 mixture of formic acid and hydrogen peroxide) was added to 5 ml of a stock solution. the resulting solution was heated to the required temperature (0–50 °c) in a water bath under stirring. after attaining the desired temperature, a sample was removed and catalyst was added to the remaining solution to initiate the reaction. stirring was continued for a further 1 h. after cooling to room temperature, the biphasic mixture was separated by decantation and the organic phase saved for analysis. oxidative desulphurization of gasoline using the formic acid/h2o2 system. in the same manner as for the oxidation of the model sulphur compounds but using actual gasoline (sulphur 391 ppm) except 20 ml of gasoline was used. after the oxidation was finished, the mixture was cooled down to room temperature and 10 ml acetonitrile (mecn) was added to extract the oxidized sulphur compounds. the observed biphasic system was separated by decantation and weighed to calculate % recovery of gasoline. (through three times reaction: 98, 97 and 95 %). 94 ali rezvani et al. determination of the total sulphur and mercaptan sulphur contents determination of the contents of total sulphur and mercaptan sulphur in the gasoline and simulated gasoline samples before and after reaction were determined by x-ray fluorescence spectroscopy using a tanaka rx-360 sh x-ray fluorescence spectrometer following the astm d-4294 and astm d-3227 standard test methods. details of the astm d-4294 method are discussed in the supplementary material to this paper. recycling of the catalyst at the end of the oxidative desulphurization of the model sulphur compounds and gasoline, the catalyst was filtered off and washed with dichloromethane. in order to determine whether the catalyst would succumb to poisoning and lose its catalytic activity during the reaction, the reusability of the catalyst was investigated. for this purpose, the desulphurization reaction of gasoline and model compounds was performed in the presence of fresh and recovered catalyst. result and discussion catalysis characterization the chemical compositions of the vanadium-containing catalysts are given in table ii. table ii. characteristic ir vibrations (as / cm–1) and elemental analysis data for the vanadium-containing polyoxmetalate catalysts composition, % mo–oc–momo–ob–mo mo–od p–oa catalyst v mo p cal. found cal. foundcal.found 2.13 3.35 44.1 46.51.291.26741 875 954 1053 h4pmo11vo40 4.31 6.91 40.4 40.61.311.33740 873 952 1052 h5pmo10v2o40 17.4 16.3 32.7 39.21.761.75732 865 947 1042 h9pv6mo6o40 ftir spectroscopy is an extensively used tool for the characterization of polyoxometalates as this technique provides finger printing in the structural elucidation of the keggin structure.12,14 the ftir spectra of different polyoxometalates salts showed the common characteristic absorption peaks ranging from 500 to 1100 cm–1 that correspond to the oxometalate anion configurations (table ii). the peak ranges (table ii) characteristic for pom are: 730–765 cm–1, octahedral corner sharing m–oc–m; 860–885 cm–1, octahedral bridge/edge sharing m–ob–m; 940–965 cm–1, terminal m–o and 1040–1070 cm–1, p–o configurations.12,14 these configurations collectively account for keggin type polyoxometalates. the oxygen atoms of the keggin structure can be subdivided into four different types, i.e., oa, inner oxygen; oc, corner-sharing oxygen; ob, edge-sharing oxygen and od, terminal oxygen. these exhibit four well-defined infrared bands that can be applied for the identification and discrimination of different heteropolyacid catalysts. the four classes of oxygen atoms can be described as follows: the central oxygen x–oa establishes a connection between the central heteroatom of the xo4 tetrahedron and the transition metal atoms of a trimetallic (n(but)4)5h4pv6mo6o40 as catalyst for desulfurization of gasoline 95 mo3 structure. the m–ob–m oxygen atoms connect two m3o13 units by corner sharing. furthermore, m–oc–m oxygen links two transition metal atoms by edge sharing of two mo6 units and finally the terminal oxygen atom m–od binds to only one transition metal atom.8,14,22 effect of the catalyst structure on the oxidative desulphurization the effect of the nature of the catalyst on the oxidative desulphurization of gasoline using formic acid/h2o2 as the oxidant is shown in tables iii and iv. the amount of each catalyst was constant throughout the series. the keggin type polyoxometalate catalyst (n(but)4)5h4pv6mo6o40 was a very active system for the oxidation of gasoline, while the other studied polyoxometalates systems were much less active. this system ((n(but)4)5h4pv6mo6o40) with a phase transfer or emulsion catalyst comprising a quaternary ammonium salt-based polyoxometalate was shown to be a very active system for oxidative desulphurization of gasoline. this quaternary ammonium keggin-type molybdovanadophosphate ((n(but)4)5h4pv6mo6o40) that has a lipophilic cation could better act as phase transfer agent and better transfer the peroxometal anion into the organic phase. that is, the oxidation reactivity of the catalysts depends on the type of countercation: ((c4h9)4n)+ > nh4+ > k+. it was shown that the order of the oxidation reactivity of the catalyst in the presence of hydrogen peroxide/formic acid was: ((n(but)4)5h4pv6mo6o40 > (n(but)4)4hpmo10v2o40 > (nh4)5h4pv6mo6o40 > k5h4pv6mo6o40 >h5pmo10v2o40 > h4pmo11vo40. from the result of table iii, the catalytic activity of r3+npm12–nvno40 (r = h, k, nh4 or n(but)4; m = mo or w; n = 0 to 6) was in the order of n = 6 >…> n = 0. the results show that the catalytic activity of ((n(but)4)5h4pv6mo6o40 was much higher than those of the other polyoxometalates. in fact, vn+ is the most strongly oxidizing element and can be readily reduced to v(n–1)+ with the concomitant oxidation of an organic substrate. the introduction of vn+ into the keggin framework shifts the catalytic activity from acid-dominated to redox-dominated. table iii. effect of different catalysts on the ods of gasoline and simulated gasoline conversion; conditions for desulphurization: 5 ml of model gasoline (200 ppm s) or 20 ml of gasoline, 0.1 mmol catalyst, 2 ml performic acid, 10 ml extraction solvent, time 1 h and temperature 40 °c entry catalyst ratio conversion, % bt/catalyst oxidant/bt thiophene bt gasoline 1 (n(but)4)5h4pv6mo6o40 100 10 98 97 97 2 (nh4)5h4pv6mo6o40 30 10 92 90 91 3 k5h4pv6mo6o40 30 15 86 85 85 4 h5pmo10v2o40 30 15 85 85 84 5 (n(but)4)4hpmo10v2o40 50 10 94 94 94 6 h4pmo11vo40 20 15 84 83 83 7 h3pmo12o40 20 15 81 81 81 96 ali rezvani et al. table iv. effect of different catalysts on the ods of gasoline; conditions for desulphurization: 20 ml of gasoline, 0.1 g catalyst, 2 ml oxidant, 10 ml of extraction solvent, time 1 h, temperature 40 °c entry catalyst total sulphur removed, % mercaptans removed, % with h2o2 without h2o2 with h2o2 without h2o2 1 (n(but)4)5h4pv6mo6o40 97 45 94 38 2 (nh4)5h4pv6mo6o40 91 40 89 30 3 k5h4pv6mo6o40 85 38 84 33 4 h5pmo10v2o40 84 37 82 31 5 (n(but)4)4hpmo10v2o40 94 41 90 36 6 h4pmo11vo40 83 34 81 30 7 h3pmo12o40 81 33 80 30 8 n(but)4br 33 16 34 15 9 none 22 – 21 – effect of catalyst dosage another factor that should be considered is the catalyst dosage. it was found that the catalyst dosage had a marked influence on the process efficiency (table v). under otherwise identical conditions, without catalyst, 24 % of the thiophene and 23 % of the benzothiophene were removed from the n-heptane phase and 23 % of the sulphur from actual gasoline in 60 min by oxidation. the percent conversions in actual gasoline in the presence of ((n(but)4)5h4pv6mo6o40 were found to be 64, 87.5 and 97 %, corresponding to catalyst amount of 0.06, 0.08 and 0.1 g, respectively. thus, the desulfurization efficiency increased rapidly with increasing catalyst dosage. table v. effect of catalyst dosage on the ods of gasoline and simulated gasoline conversion; conditions for desulphurization: 20 ml of gasoline, catalyst, (n(but)4)5h4pv6mo6o40, 2 ml oxidant, 10 ml of extraction solvent, time 1 h, temperature 40 °c entry amount of catalyst, g reactant thiophene benzothiophene actual gasoline 1 0 24 23 22 2 0.02 38 36 35 3 0.04 45 43 42 4 0.06 67 65 64 5 0.08 89 87 87 6 0.1 98 97 97 7 0.11 98 97 97 8 0.12 98 97 97 influence of quaternary ammonium cation on the catalytic activity countercation with quaternary ammonium salts with lipophilic cation could act as a phase transfer agent and could transfer the peroxometal anion into the organic phase. an amphiphilic catalyst with a proper quaternary ammonium cat (n(but)4)5h4pv6mo6o40 as catalyst for desulfurization of gasoline 97 ion could form metastable emulsion droplets in gasoline with an aqueous h2o2 solution, demonstrating high oxidative activity, and could be separated after reaction through centrifugation. for example, [n(c4h9)4]+ is a proper quaternary ammonium cation (tables iii–v). other cations were tested and it was found that [n(c4h9)4]+ forms metastable emulsion droplets in gasoline more readily than do nh4+ and k+. effect of temperature on the oxidative desulfurization of gasoline or simulated gasoline the reaction was carried out at different temperatures under the same conditions using ((n(but)4)5h4pv6mo6o40 as the catalyst and formic acid/h2o2 as the oxidant. the results, given in table vi, show that yields of the products are a function of temperature. the percent conversions of sulphur in the solutions of the model compounds and in actual gasoline increased with temperature and time. the percent conversion of sulphur in the simulated fuel at 40 °c was higher than that at 30 °c. at 40 °c in 60 min, 97 % conversion of sulphur was obtained. table vi. effect of different temperatures on the ods of gasoline and simulated gasoline conversion; conditions for desulphurization: 10ml simulated gasoline or 20 ml of gasoline, 0.1 g catalyst, 2 ml oxidant, 10 ml of extraction solvent, time 1 h entry temperature, °c reactant thiophene benzothiophene actual gasoline 1 25 82 81 80 2 30 86 85 84 3 35 90 88 87 4 40 98 97 97 5 45 98 97 97 6 50 97 96 97 effect of different oxidation system on the oxidative desulphurization of gasoline effect of oxidation system on the oxidative desulfurization of gasoline was studied (table vii). hydrogen peroxide, kmno4 and k2cr2o5 were selected as oxidizing agents, which were used in the presence of an organic or inorganic acid, i.e., formic acid, acetic acid, oxalic acid, benzoic acid, h2so4 and h2co3 to acidify the system. the results in table vii showed that in the presence of the inorganic acids, h2so4 and h2co3, the oxidation reactivity was lower than in the presence of the organic acids. in actual gasoline, h2so4 and h2co3 cannot dissolve; thus, the sulphur removal from gasoline by inorganic acid/h2o2 was lower than the removal by the organic acid/h2o2 systems. effect of the amount of formic acid effect of the amount of formic acid on the oxidative desulphurization of different sulphur compounds was studied and the results are given in table viii. in 98 ali rezvani et al. table vii. effect of different oxidation system on the ods of gasoline; conditions for desulphurization: 20 ml of gasoline, 0.1 g ((n(but)4)5h4pv6mo6o40, 2 ml oxidant, 10 ml of extraction solvent, time 1 h, temperature 40 °c total sulphur removed, % mercaptans removed, % acid oxidant entry 97 94 formic acid h2o2 1 97 93 acetic acid h2o2 2 92 89 oxalic acid h2o2 3 91 87 benzoic acidh2o2 4 79 80 h2so4 h2o2 5 78 78 h2co3 h2o2 6 85 86 – h2o2 7 74 75 formic acid kmno4 8 73 74 oxalic acid kmno4 9 74 73 h2so4 kmno4 10 76 76 – kmno4 11 74 76 formic acid k2cr2o5 12 74 73 oxalic acid k2cr2o5 13 73 74 h2so4 k2cr2o5 14 76 77 – k2cr2o5 15 table viii. effect of formic acid amount on the ods of gasoline and simulated gasoline conversion; conditions for the desulphurization: 20 ml of gasoline or 10 ml simulated gasoline, 2 ml h2o2/formic acid as oxidant, 0.1 g (n(but)4)5h4pv6mo6o40, 2 ml oxidant, 10 ml of extraction solvent, time 1 h entry formic acid/sulphur compound mole ratio reactant thiophene benzothiophene actual gasoline 1 0.25 56 54 53 2 0.5 69 67 66 3 0.75 86 85 85 4 1.0 98 97 97 5 1.25 97 96 95 6 1.5 97 95 95 the formic acid catalyzed reaction, the formic acid can interact with sulphur without any steric hindrance from the alkyl groups. therefore, the reactivity trend obtained in the formic acid catalyzed reactions reflects the intrinsic oxidation reactivity of the thiophenes. the % sulphur removal of the simulated gas oil increased with increasing formic acid. it could be seen that a formic acid/h2o2 mole ratio of 1.0 (98 % conversion of thiophene) was better than the other mole ratios. therefore, in all other experiments, this formic acid/thiophene mole ratio was used. in gasoline mixed with formic acid/h2o2 (performic acid), the oxidative reaction occurred below 50 °c under atmospheric pressure. this was followed by liquid//liquid extraction to obtain a gasoline with a low sulphur and an extract with a high sulphur content. finally, the low sulphur gasoline may require additional treatment. the extraction solvent was then removed from the extract (n(but)4)5h4pv6mo6o40 as catalyst for desulfurization of gasoline 99 for reuse and the concentrated extract was made available for further processing to remove sulphur and to produce hydrocarbons. reusability of the catalyst the catalyst from the first desulphurization was recovered from the reaction mixture by filtration, washed with dichloromethane and used for the next desulphurization. this was repeated a further three times. the results of the effectiveness of the reused catalyst are given in table ix, from which it could be seen that the catalyst largely retained its activity on recycling. table ix. reuse of the catalyst on the ods of thiophene; conditions for desulphurization: 5 ml of model gasoline (200 ppm s), 0.1 mmol (n(but)4)5h4pv6mo6o40, 2 ml formic acid/h2o2, 10 ml extraction solvent, time 1 h, temperature 40 °c isolated yield, % entry 98 1 96 2 96 3 95 4 94 5 general remark concerning the desulfurization process a model gasoline was made by adding t and bt into n-heptane solvent, with a total sulphur concentration of 200 mg l–1. the organic sulphur compounds were mixed with formic acid/h2o2 and ((n(but)4)5h4pv6mo6o40 and then the oxidation reaction occurred at 40 °c under atmospheric pressure. this was followed by a liquid extraction (acetonitrile) to obtain gasoline with a low sulphur content. many oxidizing agents have been reported in ods processes, whereby h2o2 was the main one. hydrogen peroxide is one of the most attractive oxidants, mainly because it is environmentally clean and easily handled. hydrogen peroxide first rapidly reacts with an organic acid to generate peracid. it should be noted that during the ods process, h2o2 was used in the presence of formic acid as oxidants because formic acid, as an organic acid, reacts with h2o2 to in situ produce peracid, which can efficiency convert organic sulphur to sulphones without the formation of a substantial amount of residual product. the role of the metal atoms in ((n(but)4)5h4pv6mo6o40, m = v or mo, was to form peroxo-metal species, which are able to activate the h2o2 and peracid molecules. ((n(but)4)5h4pv6mo6o40 accepted the active oxygen from the oxidant h2o2 to form new oxoperoxo species mediates. the cation with the carbon chain transferred oxoperoxo species to the substrates (t or bt) and enabled the oxidation reaction to be accomplished completely. 100 ali rezvani et al. conclusions the system with ((n(but)4)5h4pv6mo6o40), a phase transfer or emulsion catalyst comprising a quaternary ammonium salt-based polyoxometalate, was shown to be a very active system for the oxidative desulphurization of gasoline. this quaternary ammonium keggin-type molybdovanadophosphate ((n(but)4)5h4pv6mo6o40), which has a lipophilic cation, can act as phase transfer agent and transfer the peroxometal anion into the organic phase. that is, the oxidation reactivities of the catalysts depend on the type of countercation: ((c4h9)4n)+ > nh4+ > k+. in the present work, the efficient oxidative desulphurization of gasoline and simulated gasoline using the formic acid/hydrogen peroxide (peracid) system was reported. the system provides an efficient, convenient and practical method for scavenging sulphur compounds in gasoline. supplementary material the preparation of the v-containing catalysts and a discussion of the astm d-4294 standard method for the determination of sulphur compounds in gasoline are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding authors on request. и з в о д (n(but)4)5h4pv6mo6o40 као ефикасан катализатор за оксидативну десулфуризацију бензина mohammad ali rezvani, mokhtar ali nia asli, liela abdollahi и mina oveisi department of chemistry, faculty of science, university of zanjan, 45371-38791 zanjan, iran испитивана је оксидативна десулфуризација (оsd) бензина и модел једињења која постоје у бензину са водник-пероксидом у присуству (n(but)4)5h4pv6mo6o40. показано је да ово полиоксометалатно једињење кегиновог типа може да уклони водоник-сулфид и меркаптане у виском приносу. овај систем омогућава ефикасан, погодан и практичан метод за елиминацију сумпорних једињења. квартернерни амонијум-полиоксометалат кегиновог типа, (n(but)4)5h4pv6mo6o40, који има липофилни катјон има боље карактерстике као агенс за фазни трансфер и може боље да преведе пероксометални анјон у органску фазу. тачније, оксидативна реактивност катализатора зависи од типа контракатјона: ((c4h9)4n) + > nh4 + > k+. (примљено 2. фебруара 2014, ревидирано11. маја, прихваћено 19. маја 2015) references 1. g. f. zhang, f. l. yu, r. wang, petrol. coal 51 (2009) 196 2. i. v. babich, j. a. moulijn, fuel 82 (2003) 607 3. j. m. campos, m. c. sanchez, p. presas, j. l. g. fierro, j. chem. technol. biotechnol. 85 (2010) 879 4. p. s. tam, j. r. kittrell, j. w. eldridge, ind. eng. chem. res. 29 (1990) 321 5. d. wang, e. w. qian, h. amano, k. okata, a. ishihara, t. kabe, appl. catal., a 253 (2003) 91 6. s. otsuki, t. nonaka, w. qian, a. ishihara, t. kabe, bull. chem. soc. jpn. 31 (1998) 1939 (n(but)4)5h4pv6mo6o40 as catalyst for desulfurization of gasoline 101 7. d. wang, e. w. qian, h. amano, k. okata, a. ishihara, t. kabe, appl. catal., a 253 (2003) 91 8. t. v. rao, b. sain, s. kafola, y. k. sharma, s. m. nanoti, m. o. garg, energy fuels 21 (2007) 3420 9. c. a. s. regino, d. e. richardson: inorg. chim. acta 360 (2007) 3971 10. s. z. liu, b. h. wang, b. c. cui, l. l. sun, fuel 87 (2008) 422 11. j. zongxuan, l. hongyinga, z. yongna, l. can, chin. j. catal. 32 (2011) 707 12. a. fallah shojaie, m. a. rezvani, m. h. loghmani, fuel process. technol. 118 (2014) 1 13. m. a. rezvani, a. fallah shojaie, f. m. zonoz, j. serb. chem. soc. 79 (2014) 1099 14. m. a. rezvani, f. m. zonoz, ind. eng. chem. 22 (2015) 83 15. m. a. rezvani, a. fallah shojaie , m. h. loghmani, catal. commun. 25 (2012) 36 16. a. fallah shojaie, m. a. rezvani, f. m. zonoz, j. serb. chem. soc. 78 (2013) 129 17. a. fallah shojaei, m. a. rezvani, m. heravi, j. serb. chem. soc. 76 (2011) 955 18. a. fallah shojaei, m. a. rezvani, m. heravi, j. serb. chem. soc. 76 (2011) 1513 19. r. harutyunyan, m. a. rezvani, m. m. heravi, synth. react. inorg., met.-org. nano -met. 41(2011) 94 20. m. a. rezvani, r. harutyunyan, m. m. heravi, synth. react. inorg. met.-org. chem. 42 (2012) 1232 21. m. sharifzadeh baei, m. a. rezvani, asian j. chem. 12 (2011) 5381 22. m. hamamoto, k. nakayama, y. nishiyama, y. ishii, j. org. chem. 58 (1993) 6421. comparison of organics and heavy metals acute toxicities to vibrio fischeri j. serb. chem. soc. 81 (6) 697–705 (2016) udc 504.054–039.7+546.47’48’56’76: jscs–4878 615.9:58.035:639.3 original scientific paper 697 comparison of organics and heavy metals acute toxicities to vibrio fischeri xuepeng yang1, ji yan1, fangfang wang1, jia xu1, xiangzhen liu2, ke ma1, xiangmei hu1 and jianbin ye1* 1school of food and biological engineering, henan provincial collaborative innovation center for food production and safety, zhengzhou university of light industry, dongfeng road, #5, zhengzhou, 450002, henan province, china and 2technology center, china tobacco henan industrial co., ltd., zhengzhou, 450000, china (received 24 november 2015, revised and accepted 15 january 2016) abstract: vibrio fischeri bioluminescence inhibition has been widely used to test acute toxicities of metals and organics contaminants. however, the differences of metals and organics acute toxicities to v. fischeri have not been compared. here, four heavy metals (zn2+, cu2+, cd2+ and cr6+) and five organics (phenol, benzoic acid, p-hydroxybenzoic acid, nitro-benzene and benzene) acute toxicities to v. fischeri were investigated. heavy metals toxicities to v. fischeri were increased along with the reaction time, while the organics toxicities kept the same level in different reaction times. in order to explain the difference, the relative cell death rate of v. fischeri was detected. in metals toxicities tests, the bioluminescence inhibition rate of v. fischeri was found to be significantly higher than the relative cell death rate (p < 0.05), while for the organics toxicities tests, the cell death rate was similar to the bioluminescence inhibition rate. these results indicated that organics acute toxicities to v. fischeri could reflect the death of cell, but metals acute toxicities to v. fischeri may not lead to the death of cell, just represent the bioluminescence inhibition. keywords: reaction time; luminescent bacteria; cell death; bioluminescence inhibition; cell death rate; microtox® test. introduction the luminescent bacterium, vibrio fischeri has been widely applied for the toxicity detection of chemicals, including organics and metals.1–3 due to its advantages of ease use, low cost and high reproducibility, this biotest has been accepted as a quick method of chemicals toxicities assessment in the environment (named as microtox® test), including wastewater effluent,4 sediment extracts5 and contaminated groundwater.6 * corresponding author. e-mail: happye1986@163.com doi: 10.2298/jsc151124011y _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 698 yang et al. the microtox® test is based on the luminescence response of the bacterium v. fischeri. the light emission of v. fischeri is an enzyme catalysis reaction, and the process could be concluded as eq. (1) and (2).7 given the principle of this bioprocess, toxics that do harmful to any of the factors (o2, aldehyde, flavin reductase, luciferase and nad(p)h) attending in this process could induce luminescence inhibition, and the toxicities to v. fischeri could be assessed by the luminescence inhibition rate. in the other hand, cells death caused by toxics will also cause the luminescence inhibition. thus, sporadically studies have issued that chemicals acute toxicities detected by the microtox® test could either reflect disturbances of biosynthetic pathways8 or death of v. fischeri.9 flavin reductase 2nad(p)h h fmn nad(p) fmnh+ + ⎯⎯⎯⎯⎯⎯⎯→ + (1) luciferase 2 2 2 fmnh o r cho fmn h o r cooh hv(490nm) + + − ⎯⎯⎯⎯⎯→ → + + − + (2) nevertheless, there have been relatively few researches comparing the difference of heavy metals and organics acute toxicities to v. fischeri. when assessing the toxicities of metals and organics by microtox® test, different reaction times were used by experienced-based,10 but the reasons remain unclear. based on the microtox® test, a small quantity of studies had demonstrated that heavy metals toxicities to v. fischeri were fickle at different reaction times,11,12 while the organics toxicities kept relatively stable.13 these results in some extent explain the reasonable discrepancy reaction time for detecting heavy metals and organics acute toxicities to v. fischeri. however, systematic comparison of heavy metals and organics acute toxicities to v. fischeri has not been carried out, and the reasons of using different reaction times still need to be further elucidated. in this study, heavy metals and organics acute toxicities to v. fischeri at various reaction times were determined by the microtox® test. the relative cell death rate was detected following the microtox® test to explain the possible reasons of the difference between metals and organics. materials and methods toxicity assay the bacterium, v. fischeri, used in this study was purchased from the institute of soil science, chinese academy of sciences, nanjing, china. the toxicity assay was performed according to national standard method of china (water quality – determination of the acute toxicity – luminescent bacteria test. gb/t 15441-1995). briefly, 5 mg freeze-dry powder of v. fischeri was revived in 1 ml chilled 3 % nacl, followed by adding 4 ml 3 % nacl. this solution was served as the working fluid for subsequent test. the toxicity assay was carried out by mixing 1.9 ml testing/control samples (solution only containing 3 % nacl) and 0.1 ml working fluid of v. fischeri solution, and the light was recorded by the model dxy-2 luminometer after different incubation times. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ different acute toxicities to v. fischeri 699 according to the microtox® test, four kinds of chemicals (k2cr2o7, znso4⋅7h2o, cuso4 and cdcl2) were selected as represent metals ions, cr 6+, zn2+, cu2+ and cd2+, respectively. five organics were also tested: phenol, benzoic acid, p-hydroxy benzoic acid, nitro-benzene and benzene. these chemicals were purchased from aladdin (http://www.aladdin-e.com/). these chemicals were selected as common contaminants in the water environment. the ec50 (chemicals concentrations that cause 50 % luminescence inhibition rate) was calculated according to previous research.14 dimethylsulfoxide (dmso) was used as the co-solvent for organics according to the method reported in the reference15. three different reaction times for metals (15, 30 and 45 min) and organics (5, 10 and 15 min) were tested in this study according to the preliminary experiments. relative cell death rate detection in order to explain the different acute toxicities of metals and organics to v. fischeri, relative cell death rate detection was conducted after the toxicity assay. following toxicity assay, the bacteria were spread on 12 cm plates containing lb medium with the modification of containing 3 % nacl and cultivated at 20±1 °c for 48 h. the colony-forming unit (cfu) was used to enumerate the viable cell number. the cell death rate (dr) was calculated with eq. (3): s c 100(1 )= − c dr c (3) cs is the cfu of testing sample, and cc is the cfu of the control sample without contacting with the toxics. all testing and control samples were carried out in quintuplicate. statistical analysis all toxicity assays were performed in quintuplicate. the statistical significant of values were calculated by student’s t-test (mean analysis) at 5 % level of probability using the spss 19.0 package software (spss international, chicago, il, usa). statistical significant difference was reported when the probability of the result assuming the null hypothesis (p) is less than 0.05. results and discussion heavy metals toxicities the toxicities of the four kinds of metals (zn2+, cu2+, cd2+ and cr6+) were detected, as they are common contamination in the environment.16,17 the toxicities of four kinds of metals were showed in fig. 1. not surprising, the lumenescence inhibition rate was increased along with the concentration of each metal. worth to note that, under a certain concentration of metals, luminescence inhibition rate was also increased along with the reaction times. ec50 was calculated by the linearity relationship of luminescence inhibition rate and the logarithm of concentration.18 the ec50 at different reaction times were summarized in table i. from 15 to 45 min, the ec50 of each metal was decreased significantly (p < 0.05). as showed in fig. 1, the luminescence inhibition rate increased gradually along with the increasing of reaction times. metals toxicities to v. fischeri were attributed to the metals ions,19 and earlier study demonstrated that the metals toxicities were mostly contributed by the ions that affected the luciferase in the _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 700 yang et al. luminescence bioprocess.20 results in the present study further supported this idea. the toxicities of these heavy metals were closely in agreement with the previous reports.21,22 it was reported that cd2+ could be adsorbed and trapped to the exo-polysaccharides on the outer layer of the luminescent bacteria.23 this may prevent the interaction between cd2+ and key enzymes, which resulted in low toxicity of cd2+ detected by only evaluating luminescence decrease in the present study. meanwhile, the lower toxicity of cr6+ in this study could result from cr6+ being reduced to cr3+, which has less toxic to the bioenzymes.24 thus, different luminescence inhibition rate at different exposure times with these metals could partly demonstrates that the decrease of light emission in v. fischeri was due to the enzyme disturbance by heavy metals, and the toxicities were related with how they affect the enzymes rather than cause the cell death. fig. 1. the luminescence inhibition rate by four different kinds of metals under various concentrations: a), znso4, b) cuso4, c) cdcl2 and d) k2cr2o4. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ different acute toxicities to v. fischeri 701 table i. toxicities of heavy metals at different contact times chemical contact time, min 15 30 45 ec50 / mg l -1 ec50 / mg l -1 pa ec50 / mg l -1 pa (zn) znso4 0.5±0.02 0.4±0.01 8.5×10 -6 0.3±0.01 4.1×10-8 (cu) cuso4 10.8±0.5 8.0±0.4 1.0×10 -5 6.2±0.3 1.1×10-7 (cd) cdcl2 31.9±1.6 23.6±1.2 1.5×10 -5 18.4±0.9 1.9×10-7 (cr) k2cr2o4 160.3±8.0 138.3±6.9 0.002 121.3±6.1 2.4×10 -5 ap value was compared to that of 15 min, represent that whether the toxicities of chemicals were significant different between different contact times. the results represent the mean ± sd organics toxicity five organics toxicities to v. fischeri were showed in fig. 2. similarly, the luminescence inhibition rate increased along with the concentration increasing of organics. the ec50 of all organics at different reaction times were summarized in table ii. data analysis showed that the ec50 of organics were close at different reaction times (p > 0.153). unlike heavy metals, reaction time was not a factor for organics acute toxicities to v. fischeri. organics toxicities to v. fischeri could due to different ionization constant,25 distinctive chemical–physical potential, chemical group, ionization, logarithm of the 1-octanol/water partition coefficient.26 previous researches had showed that the different atom and structure can result in much difference in the toxicities of the organics.26–28 but how they affect the v. fischeri cells has been rarely interpreted. sporadically studies issued that organics involved in the interaction with cell surface receptors, disruption of cell membrane function, or reaction with cellular components could be the reason for the luminescence inhibition of v. fischeri.29,30 the harmful that organics do to v. fischeri seem to be irreversible, and could cause the cell dead in short time. combined with results in this study, the reason of luminescence inhibition by organics could attribute to the cells death. the relative cell death detection in order to identify whether the luminescence inhibition was induced by the cells death, the relative cell death detection was carried out after microtox® test. table iii showed the relatively death rate of v. fischeri at different luminescence inhibition rate (20, 50 and 90 %). at luminescence inhibition of 20 and 50 %, the cell death rate was close to the luminescence inhibition rate after exposure to organics. the results suggested that the organics acute toxicities to v. fischeri could reflect the death of cells. however, to heavy metals, the relatively death rate of v. fischeri was significantly lower than the luminescence inhibition rate, indicated that heavy metals acute toxicities to v. fischeri may not reflect the cell death, and only caused partly cell death after contacting with the bacteria. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 702 yang et al. fig. 2. the luminescence inhibition rate by five different kinds of organics under various concentrations: a), phenol, b) benzene, c) benzoic acid, d) nitrobenzene and e) p-hydroxy benzoic acid. the different reasons of luminescence inhibition between heavy metals and organics could partly explain these results. the heavy metals may combine with some important enzymes involved in the luminescence bioprocess without causing the cells death. organics may cause damage to the cells membrane and _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ different acute toxicities to v. fischeri 703 induce cells death after contacting for a certain time. of course, after contacting with higher concentrations of organics and heavy metals, such as at the concentrations of chemicals that caused 90 % luminescence inhibition, some irreversible effect on bacteria growth happened and the cell death rate was almost 90 % for both organics and heavy metals (table iii). table ii. toxicities of aromatics at different contact times chemical contact time, min 15 30 45 ec50 / mg l -1 ec50 / mg l -1 pa ec50 / mg l -1 pa phenol 111.5±5.8 110.9±5.5 0.871 107.9±5.0 0.324 benzene 26.5±1.3 26.1±1.3 0.640 25.8±1.2 0.402 benzoic acid 10.9±0.5 10.7±0.5 0.545 10.5± 0.5 0.241 nitro-benzene 61.5±3.1 59.7±2.9 0.371 57.8±5.7 0.238 p-hydroxy benzoic acid 6.3±0.3 6.1±0.5 0.931 6.0±0.3 0.153 ap value was compared to that of 5 min, represent that whether the toxicities of chemicals were significant different between different contact times. the results represent the mean ±sd table iii. the relative cell death rate (dr / %) of v. fischeri at different luminescence inhibition rate; the results represent the mean ±sd chemical luminescence inhibition rate, % 20 50 90 zn (znso4) 8.9±0.2 33.8±0.3 91.3±0.6 cu (cuso4) 7.6±0.1 30.1±1.2 90.7±1.1 cd (cdcl2) 10.5±0.4 32.2±0.7 91.5±1.2 cr (k2cr2o4) 7.9±0.1 29.8±0.6 92.6±2.2 phenol 17.5±1.5 45.6±1.2 89.9±1.4 benzoic acid 18.6±1.1 47.6±2.3 93.5±3.2 nitrobenzene 19.7±3.2 46.5±0.8 92.6±1.3 p-hydroxybezoic acid 17.5±1.4 48.1±1.4 91.5±0.9 benzene 16.8±0.9 50.2±1.1 91.4±1.2 conclusion in summary, systematic comparison of heavy metals and organics acute toxicities to v. fischeri is reported in this study. metals toxicities detected by luminescence inhibition rate was increased along with the reaction time and was significant higher than the cell death rate, while organics toxicities kept similar at different reaction times and were consistent with the cell death rate. the discrepancy results indicated that organics acute toxicities to v. fischeri could represent the cell death, but luminescence inhibition by metals could due to the disturbing of luminescence bioprocess and may not necessary cause the cell death. acknowledgments. the project was supported in part by doctor support grants of zhengzhou university of light industry, china. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 704 yang et al. и з в о д поређење токсичности органских једињења и метала према vibrio fischeri xuepeng yang1, ji yan1, fangfang wang1, jia xu1, xiangzhen liu2, ke ma1, xiangmei hu1 и jianbin ye1 1 school of food and biological engineering, henan provincial collaborative innovation center for food production and safety, zhengzhou university of light industry, dongfeng road, #5, zhengzhou, 450002, henan province, china and 2 technology center, china tobacco henan industrial co., ltd., zhengzhou, 450000, china инхибиција биолуминисценције vibrio fischeri широко се примењује за испитивање акутних токсичности метала и органских загађујућих материја. међутим, до сада нису поређене разлике у токсичности ових материја према v. fischeri. у овом раду су испитане акутне токсичности четири тешка метала (zn2+, cu2+, cd2+ и cr6+) и пет органских једињења (фенол, бензоева киселина, p-хидроксибензоева киселина, нитробензен и бензен) према v. fischeri. токсичности тешких метала су се повећавале са реакционим временом, док је токсичност органских једињења остајала на истом нивоу при различитим реакционим временима. како би се објаснила ова разлика, мерена је релативна брзина умирања ћелија v. fischeri. у тестовима токсичности метала, брзина инхибиције биолуминисценције код v. fischeri била је значајно већа него релативна смртност ћелија (p < 0,05), док је у тестовима токсичности органских једињења, смртност ћелија била слична брзини инхибиције биолуминисценције. ови резултати дају индикацију да акутне токсичности органских једињења према v. fischeri одражавају смрт ћелије, али акутне токсичности метала не морају водити до смрти ћелије, већ само представљају инхибицију биолуминисценције. 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altschuh, r. bruggemann, chemosphere 30 (1995) 2397 30. k. e. gustavson, a. svenson, j. m. harkin, environ. toxicol. chem. 17 (1998) 1917. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis and characterization of palladium(ii) complexes with glycine coumarin derivatives} j. serb. chem. soc. 81 (12) 1383–1392 (2016) udc 546.982+547.587.51+547.466.22: jscs–4935 548.7:66.094.941 original scientific paper 1383 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. synthesis and characterization of palladium(ii) complexes with glycine coumarin derivatives danijela lj. stojković1#, alessia bacchi2, davide capucci2, milica r. milenković3#, božidar čobeljić3#, srećko r. trifunović1#, katarina anđelković3#, verica v. jevtić1#, nenad vuković1, milena vukić1 and dušan sladić3*# 1department of chemistry, faculty of science, university of kragujevac, radoja domanovića 12, 34000 kragujevac, serbia, 2dipartimento di chimica, university of parma, parco area delle scienze 17 a, i 43124 parma, italy and 3faculty of chemistry, university of belgrade, studentski trg 12–16, 11000 belgrade, serbia (received 15 september, revised 25 september, accepted 27 september 2016) abstract: a pd(ii) complex with methyl 2-([1-{2,4-dioxochroman-3-ylidene}ethyl]amino)acetate was synthesized. the structures of both the ligand and its pd(ii) complex were determined by elemental analysis, and ir and nmr spectroscopy. recrystallization of the pd(ii) complex from dmf/water solution resulted in its hydrolysis and the formation of the dimethylamine (2-[{1-(2,4 -dioxochroman-3-ylidene)ethyl}amino]acetato)palladium(ii) complex, the structure of which was determined by elemental analysis, ir, 1hand 13c-nmr spectroscopy and x-ray analysis. keywords: coumarin-derived ligands; crystal structure; mechanism of hydrolysis. introduction coumarins (derivatives of 2h-1-benzopyran-2-one) are of great interest in medicinal chemistry because of their wide range of pharmacological activity.1–6 metal complexes with coumarin derivatives have been investigated because of their anticoagulant,7 antimicrobial8 and antitumor activities.9–11 among the complexes with coumarin derivatives, pd(ii) complexes have attracted considerable attention because of their significant antitumor activity.12–15 following these findings, in this work the synthesis of methyl 2-([1-{2,4-dioxochroman-3-ylidene}ethyl]amino)acetate (hl1) and its chlorido pd(ii) complex (1) are reported. attempts to obtain monocrystals of complex 1 from dmf/water solution resulted in its hydrolysis and the formation of dimethylamine(2-[{1-(2,4-dioxochroman * corresponding author. e-mail: dsladic@chem.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc160915087s 1384 stojković et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. -3-ylidene)ethyl}amino]acetato) palladium(ii) complex (2). all the obtained compounds were characterized by elemental analysis, and ir and nmr spectroscopy. the crystal structure of complex 2 was determined by x-ray analysis. experimental materials and methods hydrochlorides of glycine and glycine methyl ester, triethylamine, methanol, ethanol, toluene, acetone and potassium tetrachloridopalladate(ii) were obtained from sigma–aldrich. 3 -acetyl-4-hydroxycoumarin was synthesized according to a previously described procedure.16 elemental analyses were performed on a vario el iii c,h,n elemental analyzer. melting points of the ligands were determined using a kofler hot stage apparatus. ir spectra were run on a perkin–elmer spectrum one ft-ir spectrometer using the kbr pellet technique (4000– –400 cm-1). 1h-nmr (200 mhz) and 13c-nmr (50 mhz) spectra of the hl1 ligand were recorded on a varian gemini 200 spectrometer (varian, palo alto, ca, usa) in cdcl3 using tms as an internal standard for 1h and 13c. the 1h-nmr (500 mhz) spectrum of complex 1 was recorded on a bruker avance 500 spectrometer in dmso-d6 using tms as an internal standard. due to the low solubility of complex 1 in dmso, it was not possible to obtain its 13c-nmr spectrum. 1h-nmr (500 mhz), 13c nmr (125 mhz) and 2d nmr spectra (cosy, hsqc) of complex 2 were recorded on a bruker avance 500 spectrometer in dmso-d6 using tms as an internal standard for 1h and 13c. analytical tlc was performed on silica gel (silica gel 60, layer 0.20 mm, alugram sil g, macherey–nagel, germany). the visualization of tlc plates was performed using a uv lamp at 254 and 365 nm (vl-4.lc, 365/254, vilber lourmat, france). characterization data are of synthesized compounds are given in supplementary material to this paper. synthesis of methyl 2-([1-{2,4-dioxochroman-3-ylidene}ethyl]amino)acetate (hl1) a mixture of 3-acetyl-4-hydroxycoumarin (0.5 g, 2.45 mmol), the hydrochloride of glycine methyl ester (2.45 mmol) and trimethylamine (0.2 g, 2.00 mmol) in methanol (50 ml) was refluxed for 2 h. the progress of the reaction was monitored by tlc (toluene:acetone volume ratio of 8:2). after completion of the reaction, evaporation of solvent to half of the volume and addition of 5 ml of water, the obtained white solid was filtered, dried and recrystallized from 96 % ethanol. synthesis of 2-([1-{2,4-dioxochroman-3-ylidene}ethyl]amino)acetic acid (h2l2) into a solution of 3-acetyl-4-hydroxycoumarin (0.5 g, 2.45 mmol) in methanol (50 ml), glycine (0.18 g, 2.45 mmol) was added. the ph of the reaction mixture was adjusted by the addition of three drops of conc. hcl. the clear, colorless solution was refluxed for 3 h. after six days, a white solid precipitated from the reaction solution. synthesis of chlorido(methyl 2-[{1-(2,4-dioxochroman-3-ylidene)ethyl}amino]acetate) palladium(ii) complex (1) potassium tetrachloridopalladate(ii) (0.05 g, 0.153 mmol) was dissolved in 10 ml of water and the same amount of enamine hl1 (0.153 mmol) dissolved in methanol (10 ml) was added. the mixture was stirred for 3 h whereby a yellow precipitate was obtained. the precipitate of complex 1 was filtered off and washed with a small amount of methanol. synthesis of dimethylamine(2-[{1-(2,4-dioxochroman-3-ylidene)ethyl}amino]acetato)-palladium(ii) complex (2) recrystallization of complex 1 from dmf/water (1:1 volume ratio) during seven days at room temperature resulted in the formation of crystals of complex 2. palladium(ii) complexes 1385 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. crystallographic structure determination single crystal x-ray diffraction data were collected using mokα radiation (λ = 0.71073 å) at t = 293 k on an apex2 diffractometer with ccd area detector. the collected intensities were corrected for lorentz and polarization factors and empirically for absorption using the sadabs program.17 structures were solved by direct methods using sir201118 and refined by full-matrix least-squares on all f2 using shelxl9719 implemented in the olex2 package.20 hydrogen atoms were introduced in the calculated positions. anisotropic displacement parameters were refined for all non-hydrogen atoms. hydrogen bonds were analyzed with shelxl9719 and use was made of the cambridge crystallographic data centre packages21 for analysis of crystal packing. the crystal data and structure determination results are summarized in table s-i of the supplementary material to this paper. results and discussion synthesis reaction of equimolar amounts of 3-acetyl-4-hydroxycoumarin and hydrochloride of glycine methyl ester in the presence of triethylamine in refluxing methanol yielded ligand hl1 (scheme 1a). ligand h2l2 was obtained in the reaction of 3-acetyl-4-hydroxycoumarin and glycine in methanol in the presence of a catalytic amount of hydrochloric acid (scheme 1b). complex 1 was synthesized in the reaction of ligand hl1 and potassium tetrachloridopalladate(ii) in a 1:1 mole ratio in water/methanol (1:1 volume ratio) solution (scheme 2). complex 2 was obtained during the recrystallization of complex 1 from dmf/water mixture. in addition, an attempt was made to synthesize complex 2 in the reaction of ligand h2l2, potassium tetrachloridopalladate(ii) and dimethylamine or its hydrochloride in a 1:1:1 mole ratio in water/methanol (1:1 volume ratio) solution. unfortunately, complex 2 could not be obtained in this way under any experimental conditions. scheme 1. synthesis of ligands hl1 and h2l2. 1386 stojković et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. scheme 2. synthesis of pd(ii) complex 1. ir spectra the ir spectrum of hl1 showed a broad band at 3406 cm–1 corresponding to the stretching vibrations of the nh group. stretching vibrations of the carbonyl group of glycine methyl ester and coumarin lactone were observed at 1748 and 1720 cm–1, respectively. in the ir spectrum of h2l2, the broad band at 3502 cm–1 corresponds to vibrations of the oh group from its carboxylic part and vibration of the nh group. this band was absent in the ir spectrum of complex 2, indicating coordination of ligand in deprotonated form. instead of this, a sharp band appeared at 3227 cm–1, corresponding to stretching vibrations of nh from coordinated dimethylamine. coordination of carboxylate resulted in a bathochromic shift of ν(c=o) from 1740 cm–1 in the spectrum of the h2l2 ligand to 1690 cm–1 in the spectrum of complex 2. a band at 1650 cm–1 originating from the imino group ν(c=n) of the uncoordinated ligand was shifted to 1660 cm–1. moreover, coordination of the deprotonated enol group of h2l2 resulted in a shift of ν(c–o) from 1224 cm–1 in the spectrum of h2l2 to 1245 cm–1 in the spectrum of complex 2. nmr spectra in the 1h-nmr spectrum of ligand hl1, the signal of the methyl protons from c2′ appeared at 2.70 ppm, while the signal of the methyl protons from the ester group (h3″) was observed at 3.86 ppm. the 1h-nmr spectrum of hl1 showed resonances at 12.51 and 14.65 ppm corresponding to the oh and nh groups of its tautomers, respectively. the observed δ value of the nh proton indicates e conformation of hl1 and intramolecular hydrogen bonding with the keto oxygen atom.22 previously reported x-ray and dft study of chromone derivatives showed that the keto tautomer with n–h…o hydrogen bonds is energetically more stable than the enol form containing o–h…n hydrogen bonds.23 based on these results, it could be assumed that the keto form is the main tautomeric form of the hl1 ligand. in the 13c-nmr spectrum of ligand hl1, the c1′ carbon showed resonance at 177.5 ppm, whereas the signal of the methyl carbon c2′ was noted at 18.9 ppm. the signals of two carbonyl groups from the 2,4-dioxochroman moiety, lactone (c2) and ketone (c4), appeared at 162.5 ppm and 182.2 ppm, respectively. palladium(ii) complexes 1387 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. in the 1h-nmr spectrum of complex 1, signals of hydrogen atoms from the oh and nh groups (12.51 and 14.65 ppm, respectively) of the ligand were absent, indicating that the ligand was coordinated in the deprotonated form. coordination of hl1 through the nitrogen atom from the imino group resulted in downfield shift of the signals of methyl h2′ and methylene h1″ hydrogen atoms. in the 1h-nmr spectrum of complex 1, the signal of the methyl group from the ester part of the ligand was shifted upfield, indicating that the ester carbonyl oxygen was involved in the coordination. coordination of the oxygen atom from c4 of the coumarin moiety resulted in an upfield shift of the h5 signal and downfield shifts of the h6, h7 and h8 signals. from the 1h-nmr spectrum of complex 2, it could be seen that the ligand h2l2 was coordinated in the deprotonated form since the signals of acidic protons were absent. the signal of the hydrogen atoms from the methylene group h1″ was shifted downfield due to coordination of the carboxylate oxygen atom. coordination of ligand through the nitrogen atom of the imino group resulted in an upfield shift of the methyl h2′ signal. the upfield shift of the h5 signal and the downfield shift of the h6, h7 and h8 signals indicate coordination of the oxygen atom at c4 in the coumarin moiety. the signals of methyl groups and nh from dimethylamine ligand appeared at 2.40 and 2.52 ppm, respectively. thus, the nmr spectral data indicated coordination of ligand h2l2 via the imine nitrogen atom, the oxygen atom from c4 and the carboxylic oxygen atom. x-ray crystallographic analysis of complex 2 in complex 2 (fig. 1), the pd(ii) cation is coordinated by one ligand molecule through the oxygen atom of the keto group from the 2,4-dioxochromane moiety, through the iminic nitrogen atom, and through the carboxylic oxygen atom; one molecule of dimethylamine completes the metal environment, where the square planar coordination of the metal cation shows two oxygen atoms and two nitrogen atoms respectively opposed. a non-coordinating water molecule fig. 1. molecular structure and atom labeling of compound 2. thermal ellipsoids are at the 50 % probability level. 1388 stojković et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. completes the asymmetric unit. a mogul21 check of the molecular geometry showed no significant deviations from the ccdc average values for all the bonding parameters, apart from the angles c4–c3–n1, o5–c6–o4 and c15–n2–c14, which are statistically smaller than the average, probably due to the mutual repulsion of c4 and o4, and to the coordination of n2. the association of molecules in the crystal is driven by intermolecular hydrogen bonds between the coordinated dimethylamine n2–h and the carboxylate o1 (n2…o1(i) = 2.997(8) å, n2–h…o1(i) = 157(5)°, i = –x+2, –y+1, –z), forming a supramolecular centro-symmetric dimer; the dimers are bridged by water molecules acting as hydrogen bond donors, interacting with the carboxylate oxygen o1 (o6··o1 = 2.84(1) å, o6–h…o1 = 142.1(9)°) and the carbonylic o4 of an adjacent molecule (o6··o4(ii) = 3.00(1)å, o6–h…o4(ii) = = 172.1(9)°, ii = x+1/2, –y+1/2, z–1/2), forming layers which expose the dimethylamine methyl groups and the water oxygens at the surfaces (fig. 2). fig. 2. crystal packing of compound 2, showing hydrogen bond layers made of dimers (in orange) bridged by water molecules. reaction mechanism considering the mechanism of the hydrolysis reaction that occurred during the recrystallization of complex 1 in the dmf/water solution, it could be assumed that the cationic complex 3 was initially formed in the substitution reaction between the chlorido ligand and a water molecule (scheme 3). in complex 3, pd(ii) acts as a lewis acid, which polarizes the ester carbonyl group and activates its carbon atom toward attack by a water molecule from the solvent. hydrolysis of the ester group of complex 3 leads to the formation of the neutral complex 4, which catalyzes the hydrolysis of the amide bond of dimethylformamide. there are two possible reaction pathways (a and b) for the hydrolysis of dimethylformamide by complex 4 (scheme 4). one of them (pathway a) involves the substitution of water molecule with carbonyl group of dimethylformamide, which is followed by an external attack of the polarized amide carbon atom by a palladium(ii) complexes 1389 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. scheme 3. mechanism of ester bond hydrolysis in complex 1. scheme 4. mechanism of amide bond hydrolysis catalyzed by complex 4. 1390 stojković et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. water molecule from the solvent. in the second possible reaction pathway (pathway b), coordination of water molecule to pd(ii) in complex 4 enhances its nucleophilicity and facilitates its attack of the amide carbon atom of uncoordinated dimethylformamide. the replacement of the water molecule in complex 4 by the dimethylamine formed in the hydrolysis reaction led to the formation of complex 2. conclusions the synthesis and structural characterization of methyl 2-([1-{2,4-dioxochroman-3-ylidene}ethyl]amino)acetate (hl1) and 2-([1-{2,4-dioxochroman-3 -ylidene}ethyl]amino)acetic acid (h2l2) are presented herein for the first time. complex 1 was synthesized in the reaction of the hl1 ligand and potassium tetrachloridopalladate(ii) (mole ratio 1:1). recrystallization of complex 1 from a dmf/water solution produced complex 2. x-ray crystallographic analysis of complex 2 showed that the pd(ii) cation was coordinated by one hl1 ligand molecule through the oxygen atom of the keto group from the 2,4-dioxochromane moiety, through the iminic nitrogen atom, and through the carboxylic oxygen atom. the square planar environment around the metal ion was completed with one molecule of dimethylamine. during the in situ synthesis starting from potassium tetrachloridopalladate(ii), ligand h2l2 and dimethylamine or its hydrochloride complex 2 were not obtained. considering the mechanism of the hydrolysis reaction that occurred during the recrystallization of complex 1 from the dmf/water solution, two possible reaction pathways (a and b) for the hydrolysis of dimethylformamide catalyzed by pd(ii) were proposed. supplementary material crystallographic data (excluding structure factors) for compound 2 were deposited with the cambridge crystallographic data centre as supplementary publication no. ccdc 1503280. copies of the data can be obtained free of charge on application to ccdc, 12 union road, cambridge cb2 1ez, uk (fax: (+44) 1223-336-033; e-mail: deposit@ccdc.cam.ac.uk). crystal data, structure refinement for 2 and the ir and nmr data for the synthesized compounds are available electronically at the pages of journal website: http:// //www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. this work was supported by the ministry of education, science and technological development of the republic of serbia (grant oi 172055 and grant oi 172016). palladium(ii) complexes 1391 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. и з в о д синтеза и карактеризација комплекса паладијума(ii) са глицинским кумаринским дериватима данијела љ. стојковић1, alessia bacchi2, davide capucci2, милица р. миленковић3, божидар чобељић3, срећко р. трифуновић1, катарина анђелковић3, верица в. јевтић1, ненад вуковић, милена вукић1 и душан сладић3 1институт за хемију, природно–математички факултет, универзитет у крагујевцу, радоја домановића 12, 34000 крагујевац, 2dipartimento di chimica, university of parma, parco area delle scienze 17 a, i 43124 parma, italy и 3хемијски факултет, универзитет у београду, студентски трг 12–16, 11000 београд синтетисан је комплекс паладијума(ii) са метил-2-([1-{2,4-диоксохроман-3-илиден}етил]амино)ацетатом. структуре како лиганда тако и комплекса паладијума(ii) одређене су елементалном анализом, ic и nmr спектроскопијом. прекристализација комплекса паладијума(ii) из смеше dmf/вода резултује у његовој хидролизи и формирању комплекса диметиламин-(2-[{1-(2,4-диоксохроман-3-илиден)етил}амино]ацетато) -паладијум(ii), чија структура је одређена елементалном анализом, ic, 1h и 13c-nmr спектроскопијом, као и рендгенском структурном анализом. (примљено 15. септембра, ревидирано 25. септембра, прихваћено 27. септембра 2016) references 1. s. emami, s. dadashpour, eur. j. med. chem. 102 (2015) 611 2. j. nawrot-modranka, e. nawrot, j. graczyk, eur. j. med. chem. 41 (2006) 1301 3. v. s. satyanarayan, p. sreevani, a. sivakumar, arkivoc 2008 (2008) 221 4. a. g. kidane, h. salacinski, a. tiwari, k. r. bruckdorfer, a. m. seifalian, biomacromolecules 5 (2004) 798 5. a. a. h. kadhum, a. a. al-amiery, a. y. musa, a. b. mohamad, int. j. mol. sci. 12 (2011) 5747 6. g. b. bubols, d. r. vianna, a. medina-remon, g. von poser, r. m. lamuela-raventos, v. l. eifler-lima, s. c. garcia, mini-rev. med. chem. 13 (2013) 318 7. i. kostova, i. manolov, i. nicolova, s. konstantinov, m. karaivanova, eur. j. med. chem. 36 (2001) 339 8. a. a. abou-hussein, w. linert, spectrochim. acta 141 (2015) 223 9. i. kostova, i. manolov, g. momekov, eur. j. med. chem. 39 (2004) 765 10. i. kostova, g. momekov, eur. j. med. chem. 41 (2006) 717 11. i. kostova, g. momekov, eur. j. med. chem. 43 (2008) 178 12. v. v. jevtić, m. pešić, g. p. radić, n. vuković, s. sukdolak, o. klisurić, a. podolski -renić, n. tanić, s. r. trifunović, j. mol. struct. 1040 (2013) 216 13. e. budzisz, m. małecka, i.-p. lorenz, p. mayer, r. a. kwiecien, p. paneth, u. krajewska, m. rozalski, inorg. chem. 45 (2006) 9688 14. e. budzisz, b. k. keppler, g. giester, m. wozniczka, a. kufelnicki, b. nawrot, eur. j. inorg. chem. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {adsorption of strontium on different sodium enriched bentonites} j. serb. chem. soc. 82 (4) 449–463 (2017) udc 546.42’33+666.32:544.723:628.316.12 jscs–4980 original scientific paper 449 adsorption of strontium on different sodium-enriched bentonites sanja r. marinović1*, marija j. ajduković1, nataša p. jović-jovičić1**, tihana m. mudrinić1, bojana m. nedić-vasiljević2, predrag t. banković1 and aleksandra d. milutinović-nikolić1# 1university of belgrade, institute of chemistry, technology and metallurgy, njegoševa 12, belgrade, serbia and 2university of belgrade, faculty of physical chemistry, studentski trg 12–16, belgrade, serbia (received 10 october, revised 19 december, accepted 21 december 2016) abstract: bentonites from three different deposits (wyoming, tx, usa and bogovina, serbia) with similar cation exchange capacities were sodium enriched and tested as adsorbents for sr2+ in aqueous solutions. x-ray diffraction analysis confirmed successful na-exchange. the textural properties of the bentonite samples were determined using low-temperature the nitrogen physisorption method. significant differences in the textural properties between the different sodium enriched bentonites were found. adsorption was investigated with respect to adsorbent dosage, ph, contact time and the initial concentration of sr2+. the adsorption capacity increased with ph. in the ph range from 4.0– –8.5, the amount of adsorbed sr2+ was almost constant but 2–3 times smaller than at ph ≈11. further experiments were performed at the unadjusted ph since extreme alkaline conditions are environmentally hostile and inapplicable in real systems. the adsorption capacity of all the investigated adsorbents toward sr2+ was similar under the investigated conditions, regardless of significant differences in the specific surface areas. it was shown and confirmed by the dubinin–radushkevich model that the cation exchange mechanism was the dominant mechanism of sr2+ adsorption. their developed microporous structures contributed to the sr2+ adsorption process. the adsorption kinetics obeyed the pseudo-second-order model. the isotherm data were best fitted with the langmuir isotherm model. keywords: sr2+; water purification; na-enriched clays; cation exchange capacity; textural properties. *,** corresponding authors. e-mail: (*)sanja@nanosys.ihtm.bg.ac.rs; (**)natasha@nanosys.ihtm.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc161010008m 450 marinović et al. introduction strontium is abundantly present in the earth’s crust in the form of minerals: srco3, srso4 and al2o3∙sro∙(sio2)6∙4h2o. in surface waters, strontium originates from the weathering of rocks or from the discharge of wastewater from industries that use strontium compounds. ionic strontium is not toxic in small concentrations, and since its concentration in water is generally low, it could be regarded as a harmless substance. the environmental impact of sr arises from its radioactive isotopes. besides the four stable sr isotopes naturally present in soil, there are also artificial, radioactive isotopes 89sr and 90sr.1,2 these radioactive isotopes occur as waste products in nuclear power plants and in the reprocessing of nuclear fuels.3 strontium is an alkaline earth metal that resembles calcium and barium in chemical properties. due to its similarity with calcium, strontium tends to accumulate in bone tissues in the same manner. in the case of radioactive strontium, accumulation in bone tissue causes radiation damage affecting human and/or animal health.4 therefore, the presence of radioactive sr in the environment should be resolved. the disposal of radioactive wastewater from commercial nuclear plants is one of the major problems in nuclear waste management.5 the adsorption of strontium from wastewater prior to its discharge to water bodies in the environment is among the most suitable solutions for pollution prevention. the adsorption of non-radioactive strontium has been investigated on different adsorbents, such as a pan/zeolite composite,6 expanded perlite,7 activated carbon,2 etc. some authors investigated the adsorption of radioactive strontium on kaolinite and montmorillonite,8 illite/smectite mixed clays9 and on magnetite and a magnetite silica composite.10 the adsorptive behavior of radioactive strontium isotopes is similar to that of non-radioactive strontium. this enables the potential adsorptive removal of radioactive strontium from aqueous solutions to be determined using experimental results and models obtained for systems containing the non-radioactive isotopes. bentonites are widely used as adsorbents for the removal of heavy metals from wastewater.11–14 they are low cost, naturally occurring, non-toxic materials, abundantly present in different parts of the world, including serbia.12,14–16 bentonites are clays rich in smectite minerals. smectites are 2:1 phyllosilicates – layered minerals the layers of which are composed of an octahedral [alo3(oh)3]6– sheet sandwiched between two opposing tetrahedral [sio4]4– sheets.17 smectites have a total (negative) layer charge between 0.2 and 0.6 per half unit cell and include the tri-octahedral smectites: hectorite, saponite, sauconite, stevensite and swinefordite, and di-octahedral smectites: beidellite, montmorillonite, nontronite and volkonskoite. beidellite and montmorillonite differ in the origin of the net structural charge. the net structural charge of beidellite is strontium adsorption on different bentonites 451 mainly located in smectite tetrahedral sheets, while that of montmorillonite is mainly located in smectite octahedral sheets.17 the present work is focused on the removal of sr2+ from aqueous solutions using three different sodium-enriched bentonites with similar cation exchange capacities (cec) but with different textural properties, particularly different specific surface areas. the bentonites selected for this work included standard source clays from the clay minerals society18 and a previously well-characterized clay from a serbian deposit.19,20 the adsorption was studied as a function of adsorbent dosage, suspension ph, adsorption time and the initial concentration of sr2+. the goal of this investigation was to confirm the applicability of the investigated bentonites as sr2+ adsorbents and to determine the most significant adsorption mechanism for sr2+. experimental materials two bentonites were supplied by the clay minerals society: a ca-montmorillonite dominant clay (stx-1) originating from the manning formation, county of gonzales, texas, usa and a na-montmorillonite dominant clay (swy-2) originating from the newcastle formation in the county of crook, wyoming, usa.18 the chemical composition (mass %) of stx-1 was: sio2, 70.1; al2o3, 16.0; tio2, 0.22; fe2o3, 0.65; feo, 0.15; mno, 0.009; mgo, 3.69; cao, 1.59; na2o, 0.27; k2o, 0.078; f, 0.084; p2o5, 0.026; s, 0.04 and co2, 0.16 and ignition loss up to 1100 °c was 6.54 mass %. the composition of swy-2 was: sio2, 62.9; al2o3, 19.6; tio2, 0.090; fe2o3, 3.35; feo, 0.32; mno, 0.006; mgo, 3.05; cao, 1.68; na2o, 1.53; k2o, 0.53; f, 0.111; p2o5, 0.049; s, 0.05 and co2, 1.33, and the ignition loss up to 1100 °c was 6.06 mass %.18 the third bentonite material (the dominant smectite mineral phase of which consisted of montmorillonite and beidellite in the ratio of approx. 1:9)21 was obtained from the ‟bogovina coal and bentonite mine”, bogovina, serbia. it had been previously thoroughly characterized19,22 and tested as a pb, cd and ni adsorbent.23 the chemical composition (mass %) of this clay was: sio2, 57.51; al2o3, 17.13; fe2o3, 7.67; mgo, 2.35; cao, 1.81; na2o, 0.75; k2o, 1.18 and co2, 0.5, and the ignition loss was 11.10 mass %.19 as previously reported, the cation exchange capacities (cec) of the bentonites obtained by the ammonium acetate method24 were 84.4, 76.4 and 63.3 mmol of monovalent cation per 100 g of clay dried at 110 °c for the texas, wyoming and bogovina originating bentonites, respectively.18,20 the samples were sieved through a 74-μm sieve. sodium enrichment of the bentonite samples was performed using the previously reported procedure20 and the obtained materials based on wyoming, texas and bogovina bentonites were denoted as na-w, na-t and na-b, respectively. sodium enrichment was performed with the goal of obtaining homoionic absorbents with na+ as the exchangeable cation, which could be more easily replaced with sr2+ in the adsorption process. srcl2∙6h2o, with 98 % purity, was obtained from carlo erba and used as received. 452 marinović et al. characterization methods the x-ray diffraction patterns of the powders of na-enriched bentonites were obtained using a philips pw 1710 x-ray powder diffractometer, equipped with a cu anode (λ = = 0.154178 nm). the point of zero charge (phpzc) was determined using a batch equilibration technique.25 suspensions each containing 50 mg of one of the bentonite samples in 20 cm3 of a 10-2 mol dm-3 nacl solution were prepared by shaking for 24 h at room temperature. the initial ph values (phi) were adjusted in the ph range from 2 to 12 by the addition of small amounts of 0.1 mol dm-3 hcl or naoh solution. the phi and phf (final ph measured after 24 h of shaking) were measured using a meterlab ph/ion meter phm240 ph-meter. the point of zero charge was determined from the phi vs. phf diagram. nitrogen physisorption isotherms were determined on a thermo finnigan sorptomatic 1990 instrument at –196 °c and the values of the textural parameters were obtained using the adp 2005 software. the samples were out-gassed at 160 °c for 20 h. textural parameters’ values were calculated according to the usual methods.26-28 the specific surface area, sbet, was calculated from the adsorption data in the p/p0 range from 0.05 to 0.26 according to the brunauer, emmett, teller method. the total pore volume, v0.98, was calculated according to the gurvitsch method. the dubinin–radushkevich method was used for the calculation of the micropore volume, while specific surface area in the micropore region was obtained using the horvath–kawazoe method. pore diameter distribution curves were obtained according to the barrett, joyner, halenda method. to distinguish between micropores and the external surface area, the t-plot method was applied. the harkins and jura relation was used as the standard reference t-curve. adsorption experiments batch-type adsorption experiments were conducted in aqueous solutions in a temperature-controlled water bath shaker (memmert wne 14 and sv 1422). adsorption was carried out with respect to contact time and initial sr2+ concentration. aliquots were withdrawn from the shaker at regular time intervals and the solution was centrifuged at 17000 rpm for 10 min (hettich eba-21). the sr2+ content in the supernatant solutions was determined by inductively coupled plasma optical emission spectrometry (icp– –oes). the strontium concentrations in the supernatants were determined using an icap 6500 duo icp (thermo fisher scientific, cambridge, uk) inductively coupled plasma optical emission spectrometer (icp-oes) with iteva operational software. the strontium calibration solutions were prepared using multi-element plasma standard solution 4, specpure® (alfa aesar, germany). strontium concentration was measured at the emission wavelength sr ii 215.284 nm. for each sample, the measurement was performed in triplicate. the relative standard deviation was lower than 0.5 %. analytical process quality control was performed using a certified reference material (epa method 200.7 lpc solution; ultra scientific, usa), which indicated a strontium recovery of 99.4 %. all experiments were performed in triplicate at 25 °c, using the same mass of adsorbent (mads = 20.0 mg) and volume of solution (v = 50.0 cm3). the amount of sr2+ adsorbed qt (mg g-1) after time t was calculated from the following mass balance relationship: 0 ads t t c c q m − = (1) strontium adsorption on different bentonites 453 where c0 and ct are the concentration (mg dm-3) of sr2+ in solution initially and after adsorption time t, respectively. results and discussion x-ray powder diffraction the xrd patterns of the powders of the na-enriched bentonites are presented in fig. 1. fig. 1. the xrd patterns of the powders of the na-enriched bentonites (s – smectite, q – quartz, f – feldspar and c – calcite). the obtained diffractograms confirmed the phase composition of bentonites with dominant smectite phase (either montmorillonite or a montmorillonite/beidelite mixture) with different associated minerals in accordance with the different origins of the bentonites. as expected, na-w contained the highest amount of feldspar among the investigated bentonites, while na-b had the highest amount of quartz.22,29 only na-b contained a detectable amount of calcite as an impurity. the xrd pattern revealed that na-exchange had been successfully performed since the position of the 001 smectite peak in all the investigated samples corresponded to the na-forms of smectite with d001 at around 1.2–1.3 nm.30 the raw bentonites from texas and bogovina were dominantly smectites of the ca -type with characteristic basal spacing of d001 = 1.5 nm. textural properties low temperature n2 physisorption measurements resulted in the isotherms presented in fig. 2. all the presented isotherms were reversible at lower equilibrium pressures, belonging to the type ii isotherms according to iupac nomenclature. type ii isotherms are characteristic for materials consisting of aggregated planar particles that form slit shape pores. such a result is in accordance with the literature.31 at equilibrium pressures above p/p0 = 0.4, a hysteresis loop of the h3 type appeared, 454 marinović et al. which indicates multilayer nitrogen adsorption and capillary condensation within the smectite mesopores of all the smectites.22,32 the adsorption isotherms of all the materials exhibited an inflection point at about p/p0 = 0.2. this indicates that no overlapping between monolayer and multilayer adsorption occurred, i.e., the formation of the second adsorption layer began only after monolayer adsorption had been completed. selected textural data are presented in table i. fig. 2. nitrogen adsorption–desorption isotherms of na-t, na-w and na-b. table i. selected textural properties; sbet – specific surface area (brunauer, emmett, teller – three parameter plot); v0.98 – total pore volume (gurvitch); vmes,bjh – mesopore volume (barrett, joyner, hallenda); dmed – median pore diameter; dmax – the most abundant pore diameter; st – specific surface area (t-plot); and smic – micropore specific surface area sample sbet m2 g-1 v0.98 cm3 g-1 vmes,bjh cm3 g-1 dmax nm dmed nm st m2 g-1 smic=sbet–st m2 g-1 na-t 113 0.2000 0.2101 3.83 9.17 83 30 na-w 52 0.0759 0.0871 4.08 4.26 31 21 na-b 98 0.0853 0.0788 3.93 3.99 43 55 all the na-enriched materials exhibited mesoand microporosity developed to different extents. while the mesoporous surface area increased in the order na-w < na-b < na-t, the increase of the surface area of micropores followed the sequence na-w < na-t < na-b. the microporous surface area should be considered as more relevant in the investigated cation exchange process, since it occurs in the interlamellar smectite region. therefore, if sr2+ adsorption on the strontium adsorption on different bentonites 455 na-enriched clays is dominantly dependent on the surface area, the efficiency of sr2+ uptake is expected to follow the sequence na-b > na-t > na-w. the effect of adsorbent dosage on the adsorption of sr2+ in order for the influence of the adsorbent dosage on the adsorption of sr2+ ions to be investigated, the mass of all investigated na-enriched bentonites was varied from 10 to 100 mg. all the experiments were performed for three hours at 25 °c with an initial sr2+ concentration of 50 mg dm–3. the influence of adsorbent dosage on the amount of sr2+ ions adsorbed on the investigated na-enriched bentonites after 180 min at 25 °c is presented in fig. 3. fig. 3. the influence of adsorbent dosage on the amount of adsorbed sr2+. as expected, the quantity of adsorbed sr2+ per unit mass of adsorbent decreased with increasing adsorbent dosage. it decreased steeply from a dosage of 10 mg to one of 20 mg, after which a plateau was attained. for dosages from 20 to 50 mg, the adsorption per unit mass did not change significantly. after the plateau, the drop in the adsorption per unit mass was more pronounced. for a dosage of 100 mg, almost complete sr2+ removal was obtained. a dosage of 20 mg was chosen because it is in the plateau of the q–m diagram, while it is low enough to meet economic requirements. the effect of ph on the adsorption of sr2+ the effect of the initial ph on the adsorption of sr2+ was investigated within the ph range 2–11 and the results are presented in fig. 4, together with the ph behavior of all the na-enriched bentonites. the diagram shows the initial ph of the suspensions of na-enriched bentonites in 0.01 m nacl (phi) vs. ph after 24 h of shaking (phf). a plateau 456 marinović et al. between ph values of 4 and 8.5 was observed and the point of zero charge (phpzc) was estimated to be 6.5 for na-t, 6.3 for na-w and 8.2 for na-b. fig. 4. the effect of ph on the adsorption of sr2+ and phi vs. phf diagram. lower than phpzc, the surface charge of the samples is positive, while above these values, the surface charge is negative. on the other hand, strontium is in the cationic form within the entire investigated ph range. the quantity of adsorbed sr2+ was very low in an extremely acidic environment, which could be explained by the repulsion between positive surface charge of the adsorbents and sr2+. in the 4–8.5 ph range, the amount of adsorbed sr2+ was higher and almost constant due to a constant ratio between the positive and negative surface charges of the adsorbents. further increases of the ph value (>8.5) led to very high amounts of adsorbed sr2+ as a consequence of the electrostatic interaction of the negative surface of adsorbents and the sr2+. further experiments were realized at an unadjusted ph (ph 6.8), which lies on the plateau in fig. 4, where the adsorption efficiency is almost constant in the ph interval from 4 to 8.5.33 unadjusted ph was also chosen because adjusting the ph would lead to an additional operation, which increases the procedure cost. moreover, extreme ph values could cause ecological problems that should be avoided. the adsorption at unadjusted ph was satisfactory (around 30 % removal of sr2+ from water) under the given experimental conditions.34 strontium adsorption on different bentonites 457 the effect of contact time on the adsorption of sr2+ in order to determine the equilibrium time for the maximum uptake of sr2+, the adsorption of sr2+ was monitored with respect to contact time. the initial sr2+ concentration was 50 mg dm–3. the effect of contact time on the amount of sr2+ adsorbed on the bentonites is presented in fig. 5. fig. 5. the effect of contact time on the adsorption of sr2+ on the three studied bentonites. for all adsorbents, adsorption rate was initially high and then, it gradually reached a plateau. all adsorbents exhibited similar t1/2 values (t1/2 – the time at which the amount of adsorbed sr2+ is equal to half of the total amount adsorbed at equilibrium). the t1/2 values were 5.8, 6.9 and 5.5 min for na-t, na-w and na-b, respectively. both parameters that describe adsorbent quality, i.e., the amount of sr2+ adsorbed per unit mass and t1/2, were similar for all the sodium -enriched bentonites. the obtained results confirmed that the degree of the development of a porous structure had no influence on the adsorption of sr2+. na-w, the material with the least developed micro and mesopore surface, even exhibited a slightly higher adsorption of sr2+. this indicates that the ion exchange mechanism was the dominant adsorption mechanism. the pseudo-first order and pseudo-second order kinetics models were tested for the adsorption of sr2+ on the na-enriched bentonites. the parameters calculated for the pseudo-first and pseudo-second order kinetics models35,36 are presented in table ii. 458 marinović et al. table ii. pseudo-first-order-kinetics and pseudo-second-order-kinetics for the adsorption of sr2+ ions on na-t, na-w and na-b; k1 is the pseudo-first order rate constant; k2 is the pseudo-second order rate constant; qeexp is the experimentally obtained value for the equilibrium adsorption capacity; qecalc is calculated value for the equilibrium adsorption capacity and r2 is the square of the correlation coefficients sample na-t na-w na-b qeexp / mg g-1 38.98 41.78 35.68 pseudo-first order kinetics model qecalc / mg g-1 3.121 2.239 1.374 k1 / min-1 0.027 0.007 0.029 r2 0.9978 0.6747 0.6759 pseudo-second order kinetics model qecalc / mg g-1 37.22 42.94 35.62 k2×102 / g mg-1 min-1 1.092 0.447 7.269 r2 0.9998 0.9998 0.9999 the square of the correlation coefficients (r2) for the pseudo-first order kinetics model were relatively low for na-w and na-b, indicating poor correlation of the data with the model. on the other hand, the r2 values for the pseudo -second order kinetics model were > 0.999 for all bentonites. although na-t exhibited a rather high r2 value for the pseudo-first order kinetics, the values of the equilibrium adsorption capacity qecalc calculated from the equation for the pseudo-first order kinetics for all samples exhibited high discrepancy from the experimentally obtained adsorption capacity, qeexp. on the other hand, for the pseudo-second order kinetics model, qecalc and qeexp were in good agreement, confirming that the adsorption of sr2+ on the studied bentonites obeyed pseudo -second order kinetics. the pseudo-second-order kinetics model for the adsorption of sr2+ on the investigated bentonites is shown in fig. 6. the effect of the initial concentration of sr2+ on their adsorption the effect of the initial concentration of sr2+ on the adsorption capacity was also investigated for four different initial concentrations of sr2+ in the concentration range from 25 to 100 mg dm–3. the obtained data were fitted using the langmuir, freundlich and dubinin–radushkevich isotherm models.37–39 the value obtained for the adsorption energy could be used to predict the type of adsorption.40 if e is smaller than 8 kj mol–1, the adsorption could be described as physisorption, while e in the range from 8−16 kj mol–1 indicates chemisorption. average values of repeated experiments for each adsorbent were fitted using the langmuir and freundlich models and the results are presented in figs. 7a and b, respectively. the calculated parameters for the investigated models are presented in table iii. strontium adsorption on different bentonites 459 fig. 6. plots of the pseudo-secondorder kinetics model for the adsorption of sr2+ on the studied bentonites. fig. 7. adsorption isotherms: a) langmuir and b) freundlich isotherm model. the langmuir model better fitted the experimental data, having r2 close to unity. it is appropriate to describe the adsorption of sr2+ on all the studied bentonites. as previously stated, the ion exchange mechanism could be regarded as the dominant adsorption mechanism. in order to verify this statement, the qmax values calculated according to the langmuir model were compared with the corresponding cation exchange capacity (cec) values.18,20 since strontium is divalent and the cec values are given for monovalent cations, the qmax values should be compared with half of the cec values. the corresponding values were as follows: for na-t, qmax was 0.526 mmol g–1 and half the cec value was 0.422 mmol g–1; for na-w, qmax was 0.543 mmol g–1 and half of the cec value was 0.382 mmol g–1 and for na-b, qmax was 0.521 mmol g–1 and half of the cec value was 0.316 mmol g–1. the obtained values of qmax exceeded the 460 marinović et al. amount of available exchangeable cations, indicating that although the ion exchange mechanism was dominant, there are other contributions to the adsorption process. bearing in mind that the increase in the surface area of the micropores followed the sequence na-w < na-t < na-b, it could be assumed that developed microporous structure contributed somewhat to the sr2+ adsorption process. table iii. parameters calculated for the langmuir, freundlich and dubinin–radushkevich isotherm models; qmax is the maximum adsorption capacity; kl is the langmuir adsorption constant; kf and n are the freundlich adsorption constants characteristic for the system; kdr is the adsorption energy constant and e is the energy of adsorption sample na-t na-w na-b langmuir isotherm qmax / mg g-1 46.1 47.6 45.7 kl / dm3 mg-1 7.31 5.21 9.49 r2 0.968 0.998 0.973 freundlich isotherm n 8.62 9.558 5.78 kf / dm3 mg-1 24.9 26.6 19.0 r2 0.689 0.901 0.880 dubinin–radushkevich kdr / mol2 kj-2 6.81·10-3 6.99·10-3 7.43·10-3 e / kj mol-1 8.57 8.46 8.20 r2 0.995 0.956 0.935 the squared correlation coefficients (r2) showed that the experimental data were in good agreement with the dubinin–radushkevich isotherm model (table iii). the calculated e values were similar for all investigated adsorbents and slightly higher than 8 kj mol–1, indicating that the type of adsorption was chemisorption. according to literature data, the obtained e values were in the range of adsorption energy (8–16 kj mol–1) characteristic for systems where ion exchange is the dominant mechanism.41 since the experiments related to sr2+ adsorption on various adsorbents reported in literature were performed under different experimental conditions, direct comparison of the obtained data is not possible. however, a few descriptive data will be given. chegrouche et. al.2 reported a maximum adsorption capacity of 0.507 mmol g–1 (44.4 mg g–1) on activated carbon, while yusan and erenturk6 obtained a maximum adsorption capacity of 0.011 mg g–1 on a pan/ /zeolite composite adsorbent. ahmadpour et al.5 investigated adsorption of sr2+ on treated almond green hull and obtained a maximum adsorption capacity of 116.3 mg g–1. considering the literature data, the adsorption of sr2+ on the investigated na-enriched bentonites showed that they could be considered as possible adsorbents for the removal of sr2+ from wastewater. strontium adsorption on different bentonites 461 conclusions three different bentonites were sodium-enriched in order to be tested as adsorbents for sr2+. the characterization of these bentonites included xrd and detailed textural analysis. the xrd analysis revealed that na-exchange had been successfully performed. the mesoporous surface area increased in the order na-w < na-b < na-t, while the increase in the surface area of the micropores followed the sequence na-w < na-t < na-b. the surface areas of the investigated bentonites differed significantly. the adsorption of sr2+ ions from aqueous solutions was performed on all three na-enriched bentonites. adsorption was realized with respect to the adsorbent dosage, ph, contact time and initial concentration of sr2+. the adsorption capacity increased with ph. in the ph range from 4–8.5, the amount of adsorbed sr2+ was almost constant but 2–3 times smaller than maximal obtained at ph ≈11. it is concluded that the adsorption was mainly governed by the ion exchange mechanism, since the bentonites with similar cec values and different specific surface areas had similar adsorption capacities. bearing in mind that the increase of the surface area of micropores followed the sequence na-w < na-t < na-b, it could be assumed that the developed microporous structure contributed somewhat to the sr2+ adsorption process. the adsorption dynamics was described well by the pseudo-second-order kinetics model. the langmuir isotherm model best fitted the experimental data. the dubinin–radushkevich isotherm was used to provide a better understanding of the adsorption mechanism and to distinguish physisorption from chemisorption. the value obtained for the adsorption energy was slightly higher than 8 kj mol–1. this indicated chemisorption and partially ion exchange as the dominant mechanisms. all the investigated adsorbents could be used for the removal of radioactive strontium, considering the fact that it has similar adsorptive behavior to its non-radioactive counterpart. moreover, according to the findings in this study, bentonite from unexploited deposits could be used as a sr2+ adsorbent. knowing the cation exchange capacity of any clay, the prediction of its adsorption potential is possible. acknowledgement. this work was supported by the ministry of education, science and technological development of the republic of serbia (project iii 45001). и з в о д адсорпција стронцијума на различитим натријумом измењеним бентонитима сања р. мариновић1, марија ј. ајдуковић1, наташа п. јовић-јовичић1 , предраг т. банковић1, тихана м. мудринић1, бојана м. недић васиљевић2 и александра д. милутиновић-николић1 1универзитет у београду – институт за хемију, технологију и металургију, центар за катализу и хемијско инжењерство, његошева 12, београд и 2универзитет у београду – факултет за физичку хемију, студентски трг 12–16, београд бентонити из три различита налазишта (вајоминг и тексас, сад, и боговина) са сличним капацитетима катјонске измене натријумски су измењени и испитани као 462 marinović et al. адсорбенси за уклањање sr2+ из водених раствора. успешност натријумске измене потврђена је рендгено-структурном анализом. текстурална својства бентонитних узорака су одређена коришћењем методе нискотемпературне физисорпције азота. добијене су значајне разлике у текстуралним својствима натријумски измењених бентонита различитог порекла. испитиван је утицај масе адсорбента, ph, времена контакта и почетне концентрације sr2+ на адсорпцију sr2+. aдсорпциони капацитет расте са порастом ph. у опсегу ph од 4 до 8,5 количина адсорбованих sr2+ је скоро константна, али 2 до 3 пута мања од максималне остварене при ph ≈11. у свим наредним експериментима коришћен је неподешени ph раствора, јер је коришћење екстремно базних условa штетно за животну средину и сходно томе неприменљиво у реалним системима. утврђено је да је адсорпциони капацитет свих адсорбенаса према sr2+ сличан, без обзира на значајне разлике у специфичним површинама. показано је да је механизам катјонске измене доминантан механизам при адсорпцији sr2+ на натријумски измењеним бентонитима, што је потврђено и моделом dubinin–radushkevich. развијена микропорозна структура такође у извесној мери доприноси процесу адсорпције sr2+. адсорпциона динамика прати кинетички модел псеудо-другог реда за све адсорбенсе, док лангмирова изотерма најбоље описује адсорпцију у испитаним системима. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice influence of bunch morphology on quality of wines produced from clones of grape variety prokupac j. serb. chem. soc. 81 (8) 883–895 (2016) udc 634.853:581.4+57.03:547.973: jscs–4894 547.918+663.251 original scientific paper 883 influence of bunch morphology on quality of wines produced from clones of grape variety prokupac jelena živković1*, katarina šavikin1, gordana zdunić1, dejan gođevac2, nebojša marković3, zoran pržić3 and nebojša menković1 1institute for medicinal plants research “dr. josif pančić”, tadeuša košćuška 1, 11000 belgrade, serbia, 2university of belgrade, institute for chemistry, technology and metallurgy, njegoševa 12, 11000 belgrade, serbia and 3university of belgrade, faculty of agriculture, nemanjina 6, 11080 belgrade, serbia (received 14 november 2015, revised 2 april, accepted 4 april 2016) abstract: wine quality depends mainly on the characteristics of the grape it is made of, and one of the attributes affecting wine composition is cluster and berry morphology. the aim of this study was to represent variability of the morphological characteristics between different clones of the autochthonous grape variety prokupac and to perform chemical evaluation of wines obtained from them. total phenolic content was generally low and it ranged from 33.0 to 114.5 mg gae/100 ml. six main anthocyanin compounds including malvidin as the main anthocyanidin were detected. malvidin 3-o-glucoside was the most abundant anthocyanin with concentration of 59.8 to 101.7 µg/ml. clones 43/5 and 43/4 yielded highest quality wines. according to the results, clonal selection makes a significant difference in prokupac wine quality. on the other hand, there is a minor dependance of wine quality parameters to variation in morphological attributes of clusters and berries (bunch weight, proportion of stem, berry and seed weight, skin, pulp and seed weight per berry). keywords: anthocyanins; clonal selection; malvidin 3-o-glucoside. introduction clonal selection is considered a very important tool for grapevine genetic improvement.1 for vitis vinifera l. (vitaceae), clones could be selected aiming for better grape quality attributes, stronger wine aroma and coloration, as well as genetic resistance to main pests and diseases. clones from one grape variety can differ in their productive traits and their ability to produce wines with different organoleptic characteristics. it has been shown that some clones have the cap * corresponding author. e-mail: jzivkovic@mocbilja.rs doi: 10.2298/jsc151114033z _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 884 živković et al. acity to produce wines with distinct color, aromatic profile and phenolic content.2,3 the main polyphenols present in the wine are phenolic acids, stilbenes, flavonols, dihydroflavonols, anthocyanins, flavanol monomers (catechins) and flavanol polymers – proanthocyanidins.4 as the phenolic acids are largely present in the berry pulp, anthocyanins and stilbenes in the berry skin, and other polyphenols (catechins, proanthocyanidins and flavonols) in the berry skin and seeds, the proportion of the different polyphenols in wines could vary according to the type of vinification.5 total phenolic content, combined with total anthocyanin, proanthocyanidin and tannin content, are important parameters to be considered for the quality of the produced wine.6 the quantity and composition of these secondary metabolites greatly depend on genetic factors, but their amount could also be affected by environmental factors and cultural practices, as well as berry morphology.7 it is widely believed by grape growers that smaller berries and lower yield produce higher quality wines due to a higher proportion of skin and seed derived compounds.8 although wine composition can be manipulated by changing berry size, growers need to balance this with the need for a high yield from well-sized berries on their vines. the southern area of serbia has a well established tradition of viticulture and winemaking since the dominant soil types and climatic conditions of the region are very beneficial for the cultivation of vines.9 prokupac is a serbian autochthonous red wine variety, mostly spread throughout southern and central parts of serbia, as well as in macedonia and bulgaria. it is characterized by the strong vigor and big yielding capacity. its bunch could be classified as medium large, cylindrical or conical in shape, bearing medium compact, round or slightly oval berries with dark blue epidermis.10 prokupac wine is refreshing and nicely red colored. the objective of this study was to represent the morphological variability among clones of the autochthonous variety prokupac and make chemical evaluation of the wines obtained from them. major groups of compounds were evaluated in total amounts, i.e., total phenolics, total anthocyanins, total proanthocyanidins, and the identification and quantification of individual anthocyanins in prokupac wines was done. the influence of grape berry morphological parameters on the wine composition, especially anthocyanin extractability was also determined. experimental wine grape sample preparation prokupac clones tested in this study have been allocated in vineyards cca. 100 years old from southern and central parts of serbia. during research period (2010–2013), 26 clones were grafted and planted on faculty of agriculture experimental field radmilovac, serbia. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ berry morphology influence wine quality? 885 ministry of agriculture of serbia recognized 12 clones as technologically superior compared to the standard variety. morphological analysis within each clone was performed by standard prostoserdov method11 on 10 clusters per 10 vines separately. firstly, weight of bunches was measured. rachis (pedicel) from each berry was carefully separated, so that as little as possible mesocarp was left on the stem. berry mass per bunch as well as mass of stems per bunch was measured using analytical balance. mass of seeds and skin of 100 berries was measured using analytical balance, and number of seeds in 100 berries was determined by counting. other parameters shown in table 1 were obtained by calculation. microvinification was performed in the laboratory of faculty of agriculture, university of belgrade. harvesting was done manually. grape crashing was done using mechanical press and crushed grapes during microvinification were stored in 10 l container. to crushed grapes 0.1 g/1 kg of sulphur was added. for microvinification process, selected saccharomyces cerevisiae red wine yeast-bdx lalemand was used (2.5 g/10kg of crushed grape). each clone was tested in triplicate. microvinification ending was detected by oeshle mostwage when 0°oe of sugar in must was indicated. wine was stored in 1 l glass bottle prior to analysis. determination of total phenolics the concentration of total phenolic compounds in wine was estimated spectrophotometrically using slightly modified folin–ciocalteu method.11 two hundred microliters of wine (5 mg/ml 50% etoh) were added to 1 ml of diluted folin–ciocalteu reagent (1:10). after 4 min, 800 μl of sodium carbonate (75 g/l) were added. the absorbance was measured after 2 h of incubation at room temperature, at 765 nm. gallic acid (0–100 mg/l) was used for calibration of a standard curve. the results were expressed as milligrams of gallic acid equivalents per gram of dry weight of fraction (mg gae/g dw). determination of total anthocyanins total anthocyanin content was analyzed according to the procedure described in european pharmacopoeia 6.0. using slight modifications.12 wine samples were diluted (1:5) in a solution of hydrochloric acid in methanol (0.1 vol. %). the absorbance was measured at 528 nm, with 0.1% v/v solution of hydrochloric acid in methanol as the compensation liquid. the percentage content of anthocyanins was calculated as a×5000/718m (a = absorbance at 528 nm; 718 = specific absorbance of cyaniding 3-glucoside chloride at 528 nm; m = mass of the wine to be examined in grams) and expressed as cyaniding 3-glucoside chloride. the results were presented as percentages. determination of total proanthocyanidins the content of total proanthocyanidin compounds in the samples was determined spectrophotometrically using p-dimethylaminocinnamaldehyde (p-dmaca) reagent with slight modifications.13 one hundred μl of wine samples were mixed with 80 μl of p-dmaca reagent, 2 ml of methanol, and a drop of glycerol. after 7 min, the absorbance was measured at 640 nm. the content of proanthocyanidins in the samples was presented as milligrams of catechin equivalents per 100 ml of sample (mg ce/100 ml). lc–ms analysis lc–ms analysis was performed on an agilent msd tof coupled to an agilent 1200 series hplc. wine samples were separated on zorbax sb-aq column (250 mm×4.6 mm, 5μm). a gradient consisting of solvent a (10 % formic acid in water) and solvent b (acetonitrile) was applied at a flow rate of 1 ml min-1 as follows: 0–1 min, 1–7 % b; 1–4 min, 7 % _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 886 živković et al. b; 4–7.5 min, 7–10 % b; 7.5–11.5 min, 10–14 % b; 11.5–15.5 min, 14–25 % b; 15.5–18.5 min, 25–40 % b; 18.5–22 min 40–75 % b; 22–25 min 75% b; 25–26 min 75–99 % b; 26–27 min, 99–1 % b. the injection volume was 10 ml. mass spectra were acquired using an agilent esi-msd tof. the drying gas (n2) flow was 12 l min -1; the nebulizer pressure was set at 3.1 bar; the drying gas temperature was 350 °c. for esi analysis, the parameters were as follows: capillary voltage, 4000 v; fragmentor, 140 v; skimmer, 60 v; oct rf v 250 v, for the negative (etoac wine fractions) and positive modes (anthocyanins). the mass range was from 100 to 2000 m/z. data processing was done using software molecular feature extractor. hplc-dad analysis analysis of anthocyanins was done using hplc agilent 1200 series with uv–vis dad for multiwavelength detection. wine samples were separated on zorbax sb-aq column (250 mm×4.6 mm, 5μm) according to the compendium of international methods oiv.14 mobile phase consisting of solvent a (h2o/hcooh/ch3cn, 87:10:3 volume ratio) and solvent b (h2o/hcooh/ch3cn, 40:10:50 volume ratio) was applied at a flow rate of 0.8 ml/min according to following gradient program: 6 to 30% b linear from 0 to 15 min, 30 to 50% b linear from 15 to 30 min, 50 to 60% b linear from 30 to 35 min, and 60 to 6% b linear from 35 to 41 min. the injection volume was 50 μl, and the column was thermostated at 40 °c. identification was possible by monitoring anthocyanins at 520 nm and by comparing their spectra and retention times with those of commercial standards. quantification was done using calibration curves of authentic standards. statistical analysis statistical analysis was carried out using the software package statistica, v. 7.0. data are presented as the mean values ± standard deviation from three independent measurements. the variation in chemical parameters was analyzed using one-way analysis of variance (anova) and differences between clones were estimated with duncan test (p < 0.05). correlations were considered statistically significant, if the p-value was less than 0.05. hierarchical cluster analysis was applied for finding relatively homogenous clusters of cases based on measured characteristics. differences between classes were tested with average euclidean distances using the ward method. results of hierarchical clustering process were represented as dendrogram. relationship between individual anthocyanins and total compounds were determined by principal component analysis (pca). results and discussion morphological characteristics of prokupac clones some of the viticultural characteristics that make distinct clones include berry and cluster morphology as well as final yield. the same characteristics are also widely recognized as factors influencing the winegrape quality15. there are numerous factors, besides genetics that can affect berry size and the composition of grapes and wines like water status, cultural practices or annual weather conditions.16 in our study, since all the samples were grown under the same conditions, the differences in morphological parameters were mainly the outcome of genetic factors. the results of basic morphological characteristics for the tested prokupac clones are presented in table i. although not statistically significant, _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ berry morphology influence wine quality? 887 minor differences were observed among clones for investigated morphological parameters. clone 42/2 had the lowest bunch weight (145.30 g) and clone 41/1 the _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 888 živković et al. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ berry morphology influence wine quality? 889 highest (224.78 g). the bunch structure is defined through percent of bunch stem (rachis) and percent of berries in a bunch. clone 42/2 also had the highest proportion of bunch stem (5.48%), but the lowest proportion of berries (94.52%) in a bunch. on the contrary, clone 43/2 had the lowest proportion of stem (3.08 %) and the highest proportion of berries (96.92 %). the seed mass was uniform (0.03 g) for most of the samples with the exception of clone 41/3 that was slightly larger (0.04 g). in relation to the values of berry structural composition expressed as skin, seed and pulp weight, the highest values were recorded for skin of clone 40/5, seeds of clones 40/8 and 43/7 and pulp of clones 40/8 and 41/3. according to the results, prokupac berries are comprised of approximately 5 % skin, 2.5 % seed and 97.5 % flesh. generally, the proportion of skin to seed is smaller than in cabernet sauvignon grapevines where relative skin mass varied between 15 and 20 % and relative seed mass between 4 and 6 %;17 or in the chardonnay variety where skin proportion in berry weight was 16%.18 for the increasing berry size, the ratio of seed weight/berry weight decreased, while we have not observed any pattern for skin weight/berry mass ratio. wine phenolic composition after microvinification process, produced wine samples of the investigated clones were further analysed using spectrophotometric methods. phenolic compounds are of considerable oenological cultivar potential and play a key role in estimating the quality of wine. the amount of total phenolics in the wines obtained from 13 clones of autochtonous grape variety prokupac are presented in table ii. the values ranged from 33.0 (clone 40/8) to 114.5 (clone 43/4) mg gae/100 ml and statistically significant (f = 55.09, p < 0.001) difference was noticed between tested clones. previously, a significant difference in total phenolic content between wines produced from cabernet sauvignon clones 169 and 685 cultivated in brazil was reported by burin et al.2 the phenolic composition is primarily dependent of the grape cultivar and oenological practices, and for young red wines it ranges between 100 and 500 mg/100 ml.19 according to previous results,20 among the wines produced from different cultivars (merlot, cabernet sauvignon, pinot noir and prokupac) the lowest phenolic content was found in the wine from the native cultivar prokupac. depending on the applied winemaking technology, total phenolic content in prokupac wines varied from 54.4–115.9 mg gae/100 ml, which is in accordance with our results. in balkan countries, the most similar results of total phenolic contents in red wines compared to our prokupac clone wines reported kallithraka et al.21 for greek red wines, where the values ranged from 62.2–320.0 mg gae/ml. šeruga et al.22 reported slightly greater values (101.2–326.4 mg gae/ml) for croatian red wines. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 890 živković et al. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ berry morphology influence wine quality? 891 the principal source of red color in wine comes from its anthocyanin content. in grape berries anthocyanins are found mainly in the skin, while pulp contains little or no anthocyanins.since anthocyanins are primarily responsible for the red wine color, they were extensively studied in order to identify the environmental impacts and genetic effects on their synthesis in the berry skin and accumulation in the wine.23–25 also, they were used for chemotaxonomic purposes with the goal of classifying red-grape varieties and the red wines made from them.26 the amounts of total anthocyanins in wines obtained from 13 clones of autochtonous grape variety prokupac are presented in table ii. same as for the total phenolics, statisticaly significant (f = 2.58, p < 0.05) difference among total anthocyanins content between wines produced from different clones was reported. the smallest amount of total anthocyanins (0.006 %) was measured in the clone 40/8 while the most value (0.015 %) was noticed in the clone 43/4 where the highest level of total phenolic content was also observed. tannins in the wine, which are mainly responsible for its bitterness and astringency are composed of proanthocyanidins localized in the grape skin and seeds. while skin proanthocyanidins are larger polymers contained primarily of epigallocatechin, seed proanthocyanidins are smaller molecules with higher proportion of galloylated subunits.16 the amounts of total proanthocyanidins in the wines obtained from investigated prokupac clones are presented in table ii. the total proanthocyanidin content of red wines averaged at 17.5 mg/100 ml,27 which is significantly lower compared to prokupac wines investigated in this study. the smallest amount was recorded in the clone 40/8 (20.41 mg catechin/100 ml) which also had the lowest amount of total phenolics. the highest amount (80.11 mg catechin/100 ml) was observed in clone 43/5, one of the richest in total phenolics. same as in the case of total phenolics and total anthocyanins, statisticaly significant (f = 142.93, p < 0.001) difference was noticed for total proanthocyanidins among tested wine samples. significant correlation was found between total phenolics and total anthocyanins as well as between total anthocyanins and total proanthocyanidins. hplc analysis the anthocyanins were tentatively identified using lc/ms and since the standard compounds were not available, the concentrations of detected compounds were expressed as aglycones. six main anthocyanin compounds (table ii) were identified and malvidin was the main anthocyanidin present. actually, malvidin 3-o-glucoside (the most abundant anthocyanin ranging from 59.8– –101.7 µg/ml), malvidin 3-o-(6-o-acetyl)hexoside and malvidin 3-o-(6-o-coumaroyl)hexoside were found. previous results showed that those are the predominant anthocyanins for vitis vinifera wines.28,29 moreover, in our investigation, other anthocyanins were also detected: delphinidin 3-o-glucoside, peoni_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 892 živković et al. din 3-o-hexoside and peonidin 3-o-(6-o-acetyl)hexoside. in the tested wine samples the group of anthocyanin monoglycosides represented the highest proportion of anthocyanins varying from 67.90 (clone 43/5) to 83.10 % (clone 42/2), followed by the acetyl derivatives varying from 11.79 (clone 43/7) to 21.46 % (clone 43/5) and p-coumaroyl glucosides at 6.21 (clone 41/6) to 10.64 % (clone 43/5). although in red wines of different grape varieties derivatives of cyanidin were detected,30,31 our prokupac wine samples did not contain that aglycon or its derivatives. variation in the amount of individual phenolics in wines obtained from the same grape variety were noticed by other authors. for example, van leeuw et al.32 showed large variability in the levels of individual phenolic compounds as well as in antioxidant capacity in 38 different wine samples obtained from clones of four main grape varieties. the typical concentration of free anthocyanins in full bodied red wines is approximately 50 mg/100 ml, but in some cases could be higher than 200 mg/100 ml.33 for our tested wine samples the content of total anthocyanins calculated by hplc analysis ranged from 78.30 (clone 43/7) to 143.80 (clone 40/6). hierarchical cluster analysis was also employed in order to investigate the relationship between the samples and produce a tree diagram.34 based on mean values for the investigated chemical parameters (tp, tpr, ta and contents of individual anthocyanin compounds) we designed a dendrogram of phenotypic differences for the examined prokupac clones. as proximity measures of average linkage between groups, we used euclidean distances. two distant clusters were detected (fig. 1). five clones were classified in cluster i (41/1, 41/2, 40/6, 43/5 and 43/4), while cluster ii containing 8 clones can be divided into two subgroups (subgroup i – 40/5, 42/2, 43/6, 41/6, 43/7 and subgroup ii – 40/8, 41/3 and 43/2). the minimum distance was observed between clones 40/5 and 42/2. according to cluster analysis regarding morphological characteristics of the tested clones, these two samples were very distant. in order to determine any relation among the investigated samples and to identify the variables that could discriminate among clones, principal component analysis (pca) was also performed (fig. 2). the obtained results showed that from nine principal components, the first two having eigenvalues greater than one are sufficient to explain 85.02 % of the total variability observed. the first principal component presented 58.19 % of the variation and was mostly due to the content of detected individual anthocyanins, which were positively correlated, while the second principal component accounted for 26.83 % of the variation and was mostly due to the content of total phenolics, total proanthocyanidins and total anthocyanins. the wines grouped on the right side of the diagram (obtained from clones 40/5, 40/8, 41/6, 42/2, 43/2, 43/6 and 43/7) were characterized by lower levels of individual anthocyanins. the vertical separation obs_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ berry morphology influence wine quality? 893 erved along pc2 is caused by lower content of total phenolics, total anthocyanins and total proanthocyanidins in wines obtained from clones 40/8, 41/1, 41/3, 41/6, 43/2 and 41/2. fig. 1. cluster dendrogram of the wines obtained from 13 clones of serbian autochthonous grape variety “prokupac” based on their chemical characteristics. fig. 2. scatterplot of the first two principal components (pc1 vs. pc2) for prokupac wine samples. correlation analysis the grape berries typically contain non-flavonoid compounds in the pulp and flavonoid compounds in the skin, seeds and stem.35 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 894 živković et al. the results of correlation analysis showed that there is a poor response of quality parameters to variation in morphological parameters. total phenolics did not show significant correlation to investigated morphological parameters. this is in accordance with the results obtained by poni et al.36 they showed that the size of a grape berry has no influence on the wine composition. on the other hand, walker et al.37 found a difference in the anthocyanin concentration and wine color only for the smallest size class. anthocyanins are located in the skin cells, in free form inside the vacuoles. they become diluted by sap released from the berry flesh upon crushing.17 in our study, low correlation was found between berry weight and total anthocyanin content in the tested wines, as well as between the skin weight per berry and the total anthocyanin content. such low correlation could be explained by the fact that during maceration, anthocyanins are remained in the berry skin due to mechanical properties of skin cell walls and low extractability.24 romero-cascales et al.38 found a higher concentration of anthocyanins in smaller monastrel berries compared to bigger ones, but these two berry categories were from different localities. similarly, wine made from smaller berries had higher concentration of anthocyanins and tannins compared to the wine obtained from intermediate or large berries. also, gil et al.16 found that total phenolic index, anthocyanins, hydroxycinnamic acids as well as the stilbene concentration increase when berry size decreases. in the same study, the smount of individual anthocyanins obtained by hplc did not show any specific trend for the wines made from different sized berries. on the other hand, for cabernet sauvignon the relationship between berry size and fruit composition parameters (including total anthocyanins) was not direct, calderon-orellana et al.39 matthews and nuzzo40 concluded that viticultural practices used to control yield in a vineyard may be more important than the yield or berry size values per se in determining the quality of the resulting grapes and wines. although the majority of flavan-3-ol monomers in wine come from the seeds, low correlation was observed between the seed weight per berry and proanthocyanidin content in the wine. due to their chemical composition, skin and pulp cell walls may adsorb proanthocyanidins. cell walls are composed of 90% polysaccharides and 10% structural proteins. due to their hydroxyl groups, aromatic and glycosidic oxygen atoms, cell wall polysaccharides are capable of forming hydrogen bonds and establishing hydrophobic interactions with some molecules, proanthocyanidins among them.41 conclusion according to our results, clonal selection makes a significant difference in prokupac wine quality. clones 43/5 and 43/4 were marked as those from which wines with the highest quality (in terms of total phenolics, total anthocyanins and _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ berry morphology influence wine quality? 895 total proanthocyanidins) were obtained. no significant differences in berry morphology were observed. very distinct differences in the quality of prokupac wines were noted that were obviously not related to morphological parameters of the prokupac clones. acknowledgements. the authors acknowledge their gratitude to the ministry of education, science and technological development of serbia for financial support, project numbers 46013, 31063 and 43007. и з в о д утицај морфологије бобица грожђа на квалитет вина произведеног од клонова сорте прокупац jелена живковић1, катарина шавикин1, гордана здунић1, дејан гођевац2, небојша марковић3, зоран пржић3 и небојша менковић1 1институт за проучавање лековитог биља „др јосиф панчић“, тадеуша кошћушка 1, 11000 београд, 2универзитет у београду, институт за хемију, технологију и металургију, његошева 12, 11000 београд и 3универзитет у београду, пољопривредни факултет, немањина 6, 11080, београд квалитет вина зависи од бројних карактеристика грожђа од ког је добијено, између осталих и од морфологије грозда и бобице. циљ ове студије био је да представи варијабилност морфолошких карактеристика различитих клонова аутохтоне сорте прокупац и да испита хемијски састав вина добијеног од њих. садржај укупних фенола био је генерално низак и варирао је од 33,0 до 114,5 mg gae/100 ml. идентификовано је шест главних антоцијана са малвидином као доминантним антоцијанидином. малвидин-3-о -глукозид је најзаступљенији међу њима са садржајем од 59,8 до 101,7 μg/ml. клонови 43/5 и 43/4 су означени као они од којих се добијају најквалитетнија вина. према нашим резултатима клонска селекција доводи до значајних разлика у квалитету вина сорте прокупац. са друге стране, мала је зависност одређиваних секундарних метаболита у вину од морфолошких карактеристика гроздова и бобица (маса грозда, удео шепурине, маса бобице и семенке и удео корe, пулпе и семенке у маси бобице). 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice nucleus-independent chemical shift profiles along the intrinsic distortion path for jahn–teller active molecules. study on the cyclopentadienyl radical and cobaltocene j. serb. chem. soc. 80 (7) 877–888 (2015) udc 547.514.72+546.732:547.521+ jscs–4766 519.677:544.112–185 original scientific paper 877 nucleus-independent chemical shift profiles along the intrinsic distortion path for jahn–teller active molecules. study on the cyclopentadienyl radical and cobaltocene ljubica andjelković1, marko perić1#, matija zlatar1 and maja gruden-pavlović2*# 1center for chemistry, ictm, university of belgrade, njegoševa 12, 11001 belgrade, serbia and 2faculty of chemistry, university of belgrade, studentski trg 12–16, 11001 belgrade, serbia (received 7 november 2014, revised 11 march, accepted 12 march 2015) abstract: the aromatic/anti-aromatic behavior of the cyclopentadienyl anion (cp-), bis(η5-cyclopentadienyl)iron(ii) (fe(cp)2), as well as of the jahn–teller (jt) active cyclopentadienyl radical (cp•) and bis(η5-cyclopentadienyl)cobalt(ii) (co(cp)2) were investigated using density functional theory (dft) calculations of the nuclear independent chemical shifts (nics). according to the nics values, pentagon ring in fe(cp)2 is more aromatic than that of the isolated cp-. the nics parameters were scanned along the intrinsic distortion path (idp) for cp• and co(cp)2 showing anti-aromaticity, which decreased with increasing deviation from the high symmetry d5h to the low symmetry (ls) c2v. changes in the nics values along the idp revealed that co(cp)2 in the ls nuclear arrangement has aromatic character, in contrast to the case of cp•. keywords: vibronic coupling; dft; aromaticity; metallocene. introduction aromaticity, an intuitive concept in chemistry and physics, is considered as a property of systems that are thermodynamically stabilized due to cyclic electron delocalization. the delocalized electronic structure of aromatic compounds yields enhanced planarity, equalized bond lengths, enhanced stability due to the resonance, favoring substitution instead of addition that would be typical for isolated double bonds, and the ability to sustain ring currents when exposed to external magnetic fields. contrary to the concept of aromaticity, the jahn–teller (jt) effect1,2 induces unequalization of bond lengths, leading to the stabilization * corresponding author. e-mail: gmaja@chem.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc141107025a _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 878 andjelković et al. of a system upon distortion. the connection of aromaticity and the jt effect is of utmost importance, since both effects have been very useful in the characterization and interpretation of the structure, stability and reactivity of many molecules. for this reason, computational chemists seek the origin, explanation and understanding of these two phenomena. according to the hückel molecular orbital (mo) theory,3,4 species with 4n + 2 π electrons are aromatic, whereas structures with 4n π electrons are defined as anti-aromatic. structures with 4n+1 π electrons with unpaired electrons in degenerate orbitals are jt active species, and are supposed to show anti-aromaticity.5–8 bearing in mind that the jt theorem states that a molecule with a degenerate ground electronic state distorts along non-totally symmetric vibrational coordinates, and in such a way removes the degeneracy and lowers the energy, the question naturally arises: how does the jt distortion influence the aromaticity/anti-aromaticity? although it is well known that different criteria of aromaticity may lead to different overall conclusions,9 nucleus-independent chemical shifts (nics) provide a widely accepted quantitative measure of aromaticity/anti-aromaticity.10,11 furthermore, a scan of the nics parameters along the intrinsic distortion path (idp)12–14 was found to be the method of choice in the analysis of the aromatic behavior of jt active species,7,8,15 since the distortion path provides direct insight into the microscopic origin, mechanism and consequences of distortion.13,14 а detailed density functional theory (dft) computational analysis was performed to investigate the aromaticity of the cyclopentadienyl anion (cp–), bis(η5 -cyclopentadienyl)iron(ii) (fe(cp)2), the cyclopentadienyl radical (cp •) and bis(η5-cyclopentadienyl)cobalt(ii) (co(cp)2). in spite of their relatively simple composition, the aromaticity of the jt active cp• and co(cp)2 have not been fully rationalized so far. therefore, the aim of this work was also to understand the influence of the jt effect on the aromatic behavior of these species. the nics were determined for the reference non-jt active species, cp– and fe(cp)2, and molecules prone to the jt effect, cp• and co(cp)2. in order to preserve chemically important features, an analysis of the full nics profile of cp• and co(cp)2 along the idp was performed. methodology all the dft calculations were realized using the amsterdam density functional program package, adf2013.01.16-18 geometry optimization of all the investigated molecules was performed using the local density approximation (lda) characterized by the vosko–willk– –nusair (vwn) parametrization,19 as well as using general gradient approximations (gga), such as bp86,20,21 pw91,22 opbe,23 and s12g,24 and hybrids b3lyp25,26 and s12h.24 all electron triple-zeta slater-type orbitals (sto) plus one polarization function (tzp) basis set were used for all atoms. all calculations were spin-unrestricted. analytical harmonic frequencies27,28 were calculated in order to ascertain that the low symmetry (ls) structures correspond to the stationary points on the potential energy surfaces. calculations of the nics values were performed at the b3lyp/6-311+g* level of theory using the gaussian 09w _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ jahn-teller effect and aromaticity 879 program package using lda optimized geometries.21,29-31 the nics parameters were calculated for ghost atoms located at the center of cpand cp•. in order to obtain the full profile of aromatic/anti-aromatic behavior, calculations of the nics parameters were performed from 0 å to 5 å, in steps of 0.5 å. in order to avoid the influence of the magnetic field of the central metal ion in metallocenes, the first nics value was calculated 1 å above the metal ion along the z-axis. for jt active species in a high symmetry (hs) nuclear arrangement, the nics values were calculated imposing hs (d5h) nuclear arrangement and ls (c2v) of electron density. the nics parameters for jt active species, cp• and co(cp)2, were scanned along the idp. intrinsic distortion path – idp the idp method12-14 is based on the fact that all the information about the vibronic coupling at the hs nuclear arrangement is also contained in the distorted ls minimum energy structure. hence, the distortion is given as a superposition of all totally symmetric normal modes in the ls point group, linking the hs configuration with the ls structure. every point on the potential energy surface can be represented by a 3n dimensional vector, n being the number of atoms, xr  , using mass-weighted generalized coordinates relative to the origin. the geometry of the ls energy minimum obtained by dft calculations was chosen to be the origin of the configuration space, ls 0r =  . within the harmonic approximation, it is possible to express xr  as a linear combination of na1 totally symmetric normal coordinates in the ls: a1 1 n x xk k k r qω = =   (1) where ωxk are weighting factors that represent the contribution of the displacements along the different totally symmetric normal coordinates to xr  ; kq  are mass-weighted totally symmetric normal coordinates, which are the eigenvectors of the hessian, obtained from the dft frequency calculations in the ls minimum energy conformation. the corresponding eigenvalues are λk. within this model, the energy of any nuclear configuration xr  , ex, relative to the ls energy minimum, is expressed as the sum of the energy contributions of all the ls totally symmetric normal modes: a1 a1 2 2 1 1 1 2 n n x k kxk k k k e e qω λ = = = =   (2) the force at any given point ( xr  ), xkf  is defined as a derivate of the energy over cartesian coordinates and in the hs point, it indicates the main driving force for the jt distortion. the total force is represented as a vector sum of the individual forces: a1 a1 tot 1/ 2 1 1 1 2 n n x xk k k xk k k f m q fω λ = = = =    (3) where m is a diagonal 3n×3n matrix with atomic masses in triplicate as elements (m1, m1, m1, m2,…, mn), and enables the calculation of the idp exactly from the hs to the ls point. the above details about idp can be found elsewhere.12-14 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 880 andjelković et al. results and discussion the planar cp– in its singlet state with d5h symmetry was optimized using several different levels of theory. all chosen exchange correlation (xc) functi onals reproduced experimental geometrical parameters with sufficient accuracy,32 table i. the fe(cp)2 molecule has two possible conformations, eclipsed d5h and staggered d5d. according to previous studies, the d5h conformation is the global minimum on the potential energy surface.33–38 the calculated bond lengths, table i, are in excellent agreement with the experimental data,39 and with earlier theoretical investigations.40 since all the functionals reproduced geometrical parameters with good accuracy, the nics were computed at the b3lyp/ /6-311+g* level, using geometries obtained with the simplest lda functional. calculated nics values for cp– and fe(cp)2 are given in table ii. previous sophisticated computational studies provide a rather satisfactory insight into the nature of the aromaticity of cp– and revealed that cp– is aromatic, which is in accordance with the herein presented results.41–46 it is important to emphasize that the nics parameters for cp– were computed at the center of the pentagon and at various distances from the center of the ring. in the case of fe(cp)2, the starting point for the nics calculation was 1 å above the central metal ion following the z-axis. the nics value calculated at 1.6 å represent the nics in the center of the pentagon ring of the cyclopentadienyl ligand. according to the results, both molecules show aromatic character, and fe(cp)2 is more aromatic, table i. selected bond lengths (å) for the stationary points of the investigated molecules, calculated with different xc functionals molecule bond lda bp86 pw91 opbe s12g b3lyp s12h exp. cp– (d5h, 1a1′) c–c 1.406 1.420 1.417 1.413 1.414 1.410 1.404 1.413 32 fe(cp)2 (d5h, 1a1′) fe–c 2.004 2.055 2.049 2.006 2.033 2.082 2.048 2.03 39 c–c 1.422 1.434 1.432 1.429 1.429 1.422 1.417 1.4339 cp· (c2v, 2b1) c–c 1.364 1.424 1.374 1.439 1.372 1.436 1.370 1.432 1.371 1.432 1.365 1.432 1.360 1.426 – cp· (c2v, 2a2) c–c 1.455 1.390 1.471 1.402 1.468 1.400 1.462 1.397 1.463 1.397 1.465 1.394 1.457 1.389 – co(cp)2 (c2v, 2b1) co–c 2.029 2.070 2.098 2.091 2.134 2.155 2.084 2.128 2.149 2.038 2.080 2.108 2.070 2.112 2.135 2.129 2.175 2.185 2.092 2.137 2.151 2.11950 c–c 1.434 1.415 1.402 1.447 1.426 1.412 1.445 1.424 1.410 1.441 1.421 1.408 1.441 1.421 1.408 1.435 1.415 1.400 1.429 1.409 1.396 1.42950 co(cp)2 (c2v, 2a2) co–c 2.090 2.047 2.022 2.150 2.111 2.083 2.143 2.104 2.076 2.101 2.056 2.031 2.129 2.089 2.062 2.184 2.153 2.118 2.149 2.115 2.083 2.11950 c–c 1.406 1.426 1.438 1.416 1.438 1.450 1.414 1.436 1.448 1.412 1.432 1.444 1.412 1.432 1.444 1.404 1.427 1.439 1.400 1.421 1.432 1.42950 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ jahn-teller effect and aromaticity 881 table ii.41–49 the isolated cp– ring has a nics value of –12.52 ppm in the center, table ii. when two cp– rings are placed at the distance which they have in the fe(cp)2 molecule (3.2 å), the nics parameter calculated at the center of one 5-membered ring was –12.13 ppm. hence, the difference between the nics parameters for the isolated cp– and for the cp– fragment in fe(cp)2 is caused by the presence of the central metal ion. table ii. calculated nics values (ppm) for cpand fe(cp)2 using lda optimized geometries at various distances (å) from the center of the molecules (for fe(cp)2, the nics calculated at 1.6 å corresponds to the nics value at the center of the cpligand) distance, å nics nicszz cp 0.0 –12.52 –15.95 1.0 –9.44 –33.70 2.0 –3.97 –17.86 3.0 –1.63 –7.87 4.0 –0.77 –3.87 5.0 –0.40 –2.13 fe(cp)2 1.0 –103.47 –65.35 1.6 –42.14 –29.40 2.0 –31.15 –29.73 3.0 –9.46 –29.07 4.0 –2.44 –13.37 5.0 –0.95 –6.38 it is worth noting that cp• and co(cp)2 have a hole and an unpaired electron, respectively, in a doubly degenerate highest occupied molecular orbital. since it was previously shown that the eclipsed conformation is more stable and the jt effect does not depend on the rotation of the rings,51 the discussion will be limited only to co(cp)2 in the eclipsed conformation. the ground electronic state of the investigated species in d5h symmetry is 2e1″, which couples with the doubly degenerate vibration, e2′. according to group theory considerations, the descent in symmetry goes from the d5h to the c2v point group, and thus, the state 2e1″ splits into 2a2 and 2b1. the average bond distances for the distorted ls structures of cp• calculated at different levels of theory are consistent, table i. the calculated bond lengths for co(cp)2 are in accordance with experiments,50 regardless of the choice of selected xc functional, table i. recently, the jt distortion in these molecules was analyzed in detail using a multideterminental dft approach and the idp method.12,14,51–53 the calculated jt parameters of cp• and co(cp)2 using a multideterminental dft approach are given in table iii. the results for cp• obtained by miller et al., who used dispersed fluorescence spectroscopy, are considered to be the benchmark (ejt = 1237 cm–1).54 the pre _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 882 andjelković et al. sent calculations at the lda level gave a value of 1244 cm–1, which is in great accordance with the experimentally obtained one, although all other xc functionals also give satisfactory results, table iii. the values of the jt stabilization energies in co(cp)2, table iii, are in agreement with the value of 1050 cm–1 estimated from its solid state epr spectra.55 for both investigated jt active molecules, the warping barrier is close to zero, independent of the level of theory (table iii). the different ground states obtained by different xc functionals (table iii) are due to the very small warping barriers, which are within the range of the accuracy of the calculation. furthermore, idp analysis gave a deeper insight into the vibronic coupling in these jt active molecules.14,53 in both molecules, it is possible to distinguish two distinct regions on the potential energy profile. in the first region, the energy changes faster, and most of the ejt is achieved after 40 % of the path. in the second region, the change of the energy is small, the adiabatic potential energy surface is flat and the molecule just relaxes towards the global minimum. three vibrations are most important for the distortion in cp: c–c stretch, c–c–c bend, and c–c–h bend. these three modes were experimentally found to be the most significant.54 in the case of co(cp)2, the out-of-plane ring deformation and c–h wagging (the out-of-plane c–h bending), are the most important for the jt distortion. although the jt effect in cp• table iii. results of the dft calculations performed to analyze the jt effect in cp•·and co(cp)2; the energies are given in ev; the jt parameters ejt and δ are given in cm -1 and rjt in (amu)1/2å molecule symmetry lda bp86 pw91 opbe s12g b3lyp s12h cp• d5h, 2e1″ –64.674 –59.989 –61.008 –60.966 –61.624 –67.948 –72.863 d5h, 2a2 –64.653 –60.031 –61.042 –61.018 –61.658 –68.369 –73.402 d5h, 2b1 –64.653 –60.031 –61.042 –61.018 –61.658 –68.369 –73.402 c2v, 2a2 –64.808 –60.192 –61.203 –61.179 –61.817 –68.578 –73.596 c2v, 2b1 –64.808 –60.192 –61.205 –61.179 –61.817 –68.578 –73.596 ejt, 2a2 1244.5 1301.8 1294.5 1300.2 1281.6 1685.7 1567.9 ejt, 2b1 1244.5 1301.0 1312.3 1301.0 1284.0 1688.1 1568.8 δ 0.0 –0.8 –17.7 –1.6 –2.41 0.0 0.8 rjt, 2a2 0.25 0.26 0.26 0.26 0.26 0.27 0.27 rjt, 2b1 0.25 0.26 0.26 0.26 0.26 0.27 0.27 co(cp)2 d5h, 2e1″ –142.290 –130.675 –133.049 –133.385 –134.525 –149.601 –161.584 d5h, 2a2 –142.261 –130.690 –133.061 –133.381 –134.528 –150.140 –162.247 d5h, 2b1 –142.261 –130.690 –133.061 –133.381 –134.528 –150.140 –162.246 c2v, 2a2 –142.362 –130.785 –133.156 –133.485 –134.626 –150.230 –162.345 c2v, 2b1 –142.362 –130.785 –133.156 –133.485 –134.626 –150.231 –162.344 ejt, 2a2 813.8 762.2 761.4 838.0 783.9 727.5 791.2 ejt, 2b1 813.8 761.4 759.8 839.6 785.6 729.1 785.6 δ 0.0 0.0 1.6 –1.6 0.0 –2.4 13.7 rjt, 2a2 0.35 0.34 0.34 0.35 0.34 0.34 0.34 rjt, 2b1 0.35 0.34 0.34 0.35 0.34 0.34 0.34 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ jahn-teller effect and aromaticity 883 and co(cp)2 has often been studied,54–56 the influence of the vibronic coupling on their aromatic character has not been investigated. the question is whether the distortion has a significant impact on the change of aromaticity. in cp• and co(cp)2, the degeneracy of the 2e1″ state is broken by the jt distortion that stabilizes the system. therefore, the nics parameters were computed at the lda optimized geometries for both the hs and ls points on the potential energy surfaces. observing the nics values at the hs point of cp•, a high anti-aromatic character is noticeable, table iv. the calculated parameters for the global minimum of cp• show that it still possesses anti-aromatic character,5 but much weaker than in the hs point, table iv. moving from the center of the pentagon ring along the z-axis, the nics indices decrease. in the case of co(cp)2, it is evident that molecule at the hs point has high anti-aromatic character, table v. in the global minimum structure, co(cp)2 shows σ aromaticity and π anti-aromaticity according to the nics and nicszz values, tables iv and v. table iv. calculated nics values (ppm) for cp• using lda optimized geometries at hs and ls geometries at various distances (å) from the center of the ring; the nics values for both ls structures are the same electronic state distance, å nics nicszz cp• (d5h) 2b1/ 2a2 0.0 86.88 273.95 1.0 72.10 217.30 2.0 21.36 61.38 3.0 6.65 18.12 4.0 2.69 7.06 5.0 1.34 3.40 cp• (c2v) 2b1/ 2a2 0.0 20.78 77.09 1.0 14.79 45.84 2.0 3.58 8.00 3.0 0.91 0.87 4.0 0.31 –0.12 5.0 0.13 –0.22 since dependence of the nics parameters on the distortion, /x jtr r   , and distances (å) from the center of the molecules gives a more detailed picture, the nics parameters were monitored along the idp path for cp• and co(cp)2, fig. 1 and 2, respectively. the nics parameters were scanned only for the 2b1 state, since those computed for the 2a2 state were almost the same. due to the nontotally symmetric electron density, both molecules in d5h possess strong antiaromatic character. near the point of electron degeneracy, i.e., near the hs nuclear arrangement, the homo–lumo gap is substantially small, thus the nics parameters have large positive values in the first region of the idp (figs. 1 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 884 andjelković et al. table v. calculated nics values (ppm) for co(cp)2 using lda optimized geometries at hs and ls geometries at various distances (å) from central metal ion. the nics value calculated at 1.7 å corresponds to the nics parameter in the center of the pentagon in co(cp)2; the nics values for both ls structures are the same electronic state distance, å nics nicszz co(cp)2 (d5h) 2b1/ 2a2 1.0 302.49 1135.98 1.7 67.59 281.61 2.0 42.20 182.76 3.0 24.86 73.78 4.0 9.80 24.48 5.0 4.04 9.32 co(cp)2 (c2v) 2b1/ 2a2 1.0 –3.91 209.45 1.7 –10.86 42.41 2.0 –11.11 21.41 3.0 –1.64 –5.91 4.0 –0.10 –5.08 5.0 –0.04 –2.92 fig. 1. schematic plot of the nics values along the idp for cp• (from d5h to c2v), 2b1/ 2a2 electronic states. and 2). going toward the global minimum point on the potential energy surface, the nics values decrease and hence, the initially strong anti-aromatic character lowers, and finally, in the case of co(cp)2, the nics indices become negative, figs. 1 and 2. it is worth noting that the negative nics values in co(cp)2 occur after 40 % of the idp, where most of the jt stabilization is attained. the driving force responsible for the removal of orbital degeneracy leads to an enlargement of the homo–lumo gap. considering cp•, for both the hs and ls nuclear arrange_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ jahn-teller effect and aromaticity 885 ment, the calculations revealed that the nics decrease gradually along the z-axis, fig. 1. moving apart along the z-axis in the hs configuration of co(cp)2, an abrupt decrease in the nics parameters was observed until a distance of 1.7 å (center of pentagon ring), fig. 2. going even further, the nics parameters show a smooth decreasing trend, as expected. fig. 2. schematic plot of the nics values along the idp for co(cp)2 (from d5h to c2v), 2b1/ 2a2 electronic states. conclusions aromaticity is one of the oldest and most fundamental concepts in chemistry. the nature of aromatic compounds is very attractive, thus these species are among the most desirable synthetic targets. since aromaticity is still awaiting comprehensive investigation, a detailed analysis of the nics parameters, as one of the most widely used and efficient magnetic criteria, was performed in cp–, fe(cp)2, cp • and co(cp)2 by the means of dft. the calculations revealed that cp– and fe(cp)2 are highly aromatic molecules. according to the nics, the cp– ring in fe(cp)2 is far more aromatic than the isolated cp–, showing the impact of the fe2+. in other words, these results would be highly attractive in applied sciences for the design of new advanced materials with desired properties, and the understanding of electronic structure, chemical bonding and properties in a moiety of aromatic species. a thorough study of the influence of the jt effect on the aromaticity is presently of the utmost importance due to the increasing development of material chemistry, and substantial differences in aromatic/anti-aromatic behavior of jt active molecules. thus, finding a method of choice to determine both chemically relevant phenomena is essential since they lead to the different reaction pathways _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 886 andjelković et al. and the connection of aromaticity and vibronic coupling demands tremendous caution. for this purpose, the monitoring of the nics along the idp was performed. similarly to the previous studies,7,8,15 the anti-aromaticity decreases with increasing deviation from the high symmetry d5h structures to the low symmetry c2v global minimum structures, confirming that the jt distortion represents a mechanism for reducing anti-aromatic character. in the ls structure of co(cp)2, the nics become even negative, revealing aromatic character, in contrast to cp•. acknowledgement. this work was financially supported by the ministry of education, science and technological development of the republic of serbia (grant no. 172035). и з в о д профил хемијских померања независних од језгра дуж својсtвеног пута дисторзије за молекуле подложне јан–телеровој дисторзији. проучавање циклопентадиенил радикала и кобалтоцена. љубица анђелковић1, марко перић1, матија златар1 и маја груден-павловић2 1центар за хемију, ихтм, универзитет у београду, његошева 12, 11001 београд и 2хемијски факултет, универзитет у београду, студентски трг 16, 11001 београд ароматичност/антиароматичност циклопентадиенил анјона (cp ), бис(η5-циклопентадиенил)гвожђа(ii) (fe(cp)2), као и, циклопентадиенил радикала (cp •) и бис(η5 -циклопентадиенил)кобалта(ii) (co(cp)2), молекула подложних јан–телеровој дисторзији, испитивана је коришћењем теорије функционала густине (dft) за израчунавање хемијских померања независних од језгра (nics). на основу израчунатих хемијских померања, петочлани прстен у fe(cp)2 је ароматичнији него изоловани cp . nics вредности, праћене дуж својственог пута дисторзије за cp• и co(cp)2, указују на антиароматичност испитиваних врста, која се смањује са снижењем симетрије од d5h до c2v. промене у nics параметрима дуж пута дисторзије показују да co(cp)2 у ниској симетрији поседује ароматични карактер, за разлику од cp•. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice jscs, vol. 80 (2015) :: author index j. serb. chem. soc. 80 (12) 1593–1596 (2015) 1593 author index abbas, m. i., 1251 aćimović, d. d., 1515 agbaba, d., 1035 aghadadashi-abhari, l., 459 akman, s., 97 alagarsamy, v., 1471 alavi, m. s., 355 aleksić, m. m., 1035 ali, s., 755 almásy, l., 367 amani, h., 1139 anačkov, g., 475 anđelković, d. t., 983 anđelković, h. d., 983 andjelković, lj., 877 andrić, v., 805 antanasijević, z. d., 421 anu, n., 253 aryan, r., 453 ashok, d., 305, 1361 auroux, a., 1297 ayla, s. s., 731 babić, n., 613 badiei, a., 1265 bahar, h., 731 bai, m., 343 bajat, b. j., 73 balan, m., 673 barvinschi, p., 485 bastami, z., 509 beitollahi, h., 789 belbachir, m., 917 belošević, s., 549 benkhaled, m., 137 beyzaei, h., 453 bicen, m., 1061 bing, s., 499 blanuša, l. j., 197 bogoeva-gaceva, g., 223 bojić-trbojević, ž., 159 borozan, z. s., 1273 božinović, n., 839 božović, d., 143 brahman, d., 35 brahmbhatt, i. d., 739 brčeski, i., 1087 buchberger, w., 589 bugarčić, z., 595 bugarski, b., 1541 bui, h. m., 115 bukhari, h. i., 755 bursić, v., 475 carlescu, i., 673 chang, j., 343 chen, m., 1051 chen, p., 889 chen, r., 171 chen, x., 1051 chovatiya, l. y., 739 ciganović, j., 1505 ciubotariu, c., 659 ciubotariu, d., 659 constantin, d. v., 1461 creanga, d., 367 cui, h., 853 cvetković, o., 575 cvetković, v., 391 cvijović-alagić, i., 1541 cvjetićanin, n., 685 čudina, o., 1481 ćujić, d., 159 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1594 journal of the serbian chemical society dakić, d., 377 damjanović, lj., 805 dehestani, m., 997 dekanski, a., 959 dekić, b., 315 dekić, v., 315 deng, w. z., 171 dikshit, k. a., 465 dimić, i., 1541 dimitrov, n. a., 179 dimkić, m., 947 divarova, v. v., 179 djahaniani, h., 459 djordjević, b., 21 djordjević, d. b., 1073, 1423 djordjević, i., 329 djošić, s. m., 237 djurković-djaković, o., 1339 dobričić, v., 1481 dodevska, m., 21 dojnov, b., 1279, 1375 draghici, c., 1461 dragoş, d., 659 dramićanin, d. m., 63 du, s. s., 171 duba, v. e., 1149 đoković, n., 575 đonlagić, j., 529 đorđević, n., 529 eich-greatorex, s., 1203 elaasser, m. m., 1251 elneairy, a. a. m., 1251 erić, a., 377 farsa, o., 605 felinger, a., 407 filimon, n. m., 1127 francuski, dj., 1481 francuski, m. b., 1481 fuliaș, a., 485 furtula, b., 1009 ganesh, a., 305, 1361 ganjali, r. m., 187 gaur, p., 639 ghulam, ali k., 755 giester, g., 867 giri, r. r., 739 goić-barišić, s. i., 819 gokula krishnan, k., 1101 golkarian, a. r., 1449 gomha, m. s., 1251 gong, a., 853 grba, n., 827 grbović, lj., 749 grgur, n. b., 903 grubišić, s., 329 gruden, m., 1399 gruden-pavlović, m., 877 grujić, m., 1279, 1375 gu, s., 853 gungor, a., 1061 gutman, i., 1009 haba, h., 137 hacihasanoglu cakmak, n., 627 hadj-ali, t., 1435 hadžić, a. p., 1273 haghdadi, m., 1139 hamidi, s., 695 han, q., 1289 harakat, d., 137 heidari, b., 779 hejazi, z. s., 971 hercigonja, r., 1297 holclajtner-antunović, d. i., 63 hou, z., 889 hrenović, j. j., 819 hui, n., 87 hussain, s., 755 huzum, c.-c., 673 ibis, c., 731 ibrić, s., 209 inanlu, h., 779 ionescu, s., 517 isaković, a., 143 isvoran, a., 1127 ivaniš, r. g., 1073, 1423 ivanović, r. e., 107 izrael živković, t. l., 1113 jabbarzadeh, m., 1449 jaćević, v., 1481 jaćimović, k. ž., 867 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ vol. 80: author index 1595 janaćković, t. dj., 1193 janevski, a., 223 janković, n., 595 janković-častvan, m. i., 1193 jiao, l., 499 jing, y., 53 jokić, m. b., 1113, 1193 jouyban, a., 695 jovančićević, b., 1087, 827 jovanović, u., 1505 kadoya, t., 159 kapetanović, v., 1035 karadžić, m. i., 1113, 279 karatas, s., 1061 karplyakova, s. n., 1149 karunakaran, c., 1411 karuthapandian, s., 1411 kašanin-grubin, m., 1087 keshtkar, n., 789 kevrešan, s., 749 khorshidi, a., 779 kijevčanin, lj. m., 933, 1073, 1423 kilár, f., 407 kiprovski, b., 475 kitte, a. s., 1061 kizilkaya, c., 1061 knežević-ušaj, s., 1339 kocić, m.g., 983 kolundžić, n., 159 komatina, m., 377 kombiah, p., 465 kong, d.-y., 1289 kőnig-péter, a., 407 kononova, n. o., 1149 korać, j., 613 korolija, n. j., 1567 kosović, m., 867 kostić rajačić, s., 839 krishnamoorthy, g., 1471 krkobabić, a., 705 krogstad, t., 1203 krstić, m., 209 kurunczi, l., 485 kusigerski, b. v., 197 lad, h. b., 739 lakshmi, v. b., 305 lamouri, s., 917, 1435 laušević, z. p., 1515 lavaud, c., 137 lazarević, s. s., 1193, 717 lazić, v., 705 ledeţi, i., 485 leito, i., 589 lekova, d. v., 179 li, d., 1391 li, h., 499 li, p., 563 li, s., 1553 li, s.-f., 563 li, s.-t., 1367 li, x., 1009 li, y.-w., 1367 liang, a., 87 lisa, g., 673 liu, h. c., 1 liu, l. z., 171 liu, l., 1051 liu, s.-l., 343 lović, d. j., 1217 maamria, l., 137 mabrouk, a. k. b., 1251 mahmoud, y., 1435 majkić-dursun, b., 947 maksimović, i., 749 maksimović, v. m., 197 mandić, m. lj., 1567 marčeta kaninski, p. m., 1515 marić stojanović, m., 805 marjanović, o., 805 marković, i., 143 marković, m. z., 63 marković, s., 595 martinović, m., 143 matić, z. i., 1339 maxa, j., 605 mehrotra, r., 639 mendoza-huizar, h. l., 767 mijin, ž. d., 903 milanovic, d. v., 1321 milčić, m., 329 milenković, k. j., 819 milićević, a., 549 milojković, s. d., 983 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1596 journal of the serbian chemical society milosavljević, s. m., 63 milošević, m., 705 milošević, s., 685 milovanović, a. j., 717 milovanović, j., 1203 mioč, b. u., 805 mišković-stanković, b. v., 237 mitrić, n. m., 237, 685, 1529 mitrović, d., 575 mitrović, p., 143 miulović, m. s., 1515 mladenović, m., 377 moghadas, h., 453 mohajeri avval, z., 187 mohtat, b., 459 momčilović, m., 1505 morcali, h. m., 97 morić, i., 391 mracec, m., 659 myhrvold, m. e., 717 nab, n., 1139 nadejde, c., 367 narendar, b., 1471 nedić, o., 959 nemoda, s., 377 neubauer, f., 827 niketić, s., 329 nikolić, d. n., 107, 197 nikolić, g., 143 nikolić, k., 1035 nikolić, m. v., 1515 nikolić, s. m., 529 nikolić, v., 1177 norouzi, p., 187 nouri, f., 1265 novaković, b. s., 867 novaković, b. t., 1529 novaković, i., 839 obradović, n., 1541 olana, n. b., 1061 olariu, t., 659 oliveira, g. b., 651 opsenica, m. d, 1339 opsenica, m. i., 839 ostafe, v., 1127 pan, y., 1553 panić, v., 1177 pantović, s., 143 paprika, m., 377 parveen, b., 755 pavić, a., 391 pavlović, k., 749 perčević, b., 1279 perić, m., 877 perić-grujić, a. a., 421 perng, s. y., 115 pernyeszi, t., 407 perunović, t., 1087 petković, a., 947 petrović, d. r., 1193 petrović, j., 1541 petrović, m. m., 73 petrović, m., 475 petrović, p. s., 1529 piroozi, f., 1311 pocajt, v. v., 421 podlipná, j., 605 polović, n., 613 pomjakushina, e., 367 popović, đ. k., 1217 popović, a., 475 popović, g. i., 1177 popsavin, m. m., 1273 pourbasheer, e., 187 prekodravac, r. j., 63 prvulović, d., 475 putić, s., 1541 putnik-delić, m., 749 qin, j.-k., 127 qomi, m., 1311 rac, v., 1297 racheva, v. p., 179 radetić, m., 705 radivojević, j., 1339 radmilović, r. v., 107 radoičić, m., 705 radović, a., 867 radović, r. i., 933, 1073, 1423 radulović, l., 143 radulović, s. n., 315 radu-popescu, a. m., 1461 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ vol. 80: author index 1597 rahimifard, m., 1265 rahul, a., 253 rajić, n., 1203 rajić, s., 1567 rajić, z. n., 717, 819 rakić, v., 1203, 1297 rakin, m., 1541 rangabhashiyam, s., 253 ranković, d., 1505 rašković, b., 613 rathi, m. j., 465 ravi, s., 305, 1361 ražić, s., 209 rezaee, r., 1311 rikalović, g. m., 279 ristić, đ. m., 421 rodić, v. m., 1481 rogan, j., 529 rosta, l., 367 rožić, s. lj., 1529 safidine, z., 917 saha, s., 35 sahayaraj, k., 465 saramet, g., 1461 savić, b. a., 1113 savović, j., 1505 sayil, c., 45 sbârcea, l., 485 scutaru, d., 673 sedláková, š., 605 selvaraju, n., 253 shahid, m., 755 shahzadi, s., 755 shirini, f., 971 shntaif, h. a., 731 shojaei, f. a., 971 shukla, n. s., 639 simić, v., 1087 simion, a., 673 sinha, b., 35 sivakumar, r., 1101 socea, b., 1461 socea, i. l., 1461 solomon, v.r., 1339 soltanpour, s., 509 song, h., 889 soreta, r. t., 1061 spasojević, đ., 209 spasojević, p., 1177 stamenković, j. l., 421 stanković, s., 391, 73 stensrød, e. r., 717 stepanović, s., 1399 stevanović, i. s., 73 stöcker, m., 717 stoiljković, m., 705, 1505 stojanović, k., 1087, 575, 827 stojanović, n., 315 stojanović, p., 315 stojanović-radić, z., 315 stojković, i., 685 stojnova, t. k., 179 subramaniam, p., 1019 sulthana, m. t., 1471 sun, w., 87 swart, m., 1399 šajnović, a., 1087, 827 šaponjić, z., 705 šerbanović, p. s., 933, 1073, 1423 šešlija, s., 1177 šobajić, s., 21 šolaja, a. b., 839, 1339 španić, d., 575 štajner, t., 1339 šućur, j., 475 tabatabaeian, k., 971 taher, a. m., 789 tan, x., 53 tang, h., 127 tasić, s. g., 1515 tasić, ž. a., 1073, 1423 tavman, a., 45 thamil selvi, n., 1019 thanikachalam, v., 1101 todorović-marković, m. b., 63 tomanović, i., 549 tomašević, b., 435, 1321 tomić, d. m., 819 tomić, d. v., 903 tomić, v. m., 73 tong, j.-b., 343 tošić, d. d., 63 tošić, m., 1567 trivić, d., 435, 1321 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1598 journal of the serbian chemical society troskot-čorbić, t., 575 trtica, m., 1505 tschentscher, r., 717 tucaković, d., 549 udrescu, l., 485 ursache-oprisan, m., 367 vasiljević, b., 391, 749 vasiljević, d., 209 veličković, s., 1177 vićovac, lj., 159 vijaya lakshmi, b., 1361 vinayagamoorthy, p., 1411 vladimirov, s., 1481 vladoiu, l. d, 1127 vlaia, v., 659 vlase, t., 485 voia, o. s., 1127 voicescu, m., 517 vojnović, s., 391 vrvić, m. m., 279 vujčić, z., 1279, 140 vujković, m., 801 vuković, m. z., 1529 vuksanović, m. j., 933 wang, j., 9, 853 wang, m., 9 wang, r., 265 wang, x., 499 wang, x., 9 wang, x.-y., 1289 wang, y., 171 wang, z., 1 worsfold, p., 589 wu, f., 9 wu, w., 9 wu, x., 853 wu, y., 171 wu, z.-x., 1391 xue, c., 87 xue, z., 499 yan, h. d., 1 yanardag, r., 627 yang, s.-c., 1553 yang, k., 171 yang, n., 265 yang, s.-c., 563 yao, s., 889 you, x. z., 171 yu, m., 1289 yucel, o., 97 zaynali, y., 355 zeghioud, h., 917, 1435 zeidabadinejad, l., 997 zeytuncu, b., 97 zhang, j. w., 171 zhang, j.-h., 563, 1553 zhao, h.-t., 127 zhao, s.-l., 563 zhao, z.-y., 1289 zhong, g.-q., 1391 zhong, s.-m., 127 zhu, w., 889 ziarani, m. g., 1265 zlatar, m., 877 živković, s. lj., 1113 životić, d., 575 župunski, a., 749 subject index of vol. 80 and list of referees in 2015 are given in the electronic form at the internet address of the journal of the serbian chemical society: http://www.shd.org.rs/jscs/vol80/no12.html end of volume 80. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice the effect of iron oxidation in the groundwater of the alluvial aquifer of the velika morava river, serbia, on the clogging of water supply wells j. serb. chem. soc. 80 (7) 947–957 (2015) udc 546.722+66.094.3+544.4:628.112 jscs–4771 original scientific paper 947 the effect of iron oxidation in the groundwater of the alluvial aquifer of the velika morava river, serbia, on the clogging of water supply wells brankica majkić-dursun*, anđelka petković and milan dimkić jaroslav černi institute for the development of water resources, jaroslava černog 80, belgrade, serbia (received 4 february, revised 27 june, accepted 3 september 2014) abstract: the oxidation of iron(ii) dissolved in groundwater and subsequent precipitation of the oxidation products on the screens and discharge pipes of water wells that tap shallow alluvial aquifers leads to the formation of well encrustations. the main goal of the presented research was to determine the reasons for the rapid clogging of water supply wells. in the particular case of the alluvial aquifer of the velika morava river, serbia, the encrustations include mostly iron-(hydr)oxides (62.6 to 76.2 wt. %). groundwater over-exploitation leads to the mixing of different geochemical zones and the formation of a redox front. during the two-year survey, the concentrations of the dissolved oxygen in the groundwater varied over a wide range from 0.1 to 7.1 mg l-1 as the result of unsuitable exploitation regime. the on site measured groundwater temperature, concentrations of dissolved oxygen and ph values, and the laboratory analysis of dissolved iron concentrations showed that iron precipitation was favorable under groundwater over-exploitation conditions. keywords: iron encrustations; groundwater over-exploitations; trnovče; redox front formation. introduction the rapid formation of encrustations inside the wells of the water supply source trnovče in serbia necessitated detailed research of the clogging process. decreasing groundwater levels and well discharges indicated over-exploitation conditions at this groundwater source. over-exploitation could be defined as the situation in which the average rate of aquifer abstraction is greater than, or close to, the average recharge rate. over-exploitation implies intensive use of groundwater, resulting in declining groundwater levels in the wells and over an extended area of the source, changes in the hydraulic gradients and aquifer recharge regime, reduced discharges of natural springs and variations in the oxic * corresponding author. e-mail: brankica.majkic@jcerni.co.rs doi: 10.2298/jsc140204089m _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 948 majkić-dursun, petković and dimkić state and groundwater quality, which usually deteriorates due to groundwater inflow from the upland. case studies of rapid well clogging under over-exploitation conditions were presented in the usa,1 germany2 and france.3 an unsuitable pumping scheme produces a general decline in the groundwater levels and disturbs the geochemical conditions of the initial system.3 under such conditions, anoxic groundwater become oxidized, while the groundwater composition and microbial ecology are changed.3,4 based on the chemical analyses of the groundwater samples, a study of data derived from microbiological analyses and assessments of the data on the static and dynamic groundwater levels inside the wells, the trnovče source was characterized as an alluvial setting where oxic and anoxic ground waters blend.5,6 high concentrations of dissolved oxygen in the upper, water unsaturated part, of the aquifer and the inflow of anoxic groundwater featuring elevated iron concentrations from deeper, saturated parts of the aquifer have created a redox front within the well screens (fig. 1), leading to a thorough precipitation of iron-(hydr)oxides.6 the conditions that lead to the formation of ferric deposits in water wells were analyzed in this study. hydrochemical data were collected during a two -year survey (2010–2011), while the discharge data were analyzed for the last ten years. fig. 1. 3d representation of groundwater levels in the wells and piezometers at the water supply source of trnovče on 1 september 2011,5 modified. legend: 1 – zero elevation of the well, 2 – well construction, 3 – well screens above the saturation zone, 4 – water saturated part of the aquifer, the thickness of which varies during the year, 5 – aquifer floor consisting of neogene clays, 6 – semi-pervious overlying stratum, 3–6 m thick, 7 – zone of blending of oxic and anoxic ground waters. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ iron oxidation in the groundwater 949 applin and zhao1 were the first to describe chemical clogging of production wells with iron compounds. the chemical oxidation of ferrous to ferric ions by oxygen and subsequent precipitation is a complex process. the oxidation of dissolved fe(ii) from the groundwater to fe(iii) tends to be gradual.7,8 the overall process involves a variety of partially oxidized low-crystalline fe(ii)–fe(iii) intermediate species in aqueous solution. these fe-intermediates could recrystallize into a variety of stable iron (hydr)oxide end-products (such as goethite) during time.9 the iron oxidation in the presence of dissolved oxygen is expressed as: fe2+ + o2 + 2h+ fe3+ + 2oh– (1) the produced fe3+ is immediately hydrolyzed: fe3+ + 3h2o  fe(oh)3 + 3h+ (2) the overall reaction that describes formation of insoluble iron encrustations2,3,8 is given as: 4fe2+ + o2 + 10h2o  4fe(oh)3(s) + 8h+ (3) as a result, encrustations appear on the well screen, in the near-well region and, in extreme cases, in the discharge pipes, pumps and equipment installed inside the well (fig. 2). fig. 2. encrustations in a discharge pipe of well bn-6 at the water supply source of trnovče (picture taken on 1 sep, 2011). the rate law of fe(ii) oxidation for mildly acid to neutral waters (ph value 6 to 8) was found to be first-order with respect to the concentrations of both fe(ii) and o2 and second-order with respect to the oh–:7–12 –d[fe]/dt = k[fe2+]p(o2)[oh–]2 (4) the reaction is rather fast. at ph less than 4, iron as fe(ii) dominates and the oxidation rate is dependent on the ph value.9 in addition, studies showed that ferrous oxidation is not dependent on ph if the value exceeds 8.9,10 many researchers7,8 showed that the formed iron-(hydr)oxides have a catalytic effect on the oxidation of dissolved iron. tamura et al.7 established an _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 950 majkić-dursun, petković and dimkić expanded rate law that includes both eq. (4) and a heterogeneous reaction at the solid iron-(hydr)oxides surface: r = k1[fe2+][o2(aq.)][h+]–2 + k2[fe(iii)][fe2+][o2][h+]–2 (5) in eq. (5), k1 is the rate constant for iron oxidation and k2 represents the product of the equilibrium constant, kads, for the adsorption of ferrous iron onto ferric oxide and the rate constant, ks, of the oxidation at the surface of the already formed precipitates.2,3 earlier research mentioned that this auto-catalytic effect gains measurable influence only if the initial concentration of fe(ii) is above 3 mg l–1.7 houben presented that only newly formed precipitates that coat older precipitates could act as catalyst and states that the catalysis is more a function of the amount of available surface sites than of the total concentration of ferric iron.8 for understanding the process of well clogging, it is also important to consider that low-crystallinity iron-(hydr)oxides, such as ferryhydrite, are more reactive than thermodynamically more stable iron oxides, such as goethite. the abiotic oxidation presented by the rate law (5) is insufficient to explain the rapid well clogging, which is often observed. in iron-deposits, large numbers of different bacteria are often found that are able to use iron for their metabolic activity. in the presence of low amounts of dissolved oxygen (0.1–1.0 mg l–1), dissolved iron can cause substantial bacteria growth, particularly iron-oxidizing bacteria such as the stalked gallionella and sheathed leptothrix.6,13–15 it is believed that these bacteria accelerate iron oxidation.13,16 experimental the in situ groundwater tests included the determination of the physicochemical parameters: redox potential, eh, groundwater temperature, concentration of dissolved oxygen, mg l-1, the conductance, μs cm-1, and ph value of the groundwater. the measurements were conducted using a multi-parameter probe (seba multiparameter dipper kll-q, seba hydrometrie, germany), with the following electrodes: opp-polymer for the redox potential and seba oxysens ii for oxygen. the multi-parameter probe was equipped with an automated groundwater level sensor that enabled sampling from the same depth in each campaign, and allowed data to be collected from various water column depths, in case of depth profile analyses. the total iron concentration was determined from an acidic solution17 using icp-oes (icp spectro genesis eop ii, spectro analytical instruments, germany). fe(ii) was analyzed in samples filtered on site (0.45 μm) and preserved with hcl.17 well discharge data were collected from individual flow meters, to monitor capacity decline over time. groundwater levels were measured with a level-meter (seba level-meter kll-t, seba hydrometrie, germany). the aggressiveness of iron-related bacteria (irb) was analyzed using the commercial bart test (irb barts, draycon bioconcept inc.). the irb aggressiveness in water samples was used to estimate the activity level of the bacteria rather than the number of cells (population commonly presented as colony forming units per ml). the irb aggressiveness was obtained from the relationship between the time lag (expressed in days) and the first _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ iron oxidation in the groundwater 951 reaction in the irb-bart™ tests. the bacteria that may be detected by this test include iron oxidizing and reducing bacteria, the sheathed iron bacteria, gallionella, pseudomonas and enteric bacteria. chemical analysis on the encrustations were performed using an inca energy-dispersion x-ray analysis (eds) instrument, model jeol jsm-6610lv, usa. the samples were placed in sterile jars and immediately refrigerated to prevent oxidation after sampling. the samples were dried at a temperature of 60 or 37 °c, if the samples contained manganese.2,6 for analytical purposes, the samples were ground into powder in an agate mortar. the powdered samples were sputter-coated with 24-carat gold. the detection limits of the applied eds measurements were 0.2 wt. %. x-ray powder diffraction (xrpd) analysis of the sample from well bn-5 was conducted using a philips pw-1710 automated diffractometer (equipped with a diffracted beam curved graphite monochromator and an xe-filled proportional counter), including a cu-tube operated at 40 kv and 30 ma. results and discussion the rate of precipitation of low-crystallinity iron-(hydr)oxides under natural conditions also depends on the groundwater retention time in the well, well pump operation and the groundwater abstraction regime. apart from the above, the rate of formation of well deposits is affected to a considerable extent by previous well regenerations and the state of “cleanliness” of the near-well region and the well screen. if regeneration was only a short-term, with poor removal of deposited iron-(hydr)oxides, precipitation will be more intensive. the in situ measured parameters are given in table i. table i. hydrochemical parameters from the trnovče water supply source well sampling date t °c ph [о2(аq.)] mg l-1 [fe2+] mg l-1 ec μs cm-1 eh mv bnz-1 3/2/2010 13.2 7.1 0.3 1.53 857 163 bnz-1 24/5/2010 13.1 7.2 1 1.16 814 181 bnz-1 9/11/2010 13.0 7.1 0.5 1.45 838 156 bnz-1 13/5/2011 13.2 7.1 0.6 1.13 862 155 bnz-1 1/9/2011 13.4 7.1 0.4 2.32 863 110 bn-5 3/2/2010 11.9 7.1 0.8 0.30 532 262 bn-5 24/5/2010 11.8 7.2 0.1 0.25 620 246 bn-5 13/5/2011 11.9 7.1 1.2 0.21 619 212 bn-5 1/9/2011 14.6 7.2 7.1 0.32 672 249 bn-6 3/2/2010 12.6 7.1 2.3 1.53 623 236 bn-6 24/5/2010 12.4 7.1 0.8 1.20 635 233 bn-6 9/11/2010 12.5 7.1 0.5 3.40 644 138 bn-6 13/5/2011 12.8 7.0 0.8 1.44 645 148 bn-6 7/11/2011 12.7 7.2 0.1 2.12 541 78 bn-9g 1/9/2011 12.3 7.1 1.1 0.49 695 280 bn-9g 7/11/2011 11.9 6.9 7.9 0.82 688 188 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 952 majkić-dursun, petković and dimkić the main question is whether the parameter values given in table i are favorable for the considerable well clogging shown in fig. 2. the groundwater at the trnovče water supply source has ph values between 6.9 and 7.2 (table i). for roughly neutral groundwater, as in the presented case study, iron oxidation is strongly ph dependent. it also means that small variation in ph values could change the amount of mass converted from ferrous to ferric iron during time. these conditions favor the rapid formation of iron-(hydr)oxide. on the other hand, the oxidation rate shows a linear dependence on o2, eq. (5).7,10,18 during the two-year survey, the concentrations of dissolved oxygen in the groundwater varied over a wide range (table i). generally, as river waters flow into the aquifer, removal of o2 may occur through oxidation of organic matter or adjacent sediments. there are several reasons for the large variations in o2 content: the setting of submergible pumps into the screen slot, the declining of static and dynamic groundwater levels into the casing interval, an unsuitable pump work scheme and aquifer recharge mode. research performed in france3 showed that the well de-watering process induced important perturbations of the physical and chemical equilibria in the water within the aquifer around the well, which led to deposition of scale materials. an introduction of the piezometric heads into the screen slots (fig. 1) leads to exposure of the groundwater to atmospheric conditions, resulting in an oxygen uptake (high values presented in table i). the formation of mixed oxic–anoxic zone, caused by an unsuitable pump work scheme and groundwater abstraction regime, also favors the rapid clogging. propagation of the clogging process in and around the well and the mixing of different geochemical zones are explained in following separate section. the concentrations of dissolved oxygen are also important for iron-related bacteria. the results of irb bart tests indicated that bacterial aggressiveness was high in 16 %, mediate in 36 % and low in 48 % of the samples. low aggressiveness means that the bacterial population is too small (or that it has very low activity), whereas mediate indicates that there is either a moderately active or modest population of the targeted bacteria. high aggressiveness was shown only by the bacterial consortium sampled on the 1st of september 2011. the most important iron-oxidizing bacteria found in water samples and encrustations, were gallionella ferruginea, lepthotrix sp. and siderocapsaceae. according to roden et al.,19 fe(ii)-oxidizing bacteria dwell in micro-aerobic environments of low oxygen concentrations (table i, values less than 1 mg l–1), but vet et al.16 concluded that gallionella spp. can grow under neutral ph and aerated conditions when chemical iron oxidation is inhibited by low water temperature (about 13 °c) and suppression of auto-catalytic iron oxidation. the concentration of iron in the groundwater is also important for the formation of encrustations. usually, drinking water has a relatively small concentration of dissolved iron. in the case of the trnovče water supply source, the aver_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ iron oxidation in the groundwater 953 age concentration of total iron was 1.79 mg l–1, while the maximum value was 18.8 mg l–1 (bnz-1, before the regeneration). the values of the dissolved iron concentrations are given in table i. the concentrations of iron in the groundwater could vary over the year, depending on the mode of aquifer charging. during the summer or drought periods, the groundwater feeds the velika morava river.20 charging of the aquifer is slower and the groundwater comes from the upland. generally, groundwater from deeper part of the aquifer is anoxic and iron is present as dissolved ferrous iron. in contact with dissolved oxygen, the fe(ii) is rapidly oxidized to fe(iii). pham and white noticed that the rate at which fe(ii) is oxidized to fe(iii) is critical in determining the fate of iron and its bioavailability in many natural waters.21 in the particular case of trnovče, this means that bacteria have to compete with rapid chemical oxidation. the values of the conductance were higher for the bnz-1 well than for the other wells. bnz-1 well is located in the hinterland while the other three wells are closer to the riverbed. the values of the redox potential also indicate the mixing of different geochemical zones. xrpd analysis detected low-crystalline (hydr)oxides and kaolinite in the sample collected from the bn-5 well (fig. 3). fig. 3. xrpd pattern of encrustation from bn-5 well. chemical analyses of the encrustations sampled from the water supply wells (bn-5, bnz-1, bn-6 and bn-9g) performed by eds revealed the presence of iron and manganese (hydr)oxides. the proportion by weight of manganese (hydr)oxides in the well deposit samples ranged from 0.3 to 21.3 wt. %, while that of iron(hydr)oxides was from 62.6 to 76.2 wt. %. in addition to these (hydr)oxides, there were also carbonates (5.4 wt. % on average), phosphates (4.3 to 7.2 wt. %) and silicate grains (clay and sand), drawn in from the alluvial matrix through the screen slots. the chemical oxidation of manganese is slower than that of iron. compared to iron, the rate of manganese oxidation is at least 106 times slower in waters with a ph value close to neutral.22 chemical oxidation of manganese accelerates with increasing ph value (ph > 8).22 the joint occurrence of lowcrystallinity manganese and iron (hydr)oxides in the deposits of trnovče wells can be explained by the microorganisms present in the environment.14 it is well known that the iron-oxidizing bacteria leptothrix sp. are also able to oxidize manganese(ii). _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 954 majkić-dursun, petković and dimkić propagation of clogging around the trnovče wells assessments of well clogging rates need to address the groundwater retention time in the near-well region (gravel pack). the bn-5 well, which exhibited the poorest performance at trnovče, was selected for detailed analysis. its drilling diameter is 500 mm, the diameter of the screen 219 mm, and the screen length 7 m. in order to explain the iron hydr(oxide) precipitation process in and around the well screens, leading to screen slot clogging in shallow alluvial aquifers, four phases can be distinguished.24 these phases illustrate the formation of ferric deposits and a discharge loss for the bn-5 well. in the first stage, prior to the development of the water supply source, the underground environment was undisturbed; the aquifer is sub-artesian and the groundwater was anoxic (fig. 4).24 in this stage, the concentration of dissolved o2 was much lower than in stages of exploitation. following the commissioning of the wells during the second stage, a cone of depression is formed and the front of the potential blending of traces of groundwater with different geochemical zones moved downwards. the initial capacity of the studied well was q = 15 l s–1 (in 1999), lasting over a relatively short time, only until 2001. the third stage of the life cycle of the well began as early as 2002, when its capacity dropped to q = 7 l s–1 (46 % of its initial capacity). in the period 2002–2008, the number of production wells at trnovče increased to 18; the dynamic groundwater level reached the upper zone of the screen (figs. 1 and 4), moving the redox front to the water-receiving part of the well. iron-(hydr)oxides were deposited on the walls of the well screen and casing. as soon as iron-(hydr)oxides were present on the screen slots, the oxidation reaction accelerated and, besides homogeneous oxidation, heterogeneous oxidation also occurred.3,8,23,24 the first regenerations at this site were conducted in 2004, combining mechanical cleaning with the chem fig. 4. stages of well clogging due to blending of oxic and anoxic ground waters24 with permission from iwa publishing. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ iron oxidation in the groundwater 955 ical action of hydrochloric acid and citric acid as an inhibitor. this procedure increased the capacity of the well to q = 12 l s–1 (fig. 5). the next regeneration of water supplying bn-5 well was undertaken in 2008, when the capacity of the well had dropped to 2 l s–1. the same method was applied and the discharge was increased to 8 l s–1. fig. 5. declining discharge of well bn-5 over 10 years of service. at stage four, iron was precipitated in the well itself and over an extended area around the well. insoluble (hydr)oxides were deposited on the grains of the pack in the near-well region, as well as further afield from the well, on alluvial sediment grains. in near-well region, coated grains were found in 2010 during drilling for piezometers near the wells bnz-1, bn-5 and bn-6. deposition of iron (hydr)oxide on sediment grains reduces porosity and change the entrance velocities in the screen slot. in connection with the problem of a general decline of the groundwater level over the wide area of the trnovče source, the pumps were occasionally stopped. during the pumping stops (from several hours to more than one month), there was sufficient time for the iron species that remained within the column of water within the well to be oxidize and the screen and casing were encrusted. this final stage of the life cycle of the well resulted in complete shutdown of the well. conclusions the exploitation wells at the trnovče water source are jeopardized by the clogging process. variations in values of chemical parameters, such as the concentration of fe(ii), dissolved oxygen and redox potential, are the result of an unsuitable pumping scheme and general declining of the groundwater levels. as the groundwater level dropped to the screen zone, the redox front shifted to the water-receiving part of the well. mixing of water containing oxygen with anoxic groundwater reaching fe(ii) led to mineral encrustations, especially iron (hydr)oxides. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 956 majkić-dursun, petković and dimkić at ph values around 7, as is the case of the trnovče water source, the chemical oxidation of fe(ii) is strongly ph dependent. small variations in the measured values could significantly change the oxidation rate. the conditions present in the groundwater are favorable for the rapid oxidation of dissolved iron. iron-oxidizing bacteria could accelerate this process. the role of bacteria in clogging process depends on the availability of dissolved iron and the concentration of dissolved oxygen. in alluvial aquifers, variable amounts of ions, catalysts, inhibitors, bacteria and minerals could be present simultaneously. the definition of an exact rate law for such a complex aquatic system is very difficult. during the precipitation, newly formed precipitates coat older ones. preliminary results showed high contents of low-crystalline iron and manganese (hydr)oxides inside the water supply wells. with time, if the regeneration methods were not proper, the early formed iron deposits will be re-crystallized into a variety of stable iron oxide end-products. this could be expected in the extended area around the well. due to the poor design of the well casings (small diameter and short tailpipe), the pumps were housed inside the screen and this additionally accelerated precipitation. iron oxidation did not occur solely inside the well; the process tended to extend into a much wider zone, beyond the well. especially vulnerable is the well pack, but the process propagates further when there are considerable fluctuations of the groundwater level in the extended zone of the trnovče water supply source. an adequate exploitation scheme could reduce these phenomena and extend the life of the wells. acknowledgment. the authors express their gratitude to the ministry of education, science and technological development of the republic of serbia for financially supporting this work, project no. tr37014. и з в о д ефекat оксидације гвожђа у подземним водама алувијалног аквифера велике мораве на капацитет бунара за водоснабдевање бранкица мајкић-дурсун, анђелка петковић и милан димкић институт за водопривреду “јарослав черни“, јарослава черног 80, 11226 београд процес оксидације двовалентног гвожђа из подземне воде на филтрима и потисним цевоводима бунара који каптирају плитке алувијалне средине, доводи до формирања бунарских талога. основни циљ овог истраживања био је утврђивање разлога брзог колмирања бунара за јавно водоснабдевање. у конкретном случају, талоге чине хидроксиди гвожђа (62,6 до 76,2 мас. %). прекомерна експлоатација бунара доводи до мешања различитих геохемијских зона и формирања редокс фронта. током две године истраживања, концентрација раствореног кисеоника у подземној води варирала је у широком опсегу од 0,1 до 7,1 mg l-1 као последица неодговарајућег експлоатационог режима. теренска мерења температуре подземне воде, концентрације раствореног кисеоника, ph вредности, као и лабораторијске анализе раствореног гвожђа, показала су да измерене _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ iron oxidation in the groundwater 957 вредности погодују таложењу гвожђа и формирању наслага, у условима прекомерне експлоатације подземних вода. (примљено 4. фебруара, ревидирано 27. јуна, прихваћено 3. септембра 2014) references 1. k. r. applin, n. zhao, groundwater 27 (1989) 168 2. g. houben, c. treskatis, water well rehabilitation and reconstruction, mcgraw-hill, new york, 2007, p. 67 3. f. larroque, m. franceschi, environ. earth sci. 64 (2010) 119 4. s. smith, a. comeskey, sustainable wells: maintenance, problem prevention and rehabilitation, crc press, taylor and francis group, boca raton, fl, usa, 2010, p. 17 5. b. majkić-dursun, lj. popović, d. miolski, o. anđelković, vodoprivreda 44 (2012) 181 (in serbian) 6. b. majkić, phd thesis, faculty of mining and geology, belgrade, 2013, p. 223 (in serbian) 7. h. tamura, k. goto, m. nagayama, corros. sci. 16 (1976) 197 8. g. houben, groundwater 42 (2004) 78 9. b. morgan, o. lahav, chemosphere 68 (2007) 2080 10. w. stumm, j. j. morgan, aquatic chemistry: chemical equilibria and rates in natural waters, 3rd ed., wiley, new york, usa, 1996, p. 686 11. n. el azher, b. gourich, c. vial, m. belhaj soulami, m. ziyad, chem. eng. process. 47 (2008) 1877 12. j. n. geroni, d. j. sapsford, app. geochem. 26 (2011) 1452 13. e. r. david, j. m. stevenson, water res. 29 (1995) 365 14. v. obradović, b. majkić-dursun, a. petković, m. dimkić, in proceedings of the 41st annual conference of the serbian water pollution control society, divčibare, serbia, 2012, p. 359 (in serbian) 15. m. dimkić, m. pušić, v. obradović, s. kovačević, water sci. tech. 65 (2012), 2206 16. w. w. j. m. de vet, i. j. t. dinkla, l. c. rietveld, m. c. m. van loosdrecht, water res. 45 (2011) 5389 17. standard methods for the examination of water and wastewater – smeww, 3500-fe b method, 21st еd., american public health association, american water works association and water environment federation, washington dc, 2005, pp. 3–77 18. h. sugimori, y. kanzaki, k. yokota, t. murakami, j. miner. petro. sci. 106 (2011) 142 19. e. e. roden, d. sobolev, b. glazer, g. w. luther, geomicrobiol. j. 21 (2004) 379 20. m. komatina, the hydrogeology of šumadija, geological and geophysical research institute, belgrade, serbia, 1976, p. 38 (in serbian) 21. a. n. pham, t. d. whaite, geochim. cosmochim. acta 72 (2008) 3616 22. s. t. martin, in environmental catalysis, v. h. grassian, ed., taylor and francis group, boca raton, fl, 2005, pp. 61–83 23. u. park, b. a. dempsey, environ. sci. technol. 39 (2005) 6494 24. c. g. e. m. (kees) van beek, cause and prevention of clogging of wells abstracting groundwater from unconsolidated aquifers, kwr water cycle research institute series, iwa publishing, london, uk, 2012, p. 115. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice adsorption of candida rugosa lipase onto alumina: effect of surface charge j. serb. chem. soc. 80 (9) 1113–1125 (2015) udc 661.183.8+577.115:541.183 jscs–4784 original scientific paper 1113 adsorption of candida rugosa lipase onto alumina: effect of surface charge lidija t. izrael živković1*#, ljiljana s. živković2#, bojan m. jokić3, andrija b. savić2 and ivanka m. karadžić1 1university of belgrade, school of medicine, institute of chemistry in medicine, visegradska 26, 11000 belgrade, serbia, 2university of belgrade, the vinča institute of nuclear sciences, 11000 belgrade, serbia and 3university of belgrade, faculty of technology and metallurgy, karnegijeva 4, 11001 belgrade, serbia (received 22 february, revised 16 april, accepted 30 april 2015) abstract: the impact of the surface charge of alumina supports on the adsorption of candida rugosa lipase was investigated in terms of the zeta potentials of the adsorption partners. the lipase adhered onto alumina with similar efficiency under both repulsive and attractive electrostatic conditions, shifting the zeta potential of the support towards that of the enzyme. the behavior was explained by a heterogeneous distribution of the surface charge of the lipase molecule. special emphasis in this study was placed on the effect of immobilization on the enzyme kinetics and principal reasons for enzyme immobilization: improvement in stability and potential for reuse. the enzyme affinity was not altered by its adsorption onto alumina, while the vmax value of the lipase decreased. the thermostability of the adsorbed lipase was improved. a significant potential for reuse was found. keywords: alumina; lipase; adsorption; immobilization; candida rugosa. introduction increasing environmental concerns have led to the replacement of conventional chemical methods with enzyme-based strategies. the utilization of enzymes as biocatalysts has become an integral part of different processes in the oil, pharmaceutical, detergent, and food industries. during the last decade, the potential of different techniques for immobilization has been studied in order to overcome instability, enhance enzyme reuse, as well as easier separation.1 among them, the most commonly used methods for enzyme immobilization are cross-linking or covalent binding, entrapment and adsorption. one of the simplest methods, with high commercial potential and wide applicability, is adsorption.2 adsorption * corresponding author. e-mail: lidija.izrael-zivkovic@mfub.bg.ac.rs # these authors contributed equally to this article. doi: 10.2298/jsc150222035i _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1114 izrael živković et al. causes little or no conformational changes of the enzyme but the linkages established between the enzyme and support are weak, so the enzyme could be easily desorbed (leaking of the enzyme from the support). the frequently used supports are inert polymers and inorganic materials.3 alumina exhibits high mechanical strength, excellent corrosion and wear resistance and good biocompatibility4 and it is among the mostly used ceramics.5 lipases are an important group of biocatalysts as they improve a variety of reactions with high specificity and selectivity.6,7 candida rugosa lipase has been widely used in stereoselective synthesis of pharmaceuticals, production of carbohydrate esters of fatty acids, biodiesel, biosensors, food and flavor making.8,9 considering the fact that c. rugosa is one of the most important enzymes in biotransformations, different protocols for its immobilization have been developed but novel and efficient methods are still required. even though common and simple for performing, adsorption is a very complex phenomenon.10 research teams have identified numerous factors that strongly influence enzyme loading and biocatalyst activity, such as support surface area, pore size distribution, pore volume, particle size, ionic strength as well as surface charge of both support and enzyme.11 in a few studies, electrokinetic measurements were conducted, yielding different results. although there is no doubt that electrostatic interactions influence the adsorption process, some authors claim that the maximal affinity of a protein towards a support surface occurs when the adsorption partners exhibit opposite surface charges,11,12 while others suggest the highest adsorption efficiency under repulsive electrostatic conditions (same surface charge),13,14 or at phs close to the isoelectric point of the protein,15 thus creating contradictory approaches. in this regard, a previous study dealt with effect of the surface charge on the adsorption of c. rugosa lipase. two inorganic supports, mesoporous silica sba -15 and macroporous zirconia were used.16 the electrostatic nature of the process was estimated based on the electrokinetic (zeta) potentials of the interacting partners. however, no direct correlation between adsorption efficiency and immobilization ph was hitherto established; the enzyme adhered similarly under both attractive and repulsive electrostatic conditions. aimed at obtaining better insight into the impact of electrostatics on the adsorption of enzymes onto supports, the idea of the current study was to minimize the influence of the support morphology. for this purpose, nonporous submicron-sized alumina was selected as the model support. the same methodology was used, i.e., changes in zeta (ζ) potential of the adsorption partners were monitored at different ph values and correlated with the amount of adsorbed lipase. the second part of the study focused on the effect of immobilization on the enzyme kinetics and the principal reasons for enzyme immobilization: improvement in stability and potential for reuse. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ lipase adsorption onto alumina 1115 experimental materials lipase from c. rugosa (lyophilized powder, type vii, nominal activity 746 u mg-1) and p-nitrophenyl palmitate (p-npp) were purchased from sigma chemical co. (st. louis, mo, usa). the support material, alumina (al2o3, akp-30 sumitomo, japan), was a high purity (>99.99 %) α-phase, with an average particle size of 0.32 µm (size distribution: d90, d50 and d10 of 0.61, 0.35 and 0.2 µm, respectively, sedigraph 5100, micrometrics)17 and a specific surface area, sp = 7 m2 g-1. all other chemicals were of analytical grade. enzyme assay the activity of free lipase was measured spectrophotometrically using an assay based on the hydrolysis of p-npp. the p-npp solution was prepared as follows: 30 mg of p-npp in 10 ml of 2-propanol was added to 90 ml of 0.05 m phosphate buffer (ph 8.0) supplemented with 200 mg of na deoxycholate and 100 mg of gum arabic, (the final p-npp concentration was 0.8 mm). the absorbance was measured at 410 nm for the first 3 min of the reaction at 25 °c. one unit (1 u) was defined as the quantity of enzyme that under the test conditions liberated 1 μmol of p-nitrophenol per min (ε = 18.5 mm-1 cm-1). the reaction mixture was composed of 900 μl of p-npp solution and 100 μl of lipase solution (lipase concentration 0.05 mg ml-1).18 activity of immobilized lipase was determined using 10 mg of immobilized preparation supplemented with 2 ml of p-npp solution (the p-npp of concentration was 0.8 mm, in phosphate buffer of ph 8.0) the reaction was performed at 25 °c for 3 min when it was stopped by the addition of 0.5 m sodium carbonate solution. the precipitate was removed by centrifugation at 10,000 rpm for 10 min. the supernatant was diluted 10-fold with deionized water and the absorbance was measured at 410 nm. one unit of enzyme activity (1 u) was defined as explained in the previous paragraph. the activity of the immobilized enzyme was calculated as u per g of support material. lipase immobilization the effect of ph on the adsorption of lipase was investigated in the ph range 5 to 9, using the following 10 mm buffer solutions: acetate buffer (ph 5.0), phosphate buffer (ph 6.0, 7.0 and 8.0) and tris-buffer (ph 7.6 and 9.0). alumina was prepared for immobilization as follows: 8 mg support ml-1 in the selected buffer was de-agglomerated for 10 min by sonication. then, a lipase solution (2 mg ml-1 in the selected buffer) was added, the resulting suspension sonicated for 10 min and immobilization realized at room temperature under mild stirring (90 rpm). after 60 min, the immobilized lipase was precipitated by centrifugation at 10,000 rpm for 10 min using a microcentrifuge (denver instruments, usa). the immobilized enzyme was washed twice to remove the excess of unbound enzyme, dried for 1 h at room temperature and used for the assay. the efficiency of immobilization was evaluated as the percent of the lipase adsorbed, which was calculated as: 1 0 100 lipase adsorbed (%) p p = (1) where p0 is the initial concentration of lipase and p1 is the concentration of immobilized lipase. the concentration of immobilized lipase was calculated as a difference between initial lipase concentration and lipase concentration in the supernatant after immobilization.19 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1116 izrael živković et al. the concentration of lipase was determined by the bradford method using bovine serum albumin as the standard.20 zeta potential measurements electrokinetic (zeta) potential measurements were performed by means of a zetasizer nano zs instrument (malvern, uk). the instrument uses the electrophoretic light scattering and laser doppler velocimetry methods for determination of particle velocity, and from it, the zeta (ζ) potential. the integrated software calculates the ζ-potential from mobility values by the henry equation, using the smoluchowski approximation.21,22 the measurements were performed at least in triplicate and the average values are presented. each measurement comprised of minimum 10 runs. only the results that met quality criteria were taken into account. aimed at determining of the isoelectric point (iep) of support, the ζ-potential of the alumina particles was measured over a wide range of ph values (4–10), using aqueous solution of nacl (0.01 m) as the inert background electrolyte.23 prior to titration, powder dispersion (1 mg ml-1) was equilibrated for 24 h under mild shaking at room temperature. the ζ-potential measurements of lipase and that of the support particles (before and after enzyme adsorption) were then performed in the selected buffers adjusted to different ph values (5–9). field emission scanning electron microscopy (fesem) field emission scanning electron microscopy (fesem), using a tescan mira3 xmu (czech republic) at 20 kv, was employed to study the morphology of the support before and after the adsorption of lipase. prior to analysis, the samples were coated with au–pd alloy using a sputter coater. determination of the kinetic parameters michaelis–menten kinetics were used to describe the dependence of enzyme activity on substrate concentration for free and immobilized lipase. the kinetic parameters, vmax and km were determined for free and adsorbed lipase using solutions of p-npp of the following concentrations: 0.1, 0.2, 0.4, 0.8, 1.2, 1.6 and 2.0 mm, according to the above-described methods for the determination of lipase activity. vmax – units of activity per milligram of protein (u mg-1) and km – substrate concentration that gives a reaction rate of vmax/2 (mm) were estimated from the experimental data using the lineweaver–burk equation: m max max 1 1 1 s k v v v   = +       (2) the y-axis intercept is 1/vmax and the slope is km/vmax. thermal stability reaction mixtures containing free or immobilized enzyme were incubated at 50 and 60 °c for various periods (20–120 min) and quickly cooled. enzyme activity was measured according to the standard protocol for free and immobilized enzyme with p-npp as the substrate. the half-life (t1/2) of the enzyme with kd as the decay constant was calculated as: t1/2 = (ln 2)/kd (3) reuse stability the potential for reuse was determined using 25 mg of immobilized enzyme packed in a small filter paper bag soaked into 3 ml of p-npp solution for 30 min at room temperature, _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ lipase adsorption onto alumina 1117 under mild stirring. after each batch reaction, the immobilized lipase was washed three times with deionized water and then reused. into 2 ml of removed supernatant solution, 2 ml of 0.5 m sodium carbonate was added to stop the reaction. the absorbance was measured at 410 nm after centrifugation at 10,000 rpm for 10 min and 10-fold dilutions. results and discussion zeta (ζ) potential and immobilization ph study there are close analogies between protein adsorption and particle deposition24,25 both being governed by short-range interactions between charged surfaces at the solid–liquid interface. the dlvo (derjuangin–landau–verbey– –overbeek) theory26 plays an essential role in the quantification of the processes. although based on simple additivity of electrostatic and van der waals forces, it is successful in predicting the basic features of colloidal stability and adsorption or deposition processes. since the electrostatic forces are dominant for attachment, the relative charges of the surfaces dictate whether the interaction is repulsive or attractive. the isoelectric points (iep) of the partners indicate the ph where the ζ potential equals zero and the range over which the interactions are favored. thus, a prediction of the process requires knowledge of the iep values for both materials. the reactions occurring at the solid–liquid interface were the subject of numerous experimental and theoretical investigations. to better understand the adsorption of proteins onto the surface of a support (e.g., the mechanism of binding, the build-up of layers, etc.), the electrokinetic ζ potential changes vs. ph were often collected.22,27–29 however, the aim of this study was not to elucidate the mechanism of c. rugosa lipase adsorption onto alumina surface by means of electrokinetic measurements, but to test the zeta potential of the adsorption partners as a diagnostic tool for the efficiency of binding of the protein to the support. the variation in zeta potential of alumina, used as support, as a function of ph is presented in fig. 1. as is evident, the isoelectric point was located at ph fig. 1. titration curve: zeta potential vs. ph for alumina powder in nacl (0.01 m). _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1118 izrael živković et al. 8.8, in good accordance with the literature.17 thus, the alumina develops positive and negative surface charges below and above this ph by protonation–deprotonation of the hydroxide groups present on the surface.30 lipase from c. rugosa is a globular glycoprotein with molecular mass of 57 kda. its basic structural characteristics are summarized in table i. with 31 acidic and 18 basic amino acids exposed on the surface,16 its iep is located at ph 4.65. above this ph, the lipase acquires a negative charge and hence the enzyme should always be negative in this study. table i. protein parameters of c. rugosa lipase, p20261 (uniprotkb) parameter value molecular mass, kda 57 dimensions, mm 5×4.2×3.3 isoelectric point (iep) 4.65 total number of amino acids 534 number of acidic amino acids 52 number of basic amino acids 39 non-polar surface, % 63 surface acidic amino acids 31 surface basic amino acids 18 α-helical structure, % 38 β-sheet structure, % 18 post-translational modifications, ptm disulfide bonds: 75–112, 283–292 glycosilation (n-acetyl-d-glucosamine): 329, 366 e.c. (brenda) 3d structure in pdb 3.1.1.3.1139. 1crl to evaluate the effect of the surface charge of the adsorption partners on the adsorption efficiency, experiments were performed below and around the iep of alumina (ph 8.8), where 2 different electrostatic scenarios were expected. the first one (ph < iepalumina) includes favorable adsorption conditions, i.e., negatively charged lipase in the presence of positively charged alumina particles. the second adsorption scenario (ph ≈ iepalumina) is related to the interaction of negatively charged lipase with uncharged alumina, thus being much less favorable. different buffers were used over the investigated ph range 5–9. the ζ-potentials of enzyme and support, before and after adsorption, were monitored in parallel, and the percent of adsorbed lipase (ηenz) related to the values of the ζ-potential are summarized in table ii. indeed, in agreement with theoretical calculations, the lipase was negatively charged in the investigated ph range, reaching the highest absolute magnitude of ζ-potential at ph values 7–9. on the other hand, the alumina particles were positive in the acetic buffer adjusted to ph 5, as expected (fig. 1). surprisingly, in the presence of phosphate buffer, a charge reversal occurred: the alumina _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ lipase adsorption onto alumina 1119 turned highly negative even at ph 6. this result was confirmed for ph values 7–8, indicating specific adsorption of phosphates onto alumina surface occurred. a significant shift in the iep towards acidic ph upon addition of po43– was also reported by zheng.31 to avoid preferential adsorption of phosphates, tris buffer was introduced, in which alumina behaved analogously as in nacl. likewise, at ph 7.6, the particles developed a positive charge, whilst at ph 9, a negative ζ-value was recorded. table ii. ζ-potentials of free lipase and alumina, before and after adsorption of lipase, and the amount of lipase adsorbed at ph values 5–9 buffer ζ-potential, mv ηenz / % lipase alumina alumina–lipase ph 5 (acetic) –4.3±0.5 +40.2±1.6 –9.4±1.1 30 ph 6 (phosphate) –9.8±1.2 –35.9±2.1 –13.7±1.4 32 ph 7 (phosphate) –12.4±0.4 –36.7±0.4 –25.0±2.3 33 ph 8 (phosphate) –14.3±1.3 –30.7±1.3 –17.3±1.4 30 ph 7.6 (tris) –14.3±0.6 +22.1±0.9 –25.4±2.2 38 ph 9 (tris) –16.7±0.8 –12±0.5 –16.4±0.8 5 albeit two electrostatic scenarios were expected, the phosphate buffer actually created the third repulsive electrostatic conditions, in which both alumina and lipase carried a negative surface charge. as presented in table ii, the percent of adsorbed lipase was comparable under both attractive and repulsive electrostatic conditions. whatever the charges of the immobilization partners were (like or dislike, as a function of the buffer used), the amount of adsorbed enzyme was moderate (around 30 %). contrarily, at ph 9 (i.e., when the ph ≈ iep of alumina), only a small percentage (5 %) of adsorbed lipase was registered. this result is logical, since a ph value in the close vicinity of the iep of alumina, when the particles carry only a negligible surface charge, strongly promoted their aggregation and sedimentation. such adsorption condition was inconvenient for the lipase to reach the surfaces of the particles and adhere. on the contrary, the alumina particles were quite well dispersed at the other investigated ph values. a ζ-potential of ca. 30 mv (positive or negative) is normally required to achieve a reasonably stable dispersion.30 an anomalous adsorption of lipase molecules onto alumina bearing the same surface charge contradicts the classical dlvo theory. however, adsorption–deposition of protein–particles under repulsive electrostatic conditions is not an unusual phenomenon.13,32,33 although the overall charge of the lipase was negative in the selected ph range, the enzyme surface consists of positively (lys, arg, his) or negatively (glu, asp) charged, and non-polar residues dispensed into a patchwork which leads to localized interactions (table i). the hetero_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1120 izrael živković et al. geneous surface charge of an enzyme always establishes attractive electrostatic conditions, whatever is the charge on the support particles. therefore, the adsorption efficiency of enzyme depends on the dispersion state of support particles, i.e., on the magnitude of the ζ-potential, not its sign, increasing enzyme availability to approach and adhere onto the surfaces of the particle. the presented results suggest that the zeta potential is a useful indicator of an adsorption process. an obvious change in the ζ-potential of the support was always registered after enzyme immobilization, indicating that the surfaces of the alumina particle were altered. negatively charged lipase shifted the ζ-potential of support towards negative values, sometimes even more negative than its own. the highest adsorption was achieved in the tris buffer of ph 7.6, therefore this buffer was chosen as suitable for lipase immobilization. lipolytic activity of immobilized enzyme in chosen system was determined to be 58 u g–1 of alumina. field emission scanning electron microscopy (fesem) adsorption of lipase onto support was also characterized by fesem (fig. 2). the powder was composed of differently sized sub-micrometer particles of irregular shapes. their surfaces appeared smooth and nonporous, corroborating well the small value of specific surface area (7 m2 g–1). at high magnification (inset), a certain number of small, differently sized piles (20–80 nm), can be randomly spotted on the particle surfaces. they could be associated with lipase aggregates, composed of several lipase molecules (5 nm×4.2 nm×3.3 nm). fig. 2. fesem micrographs of alumina before and after (inset) lipase adsorption. the rather moderate adsorption of enzyme achieved (ca. 30 %) could be related to the poorly developed surface geometry of the alumina particles, not favorable for lipase adhesion. a much higher percent adsorption of c. rugosa lipase was reported in a previous work (ca. 80 % onto silica), which was due to mesoporous and fibrous morphology of sba-15.16 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ lipase adsorption onto alumina 1121 therefore, besides electrostatics, the morphology of the support seems to plays an important role in the adsorption and should also be considered. it could be a determining factor of the efficiency of enzyme adsorption, once the optimum dispersion conditions were provided. effect of immobilization on the catalytic properties of the enzyme catalytic properties of an enzyme can be modified by immobilization. these changes may be due to conformational alterations within the enzyme. the kinetic behavior of an immobilized enzyme could differ significantly from that of the same enzyme in free solution. immobilization can also greatly affect the stability of an enzyme. this is primarily due to the physical prevention of large conformational changes within the protein structure. the catalytic action of the lipase depends on the interfacial activation that occurs when the lipase binds to a lipid interface via the opening of the α-helical lid that covers the active site. there are several kinetic models for lipase activity, ranging from michaelis–menten kinetics and the first order ping pong bi bi mechanism34 to more complicated ones taking into consideration the differences between the interfacial and bulk concentration35 and also the time of solution penetration into support with immobilized lipase.36 although michaelis–menten mechanism assumes enzyme reaction in the medium where enzyme and substrate must be part of the same phase and does not take into account the activation of the lipase, it is still commonly used as a simplified model for explanations and the determination of the kinetic parameters. free lipase and lipase immobilized onto alumina obeyed michaelis–menten kinetics. two parameters, vmax and km, for the free and immobilized c. rugosa lipase were determined and compared. the vmax, defined as the highest possible rate of enzyme activity, occurs when the enzyme is saturated with the substrate, and reflects the intrinsic catalytic character of the enzyme. km is defined as the substrate concentration that gives a reaction rate of 0.5vmax, implying an affinity between enzyme and substrate. values of kinetic parameters could not be directly compared with those previously reported for free and immobilized enzyme if the assay conditions (temperature, reaction system used) were not the same, but the trend of the modification of the catalytic property is comparable. vmax and km were calculated from the experimental data using the lineweaver–burk equation. in this study, the km value of the immobilized lipase was similar to that of free lipase, 0.264 vs. 0.241 mm, suggesting that enzyme affinity towards the substrate was not altered by its adsorption onto alumina. similar results were reported for c. rugosa lipase immobilized on zirconia, magnetized dacron and niobium oxide,16,37,38 since adsorption causes little or no conformational change of the enzyme or destruction of its active site.39 the vmax value for the free lipase was 172.0 u mg–1 that decreased after immobilization onto _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1122 izrael živković et al. alumina to 28.8 u mg–1, which might be a consequence of increased rigidity of the enzyme. a reduced maximal velocity of immobilized enzymes was reported for c. rugosa lipase adsorbed onto silica, zirconia and niobium oxide.16,38 thermal stability one of the advantages of enzyme immobilization is its improved stability. thermal stability of the immobilized lipase was evaluated, as shown in fig. 3. immobilized lipase was incubated for 2 h at 50 and 60 °c and the lipase activities were measured at 20 min intervals. (a) (b) fig. 3. thermal stability of free () and immobilized lipase () at: a) 50 and b) 60 °c. after 2 h of incubation at 50 °c, the remaining activity of free lipase was 31 %, but the remaining activity of immobilized lipase was 73 % (fig. 3a). in terms of the half-life at 50 °c, the t1/2 of lipase increased after immobilization onto alumina 5.6 times (from 32 min to 180 min). after 1 h of incubation at 60 °c, the free lipase was not active at all while 60 % of its activity remained when immobilized (fig. 3b). even after 2 h, the activity of the lipase immobilized onto alumina was significantly preserved (nearly 40 %). in terms of half-life at 60 °c, the t1/2 of the lipase increased after adsorption 5.8 times (from 14 to 81 min). thermal stability of lipase on support was significantly improved. improvement in thermal stability seems to be a result of restricted movement of the protein after adsorption, which prevents conformation changes and unfolding. reuse stability one of the most useful advantages of immobilization of an enzyme is its reusability. adsorption on a matrix as an immobilization method is usually considered a method with poor reuse potential. however, the remaining activity of the enzyme on alumina was higher than 80 % after seven reuses and almost 50 % _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ lipase adsorption onto alumina 1123 after 11 cycles of use (fig. 4). as the strength of protein–support interactions can be rated from the ability of the enzyme to resist removal – leaching, this result suggests a significant potential of lipase immobilized on alumina to be reused in biotechnological processes. fig. 4. effect of reuse on the activity of immobilized lipase. conclusions in this study, c. rugosa lipase was successfully adsorbed onto alumina as a support. the lipase adhered under both repulsive and attractive electrostatic conditions with comparable efficiency. patchwork surface with mixed positive and negative charges was supposed to be responsible for this behavior. the zeta potential was proven an indicator of protein binding, i.e., a shift in the zeta potential of the support towards that of enzyme was always registered. the morphology of the support also seems to play an important role in the adsorption process. kinetic constants provided clear evidence that the enzyme affinity was not altered by its adsorption onto alumina, while vmax of the lipase decreased 6-fold after immobilization. the thermostability of the adsorbed lipase was improved more than 5 times at 50 and 60 °c. the results suggest a significant potential for reuse of the lipase immobilized onto alumina. acknowledgment. this research was financed by the ministry of education, science and technological development of the republic of serbia (projects nos. iii 43004 and iii 45012). и з в о д адсорпција липазе из candida rugosa на глиници: утицај површинског наелектрисања лидија т израел живковић1, љиљана с живковић2, бојан м. јокић3, андрија б. савић2 и иванка м. караџић1 1универзитет у београду, медицински факултет, институт за хемију у медицини, вишеградска 26, 11000 београд, 2универзитет у београду, институт за нуклеарне науке винча, 11000 београд и 3универзитет у београду, технолошко–металуршки факултет, карнегијева 4, 11000 београд у овом раду испитиван је утицај површинског наелектрисања глинице на адсорпцију candida rugosa липазе на основу зета потенцијала оба учесника процеса. липаза се адсорбује у условима и електростатичког привлачења и одбијања, при чему се вредност зета потенцијала носача помера ка вредности истог ензима. овакво понашање је објаш_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1124 izrael živković et al. њено хетерогеном расподелом површинског наелектрисања молекула липазе. посебна пажња је посвећена проучавању утицаја имобилизације на кинетичке параметре липазе. афинитет ензима није промењен након адсорпције, али је максимална брзинa смањена. термостабилност адсорбоване липазе је побољшана. потврђена је и могућност вишеструке употребе имобилизоване липазе. 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(2011) 216435 39. s. c. wu, y. k. li, j. mol. catal., b 54 (2008) 103. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis of vinyldihydropyrane by cooperative catalysis} j. serb. chem. soc. 81 (12) 1335–1343 (2016) udc 547.361’811+542.913+544.4: jscs–4931 66.095.25:547.1–41 original scientific paper 1335 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. synthesis of vinyldihydropyran by cooperative catalysis bojan vulović1**, ivana marić1#, radomir matović2 and radomir n. saičić1* 1faculty of chemistry, university of belgrade, studentski trg 16, p. o. box 51, 11158 belgrade 118, serbia and 2ictm, center for chemistry, university of belgrade, njegoševa 12, belgrade, serbia (received 2 november, revised 10 november, accepted 16 november 2016) abstract: δ5-unsaturated aldehydes with a suitably positioned allylic halide, or phosphate, leaving group undergo doubly-catalyzed cyclization to give dihydropyran derivatives. the cyclization proceeds under the synergetic action of diazabicycloundecene and pd(pph3)4. this type of transformation was also accomplished with an aryl ketone. keywords: dihydropyran; cooperative catalysis; cyclization; organopalladium reagents. introduction catalytic methods are the most important tools in the armamentarium of organic synthesis. traditionally, organotransition metal catalysis1 and organocatalysis2 were considered as two distinct, separate and mutually exclusive approaches. however, within the last decade, the scientific community has witnessed a merger of these two approaches into a sophisticated catalytic procedure named cooperative catalysis3 (also known as dual catalysis, or tandem catalysis; terminological classification has recently been proposed).4 with a proper design of the reaction sequence and the appropriate choice of catalysts, a rapid increase in complexity is possible within a single synthetic step, which contributes greatly to the efficiency of the synthetic procedure. alternatively, under conditions of cooperative catalysis, the double activation of the reaction partners allows reactions that are otherwise difficult, or impossible, to proceed under “conventional” reaction set-ups. a prominent example of cooperative catalysis is a combination of enamine catalysis with palladium catalysis. the first example of intermolecular carbon– *,** corresponding authors. e-mail: (*)rsaicic@chem.bg.ac.rs; (**)bvulovic@chem.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc161102103v 1336 vulović et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. –carbon bond formation, described by cordova and coworkers, involved the enamine-catalyzed allylation of cyclohexanone with in situ created π-allylpalladium complex (scheme 1, example 1).5 this was followed by the intramolecular variant, which gave rise to fiveand six-membered rings (scheme 1, example 2).6 in this variant, a secondary amine and a pd-complex simultaneously reacted with the aldehyde and allylic parts of the substrate, respectively, thus creating two reactive species within the same molecule (the enamine and π-allylpalladium complex), which gave rise to a ring closure. further improvements allowed the cyclization to proceed in a catalytic asymmetric fashion, with excellent levels of asymmetric induction.7 however, attempts to apply this protocol to the synthesis of vinylcyclobutane analogues resulted in heterocyclization and the vinyldihydropyrane derivative 1 was formed (scheme 1, example 3). this compound could be a useful intermediate in the syntheses of sugars. to the best of our knowledge, such a reaction (double-activated aldehydes can cyclize) had not hitherto been reported in literature,8 but no examples of simple aldehydes giving enol-ethers are known, and hence, it was decided to examine the reaction in detail. pd(pph3)4 (cat) eto2c co2et br ohc eto2c co2et pd pyrrolidine (cat), et3n thf, rt n ohc eto2c co2et o pd(pph3)4 (cat) pyrrolidine (cat) dmso, rt + oac on + pd pd(pph3)4 (cat)cho eto2c co2et br pyrrolidine (cat), et3n thf, rt eto2c co2et ohcx o eto2c co2et (1) (2) (3) previous work: previous work: this work: 1 scheme 1. the aim of this study: previous works and the present work. results and discussion the most often used and the most easily accessible precursors of π-allylpalladium complexes are halides, acetates and phosphates.9 therefore, it was decided to prepare all three types of precursors. their syntheses were accomplished as presented in scheme 2. malonic ester 2, the preparation of which was synthesis of vinyldihydropyran 1337 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. described earlier,10 was alkylated with difunctional halides 3–5, to give esters 6–9, respectively. acetates 7 and 9 were converted into the corresponding phosphates 8 and 10 upon treatment with diethylphosphoryl chloride; both eand z-isomer were prepared. upon treatment with 3 m hydrochloric acid, acetals 6–10 were then hydrolyzed into the corresponding aldehydes 11–15. scheme 2. synthesis of the cyclization precursors. initial experiments were performed with bromide 11. tetrakis(triphenylphosphine)palladium (pd(pph3)4) was used as a metal catalyst, whereas optimization of the reaction conditions involved screening of the base (triethylamine (et3n), n,n-diisopropylethylamine (dipea), 1,8-diazabicyclo[5.4.0]undec-7-ene (dbu), 1,5-diazabicyclo[4.3.0]non-5-ene (dbn), the phosphazene base 2-tert -butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine (bemp)) and solvent (tetrahydrofuran (thf), dimethylformamide (dmf), dimethyl sulfoxide (dmso) and acetonitrile (ch3cn)). the best result was obtained using 5 mol % of pd(pph3)4 in the presence of 1 equivalent of dbn as the base, in a solvent mixture thf/dmso, at room temperature; under these conditions the reaction was complete within 1 h and afforded the targeted dihydropyrane derivative 1 in 36 % yield (in one experiment, product 1 was obtained in 56 % yield, but subsequently, this result could not be repeated; scheme 3). substrates with phosphate leaving groups (13 and 15) behaved similarly, affording the product in identical yield, albeit the reaction was somewhat faster (with respect to bromide 11). the geometry of the alkene bond was without importance for the reaction outcome, as the eand the z-isomers (13 and 15) gave identical results. no side reactions were observed; in addition to the starting compounds, dihydropyran 1 was the only spot that could be noticed on tlc. therefore, it remains unclear what happened with a part of the material and why the yield of 1 was modest. 1338 vulović et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. acetate precursors (12 and 14) did not react at room temperature. when left over night at 50 °c, both 12 and 14 gave the corresponding decarbethoxylated products 16 (64 %) and 17 (32 %), with retention of geometry of the alkene bond. it is well known that decarbethoxylations in dmso require relatively harsh reaction conditions (i.e., temperatures higher than 140 °c)11 and therefore, it was surprising to find that this reaction occurred at 50 °c. the possible mechanistic explanation, presented in scheme 4, involves anchimeric assistance: if the formation of the π-allylpalladium complex is slow (which is apparently the case with the acetate precursors), enolate 18 attacks the ester group intramolecularly and creates the enol-lactone 19. this species then hydrolyses to give the mono-acid 20 that could undergo decarboxylation at relatively low temperatures. scheme 3. cyclization reactions of various substrates. o oac h eto2c co2et oac o co2et eto o o oac co2et o h2o oac co2et o b o co2et oac -co2 12 or 14 18 19 20 16 or 17 ho2c scheme 4. mechanism of the side reaction with acetates. to find out whether ketones could be used as substrates for the reaction, substrate 22 was prepared, as shown in scheme 5. the phenyl substituent was synthesis of vinyldihydropyran 1339 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. expected to both direct (only one side of the ketone is available for deprotonation) and facilitate enolization (through conjugation in the styrene-like enolate). when submitted to the reaction conditions, 22 afforded the corresponding dihydropyran derivative 23 in 38 % yield. here again, no other products could be detected by tlc, or isolated during the purification of the reaction mixture. scheme 5. synthesis of the ketone precursor and its cyclization. an attractive possibility would be to perform the reaction with chiral ligands on palladium and obtain a chiral product in a catalytic asymmetric reaction. unfortunately, substituting axially chiral ligands (binap, biphep) for pph3 resulted in no reaction at all. a plausible reaction mechanism is represented in scheme 6. whereas dbn effects enolization of the starting aldehyde, pd(pph3)4 creates π-allylpalladium complex within the same molecule. the doubly activated species 24 undergoes cyclization to 1 and expels the pd complex that starts a new catalytic cycle. scheme 6. the proposed reaction mechanism. to conclude, aldehydes and ketones with a suitably positioned allylic leaving group undergo pd-catalyzed 6-exo-heterocyclization to give vinyldihydropyran derivatives. further studies are needed in order to establish the scope and limitations of the reaction, and to accomplish it with asymmetric induction. experimental all chromatographic separations were performed on silica, 10–18, 60 å, icn biomedicals.12 petroleum ether refers to a fraction with a boiling point range 68–72 °c. standard 1340 vulović et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. techniques were used for the purification of reagents and solvents.13 the nmr spectra were recorded on a varian gemini 200 (1h-nmr at 200 mhz and 13c-nmr at 50 mhz), and on bruker avance iii 500 (1h-nmr at 500 mhz and 13c-nmr at 125 mhz) instruments. chemical shifts are expressed in ppm (δ) using tetramethylsilane as an internal standard, coupling constants (j) are in hz. the ir spectra were recorded on a nicolet 6700 ft instrument, and are expressed in cm-1. mass spectra were obtained on an agilent technologies 6210 tof lc/ms instrument (lc: series 1200). analytical and spectral data of the synthesized compounds are given in supplementary material to this paper. (e)-diethyl 2-(4-bromobut-2-en-1-yl)-2-(2,2-diethoxyethyl)malonate (6). this compound was prepared according to the procedure described earlier.10 (e)-diethyl 2-(4-acetoxybut-2-en-1-yl)-2-(2,2-diethoxyethyl)malonate (7). sodium hydride (195 mg, 8.0 mmol) was added to a cold (0 °c) solution of compound 2 (840 mg; 3.04 mmol) in thf (10 ml) and the mixture was stirred for 15 min under an argon atmosphere. compound 4 (1.40 g; 7.25 mmol) was added and the reaction mixture was stirred for 2.5 h, during which time the mixture was allowed to reach room temperature. the reaction mixture was partitioned between water and dichloromethane, the organic layer was washed with water and saturated aq. nahco3, dried over anh. mgso4 and concentrated under reduced pressure. purification of the residue by dry-flash chromatography (sio2; eluent: petroleum ether/etoac = = 9/1) afforded 1.19 g (98 %) of the title compound 7, as a pale yellow liquid. (e)-diethyl 2-(2,2-diethoxyethyl)-2-{4-[(diethoxyphosphoryl)oxy]but-2-en-1-yl}malonate (8). part 1: hydrolysis of 7 and preparation of (e)-diethyl 2-(2,2-diethoxyethyl)-2-(4-hydroxybut-2-en-1-yl)malonate. a solution of compound 7 (802 mg; 2.06 mmol) and k2co3 (285 mg; 2.06 mmol) in a solvent mixture meoh/h2o (1:1; 16 ml) was stirred for 5 h at 0 °c, then diluted with dichloromethane (120 ml). the organic extract was washed with water (2×30 ml), dried over anh. mgso4 and concentrated under reduced pressure. purification by dry flash chromatography (sio2, eluent: petroleum ether/etoac = 4/1 to 1/1) afforded 28 mg of the starting compound 7, followed by 661 mg (96 %) of (e)-diethyl 2-(2,2-diethoxyethyl) -2-(4-hydroxybut-2-en-1-yl)malonate, as a pale-yellow oil. part 2: phosphorylation of (e)-diethyl 2-(2,2-diethoxyethyl)-2-(4-hydroxybut-2-en-1-yl)malonate. diethyl chlorophosphate (250 μl; 1.73 mmol) was added to a cold (0 °c) solution of (e)-diethyl 2-(2,2-diethoxyethyl)-2-(4-hydroxybut-2-en-1-yl)malonate (600 mg; 1.73 mmol), et3n (362 μl; 2.60 mmol) and 4-(dimethylamino)pyridine (dmap, 10.6 mg; 0.09 mmol) in thf (5.6 ml) and the reaction mixture was stirred for 8 h. the reaction mixture was diluted with dichloromethane (100 ml), washed with water (2×70 ml), dried over anh. mgso4 and concentrated under reduced pressure. purification of the residue by dry-flash chromatography (sio2, eluent: petroleum ether/etoac = 9/1) afforded 505 mg (60 %) of the title compound 8, as a pale-yellow liquid. (z)-diethyl 2-(4-acetoxybut-2-en-1-yl)-2-(2,2-diethoxyethyl)malonate (9). according to the procedure for compound 7, starting from compounds 2 (1.1 g; 4.01 mmol) and 5 (1.9 g; 9.37 mmol), the title compound 9 was obtained (1.48 g; 95 %) as a colorless liquid. (z)-diethyl 2-(2,2-diethoxyethyl)-2-{4-[(diethoxyphosphoryl)oxy]but-2-en-1-yl}malonate (10). part 1: hydrolysis of 9 and preparation of (z)-diethyl 2-(2,2-diethoxyethyl)-2-(4-hydroxybut-2-en-1-yl)malonate. according to the procedure for the hydrolysis of compound 7, starting from compound 9 (579 mg; 1.5 mmol), 348 mg (67 %) of (z)-diethyl 2-(2,2-diethoxyethyl)-2-(4-hydroxybut-2-en-1-yl)malonate was obtained, as a colorless viscous liquid. synthesis of vinyldihydropyran 1341 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. part 2: phosphorylation of (z)-diethyl 2-(2,2-diethoxyethyl)-2-(4-hydroxybut-2-en-1-yl)malonate. according to the procedure for the phosphorylation of (e)-diethyl 2-(2,2-diethoxyethyl)-2-(4-hydroxybut-2-en-1-yl)malonate, starting from (z)-diethyl 2-(2,2-diethoxyethyl)-2-(4-hydroxybut-2-en-1-yl)malonate (250 mg; 0.72 mmol), the title compound 10 was obtained (250 mg; 72 %) as a colorless, viscous liquid. (e)-diethyl 2-(4-bromobut-2-en-1-yl)-2-(2-oxoethyl)malonate (11). 3 m hcl (1.8 ml) was added dropwise to a solution of compound 6 (250 mg; 0.61 mmol) in thf (1.5 ml), and the reaction mixture was stirred at r.t. for 1 h. the reaction mixture was neutralized by the addition of saturated aq. nahco3 (10 ml), extracted with etoac (3×20 ml), dried over anh. mgso4 and concentrated under reduced pressure. purification by column chromatography (sio2, eluent: petroleum ether/etoac = 9/1) afforded 110 mg (60 %) of the title aldehyde 11, as a colorless oil. the compound is unstable, and was immediately used for the cyclization step. (e)-diethyl 2-(4-acetoxybut-2-en-1-yl)-2-(2-oxoethyl)malonate (12). according to the procedure for the preparation of 11, starting from compound 7 (89 mg; 0.23 mmol), 36 mg (50 %) of the title compound 12 was obtained, as a colorless oil (purification by column chromatography, sio2, eluent: petroleum ether/etoac = 9/1). the compound is unstable, and was immediately used for the cyclization step. (e)-diethyl 2-{4-[(diethoxyphosphoryl)oxy]but-2-en-1-yl}-2-(2-oxoethyl)malonate (13). according to the procedure for the preparation of 11, starting from compound 8 (50 mg; 0.10 mmol), 41 mg (75 %) of the title compound 13 was obtained, as a colorless oil (purification by column chromatography, sio2, eluent: petroleum ether/etoac = 9/1). the compound is unstable, and was immediately used for the cyclization step. (z)-diethyl 2-(4-acetoxybut-2-en-1-yl)-2-(2-oxoethyl)malonate (14). according to the procedure for the preparation of 11, starting from compound 9 (156 mg; 0.40 mmol), 30 mg (35 %) of the title compound 14 was obtained, as a colorless oil (purification by column chromatography, sio2, eluent: petroleum ether/etoac = 9/1). the compound is unstable, and was immediately used for the cyclization step. (z)-diethyl 2-{4-[(diethoxyphosphoryl}oxy]but-2-en-1-yl}-2-(2-oxoethyl)malonate (15). according to the procedure for the preparation of 11, starting from compound 10 (45 mg; 0.09 mmol), 21 mg (54 %) of the title compound 15 was obtained, as a colorless oil (purification by column chromatography, sio2, eluent: toluene/etoh = 98/2). the compound is unstable, and was immediately used for the cyclization step. diethyl 2-vinyl-2,3-dihydro-4h-pyran-4,4-dicarboxylate (1). the cyclization procedure. pd(pph3)4 (2.6 mg; 5 mol %) was added to a solution of compound 11 (15 mg; 0.04 mmol) in thf (0.5 ml), followed by the addition of dbn (5.3 μl) and the reaction mixture was stirred at r.t. for 10 min under an argon atmosphere. dmso (0.5 ml) was added and the reaction mixture was stirred at r.t. for 1 h, when tlc indicated the complete consumption of the starting material. the reaction mixture was concentrated under reduced pressure and the residue was purified by dry flash chromatography (sio2, eluent: petroleum ether/etoac = = 9/1) to give 4.1 mg (36 %) of the title dihydropyrane derivative 1, as a colorless oil. (e)-ethyl 6-acetoxy-2-(2-oxoethyl)hex-4-enoate (16). de-ethoxycarbonylation of compound 12. according to the procedure for the synthesis of compound 1, save that the reaction was performed at 50 °c for 7 h, starting from compound 12 (16.5 mg; 0.05 mmol), after purification by dry flash chromatography (sio2, eluent: petroleum ether/etoac = 9/1), the title compound 16 (10 mg, 64 %) was obtained as a colorless, viscous liquid. (z)-ethyl 6-acetoxy-2-(2-oxoethyl)hex-4-enoate (17). de-ethoxycarbonylation of compound 14. according to the procedure for the synthesis of compound 1, save that the reaction was performed at 50 °c, for 17 h. starting from compound 14 (12.0 mg; 0.04 mmol), after 1342 vulović et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. purification by dry flash chromatography (sio2, eluent: benzene/etoac = 9/1), the title compound 17 (3 mg, 32 %) was obtained as a colorless, viscous liquid. diethyl 2-(2-oxo-2-phenylethyl)malonate (21). to a suspension of nah (0.18 g, 0.75 mmol) in thf (10 ml) was added diethyl malonate (1.2 g, 0.75 mmol) in thf (15 ml) at 0 °c, followed by the addition of 2-bromo-1-phenylethan-1-one (1.25 g, 0.75 mmol). the mixture was stirred at room temperature overnight and then saturated aqueous solution of nh4cl was added. the aqueous layer was extracted with et2o, the combined organic extract was washed with water and brine, dried over anh. na2so4, and concentrated in vacuo. the residue was purified by column chromatography (sio2; eluent: petroleum ether/ethyl acetate = = 8/2) to afford 1.79 (86 %) of the title compound 21, as a colorless oil. diethyl (e)-2-(4-bromobut-2-en-1-yl)-2-(2-oxo-2-phenylethyl)malonate (22). a solution of compound 21 (0.278 g, 1 mmol) in thf (7 ml) was added to a stirred suspension of sodium hydride (0.028 g, 1.2 mmol) in thf (5 ml), at 0 °c. after 15 min, (e)-1,4-dibromo -2-butene (0.535 g, 2.5 mmol, 2.5 equiv.) was added in one portion, and the reaction mixture was stirred for 2 h at 0 °c. the reaction mixture was diluted with dichloromethane (30 ml), washed with water (4×15 ml), saturated aq. nahco3 (15 ml), dried over anhydrous na2so4, filtered and concentrated under reduced pressure. purification by dry flash chromatography (sio2; eluent: petroleum ether/ethyl acetate = 9/1) afforded 0.360 g (88 %) of the title compound 22 as a colorless, viscous oil. diethyl 6-phenyl-2-vinyl-2,3-dihydro-4h-pyran-4,4-dicarboxylate (23). a solution of compound 22 (0.043 g, 0.104 mmol), pd(pph3)4 (6 mg, 5.2 μmol, 5mol %), dbn (7.7 mg, 0.062 mmol, 60 mol %) in a solvent mixture thf/dmso (1/1, 1 ml) was stirred at r. t. under an argon atmosphere. the reaction was monitored by tlc (eluent: petroleum ether/ethyl acetate = 9/1). after 2 h, the reaction mixture was diluted with dichloromethane (40 ml), washed with water (4×15 ml), dried over anhydrous na2so4, filtered and concentrated under reduced pressure. purification of the residue by dry flash chromatography (sio2; eluent: petroleum-ether/ethyl acetate = 9/1) afforded 10.2 mg (38 %) of the title compound 23, as a colorless oil. supplementary material analytical and spectral data of the synthesized compounds are available electronically at the pages of the journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgment. this work was supported by the ministry of education, science and technological development of the republic of serbia (project no. on172027). и з в о д синтеза винилдихидропирана кооперативном катализом бојан вуловић1, ивана марић1, радомир матовић2 и радомир н. саичић1 1хемијски факултет, универзитет у београду, студентски трг 16, п. пр. 51, 11158 београд и 2ихтм центар за хемију, универзитет у београду, његошева 12, београд δ5-незасићени алдехиди, који у погодном положају поседују одлазећу групу као што је алилни бромид или фосфат, подлежу двоструко катализованој циклизацији и дају дериват дихидропирана. циклизација се одвија под синергичким дејством диазабициклоундецена и тетракис(трифенилфосфин)паладијума. ова трансформација остварена је и на арил-кетону. примљено 2. новембра, ревидирано 10. новембра, прихваћено 16. новембра 2016) synthesis of vinyldihydropyran 1343 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. references 1. a) j. f. hartwig, organotransition metal chemistry from bonding to catalysis, university science books, mill valley, ca, usa, 2010; b) m. beller, c. bolm, transition metals for organic synthesis: building blocks and fine chemicals, 2nd ed., vols. 1 and 2, wiley–vch, weinheim, germany, 2004 2. a) p. i. dalko, enantioselective organocatalysis: reactions and experimental procedures, wiley–vch, weinheim, germany, 2007; b) a. berkessel, h. groger, asymmetric organocatalysis from biomimetic concepts to applications in asymmetric synthesis, wiley–vch, weinheim, germany, 2005; c) h. pellissier, tetrahedron 63 (2007) 9267 3. review articles on cooperative catalysis: a) z. shao, h. zhang, chem. soc. rev. 38 (2009) 2745; b) c. zhong, x. shi, eur. j. org. chem. 2010, 2999; c) z. du, z. shao, chem. soc. rev. 42 (2013) 1337; d) s. m. inamdar, v. s. shinde, n. t. patil, org. biomol. chem. 13 (2015) 8116; e) s. afewerki, a. cordova, chem. rev. 116 (2016) 13512 4. n. t. patil, v. s. shinde, b. gajula, org. biomol. chem. 10 (2012) 211 5. i. ibrahem, a. cordova, angew. chem. int. ed. 45 (2006) 1952 6. f. bihelovic, r. matovic, b. vulovic, r. n. saicic, org. lett. 9 (2007) 5063; additions and corrections: org. lett. 9 (2007) 5649 7. b. vulovic, f. bihelovic, r. matovic, r. n. saicic, tetrahedron 65 (2009) 65, 10485; corrigendum: 66 (2010) 3275 8. a) j. tsuji, y. kobayashi, h. kataoka, t. takahashi, tetrahedron lett. 21 (1980) 1475; b) b. m. trost, l. n. jungheim, j. am. chem. soc. 102 (1980) 7910; c) y. huang, x. lu, tetrahedron lett. 28 (1987) 6219 9. v. maslak, z. tokic-vujosevic, r. n. saicic, tetrahedron lett. 50 (2009) 1858 10. a. ivković, r. matović, r. n. saičić, j. serb. chem. soc. 67 (2002) 141 11. a. p. krapcho, synthesis 1982 (1982) 805 12. for descriptions of the technique of dry flash chromatography, see: a) l. m. harwood, aldrichim. acta 18 (1985) 25; b) vogel’s textbook of practical organic chemistry, longman scientific & technical, 5th ed., london, 1989, p. 220; c) for an account which includes some improvements of the separation technique, see: d. s. pedersen, c. rosenbohm, synthesis 2001, 2431 13. w. l. f. armarego, c. l. l. chai, purification of laboratory chemicals, 6th ed., elsevier, oxford, 2009. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {environmentally benign copper nanoparticles supported on walnut shell as a highly durable nanocatalyst for the synthesis of propargylamines} j. serb. chem. soc. 82 (0) 1211–1221 (2017) udc 546.56+539.12:544.478.1: jscs–5034 547.213’233+544.6.076.32:504.54 original scientific paper 1211 environmentally benign copper nanoparticles supported on walnut shell as a highly durable nanocatalyst for the synthesis of propargylamines fariba saadati1*, vahid leghaei1 and asghar zamani2 1department of chemistry, faculty of science, university of zanjan, p.o. box 45195-313, zanjan, iran and 2department of nanotechnology, faculty of science, urmia university, urmia 57154, urmia, iran (received 21 december 2016, revised 21 june, accepted 29 june 2017) abstract: this study was an attempt to prepare copper nanoparticles and to determine their catalytic activity employing walnut shell (ws) residue as a green support and a reusable material. the nanocatalyst was characterized though inductively coupled plasma optical emission spectrometry (icp-oes), n2-sorption analysis, x-ray powder diffraction (xrd), energy-dispersive x-ray spectroscopy (eds), and transmission electron microscopy (tem). the catalytic performance of cunps@ws was then studied in the preparation of propargylamines via a three-component reaction. the catalyst could be recovered and reused without a significant decrease in its activity over eight reaction cycles. the simple methodology described herein is an economic, ecofriendly and practical method accompanied by higher yields. keywords: walnut shell; nanoparticles; reused material; copper; propargylamine. introduction the waste production on the planet earth is growing in mass and toxicity in consequence of economic development and population growth.1 a solution to this problem is moving toward zero waste as a suitable approach to waste management that focuses on minimizing waste, reducing superfluous consumption, extending recycling, and the manufacture of materials that could be reused, reprocessed, or recycled.2 nowadays, there is growing concern about environmental problems resulting from chemical processes and materials, which has drawn much attention to the preparation of valuable and practical materials from residues as a relatively novel area.3 a solution to the problem of waste from chemical processes in the industry is replacement of classical stoichiometric reagents with efficient catalytic * corresponding author. e-mail: saadati@znu.ac.ir https://doi.org/10.2298/jsc161221081s ________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 1212 saadati, leghaei and zamani alternatives. catalysts, as the most beneficial and practical substances, would be an appropriate subject for the application of reused waste in the context of green chemistry.4 the design of catalysts that have the advantages of being reusable, biodegradable and renewable, and which are readily available, cheap and of low toxicity is extremely challenging. in recent years, heterogeneous catalysts containing metal nanoparticles have found a wide range of applications due to their high efficiency, emanating from their large surface-to-volume ratio, and because they are potentially environmentally friendly.5 transition metal nanoparticles have already proven their utility in a multitude of industrial applications. in this regard, catalysis by copper is an area of substantial scientific exploration, especially with respect to developing new environmentally friendly catalysts.6–10 versatile heterogeneous copper catalysts immobilized by various supports, such as hydroxyapatite,11 zeolite,12 magnetic nanoparticles,13 carbon nanotubes,14 silica,15 mesoporous silica,16 titania,17 clay,18 graphene,19 functionalized polystyrene,20 and metal–organic framework derived nanoporous carbon21 have been efficiently used to access propargylamines, which are key intermediates in the preparation of several biologically active nitrogen compounds.22–26 however, most of the supported catalysts have some disadvantages, such as low catalytic efficiency, complex preparation methods, disordered distribution of the active sites, and environmental problems. one of the most significant advances to increasing environmental consciousness has been the immobilization of reagents and catalysts on biomaterial supports because of their renewability and biodegradability. various natural polymers, such as cellulose,27,28 agarose,29 chitosan30 and starch,31 have been used as supports and stabilizers for copper compounds. as part of ongoing interest in the development of new preparative routes in the use of recyclable and degradable heterogeneous catalysts based on biomaterials, herein a new approach to the synthesis, characterization, and application of copper nanoparticles supported on walnut shell (cunps@ws) was developed with green chemistry in mind. walnut shell, as an agricultural by-product, is available in abundance from the walnut-processing industry and is used in abrasion,32 oil removal33 and adsorption.34–36 copper nanoparticles supported on widely available, economic, renewable, biocompatible, and biodegradable matrices could be used as a green catalyst with potential industrial applications, particularly in organic synthesis. it is shown that the resulting nanocomposite, cunps@ws, has a catalytic effect on the reaction of various aldehydes, secondary amine, and the terminal alkyne in the generation of propargylamines via the a3 coupling reaction. ________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ environmentally benign cu nanoparticles on walnut shell as catalyst 1213 experimental materials all chemicals were purchased from sigma–aldrich or merck chemical companies. deionized water was used to prepare the chemical solutions and for rinsing. purification was performed via column chromatography on silica gel 60 (n-hexane/ethyl acetate as eluent). for thin-layer chromatography (tlc), silica gel 60 f254 plates were used to follow the reaction progress. instruments and measurements inductively coupled plasma optical emission spectrometry (icp-oes) was used to determine metal concentrations. fourier transform infrared (ftir) spectra were obtained using a bruker vector 22 ftir spectrophotometer under ambient conditions in kbr/nujol mull in the wavenumber range 400–4000 cm-1. the powder x-ray diffraction (xrd) patterns were obtained using a bruker d8 advance x-ray powder diffractometer based on the cukα line and in increment 2θ scanning steps of 0.07°. nitrogen physisorption analysis was performed at the normal boiling point of liquid nitrogen using a belsorp-max system. scanning electron microscopy (sem) and energy-dispersive x-ray spectroscopy (eds) were realized using a tescan vega system. for sample preparation, the powder samples were deposited on a carbon tape, and then mounted on an aluminum stub. the size and morphology of the particles were determined using a philips cm120 electron spectroscopic microscope operated at 120 kv. 1hand 13c-nmr spectra were recorded at 250 and 62.9 mhz, respectively, in cdcl3 using a bruker avance 250 mhz nmr spectrometer. preparation of cunps@ws walnut shells, collected from local fruit, were crushed and the resulting product was sieved to the desired particle size of < 88 μm. in order to eliminate dust and soluble components, the residues were subsequently washed with deionized water and organic solvents. the prepared walnut shells were dried in an oven at 65 °c for 24 h and then stored in a desiccator. in the next step, walnut shells (1.0 g) in deionized water (10.0 ml) were placed in a schlenk flask under an argon atmosphere. the suspension was then subjected to sonication at 60 w for 45 min. then, cu(ch3coo)2·h2o (0.15 mmol, 0.03 g) dissolved in 2.0 ml of h2o was added gently to the suspension, and the slurry was stirred for 4 h at 750 rpm at room temperature. then, a solution of nabh4 (0.45 mmol, 0.017 g) in water (25 ml) was added dropwise under an argon atmosphere, and the reaction mixture was stirred at 45 °c for 12 h. finally, the material was collected by filtration, washed with deionized water, and dried under vacuum overnight. general synthetic procedure for the a3 coupling reaction of aldehyde, amine, and the terminal alkyne a mixture of aldehyde (1.0 mmol), secondary amine (1.2 mmol), terminal alkyne (1.5 mmol) and the nanocatalyst (0.3 g, 3 mol %) in 2.0 ml of acetonitrile was placed in a roundbottomed flask. the reaction mixture was stirred at 80 °c for 20 h under an argon atmosphere. after completion of the reaction, the catalyst was filtered, and the solvent was evaporated off under reduced pressure. the residue was purified via column chromatography on silica gel (n-hexane/ethyl acetate) to obtain the corresponding propargylamine. all products were verified based on their spectral analysis. ________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 1214 saadati, leghaei and zamani results and discussion initially, walnut shell that is readily available in iran37 was prepared. the resulting support was then mixed with an appropriate concentration of copper(ii) acetate. next, the prepared slurry was reduced with sodium borohydride to afford the corresponding cunps@ws. the nanocatalyst was then characterized using multiple techniques to obtain insight into the structural features of the material. the icp-oes of cunps@ws shows the presence of 0.1 mmol g–1 of copper. the porosity data of the ws and cunps@ws samples are given in table i. the liquid nitrogen adsorption–desorption analysis of the samples demonstrated that walnut shell has a brunauer–emmett–teller (bet) surface area of 10.09 m2 g–1 and a total pore volume of 0.029 cm3 g–1 prior to the immobilization of nanoparticles. the average pore diameter is around 11.42 nm. the introduction of cu species into the biomaterial matrix reduces its total pore volume and average pore diameter. this is in agreement with porosimetry results: a reduction to 0.020 cm3 g–1 in the pore volume and an average pore diameter of approximately 5.46 nm. moreover, the surface area is around 14.80 m2 g–1, which is more than that of the parent walnut shell (figs. s-1 and s-3 of the supplementary material to this paper). nitrogen adsorption–desorption analysis of the catalyst exhibited a type iii isotherm, which is generally observed for nonporous materials38 (figs. s-2 and s-4 of the supplementary material). table i. nitrogen adsorption–desorption characterization of the ws and cunps@ws samples entry catalyst as / m2 g-1 diameter, nm total pore volume, cm3 g-1 1 ws 10.09 11.42 0.029 2 cunps@ ws 14.80 5.46 0.020 the ftir spectra of ws and cunps@ws are shown in fig. 1. a comparison of the spectra showed that all the vibrations of the support remained. the vibration peak at 3420 cm–1 could be assigned to the vibration range of intraand inter-molecular hydrogen bonded oh-groups. the vibration peaks associated with c−h, c=o, and c−o stretching were observed at 2902, 1743, and 1023 cm–1, respectively. in addition, the peaks appearing at around 1600 and 1506 cm–1 may be attributed to aromatic skeleton vibrations of lignin. a comparison of the spectra revealed that many characteristic bands shifted, or the signal intensity increased when the walnut shell powder was loaded with copper. these changes in ftir spectra confirm the role of functional groups in metal binding. the xrd pattern of the nanocatalyst exhibited the characteristic diffraction peaks of cu at 2θ positions of 34.7 and 42.1° (fig. 2). the diffraction peaks are characteristic peaks of cu2o (jcpds no. 05-0667) and cu (jcpds no. 04-0836), which indicate the presence of both cu(0) and cu(i) species in the sample. reduction using a strong reducing agent nabh4 yielded both cu(0) and cu2o.28 the ________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ environmentally benign cu nanoparticles on walnut shell as catalyst 1215 broad peak appearing at around a 2θ value of 22.1° is attributable to cellulose (jcpds no. 030289).27 fig. 1. ftir spectra of: a) the ws biomaterial and b) the cunps@ws nanocatalyst. cu 2 o (111) cu (111) fig. 2. x-ray diffraction pattern of cunps@ws. furthermore, the eds analysis of the cunps@ws catalyst showed carbon, oxygen, calcium and sodium peaks, which were derived from the walnut shell. as could be seen, the presence of the copper peaks proved that cu had been successfully loaded onto the biomaterial (fig. 3). the tem image of cunps@ws confirmed the presence of the cunps with an average diameter of 10–15 nm on the support (fig. 4). to study the catalytic performance of the cunps@ws nanocatalyst in the a3 coupling reaction, a model reaction of benzaldehyde, piperidine, and phenylacetylene was further optimized on the reaction parameters such as the solvent, temperature, and catalyst loading. the effect of the solvent was studied under reflux conditions. the coupling reaction in ch3cn at 80 °c was found to produce the best yield (table ii, entry ________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 1216 saadati, leghaei and zamani 4), the use of other solvents, such as chcl3, ch2cl2, and thf, produced lower yields under the same reaction conditions (table ii, entries 1–4). the effect of the reaction temperature was examined and it was found that reducing the temperature resulted in lower yields (table ii, entries 4 and 5). a study of the three variations of catalyst loading (1–3 mol %) revealed that increasing the loading of the catalyst significantly enhanced the yield (table ii, entries 4, 6 and 7). moreover, no detectable product was found for the reaction in the presence of walnut shell alone, i.e., not bearing copper nanoparticles, as the catalyst (table ii, entry 8). fig. 3. energy-dispersive x-ray spectrum of cunps@ws. fig. 4. transmission electron microscopy images of cunps@ws. based on the optimized conditions, the scope and limitations of the novel nanocatalyst were examined for a variety of aldehydes and amines possessing different functional groups. as shown in table iii, the reaction with various alde________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ environmentally benign cu nanoparticles on walnut shell as catalyst 1217 hydes, such as aliphatic, aromatic and heterocyclic aldehydes, and cyclic amines, such as morpholine and piperidine, were found to proceed well, and propargylamines were produced in high yields without the generation of any traceable side product. table ii. optimization conditions for the three-component reaction of benzaldehyde, phenylacetylene, and piperidine using cunps@ws as a nanocatalyst; reaction conditions: benzaldehyde (1 mmol), piperidine (1.2 mmol), phenylacetylene (1.5 mmol), catalyst (x mol %), 2 ml solvent, ar atmosphere, 20 h entry x / mol % solvent t / °c yielda, % 1 3 ch3cl 60 20 2 3 ch2cl2 40 40 3 3 thf 66 45 4 3 ch3cn 80 91 5 3 ch3cn 45 40 6 2 ch3cn 80 70 7 1 ch3cn 80 57 8 –b ch3cn 80 – aisolated yield; bcatalyst: walnut shell (0.3 g) notably, this nanocatalyst had an equal effect on aromatic aldehydes bearing either electron-withdrawing or electron-donating groups, such as methoxy-, methyl-, bromoand chloro-groups, and the respective propargylamines were produced in good to excellent yields. moreover, heterocyclic aldehyde and 1-naphthaldehyde successfully reacted with piperidine and phenylacetylene to provide the corresponding coupling products (table iii, entries 13 and 15). it is important to note that even an aliphatic aldehyde produced the corresponding product in high yield (table iii, entry 16). these results strongly confirm the outstanding performance and generality of the cunps@ws nanocatalyst in terms of the catalysis of the three-component reactions. the recoverability and reusability of the nanocatalyst were also examined via isolating the catalyst from the reaction of benzaldehyde, piperidine, and phenylacetylene under standard reaction conditions (fig. 5). for this purpose, after the completion of the reaction, the reaction mixture was filtered and the catalyst was washed with acetonitrile. the catalyst was recovered and reused at least eight times, with its activity slowly decreasing over the repeated runs under ________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 1218 saadati, leghaei and zamani the same reaction conditions as before. the results proved the good durability, reusability, and recoverability of the cunps@ws nanocatalyst. table iii. three-component reactions of aldehydes, amines, and phenylacetylene in the presence of the cunps@ws nanocatalyst; conditions: aldehyde (1 mmol), amine (1.2 mmol), phenylacetylene (1.5 mmol), catalyst (3 mol %), acetonitrile (2 ml), 80 °c, ar atmosphere, 20 h entry r1 r22nh yield a, % 1 c6h5 piperidine 91 2 c6h5 morpholine 85 3 4-och3-c6h4 morpholine 89 4 4-ch3-c6h4 morpholine 90 5 4-ch3-c6h4 piperidine 86 6 4-ch3-c6h4 pyrrolidine 76 7 3-ch3-c6h4 piperidine 65 8 4-br-c6h4 piperidine 90 9 4-cl-c6h4 piperidine 83 10 2-cl-c6h4 piperidine 89 11 2-cl-c6h4 morpholine 87 12 2,4-cl2-c6h3 piperidine 84 13 1-naphtyl piperidine 90 14 1-naphtyl morpholine 83 15 2-furanyl piperidine 85 16 hexyl morpholine 87 aisolated yield fig. 5. reusability of the cunps@ws nanocatalyst in the three-component reaction of benzaldehyde, piperidine, and phenylacetylene. in another experiment, a filtration test was performed to determine the possibility of weakening the interaction between cu nanoparticles and walnut shell. ________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ environmentally benign cu nanoparticles on walnut shell as catalyst 1219 to this end, the above-mentioned model reaction was performed under the same conditions. after 10 h and before the complete consumption of all the starting materials, the reaction mixture was filtered. a conversion percent of 43 % was obtained at this stage. then, the reaction was allowed to continue for another 10 h. this brought about a conversion of 60 %, indicating negligible leaching. the leaching of copper species to reaction medium was 0.18 % as determined by icp analysis. in a final experiment, the catalytic performance of cunps@ws was compared with that of a number of previously reported copper nanoparticles containing heterogeneous catalysts in the synthesis of propargylamine from benzaldehyde, piperidine, and phenylacetylene (table iv). the recycling properties of the nanocatalyst introduced in the present study were found to be superior to those of most of the previously reported catalytic systems. moreover, in most other cases, the reaction was realized at a temperature higher than that employed for the present study. these findings confirmed the high efficiency of the present nanocatalyst in the a3 coupling reaction. table iv. a comparison of the cunps@ws nanocatalyst with the previously reported copper nanoparticles catalytic systems in the synthesis of propargylamine from benzaldehyde, piperidine, and phenylacetylene entry catalyst conditions recycling times yield, % ref. 1 cunps mecn, reflux, n2, 6h 4 94 39 2 sio2–nhc–cuia neat, 70 °c, n2, 4 h 5 91 40 3 cunps/tio2 neat, 70 °c, 7 h 4 90 17 4 cu/ncntb thf, 70 °c, n2, 7 h 4 85 14 5 cu0–montc toluene, 110 °c, 6 h 3 94 18 6 cu/gd toluene, 100 °c, ar, 24 h – 84 19 7 cunps@mof-5–ce toluene, 110 °c, n2, 6 h – 96 21 8 cunps@ws mecn, 80 °c, ar, 20 h 8 91 this work asilica-immobilized n-heterocyclic carbenes cu(i) complexes; bcopper nanoparticles stabilized on nitrogen -doped carbon nanotubes; ccopper nanoparticles stabilized on modified montmorillonite; dcopper nanoparticles supported on graphene; ecopper nanoparticles supported on metal–organic framework derived nanoporous carbon conclusions the present study used empirical findings to show the potential of walnut shell residue as a representative of biomaterials and green supports. the copper nanoparticles supported on walnut shell showed superior catalytic activity in the three-component coupling reaction of a wide range of aromatic, aliphatic, and heterocyclic aldehydes with secondary amines, and a terminal alkyne. the catalyst was reused and recycled for eight consecutive runs. this new material offers the advantages of being renewable, biodegradable, and stable under the reaction conditions. considering the results, the stated simple methodology could be extended for the preparation of a large number of new nanocatalysts. ________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 1220 saadati, leghaei and zamani supplementary material additional data and the results of the characterization of the products are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgment. this work was supported by the research council of university of zanjan (grant no. 6613). и з в о д еколошки повољан поступак синтезе пропаргиламина употребом стабилног нанокатализатора добијеног из наночестица бакра на спрашеној љусци ораха као носачу fariba saadati1, vahid leghae1 и asghar zamani2 1department of chemistry, faculty of science, university of zanjan, p.o. box 45195-313, zanjan, iran и 2department of nanotechnology, faculty of science, urmia university, urmia 57154, urmia, iran током истраживања припремљене су наночестице бакра (cunps) на спрашеној љусци ораха (ws) као еколошки прихватљивог носача, и испитана је њихова каталитичка активност. нанокатализатор је окарактерисан индуктивно-куплованом оптичком емисионом спектрометријом (icp-oes), n2-сорпционом анализом, рендгенском анализом праха (xrd), енергетски дисперзионом рендгенском спектроскопијом (eds), и трансмисионом електронском микроскопијом (тем). каталитичке способности cunps@ws су испитане у синтези пропаргиламина у трокомпонентним реакцијама. катализатор може да се регенерише и поново користи без значајнијег пада каталитичке активности током осам реакционих циклуса. приказани поступак је једноставан, економичан, еколошки прихватљив, практичан и даје висок принос производа. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice an apparatus proposed for density measurements in compressed liquid regions at pressures of 0.1–60 mpa and temperatures of 288.15–413.15 k j. serb. chem. soc. 80 (8) 1073–1085 (2015) udc 547.216+547.533+547.412.1:532.14+ jscs–4781 542.3:537.11 original scientific paper 1073 an apparatus proposed for density measurements in compressed liquid regions at pressures of 0.1–60 mpa and temperatures of 288.15–413.15 k gorica r. ivaniš#, aleksandar ž. tasić*, ivona r. radović#, bojan d. djordjević#, slobodan p. šerbanović# and mirjana lj. kijevčanin# faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11120 belgrade, serbia (received 27 november 2014, revised 16 march, accepted 17 march 2015) abstract: in this work, an apparatus for density measurements in the compressed liquid regions is presented. this installation is based on the use of a dma hp density measuring cell and a dma 5000 densimeter (both instruments are products of anton paar, gratz, austria). calibration of the dma hp cell was performed by applying the classical method in which a vacuum, water and n-decane were recommended to be used as calibration fluids. to test the capabilities of the set-up, the densities of n-hexane, toluene and dichloromethane were measured in the temperature interval 288.15 to 413.15 k and the pressure range 0.1–60 mpa. the obtained results were compared with the corresponding values found in the literature. depending on the literature selected for comparison (the temperature and pressure ranges available), the average absolute percentage deviations were for n-hexane, 0.03–0.10 %; for toluene, 0.04–0.08 % and for dichloromethane, 0.02–0.03 %. a deeper insight into the results of this work showed that most of them were in good agreement with the literature values; higher discrepancies were evidenced in the vicinity of the ends of the temperature and pressure ranges. keywords: high pressure; elevated temperature; density; n-hexane; toluene; dichloromethane. introduction it is well known that density represents one of the most important properties of fluids, from both the theoretical and practical points of view. namely, accurate density data can help to clarify molecular structure of pure liquid substances and of their mixtures at defined temperatures, pressures and composition. some essential liquid properties that can be derived from density * corresponding author. e-mail: tasic33@gmail.com # serbian chemical society member. doi: 10.2298/jsc141127026i _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1074 ivaniš et al. measurements are thermomechanical coefficients, such as the isothermal compressibility, κt, and the isobaric thermal expansion coefficient, αp. based on these properties, the internal pressure, important for studying attractive and repulsive forces present in liquids, can also be determined. by coupling the mentioned isothermal and isobaric properties, κt and αp, and by using the isentropic compressibility κs (that is related to the thermodynamic speed of sound), some additional properties can be calculated; in this respect, the isobaric heat capacity and the difference in the isobaric and isothermal heat capacity (cp–cv) could be mentioned. from the practical point of view, density data are of utmost importance in the development of new and the testing of existing equations of state; these equations have significant value in the design and operation of production plants in chemical and related process industries. in our group, over a longer period, some important physical/thermodynamic and transport properties have been investigated; the obtained results were correlated and several modern prediction methods were tested. mostly, pure non-electrolyte substances and mixtures were investigated, although systems with ionic liquids were also included.1–5 these activities were performed under ambient pressure and in the temperature range up to 333.15 k. in this work, the results of an effort to extend the research to the conditions of elevated temperatures and high pressures are presented. in this respect, the proposed apparatus, based on the principle given by gardas et al.,6 was developed and constructed. the classical method of lagurette et al.,7 that was recently adjusted by comuñas et al.8 for use in broad ranges of temperature and pressure, was chosen for calibration. a vacuum and water were used as calibration fluids, except under conditions where water was no longer in liquid state, when the calibration fluids were vacuum and n-decane. the proposed set-up was tested using n-hexane, since it was suggested as a model substance for compressed simple liquids.9–12 in addition, toluene and dichloromethane were also selected for testing the employed device and the experimental procedure followed. experimental materials n-hexane, n-decane and toluene were purchased from merck with purities of ≥99.0, ≥99 and ≥99.9 mass %, respectively. sigma–aldrich supplied dichloromethane with a purity of ≥99.9 mass %. the purities of the used substances were checked by comparing their measured densities at atmospheric pressure and at various temperatures with the corresponding literature values (table s-i of the supplementary material to this paper) and they were in a good agreement, within 0.2, 0.5 and 0.7 kg m-3 for n-hexane, toluene and dichloromethane, respectively. nitrogen 5.0 was provided by messer tehnogas, ad, with a purity of >99.999 mass %. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ density measurements at high pressures 1075 the double deionized millipore water, provided by veolia ionpro-lx mkii system, was used as a calibration fluid. the treated water has a specific conductivity of 2.5 µs cm-1 and a specific resistance of 18 mω cm. apparatus – description of the set-up the compressed liquid densities at various temperatures and pressures were experimentally studied, employing an anton paar dma hp density-measuring cell for high pressures and temperatures, connected to an anton paar dma 5000 vibrating tube densimeter, as shown schematically in fig. 1. fig. 1. scheme of the apparatus for measuring densities at high pressures and elevated temperatures. as mentioned in the introductory section, the proposed apparatus has been based on the principle proposed by gardas et al.,6 although some additional parts of equipment have been brought into the system to construct the set-up. the apparatus was used in broad ranges of temperature and pressure, varying between 288.15–413.15 k and 0.1–60 mpa, respectively. each selected temperature was controlled with an integrated peltier thermostat and the expanded uncertainty (coverage factor k = 2) for the temperature was 0.01 k. a pressure generator, model 50-6-15, from high pressure equipment co. (hip), was used to adjust and control the pressure in the system; acetone was used as a hydraulic fluid, as proposed in the literature.6 the pressure in the system was measured using a pressure transducer wika, s-10, alexander wiegand gmbh & co. the transducer was calibrated up to 60 mpa and the expanded uncertainty (k = 2) for the pressure was 0.05 mpa. the period of tube vibration, τ was read on the display of the dma 5000 densimeter with a digital counter; the vibrating period was displayed to seven significant digits. all tubing (1/16″ and 1/8″ o.d.), the high-pressure valves and other high-pressure fittings shown in fig. 1 are products of hip co, usa. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1076 ivaniš et al. a syringe pump, enabling precise flow control during charging of the sample fluid, was used in order to avoid the formation of micro-bubbles in the capillary tube of the densimeter. the assembly presented in fig. 1 incorporates additional pieces of equipment, some of them are mentioned here: the duoseal vacuum pump, welch model 1400, capable to reach an ultimate vacuum down to ca. 0.133 pa (0.001 torr); the vacuum was indicated by a pirani– –penning instrument; a kgw isotherm cold trap was situated near the entrance of the pump; in addition, a compressed nitrogen bottle was included in the installation. the stainless steel spiral buffer tube (1/16" o.d. and ca. 1.5 m long) guaranties the absence of diffusion of the hydraulic liquid contained in the densimeter cell.6 transferring of the collected data (period of oscillation of u-tube, cell temperature and pressure) from the dma hp to the dma 5000 was enabled by connecting the s-bus interface of the dma hp and dma 5000. the apsoftprint software program (a microsoft excel add-in) was employed to read out and transfer the measured values to a pc. loading of the set-up with the pressurizing fluid to prepare the apparatus for experimental runs, the pressurizing fluid should be introduced into the appropriate parts: the tubes (and fittings), connecting valves v2 through v5 (fig. 1), and those that join the high-pressure generator (hpg) with the pressure transducer. the hpg must also be filled. to remove air from the parts of the installation described, the valves v3 and v5 are opened, enabling access to the vacuum pump (valve v2 is closed). after several hours of evacuation, valve v5 is closed. then, a hamilton multilayer silicon rubber septum, being a part of the pressurizing fluid reservoir (not shown in detail in fig. 1), is penetrated by a needle of a syringe containing degassed acetone. in this way, acetone will occupy the entire evacuated space. the measurement procedure before starting any run, the part of circuit between the syringe pump and valve v2 (v2 and v4 were closed) was cleaned by successive use of ethanol and acetone to remove residues from the previous sample; then dry nitrogen gas was circulated through this part of the system for an appropriate period. after a slight release of the connection between the buffer tube and the entrance of the valve v2/v4, the syringe pump was started, dispensing the sample between the valves v1 and v2, until several drops of the fluid had been withdrawn from the apparatus; then the pump was stopped and the mentioned connection tightened again. since the tubes between the valves v1 and v2 were filled with the sample, the valve v1 was closed. hence, the sample was introduced into the system by the described operation. now, when the desired conditions of temperature and pressure in the cell were stable, the vibration period of the u-tube could be determined. in the present work, isothermal measurements were performed; at each selected isotherm, the pressure was imposed starting from its initial (lowest) value and elevating it towards the maximum value. hysteresis effects were checked at every isotherm. then, the temperature of the cell was changed and measurements at a new isotherm were performed. calibration since the vibrating tube densimeter does not generate directly the density values, it is necessary to calculate the density from the measured period of the oscillation (under a defined temperature t and pressure p). _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ density measurements at high pressures 1077 based on the model of the performance of vibrating tube established by kratky et al.,13 the period of vibration τ(t,p) can be related to the sample density ρ(t,p) by a linear function that incorporates two apparatus parameters a(t,p) and b(t,p): ( ) ( ) ( ) ( )2, , , ,t p a t p t p b t pρ τ= − (1) according to the approach of lagourette et al.,7 who hypothetically assumed that the parameter a depends only on temperature while the parameter b remains dependent upon both temperature and pressure, eq. (1) could be rewritten in the form: ( ) ( ) ( ) ( )2, , ,t p a t t p b t pρ τ= − (2) use of this equation considerably simplifies the calibration of the densimeter (i.e., the determination of its parameters a(t) and b(t,p)). this method has to be performed by measuring the oscillating period of the evacuated tube over the entire temperature range of interest. in addition, the period of the tube full of the chosen reference fluid, having the certified density, has to be measured over the entire temperature and pressure ranges of the experimental significance. lagourette et al.7 performed the calibration using the accurate density values for water of kell and whalley14 in the temperature and pressure ranges 293.15– –373.15 k and 0.1–40 mpa, respectively. comuñas et al.8 adjusted the calibration procedure of lagourette et al.7 in order to make it suitable for the new equipment (anton paar dma hpm), enabling measurements in broad ranges of temperature and pressures up to 403.15 k and up to 140 mpa, respectively. thus, the procedure of comuñas et al.8 was employed to calibrate the anton paar dma hp densimeter included in fig. 1. in that sense, the period of the oscillation of the evacuated tube was measured over the entire temperature range indicated previously. for the specific volume of water, fisher and dial15 selected the tumlirz equation in the form given in the analysis of eckert:16 0 λ ∞= + +p v v p p (3) where vp / cm 3 g-1 is the specific volume as a function of pressure and temperature; v∞ / cm 3 g-1, λ / bar cm3 g-1 and p0 / bar, are the pure water parameters. for these parameters, analytical functions were obtained15 that, in conjunction with eq. (3), accurately represent the high precision data of kell and whalley17 (within 8 ppm from 273.15–373.15 k and within 15 ppm from 373.15–423.15 k, up to 100 mpa): v∞ = 0.6980547 – 0.7435626×10-3t + 0.3704258×10-4t2 – 0.6315724×10-6t3 + 0.9829576×10-8t4 – 0.1197269×10-9t5 + 0.1005461×10-11t6 – (4) 0.5437898×10-14t7 + 0.169946×10-16t8 – 0.2295063×10-19t9 λ = 1788.316+21.55053t – 0.4695911t 2 + 3.096363×10-3t3 – 0.7341182×10-5t4 (5) p0 = 5918.499 + 58.05267t – 1.1253317t 2 + 6.6123869×10-3t3 – 1.4661625×10-5t4 (6) finally, the specific volume values for water, supplied in this way, were recalculated to density. accordingly: a) at 0.1 ≤ p ≤ 60 mpa and 288.15 ≤ t ≤ 363.15 k, calibration of the cell was realized using the data collected as described above and by employing eq. (7),8 obtained by applying eq. (2): _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1078 ivaniš et al. ( ) ( ) ( ) ( ) ( )( ) ( ) 2 2 water water water 2 2 vacuumwater , , , , , 0.1 mpa , 0.1 mpa t p t p t p t p t t t τ τ ρ ρ ρ τ τ  − = +   −   (7) here, ρwater(t,p) is the density of water at t and p; ρwater(t,p) represents the period of the oscillation of the vibrating tube full of water at t and p; τvacuum (t) stands for the period of the evacuated tube at temperature t. b) at p = 0.1 mpa and t ≥ 373.15 k: under these conditions, water is no longer appropriate to be employed as the reference fluid because it exists in the gaseous state. in the present work, n-decane was used, as proposed by comuñas et al.8 hence, the data given in the literature18 was employed; under these circumstances, the following relation was used instead of eq. (7), as shown in reference:8 ( ) ( ) ( ) ( )( ) ( ) 2 2 -decane -decane 2 2 vacuum-decane ,0.1 mpa ,0.1 mpa ,0.1 mpa , 0.1 mpa 1 ,0.1 mpa n n n t t t t t t τ τ ρ ρ τ τ  − = +  −   (8) c) at p > 0.1 mpa and t ≥373.15 k, eq. (9) given by comuñas et al.8 was used in the present work: ( ) ( ) ( ) ( ) ( )( ) ( ) 2 2 water water -decane 2 2 vacuum-decane , , , , , 0.1 mpa , 0.1 mpa n n t p t p t p t p t t t τ τ ρ ρ ρ τ τ  − = +   −   (9) by employing the described calibration procedure in the temperature and pressure ranges of interest (288.15–413.15 k and 0.1–60 mpa, respectively), numerous measurements, distributed on 16 isotherms were performed. the parameters a(t) and b(t,p) of eq. (2) were determined using eqs. (10) and (11), respectively, which were clearly presented in the explicit form by segovia et al.:19 ( ) ( )( ) ( ) ref1 2 2 vacuumref1 , 0.1 mpa , 0.1 mpa t a t t t ρ τ τ = − (10) ( ) ( ) ( )( ) ( ) ( ) 2 ref2 ref1 ref22 2 vacuumref1 , , , 0.1 mpa , , 0.1 mpa t p b t p t t p t t τ ρ ρ τ τ = − − (11) where ρref1 and ρref2 are the densities of reference fluids 1 and 2, respectively, and τref1 and τref2 are the oscillation period of the u tube full of reference fluid 1 and 2, respectively. for the interval: 0.1 ≤ p ≤ 60 mpa and 288.15 ≤ t ≤ 363.15 k, both reference fluids are water. at p = 0.1 mpa and t ≥ 373.15 k, ref1 and ref2 refer to n-decane and at p > 0.1 mpa and t ≥ 373.15 k, reference fluid 1 is n-decane while reference fluid 2 is water. as could be seen from fig. 2a, the values of a(t) decreased linearly with increasing temperature, as expected according to the conclusions of lagourette et al.7 in addition, these authors remarked that the ratio of the calibration parameters a(t)/b(t,p) is practically independent of pressure. this observation was appraised by a number of authors who employed various calibration fluid pairs.19-21 in each of these cases, it was shown that the ratio a(t)/b(t,p) decreased slightly with increasing pressure. the dependence of the ratio of the calibration parameters on pressure at the selected isotherms chosen in the present work is demonstrated is fig. 2b. the calculated expanded uncertainty of the density measurements with a coverage probability of 95 % (coverage factor k = 2) is 1.7 kg m-3 in the temperature interval 288.15– –363.15 k, and 2.7 kg m-3 at temperatures 373.15–413.15 k. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ density measurements at high pressures 1079 (a) (b) fig. 2. the calibration parameters: a) dependence of a(t) of the temperature, 288.15–413.15 k, b) the ratio of the a(t)/b(t,p) vs. pressure at different temperatures: () 288.15, () 303.15, () 318.15, () 333.15, () 343.15, () 353.15, () 363.15, () 373.15, () 393.15 and () 413.15 k. the influence of viscosity of the substances on density was also studied. a personal correspondence with the supplier anton paar provided the information necessary for the calculation of density correction due to damping effects on the vibrating tube. assuming that temperature has a greater impact on viscosity than pressure and that the viscosity decreases with increasing temperature, literature data22-24 for the viscosity at lower temperatures from the interval of interest in this work were used for the calculation. the calculated differences between the densities presented in this work and those corrected because of the viscosity effect for all three examined substances were under 0.03 kg m-3, which was significantly lower than the calculated value of the expanded uncertainty for density and hence the viscosity influence on density were neglected. results and discussion with the intention of checking the quality of the calibration parameters obtained in this study, the densities of n-hexane, toluene and dichloromethane, were experimentally determined and compared with some corresponding literature data. the vibration periods of the u tube within the dma hp densimeter full of samples, n-hexane, toluene or dichloromethane, were measured in the temperature range 288.15–413.15 k, at 16 isotherms, and at pressures up to 60 mpa. the densities of the measured substances were calculated by application of eqs. (3)–(9) and the obtained values are presented in tables s-ii–iv of the supplementary material to this paper. the following criteria, the absolute average percentage deviation (aad), the percentage maximum deviation (md) and the average percentage deviation (bias) were used to evaluate the quality of the agreement of the obtained experimental results with the corresponding literature values: _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1080 ivaniš et al. exp lit exp 1 100 n i i i i aad n ρ ρ ρ= − =  (12) exp lit exp max 100 i i i md ρ ρ ρ  − =     ; i = 1,n (13) exp lit exp 1 100 n i i i i bias n ρ ρ ρ=  −  =      (14) where n stands for the number of experimental points, ρexp denotes the experimental density and ρlit is the density value from the selected literature. n-hexane the measured data for n-hexane (table s-ii) were compared with the densities given in the work of troncoso et al.25 in the temperature range 288.15– –313.15 k and at pressures up to 40 mpa. the comparison presented in fig. 3a shows that the present measurements are in accordance with the tabulated data of troncoso et al. (aad = 0.03 % (less than 0.2 kg m–3), md = 0.09 % and bias = = –0.02 %). the agreement with the values of daridon et al.26 is moderate in the presented region (aad = 0.08 % (less than 0.6 kg m–3), md = 0.22 % and bias = –0.08 %). (a) (b) fig. 3. comparison of the experimental densities of n-hexane with literature values: a) troncoso et al.:11 (), 288.15, () 293.15, () 298.15, () 303.15, () 308.15 and () 313.15 k, and daridon et al.:26 () 293.15, () 303.15 and () 313.15; b) daridon et al.:26 () 293.15, () 303.15, () 313.15, () 323.15, () 333.15, () 343.15, () 353.15, () 363.15 and () 373.15 k. the obtained experimental results were compared with the tabulated data of daridon et al.26 in the temperature interval 293.15–373.15 k and at pressures up _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ density measurements at high pressures 1081 to 60 mpa. the calculated deviations were: aad = 0.10 % (less than 0.7 kg m–3), md = 0.29 % and bias = –0.01 %, as shown in fig. 3b. in addition, the measurements were compared with the density data of sanmamed et al.27 in the regions: 288.15–323.15 k and up to 60 mpa. the obtained values of the criteria were: aad = 0.05 % (less than 0.4 kg m–3), md = 0.17 % and bias = –0.04 % that could be considered as acceptable. in conclusion, the density measurements are in very good agreement with those of troncoso et al.25, as well as in accordance with those of sanmamed et al.27 on the other hand, the agreement with the tabulation of daridon et al.,26 which were based on speed of sound measurements, are less good than expected, but should be emphasized that the densities reported in the literature26 belong to a much wider temperature interval. toluene the experimental densities of toluene, given in table s-iii, were compared with those obtained from the equations proposed by cibulka and takagi28 over the entire temperature and pressure ranges of interest in the present work. the aad of the comparison was 0.04 % (less than 0.3 kg m–3), the md was 0.12 % and the bias was –0.04 %; hence, it could be concluded that the results obtained in the present study are in good agreement with those calculated by the procedure proposed in the literature.28 this fact could be noticed by inspecting fig. 4. density data of the present work were also compared with the data calculated from the fit given by assael et al.29 in the temperature range 288.15– 373.15 k and up to 60 mpa. the values of the criteria for this comparison were: aad = 0.08 % (less than 0.7 kg m–3), md = 0.15 % and bias = –0.08 %. it should be emphasized that the dispersion of the present data corresponded to that given in fig. 2 of reference.29 fig. 4. comparison of the experimental densities with those obtained using the equation reported by cibulka et al.28 for toluene at various temperatures: () 288.15, () 303.15, () 318.15, () 333.15, () 343.15, () 353.15, () 363.15, () 373.15, () 393.15 and () 413.15 k. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1082 ivaniš et al. in order to assess the obtained densities, an additional comparison was made. namely, segovia et al.19 gave a set of density data at various temperatures and pressures (288.15–393.15.15k and up to 60 mpa). these data were fitted to the modified tammann–tait equation for comparison with the densities given in the present work: ref ref ( ) 1 ln ( ) b t p c b t p ρ ρ =  + −  +  (15) where ρref is density at the reference pressure, pref, which was chosen to be 0.1 mpa. the parameter c was treated as temperature independent and b(t) was obtained using the polynomial expression: 2 0 ( ) ii i b t b t = =  (16) the values of the individual criteria were: aad = 0.05% (less than 0.4 kg m–3), md = 0.09 % and bias = –0.04 %. by reviewing the values of the criteria mentioned above, it could be noticed that the densities reported herein are in close agreement with the corresponding results appearing in the literature. dichloromethane gonçalves et al.22 presented a correlation for calculating the densities of dichloromethane in broad ranges of temperature and pressure that was used for comparison with the present data measured in the temperature interval 288.15– –413.15 k and under pressures of up to 60 mpa (presented in table s-iv). deviations between these two density sets were: aad = 0.03 % (less than 0.4 kg m–3), md = 0.13 % and bias = –0.002 %, indicating good agreement that is visible in fig. 5. in the work of lugo et al.,20 a density data set for dichloromethane was presented, covering temperature and pressure ranges of 293.15–353.15 k and 0.1–25 mpa. the present measurements were compared with the density values presented therein; the following deviations were reached: aad = 0.03 % (less than 0.4 kg m–3), md = 0.04 % and bias = –0.03 %, showing good agreement between the two sets of data. in addition, the densities of dichloromethane given by demiriz30 in broad temperature and pressure ranges were employed to assess the densities from the present work. these data31 were fitted to the tammann–tait equation, according to eqs. (15) and (16) and in this case pref was 1 mpa in the ranges: 270–430 k and up to 60 mpa. the densities obtained in the present work agreed quite well _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ density measurements at high pressures 1083 with those estimated using this fit. the deviations achieved were: aad = 0.03 % (less than 0.5 kg m–3), md = 0.30 % and bias = –0.03 %. values of the criteria given in this section could be considered as good and acceptable compared to those reported by the other authors. fig. 5. comparison of experimental densities with those obtained using the equation reported by gonçalves et al.24 for dichloromethane at various temperatures: () 288.15, () 303.15, () 318.15, () 333.15, () 343.15, () 353.15, () 363.15, () 373.15, () 393.15 and () 413.15 k. conclusions in this work, an apparatus proposed for compressed liquids density measurement over broad ranges of temperature and pressure was described. the anton paar dma hp and anton paar 5000 represent the central parts of the set-up. this variable volume apparatus makes use of a buffer tube, as proposed by gardas et al.6 the dma hp was calibrated by applying the method of comuñas et al.8 in the temperature and pressure ranges: 288.15 to 413.15 k and 0.1–60 mpa, respectively. the present density measurements for n-hexane, toluene and dichloromethane were compared with the corresponding literature values. this assessment showed good quality of the measurements performed. thus, it could be expected that the recommended apparatus could be reliably applied for measuring the density of a number of other compressed liquids in broad ranges of temperatures and pressures. supplementary material the experimental values of the densities of n-hexane, toluene and dichloromethane at different temperatures (288.15–413.15 k) and pressures (0.1–60 mpa), as well as at atmospheric pressure, are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. it is our pleasure to thank professor abel ferreira (faculdade de cięncias e tecnologia, universidade de coimbra, portugal) for valuable discussion. furthermore, we highly appreciate the anton paar gmbh, graz, austria for donating a dma hp density-measuring cell to our laboratory, on the occasion of the fortieth anniversary of the vibrating tube densimeter invention. the authors gratefully acknowledge the financial support _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1084 ivaniš et al. received from the research fund of ministry of education, science and technological development of the republic of serbia (project no 172063) and the faculty of technology and metallurgy, university of belgrade. и з в о д апаратура предложена за мерење густина у области компримоване течности од 0,1 до 60 mpa и на температурама од 288,15 до 413,15 k горица р. иваниш, александар ж. тасић, ивона р. радовић, бојан д. ђорђевић, слободан п. шербановић и мирјана љ. кијевчанин технолошко-металуршки факултет, универзитет у београду, карнегијева 4, 11120, београд у овом раду је предложена апаратура за мерење густине у области компримованих течности. инсталација се заснива на коришћењу ћелије за мерење густине dma hp и густиномера dma 5000 (произвођач оба производа је anton paar, грац, аустрија). калибрација ћелије dma hp је извршена применом класичне методе која препоручује употребу вакуума, воде и n-декана као калибрационих флуида. у циљу провере рада приказане апаратуре мерене су густине n-хексана, толуена и дихлорметана у температурном интервалу 288,15 до 413,15 к и опсегу притиска од 0,1 до 60 mpa. добијени резултати су упоређени са одговарајућим вредностима из литературе. у зависности од одабране литературе, односно опсега температуре и притиска, добијена су следећа средња апсоултна процентуална одступања: за n-хексан, 0,03–0,10 %; за толуен, 0,04–0,08 %, а за дихлорметан, 0,02–0,03 %. детаљнији увид у резултатe овог рада показује да је већина њих у доброј сагласности са вредностима из литературе; веће разлике су приметне у близини крајева интервала температуре и притиска. 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sanmamed, a. dopazo-paz, d. gonzález-salgado, j. troncoso, l. romaní, j. chem. thermodyn. 41 (2009) 1060 28. i. cibulka, t. takagi, j. chem. eng. data 44 (1999) 411 29. m. j. assael, h. m. t. avelino, n. k. dalaouti, j. m. n. a. fareleira, k. r. harris, int. j. thermophys. 22 (2001) 789 30. a. m. demiriz, phd thesis, university of bochum, bochum, germany, 1986. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice synthesis and antimicrobial activity of azepine and thiepine derivatives j. serb. chem. soc. 80 (7) 839–852 (2015) udc 547.71.8+547.639.5+546.98+ jscs–4763 542.913:615.281/.282 original scientific paper 839 synthesis and antimicrobial activity of azepine and thiepine derivatives nina božinović1#, irena novaković2#, slađana kostić rajačić2#, igor m. opsenica1*# and bogdan a. šolaja1**# 1faculty of chemistry, university of belgrade, studentski trg 16, p. o. box 51, 11158, belgrade, serbia and 2institute of chemistry, technology, and metallurgy, university of belgrade, njegoševa 12, 11000 belgrade, serbia (received 16 january, revised 2 february, accepted 6 february 2015) abstract: a series of new pyridobenzazepine and pyridobenzothiepine derivatives was synthesized by pd-catalyzed formation of c–n and c–s bonds. all synthesized compounds were tested for their in vitro antimicrobial activity. the pyridobenzazepine derivatives showed better antibacterial and antifungal activity than the corresponding dipyridoazepine analogue. among the synthesized azepines, derivative 8 displayed potent activity against the tested bacteria (mic ranged 39–78 µg ml-1), while azepine 12 showed promising antifungal activity (mic ranged 156–313 µg ml-1). the synthesized thiepine derivatives exhibited weak antibacterial activity, but showed pronounced antifungal activity. keywords: azepines; thiepines; heterocycles; palladium; antibacterials; antifungal. introduction the tricyclic moieties of 5h-dibenz[b,f]azepine (1)1 and dibenzo[b,f]thiepine (2)2 are important heterocyclic pharmacophores in a number of drugs. carbamazepine (3) and opipramol (4) belong to the dibenzazepine group of heterocyclic compounds. carbamazepine (3) is an anticonvulsant used to treat seizures, nerve pain and bipolar disorder,1a while opipramol (4) is a tricyclic antidepressant (tca) and is used to treat generalized anxiety disorders.1b the dibenzothiepine zotepine (5) is an atypical antipsychotic, and it is used to treat schizophrenia (fig. 1).3 over the past few decades, several different strategies were developed for the synthesis of 5h-dibenz[b,f]azepines4 and dibenzo[b,f]thiepines.2,4e,5 the use of palladium-catalyzed reactions is an efficient procedure for the synthesis of 5h *,** corresponding authors. e-mails: (*)igorop@chem.bg.ac.rs; (**)bsolaja@chem.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc150116013b _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 840 božinović et al. -dibenz[b,f]azepines,6 and methods based on the double n-arylation reaction are of particular relevance.7 hitherto, only one method employing a pd-catalyzed reaction for the construction of the dibenzothiepine core has been reported.8 the mizoroki–heck cyclisation of the corresponding diaryl thioether was used for the synthesis of dibenzothiepine 2. fig. 1. tricyclic 5h-dibenz[b,f]azepines and dibenzo[b,f]thiepines. recently, a simple and efficient pd-catalyzed method was developed for the synthesis of 5h-pyrido[4,3-b:3',4'-f]benzazepine and 5h-dipyrido[4,3-b][1]azepine compounds (scheme 1).9 scheme 1. pd-catalyzed synthesis of 5h-pyrido[4,3-b][1]benzazepine and 5h-dipyrido[4,3-b:3',4'-f]azepine compounds. the protocol is based on a pd-catalyzed double amination reaction of the corresponding stilbenes. additionally, as an expansion of the methodology, for the first time pd-catalyzed formation of c–s bonds was applied to the ring closure of a thiepine derivatives from the corresponding stilbene precursors and an s-nucleophile (scheme 1). formerly, the synthesized azepines and thiepines are shown in fig. 2. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis and antimicrobial activity of azepine and thiepine derivatives 841 herein, the synthesis of some new pyridobenzazepine and pyridobenzothiepine derivatives using the previously described methodology is presented. all synthesized compounds were evaluated for their in vitro antimicrobial activity against eight bacterial and three fungal pathogenic strains. fig. 2. structures of the synthesized azepine and thiepine derivatives. results and discussion chemistry the wittig reaction between phosphonium salt 229 and aldehydes 1910 and 2111 provided the corresponding z-stilbenes 24 and 25, respectively, in high yield. for the preparation of ethylene derivative 26, commercially available 2-bromo-5-fluorobenzaldehyde 23 was used (scheme 2). scheme 2. the synthesis of z-stilbenes 24–26. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 842 božinović et al. the syntheses of new iminostilbene compounds 27–30 were achieved using the previously described reaction conditions for pd-catalyzed double amination reactions (scheme 3).9 scheme 3. the synthesis of new pyridobenzazepine and dipyridoazepine compounds. the reactions of z-stilbenes 6, 7 and 24–26 with potassium thioacetate (1.2 equiv.) in the presence of a catalyst composed from pd2(dba)3 (5 mol %) and dppf (10 mol %) under microwave-mediated heating afforded the thiepine derivatives 16, 17 and 31–33 in moderate yields (scheme 4). it should be noted that higher proportions of ksac (2.4 eqiuv.) resulted in significantly better yields of 31 and 32, whereas the yield of thiepines 16 and 33 did not improve. in the case of stilbene 7, the higher load of ksac resulted in a complex reaction mixture. scheme 4. the synthesis of thiepine derivatives. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis and antimicrobial activity of azepine and thiepine derivatives 843 in the next synthetic step, the thiepine compound 31 was chemically transformed into its tetrahydro and biphenyl derivatives 34 and 35, respectively. after n-methylation, and nabh4 reduction, the tetrahydro derivative 34 was obtained in 66% yield. the suzuki–miyaura reaction on thiepine 31 with phenylboronic acid gave derivative 35 in moderate yield (scheme 5). the coupling reaction was performed with the catalytic system pd(oac)2/sphos–k3po4 in toluene. these transformations of thiepine 31 opened up new possibilities for the preparation of structurally diverse substituted thiepines. scheme 5. the transformations of thiepine compound 31. antimicrobial activity the synthesized azepine derivatives were screened for their antibacterial and antifungal activities against five gram-negative bacteria (escherichia coli, proteus hauseri, pseudomonas aeruginosa, salmonella enterica subsp. enterica serovar enteritidis and klebsiella pneumoniae), three gram-positive bacteria (staphylococcus aureus, micrococcus luteus atcc 10240 and m. luteus atcc 4698) and three fungal strains (candida albicans, saccharomyces cerevisiae and aspergillus brasiliensis). amikacin (amk) and chloramphenicol (chl) were used as standard antibacterials, and nystatin (nyt) and fluconazole (flc) were used as antifungal reference compounds. the minimum inhibitory concentration (mic) was determined as the lowest concentration of the compound that resulted in inhibition of bacterial, respectively fungal growth, using a broth microdilution method. the results of antibacterial activities of azepine derivatives are given in table i. the azepines 8–15 and 27–30 exhibited lower antibacterial activity with respect to amikacin (table i). compound 8 was more potent than chloramphenicol against three gram-negative bacteria (e. coli, p. hauseri and p. aeruginosa) and one gram–positive bacteria (m. luteus atcc 4698). all pyridobenzazepine derivatives (8, 10, 12, 14 and 27) showed higher inhibitory activity than the corresponding dipyridoazepine analogues (9, 11, 13, 15 and 28) against all bacteria. azepine 8 with an n,n-dimethyl substituent was 4 times more potent than the corresponding n,n-diethyl substituted analogue 10. additionally, 27, which incorporates the side chain nitrogen in pyrrolidine ring was more potent than 10, but less active than 8. the results of the antibacterial screening for compounds 8 and 14 revealed that the introduction of an oxygen _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 844 božinović et al. atom in the side chain significantly decreased the antibacterial activity. the substituted azepines 29 and 30 showed lower antibacterial potency than 8 against all the screened bacteria. table i. antibacterial minimal inhibitory concentrations (mic / µg ml-1) of the azepine derivatives cmpd. gram-negative bacteria gram-positive bacteria e. coli p. hauseri p. aeruginosa s. enterica k. pneumoniae s. aureus m. luteus atcc 10240 m. luteus atcc 4698 8 39 78 78 78 78 39 78 39 9 1250 1250 1250 1250 625 1250 1250 2500 10 156 313 313 625 625 156 313 313 11 1250 1250 1250 1250 1250 1250 1250 1250 12 39 313 313 313 313 39 156 78 13 1250 2500 1250 1250 1250 2500 1250 2500 14 313 625 625 625 625 313 625 313 15 1250 1250 1250 1250 1250 1250 1250 1250 27 78 156 313 313 313 156 156 156 28 625 625 625 625 625 625 625 625 29 78 156 313 313 313 156 313 156 30 156 313 313 313 625 313 313 313 amk 5 7 50 8 8 11 2 2 chl 62 125 250 43 62 15 31 125 finally, among the synthesized azepines, derivative 8 was the most active one and showed a broad spectrum of antibacterial activity (mic ranged 39–78 µg ml–1). the minimum inhibitory concentrations (mic) of the synthesized azepines against three fungal strains are presented in table ii. compounds 12 and 27 showed excellent activity (mic = 156 µg ml–1) against c. albicans and s. cerevisiae; they were more potent than nystatin and fluconazole. compounds 12 and 27 are 16 times more active than nystatin against c. albicans, and 8 times more potent against s. cerevisiae than nystatin. in addition, compound 8 was more potent than nystatin and fluconazole against s. cerevisiae, while derivative 29 was more active than the reference compounds against the c. albicans strain. compounds 12 and 30 showed a four-fold greater potency (mic = 313 µg ml–1) than nystatin in inhibiting the growth of the a. brasiliensis strain, but were less active when compared to fluconazole. again, as with the antibacterial activity, it was observed that the pyridobenzazepine derivatives (8, 10, 12, 14 and 27) showed better antifungal activity than the corresponding dipyridoazepine analogues (9, 11, 13, 15 and 28). the synthesized thiepines were screened for their antibacterial and antifungal activities against four gram-negative bacteria (e. coli, p. hauseri, p. aeruginosa and salmonella enterica subsp. enterica serovar enteritidis), four gram _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis and antimicrobial activity of azepine and thiepine derivatives 845 -positive bacteria (clostridium sporogenes, s. aureus, m. luteus atcc 10240 and kocuria rhizophila) and three fungal strains (c. albicans, s. cerevisiae and a. brasiliensis), using a disk diffusion method. amikacin (amk) was used as the standard antibacterial drug, and nystatin (nyt) was used as the antifungal reference compound. the antimicrobial activity was evaluated based on the diameter of the zone of inhibition. table ii. antifungal minimal inhibitory concentrations (mic / µg ml-1) of the azepine derivatives cmpd. c. albicans s. cerevisiae a. brasiliensis 8 2500 156 1250 9 2500 1250 1250 10 625 625 625 11 1250 1250 1250 12 156 156 313 13 2500 1250 1250 14 625 313 625 15 1250 1250 1250 27 156 156 625 28 625 625 1250 29 156 313 625 30 313 313 313 nyt 2500 1250 1250 flc 313 313 156 the results of antimicrobial activities of thiepine derivatives (table iii) revealed that all the tested thiepines displayed weak antibacterial activity with inhibition zones of 10–20 mm. table iii. antibacterial activity expressed as inhibition diameter zones in millimetres (mm) of thiepine derivatives cmpd.a gram–negative bacteria gram–positive bacteria e. coli p. hauseri p. aeruginosa s. enterica c. sporogenes s. aureus m. luteus atcc 10240 k. rhizophila 16 n.a.b n.a. n.a. n.a. n.a. n.a. n.a. n.a. 17 14 10 n.a. 10 16 n.a. n.a. n.a. 31 14 n.a. n.a. n.a. 20 n.a. n.a. 10 32 14 10 n.a. 10 14 n.a. n.a. n.a. 33 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 34 10 14 n.a. n.a. 10 n.a. n.a. n.a. 35 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. amkc 26 26 25 25 24 24 27 23 a compounds concentration 1 mg disk-1; b n.a.: no activity (inhibition zone <10 mm); c amk concentration 30 μg disk-1 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 846 božinović et al. on the other hand, with the exception of 31, all compounds exhibited pronounced antifungal activity against the three fungal strains (table iv). the investigation of antifungal screening revealed that at a concentration of 1000 µg disk–1, compounds 16, 17, 32, 33, 34 and 35 were very potent, and showed complete growth inhibition against the c. albicans and s. cerevisiae strains. among synthesized thiepines, compound 32 showed excellent antifungal activity particularly on the c. albicans strain with an inhibition zone of 50 mm at 250 µg disk–1, 28 mm at 125 µg disk–1 and 14 mm at 62.5 µg disk–1 concentrations. in addition, compound 32 at a concentration 125 µg disk–1 displayed moderate activity against the a. brasiliensis and s. cerevisiae strains (growth inhibition zones 12–18 mm). all the tested thiepine derivatives were completely inactive against the fungal strains at a concentration 31.3 µg disk–1. table iv. antifungal activity of the thiepine derivatives expressed as diameter of the inhibition zones in millimetres (mm) cmpd. c. albicansa s. cerevisiaeb a. brasiliensisa concentration, µg disk-1 1000 500 250 125 1000 500 250 125 1000 500 250 125 16 c.i.c 12 10 n.a.d c.i. 18 15 11 18 14 12 10 17 c.i. c.i. 30 n.a. c.i. 20 16 12 c.i. 16 12 10 31 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a. 32 c.i. c.i. 50 28 c.i. 30 22 18 c.i. 30 20 12 33 c.i. 14 n.a. n.a. c.i. 22 16 12 12 10 n.a. n.a. 34 c.i. c.i. 10 n.a. c.i. 26 18 n.a. c.i. 10 n.a. n.a. 35 c.i. 14 n.a. n.a. c.i. 16 10 n.a. 12 n.a. n.a. n.a. anystatin concentration 30 μg disk-1, 30 mm including disk; bnystatin concentration 30 μg disk-1, 54 mm including disk; ccomplete inhibition; dno activity (inhibition zone <10 mm) experimental instrumentation microwave reactions were performed in a biotage initiator 2.5 microwave reactor. melting points were determined using a boetius pmhk apparatus (carl zeiss, germany) and are not corrected. the ir spectra were recorded on a perkin-elmer spectrophotometer ftir 1725x. the nmr spectra were recorded on a bruker ultrashield advance iii spectrometer (500 mhz) using tms as the internal standard. the chemical shifts are expressed in ppm (δ) values and coupling constants (j) in hz. the esi–ms (hrms) spectra were acquired on an agilent technologies 1200 series instrument equipped with a zorbax eclipse plus c18 column and a dad detector in combination with a 6210 time-of-flight lc/ms instrument in the positive ion mode. the samples were dissolved in meoh. gc/ms spectra were acquired on an agilent technologies 7890a instrument equipped with a db-5 ms column and 5975c msd and fid detector. lobar lichroprep si 60 or lichroprep rp-18 columns (merck, germany), coupled to a waters ri 401 detector, were used for preparative column chromatography. thin-layer chromatography was performed on pre-coated merck silica gel 60 f254 and merck rp-18 f254 plates. the solution meoh (nh3) stands for a combination meoh/ /nh3 aq. = 9:1. the compounds were analyzed for purity using an agilent 1200 hplc system _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis and antimicrobial activity of azepine and thiepine derivatives 847 equipped with quat pump (g1311b) and dad detector 1260 vl (other details are presented in the supplementary material to this paper). all compounds were >95 % pure. chemistry (2-bromo-5-chlorophenyl)methanediyl diacetate. a mixture of 1-bromo-4-chloro-2 -methylbenzene (1.0 g, 4.9 mmol), acetic anhydride (6.4 ml), acetic acid (5.0 ml) and concentrated sulphuric acid (1.5 ml) was cooled to 0 °c in an ice bath. then an acetic acid (5.0 ml) solution of cro3 (1.8 g, 18.0 mmol) was dropwisely added into the stirred mixture over 1.5 h. the mixture was stirred for the next 2 h at 0 °c. the product was filtered, washed with water (50 ml) and dried under reduced pressure. 2-bromo-5-chlorobenzaldehyde (19). (2-bromo-5-chlorophenyl)methanediyl diacetate (1.5 g, 4.7 mmol) was refluxed in meoh–h2o (15 ml, 1/1 v/v) containing h2so4 (1.6 ml) for 30 min. the reaction mixture was then diluted with h2o (15 ml) and extracted with etoac (3×20 ml). the combined organic layers were washed with h2o (20 ml) and brine (20 ml), dried over na2so4 and concentrated under reduced pressure. the residue and 1 m hydrochloric acid (4.0 ml) were heated for 3 h in thf (15 ml) under reflux. the solvent was removed under reduced pressure. the residue was purified by column chromatography (sio2, hexane/etoac = 95/5). yield: 0.84 g, 83 %. 2-bromo-5-methoxybenzaldehyde (21). to the solution of meta-anisaldehyde (3.03 g, 22.3 mmol) in acoh (5.0 ml), br2 (1.4 ml, 26.8 mmol, 1.2 eq.) was drop wisely added, and the reaction mixture was stirred for 36 h at room temperature. upon completion, the reaction was quenched with a saturated solution of na2so3 (25 ml), then poured into water (10 ml), and extracted with etoac (3×25 ml). the combined organic layers were washed with water (3×20 ml) and brine (15 ml), dried (na2so4), and concentrated to give the desired 2-bromo -5-methoxybenzaldehyde (21, 3.99 g, 83 %). 3-[(z)-2-(2-bromo-5-chlorophenyl)ethenyl]-4-chloropyridine (24). to a suspension of phosphonium salt 22 (0.55 g, 1.2 mmol) in thf (12 ml) kot-bu (0.16 g, 1.4 mmol) was added. after 30 min, a solution of 2-bromo-5-chlorobenzaldehyde (0.26 g, 1.2 mmol) in thf (3 ml) was added over 5 min. the reaction mixture was stirred at room temperature for 16 h, when it was quenched with sat. aqueous soln. of nahco3. the aqueous phase was separated and extracted with etoac (3×25 ml). the organic extracts were combined, dried over na2so4, concentrated under vacuum and purified by column chromatography (sio2, hexane/etoac = 95/5) to yield the z-isomer (270 mg, 69 %). 3-[(z)-2-(2-bromo-5-methoxyphenyl)ethenyl]-4-chloropyridine (25). to a suspension of phosphonium salt 22 (0.14 g, 0.30 mmol) in thf (1.6 ml) was added kot-bu (40 mg, 0.36 mmol). after 30 min, a solution of 2-bromo-5-methoxybenzaldehyde (65 mg, 0.30 mmol) in thf (2 ml) was added over 5 min. the reaction mixture was stirred at room temperature and after 18 h, it was quenched with nahco3. the aqueous phase was separated and extracted with etoac (3×10 ml). the organic extracts were combined, dried over na2so4, concentrated under vacuum and purified by preparative column chromatography (rp, meoh/h2o = = 8:2) to yield the z-isomer (60 mg, 82 %). 3-[(z)-2-(2-bromo-5-fluorophenyl)ethenyl]-4-chloropyridine (26). to a suspension of phosphonium salt 22 (0.45 g, 0.96 mmol) in thf (12 ml) was added kot-bu (0.13 g, 1.2 mmol). after 30 min, a solution of 2-bromo-5-fluorobenzaldehyde (0.19 g, 0.96 mmol) in thf (2 ml) was added over 5 min. the reaction mixture was stirred at room temperature and after 18 h, it was quenched with saturated aqueous solution of nahco3 (15 ml). the aqueous phase was separated and extracted with etoac (3×20 ml). the organic extracts were com_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 848 božinović et al. bined, dried over na2so4, concentrated under vacuum and purified by preparative column chromatography (rp, meoh/h2o = 8:2) to yield compound 26 (196 mg, 65 %). general procedure for pd-catalyzed amination a reaction tube containing a stirring bar was evacuated and backfilled with argon. the tube was then charged with pd(oac)2 (5 mol %), johnphos (10 mol %) and naot-bu (2.8 eq.) and filled with argon. toluene was added. after stirring at room temperature for 5 min, an aryl halide (1 eq.) and amine (3 eq.) were added, the tube was filled with argon and capped. reaction mixture was heated to 100 °c and stirred at the same temperature. products were purified by preparative column chromatography: sio2, ch2cl2/meoh(nh3) = 9/1. 5-[3-(pyrrolidin-1-yl)propyl]-5h-pyrido[4,3-b][1]benzazepine (27). following the general procedure, a mixture of 3-[(z)-2-(2-bromophenyl)ethenyl]-4-chloropyridine (24 mg, 0.080 mmol), 3-(pyrrolidin-1-yl)propan-1-amine (31 µl, 0.24 mmol), sodium tert-butoxide (22 mg, 0.23 mmol), pd(oac)2 (0.9 mg, 5 mol %), johnphos (2.4 mg, 10 mol %) and toluene (1.5 ml) was stirred at 100 °c for 48 h. yield: 17 mg, 68 %. 5-[3-(pyrrolidin-1-yl)propyl]-5h-dipyrido[4,3-b:3′,4′-f]azepine (28). following the general procedure, a mixture of 3,3′-(z)-ethene-1,2-diylbis(4-chloropyridine) (20 mg, 0.080 mmol), 3-(pyrrolidin-1-yl)propan-1-amine (31 µl, 0.24 mmol), sodium tert-butoxide (22 mg, 0.23 mmol), pd(oac)2 (0.9 mg, 5 mol %), johnphos (2.4 mg, 10 mol %) and toluene (1.5 ml) was stirred at 100 °c for 24 h. yield: 17 mg, 69 %. 3-(8-chloro-5h-pyrido[4,3-b][1]benzazepin-5-yl)-n,n-dimethylpropan-1-amine (29). following the general procedure, a mixture of 3-[(z)-2-(2-bromo-5-chlorophenyl)ethenyl]-4 -chloropyridine (26 mg, 0.080 mmol), 3-(dimethylamino)-1-propylamine (30 µl, 0.24 mmol), sodium tert-butoxide (22 mg, 0.23 mmol), pd(oac)2 (0.9 mg, 5 mol %), johnphos (2.4 mg, 10 mol %) and toluene (1.5 ml) was stirred at 100 °c for 48 h. yield: 18 mg, 70 %. 3-(8-methoxy-5h-pyrido[4,3-b][1]benzazepin-5-yl)-n,n-dimethylpropan-1-amine (30). following the general procedure, a mixture of 3-[(z)-2-(2-bromo-5-methoxyphenyl)ethenyl] -4-chloropyridine (26 mg, 0.080 mmol), 3-(dimethylamino)-1-propylamine (30 µl, 0.24 mmol), sodium tert-butoxide (22 mg, 0.23 mmol), pd(oac)2 (0.9 mg, 5 mol %), johnphos (2.4 mg, 10 mol %) and toluene (1.5 ml) was stirred at 100 °c for 48 h. yield: 10 mg, 40 %. general procedure for the synthesis of the thiepine derivatives a reaction tube containing a stirring bar was evacuated and backfilled with argon. the tube was charged with tris(dibenzylideneacetone)dipalladium (pd2dba3, 5 mol %), dppf (10 mol %), naot-bu (1.2 eq.), aryl halide (1 eq.) and kscoch3 (1.2 eq.) and evacuated and backfilled with argon. the flask was capped with a rubber septum, and toluene was added. the reaction mixture was heated in a biotage initiator 2.5 microwave at 170 °c for 60 min. after completion of the reaction, the mixture was cooled to room temperature. the products were purified by column chromatography: sio2, hexane/etoac = 8/2. [1]benzothiepino[3,2-c]pyridine (16). following the general procedure, a mixture of 3-[(z)-2-(2-bromophenyl)ethenyl]-4-chloropyridine (35 mg, 0.12 mmol), kscoch3 (16 mg, 0.14 mmol), sodium tert-butoxide (14 mg, 0.14 mmol), pd2dba3 (5.4 mg, 5 mol %), dppf (6.6 mg, 10 mol %) and toluene (1.5 ml) was heated in a biotage initiator 2.5 microwave at 170 °c for 60 min. yield: 13 mg, 51 %. pyrido[3′,4′:6,7]thiepino[3,2-c]pyridine (17). following the general procedure, a mixture of 3,3′-(z)-ethene-1,2-diylbis(4-chloropyridine) (30 mg, 0.12 mmol), kscoch3 (16 mg, 0.14 mmol), sodium tert-butoxide (14 mg, 0.14 mmol), pd2dba3 (5.4 mg, 5 mol %), dppf (6.6 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis and antimicrobial activity of azepine and thiepine derivatives 849 mg, 10 mol %) and toluene (1.5 ml) was heated in a biotage initiator 2.5 microwave at 170 °c for 60 min. yield: 15 mg, 60 %. 8-chloro[1]benzothiepino[3,2-c]pyridine (31). following the general procedure, a mixture of 3-[(z)-2-(2-bromo-5-chlorophenyl)ethenyl]-4-chloropyridine (29 mg, 0.088 mmol), kscoch3 (12 mg, 0.11 mmol), sodium tert-butoxide (10 mg, 0.11 mmol), pd2dba3 (4.0 mg, 5 mol %), dppf (4.9 mg, 10 mol %) and toluene (1.1 ml) was heated in a biotage initiator 2.5 microwave at 170 °c for 60 min. yield: 6.8 mg, 32 %. 8-methoxy[1]benzothiepino[3,2-c]pyridine (32). following the general procedure, a mixture of 3-[(z)-2-(2-bromo-5-methoxyphenyl)ethenyl]-4-chloropyridine (60 mg, 0.18 mmol), kscoch3 (25 mg, 0.22 mmol), sodium tert-butoxide (21 mg, 0.22 mmol), pd2dba3 (8.5 mg, 5 mol %), dppf (10 mg, 10 mol %) and toluene (2.3 ml) was heated in a biotage initiator 2.5 microwave at 170 °c for 60 min. yield: 8.4 mg, 19 %. 8-fluoro[1]benzothiepino[3,2-c]pyridine (33). following the general procedure, a mixture of 3-[(z)-2-(2-bromo-5-fluorophenyl)ethenyl]-4-chloropyridine (30 mg, 0.096 mmol), kscoch3 (13 mg, 0.12 mmol), sodium tert-butoxide (11 mg, 0.12 mmol), pd2dba3 (4.4 mg, 5 mol %), dppf (5.3 mg, 10 mol %) and toluene (1.2 ml) was heated in a biotage initiator 2.5 microwave at 170 °c for 60 min. yield: 7.6 mg, 35 %. 8-chloro-2-methyl-1,2,3,4-tetrahydro[1]benzothiepino[3,2-c]pyridine (34). a solution of 8-chloro[1]benzothiepino[3,2-c]pyridine (17 mg, 0.068 mmol) in mecn (3 ml) was refluxed with an excess of methyl iodide (25 µl, 0.27 mmol, 4 eq.). after 2 h, the solvent was removed under reduced pressure. the resulting yellow solid was dissolved in dry methanol (3 ml) and nabh4 (6.0 mg, 0.13 mmol) was added under an inert atmosphere at room temperature. after 15 min, the meoh was removed under reduced pressure. the crude residue was dissolved in etoac and washed with h2o. the organic layer was dried over anhydrous na2so4, concentrated under vacuum and purified by column chromatography (sio2, etoac/ /meoh = 1:1) to yield the product (11.8 mg, 66 %). 8-phenyl[1]benzothiepino[3,2-c]pyridine (35). a reaction tube containing a stirring bar was evacuated and backfilled with ar. the tube was then charged with pd(oac)2 (0.9 mg, 5 mol %), sphos (3.2 mg, 10 mol %), phenylboronic acid (12 mg, 0.095 mmol, 1.2 eq.) and anhydrous k3po4 (34 mg, 0.16 mmol, 2.0 eq.). the tube was capped with a rubber septum and filled with argon. dry toluene (1.0 ml) was added through the septum and the resulting mixture was stirred at room temperature for 2 min. 8-chloro[1]benzothiepino[3,2-c]pyridine (20 mg, 0.079 mmol) was added and the tube was sealed. the reaction mixture was heated at 100 °c for 18 h. the reaction mixture was allowed to cool to room temperature. the product was purified by column chromatography (sio2, hexane/etoac = 8/2). yield: 11.5 mg, 50 %. antimicrobial evaluation microbroth dilution method. the antimicrobial activity was evaluated using a broth microdilution method according to nccls (national committee for clinical laboratory standards (2000) approval standard document m7-a5, villanova, pa, usa). the following gram-negative bacterial strains used were: escherichia coli (atcc 25922), proteus hauseri (atcc 13315), pseudomonas aeruginosa (atcc 9027), salmonella enterica subsp. enterica serovar enteritidis (atcc 13076) and klebsiella pneumoniae (atcc 10031). the gram -positive bacterial strains used were: staphylococcus aureus (atcc 6538), micrococcus luteus (atcc 10240) and m. luteus (atcc 4698). the employed fungal species were: candida albicans (atcc 10231), saccharomyces cerevisiae (atcc 9763) and aspergillus brasiliensis (atcc 16404). mic determination was performed by a serial dilution method in sterile 96-well microtitre plates. fresh mueller–hinton broth (for bacteria) and sabouraud dextrose _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 850 božinović et al. broth (for fungi) were used. stock solutions of the compounds were prepared in dimethyl sulphoxide (dmso), and then serial dilutions of the compounds were made in the concentration range from 10,000 to 4.9 µg ml-1. amikacin (amk) and chloramphenicol (chl) were used as positive controls for the bacteria, while nystatin (nyt) and fluconazole (flc) were used as positive controls for the fungi. the solvent (dmso) served as negative control. in each well of the plate, ten microlitres of bacterial cultures (106 cells ml-1) for antibacterial activity and 10 ml of fungal cultures (105 spores ml-1) were inoculated. the microtiter plates were incubated at 37 °c for 24 h for the bacteria or at 28 °c for 48 h for the fungi. the mic was determined as the lowest concentration that resulted in inhibition of bacterial or fungal growth. disk diffusion method. antimicrobial activity was evaluated using a disk diffusion method according to nccls (national committee for clinical laboratory standards (1997) approval standard document m2-a6 performance standards for antibacterial disk susceptibility test, wayne, pa, usa). antibacterial activity. the antibacterial activity was evaluated using four different strains of gram-negative bacteria: e. coli (atcc 25922), p. hauseri (atcc 13315), p. aeruginosa (atcc 9027) and s. enterica subsp. enterica serovar enteritidis (atcc 13076), and four different strains of the gram-positive bacteria: clostridium sporogenes (atcc 19404), s. aureus (atcc 6538), m. luteus (atcc 10240) and kocuria rhizophila (atcc 9341). the determination of antibacterial activity was performed using the disk diffusion method. in each petri dish (90 mm diameter), 22 ml of nutrient agar and 100 µl of bacterial suspension were added. the test substances were dissolved in ch2cl2 (1 mg 100 µl -1) and then 100 µl of solution was applied to a filter paper disk (8 mm in diameter) and the solvent was evaporated. the loaded disks were placed on the surface of the medium and left for 30 min at room temperature for compound diffusion. amikacin 30 µg per filter paper disk (8 mm in diameter) was used as the positive control, while the disks of the same diameter impregnated with 100 µl of ch2cl2 were used as the negative control. the plates were incubated for 24 h at 37 °c. the zones of inhibition were recorded in millimetres. antifungal activity. the antifungal activity was tested against three different strains: c. albicans (atcc 10231), s. cerevisiae (atcc 9763) and a. brasiliensis (atcc 16404). sabouraud dextrose agar was prepared according to the manufacture’s instruction. into each sterile petri dish (90 mm diameter), 22 ml of previously prepared agar suspension was poured and 100 µl of fungi was added. the test compounds were dissolved in ch2cl2 and applied on filter paper disk (8 mm in diameter) at final concentrations 1000, 500, 250, 125, 62.5 and 31.3 µg/disk. nystatin (30 µg disk-1) was used as a positive control while a disk impregnated with ch2cl2 was used as the negative control. petri dishes were incubated for 48 h at 28 °c. the zone of inhibition was measured in millimetres, including the disk. conclusions new pyridobenzazepine and pyridobenzothiepine derivatives were synthesized using a methodology for pd-catalyzed formation of c–n and c–s bonds.9 additionally, the successful transformations of thiepine 31 to tetrahydro and biphenyl derivatives opened up new possibilities for the preparation of structurally diverse substituted derivatives. all newly and previously synthesized compounds were evaluated for their in vitro antimicrobial activity against eight bacterial and three fungal pathogenic strains. all pyridobenzazepine derivatives _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis and antimicrobial activity of azepine and thiepine derivatives 851 showed better antibacterial and antifungal activity than the corresponding dipyridoazepine analogues. among the synthesized azepines, derivative 8 was the most active and showed a broad spectrum of antibacterial activity (mic ranged 39–78 µg ml–1). the synthesized thiepine derivatives exhibited weak antibacterial activity but, on the other hand, with the exception of 31, all thiepines showed pronounced antifungal activity. supplementary material analytical and spectral data of the compounds, as well as copies of the corresponding 1h-nmr and 13c-nmr spectra of the products and hplc purity chromatograms are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgments. this research was financially supported by the ministry of education, science and technological development of the republic of serbia (grant no. 172008) and the serbian academy of sciences and arts. и з в о д синтеза и антимикробна активност азепинских и тиепинских деривата нина божиновић1, ирена новаковић2, слађана костић рајачић2, игор м. опсеница1 и богдан а. шолаја1 1хемијски факултет, универзитет у београду, студентски трг 16, п. пр 51, 11158, београд и 2институт за хемију, технологију и металургију, универзитет у београду, његошева 12, 11000 београд синтетисана је серија пиридобензазепинских и пиридобензотиепинских деривата формирањем c–n и c–s веза помоћу катализатора на бази pd и испитана је њихова in vitro антимикробна активност. синтетисани пиридобензазепински деривати показују већу антибактеријску и антифунгалну активност у поређењу са одговарајућим дипиридоазепинским дериватима. азепин 8 показао је највећу антибактеријску активност (mic у опсегу 39–78 μg ml-1), а азепин 12 показао се као најактивнији дериват према испитаним сојевима гљива (mic у опсегу 156–313 μg ml-1). синтетисани тиепински деривати имају слабу антибактеријску активност, али са друге стране имају добру антифунгалну активност. (примљено 16. јануара, ревидирано 2. фебруара, прихваћено 6. фебруара 2015) references 1. a) b. leduc, in foye's principles of medicinal chemistry, 6th ed.; t. l. lemke, d. a. williams, eds., lippincott williams & wilkins, philadelphia, pa, 2007, p. 521; b) k. c. miles, emergency medicine: a comprehensive study guide, 6th ed., j. e. tintinalli, g. d. kelen, j. s. stapczynski, eds., mcgraw–hill, new york, ny, 2004, p. 1025 2. m. protiva, j. heterocycl. chem. 33 (1996) 497, and references cited therein 3. i. ueda, y. sato, s. maeno, s. umio, chem. pharm. bull. 26 (1978) 3058 4. a) l. j. kricka, a. ledwith, chem. rev. 74 (1974) 101, and references cited therein; b) a. knell, d. monti, m maciejewski, a. baiker, appl. catal., a 121 (1995) 139; c) g. p. tokmakov, i. i. grandberg, tetrahedron 51 (1995) 2091; d) e.-c. elliott, e. r. bowkett, j. l. maggs, j. bacsa, b. k. park, s. l. regan, p. m. o’neill, a. v. stachulski org. lett. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 852 božinović et al. 13 (2011) 5592; e) t. matsuda, s. sato, j. org. chem. 78 (2013) 3329; f) e.-c. elliott, j. l. maggs, b. k. park, p. m. o’neill, a. v. stachulski, org. biomol. chem. 11 (2013) 8426 5. a) h. shirani, t. janosik, j. org. chem. 72 (2007) 8984; b) h. shirani, j. bergman, t. janosik tetrahedron 65 (2009) 8350; c) m. saito, t. yamamoto, i. osaka, e. miyazaki, k. takimiya, h. kuwabara, m. ikeda, tetrahedron lett. 51 (2010) 5277 6. a) l. a. arnold, w. luo, r. k. guy, org. lett. 6 (2004) 3005; b) d. tsvelikhovsky, s. l. buchwald, j. am. chem. soc. 132 (2010) 14048; c) n. della ca', g. maestri, m. malacria, e. derat, m. catellani, angew. chem. int. ed. 50 (2011) 12257; d) m. tian, a. abdelrahman, s. weinhausen, s. hinz, s. weyer, s. dosa, a. el-tayeb, c. e. müller, bioorg. med. chem. 22 (2014) 1077 7. a) c. song, d. b. walker, t. m. swager, macromolecules 43 (2010) 5233; b) s. sinning, m. musgaard, m. jensen, k. severinsen, l. celik, h. koldsø, t. meyer, m. bols, h. h. jensen, b. schiøtt, o. wiborg, j. biol. chem. 285 (2010) 8363; c) h. christensen, c. schjøth-eskesen, m. jensen, s. sinning, h. h. jensen, chem. eur. j. 17 (2011) 10618; d) x. zhang, y. yang, y. liang, tetrahedron lett. 53 (2012) 6406; e) j. tsoung, j. panteleev, m. tesch, m. lautens, org. lett. 16 (2014) 110 8. t. h. jepsen, m. larsen, m. jørgensen, m. b. nielsen, synlett 23 (2012) 418 9. n. božinović, i. opsenica, b. a. šolaja, synlett 24 (2013) 49 10. s. tu, l.-h. xu, l.-y. ye, x. wang, y. sha, z.-y. xiao, j. agric. food chem. 56 (2008) 5247 11. s. a. snyder, t. c. sherwood, a. g. ross, angew. chem. int. ed. 49 (2010) 5146. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {interactions of cytotoxic amino acid derivatives of tert-butylquinone with dna lysozyme} j. serb. chem. soc. 81 (12) 1345–1358 (2016) udc 547.567+575.852:539.196:535.371: jscs–4932 577.213.3 original scientific paper 1345 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. interactions of cytotoxic amino acid derivatives of tert-butylquinone with dna and lysozyme jovana p. vilipić1, irena t. novaković2**#, mario v. zlatović3#, miroslava t. vujčić2#, srđan j. tufegdžić2# and dušan m. sladić3*# 1innovation center of the faculty of chemistry, university of belgrade, studentski trg 12–16, belgrade, serbia, 2institute of chemistry, technology and metallurgy, center for chemistry, university of belgrade, njegoševa 12, belgrade, serbia and 3faculty of chemistry, university of belgrade, studentski trg 12–16, belgrade, serbia (received 25 july, revised 10 november, accepted 14 november 2016) abstract: the interactions of nine amino acid derivatives of tert-butylquinone with biomacromolecules were studied. sodium dodecyl sulphate (sds) gel electrophoresis and mass spectrometry confirmed the absence of modifications of lysozyme by any of the synthesized compounds. spectrophotometric studies demonstrated hyperchromism, i.e., the existence of interactions between the quinones and calf thymus dna (ct-dna). determination of the binding constants by absorption titration indicated weak interactions between the quinone derivatives and ct-dna. the quenching of fluorescence of the intercalator ethidium bromide (eb) from the eb–ct-dna system and of the minor groove binder hoechst 33258 (h) from the h–ct-dna system by the synthesized derivatives indicated interactions of the compounds and ct-dna. circular dichroism (cd) spectra demonstrated a non-intercalative binding mode of the quinone derivatives to ct-dna. molecular docking results confirmed binding to the minor groove. the electrophoretic pattern showed no cleavage of the puc19 plasmid in the presence of any of the synthesized compounds. the ability of the derivatives to scavenge radicals was confirmed by the 2,2-diphenyl-1-picrylhydrazyl (dpph) test. all the presented results suggest that the dna minor groove binding is the principal mechanism of action of the examined amino acid derivatives. keywords: quinone; dna binding; minor groove; fluorescence. introduction understanding the mechanism of interactions of various anticancer drugs with biomacromolecules is a crucial aspect of drug discovery. investigation of * corresponding authors. e-mail: (*)dsladic@chem.bg.ac.rs; (**)irenan@chem.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc160725101v 1346 vilipić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. the interactions of pharmacologically active quinones with biomacromolecules is a challenging task, because of their complex mechanism of action. quinones are highly reactive compounds and the general mechanism of their action involves three processes: first, the redox couple quinone/hydroquinone can generate reactive oxygen species that damage biomacromolecules and inhibit electron transport in mitochondria and oxidative phosphorylation; second, quinones can be alkylated by cellular nucleophiles, such as proteins, enzymes and dna molecules; third, compounds with a quinone moiety can non-covalently interact with dna molecules.1–7 most of the clinically useful quinone alkylating antitumor agents are active only after bioreductive activation. the term bioreductive activation includes a series of mechanisms of reduction of anticancer agents to reactive intermediates that can later undergo nucleophilic addition.8–11 in this way, some degree of selectivity is attained. in a previous study, besides very active avarone derivatives with amino acids, nine amino acid derivatives of the avarone mimic tert-butylquinone were synthesized.12 for the derivatisation, amino acids with non-polar aliphatic and aromatic residues including one d-amino acid, were selected. the derivatives showed moderate cytotoxic activity and a good selectivity on a panel of cancer cell lines. in this paper, the mode of action of the synthesized derivatives at the molecular level on linear and circular dna, and a model protein were investigated. experimental materials and methods lysozyme from hen egg white was obtained from sigma–aldrich, usa. an eppendorf mini spin plus centrifuge was used. sodium dodecyl sulphate–polyacrylamide (sds–pa) gel electrophoresis was performed using a hoefer se 600 ruby vertical electrophoresis system. mass spectra were recorded on a linear ion trap – orbitrap hybrid mass spectrometer (ltq orbitrap xl) with a heated electrospray ionization probe, hesi (thermo fisher scientific, bremen, germany). uv–vis spectra were collected on a cintra 40 uv/visible spectrometer. fluorescence spectra were recorded using a lumina fluorescence spectrometer (thermo fisher scientific, finland) equipped with a 150 w xenon lamp. the splits on the excitation and emission beams were fixed at 10 nm. calf thymus dna (ct-dna) was purchased from serva, heidelberg. agarose was obtained from amersham pharmacia-biotech, inc, usa. a he 33 mini submarine electrophoresis unit (hoefer, usa) with an eps 300 power supply was used for the agarose gel electrophoresis. the stained gel was illuminated under a uv transilluminator (vilber lourmat, france) at 312 nm and photographed with a nikon coolpix p340 digital camera through a deep yellow 15 filter, tiffen, usa. the plasmid puc19 was purchased from sigma–aldrich, usa. a thermo scientific appliskan (thermo fisher scientific, finland) was used for measuring absorbance at 517 nm for determining the dpph scavenging activity. details related to mass spectrometry, uv–vis spectroscopic and fluorescence measurements are given in supplementary material to this paper. interactions of quinones with biological macromolecules 1347 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. preparation of the quinones the amino acid derivatives of tert-butylquinone were synthesized as described previously.12 modification of lysozyme modifications of lysozyme were performed in 20 % dimethyl sulfoxide (dmso), 20 % methanol and distilled water, respectively. the quinones were dissolved in 240 μl of dmso, methanol and water, respectively (final concentration 2 mg ml-1) and lysozyme was dissolved in 960 μl of water (final concentration 5 mg ml-1). the reaction mixtures contained 50 mm nahco3 and the ph value of the mixture was 8.5. the reaction was performed at r.t. with stirring for 48 h. during the reaction, the formation of a precipitate was observed when dmso and methanol were used. the precipitate did not contain free quinones. the mixture was centrifuged at 14000 rpm and the supernatant was desalted by dialysis against distilled water. concerning the precipitate, three volumes of water were added and after centrifugation, the resulting solution was dialysed against distilled water. modifications of lysozyme were also performed by the same procedure without nahco3, in aqueous buffer solutions (100 mm phosphate buffer ph 7.4, 100 mm acetate buffer ph 4.5 or 100 mm ammonia buffer ph 10). under these conditions, no precipitate was formed. the formation of a possible conjugate was checked using sds electrophoresis and mass spectrometry. sds pa gel electrophoresis sds polyacrylamide gels were made by a standard procedure.13 the concentration of the running gel was 10 % and of the stacking gel, 4 %. each sample (25 μl) was applied to the gel. the voltage was held constant (80 v) until the samples entered the running gel. for separation through the running gel, the voltage was raised to 150 v. dna binding experiments all buffer solutions were prepared in deionised water and filtered through 0.2 μm filters, nalgene, usa. stock solutions of the tested compounds were prepared in dmso at a concentration of 10 mm. calf thymus dna (ct-dna, lyophilized, highly polymerized, purchased from serva, heidelberg, germany) was dissolved in tris buffer (20 mm tris–hcl, ph 7.5) overnight at 4 °c. this solution was stored at the same temperature and was stable for several days. the ratio of the uv absorbance at 260 and 280 nm (a260/a280 of 1.94) indicated that the aqueous solution of ct-dna was sufficiently free of protein. the concentration of ct-dna (3.12 mg ml-1) was determined from the uv absorbance at 260 nm using an extinction coefficient ε260 = 6600 m-1 cm-1.14 cd studies the cd spectra of ct-dna (0.5 mm) alone and with two concentrations of the derivatives (0.25 and 0.50 mm) were recorded using a jasco j-815 circular dichroism spectrometer. the temperature of the sample was kept constant at 25 °c. all cd spectra were recorded in the range from 220 to 310 nm at a scan speed of 200 nm min-1 with a spectral bandwidth of 0.1 nm. the average of three scans was taken in all the experiments. the background spectrum of the bicarbonate solution (40 mm, ph 8.4) was subtracted from the spectra of dna and dna‒derivative complexes. 1348 vilipić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. dna cleavage experiments the plasmid puc19 (2686 bp) used for the dna cleavage experiments was prepared by its transformation in chemically competent cells of escherichia coli strain xl1 blue. amplification of the clone was realised according to the protocol for growing e. coli culture overnight in lb medium at 37 °c.15 purification was performed using qiagen plasmid plus maxi kit. dna was eluted in 10 mm tris–hcl buffer (ph 7.9) and stored at –20 °c. the concentration of plasmid dna (460 ng μl-1) was determined by measuring the absorbance of the dna-containing solution at 260 nm. one optical unit corresponds to 50 μg ml-1 of double -stranded dna. the cleavage reaction of supercoiled puc19 by the different compounds (final concentration 1 mm) was investigated by incubation of 460 ng of plasmid in a 20 μl reaction mixture in 40 mm bicarbonate solution (ph 8.4) at 37 °c for 90 min. the reaction mixture was vortexed from time to time. the reaction was terminated by short centrifugation at 10000 rpm and the addition of 5 μl of loading buffer consisting of 0.25 % bromophenol blue, 0.25 % xylene cyanol ff and 30 % glycerol in tae buffer (40 mm tris–acetate, 1 mm edta, ph 8.24). the samples (20 μl) were analysed by electrophoresis on 1 % agarose gel prepared in tae buffer, ph 8.24. the electrophoresis was performed at a constant voltage (80 v) until the bromophenol blue had passed through 75 % of the gel. after electrophoresis, the gel was stained for 30 min by soaking it in an aqueous ethidium bromide solution (0.5 μg ml-1). molecular docking for dna docking simulations, the crystal structure of dodecamer d(cgcgaattcgcg)2, pdb id: 3u2n,16 was obtained from the protein data bank (http://www.rcsb.org/pdb). all water molecules and ions, as well as ligands were removed using autodock tools 1.5.6.17,18 quinone molecules were prepared using maestro 10.4 from the schrodinger suite 2015 -4.19 all possible tautomer structures at ph 7.00±2.00 were generated using epik 3.4 from the schrodinger suite 2015-420 and their charges were determined in jaguar 9.021 using the hf/6 -31g* method. these charges were used further in docking simulations. the docking simulations were realised in autodock vina 1.1.2.22 the grid box size was set to 25×28×45 å3 and exhaustiveness to 500. discovery studio visualizer 4.5 was used for visualization of the interactions.23 dpph radical scavenging activity details of the dpph radical scavenging activity test are given in the supplementary material. results and discussion the amino acid derivatives of tert-butylquinone (scheme 1) were obtained by nucleophilic addition of an amino acid to the quinone, as previously described.12 for investigation of the interactions of quinones with proteins, lysozyme was chosen in a previous study as the enzyme model, since the amino groups of lysine in lysozyme were shown to react by michael addition with the quinone moiety of similar quinones, including alkylamino derivatives.24 the modification reaction was monitored by sds electrophoresis and mass spectrometry. the interactions of quinones with biological macromolecules 1349 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. results on the sds electrophoregram (fig. 1) showed that the synthesized amino acid derivatives did not modify lysozyme. modification of lysozyme was not observed under any of the applied conditions. scheme 1. structures of the investigated compounds. fig. 1. sds–page of lysozyme (lys) and modifications of lys with derivatives 1a–i in 20 % dmso. in the mass spectra obtained for both the supernatants and precipitates only the peak of unmodified lysozyme at m/z = 14305 (e.g. 1431.499 for z = 10) was 1350 vilipić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. observed. the obtained results showed that synthesized derivatives of tert-butylquinone did not modify lysozyme (fig. s-1 of the supplementary material to this paper). explanation for this behaviour is the steric bulk of tert-butylquinone, as well as the rapid establishment of tautomeric equilibrium, and consequently, the absence of the stable quinone structure12 that is necessary for attack of protein nucleophiles.24–26 it could be concluded that these compounds cannot be alkylated by the nucleophilic groups of the model protein. dna is one of the principal targets of many different classes of drugs that are in clinical use, from anticancer and antiviral drugs to antibiotics. there are two main types of interactions between dna and low molecular weight molecules, covalent and non-covalent interactions. due to the rapid interconversion of the tautomeric forms and the assumed difficulty of formation of covalent bonds between the derivatives and biomacromolecules, as evidenced with lysozyme, which contains several highly nucleophilic lysine residues, the focus of this work was on the three most important non-covalent modes of drug–dna binding: intercalation, groove binding and external binding. uv–visible absorption spectroscopy is one of the simplest, most common techniques for studying the stability of dna and its interactions with small molecules. when a small molecule interacts with dna, a complex is formed, with changes in the absorbance and/or the positions of peaks. representative uv absorption spectra of derivative 1h at different concentrations, both in the absence and presence of a fixed concentration of ct-dna, are shown in fig. 2. fig. 2. uv–vis absorption spectra of different concentrations (50, 100 and 200 µm, from bottom to top) of 1h before and after interaction with ct-dna. interactions of quinones with biological macromolecules 1351 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. in the absence of dna, the absorption spectra of the derivatives showed absorbance maxima in the range of 276–283 nm. hyperchromism was observed upon addition of dna. these results indicated an interaction between the derivatives and dna molecules. in comparison with the spectrum of ct-dna, in all absorption spectra, (the exception is the proline derivative 1f), a bathochromic shift was noted. as small red shifts were followed by hyperchromism with all derivatives (table i), it could be concluded that the examined amino acid derivatives bind to the double-stranded ct-dna through non-covalent interactions,27 probably by groove binding and/or electrostatic interactions rather than intercalation. table i. hyperchromism induced by the synthesized derivatives 1a–i in 50 µm solution. the induction in % was calculated using expression dna+d dna d dna+d100( ) /a a a a− − , where adna, ad and adna+d refer to the absorbance of dna, derivatives in 50 µm solution and complex dna–derivative, respectively, at 260 nm compound 1a 1b 1c 1d 1e 1f 1g 1h 1i % 26.71 8.55 5.18 8.04 1.12 27.68 16.37 2.20 7.03 in order to obtain information on the stability of the derivative/ct-dna complex, spectroscopic titrations of solutions of ct-dna were performed (the results are given in fig. s-2 of the supplementary material). the obtained values of binding constants (table s-i of the supplementary material) are notably lower than those reported for intercalators, which indicates non-intercalative binding interactions between the quinone derivatives and ct-dna.28 various well-known dna binding dyes are used to establish the type of drug–dna interactions. ethidium bromide is extensively used as a fluorescence probe that binds to dna in an intercalative fashion. in case of groove binders, competitive displacement studies are done using a hoechst dye that binds to the minor groove of double-stranded b-dna and is highly specific for at-rich sequences. in order to investigate further the binding mode of tert-butylquinone derivatives to ct-dna, fluorescence analyses were performed with ethidium bromide (eb) and hoechst 33258 (h). the results of the competitive fluorescence study of the representative derivative 1h with the intercalator eb are given in fig. 3. the fluorescence intensity of the band at 600 nm of the eb–ct-dna system decreased significantly with the increasing concentration of all the quinone compounds (table ii). the greatest decreases in the fluorescence intensity were observed for the alanine (1b), d-valine (1d), phenylalanine (1h) and tryptophan (1i) derivatives at the highest used concentrations. the fluorescence emission spectrum for the proline derivative 1f revealed the smallest reduction in the fluorescence intensity, which is consistent with a previous study on the effect of pro1352 vilipić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. line in displacing dna-bound ethidium bromide, in which fluorescence emission data of proline revealed a marginal displacement in contrast to other amino acids.29 fig. 3. displacement of ethidium bromide bound to ct-dna by derivative 1h: emission spectra of eb alone (bottom line), eb bound to ct-dna (top line) and quenching of eb–ct-dna system by derivative 1h at increasing concentrations (50, 100 and 200 µm solutions, from top to bottom). table ii. the decrease of fluorescence of eb–ct-dna complex upon addition of 1a–i (200 µm solution). the decrease in % was calculated using the expression fdna+eb fdna+eb+d feb fdna+eb100( ) /− −i i i i , where ifdna+eb, ifdna+eb+d and ifeb refer to the fluorescence intensity of complex eb–dna, eb–dna–derivatives and eb, respectively, at 600 nm compound 1a 1b 1c 1d 1e 1f 1g 1h 1i % 21.80 23.87 17.85 29.86 19.74 6.59 20.29 26.79 22.64 according to the results and structural differences between the most active derivatives, it could be concluded that the derivatives did interact with dna, which led to a decrease in fluorescence intensity, but the results could hardly be considered evidence of their intercalation, especially in view of the uv spectral data that showed hyperchromism and not hypochromism. it is known from the literature that an indirect displacement can occur whereby the compounds induce structural changes in dna and hence reduce the binding affinity for eb.30 binding of hoechst 33258 to ct-dna was followed by excitation at 350 nm with the maximum in fluorescence at 444 nm. the fluorescence intensity at 444 nm of the h–ct-dna complex decreased with increasing concentration of most of the quinone compounds (fig. 4, table iii). these results suggest minor groove binding of the amino acid derivatives. it seems that minor groove binding interactions of quinones with biological macromolecules 1353 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. might be of importance for their action on tumour cells since the three compounds that do not displace h from the complex are among the least active derivatives (ic50 never below 30 μm for any tested cell line). these exceptions were 1a, 1e and 1f, which induced an increase in the fluorescence of the h–ct-dna complex. a possible explanation for the increase in fluorescence is the formation of a ternary complex ct-dna–dye–quinone. such behaviour was shown in literature31 for argininamide with an oligonucleotide aptamer. this external binding of 1a and 1f induced a high hyperchromism in the uv–vis spectra, while binding of leucine derivative 1e was weak because of its bulky isobutyl group. fig. 4. displacement of hoechst 33258 bound to ct-dna by derivative 1h: emission spectra of hoechst 33258 alone (bottom line), h bound to ct-dna (top line) and quenching of h– ct-dna system by derivative 1h at increasing concentrations (50, 100 and 200 µm, from top to bottom). table iii. decrease in the fluorescence of the h–ct-dna complex upon addition of 1a–i (200 µm solution). the decrease was calculated using the expression fdna+h fdna+h+d fh fdna+h100( ) /− −i i i i , where ifdna+h, ifdna+h+d and ifh refer to the fluorescence intensity of complex h–dna, h–dna–derivatives and h, respectively, at 444 nm compound 1a 1b 1c 1d 1e 1f 1g 1h 1i % –25.55 19.52 53.83 23.76 –5.50 –37.92 31.16 30.44 33.89 cd spectroscopy is very sensitive to the changes in the secondary structure of biomacromolecules and, therefore, is widely used for detection of changes in secondary structure of dna upon interactions with small molecules,32 and non -covalent dna–drug interactions resulting in altered cd spectral properties.33,34 1354 vilipić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. the cd spectrum of ct-dna displays a positive band around 275 nm due to base stacking and a negative band around 245 nm due to the right-handed helicity of the b-dna form.35 intercalation significantly changes the intensities of both the bands, stabilizing the right-handed b conformation of dna, while groove binding and electrostatic interactions of small molecules show low or no disturbances.36 as shown in a representative cd spectrum of 1h (fig. 5), after its addition to ct-dna, there were no significant changes in the cd spectrum. these results provide evidence that the derivatives do not bind to ct-dna by intercalation, but rather via groove binding. fig. 5. effect of 1h on cd spectra of ct-dna. cd spectra of ct-dna (0.50 mm) in bicarbonate solution (40 mm, ph 8.4) with varying concentrations of 1h. each spectrum was obtained at 25 °c with 1 mm path length cell. for the support of the evidence for minor groove binding, molecular docking was performed with the dna sequence d(cgcgaattcgcg)2, pdb id: 3u2n.16 the calculations suggested binding to the minor groove. no intercalating structures were found. the pattern of binding is presented in fig. 6. the established interactions between molecules and dna nucleotides are predominantly hydrogen bonds and some carbon–hydrogen bonding and π–alkyl interactions. based on the previous results in which it was shown that several types of avarone derivatives cleaved plasmid dna,37 the capability of the synthesized derivatives to cleave puc19 plasmid was examined using electrophoretic analysis. dmso did not show any effect on plasmid dna under the applied experimental interactions of quinones with biological macromolecules 1355 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. conditions. electrophoretic pattern showed that there was no cleavage of puc19 with any of the synthesized compounds under the applied conditions (fig. 7). fig. 6. binding of compound 1h to the minor groove. fig. 7. agarose gel electrophoretic analysis of plasmid puc19 without (p) and with derivatives 1a–i (1 mm). these results differed from previous results with quinone compounds,37 which showed a significant cleavage of plasmid dna. the cleavage of dna molecules by quinones in vitro could be explained by the presence of ros, which leads to single-strand and/or double-strand breaks of the plasmid chain. quinones have the ability to generate oxygen radicals through semiquinone intermediates formed either after nucleophilic attack on the quinone moiety or after reduction and subsequent reoxidation of hydroquinone or semiquinone into quinone. as the investigated amino acid derivatives interchange rapidly between tautomers and are not susceptible to nucleophilic attack and since the experimental conditions were not reducing, the lack of cleavage could be expected. considering the observation that the derivatives do not generate radicals in vitro, their ability to scavenge radicals was investigated by the dpph test. the results of scavenging activity of amino acid derivatives of tert-butylquinone are listed in table iv. all the derivatives showed some antioxidant activity, although weaker than ascorbic acid. the activity could be ascribed to the amino acid moiety. amino acids were shown to react with dpph radical by two mechanisms, namely oxidative decarboxylation, initiated by abstraction of hydrogen from the 1356 vilipić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. carboxylic group and oxidative deamination, initiated by hydrogen abstraction from the nh group.38 based on the results presented here, which showed that the proline derivative 1f was the most active compound, and that there are no other clear-cut structure‒activity relationships, oxidative decarboxylation is the principal reaction pathway of this series of compounds. table iv. antioxidant activity (ic50 / mm) of compounds 1a–i compound 1a 1b 1c 1d 1e 1f 1g 1h 1i ascorbic acid ic50 1.57 2.87 1.25 8.89 1.27 0.34 1.37 1.02 2.37 0.07 conclusion in this paper, the mode of biological action of amino acid derivatives of tert -butylquinone was investigated at the molecular level. it was observed that the synthesized derivatives, because of the nature of their substituents, have an atypical mode of action for quinones. neither addition of nucleophilic groups of proteins nor dna strand nicking was observed. it could be concluded that the preferential mechanism of action of the investigated series of biologically active compounds with the quinone moiety, the structural elements of which allowed rapid interconversion of tautomeric forms, is a non-covalent interaction with dna molecules, namely minor groove binding. this mechanism of action, with less diverse effects at the molecular level, probably leads to the observed low toxicity of these compounds to the tested normal cell line,12 making them a promising lead for further anticancer drugs. supplementary material additional experimental details and mass spectra of the unmodified lysozyme and lysozyme after treatment with 1h are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding authors on request. acknowledgements. the authors acknowledge the financial support of the ministry of education, science and technological development of the republic of serbia (grant no. 172055). the authors acknowledge the support of the fp7 regpot project fcub era ga no. 256716. the ec does not share responsibility for the content of this article. и з в о д интеракције цитотоксичних аминокиселинских деривата tert-бутилхинона са dna и лизозимом јована п. вилипић1, ирена т. новаковић2, марио в. златовић3, мирослава т. вујчић2, срђан ј. туфегџић2 и душан м. сладић3 1иновациони центар хемијског факултета, универзитет у београду, студентски трг 12–16, београд, 2институт за хемију, технологију и металургију, центар за хемију, универзитет у београду, његошева 12, београд и 3хемијски факултет, универзитет у београду, студентски трг 12–16, београд испитане су интеракције девет аминокиселинских деривата tert-бутилхинона са биомакромолекулима. sds гел-електрофорезом и масеном спектрометријом потврђено interactions of quinones with biological macromolecules 1357 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. је одсуство модификација лизозима било којим од синтетисаних једињења. спектрофотометријска испитивања су показала појаву хиперхромизма, као последицу постојања интеракција између хинона и ct-dna. константе везивања одређене апсорпционом титрацијом указују на слабе интеракције између хинонских деривата и ct-dna. синтетисани деривати смањују интензитет флуоресценције етидијум-бромида и боје hoechst 33258 у комплексу са dna, што потврђује постојање интеракција са dna из тимуса говечета (ct-dna). cd спектри указују на изостанак интеркалације хинона у исту. резултати молекулског моделовања потврђују везивање деривата у малу бразду. електрофоретска испитивања су показала да није дошло до цепања плазмида puc19 у присуству било ког испитиваног деривата. способност деривата да хватају слободне радикале потврђена је dpph тестом. сви приказани резултати указују на то да је механизам дејства аминокиселинских деривата везивање у малу бразду. 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m. a. husain, z. yaseen, s. u. rehman, t. sarwar, m. tabish, febs j. 280 (2013) 6569 34. g. zhang, x. hu, j. pan, spectrochim. acta, a 78 (2011) 687 35. m. p. uma, m. j. palaniandavar, j. inorg. biochem. 98 (2004) 219 36. y. li, g. zhang, j. pan, sens. actuators, b 191 (2014) 464 37. m. t. vujčić, s. tufegdžić, i. novaković, d. djikanović, m. j. gašić, d. sladić, int. j. biol. macromol. 62 (2013) 405 38. g. ionita, v. e. sahini, g. semenescu, p. ionita, acta chim. slov. 47 (2000) 111. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice phenolic and mineral profile of balkan indigenous apple and pear cultivars j. serb. chem. soc. 81 (6) 607–621 (2016) udc 582.734.3:54.004.12:547.56–3+543(497) jscs–4871 original scientific paper 607 phenolic and mineral profile of balkan indigenous apple and pear cultivars jelena živković1*, katarina šavikin1, gordana zdunić1, biljana dojčinović2 and nebojša menković1 1institute for medicinal plants research ‘’dr. josif pančić’’, tadeuša košćuška 1, 11000 belgrade, serbia and 2university of belgrade, institute of chemistry, technology and metallurgy, njegoševa 12, 11000 belgrade, serbia (received 26 october 2015, revised 16 february, accepted 17 february 2016) abstract: the aim of this study was the determination of phenolic compounds, and mineral nutrients in the pulp and peel obtained from balkan indigenous apple and pear cultivars. the phenolic composition, assessed by hplc-dad and spectrophotometric methods varied significantly between the cultivars for both peel and pulp. among the apples, the mekica cultivar had the highest total phenolic content while šećerlija and zlatna parmenka had the lowest. in the case of the pear samples, the maximum total phenolic content was found in crna takiša while the minimum content was recorded for bela arapka. in most of the investigated samples, chlorogenic acid in apples and arbutin in pears were the major detected polyphenolic compounds. with regard to the mineral analysis, k was the most abundant followed by p, mg, ca and s. iron was the dominant microelement in apple peel and pulp samples, while in pear samples the principal microelement was b. the obtained results provide detailed information on the chemical composition of the tested apple and pear cultivars and thereby, could encourage their wider cultivation and consumption. keywords: cultivar, autochthonous, apple, pear, peel. introduction due to excessive and widespread agricultural modernization, many autochthonous cultivars have been neglected and substituted with more productive and new international cultivars.1 accordingly, the global diet currently relies on a reduced number of species and varieties. it is becoming homogenized and separated from serbian cultural food traditions. this not only endangers the preservation of biodiversity, but could also “open the door” to overall epidemics of certain pests and pathogens.2 * corresponding author. e-mail: jzivkovic@mocbilja.rs doi: 10.2298/jsc151026023z _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 608 živković et al. on the contrary, indigenous cultivars represent the local germplasm cultivated mainly in the marginal areas. generally, they are resilient to the local environment and represent a favorable source for crop genetic variability, resistance to biotic and abiotic stresses, as well as for phenological and quality characteristics. since traditional cultivars mostly do not meet transport and storage demands, they are not cultivated for large-scale production.3 therefore, although indigenous cultivars could be more nutritious than newer ones, they are economically less important.4 owing to its geographical location, the republic of serbia has advantageous natural conditions and areas for planting a large number of fruit species and cultivars.5 indigenous malus and pyrus varieties are found in serbia mainly on individual farms in hilly–mountainous regions. due to the ageing of orchards and the abandonment of agricultural practices, there is a great possibility that certain genotypes will disappear in the future.6 increased consumption of fruits has been recommended as one of the essential components of a healthy diet for the prevention of chronic diseases.7 there are numerous reports on health-promoting benefits of phenolic compounds present in apples and pears,8,9 and as a result of a recent study,10 there is a new claim “an apple or pear a day helps keep the stroke at bay”. studies dealing with the phenolic composition of apples and pears are often restricted to a few cultivars3,11 that are very popular with customers. little attention has been given to autochthonous apple and pear cultivars,12,13 while there are only few reports,14 on the chemical profile of those grown in serbia. besides phytochemicals, an important part of the nutritional information of fruits is their mineral content. the significant impact of metal ions on human health is demonstrated by the fact that the function of more than one-third of all human proteins depend on them.15 as with polyphenols, the cultivar significantly influences the mineral content of the fruits.16 the aim of this study was the chemical analysis of phenolic compounds mineral nutrients in the pulp and peel obtained from serbian indigenous apple and pear cultivars. this work is important for an understanding of their nutritional potential and for a consequential expansion of their cultivation and use. experimental chemicals chlorogenic acid (95 %, titration), rutin hydrate (94 %, hplc), hyperoside (97 %, hplc), isoquercitrin (90 %, hplc), phloridzin (99 %, hplc), gallic acid (97.5 %, titration), arbutin (96 %, hplc) and 4-(dimethylamino)cinnamaldehyde (p-dmaca, ≥98.0%, hplc) were purchased from sigma aldrich (st. louis, mo, usa), and quercitrin (98.5 %, hplc) from extrasynthese (genay, france). acetonitrile (99.8 %) and formic acid (98–100 %) were supplied by sigma–aldrich (steinheim, germany). water was deionized by using a milli-q system (millipore, bedford, ma, usa). all other reagents were of analytical grade. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ chemical profile of autochthonous fruits 609 plant material the apple and pear cultivars investigated in this study were harvested in two regions in serbia: ljig in central serbia (apples kolačara, streknja, šećerlija, zlatna parmenka and senabija) and on zlatibor mountain in the western part of serbia (apples masnjača, mekica, kožara, kraljica and šarenka, as well as pear cultivars bela arapka, bronzara, bela takiša, crna takiša). samples were collected at the full maturity stage during 2014. ten apples from each cultivar were picked randomly, placed in polyethylene bags and transported to the institute for medicinal plant research. in order to obtain uniform sample, damaged fruits were removed. after rinsing with water, the seeds and stems were removed, and the peel was mechanically separated from the pulp. peel and pulp were blended in a philips microblender hr2860 to obtain a paste that was further lyophilized. the obtained lyophilized samples (one per each cultivar) were stored at 4 °c until further analysis. extraction procedure ultrasound-assisted extraction was carried out at room temperature. two grams of lyophilized pulp and peel samples were extracted with 25 ml of 70 % methanol for 30 min in a bandelon sonorex rk52 ultrasonic bath (bandelon electronic, munich, germany) operating at room temperature. after filtration, the obtained extracts were used for the determination of the total phenolic content, the total proanthocyanidins content and hplc analysis. total phenolic content determination of total phenolic content (tpc) was conducted according to a previously described method.17 an aliquot of sample (20 μl) was mixed with 1580 μl of distilled water, 100 μl of folin–ciocalteu reagent and 300 μl of 20 % na2co3, followed by incubation for 2 h at room temperature. after incubation, the absorbance was recorded at 765 nm. the contents of total phenolics were calculated using a standard curve for gallic acid and are expressed as milligrams of gallic acid equivalents per 1 g of dry sample (mg gae g-1 dry weight (dw)). total proanthocyanidin content the content of total proanthocyanidin compounds (tpr) in the samples was determined spectrophotometrically using p-dmaca method with slight modifications.18 the investigated extracts were mixed with 80 μl of p-dmaca reagent (2 ml), methanol (25 ml) and a drop of glycerol. after 7 min, the absorbance at 640 nm was measured. the contents of proanthocyanidins in the samples are expressed as milligrams of catechin equivalents per 1 g of dry sample (mg ce g-1 dw). the p-dmaca reagent was prepared immediately before use, and contained 1 % (w/v) p-dmaca in a cold mixture of methanol and hcl (4:1). hplc analysis phenolic compounds in the tested extracts were determined by comparing the retention times and absorption spectra (200–400 nm) of unknown peaks with those of pure standards injected under the same conditions. the standards used were chlorogenic acid, phloretin 2′-o -glucoside (phloridzin), quercetin 3-o-galactoside (hyperoside), quercetin 3-o-rutinoside (rutin), quercetin 3-o-rhamnoside (quercitrin) and quercetin 3-o-glucoside (isoquercetine). the hplc-dad analysis was performed on an agilent 1200 series hplc (agilent technologies, palo alto, ca, usa) equipped with lichrospher® 100 rp 18e column (5 µm, 250 mm×4 mm). mobile phase a was formic acid in water (1 %) and mobile phase b was acetonitrile. the injection volume was 30 µl, and the flow rate 1 ml min-1 with a gradient program as follows: 5–15 % b 0–5 min, 15–20 % b 5–8 min, 20 % b 8–12 min, 20–30 % b 12–15 min, 30 % b 15–17 min, 30–35 % b 17–20 min, 35 % b 20–22 min, 35–100 % b 22– _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 610 živković et al. –25 min. the stop time of the analysis was 25 min and column temperature was 25 °c. for the quantitative analysis of phenolic compounds, a calibration curve was obtained by injection of known concentrations (5–400 µg ml-1) of different standard compounds: arbutin (y = = 11560.8x – 0.5, r2 = 1), chlorogenic acid (y = 83506.1x–58.1, r2 = 0.998), quercetin 3-o -rutinoside (y = 26573.7x + 70.4, r2 = 0.999), quercetin 3-o-galactoside (y = 64124.3x + + 172.7, r2 = 0.997), quercetin 3-o-glucoside (y = 69710.5x+15.7, r2 = 0.999), quercetin 3-o-rhamnoside (y = 57551.6x + 46.7, r2 = 0.999) and phloretin 2′-o-glucoside (y = 58862.7x + 51.9, r2 = 0.999). quantification was performed based on dad results, using 280 nm for chlorogenic acid and phloretin 2′-o-glucoside and 350 nm for the flavonoid compounds. the investigated samples were analyzed in triplicate. the acceptable level of method precision was shown by the percentage relative standard deviation (rsd%) lower than 5 %. determination of the mineral elements the contents of mineral elements were determined as described previously by pavlović et al.19 lyophilized peel and pulp samples were subjected to microwave digestion using an ethos 1 microwave system (advanced microwave digestion system, milestone, italy). one gram of apple or pear sample, 1.0 ml of 30 % h2o2, and 7.0 ml of concentrated ultrapure hno3 (69.0–70.0 %)were mixed and transferred into the microwave digestion vessel. after the effervescence had subsided, the sample was cooled for 5 min, transferred into a clean volumetric flask, and diluted to 25 ml with ultrapure h2o. a blank was prepared in the same way. all analyses were performed in triplicate on a thermo scientific icap 6500 duo icp (thermo fisher scientific, cambridge, uk). statistical analysis the phenolic composition analyses were performed in triplicate and the data are presented as mean ± standard deviation. differences between the group means and their significance were verified using one-way anova. statistical significance was set at p < 0.05. on the other hand, the data for the elemental analysis were obtained from one measurement. results and discussion phenolic composition the phenolic concentration is commonly linked with nutritional and sensory attributes of fruits.20 apples and pears are one of the rare food types with precise data about their phenolic composition. the polyphenolic profiles of all apple cultivars are practically the same but concentrations may range from 0.1 to 5 g of total polyphenols per kg fresh weight and may be as high as 10 g kg–1 in certain varieties of cider apples.21 in the case of pears, the situation is similar, there are reports for tpc ranging from 3.02 to 4.58 g per kg fresh weight.22 in the present investigation, the content of total phenolics (tpc) and total proanthocyanidins (tpr) showed significant variation among the tested cultivars (tables i and ii). the tpc in apple and pear peel samples determined by the folin–ciocalteu assay ranged from 2.82 to 12.24 mg gae g–1 dw and from 3.81 to 8.11 mg gae g–1 dw, respectively. in the case of apple and pear pulp, tpc varied between 1.55 to 5.95 mg gae g–1 dw and 1.38 to 3.50 mg gae g–1 dw, respectively. among the apples, the mekica cultivar had the highest tpc while šećerlija and zlatna parmenka had the lowest. in the case of pear samples the max _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ chemical profile of autochthonous fruits 611 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 612 živković et al. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ chemical profile of autochthonous fruits 613 imum tpc was found for bela arapka while the minimum tpc was recorded for crna takiša. the present findings are consistent with those of other researchers. for example huber and rupasinghe,23 reported that tpc for red delicious, empire and royal gala peel extracts were 3.80, 4.82 and 4.84 mg g–1 dw, respectively. regarding commercial apple cultivars, alarcon-flores et al.24 showed that the pink lady variety had the highest concentration of phenolic compounds (4.11 mg g–1 dw) followed by ambrosia (4.01 mg g–1 dw). for peel samples of ten chinese pear cultivars, li et al.25 showed variations in the tpc from 2.64 to 11.21 mg gae g–1 dw. the results from the present study were also comparable with the results obtained for three pakistan pear varieties, nakh, nashpati and bartlett, for which the tpc ranged from 3.81 to 8.11 mg gae g–1 dw in peel and from 3.34 to 3.56 mg gae g–1 dw in pulp.26 in the same investigation, the tpc was approximately 6–20 times lower in pulp samples compared to peel. in the present study, the tpc was generally 2–3 fold higher in peel compared to pulp samples, in both apples and pears. as important bioactive constituents, the tpr content was also determined, and found to range from 0.71 to 7.62 mg catechins g–1 dw and from 0.77 to 3.84 mg catechins g–1 dw in apples peel and pulp, respectively, and from 0.40 to 5.17 mg catechins g–1 dw and from 0.11 to 1.44 mg catechins g–1 dw in pear peel and pulp, respectively, (tables i and ii). it could be observed that the tpr content was generally lower in pear peel and pulp samples than in the investigated apple samples. in addition, in comparison with the pulp samples, the tpr content was higher in the peel samples. although the obtained values for the tpr content were high, it was difficult to compare the results with existing literature data since different methodologies for quantification were used. for example, the tpr content in the cultivar champion determined by hplc analysis was 1.65 mg g–1 dw.13 chemical analysis of the individual phenolic compounds in the investigated pear and apple samples was conducted using the hplc-dad technique and the results are presented in table i. based on retention time and spectrum information, seven phenolic compounds were identified, i.e., one phenolic acid (chlorogenic acid), one phenolic glucoside (arbutin), four flavonols (quercetin 3o-rutinoside, quercetin 3-o-galactoside, quercetin 3-o-rhamnoside and quercetin 3-o-glucoside) and one dihydrochalcone (phloretin 2′-o-glucoside). for each individual phenolic compound, anova evaluated significant differences in dependence on the variety considered. among the identified phenolic compounds, in peel and pulp samples obtained from apples collected in ljig, chlorogenic acid was the dominant compound (ranging from 93.68 to 601.62 μg g–1 dw in peel and from 336.50 to 652.14 μg g–1 dw in pulp). the situation was similar with pulp samples of apples collected from zlatibor mountain (148.42 to 1530.99 μg g–1 dw) while for the peel samples (with the exception of the cultivar kraljica) the situation was slightly different. namely, phloretin 2′-o-glucoside (in _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 614 živković et al. kožara peel) or quercetin 3-o-rhamnoside (masnjača, mekica, kraljica and šarenka peel) were prevalent compounds followed by chlorogenic acid (from 147.68 to 688.69 μg g–1 dw). in general, the content of chlorogenic acid was significantly higher in apple pulp compared to apple peel. the exceptions were cultivars zlatna parmenka and streknja, in which the content was approximately 1.5 fold higher in peel, and masnjača, in which, chlorogenic acid was equally present in peel and pulp. for some cultivars (streknja, zlatna parmenka, kožara and kraljica), the content of chlorogenic acid was quite high compared with the results obtained by other authors for some commercial cultivars. awad and de jager27 reported values between 160 and 330 μg g–1 dw in peel of jonagold cultivar and lata et al.11 values 260 and 280 μg g–1 dw for peel samples of the cultivars elstar and granny smith, respectively. in addition, the obtained values for streknja, kožara, šećerlija and kraljica peel are much higher than those reported in previous studies for peel samples of fuji, royal and pink lady when the content of chlorogenic acid ranged from 280 to 430 μg g–1 dw.24 among apple phenolics, chlorogenic acid is one of the most important substrates for polyphenol oxidase (ppo); its oxidation generates pigments that can co-oxidize other substances.28 therefore, apple cultivars with a low concentration of chlorogenic acid would be more appropriate for producing apple juice, to minimize enzymatic browning, and to control the stability of the final product. for this reason, it could be stated that the cultivars mekica and kraljica are less suitable for producing apple juice. arbutin and chlorogenic acid were detected as the major phenolic compounds in case of pear peel and pulp samples. such results were consistent with previous data.29 in peel and pulp samples of bela and crna takiša, arbutin was the main phenolic compound, as well as in pulp samples obtained from the cultivars bela arapka and bronzara, while chlorogenic acid prevailed in their peel samples. in a study conducted by li et al.,25 arbutin was the dominant compound in 11 chinese cultivars in peel (ranging from 323.3 to 6982.0 µg g–1 dw) and in pulp (ranging from 92.8 to 2077.0 µg g–1 dw). quercetin glycosides were the only flavonols found in analyzed apple and pear samples. in some cultivars, they were the most abundant phenolic group mainly identified in the peel of the investigated apple and pear cultivars, while they were present only in traces in the pulp. significant differences in the contents of these compounds were found among the tested cultivars. the exception was quercetin 3-o-rhamnoside with a content of around 100 µg g-1 dw in cultivars masnjača, mekica, kožara and šarenka. among the apple peel samples, the highest contents of individual flavonols were detected in mekica, while in the case of the pear samples, bela arapka was the cultivar with the highest content. senabija among apples and bronzara and crna takiša among pears had the lowest levels of these compounds. in most of the pear and apple samples, quercetin _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ chemical profile of autochthonous fruits 615 3-o-rhamnoside was the dominant flavonol, the exception being the apple cultivar kolačara, in which quercetin 3-o-glucoside was dominant. the quercetin 3-o-rhamnoside contents in apple peel samples of the studied cultivars (from traces to 2129 μg g–1 dw) were comparable with previously obtained results for cultivars jonagold (2660 μg g–1 dw) and elstar (580 μg g–1 dw).8 on the other hand, the levels of quercetin 3-o-glucoside (5 to 344 μg g–1 dw) were lower compared with those of the commercial cultivars jonagold and elstar for which values of 530 and 700 μg g–1 dw were found.8 in the case of the apple pulp samples, the contents of quercetin 3-o-rhamnoside in masnjača, mekica, kožara and šarenka pulp (≈100 µg g–1 dw) were significantly higher compared to the commercial cultivars royal, fuji, golden and pink lady that contained from 2.7 to 28.6 µg g–1 dw.24 in terms of the content of flavonols in pears, the peel of bela arapka was the richest one, followed by the peel sample of bela takiša. macro and microelement analysis besides phytochemicals, an important part of nutritional information is the concentration of essential elements. minerals play a key role in different physiological functions of the body, especially in regulation processes. numerous factors, such as soil type and conditions as well as the variety, may cause variations in mineral contents of fruits. in this study, the content of most of the investigated elements was significantly different depending on the species, cultivar, and part of the fruit examined (tables iii and iv). generally, peel samples showed a higher mineral content compared to pulp. these results are in accordance with previous results.30,31 the most common mineral in the tested apple and pear samples was potassium with contents ranging from 4.12 to 8.86 and from 6.27 to 8.36 mg g–1 dw in apple and pear peel, respectively. in pulp, the potassium content was lower varying from 3.86 to 7.49 and from 4.59 to 6.59 in apple and pear samples, respectively. the concentration of k was the highest in the cultivar šećerlija among the apples and regarding the pears, the cultivar bela takiša was the richest in this element. remarkable contents were also observed for p, mg, ca and s. these data are consistent with the results obtained by other authors.31,32 the highest content of p and s in peel and pulp was obtained for the cultivar šećerlija, while the concentration of ca was highest in zlatna parmenka. the present results for macroelements were in agreement with those reported by manzoor et al.31 for cultivars golden delicious and red delicious. on the other hand, the contents of mg and ca determined in the present investigation were lower than those reported by ekholm et al.16 for apple peel (0.7 and 0.5 mg g–1 dw, respectively) and pulp (0.5 and 0.4 mg g–1 dw, respectively). considering the pear samples, the highest contents of ca, mg and p were recorded in crna takiša cultivar, while bela arapka was the one richest in p. the contents of ca in the _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 616 živković et al. pear peel samples were remarkably higher compared to those found in the apple peel samples. table iii. the concentration of macroelements (expressed as mg g-1 dw) in the apple and pear samples (n = 1); * samples from ljig, ** samples from zlatibor sample mineral ca k mg p s apple cultivar kolačara* peel 0.28 7.42 0.39 0.68 0.29 kolačara* pulp 0.17 6.09 0.24 0.44 0.17 streknja* peel 0.29 7.32 0.46 0.82 0.33 streknja* pulp 0.17 6.05 0.21 0.39 0.16 šećerlija* peel 0.36 8.86 0.50 1.09 0.43 šećerlija* pulp 0.28 7.49 0.31 0.58 0.22 zlatna parmenka* peel 0.69 4.87 0.48 0.56 0.24 zlatna parmenka* pulp 0.36 5.69 0.25 0.54 0.17 senabija* peel 0.33 7.43 0.45 0.69 0.30 senabija* pulp 0.27 6.71 0.30 0.41 0.16 masnjača** peel 0.37 6.68 0.65 0.99 0.36 masnjača** pulp 0.13 5.03 0.23 0.50 0.24 mekica** peel 0.59 6.24 0.60 0.79 0.38 mekica** pulp 0.28 3.86 0.18 0.38 0.13 kožara** peel 0.28 4.12 0.90 0.31 0.24 kožara** pulp 0.07 5.46 0.30 0.22 0.11 kraljica** peel 0.28 7.19 0.62 0.78 0.36 kraljica** pulp 0.09 5.40 0.22 0.63 0.24 šarenka** peel 0.49 7.28 0.42 0.74 0.29 šarenka** pulp 0.22 5.45 0.17 0.33 0.10 pear cultivar bela takiša** peel 0.52 8.36 0.42 0.72 0.23 bela takiša** pulp 0.21 6.59 0.30 0.75 0.18 bela arapka** peel 1.29 8.11 0.58 0.85 0.42 bela arapka** pulp 0.24 6.34 0.31 0.72 0.26 bronzara** peel 0.50 6.27 0.44 0.59 0.35 bronzara** pulp 0.17 4.59 0.25 0.56 0.19 crna takiša** peel 1.43 7.39 0.56 0.95 0.27 crna takiša** pulp 0.36 5.66 0.33 0.84 0.23 trace elements with contents higher than 1 µg g–1 dw were al, b, ba, cu, fe, mn, na and zn, while the other microelements were lower in the investigated samples. according to the obtained data, fe was the dominant microelement in the apple peel and pulp samples, while b was the principal microelement in the pear samples. appreciable amounts of fe were recorded in the peel samples of kolačara and šećerlija. the iron values in the investigated apple samples were lower compared to some commercial ones.31 on the other hand, they were comparable with the values obtained for romanian autochthonous cultivars when the _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ chemical profile of autochthonous fruits 617 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 618 živković et al. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ chemical profile of autochthonous fruits 619 fe content ranged from 2.45 to 3.64 μg g–1 dw.33 the content of b was highest in apple cultivars šećerlija and kraljica, while the cultivar crna takiša exhibited the highest b content (11.50 and 8.15 µg g–1 dw in peel and pulp samples, respectively) among the pears. according to the obtained data, the al content was higher in the fruit samples from ljig compared to that in the samples collected on zlatibor mountain. while the lowest al content in apple peel was observed for the cultivar šarenka, the cultivar zlatna parmenka was statistically superior as a source of al. for zn and cu, the data showed that pear samples were a better source compared to apples. the investigated non-nutritive toxic elements that are known to have deleterious effects even in small quantities (below 100 ppm) were as, sb, cd, pb and se.34 se was below the limit of detection in all the investigated samples, as were sb in the pear samples and cd in the apple samples. the content of the remaining elements was generally higher in the peel of the fruits than in the pulp for both apples and pears. the shown variability in the mineral contents could be ascribed to differences in cultivation conditions, such as soil fertility, ph, climate and seasonal variations. however, the differential capacity of the cultivars to absorb metal ions from soils and/or the specific capacity of redistribution within the overall plant could be other reasons for the exhibited variability. conclusions the results of the present study provide valuable data regarding the phenolic and mineral contents of balkan apple and pear cultivars. the concentrations of the identified polyphenols and minerals were significantly different depending on the apple or pear cultivar for both peel and pulp. although it is difficult to directly compare the content of apple phenolics between different studies due to variations caused by different growth periods, geographic locations, genetic diversity, methodology and many other factors,11 the cultivars mekica, šarenka (among the apples) and bela arapka (among the pears) could be considered as rich sources of phenolics according to the results of this study. based on these data, mentioned cultivars show high potential for juice production while the cultivars senabija and šećerlija, due to their lower phenolic content, are recommended for direct consumption. balkan autochthonous apple and pear varieties could be considered as a good source of dietary minerals. acknowledgements. the authors acknowledge their gratitude to the ministry of education, science and technological development of the republic of serbia for financial support, project no. 46013. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 620 živković et al. и з в о д фенолни и минерални профил аутохтоних варијетета јабука и крушака са балкана јелена живковић1, катарина шавикин1, гордана здунић1, биљана дојчиновић2 и небојша менковић1 1институт за проучавање лековитог биља „др. јосиф панчић“, тадеуша кошћушка 1, 11000 београд и 2универзитет у београду, институт за хемију, технологију и металургију, његошева 12, 11000 београд циљ ове студије била је хемијска анализа фенолних једињења, као и минералних састојака присутних у узорцима коре и меса балканских аутохтоних сорти јабука и крушака. фенолне компоненте одређене применом hplc-dad и спектрофотометријских техника значајно су варирале у зависности од варијетета у узорцима коре и меса. међу јабукама варијетет мекица имао је највећи садржај укупних фенола, док је у варијететима шећерлија и златна парменка он био најнижи. у случају крушака црна такиша је била богат извор ове групе једињења, док је у варијетету бела арапка забележена најнижа концентрација. у већини испитиваних варијетета хлорогенска киселина (код јабука) и арбутин (код крушака) била су доминантна фенолна једињења. анализом елемената показано је да је k најзаступљенији, следе p, mg, ca и s. гвожђе је доминантан микроелемент у кори и месу јабука, док је у узорцима крушака то b. добијени резултати дају детљну информацију о хемијском саставу испитиваних варијетета и на тај начин стимулишу њихово шире гајење и примену у исхрани. 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manzoor, f. anwar, n. saari, m. ashraf, molecules 17 (2012) 390 32. i. j. cindrić, i. krizman, m. zeiner, š. kampič, g. medunić, g. stingeder. food chem. 135 (2012) 2675 33. o. cedar, d. simedru, o. micu, m. miclean, e. levei, a. hoaghia, e. luca, agr.-sci. pract. 91 (2014) 3 34. a. dahelean, d. a. magdas, sci. world j. 2013 (2013), article id 215423. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice effect of transition metal cations on the commensurate freezing of n-hexane confined in micropores of zsm-5 j. serb. chem. soc. 80 (10) 1297–1309 (2015) udc 547.216+541.183+549.67: jscs–4798 536.51:536.75 original scientific paper 1297 effect of transition metal cations on the commensurate freezing of n-hexane confined in micropores of zsm-5 radmila hercigonja1*, vladislav rac2, vesna rakić2 and aline auroux3 1faculty of physical chemistry, university of belgrade, 11000 belgrade, studentski trg 12, serbia, 2faculty of agriculture, nemanjina 6, 11080 belgrade-zemun, nemanjina 6, serbia and 3université lyon 1, cnrs, umr 5256, ircelyon, institut de recherches sur la catalyse et l’environnement de lyon, 2 avenue albert einstein, f-69626 villeurbanne, france (received 3 february, accepted 21 april 2015) abstract: besides its importance concerning fundamental studies on gas adsorption in narrow pores, investigation of the commensurate freezing of a fluid within a zeolite is of practical importance in the application of zeolites in the processes of adsorption, separation and catalysis. in this work, the adsorption of n-hexane on hzsm-5 and its transition metal ion-exchanged modified forms was studied at 303 k by means of microcalorimetry. the thermal molar entropies changes of adsorption were calculated and thereby, the freezing-like behaviour of n-hexane inside the structure of the zeolite as a confinement media was noticed. this effect is governed by the attractive interactions between n-hexane molecules and the pore walls, and is influenced by the length of the pores and the nature of the charge-balancing cations. among the investigated zeolites, a solid-like phase of n-hexane in the pores of zeolites with fe(ii) ions was the most similar to solid bulk n-hexane, while cu(ii) ions contributed to the lowest ordering of the obtained solid-like n-hexane phase. keywords: confinement media; adsorption; entropy; microcalorimetry; zsm-5. introduction the confinement of fluids in limited spaces, such as narrow pores, is a very interesting phenomenon. the behaviour of fluid confined in a pore is influenced by many factors, such as pore size and geometry, and the atomic structure of the pore surface. consequently, its properties are distinctly different from those of bulk phase. for example, a confined fluid can have a higher density or can be in a different aggregation state from its analogue under normal conditions. there are many experimental and theoretical studies reporting that the phase behaviour of * corresponding author. e-mail: radah@ffh.bg.ac.rs doi: 10.2298/jsc150203032h _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1298 hercigonja et al. different fluids under extreme confinement is qualitatively different from that of the bulk.1–13 in the confinement medium, the presence of fluid–pore wall and fluid–fluid forces can lead to interesting surface-driven phase changes. these include new types of phase transitions not found in the bulk phase and shifts in transitions. in a bulk system, freezing is considered a first-order phase transition accompanied by an infinitely sharp change in a suitable order parameter, usually density or composition. in a confinement medium, the freezing phase transition is possible but with evident effects of confinement. the freezing temperature of a fluid confined in pores is determined by the bulk freezing temperature, pore wall–solid and pore wall–fluid surface tensions, the molar volume of the liquid, the latent heat of melting in the bulk and pore width.14 a decrease or increase in the freezing temperature due to confinement is strongly affected by the strength of the attractive forces between the fluid molecules and the pore walls.14,15 for repulsive or weakly attractive pore walls, the shift in the freezing temperature is negative.16,17 for highly attractive adsorbents where the adsorbate–pore wall interactions are strong compared to the adsorbate–adsorbate interactions, an increase in freezing temperature over the bulk value is observed.15,18,19–22 zeolites are regular crystalline solid microporous materials with peculiar structural characteristics (a three-dimensional lattice with well defined pores, high internal surface area and curvature, high ion-exchange capacity and remarkable thermal stability). zeolites are extensively used in the chemical industry as catalysts and for the separation of gases, particularly hydrocarbons.23–25 therefore, the adsorption of hydrocarbons, especially n-alkanes, has been widely studied.4,6,26–29 according to the molecular dimensions of their pores, zeolites may be considered as confinement media and the effect of confinement on fluids adsorbed in zeolites may be expected. for example, investigations of hexane and heptane adsorption at room and at slightly higher temperatures on silicalite-1 and zsm zeolite revealed anomalous behaviour of these two hydrocarbons compared to other alkanes.2–6 an explanation was given for the first time by smit and maesen.30 their interpretation is that the adsorption of straight chain hydrocarbons on silicalite leads to a phase transition of the hydrocarbons inside the pores of the silicalite. bearing in mind that silicalite-1 and zsm have two types of channels, straight and zigzag (sinusoidal) connected via intersections, they showed that phase transition occurred when the lengths of the adsorbed molecules were similar to the length of the channels. indeed, the length of n-hexane and heptane molecules are 1.03 and 1.16 nm, respectively, while the length between the centres of channel intersections of the silicalite structure is 1.2 nm, i.e., they are comparable. under the above conditions, fluid can freeze in a configuration that is commensurate with the pore structure.30–33 phase transitions of hexane and heptane between the gas, liquid and solid phases in the pores of silicalite-1 and zsm are generally accepted and were the subject of many studies.34–41 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ commensurate freezing of n-hexane in micropores of zsm-5 1299 commensurate adsorption of hydrocarbons (e.g., p-xylene, n-hexane, n-heptane, benzene, etc.) was found in several different types of zeolites, for instance, mfi, itw, eri, cha, lta, afx and silicalites.42–46 evidence for freezing of n-hexane molecules inside the channels of zsm can be seen from the adsorption isotherms, which can show a step or kink when half of the maximum loading (about 4 molecules per unit) is achieved; such isotherms were obtained for adsorption measurements at temperatures above 338 k.4,6,30,47–49 lohse et al.50 did not provide evidence for a kink at half of the loading because the temperature of adsorption (298 k) was too low. additionally, meansen30 showed in a simulation that the kink becomes more pronounced with increasing temperature. the adsorption isotherms measured by zhu et al.51 showed a kink in the temperature region 338–373 k, but not at 303 k. the volumetric adsorption isotherms of n-hexane on hzsm-5 zeolite and its transition metal modified forms were reported,52 but they did not show a kink since a low temperature of adsorption (303 k) was applied. in addition to isotherms, temperature programmed desorption (tpd) profiles also gave evidence of commensurate freezing of n-hexane in the zigzag channels of zeolite. the tpd studies showed that among the linear alkanes, hexane and heptane behave distinctly differently.28,32,44,45,48,53–57 while other linear alkanes showed a single desorption step, n-hexane and especially n-heptane exhibited two-step desorption profiles. the first desorption peak of n-hexane and nheptane occurred at lower temperatures than expected based on the chain length of these two n-alkanes. this low temperature peak corresponds to desorption from the zigzag channels and should be caused by a relatively high gain in entropy upon desorption compared to the other n-alkanes. the relatively high gain in entropy upon desorption can only be the result of a low entropy value in the constrained position of the adsorbed n-hexane and n-heptane molecules at high loadings. partial desorption then allows a rearrangement of the adsorbed n-hexane or n-heptane molecules, resulting in an ordering similar to the ordering of the other n-alkanes with normal entropy values. the high temperature peak corresponds to desorption from the straight channels and it occurs at temperatures that are in accordance with the chain length of the n-alkane. it was reported58 that the tpd profiles of n-hexane sorbed on zsm-5 were composed of two well-defined peaks in the temperature region from 300 to 550 k, consistent with the results of other researchers.28,32,44,54,56 accordingly, this indicated that the phase transition of freezing occurred when n-hexane was adsorbed on the samples of zsm used in the present study. in the last decade, among the transition metal ion-containing zeolites, the fe-, cuand mn-mfi zeolites have received much attention because they are catalytically active in some important reactions, such as n2o and no decomposition, reduction of nox to n2 in the reaction with various hydrocarbons59–61 and catal_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1300 hercigonja et al. ytic cracking of hydrocarbons.62–64 many experimental results indicate that the physical adsorption of n-alkane reactants contributes to the kinetics of catalytic reactions.65–67 in this work, the phase transition of commensurate freezing of n-hexane confined in microporous of hzsm-5 and in its forms modified by different charge balancing cations: cu(ii), mn(ii) and fe(ii), was investigated. it is known that attractive adsorbate–pore wall interactions in zeolite partly originate from the charge balance cation–adsorbate interactions, and hence, an influence of transition metal cations on the adsorption of n-hexane and on its commensurate freezing could be expected. recently, molecular dynamics (md) simulation studies31 clearly showed that the behaviour of n-hexane at a loading of 4 mol per unit cell (u.c.) in silicalite-1 should not be ascribed to an enthalpy effect but to an entropy change of the system approaching 4 mol (u.c.)–1, which was in agreement with the results of smit et al.1 therefore, in order to investigate the influence of the nature of the charge balancing cation on the commensurate freezing of n-hexane inside the zeolite structure, one physical property, thermal molar entropy, was studied, which was chosen for two reasons: first, the appearance of a low temperature peak in tpd profile, as confirmation that commensurate freezing is associated with the entropy change, and secondly, the property in relation to the molar entropy of liquid and solid n-hexane may cast insight into the freedom of the n-hexane molecules within the zeolite and thus, could provide evidence for the occurrence of commensurate freezing. experimental the parent self-produced nazsm-5 (si/al = 20) was synthesized hydrothermally. hzsm-5 and under-exchanged forms of hzsm-5 containing cu(ii), fe(ii) or mn(ii) cations (monoor bi-metallic) were obtained by appropriate common wet ion-exchange procedures, fully described previously.22 the crystallinity of the parent nazsm-5, hzsm-5 and ion -exchanged forms was proved by x-ray diffraction. the measurements were performed on a bruker (siemens) d5005 diffractometer at room temperature using cukα radiation (0.154 nm), 2θ from 3 to 80°, in 0.02° steps with 1 s per step, and the results showed that the structure of the zsm-5 zeolite remained unchanged during the ion-exchange process. xrd analysis proved that the structure was also not changed by adsorption of n-hexane. the differential heats and the isotherms of n-hexane adsorption were collected using a coupled microcalorimetric–volumetric line, using the procedure fully described elsewhere.23 briefly, the heats of adsorption were measured in a heat-flow microcalorimeter (c80, setaram) linked to a glass volumetric line that permitted the admission of successive known doses of adsorbed gas, until a final equilibrium pressure of 66 pa. subsequently, the sample was pumped, the desorption peak was recorded and re-adsorption was performed at the same temperature. before the adsorption, the samples were pre-treated in vacuum (10-3 pa) overnight at 673 k, while the adsorption temperature was maintained at 303 k. in order to clarify the states of cation species in the investigated samples, uv–vis diffuse reflectance spectra were recorded, in 190–1000 nm spectral region, using a perkin elmer lambda 35 uv–vis spectrometer equipped with a diffuse reflectance accessory. the powder samples were placed in the sample cup and baso4 was used as a reference. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ commensurate freezing of n-hexane in micropores of zsm-5 1301 the thermal entropy of sorbed n-hexane was calculated from the values of the entropy change of adsorption, δs0, obtained from calorimetric measurements.52 the obtained values of entropy change of adsorption are in good agreement with the values from the literature.44,56,68,69 the following expression was used to calculate δs0 from the microcalorimetry data: 0 0 0 δδ ln p h s r p t = + (1) where t is the adsorption temperature, p0 is the standard pressure, p is the equilibrium pressure at temperature t and δh0 is the standard enthalpy change in the adsorption process. these values were obtained from microcalorimetry as differential heats of adsorption. the entropy change accompanying the adsorption process can be expressed as: 0 0 *g sδs s s= − (2) where 0gs is the molar entropy of n-hexane vapour at standard pressure p0 and temperature t, while *ss is the differential molar entropy of adsorbed n-hexane. the differential molar entropy, is a finite, positive quantity that may be separated into thermal and configurational entropy components.70 in this case, *ss can be represented by: *s c ths s s= + (3) where sc and sth are the differential molar configurational and differential molar thermal entropies, respectively. when the density of the molecules adsorbed in the channels of the zeolite is high, it is generally assumed that these molecules attempt to pack closely together and take up the walls of the anion framework. with such a localized sorption process, sc, may be calculated with the equation: c ln 1 x s r x = − (4) where 0/x w w= (w is the amount adsorbed at equilibrium pressure p, and w0 is the total adsorption capacity at 303 k).71 subtraction of eq. (4) from *ss gives sth: *th s ln 1 x s s r x = − − (5) results and discussion the values of differential molar entropy, differential molar configurational and differential molar thermal entropies were calculated for the adsorption of n-hexane with different loadings (expressed as n, number of molecules adsorbed per unit cell), on both parent hzsm-5 and its cation modified forms using eqs. (2)–(5). the profiles of differential molar thermal entropies plotted vs. the amount adsorbed are shown in fig. 1. the profiles of the sth values vs. loading gave evidence that the differential molar thermal entropies of the n-hexane adsorbed on zeolite was dependent on the nature of the environment of the adsorbate molecules, i.e., the porous zeolite lattice with its charge balance cations. it can be seen from fig. 1 that the sth of parent hzsm-5 was altered by the incorporation of transition metal cations in its _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1302 hercigonja et al. lattice. furthermore, the presence of transition metal cations increased the adsorption capability of hzsm-5 zeolite from 4 (obtained in the case of hzsm-5) to 6 molecules of n-hexane per unit cell (found for zeolites with transition metal cations). different numbers (from 2 to 8) of sorbed n-hexane molecules per unit cell were reported in the literature,4,32,72 while the theoretical loading limit for zsm-5 was 8 hexane molecules per unit cell.73 it could be inferred that the charge balance cations influence the values of sth since they result from n-hexane attractive interactions with the zeolite pore wall, originating from the macroanion framework and extra-framework charge-balancing cations. fig. 1. differential molar thermal entropies (sth) as a function of loading n (molecules per unit cell) of n-hexane. in a study of n-hexane adsorption on zsm, several facts must be taken into account. first, the length of the n-hexane molecule is almost the same as the distance between channel intersections and hence, these molecules cannot be wholly located in the channel segments and the possibility of adsorbate–adsorbate interaction at the channel intersections always exists at high surface loadings. in fact, adsorbate–adsorbate interaction are enhanced for a loading of 4 n-hexane molecules per unit cell; while for even higher values (4–8 molecules per unit cell), the adsorbed molecules arrange in such way that additional side-on interactions can occur; while with further increase in the n values, closer packing of n-hexane molecules occurs and thus, repulsion interactions become significant.4,47,74,75 for all the investigated zeolites, the sth vs. loading profiles can be divided into three regions of loading. it could be seen from fig. 1 that these regions are: _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ commensurate freezing of n-hexane in micropores of zsm-5 1303 n < 2, 2 < n < 6 and n > 6. the sth values found for n-hexane adsorption on hzsm-5, cuzsm-5 and cumnzsm-5 decreased sharply in the initial, micro -pore-filling region (n < 2). this is probably due to the fact that the first molecules entering the micropores occupy the most favourable sites, which results in their fast ordering and fast loss in sth. in the case of adsorption on fezsm-5, the sth values decreased very slowly up to n < 2. however, it is important to notice that in this low-surface coverage region, the sth values measured for n-hexane adsorption on mnzsm-5, femnzsm-5 and fecuzsm-5 showed increased values with a more or less marked maxima. barrer et al.70,76 reported that strong energetic heterogeneity is reflected in a maximum in the entropy curves against coverage for low adsorbate uptakes. indeed, it seems that on these samples, the ordering of adsorbed molecules occurred as a result of interaction with surface active sites. after the maximum values of sth were reached on mnzsm-5 and on the samples containing fe(ii) cations (fezsm-5, femnzsm-5 and fecuzsm-5), the sth values remained almost constant in the medium surface coverage region (2 < n < 6), thus indicating highly localized adsorption. in contrast, the sth values of hzsm-5 and the zeolites containing cu(ii) cations (cuzsm-5 and cumnzsm5) slightly decreased from the constant values, thus indicating that there were some deviations of the localized sorption. for high surface coverages (n ˃ 4 for hmnzsm-5 and n ˃ 6 for all other samples), the sth values increased sharply as a result of repulsion interactions among the closely packed n-hexane molecules. at low surface coverage, the adsorbate molecules can move freely in the zigzag channels and thus fill one part of the intersections for some time. as a consequence, further adsorption would become restricted, since the intersections are blocked while the straight channels are too short to accommodate n-hexane molecules. to fill the zeolite completely, the molecules adsorbed in a zigzag channel have to be confined in their position, which leads to a loss in entropy.8 it is accepted that when a coverage of half a loading per unit cell is achieved, the intersections are blocked. then, the adsorbed molecules in the zigzag zeolite channels undergo phase transition, which is known as commensurate freezing. the sth values of n-hexane adsorbed that are achieved at half-loading (≈3 molecules per unit cell) may be compared with the entropy of liquid n-hexane at 303 k (300 j mol–1 k–1) and the sum of the vaporization entropy change (94 j mol–1 k–1) and the fusion entropy change of n-hexane (72 j mol–1 k–1) at 178 k.77 according to eq. (3), the values of the differential molar entropy at half-loading enable the derivation the sth part of the respective differential molar entropy since c 0s = . these values of sth are listed in table i. the sth part of the respective *ss obtained for the configurational entropy c 0s = of n-hexane adsorbed within the channels of fezsm-5, femnzsm-5 and fecuzsm-5 were between 55 and 62 j mol–1 k–1; values sufficiently lower than the sum of the vaporization entropy change and the fusion entropy change of n-hex _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1304 hercigonja et al. table i. the sth part of respective *ss (for c 0s = ) zeolite *ss (= ths ) / j mol-1 k-1 ( c 0s = ) cumnzsm-5 134 cuzsm-5 136 mnzsm-5 98 femnzsm-5 60 fezsm-5 55 fecuzsm-5 62 ane at 178 k to suggest that degree of ordering of the molecules adsorbed in the pores of these zeolites was similar to that in solid n-hexane. similar results were found for n-hexane adsorbed on mnzsm-5. in this case, the sth part of the respective *ss , for c 0s = equalled 98 j mol–1 k–1, which is more than the sth found in the case of the zeolites containing fe(ii) cations, but still less than the sum of the vaporization entropy change and the fusion entropy change of n-hexane at 178 k. the sth parts of the respective *ss obtained for c 0s = within the channels of cumnzsm-5 and cuzsm-5 were practically equal (134 and 136 j mol–1 k–1) and also less than the sum of the vaporization entropy change and the fusion entropy change of n-hexane at 178 k. obviously, the presence of mn(ii), cu(ii) and especially fe(ii) cations changes the attractive interactions between n-hexane molecules and the pore walls in such a way to enable the adsorbed n-hexane molecules to be arranged as in a solid-like state of n-hexane. it is noteworthy that the sth values obtained for the samples containing fe(ii) cations were about 100 j mol–1 k–1 lower than the sum of the vaporization entropy change and the fusion entropy change of n-hexane at 178 k, while the sth values for the samples containing cu(ii) ions were only about 20 j mol–1 k–1 lower than the above mentioned sum, indicating a higher ordering of the hexane molecules in the zeolites containing fe(ii) cations than inside those containing cu(ii) cations. the general conclusion, based on the values of sth, is that a phase transition from gaseous n-hexane to a solid-like structure of n-hexane, i.e., commensurate freezing of n-hexane, occurred in the pores of zsm-5 containing transition metal ions. the commensurate freezing occurred at 303 k, well above the temperature of the bulk freezing of n-hexane (178 k). increasing of the freezing temperature is a characteristic of fluids confined in a confinement medium with highly attracting pore-walls, as is the case with the investigated zeolites. in order to better illustrate the influence of the extra framework cations on decreasing sth, the difference between sth and 0gs was calculated and plotted as 0 th g( )s s− vs. loading (fig. 2). at the very beginning of the sorption, the entropy loss followed the order: fecuzsm-5 > femnzsm-5 > fezsm-5 > mnzsm-5 > hzsm-5 > cuzsm-5 > cumnzsm-5. at half of the maximum loading, the entropy loss followed the order: fezsm-5 > fecuzsm-5 = femnzsm-5 > hzsm-5 > mnzsm-5 > _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ commensurate freezing of n-hexane in micropores of zsm-5 1305 cuzsm-5 = cumnzsm-5, while at the maximum loading (n = 6), the entropy loss followed the order: fezsm-5 > fecuzsm-5 > femnzsm-5 > mnzsm-5 > cumnzsm-5 > cuzsm-5. fig. 2. dependence of the entropy loss 0th g( )s s− of adsorbed n-hexane on loading n. the maximum entropy loss was achieved for n-hexane adsorption in the zeolites with fe(ii) cations and the minimum entropy loss was found for n-hexane adsorption in zeolites with cu(ii) cations. the rigid zeolite structure is the same in all these samples, which means that the presence of the charge-balancing cations influences the distribution and ordering of n-hexane molecules inside the zeolite channels, and thus the sth values. it is important to note that27 al mas nmr experiments showed that the hzsm-5 investigated in this work does not contain extra framework al, which means that some steric hindrance for n-hexane adsorption was not to be expected.78 the different values of the sth part of respective *ss (for c 0s = ) of the samples with different charge balancing cations indicate that the created solid-like phase was not always the same and depended on the type of the extra framework cations. cu(ii), mn(ii) and fe(ii) have the same charge, but different ion radii (cu: 0.73 nm, mn: 0.82 nm and fe: 0.78 nm) and hence, they create different electrostatic interactions with n-hexane molecules. in addition, cu(ii), mn(ii) and fe(ii) have different electron configurations, i.e., (mn(ii) possesses five and fe(ii) four incomplete d-orbitals, while cu(ii) has only one unpaired electron in the 4s1 orbital). the electrons of uncompleted orbitals can form bonds with oxygen atoms from the lattice, as well with _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1306 hercigonja et al. adsorbed n-hexane molecules. if these electrons “react” with the oxygen of the lattice, the reaction of n-hexane with oxygen atoms is reduced, while the cations partly lose the possibility of interaction with n-hexane. both effects cause an increase in sth. however, the interaction of spin unpaired electrons with n-hexane molecules produce the opposite effect, i.e., a decrease in sth. the low sth obtained for samples with mn(ii) and fe(ii) cations shows that mn(ii) and fe(ii) present in zeolite lattice coordinated the electrons of adsorbed n-hexane easier than the electrons of the oxygen atoms from the lattice. therefore, the sth of the samples containing mn(ii) and fe(ii) ions are influenced both by ion-induced dipole interactions, while the additional interactions originate from the possibility of mn(ii) and fe(ii) to behave as electron acceptors. on the contrary, considering that cu(ii) ions have only one unpaired electron in the 4s1 orbital, the sth value of samples possessing cu(ii) ions is mostly determined by ion-induced dipole interactions through electrostatic and dispersive forces. if all the interactions achieved between n-hexane molecules and the samples possessing fe(ii) and mn(ii) ions are compared, it could be concluded that fe(ii) ions can achieve stronger electrostatic (its ionic radius is smaller), but weaker additional interactions (fe(ii) possesses four uncompleted d-orbitals, mn(ii) five). based on the values of sth, it seems that both these interactions are stronger in the samples containing fe(ii) cations, which led to better ordering of the adsorbate and finally to the formation of a solid-like phase in the course of a phase transition known as commensurate freezing. conclusions the results obtained in this work show that the phase transition of commensurate freezing of n-hexane occurred during the adsorption of n-hexane into the parent hzsm-5 and its transition metal modified forms: cuzsm-5, mnzsm-5, fezsm-5, cumnzsm-5, femnzsm-5 and fecuzsm-5. freezing of n-hexane, the molecules of which “fit” the zigzag channels of zsm-5, occurred at 303 k, which is well above the freezing temperature of bulk n-hexane (178 k). the positive shift in the freezing temperature was affected by the strong attractive forces between the n-hexane molecules and pore walls of the zeolite, originating from the presence of charge balancing cations. the results showed that above the circumstances of reduced dimensionality, such is in the case of zeolite lattice, the type (size and charge) and nature (electron configuration) of the charge balancing cation determine the ordering of the solid like phase of n-hexane obtained in the phase transition of commensurate freezing. among the investigated zeolites, the solid-like phases obtained in zeolites possessing fe(ii) cations (fezsm-5, femnzsm-5 and fecuzsm-5) were the most similar to the solid n-hexane phase. obviously, the presence of fe(ii) as the charge balancing cation facilitated the ordering of the adsorbed n-hexane molecules inside the zeolite. the smallest _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ commensurate freezing of n-hexane in micropores of zsm-5 1307 ordering of the solid like phase of n-hexane obtained in the process of commensurate freezing was in the zeolites with cu(ii) as the charge balancing cations (cumnzsm-5 and cuzsm-5). acknowledgement. the authors acknowledge the support from the ministry of education, science and technological development of the republic of serbia (project no. 172018). и з в о д утицај катјона прелазних метала на “мржњење” хексана у ограниченом простору микропора zsm-5 зеолита радмила херцигоња1, владислав рац2, весна ракић2 и aline auroux3 1факултет за физичку хемију, универзитет у београду, студентски трг 12, 11 000 београд, 2пољопривредни факултет, универзитет у београду, немањина 6, 11080 београд-земун и 3université lyon 1, cnrs, umr 5256, ircelyon, institut de recherches sur la catalyse et l’environnement de lyon, 2 avenue albert einstein, f-69626 villeurbanne, francе commensurate freezing је назив за појаву фазног прелаза првог реда (мржњења) гасова на температурама које су знатно изнад температуре сублимације слободног гаса, у условима када се гас адсорбује у уским порама чије димензије одговарају димензијама молекула гаса. поред значаја у основним испитивањима адсорпције гасова, испитивање “мржњења” флуида унутар зеолита као микропорозног материјала је од практичног значаја у примени зеолита у процесима адсорпције, раздвајања компонената и катализи. у овом раду је проучавана адсорпција n-хексана из гасне фазе на zsm-5 зеолиту, као и његовим облицима модификованим јонима прелазних метала, применом микрокалориметрије на температури од 303 k. zsm-5 зеолит има два типа канала, праве и синусоидалне (цик-цак) који се међусобно секу. дужина молекула n-хексана (1,03 nm) одговара растојању између центара пресека правих и цик-цак канала зеолита (1,2 nm), тако да под овим условима n-хексан може да се адсорбује на такав начин да је његово кретање толико ограничено да може да се схвати као замрзавање у каналима зеолита. ова појава је у литаратури названа commensurate freezing и повезана је са променом ентропије система, па је у раду на основу израчунатих вредности промене термалне моларне ентропије адсорпције потврђено да је дошло до формирања структуре n-хексана која личи на чврсти n-хексан. такође је показано да наведена фазна промена n-хексана зависи од природе катјона прелазних метала (електронске конфигурације, димензија и наелектрисања) који компензују негативно наелектрисање решетке. међу испитиваним зеолитима, у оном са fe(ii) јонима је постигнут највећи степен уређености молекула nхексана тако да је у том случају он у стању које је најсличније чврстом док је у зеолиту са cu(ii) јонима постигнута његова најмања уређеност. 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snurr, g. baron, j. phys. chem., b 107 (2003) 10760 74. p. titus, j. phys. chem., c 115 (2011) 3346 75. k. m. a. de meyer, m. kurt, s. chempath, j. denayer, j. martens, a. johan, r. snurr, g. baron, j. phys. chem., b 107 (2003) 10760 76. r. barrer, r. gibbons, trans. faraday soc. 59 (1963) 2875 77. nist chemistry webbook, http://www.webbook.nist.gov/chemistry/ 78. h. zou, m. li, j. shen, a. auroux, j. therm. anal. calorim. 72 (2003) 209. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice electrochemical determination of ascorbic acid at p-phenylenediamine film–holes modified glassy carbon electrodes j. serb. chem. soc. 80 (9) 1161–1175 (2015) udc 547.475.2+543.55+544.6.076.32– jscs–4788 033.5–039.6 original scientific paper 1161 electrochemical determination of ascorbic acid at p-phenylenediamine film–holes modified glassy carbon electrodes bikila nagasa olana, shimeles addisu kitte and tesfaye refera soreta* department of chemistry, college of natural sciences, jimma university, p. o. box 378, jimma, ethiopia (received 4 november, revised 30 december 2014, accepted 19 january 2015) abstract: in this work, the determination of ascorbic acid (aa) at a glassy carbon electrode (gce) modified with a perforated film produced by reduction of diazonium generated in situ from p-phenylenediamine (pd) is reported. holes were intentionally created in the modifier film by stripping pre-deposited gold nanoparticles. the modified electrodes were electrochemically characterized using common redox probes: hydroquinone, ferrocyanide and hexamineruthenium(iii). the cyclic voltammetric and amperometric responses of aa using the modified electrodes were compared with those of a bare gce. the bare gce showed a linear response to aa in the concentration range of 5 mm to 45 mm with detection limit of 1.656 mm and the modified gce showed a linear response to aa in the concentration range from 5 to 45 µm with detection limit of 0.123 μm. the effects of potential interferents on amperometric signal of aa at the modified gce were examined and found to be minimal. the inter-electrode reproducibility, stability, and accuracy were determined. the modified electrode showed excellent inter-electrode reproducibility, accuracy and stability. the modified electrode reported is a promising candidate for use in the electro-analysis of aa. keywords: diazonium; p-phenyldiamine; gold nanoparticles; ascorbic acid; glassy carbon electrode. introduction ascorbic acid (aa) naturally occurs in a wide number of foods, such as fruits and vegetables. it is a water-soluble organic compound involved in many biological processes. it is known for its reductive properties that make it useful as an antioxidant agent in foods and drinks. moreover, pharmaceuticals often contain aa as a supplementary source to human diets as a free-radical scavenger. it * corresponding author. e-mail: tesfaye.refera@ju.edu.et doi: 10.2298/jsc141104006o _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1162 olana, kitte and soreta has been used for the prevention and treatment of the common cold, mental illness, scurvy and cancer.1 however, the intake of excess aa can lead to undesirable health effects, such as gastric irritation, excessive oxidative stress, diabetes mellitus, liver disease2 and renal problems.3 excessive quantities of aa in food may result in the inhibition of the occurrence of natural processes and hence may contribute to taste deterioration.4 aa is a labile substance that easily degrades due to interaction with enzymes and atmospheric oxygen. excessive heat, exposure to light, and interaction with heavy metal cations can accelerate the oxidation of aa.5 due to its susceptibility to oxidation, the analysis of the level of aa in foodstuffs and beverages helps to indicate their quality. hence, the level of aa has to be carefully monitored to estimate the relative variation of aa from manufacture, storage up to consumption. for this reason, there is a necessity for an easy-to-use, inexpensive method for the detection of aa in food, beverages and pharmaceuticals.6 many analytical methods have been reported in the literature for the determination of aa.7,8 electrochemical techniques are known to offer some benefits such as fast analysis, low cost, higher sensitivity and accuracy. however, the major problem frequently encountered in the electro-analysis of aa is the effect of interferents caused by substances with similar redox potentials at conventional electrodes, which results in poor selectivity. in the presence of co-existing oxidizable species, the determined amount of vitamin c could be overestimated.9–12 thus, it is difficult to detect specifically one substance in the presence of others substances in real biological samples at conventional electrodes. aa exists in the anionic form at physiological ph values. based on this property; different techniques were developed to detect aa selectively. modification of the working electrode with modifiers such as tetrabromo-p-benzoquinone,13 electronically conductive anion exchange polymers based on polypyrrole14 and polyaniline15 showed promising applications in the fabrication of sensors for sensitive and selective detection of aa. the most distinguishing feature of chemically modified electrodes is their modification by a selected substance that is coated onto the electrode surface thereby imparting certain desirable properties to the electrode. the use of nanomaterials for nanostructuring of an electrode surface has aroused the interest of analysts16 because nanostructured materials can be tailored to improve the selectivity and sensitivity of sensors. further investigation of these new materials in the fabrication chemically modified electrodes is required to exploit the systems. in this work, a glassy carbon working electrode was modified with electronucleated gold nanoparticles and passivated with an organic film by grafting diazonium obtained from p-phenylenediamine. holes were formed on the electrodeposited film by stripping the nucleated gold nanoparticles. improvement in the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ electrochemical determination of ascorbic acid 1163 selectivity and sensitivity of the electrode surface modified using the developed method for the determination of aa was demonstrated. experimental chemicals ascorbic acid (99 %, finkem), p-phenylenediamine (100 %, aldrich), sodium nitrite (96 %, wardle), potassium nitrate (99 %, nice), potassium teterachloroaurate (99.99 %, aldrich), 2-mercaptoethanol (100 %, aldrich), hydrochloric acid, (37 %, riedel-de haen), sulfuric acid, (98 %, merck), hydroquinone (99 %, kiran), potassium hexacyanoferrate(iii) (97 %, labmerk chemicals), hexaminerutheniumchloride(iii), (98 %, aldrich), potassium iodide (99 %, nice), iodine resublimed (99.5 %, nice), potassium chloride (99 %, finkem), sodium citrate dihydrate (99 %, finkem), citric acid (99 %, wardle), sodium acetate trihydrate (99.8 %, chem. rein), glacial acetic acid (100 %, bdh laboratory), potassium hydrogen phosphate (98 %, finkem) and potassium dihydrogen phosphate (99 %, nice) were of analytical grade and used as received. the vitamin c tablet 500 mg [batch number 11202023, ethiopian pharmaceuticals] was purchased from a local drug store. double distilled water was used to prepare all solutions. instrumentation cyclic voltammetry (cv) and amperometric experiments were performed using basi epsilion ec-version 1.40.67 voltammetric analyzer (bio-analytical systems, usa) controlled with basic epsilon software. a conventional three-electrode setup was used with a glassy carbon electrode (3 mm diameter, basi, mf 2012) as the working electrode and a platinum wire counter electrode (basi, mw 1032). an ag/agcl electrode (basi, mf 2079) served as the reference electrode. all potentials were reported with respect to this reference electrode. for stirring the electrolytes in the cell, a small magnetic bar was used in the basi c3 cell stand at 500 rpm. methods electrode preparation. prior to electrode modification, a bare glassy carbon electrode (gce) was polished with polishing paper and then further polished to a mirror finish with alumina slurries (0.3 micron, basi) and rinsed thoroughly with distilled water. the procedure reported by soreta et al.17 was used for electrochemical conditioning of the employed electrodes. fabrication of pd film–hole modified gce. pd represents the aryldiazonium generated from p-phenylenediamine. fabrication of the pd film–hole modified electrode was undertaken in several steps. the major steps were sequential electronucleation of gold nanoparticles (aunps) on the gce (three rounds), grafting of a diazonium film from p-phenyldiamine (pd) on a gce modified with aunps and stripping of the nucleated aunps. i. electrodeposition of aunps. sequential electronucleation of gold nanoparticles on a gce was performed following the procedure reported by soreta et al.18 sequential electronucleation was used to increase the number of gold nanoparticles deposited on the gce surface while preventing the growth of the already nucleated particles so that they remain in the nano-size range. ii. grafting of diazonium film generated from p-phenylenediamine (modifier film). the aunps-modified gce was covered with in situ generated p-phenylenediamine diazonium cations based on literature information.19,20 briefly, 5 ml solution of 3 mmol l-1 p-phenylenediamine in 0.5 mol l-1 hcl and 5 ml of 3 mmol l-1 sodium nitrite in 0.5 mol l-1 hcl _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1164 olana, kitte and soreta were kept separately in an ice jacketed beaker for 1 h. then, 2 ml of the nano2 solution was added to 2 ml of the p-phenylenediamine solution under stirring at room temperature and cv was used to graft the phenylenediamine film onto the aunps nucleated gce within potential window from 0.6 to –0.2 v at a scan rate of 0.1 v s-1 for 3 cycles. it was reported that an aryldiazonium film could be grafted on both carbon and gold particles surfaces.21 iii. electrochemical formation of random holes on the modifier film. this is the only new step introduced in the fabrication of the modified electrode. after the modifier film had been grafted on the gce on which gold nanoparticles were electronuclated in three rounds, the deposited aunps were stripped off by running three cv scans in the potential range of 0 to 1400 mv in 0.1 mol l-1 kcl. this step is used to create holes (could be in the nanometer diameter size) on the modifier film electrode. the size of the formed holes presumably reflected the size of the nanoparticle that was deposited on the surface of the gce. an as such prepared modified electrode is refers to as a pd film–hole modified gce. fabrication of a pd film modified gce this modified electrode is different from the pd film–hole modified electrode. the main difference is that gold nanoparticles were not deposited on the polished and electrochemically conditioned gce and hence there was no aunps striping step. this electrode was prepared by just grafting the in situ prepared diazonium cation from p-phenylenediamine onto the bare gce surface by running cv within a potential window from 0.6 to –0.2 v at a scan rate of 0.1 v s-1 for three scans. electrochemical characterization of the modified gces the prepared modified electrodes were electrochemically characterized by cv using common redox probes: hydroquinone, hexamineruthenium chloride ru(nh3)6cl3 and ferrocyanide k3fe(cn)6. the selection of the redox probes was intentional so that hydroquinone represented molecular probes, while hexamineruthenium chloride ru(nh3)6cl3 represented cationic probes and ferrocyanide k3fe(cn)6 anionic probes in aqueous solution. the voltammetric signals of these probes at the modified gces were compared to the signals of those of the bare gce. preparation of aa solutions a 2 mmol l-1 stock solution of aa was prepared in 0.1 mol l-1 acetate buffer solution (ph 5) and aa solutions of other concentrations were prepared by appropriate dilution of the stock solution in acetate buffer (ph 5). real sample solutions, preparation and analysis for real sample analysis, orange fruit, which was obtained from a local market in jimma, ethiopia, and vitamin c tablets, purchased from local drug stores in jimma, ethiopia, were used. fresh orange juice was obtained by squeezing orange fruit into a glass beaker. then, after filtering the juice through a filter paper to remove the fiber and pulp, 1 ml was diluted with 5 ml of 0.1 mol l-1 sodium acetate buffer (ph 5). vitamin c tablet solution was prepared according to a literature procedure.22 briefly a weighed tablet was crushed with a pestle and mortar, and the powder dissolved in 20 ml of distilled water and then 10 ml of 1 mol l-1 h2so4 was added. from this solution, 9.3 ml were taken and diluted with 20 ml sodium acetate buffer. the concentration of aa in orange fruit and vitamin c tablet was determined by standard iodimetric titration. amperometric analysis was performed three times at 0.237 v at the pd film–hole modified gce and average results of the three measurements were taken. concentration of aa was determined by the standard addition method. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ electrochemical determination of ascorbic acid 1165 study of the effect of ph on the oxidation peak current of aa effect of ph of the supporting electrolyte (buffer) on oxidation peak current of aa was studied within range 2 to 9. citrate buffer of 0.1 mol l-1 was used to study effect of ph within the range of 2 to 4, 0.1 mol l-1 acetate buffer was used within range of 4.5 to 6 and 0.1 mol l-1 phosphate buffer of was used within the range 6 .5 to 8.5. dilute naoh or hcl were used to adjust the ph of the buffer solutions. results and discussion electrode fabrication grafting of a pd film onto a gce. in situ generated diazonium from p-phenylenediamine was electrochemically grafted onto a gce surface and the resulting film was electrochemically characterized using common redox probes. the cv of the pd film grafted onto a bare gce is shown in fig. 1a. a broad, irreversible cathodic peak was observed in the first cycle. in the subsequent scans, the reduction peak current decreases due to the insulation effect of the grafted surface film. fig. 1. a) cvs of the grafting of a diazonium film from 3 mmol l-1 pphenylenediamine in 0.5 mol l-1 hcl at a bare gce in 2 cycles (1 and 2 representing first and second cycles, respectively); b) cvs for the striping of the electronucleated gold nanoparticles from a gce surface in 0.1 mol l-1 kcl in 3 cycles (1, 2 and 3 representing first, second and third cycles, respectively). in all cases, the scan rate was 100 mv s-1. electronucleation of gold nanoparticles and stripping of the particles gold nanoparticles were sequentially electronucleated from a solution of 0.1 mmol l–1 kaucl4 in 0.5 mol l–1 h2so4 following the procedure reported by soreta et al.18 the nucleation of gold nanoparticles was confirmed by linear a voltammetric scan from 1.4 to 0 v, when the characteristic gold oxide reduction peak appeared at 0.953 v. after three rounds of gold nanoparticles electronucleation, the electrode was passivated by electro-grafting of diazonium in situ generated from p-phenylenediamine. pores on the modifier film were intentionally _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1166 olana, kitte and soreta created by stripping the deposited au nps by running three cv scans in the potential range of 0 to 1400 mv in 0.1 mol l–1 kcl (fig. 1b). as depicted fig. 1b, the anodic peaks at around 1.2 v, associated with gold stripping, decreased as the number of scans increases. the gold was stripped by electro-oxidation due to the presence of excess chloride that encourages the oxidation of gold to form its water-soluble chlorocomplex. electrochemical characterization of the modified gces cv of hydroquinone (hq). the cv of hq at the bare gce and at the pd film–hole modified gce are depicted in fig. 2, curves a and b, respectively. the anodic peak currents are comparable for the two electrodes except the peak current was slightly higher and the peak potential slightly shifted anodically at the modified electrode. when the voltammogram of hydroquinone on the pd film modified gce (fig. 2, curve c), was compared with the two former cases, the following differences were registered, i.e., the anodic peak current was lower and shifted to a higher anodic potential. however, the modified pd–film could not prevent hydroquinone from interacting with the electrode. hydroquinone is a molecular redox probe and diffusion of the probe towards the electrode surface was not very influenced by the surface charge of the electrodes that was developed due to the presence of modifier molecules on the gce, i.e., the modifier film does not effectively block the approach of hydroquinone to the electrode. fig. 2. cv of 10 mmol l-1 hq in 0.1 mol l-1 naclo4 at: a) bare gce, b) pd film–hole modified gce and c) pd film modified gce. the scan rate was100 mv s-1 in all cases. cv of hexamineruthenium(iii) the cvs of ru(nh3)6cl3 at the bare and the modified gces are depicted in fig. 3. electrochemical response of ru(nh3)6cl3 was significantly suppressed at the pd film–hole modified gce (fig. 3, curve c) relative to that at the bare gce (fig. 3, curve a). at the pd film modified gce, redox peak of ru(nh3)63+ was significantly diminished (fig. 3, curve b). from the voltammograms, it was _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ electrochemical determination of ascorbic acid 1167 concluded that the modified electrodes were positively charged as the signal for the cationic redox probe was significantly diminished due to its repulsion from the surface of the electrode. fig. 3. cvs of 10 mmol l-1 ru(nh3)6cl3 in 0.1 mol l-1 kno3 at: a) bare gce, b) pd film modified gce and c) pd film–hole modified gce. the scan rate was 50 mv s-1 in all cases. cv of hexacyanoferrate the cvs of k3fe(cn)6 at the modified gces (fig. 4, curves a and b) were compared to that at the bare gce (fig. 4, curve c). the redox peaks for the modified gces were significantly higher than that for the bare gce. comparing the cv response of k3fe(cn)6 at the pd film–hole modified gce (fig 4, curve a) to that at the pd film gce (fig 4, curve b), the redox peak current of the probe at the modified gce was found to be higher. from this observation, it was concluded that the modification imparted a positive charge on the surface of the organic film and hence the ferrocyanide approached the electrode surface not only by diffusion, but also by electrostatic interaction between the positively charged pd film and the negatively charged ferricyanide. the presence of holes on the pd film–hole modified gce could be responsible for the extra enhancement for the ferrocyanide signal due to the three-dimension diffusion of the anionic redox probes towards nanoelectrodes. the produced holes could change diffusion of fig. 4. cv of 10 mmol l-1 k3fe(cn)6 in 0.1 mol l-1 kcl at: a) the pd film-hole modified gce, b) the pd film modified gce and c) the bare gce. the scan rate was 50 mv s-1 in all cases. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1168 olana, kitte and soreta ions from planar to three dimensional.23,24 compton and his coworkers25 demonstrated that modifying an electrode with porous layers of a conducting material could affect voltammetric peaks because of a change in the mass-transport mode from planar diffusion to one with a thin-layer character. the observation from the studied redox probes indicated that the modified pd–film was positively charged and repelled ru(nh3)63+ but strongly attracted the fe(cn)63– probe. from this, it was decided to use the modified electrode for electro-analysis of the negatively charged analyte. the advantage could be twofold: enhancement in the signal of the analyte and improvement in the selectivity as cationic interferents would not approach the modified electrode surface. with this in mind, the pd film–hole modified gce was used for the electro-analysis of aa. cv of ascorbic acid (aa) at the pd film–hole modified gce the cv curves of aa at the bare gce and the pd film–hole modified gce over a wide range of potentials are depicted in fig. 5. it is clearly presented that the oxidation peak current of aa is enhanced and shifted to a lower potential at the pd film–hole modified gce relative to those at the bare gce. the electro oxidation of aa at a bare gce generally occurs at a relatively high oxidation potential, indicating a slow electron transfer rate.26 fig. 5. cvs of 2 mmol l-1 aa in 0.1 mol l-1 acetate buffer (ph 5) at: a) pd film–hole modified gce and b) bare gce; in all cases scan rate 100 mv s-1. the oxidation of aa (c6h8o6) involves a two-electron and two-proton irreversible reaction to produce dehydroascorbic acid (c6h6o6).27 from the voltammograms, the oxidation potential of aa is close to 0.8 and 0.237 v for bare and pd film–hole modified gce, respectively. these potentials for were selected for the amperometric determination of aa at the studied electrodes. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ electrochemical determination of ascorbic acid 1169 effect of the ph of the supporting electrolyte on the electro-analysis of aa the ph of the electrolyte is one of the important parameters that could influence the response of the electrode in the analysis of aa. the ph is an important parameter that controls the surface charge of the modifier film and the state in which aa could be available in the solution. the ph of the supporting electrolyte was varied within the range of 2 to 8.5 to study the effect of ph on oxidation peak current of aa at the pd film–hole modified gce. oxidation peak current of aa was found to increase as the ph was changed from ph 2 to 5 (fig. 6, curve a). a further increase in ph of the buffer led to a decrease in the response of aa. this observation is in agreement with the proposed interaction model. in the ph range in which the modifier film can be made positive film and the aa in its anionic form, the interaction of the modified gce surface with aa enhances the redox signal. at higher ph conditions, the film might develop a negative charge (due to adsorption free hydroxyl ions) and cause the anionic form of aa to be repelled. thus, ph 5 is the condition that favors the formation of cationic film and anionic form of aa. hence, a ph value of 5 was selected as optimum condition for the electro-analysis of aa at the pd film–hole modified gce. for the sake of comparison, similar study on the effect of ph on the oxidation peak current of aa at bare gce was conducted. the oxidation peak current for aa consistently decreased with increasing ph (fig. 6, curve b). a comparison of the two results clearly demonstrated the difference in surface property between the modified and the unmodified gce. fig. 6. oxidation peak current of 2 mol l-1 aa in different ph supporting electrolytes at: a) the pd film–hole modified gce and b) the bare gce. amperometric determination of aa in this work, amperometric measurements were performed and the results compared for the determination of aa at the two electrodes, i.e., the pd film– –hole modified gce and the bare gce. for the pd film–hole modified gce, the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1170 olana, kitte and soreta amperometric measurement was performed at 0.237 v while for bare the gce, it was at 0.8 v (the potential at which the highest oxidation peak current was observed). the amperometric response of both the modified and the bare gce, for successive additions of aa, increased stepwise with increasing concentration of aa in 0.1 mol l–1 acetate buffer (ph 5). the bare gce showed a linear response to aa in the concentration range from 5 to 45 mmol l–1 with detection limit of 1.656 mmol l–1 and a correlation coefficient of 0.995 (fig. 7a). the pd film– –hole modified gce showed a linear response to aa in the concentration range from 5 to 45 µmol l–1 with a detection limit of 0.123 μmol l–1 and a correlation coefficient of 0.998 (fig. 7b). the average of three measurements for each concentration was calculated to plot the calibrations curves. the limit of detection (lod = 3 σ /slope) at the bare gce and the pd film–hole modified gce were 1.656 mmol l–1 and 0.123 μmol l–1, respectively. the pd film–hole modified gce had improved characteristics, such as, detection of a lower concentration of aa and better reproducibility of the signal for the studied concentrations. thus, the observed attributes are encouraging for the potential application of the modified electrode as an electrochemical sensor for the determination of aa. fig. 7. amperometric calibration curve for determination of aa in acetate buffer (ph 5) at: a) bare gce; b) pd film–hole modified gce. effect of interferents the influence of compounds, such as caffeine (caf), starch (sta), which could coexist in the pharmaceutical dosages, vitamin c, glucose (glu), citric acid (ca) and tartaric acid (ta), which may co-exist in fruit juices,28–30 and compounds such as glu, dopamine (da) and uric acid (ua), which co-exist in human fluid,31 may interfere with the determination of aa. amperometric signal for aa in the presence of the above possible interfering substances was studied at a fixed concentration of 1 mmol l–1 aa and 1 mmol l–1 each interferent at _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ electrochemical determination of ascorbic acid 1171 the bare gce at 0.8 v (fig. 8a). for the pd film–hole modified gce, amperemetric signal of aa in the presence of the above possible interfering substances was studied at a fixed concentration of 1 mmol l–1 aa and 200-fold excesses of interfering species at 0.237 v (fig. 8b and c). fig. 8. the amperometric response of: a) 1 mmol l-1 aa and 1 mmol l-1 interfering species at a bare gce at 0.8 v; b) 1 mmol l-1 aa and 200-fold excesses of interfering species at a pd film–hole modified gce at 0.237 v; c) the amperometric response of 1 mmol l-1 aa and 200-fold excesses of interfering species to aa at a nanohole pd grafted gce at 0.237 v. as can be seen from fig. 8, the influence of even very high concentrations of the studied potential interferents on the amperometric response of aa at the pd film–hole modified gce was found to be minimal. this could be due to a lowering of the oxidation potential of aa at the modified electrode compared to at the bare gce and the repulsion by the modifier film of species, such as da, that are available in cationic form. inter-electrode reproducibility and stability tests the inter-electrode reproducibility was investigated for pd film–hole modified gce by preparing five electrodes under the same conditions. amperometric measurement at 0.237 v for 2 mmol l–1 aa at five different electrodes, prepared _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1172 olana, kitte and soreta with the same electrode modification strategy, was used to estimate the reproducibility. the reproducibility expressed by the relative standard deviation was found to be 5.18 % (n = 5) for pd film–hole modified gce, thereby showing the good reproducibility of the modified electrode. the stability of the pd film–hole modified electrode was studied by comparing the current response of a freshly prepared pd film–hole modified gce with the response of electrodes after storage for 28 days in 0.1 mol l–1 acetate buffer (ph 5) at room temperature. for 2 mmol l–1 aa, the modified electrodes retained 98 % of the initial current response. the result showed that the pd film– –hole modified gce has a good stability and long life. comparison of the pd film–hole modified gce with previously reported methods the detection limit of the pd film hole modified gce is compared in table i with those of previously reported modified electrodes. as can be seen from table i, the electrode modification strategy reported herein resulted in an electrode with a better detection limit than most of those previously reported. table i. comparison of the modified electrode with previously reported modified electrodes for the determination of ascorbic acid electrode detection limit μm ref. bi2o2 microparticles modified gce 8.1 32 tiron modified gce 1.79 33 gce modified with carbon-spheres (linear range 2–300 μm) 0.60 34 gce modified with a nickel(ii)-bis(1,10-phenanthroline) complex (linear range 10–630 μm) 4.0 35 quaternized carbon nanotubes/ionic liquid–polyaniline composite film modified gce (linear range 20 nm–4 μm) 0.25 36 nitrogen doped porous carbon nanopolyhedra (linear range 80–2000 μm) 0.74 37 graphene modified gce (linear range 10.0–1000 μm) 1.20 38 gold electrode modified with a flower-like gold nanostructure (linear range 60–500 μm) 10 39 over oxidized p-aminophenol polymer film on gce 1.0 40 gce modified with poly(ethylene oxide) 50 41 methionine modified carbon paste electrode 5.0 42 electropolymerized aniline on gces 1.0 15 pd film–hole modified gce 0.123 this work real sample analysis to demonstrate the applicability of the pd film–hole modified gce for real sample analysis, orange fruit and vitamin c tablet samples were analyzed (table ii). the bulk concentration of aa was first determined by titration. the concentration of aa obtained in orange fruit and vitamin tablet were 23.33±0.01 mg per _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ electrochemical determination of ascorbic acid 1173 100 ml and 504.97±0.03 mg per tablet, respectively. then the aa was determined by amperometry using pd film–hole modified gce. the relative error for pd film–hole modified gce was 0.04 % for the determination of aa in orange and 0.37 % for the determination of aa in vitamin c tablet. the data presented clearly showed that the method reported herein is accurate. table ii accuracy test for the modified electrode against the standard titration method working electrode concentration of aa, mean ± sd, n = 3 for each sample in orange fruit, mg/100ml in vitamin c tablet, mg/tablet pd film–hole modified gce 23.34±0.17 506.87±0.39 standard titration method 23.33±0.01 504.97±0.03 conclusions in this work, the fabrication and electrochemical characterization of pd film–hole modified gce was reported. the modified gce was demonstrated for the amperometric determination of aa. the pd film–hole modified gce was found to have very good selectivity towards aa and high sensitivity for the determination of aa and could be applied in different matrices. the electrode modification strategy could be used as a means for the selective determination of anionic analytes in the presence of cationic interfering species. further study is required to understand fully the reported surface modification strategy and to explore different modifying films and other important analytes. acknowledgements. we would like to acknowledge the department of chemistry, jimma university, ethiopia, for providing the laboratory facilities. financial support from jimma university school of graduate studies is also acknowledged. и з в о д електрохемијско одређивање аскорбинске киселине на електроди од стакластог угљеника модификованој перфорираним филмом p-фенилендиамина bikila nagasa olana, shimeles addisu kitte и tesfaye refera soreta department of chemistry, college of natural sciences, jimma university, p. o. box 378, jimma, ethiopia у раду је приказано одређивање аскорбинске киселине (аa) на електроди од стакластог угљеника која је модификована перфорираним филмом формираним редукцијом диазонијум јона генерисаним in situ из p-фенилендиамина. перфорације филма постигнуте су растварањем претходно исталожених наночестица злата. модификоване електроде су електрохемијски карактерисане коришћењем уобичајених редокс реакција хидрохинона, јона гвожђе(ii)-цијанида и јона рутенијум(ii)-хексамина. струје оксидације аa одређене цикличном волтаметријом и хроноамперометријом на модификованим електродама су упоређене са струјама на немодификованој електроди од стакластог угљеника. немодификована електрода је показала линеаран одговор у опсегу концентрација аa од 5 до 45 mm уз границу детекције од 1,656 mm, док је модификована електрода показала линеаран одговор у опсегу концентрација аa од 5 до 45 μm уз границу детекције од 0,123 μm. на модификованој електроди је испитан утицај суп_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1174 olana, kitte and soreta станци које могу да ометају амперометријски сигнал аa и нађено је да је он минималан. такође је утврђено да су репродуктивност самих модификованих електрода, њихова стабилност и тачност одлични. mодификована електрода приказана у овом раду има потенцијалну примену за електроаналитичко одређивање аa. (примљено 4. новембра, ревидирано 30. децембра 2014, прихваћено 19. јануара 2015) references 1. o. arrigoni, m. c. de tullio, bba-gen. subjects 1569 (2002) 1 2. k. j. stutts, p. m. kovach, w. g. kuhr, r. m. wightman, anal. chem. 54 (1983) 1632 3. a. hodgkinson, oxalic acids in biology and medicine, academic press, london, 1977 4. j. wawrzyniak, a. ryniecki, w. zembrzuski, acta sci. pol. technol. aliment. 42 (2005) 5 5. h. s. burdurlu, n. koca, f. karadeniz, j. food eng. 74 (2006) 211 6. h. m. nassef, l. civit, a. fragoso, c. k. o'sullivan, analyst 133 (2006) 1736 7. a. m. pisoschi, a. pop, a. i. serban, c. fafaneata, electrochim. acta 121 (2014) 443 8. a. hossu, v. magearu, rom. biotechnol. lett. 9 (2004) 1497 9. z. h. sheng, x. q. zheng, j. y. xu, w. j. bao, f. b. wang, x. h. xia, biosens. bioelectron. 34 (2012) 125 10. a. t. markas, j. gilmartin, p. hart, analyst 120 (1995) 1029 11. l. f. xiao, j. chen, c. s. cha, j. electroanal. chem. 495 (2000) 27 12. m. ardakani, a. talebi, h. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {zinc, copper and nickel complexes of a macrocycle synthesized from pyridinedicarboxylic acid: a spectroscopic, thermal and theoretical study} j. serb. chem. soc. 82 (6) 665–680 (2017) udc 546.472’562’742+547.821+547.595: jscs–4995 519.677:543.57:615.28–188 original scientific paper 665 zinc, copper and nickel complexes of a macrocycle synthesized from pyridinedicarboxylic acid: a spectroscopic, thermal and theoretical study esmaiel soleimani, sayed ali naghi taheri* and mohsen sargolzaei faculty of chemistry, shahrood university of technology, shahrood, iran (received 6 december 2016, revised 13 march, accepted 31 march 2017) abstract: the metal(ii) ion complexes of a pentadentate macrocycle 1, namely 3,12-dioxa-6,9,18-triazabicyclo[12.3.1]octadeca-1(18),14,16-triene-2,13-dione are synthesized. this macrocycle is prepared from the reaction of dipotassium salt of 2,6-pyridinedicarboxylic acid with ethylenediamine and 1,2-dibromoethane. the reaction of 1 (l) in methanol with mcl2.xh2o gave complexes with the general formula [m(l)cl2] (where m= ni(ii) 2, cu(ii) 3 and zn(ii) 4, respectively). the analysis of ir, 1hand 13c-nmr spectral data of all complexes propose that 1 is bonded to metal(ii) ions through a nitrogen atom of pyridine ring, two nitrogen atoms of amine groups and two oxygen atoms of ester moieties. the thermal analysis indicated that there are no water molecules of hydration or coordinated in the structure of the complexes. among these complexes, the cu(ii) 3 complex demonstrated good antibacterial and antifungal activities. the molecular geometry, aim atomic charge and frontier molecular orbitals of the compounds are investigated theoretically using dft method. based on the theoretical data of these complexes represented, a bipyramidal pentagonal arrangement can be envisaged in such a way that the n3o2 pentadentate donor sites form the planar pentagonal base of the bipyramid and the two cl atoms occupy the vertexes. keywords: macrocycle ligand; pyridinedicarboxylic acid; thermal analysis; molecular modeling; dft calculation; antimicrobial studies. introduction macrocycles can be defined as cyclic organic framework with several potential donor atoms which surround a central metallic ion as a coordination center. macrocycles have received a continuous and prolonged attention due to their wide applications as catalysts, optic and magnetic materials.1–3 the various roles performed by the naturally occurring macrocycles in biological systems are well known. the synthesis of macrocycle complexes is very * corresponding author. e-mail: sayedalinaghitaheri@yahoo.com doi: 10.2298/jsc161206039s 666 soleimani, taheri and sargolzaei important due to their application as dyes, pigments, and mri contrast agents.4,5 some macrocycle complexes have been reported to display antifungal, antibacterial and anti-inflammatory activities.6,7 a series of macrocycle complexes of cd(ii), zn(ii), cu(ii), ni(ii) and co(ii) obtained from the condensation reaction of 2,6-diaminopyridine and isatin has been investigated.8 these complexes displayed significant antibacterial activity against some selected bacterial strains. abdallah and co-workers synthesized tetradentate macrocycle ligand and its complexes of n2o2 donor atoms with fe(iii) and cr(iii) ions.9 their studies cover the thermal and antimicrobial behaviour of the complexes. thermal analysis indicated that all complexes have low stability due to the hydration water. all compounds demonstrated an outstanding biological activity against some bacteria (s. aureus and e. coli) and fungi (c. albicans and a. flavus). some other macrocycle complexes of co(ii), ni(ii), cu(ii) and zn(ii) have been synthesized from condensation of glyoxal and l-leucine in ethanol.10 the fluorescence and absorption spectroscopic studies indicated that all the complexes display a significant binding to calf thymus dna. mohamed and co-workers prepared tridentate macrocycle and its mn(ii), fe(iii), co(ii), ni(ii), cu(ii) and zn(ii) complexes from triazine ligand with nno donation sites towards metal ions.11 the coordination sites of triazine ligand namely the two nitrogen atoms and oxygen atom are useful as they will form a cavity to bind the metal ions. therefore they can be used as analytical reagents for determination of metal ions. the triazine ligand and all its complexes are found to have biological activity against the desert locust schistocerca gregaria (orthoptera, acrididae). the thermal analysis showed neither water molecules of coordination nor hydration in the structure of these complexes. the macrocycle complexes of co(ii), ni(ii), cu(ii) and zn(ii) prepared from the condensation of carbohydrazide and isatin has been investigated .12 these complexes displayed excellent antibacterial behaviour against some pathogenic bacteria. the synthesis and characterization of a new series of macrocycle complexes have also been reported through template condensation reaction of 1,8diaminonaphthalene and dimedone in presence of divalent transition metal ions.13 the in vitro antimicrobial tests of these complexes showed some notable antibacterial activity against the gram-positive (s. aureus and b. subtilis) bacteria and yeast (c. albicans). pyridinedicarboxylate can be an important starting material for producing macrocycle ligand owing to its ability to form strong covalent bonds. the non -adjacent positions of the two carboxylic acid groups on the aromatic ring in pyridinedicarboxylate leads to either an oligomer or a polymeric chain structure. the nitrogen atom of the pyridine may also act as a potential site for coordin macrocyclic complexes derived from pyridinedicarboxylic acid 667 ation.14 an excellent feature of these macrocycles is their multifarious biological activity.15 they are present in many natural products, as an oxidative degradation product of vitamins, coenzymes and alkaloids, and are also an important component of fulvic acids. pyridinedicarboxylate complexes of iron are well known electron carriers in various biological models and are diagnosed as specific molecular tools for dna splitting.16 in continuation of our previous work,17,18 and due to versatile biological and chemical properties of macrocycle complexes derived from 2,6-pyridinedicarboxylate, we are reporting the synthesis and characterization of a new macrocycle derived from the reaction of this dicarboxylate with 1,2-dibromoethane and ethylenediamine, together with its complexes of three metal(ii) ions. these complexes were also screened for their antimicrobial activity against some bacteria and fungi. the thermal analyses were carried out in an inert atmosphere of nitrogen gas. thermo-gravimetric studies of macrocycle and its complexes were performed to obtain information about their thermal stability, and to decide about the presence of the water molecules and the decomposition steps of their structures. moreover, the thermal analysis technique that provides extremely sensitive measurements of heat changes can be applied widely in pharmaceutical development. the theoretical studies of molecular and electronic structures of the macrocycle and its metal complexes have also been carried out. molecular orbital calculations were conducted by density functional theory (dft) at b3lyp level with standard 6-31g(d,p) and lanl2dz basis sets. the calculations were performed to obtain the optimized molecular geometry, charge density distribution, highest occupied molecular orbital (homo) and lowest unoccupied molecular orbital (lumo) of the macrocyclic ligand and its complexes. experimental phosphorus pentoxide (aldrich), dmso (aldrich), nicl2⋅6h2o, cucl2⋅2h2o and zncl2⋅2h2o (merck), methanol (merck), 2,6-pyridinedicarboxylic acid (aldrich), 1,2-dibromoethane (merck), ethylene diamine (merck) and potassium were used as received. physical and spectral measurements the 1hand 13c-nmr spectra were recorded in dmso on a brucker av300 nmr spectrometer with the tms as an internal reference. the elemental analyses were performed on a heraeus carlo erba 1108 elemental analyzer. the electronic spectra (10-3 mol dm-3 in dmso) were obtained with a shimadzu uv-160 spectrophotometer. the ir spectra (4000–400 cm-1) were recorded on a perkin-elmer model 377 spectrometer using kbr pellets. magnetic susceptibility measurements were done with a 155 allied research vibrating sample magnetometer at room temperature. molar conductivities (10-3 mol dm-3 in dmso) were measured with cmd 8500 laboratory conductometer. melting points were measured using an electrothermal buchi 512 melting point apparatus. the metals were determined by spectrophotometric method using an aa-670 shimadzu atomic absorption flame emission spectrophotometer. thermal analyses (tg and dtg) were carried out on a sdt q 600/v8.3 build 101 668 soleimani, taheri and sargolzaei thermal analyzer with a heating rate of 10 °c/min using n2 atmosphere (20 ml/min). the samples were heated up at atmospheric pressure in a temperature range of 25–800 °c. physical and spectral data of the compound are given in supplementary material to this paper. synthesis of macrocycle 1 methanolic solution of potassium (0.276 g, 12 mmol; 5 ml) was added to the solution of 2,6-pyridinedicarboxylic acid (1.060 g, 6 mmol; 30 ml). the solution was refluxed for 2 h until a pale yellow solution was obtained. then a solution of 1,2-dibromoethane (2.256 g, 12 mmol; 10 ml methanol) was added slowly to this solution. a white precipitate forms immediately which dissolves and gives a clear solution on further refluxing for 4 h. ethylenediamine (0.360 g, 6 mmol) was added, and was further refluxed for 6 h to ensure the completion of the reaction. on cooling the solution, a white precipitate was obtained. it was filtered, washed with methanol and dried over p4o10 in vacuo to give (1.473 g, 88 %) of macrocycle 1. elemental analysis, found % (calculated %): c 56.07 (55.91), h 5.88 (6.09), n 15.27 (15.05). the molecular formula compatible with this analysis is (c13h17n3o4). the compound was decomposed at 234 °c. synthesis of the ni(ii) complex 2 the macrocycle (l) 1 (530 mg, 2 mmol) was dissolved in 15 ml of hot methanol. a solution of nicl2⋅6h2o (475 mg, 2 mmol) in the same solvent (20 ml) was then gradually added to the solution of 1 with stirring to obtain an immediate precipitation accompanied by a visible color change. the mixture was then refluxed for a further 8 h on a hot plate to ensure the completion of the reaction. the green precipitate was then filtered off, washed with methanol and dried over p4o10 in vacuo to give (695 mg, 85 %) of complex 2. elemental analysis, found % (calculated %): c 38.33 (38.17), h 4.04 (4.16), n 10.12 (10.27), ni 14.14 (14.36). its molecular formula was suggested to be (c13h17cl2nin3o4), and was decomposed at 261 °c. synthesis of the cu(ii) 3 and zn(ii) 4 complexes the same procedure as explained above, was used for the synthesis of the cu(ii) 3 and zn(ii) 4 complexes. the cu(ii) complex 3 was obtained as a blue precipitate with a yield of 86 %. elemental analysis, found % (calculated %): c 37.93 (37.73), h 4.02 (4.11), n 10.02 (10.16), cu 15.19 (15.37). its molecular formula was suggested to be c13h17cl2cun3o4, and was decomposed at 251 °c. the zn(ii) complex 4 was obtained as a yellow pale precipitate with a yield of 80 %. elemental analysis, found % (calculated %): c 37.93 (37.59), h 4.02 (4.09), n 10.02 (10.12), zn 15.19 (15.66). its molecular formula was suggested to be c13h17cl2znn3o4, and was decomposed at 297 °c. biological activity monitoring the antifungal and antibacterial activities of 1 and its complexes were examined in vitro against two fungi (s. cerevisiae and c. albicans) and two gram positive (b. subtilis and s. aureus) and two gram-negative (e. coli and p. aeruginosa) bacteria by the disc diffusion method. nystatin and ofloxacin were used as standards for comparison of the antifungal and antibacterial activities, respectively. the test organisms were grown on nutrient agar for antibacterial and potato dextrose agar medium for antifungal in petri dishes. the compounds were prepared in dmso and soaked in a filter paper disc of 5 mm diameter and 1.0 mm thickness. the discs were placed on the pre macrocyclic complexes derived from pyridinedicarboxylic acid 669 viously seeded plates and incubated at 37 °c and the diameter of inhibition zone around each disc was measured after 72 h for antifungal and 24 h for antibacterial activities. all experiments were performed as triplicate. computational details full-unconstrained geometry optimizations of 1 and its complexes were carried out at gradient corrected dft using becke’s three parameter hybrid method and the lee–yang–parr correlation functional (b3lyp) combined with 6-31g(d,p) and lanl2dz basis sets using gaussian 03w in gas phase.19–21 the studied complexes are characterized as minima (no imaginary frequency) in their potential energy surface. molecular frontier orbitals: homo and lumo, and the optimized structures were projected with gaussian view.22 using homo and lumo orbital energies, the ionization energy and electron affinity can be expressed as: ie = –ehomo, ea = –elumo, respectively. the hardness, η and chemical potential, μ were described by the following relations: η = (ie–ea)/2 and μ = –(ie+ea)/2, respectively.23 electronegativity can be expressed as: χ = –μ. resulte and discussion the macrocycle 1, namely, 3,12-dioxa-6,9,18-triazabicyclo[12.3.1]octadeca -1(18),14,16-triene-2,13-dione, was synthesized by the method described in our previous works.17,18 first, the dipotassium salt of 2,6-pyridinedicarboxylic acid was produced from its reaction with potassium methoxide. the interaction of this dipotassium salt with 1,2-dibromoethane in methanolic solution, subsequently produces bis(2-bromoethyl)-2,6-pyridinedicarboxylate intermediate which reacts further with ethylenediamine to give the macrocycle 1 (scheme 1). n cook cook + 1) brch2ch2br 2) nh2ch2ch2nh2 ch3oh n o hn o hn o o scheme 1. reaction for preparation of the macrocycle 1. secondly, the complexes were synthesized from the reaction of 1 (l) with mcl2.xh2o in meoh, giving the compounds that have the general formula [m(l)cl2] (where m = ni(ii) 2, cu(ii) 3 and zn(ii) 4, respectively, scheme 2). unfortunately, our attempts to obtain single crystals of all the complexes have not been successful. therefore 1 and its complexes were characterized on the basis of magnetic susceptibility measurement, electronic spectra data, 1h and 13c-nmr, and ir spectra, elemental and thermal analyses. the molar conductance of complexes 2–4 were obtained to be 27, 35 and 23 ω–1 cm2 mol–1, respectively. this revealed that all of the three complexes are non-electrolyte.24 so based on elemental analysis and molar conductance data, all 670 soleimani, taheri and sargolzaei complexes have [m(l)cl2] formula in which two chloride ions are placed on coordination layer. n o hn o hn o o + mcl2.xh2o m= ni, cu, zn n o n o n o o m cl cl h h ab c d e f h i k lm n g scheme 2. reaction for preparation of the complexes [m(l)cl2], and its formula structure. a single sharp band was observed at 3245 cm–1 in ir spectrum of 1 that can be assigned to stretching of ν(n–h) secondary amine vibration,25 although its position was found to be lower by 20–30 cm–1 in its complexes. this behavior suggests that nitrogen atom of the secondary amines coordinated to the metal(ii) ions.26 a medium band was observed at 1598 cm–1 which was attributed to stretching of ν(c=n) of the pyridine ring. this band was shifted to lower frequencies at 1582, 1580 and 1583 cm–1 in complexes 2–4, respectively. this observation means that nitrogen atom of the pyridine ring takes part in coordination.27,28 the band at 1275 cm–1 due to ν(c–o) of the ester group of 1 that was shifted to lower frequencies at 1258, 1260 and 1262 cm–1 in complexes 2–4, respectively. this suggests that the oxygen atom of the ester groups are also coordinated to metal(ii) ions.29 these facts are further supported by the appearance of new bands at around 475 and 459 cm–1 in all complexes, which were attributed to the ν(m–o) and ν(m–n) stretching vibrations, respectively.28 the two strong bands at 1720 and 1685 cm–1 due to asymmetric and symmetric frequencies of ν(c=o) in 1, were observed to shift to 1715–1725 and 1678–1685 cm–1 in the spectra of the complexes 2–4, respectively.30 since no considerable change were observed for ν(c=o) of 1 and its complexes, it was concluded that the oxygen atoms of the carbonyl in the carboxylic group were not involved in the coordination to the metal ions. the 1h-nmr spectrum of 1 displayed a triplet (1h, hc) and a doublet (2h, hb, hd) of relative intensities 1:2 centered at 8.18 and 7.86 ppm, which is in good agreement with the protons of the pyridine ring.31 two sets of peaks were observed as triplets at 3.82 ppm (4h, hh, hl) and 2.64 ppm (4h, hg, hm) which correspond to ch2 protons adjacent to the nitrogen atoms (–ch2n) and the oxygen atoms (–ch2o) in the macrocycle ring, respectively. the singlet peak at 3.41 ppm (4h, hi, hk) is due to the –nch2–ch2n– moiety of 1.32 these peaks were all changed slightly in 4 which indicates that the oxygen and nitrogen atoms attached to ch2 participate in coordination with the macrocyclic complexes derived from pyridinedicarboxylic acid 671 metal ions. a broad signal obtained at 4.82 ppm (2h) is due to the n–h proton of secondary amine group of 1 which shifts in coordination with zn(ii) ion.33 these are confirmed by the 13c-nmr spectra, which show seven signals for the 13 carbon atoms of 1 skeleton. the 13c-nmr spectrum of 1 displayed two resonances due to two different carbons of the methylene groups at 75.66 ppm (cg, cm) adjacent to the oxygen (o–ch2) and at 63.83 ppm (ch, cl) adjacent to the nitrogen atom (n–ch2).34 these resonances were shifted up-field to 71.24 and 58.76 ppm in 4, respectively, which indicates coordination of oxygen and nitrogen donor atoms to the m(ii) ions. the resonances of the two equivalent methylenes of the ethylenediamine moiety of 1 appear at 48.72 ppm (ci, ck).35 this peak is shifted to 45.25 ppm in 4. the shielding effects on this signal in 4 suggest coordination of nitrogen donor atoms of the macrocycle ring to m(ii) ions. the peaks obtained at 124.68 ppm (cc), 136.26 ppm (cb, cd) and 155.12 ppm (ca, ce) of 1 were attributed to three types of carbons of pyridine ring,36,37 and also the peak at 176.38 ppm (cf, cn) is due to carbon of the carbonyl of the ester groups.38 carbon signal adjacent to the nitrogen atom of pyridine ring (ca, ce) changed slightly in its position in the spectrum of 4. therefore, this may be attributed to participation of the nitrogen atom of the pyridine ring in the coordination. the electronic spectra of 1 and its complexes show an absorption band in the region 255–265 nm that can be attributed to π→π* intra-ligand transfer of the pyridine ring incorporated in the skeleton of 1.39,40 in addition to the above band, two other bands were also observed for complexes 2 and 3. these two bands were at 778 nm (12853 cm−1) and 470 nm (21276 cm−1) for 2 that can be attributed to ν1: 1a'1→1e'2 and ν2: 1a'1→1e"1 transitions in a pentagonal bipyramidal geometry around ni(ii) ion, respectively.41 since 2 is diamagnetic, so ground state configuration is (e"1)4(e'2)4 and partial term of the electronic ground state is 1a'1. the two bands at 655 nm (15267 cm–1) and 522 nm (19157 cm–1) for 3 were assigned to the 2a'1→2e'2 and 2a'1→2e"1 transitions in a pentagonal bipyramidal geometry around cu(ii) ion, respectively.41 the complex 4 is diamagnetic. as it is expected due to the d10 electronic configuration of zn(ii) ion, electronic spectra of 4 did not show any d–d transition. by analogy to those described for the complexes 2 and 3 containing n3o2 donor atoms in 1 and two chloride ions, one can suggest pentagonal bipyramidal geometry for 4. thermal analysis thermal gravimetric analysis offers information about the thermal stability of the complex as well as whether the water molecules are in the inner or outer coordination sphere of the central metal ion.42 the tg and dtg data for macro672 soleimani, taheri and sargolzaei cycle 1 and complexes 2–4 which were recorded under a nitrogen atmosphere are given in table i. table i. the thermogravimetric data (tg and dtg) of macrocycle 1 and its complexes compound tg range, °c dtgmax, °c mass loss (calcd.), % assignments (c13h17n3o4) (1) 198–280 281–335 234 308 41.5 (40.8) 58.5 (59.1) – c6h14n2 – c7h3o4 [ni(c13h17n3o4)cl2] (2) 262–340 341–534 282 415 18.5 (17.8) 63.5 (63.2) 18.0 (18.3) – 2 hcl – macrocycle moiety, c13h15n3o3 residue: nio [cu(c13h17n3o4)cl2] (3) 268–325 326–404 292 358 19.0 (17.6) 63.5 (63.1) 17.5 (19.3) – 2 hcl – macrocycle moiety, c13h15n3o3 residue: cuo [zn(c13h17n3o4)cl2] (4) 285–355 356–495 310 472 18.5 (17.6) 63.0 (62.9) 18.5 (19.5) – 2 hcl – macrocycle moiety, c13h15n3o3 residue: zno the macrocycle 1 and its complexes decomposed in two stages. the first step decomposition of 1 occurred at the temperature range 198–280 °c which is associated with a dtg peak at 234 °c. this corresponds to the loss of organic moiety (c6h14n2) with a mass loss of 41.5 % (calcd. 40.8 %). the second step of the decomposition happened in the range of 281–335 °c relating to a dtg peak at 308 °c. this correlates with the loss of pyridine-2,6-dicarboxylate moiety (c7h3no4) with a mass loss of 58.5 % (calcd. 59.1 %). it can be noticed that tg curve of complexes displays no mass loss up to 262 °c, indicating the absence of water molecules in the coordination sphere,42 and also confirms the stability of the complexes up to 262 °c. the thermogram of 3 represents two decomposition steps. the first step of the decomposition at the temperature range 268–325 °c is associated with a dtg peak at 292 °c corresponding to the loss of two chloride ions as hcl with a mass loss of 19.0 % (calcd. 17.6 %). the second decomposition step occurred in the range of 326–404 °c and showed a dtg peak at 358 °c which corresponds to the loss of 1 moiety with a mass loss of 63.5 % (calcd. 63.1 %). at the end of decomposition process, the resulting residue is cuo with a mass of 17.5 % (calcd. 19.3 %). the thermograms of complexes 2 and 4, which are similar to 3, display two decomposition steps. complexes 2 and 4 were stable up to 262 and 285 °c, respectively. the first step of the decomposition in the range 262–340 °c for 2 and 285–355 °c for 4 is associated with dtg peaks at 282, and 310 °c. this shows the loss of two hcl with a mass loss of 18.5 and 18.5 % (calcd. 17.8, and 17.6 %), respectively. macrocyclic complexes derived from pyridinedicarboxylic acid 673 the second step of the decomposition occurred in the range of 341–534 °c for 2 and 356–495 °c for 4 relating to dtg peaks at 415 and 472 °c. this can be attributed to the loss of 1 moiety with a mass loss of 63.5 and 63.0 % (calcd. 63.2 and 62.9 %), respectively. after these decomposition steps, what is left is nio and zno with a mass of 18.0 and 18.5 % (calcd. 18.3 and 19.5 %), respectively. the thermal properties (tg and dtg curves) of complexes 2–4 exhibited the absence of hydrated or coordinated water molecules. therefore the results of thermal analyses of complexes showed good agreement with the molecular formula [m(l)cl2] as suggested from the elemental analyses. the macrocycle 1 and its complexes have been screened for both antibacterial and antifungal activities. the results of the microbial screening of 1 and its complexes are given in table ii. table ii. antimicrobial activity of macrocycle 1 and its complexes; diameter of growth of inhibition zone, mm compound microbe s. aureus b. subtilis e. coli p. aeruginosa s. cerevisiae c. albicans macrocycle (1) 4 5 4 4 – – [ni(l)cl2] (2) 14 13 12 16 – – [cu(l)cl2] (3) 30 27 31 30 25 18 [zn(l)cl2] (4) – – 18 15 8 14 ofloxacin 36 34 38 36 – – nystatin – – – – 19 24 the antimicrobial monitoring data displayed that macrocycle 1 does not exhibit any activity. the complex 2 represented antibacterial activities against gram positive bacteria (b. subtilis and s. aureus) and gram negative bacteria (e. coli and p. aeruginosa), while complex 4 demonstrated antibacterial activity towards gram negative bacteria (e. coli and p. aeruginosa), and antifungal activity against fungi (s. cerevisiae and c. albicans). the complex 3 also exhibits good activities against gram-positive and gram-negative bacteria and fungi, albeit it has lower antimicrobial activity compared to standard antibiotic ofloxacin and antifungal drug nystatin. the results from this investigation have also demonstrated that coordination of metals to macrocycle serves to amend the antimicrobial activity of the ligand. the increased activity of the metal complexes can be described based on chelating theory. the other factors such as conductivity, solubility and bond distance between the metal and ligand also increase the activity.43 molecular modeling molecular orbital geometry optimization permits a quantitative discussion about not only the geometry, but also the ground electronic properties of the ligands and their complexes. electronic and geometric structures of the inves674 soleimani, taheri and sargolzaei tigated macrocycle and its complexes were computed by the optimization of their bond angles, dihedral angles and bond lengths. the optimized molecular structures with minimum energies and its atom numbering obtained from the quantum chemical calculations for 1 and its complex 3 are shown in figs. 1 and 2, respectively. the selected bond lengths and bond angles of 1 and its complexes are listed in table s-i of the supplementary material. fig. 1. the optimized geometry of the macrocycle 1. fig. 2. the optimized geometry of complex 3. as can be seen, based on the coordination number seven, and the bond angles close to 72° in equatorial plane, all complexes have distorted pentagonal macrocyclic complexes derived from pyridinedicarboxylic acid 675 bipyramidal geometry. the two axial positions are occupied by chloride ions, while the pyridine nitrogen atom, the two nitrogen atoms of amino groups and the two oxygen atoms of ester moieties of 1, occupy the equatorial positions. the n1–cu–o11, n1–cu–o12, n15–cu–o11, n16–cu–o12 and n15–cu– –n16 bond angles in 3 have values of 71.48, 71.48, 71.27, 72.40 and 73.30°, respectively. the bond angle cl39–cu–cl40 is found to be 177.26°. these results indicated that 3 has distorted pentagonal bipyramidal geometry. the bond lengths of the adjacent atoms attached to the central atom are slightly increased. these behaviors are caused by macrocycle 1 atoms participating in the coordination process of the metal ions. for example the n1–c2, o12–c8, n16–c19, n15–c17 and o11–c13 bonds in 3 are slightly increased to be 1.357, 1.384, 1.482, 1.486 and 1.462 å, respectively. the other bond distances in 1 are either affected slightly or not affected at all by the coordination to the metal ion. the bond distances of the coordinating atoms with the central metal atom in 3 are found cu–n1 = 2.160, cu–o1 = 2.424, cu–o12 = 2.428, cu–n15 = 2.340, cu–n16 = 2.340 å in equatorial, and cu–cl39 = 2.512, cu–cl40 = = 2.514 å in axial positions. the two cu–namine bond distances (cu–n15 = 2.340 and cu–n16 = 2.340 å) are longer than the cu–npy distance (cu–n1 = 2.160 å). it seems pyridine nitrogen atom is a stronger donor than nitrogen of amine group. these values are in good agreement with the observed x-ray data for cu(ii) complexes with n3o2 macrocycle including pyridine moiety and chloride ion.44–46 the calculations showed that 2 and 4 have similar geometry to that of 3. the n1–m–o11, n1–m–o12, n15–m–o11, n16–m–o12 and n15–m–n16 bond angles in 2 and 4 have values 70.77–73.71, 70.13–76.08, 71.20–72.25, 72.21– –78.25 and 71.70–74.87°, respectively. the bond angles cl39–m–cl40 are also found to be 178.90 and 176.38° for 2 and 4, respectively. therefore, these results indicate that the complexes 2 and 4 have distorted pentagonal bipyramidal geometry. energy of some bonding molecular orbital for macrocycle ligand 1 and its complexes are shown in table s-ii of the supplementary material. binding energy is more stable in complexes compared to macrocycle ligand 1. for example energy of n1–c2 bond in macrocycle 1 is found to be –0.848 a.u., and for its complexes 2–4 to be –0.880, –0.874 and –0.848 a.u., respectively. also it can be seen that the energy of all bonds is more stable for complexes. so it demonstrates that complexes are more stable than macrocycle 1. the most important orbitals in a molecule are the frontier molecular orbitals (fmos), called homo and lumo. the determination of energies of the homo (π-donor) and lumo (π-acceptor) are significant parameters in quantum chemical calculations. the homo is the orbital that primarily acts as an electron donor and the lumo is the orbital that largely acts as the electron acceptor. 676 soleimani, taheri and sargolzaei the calculated energies (ev) of important molecular orbitals containing homo and lumo, electronegativity (χ), chemical potential (μ), global hardness (η) for 1 and its complexes are listed in table s-iii of the supplementary material. the homo and lumo orbitals for macrocycle 1 and its complexes are also shown in figs. s-1 and s-2 of the supplementary material, respectively. the ehomo and elumo and their neighboring orbitals are all negative, which demonstrate that the prepared complexes are stable.47,48 the homo–lumo energy gap of 1 is small, which means that the charge transfer occurs easily. the lower homo energy values show that the ability of the molecules donating electrons is weaker. on the other hand, the higher homo energy implies that the molecule is a good electron donor. lumo energy presents the ability of a molecule to receive an electron.48,49 the orbital energy level analysis for complexes 2–4 display that ehomo values are –6.232, –6.125 and –5.558 ev, respectively, while elumo values are –2.820, –3.225 and –2.80 ev, respectively. as a result, 4 is a better electron donor, while 3 is a better electron acceptor. the homo of 1 is concentrated on two nitrogen atoms of amine groups, while lumo is concentrated on pyridine ring atoms and carboxyl groups. calculations showed that contribution of the central atomic orbitals in the formation of homo (18 % cu) and lumo (58 % cu) for 3 is greater than homo (7 % ni and 4 % zn) and lumo (2 % ni and 1 % zn) for complexes 2 and 4. the contribution of the chloride ion in the formation of homo for 4 (94 % zn) is greater than homo for complexes 2 (3 % ni) and 3 (12 % ni). this behavior can be attributed to the stronger interaction of the hard base chloride ion with harder acid zn2+ than ni2+ and cu2+. aim atomic charge calculation has a significant role in the application of quantum chemical calculations of molecular systems; this is due to the fact that the atomic charges affect some properties of molecular systems including dipole moment, and molecular polarizability.50,51 the selected net charges at the atomic sites of 1 and its complexes are given in table s-iv of the supplementary material. the charges on two of the hydrogen atoms (h24 and h38) are greater than other hydrogen atoms in the compounds. this is because they are connected to nitrogen atoms (n15 and n16) of amine groups. all carbon atoms of 1 have negative charges, except the carbon atoms of carboxyl groups (c7 and c8) that have highly positive charges. these carbon atoms of the carboxyl group (c7 and c8) together with the carbon atoms attached to the pyridine nitrogen (c2 and c6) are positively charged in the complexes, but the other carbon atoms have still negative charges. the negative charges of the oxygen atoms (o11 and o12) of the carboxyl groups and the nitrogen atoms (n15 and n16) of amine groups increased in coor macrocyclic complexes derived from pyridinedicarboxylic acid 677 dination with the metal ion. the charges on o11, o12, n15 and n16 atoms for 1 are –0.816, –0.825, –0.718 and –0.712, respectively, while the charges for the atoms in 3 are –0.928, –0.937, –0.948 and –0.947, respectively. the charge of the ni2+ in the free state is 2.0. it is seen that the positive charge of the metal ion decreases to 1.027 in 2, which indicates that the transfer of electrons from 1 to the metal ion has occurred, and the coordination bonds have formed. our calculations show that a total of 0.580 is transferred to cu(ii) during the coordination process, and the net charge on the cu(ii) reduces to 1.420. similar behaviour in charge transfer is noted in case of 4. the charge on central metal ion reduces to 1.405 after coordination. the charge of the chloride ion in the free state is –1.0. it is seen that the negative charge of the chloride ion decreases to –0.679 in 2, which indicates that the transfer of electrons from chloride ion to the metal ion has occurred, and the coordination bonds have formed. our calculations show that a charge equal to –0.350 from chloride ion is transferred to cu(ii) during the coordination process, and the net charge on chloride ion reduces to –0.650. similar behavior of charge transfer is noted in the case of 4. the charge on the chloride ion after coordination reduces to –0.606. thus it can be concluded from the distribution of electric charges on the atoms of the complexes that much electron transfer occurs from the macrocycle to the metal ions, and therefore a strong interaction occurs between 1 with metal(ii) ions. conclusions the macrocycle 1, namely, 3,12-dioxa-6,9,18-triazabicyclo[12.3.1]octadeca -1(18),14,16-triene-2,13-dione, was prepared via the reaction of dipotassium salt of 2,6-pyridinedicarboxylic acid with ethylenediamine and 1,2-dibromoethane. the complexes of 1 were prepared from its reaction with mcl2⋅xh2o. the complexes have the general formula [m(l)cl2] (where m = ni(ii) 2, cu(ii) 3 and zn (ii) 4, respectively). analyses of the nmr and ir spectral data and molar conductivity measurements propose that 1 is bonded to metal(ii) ions through a nitrogen atom of pyridine ring, the two nitrogen atoms of amine groups and the two oxygen atoms of ester moieties. thermal gravimetric analyses displayed that these complexes can be stable up to 262 °c. the complex 2 exhibited antibacterial activities against gram-positive bacteria (b. subtilis and s. aureus) and gram-negative bacteria (e. coli and p. aeruginosa), while 4 showed antibacterial activity against gram-negative bacteria (e. coli and p. aeruginosa), and antifungal activity against fungi (s. cerevisiae and c. albicans). the complex 3 demonstrated good activities towards tested bacteria and fungi. 678 soleimani, taheri and sargolzaei the optimized structures of 1 and its complexes were computed by theoretical dft method, and all the complexes have distorted pentagonal bipyramidal geometry. the homo and lumo energies were also determined. the charges on the atoms of compounds have also been calculated, and it has been found that much electron transfer occurs from 1 to the central atoms in the complexes. this behaviour confirms strong interaction of 1 with metal(ii) ions. supplementary material physical and spectral data of the compound are available at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgments. the authors would like to thank the research council of shahrood university of technology, iran, for the financial support of this work. authors also thank dr. n. farrokhi from biology department, shahrood university of technology for microbiology measurements. и з в о д комплекси цинка, бакра и никла са макроцикличним лигандом који је синтетисан из пиридиндикарбоксилне киселине: спектроскопска, термална и теоријска испитивања esmaiel soleimani, sayed ali naghi taheri и mohsen sargolzaei faculty of chemistry, shahrood university of technology, shahrood, iran синтетисани су метал(ii) комплекси са макроцикличним пентадентатним лигандом 3,12-диокса-6,9,18-триазабицyкло[12.3.1]октадека-1(18),14,16-триен-2,13-дионом (l, 1). овај макроциклични лиганд је добијен у реакцији калијумове соли 2,6-пиридиндикарбоксилне киселине са етилендиамином и 1,2-диброметаном. у реакцији 1 (l) са mcl2⋅xh2o у метанолу настају комплекси опште формуле [m(l)cl2] (m = ni(ii) 2, cu(ii) 3 и zn(ii) 4). на основу ir и nmr (1h и 13c) спектроскопских мерења нађено је да је у свим комплексима лиганд 1 координован за испитиване јоне метала преко атома азота из пиридина, два атома азота аминских група и два атома кисеоника из естарске групе. на основу термалне анализе нађено је да у структури испитиваних комплекса не постоје координовани или хидратисани молекули воде. од свих синтетисаних комплекса, само је комплекс бакра(ii) 3 показао добру антибактеријску и антифунгалну активност. геометрија комплекса, маликеново (mulliken) наелектрисање атома и молекулске орбитале [m(l)cl2] комплекса испитивани су применом dft методе. на основу теоријских израчунавања за испитиване комплексе претпостављена је пентагонална бипирамидална геометрија са n3o2 хромофором у планарној пентагоналној основи бипирамиде са два хлоридо лиганда у њеном аксијалном положају. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice light-induced oxidative transformation of diphenylamine on zro2. synergism by zno and zns j. serb. chem. soc. 80 (11) 1411–1421 (2015) udc 547.551.2+544.526.5:546.831–31: jscs–4807 546.47–31+546.47’22 original scientific paper 1411 light-induced oxidative transformation of diphenylamine on zro2. synergism by zno and zns chockalingam karunakaran*, swaminathan karuthapandian** and pazhamalai vinayagamoorthy department of chemistry, annamalai university, annamalainagar 608002, tamilnadu, india (received 24 august, revised 13 december, accepted 15 december 2014) abstract: diphenylamine (dpa) in ethanol on the surface of zro2 undergoes photoinduced oxidative transformation yielding n-phenyl-p-benzoquinonimine (pbqi). the light-induced transformation on zro2 enhanced with dpa concentration, zro2-loading, airflow rate and photon flux. the formation of pbqi on zro2 is larger on illumination at 254 nm than at 365 nm. the zro2 is reusable without any treatment. the mechanism of light-induced oxidative transformation of dpa on zro2 is discussed with an appropriate kinetic law. zno and zns enhance the uv light-induced transformation of dpa on zro2, indicating synergism. keywords: photooxidation; wide band gap; sub-band gap illumination; photocatalysis. introduction semiconductor-photocatalyzed selective organic oxidative transformations have gained interest because of their environmental benign nature and the reviews by lang et al.,1 palmisano et al.2 and shiraishi and hirai3 present the different kinds of organic transformations performed photocatalytically. tio2 -based materials are widely used as photocatalysts for organic synthesis.1–3 like tio2, zro2 is a nontoxic and photostable iv–vi semiconductor but with a larger band gap energy (≈5 ev). however, this large band gap enables the photocatalytic splitting of water4 and reduction of co2.5,6 the band gap of combustion synthesized zro2 is not large (3.5 ev) and madras and coworkers7 reported degradation of dyes with illumination at 365 nm; defects in the crystal lattice are the likely reason for the decreased band gap. here, for the first time, the photoinduced oxidative transformation of diphenylamine (dpa) on untreated zro2, * corresponding author. e-mail: karunakaranc@rediffmail.com ** present address: department of chemistry, vhnsn college, virudhunagar 626001, tamilnadu, india. doi: 10.2298/jsc140824122k 1412 karunakaran, karuthapandian and vinayagamoorthy which is reusable, is reported. dpa is used in post-harvest treatment of apple and pear8 and the photosensitized oxidation of dpa is known; cyanoanthracenes9 and benzophenone10 are some of the photosensitizers used. the unsensitized photo-oxidation of dpa to n-phenyl-p-benzoquinonimine (pbqi) is slow.11 the chemical transformation of dpa was studied in the absence and presence of zro2 and the difference in the rates provides the rate of pbqi formation on zro2. the oxidative transformation on zro2 surface was investigated under uv light and under natural sunlight at different experimental conditions to obtain the kinetic law and to elucidate the reaction mechanism. zno is a promising ii–vi semiconductor photocatalyst with a band gap of 3.2 ev and large excitonic binding energy (60 mev) at room temperature. although the band gap energy of zno is not different from that of tio2 and the conduction band (cb) energy levels of the two semiconductors do not differ significantly and so are the valence band (vb) edges, there are reports that zno is a better photocatalyst than tio2.12 zns is also a ii–vi semiconductor but with a wide band gap (≈3.6 ev). semiconductor mixtures are reported to enhance photocatalytic mineralization of organic molecules13,14 and here the photoconversion of dpa to pbqi on zro2 was enhanced on mixing zno or zns with zro2. experimental materials and measurements zro2, zns and zno (merck) were used as received and their specific surface areas, obtained by bet method, were 15.1, 7.7 and 12.2 m2 g-1, respectively.15 the mean particle sizes (t) of zro2, zns and zno, obtained using the formula t = 6/ρs, where ρ is the material density and s is the specific surface area, are 68, 190 and 87 nm, respectively. the uv–visible diffuse reflectance spectra (drs) of the semiconductors were obtained using a shimadzu uv-2600 spectrophotometer with an isr-2600 integrating sphere attachment. the kubelka– –munk (km) plots provide the band gaps of zns and zno as 3.57 and 3.15 ev, respectively. potassium tris(oxalato)ferrate(iii), k3[fe(c2o4)3]·3h2o, was prepared using a standard procedure.16 dpa, ar (merck) was used as received. the infrared spectra were recorded on a nicolet is5 ft-ir spectrometer. commercial ethanol was purified by distillation with calcium oxide. uv light-driven transformation the uv light-driven transformation on zro2 was performed in a multilamp photoreactor equipped with eight 8 w mercury uv lamps (sankyo denki, japan) emitting at 365 nm. the lamps were shielded by a highly polished anodized aluminum reflector. four cooling fans mounted at the bottom of the reactor dissipated the generated heat. a borosilicate glass tube of 15-mm inner diameter was used as the reaction vessel and was placed at the center of the photoreactor. the uv light-induced reaction was also studied with a micro-photoreactor fixed with a 6 w 254 nm low-pressure mercury lamp and a 6 w 365 nm mercury lamp. quartz and borosilicate glass tubes were employed as reaction vessels for 254 and 365 nm lamps, respectively. the light intensity (i) was measured by ferrioxalate actinometry. the volume of the reaction solution was always maintained as 25 ml in the multilamp photoreactor and 10 ml in the micro-photoreactor. air was bubbled through the solution at a flow rate measured by photooxidation of diphenylamine on zro2 1413 the soap bubble method. the uv–visible spectra were obtained with a hitachi u-2001 uv– –vis spectrophotometer. the solution was diluted 5-times to lower the absorbance to the beer–lambert law limit. the pbqi formed was estimated from its absorbance at 450 nm. sunlight-driven transformation the sunlight-induced transformation on zro2 was performed under clear sky in summer (march–july) at 11.30 am–12.30 pm. the solar irradiance (440 w m-2) was measured using a global pyranometer, supplied by industrial meters, bombay, india. ethanolic solutions of dpa of the required concentration were prepared afresh and taken in wide cylindrical glass vessels of uniform diameter. the entire bottom of the vessel was covered with zro2 powder. air was bubbled using a micro-pump without disturbing the zro2 bed. the volume of dpa solution was 25 ml and the loss of solvent because of evaporation was compensated periodically. the formed pbqi was estimated spectrophotometrically. results and discussion uv light-induced oxidative transformation on zro2 the uv light-promoted oxidative transformation of dpa in ethanol on zro2 surface was realized by bubbling air in a multilamp photoreactor fixed with uv lamps emitting at 365 nm. the uv-visible spectra of the dpa solution recorded at different illumination times show the formation of pbqi (λmax = 450 nm). the time spectra are displayed in fig. 1. the illuminated solution is epr silent showing the absence of the formation of diphenylnitroxide. in addition, a thin layer chromatographic experiment revealed the formation of a single product. the illuminated dpa solution was evaporated to dryness after recovery of the particulate zro2 and the solid was dissolved in chloroform to develop the chromatogram on a silica gel g-coated plate using benzene as eluent. the pbqi formed was estimated from its absorbance at 450 nm using the reported molar absorptivity.17,18 the linear increase of the concentration of pbqi with illumination time, as seen in the inset to fig. 1, provides the pbqi formation rate and the fig. 1. uv light-induced pbqi formation with zro2 in ethanol: the uv-visible spectra of reaction solution (5-times diluted) at 0, 30, 60, 90 and 120 min (increasing a); [dpa] = = 20 mm, zro2loading = 1.0 g, airflow rate = 7.8 ml s-1, i = 13.7×10-24 j l-1 s-1, volume of reaction solution = 25 ml; inset: linear increase of formed pbqi with illumination time. 1414 karunakaran, karuthapandian and vinayagamoorthy rates were reproducible to ±6 %. the photoformation of pbqi by direct photooxidation of dap in the absence of zro2 was slow11 and the rate of pbqi formation on zro2 was obtained by measuring the rates of pbqi formation in the presence and absence of zro2. the enhancement of pbqi formation on zro2 with concentration of dpa is displayed in fig. 2. the observed enhancement conforms to langmuir–hinshelwood (lh) kinetics with respect to the dpa concentration. the rate of surface reaction increased with zro2 loading in the dpa solution and the rate reached a limit at a high zro2-loading, as could be seen in fig. 3. a study of pbqi formation on zro2 as a function of the airflow rate showed enhancement of the surface reaction by oxygen and the rate dependence on the airflow rate conformed to the lh kinetic law, as could be seen in fig. 4. fig. 2. light-induced pbqi formation on zro2 as a function of dpa concentration; zro2-loading = 1.0 g, volume of reaction solution = 25 ml; uv: λ = 365 nm, i = = 13.7×10-24 j l-1 s-1, airflow rate = 7.8 ml s-1; solar: bed area = 11.36 cm2, airflow rate = 4.6 ml s-1. fig. 3. photoinduced pbqi formation on zro2 at different zro2-loading; [dpa] = 5.0 mm, airflow rate = 7.8 ml s-1, λ = 365 nm, i = 13.7×10-24 j l-1 s-1, volume of reaction solution = 25 ml. fig. 4. photoformation of pbqi on zro2 as a function of airflow rate; [dpa] = 5.0 mm, zro2-loading = 1.0 g, volume of reaction solution = 25 ml; uv: λ = 365 nm, i = 13.7× ×10-24 j l-1 s-1; solar: bed area = 11.36 cm2. photooxidation of diphenylamine on zro2 1415 the formation of pbqi on zro2 was also determined without bubbling air but the solution was not deoxygenated. the dissolved oxygen itself enabled the light-induced surface reaction, but the reaction was slow. pbqi formation on zro2 was studied at different intensities of illumination. the chemical transformation was studied with two, four and eight lamps and the angles sustained by adjacent lamps were 180, 90 and 45°, respectively. the dependence of the surface reaction rate on photon flux is displayed in fig. 5. pbqi was not formed in the absence of illumination. a study of pbqi formation on zro2 under uv-a and uv-c light, using a 6 w 365 nm mercury lamp (i = 10.0×10-24 j l–1 s–1) and a 6 w 254 nm low-pressure mercury lamp (i = 4.09×10-24 j l–1 s–1), separately in a micro-photoreactor under identical conditions showed that uv-c light was more efficient than uv-a light in inducing the organic transformation on zro2. the rate of pbqi formation with uv-a and uv-c light were 9.2 and 20.6 nm s–1, respectively ([dpa] = 5.0 mm, zro2 suspended: 1.0 g, airflow rate = = 7.8 ml s–1). the zro2 retained its activity on usage. reuse of zro2 showed sustainable light-induced pbqi formation. the azide ion (5 mm), a singlet oxygen quencher, failed to suppress pbqi formation, showing the absence of an involvement of singlet oxygen in the light-induced organic transformation on zro2. this finds literature support; fox and chen19 ruled out the possibility of singlet oxygen in the tio2-photocatalyzed olefin-to-carbonyl oxidative cleavage. fig. 5. influence of photon flux on zro2-promoted pbqi formation; [dpa] = 5.0 mm, zro2-loading = 1.0 g, airflow rate = 7.8 ml s-1, λ = 365 nm, volume of reaction solution = 25 ml. sunlight-induced oxidative transformation on zro2 the zro2-mediated oxidative transformation of dpa into pbqi also occurs under natural sunlight. the uv–visible spectrum of sun-shined dpa solution in ethanol in the presence of zro2 and air was similar to that with uv light (λmax = = 450 nm). furthermore, the sun-shined solution was epr silent revealing the absence of diphenylnitroxide. in addition, tlc analysis shows the formation of a single product. determination of the solar irradiance (w m–2) showed fluctuation of the sunlight intensity during the experiment, even under a clear sky. hence, the solar experiments under different reaction conditions were performed in a set 1416 karunakaran, karuthapandian and vinayagamoorthy to maintain the quantum of sunlight incident on a unit area the same. this enabled comparison of the solar results. a pair of solar experiments performed simultaneously under identical reaction conditions yielded results within ±6 %, which was also the case on different days. the effect of the operational parameters on the solar-promoted oxidative transformation was studied by performing the given set of experiments simultaneously and the results displayed in each figure represent identical solar irradiance. the rate of pbqi formation was obtained by shining the dpa solution on zro2 bed for 60 min. the dependence of pbqi formation rate on the concentration of dpa is shown in fig. 2. the observed increase of pbqi formation with dpa concentration is characteristic of the lh kinetic law. the double reciprocal plot of the pbqi formation rate versus dpa concentration was a straight line with a positive y-intercept (figure not shown), which confirmed the lh kinetic model. the rates of pbqi formation on zro2 at different airflow rates are shown in fig. 4. the observed enhancement of the pbqi formation by oxygen revealed that the surface reaction also conformed to lh kinetics with respect to oxygen. the double reciprocal plot of reaction rate versus airflow rate was linear with a finite y-intercept (figure not presented). the pbqi formation on zro2 was measured without bubbling air but the solution was not deoxygenated. the dissolved oxygen was sufficient to effect the chemical transformation on zro2 during the experimental period. however, the transformation was slow. the pbqi formation on zro2 increased linearly with the apparent area of the zro2-bed, as could be seen in fig. 6. the oxidative transformation did not occur in the absence of sunlight. zro2 does not lose its activity on usage. reuse of zro2 showed sustainable activity. fig. 6. dependence of sunlight-driven pbqi formation rate on zro2-bed area; [dpa] = 5.0 mm, zro2-loading = 1.0 g, airflow rate = 4.6 ml s-1, volume of reaction solution = 25 ml. mechanism the drs of zro2 is presented in fig. 7, from which it could be observed that the absorption edge of the employed pristine zro2 was 320 nm. illumination of zro2 with light of wavelength 365 nm is energetically unviable to bring about photooxidation of diphenylamine on zro2 1417 band gap excitation and hence the operation of the usual semiconductor-photocatalysis mechanism is ruled out. dpa is likely to be adsorbed on the surface of zro2. the ft-ir spectra of dpa and dpa adsorbed on zro2 are displayed in fig. 8. the shift of the >n–h stretching vibrational frequency from 3408 to 3404 cm–1 and bending vibrational frequency from 1595 to 1591 cm–1 indicate binding of dpa with zro2 through the amine hydrogen. the drs of dpa-adsorbed zro2 shows a shift of the absorption edge to the visible region (413 nm; fig. 7). this absorption is likely due to electronic excitation of the adsorbed dpa. the excited electron may move to zr4+ resulting in the formation of the radical cation ph2nh •+. the reduced form of zr4+ (i.e., zr3+) may lose an electron to the adsorbed molecular oxygen yielding superoxide radical ion (o2 •–). the reaction of the formed radical cation with the superoxide radical ion may yield the product pbqi. fig. 7. drs of bare zro2 and dpa-adsorbed zro2. fig. 8. ft-ir spectra of dpa and dpa-adsorbed zro2. ali et al.20 studied the photodegradation of methylene blue on zno films, deposited on glass slides by either the hydrothermal method or the magnetron sputtering technique, with uv-c light and the results conformed to the lh kinetic law. leaching of zinc due to photocatalysis was reported under oxygen limited conditions but the zno films were observed to be more stable under oxygen-rich conditions. it was suggested that the oxygen from the zno lattice 1418 karunakaran, karuthapandian and vinayagamoorthy was removed and used in the radical initiation and propagation phases of the photocatalysis under oxygen-limited conditions. that is, the observed deformation of the zno lattice over time was ascribed to the use of lattice oxygen in the photocatalytic process. oxygen-rich conditions either minimize the release of or replace the last zno lattice oxygen and the authors proposed the operation of a mars van krevelen (mvk) type mechanism in the photocatalytic degradation of methylene blue under oxygen-limited conditions. in a similar study with uv-a and uv-c light, the same authors suggested the predominance of the mvk mechanism under uv-c illumination.21 however, delmon22 stated that the reoxidation step in the mvk mechanism is often too slow and consequently the catalysts become reduced in the corresponding reduction–oxidation cycle. that is, the photocatalytic activity is decreased remarkably on reuse. furthermore, irrespective of the operative mechanism, leaching of zinc or a small decrease in the photocatalytic activity of zno on repeated reuse is well known.23,24 however, zro2 is chemically unreactive and hence operation of a mvk type mechanism in the present study is unlikely. in addition, the fact that zro2 shows sustainable photocatalytic activity (pbqi formation rate was not lowered on reuse of zro2) does not support the operation of a mvk type mechanism in the title reaction. serpone and co-workers25–28 stated that both the lh and eley–rideal (er) models are applicable for semiconductor photocatalytic reactions. the lh model presents the adsorption of both the reactant molecules on the surface29 while the er model represents adsorption of one of the reactants on the surface; the other reactant molecule interacts with adsorbed reactant molecule to form the product. the lh model requires saturation kinetics with respect to both the reactants whereas the er model demands saturation kinetics with respect to one of the reactants and first order dependence of the reaction rate with respect to the other reactant. the studied photocatalyzed reaction on zro2 surface shows saturation kinetics with respect to dpa as well as oxygen (figs. 2 and 4) and hence operation of the er reaction mechanism is ruled out. although bansal and sidhu30 stated that singlet oxygen is the oxidant in dye-sensitized photooxidation of dpa, this has been ruled out in the present photocatalytic transformation as azide ion does not suppress the formation of pbqi (vide supra); de lasa et al.31 also proposed the formation of reactive species superoxide radical anion in semiconductor photocatalyzed reactions. kinetic law the heterogeneous photoinduced reaction occurring in a continuously stirred tank reactor (cstr) conforms to the kinetic law:32 rate of pbqi formation on zro2 = [ ] [ ]( ) ( ) 1 2 1 2 dpa 1 dpa 1 kk k sic k k γ γ+ ⋅ + (1) photooxidation of diphenylamine on zro2 1419 where k1 and k2 are the adsorption coefficients of dpa and o2 on the illuminated zro2 surface, k is the specific rate of oxidation of dpa on the zro2 surface, γ is the airflow rate, s is the specific surface area of zro2, c is the amount of zro2 suspended per liter and i is the light intensity. the data-fit to the lh kinetic curve, drawn using a computer program,32 confirmed the kinetic law (figs. 2 and 4). the linear double reciprocal plots of surface reaction rate versus the dpa concentration and the airflow rate supports the lh kinetic law. the data-fit provides the adsorption coefficients k1 and k2 as 76 l mol–1 and 0.039 ml–1 s, respectively, and the specific reaction rate k as 2.75×109 mol l m–2 j–1. however, the rate of pbqi formation on zro2 surface fails to increase linearly with zro2-loading. this is because of the high zro2 loading. at high zro2 loadings, the surface area of the zro2 exposed to illumination does not correspond to the weight of zro2. the quantity of zro2 used is beyond the critical amount corresponding to the volume of the reaction solution and reaction vessel; the whole quantity of zro2 is not exposed to light. the photoinduced transformation lacks linear dependence on the illumination intensity; a lower than first power dependence of a surface-photoreaction rate on the light intensity at high photon flux is well known.33 synergism by zno and zns vectorial transfer of electrons and holes from one semiconductor to another is possible in semiconductor mixtures under band gap-illumination. this charge separation enhances the photocatalytic activity.13,14 however, what was observed in this study was enhanced phototransformation due to the presence of the semiconductor zno or zns nanoparticles with zro2 nanoparticles; the wavelength of illumination could effect the band gap excitation of zno and zns but not zro2. the enhanced formation of pbqi with zro2 mixed with zno or zns is displayed in fig. 9; the two nanoparticles were kept under suspension and under continuous motion by bubbling air through the illuminated solution. aggregation of nanoparticles under suspension is known.34 the particle size distribution of zro2, zno and zns under suspension, determined by the light scattering method, are presented in fig. 10. examination of fig. 10 along with the size of the particles obtained from the xrd and bet methods revealed aggregation of the nanoparticles. as observed in the individual zro2, zno and zns suspensions, aggregation in the zro2–zno and zro2–zns mixtures under suspension is likely, and both zro2 and zno or zns nanoparticles are likely to be present in the aggregate. this may lead to transfer of the generated hole from the illuminated zno or zns to the dpa molecule adsorbed on zro2 surface, resulting in enhanced photooxidation. the densities and particle sizes of zro2, zno and zns are different and this may be a reason for not observing maximum enhanced photo-oxidation at 50 % composition. 1420 karunakaran, karuthapandian and vinayagamoorthy fig. 9. enhanced pbqi formation on mixing zro2 with zno or zns; [dpa] = 5.0 mm, nanoparticles-loading = 1.0 g, airflow rate = 7.8 ml s-1, λ = 365 nm, i = 13.7×10-24 j l-1 s-1, illumination time = 30 min, volume of reaction solution = 25 ml. fig. 10. particles aggregation. conclusions zro2 mediates photoinduced oxidative transformation of dpa to pbqi. the formation of pbqi on zro2 enhances with dpa concentration, airflow rate and photon flux and conforms to the langmuir–hinshelwood kinetic law. the pbqi formation on zro2 is greater with uv-c light than with uv-a light. zro2 mixed with zno or zns affords more pbqi than the individual nanoparticles due to synergism. acknowledgements. prof. c. karunakaran is thankful to the council of scientific and industrial research (csir), new delhi for the emeritus scientist scheme (21(0887)/ /12/emr-ii). и з в о д фото-индукована оксидативна трансформација дифениламина на zro2. синергизам zno и zns c. karunakaran, s. karuthapandian и p. vinayagamoorthy department of chemistry, annamalai university, annamalainagar 608002, tamilnadu, india дифениламин (dpa) у етанолу на површини zro2 подлеже фото-индукованој оксидативној трансформацији дајући n-фенил-p-бензохинонимин (pbqi). фото-индукована трансформација на zro2 се повећава са порастом [dpa], количине zro2, брзине протока ваздуха и флукса фотона. стварање pbqi на zro2 је веће при озрачивању на 254 nm него на 365 nm. zro2 се може поново користити без икаквог третмана. мехнизам фотоиндуковане оксидативне трансформације dpa на zro2 је разматран применом одгова photooxidation of diphenylamine on zro2 1421 рајућег кинетичког закона. zno и zns повећавају uv фото-индуковану трансформацију dpa на zro2 указујући на синергизам. 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lasa, b. serrano, m. salaices, photocatalytic reaction engineering, springer, new york, 2005, p. 2 32. c. karunakaran, s. senthilvelan, curr. sci. 88 (2005) 962 33. l. vincze, t. j. kemp, j. photochem. photobiol., a 87 (1995) 257 34. m. li, m. e. noriega-trevino, n. nino-martinez, c. marambio-jones, j. wang, r. damoiseause, f. ruiz, e. m. v. hock, environ. sci. technol. 45 (2011) 8989. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {enhancing and assessing students' systems thinking skills by applying systemic synthesis questions in organic chemistry course} j. serb. chem. soc. 81 (12) 1455–1471 (2016) udc 547:377.1+378.4:001.38 jscs–4941 original scientific paper 1455 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. enhancement and assessment of students’ systems thinking skills by application of systemic synthesis questions in the organic chemistry course tamara n. hrin*#, dušica d. milenković#, mirjana d. segedinac# and saša horvat faculty of sciences, university of novi sad, trg d. obradovića 3, 21000 novi sad, serbia (received 11 august, revised 28 september, accepted 27 october 2016) abstract: many studies in the field of science education have emphasized the fact that systems thinking is a very important higher-order thinking skill that should be fostered during classes. however, more attention has been dedicated to the different ways of the assessment of systems thinking skills, and less to their enhancement. taking this into consideration, the goal of this study was not only to validate the systems thinking skills of secondary school students, but also to help students in the complex process of their development. with this goal, new instructional and assessment tools – systemic synthesis questions [ssynqs], were constructed, and an experiment with one experimental (e) and one control (c) group was conducted during organic chemistry classes. namely, the instructional teaching/learning method for both e and c groups was the same in processing new contents, but different on classes for the revision of the selected organic chemistry contents. the results showed that students exposed to the new instructional method (e group) achieved higher performance scores on three different types of systems thinking than students from the c group, who were taught by the traditional method. the greatest difference between the groups was found in the most complex dimension of systems thinking construct – in the ii level of procedural systems thinking. along with this dimension, structural systems thinking and the i level of the procedural systems thinking were also observed. keywords: assessment tool; instructional tool; secondary chemistry education; systemic approach; higher-order thinking. introduction in science education literature, the central goal of fostering students’ higher -order thinking skills (hots),1–3 or higher-order cognitive skills (hocs)4,5 has been widely accepted. even though it is challenging to provide an exact defin * corresponding author. e-mail: tamara.hrin@dh.uns.ac.rs # serbian chemical society member. doi: 10.2298/jsc160811097h 1456 hrin et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. ition of hots, resnick6 noted their non-algorithmic, multiple and complex nature. according to this notation, it is expected that hots include a wide range of different skills. hence, these skills include problem solving,5,7 decision making,3,5,7 critical thinking,5,7 the ability to ask complex questions,1,3,7 generating argumentation1,3 and systems thinking.3,7 in our opinion, a very detailed definition of systems thinking is that proposed by salisbury.8 this author defined systems thinking as the ability to think about all the concepts and relations that exist within a system, in order to effectively structure these relations. furthermore, evagorou et al.9 simplified this definition, describing systems thinking as the ability of an individual to understand and interpret complex systems. in addition, system could be considered as an entity that exists and functions through the interactions of its parts.10 the term system is very broad, and appears in several domains: social systems, technological systems and natural systems. in recent years, it has been recognized that many phenomena around us are examples of complex natural systems (e.g., ecosystems, hydro-cycle, metabolism, mixtures, organic compounds, etc.). for this reason, the understanding of complex systems and systems thinking has received growing attention in science education.9,11 yet, the real problem was stated when researchers recognized students’ limited understanding of complex systems.12 in order to solve this problem, gulyaev and stonyer13 proposed building an integrated curricula and developing an instructional method based on a mapping model. however, it should be mentioned that science education literature offers just a few empirical studies about how students could become good systems thinkers,9 and how systems thinking skills could be promoted through teaching. within this goal, computer simulations have often been used. regarding elementary school students, evagorou et al.9 examined the impact of simulation on the students’ systems thinking skills in one science (biology) topic, i.e., the eco -system of a marsh. additionally, the same instructional method was effective in developing the students’ systems thinking skills in secondary school biology lessons, however only in combination with a specific lesson within the context of education for sustainability development (esd).14 along with simulations, a specifically designed computer game seemed to be effective in biology classes too.15 furthermore, a slightly different approach to promoting the students’ systems thinking skills was proposed by assaraf and orion10 in earth science classes. these authors perceived the need for transforming the traditional classroom setting into specifically designed classes – a multidisciplinary-based study program. these classes should include a knowledge-integration activity, as well as an outdoor learning activity. according to the previously mentioned studies, systems thinking is a very important hots that students should develop,9,16 but also very difficult to measure.16 brandstädter et al.17 stated that, in addition to video analysis, the enhancing and assessing students’ systems thinking skills 1457 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. most widely used tools for assessing students’ systems thinking skills are questionnaires and interviews. this statement confirms a study by assaraf and orion,10 who used several different qualitative and quantitative research tools along with questionnaires and interviews. for example, by analyzing drawings and word associations of students, these two authors concluded that students possess an incomplete picture of the water cycle, as well as many misconceptions. on the other hand, observing the results of preand post-test analysis of concept maps, assaraf and orion10 noted the improvement of students in their ability to identify relations among concepts. similar results were reported in a paper by brandstädter et al.,17 who suggested computer and paper-pencil concept maps as adequate instruments for analyzing students’ systems thinking. however, these authors took a step forward, observing two-dimensional way of systems thinking – structural and procedural systems thinking. the first dimension, or structural systems thinking, refers to analyzing the basic structure of the system – relevant concepts and their relationships. on the other hand, second dimension, or procedural systems thinking, refers to the understanding of dynamic and cyclic processes that occur within systems’ elements and subsystems.17 the importance of this research was observed by dauer et al.18 who applied boxand-arrow (similar to concept maps) as an instructional and assessment tool in biology courses. even though the results of this study indicated that students improved their structural systems thinking ability, the authors did not examine the students’ procedural systems thinking ability. one of the latest papers is that by vachliotis et al.,19 who examined secondary school students’ systems thinking skills in an organic chemistry domain. for this purpose, the authors constructed and evaluated fill-in-the blank systemic assessment questions (saqs). saqs were introduced by fahmy and lagowski20,21 a few years later than their precursors – systemic diagrams.22,23 systemic diagrams and saqs take a central role within the context of systemic approach to teaching and learning chemistry (satlc), as basic instructional, or/and assessment tools. according to vachliotis et al.,24 systemic diagrams and saqs belong to the broader group of concept mapping techniques. however, while novak et al.25 highlighted hierarchical structure as the main characteristic of concept maps, systemic diagrams and saqs do not possess hierarchical levels of concepts in their structures.24 on the contrary, systemic diagrams and saqs present closed conceptual structures,21 in which all relations between concepts are made explicit to the learners.23 namely, emphasizing cyclic interrelations among new concepts and those previously acquired, systemic diagrams and saqs could provide more information to students than concept maps. however, the main differences between systemic diagrams and saqs should be mentioned. on the one hand, systemic diagrams represent all relevant concepts within a specific teaching unit, and therefore contain a large number of 1458 hrin et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. concepts. on the other hand, saqs contain a smaller number of concepts compared to systemic diagrams, as they were designed to effectively assess meaningful understanding and systems thinking after students have become familiar with a particular teaching theme.26,27 as was previously mentioned, in saqs, all concepts are directly or indirectly interrelated forming a closed structure,21 which could follow various geometric shapes. fahmy and lagowski20 proposed triangular, quadrilateral, pentagonal, hexagonal saqs, depending on the number of concepts included in the structures. an example of a common form of pentagonal saq with five concepts is presented in fig. 1. panels a–e represent a selected collection of concepts (e.g. chemical compounds), while x, y, z, q, and w highlight the relation between concepts (e.g. reagents, light, catalyst, temperature, pressure). fig. 1. common form of pentagonal saq. besides their geometric shape, saqs could also be distinguished by their type. fahmy and lagowski20,21 proposed various types of saqs, i.e., systemic multiple-choice questions (smcqs), systemic true/false questions (stfqs), systemic sequencing questions (ssqs), systemic matching questions (smqs), systemic synthesis questions (ssynqs), and systemic analyzing questions (sanqs). in the present study, ssynqs, as new instructional and assessment tools in systems thinking literature, were included. however, some changes were made to the original version. in fahmy and lagowski’s21 version of ssynqs, all concepts are proposed in the statement of a particular task and students should develop (build) a diagram by positioning these concepts in the correct fields. namely, they needed to determine the correct relations between concepts. on the other hand, the herein presented ssynqs were designed in a way that required recognizing, or identifying new concepts by observing the defined relations between them. henceforth, students were faced with unfilled, or partially filled ssynqs (the initial concept was presented) in order to fill in the concepts that were missing. it should be noted that fill-in-the-blank saqs were previously characterized as valid and reliable tools for assessing students’ meaningful and conceptual understanding of organic chemistry concepts by vachliotis et al.19,24 in their latest paper, these authors19 included an additional variable in the research, i.e., students’ systems thinking skills. to the best of our knowledge, this is the only enhancing and assessing students’ systems thinking skills 1459 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. study that considered students’ systems thinking skills in the chemistry educational process. however, this study19 only considered the assessment of students’ systems thinking skills, and it is still unknown how these skills could be promoted in the classroom. methodology study objective the main objective of this study was to examine the impact of ssynqs on the learning gains of secondary school students. such learning gains were observed through an assessment of experimental and control groups students’ systems thinking skills in the domain of organic chemistry. from the defined research objective, two research questions arise: 1. which test questions (conventional and/or ssynqs) could effectively assess the ability of secondary school students for structural and procedural systems thinking? 2. which type of instructional method (traditional or the systemic approach by applying ssynqs) could more effectively provide an enhancement of students’ structural and procedural systems thinking skills? sample the study sample comprised 119 high school students (61 males and 58 females; four classes) of mixed ability and socio-economic status. the experiment was conducted during the second semester of the 2012/2013 school year, and focused on third-grade students (11th grade in the anglo-american educational system; 17–18-year-old) who attended the same study stream: science and mathematics, in one high school in an urban region in serbia. the participants of the study also included two chemistry teachers who have a master’s degree in teaching chemistry, and similar experience working with secondary school students (10 years of experience in teaching chemistry). each of the teachers taught two classes. in order to achieve the aim of this study, a quasi-experimental research design involving non-random assignment to two parallel groups, one experimental (e) and one control (c) was conducted. it should be clarified that the instructional method for both e and c groups was the same in the processing new organic chemistry contents, but different in revision classes. namely, the e group students were treated by applying ssynqs, while c group was treated by applying the traditional method of teaching and learning the same material as the e group, however they did not receive any training in the systemic approach. such a research design with post-test only is involved in many studies in science education literature.26-29 students were allocated to the different instructional conditions according to the average chemistry grades achieved at the end of previous school years (first and second school years). the average chemistry grade was 4.38 (sd = 0.63) in the e group and 4.42 (sd = 0.70) in the c group, in a five-point grading system. group equalization based on average grades instead of initial testing was chosen to avoid the hawtorne effect.30,31 since the collected data was not normally distributed (shapiro–wilk test: e group: w = 0.83, p = = 0.000; c group: w = 0.79, p = 0.000), application of the non-parametric mann–whitney test was chosen to compare the medians of the e and c groups. the obtained results showed that there was no statistically significant difference between e (mean rank, mr = 58.77, sum of ranks, sr = 3820.00) and c (mr = 61.48, sr = 3320.00) groups before the conducted research; for u = 1675.00, the p value was greater than 0.05 (p = 0.653). thus, the formed e group contained 65 students, while the c group contained 54 students. 1460 hrin et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. context of the study first, it should be mentioned that prior to the present study, a pilot study was performed with two groups (one e and one c) and one teacher. the design of the pilot study was similar to that of the present study, and it was conducted in the first semester of the 2012/2013 school year, with smaller sample of participants (two classes), who followed the organic chemistry course: “hydrocarbons and their halogen derivatives”. the main objective of the pilot study was to investigate initially the efficiency of ssynqs as assessment and instructional tools, examining the students’ performance because of their meaningful learning,26 as well as perceived mental effort.27 in the present study (second semester of the 2012/2013 school year) students followed the organic chemistry course: “organic compounds with oxygen”. one teaching theme “carboxylic acids and their derivatives” was chosen as the material for this experiment, which was processed in five school classes (one class lasted 45 minutes). in accordance with the curriculum regulations of the third grade of the science-oriented and general high school, as well as with the recommended textbook, the following contents were processed: 1) general properties of carboxylic acids, 2) classification of carboxylic acids, 3) preparation of carboxylic acids and their physical properties, 4) chemical properties of carboxylic acids and 5) the function and application of carboxylic acids. the main objective of the phase of the conducted experiment was to familiarize students with the basic features of the aforementioned teaching units. namely, the transfer of information and accumulation of students’ knowledge were realised through the teacher’s lectures, discussion sections, and laboratory work (traditional method of teaching). during this period, all students were treated the same, relying on the recommended textbook and course material. after the completion of this phase, the students were divided into e group (two classes) and c group (two classes). hence, the e group students were taught by one chemistry teacher, while c group students were thought by the other chemistry teacher. one teacher was chosen for both e group classes for of several reasons. firstly, the selected teacher has already participated in the prior study, and as such, was experienced in the systemic approach. namely, the teacher was familiar with basic features of systemic approach and its applications in organic chemistry. apart from this, özmen et al.32 recognized that a teacher could have “experimenter bias”, which could be explained by the fact that the teacher could be more enthusiastic about the experiment, and thus be more interested in the e than in the c group. for this reason, an experiment with the random assignment of the teachers to one e class and one c class was not chosen. additionally, it is important to note that one author of this paper, who was a research assistant at the faculty of sciences, attended all the classes of both teachers in order to provide the same teaching quality. the new teaching and learning environment it should be mentioned that the revision of the selected organic chemistry contents (phase 2 of the study) was conducted during two school classes (90 min) in both e and c groups, so the length of time to study and revise was the same in both groups. for the purpose of the experiment, learning sheets with five unfilled and partially filled ssynqs were prepared, and presented to the teacher prior to their implementation in the classes. the ssynqs consisted of four to eight main concepts, and one example of the ssynqs with six concepts (main fields) is presented in fig. 2. before receiving learning sheets, the students were introduced to the operational definitions of terms applied in ssynqs, i.e., concepts, main relations, and cross-links. additionally, before solving each ssynq, they should read the instruction (presented in a text enhancing and assessing students’ systems thinking skills 1461 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. above the ssynq). by this, they could perceive the important relations between undefined concepts. furthermore, the students were encouraged to participate actively in the classes, answering the teacher’s questions and filling in the empty fields in the ssynqs with structural formulas and iupac-names of the required organic compounds. in this process, the teacher used a power-point presentation to ensure that all the students could see the correct answer at the end, which was presented via a video projector. the main goal of the experimental classes was to achieve a global view of selected chemical contents, connecting one class of organic compounds with others, previously learnt (e.g., carboxylic acids and their salts with alkanes and alkyl halides; fig. 2). fig. 2. example of an ssynq with six concepts that was applied in the experiment. on the other hand, during phase 2, c group students continued with the application of the traditional method of teaching and learning of organic chemistry. thus, students in the c group revised the same material as the e group, but they did not receive any training in the systemic approach. namely, under the controlled condition (one author was present) they solved specifically constructed conventional tasks (e.g. multiple-choice questions, completion type questions). according to this, they receive instruction on the main systems thinking language: “links” that represent the influence of one concept on others. more precisely, such links highlighted the conditions (temperature, pressure, catalyst) and direction in which one specific concept could be transformed to the other. after completion of each task, the students received feedback about their response to a particular problem (task). research instrument the instrument for assessing students’ systems thinking skills (a specifically designed test of knowledge) consisted of 10 tasks. these tasks were divided into two main categories: ssynqs and conventional questions, which were previously used as valid and reliable tools for assessing systems thinking.14,15,17 by application of additional assessment tools along with ssynqs, a disadvantage position of the c group students was prevented. conventional questions were designed following several question types: matching question (q1), completion type questions (q2, q10ii), multiple-choice questions (q3i, q5, q7, q10i), and open-response questions (q6, q9). additionally, ssynqs were presented by the numbers q4 and q8. for example, one open-response question (q6) was defined as: “after finishing reaction between an aliphatic compound a with a molecular formula c5h10o, and 1462 hrin et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. potassium dichromate solution acidified with dilute sulphuric acid, the orange solution turns green. the obtained substance b reacts with the sodium bicarbonate, forming a gas as one of the reaction products, which does not help burning (lighted wooden splint is extinguished). draw structures and write iupac names for compounds a and b, if both compounds contain one chiral centre.” on the other hand, an ssynq (q4, fig. 3) was defined as: “write the structure of ethanoic acid in field a, and then, in the field b, write the structure and the iupac name of the compound that can be obtained by the reaction of ethanoic acid and calcium hydroxide. compound b is further subjected to thermal decomposition to produce compound c. compound c can be oxidized by hot nitric acid yielding two products: ethanoic acid and compound d. compound d is reduced with lithium aluminium hydride to give compound e, which further reacts with compound f (obtained by a reaction of ethanoic acid and phosphorus trichloride) giving the final product g.” the correct answer for q4 is presented in fig. 3. fig. 3. solved test question q4 presented as an ssynq. scoring of the performance on the test questions was performed based on the nature of the questions. namely, multiple-choice questions were scored with one point, while in completion, open-response, matching questions, and [ssynqs], each individual requirement was scored with 0.5 points. in an ssynq, 0.5 points were scored for each concept presented in the right field in a diagram by correct structural formula or iupac name, or with one point if such concept was presented by both the correct structural formula and iupac-name. the maximum performance score in the test was 28 points. quality assurance of the applied instrument. the credibility of the applied instrument was assured by considering the test validity and reliability. the content validity as a pre-test assurance parameter was estimated through the work of an expert team, composed of two university professors and two research assistants in the field of chemistry teaching, and one enhancing and assessing students’ systems thinking skills 1463 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. high school chemistry teacher. they concluded that the test was valid as it was devised following the curriculum regulations of the selected chemistry course, as well as the recommended textbook. additionally, the clarity and meaningfulness of the test question requirements, the variety of question types, the length of sentences and the applied terminology were considered. validity as a post-test assurance parameter was estimated considering the statistical results of the applied test, in accordance with lopez et al.33 in this analysis, the relation (pearson’s correlation coefficient) between ssynqs scores and performance scores on conventional questions was considered. after conducting a correlation analysis of the obtained results, the pearson’s coefficient of 0.774 (p < 0.05) showed, according to dunn,34 a strong positive, statistically significant correlation between the students’ performance on [ssynqs] and conventional questions. this parameter confirmed the validity of the test. the reliability was estimated by two different methods: i) internal consistency and ii) the split-half method. the reliability of internal consistency was calculated using the cronbach α coefficient, which was found to be 0.925 (table i). additionally, the split-half method took into consideration several different coefficients (e.g. cronbach’s α for each part, the speaman–brown coefficients, etc., table i), which ranged from 0.855 to 0.922. the results of both methods showed that the applied test has excellent reliability. table i. reliability calculations for the applied instrument internal consistency cronbach’s α 0.925 split-half method cronbach’s α for part 1 0.865 cronbach’s α for part 2 0.861 correlation between parts 0.855 spearman–brown coefficient for equal lengths 0.922 spearman–brown coefficient for unequal lengths 0.922 guttman split-half coefficient 0.921 results and discussion assessing students’ systems thinking skills: analysis of the proposed model in order to answer the first research question, exploratory factor analysis (efa) with maximum likelihood (ml) extraction was used. it should be mentioned that this study put 99 data into the factor analysis as not all students completed the test. before developing intervention and test items, it was hypothesized that the two-factor model (structural and procedural system thinking) proposed by brandstädter et al.,17 would explain the students’ responses to the set of 10 test questions. however, a chi-square measure of goodness-of-fit (χ2 = 58.99, df = 26, p = 0.000) rejected the hypothesis that two factors were adequate to explain the model. thus, an alternative three-factor model was estimated. the bartlett’s test of sphericity (χ2 = 682.10, df = 45, p = 0.000; p < 0.05) and kaiser–mayer–olkin measure of sampling adequacy (kmo = 0.89; kmo > 0.50) indicated the suitability of the efa for the obtained data, and a chi-square measure of goodness -of-fit (χ2 = 28.78, df = 18, p = 0.051; p > 0.05) indicated that systems thinking 1464 hrin et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. (st) could be represented through the three factors. the three-factor model explained 76.97 % of the variance. the first factor was labelled as “structural st”, the second factor as “i level of procedural st”, and the third as “ii level of procedural st”. the different factors are explained below. in order to determine the factor to which each test question is associated, the factor loadings were considered. the factor loadings were interpreted according to the tabachnick and fidell35 model, by which the lowest acceptable value for factor loadings is 0.32. it should be mentioned that after conducting the efa, all test questions were associated with the first factor, however, some of the questions were also associated with the second or the third factor (table ii). in the present case, it was not a surprising fact that all questions were loaded on the first factor as the test of knowledge was constructed to tap into the high school students’ st skills, after applying selected instructional methods. additionally, according to brandstädter’s et al.17 definition of structural st, students should have developed the ability to solve any of the ten test questions, identifying relevant elements (concepts) of the selected subsystem (“carboxylic acids and their derivatives”) of one isolated system (“organic compounds with oxygen”), and observe their interrelationships. the defined structural st skills substantially correspond to the second identification step of the vachliotis’s et al.19 st construct. table ii. the main factors and factor loadings for the test questions question number first factor “structural st” second factor “i level of the procedural st” third factor “ii level of the procedural st” q1 0.684 0.317 0.383 q2 0.688 0.243 0.416 q3 0.674 – 0.110 q4 0.845 0.533 – q5 0.847 –0.531 – q6 0.610 0.519 – q7 0.603 – 0.269 q8 0.786 – 0.486 q9 0.503 0.129 – q10 0.610 – 0.243 additionally, to solve questions q1, q2, q4, q6 and q8 (table ii) the structural st was not sufficient. for this reason, a one-factor model was not chosen, and these questions were associated with another factor. in literature, some authors noted that it is acceptable that some questions are loaded on two factors.19,24 for example, q4 and q6 were also associated with the second factor (“i level of the procedural st”), while q1, q2 and q8 were associated with the third factor (“ii level of the procedural st”). in order to explain the way in which some of the questions could evaluate lower or higher levels of students’ skills of enhancing and assessing students’ systems thinking skills 1465 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. the procedural st, the systems and subsystems that included these questions were examined. as can be seen from table iii, q4 and q6 have included one system (“organic compounds with oxygen”) and several subsystems (q4: “carboxylic acids and their derivatives”, “carbonyl compounds”, “alcohols”; q6: “carboxylic acids and their derivatives”, “carbonyl compounds”). hence, in order to solve these questions, the students should interconnect several larger parts (subsystems), constituting a meaningful whole – conceptual system of interest. according to this, in our opinion, the i level of the procedural st could actually be the third identification step of the st construct developed by vachliotis et al.19 table iii. representation of associated systems and subsystems included in the test questions; system 1: organic compounds with oxygen question number subsystems of system 1 system 2 subsystems of system 1 q1 carboxylic acids and their derivatives inorganic acids binary acids; oxyacids q2 carboxylic acids and their derivatives hydrocarbons cyclic alkyl halides q3 carboxylic acids and their derivatives q4 carboxylic acids and their derivatives; carbonyl compounds; alcohols q5 carboxylic acids and their derivatives q6 carboxylic acids and their derivatives; carbonyl compounds q7 carboxylic acids and their derivatives q8 carboxylic acids and their derivatives; alcohols hydrocarbons alkanes; alkenes; alkyl halides q9 carboxylic acids and their derivatives q10 carboxylic acids and their derivatives on the other hand, the remaining three test questions, q1, q2 and q8, showed not only interconnection of several subsystems of one isolated system, but also interconnection of more than one system, i.e., two associated conceptual systems. thus, q1 included the additional system “inorganic acids”, while q2 and q8 included “hydrocarbons” (table iii). according to this, in order to solve the test questions associated with the third factor (ii level of the procedural st), the students should develop the ability to identify concepts and relationships of several subsystems of more than one system. in the paper by vachliotis et al.,19 such st skills were not mentioned. henceforth, after discussing the efa results, all the test questions that were associated only with the first factor were considered as assessment tools for students’ structural st skills in the further analysis. additionally, the test questions that were associated with the second or third factor along with the first factor were considered as assessment tools for the i or ii level of students’ procedural st skills. the stability of the three-factor model was additionally confirmed by 1466 hrin et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. calculating the cronbach alpha of the separated subscales (α1 = 0.843; α2 = = 0.707; α3 = 0.871). students’ performance in the three types of sts after determining which test questions could evaluate the students’ ability of structural, or i and ii level of procedural st skills, the next aim of this research was to investigate the significance of the differences in the performance of the e and c group students in the three different types of sts. the basic statistical parameters obtained for these variables are summarized in table iv, including the mean scores (m), the difference in the mean scores between the e and c groups (∆m), the standard deviations (sd), the minima and maxima, and the ranges. the mean scores indicated that the e group achieved significantly higher scores in comparison to the c group, observing the performance in each of the three types of sts. from table iv, it could be seen that the largest difference in performance between the groups was detected in the ii level of the procedural st, while the lowest difference appeared in the category of the structural st. in particular, it was found that implementation of ssynqs as instructional tools increased the students’ structural st ability by 27.81 percentage points, the i level of the procedural st ability by 30.74 percentage points, and the ii level of the procedural st by 33.21 percentage points. these differences are examined and discussed in detail in the following section. table iv. descriptive statistics for the students’ performance in the three different types of sts parameter performance in the structural st performance in the i level of the procedural st performance in the ii level of the procedural st e c e c e c m / % 86.25 58.43 84.44 53.70 58.17 24.96 ∆m / % 27.81 30.74 33.21 sd 14.71 34.93 15.54 37.68 26.40 24.09 minimum 50.00 0.00 33.33 0.00 12.50 0.00 maximum 100.00 100.00 100.00 100.00 100.00 100.00 range 50.00 100.00 66.67 100.00 87.50 100.00 in order to examine the statistically significant differences in the final examination scores between the two groups, the mann–whitney test with two independent samples (level of significance at p < 0.05), was applied. the statistical calculations were realised by application of the nonparametric tests from the statistical package imb spss statistics, version 19. for this analysis, the students’ performances in the three types of sts were defined as dependent variables. it should be noted that the mann–whitney test was used after determining that the collected data did not satisfy the requirements of a normal distribution, which was confirmed using then shapiro–wilk test, as well as observing the enhancing and assessing students’ systems thinking skills 1467 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. values for the standardized skewness and kurtosis. while the standardized kurtosis was within the +3 and –3 criterion35 for all the examined situations, the standardized skewness did not meet an acceptable range in several of the situations. the values of standardized skewness for the performance of the e group students on structural st (ss = –3.15) and on the i level of the procedural st (ss = –4.71) highlighted the fact that the students’ performances were significantly shifted towards higher scores. on the other hand, the same parameter for the performance of the c group students on the ii level of procedural st (ss = = 3.04) indicated the presence of too many low scores. in addition, a heterogeneity of the variance between the groups was also detected (levene’s test: f(5, 291) = 31.15, p = 0.000, p < 0.05). the results of the applied mann–whitney test, presented in table v, showed that the e group had higher mean ranks that the c group for each of the three dependent variables. the assumption that the group with the highest mean ranks should have a greater number of higher scores was further confirmed with p-values and z-values. the p-values resulting from this test were lower than 0.05, while the obtained z-values were lower than the critical value, zc = –1.96 (corder and foreman36), observing the students’ performance on the structural st (z = = –4.31, p = 0.000), on the i level of the procedural st (z = –4.35, p = 0.000), and on the ii level of the procedural st (z = –5.79, p = 0.000). these parameters showed that the differences in the performance on the three types of st of the e group, with respect to the c group, were statistically significant. in order to provide a clear description of the size of the observed statistically significant effects, using the z-values the effect size index (r) was calculated for all dependent variables, following the formula:37 r=z/√n (1) henceforth, using the r benchmarks provided by cohen,38 the effect sizes were evaluated. while medium effect sizes were determined for the performance in the structural st (r = –0.43), and the i level of the procedural st (r = –0.44), the students’ performance in the ii level of the procedural st was characterized by a large effect size (r = –0.58). table v. results of the mann–whitney test for students’ performance in different types of sts; p = 0.000 performance group mean rank sum of ranks u z structural st e (n=48) 62.74 3011.50 612.50 –4.31 c (n=51) 38.01 1938.50 i level of the procedural st e (n=48) 62.94 3021.00 603.00 –4.35 c (n=51) 37.82 1929.00 ii level of the procedural st e (n=48) 67.21 3021.00 398.00 –5.79 c (n=51) 33.80 1929.00 1468 hrin et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. from the results obtained in the mann–whitney test, it could be stated that students who applied ssynqs during learning the selected organic chemistry topic (“carboxylic acids and their derivatives”) outperformed the students from the c group. the e group students managed to develop procedural as well as structural st ability in a much more effective way than the students who were taught by the traditional method. apart from this, the calculated effect sizes indicated that the most notable difference between the groups appeared in the highest level of st. this could be explained by the fact that instruction via ssynqs brought students to a level in which they could not only identify the initial concepts (organic compounds) and simple relations, but also effectively “transform” such concepts within the selected system. according to assaraf and orion,10 this implies improved ability of students to include correct concepts into cycles (subsystems), identifying interrelations among them, and organize them into a complex system. however, the important question that follows is which characteristics of the applied instructional method provided such benefits, in comparison to the traditional instructional method. perhaps, the right answer lies in the structure characteristics of [ssynqs]. observing the structure and design of [ssynqs], these instructional tools could be characterized as completion of problems. van merriënboer and sweller39 defined completion problems within cognitive load theory (clt) as problems (tasks) presented in a more or less completed (partially completed) form, in which students need to find the remaining concepts for themselves. in their study, mihalca et al.40 found that completion of problems, as instructional tools, stimulate students to process the solution more deeply, and enables them to construct complex cognitive schemas. according to van merriënboer et al.,41 “schemas provide a bridge between what learners already know and what they need to know to perform the learning task”. finally, the ability to make such relations and networks within the selected system is the key characteristic of good system thinkers, and is highly relevant and appreciated in science education. conclusion and implications the first aim of this study was to examine the effectiveness of [ssynqs], as new evaluation tools in systems thinking literature, in assessing the st skills of secondary school students. considering the paper by brandstädter et al.,17 it was expected that a two-dimensional model of st, structural and procedural sts, would be found and then analyzed. contrary to expectations, a three-dimensional model was found where the [ssynqs] were effective in evaluating the i and ii level of the procedural st skills of students. these findings could be considered as valuable for future research, in which some another types of saqs should be constructed and examined as tools for assessing different aspects of systems thinking construct. enhancing and assessing students’ systems thinking skills 1469 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. with respect to the second suggested application of [ssynqs], the obtained results created a clear picture of [ssynqs] as effective instructional tools. students from the e group managed to achieve higher performance scores on each of the three different types of sts, compared to the students from the c group. in addition, the highest difference between groups was found in the most complex dimension of st – the ii level of the procedural st. these findings are in agreement with those of hung,16 who stated that, in order to develop deep understanding of a selected complex system, students should develop the ability to integrate fully their knowledge. in this point, the traditional instructional method is usually not convenient. significance of the study. this study is significant in the area of science (chemistry) education because, to the best of our knowledge, this is the first paper that highlights a good way of how students’ st skills could be promoted in chemistry classes. additionally, [ssynqs] were introduced and examined for the first time as new instructional and evaluation tools in the area of st. by this, the results obtained in this study contribute to the growing body of both theory and empirical practice in st literature, in the field of science education. limitations of the study. limitations of this study could be observed from a methodological standpoint, since this study included students from only one high school study stream: science and mathematics. additionally, only organic chemistry contents were investigated. implications for future research. future research with another sample (e.g., another high school study stream, or another secondary school profile) would assist to confirm the results, or to obtain new ones. in addition, a more extensive coverage of organic chemistry material should have been tested. apart from that, this research could be applied to different chemistry domains, such as general, inorganic chemistry, or biochemistry. however, the main future research attention should be focused on analyzing the possible gender differences, as well as differences in students’ cognitive load (invested mental effort) in the defined dimensions of st. by that, the connection between systems thinking constructs and the cognitive load theory would be posted. such results have not been yet presented in science education literature. acknowledgement. this work was supported by the ministry of education, science and technological development of the republic of serbia under grant no. 179010. 1470 hrin et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. и з в о д процена и побољшање ученичких вештина системског мишљења применом системичких задатака синтезе у органској хемији тамара н. хрин, душица д. миленковић, мирјана д. сегединац и саша хорват природно–математички факултет, универзитет у новом саду, трг д. обрадовића 3, 21000 нови сад многа истраживања у пољу образовања у природним наукама истичу чињеницу о важности системског мишљења, као вештине мишљења вишег реда које треба неговати у наставном процесу. међутим, већа пажња је посвећена различитим методама процене системског мишљења, а мења његовом побољшању. узимајући ово у обзир, циљ истраживања био је не само да се процене вештине системског мишљења код ученика средњошколског узраста, већ да се помогне ученицима у комплексном процесу његовог развоја. са тим циљем, конструисани су нови инструкциони алати и алати евалуације – системички задаци синтезе [ssynqs], након чега је спроведен експеримент на часовима органске хемије, са једном експерименталном (е) и једном контролном (к) групом. наиме, инструкциони метод учења и обучавања за е и к групу је био исти код обраде новог градива, али се разликовао на часовима понављања одабраних садржаја органске хемије. резултати су показали да ученици подвргнути новом инструкционом методу (е група) остварују боља постигнућа на три различите димензије системског мишљења од ученика к групе који су обучавани традиционално. највећа разлика се појавила у најкомплекснијој димензији конструкта системског мишљења – у ii нивоу процедуралног системског мишљења. поред ове димензије, структурално системско мишљење и i ниво процедуралног системског мишљења су такође размотрени. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice ruo4-mediated oxidation of secondary amines. part 1. are hydroxylamines the main intermediates? j. serb. chem. soc. 81 (5) 475–486 (2016) udc 546.96+542.943+547.233.2:546.172–41 jscs–4861 original scientific paper 475 ruo4-mediated oxidation of secondary amines. part 1. are hydroxylamines the main intermediates? cristina a. florea* and horia petride romanian academy, “costin d. nenitzescu” center of organic chemistry, spl. independenţei 202-b, ro-060023 bucharest, romania (received 15 december 2015, revised 13 january, accepted 14 march 2016) abstract: the ruo4-catalyzed oxidation of secondary amines bn–nh–ch2r (1a and b; r=h, me) gave mainly amides, but minute amounts of nitrones phch=n(o)–ch2r (9a and b) and traces of bn–n(oh)–ch2r (r=h, 4a) were also detected. in the presence of cyanide, up to 22 reaction products were identified, but mainly α-aminonitriles. comparison of the oxidation products of 1a and b with those of 4a and b and 9a and b, and bn–n(o)=chr (10a and b) showed that 4a and b cannot be the main reaction intermediates formed from 1a and b. keywords: oxidation; secondary amines; ruthenium tetraoxide; hydroxylamines; nitrones. introduction ruthenium-based catalysts, ruthenium tetraoxide (ruo4) included, have been extensively used to oxidize a wide variety of organic compounds, such as aliphatic and aromatic hydrocarbons, alkenes, alkynes, alcohols, ethers, amines, or amides.1–11 the versatility, specificity, the high yields obtained in many cases, and the environmentally friendly reaction conditions have strongly recommended ruthenium-containing catalysts be successfully utilized in fine and green chemistry.1–10 in the case of tertiary amines of the rch2–nr1r2 type, earlier papers12–16 indicated that ruo4 attacks the n–ch2 positions to afford amides (rco–nr1r2) and for about 30 years, this reaction was viewed as a highly regioselective oxidation. however, the ruo4-mediated oxidation of tertiary amines containing different types of n–ch2 groups, such as n-benzylazacycloalkanes,17,18 n,n-dialkylbenzylamines19 or n-benzyl-2-azanorbornane derivatives,20,21 caused doubts on the previously claimed high regioselectivity. in the case of n,n-dialkylbenzylamines, the oxidation compounds were derived from both reactive n–ch2 sites * corresponding author. e-mail: antonetaflorea@yahoo.com doi: 10.2298/jsc151215029f 476 florea and petride (i.e., benzylic and alkylic). in addition, some n-oxides were also formed, indicating the existence of a third, minor oxidative route. in the presence of the cyanide ion, only the first two routes were followed. formation of two kinds of n-αcyano derivatives suggested the transient existence of iminium cations as intermediates. the statistically corrected alkyl/benzyl regioselectivity of the oxidation reaction was relatively poor (4.1 for n,n-dimethyland 2.1 for n,n-diethylbenzylamine).19 in continuation of previous work on the ruo4-mediated oxidation of tertiary amines,17–19 it was considered useful to extend the study on secondary amines, more precisely on n-methyl(1a) and n-ethylbenzylamine (1b). by analogy with the behavior of tertiary amines presented before, the secondary amines 1a and b could suffer oxidative attack at three positions: 1) benzylic, 2) alkylic, and/or 3) at the nitrogen atom (scheme 1). depending on the followed path, the first oxidation step should give n-alkyl imines 2a and b, n-benzyl imines 3a and b, and/or hydroxylamines 4a and b, respectively. in this paper, it was analyzed whether 4a and b could be the main reaction intermediates. bn-nh-ch2r phch=n-ch2r benzyl 2a-b 1a-b bn-n=ch-r 3a-b attack at attack at n attack at alkyl 4a-b bn:benzyla: r=h b: r=me bn-n(oh)-ch2r scheme 1. oxidative routes for 1a and b. experimental instrumentation the ft-ir spectra were registered on a bruker vertex 70 instrument, equipped with a diamond crystal atr. the nmr spectra were recorded with a varian unity inova 400 spectrometer, operating at 400 mhz (1h) and 100 mhz (13c). the mass spectra were obtained using a gc 6890 agilent technologies gas chromatograph coupled with a ms 5975 b quadrupole mass spectrometer, using the standard 70 ev ionization energy. nmr (1h and 13c) and ms data for 1, 2 and 4–28 are given in supplementary material to this paper. materials the formulae of all substrates and reaction products 1, 2 and 4–28 are given in scheme 2. compounds 1a, 2c, hydrated ruo2 (all from aldrich), 1b, 5c, 6c, 7a, 11–13, 26, benzoic acid, naio4 (all from merck) and the organic solvents (from chemical co., iaşi, romania) were used as purchased, except for chcl3 and ch2cl2, which were stored over anhydrous na2co3 and filtered prior to use. derivatives 2a and b, 6a and b, 7b, 8a–e, 9a and b, 14, 15a and b, 16a and b, 17a–e, 18a and b, 19a and b, 20a and b, 21a and b, 22a and b, 23a and b, 24a and b and 25a and b were prepared and characterized previously.19,22 compounds 4a and oxidation of secondary amines 477 b,23 9c,24,25 10a,26 10b,27 15c,28 2729 and 2830 are all known from the literature and were prepared according to the indicated procedures. the purity of all non-commercial chemicals was checked by ft-ir, nmr and gc–ms. nmr and ms spectroscopies were extensively used to identify the oxidation products. even though the nmr characteristics of most of compounds depicted in scheme 2 have been already reported19,22 or are widely known (i.e., 5c and 11–13), it was judged useful to present the corresponding nmr (and ms) spectral data of all these compounds (1, 2 and 4–28) as supplementary material. bn: benzyl 8, 17 r r1 a h h b me me c me h d h ph e me ph bn-nh-ch2r 1a-b ruo4/naio4 (nacn) imines (2), hydroxylamines (4) amides i (6, 7), amides ii (8) nitrones (9, 10) others i (11-14) nitriles i (15, 16), nitriles ii (17) cyanoimines (18-20) cyanonitrones (21, 22) others ii (26-28) formamidines & related compounds (23-25) aldehydes (5) 2a-c: ph-ch=n-ch2r 4a-b: bn-n(oh)-ch2r 5a-c: r-cho 6a-c: phco-nh-ch2r 7a-b: bn-nh-co-r 8a-e: bn-n(ch2r)-co-r 1 9a-c: ph-ch=n(o)-ch2r 10a-b: bn-n(o)=ch-r 12: phcn 11: bnnh2 14: (bn-nme-)2ch2 13: phconh2 15a-c: ph-ch(cn)-nh-ch2r 17a-e: bn-n(ch2r)-ch(cn)-r 1 18a-b: ph-c(cn)=n-ch2r 19a-b: bn-n=c(cn)-r 20a-b: ph-ch=n-ch(cn)-r 21a-b: ph-c(cn)=n(o)-ch2r 22a-b: bn-n(o)=c(cn)-r 28: bn-nh-oh 26: ph-ch=n-oh 27: bn-n=n(o)-bn 23a-b: bn-n(ch2r)-c(=nh)-cn 24a-b: bn-n(ch2r)-cn 25a-b: bn-n(ch2r)-conh2 16a-b: bn-nh-ch(cn)-r 1, 2, 4-7, 9, 10, 15, 16, 18-25 r a h b me c ph scheme 2. oxidation products of 1a and b. oxidation by ruo4/naio4 (± nacn). general procedure to a heterogeneous mixture of chcl3 (5 ml) and aqueous solution of naio4 (0.4 m; 10 ml, 4 mmol) was added solid ruo2·xh2o (10–15 mg), followed by the substrate (1 mmol), 478 florea and petride previously dissolved in chcl3 (5 ml) (reaction conditions a). in the case of the cyano-trapping experiments (reaction conditions b), to the nacn solution [196 mg (4 mmol) in water (10 ml)] was added ruo2, the substrate (1 mmol in 10 ml of chcl3), and the co-oxidant naio4 solution (10 ml, as before), in this order. several other organic solvents (ch2cl2, me2co3 and acoet) were tried with no significant differences. in all cases the heterogeneous reaction mixture was magnetically stirred at room temperature for 3–5 h and then worked up as described in a previous paper.22 identification of the various reaction products was made by comparison of the nmr and gc–ms spectra before and after the addition of pure compounds into the analyzed samples. 1,4-dimethoxybenzene (dmb) was employed as an internal standard for quantitative analyses.22 work-up losses were determined on synthetic mixtures and then used to correct the experimentally found amounts. these corrected amounts were used to calculate the yields given in table i (see below). oxidation of 1a and b by h2o2/na2wo4 hydrogen peroxide (30 %; 1 ml, 9.8 mmol) was added dropwise into a magnetically stirred methanolic solution (10 ml) of 1a (0.53 ml, 4 mmol) and na2wo4·2h2o (132 mg, 0.4 mmol), maintaining the temperature within 25–30°c with a water bath. the mixture was stirred at room temperature for another 4–5 h and then filtered. the clear filtrate was evaporated to dryness, the residue taken up in chcl3, the solution filtered again, and the organic solvent eliminated in vacuo. the obtained liquid residue was analyzed by nmr, as such and with a known amount of dmb. besides benzaldehyde and other compounds, 9a and 10a resulted in about 32 and 17 % yield, respectively. treatment of this oxidation residue with lialh4/ether23 transformed both nitrones into hydroxylamine 4a. analogously, 9b (≈31 %) and 10b (≈25 %) were formed from 1b; reduction of the nitrones gave 4b. results and discussion the oxidations were performed in the absence/presence of cyanide (aand b-conditions, respectively) and the corresponding results are shown in table i. the yield of benzaldehyde 5c in table i actually refers to the yield of 5c+benzoic acid. this is allowed because benzoic acid is always derived from 5c, merely by air oxidation during the reaction mixture work-up. to gain in simplicity, the desired entries (let them be x and y) of table i will be cited as ti-x,y throughout this paper. oxidation of amines 1a–b. reaction products the various components of the oxidation mixtures obtained from 1a–b in the absence of cyanide (ti-1,2) can be divided into imines (2a–c), hydroxylamines (only 4a was detected), aldehydes (only 5c was detected as such), amides (6–8), nitrones (9), benzonitrile (12), and benzamide (13). in the case of 1a, diamine 14 was also present. some reaction products contain more carbon atoms than in the starting amines. whatever the following oxidation route, they cannot be primary reaction products. thus, amides 8a–e (amides ii in scheme 2) should be formed in two steps, as depicted in scheme 3: i) the condensation of the appropriate amine 1 with aldehyde 5 affords the intermediate 29, which ii) is oxidized further at the oxidation of secondary amines 479 oh level.31,32 besides oxidation to 8a, the hemiaminal 29a could also react with 1a to give the diamine 14. it should be noted that 8a and 8d resulted from 1a (ti -1), but 8b, 8c, and 8e from 1b (ti-2). according to scheme 3, this implies the formation of aldehydes 5a and 5c during the oxidation of 1a and of 5a, 5b, and 5c during the analogous reaction of 1b. table i. oxidation of selected compounds entry no. compd. (conversion, %)a reaction productsb (yield, %)c 0 1 2 a) oxidations in the absence of cyanided 1 1a (55) 2a (2.2), 2c (8.6), 4a (0.2), 5c (8.0), 6a (2.2), 6c (1.8), 7a (3.8), 8a (30.0), 8d (7.4), 9a (2.4), 12 (2.0), 13 (0.6), 14 (1.3) 2 1b (81) 2b (2.1), 2c (4.1), 5c (34.3), 6b (3.9), 6c (3.1), 7a (1.7), 7b (4.1), 8b (3.5), 8c (15.2), 8e (3.8), 9b (0.3), 12 (7.4), 13 (0.5) 3 4a (100) 5c (55.0), 6a (1.9), 7a (1.8), 12 (7.7), 13 (4.1), 26 (2.3), 27 (1.4) 4 4b (100) 5c (40.8), 6b (3.6), 7b (4.0), 12 (8.6), 13 (10.9), 26 (4.7), 27 (2.8) 5 9a (100) 5c (75.6), 6a (0.8), 12 (0.4), 13 (0.8) 6 9b (100) 5c (78.1), 6b (0.6), 12 (9.6), 13 (0.3) 7 10a (100) 5c (48.7), 7a (4.6), 9c (0.6), 12 (7.1), 13 (5.8), 26 (5.7), 27 (3.0) 8 10b (100) 5c (50.1), 7b (4.0), 9c (0.5), 12 (7.0), 13 (6.0), 26 (3.1), 27 (1.8) 9 28 (100) 5c (10.7), 9c (4.0), 12 (4.3), 13 (0.5), 26 (34.7), 27 (8.7) b) oxidations in the presence of cyanided 10 1a (13)e 2c (1.2), 5c (0.9), 6a (0.4), 7a (0.7), 8a (1.8), 8d (8.9), 11 (2.5), 12 (0.9), 15a (6.3), 16a (2.2), 17a (18.2), 17d (15.3), 18a (0.6), 20a (0.3), 21a (0.4), 22a (0.8), 23a (2.9), 24a (1.0), 25a (4.5) 11 1b (37)e 2b (5.2), 2c (1.8), 5c (0.5), 6b (0.5), 7b (0.6), 8b (1.0), 8c (0.6), 8e (2.7), 9b (7.8), 12 (1.5), 15b (15.6), 16b (10.9), 17b (9.9), 17c (1.0), 17e (8.1), 18b (0.8), 20b (0.5), 21b (0.5), 22b (0.9), 23b (4.2), 24b (0.9), 25b (8.0) 12 4a (100) 5c (2.5), 6a (0.7), 7a (2.7), 9a (28.8), 9c (1.2), 12 (4.0), 13 (6.0), 18a (7.3), 21a (7.6), 22a (2.0), 26 (0.3) 13 4b (100) 5c (1.8), 6b (0.3), 9b (39.4), 10b (25.8), 12 (8.8), 13 (3.6), 18b (1.1), 21b (3.0), 22b (5.8) 14 9a (20) 5c (34.7), 6a (1.2), 12 (6.9), 18a (22.0), 21a (9.7) 15 9b (22) 5c (37.5), 6b (1.5), 12 (6.0), 18b (20.2), 21b (8.1) 16 10a (100) 5c (14.0), 7a (2.5), 9c (5.0), 12 (8.7), 13 (2.9), 22a (11.9), 26 (2.9), 27 (3.6) 17 10b (93) 5c (12.3), 7b (1.5), 9c (5.1), 12 (8.3), 13 (5.9), 22b (12.1), 26 (4.3), 27 (2.0) aconversion calculated against the reacted substrate; bformulae in scheme 2; cyields (mole ratios of product/ /reacted substrate) were calculated from the nmr and ms spectra, regardless of the stoichiometry; dreaction conditions (for 1 mmol of substrate): a – ruo2⋅xh2o(10–15 mg), naio4 (4 mmol), chcl3/water = = 10/10 (ml/ml), room temperature, 3–5 h; b – as in a, but nacn (4 mmol) in water (10 ml) was also added; eliterature data22 480 florea and petride secondary reaction products could also be the derivatives 2c and 6c, their existence implying the transient formation of benzylamine 11. thus, benzamide 6c might be formed by a sequence similar to that depicted in scheme 3 (i.e., 11+5c → bn–nh–ch(oh)–ph → 6c). moreover, unlike 29, the generated hemiaminal has an n–h proton (in italics) thus allowing the concurrent dehydration to imine 2c. as presented in a forthcoming paper,33 compounds 5c (some of it), 12 and 13, were also derived from benzylamine. it is worth mentioning that the origin of 6a and b and 7a and b (amides i in scheme 2) will be clarified elsewhere.33 bn-n(ch2r)-co-r 1 ox 8a-e 8, 17, 29 r r1 a h h b me me c me h d h ph e me ph bn-n(ch2r)-ch(oh)r 1 hcn 14 ox: oxidation + 1a (r=r1=h) (bn-nme-)2ch2 bn-nh-ch2r + r 1-cho 1a-b 5a-c 29a-e bn-n(ch2r)-ch(cn)r 1 30a-c1a-b 17a-e -h2o bn-nh-ch2r + r 1-ch(oh)-cn 1, 5, 30 r/r1 a h b me c ph scheme 3. formation of 8, 14 and 17. these considerations are also supported by the results obtained in the presence of cyanide (ti-10,11). in this case, along with some compounds encountered in the corresponding cyanide-free reactions, most of the starting amine was consumed in the formation of new, cyano-containing products: nitriles 15–17, cyanoimines 18 and 20, cyanonitrones 21 and 22, as well as derivatives 23–25. identification of benzylamine 11 in the oxidation mixture derived from 1a (ti-10) confirmed the preceding deductions on its formation. as already reported,22 formamidines 23 and their hydrolysates 24 and 25 are side products, which do not involve oxidation of 1a and b. therefore, they are not important for the present discussion. in general, all nitriles (15–17) could be formally derived by substituting the c=o function in the analogous amides 6–8 with the ch–cn group. as depicted in scheme 3, nitriles 17a–e might result from 1a and b and cyanohydrins 30a–c, oxidation of secondary amines 481 formed in turn from 5a–c. it is well known that cyanohydrins result easily by adding an aldehyde (or aliphatic ketone) to a cyanide-containing medium. compounds 18 and 20–22 are not primary oxidation products of 1a and b. as presented below, some of them might derive from the oxidation of 4a and b, via 9a and b and 10a and b. the existence of nitrones 9a and b (ti-1,2,11) implies the transient formation of 4a and b in all oxidations of 1a and b, respectively. in fact, it is well known that nitrones are usually prepared by oxidation of hydroxylamines under various conditions.34–36 it is also known that secondary amines can give nitrones directly, especially under tungsten catalysis,37 but low -valent ruthenium compounds can also be used under certain conditions (for instance, with catalytic tetra-n-propylammonium perruthenate).38 the possible oxidation of imines to nitrones (such as 2a–c → 9a–c) can be excluded in the present case, because it requires peracids.39 the oxidation of 1a and b was performed with the h2o2/na2wo4 system and the expected mixtures of nitrones 9a+10a and 9b+10b, respectively, were obtained. the cumulated yields of about 50 % (see experimental) were in contrast to the very modest values obtained under ruo4-catalysis (ti-1,2,10,11). this raises doubts about the implication of nitrones (and by extension of hydroxylamines) as the main intermediates during the ruo4-oxidation of 1a and b. a detailed comparison of oxidation products would clarify this supposition. oxidation of hydroxylamines 4a and b and related nitrones under cyanide-free conditions (ti-3,4), 4a and b gave mainly benzaldehyde (5c) and small amounts of 6a+7a/6b+7b, 12 and 13. in both cases, two new compounds appeared, which were identified as benzaldoxime (26) and 1,2-bis(phenylmethyl)diazene 1-oxide (27). in the presence of cyanide (t1-12), the oxidation of 4a gave mainly nitrone 9a, but cyano-containing derivatives 18a, 21a, and 22a were also formed. more informative was the behavior of 4b (t1-13). analogously with 4a, compounds 9b, 18b, 21b, and 22b were present, but accompanied by another nitrone, 10b. the two nitrones, 9b and 10b, accounted for 65 % of the reacted 4b. the formation of the nitrones 9b and 10b suggested that 4b was attacked at both n-α-positions, namely benzylic and alkylic, respectively (scheme 4). this is probably the first reaction step in all ruo4-mediated oxidations of 4a and b, but, usually, only the most stable nitrones (under the considered conditions) are observed. the behavior of all four nitrones was verified and it was found that the n-alkylic ones (9a and b) were completely transformed under conditions a (conversion of 100 % in ti-5,6), but only partially under conditions b (≈21 % in ti-14,15). at the same time, the n-benzyl nitrones 10a and b were completely transformed under both a (ti-7,8) and b conditions (ti-16,17), meaning that they are less stable than their n-alkyl analogs 9a and b. 482 florea and petride 21a-b 9a-b 4a-b ph-cho + ho-nh-ch2r bn:benzyl ox: oxidation x: oh, cn (x=oh) 5c +hx (x=cn) (x=oh) 22a-b bn-nh-oh + ohc-r 28 5a-b 9c + 5c ph-ch=n(o)-ch2r bn-n(oh)-ch2r ph-c(cn)=n(o)-ch2r bn-n(o)=c(cn)r ph-ch=n(o)-bn 10a-b 7a-b bn-nh-co-r +hx -h2o bn-n(o)=ch-r r a h b me (x=oh) tautom. 6a-b phco-nh-ch2r x=oh: 36a-b x=cn: 19a-b bn-n=cx-r x=oh: 34a-b x=cn: 35a-b bn-n(oh)-chx-r x=oh: 31a-b x=cn: 32a-b ph-chx-n(oh)-ch2r (x=cn) 27 +h2o bn-n=n(o)-bn x=oh: 33a-b x=cn: 18a-b ph-cx=n-ch2r -h2o (x=oh) 5c37 phcho + h2n-oh 26 bn-no ph-ch=n-oh (bn-no)2 (37)2 ox ox ox ox ox + 37 scheme 4. oxidation of 4a and b, 9a anb b, 10a and b and 28. the behavior of 9a and b and 10a and b is more consistent with hydrolysis than with oxidation, at least in the first step. for instance, 9a and b gave large (≈77 %) or medium (≈33 %) amounts of benzaldehyde (5c) under conditions a and b, respectively. the imagined formations of the corresponding intermediates 31a and b and 32a and b are presented in scheme 4. under a-conditions, 31a and b can be either broken to a mixture of 5c + an aliphatic hydroxylamine, or dehydrated to 33a and b, the enolic form of the most stable benzamides 6a and b. under b-conditions, the intermediates 32a and b can suffer either oxidation to cyanonitrones 21a and b, or dehydration to the corresponding cyanoimines 18a and b. actually, it is well known that, under non-oxidative conditions, imines 18a and b can easily be obtained from 9a and b and sodium cyanide in aqueous ethanol.40 a similar reaction scheme was imagined also for 10a and b (scheme 4). nucleophilic addition of hx affords 34a and b (x=oh) or 35a and b (x=cn). analogously to 31a and b, the intermediates 34a and b can give 28+5a and b oxidation of secondary amines 483 and/or amides 7a and b (via 36a and b). similarly to 32a and b, the intermediates 35a and b could be the sources of cyanonitrones 22a and b and/or cyanoimines 19a and b. however, no explanation can offered for the absence of 19a and b. turning back to the reactions of 10a and b (ti-7,8), it was noted that about 50 % of the substrate was consumed towards benzaldehyde 5c. why 5c and not benzylhydroxylamine 28, the logical output of hydrolyzed 10a and b, as expected from scheme 4? to understand these transformations, the oxidation of 28 was studied separately. in the reaction mixture (ti-9), 5c, nitrone 9c, benzaldoxime 26, and the azoxy compound 27 were found; a similar composition was obtained under b-conditions (absent in table i). remember that all these compounds were also present in the oxidation mixtures of 10a and b. the reaction was interpreted as depicted in scheme 4, where 28 is oxidized first to the nitroso derivative 37, which is in equilibrium with its dimer (37)2. the key intermediate 37 could give 26 (by tautomerization), but also 27 by condensation with unreacted 28. benzaldehyde 5c, generated from 26 by hydrolysis, could be the source of nitrone 9c, after condensation with 28. the dimer (37)2 was not synthesized, but a rapid nmr scan of the analyzed sample showed the presence of a transient compound with nmr characteristics (a ch2–singlet at 5.37 ppm (63.1 ppm) long-range coupled with peaks at 129.4 (ortho-arom. ch) and 134.1 ppm (ipso-arom. c), similar to those given in the literature for 1,2-bis(phenylmethyl)diazene 1,2-dioxide ((37)2).41,42 moreover, in the absence of 28 and directly in the nmr tube, it gave 26 quantitatively, in a few hours. this is not surprising, since transformations such 37 → 26 are well known for about 125 years; the reaction is catalyzed by acids or bases.43,44 the presence of 9c, 26 and 27 in an oxidation mixture might be considered as a fingerprint for the previous existence of nitrones 10a and b. the fact that some of them are absent in the reaction mixtures of 4a and b (ti-3,4,12) is probably due to their relative amounts, presumably too small to be detected. the same reason could be invoked for entry 13, when all three compounds were missing, probably because too little 10b underwent hydrolysis. it is now clear that the behavior of 4 is the sum of that of corresponding nitrones 9 and 10. comparison of results now it is possible to compare the reaction products formed from 1a and b with those derived from 4a and b. in these comparisons, compounds 8a–e (amides ii in scheme 2), 14 and 17a–e (nitriles ii) were not considered, as they could not result from 4a and b, because their formation requires the presence of amines 1a and b. however, if 4a and b are the main reaction intermediates, their oxidative outputs should explain the formation of benzylamine 11, as well as of aldehydes 5a+5c and 5a+5b+5c, respectively. 484 florea and petride comparison of the reaction products derived from 1a (ti-1,10) with those resulting from 4a (ti-3,12) and/or 9a+10a (ti-5,14 + ti-7,16) shows that 2a, 2c, 6c, 11, 15a, 16a and 20a were derived from 1a only. on the contrary, 9c, 26 and 27 resulted from 4a only. similar observations could be made comparing the outputs of 1b (ti-2,11) with those of 4b (ti-4,13) and 9b+10b (ti-6,15 + ti-8,17): 2b, 2c, 5a, 6c, 15b, 16b and 20b came from 1b only, but the unique source of 9c, 26 and 27 was 4b (or 9b+10b). this means that, under conditions a, the oxidation of 4a and b cannot explain the formation of imines 2a and b and benzylamine (source of 2c and 6c). in the case of 4b, it must add also the lack of any species implying formaldehyde (5a), used by 1b to generate 8c, as in scheme 3. moreover, under conditions b, the oxidation of 4a and b does not imply the formation of nitriles 15a and b and 16a and b (the hcn-trapped forms of 2a and b and 3a and b, respectively).33 at the same time, although 18+21+22 are generated from hydroxylamines 4 as well as from amines 1, the cyanoimines 20 are formed only from 1. this causes doubts regarding the route followed to generate all cyanoderivatives 18 and 20–22. actually, it will be shown33 that during the oxidation of 1a and b, the real source of 18+20+21+22 is just the oxidation of 15a and b and 16a and b. at the same time, no 9c, 26, nor 27 seemed to be ever formed from 1a and b. all these considerations do not favor 4a and b as the main intermediates during the ruo4-mediated oxidation of 1a and b. however, this route does exist, but its minor role consists only in the formation of small amounts of nitrones 9a and b and 10a and b. conclusions hydroxylamines bn–n(oh)–ch2r (4a and b; r=h and me) and nitrones phch=n(o)–ch2r (9a and b) are present in minute amounts in the ruo4-mediated oxidation mixtures derived from the secondary amines bn–nh–ch2r (1a and b). careful comparative analysis of the complex reaction mixtures showed that 4a and b, 9a and b, as well as nitrones bn–n(o)=ch–r (10a and b) cannot be the main oxidation intermediates of 1a and b. supplementary material nmr (1h and 13c) and ms data for 1, 2 and 4–28 are available electronically from http://www.sbd.org.rs/jscs/ or from the corresponding author on request.. oxidation of secondary amines 485 и з в о д оксидација секундарних амина уз посредовање ruo4. део 1. да ли су хидроксиламини главни интермедијери? cristina a. florea и horia petride romanian academy, “costin d. nenitzescu” center of organic chemistry, spl. independen�ei 202-b, ro-060023 bucharest, romania оксидација секундарних амина bn–nh–ch2r (1a и b; r=h, me) ruo4 катализатором даје као главне производе амиде, али и незнатне количине нитрона phch=n(o)–ch2r (9a и b) и bn–n(oh)–ch2r (r=h, 4a) у траговима. у присуству цијанида идентификована су 22 производа, углавном α-аминонитрили. поређењем производа оксидације 1a и b са производима оксидације 4a и b, 9a и b и bn–n(o)=chr (10a и b) закључено је да производи 4a и b не могу бити главни реакциони интермедијери који се формирају из полазних једињења 1a и b. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice j. serb. chem. soc. 81 (7) 847–849 (2016) udc 546.3:542.8:541.135:544.6(02) jscs–4890 book review book review morphology of electrochemically and chemically deposited metals authors: konstantin i. popov, stojan s. djokić, nebojša d. nikolić and vladimir d. jović published by springer international publishing, 2016. hardcover isbn: 978-3-319-26071-6 and e-book isbn: 978-3-319-26073-0 jasmina s. stevanović* institute of chemistry, technology and metallurgy, university of belgrade, serbia (received 26 june, accepted 27 july 2016) the book titled morphology of electrochemically and chemically deposited metals by popov, djokić, nikolić and jović is published on 368 pages with 226 figures, 436 equations, 8 tables and 460 references. it consists of 9 chapters: 1. the cathodic polarization curves in eelectrodeposition of metals; 2. mechanisms of formation of some forms of electrodeposited pure metals; 3. current distribution in electrochemical cells; 4. electrodeposition at a periodically changing rate; 5. electrodeposition of metals with hydrogen evolution; 6. electrochemically produced metal powders; 7. electrodeposited alloys and multilayered structures; 8. electrodeposited alloy powders; 9. chemical deposition of metals and alloys from aqueous solutions. at first glance, it might be concluded that this book is exclusively written for the academic community, but this is simply not true. any successful technological development cannot be achieved without the strong fundamental background, and this book offers numerous examples of scientific investigations and possible technological applications. chapters 1–8 (written by popov, nikolić and jović) are a result of long-lasting investigations performed at world-famous belgrade electrochemical school, which already contributed immensely on the investigation of metal deposits morphology and the mechanism of formation. chapter 9 (written by djokić) was completed at university of alberta, although i have to reveal that the author of this chapter is also an “offspring” of belgrade electrochemical school. influence of various parameters and regimes of electrolysis, as well as various methods of chemical deposition have been systematically * e-mail: jaca@tmf.bg.ac.rs _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 848 stevanović researched over time period of almost fifty years, and as a result, we have today this book devoted to morphologies of electrochemically and chemically deposited metals and alloys. morphology of electrochemically and chemically deposited metals is, to some extent, a continuation of the book fundamental aspects of electrometallurgy by k. i. popov, s. s. djokić and b. n. grgur, kluwer academic/plenum publishers, new york, 2002), but it brings a significant novelty and hence differences in relation to it, as a consequence of intensive investigations performed in the last fifteen years. for example, the effect of hydrogen evolution on metal electrodeposition at constant and at periodically changing rate (chapter 5), novel approach to investigation of the pure metal powders production by electrolysis (chapters 6), electrodeposition of alloys and multilayered structures (chapter 7) and alloy powders formation by electrolysis (chapter 8) represent completely new and original contributions that make a difference between this book and the previously published. at the same time, some of the chapters given in this book (chapters 3, 4 and 9) are also supplemented and rearranged in relation to the book fundamental aspects of electrometallurgy from 2002. its particular concept is why this book differs from all the other books on the same topic. morphology of electrochemically and chemically deposited metals, being primarily devoted to morphologies of electrochemically and chemically deposited metals and alloys, represents impressive collection of microphotographs of electrodeposited crystals in all their variety. mathematical models, along with these microphotographs explain all known electrodeposited phenomena in detail. i would like to especially stress concept of chapters 1 and 2. in the chapter 1, all forms of polarization curve equations are considered and experimentally confirmed. correlation between the polarization characteristics and the type of electrodeposition control gives completely new insight into the mechanism of electrodeposition processes. on the other hand, mechanisms of formation of almost all existing morphological forms that could be obtained by electrodeposition of pure metals are given in chapter 2, electrodeposition of alloys and multilayered structures, as well as mechanism of the electrodeposition of alloy powders are very graphically presented in chapters 7 and 8. finally, chapter 9 gives impressive review of all the phenomena related to the processes of chemical deposition of metals and alloys. finally, i have to point out the fact that journal of the serbian chemical society is not selected by chance to present this book review. almost 45 papers published on the subject matter in the journal of the serbian chemical society in the last fifty years were used for writing this book. furthermore, chapter 3 is almost entirely written on the basis of the results previously published in the journal. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 849 book review и з в о д приказ књиге: morphology of electrochemically and chemically deposited metals (морфологија електрохемијски и хемијски таложених метала) аутори: константин и. попов, стојан с. ђокић, небојша д. николић и владимир д. јовић јасмина с. стевановић институт за хемију, технологију и металургију, универзитет у београду, београд дат је основни приказ књиге која има 9 поглавља са 368 страна, 226 слика, 436 једначина, 8 таблица и 460 литературних навода. књига је синтеза скоро педесетогодишњих истраживања у области електрохемијског таложења метала и обухвата, поред осталих, око 45 радова публикованих током наведеног периода у journal of the serbian chemical society. (примљено 26. јуна, прихваћено27. јула 2016) _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {prediction of osmotic coefficients for ionic liquids in various solvents with artificial neural network} j. serb. chem. soc. 82 (4) 399–409 (2017) udc 532.7:544.275–128+54–145.2:510.644 jscs–4975 original scientific paper 399 prediction of osmotic coefficients for ionic liquids in various solvents with artificial neural network yu cao1, shun yao2, xianlong wang3,4, tian yao2 and hang song2* 1college of life science & biotechnology, mianyang normal university, mianyang 621000, china, 2department of pharmaceutical and biological engineering, sichuan university, chengdu 610065, china, 3school of life science and technology, university of electronic science and technology of china, chengdu 610054, china and 4department of chemistry, bryn mawr college, 101 n merion ave, bryn mawr, pa 19010, usa (received 17 september, revised 23 december 2016, accepted 9 january 2017) abstract: the relationship between the structural descriptions and osmotic coefficients of binary mixtures containing sixteen different ionic liquids and seven kinds of solvents has been investigated by back propagation artificial neural network (bp ann). the influence of temperature on the osmotic coefficients was considered and the concentrations of ionic liquids were close to 1 mol kg-1, except in acetonitrile. multi linear regression (mlr) was used to choose the variables for the artificial neural network (ann) model. a three layer bp ann with seven variables containing structural descriptions of the ionic liquids and the character of the solvent as input variables was developed. compared with experimental data, the osmotic coefficients calculated using the ann model had a high squared correlation coefficient (r2) and a low root mean squared error (rmse). keywords: binary mixture; back propagation; artificial neural network; multi linear regression. introduction ionic liquids (ils), as a type of widely applied green solvents, have aroused popular interest of academic community. their excellent properties, such as chemical stability, thermal stability, low vapor pressure, high electronic conductivity, etc.1 determine the extensive application of ionic liquids in different fields.2 currently, ils have been successfully used in the extraction of bioactive components from natural products3 and extraction of biological substances, such as proteins, amino acids, dna, etc.4 the use of an extraction solvent demands an understanding of its physical chemistry. in these properties, the osmotic coefficient is thought to be very important for the selection of an extraction solvent.5 * corresponding author. e-mail: hangsong@scu.edu.cn doi: 10.2298/jsc160725013d 400 cao et al. moreover, ionic liquids are thought to be designable solvents because their synthesis is relatively simple. a model that could predict the osmotic coefficients of ionic liquids would aid in the design of new and excellent ionic liquids as extraction solvents. a prediction model that could guide the chemical synthesis of ionic liquids is required by chemists. recently, the osmotic coefficients of ionic liquids in some ordinary solvents, such as water, alcohols, benzene, acetonitrile, etc., have been studied by experimental and theoretical methods.6–8 however, compared with other physicochemical and related properties of ionic liquids (see fig. 19–19), theoretical models for estimating the osmotic coefficients of ionic liquids are relatively little researched at present. fig. 1. reported calculation methods in the study of some properties of ionic liquids. for aqueous solutions, the osmotic coefficients could be calculated theoretically by the pitzer equations or by the three-characteristic parameter correlation (tcpc) model.20 in addition, the earlier and more popular pitzer model was successfully used to correlate the osmotic coefficients of ionic liquid dissolved in water and acetonitrile.5,21 furthermore, karimzadeh et al.19 used monte carlo simulations to compute the osmotic coefficient of aqueous solutions of ionic liquids and reviewed computational models in the investigation of binary mixtures containing ionic liquids. within the methods concerning the investigation of ionic liquids, the quantitative structure–property relationship (qspr) model has been widely used in the study of the properties of ils and was proved to be an ideal method to fit and predict related data.22 the artificial neural network (ann) method that is good at predictions is a very important algorithm to develop a qspr model and was successfully adopted to study the electronic conductivity of ionic liquids in a previous work.18 in this context, a qspr model based on the ann method was set prediction of ils osmotic coefficients 401 up to correlate the structural characteristics and osmotic coefficients of seven solvents containing sixteen different ionic liquids. computational details dataset and descriptors 84 sets of osmotic coefficient data for 16 types of ionic liquids in 7 kinds of solvents (chemical names and acronyms for the ils are provided as table i) at different temperatures were collected from the ionic liquid database23 and other references.6,8,24,25 table i. acronyms used for the studied ils name acronym 1-methyl-3-methylimidazolium methyl sulfate [mmim] [meso4] 1-butyl-3-methylimidazolium methyl sulfate [bmim][meso4] 1-ethyl-3-methylimidazolium ethyl sulfate [emim] [etso4] 1-ethyl-3-methylpyridinium ethyl sulfate [empy][etso4] 1-butyl-3-methylimidazolium octyl sulfate [bmim][octso4] 1-ethyl-3-methylimidazolium chloride [emim][cl] 1-butyl-3-methylimidazolium chloride [bmim][cl] 1-hexyl-3-methylimidazolium chloride [hmim][cl] 1-ethyl-3-methylimidazolium bromide [emim][br] 1-propyl-3-methylimidazolium bromide [pmim][br] 1-n-butyl-3-methylimidazolium bromide [nbmim][br] 1-pentyl-3-methylimidazolium bromide [pnmim][br] 1-hexyl-3-methylimidazolium bromide [hmim][br] 1-butyl-3-methylimidazolium tetrafluoroborate [bmim][bf4] 1-octyl-3-methylimidazolium tetrafluoroborate [omim][bf4] 1-butyl-3-methylimidazolium hexafluorophosphate [bmim][pf6] the 3d structural coordinates of ionic liquids were obtained from the cambridge structural database (csd).26 to reflect the prediction capability of model, the data points of the same solvent containing the same ionic liquid were not divided into different datasets. the 84 sets of data were divided into three datasets, i.e., a training set, a validation set and a test set containing 46, 24 and 30 % of the total data, respectively (table ii). table ii. the solvents with ionic liquids and data sets; training set: entry 1–15, validation set: entry 16–24, test set: entry 25–36 entry solvent il concentration, mol kg-1 temperature, k osmotic coefficient 1 1-propanol [bmim][meso4] 1.0071 323.15 0.629 2 1-propanol [empy][etso4] 0.9272 323.15 0.605 3 2-propanol [mmim] [meso4] 0.9721 323.15 0.461 4 ethanol [bmim][cl] 1.096 313.15 0.742 1.096 333.15 0.63 5 water [bmim][meso4] 0.9552 298.15 0.698 0.9552 308.15 0.716 0.9552 318.15 0.732 0.9552 328.15 0.76 402 cao et al. table ii. continued entry solvent il concentration, mol kg-1 temperature, k osmotic coefficient 6 water [emim][cl] 1.0472 313.15 0.861 1.0472 333.15 0.812 7 water [bmim][bf4] 0.9322 298.15 0.523 7 water [bmim][bf4] 0.9322 308.15 0.545 0.9322 318.15 0.564 0.9322 328.15 0.589 8 water [empy][etso4] 1.0336 313.15 0.813 1.0336 333.15 0.784 9 water [pmim][br] 1.0292 298.15 0.702 1.0292 308.15 0.714 1.0292 318.15 0.717 1.0292 328.15 0.724 10 benzene [bmim][octso4] 1.1483 298.15 0.018 1.1483 303.15 0.016 1.1483 308.15 0.027 1.1483 313.15 0.039 11 methanol [bmim][octso4] 1.1872 298.15 0.373 1.1872 303.15 0.466 1.1872 308.15 0.579 1.1872 313.15 0.71 12 1-propanol [omim][bf4] 1.0008 298.15 0.045 1.0008 303.15 0.097 1.0008 308.15 0.165 1.0008 313.15 0.201 13 acetonitrile [bmim][pf6] 0.1015 318.15 0.3139 14 methanol [omim][bf4] 1.2766 298.15 0.327 1.2766 303.15 0.401 1.2766 308.15 0.481 1.2766 313.15 0.543 15 water [emim][br] 0.4422 298.15 0.85881 16 1-propanol [bmim][octso4] 1.0548 298.15 0.057 1.0548 303.15 0.202 1.0548 308.15 0.319 1.0548 313.15 0.417 17 2-propanol [emim] [etso4] 0.9702 323.15 0.575 18 ethanol [bmim][meso4] 0.9851 323.15 0.496 19 ethanol [empy][etso4] 0.982 323.15 0.498 20 water [nbmim][br] 0.9713 318.15 0.7125 21 water [mmim] [meso4] 0.9792 313.15 0.896 0.9792 333.15 0.87 22 benzene [omim][bf4] 1.4487 298.15 0.026 1.4487 303.15 0.036 1.4487 308.15 0.075 1.4487 313.15 0.173 prediction of ils osmotic coefficients 403 table ii. continued entry solvent il concentration, mol kg-1 temperature, k osmotic coefficient 23 water [hmim][cl] 1.0049 313.15 0.765 1.0049 333.15 0.732 24 water [hmim][br] 1.1189 298.15 0.384 1.1189 308.15 0.397 24 water [hmim][br] 1.1189 318.15 0.413 1.1189 328.15 0.429 25 1-propanol [emim] [etso4] 0.9772 323.15 0.596 26 2-propanol [bmim][meso4] 0.9823 323.15 0.523 27 2-propanol [empy][etso4] 0.9707 323.15 0.594 28 ethanol [emim] [etso4] 0.9888 323.15 0.504 29 ethanol [mmim] [meso4] 0.9868 323.15 0.449 30 ethanol [hmim][cl] 1.0138 313.15 0.677 1.0138 333.15 0.5679 31 water [pnmim][br] 1.0258 298.15 0.618 1.0258 308.15 0.637 1.0258 318.15 0.632 1.0258 328.15 0.649 32 water [emim] [etso4] 0.8919 298.15 0.706 0.8919 308.15 0.721 0.8919 318.15 0.73 0.8919 328.15 0.747 33 ethanol [omim][bf4] 1.0646 298.15 0.107 1.0646 303.15 0.197 1.0646 308.15 0.255 1.0646 313.15 0.33 34 1-propanol [mmim] [meso4] 0.9815 323.15 0.454 35 acetonitrile [bmim][bf4] 0.1043 318.15 0.3139 36 ethanol [bmim][octso4] 0.7636 298.15 0.036 0.7636 303.15 0.061 0.7636 308.15 0.106 0.7636 313.15 0.137 the cations and anions of the ionic liquids were optimized using the b3lyp method27,28 with the 6-31g(d) basis set, respectively. the optimization calculations were performed with the gaussian03 program29 and quantum mechanics descriptors, such as energy (e), volume (v), the highest occupied molecular orbital energy (ehomo), the lowest unoccupied molecular orbital energy (elumo) of the cations and anions, were considered. the molecular connectivity index, which is thought to be helpful in the investigation of the physical properties,30 was considered and chi index of zero order (0x), first order (1x) and second order (2x) of the ions were also calculated. all descriptors of the ions are listed in table iii. the solvents were described by their dielectric constant. the osmotic coefficients of ionic liquids are influenced by temperature and their concentration in solvents. in this context, the concentration of ionic liquids was kept close to 1 mol kg-1, except for acetonitrile, because the osmotic coefficient is much larger in this solvent than in the other solvents and related experimental data are lacking. to predict the osmotic coefficients of ionic liquids at different 404 cao et al. temperatures, temperature must be considered as a variable in the related models. to decrease the redundancy existing in the descriptor data matrix, the correlation among related descriptors and their correlation with the osmotic coefficient of the ils were examined and collinear descriptors (i.e., |r| > 0.9) were detected.31 among the collinear descriptors, those showing high correlation with the osmotic coefficient were retained and the others were removed from the data matrix. table iii. data on the calculated descriptors of cations and anions ion e / ha v cm3 mol-1 ehomo ev elumo ev 0x 1x 2x [mmim]+ –305.2307599 79.190 –0.43626 –0.18467 4.5875126942.176597520 1.663519480 [bmim]+ –423.1786802 131.637 –0.42798 –0.17697 6.1106756493.752718471 2.226796000 [emim]+ –344.5497093 91.159 –0.43158 –0.1805 5.2946194752.752718471 1.828497838 [empy]+ –366.6178078 106.616 –0.44336 –0.23357 5.9247560003.211203000 1.979076000 [hmim]+ –501.8072113 176.128 –0.40700 –0.17572 7.5248892004.752718000 2.933903000 [pmim]+ –383.8648154 98.590 –0.42903 –0.17839 5.4035690003.252718000 1.873243000 [nbmim]+ –423.1828425 122.342 –0.42344 –0.17091 7.0875130003.452991000 3.427070000 [pnmim]+ –462.4932894 101.983 –0.42303 –0.17662 6.8177824304.252718471 2.580349390 [omim]+ –580.4349008 166.481 –0.37963 –0.17545 8.9391028005.752718000 3.641010000 [meso4]–738.9948944 72.761 –0.06002 0.25643 3.0802070001.090822000 0.495772000 [etso4]–778.3139770 102.525 –0.06373 0.23851 3.7873135391.678356102 0.834257000 [octso4]–1014.197168 149.131 –0.06399 0.14990 8.0299542304.678356102 3.549402980 [cl]–460.2522333 22.518 0.01479 0.64337 0.00 0.00 0.00 [br]–2571.7613390 35.840 0.21400 0.50035 0.00 0.00 0.00 [bf4]–424.4990828 39.883 –0.11504 0.45846 2.2189646731.069044968 0.606092000 [pf6]–940.6433857 60.874 –0.15668 0.28100 2.7677868381.133893419 1.071429000 prediction models the retained descriptors, temperature and concentration were selected as input variables of the multilinear regression model (mlr) model. to decrease the number of input variables for an ann model, the descriptors usually need to be selected. the mlr model was thought to be a simple selection method that was successfully used in other studies.18,32 all the 84 sets of data were used to study the mlr relationship between the descriptors and osmotic coefficients by statistical products and service solutions (spss) software. the descriptors contributing the most were selected according to the squared correlation coefficient (r2) of mlr model. the descriptors selected by mlr model were collected as input variables for the back propagation (bp) ann model. a brief description of the ann has already been given:33 “a computational neural network consists of simple processing units called neurons. the strength of the neurons is determined by the weights (adjusted) that are first summed (combined) and then passed through a transfer function to produce the output for that neuron.” the bp algorithm means that the determination of the weight change is based on the error of the output unit. the fundamental theory and formulas of bp ann and transfer functions can be found elsewhere.33,34 the experimental data of the osmotic coefficients were the output variable. a three-layer bp ann model usually used to deal with data was set up. all parameters were first normalized to a scale of 0 to 1 before training, validation or testing to avoid numerical overflows during the ann processing. the initial weights of the training network and momentum factor were random. then different transfer functions, learning rates and num prediction of ils osmotic coefficients 405 ber of neurons were inputted to calculate the osmotic coefficient of the ils in the training sets by the bp ann method. the ann algorithms were implemented in matlab programming language. results and discussion descriptor selected by mlr method the collected descriptors were the independent variables and the osmotic coefficient was the dependent variable. the good correlations with the experimental osmotic coefficient were selected based on the squared correlation coefficient (r2). after discarding the descriptors without effective influence on the squared correlation coefficient, the model with the least number of descriptors and a high r2 was developed. the correlation obtained for 84 data points of the osmotic coefficient of the ils was presented by a seven-parameter equation as follows: 1 2 3 4 5 6 7 0.496 0.00459 0.00148 0.0440 0.00376 0.202 0.268 0.212 = + + − + + + − + y x x x x x x x (1) r2 = 0.774, f = 37.199, adjusted r2 = 0.753 standard error = 0.129 where y is the predicted osmotic coefficient of the ionic liquids and x1 to x7 represent the descriptors of ionic liquids and solvents. the mlr model was developed to predict the osmotic coefficient of the ils as follows: x1 is the dielectric constant of the solvents; x2 is the energy of the cations; x3 is the anion chi index of second order; x4 is the temperature; x5 is the concentration of the ils in solution; x6 and x7 are the energies of the homo and lumo of the anion, respectively. the root mean squared error (rmse), which is calculated as below, was used to measure the difference between the actual and the estimated values. ( )2exp calc 1 n i i i y y rmse n = − =  (2) where i represents the i-th sample, yiexp is an experimental value, yicalc is the corresponding value predicted by the model and n is the number of samples in the dataset.35 the rmse of the above mlr model was 1.0014, which showed that the calculated osmotic coefficient of the ionic liquids had large deviations from the experimental data. the mlr model to predict the osmotic coefficient of the ionic liquids did not have enough precision to guide in the design of new ionic liquids. however, the descriptors that enabled the mlr model to have high r2 values in eq. (1) may be major factors influencing the osmotic coefficient. 406 cao et al. to avoid chance correlations, the y-randomization test was applied to the mlr model. the calculation procedure was repeated twenty-five times after shuffling the y vector randomly. the y-randomization results were that the highest r2 within 25 time-calculations was 0.151, and the average r2 of 25 time -calculations was 0.077. the low r2 values suggest minor risk of chance correlations. bp ann model the descriptors selected by the mlr model were used as input variables for the bp ann model, and the osmotic coefficient was the output variable. a threelayer ann, which was usually used in qspr models because of the good prediction capability, was chosen in this study. after optimization for the model several times, the number of neurons in the hidden layers was 7 and the learning rate was 0.06. a tan-sigmoid transfer function within the hidden layer and a linear transfer function within the output layer were selected for the ann model. multiple calculations were performed in order to obtain the global best results. finally, the developed ann model that achieved the goal of the training set and had good performance for the validation set was selected as the prediction model for the osmotic coefficient of ils. the test set without contribution in model development steps was calculated to test the prediction capability of the ann model. the osmotic coefficients calculated by the ann model are displayed in fig. 2. fig. 2. the plots of the osmotic coefficient calculated by the ann model versus the experimental values. the dotted line presents y = x. prediction of ils osmotic coefficients 407 the r2 of the training set, the validation set and the test set were 0.9733, 0.9478 and 0.9102, respectively. the rmse of the training set, the validation set and the test set were 0.0428, 0.0658 and 0.0726, respectively. the results in fig. 2 suggest that the osmotic coefficient calculated by the ann model were similar to the experiment data. the training set was used to construct the ann model, the high r2 and small rmse suggested that the ann model was able to fit the osmotic coefficient of ionic liquids. the validation set was used to determine the parameters of the ann model. the data of test set did not appear in training and validation sets and did not participate in the construction of the ann model. thus, the test set could examine the prediction ability of the ann model. the small rmse of the test set suggested that the ann model could be used to predict the osmotic coefficient of ionic liquids. variable analysis the value of the variation inflation factor (vif), which is thought to reflect the statistic significance of a model,35 was calculated using the following equation: 2 1 1 = − vif r (3) where, r is the correlation coefficient of the multiple regression equation between one descriptor and the others. the vif value of the seven variables employed in the mlr and ann models were 2.025, 2.532, 3.257, 1.453, 1.748, 4.000 and 1.261, respectively. for each descriptor, its vif value was less than five, which indicated the model had obvious statistical significance. in the seven variables, except temperature and concentration, the other five variables were dielectric constant of solvents, the energy of cations, the anion chi index of second order, and the energy of the homo and lumo of the anion, respectively. there were three descriptors related with the anion: the chi index belonging to molecular topology information, which affects the combination of atoms in a molecule, and the energies of the homo and lumo, which is related to the reaction activity of molecule. there was only one descriptor of the cation, which affects the stability of ions in the model. conclusions in this study, an ann model was established based on mlr analysis to fit and predict the osmotic coefficient of seven solvents containing various ils at different temperatures. the ann model with r2 = 0.9733 and rmse = 0.0428 for the training set showed its good fitting capability. the predictive capability of the ann model was proven by r2 = 0.9102 and rmse = 0.0726. the selection of the descriptors by the mlr method were investigated and proved by good results. the analysis of the descriptors reflected the model had obvious statistical significance. 408 cao et al. acknowledgments. preparation of this paper was supported by the educational commission of sichuan province, china (no. 16zb0319) and the scientific research foundation of mianyang normal university (no. qd2014a008). и з в о д предвиђање осмотских коефицијената за јонске течности у различитим растварачима помоћу вештачких неуронских мрежа yu cao1, shun yao2, xianlong wang3,4, tian yao2 и hang song2 1college of life science & biotechnology, mianyang normal university, mianyang 621000, china, 2department of pharmaceutical and biological engineering, sichuan university, chengdu 610065, china, 3school of life science and technology, university of electronic science and technology of china, chengdu 610054, china и 4department of chemistry, bryn mawr college, 101 n merion ave, bryn mawr, pa 19010, usa релација узмеђу структурних описника и осмотских коефицијената бинарних смеша које садрже шеснаест различитих јонских течности и седам врста растварача истраживана је са унатраг напредујућим вештачким неуронским мрежама (bp ann). узет је у обзир утицај температуре на осмотске коефицијенте а концентрација јонских течности је била близу 1 mol kg-1 изузев у ацетонитрилу. мултилинеарна регресија (mlr) коришћена је за избор варијабли за модел вештачке неуронске мреже (ann). развијен је трослојни bp ann са седам варијабли које садрже структурне описнике јонских течности и карактера растварача као улазне варијабле. поређењем са експерименталним подацима, осмотски коефицијенти израчунати са ann моделом имају висок корелациони коефицијент (r2) и малу просечну квадратну грешку (rmse). 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {aminobenzene sulfonic acid-functionalized carbon nanotubes on a glassy carbon electrode for probing traces of mercury(ii)} j. serb. chem. soc. 82 (1) 73–82 (2017) udc 547.466’581.2’269.3+681.586.74: jscs–4948 628.193:546.492 original scientific paper 73 aminobenzene sulfonic acid-functionalized carbon nanotubes on glassy carbon electrodes for probing traces of mercury(ii) jing lv1,2, ying tang2, liumei teng2, dianyong tang2 and jin zhang2* 1college of chemistry and chemical engineering, southwest university, chongqing 400715, p. r. china and 2chongqing key laboratory of environmental materials & remediation technologies, chongqing university of arts and sciences, chongqing 402160, p. r. china (received 19 april, revised 13 august, accepted 3 october 2016) abstract: this work reports on a new electrochemical sensing strategy for quantitative monitoring of inorganic mercury(ii) in contaminated water by immobilization of poly(p-aminobenzene sulfonic acid)-functionalized singlewalled carbon nanotubes on a glassy carbon electrode. the electrochemical behavior of the resulting sensor was assessed and optimized. under optimal conditions, the sensor displayed good electrocatalytic responses to hg2+ reduction ranging from 1.0 μmol l-1 to 0.5 mmol l-1 with a detection limit of 0.5 μmol l-1. the selectivity against other potentially interfering ions and the applicability in contaminated water samples were also evaluated. keywords: electrochemical sensor; p-aminobenzene sulfonic acid; single-wall carbon nanotubes (swcnts); contaminated water. introduction mercury, a well-known global pollutant, is one of the most toxic elements.1 typically, its bioavailability, toxicity and bioaccumulation in living organisms are dependent on its chemical form.2 inorganic mercury(ii) (hg2+) is one of the most common and stable forms due to its solubility in water. soluble hg(ii) can accumulate in vital organs through the food chain and cause severe damage to the brain, nervous system, kidneys, heart and endocrine system.3 thus, sensitive monitoring of hg(ii) is very important in the analysis of food safety and environmental surveys. nowadays, different methods and strategies based on various signal-generation principles have been developed for hg(ii) quantification, such as atomic fluorescence spectrometry,4,5 atomic absorption spectrometry,6 inductively coupled plasma-mass spectrometry,3,7 inductively coupled plasma atomic emis * corresponding author. e-mail: zhangjin@cqwu.edu.cn doi: 10.2298/jsc160419090l 74 lv et al. sion spectrometry,8 high-performance liquid chromatography,9 ion chromatography,10 colorimetric detection11 and sensing strategy. despite the high sensitivity of conventional methods, they have some limitations, such as high operating costs, inadequate detection limits, or expensive instrumentation. to this end, an alternative strategy that combines the high-efficiency signal-transduction methods with simple and low-cost signal-generation tags would be adventageous.12 electrochemical sensors hold great potentials for this purpose because of their high sensitivity, simple and convenient operation, and low cost. the electron transfer between the solution and the underlying electrode not only provides the model for the mechanistic study of electron transport, but also enables the construction of highly sensitive reactors. therefore, for the development of a high-efficiency electrochemical sensor for the detection of trace hg(ii), the exploration of suitable materials for the fabrication of electrochemical sensors is of great interest. carbon nanotubes (cnts) can effectively accelerate some electron-transfer reactions between electrodes and electro-active species, increase the accessible surface area, and enhance electrocatalytic activity. due to their large specific surface area, excellent electrochemical durability and electrical conductivity, cnts have gained widespread attention.13 carbon nanotubes modified electrodes have been used to detect the biomolecules.14–16 the surface of singlewalled carbon nanotubes (swcnts) is comparatively pure due to their great chemical inertness. swcnts have good adsorption and electro-catalytic properties after carboxylation. many researchers have applied swcnt or functionalized swcnt to analyze organic compounds and selected metals.17–19 moreover, many carbon-based electrodes surface or bulk-modified with swcnts have been used for monitoring and analysis, such as carbon paste electrodes17 and carbon-fiber electrode.20 despite their large volume-to-surface ratio and high conductivity, carbon nanotubes alone cannot exhibit good redox characteristics during electrochemical measurements. inspiringly, recent research was aimed at the development of innovative and powerful functionalized nanomaterials with organic molecules, controlling and tailoring their properties in a very predictable manner to meet the needs of specific applications. the poly(p-aminobenzene sulfonic acid) [poly(p-absa)]-modified electrode has several advantages, such as well-filmed, long stability, little interference, and good electrical conductivity. the detection of organic acids by the poly(p-absa)-modified electrode was reported.21,22 to the best of our knowledge, however, there are no reports focusing on poly(p-absa)-functionalized single-walled carbon nanotubes for electrochemical detection of hg(ii). herein, a simple and feasible electrochemical sensing protocol for the sensitive detection of hg(ii) based on poly(p-absa)-functionalized swcnts is reported. the p-absa monomer was electrochemically polymerized onto a swcnts-modified glassy carbon electrode. the assay was realized based on electrochemical sensor for mercury detection 75 organic–inorganic hybrid nanostructures toward the catalytic reduction of hg(ii). the aim of the present work was to construct a high-efficiency and cost-effective electrochemical sensor for the quantitative monitoring of hg(ii) in environmental samples. experimental materials and apparatus carboxlylated single-walled carbon nanotubes (swcnt) were purchased from boyegaoke new mater. technol. co., ltd. (beijing, china). hg2+ standards of different concentrations were prepared by dissolving hgcl2 in distilled water. n,n-dimethylformamide (dmf) and anhydrous p-aminobenzene sulfonic acid (p-absa) were procured from chengdu cdkelong chem. re. co., ltd. (chengdu, china). different ph phosphate-buffered saline (pbs) solution containing 0.9 % kcl were prepared by mixing 0.1 mol l-1 kh2po4 and 0.1 mol l-1 na2hpo4. all other chemicals were of analytical grade and used as received. doubledistilled water was used throughout this study. all electrochemical measurements, including cyclic voltammetry (cv) and anodic stripping voltammetry (asv), were performed using a chi 660d electrochemical workstation (shanghai ch instruments co., ltd., china) with a conventional three-electrode system, including a modified glassy carbon disk electrode (gce, d = 4 mm) as the working electrode, a saturated calomel electrode (sce) as the reference electrode, and a platinum-wire counter electrode. the morphology of the modified electrode was obtained using a fei quatan 250 scanning electron microscope (fei company, usa). the comparative measurement was performed using a purkinje t9 uv–vis spectrophotometer (purkinje general instrument co., ltd., china) fabrication of the hg2+ electrochemical sensor prior to the experiment, 1.0 mg of swcnts was dispersed into 1.0 ml dmf by for 20 min to obtain a homogenous swcnt suspension. a glassy carbon electrode was carefully polished initially with 0.3 µm and 0.05 µm aluminum oxide, then rinsed in acetone, absolute ethanol and water with sonication for 5 min to obtain a mirror-like surface, and then dried at room temperature. then 4 µl of the above-prepared swcnt suspension was cast onto the cleaned gce using a microsyringe and dried under an infrared lamp (the resulting electrode is denoted as swcnt/gce). after gentle washing with distilled water, p-absa was electrochemically polymerized onto the swcnt/gce in ph 7.0 pbs containing 2.0 mmol l-1 p-absa. polymer growth was achieved within an applied potential ranging from between −1.5 and 1.5 v at 100 mv s-1, and continuously scanning for 10 cycles. after washing with distilled water, the sensor (the resulting electrode is denoted poly(p-absa)–swcnt/gce) was used for the detection of hg2+. for comparison, a poly(p-absa)-modified gce (the resulting electrode is denoted poly(p-absa)–gce) was prepared using the same electropolymerization procedure. electrochemical measurement for target hg2+ before the determination, the poly(p-absa)–swcnt/gce was subjected to scanning cyclic voltammetry until a steady-state cyclic voltammogram was obtained in ph 4.0 pbs. afterwards, hg2+ standards with different concentrations were added into ph 4.0 pbs. meanwhile, the electrochemical signal was monitored and recorded based on anodic stripping voltammetry by accumulating hg2+ at –1.0 v for 5 min. the peak current (ip) was evaluated relative to the baseline. 76 lv et al. results and discussion electropolymerization of p-absa on the swcnt/gce the cyclic voltammograms of the polymerization on the swcnt-modified gce are shown in fig. 1a. the swcnts possessed many carboxyl groups, and p-absa could easily combine to swcnts by hydrogen bonds.23 as seen from the voltammograms, two pairs of redox peaks (peaks a and c and peaks b and d) were observed. during the first scanning run, the p-absa initially lost an electron, and then a one-electron oxidation of the amino group turns it into its corresponding cation radical, thus resulting in the formation of the anodic peak at 0.25 v (peak a). in addition, the corresponding cathodic peak was observed at –0.1 v (peak c) on the reverse scan, indicating that the cation radical lost a hydrogen ion and generated the p-absa radical. thereafter, the radical dimerizes to form 4,4′-disulfohydrazobenzene. with continuous scanning, a cathodic peak at –0.4 v (peak d) was obtained. the reason was attributed to the fact that 4,4′-disulfoazobenzene lost two hydrogen ions and continued to loose two electrons to produce 4,4′-disulfoazobenzene dimers,24 thus causing another anodic peak at 0.9 v (peak b). moreover, according to fig. 1a, the peak currents increased with successive potential scans, indicating that deposition of p-absa on the swcnt/ /gce occurred. furthermore, the sem technique was used to investigate the morphologies of modified electrodes (fig. 1b). indeed, the swcnts layers formed on gce were randomly distributed along the electrode and a thin film of poly(p-absa) could be seen on the swcnts layers. fig. 1. a) cyclic voltammograms of p-absa on swcnt/gce during the electropolymerization in ph 7.0 pbs containing 2.0 mmol l-1 p-absa at 100 mv s-1 (insets: the corresponding voltammometric curves), b) sem image of poly(p-absa)–swcnt/gce (inset: sem image of clean bare gce). (b) electrochemical sensor for mercury detection 77 electrochemical behavior of mercury(ii) on differently modified electrodes to investigate the amplification efficiency of the as-prepared poly(p -absa)–swcnt/gce, a comparative study using different working electrodes (gce, poly(p-absa)–gce, swcnt/gce and poly(p-absa)–swcnt/gce) was performed for the detection of 10 μmol l–1 hg2+ in ph 4.0 pbs by asv. curve a in fig. 2 is the baseline. as seen from curve b in fig. 2, no obvious peak current could be observed at the bare gce. in contrast, both poly(p-absa)– –gce (curve c) and the swcnt-modified gce (curve d) could increase the voltammometric response toward hg2+. however, the peak potentials at two modified electrodes were different. this could be attributed to the fact that because of its two groups (amino and sulfo groups) in a counterpoint, strong steric and electron effects prevented the p-absa from completely forming long end to end chains, which limited the long-distance transfer of electrons. however, the unique structure of swcnts determined the properties, i.e., the high surface areas, strong electrical conductivity and adsorption could improve the electrochemical response of the mercury ions. more significantly, when poly(p -absa) and swcnt were simultaneously modified onto the gce, the asv peak current greatly increased (curve e). relative to poly(p-absa)–gce and swcnt/gce, the use of poly(p-absa)–swcnt/gce could increase the peak current by about 250 and 200 % , respectively. the sidewalls of swcnts, having many delocalized π electron systems, can form π–π stack with p-absa dimers by π–π interaction. meanwhile, the carboxyl groups of swcnts and the amino groups of p-absa can form a conjugated system, which could accelerate electron transfer. fig. 2. asv curves at (a) baseline, (b) gce, (c) poly(p-absa)–gce, (d) swcnt/gce and (e) poly(p-absa)–swcnt/gce in ph 4.0 pbs containing 10 μmol l-1 hg2+. optimization of experimental conditions the following conditions (ph, stripping potential, stripping time, amount of swcnt, and number of polymerization cycles of p-absa) were optimized to 78 lv et al. obtain an optimal analytical performance. the effect of the ph of the buffer solution on the electrochemical signal of the hg2+ sensor using 10 μmol l–1 hg2+ as an example is shown in fig. 3a. the results indicated that the current initially increased with the increasing ph from 3.0 to 4.0, and then decreased. the optimal current was obtained at ph 4.0. therefore, pbs of ph 4.0 was selected as the optimum supporting electrolyte for the detection of hg2+. fig. 3. effects of: a) ph of pbs, b) stripping potential, c) stripping time, d) amount of swcnt and e) number of polymerization cycle of p-absa on the electrochemical responses of the hg2+ sensor (10 μmol l-1 hg2+ was used in all cases). the dependence of the stripping peak current on the stripping potential was examined over the range from 0 to –1.4 v. as shown in fig. 3b, the current electrochemical sensor for mercury detection 79 increased rapidly when the potential became more negative. however, the optimal current was obtained at the potential of –1.0 v. thus, a stripping potential of –1.0 v was selected in this work. at this stripping potential, the effect of the stripping time on the peak current was investigated (fig. 3c). a maximum peak current was achieved at the deposition time of 5 min; therefore, this time was selected for the preparation of the sensor. the dosage of swcnt for the preparation of hg2+ sensor could prominently influence the stripping current. as shown in fig. 3d, the peak currents increased with increasing swcnt dosage. however, when the dosage of the swcnts suspension was greater than 4 μl, the peak potential shifted. the small surface area of the electrode gave rise to the instability of operation and was counterproductive. thus, 4 μl dosage of the swcnts suspension was used in the preparation of modified electrode. in addition, we have also monitored the effect of polymerization cycle number of p-absa on the electrochemical response of the as-prepared sensor. as seen from fig. 3e, the maximum current was achieved after 10 cycles. interference to investigate the selectivity of the as-prepared poly(p-absa)–swcnt/ /gce, the system was challenged against other metal ions, i.e.., na+, k+, mg2+, ca2+, al3+, cd2+, ni2+, mn2+and pb2+. possible interference with the stripping peak currents of 10 μmol l–1 hg2+ was investigated by the addition of the possible interfering ions (100 μmol l–1) to a solution containing 10 μmol l–1 hg2+ under the optimized conditions. according to the results (fig. 4), no interference was caused by the investigated metal ions (signal change below 5 %); hence, the proposed method offers a very high selectivity. fig. 4. effects of possible interfering ions (100 μmol l-1) to a solution containing 10 μmol l-1 hg2+. 80 lv et al. dose response of poly(p-absa)–swcnt/gce toward the target hg2+ ions under the optimal conditions, different concentrations of hg2+ were determined by the poly(p-absa)–swcnt/gce with asv measurement in ph 4.0 pbs. the peak current linearly increased with increasing hg2+ concentration in the range of 1.0 μmol l–1 to 0.5 mmol l–1 (fig. 5). the detection limit was found to be 0.5 μmol l–1. the correlation coefficient was 0.9939, and the linear regression equation was: ip / μa = –0.0534chg2+ / μmol l–1 – 25.220 (1) the reason for the relatively large intercept in the regression equation mainly arose from the electro-active poly(p-absa)–swcnt immobilized on the gce. fig 5. a) asv responses of the poly(p-absa)–swcnt/gce toward different concentration hg2+ in ph 4.0 pbs (from top to bottom): 1.0, 5.0 and 10.0 μmol l-1; 0.1, 0.15, 0.2, 0.3, 0.4 and 0.5 mmol l-1, and b) the corresponding calibration curve. reproducibility and stability the reproducibility and stability of the poly(p-absa)–swcnt/gce were evaluated under the optimal conditions. the obtained results indicated that the relative standard deviation (rsd) of using the same sensor for the determination of 10 μmol l–1 hg2+ was 2.3 % (n = 8), while that of using the different sensors was 3.9 % (n = 5). the stability of the sensor was also studied after storage of several days. the results revealed that the asv peak currents decreased by 0.9, 1.6, 5.8 and 7.1 % after 4, 7, 10 and 20 days, respectively. thus, the reproducibility and stability of the sensor were acceptable. analysis of real samples with great reproducibility and stability in buffer, the mercuric sensor was further tested for the determination of hg2+ in spiked water. the obtained results were compared with those of colorimetry using dithizone and a uv–vis spectrophotometer. the method was performed according to the national standards of electrochemical sensor for mercury detection 81 the people's republic of china (gb5750-85), which is of high accuracy. the comparative results are presented in table i. each sample was determined 3 times. the detection results of the two methods were basically the same. the rsds were 1.1–5.8 %, verifying the practical applicability of the method. table i. comparison of the obtained results for the determination of hg2+ in spiked water between the poly(p-absa)–swcnt/gce sensor and the uv-vis-based method sample no. added hg2+, μmol l-1 chg2+ / μmol l-1 detected by the sensor chg2+ / μmol l-1 detected by uv rsd / % 1 17.50 17.65±0.10 17.71±0.23 2.8 2 17.00 17.13±0.07 17.06±0.16 5.5 3 17.50 17.58±0.19 17.61±0.14 1.1 conclusions the electrochemical behavior of hg(ii) on the surface of the chemically modified electrode was studied by asv. the new electrochemical sensor revealed excellent electrocatalytic activity through the prominent enhancement of the signal current of hg(ii). the results showed that the presence of the swcnts and poly(p-absa) on the glassy carbon electrode could significantly improve the electrical conductivity. in addition, the electrochemical sensor was suited to sensitive voltammometric detection of hg(ii) in the presence of other metal ions. furthermore, the prepared sensor has good stability and reproducibility. the modified electrode was used in real samples. hence, a new electrochemical method was developed for the sensitive determination of hg(ii). the designed method was precise, rapid and simple to perform. it is of good utility for further sensor development. acknowledgements. this work was supported by the chongqing science & technology commission, china (grant no. cstc2015jcyjbx0126) and key laboratory of analysis & detection for food safety (fuzhou university), ministry of education (grant no.fs-1401). и з в о д угљеничне наноцеви на електроди од стакластог угљеника функционализоване аминобензен-сулфонском киселином за одређивање трагова hg(ii) јона jing lv1,2, ying tang2, liumei teng2, dianyong tang2 и jin zhang2 1 college of chemistry and chemical engineering, southwest university, chongqing 400715, p. r. china и 2 chongqing key laboratory of environmental materials & remediation technologies, chongqing university of arts and sciences, chongqing 402160, p. r. china у овом раду је презентована нова стратегија, базирана на сензорима, за електрохемијску квантитативну анализу hg(ii) јона у загађеним водама имобилизацијом једнослојних угљеничних наноцеви функционализованих аминобензен-сулфонском киселином на електроди од стакластог угљеника. испитано је и оптимизовано електрохемијско понашање добијеног сензора. под оптималним условима сенсор даје добар електрокаталитички одговор на редукцију hg(ii) јона у опсегу 1,0 μmol l-1 до 0,5 mmol l-1 са 82 lv et al. детекционим лимитом од 0,5 μmol l-1. евалуирана је такође и селекивност у односу на потенцијално ометајуће јоне, као и применљивост у анализи загађених узорака вода. (примљено 19. априла, ревидирано 13. августа, прихваћено 3. октобра 2016) references 1. h. seiler, a. sigel, h. sigel, handbook on metals in clinical and analytical chemistry, crc press, boca raton, fl, 1994 2. p. krystek, r. ritsema, appl. organomet. chem. 18 (2004) 640 3. z. gu, m. zhao, y. sheng, l. a. bentolila, y. tang, anal. chem. 83 (2011) 2324 4. k. leopold, l. harwardt, m. schuster, g. schlemmer, talanta 76 (2008) 382 5. j. sneddon, advances in atomic spectroscopy, elsevier science, amsterdam, 2002 6. d. p. torres, v. l. frescura, a. j. curtius, microchem. j. 93 (2009) 206 7. m. wang, w. feng, j. shi, f. zhang, b. wang, m. zhu, b. li, y. zhao , z. chai, talanta 71 (2007) 2034 8. y. wu, y. i. lee, l. wu, x. hou, microchem. j. 103 (2012) 105 9. h. kodamatani, a. matsuyama, k. saito, y. kono, r. kanzaki, t. tomiyasu, anal. sci. 28 (2012) 959 10. q. liu, microchem. j. 95 (2010) 255 11. x. xue, f. wang, x. liu, j. am. chem. soc. 130 (2008) 3244 12. k. s. guha, r. j. mascarenhas, t. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {solubility and degradation of paracetamol in subcritical water} j. serb. chem. soc. 82 (1) 99–106 (2017) udc 615.2paracetamol+546.212:542.92: jscs–4950 544.351.3 original scientific paper 99 solubility and degradation of paracetamol in subcritical water zuhal emi̇re, erdal yabalak*, özkan görmez and ahmet murat gi̇zi̇r mersin university, faculty of arts and science, department of chemistry, çiftlikköy campus, tr-33343, mersin, turkey (received 20 may, revised 29 august, accepted 16 september 2016) abstract: in this study, solubility and degradation of paracetamol were examined using subcritical water. the effect of temperature and static time was investigated during the solubility process in subcritical water at constant pressure (50 bar). the experimental results showed that temperature and static time have crucial effects on the degradation and solubility degrees. the maximal solubility of paracetamol was obtained at 403 k as (14.68±0.74)×103. an approximation model for the solubility of paracetamol was proposed. o2 and h2o2 were used for the degradation of paracetamol. the maximum degradation degree was found as 68.66±1.05 % and 100±0.00 % using o2 and h2o2, respectively. keywords: subcritical water medium; approximation model; paracetamol; degradation; solubility. introduction in recent times, pharmaceuticals have been detected in surface water, ground water, drinking water and sewage effluents due to their intensive usage.1,2 the presence of pharmaceuticals in drinking water and aquatic environments endangers human health and causes various health problems.3 thus, the treatment of water contaminated with pharmaceuticals, such as anticonvulsants, antipyretics, antidepressants, cytostatic drugs, chemotherapy agents and antibiotics, has become a serious problem.4–6 paracetamol (acetaminophen, n-(4-hydroxyphenyl)acetamide) is a major analgesic and antipyretic agent and is widely used as an intermediate agent in the azo dyes industries and photographic chemicals worldwide.7,8 although paracetamol, used as fever reduction drug and for pain relief, is relatively safe at lower doses, it has various hazards, such as gastrointestinal disease, liver failure, hepatotoxic potential, and centrilobular necrosis in the liver.9,10 * corresponding author. e-mail: yabalakerdal@gmail.com doi: 10.2298/jsc160520079e 100 emi̇re et al. paracetamol has been found in sewage treatment plant with concentration up to 6 and 10 ppb in natural waters.7,11,12 thus, efficient degradation techniques are required to avoid potential risks of contaminated waters polluted by pharmaceuticals, particularly by paracetamol. conventional methods, such as chemical coagulation, biological remediation, adsorption and advanced oxidation processes, have been applied for the treatment of wastewaters containing various contaminants.13–15 moreover, subcritical water oxidation, with or without oxidants, is an effectual method for the degradation of hazardous compounds that exist in aquatic environments.16,17 using subcritical water with oxidizing agents, such as oxygen, permanganate, hydrogen peroxide, etc., various potential pollutants that are difficult to oxidize with conventional methods can be efficiently oxidized.16–18 hydrogen peroxide is a relatively innocuous oxidation agent that decomposes to o2 and h2o at room temperature. it produces reactive hydroxyl radicals (ho•) which are able to efficiently degrade most organic pollutants present in wastewaters.19,20 although oxygen is not as effective as hydrogen peroxide, it has been employed in many studies concerned with wastewater treatments due to its environmentally friendly nature.21 thus, hydrogen peroxide, and oxygen were used as oxidizing agents in the present study. in addition, the solubility of pharmaceuticals is essential for their removal from contaminated sites. dissolving hazardous organic compounds in subcritical water appears to be an effective method for remediating contaminated water.22 many literature studies focused on using several methods for determining the solubility of paracetamol in various solvents.7 these methods were often toxic and required an additional solvent removal step. herein, subcritical water was used as a unique alternative and environmentally friendly method that offers many advantages.23 in the literature, there are several investigations about the solubilities of pharmaceuticals in single and binary solvent mixtures, which is important for pharmaceutical industry. approximation model equations are useful to predict the solubilities of the pharmaceuticals over a wide range of temperatures. these equations are obtained using experimental data for the pharmaceuticals. various physicochemical parameters affect the approximation models, such as pressure, ph, salt and co-solvent concentration, dissolution enthalpy, entropy, etc. moreover, the decomposition temperature of pharmaceuticals obtained through these models seems to be important.14,24,25 thus, the mentioned models are recommended as useful for determining appropriate operating parameters by performing a limited number of experiments in decontamination treatments. paracetamol was degraded using o2 and h2o2 in subcritical water. the solubility characteristics of paracetamol in subcritical water were investigated. in addition, an approximation model for the solubility of paracetamol was obtained using reported models for organic molecules.23 solubility and degradation of paracetamol 101 experimental reagents and apparatus paracetamol was supplied by sigma–aldrich, glacial acetic acid and h2o2 (35 vol. %, ρ = 1.13 g cm-3) were from merck, and methanol from j. t. baker. deionized water (18 mω cm at 25 °c) was obtained from a millipore milli-q advantage a10. the hplc column ace 5 c18 (250 mm×4.6 mm id) was supplied by mac-mod (usa). an empty hplc column (150 mm×4.6 mm i.d.) was used as an extraction cell. a teledyne isco 260 d series syringe pump system (usa) was used for delivering water and providing pressure. o2 and n2 gases were supplied from linde gas (turkey). an agilent 1200 model hplc apparatus was used for the hplc analyses. solubility experiments procedures a syringe pump system and a self-made oven system were used for the solubility experiments, as described in a previous work.26 an empty cylindrical hplc column was filled with 0.25 g of paracetamol. both ends of the column were covered with 0.45 µm mesh size frits and the cell was tightened to prevent leakage. the filled column was placed in a teknosem tf r400 model oven. the syringe pump system was used to deliver water in the pressure mode fixed to 50 bar. fractions (3 ml) were collected at 5, 10, 15 and 30 min after 60 min equilibration at each temperature. solubility experiments were performed in triplicate to ensure accuracy of the experimental solubility data and were performed at six different temperatures ranging from 293 to 433 k. degradation experiments procedures a self-made system, a so-called reactor, was used for the degradation experiments, as shown in a previous work.27 the reactor was filled with 120 ml of 5 ppm aqueous paracetamol solution. degradation experiments were performed at four different temperatures ranging from 373 to 433 k. the samples were collected at 5, 15, 30 and 60 min. the pressure was maintained at 30 bar with nitrogen to keep the water in liquid form, and 0.035 ml of h2o2 were used as oxidizing agent in each degradation experiment. in the other batch of degradation experiments, the pressure was maintained at 30 bar with oxygen, which also acted as an oxidizing agent in this case. hplc analysis paracetamol was analyzed using a mobile phase consisting of a mixture of 28 vol. % of methanol and 3 vol. % of glacial acetic acid in water at a flow rate of 1.5 ml min-1 at ambient temperature. a uv-dad detector was used at a wavelength of 275 nm. paracetamol standards were prepared in deionized water. results and discussion effect of temperature on solubility experimental mole fraction solubilities (x2) of paracetamol in subcritical water obtained at different temperatures are summarized in table i. it is clearly seen that the temperature has a significant effect on the solubility as stated in previous studies.15,28 however, the mole fraction solubility of paracetamol decreased dramatically to x2 < 10 at 413 k due to possible degradation of paracetamol at higher temperature. 102 emi̇re et al. table i. comparison of experimental mole fraction solubility (x2×10 3) of paracetamol in subcritical water obtained in a static time (t) of 30 min according to eqs. (1)–(6) defined later t / k exp.a x2±sd (30 min) eq. (1) eq. (2) eq. (3) eq. (4) eq. (5) eq. (6) 293 1.52±0.01 1.52 1.52 1.52 1.52 1.52 1.52 298 1.77±0.00 1.69 2.29 0.25 4.91 1.69 1.74 303 2.06±0.00 1.88 2.55 0.31 5.97 1.88 1.98 373 10.06±1.37 6.10 8.27 2.83 52.75 6.55 9.44 383 10.92±1.13 6.97 10.76 3.58 67.49 7.71 11.39 393 11.37±0.85 7.91 14.35 4.47 85.26 9.09 13.65 403 14.68±0.74 8.91 19.72 5.48 106.47 10.73 16.26 athe experiments were performed in triplicate effect of static time on solubility the highest mole fraction solubility of paracetamol was obtained at 403 k as 12.78±0.51, 14.36±0.59, 14.44±0.46 and 14.68±0.74, in static times of 5, 10, 15 and 30 min, respectively. the static time is an effectual parameter for the solubility of organic molecules, as stated in previous papers.23 it was found that a static time above 10 min had a minor effect on the rates so that an adequate time was provided to the system to attain equilibrium. approximation models miller et al. proposed a new equation (eq. (1)) for the solubility of polycyclic aromatic hydrocarbons in subcritical water.13 the mole fraction solubility at elevated temperatures can be predicted using the mole fraction solubility obtained at ambient temperature (t0) as shown in eq. (1):13 ( ) ( )02 2 0ln ln t x t x t t  ≈     (1) where x2(t) demonstrates the mole fraction solubility at any temperature (t), x2(t0) demonstrates the ambient mole fraction solubility and t0 demonstrates ambient temperature. the authors developed the equation by adding a cubic term to the base equation, eq. (1), as is shown in eq. (2): ( ) ( ) 3 0 0 2 2 0ln ln 15 1 t t x t x t t t    = −     +    (2) mathis et al. developed eq. (3), which was employed for determining the solubility of liquid apolar organic compounds in subcritical water, as follows:14 3 0 0 2 2 0 0 ln ( ) ln ( ) 2 1 t t t x t x t t t  − = + −       (3) solubility and degradation of paracetamol 103 kayan et al. developed an approximation model, eq. (4), for the solubility of benzoic and salicylic acids, which yabalak et al. successfully applied for the solubility of sebacic acid:15,23 ( ) 02 2 0ln 1.85 1 )ln ( t x t x t t  = −    (4) kapalavavi et al. developed a new model, eq. (5), for the solubility of paraben in subcritical water:28 ( ) 0 02 2 0 0.5(ln ln 11 ) t t x t cx t t t  + − −  =   (5) as is shown in table i, none of the five equations could correctly predict the mole fraction solubility of paracetamol. thus, a new modified approximation model (eq. 6) was obtained, which correctly predicts the mole fraction solubility of paracetamol: ( ) 02 2 0 0 0 8 / 5ln ln tt x t x t t t t =  − +     (6) the developed model ensures a comprehensive prediction for the solubility of paracetamol at most of the temperatures compared with experimental ones, as illustrated in table i. degradation of paracetamol with o2 the degradation process was performed in subcritical water medium under o2 atmosphere. using o2 in subcritical water medium offers a powerful and eco -friendly method that is widely used in the water recycling process and other environmental treatments.29,30 dissolving oxygen in subcritical water initiates the formation of hydroxyl and other reactive radicals that participate in the reaction process, thereby increasing the degradation degree.31 degradation experiments were realized at four selected temperatures, specifically at 373, 393, 413 and 433 k, as demonstrated in table ii. the degradation degree of paracetamol during 60 min were determined as 52.20±1.42 % without using oxidant at 433 k, and 68.66±1.05 % using the same conditions but under 30 bar of o2 pressure. table ii. effect of temperature on the degradation degrees of paracetamol using o2 in 60 min t / k 433 (without o2) 373 393 413 433 degradation, % 52.20±1.42 10.13±1.98 42.62±1.88 64.11±1.71 68.66±1.05 104 emi̇re et al. the effect of static time on degradation degree was investigated at four selected times (5, 10, 30 and 60 min), as shown in table iii. increasing the static time from 5 to 60 min increased the degradation degree of paracetamol from 64.45±1.11 % to 68.66±1.05 % at 433 k. high static time might have enhanced the interaction of o2 and other radicals formed by o2 with paracetamol, thereby increasing the degradation degree. table iii. effect of static time on the degradation degrees of paracetamol using o2 at 433 k t / min 60 (without o2) 5 10 30 60 remaining amount, ppm 2.18±0.08 1.77±0.06 1.68±0.09 1.62±0.07 1.57±0.05 degradation degree, % 52.20±1.42 64.45±1.11 66.31±1.81 67.61±1.31 68.66±1.05 degradation of paracetamol with hydrogen peroxide hydrogen peroxide is a unique alternative oxidant to air or pure oxygen in degradation of organic compounds to carbon dioxide and water.32,33 while h2o2 is reduced to h2o and o2 at room conditions, both of h2o and o2 do not have any effect on degradation degrees.34,35 it decomposes to form highly reactive hydroxyl radicals in subcritical water medium.36,37 once hydroxyl radicals are produced, they initiate chain reactions in which reactive radicals are formed.38 generally, temperature and static time play an important role in the degradation process.16 high temperature enhances the decomposition rate of h2o2 to the aforementioned reactive radicals, thereby increasing the degradation rate of the target molecules. adequate static time is essential for the effective interaction of oxidative species and the target molecules.16 degradation experiments were performed at four selected temperatures, i.e., 373, 393, 413 and 433 k, as shown in table iv. the degradation degree of paracetamol in 60 min was determined as 52.20±1.42 % without using an oxidant at 433 k and 100±0.00 % under the same conditions using 0.035 ml h2o2, under nitrogen pressure fixed at 30 bar, as shown in table iv. table iv. effect of temperature on the degradation degrees of paracetamol using h2o2 t / k 373 393 413 433 degradation degree, % 98.28±1.94 100±0.00 100±0.00 100±0.00 in addition, increasing static time from 5 to 60 min increased the degradation degree of paracetamol from 93.81±0.14 % to 98.28±1.94 % at 373 k, as shown in table v. table v. effect of static time on the degradation degree of paracetamol using h2o2 at 373 k t / min 5 10 30 60 remaining amount, ppm 0.31±0.01 0.30±0.01 0.21±0.02 0.09±0.10 degradation degree, % 93.81±0.14 94.07±0.11 95.73±0.30 98.28±1.94 solubility and degradation of paracetamol 105 conclusions this study demonstrated that subcritical water has a remarkable effect on the degradation and solubility of paracetamol. it was found that the static time is an effective parameter along with temperature in both the degradation and solubility processes. the experimental solubility results show a good consistency with the developed approximation model (eq. (6)). the mole fraction solubility of paracetamol was determined as (14.68±0.74)×103 at 30 min static time and 403 k. furthermore, o2 has a significant impact on the degradation degrees of paracetamol, enhancing the degrees by up to 18 %. while 100 % degradation was obtained using h2o2, the attained degradation of 68 % using o2 should not be underestimated due to its environmentally friendly nature. acknowledgements. this work was funded by the mersin university research fund (project no. bap-fbe kb (ze) 2014-1 yl). the authors thank sengul aksu and umut ufuk demi̇rhan (meu. linguistics department) for regulation of the english grammar. и з в о д растворљивост и разградња парацетамола у подкрититичној води zuhal emi̇re, erdal yabalak, , özkan görmez, a. murat gi̇zi̇r mersin university, faculty of arts and science, department of chemistry, çiftlikköy campus, tr-33343, mersin, turkey испитани су растворљивост и разградња парацетамола коришћењем подкритичне воде. проучен је утицај температуре и времена стајања на процес растварања при константном притиску (50 bar). експериментални резултати показују да температура и време стајања имају пресудан утицај на разградњу и растворљивост. максимална растворљивост од (14.68±0.74)×103 за парацетамол постигнута је на 403 k. предложен је апроксимативан теоријски модел за растворљивост парацетамола. при разградњи парацетамола коришћени су кисеоник и h2o2. максимални степен разградње од 68,66±1,05 и 100±0,00 % постигнут је разградњом помоћу o2, односно h2o2. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {microwave assisted synthesis of substituted 4-chloro-8-methyl-2-(1,3-diphenyl-1h-pyrazol-4-yl)-1,5-dioxa-2h-phenanthren-6-ones and their antimicrobial activity} j. serb. chem. soc. 82 (2) 117–125 (2017) udc 547.814.1+547.587.51+547.772.2: jscs–4952 537.5–962:615.281–188 original scientific paper 117 microwave assisted synthesis of substituted 4-chloro-8-methyl-2 -(1,3-diphenyl-1h-pyrazol-4-yl)-1,5-dioxa-2h-phenanthren-6 -ones and their antimicrobial activity dongamanti ashok1*, bachi reddy vanaja1, mdderla sarasija2 and b. vijaya lakshmi1 1green and medicinal chemistry laboratory, department of chemistry, osmania university, 500007 hyderabad, india and 2department of chemistry, satavahana university, karimnagar, 505001 telangana, india (received 9 february, revised 5 december, accepted 14 december 2016) abstract: due to the potential antimicrobial activity of pyranochromenones and pyrazolines moieties, hybrid compounds containing both substituted 4-chloro -8-methyl-2-(1,3-diphenyl-1h-pyrazol-4-yl)-1,5-dioxa-2h-phenanthren-6-ones (4a–g), were synthesized from substituted (e)-1-(7-hydroxy-4-methyl-8 -coumarinyl)-3-(1,3-diphenyl-1h-pyrazol-4-yl)-2-propen-1-ones (3a–g) in good yield using the vilsmeier reaction, by the microwave-assisted method. the structures of all the compounds were established based on their analytical and spectral data. all the synthesized compounds were tested in vitro for their antibacterial and antifungal activities. some of the compounds showed very good activity compared to standard drugs against all the tested pathogenic bacteria and fungi. keywords: antimicrobial activity; chromene; coumarin; microwave irradiation; pyrazolines; vilsmeier reagent. introduction the coumarin and chromene core moieties are important six-membered oxygen heterocyclic motifs embedded in several natural products and drugs. these systems are widely distributed in nature, and their derivatives have been shown to exhibit significant pharmacological activities.1 chromenes and fused chromenes are biologically important compounds due to their antibacterial,2 antifungal,3 antitumor4 and antiviral5 activities. coumarin derivatives were reported to exhibit anti-inflammatory,6,7 antimicrobial,8 antioxidant,9 anticancer10 and chemoprophylactic11 activities. hybrid compounds containing both coumarin and chromene moieties, called pyranochromenone, due to the combined effect, * corresponding author. e-mail: ashokdou@gmail.com doi: 10.2298/jsc160209001a 118 ashok et al. may exhibit better biological activity. the compounds embedded with pyranochromenone (fig. 1), soulattrolide, inophyllum g-1, cordatolide a and oblongulide were reported by patil et al. to have potential application for the treatment of hiv.12 fig. 1.representative examples of pyranochromenones that exhibit anti-hiv activity. compounds with the backbone of chalcones were reported to possess various biological activities, such as antimicrobial, anti-inflammatory, analgesic, antiplatelet, anti-ulcerative, antimalarial, anticancer,13 antiviral, antileishmanial, antioxidant,14 antitubercular,15 antihyperglycemic, immunomodulatory, inhibiters of chemical mediators release,16 inhibitors of leukotriene b4,17 inhibiters of tyrosinase18 and inhibitors of aldose reductase,19 estrogenic activities.20 pyrazolines were found to possess antimicrobial,21 antibacterial,22 anti-amoebic,23–24 antidepressant,25 anticonvulsant,26 anti–inflammatory27–28 and antitumor activities. the recent literature is enriched with progressive findings concerning the synthesis and pharmacological properties of pyrazolines.21–28 microwave irradiation has gained popularity in the past decade as a powerful tool for rapid and efficient synthesis of a variety of compounds because of selective absorption of microwave energy by polar molecules.29 the application of microwave irradiation provides enhanced reaction rates and improved products in the field organic synthesis and is quite successful in the formation of a variety of carbon–heteroatom bonds. our research group has been making considerable efforts in the design and realization of innovative synthetic protocols in organic synthesis adopting a more eco-sustainable approach.30–32 results and discussion the synthetic route to compounds 4a–g is shown in scheme 1. compounds 3a–g were synthesized according to a previous work.33 the condensation of 8-acetyl-7-hydroxy-4-methyl coumarin (1) with 1-aryl-3-phenyl-1h-pyrazole-4 -carbaldehydes (2a–g) in the presence of piperdine under microwave irradiation gave (e)-1-(7-hydroxy-4-methyl-8-coumarinyl)-3-(1,3-diphenyl-1h-pyrazol-4 -yl)-2-propen-1-ones (3a–g). subsequently these chalcones 3a–g on reaction with vilsmeier reagent (dmf/pocl3) yielded substituted 4-chloro-8-methyl-2-(1,3 -diphenyl-1h-pyrazol-4-yl)-1,5-dioxa-2h-phenanthren-6-ones (4a–g). however, synthesis and microbial activity of pyranochromenone–pyrazoline hybrids 119 as the vilsmeier reagent (dmf/pocl3) serves for formylation of electron-rich aromatic rings, it was highly efficient for intramolecular cyclization of 2′-hydroxychalcones to 4-chloro-2h-chromenes.34-36 initially, the reaction was performed at room temperature but no product was formed. optimum results were obtained when the temperature was maintained at 90–100 °c by taking 6 equivalents of pocl3. in case of the microwave irradiation method, optimum results were obtained by irradiating at 160 w for 4–5 min. the crude products were purified using column chromatography to afford the pure products. scheme 1. synthetic route to substituted 4-chloro-8-methyl-2-(1,3-diphenyl-1h-pyrazol-4-yl) -1,5-dioxa-2h-phenanthren-6-ones; a, r1=h, r2=h; b, r1=och3, r2=h; c, r1=och3, r2= och3; d, r1=ch3, r2=h; e, r1=f, r2=h; f, r1=cl, r2=h; g, r1=br, r2=h. it was observed that the yields were better when the microwave irradiation method was used rather than the conventional heating method, table i. it is known that microwave irradiation is used for a variety of organic reactions due to table i. comparisons of the yields of the synthesized compounds 4a–g compd. conventional method mwi t / h yield, % t / min yield, % 4a 5 54 4 81 4b 5 61 4.5 89 4c 6 64 4 87 4d 6 57 5 88 4e 5.5 58 4.5 88 4f 5.5 61 4 90 4g 3 54 2 86 120 ashok et al. short reaction times, cleaner reactions, easier work-up and good yield. all the newly synthesized compounds were characterized using spectral analysis. the results are given in the supplementary material to this paper. the ir spectra of intermediate compounds 3a–g showed characteristic peaks of functional group c=n stretching between 1576–1598 cm–1, c=o stretching (of chalcone) between 1633–1654 cm–1 and oh stretching between 3436–3448 cm–1. for the final derivatives 4a–g, characteristic bands were present in each spectra in wave number (cm–1) ranges: 703–721, 1078–1086, 1654–1665 and 1725–1736, which correspond to c–cl, c–o–c, c=n and c=o stretching, respectively. the 1h-nmr spectra of chalcones 3a–g showed characteristic signals in the δ (ppm) ranges: 8.03–8.05, 8.15–8.18 and 8.60–8.64, corresponding to hα, hβ and the triazole h, respectively. in case of compounds 4a–g, the protons, h2, h7, h3 and h10 appeared as doublets in the δ ranges 5.96–5.96, 6.11–6.20, 6.18–6.21 and 6.83–6.89, respectively. in the 13c-nmr spectra, the carbonyl carbon of the chalcones 3a–g appeared in the δ 192.4–196.1. in the case of the final compounds 4a–g, c2 appeared in δ range 75.5–78.0. the rest of the carbons appeared in their usual regions, which supports the formation of compounds 3a–g and 4a–g. in the mass spectra of 3a–g and 4a–g, the molecular ion peaks, observed as [m+1] peaks, confirmed the molecular weights of the compounds. antibacterial activity all the compounds were screened for their antibacterial activity against staphylococcus aureus, bacillus subtilis, pseudomonas aeruginosa and escherichia coli using ampicillin as the standard drug (table ii). the activity was determined using the cup plate agar diffusion method by measuring the zone of inhibition in mm. the compounds were screened at a concentration of 50 μg ml–1 in dmso. from the screening studies, it was evident that the synthesized compounds 4b and 4c showed good antibacterial activity against all the tested organisms. it was further observed that the electron rich compound 4c, with both aryl rings possessing a methoxy substituent showed the best activity, closely followed by 4b, table ii. antibacterial activity (zone of inhibition, mm) of compounds 4a–g compd. gram-positive bacteria gram-negative bacteria s aureus b. subtilis p. aeruginosa e. coli 4a 20 7 5 22 4b 30 15 12 30 4c 32 16 13 33 4d 18 10 6 18 4e 22 11 8 25 4f 10 8 2 23 4g 28 11 9 28 ampicillin 30 12 10 30 synthesis and microbial activity of pyranochromenone–pyrazoline hybrids 121 which has only one methoxy substituent. this led to the conclusion that electron rich chalcones showed higher activity. furthermore, changing in the halogens from f to cl or br did not provide any significant change in the levels of activity against the bacteria. antifungal activity all the compounds were screened for their antifungal activity against aspergillus nigerzeae, penicillium italicum and fusarium oxysporum using grieseofulvin as the standard drug (table iii). the activity was determined using the cup plate agar diffusion method by measuring the zone of inhibition in mm. the compounds were screened at a concentration of 50 μg ml–1 in dmso. from the screening studies, it is evident that the synthesized compounds 4a, 4c and 4g showed good antifungal activity against all the tested organisms. the unsubstitiuted compound 4a showed the highest activity against the fungi. the highly electron rich compound 4c showed activity which was comparable to that of 4a, but in general, substituents on the pyrazole were detrimental to the observed activity. among the halogen derivates, the bromo substituent (4g) showed significantly higher activity than the chloro (4f) and fluoro (4e) substituted compounds. table iii. antifungal activity (zone of inhibition, mm) of compounds 4a–g cmpd. fungi a. nigerzeae p. italicum f. oxysporum 4a 17 24 26 4b 8 14 19 4c 14 20 26 4d 7 15 18 4e 10 12 12 4f 8 11 12 4g 13 21 24 grieseofulvin 12 20 25 experimental materials all the used materials were obtained commercially, mostly from sigma–aldrich, and were used without further purification. equipment melting points were determined in open capillaries and are uncorrected. the purity of the compounds was checked by tlc on silica gel 60 f254 (merck). the 1h-nmr and 13c-nmr spectra were recorded on a bruker avance ii 400 spectrometer using tms as an internal standard. the ir spectra were recorded in kbr on a shimadzu ftir 8400s spectrophotometer. the high-resolution electron spray ionization mass spectra (esi-hrms) were recorded 122 ashok et al. on a micromass q-tof (esi-hr-ms) mass spectrometer. the microwave reactions were performed in the milestone multisynth microwave system. general procedure for the synthesis of substituted of (e)-1-(7-hydroxy-4-methyl-8-coumarinyl)-3-(1-phenyl-3-phenyl-1h-pyrazol-4-yl)-2-propen-1-one analogues 3a–g a mixture of 8-acetyl-7-hydroxy-4-methyl coumarin 1 (1 mmol), substituted 1-(phenyl) -3-phenyl-1h-pyrazole-4-carbaldehyde (2a–g, 1 mmol) in ethanol (2 ml) and few drops of piperidine was taken in a glass vial equipped with a cap and then subjected to microwave irradiation at 100 watts, by maintaining 80 °c for 10 to 15 min. the progress of the reaction was monitored by tlc. after completion of the reaction, the mixture was diluted with cold water and acidified with dil. hcl. the precipitate that formed was filtered, dried and recrystallized from ethanol to afford pure chalcone. general procedure for the synthesis of substituted 4-chloro-8-methyl-2-(1,3-diphenyl-1h-pyrazol-4-yl)-1,5-dioxa-2h-phenanthren-6-ones 4a–g a) conventional method a. dmf (5 ml) was taken in a round-bottomed flask and cooled to 0–5 °c. pocl3 (0.006 mol) was added drop wise under stirring. the mixture was stirred at 0–5 °c for 15 min and then a substituted (e)-1-(7-hydroxy-4-methyl-8-coumarinyl)-3-(1,3 -diphenyl-1h-pyrazol-4-yl)-2-propen-1-one (3a–g, 0.001 mol) solution in 3 ml of dmf was added at 0–5 °c and the temperature was maintained at 0–5 °c for 30 min. the reaction mixture was then heated in a water bath at 90 °c for 6–8 h. after completion of the reaction (monitored by tlc, etoac:hexane in 1:4 volume ratio), the reaction mixture was poured into ice–water and neutralized with 10 % naoh solution and extracted with chloroform (2×20 ml). the combined organic layer was washed with 10 ml water and dried over anhydrous magnesium sulfate. the solvent was evaporated and the residue was purified by silica gel column chromatography to afford pure product 4a–g. b) microwave method b. dmf (5 ml) was taken in a round-bottomed flask and cooled to 0–5 °c. pocl3 (0.006 mol) was added drop wise under stirring. the stirring at 0–5 °c was continued for 15 min and then a substituted (e)-1-(7-hydroxy-4-methyl-8-coumarinyl)-3-(1,3diphenyl-1h-pyrazol-4-yl)-2-propen-1-one (3a–g, 0.001 mol) solution in 3 ml of dmf was added at 0–5 °c. the temperature was maintained at 0–5 °c for 30 min. the reaction mixture was placed in a microwave oven and subjected to microwave irradiation at 160 w by maintaining 90 °c for 4–5 min. the progress of reaction was monitored by tlc (etoac:hexane in 1:4 volume ratio). after completion of the reaction, the mixture was poured into ice water, neutralized with 10 % naoh solution and extracted with chloroform (2×20 ml). the combined organic layer was washed with 10 ml water and dried over anhydrous magnesium sulfate. the solvent was evaporated and the residue was purified by silica gel column chromatography to afford pure product 4a–g. biological assay antibacterial activity. the synthesized novel compounds 3a–g and 4a–g were screened for their antibacterial activity against different types of bacterial strains, i.e., the gram-negative bacterial strains p. aeruginosa and e. coli and the gram-positive bacterial strains b. subtilis and s. aureus at a concentration of 50 µ ml-1. the cultures were diluted with 5 % autoclaved saline and the final volume was made with a concentration of approximately 105–106 cfu ml-1. the synthesized compounds were diluted in acetone for antibacterial biological assays. for the agar disc diffusion method,37 the liquid form of the test compound was soaked on to the disc and then allowed to air dry, so that synthesis and microbial activity of pyranochromenone–pyrazoline hybrids 123 the disc became completely saturated with test compound. the saturated chemical discs were introduced onto the upper layer of the medium evenly loaded with the bacteria. the discs dipped in different chemical samples were placed over the evenly spread bacterial nutrient media and incubated at 37 °c for 24 to 48 h for better inhibition of bacteria. the zones of inhibition were measured after 24 to 48 h. all the experiments were carried out in triplicates and the results were expressed as zone of inhibition in mm. the zones of inhibition of synthesized compounds 4a–g were compared with the zone of inhibition of standard antibiotic concentration of ampicillin (50 µg/ml). the antibacterial activity was evaluated and the results are presented in table i. antifungal activity. the antifungal activity of synthesized compounds 4a–g was tested against three pathogenic fungi, namely fusarium oxysporum, aspergillus niger, and penicillium italicum, by the poison plate technique at a concentration of 50 µg ml-1. the three kinds of fungi were incubated in potato dextrose agar (pda) at 25±1 °c for 5 days to obtain new mycelium for the antifungal assay, then a mycelia as disks of approximately 0.45 cm diameter cut from the culture medium were picked up with a sterilized inoculation needle and inoculated in the center of the pda plate. test compounds were dissolved in acetone (10 ml) then added to the medium pda (90 ml). the final concentration of compounds in the medium was adjusted to 50 µg ml-1. the inoculated plates were incubated at 25±1 °c for 5 days. acetone was diluted with sterilized distilled water and used as control, while grieseofulvin (50 µg ml-1) was used as standard control for each treatment. three replicates of the experiments were performed. the radial growth of the fungal colonies was measured on the sixth day. conclusions a new series of compounds (4a–g) were synthesized under conventional and microwave irradiation conditions. using the microwave irradiation method, the reactions were completed in a shorter time with better yields as compared to the conventional method. all the new compounds were screened for their antimicrobial activity. it was observed that compounds 4b and 4c exhibited a broad spectrum of antibacterial activity, and compounds 4a, 4c and 4g showed a broader spectrum of antifungal activity against all the tested strains as compared to the standard drugs with their respective concentrations. supplementary material analytical and spectral data of the compounds are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgments. we are thankful to the head of the department of chemistry, osmania university, hyderabad, india, for providing the laboratory facilities and the director, cfrd, osmania university, hyderabad, for providing spectral analysis facilities. 124 ashok et al. и з в о д синтеза озрачивањем микроталасима супституисаних 4-хлор-8-метил-2-(1,3-дифенил-1h-пиразол-4-ил)-1,5-диокса-2h -фенантрен-6-она и њихова антимикробна активност dongamanti ashok1, bachi reddy vanaja1, mdderla sarasija2 и b. vijaya lakshmi1 1 green and medicinal chemistry laboratory, department of chemistry, osmania university, 500007 hyderabad, india и 2department of chemistry, satavahana university, karimnagar, 505001 telangana, india због потенијалне антимикробне активности пиранохроменонских и пиразолинских једињења, вилсмајеровом реакцијом су синтетисани хибридни деривати супституисаних 4-хлор-8-метил-2-(1,3-дифенил-1h-пиразол-4-ил)-1,5-диокса-2h-фенантрен-6-она (4a– –g), полазећи од супституисаних (e)-1-(7-хидрокси-4-метил-8-кумаринил)-3-(1,3дифенил-1h-пиразол-4-ил)-2-пропен-1-она (3a–g), у добром приносу, применом микроталаса. структура свих једињења утврђена је на основу аналитичких и спектралних података. одређена је in vitro антибактеријска и антифунгална активност свих синтетисаних једињења. поједини деривати показују веома добру активност према свим испитиваним бактеријама и гљивицама, у поређењу са стандардним лековима. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice green one-pot, four-component synthesis of spiro[indoline-3,4′-pyrano[2,3-c]pyrazole] derivatives using amino-functionalized nanoporous silica sba-15 under solvent-free conditions j. serb. chem. soc. 80 (10) 1265–1272 (2015) udc 547.75’81’77:542.913+544.351– jscs–4794 145.82+546.284–31 original scientific paper 1265 green one-pot, four-component synthesis of spiro[indoline-3,4′ -pyrano[2,3-c]pyrazole] derivatives using amino-functionalized nanoporous silica sba-15 under solvent-free conditions ghodsi mohammadi ziarani1*, mahshid rahimifard1, fatemeh nouri1 and alireza badiei2 1department of chemistry, alzahra university, vanak square, p. o. box 19938939973, tehran, iran and 2school of chemistry, college of science, university of tehran, tehran, iran (received 30 september 2014, revised 18 may, accepted 24 may 2015) abstract: propylamine functionalized nanoporous silica (sba–pr-nh2) was used as an efficient heterogeneous solid basic nanoreactor in the synthesis of 6′-amino-1′h-spiro[indoline-3,4′-pyrano[2,3-c]pyrazol]-2-one derivatives 5 through a one-pot, four-component condensation of isatin derivatives 1, activated methylene reagents 2, hydrazine hydrate 3 and β-keto esters 4 under solvent-free conditions at room temperature. keywords: amino-functionalized nanoporous silica; solvent-free; nanoporous silica; four components; one-pot; spiro indole; pyranopyrazole. introduction the indole ring is the core structure of many alkaloids, natural products and medicinal agents.1 compounds containing this moiety present a variety of antibacterial and antifungal activities.2 in addition, it was reported that substitution of the indole ring with heterocycles at the 3-carbon position significantly improves biological properties.3 the resulting spirooxindoles are found in various pharmaceutical components and natural products (scheme 1).4 heterocyclic compounds consisting of pyrano[2,3-c]pyrazoles with numerous biological properties, such as anticancer,5 antibacterial,6 antimicrobial,7 antiinflammatory,8 chk1 kinase inhibitors,9 antifungal10 and molluscicidal activity11 occupy a special place in medicinal chemistry. thus, considerable attention has been focused on the development of new modified methods for their synthesis. spiroindoline-pyranopyrazole derivatives can be obtained through various synthetic methods. shestopalov et al. reported a four-component reaction for their synthesis by condensation of isatin, hydrazine, malononitrile and β-keto * corresponding author. e-mails: gmziarani@hotmail.com; gmohammadi@alzahra.ac.ir doi: 10.2298/jsc140930045m _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1266 ziarani et al. esters using triethylamine in ethanol under reflux conditions.12 this reaction can also be affected using different catalysts, such as piperidine,13,14 l-proline,15 [dmbsi]hso4,16 mn(bpyo)2/mcm-41,17 bmim(oh)/chitosan,18 uncapped sno2 quantum dots,19 4-(dimethylamino)pyridine20 and meglumine.21 another common synthetic method for the synthesis of this class of compounds is a three -component condensation of pyrazolone, malononitrile and isatin in the presence of catalysts such as triethylamine,22 zns nanoparticles,4 l-proline,23 k2co3,24 triethanolamine25 and nahco3.26 however these methods have some disadvantages, such as long reaction times, expensive and non-reusable catalysts and hard work-up or catalyst removal. scheme 1. compounds with spirooxindole skeleton. in recent years, mesoporous materials especially mesoporous silica, such as sba-15 (santa barbara amorphous), have attracted considerable attention. sba -15 is a unique inorganic solid support with high surface area, large pore size and high thermal stability.27 grafting various organic compounds on the surface of sba-15 could improve the catalytic activity of the silica surface. amino functionalized sba-15 (sba–pr-nh2) was proved to be an efficient heterogeneous mesoporous solid base catalyst that could be used in the synthesis of various heterocyclic compounds.28–30 in this work, an attempt was made to develop a modified methodology in the synthesis of spiro[indoline-3,4′-pyrano[2,3-c]pyrazole] derivatives 5 using the green solid heterogeneous base nanocatalyst sba– –pr-nh2 under solvent free conditions at room temperature via a one-pot four -component condensation of isatin derivatives 1, activated methylene reagents 2, hydrazine hydrate 3 and β-keto esters 4. results and discussion this report is devoted to the study of the four component condensation of isatin derivatives 1, activated methylene reagents (malononitrile or ethyl cyanoacetate) 2, hydrazine hydrate 3 and β-keto esters 4 catalyzed by nanoporous base catalyst of sba–pr-nh2 under solvent-free conditions at room temperature (scheme 2). in initiation of this study, various conditions employing different solvents, such as ethanol or water, and a solvent-free system with or without catalyst at room temperature were evaluated. among the tested conditions, the best _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis of spiro[indoline-3,4′-pyrano[2,3-c]pyrazole] using sba–pr-nh2 1267 result was obtained in the presence of sba–pr-nh2 using the solvent-free system at room temperature (table i). scheme 2. synthesis of 6′-amino-1′h-spiro[indoline-3,4′-pyrano[2,3-c]pyrazol]-2-one derivatives 5 in the presence of sba–pr-nh2. table i. the optimization of the reaction conditions in the synthesis of 5a at room temperature yield, % t / h solvent catalyst entry 50 8 etoh – 1 50 3 h2o – 2 70 2 etoh sba–pr-nh2 3 80 0.25 – sba–pr-nh2 4 under the optimized conditions, various 6′-amino-1′h-spiro[indoline-3,4′ -pyrano[2,3-c]pyrazol]-2-one derivatives 5a–f were synthesized in the presence of sba–pr-nh2 using several isatin derivatives 1a–d, activated methylene reagents 2a and b, hydrazine hydrate 3 and β-keto esters 4a–c. results are summarized in table ii. under these conditions, the reactions were realized easily to produce spiroindoline-pyranopyrazole derivatives in good yields. it should be noted that the presence of halogens on the reacting isatins (entries 3 and 5) decreased the reaction time in comparison to that for to isatin (entry 1). it may be related to inductive withdrawing effects of halogens on the carbonyl group of isatin. on the other hand, replacing malononitrile with ethyl cyanoacetate increased the reaction time, which was attributed to the competing formation of the knoevenagel adduct of isatin and the activated methylene reagents. table ii. four-component synthesis of spiro[indoline-3,4′-pyrano[2,3-c]pyrazole] derivatives 5a–f in the presence of sba–pr-nh2 literature m.p., °c m.p. range, °c yield % t min product r5 r4 r3 r2 r1 entry 279–28026 278–280 80 15 5a meet cn h h 1 279–28026 278–280 87 15 5a meme cn h h 2 282–28326 281–283 85 10 5b meet cn h br 3 not reported 268–270 78 15 5c meet cn ch2ph h 4 297–29815 296–298 83 10 5d meet cn h cl 5 280–28113 279–281 85 15 5e ph et cn h h 6 281–28231 280–282 80 20 5f meet co2et h h 7 281–28231 280–282 83 25 5f memeco2et h h 8 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1268 ziarani et al. the reusability of the catalyst was investigated under the optimized conditions for the synthesis of the model compound 5a. as shown in table iii, the recycling process was completed four times with no significant decrease in the catalyst activity. the yields for the four runs were found to be 80, 78, 78 and 76 %, respectively. table iii. synthesis of the spiroindoline–pyranopyrazole 5a with recycled sba–pr-nh2 parameter 1st run 2nd run 3rd run 4th run time, min 15 15 20 20 yield, % 80 78 78 76 a possible mechanism for synthesis of 6′-amino-1′h-spiro[indoline-3,4′-pyrano[2,3-c]pyrazol]-2-one derivatives 5 is presented in scheme 3. the initiation step begins with the two-component condensation of hydrazine hydrate 3 and β-keto esters 4 to afford the 5-alkyl-2,4-dihydro-3h-pyrazol-3-one 7, which was deprotonated by sba–pr-nh2. then a fast knoevenagel condensation occurred scheme 3. plausible mechanism. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis of spiro[indoline-3,4′-pyrano[2,3-c]pyrazole] using sba–pr-nh2 1269 between isatin derivatives 1 and activated methylene reagents 2. michael addition of 8 to 10 afforded compound 11, which was followed by enol–keto tautomerization to yield intermediate 12. addition of hydroxyl to cyano group provided compound 13. tautomerization of compound 13 yielded the desired product 5 (scheme 3). several varying conditions have been reported in the literature for the synthesis of 6′-amino-1′h-spiro[indoline-3,4′-pyrano[2,3-c]pyrazol]-2-one derivatives 5, as given in table iv. table iv. comparison of several conditions year yield, % t / min conditionssolvent catalyst entry 200912 76–85 5–30 reflux etoh n(et)3 1 201014 80-97 300 r.t. h2o piperidine 2 201213 73–93 60 ultrasoundetoh piperidine 3 201416 88–96 1–2 60 °c – [dmbsi]hso4 4 201517 89–92 18–24 reflux h2o mn(bpyo)2/mcm-41 5 201418 89–93 150–210r.t. bmim(oh)/chitosan 6 201419 90–93 120–150r.t. h2o uncapped sno2 quantum dot7 201420 75–85 60 60 °c etoh 4-(dimethylamino)pyridine 8 201321 90–93 27–35 r.t. etoh/h2omeglumine 9 201315 83–92 10–30 80 °c h2o l-proline 10 present work 78–87 10–25 r.t. – sba–pr-nh2 11 in the current method, the basic nanoreactor with hexagonal platelet morphology, several reusabilities, ease of handling and removal from the reaction medium could make it an economic and efficient green solid heterogeneous nanocatalyst for this synthesis. furthermore, the short reaction time, solvent-free conditions, room temperature and simple procedure are other advantages of this method. structure of the catalyst the surface of the catalyst was analyzed by different methods, such as tga, ft-ir and others, which demonstrated that the organic groups (propylamine) were immobilized into the pores.29 the same ordered mesoscopic structured silica with (100), (110) and (200) reflections in the low-angle xrd patterns of sba-15 and sba–pr-nh2 indicated a two-dimensional hexagonal symmetrical array of nano-channels. this means that the structural integrity of sba-15 was not affected during the functionalization reaction. moreover, the tem image of sba–pr-nh2 confirmed the parallel channels were similar to the configuration of the pores in sba-15. this indicated that during grafting of the aminopropyl-triethoxysilane (aptes) groups, the pores of sba-15 did not collapse.29 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1270 ziarani et al. fig. 1. functionalized sba-15. experimental materials and methods the chemical compounds used in this work were all obtained from merck and were employed without further purification. the ir spectra were recorded from kbr disks using a fourier-transform (ft)-ir bruker tensor 27 instrument. the melting points were measured using the capillary tube method with an electrothermal 9200 apparatus. 1hand 13c-nmr were run on a bruker dpx at 400 or 250 mhz using tms as an internal standard. the mass spectra were obtained on an agilent 5973 ms detector. the physical, analytical and spectral data of compounds 5a–f are given in the supplementary material to this paper. synthesis and functionalization of sba-15 the nanoporous compound sba-15 was synthesized and functionalized according to a previous report. the triblock copolymer pluronic p126 was used as the directing agent for the preparation of sba-15 as nanoporous silica.32,33 functionalization of sba-15 was performed through post-grafting of calcined sba-15 with (3-aminopropyl)triethoxysilane (aptes, fig. 1).29 general procedure for the synthesis of the 6′-amino-1'h-spiro[indoline-3,4′-pyrano[2,3-c]pyrazol]-2-one derivatives a suspension of sba–pr-nh2 (0.02 g), isatin derivatives 1 (1 mmol), methylene reagent (malononitrile or ethyl cyanoacetate) 2 (1 mmol), hydrazine hydrate (80 %) 3 (1.4 mmol, 0.07 g) and β-keto ester 4 (1 mmol) was stirred at room temperature under solvent-free conditions for an appropriate time as indicated in table ii. upon completion of the reaction as monitored by tlc (thin layer chromatography), the solid product was dissolved in hot ethyl acetate and the insoluble catalyst was removed by filtration. the filtrate was cooled to room temperature to yield pure crystals of a spiro[indoline-3,4′-pyrano[2,3-c]pyrazole] derivative 5. conclusion in conclusion, amino-functionalized sba-15 could serve as an efficient heterogeneous solid basic nanocatalyst for the synthesis of 6′-amino1′h-spiro[indoline-3,4′-pyrano[2,3-c]pyrazol]-2-one derivatives 5 at room _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis of spiro[indoline-3,4′-pyrano[2,3-c]pyrazole] using sba–pr-nh2 1271 temperature and under solvent-free conditions. this procedure offers several advantages, such as short reaction times, mild reaction conditions, high yield of products, easy workup procedure, and reusability of the catalyst. supplementary material physical, analytical and spectral data of compounds 5a–f are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. we gratefully acknowledge the financial support from the research council of alzahra university and the university of tehran. и з в о д зелена синтеза деривата спиро[индолин-3,4'-пирано[2,3-c]пиразола], у једном реакционом кораку, употребом амино функционализованих нано-честица силика-гела без растварача ghodsi mohammadi ziarani1, mahshid rahimifard1, fatemeh nouri1 и alireza badiei2 1 department of chemistry, alzahra university, vanak square, p. o. box no. 19938939973, tehran, iran и 2 school of chemistry, college of science, university of tehran, tehran, iran нано-честице силика-гела функционализоване пропиламином употребљене су као ефикасан хетерогени нанореактор у синтези деривата 6'-амино-1'h-спиро[индолин-3,4'пирано[2,3-c]пиразол]-2-она 5 у четворокомпонентној реакцији кондензације деривата изатина 1, активних метиленских реагенаса 2, хидразин-хидрата 3 и β-кeтo-естара 4 без присуства растварача, на собној температури. 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mohammadi ziarani, n. h. mohtasham, n. lashgari, a. badiei, m. amanlou, r. bazl, j. nanostructure chem. 2 (2013) 489 31. e. a. a. hafez, f. m. abdul galil, s. m. sherif, m. h. elnagdi, j. heterocycl. chem. 23 (1986) 1375 32. a. shahbazi, h. younesi, a. badiei, chem. eng. j. 168 (2011) 505 33. a. badiei, h. goldooz, g. mohammadi ziarani, appl. surf. sci. 257 (2011) 4912. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice j. serb. chem. soc. 81 (12) 1473–1483 (2016) 1473 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. contents of volume 81 number 1 organic chemistry s. m. bukhari, a. j. feuerherm, h. m. tunset, s. m. isaksen, m. sæther, t. h. k. thvedt, s. v. gonzalez, r. schmid, a. brunsvik, e. fuglseth, b. zlatković, b. johansen and n. simić: a novel compound isolated from sclerochloa dura has anti-inflammatory effects .......................................................................................................................... 1 h. mao, h. liu, y. tu, z. zhong, x. lv and x. wang: one-pot preparation of carbamoyl benzotriazoles and their applications in the preparation of ureas, hydrazinecarboxamides and carbamic esters ....................................................................................... 13 y.-w. li and c.-l. ma: first and efficient synthesis of 4-[((3,4-dihydroxybenzoyl)oxy)methyl]phenyl β-d-glucopyranoside, an antioxidant from origanum vulgarem (short communication) ............................................................................................... 23 r. kazemi-rad, j. azizian and h. kefayati: electrocatalytic multicomponent assembling of aminouracils, aldehydes and malononitrile: an efficient approach to 7-aminopyrido[2,3-d]pyrimidine-6-carbonitrile derivatives .................................................... 29 biochemistry and biotechnology s. đ. bučko, j. m. katona, lj. m. popović, ž. g. vaštag and l. b. petrović: functional properties of pumpkin (cucurbita pepo) seed protein isolate and hydrolysate ........... 35 inorganic chemistry m. minato, t. ito and j.-g. ren: coordination studies of 1,2-bis(diphenylphosphino)ethane with di-μ-hydroxo dinuclear complexes of tungsten(iv) and molybdenum(iv) ................................................................................................................... 47 n. m. hosny, r. ibrahim and a. a. el-asmy: spectral, thermal, optical and biological studies on (e)-4-[(2-hydroxyphenyl)imino]pentan-2-one and its complexes .............. 57 theoretical chemistry m. haghdadi, s. soghra mousavi and h. ghasemnejad: stepwise or concerted? a dft study on the mechanism of ionic diels–alder reactions of chromanes ....................... 67 i. gutman: stability of bicalicene isomers – a topological study ....................................... 81 chemical engineering m. ali rezvani, m. ali nia asli, l. abdollahi and m. oveisi: (n(but)4)5h4pv6mo6o40 as an efficient catalyst for the oxidative desulphurisation of gasoline ........................ 91 number 2 organic chemistry m. r. simić, a. b. damjanović, m. d. kalinić, g. d. tasić, s. m. erić, j. a. antić-stanković and v. m. savić: synthesis, cytotoxicity and computational study of novel 1474 journal of the serbian chemical society available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. protoberberine derivatives ........................................................................................... 103 a. amini manesh and k. azarin: novel use of biocl as an efficient and selective reagent for cleavage of 2,4-dinitrophenylhydrazones to carbonyl compounds ........................ 125 c. m. al matarneh, c. i. ciobanu, i. i. mangalagiu and r. danac: design, synthesis and antimycobacterial evaluation of some new azaheterocycles with the 4,7-phenanthroline skeleton. part vi (short communication) ............................................................. 133 biochemistry and biotechnology j. yan, j. yang, r. yang, h. he, q. liu and h. lin: formation of conjugated double bonds in soybean oil with iodine as a catalyst ............................................................. 141 inorganic chemistry m. lashanizadegan, s. shayegan and m. sarkheil: copper(ii) complex of (±)trans-1,2cyclohexanediamine azo-linked schiff base ligand encapsulated in the nanocavity of zeolite-y for the catalytic oxidation of olefins ....................................................... 153 m. birdeanu, g. fagadar-cosma, i. sebarchievici, a.-v. birdeanu, b. taranu, i. taranu and e. fagadar-cosma: zn(ta1–xnbx)2o6 nanomaterials. synthesis, characterization and corrosion behaviour .................................................................................. 163 theoretical chemistry m. andoni, m. medeleanu, m. ştefănuţ, a. căta, i. ienaşcu, c. tănasie and r. pop: theoretical determination of the redox electrode potential of cyanidin ...................... 177 w. he, x. tan, n. wang and h. zhang: theoretical study on the mechanism of the cycloaddition reaction between ketenimine and hydrogen cyanide ............................ 187 physical chemistry s. s. lazarević, i. m. janković-častvan, b. m. jokić, dj. t. janaćković and r. d. petrović: sepiolite functionalized with n-[3-(trimethoxysilyl)propyl]ethylenediamine triacetic acid trisodium salt. part ii: sorption of ni2+ from aqueous solutions ...................................................................................................................... 197 analytical chemistry l. jiao, s. bing, x. zhang and h. li: interval partial least squares and moving window partial least squares in determining the enantiomeric composition of tryptophan using uv–vis spectroscopy ........................................................................................ 209 number 3 organic chemistry m. m. milosavljević, i. m. vukićević, s. ž. drmanić, j. b. nikolić, a. d. marinković, s. s. krstić and s. d. petrović: simple one-pot synthesis of thioureas from amines, carbon disulfide and oxidants in water ........................................................................ 219 k. battula, s. narsimha, v. nagavelli, p. bollepelli, m. srinivasa rao: synthesis and antimicrobial evaluation of some novel thiomorpholine derived 1,4-disubstituted 1,2,3-triazoles .............................................................................................................. 233 biochemistry and biotechnology j. wang and x.-h. zhao: degradation kinetics of fisetin and quercetin in solutions affected by medium ph, temperature and co-existing proteins ................................... 243 inorganic chemistry i. kostić, t. anđelković, d. anđelković, r. nikolić, a. bojić, t. cvetković and g. nikolić: interaction of cobalt(ii), nickel(ii) and zinc(ii) with humic-like ligands studied by volume 81: contents 1475 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. the esi-ms and ion-exchange methods ...................................................................... 255 theoretical chemistry b. furtula, i. gutman and k. ch. das: on atom–bond connectivity molecule structure descriptors ................................................................................................................... 271 d. farmanzadeh and m. najafi: novel trolox derivatives as antioxidants: a dft investigation ......................................................................................................................... 277 electrochemistry k. i. popov, p. m. živković, b. jokić and n. d. nikolić: the shape of the polarization curve and diagnostic criteria for control of the metal electrodeposition process ........ 291 polymers e. c. buruiana and m. murariu: copolymers based on n-acryloyl-l-leucine and urea methacrylate with pyridine moieties ........................................................................... 307 materials m. vaida, n. duteanu and i. grozescu: the effects of doping on the structural, optical and electric properties of zn4sb3 material ................................................................... 323 environmental m. milanović, j. sudji, n. grujić letić, j. radonić, m. turk sekulić, m. vojinović miloradov and n. milić: seasonal variations of bisphenol a in the danube river by the municipality of novi sad, serbia .......................................................................... 333 number 4 organic chemistry j. z. penjišević, v. v. šukalović, d. b. andrić, g. m. roglić, i. t. novaković, v. šoškić and s. v. kostić-rajačić: synthesis, biological evaluation and docking analysis of substituted piperidines and (2-methoxyphenyl)piperazines ........................................ 347 i. e. orhan, f. tosun and k. skalicka-woźniak: cholinesterase and tyrosinase inhibitory, and antioxidant potential of randomly selected umbelliferous plant species and the chromatographic profile of heracleum platytaenium boiss. and angelica sylvestris l. var. sylvestris .......................................................................................................... 357 inorganic chemistry m. sarkheil and m. lashanizadegan: schiff base ligand derived from (±)trans-1,2 -cyclohexanediamine and its cu(ii), co(ii), zn(ii) and mn(ii) complexes: synthesis, characterization, styrene oxidation and a hydrolysis study of the imine bond in the cu(ii) schiff base complex ............................................................................... 369 theoretical chemistry j.-b. tong, m. bai and x. zhao: application of an r-group search technique in the molecular design of hiv-1 integrase inhibitors .......................................................... 383 physical chemistry h. heli, n. sattarahmady and f. pourbahman: synthesis of copper nanoshales from a triton™ x-100/cyclohexane/water ternary microemulsion system (short communication) ........................................................................................................................ 395 analytical chemistry a. m. tasić, i. d. sredović ignjatović, lj. m. ignjatović, i. b. anđelković, m. p. antić and lj. v. rajaković: investigation of different extraction procedures for the 1476 journal of the serbian chemical society available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. determination of major and trace elements in coal by icp-aes and ion chromatography ...................................................................................................................... 403 polymers t. tomković, f. radovanović, b. grgur, a. nastasović, d. vasiljević-radović and a. onjia: novel negatively charged membrane adsorbers made using a combination of photopolymerization and immersion precipitation ...................................................... 419 materials c. s. ciobanu, c. l. popa and d. predoi: cerium-doped hydroxyapatite nanoparticles synthesized by the co-precipitation method ................................................................ 433 environmental g. andrejić, t. rakić, j. šinžar-sekulić, n. mihailović, j. grubin, b. stevanović and g. tomović: assessment of heavy metal pollution of topsoils and plants in the city of belgrade ...................................................................................................................... 447 t. đorđević, m. kašanin-grubin, g. gajica, z. popović, r. matić, lj. josić, m. milenković, a. lazarević and b. jovančićević: fruška gora mountainous environments – assessing the impact of geological setting and land use on soil properties ................. 459 euchems news ................................................................................................................... 469 number 5 organic chemistry c. a. florea and h. petride: ruo4-mediated oxidation of secondary amines. part 1. are hydroxylamines the main intermediates? .................................................................... 475 biochemistry and biotechnology j. trifunović-macedoljan, n. pantelić, a. damjanović, s. rašković, m. nikolić-đurović, g. pudar, m. jadranin, i. juranić and z. juranić: lc/dad determination of biogenic amines in serum of patients with diabetes mellitus, chronic urticaria or hashimoto's thyroiditis ............................................................................................... 487 dj. malenčić, b. kiprovski, v. bursić, g. vuković, n. hristov and a. kondić-špika: whole grain phenolics and antioxidant activity of triticum cultivars and wild accessions ............................................................................................................................ 499 inorganic chemistry e. g. bakirdere, m. f. fellah, e. canpolat, m. kaya and s. gür: design, synthesis, characterization, and antimicrobial activity of the new 2-{(e)-[(4-aminophenyl)imino]methyl}-4,6-dichlorophenol and its complexes with co(ii), ni(ii), cu(ii) and zn(ii): an experimental and dft study ..................................................................... 509 theoretical chemistry y. zhang, j. li and l. wang: effect of principal and secondary ligands on the electronic structures and spectra of a series of ruthenium(ii) complexes .................................... 521 physical chemistry s. zeighaminezhad and k. majlesi: complexation of molybdenum(vi) with methyliminodiacetic acid in different water + methanol solutions ........................................ 541 electrochemistry n. d. nikolić, p. m. živković, s. i. stevanović and g. branković: relationship between the kinetic parameters and morphology of electrochemically deposited lead ............. 553 volume 81: contents 1477 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. analytical chemistry u. m. gašić, d. m. stanković, d. č. dabić, d. m. milojković-opsenica, m. m. natić, ž. lj. tešić and j. j. mutić: analytical possibilities for the relative estimation of the antioxidative capacity of honey varieties harvested in different regions of serbia (short communication) ............................................................................................... 567 polymers l. b. petrović, j. r. milinković, j. l. fraj, s. đ. bučko and j. m. katona: an investigation of chitosan and sodium dodecyl sulfate interactions in acetic media ............. 575 materials n. đorđević, a. d. marinković, j. b. nikolić, s. ž. drmanić, m. rančić, d. v. brković and p. s. uskoković: a study of the barrier properties of polyethylene coated with a nanocellulose/magnetite composite film ..................................................................... 589 number 6 biochemistry and biotechnology j. živković, k. šavikin, g. zdunić, b. dojčinović and n. menković: phenolic and mineral profile of balkan indigenous apple and pear cultivars ................................................ 607 x.-b. zhu, l. pan, w. wei, j.-q. pen, y.-w. qi and x.-l. ren: changes in the content of water-soluble vitamins in actinidia chinensis during cold storage ............................. 623 theoretical chemistry x. lu and j. ming: ab initio study of the mechanism of formation of a spiro si-heterocyclic ring compound involving ge from h2ge=si: and acetaldehyde ....................... 633 analytical chemistry t. n. simonova, v. a. dubrovina and a. b. vishnikin: speciation of chromium through aqueous two-phase extraction of complexes of cr(iii) with 4-(2-pyridylazo)resorcinol and cr(vi) with 1,5-diphenylcarbazide .............................................................. 645 ž. n. todorović, lj. v. rajaković and a. e. onjia: interpretative optimization of the isocratic ion chromatographic separation of anions .................................................... 661 polymers m. nechifor: aromatic polyesters with photosensitive side chains: synthesis, characterization and properties .............................................................................................. 673 materials d. frem: some nitrogen-rich heterocycles derivatives as potential explosives and propellants: a theoretical study ........................................................................................ 687 environmental x. yang, j. yan, f. wang, j. xu, x. liu, k. ma, x. hu and j. ye: comparison of organics and heavy metals acute toxicities to vibrio fischeri .................................................... 697 t. zeremski, j. vasin, s. milić, p. sekulić, š. hansman and v. bursić: occurrence and distribution of the cyclodiene-type organochlorine pesticides in soils of vojvodina province, serbia .......................................................................................................... 707 hystory of and education in chemistry d. d. milenković, m. d. segedinac, t. n. hrin and s. horvat: the impact of instructional strategy based on the triplet model of content representation on elimination of students’ misconceptions regarding inorganic reactions ............................................. 717 1478 journal of the serbian chemical society available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. number 7 organic chemistry m. g. gündüz, e. albayrak, f. i̇şli, g. s. ö. fi̇ncan, ş. yıldırım, r. şimşek, c. şafak, y. sarıoğlu, s. ö. yıdırım and r. j. butcher: synthesis, structural characterization and myorelaxant activity of 4-naphthylhexahydroquinoline derivatives containing different ester groups ....................................................................................................... 729 biochemistry and biotechnology h. cheng, y. li, x. yin, m. subirade and l. liang: the β-casein–resveratrol complex: physicochemical characteristics and implications for enhanced nutrition .................. 739 inorganic chemistry t. a. lastovina, a. p. budnyk, g. a. khaishbashev, e. a. kudryavtsev and a. v. soldatov: copper-based nanoparticles prepared from copper(ii) acetate bipyridine complex ....................................................................................................................... 751 theoretical chemistry d. s. arputhara, p. v. nidhin and p. srinivasan: ab initio prediction of the polymorphic structures of pyrazinamide – a validation study ......................................................... 763 physical chemistry b. a. vidoeski, s. p. jovanović, i. d. holclajtner-antunović, d. v. bajuk-bogdanović, m. d. budimir, z. m. marković and b. m. todorović marković: raman study of the interactions between highly ordered pyrolytic graphite (hopg) and polyoxometalates: the effects of acid concentration ............................................................... 777 thermodynamics d. soldatović, n. grozdanić, z. višak, i. radović and m. kijevčanin: effects of solid poly (ethylene glycols) addition to the solutions of aniline or n,n-dimethylaniline with water: experimental measurements and modelling ............................................. 789 materials m. r. kaluđerović, j. p. schreckenbach and h.-l. graf: biocompatibility of the titaniumbased implant surfaces: еffect of the calcium dihydrogen phosphate on osteoblast cells ............................................................................................................................. 799 environmental m. savić biserčić, l. pezo, i. sredović ignjatović, lj. ignjatović, a. savić, u. jovanović and v. andrić: ultrasound and shacking-assisted water-leaching of anions and cations from fly ash ..................................................................................................... 813 b. p. dojčinović, b. m. obradović, m. m. kuraica, m. v. pergal, s. d. dolić, d. r. inđić, t. b. tosti and d. d. manojlović: application of non-thermal plasma reactor for degradation and detoxification of high concentrations of dye reactive black 5 in water ........................................................................................................................... 829 book review j. s. stevanović: morphology of electrochemically and chemically deposited metals, authors: konstantin i. popov, stojan s. djokić, nebojša d. nikolić and vladimir d. jović (book review) .................................................................................................... 847 volume 81: contents 1479 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. number 8 organic chemistry d. ashok, v. h. rao and r. kavitha: microwave assisted synthesis of 2-(2-(tetrazolo[1,5 -a]quinolin-4-yl)-2,3-dihydro-1h-1,5-benzodiazepin-4-yl) substituted phenols and evaluation of their antimicrobial activity .................................................................... 851 k. singaram, d. marimuthu, s. baskaran and v. ramaswamy: synthesis and antimicrobial activity of new 3,5-diarylidene-4-piperidone derivatives ................................. 859 biochemistry and biotechnology j. lazic, j. spasic, dj. francuski, z. tokic-vujosevic, j. nikodinovic-runic, v. maslak and l. djokic: importance of n-terminal proline for the promiscuous activity of 4-oxalocrotonate tautomerase (4-ot) ......................................................................... 871 j. živković, k. šavikin, g. zdunić, d. gođevac, n. marković, z. pržić and n. menković: influence of bunch morphology on quality of wines produced from clones of grape variety prokupac ......................................................................................................... 883 inorganic chemistry lj. e. mihajlović-lalić, lj. damjanović, m. šumar-ristović, a. savić, t. j. sabo, v. dondur and s. grgurić-šipka: cytotoxic pt(iv) and ru(ii) complexes containing a biologically relevant edda-type ligand: a comparative study of their thermal properties .......................................................................................................................... 897 physical chemistry r. m. alosmanov: adsorption of arsenazo iii by phosphorus-containing polymer sorbent .... 907 analytical chemistry r. đurović-pejčev, t. đorđević and v. bursić: determination of multi-class herbicides in soil by liquid–solid extraction coupled with headspace solid phase microextraction method ........................................................................................................................ 923 polymers j. leng, y. pan, s. yang, s. wang and a. adebayo: research of the relationship between the chain length of the dialkyl phosphate ester and the oil-based fracturing fluid viscosity ...................................................................................................................... 935 environmental z. veličković, n. ivanković, v. striković, r. karkalić, d. jovanović, z. bajić and j. bogdanov: ultrasound and shacking-assisted water-leaching of anions and cations from fly ash ................................................................................................................. 947 h. b. manh: modeling the removal of sunfix red s3b from aqueous solution by electrocoagulation process using artificial neural network .................................................... 959 number 9 organic chemistry d. setamdideh: one-pot green synthesis of isoxazol-5(4h)-one derivatives using dowex1-x8oh in water .............................................................................................. 971 d. r. brkić, a. r. božić, v. d. nikolić, a. d. marinković, h. elshaflu, j. b. nikolić and s. ž. drmanić: solvatochromism of isatin based schiff bases: an lser and lfer study ............................................................................................................................ 979 1480 journal of the serbian chemical society available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. biochemistry and biotechnology s. dmitrović, m. skorić, j. boljević, n. aničić, d. božić, d. mišić, v. filipović and d. opsenica: elicitation effects of a synthetic 1,2,4,5-tetraoxane and a 2,5-diphenylthiophene in shoot cultures of two nepeta species ...................................................... 999 m. košutić, j. filipović, l. pezo, d. plavšić and m. ivkov: physical and sensory properties of corn flakes with added dry residue from wild oregano distillation ......................... 1013 physical chemistry a. vashurin, i. kuzmin, v. mayzlish, m. razumov, o. golubchikov and o. koifman: kinetics and mechanism of the oxidation of dithiocarbamic acids in the presence of co(ii) phthalocyaninetetacarboxylic acid ................................................................... 1025 analytical chemistry s. agatonovic-kustrin, m. lebete, m. e. brown, d. w. morton and b. d. glass: chemical characterization of the photodegradation products of midazolam complexes with randomly methylated-β-cyclodextrin by hplc and lc–ms/ms ............ 1039 polymers f. jitaru, a. l. chibac, g. epurescu, i. ion and t. buruiana: polymer–graphene composites by the photocuring of a system containing benzophenone macromer ............. 1057 materials a. khorshidi, s. shariati and m. abootalebi: synthesis of bisand tris(indolyl)methanes catalyzed by an inorganic nano-sized catalyst followed by dehydrogenation to hyperconjugated products ....................................................................................... 1071 environmental a. m. tasić, i. d. sredović ignjatović, lj. m. ignjatović, m. a. ilić and m. p. antić: comparison of sequential and single extraction in order to estimate the environmental impact of metals from fly ash .......................................................................... 1083 m. m. aonyas, b. p. dojčinović, s. d. dolić, b. m. obradović, d. d. manojlović, m. d. marković and g. m. roglić: degradation of anionic surfactants using the reactor based on dielectric barrier discharge ........................................................................... 1099 number 10 organic chemistry f. bagheri and a. olyaei: a novel approach toward the synthesis of some new tridentate schiff bases from anil-like compounds ....................................................................... 1111 m. atbakar, o. topbastekin and n. ocal: reductive heck reactions of n-arylamino -substituted tricyclic imides ........................................................................................ 1121 biochemistry and biotechnology t. živanović radnić, k. simić-pašalić, m. šefik bukilica, s. misirlić denčić, a. m. isaković, t. stojković, n. petronijević, n. damjanov and j. vojinović: alfacalcidol modulates oxidative stress parameters in the peripheral blood of patients with active rheumatoid arthritis ............................................................................................................... 1127 d. jančić, v. todorović, z. basić and s. šobajić: chemical composition and nutritive potential of cichorium intybus l. leaves from montenegro ....................................... 1141 inorganic chemistry s. saha, a. das, k. acharjee and b. sinha: synthesis, characterization and antibacterial studies of mn(ii) and co(ii) complexes of an ionic liquid tagged schiff base ........... 1151 volume 81: contents 1481 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. physical chemistry m. faraji and a. farajtabar: solvatochromism of naringenin in aqueous alcoholic mixtures ...................................................................................................................... 1161 analytical chemistry v. dobričić, n. bubić pajić, b. marković, s. vladimirov, s. savić and g. vuleta: development and validation of an lc–ms/ms method for the determination of adapalene in pharmaceutical forms for skin application........................................................ 1171 polymers m. mouffok, a. mesli, i. abdelmalek and e. gontier: effect of the formulation parameters on the encapsulation efficiency and release behavior of p-aminobenzoic acid-loaded ethylcellulose microspheres ..................................................................... 1183 environmental k. a. taleb, j. d. rusmirović, m. p. rančić, j. b. nikolić, s. ž. drmanić, z. s. veličković and a. d. marinković: efficient pollutants removal by amino-modified nanocellulose impregnated with iron oxide ................................................................................ 1199 x. wang, m. q. yao, l. liu, y. cao and j. bao: degradation of chlorpyrifos in contaminated soil by immobilized laccase ........................................................................ 1215 number 11 organic chemistry n. božinović, b. a. šolaja and i. m. opsenica: microwave-assisted synthesis of azepines via nucleophilic aromatic substitution......................................................................... 1225 biochemistry and biotechnology y. n. mitrev, a. y. mehandzhiyski, d. i. batovska, a. liese and b. galunsky: original enzyme-catalyzed synthesis of chalcones: utilization of hydrolase promiscuity ........ 1231 b. kiprovski, đ. malenčić, s. đurić, m. bursać, j. cvejić and v. sikora: isoflavone content and antioxidant activity of soybean inoculated with plant-growth promoting rhizobacteria ........................................................................................................................ 1239 inorganic chemistry o. f. adekunle, r. butcher, o. bakare and o. a. odunola: synthesis, characterization, electrochemical studies and x-ray structures of mixed-ligand polypyridyl copper(ii) complexes with the acetate ......................................................................................... 1251 theoretical chemistry x. wang, j. li, l. wang, w. wu, z. du and w. luo: dft/td-dft study on the spectroscopic properties of zinc(ii), nickel(ii), and palladium(ii) metal complexes with a thiourea derivative ...................................................................................................... 1263 analytical chemistry l. u. r. chiavelli, l. c. figueiredo, r. t. r. almeida, t. claus, s. a. maruyama and w. f. costa: validation and application of a gc–ms method for the determination of haloacetic acids in drinking water ............................................................................... 1273 thermodynamics b. k. pandit, a. sarkar and b. sinha: solution thermodynamics of sodium pyruvate in aqueous glycine solutions at t 298.15–313.15 k ........................................................ 1283 1482 journal of the serbian chemical society available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. materials i. janković-častvan, s. lazarević, d. stojanović, p. živković, r. petrović and đ. janaćković: pvb/sepiolite nanocomposites as reinforcement agents for paper ................... 1295 metal materials and metallurgy v. m. maksimović, m. m. stoiljković and a. d. čairović: some consequences of repeated casting of co–cr dental alloy ...................................................................................... 1307 environmental s. sarri, p. misaelides, d. zamboulis, f. noli, j. warchoł, f. pinakidou and m. katsikini: chromium(vi) removal from aqueous solutions using a polyethylenimine–epichlorohydrin resin .............................................................................................................. 1321 number 12 organic chemistry b. vulović, i. marić, r. matović and r. n. saičić: synthesis of vinyldihydropyran by cooperative catalysis ................................................................................................... 1335 biochemistry and biotechnology j. p. vilipić, i. t. novaković, m. v. zlatović, m. t. vujčić, s. j. tufegdžić and d. m. sladić: interactions of cytotoxic amino acid derivatives of tert-butylquinone with dna and lysozyme ..................................................................................................... 1345 a. n. margetić and z. m. vujčić: immobilization of cell wall invertase in a polyacrylamide hydrogel for invert sugar production ................................................................ 1359 m. mihailović, j. trbojević-ivić, k. banjanac, n. milosavić, d. veličković, m. carević and d. bezbradica: immobilization of maltase from saccharomyces cerevisiae on thiosulfonate supports ................................................................................................. 1371 inorganic chemistry d. lj. stojković, a. bacchi, d. capucci, m. r. milenković, b. čobeljić, s. r. trifunović, k. anđelković, v. v. jevtić, n. vuković, m. vukić and d. sladić: synthesis and characterization of palladium(ii) complexes with glycine coumarin derivatives ........ 1383 theoretical chemistry m. kojić, m. petković and m. etinski: unrevealing mechanism of the thermal tautomerization of avobenzone by means of quantum chemical computations .................. 1393 physical chemistry d. veselinović and z. velikić: the ph measurement with glass electrode in an electromagnetic field ............................................................................................................. 1407 analytical chemistry n. perišić, m. marić-stojanović, v. andrić, u. b. mioč and lj. damjanović: physicochemical characterisation of pottery from the vinča culture, serbia, regarding the firing temperature and decoration techniques ............................................................. 1415 thermodynamics a. m. stanimirović, e. m. živković, d. m. majstorović and m. lj. kijevčanin: transport properties of binary liquid mixtures – candidate solvents for optimized flue gas cleaning processes ....................................................................................................... 1427 volume 81: contents 1483 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. environmental a. m. tasić, i. d. sredović ignjatović, lj. m. ignjatović, d. đuranović and m. p. antić: aqueous extraction of anions from coal and fly ash followed by ion-chromatographic determination .............................................................................................. 1441 history of and education in chemistry t. n. hrin, d. d. milenković, m. d. segedinac and s. horvat: enhancement and assessment of students’ systems thinking skills by application of systemic synthesis questions in the organic chemistry course ................................................................... 1455 contents of volume 81 ....................................................................................................... 1473 author index ....................................................................................................................... 1485 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice interval partial least squares and moving window partial least squares in determining the enantiomeric composition of tryptophan using uv–vis spectroscopy j. serb. chem. soc. 81 (2) 209–218 (2016) udc 547.757:541.72:543.422.6 jscs–4840 original scientific paper 209 interval partial least squares and moving window partial least squares in determining the enantiomeric composition of tryptophan using uv–vis spectroscopy long jiao1,2*, shan bing1, xiaofeng zhang1 and hua li2 1college of chemistry and chemical engineering, xi’an shiyou university, xi’an 710065, china and 2college of chemistry and materials science, northwest university, xi’an 710069, china (received 27 february, revised 2 august, accepted 3 august 2015) abstract: the application of interval partial least squares (ipls) and moving window partial least squares (mwpls) for the enantiomeric analysis of tryptophan (trp) was investigated. a uv–vis spectroscopic method for determining the enantiomeric composition of trp was developed. the calibration model was built using partial least squares (pls), ipls and mwpls, respectively. leave-one-out cross validation and external test validation were used to assess the prediction performance of the established models. the validation result demonstrated the established full-spectrum pls model is impractical for quantifying the relationship between the spectral data and enantiomeric composition of l-trp. on the contrary, the developed ipls and mwpls models are both practicable for modeling this relationship. for the ipls model, the root mean square relative error (rmsre) values of the external test validation and leave-one-out cross validation were 4.03 and 6.50, respectively. for the mwpls model, the rmsre values of the external test validation and leave -one-out cross validation were 2.93 and 4.73, respectively. obviously, the prediction accuracy of the mwpls model was higher than that of the ipls model. it was demonstrated that uv–vis spectroscopy combined with mwpls is a commendable method for determining the enantiomeric composition of trp. mwpls was superior to ipls for selecting the spectral region in the uv–vis spectroscopy analysis. keywords: uv–vis spectroscopy; interval partial least squares; moving window partial least squares; enantiomeric composition; tryptophan. introduction enantiomeric pure products are required in various fields because enantiomers may show great differences in their chemical and biological reactions.1,2 at * corresponding author. e-mail: mop@xsyu.edu.cn doi: 10.2298/jsc150227065j _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 210 jiao et al. present, determining the enantiomeric composition of chiral compounds has gained much attention. spectroscopic methods, including uv–vis,3–9 nir10,11 and fluorescence12–14 have been successfully used to determine the enantiomeric composition of chiral compounds. it was shown that a quantitative relationship between the enantiomeric composition and the spectra of chiral compounds exist.3–14 multivariable calibration methods are required to reveal this relationship. the partial least squares (pls) method was usually used to develop the calibration model. the models reported in these studies were all full-spectrum pls models, which were developed using the whole spectrum as the independent variable. however, a spectrum often contains spectral regions related to non-modeled information originating from unwanted constituents, background variations, random interferences and interactions. these spectral regions may degrade the calibration model and should not be used in model building.15–17 thus, selection of the spectral region is often required in multi-component spectroscopic analysis in order to eliminate the influence of non-modeled spectral regions and build well-fitted calibration models.17–21 many studies have shown that spectral region selection can lead to significant improvement of the prediction performance over full-spectrum calibration models.15–24 it seems spectral region selection could improve the accuracy of the calibration model when spectroscopic methods are used to determine the enantiomeric composition of chiral compounds. however, spectral region selection has not hitherto been applied to the spectroscopic determination of enantiomeric composition. interval partial least squares (ipls) and moving window partial least squares (mwpls) are two local modeling methods which have been commonly used to select spectral regions in spectroscopy analysis.16–18,22–35 tryptophan (trp) is an essential amino acid for humans. the biological function of l-trp is quite different from d-trp. thus, a method for the enantiomeric determination of trp is desirable. therefore, in the present study, ipls and mwpls were applied to the determination of the enantiomeric composition of trp. uv-vis spectroscopy was used to measure the spectra of trp enantiomers. an ipls model and a mwpls model were developed and investigated. experimental reagents l-trp and d-trp (enantiomeric pure) were purchased from sigma–aldrich co. llc (usa). bovine serum albumin (bsa, fraction v) was purchased from sino-american biotechnology co. (shanghai, china). water from a milli-q synthesis a10 system (merck group, germany) was used to prepare the samples. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ determining enantiomeric compostition of tryptophan with mwpls 211 spectra the uv–vis spectra of the prepared samples were collected with an agilent 8453 uv– –visible spectrophotometer (agilent, u.s.a.) over the wavelength range from 190 nm to 789 nm. quartz cells of 1.0 cm path length were used. all the spectra were blank corrected. data and software the root mean square relative error (rmsre) was employed to assess the prediction performance of the obtained models. it is defined as: 2 1 1 ( ) n i i rmsre re n = =  (1) where rei denotes the relative error of prediction for the ith sample and n is the number of samples. pls toolbox 5.5 (eigenvector research inc., u.s.a.) was used to perform pls. ipls and mwpls were conducted using itoolbox (programmed by prof. nørgaard, kvl, denmark; shared at http://www.models.kvl.dk/itoolbox). the independent variable matrix (x matrix) of pls, ipls and mwpls was generated by assembling the spectral data of each sample as a row vector. the dependent variable (y matrix) of each model is composed by the mol fraction of l-trp. the x and y matrix were mean-centered in the procedure of pls, ipls and mwpls. results and discussion spectra of the samples twenty samples were prepared. each sample contained 0.01 mmol l–1 trp and 0.02 mmol l–1 bsa. the enantiomeric composition of the 20 samples was varied from mol fraction 0.12 to 0.88 of l-trp. the mol fractions of l-trp in these samples are listed in table i. in the prepared samples, bsa formed two different guest–host complexes with the enantiomers of trp. the complex formed from l-trp is diastereomeric to the complex formed from d-trp. hence, the two complexes were different in their uv–vis spectra, as shown in fig. 1. correspondingly, the twenty prepared samples should differ in their uv–vis spectra because they differ in their enantiomeric composition. the uv–vis spectra of the twenty samples are shown in fig. 2, from which the slight differences could be seen. these spectral differences are actually related to the enantiomeric composition of trp. however, the differences are so small that a quantitative relationship between the spectra and the enantiomeric composition of trp could not be modeled using the classical least squares method. thus, pls, ipls and mwpls were used to build calibration models between the spectral data and the enantiomeric composition of trp. full-spectrum pls model pls was used to build the calibration model between the uv–vis spectra and the mol fraction of l-trp. the spectral data of the whole wavelength range and the mol fraction of l-trp were used as independent variable and dependent _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 212 jiao et al. table i. result of external test validation and leave-one-out cross validation; the samples marked by an asterisk are the samples in group ii (see text) no. actual mole fraction predicted mole fraction relative error, % pls ipls mwpls pls ipls mwpls 1 0.12 0.249 0.128 0.128 107.50 6.67 6.67 2 0.16 0.126 0.151 0.152 –21.25 –5.63 –5.00 3* 0.20 –0.155 0.207 0.205 –177.50 3.50 2.50 4 0.24 1.100 0.217 0.240 358.33 –9.58 0.00 5 0.28 0.631 0.242 0.270 125.36 –13.57 –3.57 6 0.32 0.099 0.348 0.348 –69.06 8.75 8.75 7* 0.36 0.740 0.352 0.368 105.56 –2.22 2.22 8 0.40 0.437 0.414 0.398 9.25 3.50 –0.50 9 0.44 0.748 0.469 0.455 70.00 6.59 3.41 10 0.48 0.597 0.511 0.509 24.38 6.46 6.04 11* 0.52 0.370 0.504 0.501 –28.85 –3.08 –3.65 12 0.56 0.654 0.577 0.564 16.79 3.04 0.71 13 0.60 0.344 0.583 0.561 –42.67 –2.83 –6.50 14 0.64 –0.093 0.635 0.605 –114.53 –0.78 –5.47 15* 0.68 0.096 0.724 0.687 –85.88 6.47 1.03 16 0.72 0.394 0.744 0.733 –45.28 3.33 1.81 17 0.76 0.915 0.766 0.781 20.39 0.79 2.76 18 0.80 0.098 0.781 0.818 –87.75 –2.38 2.25 19* 0.84 0.584 0.810 0.805 –30.48 –3.57 –4.17 20 0.88 0.276 0.805 0.824 –68.64 –8.52 –6.36 190 390 590 790 0 4 λ in te ns ity , a . u . / nm 300 324 0.04 0.12 λ in te ns ity , a .u . / nm fig. 1. uv–vis spectra of two solutions containing 0.01 mmol l-1 trp and 0.02 mmol l-1 bsa; solid line: d-trp+bsa; dashed line: l-trp+bsa. inset: spectra from 300 to 324 nm. variable, respectively. this model was a full-spectrum pls model. then, leave -one-out cross validation36 and external test validation37 were employed to assess _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ determining enantiomeric compostition of tryptophan with mwpls 213 the prediction performance of this model. the prepared 20 samples were split into two groups: group i and group ii. group i comprised 15 samples. group ii comprised 5 samples (marked by an asterisk in table i). 190 390 590 790 0 4 λ in te ns ity , a .u . / nm 300 324 0.04 0.12 λ in te ns ity , a .u . / nm fig. 2. uv–vis spectra of the twenty prepared samples. inset: spectra from 300 to 324 nm. first, the external test validation was performed. the pls model was generated by using group i as the calibration set. the obtained model contained four latent variables. group ii was used as the external test set. the mole fraction of l-trp of the 5 samples in group ii was predicted using the obtained pls model. the prediction result is listed in table i. for the 5 samples, the prediction rmsre was 101.77. subsequently, the leave-one-out cross validation was performed. the mole fraction of l-trp in group i was predicted in turn. the used pls model still included four latent variables. the prediction result is listed in table i. for the 15 samples, the rmsre of prediction reached 114.48. it could be seen from table i that the predicted mole fractions were not in agreement with the actual values. in particular, the prediction results for samples 3, 4 and 14 were completely unreasonable. the results of the two validations demonstrate the full-spectrum pls model could not model a quantitative relationship between the spectral data and the mole fraction of l-trp. some regions in the spectra may contain non-modeled information and should therefore, be excluded from the model. thus, spectral region selection should be helpful in the building of a reasonable calibration model. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 214 jiao et al. ipls model ipls was used to perform spectral region selection and develop a calibration model. the prediction accuracy of the established ipls model was evaluated by external test validation and leave-one-out cross validation. in the external test validation, group i was used as the calibration set to build the ipls model. the full spectrum was split into 19 intervals. the wavelength range of these intervals were 190−221, 222−253, 254−285, 286−317, 318−349, 350−381, 382−413, 414−445, 446−477, 478−509, 510−541, 542−572, 573−603, 604−634, 635−665, 666−696, 697−727, 728−758 and 759−789 nm. the results of ipls are shown in fig. 3. according to fig. 3, the optimal calibration model was obtained at the fourth interval (the wavelength range 286−317 nm) coupled with seven latent variables. this local model is the ipls model was required. then, it was used to predict the mol fraction of l-trp of group ii. the results are presented in table i. the rmsre of the prediction was 4.03 for the five samples. in the leave-one-out cross validation, the used ipls model was again developed using the spectral data for 286−317 nm and it included seven latent variables. the prediction results are presented in table i. the rmsre of this prediction was 6.50 for the 15 samples. as shown in table i, the predicted mole fractions were almost in agreement with the actual values, which showed that ipls was practicable for modeling the quantitative relationship between the fig. 3. root mean square error of cross validation (rmsecv) plot obtained from ipls. vertical bar is the rmsecv for each local model. the figure in each bar is the optimal number of latent variables for each local model. the dashed line gives the mean spectrum of the 20 samples. the dotted line is the rmsecv of the full-spectrum pls model. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ determining enantiomeric compostition of tryptophan with mwpls 215 spectral data and the mol fraction of l-trp. obviously, selection of the spectral region is a crucial step to build a feasible calibration model. ipls was superior to pls for building the calibration model, because it involves the procedure for the selection of the spectral region. although the obtained ipls model was acceptable, it was still worthwhile to search for a better calibration model because the prediction error of samples 5 was somewhat large. ipls can only divide the spectrum into a series of equidistant intervals at certain positions. the spectral intervals built by ipls may still contain non-modeled information and so was not the best region for building the model. if a more informative spectral region could be found to build the model, a better calibration model should be obtained. mwpls model mwpls finds the optimal spectral region by moving the window through the total spectral range. it can inspect more potential spectral regions than ipls. thus, a mwpls model was developed in order to improve the obtained calibration model. the prediction performance of the obtained mwpls model was still assessed by external test validation and leave-one-out cross validation. in the external test validation, group i was used as the calibration set to build the model. the window size was set to 31 nm. the results of mwpls are illustrated in fig. 4. in terms of the rmsecv, the optimal calibration model was the local model built at the position 306 nm, i.e., the established mwpls model was actually the local pls model developed from the spectral region 291−321 nm. this model includes five latent variables. the mol fraction of l-trp of group ii was then predicted using this model. the results are listed in table i. the rmsre of the prediction was 2.93 for the five samples. in the leave-one-out cross validation, the used mwpls model was also developed from the spectral region of 291−321 nm and it included five latent variables. the prediction results are presented in table i. for the 15 samples, the rmsre of the prediction was 4.73. as shown in table i, the predicted mol fractions were in good agreement with the actual values. the results presented above demonstrate mwpls to be a practicable method for modeling the quantitative relationship between the spectral data and the mol fraction of l-trp. as expected, the prediction accuracy of the obtained mwpls model was higher than that of the ipls model. it was shown that using mwpls was better than using ipls for the determination of the mol fraction of l-trp. mwpls is a more promising method for analyzing uv–vis spectrum. obviously, using the mwpls selected spectral region instead of the ipls selected spectral region as the independent variables substantially improved the prediction accuracy of the calibration model. it was demonstrated that the selection of the _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 216 jiao et al. spectral region significantly influences the calibration results of pls in the analysis of uv–vis spectroscopic results. inspecting more regions of the spectra is helpful in obtaining better calibration results. fig. 4. rmsecv plots obtained from mwpls. the solid line gives the rmsecv for all the local models. the dotted line represents the rmsecv for the full-spectrum pls model. the dashed shows the mean spectrum of the 20 samples. conclusions a uv–vis spectroscopic method for the determination of the enantiomeric composition of trp was developed. the result of external test validation and leave-one-out cross validation demonstrated that both ipls and mwpls are practicable for modeling the quantitative relationship between the spectral data and the mol fraction of l-trp. on the contrary, full-spectrum pls is unable to model this relationship. it was shown that the selection of the spectral region is an indispensable step to determine the enatiomeric composition of trp by uv– –vis spectroscopy. the mwpls model showed higher prediction accuracy compared with the ipls model. it was demonstrated that a more suitable spectral region for building the calibration model was found by mwpls, leading to the establishment of a better calibration model. obviously, mwpls was better than ipls for determining the enatiomeric composition of trp. since the results of pls, ipls and mwpls were quite different, it was shown that the selection of the spectral region has a significant influence on the calibration result in uv–vis spectro_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ determining enantiomeric compostition of tryptophan with mwpls 217 scopic analysis. mwpls is superior to ipls for selecting the spectral region in uv–vis spectroscopy analysis. the combination of uv–vis spectroscopy and mwpls is an easy-to-use and commendable method for determining the enantiomeric composition of trp at low concentration levels. the enantiomeric composition of trp can be accurately determined provided there is 0.01 mmol l–1 trp in the sample. although this method was only applied to trp in the present study, it should be a promising method for other chiral compounds. acknowledgements. the work was supported by the national natural science foundation of china (nos. 21305108 and 21375105), the natural science basic research plan in shaanxi province of china (program no. 2014jm2039) and the innovative research team of xi’an shiyou university (no. 2013qnkycxtd01). we greatly appreciate dr. yunxia wang (northwest university, china) for her cooperation and prof. l. nørgaard (kvl, denmark) for sharing the itoolbox program at the website of kvl. и з в о д методе парцијалних најмањих квадрата уз фиксну и мобилну селекцију интервала у одређивању енантиомерног састава триптофана применом uv–vis спектроскопије long jiao1,2,, shan bing1, xiaofeng zhang1 и hua li2 1college of chemistry and chemical engineering, xi’an shiyou university, xi’an 710065, china и 2college of chemistry and materials science, northwest university, xi’an 710069, china испитивана је примена методе парцијалних најмањих квадрата (pls) уз фиксну и мобилну селекцију спектралних интервала (ipls и mwpls, редом) у анализи енантиомера триптофана (trp). развијена је uv–vis спектроскопска метода за одређивање енентиомерног састава trp. добијени су калибрациони модели применом pls, ipls и mwpls приступа. методом унакрсне валидације појединачних изостављања (leave-one -out) и валидацијом на екстерном сету објеката процењена је поузданост предвиђања добијених модела. резултати валидације показују непрактичност примене pls приступа у моделовању целог спектралног опсега, у циљу одређивања енантиомерног састава l-trp на основу спектралних података. насупрот томе, оба новоразвијена приступа, ipls и mwpls, су погодна за моделовање. у ipls моделу, релативна грешка изражена као корен средње вредности квадрата одступања измерених од предвиђених вредности (rmsre) у случају екстерне калибрације и унакрсне валидације појединачних изостављања износе 4,03 и 6,50, редом. за mwpls модел, rmsre теста екстерне калибрације и унакрсне валидације износе 2,93 и 4,73, редом. очигледно је да је предвиђена тачност mwpls модела виша него код ipls модела. показано је да је uv–vis спектроскопија комбинована са mwpls погодна за одређивање енантиомерног састава trp. mwpls модел је супериорнији у односу на ipls за селекцију спектралне области и uv–vis спектроскопску анализу. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice synthesis, structural characterization and myorelaxant activity of 4-naphthylhexahydroquinoline derivatives containing different ester groups j. serb. chem. soc. 81 (7) 729–738 (2016) udc 547.822.1.004.12+542.913.548.7: jscs–4881 547.831.5:547.26’11 original scientific paper 729 synthesis, structural characterization and myorelaxant activity of 4-naphthylhexahydroquinoline derivatives containing different ester groups mi̇yase gözde gündüz1*, emi̇ne albayrak1, fatma i̇şli̇2, gökçe sevi̇m öztürk fi̇ncan3, şeni̇z yildirim4, rahi̇me şi̇mşek1, ci̇hat şafak1, yusuf sarioğlu3, sema öztürk yidirim5 and ray j. butcher6 (received 6 december 2015, revised 24 march, accepted 29 march 2016) 1department of pharmaceutical chemistry, faculty of pharmacy, hacettepe university, ankara 06100, turkey, 2department of rational drug use and drug supply management, turkish medicines and medical devices agency, ankara 06520, turkey, 3department of pharmacology, faculty of medicine, gazi university, ankara 06500, turkey, 4department of pharmacology, faculty of medicine, kırıkkale university, kırıkkale 71450, turkey, 5department of physics, faculty of sciences, erciyes university, kayseri 38039, turkey and 6department of chemistry, howard university, washington dc 20059, usa abstract: the present study reports the synthesis, structural characterization and myorelaxant activity evaluation of a series of 16 novel 4-naphthylhexahydroquinoline derivatives. the compounds were achieved by one-pot microwave-assisted method via a modified hantzsch reaction. the structures of the compounds were confirmed by various spectral methods, such as ir, 1d and 2d nmr techniques and mass analysis. x-ray studies of compound 10 provided further evidence for the proposed structure. to evaluate their myorelaxant activities, the emax and pd2 values of the compounds and nifedipine were determined on isolated rabbit gastric fundus smooth muscle strips. the obtained results indicated that the introduction of long chain alkyl groups, such as the 2-methoxyethyl or 2-(methacryloyloxy)ethyl moiety, to the ester group led to the most active compounds. keywords: 1,4-dihydropyridine; synthesis; myorelaxant activity; crystal structure, structure elucidation. introduction dihydropyridines (dhps) represent low molecular weight heterocyclic compounds based on a pyridine core. although theoretically five isomeric dhps could exist, the most recognized ones have the 1,4-dihydro structure.1 1,4-dihydropyridines are one of the most important chemical classes introduced into bio * corresponding author. e-mail: miyasegunduz@yahoo.com doi: 10.2298/jsc151206035g _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 730 gündüz et al. logical sciences and are common in many commercialized drugs. these compounds mainly present a well-known capacity as calcium channel blockers, thus acting as vital drugs against cardiovascular diseases, particularly hypertension and angina pectoris.2 although dhps were primarily developed as cardiovascular agents, medicinal chemists decorated the 1,4-dhp nucleus and achieved diverse activities at several receptors, channels and enzymes with different medical applications, such as antitubercular, antioxidant, antitumor, antithrombotic, antimicrobial, antidiabetic, antidyslipidemic and anticonvulsant.3 since their introduction into clinical medicine, 1,4-dhps have been one of the most studied class of drugs and many modifications have been performed on the structure of nifedipine, the prototype of dhps, in order to enhance calcium modulating effects and obtain structure–activity relationships (sar). according to sar studies, a 1,4-dhp ring with an unsubstituted nitrogen and pseudoaxial oriented aryl ring substituent at c-4 are essential for activity.4 ester functionalities at c-3 and c-5 position are of utmost importance to modulate the activity and tissue selectivity. it was proved that modification of the ester moiety plays a key role in the ability of condensed 1,4-dhps to block calcium currents.5 fused dhps, such as hexahydroquinolines, that could be obtained by introducing the dhp ring into condensed ring systems, are active derivatives exhibiting calcium antagonistic effects.6 the classical method for the synthesis of 1,4-dhps is a one-pot hantzsch reaction, which proceeds effectively and involves dehydrative coupling of an aldehyde, two equivalents of a 1,3-dicarbonyl compound and ammonia.7 depending on the reagents and reaction conditions, long reaction times, unexpected products or low yields can be obtained.8 although most of the efforts focused on improving the reaction using various catalysts, such as fe3o4, cobalt and ytterbium,9–11 microwave (mw) irradiation has recently gained great popularity as an energy source for hantzsch reactions because of its ability to reduce reaction times, to improve yields and to simplify the work-up processes.12 in the present study, sixteen dhp derivatives in which substituted cyclohexane rings were fused to the dhp ring under microwave irradiation were synthesized and how different ester groups and the naphthyl moiety attached to this backbone affected the myorelaxant activities of these compounds was investigated to obtain additional information to enrich the classical sar studies. experimental general all chemicals used in this study were purchased from aldrich and fluka (steinheim, germany). the reactions were performed in a discover microwave apparatus (cem). thin layer chromatography (tlc) was run on merck aluminum sheets (darmstadt, germany), silica gel 60 f254, mobile phase ethyl acetate–hexane: (1:1) and ultraviolet (uv) absorbing spots were detected by short wavelength (254 nm) uv light (camag uv cabinet, wiesloch, _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ myorelaxant condensed 1,4-dihydropyridine derivatives 731 germany). melting points were determined on a thomas hoover capillary melting point apparatus (philadelphia, pa, usa) and are uncorrected. infrared spectra were recorded on a perkin–elmer spectrum bx ft-ir instrument (beaconsfield, uk) and are reported in cm-1. the 1h-nmr spectra were obtained in dimethyl sulfoxide (dmso) solutions on a varian mercury 400, 400 mhz high performance digital ft-nmr spectrometer (palo alto, ca, usa). 13c-nmr and cosy (2d-1h–1h homonuclear correlation spectrum) spectra were recorded on the same instrument. the chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane. the x-ray crystallographic analysis was realized on an agilent xcalibur (ruby, gemini) diffractometer. the esi-ms spectra were measured on a micromass zq-4000 single quadruple mass spectrometer. elemental analyses were performed on a leco chns-932 elemental analyzer (philadelphia, pa, usa). analytical, physical and spectral data of the synthesized compounds are given in supplementary material to this paper. chemistry the general procedure for the preparation of alkyl 2,6,6-trimethyl-4-(1-naphthyl/2-naphthyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylates (compounds 1–16) was as follows: a one-pot four component mixture of 2 mmol 4,4-dimethyl-1,3-cyclohexanedione, 2 mmol of 1or 2-naphthaldehyde, 2 mmol of an appropriate alkyl acetoacetate and 10 mmol of ammonium acetate was placed into a 35-ml microwave pressure vial and heated under microwave irradiation (power 50 w, maximum temperature 120 °c) for 5 min. in 5 ml ethanol. after completion of the reaction, monitored by tlc, the reaction mixture was poured into ice–water and the obtained precipitate was filtered and crystallized from ethanol–water. the synthetic route used to synthesize the target compounds is outlined in fig. 1. fig. 1. synthesis of compounds 1–16. x-ray crystallography computing details. data collection: crysalis pro,13 cell refinement: crysalis pro,13; data reduction: crysalis pro.13 program(s) used to solve and refine the structure: shelxs97.14 program(s) used for molecular graphics and to prepare the material for publication: shelxtl.14 refinement. carbon-bound h-atoms were placed in calculated positions (c–h, 0.93–0.98 å) and were included in the refinement in the riding-model approximation, with uiso(h) = = 1.2ueq(c) or 1.5 ueq(c-methyl). a rotating-group model was applied for the methyl groups. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 732 gündüz et al. the n-bound h-atoms were located in a difference fourier map but were refined with a distance restraint: n–h = 0.83 (1) å with uiso(h) = 1.2ueq(n). pharmacology new zealand white rabbits, weighing 2.5–3 kg, were used in this study. the rabbits were sacrificed with i.v. injection of sodium pentobarbital (30–40 mg kg-1), followed by removal of the stomach through abdominal incision. the fundal part of the stomach was then dissected parallel to the longitudinal muscle wall. one muscle strip about 15–20 mm length and 2 mm width was obtained and allowed to equilibrate for 60 min in 20 ml organ baths filled with calcium (ca2+) free krebs–henseleit solution (khs). the composition of the krebs solution was as follows (in mmol l-1): nacl 118; kcl 4.7; nahco3 25; mgcl2 0.54; nahpo4 0.9; glucose 11. the solution was gassed with 95 % o2 and 5 % co2 during the study and temperature was maintained at 37 °c by a thermoregulated water circuit. the ph of the saturated solution was 7.4. each strip was connected to a force transducer (fdt 10-a, may iobs 99, commat iletisim co., ankara, turkey) for measurement of the isometric force, which was continuously displaced and recorded on an online computer via a four-channel transducer data acquisition system (mp30b-ce, biopac systems inc., santa barbara, ca) using software (bsl pro v. 3.6.7, biopac systems inc.) which also had the capacity to analyze the data. after mounting, each strip was allowed to equilibrate with a basal tension of 1 g for 60 min. ca2+ free khs was replaced with fresh solution every 15 min during this time. nω-nitro-l -arginine methyl ester hydrochloride (l-name, a nitric oxide synthase inhibitor, 10-4 m), indomethacin (cox inhibitor, 10-5 m), tetraethylammonium chloride (ca2+-activated k+ channel blocker, 10-4 m), glibenclamide (atp-sensitive k+ channel blocker, 10-6 m) and guanethidine (adrenergic nerve blocker, 10-6 m) were added into the organ bath 20 min before the compounds were added in order to eliminate the effects of nitric oxide, cyclooxygenase, ca2+-activated k+ channel, atp sensitive k+ channel and adrenergic pathways, respectively. after the smooth muscle strips of rabbit gastric fundus had been placed in a high k+ -containing (80 mm) solution, 2.5 mm ca2+ was added to the organ bath to develop contraction. concentration–relaxation responses for the compounds 1–16 (10-8–3×10-4 m) and nifedipine (10-9–10-6 m) were obtained by adding these into the bath in a cumulative manner. a cumulative concentration–response curve was constructed in a stepwise manner after the response to the previous concentration had reached a plateau. the relaxant effects of the compounds and nifedipine were expressed as percentage of the precontraction with 2.5 mm ca2+ in the high k+ containing solution. dmso, used in activity studies as solvent, was also tested. to evaluate the effects of the compounds, the maximum response (emax) values of compounds and nifedipine were established at 3×10-4 m and 10-6 m concentrations, respectively and pd2 values (the negative logarithm of the concentration for the half-maximal response (ec50)) were calculated, as predicted from the scatchard equation for drug–receptor interaction. agonist pd2 values (apparent agonist affinity constants) were calculated from each agonist concentration–response curve by linear regression of the linear part of the curve and taken as a measure of the sensitivity of the tissues to each agonist. while emax is the parameter for efficacy, pd2 is the parameter for potency. all data are expressed as mean ± standard error. statistical comparison between groups were performed using general linear models by the scheffe f-test and p values less than 0.05 were considered to be statistically significant. the study was approved by the gazi university ethics committee. procedures involving animals and their care were conducted in conformity with international laws and policies. l-name hydrochloride, indomethacin, guanethidine, nifedipine, glibenclamide, and tetraethylammonium chloride were supplied by sigma. while l-name, tetraethylammonium _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ myorelaxant condensed 1,4-dihydropyridine derivatives 733 chloride and guanethidine were dissolved in distilled water, indomethacin, glibenclamide, nifedipine and the compounds were dissolved in dmso. results and discussion chemistry a series of condensed 4-naphthyl-1,4-dhp derivatives were prepared via a modified hantzsch reaction. in order to obtain the target compounds, 4,4 -dimethyl-1,3-cyclohexanedione, 1-naphthaldehyde/2-naphthaldehyde, an appropriate alkyl acetoacetate were heated in the presence of excess ammonium acetate under microwave irradiation in ethanol, which was classified as an excellent microwave-absorbing solvent.15,16 the appearance of the products was monitored by tlc and the reaction time was determined as 5 min., which is quite a short time compared to conventional heating for the hantzsch reaction.17,18 the structures and chemical characteristics of the synthesized compounds are reported in table s-i of the supplementary material. the structures of the synthesized compounds were elucidated by spectral methods (ir, 1h-nmr, 13c-nmr, cosy, x-ray analysis and mass spectra) and confirmed by elemental analysis. in the ir spectra, characteristic n–h, c=o (ester) and c=o (ketone) stretching bonds were observed. in the 1h-nmr spectra, the protons of the methyl substituents at the 6-position of the hexahydroquinoline ring were observed separately and as singlets at 0.73–0.99 ppm. the methylene groups of the same ring were at 1.57–2.57 ppm. the methine protons of the 1,4-dhp ring were seen as a singlet at 4.96–5.63 ppm. the aromatic protons of the naphthyl and phenyl rings were at 6.91–8.73 ppm, while the n–h protons of the dhp ring were seen at 9.02–9.21 ppm. in the 13c-nmr spectra, the number of the signals fitted exactly the number of carbon atoms. the correlations between the interacting protons of compound 2 were determined by cosy. the correlations between h-7 and h-8, ch2 and ch3 protons in the ester side chain and h-4 and the aromatic protons were observed. these correlations are demonstrated in fig. 2 and the cosy spectrum is provided as supplementary material. based on this information; the structure of compound 2 was conclusively identified as ethyl 2,6,6-trimethyl-4-(1-naphthyl)-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate. the mass spectra of the compounds were recorded via the electrospray ionization technique. the quasimolecular ions created by the addition of sodium ion [m+na]+ and also of a hydrogen cation [m+1+na]+ were observed in the spectra of all compounds. cleavage of the ester group and the naphthyl ring from the parent molecule were the next most observed fragmentations. elemental analysis results were within ±0.4 % of the theoretical values for all compounds. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 734 gündüz et al. fig. 2. cosy correlations of compound 2. x-ray analysis of compound 10 the three-dimensional structure of ethyl 2,6,6-trimethyl-4-(2-naphthyl)-5 -oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (10) was evaluated by x-ray crystallography (fig. 3). fig. 3. x-ray molecular structure of compound 10 with the atom-numbering scheme used in the crystallographic analysis. the crystal data and a summary of intensity data collections and structural refinements, selected bond lengths, bond angles and torsion angles are given in the supplementary material. in compound 10 (fig. 3), the naphthalene ring is almost planar with a maximum deviation from the mean plane of –0.029(2) å for atom c(17). the cyclohexene rings adopt a sofa conformation and are puckered with puckering parameters19 of qt = 0.4383(17) å, θ = 50.6(2)°, φ = 127.6(3)°. the values of the bond lengths20 and angles in the title compound are within the normal ranges and are comparable with those of related relationships.21–23 the x-ray crystallographic data of the title compound demonstrated that there are intraand intermolecular hydrogen bonds. in the crystal, molecules are linked by pairs of intermolecular n–h…o hydrogen bonds, forming dimers with r12(6) ring motifs,24,25 and _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ myorelaxant condensed 1,4-dihydropyridine derivatives 735 these dimers are connected by n–h…o hydrogen bonds, generating one-dimensional chains along [011] (supplementary material). pharmacology the maximum relaxant effects (emax) and pd2 values of compounds 1–16, dmso and nifedipine on isolated strips of rabbit gastric fundus smooth muscle are given in table i. table i. maximum relaxant responses (emax) and pd2 values of the compounds, nifedipine and dmso on strips of rabbit gastric fundus smooth muscle; the relaxation is expressed as the percentage of the precontraction induced by 2.5 mm ca2+. the negative logarithm of the concentration for the half-maximal response (pd2) and emax values represent mean value ± sem; p < 0.05 compared with the control responses, n = 6 compound emax pd2 1 81.20±10.26 5.42±0.71 2 82.78±8.98 5.53±0.65 3 77.42±4.71 4.63±0.37 4 45.52±9.10 4.92±0.66 5 53.32±7.53 5.00±0.57 6 93.54±9.31 5.56±0.67 7 91.83±5.29 4.95±0.42 8 43.17±5.97 5.39±0.47 9 63.93±5.90 4.52±0.47 10 42.30±4.67 5.31±0.37 11 50.42±10.01 4.37±0.70 12 36.88±5.96 6.09±0.47 13 48.90±10.41 5.37±0.71 14 92.68±8.15 4.67±0.61 15 91.50±5.94 5.07±0.47 16 21.94±3.68 6.34±0.26 nifedipine 100.00±2.18 8.33±0.04 dmso 11.35±2.25 6.31±0.05 tissues were pretreated with indomethacin, guanethidine, l-name, tetraethylammonium chloride and glibenclamide to investigate whether the relaxation induced by the compounds occurred through cyclooxygenase, the adrenergic system, nitric oxide pathways, ca2+-activated k+ channel and atp-sensitive k+ channel, respectively. pretreatment of the strips with indomethacin, guanethidine, l-name, tetraethylammonium chloride and glibenclamide confirmed that cyclooxygenase, adrenergic and nitric oxide pathways, ca2+-activated k+ channel and atp-sensitive k+ channel played no roles on the relaxations evoked by these substances. the results of this study indicate that all of the compounds (10–8–3×10–4 m) and nifedipine (10–9–10–6 m) produced concentration-dependent relaxation on the rabbit gastric fundus smooth muscle strips that were statistically significant _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 736 gündüz et al. from the control relaxations produced by dmso. the compounds and nifedipine exerted concentration-dependent relaxation responses on the gastric fundus smooth muscle strips precontracted with ca2+ (2.5 mm) with the efficacy order: nifedipine ≥ 6 ≥ 14 ≥ 7 = 15 > 2 ≥ 1 ≥ 3 > 9≥ 5 ≥ 11 ≥ 13 ≥ 4 ≥ 8 ≥ 10 ≥ 12 > 16. the efficacy of compounds 6, 7, 14 and 15 were found to be the same as nifedipine. the obtained results suggested that myorelaxant effects of the compounds seem to exert their effects by blocking ca2+ channels as does nifedipine. given that the main difference between these compounds is their ester groups, it follows that the ester moiety plays a key role in the ability of these compounds to block calcium channels. increasing the side chain length of the ester group mediated an increase whereas introducing a ring structure at the same locus did not lead to a significant improvement in blocking the activity. the introduction of a 2-(methacryloyloxy)ethyl or 2-methoxyethyl group as the side chain of the ester group resulted in a series of highly active compounds. two methyl groups at the 6-position of the hexahydroquinoline ring are present in all compounds and therefore they are not the most critical components for the preferential activity. when the obtained results are analyzed in terms of the substitution position of the naphthyl ring at the c-4 position of dhp, generally the 1-naphthyl derivatives possessed better activities. although all compounds are potent myorelaxant agents on the gastric fundus smooth muscle strips, introduction of a naphthalene substituent into the 4-position of the 1,4-dhp nucleus decreased the myorelaxant effect of the compounds compared to nifedipine. this could suggest that the substitution of an o-nitrophenyl ring by a naphthyl ring increases the size of the molecules and may have a negative effect on the ability of these compounds to show their effects. as a result, the naphthyl ring could be a good choice as the aromatic substituent at the c-4 position of dhp only in combination with long chain alkyl esters. conclusions an easy, very rapid and convenient method for the preparation of condensed 1,4-dhps under mw irradiation was reported. the target compounds were achieved by the reaction of 4,4-dimethyl-1,3-cyclohexanedione, 1-naphthaldehyde/2-naphthaldehyde, an appropriate alkyl acetoacetate and ammonium acetate in ethanol. this method also offers a reduction of solvent use and reaction time in addition to higher yields. the obtained pharmacological results showed that all the synthesized compounds had relaxing effects on isolated rabbit gastric fundus smooth muscle, possibly due to the blockade of the ca2+ channels, similar to the action of nifedipine. the introduction of long chain alkyl groups, such as the 2-methoxyethyl or 2-(methacryloyloxy)ethyl moiety to the ester group led to the most active com_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ myorelaxant condensed 1,4-dihydropyridine derivatives 737 pounds, suggesting that two hydrogen bond acceptor groups might be required for the calcium channel blocking activity. it was also proved that there is no contribution of cyclooxygenase, adrenergic and nitric oxide pathways, atp-sensitive k+ channels and ca2+-activated k+ channels to the myorelaxant effects of the compounds. as a result, further investigations are required to ascertain the ca2+ channel blockage effects of the compounds. supplementary material data on the characterization of the synthesized compounds are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the authors gratefully acknowledge the financial support provided by the scientific research fund of hacettepe university, turkey through project 013.d03.301.001. rjb wishes to acknowledge the nsf–mri program (grant che-0619278) for funds to purchase the diffractometer and the howard university nanoscience facility for access to liquid nitrogen. и з в о д синтеза, структурна карактеризација и миорелакснтна активност деривата 4-нафтилхексахидрохинолина који садрже различите естарске групе miyase gözde gündüz1, emine albayrak1, fatma i̇şli2, gökçe sevim öztürk fincan3, şeniz yildirim4, rahime şimşek1, cihat şafak1, yusuf sarioğlu3, sema öztürk yidirim5 и ray j. butcher6 1 department of pharmaceutical chemistry, faculty of pharmacy, hacettepe university, ankara 06100, turkey, 2 department of rational drug use and drug supply management, turkish medicines and medical devices agency, ankara 06520, turkey, 3 department of pharmacology, faculty of medicine, gazi university, ankara 06500, turkey, 4 department of pharmacology, faculty of medicine, kırıkkale university, kırıkkale 71450, turkey, 5 department of physics, faculty of sciences, erciyes university, kayseri 38039, turkey и 6 department of chemistry, howard university, washington dc 20059, usa приказана је синтеза, структурна карактеризација и миорелаксантна активност серије од 16 нових деривата 4-нафтилхексахидрохинолина. једињења су синтетисана у једном реакционом кораку под микроталсним озрачивањем, модификованом ханчовом реакцијом. структуре једињења одређене су на основу спектралних података иц, 1d и 2d nmr спектроскопије и масене спектрометрије. анализом рендгенске структуре монокристала деривата 10 додатно је потврђена предложена структура једињења. током испитивања миорелаксантне активности одређене су emax и pd2 вредности испитиваних једињења и нифедипина, на исечцима глаткомишићних ћелија желудачног дна зеца. добијени резултати указују на допринос алкил-група дугог ланца, као што су 2-метоксиетил или 2-(метакрилоилокси)етил-естара, доброј активности. (примљено 6. децембра 2015, ревидирано 24. марта, прихваћено 29. марта 2016) references 1. n. edraki, a. r. mehdipour, m. khoshneviszadeh, r. miri, drug discovery today 14 (2009) 1058 2. g. w. zamponi, j. striessnig, a. koschak, a. c. dolphin, pharmacol. rev. 67 (2015) 821 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 738 gündüz et al. 3. e. carosati, p. ioan, m. micucci, f. broccatelli, g. cruciani, b. s. zhorov, a. chiarini, r. budriesi, curr. med. chem. 19 (2012) 4306 4. p. ioan, e. carosati, m. micucci, g. cruciani, f. broccatelli, b. s. zhorov, a. chiarini, r. budriesi, curr. med. chem. 18 (2011) 4901 5. c. bladen, m. g. gunduz, r. simsek, c. safak, g. w. zamponi, pfluegers arch. 466 (2014) 1355 6. r. simsek, g. s. ozturk, i. m. vural, m. g. gunduz, y. sarioglu, c. safak, arch. pharm. 341 (2008) 55 7. v. g. santos, m. n. godoi, 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {aqueous extraction of anions from coal and fly ash followed by ion-chromatographic determination} j. serb. chem. soc. 81 (12) 1441–1453 (2016) udc 552.574+66.046.59:66.061: jscs–4940 543.544–128.2 original scientific paper 1441 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. aqueous extraction of anions from coal and fly ash followed by ion-chromatographic determination aleksandra m. tasić1, ivana d. sredović ignjatović2, ljubiša m. ignjatović1, danijel đuranović1 and mališa p. antić2* 1university of belgrade, faculty of physical chemistry, studentski trg 12–16, 11000 belgrade, serbia and 2university of belgrade, faculty of agriculture, nemanjina 6, 11080 belgrade, serbia (received 23 february, revised 6 september, accepted 23 september 2016) abstract: three different techniques were applied for the aqueous extraction of anions from coal and fly ash: rotary mixerand ultrasonic-assisted extraction with different duration time, and microwave-assisted extraction at different temperatures. validation showed that the ion-chromatographic method was suitable for the analysis of anions in coal and fly ash extracts. the variations in the amounts of anions using different extraction times during rotary-assisted extraction were minimal for all investigated anions. the efficiency of ultrasound-assisted extraction of anions from coal depended on the sonication time and was highest at 30 min. the ultrasound-assisted extraction was less efficient for the extraction of anions from fly ash than rotary-assisted extraction. increase of temperature in the microwave-assisted extraction had a positive effect on the amounts of all anions extracted from coal and sulphate from fly ash, while the amounts of fluoride and chloride in fly ash extracts decreased. the microwave-assisted extraction of coal at 150 °c was compared with standard astm methods, and results were in good agreement only for chloride. changes in the ph value and conductivity during ultrasound-assisted extraction were measured in order to explain changes on the surface of coal particles in contact with water and different processes that occur under environmental conditions. keywords: coal; fly ash; anions; extraction; ion chromatography. introduction coal preparation, as well as disposal of coal fly ash, can lead to introduction of various materials into the environment through leaching process and cause water, air and soil pollution. some novel research indicated that chlorine and fluorine in fly ash are enriched on the surface and that both halogens on the ash * corresponding author. e-mail: mantic@agrif.bg.ac.rs doi: 10.2298/jsc160223086t 1442 tasić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. surface consist of organic and inorganic functionalities.1,2 the monitoring of sulphur, phosphorus and the halogens is of great importance for a number of reasons: with the exception of bromine, all of these elements are essential for the life of humans, animals and many plants; however, in elemental form and/or in certain compounds, they are highly toxic. all of the above-mentioned elements are vastly used in the chemical industry and, consequently, their effects on human health and the environment are important.3–5 the quantitative determination of halogens and sulphur in solid samples usually involves sample preparation (in order to bring the analytes into solution) followed by analytical determination. sample decomposition is a critical step in halogen analysis. halogens are volatile and readily lost when the sample is treated; furthermore, the reagents used for sample digestion may interfere with the determination process itself. the alternative to decreasing the time spent in performing analyses is the employment of ultrasonic baths or microwave ovens in the digestion or extraction process. the details related to coal, coal fly ash and extraction processes are given in supplementary material to this paper. the aim of this study was the extraction of fluoride, chloride and sulphate from coal and coal fly ash under aqueous conditions. different extraction techniques were employed to extract anions from coal and the efficiencies of those techniques were investigated by ion chromatographic determination. in addition to the comparison of the efficiency of various types of extractions, the influence of extraction time was investigated with respect to the rotary and ultrasonic extraction, as well as, the influence of temperature during microwave-assisted extraction. deionised water was used as a soft extraction solvent because of its hydrolytic and dissolving effect on some coal and fly ash constituents. trace elements extracted by water are relatively labile and thus may be potentially bioavailable. this phase contains water-soluble species consisting of free ions and ions complexed with soluble organic matter and other constituents. it constitutes the most mobile and potentially the most available metal, non-metal and metalloid species.6 studies of drainage waters from coal mines and fly ash dumps showed that, depending on the ph value, they can contained ca, mg, fe, al, si and trace elements, such as mn, zn, ni and co, and that the most important anions are sulphate, hydrogen carbonate and carbonate.7–9 experimental materials and reagents composite samples (coal and fly ash) from kostolac coal field, serbia (see supplementary material for details) and power plant were ground, homogenized and sieved through a sieve with 0.212 mm openings, corresponding to us standard mesh 70 and tyler standard mesh 65. the samples were oven-dried at 105 °c overnight prior to use. extraction of anions from coal and fly ash 1443 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. the working calibration solutions were prepared by serial dilution (10, 20, 50, 100 and 200 times) of the stock standard solution using deionised water (for details on reagents used, see supplementary material). instrumentation an overhead mixer reax 20/8 (carl roth, germany) rotary mixer and a transsonic t 760 dh (elma, germany) ultrasonic bath with an ultrasonic frequency of 40 khz and an effective ultrasound power of 170 w were used . the type of the microwave digestion system used for coal sample preparation was ethos 1, advanced microwave digestion system (milestone, italy), equipped with 10 ptfe containers. the capacity of the containers was 100 cm3 each, with a maximum pressure and temperature of 10 mpa and 240 °c, respectively. ion chromatography (ic) measurements were realised using a metrohm 761 compact ic system (metrohm, switzerland). the a metrosep a supp 1 ion-exchange column, with polystyrene–divinylbenzene copolymer as the carrier material (particle size 7 μm, column dimensions 4.6 mm×250 mm) was used for the anion separation, followed by the metrohm suppressor module and then a thermostated conductivity detector. as eluent 3.0 mmol dm-3 na2co3 was used at a flow rate of 1.0 cm3 min-1. the full-scale range was 50 μs and the injected sample volume was 20 μl for each probe. extraction procedures the extraction mixtures were prepared by mixing coal or fly ash sample with deionised water in the mass/volume ratio of 1 g:10 cm3. the extracted anions were identified and determined using ion chromatography. extractions were performed using a rotary mixer (rae) in which the suspension was processed for the following extraction times: 30, 60, 90, 120 and 180 min by mixing at 10 rpm at room temperature (20 °c). the second technique involved the use of an ultrasonic bath (ultrasound-assisted extraction, uae) with the mixtures being positioned at the same place in the bath and at the same initial water temperature of 17 °c. extractions were made during extraction times of 10, 20, 30, 40 and 50 min. the third technique involved the use of a microwave system (microwave-assisted extraction, mae) at the following temperatures: 50, 100 and 150 °c. the given temperature was achieved in 15 min for each extraction cycle, while the extraction itself lasted 15 min. subsequently, the samples were cooled to room temperature. five replicate extractions of each sample were made and the results presented are the average value. a blank was prepared for each of the employed extraction procedure. when the extraction processes were finished, each of the extraction mixtures was first centrifuged and then filtered through a 0.22 μm pore size membrane filter. the extracts were preserved at 4 °c in a laboratory refrigerator for further analyses. results and discussion validation of ic measurements are given in supplementary material to this paper. determination of anions in coal the important inorganic anions in coal (fluoride, chloride and sulphate) were found as coal inorganic constituents and as pollutants. the chromatogram of a standard solution and a typical chromatogram for the coal extract are shown in fig. 1. 1444 tasić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. the sulphate peak is well distinguished from the fluoride and chloride peaks at a considerably longer retention time of 13.24 min. all the analysed coal samples contained trace levels of fluoride, and therefore fluoride was detected in the aqueous extracts of the coals. under the conditions used for the ic measurements (fig. 1), fluoride is distinguished from acetate, which is also present in the extracts. the identification of acetate, to which the peak 2 on fig. 1 (lower) is assigned, was made by spiking the measured extract with sodium acetate solution. the assignment of the fluoride peak is based on the confirmation of retention time by spiking the samples extract with a known quantity of fluoride, and by detection and quantification of fluoride using an ion selective electrode (ise).10 the quantification of the fluoride content in the extracts using ise was in good agreement with the results obtained by ic. the level of extracted fluoride appears to be greater for low-rank coals. fluorine in coal is mostly associated with mineral matter, usually fluorapatite and clays, but fluorite and complex silicates, such as tourmaline, may also be present.11 these minerals are finely dispersed throughout coal and it is feasible that the extraction process enables fluoride to be leached from these sparingly soluble materials. the results of the determinations of fluoride, chloride and sulphate in the coal extracts obtained using rae and uae are presented in table i. fig. 1. ion chromatograms of: (upper) standard solution (1 f-, 2 cl-, 3 no3-, 4 br-, 5 po43-, 6 so42-) and (lower) coal extract provided by rotary-mixing (1 f-, 2 ch3coo-, 3 cl-, 4 so42-). extraction of anions from coal and fly ash 1445 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. from the results listed in table i, fluoride concentration was almost constant with time during rotary-assisted extraction. considering ultrasound-assisted extraction of fluoride, the obtained results indicated that after initial increase, the concentration of fluoride in extracts did not change significantly with time. the extraction of fluoride by means of rae yielded results that were, on average, comparable to the results realized using uae. in the case of both extraction techniques, there was no positive correlation between the extraction time and the amount of extracted fluoride. table i. the results of the determination of anions (μg g-1) after rae/uae (n = 5, uncertainty as standard deviation) extraction time, min fluoride chloride sulphate rae 30 20.30±0.57 17.00±0.76 3014±89 60 19.50±0.13 19.08±0.12 2822±37 90 20.36±0.18 19.82±0.02 2976±26 120 20.20±0.01 18.60±0.42 3011±2 180 20.50±0.05 21.49±0.01 3016±2 uae 10 17.90±0.73 13.58±0.34 2409±89 20 20.42±0.30 19.55±0.21 3330±30 30 20.88±1.07 23.98±0.28 3622±16 40 21.75±0.07 20.44±0.15 3402±22 50 20.74±0.33 20.03±0.10 3080±76 the rae of chloride (table i) demonstrated that prolongation of the extraction time led initially to a slight increase in extracted amount between 30 and 180 min. the results presented in table i indicated that prolongation of the uae extraction time led initially to an increase in number of extracted ions, first by 43 % and finally by 22 % when the extraction was extended from 20 to 30 min. the highest chloride amount was extracted from coal after 30 min of ultrasound -assisted extraction. when compared to rae, the uae was more efficient for the extraction of chloride. the physical and chemical effects of ultrasound do not come from the direct interaction of sound with molecular species. when sound passes through a liquid, the formation, growth, and finally implosive collapse of bubbles occur. very high effective temperatures (which increase solubility and diffusivity) and pressures (which favours penetration and transport) at the interface between an aqueous solution subjected to ultrasonic energy and a solid matrix, combined with the oxidative energy of radicals (hydroxyl and hydrogen peroxide) created during sonolysis, result in high extractive power.12 the phenomenon of ultrasonic cavitation caused by implosion of cavitation bubbles and subsequent local pressure increases and elastic shock waves has been used for coal and fly ash extraction.13,14 1446 tasić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. the results shown in table i indicated that increasing the extraction time had no significant influence on the rae of sulphate. the uae of sulphate (table i) demonstrated that maximum extracted concentration was obtained after 30 min, and with further increase in the extraction time, the concentration of sulphate decreased. the coal samples from kostolac coalfield had up to 2 % of sulphur, and the extracted amount was small, meaning that most of the sulphur was insoluble in water.15 this is expected, since sulphur in coal exists as inorganic and organic sulphur components. the major inorganic sulphur compound in coal is pyrite; organic sulphur compounds are diverse and contain mainly thiol, sulphide, disulfide and thiophene groups.15,16 changes in ph value and conductivity of the extraction suspension as a function of sonication time are shown in fig. 2. the results showed that the conductivity of the suspension increased during sonication until 30 min, partly due to an increase in the number of total charged particles in the suspension. fig. 2. changes in the ph value and conductivity of the extraction suspension as a function of sonication time (at 23.6 °c). contrarily, a sharp decrease of ph value in the extract was observed for the same sonication time, which implies that hydrogen ions are released to the solution. with prolongation of the sonication time, the conductivity and ph value in the extract changed in the opposite manner. along with anions, various cations released into the solution also caused the changes in the ph value and conductivity, which could further influence the adsorption and desorption processes to some extent.17 these processes are mainly related to competitive cation interact extraction of anions from coal and fly ash 1447 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. ions for the sorbing phase and the exchange of sorbed cations with those from solution influenced by ultrasound. the results of determination of fluoride, chloride and sulphate anions in water leachate from coal using the microwave-assisted technique are shown in fig. 3. the concentration of extracted fluoride, increased with increasing extraction temperature. the mae was the most efficient for fluoride extraction, giving at 100 °c an extracted amount that was 1.50 times higher than the ones obtained by rae and uae. the mae at 150 °c resulted in the highest amount of extracted fluoride compared to any of the other extraction techniques used: the amount of extracted fluoride was 2.40 times higher than the average amounts obtained by employing an ultrasonic bath and rotary mixer for all extraction times. the interaction of microwave energy with samples and reagents causes both ionic migration and dipole rotation, resulting in fast heating of the suspension with the consequent acceleration of the physico-chemical processes.18 the use of microwave energy for coal sample preparation and extraction of elements from coal has increased in recent years mainly due to the heating mechanism, resulting in a shortening of the extraction and digestion times, as well as in a decreased consumption of the extraction agent.19 50 100 150 0 10 20 30 40 50 2500 3000 3500 4000 c on ce nt ra tio n, μ g g1 temperature, °c f cl so 4 2 fig. 3. change in the extracted amounts of fluoride, chloride and sulphate (μg g-1) obtained by mae as a function of extraction temperature. the concentration of chloride extracted by the mae at 100 °c was about 1.1 times higher than the highest concentration obtained by the other two techniques. when the temperature was increased to 150 °c, the obtained concentration was about 1.2 times higher than the highest concentration resulting from the use of 1448 tasić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. rae or uae. the extraction behaviour of chloride depends on temperature and increasing leaching temperatures causes more chlorine to be leached. the results for mae (fig. 3) demonstrated that significantly increased amounts of extracted ions occurred with increasing extraction temperature. the use of mae at a temperature of 150 °c gave an almost 1.3 time higher amount of sulphate than the average amount obtained by uae. compared to the all the other investigated techniques, mae at 150 °c was more efficient for the extraction of sulphate ions. the average value obtained by the mae was higher than the one obtained by the uae after the extraction time of 30 min, which gave the highest extracted amount. considering the mae technique, there was, as expected, an obviously positive correlation between the number of extracted ions and the extraction temperature (fig. 3). the variations in the extracted amount resulting from the use of different extraction techniques were minimized in the case of fluoride. the results of extracted anions from the coal sample using the mae technique showed that an increase in temperature produced higher amounts of extracted anions. the microwave-assisted procedures were faster than the rotary mixer and ultrasonic bath procedures. the precision of all techniques was very good with the majority of the rsd values below 5 %, thereby demonstrating the good reproducibility of all techniques and indicating that the microwave-assisted procedures offer comparable data quality in terms of reproducibility to the rea and uae techniques. the amounts of extracted fluoride and chloride by the mae at 150 °c were compared to anion content determined according to standard methods.21,22 the results are given in table ii as recovery values, calculated as the found to declared concentration ratio. in the case of fluoride, the recovery was satisfactory, but not good. fluoride has been identified as an ecologically important trace element, and the extracted quantity corresponded to water soluble and exchangeable fluorine. the leaching behaviour of fluorine is ph-dependent: a lower ph value led to fluorine release from the mineral matrix; a higher ion strength or ph value of leaching solution tended to free more fluorine into the aqueous solution.22 the obtained results correspond to the leaching of fluoride under mild ph conditions. on the other hand, fluorine in coal is mostly present as stable inorganic compounds, which means that the proposed extraction method is not suitable for stable inorganic forms of fluorine, such as caf2, mgf2 or ca10(po4)6(oh)f.11 the good recovery for chlorine indicated that the total chlorine content in the analyzed coal was extracted by mae at 150 °c. the results of leaching showed that chlorine occurs in the investigated coal in various soluble mostly mineral forms. the sulphate content in coal in the absorption solution was also determined after oxygen bomb combustion. low recovery for sulphate was expected, since the combustion method provides total sulphur analysis, regard extraction of anions from coal and fly ash 1449 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. less the form of the sulphur in coal. the sulphate concentration determined by mae at 150 °c corresponds to the soluble sulphate in coal. these results indicated that extracted sulphate was present in the analysed coal in less soluble and/or accessible forms than was chloride. table ii. recovery of fluoride, chloride and sulphate determined by mae at 150 °c anion declareda, µg g-1 found, µg g-1 recovery, % f67.60 48.08 71.0 cl26.62 27.44 103 so42 62043 3982 6.40 adetermined by the astm standard methods when results for total fluoride, chloride and sulphate are compared to those obtained by uae for 30 min, the recoveries were 31.0, 90 and 4.96 %, respectively. these indicated that recovery was good for chloride, meaning that uae could be successfully applied for chloride determination. previous leaching experiments for chloride showed that about 50 % of chloride could be removed from particles of grain size less than 45 µm, and 30 % from 100–500 µm granulations.23 the effect of ultrasound on chloride leaching from coal was proven since the efficiency was three times higher for the particles used in the present experiments (smaller than 212 µm). the recovery of uae leaching of sulphate was in accordance with the results of other authors who investigated the effect of ultrasound on sulphur removal from coal.24 the availability and mobility of anions occurring in coal depended on the physicochemical form of the elements. all three procedures showed relatively good levels of accuracy. applying these techniques to the extraction of anions from coal samples combined with different extraction solvents, e.g. dilute acids or salts solutions, could be considered and requires further investigation. determination of anions in fly ash the results of the determinations of fluoride, chloride and sulphate in the coal fly ash extracts obtained using rae and uae are presented in table iii. fluoride extraction using rae showed slight differences between obtained concentrations with time. the maximum extracted quantity was obtained after 60 min of extraction, and further increase of extraction time led to decreases in the concentration. in the case of uae, the concentration of fluoride after 20 min of extraction was 10 % lower than after 10 min of extraction, probably due to resorption, but subsequently increased and after 50 min, it was the same as after 10 min of extraction. the extraction of fluoride by uae was almost efficient as rae for all investigated times except for 60 minutes of rae. extraction of chloride by rae and uae followed a similar trend. after the initial extraction, with increasing time, the chloride concentration fell, and then 1450 tasić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. rose to a constant level. the only difference was for the highest investigated time of extraction. for the rotary-assisted extraction, the maximum chloride concentration was achieved after 180 min. for the ultrasound-assisted extraction, the concentration of chloride extracted after 50 min was the same as that after 10 min extraction. in general, rae was more efficient for chloride extraction from fly ash than uae. table iii. the results of the determination of anions in fly ash (µg g-1) by rae/uae (n = 5, uncertainty as standard deviation) time, min fluoride chloride sulphate rae 30 29.12±0.04 13.85±0.04 6250±40 60 33.26±0.09 12.48±0.07 7234±100 90 29.42±0.90 14.85±0.30 6893±70 120 26.48±1.50 15.58±0.70 6813±50 180 28.66±0.80 18.14±0.90 6965±55 uae 10 30.38±1.20 14.52±0.55 6017±62 20 27.31±0.80 12.76±0.35 6467±97 30 29.62±0.40 13.16±0.20 5690±85 40 29.60±0.85 13.95±0.50 7762±80 50 30.70±0.58 14.12±0.20 7851±44 the concentration of sulphate in the extract increased until 60 min of rae, and then reached a maximum. further prolongation of the extraction time led to a decrease in the sulphate concentration, which then remained almost constant. the amount of sulphate anions extracted by uae gradually increased, but after 30 min, it dropped sharply. subsequently, the amount of sulphate sharply increased with extraction time and was then almost constant. comparing to the extraction of the anions from coal, the concentrations obtained by rae and uae indicated that fluoride condensed on fly ash.1,25 this was expected since fluorine volatilizes during coal combustion. on the other hand, after combustion, the fluoride in fly ash was present in soluble forms. the amounts of chlorine extracted from fly ash using rae and uae were lower than those obtained from coal. during combustion, depending on the conditions, chlorine in coal can form compound with various metals, which are less soluble.25 the sulphate extraction indicated that after combustion, sulphur in fly ash was in soluble forms since the pyrite in coal converts to sulphate. due to its dominant surface association in fly ash and the marked solubility of most sulphate-bearing compounds, sulphur is the major soluble element in fly ash, along with ca. under environmental conditions, reduced sulphur species are generally present in negligible quantities and sulphate is the dominant species.25 extraction of anions from coal and fly ash 1451 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. the amount of fluoride and chloride extracted using mae (table iv) decreased with increasing temperature. this was contrary to results obtained for the mae of coal. the reason could be the volatility of fluorine and chlorine compounds table iv. the results of the determination of anions in fly ash (µg g-1) after mae (n = 3, uncertainty as standard deviation) temperature, °c fluoride chloride sulphate 50 41.36±0.56 35.48±3.70 7731±135 100 35.60±0.90 22.94±3.60 8043±158 150 33.80±2.10 19.54±1.70 8164±114 present in fly ash. the microwave-assisted extraction of fluoride and chloride at 50 °c was more efficient than rae and uae. the concentration of sulphate in the extracts obtained by mae increased with increasing temperature. the extraction of sulphate from coal and fly ash had a similar trend. when temperature changes from 50 to 100 °c, more sulphate was extracted than for a further temperature increase of 50 °c. conclusions from the obtained results, it can be concluded that the techniques employed in this research as preparation steps of coal and fly ash samples are significant for estimating the concentration of trace elements and pollutants that could possibly be released into the environment. the results of ion measurements in the extraction solutions of coal by ic were reliable with standard deviations of less than 5 %. the fluoride content extracted using a rotary mixer and an ultrasonic bath were very similar and much lower than the quantity extracted in the microwave-assisted extraction at the highest investigated temperature (150 °c). the ultrasound-assisted extraction of chloride was more efficient than the rotary mixing extraction, and comparable with the microwave-assisted extraction. sulphate extraction using the ultrasound bath was slightly more efficient than by the rotary mixer, and the assistance of the microwave system did not influence the sulphate extraction significantly. the results of ultrasonic testing indicated that highest extracted quantities of chloride and sulphate were obtained after 30 min. the measurements of ph value and conductivity in the extracts suspensions showed that these parameters changed in the opposite manner after 30 min of ultrasound-assisted extraction. it was concluded that not only the extraction of anions influence these changes but captions extraction as well. comparing the microwave-assisted extraction at 150 °c to standard astm methods, it was found that recovery for fluoride was good, but not satisfactory, for chloride good and for sulphate very low. this led to the following conclusion: a) chloride in coal is present in various soluble mostly mineral forms; b) the applied temperature was not suitable for stable inorganic forms 1452 tasić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. of fluoride, and c) sulphate in the analyzed coal was present in sparingly soluble inorganic forms and insoluble organic forms. in general, the microwave-assisted procedure showed good performance for coal extraction, safety and high throughout, but further investigations are required to improve the accuracy and precision of the analysis. in general, the rotary-assisted extraction was more efficient for extraction of fluoride and chloride from fly ash than ultrasoundassisted extraction. the optimum extraction time for fluoride and sulphate using rotary-assisted extraction was 60 min, and for chloride, 180 min. increasing the temperature for microwave-assisted extraction led to increasing sulphate concentrations in the extract, while concentration of fluoride and chloride decreased. comparing to the extraction of anions from coal, the extraction from fly ash indicated that fluoride, chloride and sulphate condensed on the fly ash. supplementary material additional data and details related to coal, its combustion and extraction processes for solid samples and reagents, as well as validation of ic measurements, are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements this study was supported by ministry of education, science and technologic development of the republic of serbia, project nos. 172030, 176006 and iii43009. и з в о д екстракција анјона из угља и лебдећег пепела водом и њихово јон-хроматографско одређивање александра м. тасић1, ивана д. средовић игњатовић2, љубиша m. игњатовић1, данијел ђурановић1 и малиша п. антић2 1универзитет у београду, факултет за физичку хемију, студентски трг 12–16, 11000 београд и 2универзитет у београду, пољопривредни факултет, немањина 6, 11080 београд представљене су три технике екстракције анјона из угља и лебдећег пепела помоћу дејонизоване воде: употребом ротационо-механичке мешалице и ултразвука при различитим екстракционим временима, и микроталасне екстракције која је извођена при различитим температурама. валидацијом је утврђено да је јонска хроматографија метода која је погодна за одређивање анјона у екстрактима испитиваних узорака. промене количине екстрахованих анјона из угља и пепела, у току времена екстракције, су биле незнатне при примени ротационо-механичког мућкања. ефикасност ултразвучне екстракције је временски зависна и највећа је за 30 min. ултразвучна екстракција анјона из лебдећег пепела се показала мање ефикасном од ротационо-механичке. пораст температуре микроталасне екстракције има позитиван ефекат на количину екстрахованих свих анјона из угља као и на сулфате из пепела, док се количина флуорида и хлорида екстрахованих из пепела са порастом температуре смањује. резултати микроталасне екстракције анјона из угља на 150 °c су упоређени са добиjеним аstm стандардним методама и у доброј су сагласности само за одређивање хлорида. промене ph вредности и проводљивости током ултразвучне екстракције су мерене у циљу објашњења промена extraction of anions from coal and fly ash 1453 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. на површини честица угља у контакту са водом и различитих процеса који се дешавају у природним условима. 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bomb combustion/ion selective electrode method, astm international, west conshohocken, pa, usa, 1996 22. x. gao, y. hu, c. li, c. dai, l. li, x. ou, y. wang, sci. total environ. 545–546 (2016) 1 23. m. golda, a. janas, d. olszewska, fuel process. technol. 92 (2011) 1230 24. b. k. saikia, a. m. dutta, l. saikia, s. ahmed, b. p. baruah, fuel process. technol. 123 (2013) 107 25. j. wang, a. tomita, energy fuels 17 (2003) 954. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice the influence of interlayer interactions on the mechanical properties of polymeric nanocomposites j. serb. chem. soc. 80 (11) 1449–1459 (2015) udc 678.017+66.017:620.3:539.196:539.3 jscs–4810 original scientific paper 1449 the influence of interlayer interactions on the mechanical properties of polymeric nanocomposites mehrdad jabbarzadeh1* and amir reza golkarian2 1department of mechanical engineering, mashhad branch, islamic azad university, mashhad, iran and 2department of mechanical engineering, science and research branch, islamic azad university, tehran, iran (received 25 october 2014, revised 10 march, accepted 18 june 2015) abstract: in this study, the influence of the type of interlayer interactions on the elastic modules of multilayer graphene sheets (gs) and nanocomposites was investigated. the modeling and investigation of mechanical properties of graphite layers were performed using the molecular mechanics (mm) method. initially, for improving the model and decreasing the amount of computations, three types of elements, i.e., a beam, a linear spring and a nonlinear spring, were used. continuing, the mechanical properties of multilayers and nanocomposites were compared using three types of interlayer interactions. initially, a nonlinear spring defined by the leonard jones potential was used to define the interlayer interactions (ordinary case). then, a linear spring with a certain stiffness, to obtain an equal linear spring and to investigate the ultimate capacity of interlayer interactions in the translation of force, by increasing the stiffness of linear springs, was employed (chemical change). then, by omitting all van der waals interactions and the creation of defects in the graphite layers, covalent interlayer interactions (using the morse potential) were created. finally, van der waals and covalent interlayer interactions were created spontaneously to study the properties of multilayers and nanocomposites (functionalization). the results were compared with other available literature data in order to validate the modeling. keywords: structural mechanics approach; graphene sheet; elastic modules; vacancy defect; functionalization. introduction graphene sheets (gs) and carbon nanotubes (cnt) have attracted the attention of many researchers and scientists due to their wonderful properties and abundant applications in industry, medicine, martial science and other fields.1–6 besides their unique mechanical, thermal and electrical properties, their significant capabilities in other fields have attracted further attention. among these * corresponding author. e-mail: jabbarzadeh@mshdiau.ac.ir doi: 10.2298/jsc141025054j 1450 jabbarzadeh and golkarian properties, their potential to be used as a reinforcement agent in polymeric composites and producing nanocomposites with exclusive properties could be mentioned. high tensile strength beside the lightening and high flexibility of these products is one of their extraordinary mechanical properties that make them unique. besides the mentioned benefits, weak interlayer interactions, which persuade researchers to demonstrate the functionalization idea or chemical changes,7–12 should be noticed. modeling approaches for these nanomaterials are needed, due to their small scales and expensive equipment in this field. one of the most attractive methods in this field is the molecular mechanic (mm) method, which was introduced for the first time by li and chou13 who used linear beam elements to simulate cnts. subsequently, many researchers devoted their time to improving this field by working on the type and quality of the employed elements. among the studies performed in this field, one by meo and rossi,14 who introduced spring elements as a suitable replacement for beam elements, could be mentioned. afterwards, georgantzinos et al.15 presented the idea of using fully nonlinear spring-based models, which were then improved further by other researchers.16–20 to date, nonlinear spring elements were commonly used to simulate interlayer interactions in nanocomposites.21–23 rafiee et al.24 studied the idea of functionalization of cnt nanocomposites using linear beam elements (introduced by the amber potential) as covalent interlayer interactions and reported that this action decreased the elastic modulus of cnts and reinforced nanocomposites. the focus of this study was the important problem that the connector between the base material and the reinforcement (cnt) agent are weak van der waals interactions, which cannot translate the maximum power of the reinforcement to the base. therefore, in this study, the effect of using different types of interlayer interactions was investigated. the most important goals were the calculation of the maximum limit of load transferring and the study of the influence of different types of functionalization (introduced by many researchers).7–12 the most important objectives investigated were: – the influence of different types of mechanical elements used to simulate monolayer gs. – the influence of four different types of interlayer interactions on the mechanical properties of multilayer gss and nanocomposites, i.e.: a) van der waals interlayer interactions using nonlinear spring elements defined by the leonard– –jones potential (ordinary case), b) linear spring elements, whose the stiffness of which increased at each step to obtain the ultimate value of translating force (chemical change), c) covalent interlayer interaction using a linear spring element defined by the morse potential energy and d) a combination of van der waals and covalent bonds. interlayer interactions in polymeric nanocomposites 1451 – a comparison between the reinforcing effect of monolayer and multilayer gss in nanocomposites. considered models considered models, theoretical background and calculations are described in supplementary material to this paper. results and discussion monolayer gs the results related to the elastic module of a monolayer gs for different types of mechanical elements are given in table i. with change in the mechanical element from linear beam to linear spring, the influence of the defined cross section area could be obtained. by replacing a nonlinear spring with a linear spring, the influence of the definition of the nonlinear behavior for the employed elements could be computed. it was observed that by a replacing a linear beam with a linear spring, the elastic module in model i increased by 11 % and in model ii increased by 17 %. this shows that by assuming a cross section area for the element, a significant change was observed in the elastic module. in addition, in this step, the time of the computations for both cases was the same. to continue, by replacing a nonlinear spring with a linear spring, the elastic module of both models increased by about 3 %. this showed that by defining nonlinear behavior for the elements, the results did not show significant changes but the time of the computations was very long, which further increased on increasing the dimensions of the sheets. therefore, a linear spring element could be introduced as a suitable element to simulate interatomic interactions of gss, because they make less error and require shorter computation times. table i. variation of the elastic module of a gs in dependence on the type of the mechanical element elastic module, tpa type of the mechanical element model i: 1.096 linear beam model ii: 1.078 lit.25: 1.025 model i: 1.217 linear spring model ii: 1.269 lit.26: 1.367 model i: 1.255 nonlinear spring model ii: 1.308 lit.18: 1.245 double-layer gss interlayer interaction: type i. gs used at this step is perfect and its elastic module is about 1.2175 tpa. by defining a second layer at a distance of 0.34 nm 1452 jabbarzadeh and golkarian from the first one and the creation of van der waals interaction between the layers with a cut-off distance of 0.38 nm, the elastic module increased to 1.2275 tpa. this result is in good agreement with the results reported by other researchers (table ii). table ii. a comparison of the results related to the elastic module of double-layer gss with van der waals interlayer interactions elastic module of a double-layer, tpa elastic module of a monolayer, tpa study 1.2275 1.2175 present study 1.2537 1.2447 golkarian and jabbarzadeh18 1.035 1.025 li and chou13 1.032 1.030 bao et al.27 interlayer interaction: type ii. the results obtained at this step (table iii) showed that at a stiffness of 300 nn nm–1, the elastic module is about 1.2263 tpa, which is in good agreement with the value 1.2257 tpa related to the previous step and showed that this stiffness is a good replacement for nonlinear van der waals interactions. it was observed that at a stiffness of about 108 nn nm–1 (330000 times stronger than 300 nn nm–1 equal to the van der waals forces), the elastic module of double-layer gs increased to 1.59 tpa, showing a 30 % increase. it could be deduced that if the maximum increase of 30 % was satisfactory and the complication of working on the power of interlayer interactions was possible, chemical work on the interlayer interactions could be helpful. table iii. variation of the elastic module of double-layer gss from the stiffness of linear springs as interlayer interactions elastic module, tpa stiffness, nn nm-1 1.2175 0 1.2176 1 1.2178 10 1.2205 100 1.2263 300 1.2452 103 1.5513 104 1.5905 106 1.5954 108 1.5955 1010 1.5955 1012 interlayer interaction: type iii. in the third step, by defining a defect in each layer, the elastic module of each layer was reduced to 1.1979 tpa; this negligible decrease is in the range of results reported by other researchers (table iv). by defining van der waals forces between two layers, the elastic module of the interlayer interactions in polymeric nanocomposites 1453 layers increased to 1.2060 tpa. if the layers were linked together by covalent bonds, the elastic module increased to 1.1988 tpa and when a combination of van der waals and covalent bonds was used, the elastic module increased to 1.2067 tpa, which shows a 0.7 % increase in comparison to defective monolayers and 0.9 % reduction if compared to the perfect ones. table iv. variation of elastic module of a monolayer gs caused by defining a defect defective elastic module, tpa perfect elastic module, tpa study 1.1979 1.2175 present study 0.990 1.032 rafiee & pourazizi24 0.77 0.79 ansari et al.28 1.036 1.042 scalante et al.29 the results showed that by defining a defect in the layers, the elastic module showed a 1.6 % reduction and that the reduction after functionalization by defining covalent and van der waals forces was 1.5 %, which is a negligible difference. from these results, it could be deduced that functionalization in order to repair existing defects or after defining the defects in the model, to form covalent interlayer interactions cannot improve the mechanical properties of the model. therefore, other ways, such as a change in the type of interlayer interaction, should be considered. nanocomposite monolayer reinforcement. interlayer interaction: type i. in this step, a gs with an elastic module of 1.2175 tpa was imported into a polymeric base with an elastic module of 3.5 gpa and they were coupled with van der waals forces. the elastic module of nanocomposite at this step was 63.7 gpa, which is in good agreement with the result of 64.2 gpa obtained from rom. in a same research performed by rafii-tabar and montazeri21 for a polymeric base with an elastic module of 3.5 gpa, the elastic module of nanocomposite was reported to be about 59.536 gpa, which is in suitable agreement with the results obtained in this study. moreover, the elastic module of the polymeric base (3.5 gpa) in this step was found to be 3.67 gpa, which was a 4.8 % increase. interlayer interaction: type ii. by using linear springs instead of van der waals forces and increasing its stiffness, no significant change was observed in the translate ratio from reinforced to polymeric base (table v). it could be seen that the maximum elastic module of the nanocomposite in a stiffness of 300 nn nm–1 (the same as when van der waals forces existed) was obtained, which was equal to the 63.68 gpa. the maximum value for the elastic module of polymeric base was about 3.678 gpa, i.e., a 1.5 % increase. interlayer interaction: type iii. by omitting all van der waals forces, defining a defect and creating three covalent bonds, the elastic module of the nanocomposite and the polymeric base were about 62.69 gpa and 3.5 gpa, respect1454 jabbarzadeh and golkarian ively. by defining, a second defect and the next three covalent bonds (bidirectional), the elastic module of the nanocomposite and of the polymeric base were 61.69 and 3.5 gpa, respectively. by defining van der waals forces (together with bidirectional covalent bonds), the elastic modules reached 61.71 and 3.6 gpa, respectively. from these results, it can be deduced that the functionalization of nanocomposites reinforced by monolayer gs does not lead to an improvement of the elastic module of the nanocomposite. table v. variation of elastic module of polymeric base according to the stiffness of a linear spring as an interlayer interaction (monolayer reinforcement) elastic module of polymeric base, gpa stiffness, nn nm-1 3.5 0 3.557 0.1 3.67 10 3.677 300 3.678 1000 3.678 104 double-layer reinforcement. interlayer interaction: type i. by importing a double-layer gs in polymeric base, the elastic module of nanocomposite increased to 64.11 gpa, which is a 0.7 % increase. in this step, the elastic module of polymeric base decreased to 3.6 from 3.67 gpa for a monolayer reinforcement. this showed that increasing the number of layers in the presence of van der waals forces as interlayer interactions was inefficient. from the results of this step and the previous steps (related to the functionalization by using monolayer reinforcement), it can be deduced that the functionalization of double-layer gs would be inefficient, because the increase in the number of layers did not improve the elastic module of the nanocomposite and also defect creation (in order to make covalent bonds) decreased the elastic module of the nanocomposite. interlayer interaction: type ii. by increasing the stiffness of all interlayer interactions including the interactions gs–gs and gs–polymeric base, the elastic module of the nanocomposite increased to 82.5 gpa, i.e., a 30 % increase (in a stiffness of 1 nn nm–1) (table vi), which is equal to the time that the elastic module of double-layer gs reached its maximum value. in addition, the elastic module of the polymeric base decreased to 3.605 gpa (in stiffness of 100 nn nm–1) from 3.67 gpa related to the use of monolayer reinforcement (table vii). this shows that increasing the number of layers and the stiffness of interlayer interactions cannot improve the elastic module of the polymeric base. some of the important points that can be deduced from the results are: 1. making a defect in the graphene layer for replacing three carbon–carbon covalent bonds instead of three weak van der waals interlayer interactions to inc interlayer interactions in polymeric nanocomposites 1455 rease the elastic module of nanocomposite or multilayers is a useless operation. this is because the decreasing effect of making a defect in a layer on the elastic module more than decreases the effect of this type of functionalization. table vii. variation of the elastic module of the polymeric base in dependence on the stiffness of the linear spring elements as interlayer interaction (double-layer reinforcement) elastic module of the polymeric base, gpa stiffness, nn nm-1 3.5 0 3.585 1 3.602 10 3.605 100 3.605 1000 2. by increasing the strength of interlayer interactions (which includes each type of functionalization or chemical changes), a maximum increasing effect of 30 % in the elastic module of nanocomposites reinforced by multilayer graphene sheets is estimated. of course, this results may have some changes in experimental studies because, in this case, the influence of many graphene layers as a multilayer gs was only investigated and the influence of some important parameters, such as the contribution, dispersion, local density, direction of the layers and some other parameters that could not be considered in the atomic mechanical modeling and appear only in real, experimental investigations. 3. this increasing effect is when the employment of multilayer graphene layers (in the experimental works most of the employed graphene layers are also multilayers, because monolayers are rarely available) and increasing the strength of interlayer interactions between a monolayer graphene sheet and a polymeric base does not lead to a significant increasing effect in the elastic module of the nanocomposite. table vi. variation of elastic module of nanocomposite according to the stiffness of the linear spring element as an interlayer interaction (double layer reinforcement) elastic module of nanocomposite, gpa stiffness, nn nm-1 63.7 0 63.68 1 63.69 10 63.74 100 64.18 1000 67.39 104 76.72 105 81.68 106 82.41 107 82.48 108 82.5 109 1456 jabbarzadeh and golkarian among theoretical studies performed in this case, the study performed recently by rafiee and rourazizi24 could be mentioned, in which they used the molecular mechanic modeling method and reinforced a polymeric cylindrical matrix with monolayer carbon nanotube and investigated the influence of this type of functionalization (making defect and replacing c–c covalent bonds instead of van der waals interlayer interactions) and reported the same results. they reported the decreasing effect of this type of functionalization on the elastic module of a cylindrical polymeric base reinforced by single layer carbon nanotube and they explained that the effect of functionalization cannot be observed on the micro scale but its improving effect may appear on the meso or macro scale. therefore, in the present study, time was devoted to investigate the effect of each type of functionalization (by increasing the strength of interlayer interactions up to its highest level) for both mono and multilayer graphene sheets. it was found that in the case of multilayers, an up to 30 % increase in the elastic module of the nanocomposite was possible by increasing the effect but in the case of monolayer reinforcements, no increase was observed in the case of graphene sheet, but not with carbon nanotubes as a general result, it could be deduced that, maybe, more attention should be paid to the interactions between the graphene layers than those between the graphene layer and the polymeric base. however, this objective cannot be strongly emphasized because, as explained previously, there are some crucial parameters that cannot be considered using this modeling approach. conclusions in this paper the influences of three types of interlayer interactions on the elastic module of multilayer gss and nanocomposites reinforced by monolayer and double-layer gs were studied. the following important cases were considered in this investigation: the influence of type of element used for simulating of gss on the elastic module and the amount of computations. the influence of type of element used for simulating interlayer interactions in double-layer gss and nanocomposites reinforced by monolayer and double -layer gs. using four types of interlayer interactions: – van der waals interlayer interaction using nonlinear spring element defined by the leonard–jones potential to validate the modeling method, – linear spring elements, the stiffness of which was increased at each step to find the ultimate value of the possible force translation ratio, – omitting all van der waals forces, defining defects in the layers and make three covalent interlayer interactions by defining each defect and interlayer interactions in polymeric nanocomposites 1457 – combination of van der waals and covalent interlayer interactions. the influence of an increase in the number of layers on the reinforcement of the nanocomposites. the maximum amount of possible force translation ratio from reinforcement to polymeric base (chemical changes). some of the important results are: – the linear spring element is in best agreement with other results and requires the lowest computations time. – making defects and replacing van der waals interlayer interactions with c–c covalent bonds cannot improve the elastic module of multilayer gss and nanocomposites. this means that the decreasing effect of making defects is more than the increasing effect of the replacement by covalent interlayer interactions. – chemical changes (functionalization) in interlayer interactions under the best conditions can lead to an increase of about 30 % in the elastic module of multilayer gss and nanocomposites reinforced by multilayer gss. – improving the elastic modules of nanocomposites due to the functionalization (reported by experimental works) is the consequence of functionalization of interlayer interactions between graphene layers not between graphene layer and polymeric base. – improving the quality of interlayer interactions cannot help to improve the elastic module of polymeric base. consequently, it could be deduced that by using multilayer gss and improving the strength of the interlayer interactions, significant increases in the elastic module of double-layer gss and also nanocomposites reinforced by multilayer gss of up to 30 % could be expected. in addition, improving the strength of the interlayer interactions or increasing the number of layers does not lead to an improvement in the elastic module of the polymeric base. supplementary material details of the considered models, their theoretical background and method of calculations are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. the authors wish to thank the islamic azad university of mashhad for financing the project “the influence of quality of interlayer interactions on the mechanical properties of polymeric nanocomposites”. 1458 jabbarzadeh and golkarian и з в о д утицај интеракција међуслоја на механичка својства полимерних нанокомпозита mehrdad jabbarzadeh и amir reza golkarian department of mechanical engineering, mashhad branch, islamic azad university, mashhad, iran проучаван је утицај типова интеракције међуслоја на еластичне модуле вишеслојног графена (gs) и нанокомпозита. моделовање и испитивање механичких својстава графитних слојева изведено је методом молекулске механике (мм). најпре су, због побољшања модела и смањења обима израчунавања, коришћена три елемента: зрак, линеарна и нелинеарна опруга, а у наставку су упоређена механичка својства вишеструких слојева и нанокомпозита применом три типа интеракције међуслоја. прво је коришћена нелинеарна опруга дефинисана leonard–jones потенцијалом да би се дефинисале интеракције међуслоја (ординарни случај). затим је примењена донекле пригушена линеарна опруга (еквивалентна линеарна опруга) и повећавањем пригушености линеарних опруга испитао крајњи капацитет интеракције међуслоја у транслацији силе (хемијска промена). уз то су, у једном случају занемарене све van der waals интеракције и настајање дефеката у графитним слојевима, те је оно доводило до ковалентних интеракција међуслоја (morse потенцијал), а у другом , van der waals и ковалентне интеракције међуслоја креиране су спонтано да би се проучила својства вишеструких слојева и нанокомпозита (функционализација). резултати су поређени са подацима из литературе ради валидизације моделовања. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice dynamics of cetyltrimethylammonium bromide-mediated reaction of phenylsulfinylacetic acid with cr(vi): treatment of pseudo-phase models j. serb. chem. soc. 80 (8) 1019–1034 (2015) udc 547.551.41+546.766+547.288.1+ jscs–4777 546.141:542.9:531.3 original scientific paper 1019 dynamics of cetyltrimethylammonium bromide-mediated reaction of phenylsulfinylacetic acid with cr(vi): treatment of pseudo-phase models perumal subramaniam1* and natesan thamil selvi2 1research department of chemistry, aditanar college of arts and science, tiruchendur-628 216, tamil nadu, india and 2govindammal aditanar college for women, tiruchendur-628 215, tamil nadu, india (received 19 september, revised 28 december, accepted 31 december 2014) abstract: the influence of cetyltrimethylammonium bromide, ctab, on the oxidative decarboxylation of phenylsulfinylacetic acid, psaa, and several metaand para-substituted psaas by cr(vi) was investigated in 95 % h2o–5 % ch3cn medium. the rate profile displayed a peculiar trend with an initial rate increase at low ctab followed by sharp rate inhibition at higher ctab concentrations. the initial rate acceleration could be explained by strong binding of so4 2on the positively charged micellar surface. the specific partitioning of psaa in the micellar phase by hydrophobic interaction and the oxidizing species hcro3 + in aqueous phase by electrostatic repulsion accounted for the rate retardation at higher ctab concentrations. the hammett plot with different substituted psaas showed excellent correlation affording negative ρ value, which supports the proposed mechanism involving the intermediate formation of sulfonium cation. the obtained ρ value in ctab medium was found to be slightly lower than that in aqueous medium. quantitative analysis of the rate data for the inhibition shown by ctab was performed using the menger–portnoy and the piszkiewicz pseudo-phase models. the binding constant for psaa with micelles was evaluated from the piszkiewicz cooperative model. keywords: phenylsulfinylacetic acid; cr(vi); ctab micellar effect; hammett correlation; piszkiewicz cooperative model. introduction surfactants and their micellar aggregates exhibit widespread applications in chemical, biochemical, pharmaceutical and industrial fields. cationic surfactants are useful as antifungal, antibacterial and antiseptic agents and have attracted much attention with reference to their interaction with dna and lipids.1 the * corresponding author. e-mail: subramaniam.perumal@gmail.com doi: 10.2298/jsc140916001s _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1020 subramaniam and thamil selvi fascinating feature of aqueous surfactant solution is their self-organization into micelles above the critical micellar concentration, cmc. this leads to micellar aggregates having an architecture in which the hydrophobic groups of surfactants occupy the interior while the hydrophilic head groups at the surface are in contact with bulk water. in the case of ionic micelles, the interface is charged giving rise to an electrical double layer with a potential difference up to hundred of millivolts between the micellar pseudo-phase and water. thus, the electrostatic potential and polarity prevailing in the interior of the aggregate differ from those of the bulk aqueous phase. the redox processes in micellar media are considered as models to obtain insight into the dynamics and mechanistic paths of several redox processes occurring in complex biological systems.2–5 micellar systems were used in a number of studies, including oxidation reactions, due to their effectiveness in altering the rate of the reactions by their inherent characteristics of their surface activity.6–11 micelles act as nano reactors that may drastically modulate the reactivity of entrapped reactants. the importance of micelles arise from the fact that micelle catalyzed reactions resemble enzyme catalyzed reactions in several aspects.12 the investigation of coupled systems composed of electron transfer and micelle-forming surfactant may contribute in a unique way to the understanding of the redox processes. ionic micelles typically catalyze the reactions of a reactive counter ion with hydrophobic substrates that bind to the micelles.13–16 for bimolecular reactions, inhibition arises from the incorporation of one reactant into the micellar pseudo-phase and exclusion of the other from it. although there are several reports in the literature on the investigation of micellar effects on the redox reactions of organic sulfur compounds,16–21 the corresponding study on sulfur-containing carboxylic acids is limited. even though phenylsulfinylacetic acid (psaa) shows a wide range of synthetic utility, no reports are available in the literature on the oxidation of psaa except a few recent works.22–25 hence, this investigation of the redox reaction between psaa and cr(vi) in the presence of cationic surfactant cetyltrimethylammonium bromide (ctab) is of great interest. the substituent and micellar effects are highlighted and the rate data in the presence of ctab were subjected to menger– portnoy and piszkiewicz kinetic pseudo-phase models. experimental preparation of phenylmercaptoacetic acids phenylmercaptoacetic acids, the precursors used for the synthesis of the psaas, were prepared from the corresponding thiophenols.26,27 the appropriate amount of thiophenol (0.05 mol) dissolved in 10 ml of 20 % sodium hydroxide solution was mixed with 4.7 g of chloroacetic acid dissolved in 10 ml of water without allowing the temperature to rise and then the mixture was heated in an oil bath at 120–130 °c for 5 h. the solution was cooled, acidified with 50 % hcl and the phenylmercaptoacetic acid obtained as solid was recrystallized from _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ ctab-mediated psaa-cr(vi) redox reaction 1021 water. the melting points of phenylmercaptoacetic acids were determined and checked with the corresponding literature values.28 preparation of phenylsulfinylacetic acids psaas and ten metaand para-substituted psaas were prepared by the controlled oxidation of the corresponding phenylmercaptoacetic acids using 30 % h2o2. 29 the temperature was maintained at 40 °c and the reaction mixture was stirred until all the h2o2 had been consumed. at the end of the reaction, a clear and colorless syrupy liquid was obtained which was kept overnight. after evaporation of water under reduced pressure, a white solid was obtained. the solid was digested with hot benzene for a few minutes and then recrystallized. for the recrystallization of paraand meta-methyl psaas, chcl3–petroleum ether (1:1) mixture was used, while ethyl acetate–benzene (1:1) solvent mixture was used for the recrystallization of psaa and the other metaand para-substituted psaas. the recrystallized psaas were dried and their melting points were determined and compared with the corresponding literature values.28 the purity was also checked by lc-ms. the recrystallized samples were stored in a vacuum desiccator and used for kinetic studies. kinetic measurements all the kinetic runs were performed under pseudo-first-order conditions by maintaining a large excess of psaa over the concentration of cr(vi). the progress of the reaction in ctab medium was monitored by following the rate of decrease of [cr(vi)] spectrophotometrically at 360 nm. in order to avoid solubility problems, the reaction was conducted in a 95 % water– –5 % ch3cn medium. several researchers used similar solvent systems to study the micellar effect in the oxidation of organic sulfides.17,21,30-32 as the reaction mixture in the presence of ctab produced turbidity with perchloric acid, in order to circumvent the solubility problem, sulfuric acid and potassium sulfate were used to maintain the h+ concentration and ionic strength, respectively. results and discussion effect of reactants on the reaction rate the kinetic study at different initial concentrations of reactants, [cr(vi)], [psaa] and [h+], at fixed concentrations of the other substances showed first -order dependence on cr(vi), psaa and h+. the constancy of the pseudo-first -order rate constant values with increasing concentration of cr(vi) in the presence of ctab (table i) and excellent linear plots of log [cr(vi)] against time are in agreement with the first-order dependence of the reaction on the cr(vi) concentration. the observed trend of the constant k1 with varying cr(vi) contable i. effect of the cr(vi) and psaa concentrations on the rate of the ctab-mediated reaction. [h+] = 0.50 mol dm-3, [ctab] = 5.0×10-2 mol dm-3, i = 0.65 mol dm-3 [psaa] / 10-2 mol dm-3 [cr(vi)] / 10-4 mol dm-3 k1 / 10 -5 s-1 k2 / 10 -3 mol-1 dm3 s-1 3.0 5.0 3.16±0.01 1.05±0.03 5.0 5.0 6.03±0.01 1.21±0.02 7.0 5.0 7.86±0.01 1.12±0.01 10 5.0 11.6±0.02 1.16±0.02 5.0 3.0 6.31±0.01 1.26±0.02 5.0 7.0 6.15±0.02 1.23±0.04 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1022 subramaniam and thamil selvi centrations is contradictory with that of the reaction in the absence of micelles, where a negative effect with concentration of cr(vi) was registerd.23 the observed constancy of the k1 values in the ctab-mediated reaction indicates that dimer formation was prevented by the surfactant molecules. the unit-order dependence on psaa was evidenced by the unit slope of the double logarithmic plot of k1 vs. [psaa] and invariant second-order rate constant values at different psaa concentrations (table i). the first-order dependence with respect to h+ in the presence of ctab is assessed from the unit slope of the log–log plot of k1 vs. [h+] and the constant values of k1/[h+] (table ii). table ii. effect of the h+ concentration and temperature on the rate of the reaction; [psaa] = = 5.0×10-2 mol dm-3, [cr(vi)] = 5.0×10-4 mol dm-3, [ctab] = 5.0×10-2 mol dm-3, solvent: 95 % h2o–5 % ch3cn (v/v) [h+] / mol dm-3 k1 a×105 s-1 k1×104/[h+] mol-1 dm3 s-1 t °c k2 b×104 mol-1 dm3 s-1 0.25 2.64±0.02 1.06±0.08 20 3.61±0.03 0.35 3.65±0.01 1.04±0.03 25 7.20±0.01 0.50 6.03±0.01 1.20±0.02 30 12.1±0.02 0.75 7.84±0.02 1.12±0.03 35 18.2±0.03 ∆h = 80.0±3.25 kj mol-1 ∆s = –37.3±11.3 j mol-1 k-1 ai = 0.80 mol dm-3; bi = 0.65 mol dm-3, [h+] = 0.50 mol dm-3 effect of temperature and activation parameters in order to evaluate the activation parameters, the reaction in ctab medium was performed at four different temperatures, viz., 20, 25, 30 and 35 °c. the second-order rate constants, k2 and the activation parameters, ∆s and ∆h were evaluated respectively from the intercept and slope of the eyring plot of log k2/t vs. 1/t and are presented in table ii. the value of the entropy of activation is useful for the interpretation of the structure of transition state. a comparison of ∆s with that of the reaction in aqueous medium (∆s = –24.5 j mol–1 k–1) shows that the reactant molecules tend to associate in a well-structured activated state in the micellar medium with less degrees of freedom. effect of variation of the ctab concentration in order to explore the role of the ctab surfactant micelles on the reaction rate, the reaction was studied as a function of the ctab concentration at constant concentrations of cr(vi), psaa and h+ at 30 °c. the effect of the surfactant ctab on the rate of the reaction and the calculated pseudo-first-order rate constants are presented in table iii. the variation of the ctab concentration showed two distinct effects on the rate of the reaction, i.e., an initial increase in rate up to 0.5×10–2 mol dm–3 followed by a steady downward trend. there was a well-defined maximum in the rate profile at 0.5×10–2 mol dm–3 of ctab. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ ctab-mediated psaa-cr(vi) redox reaction 1023 table iii. effect of the ctab concentration on the rate of the reaction. [psaa] = 5.0×10-2 mol dm-3, [cr(vi)] = 5.0×10-4 mol dm-3, [h+] = 0.50 mol dm-3, solvent: 95 % h2o–5 % ch3cn (v/v), i = 0.65 mol dm -3 [ctab] / 10-2 mol dm-3 k1 / 10 -5 s-1 0 8.35±0.02 0.10 8.72±0.01 0.25 9.37±0.02 0.50 10.9±0.04 0.75 10.2±0.07 1.0 10.0±0.02 2.0 8.22±0.04 3.0 7.13±0.03 5.0 6.03±0.01 7.0 4.93±0.02 9.0 3.82±0.02 10 3.31±0.05 12 2.50±0.03 substituent effect the study of the influence of substituents on the rate of a reaction often provides insight into the nature of the transition state and mechanism. to probe the reaction mechanism in the cationic micellar medium, the substituent effect was studied with several metaand para-substituted psaas at 30 °c in the presence of ctab. the investigation showed that the rate of the reaction was accelerated by electron-donating groups and retarded by electron-withdrawing groups present in the phenyl ring of psaas. this indicates that electron-donating substituents enhance the nucleophilicity of the sulfur and facilitate the attack by the oxidizing species in the rate-determining step. a hammett plot (fig. 1) drawn for the kinetic data (table iv) obtained in ctab medium at 30 °c shows excelfig. 1. hammett plot for ctab mediated reaction at 30 °c; ρ = = –0.864±0.03; r = 0.996. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1024 subramaniam and thamil selvi lent correlation with the σ values affording negative ρ value (–0.864), which is lower in magnitude than that of the reaction in the absence of ctab. this showed that the reaction in ctab micellar medium was less sensitive to substituent effects, which is against the reactivity–selectivity principle. however, the obtained ρ value is comparable with that of the reaction in the presence of sodium dodecyl sulfate (sds).25 the observed negative reaction constant supports the generation of an electron deficient sulfur center in the transition state and the linear relationship proves the operation of same mechanism in all the substituted psaas. table iv. second-order rate constants for the reactions of substituted psaas with cr(vi) in ctab medium; [x-psaa] = 3.0×10-2 mol dm-3, [cr(vi)] = 5.0×10-4 mol dm-3, [h+] = 0.50 mol dm-3, i = 0.65 mol dm-3, t = 30 °c cmpd. no. x k2 / 10 -4 mol-1 dm3 s-1 k2 ctab/k2 aq. 1 m-br 5.35±0.10 0.054 2 m-cl 6.12±0.05 0.059 3 m-f 5.66±0.04 0.046 4 p-cl 8.04±0.02 0.047 5 p-br 7.47±0.02 0.041 6 p-f 9.88±0.01 0.041 7 h 12.1±0.02 0.039 8 m-ch3 13.3±0.03 0.032 9 p-ch3 17.2±0.02 0.032 10 p-oc2h5 19.4±0.05 0.033 11 p-och3 20.1±0.02 0.029 product analysis the organic product of the reaction was identified from lc-ms and ft-ir spectral studies as (methylsulfonyl)benzene. the recorded lc-ms spectrogram shows that the product eluted at a retention time of 1.829 min ionized in the atmospheric pressure chemical ionization (apci) (+) mode at m/z 157 with a relative abundance of 86 %, which corresponds to the mass of methyl phenyl sulfone (m/z = 156). the ir spectrum of the product showed strong absorptions for the symmetric and asymmetric stretching vibrations of the >so2 group at 1148 and 1290 cm–1, respectively. the final product of cr(vi) was identified as cr(iii) from the absorption spectrum of the reaction mixture after completion of the reaction, which displayed two absorption peaks in the region 410 and 580 nm (fig. 2) corresponding to the d–d transitions of cr(iii). this finding is in contradiction with the results observed in the reaction without micelles when a blue shift was observed in the absorption peaks of cr(iii).23 it was assumed that in the absence of ctab, cr(iii) formed a complex with the organic product, methyl phenyl sulfone. in a ctab micellar medium, methyl phenyl sulfone may be solubilized deep in the micelle by hydrophobic interaction while the cr(iii) ion _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ ctab-mediated psaa-cr(vi) redox reaction 1025 existed in the aqueous phase as the result of electrostatic repulsion. thus, the methyl phenyl sulfone became inaccessible for complexation with and hence there was no shift in the characteristic peaks of the cr(iii) ion in the absorption spectrum of the product. fig. 2. absorption spectrum of: a) free cr(iii) ion, b) the reaction mixture in ctab after completion and c) the reaction mixture without ctab after completion. reaction mechanism in micellar media the kinetic results reveal that the redox reaction of psaa and cr(vi) follows the rate law: + d[cr(vi)] = [psaa][h ][cr(vi)] d k t − (1) from the kinetic evidence, it is clear that the redox reaction between psaa and cr(vi) in ctab micelle follows unit-order with respect to cr(vi), psaa and h+, as observed in the absence of micelles. based on these kinetic observations, the substituent effect and the formation of methyl phenyl sulfone as the product, it is concluded that the same reaction mechanism as in aqueous medium,23 was operative in ctab micellar medium (scheme 1). scheme 1 satisfactorily explains the retardation effect observed in cationic micellar medium. the positive charge on the cationic micelle, ctab prevents the approach of the protonated species of cr(vi) and also disfavors the formation of positively charged sulfonium ion intermediate (i) in the rate determining step by coulombic forces. the observed micellar effect thus favors the proposed mechanism involving the formation of positively charged sulfonium ion intermediate because of nucleophilic attack of sulfur atom of psaa on cr of hcro3+. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1026 subramaniam and thamil selvi scheme 1. mechanism of oxidative decarboxylation of phenylsulfinylacetic acid. to afford spectral evidence for the mechanism of the reaction of psaa with cr(vi) in micellar media, the reaction mixture in the presence of ctab was scanned in the uv–vis region at different time intervals (fig. 3). the uv–vis absorption spectra of the reaction mixture in ctab micellar medium displayed a similar pattern of absorption peaks to that in the absence of micelles, which may be taken as positive evidence for the existence of the same type of intermediate both in the aqueous and micelle-mediated reactions. the change in the absorption spectra of cr(vi) with psaa, the significant hyperchromic shift and the widen fig. 3. variation of absorbance of the reaction mixture with time. [psaa] = 5.0×10-2 mol dm-3, [cr(vi)] = 5.0×10-4 mol dm-3, [h+] = 0.50 mol dm-3, [ctab] = 5.0×10-2 mol dm-3. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ ctab-mediated psaa-cr(vi) redox reaction 1027 ing of the peak in the region 200 to 300 nm for the reaction mixture (fig. 3) also confirmed the existence of the cr(vi)–psaa complex (i) having a direct s–cr bond, as in the case of the reaction in the absence of ctab. the uv–vis absorption spectrum of cr(vi) in ctab medium showed a hyperchromic shift in the region of 200 to 300 nm and a hypochromic shift near 350 nm. furthermore, a red shift of the wavelength from 351 to 360 nm was observed in the spectrum of cr(vi) in ctab medium, which indicates that the absorption spectrum of cr(vi) was affected by the surfactant ctab (fig. 4). although this spectrum appears to be similar to the one in sds medium,25 a notable difference in sds medium was the observation of hyperchromic shifts in both the peaks of cr(vi). the similarity of absorption spectra of the reaction mixture in both sds and ctab media demonstrated the involvement of the same transition state intermediate in both cases. fig. 4. absorption spectra of: a) cr(vi), b) cr(vi) in ctab and c) reaction mixture; [psaa] = 5.0×10-2 mol dm-3, [cr(vi)] = 5.0×10-4 mol dm-3, [h+] = 0.50 mol dm-3, [ctab] = 5.0×10-2 mol dm-3. interpretation of the micellar effect the effect of ionic micelles on the reaction rate of bimolecular reactions is mainly due to the association or incorporation of reactants through electrostatic or hydrophobic interactions within the small volume of the self-assemblies.12 in the present cationic micellar medium, the positively charged head groups of ctab prevented the approach of the oxidizing species hcro3+ by electrostatic repulsion and so the oxidizing species remained in aqueous phase. the other reactant psaa that is neutral preferably partitioned in the micellar pseudo-phase of ctab by hydrophobic interaction, and hence, became unavailable for the reaction with hcro3+ in aqueous phase. thus, the overall retarding effect obs_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1028 subramaniam and thamil selvi erved with increasing ctab concentration above 0.5×10–2 mol dm–3 was due to preferential partitioning of psaa and hcro3+ in the micellar pseudo-phase and aqueous medium, respectively, followed by a decrease in stoichiometric concentration of both reactants in both the phases. consequently, the reaction rate in both aqueous and micellar phases decreased that led to an overall rate retardation. moreover, during the course of the reaction, a positive charge developed on the sulfur due to electron transfer from the sulfur atom of psaa to cr(vi), which was prevented at higher concentrations of ctab. the increase in rate constant at low concentrations of ctab up to 0.5×10–2 mol dm–3 is interesting and this may be due to a specific salt effect. as sulfuric acid and potassium sulfate are used, respectively, to maintain the h+ concentration and ionic strength in the ctab medium, the reaction mixture contained excess of so42–. the trend of binding efficiency of counter ions with micelles follows the hofmeister series.33 as so42– is positioned in the place of higher order in the hofmeister series, the so42– has a high probability of binding with the positively charged ctab. it was shown that strong binding of so42– to the micellar surface of cetyltrimethylammonium ion yields micelle properties which differ considerably from those with other counter ions.34 among the homologous of ctax surfactants, x = cl–, br–, so42–, cta sulfate is the only one that exhibits a positive enthalpy of micellization as a result of the strong and specific affinity of so42– for the micelle interface.35 furthermore, the micelle size and reactivity of ctab aggregates were found to be affected by divalent counter ions, such as so42–, as a result of strong binding.36,37 based on these facts, it is assumed that complete neutralization of the positive charges on the ctab micellar surface may occur at low concentrations of ctab, i.e., up to 0.5×10–2 mol dm–3, as a result of strong binding of so42–. under such condition, inclusion of hcro3+ into ctab micellar phase is possible. thus, the accelerating effect observed in the region of low concentrations of ctab may be due to increasing stoichiometric concentrations of both reactants in micellar phase, which favors the formation of the sulfonium ion intermediate. it is worth mentioning here that kabir-ud-din et al.38 observed a substantial decrease in rate in the oxidation of oxalic acid by cr(vi) in ctab medium in the presence of added salt, na2so4. they explained the result based on the strong binding of so42– to ctab, which prevents the inclusion of the reactive species hcro4–. treatment of pseudo-phase models various models have been proposed to describe the variation of reaction rate in the presence of micelles. among them, the pseudo-phase model is the most commonly used model to interpret the catalytic/inhibitory activity of micelles and to calculate binding parameters. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ ctab-mediated psaa-cr(vi) redox reaction 1029 menger–portnoy model the observed rate inhibition in the ctab medium could be analyzed by considering the distribution pattern of reactants between the micellar and aqueous phases by application of the pseudo-phase kinetic model proposed by menger and portnoy and its modified forms.39–42 this model considers the partitioning of the substrate between the aqueous and micellar pseudo-phases as given in scheme 2 proposed by menger and portnoy.39 scheme 2. menger–portnoy model. from scheme 2, the observed rate constant, kψ, is given by: w m s n s n [d ] 1 [d ] + = +ψ k k k k k (2) where dn, s and sdn represent micellar surfactant, free substrate and associated substrate, respectively, and kw and km are the pseudo-first-order rate constants in aqueous and micellar phases, respectively, ks is the binding constant of the substrate with the surfactant and [dn] is the concentration of the micellar surfactant, which is related to the stoichiometric concentration of the surfactant, [d]t and critical micelle concentration, cmc as [dn] = [d]t – cmc. the cmc value of ctab was taken from the literature as 9.2×10–4 mol dm–3.43,44 the applicability of menger–portnoy model (scheme 2) for the observed inhibitory effect of ctab in the oxidative decarboxylation of psaa was tested by modifying eq. (2) as: w w m w m s n 1 1 1 ( ) ( ) ( ) [d ] = + − − −ψk k k k k k k (3) the modified eq. (3) was successfully applied to various micellar inhibition reactions by different researchers.45–47 using the rate data, the values of 1/(kw–kψ) were plotted against 1/[dn] in the concentration range 2.0×10–2–12×10–2 mol dm–3. the menger–portnoy model is applicable to micellar inhibition only if the plot of 1/(kw–kψ) vs. 1/[dn] is linear with a positive slope and intercept.48 in the present case, although the plot of 1/(kw–kψ) vs. 1/[dn] was linear, the plot afforded a negative intercept, showing its inadequacy to explain the micellar effect. according to rajasekaran et al.,49 as double reciprocal plot is involved in this model, there exists some uncertainty in the intercept. in order to remove this uncertainty, they modified eq. (3) to: _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1030 subramaniam and thamil selvi w w m w s n ( ) ( ) ( ) [d ] − − = − + −ψψ k k k k k k k (4) the plot of (kψ–kw) vs. (kψ–kw)/[dn] should be linear and from the slope and intercept of the plot, the rate constant for the micellar phase, km, and the substrate–micelle binding constant, ks could be evaluated. however, for the present reaction, the plot of (kψ–kw) vs. (kψ–kw)/[dn] was non-linear (fig. 5), which indicates the inapplicability of the menger–portnoy model to the inhibition of this reaction by ctab. fig. 5. validity of menger–portnoy model. piszkiewicz cooperative model many micellar reactions were also explained by the cooperative model developed by piszkiewicz,50–52 which is analogous to the hill model applied to enzyme-catalyzed reactions. this model was developed for micelle-catalyzed reactions showing a maximum rate followed by inhibition, as was observed in the present case. this model relates the cooperativity between a neutral species of the reaction and a surfactant to form the reactive micelles, as in scheme 3 that was proposed by piszkiewicz.50 from scheme 3, the observed rate constant, kψ could be expressed as a function of the concentration of surfactant: m d w d [d] [d] + = + n ψ n k k k k k (5) equation (5) can be rearranged to: w d m ( ) log log[d] log ( ) − = − − ψ ψ k k n k k k (6) _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ ctab-mediated psaa-cr(vi) redox reaction 1031 where, km is the rate constant at maximum surfactant concentration within the given range and km ≈ 0 if the reaction is inhibited by surfactant.19,49,53 n is the index of cooperativity that is a measure of the association of additional surfactant molecules to an aggregate in the whole surfactant concentration range, kd is the dissociation constant of micellized substrate back to its free components and its inverse is k that is the association constant of the micelle–substrate complex. the advantage of the eq. (6) is that it does not require knowledge of the cmc value of the surfactant used. although eq. (6) is generally used for micelle-catalyzed reactions, it was also applied to micellar inhibition reactions with certain modifications.19,54–57 since ctab retards the rate to a significant extent at high concentrations, it is assumed that the incorporation of positively charged oxidizing species, hcro3+ into the positively charged micelle is negligible. thus at higher ctab concentrations, hcro3+ is mainly solubilized in the aqueous phase and hence, it is presumed that km ≈ 0. under these conditions, eq. (6) becomes: w d ( ) log log[d] log − = −ψ ψ k k n k k (7) in the present reaction, the plot of log [(kw–kψ)/kψ] vs. log [ctab] was found to be linear (fig. 6, r = 0.996) in the rate retardation region of ctab, 3.0×10–2–12×10–2 mol dm–3. the linearity of the plot obtained with the micelle support the positive cooperativity between psaa and micelles to form reactive micelles, which indicates that the psaa molecules are included into the micellar phase. the piszkiewicz parameters, n and kd determined, respectively, from the slope and intercept of the linear plot were n = 1.86 and kd = 9.11×10–3. the association constant, k computed from the reciprocal of kd was 109.82, which fig. 6. applicability of piszkiewicz cooperative model. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1032 subramaniam and thamil selvi indicates binding of psaa to the micelles. the value of n greater than unity indicates positive cooperativity, i.e., the binding of the first molecule of the substrate makes it easier for subsequent molecules to bind. furthermore, the value of n is far less than the aggregation number (20 to 100) of the surfactants, which indicates the formation of catalytically productive sub-micellar aggregates.38 conclusions the ctab mediated redox reaction of psaa with cr(vi) displayed two different effects, an initial increase in the rate constant followed by a sharp decrease with increasing concentration of ctab. the observed trends of the rate constant were explained in terms of specific binding of so42– on the micellar surface and interactions such as electrostatic and hydrophobic. the kinetic data were treated for micellar effects with the menger–portnoy and piszkiewicz pseudo-phase models. the results obtained from the kinetic studies were better fitted with the piszkiewicz model and hence, the binding constant for psaa with ctab micelles was evaluated using this model. и з в о д динамика реакције фенилсулфинилацетатне киселине са cr(vi) у присуству цетилтриметиламонијум-бромида: третман псеудо-фазним моделима perumal subramaniam 1 и natesan thamil selvi 2 1research department of chemistry, aditanar college of arts and science, tiruchendur-628 216, tamil nadu, india и 2govindammal aditanar college for women, tiruchendur-628 215, tamil nadu, india утицај цетилтриметиламонијум бромида (ctab) на оксидативну декарбоксилацију фенилсулфинилацетатне киселине (psaa) и неколико метаи пара-супституисаних фенилсулфинилацетатних киселина (psaa-а) са cr(vi) је испитиван у раствору 95 % h2o–5% ch3cn. почетна брзина реакције расте при малим [ctab], а затим долази до наглог пада брзине реакције при већим [ctab]. раст почетне брзине може бити објашњен јаком везом између so4 2– и позитивно наелектрисане мицеларне површине. специфична расподела psaa у мицеларној фази услед хидрофобних интеракција, односно оксидујуће врсте hcro3 + у воденој фази услед електростатичког одбијања, доводе до успоравања брзине при већим [ctab]. hammett дијаграм са различитим супституисаним psaa-а је показао одличну корелацију негативне вредности ρ што подржава предложени мехнизам који укључује формирање интермедијера сулфонијум катјона. добијена вредност ρ у ctab средини је нешто нижа у поређењу са вредношћу добијеном за водену средину. квантитатвина анализа података за инхибицију брзине реакције од стране ctab је урађена применом menger– portnoy и piszkiewicz псеудо-фазног модела. константа везивања psaa за мицелу је одређена на основу piszkiewicz кооперативног модела. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice an investigation of chitosan and sodium dodecyl sulfate interactions in acetic media j. serb. chem. soc. 81 (5) 575–587 (2016) udc 541.64:547.459.5+546.33’226+ jscs–4869 544.6.018.47–036.5:537.363 original scientific paper 575 an investigation of chitosan and sodium dodecyl sulfate interactions in acetic media lidija b. petrović#, jelena r. milinković*, jadranka l. fraj, sandra đ. bučko and jaroslav m. katona university of novi sad, faculty of technology, bulevar cara lazara 1, 21000 novi sad, serbia (received 19 november 2015, revised 5 february, accepted 9 march 2016) abstract: a polymer/surfactant association is a cooperative phenomenon in which the surfactant binds to the polymer in the form of aggregates, usually through electrostatic or hydrophobic forces. as is already known, polyelectrolytes may interact with oppositely charged surfactants through electrostatic attraction that results in polymer/surfactant complex formation. this behavior could be desirable in wide range of application of polymer/surfactant mixtures, such as improving colloid stability, gelling, emulsification and microencapsulation. in the present study, surface tension, turbidity, viscosity and electrophoretic mobility measurements were used to investigate the interactions of the cationic polyelectrolyte chitosan (ch) and the oppositely charged anionic surfactant sodium dodecyl sulfate (sds) in buffered water. obtained results show the presence of interactions that lead to ch/sds complexes formation at all investigated ph and for all investigated polymer concentrations. mechanisms of interaction, as well as characteristics of formed ch/sds complexes, are highly dependent on their mass ratio in the mixtures, while ph has no significant influence. keywords: polymer/surfactant complexes; cationic polyelectrolyte; anionic surfactant; surface tension; viscosity; electrophoretic mobility. introduction the field of polymer/surfactant interactions has attracted a lot of attention both from the theoretical and practical points of view. although the subject has been studied over a period of several decades, the perspective of the application of polymer/surfactant mixtures in the chemical, food and pharmaceutical industries determines its further extensive research.1 the effects of polymer/surf * corresponding author. e-mail: jelenamilinkovic@tf.uns.ac.rs # serbian chemical society member. doi: 10.2298/jsc151119024p 576 petrović et al. actant interactions are useful in practice to achieve emulsification, colloidal stability, viscosity enhancement, gel formation and solubilization.2–4 polymer/surfactant interactions in the mixtures can be relatively weak, between the polymer chain and the surfactant head group, or strong, electrostatic interactions between oppositely charged polymers and surfactant head groups. however, hydrophobic interactions between the polymer and the surfactant chains are also always present and, in some systems, could be the predominant attractive forces. in particular, oppositely charged polymer/surfactant systems that includes polyelectrolytes and amphiphilic polyelectrolytes, have been widely studied by means of variety of techniques, such as tensiometry,5,6 conductometry,7–9 isothermal titration calorimetry,10,11 neutron refractometry6 and some others. strong interactions within these systems, usually caused by electrostatic attraction and hydrophobic interaction, lead to the formation of complexes with various structures and characteristics and, in some cases, to coacervate formation and phase separation.8,12 the interaction in oppositely charged polymer/surfactant systems is generally accepted as an ion-exchange process in which the electrostatic forces of interaction are reinforced by aggregation of the alkyl chains of the bound surfactant molecules.10,13 chitosan (ch) is a nontoxic, natural, biodegradable polysaccharide that currently receives a great deal of attention for applications in food and pharmaceutical products. some authors have indicated the ability of ingested ch to decrease oil absorption or lipase activity in the digestive tract of human or animal, which enables its application in functional food products.14,15 in addition, due to its antibacterial and antiviral activities, biocompatibility and bio-adhesive nature, ch has been used for different biomedical applications.7,16–20 ch is a linear copolymer composed of n-acetyl-d-glucosamine and d-glucosamine units that is usually obtained by partially or fully n-deacetylation of chitin.21 the solubility, rheological properties and surface adsorption of aqueous ch solutions depend on the average molecular weight, degree of deacetylation, and the distribution of the acetyl and amino groups along the polymer chain, as well as on the ph and ionic strength of the solution.15,22 namely, ch is readily soluble in dilute acidic solutions below ph 6 due to the protonation of the amino groups that have a pka value of 6.3.23 at low ph values, these amines are protonated, become positively charged and make ch a water soluble cationic polymer. on the other hand, at ph values higher than 6, ch is less charged (protonation of amines is restricted) and it becomes insoluble. when positively charged, the amine groups are promising sites for electrostatic interactions with oppositely charged surfactants and polyelectrolytes, thus inducing numerous changes in their aqueous mixtures.24,25,27 such changes in the bulk solution, as well as at interfaces, could be useful for various applications of ch/surfactant and ch/polyelectrolyte mixtures, such as emulsification and microencapsulation. interactions between chitosan and sds 577 the aim of this study was to investigate the interaction mechanisms between the cationic polysaccharide ch and sodium dodecyl sulfate (sds), by determining the surface and bulk properties of their aqueous mixtures. sds is a well studied anionic surfactant that commonly interacts with both non-ionic and ionic polymers through different mechanisms.3,4,24 previous studies showed that chitosan can interact with anionic surfactants to form either soluble or insoluble surfactant/polymer complexes.8,9,11 it was proposed that ch/sds complexes are stabilized by a combination of electrostatic, ion–dipole and hydrophobic interactions and can be formed even when the surfactant concentration is below its critical micelle concentration. in this study, a wide variety of methods, including surface tension, electrophoretic mobility, turbidity and viscosity measurements, were used to provide more information about ch/sds interactions. the influence of ph and ch concentration on the behavior of ch/sds complexes at interfaces and in the bulk was investigated in detail. in addition, the study gives information about the optimal mass ratio of ch and sds that may be used as a material for microcapsule formation suitable for food and pharmaceutical applications. experimental materials low molecular weight ch was obtained from sigma–aldrich (china). the degree of deacetylation of the ch, determined by potentiometric titration according to the procedure described by yuan et al.,28 was found to be 81.8 %. sds, purity >99 %, was purchased from merck (germany). in all experiments, buffered water was used as the solvent, and the ph was adjusted using an aqueous 0.2 m solution of acetic acid (zorka-pharma, serbia) and an aqueous 0.2 m solution of sodium acetate (centrohem, serbia). preparation of solutions the experiments were performed at ph 4, 5 and 6. the ph values were measured using an 827 lab ph-meter (metrohm, switzerland). stock solutions of 1 mass % ch were prepared by dissolving the required amount of polymer in buffered water of the chosen ph under stirring and after relaxation at room temperature for 24 h, the ph value of the solution was checked. sds stock solutions (2 mass %) were prepared using the same procedure. ch/sds mixtures were prepared by mixing the required volumes of the stock solutions. turbidity measurements of ch/sds mixtures were performed at a ch concentration of 0.1 mass %. for the surface tension measurements, the concentrations of sds were varied from 0.00001 up to 1 mass %, while the ch concentrations were kept constant at 0.005, 0.01 or 0.05 mass %. electrophoretic mobility measurements of ch/sds mixtures were performed on a series of mixtures at a ch concentration of 0.01 mass %. viscosity measurements were performed on a series of mixtures containing 0.01, 0.05, 0.1, 0.2, 0.5 mass % ch. the mixtures were left for 24 h at room temperature and then the ph value of the solutions was checked. turbidity measurements the changes in the turbidity of ch/sds mixtures were determined by measuring the transmittance at 600 nm in 1 cm path length cuvette against buffered water at room temperature using a halo db-20s uv–vis spectrophotometer (dynamica, uk). transmittance of 578 petrović et al. the mixtures where phase separation occurred was measured in the supernatant phase. turbidity of the samples was calculated as: turbidity = 100 – transmittance (%) all measurements were performed in triplicate. electrophoretic mobility measurements the electrophoretic mobility of ch/sds complexes at ph 4 was determined using zetasizer nano zs (malvern instruments, uk). the viscosity (0.88 mpas) and refractive index (1.33) of the solvent at 25 °c were used for data analysis. all measurements were performed in triplicate. surface tension measurements surface tension measurements were realized on a sigma 703d tensiometer (ksv instruments, finland) using the du noüy ring method at 30±0.1 °c. prior to the measurements of surface tension, the ring was immersed in the liquid (below the surface) and the surface was left for 15 min to equilibrate. surface tension of buffered water at ph 4, 5 and 6 that was used as a solvent, was 69.55 mn m-1 (measured under the same conditions), meaning that the pure electrolyte solution revealed no surface activity. the reported values of the surface tension were average values of at least three measurements. viscosity measurements viscosity measurements were performed on cannon capillary viscometer 50 e599 (cannon instrument company, usa) immersed in thermostatic bath at 30±0.1 °c. flow time for solvents at ph 4 was 198.71 s. the flow times of the ch/sds mixtures were measured and the results are expressed as ηrel vs. sds concentrations, where ηrel is given by: rel 0 t t η = where t and t0 are the flow times for the solution and pure solvent, respectively. the viscosity of the supernatant phase was measured for mixtures where phase separation occurred. for each solution, three viscosity measurements were taken and the average values were calculated. results and discussion phase separation in ch/sds mixtures as already mentioned, interactions are most pronounced in systems consisting of a polyelectrolyte and an oppositely charged surfactant and they are often accompanied by phase separation. considering that ch and sds bear opposite charges in buffered water, at ph lower than 6, the investigations commenced with the preparations mixtures containing 0.1 mass % ch with different concentrations of sds at ph 4. the ch/sds mixtures 24 h after mixing are presented in fig. 1a. it was noticed that introducing sds to the ch solution at concentrations lower than 0.01 mass % did not induce any visually observable changes in the mixtures. with further increase in sds concentration, the interactions between chitosan and sds 579 fig. 1 phase separation (a) and the turbidity measured at 600 nm (b) of 0.1 % ch mixtures containing various sds concentrations at ph 4. mixtures become turbid and phase separation occurred at an sds concentration of 0.15 mass %, i.e., insoluble ch/sds complexes precipitated at the bottom as a coacervate phase (fig. 1a). in order to quantify the visually observed changes, the turbidity of the ch/sds mixtures at ph 4 were measured and the results are presented in fig. 1b. the solution of chitosan in water at ph 4 was clear (turbidity 0.80 %) and mixtures of ch containing less than 0.005 mass % sds did not show changes in turbidity. however, further addition of sds caused an increase in the turbidity of the mixtures. namely, the gradual binding of the negatively charged polar heads of the sds molecules onto the positively charged amino groups on the ch molecules led to the formation of complexes that were less charged and thus, less soluble in water. the turbidity reached a maximum at an sds concentration of 0.05 mass % and decreased after this concentration, which could be due to the beginning of the phase separation process. the minimum in turbidity of the supernatant phase (marked with arrow in fig 580 petrović et al. 1b), i.e., complete precipitation of the complexes occurred at 0.2 mass % sds (ch/sds mass ratio 1:2), which could be explained by their neutrality. after this concentration, further addition of sds led to an increase in the turbidity of the supernatant phase. the increase in the dispersibility of the coacervate phase might be due to an excess of complexes with a negative charge caused by the hydrophobic binding of sds molecules. electrophoretic mobility of ch/sds mixtures as is known, an electrical charge on particles influences their electrophoretic mobility. measurements of electrophoretic mobility were performed in order to confirm the assumption that the precipitation of ch/sds complexes occurred because of changes in their electrical charge. the results are presented in fig. 2. fig. 2 electrophoretic mobility of ch/sds mixtures at 25 °c and ph 4 as a function of the sds concentration and the ch:sds mass ratio. the chitosan concentration was 0.01 mass %. as a cationic polyelectrolyte, ch molecules bear net positive charges in aqueous solution under acidic condition, at ph values lower than 6. this was confirmed by electrophoretic mobility measurements since electrophoretic mobility of 0.01 mass % ch solution at ph 4 was 2.84 μm cm v–1 s–1. the addition of oppositely charged sds to the ch solution induced the formation of complexes that bore a net positive charge, but it decreased the absolute value due to the gradual binding of sds by electrostatic attraction. this gradual decrease in charge caused a decrease in the solubility of the ch/sds complexes and, consequently, turbidity occurred (fig. 1b). after reaching an sds concentration of interactions between chitosan and sds 581 0.01 mass %, the charge of the complexes decreased rapidly and at an sds concentration of 0.022 mass %, i.e., at a ch/sds mass ratio of around 1:2, they became neutral. as the result, precipitation of the coacervate occurred in the mixture causing a decrease in turbidity. further addition of sds led to charge inversion and the total charge of the complexes tended towards an excess of negative charge. electrostatic repulsion between complexes caused their slight redispersion from the coacervate phase and turbidity resulted (fig. 1b). since tensiometric and viscometric measurements give important information about system behavior at the interface and in the bulk, these two techniques were used for detail examination of ch/sds interaction mechanisms. surface tension measurement of ch/sds mixtures in the mixtures of polyelectrolytes and oppositely charged surfactants, interactions are strong and predominantly caused by electrostatic attraction between the charged groups of polymer chains and head groups of surfactant molecules. in this manner, they often show very different surface tension behavior than that of weak interacting system, such as hydrophobic interactions in nonionic polymer/surfactant systems.3,4,6 the surface tensions of pure sds and ch/sds mixtures in buffered water at 30 °c and ph 4 are presented in fig. 3. fig. 3. surface tensions of pure sds (-○-) and ch/sds mixtures (-∆-) as functions of the sds concentration and the ch:sds mass ratio at 30 °c and ph 4. the chitosan concentration was 0.01 mass %. 582 petrović et al. surface tension of pure ch solution in buffer was 69.15 mn m–1, indicating the absence of surface activity of the polymer, as is case of most strong polyelectrolytes.6–8 simultaneously, sds induced a very large reduction in the surface tension at very low concentrations up to the critical micelle concentration (cmc), 0.18 mass %, after which the surface tension was practically constant. this large reduction in surface tension was due to the strong adsorption of sds molecules at the liquid–air surface, typical for most surfactants in aqueous solutions.1 as observed in fig. 3, changes in the surface tension of ch/sds mixtures showed discontinuities at characteristic sds concentrations, which correspond to changes in the turbidity and electrophoretic mobility. the overall pattern was different from those published by onesippe and lagerge, as they reported a gradual decrease in surface tension in the region of interaction with increasing concentration of sds.8,24 the interaction between ch and sds at the surface was detected at very low concentrations of surfactant. namely, a pronounced lowering of the surface tension in the ch/sds mixture occurred at 0.0001 mass % sds, indicating the formation of surface-active complexes. with increasing sds concentration, the surface tension of the ch/sds mixtures significantly decreased until the first discontinuation, t1, was reached at an sds concentration of 0.001 mass % (ch/sds mass ratio 10:1). at this mass ratio, as may be seen from figs. 1b and 2, the turbidity in the mixture occurs, indicating that the complexes, although positively charged, less soluble but still surface active. further addition of sds did not lead to a change in surface tension until the second break point, t2, was achieved at 0.02 mass % sds (ch/sds mass ratio 1:2). in the sds concentration region between t1 and t2, the positive charge on the ch/sds complexes decreased, the turbidity increased and precipitation of the coacervate occurred, but the surface tension still remained low. such behavior could be attributed to the formation different kinds of ch/sds complexes that were formed in the bulk, amphiphilic in their nature and extremely surface active. after this concentration, further addition of sds caused a significant decrease in surface tension until t3, when the value was lower than that for the pure surfactant solution at the cmc. once micelles of sds abound in solution, ch molecules are stripped from the interface and the surface tension becomes essentially equal to that of a micellar, polymer -free surfactant solution.3 at this concentration the surface was saturated only with sds molecules and further increase in sds concentration led to the formation of regular sds micelles in the bulk, i.e., surface tension remained constant. the influence of ch concentration on surface tension behavior of the ch/sds mixtures is presented in fig. 4. although the overall pattern looks similar to the one observed for weakly interacting systems, there are some important differences.6 namely, the initial pronounced decrease in surface tension at low sds concentration and the position interactions between chitosan and sds 583 fig. 4. surface tension of 0.005 (-□-), 0.01 (-∆-) and 0.05 mass % (-○-) ch mixtures with sds vs. the sds concentration at 30 °c and ph 4. of t1 were dependent on the ch concentration; t1 occurred at higher sds concentrations with increasing ch concentration. moreover, t3, i.e., the cmc for the mixed ch/sds system appeared to be similar to, or lower than, that for pure sds, in contrast to the behavior for weakly interacting systems, when the cmc of the mixture was generally higher.1 such behavior could be attributed to the formation of polymer/surfactant complexes that differed in their structure depending on the concentration of surfactant, as already reported for some related polyelectrolyte/surfactant systems.6,29 it could be assumed that at low sds concentration, below t1, single sds molecules were bound to the ch amino groups via electrostatic attraction and surface active ch/sds monomer complexes were formed. after reaching a certain sds concentration at t1, the cooperative adsorption of sds molecules in form of some sort of aggregate on the ch chain started in the bulk. such bulk complexes do not affect the ch/sds adsorbed layer at the interface and as a result, the surface tension remained unchanged. the t1–t2 plateau appeared at characteristic polymer/surfactant mass ratios, between ch/sds mass ratio 10:1 and 1:2. electrophoretic mobility measurements showed that the ch/sds complexes bear a net positive charge in the sds concentration region between t1 and t2, although a gradual decrease in the absolute value was observed (fig. 2). considering this, the occurrence of turbidity and the gradual precipitation of the bulk ch/sds complexes, which were observed in the t1–t2 region, could be attributed to a reduction in charge and the simultaneous increase in hydrophobicity. at a ch/sds mass ratio of 1:2, the complexes were almost neutral in their charge (µ = 0.37 μm cm v–1 s–1) and fully precipitated as a coacervate phase. a further increase in the sds concentration led to a gradual 584 petrović et al. deployment of the sds molecules at the interface, which resulted in a significant decrease in the surface tension. at t3, the phase interface was saturated with sds molecules and after this concentration, regular sds micelles were formed in the bulk. simultaneously, the binding of sds molecules to the ch/sds complexes continued via hydrophobic interactions and the total charge of the complexes tended towards an excess of negative charges (fig. 2). considering that the ph value may affect the interactions in polyelectrolyte/ /surfactant systems, changes in the surface tension of ch/sds solutions at different ph values were examined. the results are presented in fig. 5. fig. 5 surface tension of ch/sds mixtures as a function of sds concentration and ch:sds mass ratio at different ph values and at 30 °c. chitosan concentration was 0.01 mass %. as can be seen from fig. 5, the ch/sds mixtures containing 0.01 mass % ch showed characteristic break points, t1, t2 and t3, at all the investigated ph values. the appearance of the curves at ph 5 and 6 appeared very similar to that at ph 4. namely, the formation of soluble ch/sds complexes started at very low sds concentrations causing a significant decrease in the surface tension. in addition, the positions of discontinuities were at the same sds concentrations: t1 appears at 0.001 mass %, t2 at 0.02 mass % and t3 at 0.1 mass % sds, indicating that, in examined range, the ph had no significant influence on the interactions. interactions between chitosan and sds 585 viscometric investigation of the ch/sds interactions in order to understand better the mechanisms of interaction between ch and sds, viscometric measurements of the ch/sds mixtures were performed. the relative viscosities of the ch/sds mixtures with various sds concentrations at ph 4 are presented in fig. 6. fig. 6. relative viscosity of ch/sds mixtures containing different ch concentration vs. sds concentration at 30 °c and ph 4. the relative viscosity of pure ch solutions at concentrations of 0.01, 0.05, 0.1, 0.2 and 0.5 mass % at ph 4 were 1.10, 1.52, 2.02, 3.10 and 7.84, respectively. the introduction of sds to the ch solutions did not affect the relative viscosity, indicating that the interactions between these two molecules occurred at the interface, which fully corresponded to the previous surface tension measurements. at sds concentration of 0.01, 0.02, 0.05 and 0.1 mass % in the mixtures containing 0.05, 0.1, 0.2 and 0.5 mass % ch, respectively, the precipitation of the ch/sds complexes started (ch/sds mass ratio of 5:1) and the viscosities of supernatant phases were measured. after these concentrations, further addition of sds caused a decrease in ηrel of the supernatant phase leading to complete precipitation of the coacervate phases at 0.02, 0.1, 0.2, 0.4, 1.0 mass % sds, respectively. these polymer-depleted phases showed a minimum in the viscosity (marked with arrow) that always appeared at a ch/sds mass ratio of 1:2. further increase in the sds concentration led to an increase in the relative viscosity of supernatant phase, indicating better dispersibility of the complexes due to their electrostatic repulsion. 586 petrović et al. it is evident that viscometric measurements fully corresponded to the turbidity, surface tension and electrophoretic mobility measurements of the ch/sds mixtures. conclusions the results presented in this paper showed that the cationic polymer, chitosan and the anionic surfactant sds interacted over a wide range of surfactant concentrations. the interactions started at very low sds concentrations by electrostatic binding of sds molecules, resulting in the formation of soluble and extremely surface active complexes. at a ch/sds mass ratio of 10:1 for all the investigated chitosan concentrations, the interactions occurred in the bulk and did not affect ch/sds adsorbed layer at the interface, i.e., the surface tension remained unchanged until reaching a ch/sds mass ratio of 1:2. the bulk complexes carried a net positive charge (a gradual decrease in the absolute value was observed) and gradually precipitated from the solution with increasing sds concentration. at a ch/sds mass ratio of 1:2, independent of the polymer concentration and ph, neutral complexes fully precipitated from the solution, while the polymer depleted phase showed a minimum in viscosity. a further increase in the sds concentration led to weak hydrophobic binding of the sds molecules to the ch/sds complexes, the total charge of the complexes tended towards an excess negative charge and, consequently, the coacervate phase became slightly redispersed. acknowledgement. this work was supported by the ministry of education, science and technological development of the republic of serbia, project no. iii46010. и з в о д испитивање интеракција између хитозана и натријум-додецил -сулфата у киселој средини лидија б. петровић, јелена р. милинковић, јадранка л. фрај, сандра ђ. бучко и јарослав м. катона технолошки факултет, универзитет у новом саду, бул. цара лазара 1, 21000 нови сад повезивање полимера и сурфактанта је кооперативни феномен где се молекули сурфактанта везују за молекуле полимера у форми агрегата, обично електростатичким или хидрофобним силама. као што је већ познато, полиелектролити ступају у интеракцију са супротно наелектрисаним сурфактантима електростатичким привлачењем, што доводи до формирања комплекса полимер/сурфактант. ово понашање смеша полимер/сурфактант има широку практичну примену, како за побољшање стабилности колоидних система, гелирање, емулговање, тако и у процесима микрокапсулације. у циљу испитивања интеракције катјонског полиелектролита хитозана (ch) и супротно наелектрисаног анјонског сурфактанта натријум-додецил-сулфата (sds) у пуферу, мерени су површински напон, вискозитет, мутнoћa и електрофоретска покретљивост. добијени резултати указују на присуство интеракције која доводи до формирања ch/sds комплекса на свим испитиваним ph вредностима. механизам интеракције, као и особине interactions between chitosan and sds 587 формираних ch/sds комплекса у највећој мери зависе од њиховог масеног односа у смеши, док ph нема значајнији утицај. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice preparation and characterization of zro2-supported fe3o4-mnps as an effective and reusable superparamagnetic catalyst for the friedländer synthesis of quinoline derivatives j. serb. chem. soc. 80 (8) 971–982 (2015) udc 546.831–31+546.723’+22+547.831: jscs–4773 542.9+544.47:544.344 original scientific paper 971 preparation and characterization of zro2-supported fe3o4-mnps as an effective and reusable superparamagnetic catalyst for the friedländer synthesis of quinoline derivatives seyyedeh zoha hejazi, abdollah fallah shojaei*, khalil tabatabaeian and farhad shirini department of chemistry, faculty of sciences, university of guilan, p. o. box 41335-1914, rasht, iran (received 23 october 2014, revised 4 march, accepted 27 march 2015) abstract: in this study, a convenient, appropriate and eco-friendly method for the synthesis of quinoline derivatives via a friedländer reaction was developed using zro2/fe3o4-mnps as an effective and reusable heterogeneous catalyst. the morphology of zro2/fe3o4-mnps was studied by the xrd, ft-ir, sem, tem and vsm techniques. green reactions, straight and easy work-up, high yields of the products and good reaction times are the benefits of this procedure. further, the catalyst could be recovered using an external magnetic field and reused at least three times without a considerable decrease in its catalytic activity. keywords: quinoline derivatives; friedländer reaction; zro2/fe3o4-mnps; heterogeneous catalyst; green procedure. introduction quinoline and its derivatives are very important intermediates in organic and medicinal chemistry that show various physiological and pharmacological activities, such as antimalarial, anti-inflammatory, anti-asthmatic, antibacterial, antihypertensive, and are tyrosine kinase inhibiting agents.1–3 these heterocycles are valuable compounds for the preparation of nanoand meso-structures with enhanced electronic and photonic properties.4,5 furthermore, quinoline derivatives have been employed in the study of bio-organic and bio-organometallic processes.6,7 due to the importance of the use of quinolines in the fields of medicinal, bioorganic, industrial and synthetic organic chemistry, there is immense interest in the development of effective procedures for their synthesis. thus, several methods, such as the skraup, doebner–von miller, friedländer and * corresponding author. e-mail: a.f.shojaie@guilan.ac.ir doi: 10.2298/jsc141023031h _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 972 hejazi et al. combes, have been mentioned in the literature for the creation of quinoline derivatives.8–11 among various methodologies suggested for the preparation of quinolines, the friedländer annulation12,13 is one of the most frequently used pathways for the synthesis of polysubstituted quinolines. this method involves an acidor base-catalyzed or thermal condensation between an aromatic 2-aminoaldehyde or ketone with a carbonyl compounds containing a reactive α-methylene group followed by a cyclodehydration.14,15 in general, friedländer reactions are carried out either by refluxing an aqueous or alcoholic solution of reactants in the presence of a base or by heating a mixture of the reactants at high temperatures ranging from 150 to 220 °c in the absence of a catalyst.16 however, under thermal or basic catalysis conditions, 2-aminobenzophenone does not react with simple ketones, such as cyclohexanone and β-keto esters.17 subsequent work showed better yields of quinolines were achieved under acid catalysis.17 several acid catalysts, such as brønsted acids including hydrochloric acid in water,18 sulfamic acid,19 sulfuric acid,20 silica sulfuric acid,21 dodecylphosphonic acid,22 peg-supported sulfonic acid,23 arylidene pyruvic acids (apas),24 oxalic acid,25 lewis acids containing zr(no3)4 or zr(hso4)4,26 zirconium tetrakis(dodecyl sulfate) zr(ds)4,27 gdcl3·6h2o,28 bicl3,29 sncl2,30 fecl3,31 y(otf)332, nicl233, ag3pw12o4034 and zncl235 have been reported for this conversion. however, most of the previously reported procedures suffer from different drawbacks, such as low yields of the products, poor selectivity, long reaction times, harsh conditions, high temperatures, usage of hazardous, corrosive and relatively expensive catalysts, tedious work-up procedures and using toxic/polar solvent leading to complex isolation and recovery procedures. moreover, the main disadvantage of a number of previous methods is that the catalyst cannot be recovered. therefore, the development of a simple, efficient and environmentally friendly method for the synthesis of quinoline derivatives is still a challenging task. recently, the applications of heterogeneous nanocatalysts have attracted remarkable attention as inexpensive, non-toxic and eco-friendly catalysts for various organic transformations under mild and convenient conditions. the catalysts have advantages over conventional homogeneous catalysts, including simple recovery from the reaction mixture by easy filtration, higher surface activity and reusability.36–38 among heterogeneous catalysts, inorganic oxides especially zirconia have different physical and chemical properties and have gained much consideration by researchers. good chemical and dimensional stability, high electrical resistivity, high refractive index, mechanical strength and toughness, biocompatibility and low cost are the origin of the interest in using the zirconia as a catalyst.39,40 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ friedländer synthesis using zro2/fe3o4-mnps catalyst 973 however, the specific surface area and thermal stability of pure zro2 is low41 and it shows weak catalytic activity in a number of chemical transformations.42 in order to enhance the catalytic performance of zirconia-based catalysts, many endeavors have been inducted by introducing various metal oxides whereupon zirconia acts as a support for the preparation of the solid catalysts.43–48 although the doping of appropriate cations with specific concentration into zirconia improves thermal stability, surface area and acidity of solid acids, the separation and recovery of mixed oxide catalysts from the reaction products are still difficult and require a large amount of separation energy and cost. for this reason, a material immobilized onto the solid support plays a fundamental role in the efficiency of the resulting supported reagent catalyst. among the different modified zirconia materials, fe3o4 magnetic nanoparticles (mnps) are widely used due to easy handling, a simple work-up procedure, nontoxicity, enhanced catalytic activity and chemical selectivity in various organic synthesis.49,50 in addition, zro2-supported fe3o4-mnps can be easily separated from the reaction media by applying an external magnetic field. in continuation of ongoing studies on metal oxides as catalysts,51 herein, a simple and eco-friendly procedure is demonstrated for the synthesis of quinoline derivatives using the friedländer heteroannulation method in the presence of zro2/fe3o4-mnps as catalyst. experimental chemicals and apparatus all materials and solvents were purchased from merck and fluka, and used without further purification. yields refer to isolated products. products were characterized by their physical constants, comparison with authentic samples, and ir and nmr spectroscopy. the ir spectra were obtained in kbr discs on a perkin–elmer model spectrum one ft-ir spectrometer. the structures of the synthesized catalysts were characterized by x-ray diffraction analysis (xrd equinox 3000, inel, france). the xrd patterns were obtained using cukα radiation (wavelength 1.54056 ǻ) at a current of 200 ma and a voltage of 40 kv in the 2θ range of 10–100 at a scanning rate of 8° min-1. the surface microscopic morphologies of the zro2-supported fe3o4-mnps were visualized by scanning electron microscopy (sem miraii tescan). the size of fe3o4-mnps was investigated by transmission electron microscopy (tem philips mc 10) with an acceleration voltage of 80 kv. a magnetic study was performed using a vibrating sample magnetometer at room temperature (vsm jdm-13). reaction progress was checked by thin-layer chromatography (tlc) with detection by uv light. the 1h-nmr spectra were obtained on a bruker drx-400 avance spectrometer, while the 13c-nmr spectra were obtained on a bruker drx-100 avance spectrometer. samples were analyzed in cdcl3 and chemical shift values are reported in ppm relative to tetramethylsilane (tms) as the internal reference. melting points were measured on an electrothermal apparatus and are uncorrected. elemental analyses were realized using a carlo erba ea1110 chns-o analyzer and the values agreed with the calculated ones. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 974 hejazi et al. preparation of the catalyst (zro2/fe3o4-mnps) firstly, 96 mg of zro2 nano particles were well dispersed in 30 ml of distilled water in a two-necked round bottom flask (100 ml) by ultrasonic irradiation for 30 min. after adding 81 mg of fecl3·6h2o, the solution was stirred vigorously for 30 min. subsequently, 120 mg fecl2·4h2o was slowly added into the mixture under stirring for 30 min. the whole process was performed under an argon atmosphere. then, 8 ml of a concentrated aqueous solution of nh3 was added into the solution dropwise over 1 h. thereafter, the mixture was stirred at 60 °c for 2 h. after cooling the solution to room temperature, the black magnetic zro2/fe3o4 -mnps were recovered by centrifugation at 6000 rpm, rinsed several times with deionized water and dried at 60 °c for 24 h. it was expected that the positive ferrous and ferric ions would be in proximity with the oxygen atoms of zro2 and that they would be converted to fe3o4-mnps after the dropwise addition of the concentrated aqueous solution of nh3 into the solution. general procedure for the synthesis of quinolines catalyzed by zro2/fe3o4-mnps a mixture of 2-amino-5-chlorobenzophenone (1 mmol), a ketone or β-diketone (1.5 mmol) and zro2/fe3o4-mnps (20 mg) in ethanol (5 ml) was stirred magnetically and heated at 70 °c. after completion of the reaction, confirmed by tlc, the catalyst was collected by magnetic separation using an external magnet and washed repeatedly with warm ethanol. the aqueous phase was filtrated and cooled to room temperature. then the solid product was collected and washed with cold water to afford the pure product. in some cases, further purification was achieved by recrystallization in ethanol to give the pure product. result and discussion catalyst characterization x-ray diffraction studies. to confirm the synthesis of the catalyst, first, the xrd patterns of the pure zro2 nanoparticles and zro2/ fe3o4 magnetic nanoparticles were studied. all the diffraction peaks in the xrd patterns of the pure zro2 imply the monoclinic phase of pure zro2 nanoparticles (m-zro2, jcpds no 24-1165). the additional diffraction peaks at 2θ 30.6, 36.5, 43.7, 53.9, 57.6, and 62.8° appearing in the xrd patterns of the zro2/fe3o4-mnps correspond to the standard xrd data for the cubic fe3o4 phase of inverse spinel crystal structure (jcpds file no. 19-0629).52 no peaks corresponding to impurities were present. the average diameter of the crystallites (d) of the synthesized catalyst was calculated using the scherrer formula and found to be 21 nm, as confirmed by tem analysis. fourier transform infrared spectroscopy. the ft-ir spectrum of pure zirconia was compared with the synthesized zro2/fe3o4-mnps. the pure zirconia spectrum exhibited bands at 734, 581 and 508 cm–1, which could be attributed to the zr–o stretching vibration of zro2. these bands were not distinctly found in the spectrum of zro2/fe3o4-mnps. they are related to the broad band at around 579 cm–1, which was assigned to the fe–o stretching vibration of fe3o4-mnps that may overlap with the zr–o peaks. moreover, the absorption bands in the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ friedländer synthesis using zro2/fe3o4-mnps catalyst 975 region 3435 and 1630 cm–1 may be related to the stretching and bending vibrations of the o–h bond due to physically adsorbed water molecules. sem and tem analysis. the morphology and size of the synthesized zro2/ /fe3o4-mnps were examined by scanning electron microscopy (sem) and transmission electron microscopy (tem). from the sem image (fig. 1a), it can be seen that the loaded fe3o4-mnps were uniform in shape and size distribution and had a porous structure. the particles sizes derived from tem analysis (fig. 1b) were in the range 5–25 nm, which is comparable with the crystallite size calculated from the x-ray spectrum. (a) (b) fig. 1. a) sem and b) tem image of zro2/fe3o4-mnps. vibrating sample magnetometer (vsm) analysis the magnetic properties of zro2/fe3o4 magnetic nanoparticles were investigated by the most common method of examining the magnetic properties of a material, using a vibrating sample magnetometer (vsm). the results showed that the synthesized nanoparticles exhibit superparamagnetic behavior at room temperature and the hysteresis loops of the samples exhibited no coercivity and retentivity. the saturation magnetization (ms) values of the synthesized catalysts were 33 emu g–1, which is mainly attributed to a high weight ratio of fe3o4 magnetic nanoparticles that were loaded onto the zro2. catalyst synthesis optimization at the beginning of the investigations on the usage of zro2/fe3o4-mnps as a green and efficient heterogeneous nanocatalyst, the reaction of 2-amino-5-chlorobenzophenone and ethyl acetoacetate (eaa) was selected as a model for determining the optimal conditions. for this reason, several reactions were performed using diverse amounts of catalyst at various temperatures in different solvents to achieve a good yield of the desired products. the results are summarized in table i. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 976 hejazi et al. table i. optimization of the conditions for the synthesis of ethyl 2-methyl-4-phenylquinoline-3-carboxylate; reaction conditions: 2-amino-5-chlorobenzophenone (1 mmol), eaa (1.5 mmol) in different solvents at different temperatures with different amount of catalyst entry catalyst [amount, mg] solvent t / °c τ / min yielda, % 1 – ethanol reflux 300 trace 2 zro2-nps [300] ethanol reflux 240 18 3 zro2/fe3o4-mnps [10] ethanol reflux 60 88 4 zro2/fe3o4-mnps [20] ethanol reflux 24 91 5 zro2/fe3o4-mnps [30] ethanol reflux 25 90 6 zro2/fe3o4-mnps [20] ethanol 25 320 trace 7 zro2/fe3o4-mnps [20] ethanol 50 300 51 8 zro2/fe3o4-mnps [20] ethanol 70 25 92 9 zro2/fe3o4-mnps [20] methanol reflux 30 86 10 zro2/fe3o4-mnps [20] acetonitrile 70 120 22 11 zro2/fe3o4-mnps [20] n-hexane reflux 120 trace aisolated yield as can be seen in table i, the best result was obtained when the reaction was performed at 70 °c, with a relative ratio of the substrate: eaa:zro2/fe3o4-mnps of 1 mmol:1.5 mmol:20 mg, respectively (table i, entry 8). in this procedure, any further increase in temperature and amount of catalyst did not lead to considerable improvement in the reaction times and yields. moreover, when the same procedure was run at room temperature, the yield of the product was poor after 320 min (table i, entry 6). it is noteworthy that without any catalyst at reflux, the product was isolated in low yield after a long reaction time (table i, entry 1). moreover, it can be seen that the pure m-zirconia as the catalyst led to a lower yield of the product (table i, entry 2). in order to examine the effect of the solvent, the reaction was explored in different solvents, i.e., ethanol, methanol, acetonitrile and n-hexane (table i, entries 8–11) and ethanol was selected as the best solvent. under the optimized conditions, the model reaction gave 92 % yield of the corresponding product after 25 min (table i, entry 8 and scheme 1). o ph nh2 oet oo n ph oet o + cl cl zro2/fe3o4-mnps (20 mg) ethanol, 70oc scheme 1. zro2/fe3o4-mnps catalyzed synthesis of ethyl 2-methyl-4-phenylquinoline-3-carboxylate. subsequently, in order to evaluate the generality of this methodology, a series of ortho-aminoaryl ketones were reacted with different 1,3-dicarbonyl compounds under the optimum conditions and the results are reported in table ii. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ friedländer synthesis using zro2/fe3o4-mnps catalyst 977 table ii. synthesis of various quinolines using zro2/fe3o4-mnps as the catalyst; reaction condition: 2-aminoaryl ketone (1 mmol), ketone or β-diketone (1.5 mmol) and zro2/fe3o4mnps (20 mg) at 70 °c in ethanol (5 ml) entry substrate ketone or β-diketone quinoline τ min yielda % m.p., °c measured reported 1 o ph nh2 oo n ph o 40 89 109–111 111–11225 2 o ph nh2 oet oo n ph oet o 30 91 98–100 100–10125 3 o ph nh2 ome oo n ph ome o 35 90 99–100 98–10053 4 o ph nh2 o o n ph o 60 86 191–194 190–19234 5 o ph nh2 o o n ph o 45 90 154–156 155–15634 6 o ph nh2 o n ph 50 89 129–131 130–13234 7 o ph nh2 o n ph 60 87 153–156 156–15734 8 o ph nh2 cl oo n ph o cl 45 90 151–153 150–15134 9 o ph nh2 cl oet oo n ph oet o cl 25 92 101–103 102–10422 10 o ph nh2 cl ome oo n ph ome o cl 30 90 134–136 133–13525 11 o ph nh2 cl o o n ph o cl 45 89 207–209 208–20934 12 o ph nh2 cl o o n ph o cl 35 92 184-186 185–18634 13 o ph nh2 cl o n ph cl 45 90 107-108 106–10725 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 978 hejazi et al. table ii. continued entry substrate ketone or β-diketone quinoline τ min yielda % m.p., °c measured reported 14 o ph nh2 cl o n ph cl 60 89 165–167 164–16534 15 o ph nh2 cl o o n ph o cl 65 87 239–241 240–24354 aisolated yield according to table ii, both cyclic and acyclic diketones such as 5,5-dimethylcyclohexanedione and acetylacetone, cyclic ketones including cyclohexanone and cyclopentanone and β-ketoesters such as eaa reacted with 2-amino-5-chlorobenzophenone and 2-aminobenzophenone to afford the corresponding quinolines. it can easily be seen that in all cases, the friedländer annulation proceeded smoothly and gave good to high yields ranging from 86 to 92 %. the reactions were remarkably clean and no chromatographic separation was necessary to obtain spectra-pure compounds. furthermore, the work-up of present method was easy and the process is beneficial in avoiding the application of strong acids, high temperatures and volatile and/or toxic reactants. all of the synthesized products are known compounds and were characterized by comparing their melting points, ir, 1hand 13c-nmr spectra with those of authentic samples. the possibility of reusing the catalyst is one of the most significant benefits of heterogeneous catalysts over homogeneous systems. thus, the recovery and reusability of zro2/fe3o4-mnps was investigated in the model reaction under the optimized condition. after completion of the reaction, the catalyst was easily separated from the reaction mixture by an external magnetic field and reused in subsequent runs. the results of continuous runs showed that the recovered catalyst could be reused three times without any appreciable decrease in its activity (table iii). the strength of catalyst is emphasized via measurement of fe ions leaching by atomic absorption spectroscopy and trace metal ions were detected in the filtrate of this reaction. table iii. reusability of the zro2/fe3o4-mnps in the model reaction (table ii, entry 8) run no. τ / min yield, % 1 25 92 2 25 92 3 27 90 4 30 88 moreover, in order to demonstrate the excellent catalytic activity of zro2/ /fe3o4-mnps and the performance of this method, some of the results obtained _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ friedländer synthesis using zro2/fe3o4-mnps catalyst 979 by the presented procedure were compared with other previously reported heterogeneous catalytic systems in the literature. as shown in table iv, the obtained results show the advantage of the present protocol in terms of catalyst amount, yields or reaction times. table iv. comparison of the result obtained for the synthesis of model reaction (table ii, entry 8) using zro2/fe3o4-mnps with other catalysts reported in the literature entry catalyst amount solvent t / °c τ / min yielda, % reference 1 (bspy)hso4/mcm-41 (70 mg) – 100 80 94 55 2 nh2so3h (5 mol %) – 70 45 89 56 3 sio2/i2 (100 mg/50 mg) – 60 120 80 57 4 amberlyst-15 (10 % w/w) c2h5oh reflux 150 87 58 5 nano-flake zno (10 mol %) – 100 120 92 59 6 zro2/fe3o4-mnps (20 mg) c2h5oh 70 25 92 this work aisolated yield two possible mechanisms exist for the friedländer synthesis of quinolines that are shown in scheme 2. based on these mechanisms and in the first step, the carbonyl group is activated by zro2/fe3o4-mnps. then, 2-amino-5-chloro substituted carbonyl compound 1 and carbonyl compound 2 react in a rate-limiting step to the aldol adduct 3. this intermediate loses water in an elimination reaction to the unsaturated carbonyl compound 4 and then loses water again in imine formation to quinoline 5. in the second mechanism, the first step is schiff base formation to 6 followed by an aldol reaction to 7 and elimination to 8. scheme 2. proposed mechanism for the synthesis of quinoline derivatives in the presence of zro2/fe3o4-mnps. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 980 hejazi et al. conclusions in conclusion, a green, one-pot, effective and environmentally friendly approach has been explained for the synthesis of quinoline derivatives via friedländer annulation using zro2/fe3o4-mnps as a convenient, mild and reusable catalyst. the remarkable benefit of the described method include good substrate generality, mild reaction conditions, high yields, good reaction times, simple experimental procedure, the use of a low cost catalyst, easy work-up, clean reaction profiles and green conditions by avoiding the usage of toxic organic solvents. furthermore, the catalyst was successfully recovered and reused at least for four runs without significant loss in its activity, which make the presented procedure an interesting alternative to previously reported methods. acknowledgement. the authors are thankful to the guilan university research council for the partial support of this work. и з в о д добијање и карактеризација zro2/fe3o4 магнетних наночестица (mnps) као ефикасног суперпарамагнетног катализатора за фридлeндерову синтезу деривата хинолина seyyedeh zoha hejazi, abdollah fallah shojaei, khalil tabatabaeian и farhad shirini department of chemistry, faculty of sciences, university of guilan, p. o. box 41335-1914, rasht, iran током истраживања развијен је приступачан и еколошки прихватљив поступак синтезе деривата хинолина фридлендеровом реакцијом, употребом zro2/fe3o4-mnps као ефикасног хетерогеног катализатора. морфологија zro2/fe3o4-mnps је испитана xrd, ft-ir, sem, tem и vsm аналитичким техникама. предности овог поступка су лака обрада реакционе смеше, висок принос производа и кратко реакционо време. употребљен катализатор се уклања спољашњим магнетним пољем и може се поново употребити најмање три пута без значајнијег смањивања каталитичке активности. током синтезе и обраде реакционе смеше избегава се употреба органских растварача што целокупан процес чини еколошки прихватљивим. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice electrochemical decolorization of the reactive orange 16 dye using a dimensionally stable ti/ptox anode j. serb. chem. soc. 80 (7) 903–915 (2015) udc 543.637–035.67+667.281+ jscs–4768 546.133.1:66.099.72+541.135 original scientific paper 903 electrochemical decolorization of the reactive orange 16 dye using a dimensionally stable ti/ptox anode dušan ž. mijin*#, vuk d. tomić and branimir n. grgur faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11020 belgrade, serbia (received 17 september, revised 5 november, accepted 6 november 2014) abstract: the electrochemical decolorization of reactive orange 16 in a chloride containing solution was studied using a dimensionally stable ti/ptox anode. different reaction parameters, agitation speed, applied current, sodium chloride concentration, and dye concentration were varied and the optimum electrolysis conditions were suggested. hypochlorous acid was suggested to be the active species in the electrochemical decolorization. moreover, the reaction was studied using uv–vis spectrophotometry, high-pressure liquid chromatography (hplc), total organic carbon (toc) and total nitrogen (tn) analyses. keywords: reactive dye; azo dye; electrolysis, hypochlorite; hypochlorous acid. introduction the wastewaters from the textile industry contain, among other chemicals, unreacted dyestuffs. even a small amount of dye in water (≈1 ppm) is highly visible and affects the water transparency, the gas solubility, and possesses potential carcinogenic and mutagenic properties.1,2 textile dyes can be classified based on the method of fixation to the textile fiber (e.g., reactive, direct, acid) or chemical structure (e.g., azo, anthraquinone, indigoid). azo dyes are synthetic organic colorants with a chromophoric azo group (–n=n–). on the one side, the azo group is attached to an aromatic or heterocyclic nucleus and on the other, to an unsaturated molecule of the carbocyclic, heterocyclic or aliphatic type. azo colorants are the most widespread and versatile class of organic dyestuffs. as published in the kirk–othmer encyclopedia of chemical technology in 2003, there are more than 10,000 color index (ci) generic names assigned to commercial colorants. approximately 4,500 of them are in use, and over 50 % belongs to the azo group of compounds. the broad * corresponding author. e-mail: kavur@tmf.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc140917107m _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 904 mijin, tomić and grgur usage range of the azo dyes is due to the number of variations in chemical structure and methods of application, which are not complex in general.3 most dyes are resistant to biodegradation or physico-chemical treatments (e.g., chemical precipitation and separation of pollutants, coagulation, elimination by adsorption, etc.).4–7 these methods are not destructive, leading to the formation of sludge, and thus transferring pollutant from one phase to another, causing a new kind of pollution. an alternative to the mentioned processes are the advanced oxidation processes (aops), which are able to decolorize and mineralize dyestuffs and other organic pollutants.8–13 different electrochemical methods, such as direct or indirect anodic oxidation, cathodic reduction or electrocoagulation, could be used for decolorization and/or degradation of dyes.14–18 in order to achieve better fixation and exhaustion of reactive, direct or substantive dyes, an electrolyte, nacl or na2so4, is usually added. the salt also acts as an electrolyte during the further electrochemical treatment.17 due to its low price and the formation of strong oxidizing electrolysis products, sodium chloride is considered as one of the best choices.18 during the electrolysis, depending on the reaction conditions, strong oxidizing species – chlorine, hypochlorous acid and/or hypochlorite are formed on the anode. at ph > 6, hypochlorous acid dissociates to hypochlorite and h+, and at ph lower than ≈3.5, hocl reacts with cl– to yield cl2. a mixture of these species is commonly called “active chlorine”.17–19 ci reactive orange 16 (ro16, m = 617.5 g mol–1), with the structural formula shown in fig. 1, was the subject of electrochemical degradation studies. the electro-oxidation of ro16 dye was studied using boron-doped diamond (bdd) and dimensionally stable anode (dsa) type electrodes in acidified 0.1 m k2so4.20 the results showed that the bdd electrode had superior characteristics for color removal and in the mineralization of organic materials present in the dye. decolorization was achieved within 90 min, following the pseudo-first order reaction kinetics with rate constants ranging from 0.02 to 0.08 min–1 for the applied current densities from 75 to 200 ma cm–2. for a commercial dsa electrode, the values of the rate constants were not obtained, because only a 10 % decrease in the initial dye concentration was observed at 200 ma cm–2 after 90 fig. 1. structure of ci reactive orange 16. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ electrochemical decolorization of reactive orange 16 dye 905 min of electrolysis.20 the same authors also investigated two different boron -doped diamond films on a ti substrate for electrochemical degradation of ro16.21 the highest doped bdd electrode at current densities higher than 75 ma cm–2 showed better efficiency for the reduction of aromaticity and azo group fracture. this tendency was confirmed by total organic carbon (toc) and chromatographic measurements.21 electrochemical decolorization of ro16 was also performed in electrochemical flow–cell, using a pt electrode.22 the influences of the process variables, i.e., flow–rate, nacl concentration, applied potential and ph, were studied. using 1.0 g dm–3 nacl as the supporting electrolyte at 2.2 v vs. rhe, the determined first order rate constant was ≈0.025 min–1, and a color removal of 93 % was achieved after 60 min electrolysis. the best toc removal of 57 % was at 1.8 v, without a membrane, indicating that the ideal conditions for color removal are not necessarily the same as those for toc.22 dimensionally stable ru(ta, sn)o2-based anodes were used for the electrochemical oxidation of different reactive dyes, including ro16. the reactions were performed in either the presence or absence of nacl. at a low chloride concentration (0.01 m), total color removal was obtained after 10 min of electrolysis, and a significant increase in total dye degradation was achieved (reaching ca. 80 %, chemical oxygen demand, cod, decrease).23 the oxidation was studied using a combination of electrochemical and photo-electrochemical methods in 0.02 m na2so4 at a current density of 50 ma cm–2 in a photo-electrochemical flow-cell.24 the photo-electrochemical method was demonstrated to be more efficient considering the specific energy consumption, toc removal and a slightly higher first order rate constant of ≈0.012 min–1 compared to the one obtained for the electrochemical method, ≈0.009 min–1. the photo bleaching of ro16 in aqueous solution using tio2 thinfilm electrodes prepared by the sol–gel method was also studied.25 the best conditions for maximal photo-electrocatalytic degradation was found to be ph > 10 for a na2so4 medium and ph < 6 for a nacl medium, but only for the low dye concentrations. in both cases, 100 % color removal was obtained after 20 min. oxalic acid was identified by hplc and uv-vis spectrophotometric methods as the main degradation product generated after 180 min of photoelectrocatalysis.25 in this paper, a study of the electrochemical decolorization of ro16 in sodium chloride-containing solutions using for the first time dsa ti/ptox is reported. the influences of the basic operational parameters, such as hydrodynamic conditions, applied current, and the initial concentrations of ro16 and nacl, were studied in order to optimize the process of electrochemical decolorization of ro16. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 906 mijin, tomić and grgur experimental materials the textile dye, ci reactive orange 16, was obtained from aldrich (dye content 50 %) and used without purification. sodium chloride was p.a. grade (merck). acetonitrile (hplc grade) was purchased from j.t. baker. deionized water was obtained from a millipore waters milli-q purification unit. electrochemical decolorization the electrochemical decolorization process was investigated in a cylindrical glass batch reactor, with an electrolyte volume of 500 cm3. the electrolyte was prepared from distilled water, nacl and ro16. a 5 cm2 dsa ti/ptox anode obtained by thermal decomposition of h2ptcl6 in 2-propanol with 1 mg cm -2 of platinum loading was used, while the cathode was 10 cm2 plate made from austenite 18cr/8ni stainless steel series 304. the electrodes with a 3-mm gap between them were immersed at the top of the electrolyte. a par m273 potentiostat/galvanostat operated in the galvanostatic mode was used for the electrolysis of solution. mixing of the electrolyte was accomplished by a magnetic stirrer. at certain times during the electrolysis, 3 cm3 of the solution was taken with a micropipette and its uv-vis spectrum was instantly recorded. the concentration of dye was followed by measuring the absorption of the solution at 493 nm using a shimadzu uv-vis model 1700 spectrophotometer. hplc analysis for hplc determination, all samples were filtered through 0.45 μm syringe filters and analyzed at 245 nm and at ambient temperature (25 °c) on a spectrasystem p4000 liquid chromatograph with a spectrasystem uv1000 detector, equipped with a reversed phase column type zorbax sb c8 (150 mm×4.6 mm i.d., 5 µm particle size). the mobile phase (flow rate 1.0 cm3 min-1) was a mixture of acetonitrile and water (30:70, v/v), 0.1 % formic acid solution. the sample injection volume was 20 μl. total organic carbon (toc) and total nitrogen (tn) analyses the toc and tn analyses during the electrolysis were performed using an elementar liquitoc ii instrument. results and discussion uv–vis spectral changes the uv–vis spectral changes during the electrolysis are shown in fig. 2. several peaks were observed in the spectrum of the ro16 at the beginning of the reaction. the peak at 295 nm corresponds to the gamma acetylated acid structure, which is a precursor in the synthesis of ro16. the peak at 254 nm is indicative of benzene ring, and the peak at 386 nm could be associated with the azo group.24 finally, the peak at 493 nm corresponds to the hydrazone form of the dye because an azo-hydrazone tautomerism is possible in the ro16 molecule.17,26 the decreases in the absorption peaks at 386 and 493 nm during electrolysis indicate fast decolorization of ro16 dye over time (fig. 2). complete decolorization was observed after ≈10 min of electrochemical treatment, insert in fig. 2. at the end of the oxidation reaction, the spectrum showed small _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ electrochemical decolorization of reactive orange 16 dye 907 peaks at 254 and 295 nm. considering that the reaction was very fast, the concentrations of hypochlorite and hypochlorous acid are very low and cannot be observed in the spectra (the absorption maximum of hypochlorite is at 292 nm and the absorption maximum for hypochlorous acid is at 236 nm).27 fig. 2. typical uv–vis spectra changes of ro16 (60 mg dm-3) in 10 g dm-3 aqueous nacl water during electrochemical decolorization, i = 100 ma, ω = 250 rpm. inset: decolorization level over time. effect of the applied current in order to investigate the influence of the current on the electrochemical decolorization of the ro16 dye, currents in the range from 50 to 250 ma were applied. the dependence of the relative dye concentration over time for different values of applied current is shown in fig, 3a. more than 95 % decolorization was achieved within ten minutes for applied currents greater than 50 ma. examining different pseudo n-orders, the first-order gave the best linearity, and pseudo firstorder decolorization rate constants, kd, min–1, were determined in accordance with the kinetic equation: d 0 ln t c k t c = − (1) from the slopes of the straight lines, fig. 3, the rate constants were determined and are shown in fig. 3b. the decolorization rate constant practically linearly increased from 0.14 to 0.33 min–1 in the investigated range of applied currents, while electrolysis voltage increased from 3.5 to 5 v. the faster reaction was achieved with current of 250 ma, but considering that the specific energy consumptions with reaction rate increase, some optimum current lies at 100–150 ma. for example, an increase of current from 100 to 250 ma increases the reac_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 908 mijin, tomić and grgur tion rate 1.5 times, while the specific energy consumption is increased 2.5 times, from 1.1 to 2.8 wh g–1, calculated using the cell voltage shown in fig. 3b. fig. 3. the logarithmic plot of the relative ro16 concentration vs. the electrolysis time for different values of applied current (marked in the figure) under the conditions m(nacl) = 10 g dm-3, m(ro16) = 60 mg dm-3, ω = 250 rpm. insets: a) the dependence of the relative ro16 concentration over time for different values of the applied current; b) the dependence of the pseudo first-order reaction rate constants and the electrolysis voltage on the values of the applied current. the influence of the hydrodynamic conditions the influence of the solution mixing (rpm of magnetic stirrer) on the ro16 decolorization is shown in fig. 4. the apparent decolorization rate constant of ≈0.24 min–1 are practically independent of the agitation speed, as shown in fig. 4. during the first five minutes of the electrolysis, the ph increased relatively fast from the initial ≈5.5 to 8, and reached a steady state value of ≈8.5 after 10 min, fig. 4b. the changes in ph provoked changes in the relative ratio of the different active chlorine species in the solution (see section: possible active chlorine species). considering that diffusion, heterogeneous or homogeneous oxidation rate was highly dependent on the hydrodynamic conditions,28 the possible explanation is that the rate determining step is an activation controlled (charge transfer) reaction. the only activation-controlled reaction could be the oxidation of the chloride anions to the solvated chlorine, given by eq. (3). effect of the initial sodium chloride concentration the influence of the initial nacl concentration on the reaction rate was investigated in the range from 10 to 30 g dm–3 and the results are shown in fig. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ electrochemical decolorization of reactive orange 16 dye 909 5. the determined reaction rate constants increased with increasing salt concentration up to 25 g dm–3, and above that concentration, a small change in the reaction rate is observed, as shown in the insert of fig. 5. fig. 4. the logarithmic plot of the relative concentration of the dye vs. the electrolysis time for different agitation speeds (marked in the figure), m(nacl) = 10 g dm-3, i = 100 ma, m(ro16) = 60 mg dm-3. insets: a) the dependence of the relative ro16 concentration over time for different agitation speeds; b) the change in the ph value during electrolysis. fig. 5. the logarithmic plot of the relative ro16 concentration vs. the electrolysis time for different sodium chloride concentrations (marked in the figure), and in 10 g dm-3 na2so4 during continuous electrolysis. inset: the dependence of the pseudo first-order reaction rate constants on the sodium chloride concentration, i = 100 ma, m(ro16) = 60 mg dm-3, ω = 250 rpm. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 910 mijin, tomić and grgur according to the faraday law, the amount of the produced active chlorine should be proportional to the passed charge (it) and the rate should remain constant independently of the chloride concentration. however, the oxygen evolution: 4oh– = o2 + 2h2o + 4e– (2) and the production of active chlorine (eqs. (3)–(5)) compete.19,29 thus, the current efficiency of active chlorine production is connected with the chloride concentration. namely, as determined by kraft et al.,19 in the concentration range of nacl from 1 to 20 g dm–3, the current efficiency of active chlorine production on a ti/ptox electrode linearly increases from ≈5 to 75 %. therefore, in the solution with a nacl concentration in the range of 10 to 20 g dm–3, the oxygen evolution reaction proceeds at a relatively high rate. in the solution with higher nacl concentrations, the current efficiency steady state conditions are attained and the amount of the active chlorine production becomes nearly constant. possible active chlorine species in order to resolve which of the active chlorine species are the most active, the following experiment was performed: a solution of 5 g nacl in 500 cm3 of water was pre-electrolyzed for 1 and 5 min at 100 ma, and then, 30 mg of ro16 dye was added. the results of these experiments are presented in fig. 5. it can bee seen that decolorization occurred practically with a similar rate as during the electrolysis with higher currents (fig. 3a). the rate in the electrolyte pre-electrolyzed during 5 min was ≈20 % faster than in the electrolyte pre-electrolyzed for 1 min. in fig. 3, the results obtained during electrolysis with current of 100 ma in a solution containing 10 g dm–3 of na2so4 and 60 mg of ro16 is also shown, because of the possibilities that anodic water oxidation could result in the formation of hydroxyl radicals, which can easily oxidize ro16.17,30 under such conditions, no activity was observed. hence, it could be concluded that hydroxyl radicals were not involved in the reaction mechanism. one more important conclusion from this experiment is that ro16 is not cathodically reduced during electrochemical degradation. the oxidation of the chloride anions to the hydrated chlorine is given by the complex overall reaction: 2cl– = cl2(aq) + 2e– (3) which is followed by the fast disproportionation of the hydrated chlorine to hocl and its dissociation to ocl–:13,19 cl2(aq) + h2o = hclo + cl– + h+, k1 = 5.1×10–4 m2 (4) hclo = clo– + h+, ka = 2.9×10–8 m (5) _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ electrochemical decolorization of reactive orange 16 dye 911 the distribution of different active chlorine species in a solution containing chloride as a function of ph can be calculated starting from the chemical equilibrium equations given above. assuming that concentration corresponds to activity and that water has unity activity, the following procedure was used starting from the mass balance equation: c(cl2,act)t = c(cl2) + c(hocl) + c(ocl–) (6) the total active chlorine concentration in solution can be calculated using the faraday law: ( ) 22,act i3 (cl ) cl 0.5 dmt n it c f η= = (7) where ηi is the current efficiency for chlorine formation. by rearranging eq. (6), the concentration of hydrated chlorine is obtained: c(cl2) = c(cl2,act)t 1 2 _ 2 )cl( )ocl( )cl( )hocl( 1 −       ++ c c c c (8) using a mathematical procedure, it can be evaluated that: 1 2 (hocl) (cl ) (h ) (cl ) c k c c c+ − = (9) and: 1 2 2 (ocl ) (cl ) (h ) (cl ) ac k k c c c − + − = (10) introducing eqs. (9) and (10) into eq. (8), the free solvated chlorine concentration as a function of ph could be calculated. once the ph dependence of the free chlorine concentration is known, it is possible to calculate, by a similar procedure, the ph dependence of the concentrations of all the other species in the solution. taking into account that for the nacl concentration of 10 g dm–3 (0.17 m), current efficiency is 0.4519 and using eq. (7), the total active chlorine concentration for 1 and 5 min of electrolysis was calculated to be 0.028 and 0.125 mm. applying the above-described mathematical treatment, the distribution of different active chlorine species in solution as a function of ph was calculated and shown in fig. s-1 (supplementary material to this paper). the ph for 1 and 5 min of electrolysis was ≈6 and 8, respectively, fig 4b. at these ph values, corresponding concentrations of hocl are 29 and 37 μm, and for ocl– ≈0 and 0.1 mm, respectively. therefore, the ≈20% higher rate in the electrolyte preelectrolyzed during 5 min than in the electrolyte pre-electrolyzed for 1 min corresponds to the ratios of hocl concentration, suggesting that hypochlorous acid is the active species in ro16 decolorization. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 912 mijin, tomić and grgur effect of the initial dye concentration the dependence of logarithm of the relative concentrations over time for different ro16 concentrations, 20–80 mg dm–3, is shown in fig. 6. as it could be seen in the insert of fig. 6, increasing the dye concentration decreased the reaction rate. this trend could be connected with the ph distribution of the suggested active hocl species. namely, for a low ro16 concentration, e.g., 20 mg dm–3, the decolorization was practically completed within 3 to 4 min and corresponding ph was lower than 7.5. at this ph value, the dominant species is practically pure hocl. increasing the dye concentration, the decolorization time and ph increased as well, which is connected with a decrease in hclo concentration and a slower reaction rate. however, the observed behavior could also be explained by taking into consideration that the overall rate of ro16 decolorization was controlled by active chlorine production, as was inferred in the previous section. it could be postulated that the rate of active chlorine production was approximately the same independently of the initial concentration of ro16, because in all the measurements current was the same, i = 100 ma, as well as the chloride concentration, m(nacl) = 10 g dm–3. consequently, in the solutions with a lower initial dye concentration, proportionally lower relative concentrations of ro16 were to be expected for the same electrolysis time (fig. 6). the concentration of generated hocl active species during the electrolysis is unknown, but from the kinetics point of view, it is reasonable to assume that it was lower in the solutions with a higher initial concentration of ro16. taking into account that the concentration of active chlorine was included in the pseudo first-order rate constants, the dependence shown in the inset of fig. 6 was anticipated. fig. 6. the logarithmic plot of the relative ro16 concentration against electrolysis time for different initial dye concentrations. inset: dependence of the pseudo first-order reaction rate constants on ro16 concentration, m(nacl) = 10 g dm-3, i = 100 ma, ω = 250 rpm. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ electrochemical decolorization of reactive orange 16 dye 913 hplc and toc/tn analyses high-performance liquid chromatography (hplc) was used to obtain a better insight in the decolorization process of the ro16 dye. as suggested by miglirioni et al.,21 the reaction was followed at 245 nm using hplc with a uv detector because the appearance of signal at this wavelength is related to the π→π* transition of conjugated systems, which is characteristic for aromatic compounds. the chromatograms of the ro16 azo dye solution before the electrochemical treatment (0 min) and after 16 min of electrochemical treatment at 100 ma are presented in fig. s-2 (supplementary material). the chromatogram for the ro16 azo dye solution before the electrochemical treatment showed four peaks related to aromatic compounds with different polarities and retention times. these aromatic peaks were named in the chromatogram from a to d. the highest intensity peak c is probably related to the ro16 dye while the less intense peaks are probably related to impurities in the 50 % ro16 dye solution. after the electrochemical treatment at 100 ma with dsa ti/ptox electrode (16 min), peak c had almost disappeared while peaks d and b were reduced. at the same time, new peaks emerged (peaks e–j) due to the reaction indicating the presence of smaller, less polar aromatic molecules. toc and tn analyses were also performed in order to assess the effectiveness of used electrochemical method in the mineralization of the ro16 dye. the uv-vis absorption spectrum (fig. 2) shows that absorption in the visible part of the spectra was almost completely removed. however, the absorption in the uv region was not removed, which indicates the presence of intermediate compounds after color removal. the toc and tn changes during the reaction are shown in fig. s-3 (supplementary material). as could be seen, ≈10% of the dye was mineralized. the obtained results confirm that the degradation of ro16 was only partial, but the rate of the achieved decolorization is much faster than in photocatalytic processes,31,32 except when h2o2 was added.33 conclusions the dimensionally stable ti/ptox anode was found to be effective in the decolorization process of ci reactive orange 16 dye solutions containing chloride. the decolorization proceeded via a pseudo-first order reaction with hypochlorous acid as the main active chlorine species. depending on the electrolysis conditions, the apparent first-order reaction constant was in the range of 0.2 to 0.5 min–1. complete decolorization within 15 min could be successfully realized with currents in the range of 100 to 200 ma (20–40 ma cm–2), sodium chloride concentrations in the range of 10 to 30 g dm–3 and low (few mg dm–3) and medium (~0.1 g dm–3) contents of dye. a negligible effect of the hydrodynamic conditions on the ro16 decolorization was recorded. the specific power consumption was estimated in the range of 1 to 3 kwh kg–1, depending on the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 914 mijin, tomić and grgur applied current. some optimum electrolysis conditions can be suggested: 10–15 g dm–3 of nacl, with anodic current density of 20–25 ma cm–2 under moderate steering. hplc analysis revealed the formation of smaller aromatic molecules, which were less polar than the starting molecule. the obtained results of toc/tn analyses confirmed that the degradation of ro16 was only partial. supplementary material the distribution of different active chlorine species in solution as a function of ph, hplc chromatograms and relative toc and tn change during electrochemical treatment of the ro 16 dye solution are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the work was supported by the ministry of education, science and technological development of the republic of serbia under the research projects: on172013 and on172046. authors express their gratitude to the puc “belgrade waterworks and sewerage“ for the toc/tn analyses. и з в о д електрохемијскo обезбојавање reactive orange 16 боје помоћу димензионо стабилне ti/ptox аноде душан ж.мији*, вук д.томић и бранимир н. гргур технолошко-металуршки факултет, универзитет у београду, карнегијева 4, 11120 београд електрохемијско обезбојавање ci reactive orange 16 је испитивано на димензионо стабилној ti/ptox аноди у растворима на бази натријум-хлорида. различити реакциони параметри, као што су брзина мешања, јачина струје, концентрација натријум-хлорида и концентрација боје, су варирани и на основу добијених резултата утврђени су оптимални реакциони услови. претпостављено је да је активна врста у реакцији обезбојавања хипохлораста киселина. реакција је праћена помоћу uv–vis спектроскопије, течне хроматографије високог притиска, као и одређивањем укупног органског угљеника и азота. примљено 17 септембра, ревидирано 5 новембра, прихваћено 6 новембра 2014) references 1. p. a. carneiro, m. e. osugi, c. s. fugivara, n. boralle, m. furlan, m. v. b. zanoni, chemosphere 59 (2005) 431 2. s. meric, d. kaptan, t. olmez, chemosphere 54 (2004) 435 3. r. j. chudgar, j. oakes, in kirk–othmer encyclopedia of chemical technology, vol. 9, 5th ed., john wiley & sons, new york, 2007, pp. 349–430 4. e. foracs, t. cserhati, g. oros, environ. int. 30 (2004) 953 5. i. k. konstantinou, t. a. albanis, appl. catal., b 49 (2004) 1 6. y. m. slokar, a. m. le marechal, dyes pigm. 37 (1998) 335 7. b. ramesh babu, a. k. parande, 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice alfacalcidol modulates oxidative stress parameters in the peripheral blood of patients with active rheumatoid arthritis j. serb. chem. soc. 81 (10) 1127–1139 (2016) udc *alfacalcidol:615.279–188:616.72= jscs–4913 002.77 original scientific paper 1127 alfacalcidol modulates oxidative stress parameters in the peripheral blood of patients with active rheumatoid arthritis tatjana živanović radnić1*, katarina simić-pašalić1, mirjana šefik bukilica1,2, sonja misirlić denčić3, anđelka m. isaković3, tihomir stojković3, nataša petronijević3, nemanja damjanov1,2 and jelena vojinović4 1institute of rheumatology, belgrade, serbia, 2faculty of medicine, university of belgrade, serbia, 3institute of medical and clinical biochemistry, school of medicine, university of belgrade, serbia and 4clinical centre, faculty of medicine, university of niš, serbia (received 6 may, revised 13 may, accepted 16 may 2016) abstract: hormone d and its analogues display immunomodulatory activities providing a beneficial effect in immunoinflammatory diseases. the aim of this study was to assess the effect of alfacalcidol treatment on superoxide dismutase (sod), catalase (cat) and glutathione peroxidase (gpx) activity and glutathione (gsh) and malondialdehyde (mda) levels in patients with active ra. sixteen patients with active ra and twenty controls were enrolled in the study. blood samples were taken before and after 12 weeks of alfacalcidol therapy (2 μg/day). oxidative stress parameters were determined spectrophotometrically and by flow cytometry assessment. disease activity was assessed using das28 score. the results revealed that alfacalcidol treatment, significantly (p = 0.04) reduced sod activity and cat activity (p = 0.001) in ra patients. the activity of gpx was significantly lower in ra patients before treatment, compared to controls (p = 0.04). after therapy, gpx activity was restored to control levels, and gsh levels were significantly reduced (p = 0.01). mda levels in patients at the beginning of the study protocol, remained significantly elevated compared to controls (p = 0.01). alfacalcidol treatment decreased mda levels in patients (p = 0.19). furthermore, 12-weeks alfacalcidol therapy, changed the response of ra patients’ pbmc to stimulation preventing the o2 production and mitochondrial membrane depolarisation. after alfacalcidol treatment, significant clinical improvement was observed. keywords: antioxidant activity; disease activity; additional therapy; beneficial; pbmc. * corresponding author. e-mail: tatjanaradnic@yahoo.com doi: 10.2298/jsc160506039z _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1128 živanović radnić et al. introduction one of the most common autoimmune diseases, rheumatoid arthritis (ra) is characterized by the persistent synovitis and systemic inflammation. in the last decade, important advances have been made in the diagnosis, clinical assessment and treatment of ra patients. however, our understanding of the underlying cellular and molecular mechanisms involved in pathogenesis of ra is still incomplete. it was shown that low vitamin d intake is associated with an elevated risk of ra development and furthermore, vitamin d level is associated with ra activity.1–3 the main source of vitamin d is from dietary and supplement intake, with the majority coming from the skin exposure to sunlight, specifically ultraviolet b (uv-b) rays. higher cumulative average uv-b exposure was associated with decreased ra risk in female nurses aged 30–55 years.4 vitamin d from the skin and diet is metabolized in the liver to 25-hydroxyvitamin d (25(oh)d), which is used to determine a patients’ vitamin d status. 25-hydroxyvitamin d is metabolized in the kidneys to its active form 1,25-dihydroxycholecalciferol or calcitriol (vitamin d3) solely biologically active form acting as a steroid hormone.5 vitamin d3, has been shown to be a pleiotropic steroid hormone involved not only in regulation of calcium homeostasis and bone turnover, but also in immunomodulation, having potent anti-inflammatory, antiproliferative, prodifferentiation and antibacterial properties in various cells and tissues.6,7 furthermore, vitamin d3 can be produced by monocytes behaving as a paracrine factor thus having potent actions on all the cellular components of the immune system.8 vitamin d3 exerts its effects via a specific vitamin d receptor (vdr), found in activated lymphocytes, thymocytes and other immunocompetent cells. the significant enrichment of vitamin d response elements at ra associated loci support the hypothesis that vitamin d3 plays a role in the development of ra.9 the contribution of oxidative stress to chronic inflammation of tissues10,11 and its involvement in pathophysiological mechanism of autoimmune diseases12 have been recently recognized. oxidative stress reflects an imbalance between the systemic production of reactive oxygen species (ros) and the antioxidant ability of the organism to readily detoxify the reactive intermediates or to repair the resulting damage. there is a strong evidence supporting the role of oxidative stress in cartilage degradation in experimental arthritis13 as well as in contribution to disease activity in ra patients.14 pro-oxidation environment in ra results in redox imbalance, evidenced by increased production of reactive oxygen, nitrogen and sulphur15 species that amplify the synovial inflammatory-proliferative response.14 however, scarce information is available about the antioxidant properties of vitamin d3 and its analogues, and some data are even controversial.16–18 therefore, the role of vitamin d3 therapy in ra patients deserves to be further explored. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. alfacalcidol antioxidant activity for rheumatoid arthritis 1129 alfacalcidol (1α(oh)d3) is a synthetic vitamin d3 analogue, exerting full biological activity of calcitriol. in a recent study by radovic et al.,19 the alfacalcidol therapy showed a beneficial effect, without any side effects, on the disease activity in 93.3 % of the patients with juvenile idiopathic arthritis (jia), also known as juvenile rheumatoid arthritis (jra), which is the most common form of arthritis in children and adolescents. this study revealed that alfacalcidol therapy induced significant changes in antioxidative enzyme activities. the goal of this study was to examine the effects of alfacalcidol therapy on the superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx) activity, glutathione (gsh) levels in erythrocytes and malondialdehyde (mda) in the plasma of patients with active ra in comparison to the healthy controls. we were also interested in the therapy effect on sensitivity of ra patients peripheral blood mononuclear cells (pbmc) to stimulation. experimental sixteen female ra patients (average age 53±13), in the outpatient clinic of the institute of rheumatology, belgrade, serbia, were consecutively enrolled in the study. controls were twenty ageand sex-matched healthy volunteers selected from the staff. all subjects signed the information consent before participation in the study. the study was conducted in compliance with the declaration of helsinki, international conference on harmonization and good clinical practice guidelines, and was approved by the ethics committee of the institute of rheumatology in belgrade, ethic committee of the university of belgrade, school of medicine, and by medicines and medical devices agency of serbia. inclusion criteria were as follows: established diagnosis of ra (acr/eular criteria 2010),20 at least six months prior to enrolment; methotrexate treatment for 12 weeks or longer with stabile dose of 10-25 mg/week for 8 weeks or longer); active disease defined as 28-joint disease activity score (das28) >3.2. if non-steroidal anti-inflammatory drugs (nsaids) are administered, patients have to be using stable dose of nsaid for at least four weeks. glucocorticoids (systemic and/or local use) were not allowed for at least one month before entering the study. the treatment protocol considered the continuation of previous therapy with the addition of alfacalcidol as oral commercial preparation at the dose of 2 μg daily during 12 weeks. clinical and laboratory assessment of disease activity (serum concentration of c-reactive protein (crp), sedimentation rate (se), number of swollen and tender joints) and safety (regular biochemistry analyses including serum total and ionized calcium concentration and urine calcium concentration as well) of the studied drug were evaluated during regular visits (before start of the therapy, in four-week intervals and after completion of the alfacalcidol therapy). disease activity was assessed using the 28-joint das28.21 blood samples for vitamin d (25(oh)d) levels of our patients were obtained after at least 8 h overnight fasting, before (week 0 = w0) and after 12-week long alphacalcidol therapy (week 12 = w12). the level of vitamin d was determined by electrochemiluminescence assay from roche diagnostics (elecsys 2010). level of vitamin d < 20 ng/ml indicates deficiency, levels 21–29 ng/ml indicate insufficiency and levels ≥ 30 ng/ml indicate sufficiency. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1130 živanović radnić et al. the activity of antioxidant enzymes sod, cat and gpx and the level of gsh were determined in isolated and lysed erythrocytes, while mda was determined in plasma, of the controls and patients, before (w0) and in ra patients after 12-week long (w12) alphacalcidol therapy using manual spectrophotometric method. hemoglobin (hb) concentration in lysed erythrocytes was measured by hematology analyzer. sod activity was assayed as the ability of supernatant obtained after hb precipitation from lysed erythrocytes, to inhibit the radicalmediated autooxidation of epinephrine (using epinephrine hydrochloride, sigma).22 obtained values were expressed as the number of units (u) per gram of hemoglobin in erythrocyte haemolyzate (ghb). the intra-assay and inter-assay cvs were 5.2 and 6.7 %, respectively. cat activity was determined based on the formation of stable complex of hydrogen peroxide with ammonium molybdate whose absorption was measured spectrophotometrically at 405 nm (using hydrogen peroxide solution 30 %, zorka pharma, ammonium molybdate, sigma– –aldrich).23 results were expressed as u/ghb, the intra-assay cv was 3.9 % and the inter -assay cv was 8.5 %. gpx activity in lysed erythrocytes was measured according to previously described spectrophotometric method24 (using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid – hepes, sigma–aldrich; ethylenediaminetetraacetic acid, disodium salt, sigma; l-glutathione reduced, sigma; β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate, sigma; tert-butyl hydroperoxide solution, sigma; glutathione reductase, sigma–aldrich) and presented as u/ghb (the intra-assay cv was 5.7 % and the inter-assay cv was 7.2 %). gsh was determined according to the method by sedlak and lindsay (using the ellman’s reagent 5,5’-dithiobis-(2-nitrobenzoic acid),25 dtnb, sigma– aldrich; tris(hydroxymethyl)aminomethane, serva, germany), and obtained values were expressed as μmol/ghb (the intra-assay cv was 3.6 % and the inter-assay cv was 7.3 %). lipid peroxidation, measured as malondialdehyde (mda) level, was determined spectrophotometrically in a reaction with thiobarbituric acid. thiobarbituric acid reacts with mda released from polyunsaturated fatty acids of plasma membrane phospholipids injured by ros, forming a yellow complex whose absorbance was measured at 533 nm (trichloroacetic acid, sigma–aldrich; 2-thiobarbituric acid, sigma).26 results were expressed as nmol of mda per ml (the intra-assay cv was 5.2 % and the inter-assay cv was 7.9 %). mitochondrial production of superoxide anion (o2 -), one of ros, as well as mitochondrial membrane potential were assessed on peripheral blood mononuclear cells (pbmcs) of controls and ra patients (w0 and w12) using flow cytometry. peripheral blood mononuclear cells (pbmcs) were obtained from venous blood of controls and ra patients (on w0 and w12). pbmcs were isolated from heparinized blood by density gradient centrifugation using lymphoprep (axis shield, norway). the pbmc were maintained at 37 °c in a humidified atmosphere with 5% co2, in hepes (20 mm)-buffered rpmi 1640 cell culture medium supplemented with 10 % fetal bovine serum (fbs), 2 mm -glutamine, and 1 % of antibiotic/antimycotic mixture (all from paa, austria). cells (2.5×105 cells/well) were incubated in 24-well plates (sarstedt, nümbrecht, germany). cells were stimulated 30 min after seeding for 4 h, with phorbol 12-myristate 13-acetate (pma) (sigma– –aldrich), final concentration 10 ng/ml and ionomycin (sigma–aldrich), final concentration 1.25 μm. each experiment contained untreated and stimulated (control, patients w0 and patients w12) cells. flow cytometry assessments were performed using facscalibur (bd biosciences, heidelberg, germany) equipped with cellquest pro software for acquisition and analysis. the light-scatter channels were set on linear gains and the fluorescence channels on the logarithmic scale. a minimum of 10,000 cells was analyzed for each condition, adjusting the _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. alfacalcidol antioxidant activity for rheumatoid arthritis 1131 threshold settings so that the cell debris was excluded from the data acquisition. the superoxide anion production was analyzed by measuring the increase in red fluorescence (fl2) intensity emitted by a superoxide-specific fluorochrome, dihydroethidium (dhe; sigma–aldrich), which was incubated with cells (20 mmol/l) for 30 min. obtained results were presented as the increase of mean fluorescence intensity (compared to 1 in non-stimulated cells). mitochondrial membrane potential was assessed using a lipophilic cation jc-1 (r&d systems), which has the property of aggregating upon membrane polarization thus forming an orange-red fluorescent compound. the dye cannot access the transmembrane space if the potential is disturbed, and remains in or reverts to its green monomeric form. the cells were stained with jc-1 as described by the manufacturer, and the green monomer and the red aggregates were detected by flow cytometry. obtained values were expressed as the green/red (fl1/fl2) fluorescence ratio, and the increase corresponded to the extent of mitochondrial depolarization. the results are presented as the increase of fl1/fl2 fluorescence ratio (compared to 1 in non-stimulated cells). statistical analysis was performed using t-test for independent samples, t-test for related samples (for parametric data) and wilcoxon and mann-whiney tests (for nonparametric data). for testing the normality of the distribution parameters, the kolmogorov–smirnov test was used. correlation between parameters was determined using spearman and pearson test. the results are presented as mean±standard deviation (sd, data with normal gaussian distribution) or median (iqr, interquartile range) for non-gaussian distribution data. results at the beginning of the study, vitamin d (25(oh)d) levels in patients were sufficient (w0, 30±11 ng/ml) and remained constant after 12-week treatment (w12, 31±13 ng/ml), indicating that alfacalcidol does not cause significant (p > > 0.05) change in patients’ 25(oh)d level. furthermore, during 12 weeks of alfacacidol treatment and follow-up period no side effects were noticed in patients with ra. serum calcium and ionized calcium levels remained within physiological range of values. although the increase in urine calcium levels was detected in some patients, obtained values remained within physiological range. parameters of antioxidant enzymes (sod, cat and gpx) activity and gsh levels in the erythrocytes and plasma mda levels, of ra patients before and after 12 weeks of alfacalcidol treatment, as well as for control group are presented in table i. the alfacalcidol treatment significantly reduced both sod (w0/w12; p = 0.04) and cat (w0/w12; p = 0.001) activity in erythrocytes of ra patients. while sod activity was reduced to the levels obtained for the healthy control group, cat activity after alfacalcidol treatment was decreased to the levels significantly lower than in the controls (p = 0.0001). however, no significant difference in cat activity in erythrocytes of ra patients before (w0) therapy in comparison to the controls was observed (w0/ctrl; p = 0.37). the activity of gpx was significantly lower in erythrocytes of ra patients before treatment (w0) compared to the controls (p = 0.04). in accordance with lower gpx values, gsh levels were significantly higher in erythrocytes of ra patients (w0) compared to _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1132 živanović radnić et al. the controls (p = 0.03). even though 12-weeks of alfacalcidol therapy restored erythrocytes’ gpx activity to control levels, no significant difference was detected between values obtained for w0 and w12 (p > 0.05). on the other hand, the 12-week long alfacalcidol therapy significantly reduced gsh levels (p = 0.01). in addition to glutathione levels and antioxidant enzyme activity, oxidative stress was further evaluated through measuring the mda levels produced in lipid peroxidation initiated by ros. in the present study, although alfacalcidol treatment decreased mda levels in patients, the difference did not reach a statistical significance (w0/w12; p = 0.19). however, mda levels in patients at the beginning of the study protocol (w0), remained significantly elevated compared to the controls (w0/ctrl; p = 0.01), suggesting the presence of oxidative stress in patients with active disease (table i). table i. the activity of antioxidant enzymes (sod, cat and gpx) and glutathione (gsh) levels in erythrocytes and mda in plasma of ra patients’ before and after twelve weeks of alfacalcidol therapy; sod – superoxide dismutase; cat – catalase; gpx – gluthation peroxidase; gsh – gluthation; mda – malondialdehyde; u – intenational unit of enzyme activity; hb – hemoglobin; ra – rheumatoid arthritis; w0 – before therapy; w12 – after 12 weeks of therapy; values presented are means ± sd or median (iqr); *p < 0.05 compared to w12; **p < 0.01 compared to w12; #p < 0.05 compared to control; ##p < 0.01 compared to control therapy sod u/ghb cat u/ghb gpx u/ghb gsh µmol/ghb mda nmol/ml control 450±110 1300±600 5.2±1.8 4.7±1.2 106±57 ra patients w0 510±100* 1500±800** 3.7±1.6# 6.0±2.1**# 163±30## w12 470±130 258 (302)## 3.9 (0.8) 3.7±1.2 149±42 in order to get further insight into the effect of 12-week long alfacalcidol treatment, we measured the intensity of superoxide anion (o2–) production and mitochondrial membrane potential in controls and patients (w0 and w12) stimulated pbmc. obtained results showed that stimulation caused the increase in dhe fluorescence intensity in control pmbc indicating the o2– production (table ii). furthermore, in patients’ pbmc at the beginning of the study (w0), superoxide production was higher in comparison to the healthy controls, but this difference failed to reach a statistical significance (w0/ctrl; p = 0.09). also, fl1/ /fl2 ratio increased in stimulated controls as well as in patients (w0), suggesting the ability of stimulation to cause depolarization of the inner mitochondrial membrane (table ii). on the other hand, alfacalcidol treatment changed the response of patients (w12) pbmc to stimulation as evidenced by the reduced superoxide production (w0/w12; p = 0.10) and the absence of the inner mitochondrial membrane depolarization (w0/w12; p = 0.001). therefore, these findings suggest the protective influence of alfacalcidol on pbmc sensitivity to oxidative stress induced by stimulation. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. alfacalcidol antioxidant activity for rheumatoid arthritis 1133 table ii. production of superoxide anion (o2-) and change of mitochondrial membrane potential in controls and patients’ stimulated pbmcs before and, in patients, after twelve weeks of alfacalcidol therapy; dhe – dihydroethidium; jc1 – lipophilic cation; pbmc – peripheral blood mononuclear cells; w0 – before therapy; w12 – after 12 weeks of therapy; values presented are means ± sd; *p < 0.05 compared to w12; **p < 0.01 compared to w12; #p < 0.05 compared to control; ##p < 0.01 compared to control therapy dhe jc1 control 1.7±0.5 1.4±0.2 ra patients w0 2.2±0.6 1.3±0.2** w12 1.5±0.5 0.8±0.2 after 12-weeks of alfacalcidol treatment, significant clinical improvement was observed. disease activity score (das28) significantly decreased from 5.8±0.9, before treatment (w0) to 4.3±1.0 (p < 0.01) after (w12) therapy. at the beginning of the study (w0), most of the patients (84 %) were in the group of highactive disease (das28 > 5.1) and 16 % of the patients were with the moderateactive disease (3.2 < das28 < 5.1). there were no patients with the low-active disease (das28 < 3.2). after treatment (w12), only 16 % of the patients remained in the group of high-active disease, 68 % of them were in moderate-active disease, and 16 % of the patients had low-active disease (das28 < 3.2), fig. 1. fig. 1. alfacalcidol therapy causes significant clinical improvement of ra patients regarding disease activity score (das28). after twelve weeks of treatment (w12), 16 % of the patients were in the group of high-active disease (das 28 > 5.1), 68 % were in moderate-active disease (3.2 < das28 < 5.1) while 16 % had low-active disease (das 28 < 3.2). also, crp levels dropped from 29±28 mg/l (w0) to 10±12 mg/l (w12) (p = = 0.02) supporting the clinical improvement evidenced by das28. significant positive correlation (r2 = 0.64, p = 0.02) was observed only between changes in cat activity (δcat) and crp level (δcrp), fig. 2. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1134 živanović radnić et al. fig. 2. alfacalcidol therapy causes significant clinical improvement of ra patients regarding disease activity score (das28). after twelve weeks of treatment (w12), 16 % of the patients were in the group of high-active disease (das28 > 5.1), 68 % were in moderate-active disease (3.2 < das28 < 5.1) while 16 % had low-active disease (das28 < 3.2). discussion herein we report no significant change in patients’ 25(oh)d levels before and after 12-weeks of alfacalcidol treatment. in accordance with this, it is of great importance to emphasize that measurement of 25(oh)d levels, as the only standardized tool to estimate vitamin d status in organism, is actually only the reflection of the balance between food and/or supplement vitamin d diet intake and its utilization in the local tissues as the active d hormone.27 superoxide anion (o2–) is believed to be one of the initiators of free radical production reactions. sod is an enzyme responsible for the conversion of o2–, an important ros, to hydrogen peroxide. in this study, alfacalcidol treatment (12 -weeks), significantly (w0/w12; p = 0.04) reduced sod activity in erythrocytes of ra patients to the levels obtained in the healthy control group. the results of the present study are similar to that published by radovic et al.,19 which showed that 12-weeks of alfacalcidol treatment in patient with juvenile idiopathic arthritis (jia), reduced sod activity to the levels even lower than in healthy control group, confirming role of vitamin d in regulation of cell redox system. one explanation could be the fact that sod production is stimulated through th1 cellular and humoral immunity that are reduced by the alfacalcidol treatment. similarly, cimen et al.28 observed higher sod levels in ra patients than in control group, suggesting that excessive free radical production might be through xanthine–xanthine oxidase system rather than an impaired antioxidant system. likewise, vijaykumar et al.29 reported increased levels of sod and gpx in their study, which could dismutate the excess superoxide radicals that are generated and diffused from the inflammatory sites due to over expression of antioxidant defense system of ra patients. however akyol et al.30 and ozkan et al.31 reported no change in sod levels between ra patients and controls. however our findings are contra_________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. alfacalcidol antioxidant activity for rheumatoid arthritis 1135 dictory to the findings of chandankhede et al.32 and desai et al.33 that showed decreased activity of antioxidant enzyme sod in ra patients compared to the controls, but the patients included in their studies were not in the active stage. furthermore, our results showed that 12-weeks long alfaclcidol tretment changed the response of ra patients’ pbmc to stimulation by preventing the o2– production and mitochondrial membrane depolarisation thus supporting the result of reduced sod activity in ra patients erythrocytes after this prolonged treatment. considering that vitamin d is a membrane antioxidant able to inhibit irondependent liposomal lipid peroxidation by stabilizing the cell membranes,34 this result indicates possible protective influence of alfacalcidol on pbmc sensitivity to oxidative stress induction upon stimulation. this could be significant since in our experiments stimulation caused increase in o2– production and depolarisation of mitochondria membrane, not only in patients before treatment (w0), in accordance with the results from bulua et al.,35 but also in healthy control pmbc as well (table ii). cat and gpx are enzymes involved in the neutralization of h2o2. while cat reaction produces oxygen and h2o2, gpx uses gsh oxidized during the reaction that produces water from h2o2. in the present study the activity of cat was higher in erythrocytes of ra patients before treatment compared to the controls. beyond the cell protection against damage caused by ros it was shown that expression of cat in vitro and in vivo affects the expression of genes influencing inflammation.36 interestingly, only for cat we have shown that 12-week therapy with alfacalcidol significantly decreased activity of this enzyme to the levels much lower compared to healthy control. this finding strongly supports alfacalcidol as additional therapy in ra, because cat demonstrated leading role in detoxifying h2o2 within erythrocytes, since this enzyme was strongly elevated in ra synovial fluid.37 significant correlation between changes of cat activity and crp level suggests important role of cat activity in ra pathogenesis. it has been shown that under conditions of severe oxidative stress gpx could be inactivated as o2– can inhibit peroxide function.38 this could explain our finding of gpx decreased activity in erythrocytes of active ra patients before treatment, compared to the controls. also cimen et al.39 found that gpx might not be playing an essential role in rheumatic events. the affinity of gpx for h2o2 is stronger than the affinity of catalase, which makes gpx more efficient at low levels of h2o2 concentrations. decreased activity of gpx reduced the utilization of gsh by erythrocytes and consequently increased levels of gsh. observed decrease of gsh in erythrocytes after alfacalcidol therapy indicated that the patients were trying to recover normal gsh levels as disease improved. in addition to glutathione and antioxidant enzymes, we have also evaluated the presence of lipid peroxidation by measuring the mda levels. our results _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1136 živanović radnić et al. showed that, in patients, mda levels were significantly elevated compared to the controls, but after alfacalcidol therapy patients’ mda levels were lower, in accordance with other studies19,40 suggesting alfacalcidol protective role in prooxidative conditions. after 12-weeks of alfacalcidol treatment, our patients experienced significant clinical improvement without side effects. only one patient showed no improvement but also without disease worsening. our results are in accordance to the study done by andjelkovic et al.8 who showed statistically significant clinical improvement in ra patients treated with alfacalcidol, in both the physician’s overall assessment and in the evaluated parameters of acute phase response. it is well known that all cells of monocyte-macrophage linage have capability to synthesize 1α-hidroxylase and locally produce d-hormone that acts as a potent immunomodulatory molecule, preventing t-cell over-stimulation and decreasing lymphocyte proliferation.41 d3 hormone shifts t-cells from th1 to th2 phenotype, preventing tissue damage associated with th1 responses.42 th17, which produces the proinflammatory cytokine il-17, is thought to play a role in the pathogenesis of autoimmune diseases, including ra. there is evidence suggesting that 1,25(oh)2d3 inhibits the secretion of th17 cells via a number of pathways, including reduction of the expression of th17 stimulatory factor il-6.43 these results indicate that 12-week long alfacalcidol therapy is meaningful approach to improve ra patients’ clinical condition as evidenced by lowering das28 and crp levels. the use of alfacalcidol instead of 1,25(oh)2d3 allows higher dosages to be used with lower risk of hypercalcemia, thus increasing its potential as a therapeutic agent.44 during treatment and follow-up period, in our study, serum and urine calcium as well as ionized calcium levels remained within physiological range. the 25(oh)d form is the most plentiful and stable metabolite of vitamin d in human serum with high affinity to bind serum vitamin d-binding protein and other albumin superfamily in the blood. therefore, the level of 25(oh)d in the serum is the best indicator of vitamin d entering the host, either by coutaneous synthesis or by ingestion in the diet. nevertheless, this form is still not a hormone; rather, it is a prehormonal form of the natural hormone and does not exert almost any biologic activity in the body.45 as a result of the negative feedback regulating the final activation step of 25(oh)d into the active 1,25(oh)2d3 by the kidneys, the oral supplements of plain vitamin d will never lead to increase of hormone d3 to the levels exerting immunomodulatory and/or antioxidative effects.44,46 conclusion the present study demonstrates that twelve weeks of alfacalcidol treatment modulates the activity of antioxidant enzymes and intensity of lipid peroxidation _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. alfacalcidol antioxidant activity for rheumatoid arthritis 1137 that further correlates with significant reduction in disease activity of ra patients. we propose that hormone d could diminish the disease activity by either decreasing ros production or expanding their neutralization by the action of antioxidant enzymes, suggesting the beneficial role of alfacalcidol as the additional therapy in ra. these data could contribute to better understanding and further research of the biological mechanisms of the disease and possibly provide new therapeutic approach. и з в о д утицај алфакалцидола на параметре оксидативног стреса у периферној крви пацијената са активним реуматоидним артритисом татјана живановић раднић1, катарина симић-пашалић1, мирјана шефик букилица1,2, соња мисирлић денчић3, анђелка м. исаковић3, тихомир стојковић3, наташа петронијевић3, немања дамјанов1,2 и јелена војиновић4 1институт за реуматологију, београд, 2медицински факултет универзитета у београду, 3институт за медицинску и клиничку биохемију, медицински факултет универзитета у београду, и 4клинички центар, медицински факултет универзитета у нишу постоје докази да хормон d и његови синтетски аналози испољавају веома важне имуномодулаторне ефекте у аутоимунским болестима. циљ ове студије је био испитивање утицаја дванаестонедељне терапије алфакалцидолом на активност антиоксидативних ензима: супероксид-дисмутазе (sod), каталазе (cат) и глутатион-пероксидазе (gpx) и ниво глутатиона (gsh) и малондиалдехида (mda) у крви болесника са активним реуматоидним артритисом (ra) и здравих контрола. у испитивање је укључено 16 rа пацијената, са активном болешћу и 20 здравих добровољаца. пацијенти су поред своје редовне терапије, добијали терапију алфакалцидолом (2μg/дан), у трајању од 12 недеља. узорци периферне крви су пацијентима узимани пре почетка терапије алфакалцидолом и након 12 недеља. параметри оксидативног стреса испитивани су спектрофотометријски и методом проточне цитометрије. активност болести је процењивана на основу das28 композитног индекса. утврђено је да терапија алфакалцидолом статистички значајно (p = 0,04) смањује активност sod и cат (p = 0,001) у еритроцитима болесника са rа. активност gpx у еритроцитима је била значајно нижа (p = 0,04) код болесника на почетку студије у односу на здраве контроле, док је ниво gsh био значајно виши (p = = 0,03). након терапијског режима, активност gpx у еритроцитима је враћена на ниво активности у групи здравих контрола, а ниво gsh у еритроцитима болесника је статистички значајно смањен (p = 0,01). ниво mda код болесника на почетку истраживања, био је значајно повишен у односу на контролну групу (p = 0,01). третман алфакалцидолом смањио је ниво mda код пацијената (p = 0,19). осим тога, дванаестонедељни третман алфацалцидолом променио је одговор pbmc пацијената на стимулацију, спречавајући о2 производњу и деполаризацију митохондријалне мембране. код болесника је након дванаестонедељног терапијског режима доказано и веома значајно клиничко побољшање. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice kinetics and mechanism of the oxidation of dithiocarbamic acids in the presence of co(ii) phthalocyaninetetacarboxylic acid j. serb. chem. soc. 81 (9) 1025–1036 (2016) udc 547.584.463.1’052.1+546.732: jscs–4905 66.094.3:544.4+541.124 original scientific paper 1025 kinetics and mechanism of the oxidation of dithiocarbamic acids in the presence of co(ii) phthalocyaninetetacarboxylic acid artur vashurin1,2*, ilya kuzmin1, vladimir mayzlish1, mikhail razumov2, oleg golubchikov1 and oscar koifman2 1ivanovo state university of chemistry and technology, ivanovo 153000, russia and 2research institute of macroheterocycles of ivanovo state university of chemistry and technology, ivanovo 153000, russia (received 5 january, revised 11 may, accepted 12 may 2016) abstract: the present work contains kinetic data of the oxidation of sodium diethyldithiocarbamate in the presence of phthalocyanine catalysts. it is shown that the nature of the peripheral substituent has a great influence on the self -association of phthalocyanines and on their catalytic activity. a mechanism of the oxidation of sodium diethyldithiocarbamate involving the formation of a triple complex of the substrate, the reduced form of the catalyst and oxygen is offered. it is also shown that the oxidation mechanism of dithiocarbamic acids is different from that for cysteine. keywords: co(ii)phthalocyanines; catalysts; oxidation; kinetics; mechanism. introduction phthalocyanine metal complexes are widely used as models of enzyme active sites responsible for aerobic catalytic oxidation, such as catalysts in the oxidation of various organic substrates, decomposition of hydrogen peroxide and other catalytic processes.1–4 aliphatic and aromatic mercaptans (rsh) are important organic sulfur compounds contained in petroleum products. mild oxidation of mercaptans allows disulfides, used in the vulcanization of rubbers and the manufacture of pharmaceuticals, of high purity to be obtained.5–7 complexes of transition metals with substituted phthalocyanines are common catalysts in the selective homogeneous oxidation of mercaptans.1 the merox process8–10 that is based on cobalt and iron complexes with phthalocyanine tetrasulfonic acid was established in 1964 and modifications of it are still used. the process proceeds according to eq. (1): * corresponding author. e-mail: negovan.asvashurin@mail.ru; asvashurin@mail.ru doi: 10.2298/jsc160105048v _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1026 vashurin et al. 2 24rsh + o 2rssr + 2h o→ (1) realization of liquid-phase merox process with high efficiency is complicated by existence of associative processes of water-soluble phthalocyanine metal complexes.10–13 increasing of catalysis efficiency in reaction (1) is complicated by a number of significant contradictions concerning the mechanisms of the process known from the literature.14–21 meanwhile, theoretical aspects of metallophthalocyanines functioning as catalysts, the mechanisms of catalysis, the influence of structure and associative state of the macrocycle in solution on the kinetic characteristics of reaction (1) are fundamentals for the chemistry of phthalocyanine catalysts. wide opportunities exist for the modification of the phthalocyanine macrocycle by the introduction of peripherial substituents differing in structure and function, the nature of central metal and the existence of processes of additional axial coordinated ligands, thereby providing the possibility of controlling the rate and selectivity of processes catalyzed by phthalocyanines. the mechanisms of homogeneous oxidation14,17,21 of rsh compounds in the presence of phthalocyanine metal complexes have the main idea of coordinative interaction between the macrocycle, the oxidant and the substrate. simultaneously, acknowledging the decreasing effect of association on the catalytic activity of metal phthalocyanines, not many authors22–25 have studied the effect of association on the oxidation mechanism, which, in our opinion, is very important. some authors14,15 proposed that the mechanism of rsh oxidation is radical–ionic, in which the oxidation degree of the central metal cation of the phthalocyanine molecule is reduced: slowiii • ii slowii • i rs + co pc rs + co pc or rs + co pc rs + co pc − − ⎯⎯⎯→←⎯⎯⎯ ⎯⎯⎯→←⎯⎯⎯ (2) rapid2rs rssr⎯⎯⎯→ (3) 2 2o oi ii iiico pc co pc co pc⎯⎯⎯→ ⎯⎯⎯→ (4) where copc is macrocycle, rs– thiolate ion and rssr disulfide. in our opinion, the implementation of this mechanism is complicated. the mechanism described above suggests the possibility for the formation of disulfides in an inert atmosphere according to reaction (2), essentially independent of the state of the coordination unit of the metallophthalocyanine molecule. according to this mechanism, the change in the catalytic activity of different derivatives of cobalt phthalocyanine should be minor. however, the data obtained for sod_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ mercaptans oxidation on phthalocyanine catalysts 1027 ium diethyldithiocarbamate (dtc) oxidation in the presence of phthalocyanine catalysts is in disagreement with this mechanism. this scheme has significant limitation for catalysis caused by the state of mercaptan as ions. dissociation of mercaptan according to eq. (5) proceeds in aqueous-alkali solutions only when the ph is >8. this imposes an additional mechanistic step. +rsh h + rs− (5) the ascertainment of the relationship between the associative state of a macrocyclic structure in solution and catalytic activity would enhance the understanding of the mechanisms of catalysis and enable recommendations to be made to obtain new liquid-phase catalytic systems based on metallophthalocyanines. an attempt to ascertain the relationship between the nature of peripheral substitution, aggregation and catalytic activity in reaction (1) for cobalt complexes with water-soluble phthalocyanines (scheme 1) is described in the present work. scheme 1 metallophthalocyanines. experimental phthalocyanines synthesis three phthalocyanines were synthesized by the template synthesis method from the corresponding phthalodinitriles,21 i.e., cobalt tetrakis[(4-carboxyphenyl)amino]phthalocyanine (copc(nh)), cobalt tetrakis[(4-corboxyphenyl)sulfanyl]phthalocyanine (copc(s)) and cobalt tetrakis[(4′-carboxyphenyl)oxy]phthalocyanine (copc(o)) (scheme 1). characterization data for the synthesized phthalocyanines are given in the supplementary material to this paper. kinetic studies the oxidation reaction of sodium diethyldithiocarbamate was chosen as a model reaction of the process (1). it proceeds according to the general scheme 2. scheme 2. oxidation of dtc. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1028 vashurin et al. the advantages of this reaction are low toxicity of the initial materials, possibility to observe the concentration of the initial and desired materials and identification of reaction products using electron absorption (uv–vis) and ir spectroscopic methods. the experiments to study the kinetics of sodium diethyldithiocarbamate (dtc) oxidation were realized in a thermostatic cell into which 650 ml of dtc was loaded. the air required for oxidation was fed into the cell via a micro compressor at a constant rate of 2 l min-1. under these conditions, the reaction occurs in the kinetic region.26 after reaching a constant temperature, the reaction mixture was mixed and sample of 2 ml was taken to determine initial concentration of dtc, then the compressor was turned on. this moment was taken as the beginning of the reaction. samples of 2 ml were taken periodically during the experiment to determine current concentration of dtc. the concentration of dtc was monitored spectrophotometrically at a wavelength of 440 nm. under conditions of constant concentrations of oxygen and catalyst, and constant ph of the solution, the rate of dtc oxidation is described by the first order kinetic equation. dc/dt = –kobsc where с is dtc concentration, t time and kobs observed rate constant, s-1. the first order kinetics was confirmed by the straightness of graphics in coordinates ln c − t and the constancy of rate constants calculated according to the equation: kobs = (1/t)∙ln(c0/cτ) where с0 is the initial concentration of dtc, and c is the concentration of dtc at time t. degree of transformation was calculated according to the equation: χrssr = (c0–cτ)/c0 results and discussion the kinetic curves for dtc oxidation in presence of сopc(nh), сopc(s), сopc(o) are presented in fig. 1. 0 800 1600 2400 3200 4000 0.0 0.4 0.8 3 2 ln (c 0/c ) time, s 1 fig. 1. kinetic curves of dtc oxidation at 298.15 k, ph 8.2 in the presence of homogeneous phthalocyanine catalysts (с = = 8×10-5 m): 1) сopc(o); 2) сopc(nh) and 3) сopc(s). the structures of the investigated compounds are distinguished by the nature of the spacer atom linking the macrocycle with the functional group of the substituent. data of fig. 1 shows that the kinetic curves of dtc oxidation differ. previously,22 it was found that in the absence of catalyst, the reaction proceeds _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ mercaptans oxidation on phthalocyanine catalysts 1029 slowly with kobs of 1.7×10–5 s–1. the totality of the data suggests the impossibility of dtc oxidation according the above mechanism (eqs. (2–4)) because oxidation is slow even in excess of oxygen and there is no disulfide formation according to eq. (2) in an inert medium. it is also matched with available literature data.17–19 meanwhile, there is information in the literature1,2 about the formation of a triple complex (eqs. (6–9)), with further rs radical cleavage involved. this mechanism is indirectly confirmed by the data of fig. 1: 1ii • irs + co pc rs co pc− ⎯⎯→ ⋅←⎯⎯ k (6) 2• i • ii2 2rs co pc + o rs co pc o −⎯⎯⎯→⋅ ⋅ ⋅←⎯⎯⎯ k (7) 3 , slow• ii • iii 22 2 2 2rs co pc o h o rs + h o co pc o − −⋅ ⋅ + ⎯⎯⎯⎯→ ⋅ ⋅k (7a) + 42h , iii 2 ii 2 2 2h o co pc o co pc + h o + 2oh − −⋅ ⋅ ⎯⎯⎯⎯→k (8) instantaneous•2rs rssr⎯⎯⎯⎯⎯⎯→ (9) according to this mechanism, the effect of the nature of a peripheral substituent on the catalytic activity of the macrocycle will definitely depend on heteroatom effects, affecting the metal cation of the phthalocyanine molecule, which in turn determines the stability of the triple complex (eqs. (7) and (7a)). the associative state and axial coordination processes of the ligands in solution were investigated to determine the laws of the peripheral substitution effect on the macrocycle. it is known that carboxyl derivatives of metallophthalocyanines dimerize in aqueous solutions due to overlapping of the π-electron systems of two macrocycles forming a π–π dimer.11,12 it is easy to observe the associative state of metallophthalocyanines using electronic absorption spectroscopy because of differences in the positions of absorption bands of monomeric and associated macrocycle forms.26,27 the electronic absorption spectra of aqueous alkali solutions of investigated macrocycles are shown in fig. 2. matching of the absorption q-band maximum and the width of the spectrum in fig. 2 with literature data11–13 indicates the dimerization of сopc(o) and сopc(s) macrocycles, whereas сopc(nh) is almost monomeric. there are significant spectral changes on addition of pyridine, an axially coordinated ligand to solutions of сopc(o) and сopc(s) phthalocyanines. an example is presented in fig. 3. the significant bathochromic shift in the absorption of the q-band (by 50 nm) and an increase in the absorption intensity indicate the dissociation of copc dimers and the formation of the monomeric form presented by the axial complex with pyridine. similar values of the stability constants of the molecular com_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1030 vashurin et al. plexes of сopc(s) and сopc(nh) with pyridine indicate similar states of the coordination units of the macromolecules. this fact suggests a weak effect of the nature of peripheral substitution on the π-electronic structure of the monomeric macrocycle. 500 600 700 800 0.0 0.1 0.2 0.3 2 3 wavelength, nm a bs or ba nc e, a .u . 1 fig. 2. uv–vis spectra of aqueousalkali solutions at 298.15 k, рн 9 (с = 5×10-5 m): 1) сopc(o); 2) сopc(nh); 3) сopc(s). 500 600 700 800 900 0.0 0.1 0.2 0.3 0.4 wavelength, nm a bs or ba nc e, a .u . (i) 3 21 0 2 4 6 8 10 0.0 0.1 0.2 3 2 1 c py 104 / mol l-1 δ a (ii) fig. 3. i) uv–vis spectra of copc(o) aqueous-alkali solution at 298.15 k, рн 9 (с = 6×10-5 m): 1) without pyridine, 2) сpy = 1.9×10-4 m and 3) сpy = 6.3×10-4 m; ii) titration curves of phthalocyanine solutions (q-band; с = 5×10-5 m) with pyridine 1) сopc(o) (λ = 663 nm), 2) сopc(s) (λ = 675 nm) and 3) сopc(nh) (λ = 730 nm). the reason for the difference in the catalytic activities becomes obvious when matching the obtained data of coordination interactions of studied macrocycles with the mechanism of the reactions (6)–(9). redistribution of the electron _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ mercaptans oxidation on phthalocyanine catalysts 1031 density within the macrocycle is due to the influence of the electronic effect of the substituent. a nitrogen atom in peripheral substituents promotes a shift of the electronic density to the macrocycle. this impedes coordination of pyridine (or substrate) and decreases the catalytic activity of the macrocycle. on the contrary, a sulfur atom promotes a shift of the electronic density from the macrocycle that increases the probability of pyridine–phthalocyanine coordination interactions and partially compensates for the deficit of electronic density caused by the association of metallophthalocyanines. the lower value of the stability constant of the copc(o) complex with pyridine (table i) is caused by the impossibility of compensation of the lack of electronic density on the central metal cation. this is explained by the lower electron-donating ability of the oxygen atom compared to the sulfur atom. table i. stability of molecular complexes with pyridine and catalytic activity in the oxidation of dtc for copc (с = 5×10-5 m) at 298.15 k, ph 9; k is the thermodynamic stability constant of py-complex, kobs is the observed constant of the dtc oxidation rate (determination of the constant is described in experimental part) and χrssr is the degree of the dtc transformation to the disulfide macrocycle k / l mol-1 kobs×105 / s -1 χrssr / % сopc(o) 1900±200 17±4 84.32 сopc(s) 3200±300 85±3 92.15 сopc(nh) 3200±300 38±6 83.61 it should be noted that uv–vis spectra of investigated macrocycle solutions do not have bands characteristic of coiiipc.17,28 this confirms the stability of initial state of co2+ and hence the highest probability of the processes (eqs. (6)–(9)). the parameters kobs and χrssr for dtc oxidation under standard conditions are given in table i. kinetic research of dtc oxidation in dependence on the ph of solution shows a lack of linear correlation between ph and effective rate constant of dtc oxidation, fig. 4. 6 7 8 9 10 11 12 15 30 45 60 75 k o bs 1 05 / s1 ph 1 2 3 fig. 4. dependence of dtc oxidation rate on ph of medium in presence of phthalocyanine catalysts (с = 4.5×10-5 m): 1) сopc(s), 2) сopc(nh) and 3) сopc(o). _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1032 vashurin et al. the maximal activity of the catalysts was reached when ph values were in the range 9 to 10. this could be caused by a shift in the balance of eq. (2) toward the thiolate ion. increasing of solution basicity did not lead to an increase in the activity, on the contrary, it decreased the effective rate constant of oxidation. this is the result of competition between rs– and oh– for the central metal cation of the phthalocyanine molecule. based on spectral data described above and the stability of the cobalt cation (сoiiрс) contained in macrocycle, it could be concluded that the stage denoted by eq. (2) could not be observed. in case of its processing, there is joint coordination of the two ligands (rs– and oh–) on one side of macrocycle accompanied by the formation of a stable axial complex due to compensation for the deficiency of electronic density on the central metal cation of the phthalocyanine molecule, which is known from the literature.29 similar changes for the sulfonic acids of cobalt phthalocyanines were previously observed.30,31 in addition, it was shown32,33 on the example of the tetrasulfonic acid of cobalt phthalocyanine and various substrates (2-mercaptoethanol and cysteine) that the increase in the reaction rate is caused by auto-oxidation processes of the substrate in alkali solutions. the highest activity of these catalysts was found in the ph range of 12–13, caused by the higher acidity of the sulfonic group compared to the carboxyl group. obviously, the maximum of reaction rate will be observed in ph range wherein the concentration of labile oxygen adducts is maximal and the balance (5) is shifted toward the formation of free thiocarbamic acid. in this case, all the catalyst is converted to an intermediate that is presented by oxidized thiocarbamic acid located in the outer coordination sphere and forms hydrogen bonds with coordinated oxygen molecules. there is a further transfer of the charge from the metal to oxygen. increasing the ph leads to rivalry between dtc coordination and dimerization processes due to intensification of the latter one. it complicates the transfer of electrons. the process of electrons transfer to cobalt sulfophthalocyanines in aqueous alkali solutions was previously studied in detail.34 there are transitions сopcii → сopci and transfer of electrons from hydroxyl ion to monomeric copc in deaerated solutions when a detergent at concentrations in the range 10–2–10–3 m is introduced into the system: ii ii cco pc + oh co p oh− −⋅ ⋅ ⋅ (10) ii i cco pc oh m co p product−⋅ ⋅ ⋅ + + (11) where m is an acceptor of oh radical. there is no such process for associated phthalocyanine structures, i.e., when no detergent is present. furthermore, it was found that the phthalocyanine may be an acceptor of oh radicals.34 for this reason, it was assumed that electron transfer might be implemented in the reaction of complex сopcii⋅⋅⋅oh– with other hydroxyl ions: _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ mercaptans oxidation on phthalocyanine catalysts 1033 (сopcii⋅⋅⋅oh–)2 + oh– → сopci⋅⋅⋅сopcii⋅⋅⋅oh– + o– + h2o сopci + o2 → сopcii + o2–, сopcii + o2 → сopciii + o2– with an excess of oxygen, there is permanent formation of o2– and oxidized forms of сopciii that impedes triple complex formation under conditions of increased content of hydroxyl ions in solution and decreases the catalytic activity of phthalocyanine. it is also characteristic of the systems studied herein. obviously, there is an exchange between dtc and ligand coordinated by phthalocyanine (scheme 3), which can be presented by solvent or molecular oxygen.35 the exchange depends essentially on the electron-donor power of the second ligand, i.e., for ions, on their basicity. scheme 3. exchange of ligands in the triple complex; x: solvent. the kinetic model may be analyzed by adopting in eqs. (6–9), а for rs–, m for coiipc, x1 for rs•∙coipc, b for o2, x2 for rs•∙coiipc∙o2–, x3 for coiiipc∙o22–, d for rs• and their concentrations for c with the corresponding index. the sum of concentrations of all complexes (cmº / mol l–1) containing catalysts is calculated according to: cmº = cm + cx1 + cx2 (12), where cm is the concentration of proper catalyst (mol l–1), cx1 the concentration (mol l–1) of rs•∙coipc and cx2 the concentration (mol l–1) of rs•∙coiipc∙o2–. hence, the function of catalyst complexation takes the form: f(m) = cm/cmº (13) cx1 = k1cmca, cx2 = k2cx1cbº, under condition of cbº >> caº and assuming that steps (6) and (7) are in quasi-equilibrium before the slow step (7a), one obtains: cx2 = k1k2cmcacbº (14), where ca is the concentration (mol l–1) of rs– and cbº is the initial concentration of oxygen (mol l–1). the rate (r / mol l–1 s–1) of the limiting stage (eq. (7а)) is calculated by the following: r = k3cx2 (15) _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1034 vashurin et al. taking into account eq. (14) it follows: r = k3k1k2cmcacbº (16) the concentration of catalyst, cm, is substituted by cmº and the final equation for calculation of the rate of dtc oxidation is given by: º º3 1 2 m b a º1 a 1 2 b a1 = + + k k k c c r c k c k k c c (17) the function f(m) is linear with increasing concentration ca. possible deviation from the linearity is caused by a decreasing of concentration cbº. equation (17) is linear in lineweaver–burk coordinates:36 º º º º ºa 3 1 2 m b 3 2 b m 3 m 1 1 1 1 = + + r c k k k c c k k c c k c (18) under the condition of k1k2cacbº << (1+ k1ca), one has: º º3 1 2 m b a 1 a1 = + k k c c r c k c k (19) the dependence ca = f(τ) in this case takes the form: º1 a º º º ºm b 1 2 a m b 1 2 1 ln τ+ = k c c c k k c c c k k (20) the function ln ca–τ has linear character. it is matched with experimental data of aerobic dtc oxidation kinetics. thus, the most plausible mechanism of dtc oxidation is the set of sequential reactions (6)–(9). it should be noted that application of the michaelis–menten kinetics and its lineweaver–burke linearization to this system was realized33 in the context of cysteine oxidation. however, the previous model ignored the formation of a triple complex. in this case, varied interpretation of mercaptans, thiocarbamic acids and cysteine oxidation mechanisms appears. this may be explained by the lack of coordination activity of dtc because of its low basicity compared to mercaptan. nonlinearity of the dependence of the dtc oxidation rate on temperature observed in the temperature range of 273.15–313.15 k is most likely caused by a change of the limiting stage at system saturation. these experimental data are consistent with known results.37 conclusion new derivatives of cobalt phthalocyanine are synthesized. their catalytic activity in aerobic oxidation of sodium diethyldithiocarbamate is studied. the influence of nature of peripheral substituent on catalytic activity of catalyst is established. the catalytic activity of phthalocyanines increases depending on the nature of the spacer bridge. the activity increases according to the following: _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ mercaptans oxidation on phthalocyanine catalysts 1035 copc(s) > copc(nh) > copc(o). the mechanism of sodium diethyldithiocarbamate oxidation limited by the stage of rs• formation is proposed. this mechanism is in good agreement with the experiment. the data obtained are fundamental for understanding the mechanism of metallophthalocyanines catalytic action in oxidation of mercaptans and further development of efficient catalysts. supplementary material characterization data for the synthesized phthalocyanines are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the synthesis of macrocycles and materials based on them was supported by the russian science foundation (project no. 14-23-00204). the catalysis research was financially supported by a grant of the president of russia for state support of young scientists and phds (project мк-2776.2015.3). извод кинетика и механизам оксидације дитиокарбаминских киселина у присуству co(ii) фталоцијанинтетракарбоксилне киселине artur vashurin1,2, ilya kuzmin1, vladimir mayzlish1, mikhail razumov2, oleg golubchikov1 и oscar koifman2 1 ivanovo state university of chemistry and technology, ivanovo 153000, russia и 2 research institute of macroheterocycles of ivanovo state university of chemistry and technology, ivanovo 153000, russia предмет овог рада је одређивање кинетичких параметара оксидације натријум -диетилдитиокарбамата у присуству фталоцијанинских катализатора. показано је да природа периферних супституената има велики утицај на самоуређење фталоцијанина и њихову каталитичку ативност. предложен је механизам оксидације натријум-диетилдитиокарбамата који укључује формирање троструког комплекса супстрата, редуковане форме катализатора и кисеоника. такође је показано да се механизам оксидације дитиокарбаминских киселина разликлује од механизма оксидације цистеина. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice temperature dependent effect of difenoconazole on enzymatic activity from soil j. serb. chem. soc. 80 (9) 1127–1137 (2015) udc 504.53.054:577.15–188:577.153+ jscs–4785 66.094.258:544.032.4 original scientific paper 1127 temperature dependent effect of difenoconazole on enzymatic activity from soil marioara nicoleta filimon1,2, sorin octavian voia3*, diana larisa vladoiu1,2, adriana isvoran1,2 and vasile ostafe1,2 1west university of timişoara, faculty of chemistry–biology–geography, department of biology–chemistry, pestalozzi, 16, timisoara, 300115, romania, 2west university of timisoara, laboratory of advanced research in environmental protection, oituz 4, timisoara 300086, romania, 3banat’s university of agricultural sciences and veterinary medicine, faculty of animal science and biotechnologies, calea aradului, 119, timisoara, 300645, romania (received 18 december 2014, revised 24 march, accepted 25 march 2015) abstract: the purpose of this study was to quantify the effect of difenoconazole (dfc) on the activity of a few enzymes commonly found in soil: dehydrogenase, urease, phosphatase and protease. three experimental variants were established: under field conditions with variable temperature (10–21 °c, variants a1–a3), under laboratory conditions with constant temperature (30 °c, variants b1–b3) and untreated soil (c variant). the commercial product “score 250ec” with 250 g dfc l-1 was used at the following concentrations: 0.037 mg dfc g-1 soil (variants a1 and b1), 0.075 mg dfc g-1 soil (variants a2 and b2) and 0.150 mg dfc g-1 soil (variants a3 and b3). the dehydrogenase, phosphatase and urease activities decreased significantly (p < 0.05) under both field (variants a1–a3) and laboratory (variants b1–b3) conditions compared to untreated soil (variant c). the protease activity was reduced in variants a1– –a3 compared to variant c and increased at the dose of 0.150 mg dfc g-1 soil in the variant b3. keywords: fungicide; soil; dehydrogenase; phosphatase; urease; protease. introduction widespread and intense application of a large number of fungicides for controlling fungal pathogens of crops promotes high productivity in the modern agriculture. the fungicides used to inhibit the growth and developments of pathogenic fungi of crops have a negative effect on soil quality through quantitative and qualitative changes in the communities of microorganisms.1–3 * corresponding author. e-mail: voia@animalsci-tm.ro doi: 10.2298/jsc141218030f _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1128 filimon et al. soil microorganisms produce a variety of exo-enzymes: ureases, invertases, dehydrogenases, cellulases, amylases, phosphatases, proteases, etc. enzyme activity can be used as a biomarker of soil fertility and an indicator of many biological processes manifested in the soil.4 fungicides applied directly to plants were also found in the soil.5,6 literature data concerning the effects of fungicides on soil reveal stimulation or inhibition of enzyme activities depending on the fungicide dose, incubation temperature and time of application,7,8 and the inorganic and organic matter content of soil, the soil type, soil tillage, content of heavy metals and other environmental factors.9–11 the fungicides captan and trifloxystrobin applied for a short-term did not affect the phosphorous cycle in soil, but their application in large doses caused inhibition of enzymes involved in the nitrogen cycle.12 chen and edwards13 emphasized the toxic effect of benomyl, captan and chlorothalonil upon the microorganisms from soil and on the nitrogen cycle: reduction of fungi and nitrifying bacteria populations and inhibition of several enzyme found in soil (nitrogenase, dehydrogenase, cellulase, phosphatase urease and protease). other fungicides, such as propiconazole and chlorothalonil, applied in the recommended doses to crops did not show inhibitory effects on urease and protease activities from soil.14 published data concerning the effect of difenoconazole on communities of microorganisms in soil and on enzyme activities showed different aspects. thus, depending on the soil type, short term application of difenoconazole had an inhibitory effect on microbial activity in unfertilized soils, but not in fertilized soils.15 another study concerning the effects of difenoconazole on the activity of microorganism community in soil based on enzymatic activities (urease, arylsulfatase, β-glucosidase, alkaline phosphatase and dehydrogenase) was conducted under laboratory conditions using different concentrations of fungicide. it reflected dose dependent effects of difenoconazole on the microbial population of the soil.16 the purpose of this study was to determine the effects of different doses of difenoconazole on soil quality during 21 days of contact with the fungicide, based on enzymatic activities (dehydrogenase, urease, phosphatase, protease), under variable (10–30 °c, in experimental fields) and constant (30 °c, in laboratory conditions) temperatures. to the best of our knowledge, this is the first study comparing the effects of difenoconazole upon some enzymes from soil at variable temperatures and a constant temperature, i.e., under field and laboratory conditions. experimental materials difenoconazole (dfc, 1-[[2-[2-chloro-4-(4-chlorophenoxy)phenyl]-4-methyl-1,3-doxolan-2-yl]methyl]-1h-1,2,4-triazole) is a fungicide that inhibits sterol demethylation and is widely used against ascomycetes, basidiomycetes and deuteromycetes. the experiments _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ difenoconazole effect on enzymatic activity 1129 were performed using a product sold on a local market under the trade name “score 250 ec”, that contains 250 g l-1 dfc. soil sampling the soil samples were collected from an experimental field located nearby timisoara city, in an area where insecticides, fungicides, herbicides or chemical fertilizers were never used. chernozem soil samples were collected from the top layer of soil (0–20 cm) from five different spots in quantities varying between 1 and 2 kg. the material was ground, sieved (2 mm) and spooned by random sampling, giving sub-samples of 20 g per polyethylene bag. the samples were preserved in a refrigerator and processed as soon as possible during the following 30 days. treatment of soil samples with fungicide three doses of dfc were prepared using distilled water: half dose hd – 0.037 mg dfc g-1 soil, normal dose nd – 0.075 mg dfc g-1 soil and double dose dd – 0.150 mg dfc g-1 soil. the three dfc doses were prepared in distilled water and then applied to the soil samples to obtain 40 % humidity. the plastic bags containing the samples were homogenized on a rotary homogenizer for 2 h in order to achieve a uniform distribution in the sample.17 the following variants were obtained: variant a with three sub-variants depending on the dfc concentration (a1 – hd, a2 – nd and a3 – dd), with soil ph 6.20, storage for 21 days under field conditions, at 10–21 °c (mean temperature 17.19 °c); variant b (b1 – hd, b2 – nd and b3 – dd), with soil ph 6.44, incubated for 21 days at 30 °c (laboratory conditions); variant c was untreated soil, with a soil ph of 6.14. biochemical analyses the following enzymatic activities were assayed: dehydrogenase, urease, acid phosphatase and protease. the enzymatic activities were determined using a t90 uv/vis spectrophotometer (pg instruments, uk). the dehydrogenase activity (da) was measured using 2,3,5-triphenyltetrazolium chloride (ttc) as substrate, monitoring the reaction product (triphenylformazane, tpf) at 485 nm. the reaction mixture containing 3 g soil sample, 0.5 ml of 3% solution of ttc, 1.2 ml tris buffer (0.1 m, ph 7.6) was kept at 37 °c for 48 h. tpf was extracted with 20 ml acetone and the absorbance of the supernatant was measured at 485 nm. the da is expressed as mg tpf g-1 soil during 48 h.18 the urease activity (ua) was determined in accordance with the method described by alef and nannipieri.19 the reaction mixture consisted of 3 g soil, 5 ml phosphate buffer (0.6 m, ph 6.8) and 2 ml toluene. after homogenization (2 min on vortex), 5 ml 3 % urea was added and the mixture was vortexed for a further 2 min. finally, the reaction mixture was incubated at 37 °c for 24 h. in the collected supernatant, the quantity of produced nh4+ was determined using nessler’s reagent. the absorbance was measured at 445 nm and the ua is expressed as mg nh4+ g -1 soil during 24 h. the phosphatase activity (pha) was estimated measuring the phenol resulting from the hydrolytic separation of phenyl phosphate into disodium phosphate and phenol catalyzed by phosphomonoesterases. for each sample, about 3 g of soil were mixed into a test tube with 10 ml of 0.5 % phenyl phosphate and incubated for 48 h at 37 °c. next, 50 ml of 0.3 % ammonium aluminum sulfate were added to each test tube and the mixture was then filtered through ash-free filter paper. from each test tube, 1 ml filtrate was transferred to an empty test tube together with 5 ml borax solution (0.1 m, ph 9.4). the mixture was brought to a _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1130 filimon et al. volume of 25 ml with distilled water and the absorbance was measured at 597 nm. pha was defined20 as mg phenol g-1 soil during 48 h. the protease activity (pa) was estimated by reaction of ninhydrin with the amino acids resulting from the hydrolysis of gelatin used as substrate. for each sample, about 3 g soil was mixed with 7 ml of 2 % gelatin and 0.5 ml toluene. the mixture was homogenized (2 min on vortex) and incubated at 37 °c for 24 h. next, 25 ml of distilled water was added and the mixture was filtered through ash-free filter paper. from each test tube, 2 ml of filtrate was transferred to an empty test tube together with 5 ml of 0.2 % ninhydrin solution and the absorbance was measured at 578 nm. the pa was defined20 as mg amino-n g-1 soil during 24 h. statistical data interpretation statistical analysis of the recorded data was performed using variance analysis and the software minitab 17.21 all data are presented as average values with standard deviation (x±sd). in order to establish the correlation coefficient, the spearman test was used. significant differences in variables were tested using mann–whitney at the 0.05 level of probability. results and discussions the enzymatic activities da, ua, pha and pa were assayed in 6 experimental variants during 21 days. the results revealed increases of enzymatic activities for some enzymes and decreases for other during the monitoring period, in relation with the incubation temperature and dfc concentration. the average values and standard deviations for da during the 21 days of experiment were determined (fig. 1). the recorded values ranged between 0.462±0.375 mg tpf g–1 soil in 48 h (variant b3) and 1.734±0.601 mg tpf g–1 soil in48 h (variant a1). as the values of da obtained in the soil samples containing dfc were lower than that registered for the control soil sample (5.847±0.501 mg tpf g–1 soil in 48 h), it could be concluded that dfc had a toxic effect on the respiration process of microorganisms from soil. the higher was the dfc concentration, the higher was the percent of reduction of da in the soil samples. for example, in variant b3, the da activity was decreased with 90.16 % in comparison with the control sample (variant c). fig. 1. average values of the dehydrogenase activity in the soil samples. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ difenoconazole effect on enzymatic activity 1131 the decrease of da for the experimental variants a and b during the 21 days of the monitoring was roughly linear with the increase of the concentration of dfc. there are significant differences between variant a and b, during the 21 days of experiment (p < 0.05). among all the enzymatic activities assayed, da was the most sensitive to the variation of the dfc concentration in the soil samples. da is considered as an ecotoxicological test for an estimation of the toxicant effects on soil microorganisms as da reflects the intensity of the respiration processes of these germs.22 the results of the present study were in good agreement with other published data. the studies conducted by muñoz-leoz et al.23 revealed that small concentrations of dfc cannot show a clear effect on da, but high concentrations of dfc applied on soil lead to significant decreases in the da. srinivasulu and rangaswamy24 reported inhibition of da of soil microorganisms due to the treatment of soil with high doses of metalaxyl and mancozeb during a period of 35 days. there are reports mentioning that at low doses some fungicides increase da when applied to soil,7,25 but when large doses were applied, the da of the soil microorganisms was reduced.26,27 beside the concentration of dfc, other factors may also affect the enzymatic activities of soil microorganisms. there are reports mentioning that the water content from soil and the temperature influence the da indirectly by interfering with the redox status of the soil.28 in the present study under field conditions (variant a), da presented a negative correlation with temperature (r = –0.243). the values of urease activity (ua) registered during the 21 days of the monitoring ranged between 318.127±16.124 mg nh4+ g–1 soil in 24 h (variant a2)1 and 169.502±27.980 mg nh4+ g–1 soil in 24 h–1 (variant a3). the average values of ua are presented in fig. 2. fig. 2 average values of the urease activity in the soil samples. the highest values of ua were obtained in variants a1 and a2, an indication of the possible utilization of dfc as a carbon and nitrogen source by some microorganisms from the soil, at temperatures varying between 10 and 21 °c. using _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1132 filimon et al. dfc as a substrate determines urease activity growth. thus, the values of ua obtained in variants a1 and a2 were higher with 23.23 and 61.92 mg nh4+ g–1 soil than the value of ua in the control soil (256.204±11.971 mg nh4+ g–1 soil in 24 h). nevertheless, at high concentrations of dfc in the soil, the ua decreased significantly (p < 0.05): by 33.84 % in variant a3 and by 29.63 % in variant b3, in comparison to the value of ua in the untreated soil. based on the differences of the values of ua in variant a and variant b during the 21-day period of monitoring, it could be concluded that temperature has a significant impact on the influence of dfc on the ua of microorganisms from the treated soil. other chemicals, such as profenofos, deltamethrin and thiram, seemed to increase ua in soil at low concentrations and to reduce it when applied at high doses.29 at high concentrations, pyrimorph reduces significantly the ua.7 qian et al.30 hypothesized that validamycin may be toxic for some species as several enzymatic activities were reduced, but the obtained higher values of ua and pha may indicate the possible use of validamycin as a carbon source by some species of microorganisms. the biomass of the microbes that can use validamycin as a carbon source increased until this source was exhausted, subsequently, the number of microorganisms from the soil would return to the normal level. as the reduction of the ua by captan and trifloxystrobin can be as high as 70 % of that of control untreated soil, it was assumed that these chemicals could modify the nitrogen cycle in the soil. this kind of modification has to be considered as repeated applications of fungicides could lead to their accumulation in the treated soil. as the negative effects on the populations of microorganism are stronger at high concentrations of dfc in soil, the importance of the optimal dose of fungicide that should be applied on soil becomes more obvious. the potential nontarget side effects of pesticides against microbial communities from soil and the reduced rates of degradation of these chemicals should be considered principally when repeated treatment of soil is performed.23 as for the da, the ua of organisms from soil treated with dfc was influenced by factors other than the dfc concentration. the correlation between ua and temperature (range 10–21 °c, variant a) was positive, although with a moderately low value for the correlation coefficient (r = +0.439). similar studies confirmed a small increase in ua at moderate temperatures.25 the time a pesticide acted on the microorganisms also affected the ua of germs from treated soils.29 the values of phosphatase activity (pha) registered during the 21 days of the experiments ranged between 2.427±0.753 (variant a3) and 4.004±1.516 mg phenol g–1 soil in 48 h (variant b3). dfc applied on soil caused a reduction of the pha of microorganisms, as all the values of the pha from the experimental variants were lower than the pha found in the control untreated soil (4.828± _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ difenoconazole effect on enzymatic activity 1133 ±0.751 mg phenol g–1 soil in 48 h). for variant a, the decrease in pha correlated almost linearly with the increase in the dfc concentration (p < 0.05). the average values of pha are presented in fig. 3. fig. 3. average values of the phosphatase activity in the soil samples. in comparison to the control sample, the lowest value of pha (49.73 %) was obtained for variant a3, when the highest dfc dose was used and the temperature ranged between 10–21 °c. the lower temperature (average = 17.19 °c) and higher dose of dfc provided the conditions for a significant decrease in the pha of the microorganisms from the treated soil. under laboratory conditions at high and constant incubation temperature (30 °c), the pha from variant b1 (the lowest applied concentration of dfc) decreased by 42.56 % compared with the control sample. in variant b3 (the highest concentration of dfc), although the pha decreased, the extent of diminution was smaller. phosphatases are the enzymes responsible for releasing of orthophosphoric acid from organic combinations with metaphosphates and pyrophosphate. in soil ecosystems, phosphatases play a critical role in the phosphate cycle, being good indicators of soil fertility.31 when phosphate is deficient in the soil, the amount of acidic phosphatase released by plant roots is increased to augment the solubilization and remobilization of phosphate, influencing the resistance of plants to stress conditions.32,33 in the short term, the phosphate cycle was not influenced by moderate doses of captan and trifloxystrobin applied on the soil.12 in small concentrations, validamycin did not influence significantly pha during the incubation period, but at high doses, an increase (29.8 %) of acidic pha was observed.30 for variant a (field conditions), a weak positive correlation (r = +0.147) between the variation of temperature and pha was detected. the values for the protease activity (pa) recorded during the 21 days of the experiments ranged between 5.948±3.843 (variant a3) and 19.824±7.354 mg amino-n g–1 soil in 24 h (variant b3). in comparison with the control, untreated soil (13.289±1.751 mg amino-n g–1 soil in 24 h), almost all other samples pre_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1134 filimon et al. sented lower values for pa. the only exception was observed in variant b3, when the pa increased by 40.24 % comparing with the control sample. the most important decrease was observed in variant a3, when the pa was reduced by 56.98 %. the average values of pa are shown in fig. 4. fig. 4. average values of the protease activity in the soil samples. the values of pa obtained for variants a have similar values for all concentrations of dfc applied to the soil, but when the soil was incubated at a high temperature (variant b), there was a significant increase of pa at the highest dose of dfc. the fact that between variant a and b there was a difference of 5.38 mg amino-n g–1 soil during the 21 days of the experiments, this could be considered as evidence for the influence of temperature on the pa. a constant incubation temperature of 30 °c seems to stimulate an increase of pa, perhaps by stimulating the overall metabolism. under field conditions, the variation of temperature had a negative influence on pa (r = –0.170). proteases play an important role in the nitrogen cycle in soil, performing the hydrolysis of large peptides with production of amino acids and small peptides.34 fungicides, such as chlorothalonil and propiconazol, have a positive influence on urease and protease activities of soil microorganisms at low and moderate doses. application of these chemicals in the recommended doses does not influence the metabolism of soil germs.14 at high doses, chlorothalonil produces a reduction of pa, in comparison with untreated soil.35 similar results were obtained in case of mancozeb10 and carbendazim.36 the increase in temperature has had a significant influence on the metabolic reactions of organisms from soil by promoting the development of communities of microorganisms resistant to dfc and supporting the use of the fungicide as a source of carbon and nitrogen. at high doses of dfc, these positive effects are overcome by the inhibition of the metabolism of organisms present in soil. as similar results were obtained in the case of other fungicides, it may be concluded that most of these types of chemicals at high and repeated doses may produce an inhibition of the metabolism of communities of microorganisms present in soil.7 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ difenoconazole effect on enzymatic activity 1135 the present study confirms that soil enzymes behave differently when exposed to dfc that can stimulate or inhibit some enzymatic activities depending on the dose and temperature. several factors may influence the enzyme activity besides the concentration of dfc, such as temperature and time of exposure to the toxicant. the degree of absorbency of fungicide in soil could reduce the contact time between some pesticides and microorganisms. biodegradation seems to be the most important mechanism for reduction the concentration of dfc in soil. at least at low doses, the microorganisms from soil can degrade chemicals from soil, including pesticides, progressively reducing their toxicity. some degradation products may act as growth factors for certain microorganisms in the soil. although the assay of the activities of enzymes from soil could be considered a good indication of soil quality and health, the levels of enzymes activities cannot be correlated with the quantities of pesticides in soil. conclusions the results indicated that dfc inhibited the enzymatic activities of dehydrogenase, urease, phosphatase and protease in the treated soil samples. depending on the dose of dfc and incubation temperature, with rare exceptions, the recorded values of enzymatic activities were significantly lower (p < 0.05) than those in untreated soil. above average increases were recorded for soil with urease at hd and nd of dfc applied to the soil sample and variable temperature (variants a1 and a2) and for protease at dd of dfc at constant temperature (variant b3). it could be concluded that the metabolism of the communities of microorganisms from dfc treated soil was affected by the dfc dose and incubation temperature. acknowledgments. this work was supported by a grant of the romanian ministry of education, cncs-uefiscdi, project no. pn-ii-ru-pd-2012-3-0220, “metabolization of difenoconazole by crop plants and fungi communities from soil”. и з в о д утицај дифеноконазола на активност ензима из земљишта marioara nicoleta filimon1,2, sorin octavian voia3, diana larisa vladoiu1,2, adriana isvoran1,2 и vasile ostafe1,2 1 west university of timişoara, faculty of chemistry–biology–geography, department of biology–chemistry, timisoara, 2 west university of timisoara, laboratory of advanced research in environmental protection, timisoara, 3 banat university of agricultural sciences and veterinary medicine, faculty of animal science and biotechnology, timisoara, romania циљ овог рада је био да се квантификује ефекат фунгицида дифеноконазолa (dfc) на активности ензима (дехидрогеназе, уреазе, фосфатазе и протеазе), који се налазе у земљишту. успостављене су три експерименталне варијанте: у теренским условима са променљивом температуром (10–21 °c, варијанте а1–а3), у лабораторијским условима са константном температуром (30 °c, варијанте б1–б3) и контролна варијанта (нетретирано земљиште, ц). комерцијални производ „score 250 еc“ са. 250 g l-1 dfc је коришћен у следећим концентрацијама: 0,037 mg g-1 dfc/маса земљишта (променљиве _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1136 filimon et al. а1 и б1), 0,075 mg g-1 (променљиве а2 и б2) и 0,150 mg g-1 (променљиве а3 и б3). активности дехидрогеназе, фосфатазе и уреазе су биле значајно смањене у теренским и лабораторијским условима применом dfc (p < 0,05) у односу на нетретиране узорке. протеазна активност је била смањена у варијантама а1–а3 у односу на варијанту ц, а повећала се у варијанти б3, када је примењена доза dfc 0,150 mg g-1 земљишта. (примљено 18. децембра 2014, ревидирано 24. марта, прихваћено 25. марта 2015) references 1. a. monkiedje, m. o. ilori, m. spiteller, soil biol. biochem. 34 (2002) 1939 2. r. m. niemi, i. heiskanen, j. h. ahtiainen, a. rahkonen, k. mäntykoski, l. welling, p. laitinen, p. ruuttunen, appl. soil ecol. 41 (2009) 293 3. m. cycon, z. piotrowska-seget, j. kozdroj, int. biodeter. biodegr. 64 (2010) 316 4. c. garbisu, i. alkorta, l. epelde, appl. soil ecol. 49 (2011) 1 5. z. h. wang, t. yang, d. m. qin, g. yong, j. ying, chin. chem. lett. 19 (2008) 969 6. a. bermúdez-couso, m. arias-estévez, j. c. nóvoa-muñoz, e. lópez-periago, b. sotogonzáleza, j. simal-gándara, water res. 41 (2007) 4515 7. d. xiong, z. gao, b. fu, h. sun, s. tian, y. xiao, z. qin, eur. j. soil biol. 56 (2013) 44 8. h. guo, g. f. chen, z. p. lu, h. zhao, h. yang, j. environ. sci. 21 (2008) 494 9. m. srinivasulu, g. j. mohiddin, m. madakka, v. rangaswamy, asian j microbiol. biotech. env. sci. 12 (2010) 141 10. n. rasool, z. a. 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2000, pp. 178–180, 189–191 (in romanian) 21. softonic, http://en.softonic.com/s/minitab-14-free-download-full-version (accessed in sep, 2015) 22. p. nannipieri, e. kandeler, p. ruggiero, in enzymes in the environment, r. g. burns, r. dick, crc press, marcel dekker, new york, 2002, p. 1 23. b. muñoz-leoz, c. garbisu, j.-y. charcosset, j. m. sanchez-pérez, i. antigüedad, estilita ruiz-romera, sci. total environ. 449 (2013) 345 24. m. srinivasulu, v. rangaswamy, int. j. environ. sci. technol. 10 (2013) 341 25. d. s. kumar, v. ajit, soil enzymology, soil biology, series 22, springer, berlin, 2011, p. 25 26. o. crouzet, i. batisson, p. besse-hoggan, f. bonnemoy, c. bardot, f. poly, soil biol. biochem. 42 (2010) 193 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ difenoconazole effect on enzymatic activity 1137 27. b. muñoz-leoz, estilita 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice one-pot synthesis of carbazole based 3-hydroxy-4hchromen-4-ones by a modified algar–flynn–oyamada reaction and their antimicrobial activity j. serb. chem. soc. 80 (11) 1361–1366 (2015) udc 547.759.32+546.76–36:542.913: jscs–4802 542.96:615.28–188 original scientific paper 1361 one-pot synthesis of carbazole based 3-hydroxy-4h chromen-4-ones by a modified algar–flynn–oyamada reaction and their antimicrobial activity dongamanti ashok*, sidda ravi, bommidi vijaya lakshmi and arram ganesh department of chemistry, osmania university, hyderabad 500 007, india (received 3 december 2014, revised 30 march, accepted 10 june 2015) abstract: a new series of 2-(9-ethyl-9h-carbazol-3-yl)-3-hydroxy-4h-chromen-4-ones were synthesized from substituted 2-hydroxyacetophenones and 9-ethyl-9h-carbazole-3-carbaldehyde using naoh and h2o2 by a modified algar–flynn–oyamada reaction. in this method, the flavonols were synthesized in good yields (70–82 %) without isolating chalcones. the structures of the compounds were established based on 1h-nmr, 13c-nmr, ft-ir and mass spectral and analytical data. all the compounds were evaluated for their antimicrobial activity against bacteria, such as staphylococus aureus, bacillus subtilis, escherichia coli and klebsiella pneumoniae, as well as fungi, such as aspergillus flavus and fusarium oxysporum. keywords: 2-(9-ethyl-9h-carbazol-3-yl)-3-hydroxy-4h-chromen-4-ones; modified algar–flynn–oyamada reaction; antimicrobial activity. introduction flavonoids constitute an important class of secondary metabolites that are widely distributed in plants. their widespread distribution in nature, their structural variability, relatively low toxicity and antioxidant activities have increased interest in flavonoids. furthermore, flavonoids possess multimodal biological activities, such as anticancer,1 antihypertensive,2 anti-inflammatory,3 antibacterial4 and antifungal5 activities. chromones are interesting structural scaffolds and have been assigned as privileged structures for drug discovery. substituted chromones were reported to show potential anticancer,6 antihistamine7 and antagonistic8 activities against leukotriene d4. on the other hand, carbazole derivatives are an important class of heterocyclic compounds that are known to possess important biological properties, such as antibacterial, antifungal,9 antitumor,10 antioxidant11 and antidiabetic12 properties. * corresponding author. e-mail: ashokdou@gmail.com doi: 10.2298/jsc141203051a 1362 ashok et al. in the light of biological importance of the chromone and carbazole scaffolds and in continuation of the ongoing search for biologically active heterocyclic molecules,13 herein, the one pot synthesis of 2-(9-ethyl-9h-carbazol-3-yl)-3-hydroxy-4h-chromen-4-ones using a modified algar–flynn–oyamada reaction (scheme 1) and the antimicrobial properties of the obtained derivatives are reported. scheme 1. synthetic route for the preparation of carbazole based 3-hydroxy-4h-chromen-4-ones (3a–i). results and discussion the original algar–flynn–oyamada reaction14 is a two step process for the synthesis of 3-hydroxy chromenones. in the first step, 2-hydroxy chalcones are formed, which on subsequent cyclisation in the second step in the presence of alkaline hydrogen peroxide yields the corresponding flavonols, whereas the modified algar–flynn–oyamada reaction15 is a one step process for the synthesis of flavonols from 2-hydroxyacetophenone and aromatic aldehydes in the presence of alkaline hydrogen peroxide (table i). in this modified version there is no need to isolate the intermediate chalcones. as a model case, 2-hydroxyacetophenone 1a was condensed with 9-ethyl-9h-carbazole-3-carbaldehyde 2 at room temperature using alkali and subsequently treated with alkaline hydrogen peroxide at room temperature to yield the flavonol derivative. it was identified as 2-(9-ethyl-9h-carbazol-3-yl)-3-hydroxy-4h-chromen-4-one 3a by ir, 1h-nmr, 13c-nmr and mass spectral data, which ruled out the formation of corresponding aurones and some other benzofuran derivatives, which were reported to form as by-products in algar–flynn–oyamada reaction. in the 1h-nmr spectra of 3a, the oh proton appeared at δ 7.08 ppm as broad singlet and h4′ proton appeared as a doublet at δ 9.03 ppm. in the 13c-nmr spectra of 3a, the carbonyl carbon appeared at δ 173.0 ppm and the n–ch2 carbon resonated at δ 37.7 ppm. the esi mass spectra of 3a showed a molecular ion peak at m/z = 356 [m+h]+. synthesis of carbazole based 3-hydroxy chromenones and antimicrobial activity 1363 table i. physical data of the synthesized compounds 3a–i compd. m.p., °c r1 r2 r3 time, h yield, %a 3a 134–136 h h h 6 72 3b 128–130 f h h 7 78 3c 178–180 cl h h 5 82 3d 190–192 br h h 8 76 3e 140–142 ch3 h h 6 80 3f 206–208 cl ch3 h 6 82 3g 212–214 cl h cl 7 80 3h 148–150 h och3 h 7 76 3i 172–174 h oc2h5 h 8 70 aisolated yield antibacterial activity the newly synthesized compounds 3a–i were screened in vitro for their antibacterial activity against two gram-positiveve bacterial strains (staphylococcus aureus (atcc 6538), bacillus subtilis (atcc 6633)) and two gram -negative bacterial strains (escherichia coli (atcc 25922), klebsiella pneumoniae (atcc 13883)) at two different concentrations 20 and 40 µg ml–1. the zone of inhibition was measured in mm and ciprofloxacin was used as a standard antibacterial substance, under similar conditions for comparison. all the synthesized compounds showed good activity against the tested microorganisms (table ii). among all, compounds 3a, 3h and 3i showed maximal zones of inhibition against the tested bacterial strains. it could be concluded that 3-hydroxy chromenones with electron releasing groups, such as methoxy, ethoxy and unsubstituted compounds, showed the maximum activity. furthermore, the antibacterial results observed for other substitutions on the phenyl ring were very similar table ii. antimicrobial activities (zones of inhibition in mm) of the synthesized compounds 3a–i concentration, µg ml-1 compd. s. aureus b. subtilis e. coli k. pneumoniae a. flavus f. oxysporum 20 40 20 40 20 40 20 40 50 50 3a 16 28 14 26 14 30 13 26 10 12 3b 11 22 6 14 05 12 7 15 5 10 3c 12 24 9 18 08 17 8 15 6 5 3d 11 20 9 17 10 17 9 19 6 4 3e 10 22 7 16 7 7 5 10 9 8 3f 11 21 9 18 9 10 7 15 11 4 3g 13 23 10 21 7 15 9 19 8 12 3h 16 29 16 29 17 32 24 35 13 16 3i 18 30 17 30 19 33 25 36 15 19 ciprofloxacin 15 28 16 30 18 35 23 35 – – amphotericin-b – – – – – – – – 12 15 1364 ashok et al. to each other. it was also concluded that changing the halogen substituent from f to cl and br does not provide any significant changes in antibacterial activity. antifungal activity the antifungal activities of the synthesized compounds 3a–i were tested against two pathogenic fungi, aspergillus flavus (atcc-9643) and fusarium oxysporum (atcc-48112), at a concentration of 50 µg ml–1 and the results were compared with those of the standard, amphotericin-b. all the compounds showed good activity against the tested fungal strains (table ii). among all the compounds, 3a, 3h and 3i showed maximal zones of inhibition against the tested fungal strains. thus, electron releasing groups on 3-hydroxy chromenone, i.e., methoxy and ethoxy substitutions and the unsubstituted compound showed the highest antifungal activities, followed by the halogen substituted compounds. experimental materials all the employed materials were obtained commercially, mostly from sigma–aldrich, and used without further purification. equipment the melting points were determined in open capillaries and are uncorrected. the purity of the compounds was checked by tlc on silica gel 60 f254 (merck). the 1h-nmr and 13c-nmr spectra were recorded on a bruker avance ii 400 spectrometer using tms as an internal standard. the ir spectra were recorded in kbr on a shimadzu ftir 8400s spectrophotometer. the mass spectra were recorded on a shimadzu lcms 2020 mass spectrometer. the elemental microanalysis was realised on a perkin elmer chn-2400 analyzer. the physical, analytical and spectral data of compounds 3a–i are given in the supplementary material to this paper. general procedure for the synthesis of 2-(9-ethyl-9h-carbazol-3-yl)-3-hydroxy-4h-chromen -4-ones to a well stirred solution of 2-hydroxyacetophenone 1a–i (1 mmol) and 9-ethyl-9h -carbazole-3-carbaldehyde 2 (1 mmol) in etoh (20 ml) was added naoh (4 mmol in 10 ml of etoh) at room temperature. the reaction mixture was further stirred for 4–5 h. after consumption of reactants (as indicated by tlc), the reaction mixture was dissolved in aqueous naoh (5 mmol in 5 ml), 3 ml of 30 % h2o2 was added dropwise and the stirring was continued for 2–3 h. after completion of reaction (monitored by tlc), the resulting light yellow reaction mixture was poured onto crushed ice and neutralized with dilute hcl. the thus-obtained light yellow solid was filtered, washed with water and dried. the crude product was purified by column chromatography on silica gel using hexane:ethyl acetate (7:3) as eluent to afford the desired products 3a–i. the respective yields are given in table i. biological assay synthesized compounds were screened for their antibacterial activities against pathogenic bacteria, i.e., s. aureus, b. subtilis, e. coli and k. pneumoniae and their antifungal activity against a. flavus and f. oxysporum. synthesis of carbazole based 3-hydroxy chromenones and antimicrobial activity 1365 the test organisms were cultured on agar slants, incubated for 24 h at 37±0.5 °c and 24– –48 h at 27±0.2 °c for the bacteria and fungi, respectively, to obtain freshly prepared cultures. the synthesized compounds were evaluated for antibacterial activity and antifungal activity against these freshly prepared strains of test organisms by the agar diffusion method and the poison plate technique, respectively. muller–hinton agar (mha) and potato dextrose agar (pda) were used as nutrient media for bacterial and fungal strains, respectively. the broth cultures were diluted with sterilized saline to bring the final size of the inoculum to approximately 105–106 cfu ml-1. the compounds were diluted in acetone, dimethyl sulphoxide (dmso) and diethyl ether for biological assays. of the three solvents, diethyl ether is found to be the best. the bacterial cultures were placed on the media and incubated at 37 °c for 24 h along with the diluted compounds introduced through discs (diameter 5 mm) dipped and placed over the nutrient media. the discs of ciprofloxacin (20–40 µg) and amphotericin-b (50 µg) were also incorporated into the medium for comparison. the same procedure was employed for determining the antifungal activity except that the culture strains of fungi were maintained on pda and spores were transferred into pda medium and the plates were incubated at 27±0.2 °c for 24–48 h. inhibition of growth of the test organisms (bacterial and fungal) in presence of the test material and the standards was measured with the help of a standard scale. the values of the inhibition zones are reported in table ii. conclusions in conclusion, an easy, facile and one-pot route for the synthesis of 2-(9 -ethyl-9h-carbazol-3-yl)-3-hydroxy-4h-chromen-4-ones in good yields is reported. in this method, there is no need to isolate the intermediate chalcones, which tremendously reduces the man power, time and cost and also improves the overall yields. the antimicrobial assay of these compounds revealed that compounds 3a, 3h and 3i showed maximal zones of inhibition against the tested microorganisms compared with the standards. supplementary material physical, analytical and spectral data of compounds 3a–i are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the authors are thankful to the head of department of chemistry for providing the laboratory facilities and the director of central facilities for research and development (cfrd), osmania university, india, for providing the ir and nmr spectral analysis. financial support for sr from csir, new delhi, is gratefully acknowledged. и з в о д синтеза карбазолских деривата 3-хидрокси-4h-хромен-4-она модификованом алгар–флин–ојимадином реакцијом и испитивање њихове антимикробне активности dongamanti ashok, sidda ravi, bommidi vijaya lakshmi и arram ganesh department of chemistry, osmania university, hyderabad 500 007, india синтетисана је серија деривата 2-(9-етил-9h-карбазол-3-ил)-3-хидрокси-4h-хромен-4-она полазећи од супституисаних 2-хидрокси-ацетофенона и 9-етил-9h-карбазол -3-карбалдехида помоћу naoh и h2o2 у модификованој алгар–флин–ојамадином реакцији. овим поступком синтетисани су флавоноли, без изоловања халкона, у добром 1366 ashok et al. приносу (70–82 %). структуре једињења утврђене су 1h-nmr и 13c-nmr техникама, ftir спектроскопијом, масеном спектрометријом и елементалном анализом. испитана је антимикробна активност добијених једињења према бактеријама staphylococus aureus, bacillus subtilis, escherichia coli и klebsiella pneumoniae као и према гљивицама aspergillus flavus и fusarium oxysporum. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {divergence of barley and oat varieties according to the content of $\beta$-glucan} j. serb. chem. soc. 82 (4) 379–388 (2017) udc 633.13+633.16:581.44:632.2:641.1 jscs–4973 short communication 379 short communication divergence of barley and oat varieties according to their content of β-glucan stefan m. marković1, nevena h. đukić1*, desimir knežević2 and suzana v. leković1 1university of kragujevac, faculty of science, r. domanovića 12, 34000, kragujevac, serbia and 2university of priština, faculty of agriculture, kosovska mitrovica, lešak, serbia (received 31 october, revised 29 december, accepted 30 december 2016) abstract: barley and oat have been promoted for years as good sources of dietary fiber, especially β-glucan. studies have shown a positive effect of barley and oat β-glucan on human health and as a result, there has been an increasing demand for β-glucans in the past decade. thus, the variability of the β-glucan content was investigated in the grain of 10 barley and 10 oat varieties. the β-glucan contents were determined by the icc standard method no 168. the content of β-glucan in the analyzed barley varieties were within the range 3.52–7.81 % and in analyzed oat varieties, the content of β-glucan were within the range of 3.15–7.28 %. among analyzed barley varieties, novosadski 314 contained the highest content of β-glucan (7.81 %), while tomba had the highest content of β-glucan (7.28%) in the analyzed oat varieties. based on the results, it could be concluded that there is genetic diversity of oat and barley varieties with respect to their β-glucan content. this fact enables varieties with a high a nutritional capacity to be selected. keywords: avena sativa; hordeum sativum; selection; variability. introduction barley and oat have been promoted for years as good sources of dietary fiber, especially β-glucan.1,2 β-1,3/1,4-glucans are polysaccharides present in the bran of some grains, such as oat and barley but to a much lesser degree in rye and wheat.3 in seeds of barley and oat, the highest concentrations of β-glucans are found in the endosperm and the subaleurone layer of the seed.4,5 numerous studies are increasingly showing a positive effect of β-glucan from barley and oat on human health. it was found that β-glucans present in barley and oat stimulate the immune system, and thus have a positive effect in the fight against infections6–8 and in the prevention of cancer.9–11 the intake of oat and barley β-glu * corresponding author. e-mail: nevena@kg.ac.rs doi: 10.2298/jsc1610310010m 380 marković et al. cans through different food products significantly decreases the total cholesterol and low-density lipoprotein cholesterol, and it may lead to improvement of lipid serum status and weight reduction.12–14 moreover, the intake of oat and barley β-glucan may affect a reduction in blood glucose levels in diabetics,15 and the digestion of starch, thereby reducing glycemic and insulin response.16–18 furthermore, β-glucans may represent an important component of food in the modification of metabolic irregularities and the prevention of metabolic syndrome.19,20 based on in vitro experiments, and animal and human clinical studies, there is considerable evidence concerning the importance of β-glucan in the treatment and prevention of allergic diseases, which opens new perspectives in the use of this group of natural substances.21 the physiological effects of β-glucans are mainly attributed to their physicochemical structure, such as molecular weight, viscosity, solubility and interaction with the gastrointestinal tract.22,23 in addition, the physicochemical characteristics, and the physiological effects of β-glucan may be affected by method of extraction, concentration of β-glucan, storage and with the food preparation method.24–26 among other things, β-glucans, which exist as indigestible polysaccharides in barley and oat, have been shown not only to have positive effects on health, but also the potential to be a possible new source of prebiotic.27,28 knowing these beneficial effects of β-glucan, it is important to determine the availability and the best potential sources of β-glucan and possibly to increase them, in order to promote their wider application and accessibility in everyday life. considering that the content of β-glucan in oat and barley could be increased by breeding and using appropriate agro-technology (effect of fertilization – application of nitrogen (n) affects positively by increasing the content of β-glucan)29–31 whereby the effect of genotype is dominant and has been confirmed,29,32 the goals of this research were to define the content of β-glucan in genetically divergent cultivars of 10 oat and 10 barley varieties, comparison of the β-glucan content in different oat and barley varieties, and identification of the varieties with higher contents of β-glucan as a modest contribution to the breeding of oat and barley. experimental plant material the plant material used for the field trials consisted of 10 varieties of spring barley (hordeum sativum j. ssp. distichum l.) and 10 varieties of spring oat (avena sativa l.). these genotypes originated from the gene bank collection of field and vegetable crops in novi sad. two rows of spring barley varieties were used for investigation, i.e., novosadski 296, novosadski 297, novosadski 300, novosadski 301, novosadski 306, novosadski 310, novosadski 312, novosadski 314, novosadski 316 and novosadski 318, and 10 varieties of spring oat, i.e., flaemingsterne, vok, mozart, juha, hannes, alden, aslak, wasa, tomba and kaempe gul. the barley and oat cultivars used for analysis were employed in increase of the production of cultivar seeds, because of their high genetic potential of yield, quality and β-glucan from barley and oat 381 adaptability. nowadays, these cultivars are used in cross hybridization and represent the core gene source in breeding programs for the creation of new enhanced cultivars. field experiment the experiment design in 3 replications was a basic plot with 5 rows of 1 m length with an inter-row spacing of 0.2 m and in row distance of 0.05 m between the seeds. sowing was performed by hand. the trial was realized in the location kragujevac (44°00′51′′ n, 20°54′42′′ e), during two growing seasons – 2011 and 2012. in the first year of the experiment, phenotypes were estimated and biotypes for each variety removed. in the second year of the experiment, the seed material was used for the determination of β-glucans. mineral fertilizers (npk 15:15:15, map – monoammonium phosphate) were applied before seeding according to the recommendations, based on the chemical properties of the soil and the available content of p, k and n reserves. the following fertilizers were applied for nutrition: urea (46 % n), kan (27 % n) and an (34 % n). the crop was protected adequately against pests and weeds by the appropriate use of pesticides and herbicides, with their efficacy being monitored. for analysis of the grain, full maturity stage of 30 plants (10 plants per replication) was used. soil characteristics the growing of barley and oat genotypes was realized on an experimental field on the soil type pseudogley having poor physical properties, an acid ph (phh2o 4.1) and the following content: humus 2.34 %, readily available phosphorus 7.8 mg 100 g-1 soil and potassium 14.2 mg 100 g-1 soil. climatic conditions during growing seasons the values of temperature and precipitation in two years of experiment were different in relation to the average values of the previous ten years (table i). during the period january/june, the average values of the temperature was 9.28 °c in the first and 9.38 °c in the second year of the experiment. in both years of the experiment, the average temperature was lower than in the long-term period (10.08 °c). the precipitation was more favorable in the year 2011, with sums of 327.9 mm, than in 2012 (378.9 mm). in the second year during the grain filling stage (april–may months), the amount of precipitation (174.6 mm) was higher than in the first year (120 mm) and in the second year, the amount of precipitation was similar to the sum of the long term precipitation (373.7 mm). table i. monthly and mean temperatures and monthly and cumulative precipitation; xm – average values of temperature and precipitation period january february march april may june xm total t / °c 2011 0.3 0.6 6.6 12.2 15.6 20.4 9.28 55.7 2012 –0.1 –4.2 8.8 12.7 16.0 23.1 9.38 56.3 2000–2010 0.9 2.4 7.6 12.0 17.2 20.4 10.08 60.5 precipitation, mm 2011 28.1 59.2 48.9 37.1 82.9 71.7 54.65 327.9 2012 107.1 54.9 24.5 69.1 105.5 17.8 63.15 378.9 2000–2010 42.8 44.7 52.5 66.6 74.9 92.2 62.28 373.7 seed sample after harvesting all the genetically divergent barley and oat varieties, 5 g of seeds were used for grinding and for analysis of their content of β-glucans. 382 marković et al. laboratory analysis the β-glucan contents were determined by the icc standard method no 168, which was adapted to measure the content of β-glucan in barley, oat and their products. particles smaller than 500 µm were used in the experiment. accuracy. in barley containing 4.0 % total β-glucan, the method was accurate to 4.0±0.1 %. processing of the results results for content of β-glucan of analyzed barley and oat varieties were obtained by spectrophotometrically reading the absorbance at λ=510 µm for each of the three test tubes (table ii). the final content of β-glucan was obtained by applying the following formula for grains of barley and oat: β-glucan (mass %) = ∆a×f×94 (or 64)×0.10w×162/180 = ∆a×f/w×8.46 (or 5.76) (1) where ∆a is the absorbance after β-glucosidase treatment (reaction); f – a factor for the conversion of absorbance values to µg of glucose: f = d d 100 (μg of -glucose) absorbance of 100 μg of -glucose and 94 is a volume correction factor (0.1 ml out of 9.4 ml was analyzed for cereal samples); 64 is a volume correction factor (0.1 ml out of 6.4 ml was analyzed for cooked, toasted of extruded cereal products); 0.10w is a factor to express the β-glucan content as a percentage of dry flour weight; w is the calculated dry weight of the sample analyzed in mg; 162/180 is a factor to convert from free d-glucose, as determined, to anhydro-d-glucose, as occurs in β-glucan33 and w is the grain weight. results and discussion the content of β-glucans in barley in the analyzed barley varieties, the contents of β-glucans ranged from 3.52 to 7.81 % (table ii). the highest content of β-glucan among analyzed barley varieties was found in the variety novosadski 314, with a concentration of 7.81 %, while the lowest concentration was found in the variety novosadski 312 (3.52 %). among the other analyzed barley varieties, high contents of β-glucan were observed in novosadski 318 (7.8 %), novosadski 310 (7.39 %), novosadski 306 (7.38 %), novosadski 297 (7.23 %) and novosadski 301 (7.18 %). a lower amount of β-glucans was found in the variety novosadski 300 (6.77 %), while the barley varieties novosadski 296 (5.85 %) and novosadski 316 (5.08 %) together with the variety novosadski 312 (3.52 %) had lower contents of β-glucan than the average of 6.6 %. based on the β-glucans contents, all the analyzed barley varieties were compared with each other and a similarity dendrogram with euclidean distances was made (fig. 1). an analysis of the dendrogram showed two clusters (groups) with mutually similar varieties (fig. 1). in the first cluster were the two barley varieties novosadski 296 and novosadski 316, with a distance of about 41 % (the degree of similarity was 59 %). the second cluster consisted of a larger number of varieties and pairs showing a high percentage of similarity (novosadski 297, β-glucan from barley and oat 383 table ii. the content of β-glucan in oat and barley varieties (mass %) analyzed oat variety β-glucan content analyzed barley variety β-glucan content flaemingsterne 4.79 novosadski 296 5.85 vok 5.60 novosadski 297 7.23 mozart 4.28 novosadski 300 6.77 juha 4.71 novosadski 301 7.18 hannes 4.25 novosadski 306 7.38 alden 3.38 novosadski 310 7.39 aslak 3.15 novosadski 312 3.52 wasa 4.71 novosadski 314 7.81 tomba 7.28 novosadski 316 5.08 kaempe gul 4.37 novosadski 318 7.80 fig. 1. dendogram of the genetic distances between the clusters of barley varieties for their content of β-glucan (the dendogram was obtained with statistica for windows, version 10). novosadski 301, novosadski 306, novosadski 310, novosadski 300, novosadski 314 and novosadski 318). in the second cluster, the greatest similarity, 99 %, was observed for the varieties novosadski 306 and novosadski 310. another pair consisted of the varieties novosadski 297 and novosadski 301 with a similarity of 97 %. with the previous pair of varieties of the second cluster, they form a small group with a distance of 8 % (degree of similarity of those pairs is 92 %). the most similar to this group according to values of β-glucans was novosadski 300 with distance in the range of 22 %. the second cluster with a distance of 22 % also contains a pair of varieties, novosadski 314 and novosadski 318, which are 97.5 % similar. the degree of similarity of the first and second cluster is 51 % 384 marković et al. (49 % distance). the variety novosadski 312 stands out because of the greatest distance (82.5 %) compared to all other barley varieties. the content of β-glucans in oat the content of β-glucan in the analyzed oat varieties ranged from 3.15 to 7.28 % (table ii). the highest content of β-glucan was found in the variety tomba, with a concentration of 7.28 %. a lower content of 5.6 % was found in the variety vok. the oat varieties in which the content of β-glucan ranged from 4 % to 5% were flaemingsterne 4.79 %, juha 4.71 %, wasa 4.71 %, kaempe gul 4.37 %, hannes 4.25 % and mozart 4.23 %. among the remaining analyzed oat varieties that have a β-glucan concentration of less than 4 % are alden (3.38 %) and aslak (3.15 %), in which the lowest concentration of β-glucan was observed. the average value of the β-glucans contents in the analyzed oat varieties was 4.65 %. based on the β-glucans contents, all the analyzed oat varieties were compared with each other and a similarity dendrogram with euclidean distances was made (fig. 2). an analysis of the dendrogram shows two clusters (groups) with mutually similar varieties (fig. 2). the first cluster consisted of two small groups of varieties, which together show a high percentage of similarity. the first group includes the varieties flaemingsterne, juha and wasa, with the highest percentage of similarity (99 %) within the first cluster. within the second group, there is a pair of varieties mozart and hannes with a degree of similarity of 98 %. the variety kaempe gul joins this pair with a distance of 8 %. the degree of similarity between the first and second group is 80 % (the distance between mentioned groups is 20 %). the second cluster includes the two oat varieties alden and aslak with a mutual distance in the range of 14 %. the degree of similarity of the first and second cluster is 50 %. the variety vok stands out from the two clusters by a distance in the range of 52 %, while the most notable variety is tomba due to the greatest distance (97 %) compared to all other varieties of oat. many studies have shown that the grains of barley and oat are characterized by higher concentrations of β-glucans compared to other grains, whereby barley has a higher content of β-glucan with respect to oat. the average concentration of β-glucan in barley is approximately in the range of 3 to 7 %, whereas the concentration of β-glucan in oat is approximately in the range of 2 to 6 %.34–36 havrlentova and kraic noted a large variation in the content of β-glucan in barley and oat varieties in all genotypes and indicated that barley and oat may be convenient sources of β-glucan.34 based of the biochemical analysis of the β-glucan content in the investigated oat and barley varieties, it could be concluded that the content of β-glucan was higher in the barley varieties than in the oat varieties, which could be observed through the average value of the β-glucan contents (6.6 % of the dry weight of the grain in the analyzed barley varieties, but only 4.65 % of the dry weight of β-glucan from barley and oat 385 the grain of the analyzed oat varieties). in the tested barley varieties, the β-glucan content ranged from 3.52 to 7.81 % of the dry grain weight, while the β-glucan content of analyzed oat varieties ranged from 3.15 to 7.28 % of the dry grain weight. the highest contents of β-glucan in the analyzed barley varieties were found in novosadski 314 (7.81 %) and novosadski 318 (7.8 %), while the highest content of β-glucan in analyzed oat varieties was observed in tomba, with a concentration of 7.28 %. fig. 2. dendogram of the genetic distances between clusters of oat varieties for their content of β-glucan (the dendogram was obtained with statistica for windows, version 10). numerous studies suggest the beneficial effects of β-glucans on human health as immunostimulants,3,7,8 they can act both anti-mutagenic and anti-carcinogenic,10,11,37 they can be used for lowering cholesterol levels and improvement of serum lipid status,38–40 they can have a positive effect on glycemic and insulin response,17,18 and among others, β-glucans can represent a new source of prebiotics.27,28 as a result of their positive effects on health, there has been an increasing demand for β-glucans in the last decade. for this reason, it is necessary to find the most affordable sources of β-glucans to enable their application and ensure their availability. the variability of the β-glucan content is controlled by both genetic and environmental factors and their interaction, which acts during the period of endosperm development.36,41,42 among the environmental factors, warm and dry weather during grain-filling results in higher levels of β-glucan, while high precipitation or irrigation are det386 marković et al. rimental.42,43 nitrogen (n) fertilizer was shown to influence the level of the biochemical constituents in the grain, and to lead to increased contents of β-glucan.29–31 considering that genotype had the greatest influence on variability than all the other factors and their interactions, the dominant effect of which has been confirmed in numerous studies,29,32,41 the selection of the proper genotype is the most crucial for desired amount of β-glucan in grain.32 specific programs of cultivation, selection and breeding are the key steps to obtain the appropriate barley and oat varieties for the production of foods rich in β-glucans and of high nutritional value.37–39 conclusions based on the obtained results, it could be concluded that genetic diversity influences the content of β-glucan in oat and barley varieties, giving the possibility of choosing varieties for the selection of lines of high nutritional capacity required to improve nutrition and to use as a source for obtaining useful preparations in the pharmaceutical industry for further implementation. the varieties that could be recommend for hybridization and breeding are barley varieties novosadski 314 and novosadski 318 and oat varieties tomba and vok, as varieties with the highest content of β-glucans. acknowledgement. this work was supported by the serbian ministry of education, science and technological development of the republic of serbia (project no. tr 31092). и з в о д дивергентност сорти јечма и овса према садржају β-глукана стефан м. марковић1, невена х. ђукић1, десимир кнежевић2 и сузана в. лековић1 1универзитет у крагујевцу, природно–математички факултет, р. домановића 12, 34000 крагујевац и 2универзитет у приштини, пољопривредни факултет, косовска митровица, лешак јечам и овас се промовишу годинама уназад као добар извор дијететских влакана, а поготову β-глукана. бројне студиje показују позитиван ефекат β-глукана јечма и овса на здравље људи и као последица јавља се све већа потражња за β-глуканима у последњој деценији. у зрну 10 сорти јечма и 10 сорти овса испитиван је садржај β-глукана. за одређивање садржаја β-глукана коришћена је icc стандардна метода бр. 168. у испитиваним сортама јечма садржај β-глукана кретао се у интервалу 3,52–7,81 %, а у испитиваним сортама овса садржај β-глукана кретао се у опсегу 3,15–7,28 %. највећи садржај β-глукана код анализираних сорти јечма поседује сорта новосадски 314 (7,81 %), док је највећи садржај β-глукана код анализираних сорти овса уочен код сорте tomba (7,28 %). на основу резултата, можемо закључити да постоји генетичка дивергентност у садржају β-глукана између испитиваних сорти овса и јечма, на основу које постоји могућност одабира сорти за селекционе линијe високог нутритивног капацитета, као и за побољшање захтева исхране. 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anker-nilssen, s. sahlstrøm, s. h. knutsen, a. k. holtekjolen, a. k. uhlen, j. cereal sci. 48 (2008) 670. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true /preservecopypage true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice changes in the content of water-soluble vitamins in actinidia chinensis during cold storage j. serb. chem. soc. 81 (6) 623–632 (2016) udc 582.688.4:577.164.1/.2+54–145.2+ jscs–4872 621.565.92 original scientific paper 623 changes in the content of water-soluble vitamins in actinidia chinensis during cold storage xian-bo zhu1,2, liang pan2, wu wei2, jia-qing pen2, yin-wei qi3 and xiao-lin ren1* 1college of horticulture, northwest a & f university, yangling, 712100, shaanxi, china, 2institute of economic crop research, shiyan academy of agricultural sciences, shiyan, 442714, hubei, china and 3school of agriculture ningxia university, yinchuan, 750021, ningxia, china (received 28 october, revised 18 december 2015, accepted 29 january 2016) abstract: the effects of cold storage on nine water-soluble vitamins in seven cultivars of actinidia chinensis (kiwifruit) were assessed using high-performance liquid chromatography. samples were collected at three time points during cold storage: one day, 30 days, and when edible. it was found that the vitamin c content in most cultivars increased with cold storage time, but there was no consistent increasing or decreasing trend for the other water-soluble vitamins across the cultivars during storage. after one day of cold storage, vitamins b1 and b2 were the most prevalent vitamins in the control (wild) fruit, while vitamins b5 and b6 were most prevalent in the hongyang and qihong cultivars. however, b12 was the most prevalent vitamin in the qihong cultivar after 30 days of cold storage. vitamins b3, b7, b9, and c were detected at the edible time point in the huayou, hongyang and jingnong-2 cultivars and in the control fruit. the vitamin contents varied significantly among kiwifruit cultivars following different durations of cold storage. out of the three durations tested, 30 days in cold storage was the most suitable for the absorption of water-soluble vitamins by a. chinensis. keywords: vitamin b; kiwifruit; cultivar; storage; vitamin c. introduction water-soluble vitamins are found in very small amounts in edible foods, but they play an essential role in the metabolism of the human body. their deficiency leads to a variety of clinical abnormalities that range from anemia to growth retardation and neurological disorders.1 vegetables and fruits are the major dietary sources for water-soluble vitamins in humans, and much research has been conducted on the vitamin content of various vegetables and fruits, such as six * corresponding author. e-mail: caihou2014@163.com doi: 10.2298/jsc151028012z _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 624 zhu et al. date palm cultivars, green leafy vegetables, rhodiola imbricata root, black garlic, and okra.2–6 changes to the water-soluble vitamin content of black garlic and six date palm cultivars were registered following thermal processing or vegetable storage.2,3,5 kiwifruits have a unique flavor and are rich in vitamin c, dietary fiber, and a variety of mineral nutrients, making them popular with consumers around the world. the most commonly cultivated types are actinidia chinensis and a. deliciosa. a. chinensis kiwifruits are endemic to china, and the chinese public generally prefers a. chinensis to a. deliciosa. although it was found that kiwifruit might contain up to nine water-soluble vitamins, little research has been performed to determine which factors influence the vitamin content and, in particular, what the ideal cold-storage period is for vitamin uptake.7 water-soluble vitamins can be measured with either liquid chromatography– –mass spectrometry (lc–ms) or high-performance liquid chromatography (hplc), though hplc tends to be the more cost-effective method.2,5,7–16 thus, in this study, the effects of cold storage on the water-soluble vitamin content of 7 a. chinensis cultivars were assessed using hplc. it is hoped that this research may provide a guide for how to maximize the uptake of water-soluble vitamins by kiwifruit. experimental plant materials seven kiwifruit cultivars were selected for this study: a. chinensis wild kiwifruit (here after referred to as control), wudang-1, hongyang, qihong, huayou, jingnong-2, and cuiyu. the control (wild) kiwifruit were picked on the qinling mountains in shaanxi province. wudang-1 kiwifruit were obtained from the institute of economic crop research center in shiyan city (32°65′n, 110°79′e), hubei, china. hongyang and qihong kiwifruit were retrieved from two well-managed orchards in meixian (34°29′n, 107°76′e), shaanxi, china. huayou, jingnong-2 and cuiyu were acquired from the kiwifruit experiment station of the northwest agricultural & forestry university in shaanxi province. all plant material were picked when the total soluble solids were 6.5–9.5 % of the harvest term and immediately transported to the laboratory, where they were rapidly pre-cooled before being stored under refrigerated conditions.17 as some cultivars took over two months in cold storage (1±0.5 °c) to soften, samples were collected from all cultivars at the picking period (day 1), after 30 days of storage (day 30), and then when they reached the edible period (ep) for assessment of the content of water-soluble vitamins. the storage times were as follows: control, 56 days; wudang-1, 61 days; qihong, 61 days; hongyang, 62 days; cuiyu, 81 days; huayou, 71 days and jingnong-2, 61 days. reagents all chemicals were of analytical reagent grade, and milli-q water was distilled and filtered through a 0.22-μm membrane filter. thiamine hydrochloride (vitamin b1), riboflavin (vitamin b2), niacin (vitamin b3), pantothenic acid (vitamin b5), pyridoxine hydrochloride (vitamin b6), d-biotin (vitamin b7), folic acid (vitamin b9), cyanocobalamin (vitamin b12), and ascorbic acid (vitamin c) were purchased from sigma–aldrich (st. louis, mo, usa). _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ water-soluble vitamins in kiwifruits 625 sodium hexanesulfonate and phosphoric acid were purchased from kermel chemical reagent co., ltd. (tianjing, china). hplc-grade methanol was purchased from thermofisher company (waltham, usa). sample preparation samples (10 g) were prepared from the pulp of five kiwifruits for each cultivar and were then homogenized. sample extracts were prepared with ultrasonic assistance in 30 ml of hcl (0.005 m) for 30 min at 30 °c. all extracts were centrifuged at 10,000×g for 5 min at 4 °c and then filtered through a 0.22-µm nylon membrane. during all these steps, the samples were protected from light. three independent replicates were prepared for each sample. determination of firmness and total soluble solids (tss) firmness was measured in five kiwifruits, and the measurements were repeated three times. a ta-xt2i texture analyzer (stable microsystems, godalming, uk) was used to determine firmness using 8 mm diameter samples. the fruits were peeled at opposite side and then the firmness was determined for the peeled position.18 the tss was assessed by juicing five kiwifruits and then using a pal-1 refractometer (atago, japan). hplc analysis a shimadzu scl-10avp hplc system was used. the mobile phase consisted of (a) methanol and (c) 0.005 m sodium hexanesulfonate (phosphoric acid was used to adjust the ph to 3.0). a c18 (ymc-pack ods-a, 4.6×250 mm, 5 μm) column was used for the determination of water-soluble vitamins. in all cases, the flow rate was 0.7 ml min-1 using isocratic elution (a:c = 3:7). the column temperature was set at 25 °c, and injection volume was 10 μl. dual wavelength recording was used detect the vitamins simultaneously under the same conditions: b5 and b7 were detected at 210 nm, while the other seven water-soluble vitamins were detected at 270nm.19 hplc chromatograms are shown in supplementary material to this paper (fig. s-1) statistical analysis ibm spss statistics software v.20 and sigmaplot 12.0 were employed for data elaboration and statistical analysis. all analyses were run in three duplicates with five kiwifruits of each cultivar used in each duplicate and the results are expressed as means ± standard deviation (sd). one-way analysis of variance (anova) was employed to assess the differences in the vitamin contents for different storage times. differences (duncan) tests were considered statistically significant at p < 0.05. results and discussion determination of firmness and total soluble solids firmness is usually used as a proxy for the degree of maturity of kiwifruit, while the total soluble solids (tss) is an important indicator of kiwifruit quality.17 when kiwifruit picked period follow 6.5≤tss≤7.5, and the firmness ≤1 n mm–1, there was no need to store. the first period followed 6.5≤tss≤7.5, the second was stored 30 days, and the last period was when the firmness was ≤1 n mm–1 (table i). when the sampled kiwifruit were separated from the vine, their firmness decreased, while tss increased in all cultivars with the duration of storage (table i). _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 626 zhu et al. table i. changes in firmness and total soluble solids (tss) during cold storage of kiwifruit; each value represents the mean ± standard deviation. different letters (a–c) within a row indicate significant differences at p < 0.05 over the three storage time points for each cultivar cultivar firmness, n mm-1 total soluble solids, % day 1 day 30 edible period day 1 day 30 edible period control 6.47±0.07a 1.58±0.06b 0.36±0.02c 6.46±0.05c 12.9±0.13a 12.0±0.11b wudang-1 6.29±0.06a 3.96±0.05b 0.89±0.02c 6.52±0.06c 11.8±0.08b 13.6±0.15a huayou 6.23±0.05a 5.30±0.07b 0.96±0.07c 6.66±0.07c 9.7±0.12b 12.5±0.12a cuiyu 7.37±0.08a 7.00±0.05a 0.71±0.03b 7.46±0.15c 11.2±0.08b 16.5±0.12a jingnong-2 4.77±0.04a 4.38±0.04b 0.44±0.01c 9.40±0.11c 14.1±0.11b 17.2±0.10a hongyang 5.33±0.06a 5.28±0.07a 0.75±0.02b 6.86±0.16c 11.0±0.10b 17.2±0.10a qihong 6.59±0.08a 4.37±0.09b 0.97±0.02c 7.12±0.15c 15.3±0.13b 20.0±0.12a changes in b1, b2, b3, and b5 in cold storage for each of the nine vitamins assessed, the water-soluble vitamin content ranged from undetectable to more than 2500 µg g–1 fresh weight (fw). out of the nine water-soluble vitamins previously reported in kiwifruit, only b9, b12 and vitamin c were detected in all cultivars across all three periods. in most cultivars, these three vitamins became more prevalent in the fruit over time. vitamin b1 was not detected on day 1 in the wudang-1 samples, while it was only detected on day 30 in the cuiyu samples and only on day 1 in the qihong fruit (fig. 1a). in the jingnong-2 cultivar, the vitamin b1 content remained constant over the three time points, indicating that cold storage had no effect on the b1 content in this cultivar. previous studies found that the b1 content increased over time, a phenomenon that was observed in the present study for the wudang-1 and hongyang cultivars. in contrast, it was found that the b1 content in the control and huayou fruit decreased with storage.3 vitamin b2 can be destroyed by light and heat; therefore, rapid extraction in a dark room was used to avoid decomposition. the vitamin b2 content in all cultivars across the three points was found to be no higher than 0.1 µg g–1 fw (fig.1b), except for in the control cultivar on day 1. the vitamin b2 content increased in huayou fruit over time, perhaps due to microbial growth.20 the b2 content decreased in the control and hongyang cultivars, while the others exhibited no significant differences across the three time points despite a low vitamin b2 content. this may be because b2 participates in physiological metabolism in storage.21 the control, wudang-1 and qihong cultivars had the highest vitamin b3 content on day 30, while the huayou, cuiyu, and jingnong-2 cultivars experienced an increase in b3 content across the three time points (fig.1 c). the maximum b3 content (over 0.2 µg g–1 fw) was found in the huayou fruit at the edible period. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ water-soluble vitamins in kiwifruits 627 fig. 1. changes in the b1 (a), b2 (b), b3 (c) and b5 (d) contents of a. chinensis during cold storage. interestingly, the vitamin b5 content increased in three cultivars over time and decreased in three cultivars over time. jingnong-2 was found to have the highest b5 content with more than 8 µg g–1 fw on day 30 (fig. 1d). changes of b6, b7, b9, b12 and vitamin c in cold storage of the seven cultivars, the vitamin b6 content was not detectable in only two cultivars: huayou on day 1 and cuiyu on day 1 and at the ep (fig. 2a). the b6 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 628 zhu et al. fig. 2. changes in the b6 (a), b7 (b), b9 (c), b12 (d) and vitamin c (e) contents of a. chinensis during cold storage. each value represents the mean ± standard deviation. different letters (a–c) within a row indicate significant differences at the p < 0.05 level over the three storage time points for each cultivar. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ water-soluble vitamins in kiwifruits 629 content in some cultivars was not detected, which could be attributed to the hydroxylation of b6 in the presence of vitamin c. in general, except for huayou, the b6 contents of the other six cultivars were lower at the edible time point than at day 30. similar results were previously found with the storage of green leafy vegetables.3 biotin (vitamin b7) is involved in amino acid catabolism, gluconeogenesis, and fatty acid synthesis. the vitamin b7 content of four cultivars increased with the duration of cold storage. jingnong-2, on the other hand, had the highest b7 content on day 30, while the other cultivars had their lowest b7 contents at that time (fig. 2b). the highest b7 content registered in this study appeared in the hongyang fruit and was nearly 20 µg g–1 fw. rapid increases in the vitamin b9 content were found from day 30 to the edible time point in the control, wudang-1 and huayou kiwifruit (fig. 2c). in the same period, a rapid decrease in the qihong cultivar was found. the highest b9 content (nearly 0.5 µg g–1 fw) was found in the control fruit at the ep. for most cultivars, the vitamin b12 content increased with increasing duration of cold storage. the exceptions were wudang-1, where b12 decreased over time, and huayou and qihong, where day 30 exhibited the highest b12 content (fig. 2d). vitamin c was found to be abundant in kiwifruit, although no regular pattern was found across the seven cultivars with cold storage. for example, for the control, wudang-1 and qihong cultivars, the lowest vitamin c content was detected on day 30, while the content decreased over time for the huayou fruit (fig. 2e). for the other cultivars, the vitamin c content increased with increasing duration of storage. for all the studied a. chinensis cultivars, day 30 was found to be the time point most suitable for absorbing water-soluble vitamins, as most of them could be detected in the seven cultivars at this time (figs. 1 and 2). although all watersoluble vitamins were detected at all three time points in the control (wild) kiwifruit, these fruit had the lowest tss content (table i), making the fruit less palatable for human consumption. previous studies found a significant depletion of vitamin c in fruit juice with cold storage.22 in the present study, however, the vitamin c content increased for many cultivars. this phenomenon was also found in pea leaves, perhaps due to the bound form of vitamin c being released at different times.3 similarly, the effects of cold storage on the other water-soluble vitamins were not similar to those seen in fruit juice; this perhaps be due to watersoluble vitamins in living plant materials having some activity.3,22 the hongyang cultivar is one of the most popular cultivars and of high economic importance in northwest china, and it was found to be rich in all watersoluble vitamins (figs. 1 and 2). it is therefore the most suitable cultivar of the seven cultivars for absorbing water-soluble vitamins. water-soluble vitamins not _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 630 zhu et al. detectable in some cultivars at some time points, which corresponded to lc–ms results.10 the b1, b2, b5 and b6 contents in some cultivars increased over time. similar results were obtained during vegetables storage, indicating that the levels of these three vitamins reflect not only degradation but also synthesis.3 some previous studies used lc–ms to detect water-soluble vitamins in green and yellow kiwifruit.7 in the present study, the contents of b6 and b9 found were lower and that of b7 were higher than the values found in previous studies, perhaps due to differences in varieties studied or cultivation environments.7 interestingly, the b7 contents found in the two red kiwifruit (hongyang and qihong cultivars) were higher than those of green kiwi, red kiwifruit may have more efficient synthetic pathways in regards to this vitamin.7 in green leafy vegetables, the contents of several water-soluble vitamins change during storage, but b7 was not one of these. in fact, sometimes b7 could not be detected at all for it is often in the bound form and not often in the free form.3 vitamin b9 was detected in all kiwifruit cultivars across the three time points. this is in contrast to stored vegetables, which do not have abundant vitamin b9.3 similarly, stored vegetables have very low vitamin b12 contents, while a. chinensis cultivars contain considerable levels of b12.3 conclusions the contents of nine water-soluble vitamins changed over time in actinidia chinensis. different cultivars exhibited different trends for different vitamins. for example, vitamin c in most cultivars increased with storage, while the contents of the other vitamins showed no similar change trends. some vitamins were undetectable in some cultivars at one or two time points. in general, more watersoluble vitamins could be detected following storage for 30 days, indicating this is the optimal duration of cold storage for the uptake of water-soluble vitamins. supplementary material hplc chromatograms of a standard mixture for the studied water-soluble vitamins are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. this work was supported by the earmarked fund for modern agroindustry technology research system of china. we would like to thank dr. jiangtao suo for material assistance. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ water-soluble vitamins in kiwifruits 631 и з в о д промена садржаја витамина растворних у води у биљци actinidia chinensis током чувања на хладном xian-bo zhu1,2, liang pan2, wu wei2, jia-qing pen2, yin-wei qi3 и xiao-lin ren1 1college of horticulture, northwest a & f university, yangling, 712100, shaanxi, china, 2institute of economic crop research, shiyan academy of agricultural sciences, shiyan, 442714, hubei, china и 3school of agriculture ningxia university, yinchuan, 750021, ningxia, china испитана је промена у садржају девет витамина растворних у води седам сорти actinidia chinensis (киви) током чувања на хладном, применом методе hplc. сакупљена су три узорка током чувања: првог дана, после 30 дана и у тренутку сазревања за јело. садржај витамина ц је порастао у већини сорти током чувања, али за друге витамине није нађена правилност у промени. након једног дана чувања на хладном, витамини б1 и б2 су били доминантни у контролном (дивљем) плоду, док су витамини б5 и б6 били доминантни у сортама hongyang и qihong. после 30 дана чувања, витамин б12 је постао главни у сорти qihong. витамини б3, б7, б9 и ц су детектовани тек у зрелим плодовима сорти huayou, hongyang, jingnong-2 и у контролној сорти. садржај витамина је јако варирао између сорти кивија у различитим периодима чувања. упоређивањем три временска периода чувања, закључено је да је, након 30 дана чувања, најповољнија комбинација витамина a. chinensis за апсорпцију. 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serrano, postharvest biol. technol. 41 (2006) 317 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 632 zhu et al. 19. s. vidović, b. stojanović, j. veljković, l. pražić-arsic, g. roglić, d. manojlović, j. chromatogr. a 1202 (2008) 155 20. m. c. sanchez-mata, m. camara, c. diez-marques, food chem. 80 (2003) 309 21. f. depeint, w. r. bruce, n. shangari, r. mehta, p. j. o′brien, chem. biol. interact. 163 (2006) 94 22. l. salvia-trujillo, m. morales-de la pena, a. rojas-grau, o. martin-belloso, j. agric. food chem. 59 (2011) 10034. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {molecular dynamic simulation study of molten cesium} j. serb. chem. soc. 82 (6) 681–694 (2017) udc 546.36:544.27:536.7+544.022: jscs–4996 532.11:66.09.001.57 original scientific paper 681 molecular dynamic simulation study of molten caesium saeid yeganegi1*, vahid moeini2 and zohreh doroodi2 1department of physical chemistry, university of mazandaran, babolsar, iran and 2department of chemistry, payame noor university, p. o. box 19395-3697, tehran, iran (received 25 july 2016, revised 5 january, accepted 8 february 2017) abstract: molecular dynamics simulations were performed to study thermodynamics and structural properties of expanded caesium fluid. internal pressure, radial distribution functions (rdfs), coordination numbers and diffusion coefficients have been calculated at temperature range 700–1600 k and pressure range 100–800 bar. we used the internal pressure to predict the metal– –non-metal transition occurrence region. rdfs were calculated at wide ranges of temperature and pressure. the coordination numbers decrease and positions of the first peak of rdfs slightly increase as the temperature increases and pressure decreases. the calculated self-diffusion coefficients at various temperatures and pressures show no distinct boundary between cs metallic fluid and its expanded fluid where it continuously increases with temperature. keywords: metal–non-metal transition; md simulation; internal pressure; rdf. introduction molten cs has been attracted considerable attention for its novel properties which makes it potentially useful in the systems which need heat pipe, lubricants and coolants at high temperature. also, it is used in space and nuclear power technology.1–4 experimental and theoretical investigations show that a momentous amount of clustering takes place in the region of the metal-nonmetal transition, when the density decreases below 1.3 g cm–3.5–7 the design and development of technological applications of cs, as a kind of new engineering material, needs a precise knowledge of the structural and dynamical features of alkali metal fluids. the metal–non-metal transition for alkali metals occurs close to critical point,8 but does not coincide with it.9 because of the experimental difficulty at high pressure and temperature (the critical point of liquidvapor transitions for cs is tc = 1924 k, pc = 9.25 mpa and dc = 0.38 g cm–3),10 the simulation can be used as a valuable method to study metallic fluids. one of the early efforts to study the structure of liquid metals was * corresponding author. e-mail: yeganegi@umz.ac.ir doi: 10.2298/jsc160725018y 682 yeganegi, moeini and doroodi performed in 195211 and the first momentous molecular dynamics (md) simulation for cs was performed in 2001.12 the diffusion and shear-viscosity coefficients and velocity auto correlation function have been reported for na, k, rb and cs near the melting point.13–15 the simple but useful expressions for the relationship between structural and surface properties of liquid metals have been presented by employing the hard-sphere description near the melting point.16,17 also, md simulations have been performed to calculate the structural and transport properties of liquid alkali metals by using reformed morse potential near the melting point.18 the embedded atom method potentials have been calculated for the alkali metals along the melting line of the metal and the discrepancy between the simulated energy, and the actual energy of the metal at high temperatures has been discussed.19 recently, thermodynamic quantities of the alkali metals20 and their cluster size distributions21 have been studied based on the static crystalline properties and using ab initio potential respectively. gupta proposed a semi-empirical potential to study the metallic fluids by molecular dynamics.22 the gupta potential function has been applied successfully by many researchers to describe the structure and energy of pb23, ni, ag, au24 and cu–au, al, zn and cd clusters.25 also, gupta potential has been used to study the melting of na and cs clusters in order to investigate the effects of the geometric and electronic magic numbers on the melting temperature as a function of cluster size.26–28 the electronic structure of alkali metal fluids strongly depends on the thermodynamic state of the system. when cs fluid expands, two transitions can occur, liquid–vapour and metal–non-metal one.9 in fact, a high-density liquid metal is a conductor, while it becomes a non-metal insulator when turned into low-density vapour. near the critical point the transition from a liquid metal to a non-metal takes place.29 this transition implies that the nature of the particle interactions must change dramatically, from metallic to a van der waals-type interaction.30 the particle interactions in dense alkali metals are dominated by the coulomb potential, whereas the interactions in the nonmetallic phase are weak van der waals forces. it is suggested that there is a relationship between rapid variation in the conductivity and density.31 the metal–non-metal transition can be investigated by comparing the measured electrical conductivity with the value calculated from the nearly-free-electron theory (nfe). this theory is often used to describe metallic behaviour and is valid when the mean free path of the conduction electron is much larger than the average distance between neighbouring atoms in the liquid. it has been shown that the nearly-free electron theory provides a good account of the conductivity for cs for densities higher than 1.3 g cm–3.29–31 according to this theory, the transition begins at 1.3 g cm–3 for cs, which is exactly the density around which the deviation from the linear isotherm thermodynamics and structural properties of molten cs fluid 683 regularity (lir) is observed.5,32 keshavarzi et al.32 demonstrated that the metal– –non-metal transition is surely neither first nor second order, and it can occur separately or simultaneously with the liquid–vapour transition (lvt). in other words, the metal–non-metal transition proceeds gradually, which is indicated by some changes in liquid properties such as lower conductivity than that predicted by nfe theory,29,30 deviation of density from the lir predictions for a single phase.5,32 there has been an increasing interest in the study of internal pressure in the elucidation of thermodynamic properties of metallic fluids.5 a general method of calculating the internal pressure is based on the thermodynamic equation of state: i t v e p p t p v t ∂ ∂   = = −   ∂ ∂    (1) e is the internal energy, [ ]/ te v∂ ∂ is the internal pressure and [ ]/ vt p t∂ ∂ is usually called the thermal pressure.33–35 according to the statistical mechanics, the total internal energy e of a fluid is the sum of potential energy, u, and the kinetic energy, k. it is assumed that any kinetic energy contribution to the internal energy will vanish on taking the derivative, since the temperature is held constant.36 the potential energy can be divided into two parts, attractive ua and repulsive ur parts37 and inserting into eq. (1) gives: ( ) ( )a r i i,a i,r , ,n t n t u k u u p p p v v    ∂ + ∂ + = = = +      ∂ ∂    (2) where pi,a is the attractive internal pressure (positive) and pi,r is the repulsive internal pressure (negative). as the density is raised at constant temperature, the repulsive pressure becomes predominant, and therefore, internal pressure decreases. on the other hand, the reason is that the resultant forces under low pressure conditions are attractive, and as the pressure increases, the repulsive forces become predominant. although some approximate approaches exist, there is no definite way to distinguish the metallic and non-metallic states. moeini5 has shown that linear regularity of simple fluid can be applied successfully for predicting the metal– –non-metal transition in alkali metals. based on this assumption they showed that internal pressure of expanded cs versus density has a maximum around 1.3 g cm–3 or 0.097 l mol–1, when before reaching this density some properties such as local contraction, electrical conductivity and magnetic susceptibility show significant changes.5,9,29,30,31 internal pressure, as well as density, of molten metal can be used to recognize the metal–non-metal transition of an expanded metallic fluid.32,33 the goal of this paper is the study of the pressure dependence of internal pressure of cs fluid 684 yeganegi, moeini and doroodi by the molecular dynamics (md) simulations to recognize the initiation of metal–non-metal transition from the bulk fluid up to the expanded fluid. md simulations were performed near metal-nonmetal transition by using gupta potential. in the present work, up to our knowledge, we demonstrate the first study to calculate internal pressure by performing classical molecular dynamics. we used the internal pressure in order to predict metal–non-metal transition and also we study the structural and dynamical properties from bulk up to creating expanded fluid of cs by calculation of radial distribution function, coordination number and diffusion coefficient. simulation details we have used gupta potential to describe the interactions in cs fluid. based on this potential, the energy between particles can be written as a sum of born-mayer-type repulsive and many-body attractive energies as:38 2 0.5 0 0 exp( 1) exp[ 2 ( 1 ]ij ij j i j i r r e a p b q r r≠ ≠    = − − − − −       (3) where rij is distance between i and j atoms and r0 is the equilibrium nearest-neighbour distance in the bulk fluid. the parameters p and q represent the repulsive interaction and the attractive interaction ranges, respectively. the gupta potential parameters for cs used in this work are: a = 0.02054 ev, b = 0.24218 ev, p = 9.62, q = 1.45 and r0 = 9.44 a.u. 24 cs and other alkali metals are simple metals, meaning that the fermi surface of these metals is spherical. they are generally considered to be monatomic simple liquids.39,40 in all our molecular dynamic simulations, we have used 428 cs atoms initially placed in a bcc array replicated in xyz directions. all md simulations were performed using dl-poly-2.18 program.41 internal pressure and radial distribution function (rdf) were calculated at wide ranges of temperature (700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 and 1600 k) and pressure (100, 200, 300, 400, 500, 600, 700 and 800 bar). at each temperature for the lowest pressure, 428 atoms were placed in the simulation cell then npt (constant particle number, pressure and temperature) simulation was conducted for 5ns with the berendsen method using 1 and 3 ps as the thermostat and barostat relaxation times to reproduce the experimental density.42 the equations of atomic motion was integrated using the verlet velocity algorithm43 with a time step of 0.005 ps and cut off distance of 12.0 å. the diffusion coefficients were calculated by nvt md simulations and berendsen thermostat for 10 ns time steps where 2 ns steps were discarded as equilibration. results and discussion internal pressure the calculated total energy and density are presented and compared with experimental densities42 in table i. the deviations expressed in percent from experimental values are shown in fig. 1. our simulations underestimate cs density for temperatures higher than 900 k while overestimating it for lower temperatures. large deviation at high temperatures and low pressures can be attributed thermodynamics and structural properties of molten cs fluid 685 to the lack of sufficient accuracy of the proposed potential energy function to describe the atomic interactions. also, the large fluctuation in density due to the formation and destruction of varying size cs clusters at low pressure and high temperatures can be the cause for the observed large deviations. table i. calculated total energy, e, calculated density, dcal, and experimental density, dexp, of vargaftik et al.42 for cs fluid at different pressures, p, and temperatures, t p / bar e / j mol-1 dcal / g cm -3 dexp / g cm -3 p / bar e / j mol-1 dcal / g cm -3 dexp / g cm -3 t = 700 k t = 800 k 100 –559.263 1.662 1.626 100 –530.899 1.579 1.57 200 –561.786 1.685 1.643 200 –534.217 1.606 1.588 300 –564.169 1.706 1.659 300 –537.114 1.630 1.605 400 –566.366 1.727 1.675 400 –539.684 1.653 1.622 500 –568.235 1.747 500 –542.02 1.675 600 –569.917 1.765 1.704 600 –544.053 1.695 1.654 700 –571.529 1.783 700 –546.062 1.715 800 –572.931 1.800 800 –547.838 1.733 t = 900 k t = 1000 k 100 –502.372 1.496 1.515 100 –473.167 1.410 1.457 200 –506.437 1.526 1.533 200 –478.377 1.446 1.477 300 –509.965 1.554 1.552 300 –482.886 1.479 1.497 400 –513.026 1.579 1.570 400 –486.624 1.508 1.516 500 –516.04 1.604 500 –490.082 1.534 600 –518.563 1.627 1.604 600 –493.05 1.559 1.554 700 –520.759 1.648 700 –495.993 1.584 800 –522.955 1.669 800 –498.4 1.606 t = 1100 k t = 1200 k 100 –443.097 1.321 1.397 100 –412.117 1.229 1.335 200 –449.943 1.365 1.419 200 –420.458 1.280 1.361 300 –455.387 1.402 1.441 300 –427.654 1.325 1.385 400 –460.106 1.435 1.463 400 –433.355 1.363 1.409 500 –464.195 1.466 500 –438.284 1.397 600 –467.863 1.494 1.504 600 –442.49 1.428 1.454 700 –471.017 1.519 700 –446.228 1.456 800 –473.914 1.544 800 –449.639 1.483 t = 1300 k t = 1400 k 100 –378.87 1.127 1.272 100 –343.006 1.017 1.204 200 –390.388 1.193 1.301 200 –359.057 1.102 1.240 300 –399.103 1.245 1.328 300 –370.038 1.164 1.271 400 –406.182 1.289 1.354 400 –378.706 1.216 1.299 500 –412.093 1.328 500 –385.809 1.260 600 –417.163 1.363 1.404 600 –391.673 1.298 1.354 700 –421.509 1.394 700 –396.79 1.332 800 –425.364 1.423 800 –401.323 1.364 t = 1500 k t = 1600 k 100 –302.376 0.888 1.131 100 –251.747 0.724 1.052 200 –325.694 1.005 1.175 200 –290.344 0.902 1.108 686 yeganegi, moeini and doroodi table i. continued p / bar e / j mol-1 dcal / g cm -3 dexp / g cm -3 p / bar e / j mol-1 dcal / g cm -3 dexp / g cm -3 t = 1500 k t = 1600 k 300 –340.413 1.083 1.211 300 –309.362 0.997 1.149 400 –351.02 1.142 1.244 400 –322.469 1.067 1.187 500 –359.314 1.191 500 –332.609 1.123 600 –366.02 1.233 1.305 600 –340.553 1.170 1.256 700 –372.188 1.272 700 –347.352 1.212 800 –377.188 1.306 800 –353.193 1.249 fig. 1. relative deviations of calculated densities from the experimental values42 from table i for cs at temperature range 700–1600 k. internal energy is generally represented by a cubic expression in the density by allowing the coefficients to depend on temperature.34 we have used a similar expression in terms of volume rather than density: 2 30e e av bv cv= + + + (4) where v = v(p,t) and a, b and c are constants characteristic of a particular substance. the fitting parameters a, b and c can be determined by fitting e versus v at constant temperature over the whole pressure range. fitted parameters are shown in table ii, while coefficients of determination (r2) for all of fittings were nearly 1. after the seminal work of parsafar34 many researcher used the equation u = u(v) for obtaining the equation of state and some linear regularities (lir).36 up to our knowledge, we obtained for the first time the coefficients of u = u(v) by fitting the calculated internal energies to volume. the obtained coefficients are strongly depended on temperature, which shows the dependence of the interparticle interaction to the thermodynamic sate of the cs. the calculated internal energy from eq. (4) and the simulated one depicted good agreement in fig. 2. the internal pressure can be obtained by the straightforward differentiation of internal energy with respect to volume at constant temperature. thermodynamics and structural properties of molten cs fluid 687 table ii. fitted parameters of equation 4, a, b, c at different temperatures t t / k a / kj mol-1 m-3 b / 107 kj mol-1 m-6 c / 1012 kj mol-1 m-9 700 –3.71 675.07 394.49 800 0.19 –24.95 23.36 900 –0.96 175.57 –93.48 1000 –0.21 52.18 –26.18 1100 –0.34 71.57 –36.16 1200 –0.34 69.14 –34.43 1300 –0.09 32.16 –16.59 1400 0.07 10.44 –6.78 1500 0.15 –0.52 –2.08 1600 0.23 –9.53 1.40 fig. 2. calculated (symbols) and simulated (lines) internal energies vs. volume for cs. the resulting internal pressures, pi, versus molar volume are shown in fig. 3. it shows that at 700–1000 k, internal pressure, pi, increases with molar volume, vm, but for 1300–1600 k, it decreases with molar volume. the isotherms of 1100 and 1200 k in fig. 3 show maxima at about 0.093 and 0.094 l mol–1 (1.43 and 1.41 g cm–3), respectively. our results are in agreement with previous experimental and theoretical results5,6,44,45 which showed that the internal pressure had a maximum at 1.3 g cm–3 (0.097 l mol–1) at 1350 k. it is well known that liquid cs at high densities shows characteristics typical of a nearly free electron (nfe) metal.6,31,44,46 however, when the density is reduced below about 1.3 g cm–3 or the temperature is increased up to 1350 k, the quantitative changes begins to develop. under this condition, the measured electrical conductivity is lower than the nfe value31 and the magnetic susceptibility47 and density fluctuation48 increase. these changes indicate that the metal–non-metal transition is developing and that it is strongly dependent on the thermodynamic state.29–31 the failure of the 688 yeganegi, moeini and doroodi 0.6 1.0 1.4 1.8 2.2 2.6 0.06 0.09 0.12 0.15 0.18 p i / m pa vm / l mol-1 700 k 800 k 900 k 1000 k 1100 k 1200 k 1300 k 1400 k 1500 k 1600 k fig. 3. calculated internal pressure pi isotherms for cs at temperature range 700–1600 k. nfe theory reflects, most probably, cluster formation and the increased importance of soft potential.5,6,31,45 it is revealed that for molar volumes higher than vmax the attractive force becomes predominant and it has strong influence on the determination of internal energy and internal pressure.6,32,48,49 marcus50,51 has shown that the internal pressure of water (contrary to most liquids, except liquid metals) increases with the temperature and exhibit a maximum at 446.15 k. he has explained this behaviour in terms of gradual change in the nature of binding between water molecules. likewise, the maximum in internal pressure vs. molar volume in fig. 2 can be understood in terms of two different bindings in the molten alkali metals.32 at the low molar volumes (high pressures and low temperatures), where the measured conductivity and thermodynamic state of the system agrees well with predictions from the nfe theory and the lir, respectively,5,6,27,32 the system is in a monoatomic state with a delocalized electron cloud over the entire system. in this state, increasing the molar volume further (decreasing pressure) leads to a smaller repulsion between ions and negative repulsive internal pressure, pi,r, which tend to increases the internal pressure. as can be seen from fig. 2 at 700 k, the internal pressure increases steadily with the increasing the molar volume. further increasing of the molar volume leads to clustering, where the electrons tend to be shared between atoms within a cluster, resulting in a molecular or polyatomic structure with an insignificant conductivity. therefore, there are two different types of bindings in the system, the metallic bonding between atoms within clusters and van der waals attractions between clusters. formation of clusters diminish the metallic bonding then decreases the attraction energy over the system where this decreases the attractive internal pressure, pi,a. keshavarzi et al.32 have shown that this transition is neither first nor second order but occurs gradually in the expanded molten cs. the max thermodynamics and structural properties of molten cs fluid 689 imum of internal pressure in fig. 3 is the point where the electron delocalization over the entire system breaks down by clustering and the system composed of the metallic and non-metallic parts. the extrapolation of results of a rather small system to bulk fluid must be done carefully. in order to check the size dependency of our results, especially the location of vmax, the internal pressures versus molar volume at 1100 k were reproduced for a larger system with 856 cs atoms in fig. 4 and compared with that of a smaller system. figure 4 revealed that the density for a larger system is shifted slightly toward smaller molar volume but the location of the maximum remains nearly unchanged. so, one can conclude that the value of vmax is independent of the system size. 2.050 2.070 2.090 2.110 2.130 0.080 0.085 0.090 0.095 0.100 0.105 p i / m pa vm / l mol-1 856 cs 428 cs vmax fig. 4. calculated internal pressure, pi, for 428 (black square) and 856 cs atoms (grey triangle) at 1100 k. fluid structure we calculated radial distribution functions (rdfs) of the expanded cs in whole studied temperature and pressure ranges using gupta potential. the positions of the first peak of rdfs, namely rmax, have been analysed. it slowly rises from 4.98 å at low temperature and high pressure, to 5.03 å at high temperature and low pressure. the rdfs at temperature range 700–1600 k and 500 bar pressure are plotted as an example in fig. 5. it shows that the intensity of the first and second peak of rdfs reduced and overspread as temperature increases. the coordination number, n, up to the first minimum of rdfs for cs fluid is plotted in fig. 6 at the studied temperature and pressure ranges. the calculated coordination number generally decreases with temperature, from 12 to 7, but its variations are strongly depends on the thermodynamic state of the system. the variations of the coordination number at high temperature and low pressure are more pronounced than in the low temperature and high pressure. 690 yeganegi, moeini and doroodi 0 0.4 0.8 1.2 1.6 2 2 4 6 8 10 12 14 r d f r / å 700 k 800 k 900 k 1000 k 1100 k 1200 k 1300 k 1400 k 1500 k 1600 k fig. 5. radial distribution functions (rdfs) for cs at p = 500 bar at temperatures from 700 to 1600 k. 6 8 10 12 600 800 1000 1200 1400 1600 1800 n t / k 800 700 600 500 400 300 200 100 fig. 6. the calculated coordination number (n) of cs at temperatures from 700 to 1600 k and pressures from 100 to 800 bar. the comparison of snapshots of the system at low and high temperatures in fig. 7 shows that, as the system expands, the average nearest neighbour-distance (rmax) remains nearly constant while the average coordination number decreases. this observation agrees well with the findings of hensel30 explained based on the formation of cs clusters. the average coordination number decreases because of an increase in the number of atoms on the surface of clusters.30 this calculations are in good agreement with the results of previous studies.7,29,47 our study shows that in high temperature, when non-metallic properties are prevailing, the coordination number of cs fluid is less than 11, while it is larger than 11 for cs fluid with metallic properties at lower temperature. thermodynamics and structural properties of molten cs fluid 691 fig. 7. snapshots of the simulation ensemble at: a) t = 700 k and p = 800 bar and b) t = 1500 k and p = 100 bar. self-diffusion coefficient the knowledge of diffusivities plays an important role in the investigation of liquid metals and fluids. numerous experimental and theoretical diffusion coefficient data exist near melting point of cs 13,14,16,18 and a few studies of liquid metals have been carried out at high temperature.29,52,53 however, for temperature and pressure ranges considered here, there is no reported diffusion coefficient for cs fluid. the diffusion coefficients were calculated for some selected points around the maximum of the pi–vm curve in fig. 3 and reported in table iii. the state of cs fluid for molar volumes lower and higher than vmax were labelled m and em, respectively. the state of the maximum points of pi–vm isotherm curves at 1100 k and 400 bar are shown also in table iii. the entries in table iii were sorted based on the diffusion coefficient increment. the inspection of table iii shows that although there is not a definite boundary between metal and non -metal regions, one can certainly predict that the compressed cs fluid with diffusion coefficient less than 9.12×10–6 cm2 s–1 possess metallic characteristic and the expanded cs with diffusion coefficient greater than 1.07×10–5 cm2 s–1 starts to show non-metallic characteristic. so one can suggest the diffusion coefficient as a simple factor to characterize the metallic state of cs fluid. calculated diffusion coefficients of metallic and non-metal cs are different by one order of magnitude, but the metal to non-metal transition takes place gradually in terms of diffusion coefficient. table iii. calculated molar volume, vm, coordination number, n, diffusion coefficient, d, and state of the cs fluid designated as metal (m) or expanded metal (em) around maximum point (max) of pi–vm in fig. 3 t / k p / bar vm / l mol -1 n d / 106 cm2 s-1 state 700 800 0.074 12.83 3.96 m 700 600 0.075 12.51 4.14 m 800 100 0.084 12.08 6.07 m 692 yeganegi, moeini and doroodi table iii. continued t / k p / bar vm / l mol -1 n d / 106 cm2 s-1 state 900 100 0.089 11.05 7.45 m 1100 800 0.086 11.99 8.55 m 1100 600 0.089 11.41 8.92 m 1000 200 0.092 11.07 9.07 m 1100 500 0.091 11.20 9.12 m 1100 400 0.093 10.58 9.45 max 1000 100 0.094 11.01 9.60 m 1100 300 0.095 10.73 10.19 em 1200 800 0.090 11.52 10.45 m 1200 700 0.091 11.12 10.46 m 1200 600 0.093 11.11 10.65 m 1100 100 0.100 10.14 10.73 em 1200 500 0.095 10.58 11.36 em 1200 400 0.097 10.44 11.42 em 1300 800 0.093 10.86 11.43 em 1400 800 0.097 10.21 13.40 em 1500 800 0.102 10.16 14.94 em 1600 700 0.110 10.00 18.09 em 1600 100 0.184 6.99 21.37 em conclusions we calculated density, internal pressure, rdfs and diffusion coefficients of cs fluid using molecular dynamics and gupta potential for wide ranges of pressure and temperature to investigate the metal–non-metal transition. the internal pressure of cs fluid exhibits a pronounced maximum when the transition from metal to non-metal begins to develop. our results showed that molten cs with coordination number more than 11 exhibits metallic behaviour, while for the expanded fluid of cs, with lower electrical conductivity than predicted by nfe theory, the coordination number is less than 11. the dynamical behaviour of cs fluid was studied by the calculation of self-diffusion coefficients. our results show that the diffusion coefficients of metallic cs are smaller than those of the expanded fluid. it is shown that the diffusion coefficient can be used to characterize the metallic and non-metallic state of cs fluid accurately, although it is not as accurate as internal pressure. и з в о д студија стопљеног цезијума молекулско-динамичком симулацијом saeid yeganegi1, vahid moeini2 и zohreh doroodi2 1 department of physical chemistry, university of mazandaran, babolsar, iran и 2department of chemistry, payame noor university, p. o. box 19395-3697, tehran, iran урађене су симулације молекулском динамиком са циљем да се проуче термодинамичка и структурна својства експандованог течног cs. унутрашњи притисак, функције радијалне расподеле (rdfs), координациони бројеви и коефицијенти дифузије израчу thermodynamics and structural properties of molten cs fluid 693 нати су у температурном опсегу 700–1600 k и опсегу притиска 100–800 bar. користили смо унутрашњи притисак за предвиђање области појављивања прелаза метал–неметал. rdf су израчунате за широке области температуре и притиска. координациони бројеви опадају, а положаји првог пика у rdf лагано расту са порастом температуре и са смањењем притиска. израчунати коефицијенти самодифузије на различитим температурама и притисцима не показују за cs јасну границу између металног и експандованог течног стања, а континуално расту са температуром. 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice volume 81 (2016) 1 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. subject index (±)trans-1,2-cyclohexanediamine, 153 (indolyl)methane, 1069 1,2,3-triazoles, 233 1,2,4,5-tetraoxanes, 999 1,2-bis(diphenylphosphino)ethane, 47 1,4-dihydropyridine, 729 1,5-diphenyl-carbazide, 645 1-methylimidazole, 1151 2,5-diphenylthiophenes, 999 2-bromoethylamine hydrobromide, 1151 3,5-diarylidene-4-piperidone, 859 4-(2-pyridylazo)resorcinol, 645 4-(benzeneazo)salicylaldehyde, 153 4,7-phenanthroline, 133 4-oxalocrotonate tautomerase (4-ot), 871 α-glucosidase, 1371 β-casein, 739 π–π interactions, 1251 ab initio, 177, 633, 763 abc index, 271 actinidia chinensis, 623 activation energy, 897 adapalene, 1171 adiabatic ionization potential, 277 adsorbers, 419 adsorption, 419, 1199 air, 219 aldehydes, 125 aldrin, 707 alfacalcidol, 1127 alkyl azides, 233 allosteric, 347 amino acid, 307, 1345 amino acid-derived ureas, 13 aminouracils, 29 ancient ceramics, 1407 anil, 1111 aniline, 789 anionic surfactants, 1097 anions, 1441 anthocyanins, 883 antibacterial, 57, 509, 1151 antifungal, 57, 509 antiinflammatory, 1 antimicrobial, 233, 851, 859 antimycobacterial, 133 antioxidant, 23, 177, 277, 357, 499, 567, 1127, 1239 antitumor, 897 apiaceae, 357 apple, 607 aqueous extraction, 1441 aqueous solution, 1321 arachidonic acid, 1 arsenazo(iii), 907 arsenic, 1199 artificial saliva, 1307 ash, 1081 assessment tool, 1455 atomic force microscopy (afm), 777 autochthonous, 607 avobenzone, 1393 azaheterocycles, 133 azepines, 1225 azide alkyne cycloaddition, 233 2 journal of the serbian chemical society available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. azolinked, 153 band gap, 57, 323 beneficial, 1127 benzodiazepine, 851, 1037 benzophenone, 1055 bicalicene, 81 binary mixtures, 1161, 1427 bioaccumulation, 947 biocatalysis, 871 biocompatibility, 799 biogenic amines, 488 bioluminescence, 697 biomaterials, 433 bismuth-oxychloride, 125 bisphenol a, 333 bond dissociation enthalpy, 277 bone sialoprotein, 799 bone, 1407 borohydride, 751 buxus sempervirens, 447 cadmium, 947 calicene, 81 carbamic esters, 13 carbamoyl benzotriazoles, 13 carbon disulfide, 219 catalysts, 1025 cationic polyelectrolyte, 575 cell death, 697 cell line sw982, 1 cell wall, 1359 cerium, 433 chalcones, 851, 1231 chicory, 1141 chitosan, 575 chlorpyrifos, 1215 cholinesterase, 357 chromans, 67 chromium(iii), 645 chromium(vi), 1321 chromium, 645, 1321 chronoamperometry, 553 cichorium intybus, 1141 cinnamoyl, 673 cis-trans-nepetalactone, 999 claisen–schmidt condensation, 1231 clonal selection, 883 co(ii)phthalocyanines, 1025 coal, 403, 1441 cobalt(ii), 255, 369, 509 cobalt, 255, 369, 509, 1151 co-base alloy, 1307 color removal, 959 composites, 589 computational study, 103 concerted, 67 conjugation, 141 cooperative catalysis, 1335 coordination studies, 47 copolymers, 307 copper polypyridyl complexes, 1251 copper(ii), 153, 369, 395, 509, 751 copper, 153, 291, 369, 395, 509, 751 copper-catalyzed, 233 corn flakes, 1013 corrosion inhibition, 163 cottrell equation, 553 coumarin-derived ligands, 1383 coumarins, 357, 1383 countercation, 91 covalent disulfide bridges, 1371 crotonisation, 859 crystal structure, 729, 1383 cucurbita pepo, 35 cultivar, 607, 623 cun4o chromophore, 1251 cyanidin, 177 cyclic conjugation, 81 cyclic hydrazines, 1121 cyclization, 1335 cycloaddition, 187 cycloimmonium salts, 133 cytotoxicity, 103, 897, 1345 decolorization, 830 degradation kinetics, 243 degradation, 830, 1215 dehydrogenation, 1069 demercaptanization, 91 density functional theory (dft), 67, 277, 509, 1263, 1393 dental alloy, 1307 design, 383 detonation, 687 diabetes mellitus, 488 dialkyl phosphate ester, 935 dibutyltin oxide, 23 dieldrin, 707 volume 81: subject index 3 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. dielectric barrier discharge reactor, 830 dielectric barrier, 1097 diels-alder, 67 diffusion, 1183 dihydropyrane, 1335 dihydropyrimidine, 1111 dinuclear complexes, 47 diphenol, 673 disease, 1127 disinfection by-products, 1273 dithiocarbamic, 1025 dna binding, 1345 docking, 347 dopamine d2 receptor, 347 doping, 323 dowex1-x8oh, 971 drinking water, 1273 drug release, 1183 dye, 907, 971 ecotoxicity, 830 edda, 897 electrical resistivity, 323 electrocatalytic transformation, 29 electrochemical studies, 1251 electrocoagulation, 959 electrode potential, 177 electrodeposition, 291, 553 electromagnetic field, 1415 electronic spectra, 1263 electronic structures, 1263 electrothermal atomic absorption spectrometry (etaas), 645 embedding-crosslinking, 1215 enantiomeric composition, 209 encapsulation, 1183 endocrine disrupter, 333 endrin, 707 environment, 459 enzymatic hydrolysis, 35 enzyme promiscuity, 1231 enzyme, 35, 1231, 1359 enzyme-catalyzed synthesis, 1231 epoxide, 419 escherichia coli, 433 ethylcellulose, 1183 ethylphosphate, 935 eupergit® c, 1371 excited states, 1393 explosives, 687 extraction, 1441 firing temperature, 1407 fisetin, 243 flavonoids, 499 flue gas cleaning, 1427 fluorescence, 1345 fly ash, 813, 1441 folic acid, 739 folin-ciocalteu method, 567 free radical, 277 frequency, 1415 ftir spectroscopy, 1407 fungal, 1215 garson’s algorithm, 959 gas chromatography, 1273 gasoline, 91 gelatinizer, 935 glass electrode, 1415 glycidyl methacrylate, 419 glycine max, 1239 glycine, 1239, 1283, 1383 glycosylation, 23 grafting, 419 grape, 883 graphene–polymer, 1055 graphite, 777 green chemistry, 971 gurney velocity, 687 h2ge=si, 633 haloacetic acids, 1273 hashimoto's thyroiditis, 488 heck, 1121 herbicides, 923 heterocycles, 1225 heteropoly acids, 777 higher-order thinking, 1455 hirshfeld surface, 763 homogenous, 1097 humic acid, 255 hybrid materials, 589 hydrazine-carboxamides, 13 hydrazones, 125 hydroarylation, 1121 hydrogel, 1359 hydrogen cyanide, 187 hydrogen peroxide, 219 hydrolase promiscuity, 1231 4 journal of the serbian chemical society available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. hydrolase, 1231 hydrolysis, 369, 1383 hydroxyapatite, 433 hydroxylamine, 475 hyperconjugated, 1069 imidazole, 187 imidazopyridine, 1111 imides, 1121 immobilization, 1215, 1359, 1371 immune-mediated, 488 impulse, 687 inorganic filler, 589 inorganic reactions, 717 instructional tool, 1455 integrase inhibitors, 383 interactions in acetic media interstellar molecule, 187 invertase, 1359 iodine, 141 ion chromatography, 403, 661, 1441 ion exchange, 255 ionic diels-alder, 67 iron oxide, 1199 isatin, 979 isocratic, 661 isoflavone, 1239 isoxazol-5(4h)-one, 971 kamlet–abboud–taft equation, 541 kamlet–taft equation, 979 keggin type polyoxometalate, 91 ketenimine, 187 ketones, 125 kinetic modeling, 1183 kinetics, 197, 907, 1025, 1321 kit-6, 1069 kiwifruit, 623 laccase, 1215 lead, 291, 553, 947 least squares, 209 lfer, 979 linoleic acid, 141 linolenic acid, 141 lipase-catalyzed synthesis, 1231 lser, 979 luminescent, 697 lysine, 871 lysozyme, 1345 macro-hardness, 1307 macroscopic, 717 magnetite, 589, 1069 mahonia aquifolium, 447 malononitrile, 29 maltase, 1371 malvidin-3-o-glucoside, 883 manganese, 11514 markers, 488 mass spectrometry, 1273 maximum permissible level, 947 mea, 1427 mechanical properties, 1295 mechanism of hydrolysis, 1383 mechanism, 1025, 1383 melting, 323 membrane, 419 mercaptan, 91 merit figures, 1273 metallic ion release rate, 1307 methylated-β-cyclodextrin, 1037 methyliminodiacetic acid, 541 michael addition, 851 microemulsion, 395 microextraction, 923 micro-hardness, 1307 microparticles, 1183 microwave, 403, 751, 851, 1081, 1225 microwave-assisted synthesis, 1225 microwave-assisted, 751 midazolam, 1037 minerals, 1141 minor groove, 1345 modeling, 1321, 1427 molecular graph, 271 molecular structure descriptor, 271 molybdenocene, 47 molybdenum(iv), 47 molybdenum(vi), 541 monoethyl phosphate, 935 morphology, 291, 553, 799 multicomponent reactions, 29 multiple species analyte/eluent, 661 multiresidue method, 923 mutagenesis, 871 myorelaxant, 729 n,n-dimethylaniline, 789 n-alkylation, 133 nanocavity, 153 volume 81: subject index 5 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. nanocellulose, 589, 1199 nanocomposites, 1295 nanoindentation, 1295 nanomaterials, 163 nanoparticles, 433, 751 nanoshales, 395 nano-sized, 1069 nanostructure, 395 naringenin, 1161 natural emulsifiers, 35 nepeta cataria, 999 nepeta pannonica, 999 neural network, 959 neurobiological, 357 nickel(ii), 197, 255, 509, 1263 nitrones, 475 nrtl model, 789 nucleophilic aromatic substitution, 1225 nutrients, 1141 o-aminophenol, 1111 oilseed proteins, 35 olefins, 153 one-pot, 13, 219, 233, 971 o-phenylenediamine, 1111 organic chemistry course, 1455 organocatalysis, 871 organochlorine pesticides, 707 organopalladium reagents, 1335 origanum vulgare, 23 orthosteric binding site, 347 osteoblast cells, 799 osteocalcin, 799 oxidation, 475, 1025 oxidative stress, 1127 palladium(ii) complexes, 1383 palladium(ii), 1263, 1383 paper, 1295 partial molar volumes, 1283 pbmc, 1127 pear, 607 peel, 607 peg 200, 1427 percarbonate, 219 pericyclic reaction, 187 pes scan, 763 pesticides, 923 ph measurement, 1415 ph, 243, 1415 p-halogenobenzoyl, 133 pharmaceutical forms, 1171 phenolics, 1239 phenylalanine ammonia-lyase (pal), 1239 phospholipase a2 inhibitor, 1 phosphorus pentoxide, 935 photochemistry, 307, 1393 photo-cross-linking, 673 photocuring, 1055 photoluminescence, 433 photopolymerization, 419 photosensitivity, 673, 739 photostability, 1037 physical-textural, 1013 physicochemical characterization, 1407 pigment, 1407 piperazines, 347 piperidines, 347 plant proteins, 35 plant-growth promoting rhizobacteria (pgpr), 1239 plants, 447, 1239 plasma, 830, 1097 platinum(iv), 897 polarization, 291 pollutants, 1199 polyacrylamide, 1359 polycaprolactone, 589 polycondensation, 673 polyethersulfone, 419 polyethylene glycol, 789, 1427 polyethylenimine–epichlorohydrin resin, 1321 polymeric sorbent, 907, 971 polymorphs, 763 polyoxometalates, 777 polyphenolics, 499 polyphosphate, 935 post-modification, 307 pottery, 1407 power, 1415 preferential solvation, 1161 proline, 871 propellants, 687 protoberberine, 103 prunus laurocerasus, 447 pseudo-binary oxides, 163 6 journal of the serbian chemical society available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. purolite® a109, 1371 pvb, 1295 pyrazinamide, 763 pyrazole, 187 pyridine, 307 pyrido[2,3-d]pyrimidine, 29 quantitative structure–activity relationship (qsar), 383 quantum chemistry, 1393 quasi-reversible, 1251 quercetin, 243 quinone, 1345 radical polymerization, 307 radical scavenging, 567 reaction mechanism, 219 reactive dyes, 830 recasting, 1307 recorded ph value, 1415 removal, 971, 1321 resolution, 661 resveratrol, 739 reverse micelle, 395 r-group, 383 rosmarinic acid, 999 ruthenium tetraoxide, 475 ruthenium(ii), 521, 897 saccharomyces cerevisiae, 1359, 1371 saccharose, 1359 salicylaldehyde, 1151 scanning electron microscope (sem), 553 schiff base, 57, 369, 509, 979, 1111, 1151 sclerochloa dura, 1 secondary amines, 475 secondary chemistry education, 1455 seed, 499 selective, 125 sem, 291 sensitivity analysis, 959 sensory properties, 1013 sepiolite, 197, 1295 sequential extraction, 1081 serbia, 1407 serum, 488 shacking-assisted, 813 silylene, 633 single-agent extraction, 1081 skin, 1171 sodium dodecyl sulfate (sds), 575 sodium dodecylbenzene-sulfonate (sdbs), 1097 sodium lauryl sulfate, 1097 sodium pyruvate, 1283 soil matrix, 923 soil, 447, 459, 707, 923, 947, 1215 solid-state method, 163 solvatochromism, 979, 1161 solvent effect, 541 solvent evaporation, 1183 solvothermal, 751 sorption, 197 soybean oil, 141 soybean, 141, 1239 spectroscopy, 521 spiro si-heterocyclic ring, 633 stability, 1359 staphylococcus aureus, 433 stepwise, 67 structure elucidation, 729 students, 1455 styrene, 67, 153, 369 submicroscopic, 717 sulfonic acid, 419 surface tension, 575 surface water, 333 surfactant, 575 systemic approach, 1455 systems thinking skills, 1455 tafel curves, 163 tannins, 499 target fishing, 103 td-dft theory, 1263 tegdme, 1427 temperature, 243 tert-butylquinone, 1345 tetradentate, 521 tetrazolo quinoline aldehyde, 851 tg−dsc, 897 therapy, 1127 thermal conductivity, 1427 thermal tautomerization, 1393 thermodynamics, 197, 1283 thermoelectric, 323 thiomorpholine, 233 thiosemicarbazone, 521 thiosulfonate supports, 1371 thiourea, 219, 1263 volume 81: subject index 7 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. titanium implants, 799 topology, 81 topomer comfa, 383 topomer search, 383 trace elements, 403 transfer factor, 947 transition metal, 509 transition-metal-free conditions, 1225 transport, 1427 triticum cultivars, 499 trolox, 277 tryptophan, 209 tungsten(iv), 47 tungstenocene, 47 two photon polymerization, 1055 two-tier test, 717 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice jscs, volume 80 (2015) :: subject index volume 80 (2015) 1 subject index 1-(2-aminoethyl)-3-methylimidazolium hexafluorophosphate, 35 1,2,4-triazol-3-thiole, 1461 1,4-naphthoquinone, 731 17β-carboxamide steroids, 1481 1h-1,2,4-triazole-3-thiol, 755 2-(9-ethyl-9h-carbazol-3-yl)-3-hydroxy -4h-chromen-4-ones, 1361 2-(dimethylamino)ethyl methacrylate, 889 2,5-dihydroxybenzenesulfonic acid salt, 605 2,7-dihydroxynaphthalene, 673 2,7-naphthalenediol, 673 2-aminopyridine, 639 2-hydroxynicotinate, 1289 3-hydroxyflavone–bovine serum albumin, 517 4-(alkylamino)coumarins, 315 absorption, 1177 acenaphthene, spectroscopic studies of, 1489 acetylcholinesterase inhibitor, 127 acetylcholinesterase, 127 acidification, 563 acinetobacter baumannii, 819 activated carbon, 407 acylhydrazinecarbothioamide, 1461 adsdpv, 1035 adsorbent, 1203 adsorption capacity, 265 adsorption, 563, 899, 1113, 1297 afm, 73 agoa, 651 alcohol dehydrogenase, 1051 alcohols, 933 aldehydes, 595 algae, 407 aliphatic polyester, 529 alkylation, 1273 allelochemicals, 475 alloy, 1217 alpinia blepharocalyx, 171 alumina, 1113 aluminum-ferric-magnesium polysilicate, 1553 alzheimer’s disease, 127 amide moiety, 1367 amine, 731 amino acids, 343, 819 aminocyclopyrachlor, 767 amino-functionalized silica, 1265 aminopeptidase n, 605 ammonia, 1203 annual work plan, 435 anode materials, 685 anode, 903 anti-angiogenic effect, 605 antibacterial activity, 705, 819, 839, 1471 antibacterial potency, 755 antibacterial studies, 779 antibacterial, 639 anticancer, 1251 antidiabetic drug, 1311 antifungal activity, 839, 1367 antifungal agents, 1367 antifungal potency, 755 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 2 journal of the serbian chemical society antiinflammatory activity, 1481 antimalarials, 1339 antimicrobial activity, 305, 315, 739, 1361, 1461 antimicrobial study, 755 antimicrobial, 1101, 1251 antioxidant activity, 21, 143 antioxidant, 127, 475 antiparasitic, 1339 antitubercular activity, 1471 application, 279 aqueous extracts, 475 aqueous rechargeable lithium batteries, 685 aromadendrin, 1051 aromatic acid, 1101 aromaticity, 877 artificial neural network, 499 ascorbic acid, 1161 asog–visco parameters, 933 aspergillus niger, 1375 aspergillus oryzae, 1 aspergillus sp., 1279 astragalus depressus, 137 asymmetric bent-core, 673 atenolol, 695 atomic force microscopy, 63 atr-ftir, 485 azacyclopropenylidene, 53 azepines, 839 azo dye, 903 aβ anti-aggregating activity, 127 backpropagation neural network, 421 binary mixtures, 933 biocatalysis, 853 biocompatibility, 1541 biodegradable, 529 bioleaching, 391 biological activity, 35, 1481 biological sample, 1311 biomarkers, 575 biomaterials, 1541 bio-oil, 717 biopesticides, 475 biosorption, 407 biosurfactant, 279 bipyridines, 739 bis-benzimidazole, 45 bismuth, 1217 bolaform skeleton, 1273 bond critical point, 997 bor, 391 bromo-2h-pyran-2-ones, 1139 broth dilution method, 739 brushite, 237 calcium phosphate, 237 camphor, 171 cancer, 1339 candida albicans, 315 candida rugose, 1113 canvas, 805 carbazole, 1361 carbidopa, 789 carbon dioxide, 265 carbon nanotubes, 789 carboxylesterase, 1 carcinogenesis, 605 carrier, 1203 cassia fistula, 115 catalase, 143 catalysts, 717 cations, 1297 cd13 activity, 605 cell membranes permeability, 749 centrifugal ultrafiltration, 1051 cephalosporins, 1035 cetyltrimethylammonium bromide, 1019 chalcones, 305 charge transfer, 997 chelates, 179 chemical mass balance, 253 chemical shift, 877 chemistry knowledge, 1567 chemistry teaching, 435, 1321 chemistry textbook, 1321 chloride solutions, 1149 chromatography, 1311 chromium(vi), 899, 1019 chromium(iii) complexes, 329 cigarette beetle, 171 clay, 529 clinical isolate, 819 clinoptilolite, 717, 819, 1203 coagulation, 115, 1553 coatings, 237 cobalt complex, 1515 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ volume 80: subject index 3 cobalt(ii) complex, 179, 1391 cobalt(ii) perchlorate, 45 cobalt, 197 cobalth-based alloy, 1541 colorimetric sensing, 779 combustion, 377 composites, 223 compressed liquid, 1073 computational electrochemistry, 1035 computational fluid dynamics model, 377 conducting polymer, 917, 1435 confinement media, 1297 conformation, 1101 consistent force field, 329 contact toxicity, 171 contamination, 827 continuous flow, 853 copolyamic acid, 1061 copper(ii) complexes, 35 copper, 391, 819, 1149 copper-based alloys, 1505 co-precipitation, 367 coronary restenosis, 143 corrosion, 73 cortienic acid, 1481 coumarin, 305, 739 crystal structure, 867, 1289, 1391 crystallization, 223 ctab micellar effect, 1019 curcumin nanocrystals, 63 curricular components, 435 curricular knowledge, 435 cv, 1035 cyclocondensation, 453 cytotoxicity, 1541, 1339 decolorization, 903 decomposition, 1391 dehydrogenase, 1127 denaturation, 613 dendrite, 107 density functional theory calculations, 595, 877, 1035, 1139, 1399, 1489 density, 933, 1073 dental cast alloy, 1541 dental, 1177 denture, 1177 desmodium styracifolium, 1051 desulfurization, 549 dextran 70, 517 dialkyl acetylenedicarboxylate, 459 diatomite, 209 diazonium, 1161 dibenzo[a,d][7]annulene, 1461 dichloromethane, 1073 dichloromethane, 1423 diels–alder reaction, 1139 diet, 21 difenoconazole, 1127 dilution, 983 dimethyl sulfoxide, 1435 dimroth rearrangement. 1251 dinuclear, 639 diphenylamine, 1411 dipyrazol-1-ylmethane, 997 discharge capacity, 685 ditetrazolium salts, 179 dithiocarbamate derivatives, 1367 dithiocarboxylate, 755 dna, 87 dobesilates, 605 drug analysis, 1311 dsc, 209, 223 dubinin–radushkevich, 407 dye removal, 115 ear edema, 1481 eastern posavina, 827 ec-sod, 143 edaravone, 627 education, 1567 edxrf, 805 eis, 73 elastic modules, 1449 electric conductivity, 685 electrochemical impedance spectroscopy, 685 electrochemistry, 903, 1161, 1515 electrodeposition, 73, 107, 197, 237 electrolysis, 903 electrospinning, 97 elevated temperature, 1073 entropy, 1297 environmental education, 1567 environmental literacy, 1567 enzymatic activity, 1127 enzymatic hydrolysis, 853 enzyme production, 1279, 1375 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 4 journal of the serbian chemical society enzyme, 143 esi, 137 esipt, 517 essential oil, 171, 465 ester hydrolase, 1 ethanol, 695 ethylenediamine, 1515 eucalyptol, 171 euchems news, 589 eutrophication, 1567 evaluation, 959 extraction efficiency, 983 extraction equilibria, 179 fabaceae, 137 fatty acid methyl ester, 9 fatty acid, 9 feed composition, 355 fermentation, 1 ferrofluid, 367 fibroblasts, 1541 flavones, 517 flocculants, 1553 flotation tailings, 391 flue gas, 549 fluidized bed, 377 fluoranthene, spectroscopic studies of, 1489 fluorescence spectroscopy, 517 folic acid, 789 formic acid, 1217 formononetin, 1051 four components, 1265 fractions of fibre, 21 free radicals, 627 freezing, 1297 freundlich isotherm, 1203 friedländer reaction, 971 fruits, 21 ft-ir spectroscopy, 613, 805 ft-ir, 209 fuel cell anode catalysts, 1217 fuel cell, 1217 fukui, 767 fumigant toxicity, 171 functionalization, 1193 functionalization, 1449 fungi, 1279, 1375 fungicide, 1127 galectin-1, 159 gas chromatography, 9 gas chromatography-mass spectrometry, 983 gauche interaction, 1101 gels, 889 general regression neural network, 421 genetic algorithms, 187, 253 geo-accumulation index, 827 geochemical study, 575 geochemistry, 1087 geogenic hazard, 827 glass fibers, 223 glassy carbon electrode, 1161 glucoamylase, 1279, 1375 gold nanoparticles, 1161 graphene sheet, 1449 green leafy vegetables, 21 green procedure, 971 green synthesis, 1265 groundwater over-exploitations, 947 groundwater, 827 hammett correlation, 1019 heavy metals, 407, 827, 1087 hepatic enzymes, 727 hepatotoxicity, 627 herbicide, 767 hesperidinase, 853 heterocycles, 595, 839 heterogeneous catalyst, 971 hexadecanoic acid, 465 high angle annular dark field microscopy, 107 high pressure, 1073, 1423 hirshfeld surface analysis, 1489 hollow-fiber micro-extraction, 1311 homobimetallic complex, 755 homodinuclear, 1391 homogeneous catalysis, 595 hplc, 1311 hplc–ms, 1051 hs-crp, 143 hybrid materials, 1061 hydrazides, 453 hydrazines, 453 hydrazones, 1251 hydrocarbons, 659 hydrofluoric acid, 651 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ volume 80: subject index 5 hydrogen bonds, 651 hydrophobic nature, 1061 hydroxyapatite, 237 hypochlorite, 903 hypochlorous acid, 903 icons, 805 illumination, 1411 imidization, 1061 immobilization, 1113 in vitro, 1541 inclusion complex,485 indolylquinones, 731 inflammation, 143 inhibition, 605 inorganic compound, 1061 inorganic polymer, 1553 insecticidal activity, 171, 475 interaction, 87 intercalation, 801 interlayer interactions, 1449 ion exchange, 1149 ion-association complexes, 179 ionic liquid, 35, 265 ipomoea carnea, 465 ir spectra, 367 ir, 755 iron encrustations, 947 iron(ii) complex, 1399 iron(iii) complex, 1399 iron, 947 isobaric thermal expansivity, 1423 isoflavane, 137 isoquercitrin, 853 isotherm, 407 isothermal compressibility, 1423 itaconic acid, 917, 1435 itaconic, 1177 italian ryegrass, 1203 jahn–teller, 877 job’s plot, 485 jouyban–acree model, 695 kerosene suspensions, 367 kinetic stability, 613 kinetics, 407 kostolac basin, 575 kremna basin, 1087 krohnke reaction, 739 lake robule, 391 langmuir, 407 lasioderma serricorne, 171 layered silicate, 529 lesson plan, 435 libs, 1505 light-induced, 1411 lignin, 717 lignites, 575 lime, 549 linear sweep voltammetry, 87 lipase, 1, 1113 liquid crystals, 673 lithium, 801 lopare basin, 827 lp(a), 143 luminescence, 1289 magnesium, 1553 magnesium-doped alumina, 1515 magnetic properties, 197 magnetite nanoparticles, 367 magnetometry, 367 maltose, 1279 manure, 1203 mass spectra, 755 md – molecular descriptor, 659 mechanistic investigation, 899 mercury electrode, 1035 mercury, 779 mesoporous, 1515 metal clusters, 1217 metal-ion aqueous batteries, 801 metallocene, 877 methane emission, 421 methods, 279 methyl methacrylate, 1177 methylation, 9 methylene blue, 563 michael addition, 305 microcalorimetry, 1297 micropores, 1297 micro-raman spectroscopy, 805 microreactor, 853 microwave irradiation, 305 microwave, 563 microwave-assisted reaction, 459 milk, 983 mixed anhydride, 1101 mixed solvent, 695 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 6 journal of the serbian chemical society model, 549 modeling, 509 modification, 717 modified algar–flynn–oyamada reaction, 1361 modified electrodes, 789 molecular distance–edge vector index, 499 molecular form, 159 molecular graph, 1009 molecular sieve, 265 monomer, 1061 monte carlo method, 659 monthly work plan, 435 morphology, 197, 223, 529 mp2 method, 53 mp2, 767 multicenter wiener index, 1009 multicomponent reactions, 595 multidrug resistance, 819 multiple linear regression, 187, 421 multivariate statistical analysis, 315 n-[3-(trimethoxysilyl)propyl]ethylenediamine triacetic acid, 1193 nanocomposites, 1449 nanofiber membrane, 97 nanofiber, 97 nanoparticles, 705, 779 nanoporous silica, 1265 nanostructures, 237 naphthenic acid fractions, 749 naphthenic acids, 749 national emission, 421 natural coagulant, 115 natural zeolite, 717, 819 neovascularization, 605 neusilin, 209 n-hexane, 1073, 1297, 1423 n-methylimidazole, 459 nmr, 137, 755 non-conventional fuel, 377 nucleophilic substitution, 731 nuts, 21 o-alkylation, 459 odoiporus longicollis, 465 oily sludge, 1553 olive oil, 1 one-pot, 1361 one-pot reaction, 453 one-pot synthesis, 1265 opbe, 1399 organic matter, 5757 organic modifier, 529 organotin chloride, 755 organotin(iv) complexes, 755 orientation imaging microscopy, 107 oxapropane, 45 oxetane ring, 1273 oxidation, 947, 1217 oxidative cyclizations, 1251 oxidative transformation, 1411 oxides, 717 oxirane, 53, 651 oxldl, 143 oxoisoaporphine–tryptamine, 127 palaeo-reconstruction, 575 palladium, 839 pani, 917, 1435 papain, 613 partial atomic charges, 329 partial least square, 343 particle, 549 pbdes, 499 pcm, 651, 767 peer review, 959 pendulone, 137 peptide drugs, 343 peptide, 343 permeation, 1061 peroxides, 1339 ph, 1515 phenanthroline, 87 phenol, 717 phenylsulfinylacetic acid, 1019 phosphatase, 1127 photocatalysis, 1411 photo-curable, 97 photoluminescence spectroscopy, 63 photooxidation, 1411 photoreduction, 705 phthalate esters, 983 physicochemical characterization, 485 physiological activity, 749 pigments, 805 piperidin-4-one oxime, 1101 piroxicam, 509 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ volume 80: subject index 7 piszkiewicz cooperative model, 1019 placenta, 159 plackett-burman design, 1529 plasma, 1505 plasmodium falciparum, 1339 platinum, 1149, 1217 p-nitrophenyl esters, polarography, 87 pollution, 827, 1087 poloxamer, 209 poly(ethylene glycol) diacrylate, 933 poly(vinyl alcohol), 97 poly(vinyl chloride), 819 polyaluminum chloride, 115 polyamide, 705 polyaniline, 917 polybrominated diphenyl ethers, 499 polyethyleneimine, 899 polymer, 819, 1553 polymerization, 899, 917 polymorphous transition, 209 polypropylene, 223 porosity, 237 powder, 197 p-phenyldiamine, 1161 pre-concentration, 1311 production, 279 propane to oxygen ratio, 355 propane oxidative dehydrogenation, 355 propylene glycol, 509 protease, 1127 proteins, 517 pseudomonas aeruginosa, 279 pseudo-phase models, 1019 publishing, 959 pulses, 1505 pxrd, 209, 485 pyranopyrazole, 1265 pyrazole-based ligand, 867 pyrazolines, 305 pyridine-2,6-dicarboxylic acid, 1391 pyrolysis, 717 qsar, 187, 343, 499, 659, 1035 qtaim, 997 questionnaire, 959 quinazolinone, 1471 quinoline derivatives, 971 radical, 877 raman spectroscopy, 63 rat, 627 raw starch, 1375 reaction mechanism, 53, 1139, 1251 reactive black 5, 115 reactive blue 19, 115 reactive dye, 903 reactive orange 16, 903 reactivity indices, 1139 reactivity, 767 receptor model, 253 redox front formation, 947 refractive index, 933 regioselectivity, 1139 repellent activity, 465 reusable catalyst, 971 rhamnolipids, 279 rhodamine b, 779 ring opening, 1273 river sediments, 827 room temperature, 1391 roughness, 237 rsk inhibitors, 187 ruthenium sulphoxide complexes, 639 ruthenium, 639 rutin, 853 s12g, 1399 salicylaldehyde, 35 salmonella enterica, 315 saponins, 137 sapt, 997 satureja montana l., 475 sba-15, 1265 scanning electron microscopy, 107, 197 scavenger, 627 schiff base, 35 sds resistance, 613 secondary school, 1321 secondary structure, 613 sedimentary rocks, 827 sediments, 1087 seldi-tof ms, 159 semi-coordination, 45 sensor, 779 sepiolite, 1193 silkworm pupae oil, 9 silver, 107, 705, 779 simulated body fluid, 237 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 8 journal of the serbian chemical society simulation, 695 single crystal xrd, 1101 sitagliptin, 1311 skeletal vibrations, 329 sodium, 801 soil, 827,1087, 1127, 1203 sol–gel, 1061, 1515 solid surfactant drug delivery systems, 209 solid–solid reaction, 1391 solid-state fermentation, 1375 solubility properties, 917 solubility, 485, 509, 695, 1435 solution casting, 529 solvent effect, 651 solvent extraction, 179 solvent-free, 1265 solvothermal, 685 sorbent, 549 source contribution, 253 source profiles, 253 spacer, 639 spectrophotometry, 179 spectroscopy, 45 spin state relaxation, 1399 spin states, 1399 spiro indole, 1265 starch, 1279 statistical design, 1515 steiner distance, 1009 steiner–wiener index, 1009 stereoselectivity, 1139 steroids, 1481 storage capacity, 801 structural mechanics, 1449 sub-band gap illumination, 1411 sulfur dioxide, 549 sulphonation, 1435 sulphoxide, 639 supercooled liquid, 499 superparamagnetic catalyst, 971 supramolecular square, 1289 surface charge, 1113 svwgm, 343 synergism, 1411 synthesis, 1367 talcum powder, 563 tea co2 laser, 1505 tem, 367 tensile, 1177 tephrosia purpurea, 465 ternary alloy coatings, 73 ternary complexes, 651 tetrahydropyrimidines, 595 tetraoxanes, 1339 textbook quality, 1321 textbooks, 1321 tga, 209 theoretical study, 53, 1035 thermal analysis, 485, 1391 thermal inactivation, 613 thermodynamic, 509, 933 thiazole derivatives, 453 thienopyridines, 1251 thiepines, 839 thioamide, 97 thiocyanation, 453 thioethers, 731 thiosemicarbazide, 1471 three-component reaction, 453, 459 titanium dioxide, 705 titanium, 237 toluene, 1073, 1423 total enrichment factor, 827 total fibre, 21 total phenols, 21 toxicity, 627 toxoplasma gondii, 1339 trans-influence, 329 transition metal complex, 867 transition metal, 1297 transmission electron microscopy, 63 trisodium salt, 1193 triticale, 1279, 1375 trnovče, 947 trophoblast cells, 159 twinning, 107 two-fluid model, 377 unifac–visco parameters, 933 urease, 1127 urine, 1311 utilization, 575 uv protection, 705 vacancy defect, 1449 validation study, 1399 valproic acid, 627 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ volume 80: subject index 9 van der waals molecular surface, 659 vanadia loading, 355 vapour pressure, 499, 659 vasculopathies, 605 vibronic coupling, 877 viscosity, 933 voltammetry, 789, 1035 wastewater, 115 water supply, 947 water, 695 weathering, 827, 1087 wells, 947 wet milling, 367 wide band gap, 1411 wiener index, 1009 x-ray diffraction, 45, 197, 367, 1481 zeolite a, 779 zeolite, 1203 zinc(ii) complex, 87, 997 zinc(ii), 1289 zinc, 87, 997, 1149 zofenopril, 485 zsm-5, 717, 1297 α-amylase, 1279, 1375 β-nucleated, 223 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice structural effects of the monomer type on the properties of copolyimides and copolyimide–silica hybrid materials j. serb. chem. soc. 80 (8) 1061–1071 (2015) udc 678–13+533.583+544.773.42/.43+ jscs–4780 546–3:544.722.132 original scientific paper 1061 structural effects of the monomer type on the properties of copolyimides and copolyimide–silica hybrid materials canan kizilkaya, merve bicen, sevim karatas and atilla gungor* marmara university, faculty of science and letters, department of chemistry, 34722, istanbul, turkey (received 14 october 2014, revised 26 february, accepted 19 march 2015) abstract: in this work, the effects of two different diamine monomers containing phosphine oxide on the thermal, mechanical and morphological properties of copolyimides and their hybrid materials were investigated. the gas separation properties of the synthesized copolyimides were also analyzed. the two different diamine monomers containing phosphine oxide were bis(3-aminophenyl]phenylphosphine oxide (bappo) and bis[4-(3-aminophenoxy)phenyl)phenylphosphine oxide (m-bappo). in the synthesis of the copolyimides, 3,3′-diaminodiphenyl sulfone (3,3′-dds) was also used as the diamine, as well as 2,2-bis(3,4-dicarboxyphenyl)hexafluoropropane dianhydride (6fda). copolyimide films were prepared by thermal imidization. furthermore, hybrid materials containing 5 % sio2 were synthesized by the sol–gel technique. fourier -transform infrared spectroscopy (ftir) and nuclear magnetic resonance spectroscopy (nmr) confirmed the expected structures. dynamic mechanical analysis (dma) demonstrated that the m-bappo-based copolyimides had lower glass transition temperatures (tg) than the bappo-based ones. the thermal decomposition temperature of the m-bappo-containing copolyimide without silica was shifted to a higher value. the moduli and strength values of the bappo diamine-containing copolyimide and its hybrid were higher than those of the m-bappo-containing materials. contact angle measurements showed their hydrophobicity. scanning electron microscope (sem) analysis showed the dispersion of the silica particles in the copolyimides. these copolyimides may be used in the coating industry. the co2 permeability and the permselectivity were the highest, among the other values found in this study, when the m-bappo-containing copolyimide in the absence of silica was used. the gas permeabilities obtained from this work were in the decreasing order: pco2 > > po2 > pn2. keywords: copolyamic acid; inorganic compound; sol–gel; imidization; hydrophobic nature; permeation. * corresponding author. e-mail: atillag_1@yahoo.com doi: 10.2298/jsc141014029k _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1062 kizilkaya et al. introduction polyimides (pis) exhibit outstanding characteristics such as high tensile modulus, high thermal stability and solvent resistance. they find applications ranging from aerospace,1 microelectronic devices, and the coating industry to separation membrane technologies.2 the thermal and mechanical properties of pis can be further improved by the introduction of silica into the pi matrix, forming hybrid organic–inorganic materials. these materials can be prepared utilizing the sol–gel process to form silica in a polyamic acid solution. films are formed from the solution by solvent casting.3 the silica nanoparticles are formed in the pi matrix via hydrolysis and polycondensation of organic silanes.4 the unique characteristics of fluorine, such as high electronegativity, low polarity, low cohesive and surface free energy, gave rise to attempts to fluorinate pis in 1972. since then, fluorine-containing diamines and dianhydrides have been introduced to prepare fluorinated pis. however, fluorinated pis have drawbacks, such as poor adhesion and low mechanical strength. phosphine oxide-containing polyimides, which show excellent adhesive properties with excellent thermal stability, enabled fluorinated pis to exhibit improved properties.5 much research has been performed on pis to investigate the relationship between chemical structure and gas transport properties. 2,2-bis(3,4-dicarboxyphenyl)hexafluoropropane dianhydride (6fda)-based pis show high gas permeability. the presence of the bulky –c(cf3)2– group provides restricted intrasegmental mobility and stiffened backbones. moreover, some of 6fda-based pis were employed to fabricate high performance membranes.6 in the present work, the effects of two different diamine monomers containing phosphine oxide on the thermal, mechanical and morphological properties of copolyimides and their hybrid materials were investigated. the gas separation properties of the synthesized copolyimides were also analyzed. the two diamine monomers with phosphine oxide were bis(3-aminophenyl)phenylphosphine oxide (bappo) and bis[4-(3-aminophenoxy)phenyl]phenylphosphine oxide (m -bappo). in the synthesis of the copolyimides, 3,3′-diaminodiphenyl sulfone (3,3′-dds) was also used as the diamine, as well as 2,2-bis(3,4-dicarboxyphenyl)hexafluoropropane dianhydride (6fda). copolyimide films were prepared by thermal imidization. furthermore, hybrid materials containing 5 % sio2 were synthesized by the sol–gel technique. experimental materials the dianhydride monomer, 2,2-bis(3,4-dicarboxyphenyl)hexafluoropropane dianhydride (6fda, >99 %) was purchased from aldrich and used as received. 3,3′-diaminodiphenyl sulfone (3,3′-dds), phenylphosphonic dichloride (98 %), n-methyl-2-pyrrolidone (nmp) and dimethylacetamide (dmac) were obtained from merck. nmp and dmac were dried over _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ properties of copolyimides and hybrids 1063 phosphorus pentoxide (p2o5) and freshly distilled under vacuum before use. 1-bromo-4 -fluorobenzene (99 %), mg powder (99.9 %), anhydrous potassium carbonate (k2co3, 99.9 %) and 3-aminophenol were provided by aldrich and used as received. tetrahydrofuran (thf) was freshly distilled under nitrogen over sodium. tetraethyl orthosilicate (teos) and 3-(glycidyloxy)propyltrimethoxysilane (gptms) were purchased from merck. some common solvents, such as chloroform, ethanol, methanol and dichloroethane, were used as received. synthesis of diamine monomers in this study, two different diamine monomers (bappo and m-bappo) were synthesized. bis(3-aminophenyl)phenylphosphine oxide (bappo) was synthesized by a three-step reaction as mentioned in the previous work.7 first, triphenylphosphine (tpp) was oxidized to triphenylphosphine oxide (tppo) by treating with h2o2. then, bis(3-nitrophenyl)phenylphosphine oxide (bnppo) was prepared by the nitration of tppo using concentrated nitric acid in the presence of sulfuric acid. finally, bnppo was subjected to hydrogenation in a high-pressure reactor (parr instrument co.) to produce bappo. bis[4-(3-aminophenoxy)phenyl]phenylphosphine oxide (m-bappo) was synthesized as described in the literature.8 first, bis(4-fluorophenyl)phenylphosphine oxide (bfppo) was synthesized by treating 1-bromo-4-fluorobenzene with phenylphosphonic dichloride in the presence of mg turnings via the grignard technique. then, m-bappo (scheme s-1 of the supplementary material to this paper) was prepared by treating 37.23 g (0.118 mol) bfppo with 26.56 g (0.243 mol) 3-aminophenol in a dmac/toluene mixture using 40.20 g (0.292 mol) k2co3 as a weak base to form the required aminophenolate nucleophile. preparation of copolyamic acid solution the copolyamic acid solution (paa) used as a copolyimide precursor was prepared in nmp as follows: the diamine monomers and dried nmp were charged into a three-necked flask equipped with a nitrogen inlet and a condenser. then, equimolar amount of the dianhydride monomers were incrementally added into the content of the flask. the concentration afforded was 20 %. the reaction mixture was stirred overnight at room temperature to obtain a viscous paa solution. in this study, 6fda-based copolyimides were synthesized from the aromatic diamines 3,3′-dds, bappo and m-bappo. silica sol preparation silica sol was prepared by the hydrolysis and condensation of teos as follows: teos (8.7 g, 0.042 mol), gotms (2.25 g, 0.010 mol) and etoh (2.21 g, 0.048 mol) were charged into a glass vial and then distilled water (1.77 g, 0.098 mol) that had been acidified by adding hcl was slowly dropped into the vial. the mixture was magnetically stirred at room temperature until a clear solution was obtained. then, the silica sol was kept at room temperature for about 1 h. preparation of copolyimide–silica hybrid materials to obtain the hybrid solution, 5 % silica sol was added dropwise into a paa solution and stirred continuously at room temperature for 4 h. this preparation procedure is illustrated in scheme s-2 of the supplementary material. after thorough stirring, the clear and viscous hybrid solutions were cast onto glass plates using a 30-μm wire gaged applicator and then thermal imidization was performed stepwise at 80, 100, 150, 200 and 300 °c for 1 h at each temperature. after the imidization was completed, the glass plates were immersed in hot water (90 °c) for 1 h to remove easily the hybrid films from the glass surfaces. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1064 kizilkaya et al. characterization the ftir spectra were recorded on a perkinelmer spectrum 100 atr ftir spectrophotometer. the nmr spectra were recorded on a varian 600 mhz spectrometer operating for 1h-nmr and 31p-nmr using cdcl3 as the solvent. thermogravimetric analyses (tga) of the hybrid free films were performed using a perkin elmer thermogravimetric analyzer pyris 1 tga model. samples were run from 30 °c to 750 °c at a heating rate of 10 °c min-1 under an air atmosphere. the glass transition temperatures of free films prepared as 10 mm×20 mm samples were obtained from an sii nanotechnology exstar 6000 model dynamic mechanical analyzer (dma). the scans were obtained at a heating rate of 5 °c min-1, from 30 to 450 °c. the limiting oxygen index (loi) values of the free hybrid films were measured using a fire testing technology (ftt) type instrument according to astm d2863-08. the mechanical properties of the free films were determined by standard tensile stress–strain tests in order to measure the moduli, tensile strength, and elongation at break. standard tensile stress–strain experiments were performed at room temperature on a material testing machine z010/tn2s, using a crosshead speed of 5 mm min-1. in order to determine the hydrophobic properties of the materials, contact angle measurements were performed with a kruss (easy drop dsa-2) tensiometer, equipped with a camera. analyses were made at room temperature by means of the sessile drop technique. for each sample, at least four measurements were made, and the average was taken. the measuring liquid was distilled water. to determine the morphological behavior, secondary electron images (sei) were applied in sems. sem imaging of the films were performed on a philips xl30 esem-feg/edax. the specimens were prepared for sem by freeze fracturing in liquid nitrogen and applying a gold coating of approximately 300 å. an sem–energy-dispersive x-ray spectroscopy (sem-eds) spectrum was recorded to verify the presence of silica in the copolyimide–silica hybrid. in addition, a gas permeation analyzer (brugger gdp-c 2000) was used to measure the pure gas permeability coefficients of the polymeric membranes to o2, n2, and co2. the pure gas permeability coefficients were measured using the constant volume and variable pressure method. the experiments were performed at 35 °c with atmospheric feed-side pressure. the samples were masked for gas permeation measurement following the technique of the koros group9 with a permeation area of 3.94 cm2. results and discussion the aim of this study was to investigate the effect of diamine monomers containing phosphine oxide on the thermal, mechanical and morphological properties of copolyimides and their hybrid materials, as well as the gas separation properties. two different diamine monomers were synthesized (bappo and m-bappo). the bappo monomer was synthesized according to a previous study.7 the synthesis route for the other monomer (m-bappo) is illustrated in scheme s-1 of the supplementary material. the chemical structure of m-bappo was identified by ftir, 1h-nmr and 31p-nmr spectroscopy. the ftir results for m-bappo (shown in fig. s-1 of the supplementary material) displayed aromatic c–h stretching at 3058 cm–1, c–h out-of-plane bending at 830 cm–1, c–h in-plane bending at 1143 and 1115 cm–1, aromatic c–c stretching at 1577 and 1486 cm–1, asymmetric c–o–c stretching at 1232 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ properties of copolyimides and hybrids 1065 cm–1, p=o stretching at 1170 cm–1, p–aryl stretching at 1436 and 996 cm–1, n–h stretching at 3445,3324 and 1594 cm–1, and c–n stretching at 1283 cm–1. in the 1h-nmr spectrum (fig. s-2 of the supplementary material) of m-bappo, proton peaks arising from the ether and amino groups were observed up-field because of the shielding effects of the electron-donating ether and amino moieties. as could be seen in fig. s-2, the protons in the amino groups appeared at 3.4–3.8 ppm as a broad peak. in addition, the other proton peaks of m-bappo were: 7.71–7.40 ppm (5h, m, o=p–c6h5,), 6.80–7.20 ppm (4h, m, o=p–c6h4–o–), 6.60–6.37 ppm (4h, m, –o–c6h4–nh2). as shown in fig. s-3 of the supplementary material, the 31p-nmr analysis provided a sharp single peak at 29 ppm for m-bappo. in this study, bappo–3,3′-dds/6fda, m-bappo–3,3′-dds/6fda copolyimides and their hybrids were prepared. the hybrid materials containing the polyamic acid (paa) solution and silica sol, which was obtained in a sol–gel reaction, were synthesized. teos and gptms acted as an inorganic networker and a coupling agent, respectively. in addition, hcl acid acted as the acid catalyst for the partial hydrolysis. the compositions of the pi–sio2 hybrids are presented in table i. hybrid formulations applied on glass plates were cured by stepwise heating at elevated temperatures. the chemical structures of the copolyimides and copolyimide–silica hybrids were shown in scheme s-3a–d of the supplementary material. the ftir spectra of the copolyimides and their hybrid copolyimides containing 5 % silica are shown in fig. s-4a–d of the supplementary material. as seen in fig. s-4, characteristic imide absorptions at 1783 and 1720 cm–1 (typical of imide carbonyl asymmetrical and symmetrical stretching, respectively) were present. the peaks at 1363 and 718 cm–1 indicated c–n–c stretching and imide ring deformation. moreover, the peaks at 1203, 1300 and 1100 cm–1 were ascribed to c–f multiple stretching bands. as shown in figs. s-4b and d, when the inorganic components were introduced into the polyimide matrix, strong absorption bands were observed in the range 1000 to 1100 cm–1 and at 423 cm–1. these bands were ascribed to the characteristic si–o–si stretching vibration and bending vibration, respectively.10,11 the spectra given in fig. table i. the composition (mass, g) of copolyimide–silica hybrid films; the paa solution was 20 wt. %. the paa solution used in the experiment was 3 g paa/15 ml solvent. this means that the paa in solid form was 3 g after removal of the solvent; silica mass: 0.15 g of silica sol was added. therefore, the silica solution used in the experiment was 0.15 g silica/3 ml solution (5 wt. %); 6fda mass: 1.777 g; 3,3′-dds mass: 0.4966 g sample bappo m-bappo silica sol used bappo–3,3′-dds/6fda 0.6160 0 0 m-bappo–3,3′-dds/6fda 0 0.9850 0 bappo–3,3′-dds/6fda/sio2 0.6160 0 0.15 m-bappo–3,3′-dds/6fda/sio2 0 0.9850 0.15 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1066 kizilkaya et al. s-4 of the supplementary material confirmed the formation of the expected structures. as seen in tables ii and iii, thermal properties of the copolyimides and hybrid copolyimides were evaluated by dynamic mechanical analysis (dma) and thermogravimetric analysis (tga). the storage modulus (e′, fig. s-5a–d of the supplementary material) and tan δ (figs. s-6–s-9 of the supplementary material) were plotted. the values of the glass transition temperatures of copolyimides and hybrid copolyimides are very important for the determination of the optimum processing and service temperatures at which the polymer preserves its desirable properties.12,13 the tg of the copolyimides increased when silica was introduced into the pi matrix. this result indicated that the coupling agent improved the interaction between the polymer matrix and the inorganic segment, which caused an increased restricting strength of silica on pi and hence an increased tg of the material. the data were collected by dma. due to the flexible etheric linkage of m-bappo, the obtained tg values were lower than those of the bappo-containing copolyimides and their hybrids. however, bappo based copolyimides exhibited higher elongation at break values than those of the m-bappo-based copolyimides. this result is likely due to the three dimensional geometry of the monomers (bappo and m-bappo). the monomer bappo alone is a tetrahedral structure so that the aminophenyl groups are in approximately 110° tetrahedral geometry. therefore, the amino groups can be in different directions when reacted with the dianhydride. the obtained copolyimide might be an asymmetric structure. on the other hand, the obtained copolyimide might be a symmetric structure (more symmetric than the bappo-based one) when the m-bappo moiety reacted with the dianhydride. the aminophenyl groups of the monomer m-bappo are in trigonal (approximately 120°) geometry, due to the etheric linkages. trigonal structures are more symmetric than tetrahedral systems. when the asymmetry exits throughout the molecular structure, the asymmetric structure shows greater elasticity (greater elongation at break). easier molecular packing properties can be observed in the presence of symmetric structures. two or three tg values were obtained due to small amounts of phase separation. this might have been possible, since the reactivities of the diamines (bappo, m-bappo and 3,3′-dds) are different when they react with the dianhydride (6fda). the introduction of the inorganic material can also cause phase separation due to differences in the reactivity. table ii. glass transition temperatures of the copolyimides and hybrid copolyimides sample t1 / °c t2 / °c t3 / °c bappo–3,3′-dds/6fda 218 272 301 m-bappo–3,3′-dds/6fda 209 245 – bappo–3,3′-dds/6fda/sio2 217 274 323 m-bappo–3,3′-dds/6fda/sio2 220 257 – _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ properties of copolyimides and hybrids 1067 table iii. thermal properties of the copolyimides and hybrid copolyimides sample 1st decomposition temperature, t1 / °c 2nd decomposition temperature, t2 / °c char wt. % loi wt. % bappo–3,3′-dds/6fda 319 523 20 46.0 m-bappo–3,3′-dds/6fda 310 537 28 48.0 bappo–3,3′-dds/6fda/sio2 350 525 15 46.7 m-bappo–3,3′-dds/6fda/sio2 330 509 7 47.4 the thermal degradation behaviors of the copolyimides and hybrid copolyimides are summarized in table iii. the polymers exhibited first weight loss at temperatures between 310 and 350 °c, which could probably be attributed to incomplete imidization. the second weight loss started between 509 and 537 °c, which corresponded to the degradation of the polymer. the incorporation of silica into the polyimide matrix may cause the production of sif4(g) in the presence of –cf3– present in the 6fda moiety.14 the char yields of the copolyimide silica hybrid materials between 7–28 % were found. the thermal decomposition temperature of the hybrids shifted to lower values because of the removal of silica as sif4(g). it was found that the thermal decomposition temperature of the m-bappo-containing copolyimide without silica was shifted to a higher value, increasing the char yield. moreover, the flame retardant properties of the copolyimide and hybrid copolyimide films were evaluated by measuring their limiting oxygen index (loi) values. from table iii, it can be seen that the loi values for the copolyimides were changed only slightly on addition of the silica particles into the pi matrix. the tensile properties of copolyimide films were examined by stress–strain measurements and the results were summarized in table iv. the moduli and the strength values of bappo diamine-containing copolyimide and its hybrids were higher than those of the m-bappo-containing materials. this situation was explained by higher free volume of m-bappo because of the extra phenyl group that resulted in a lowering of the moduli. in other words, the more space the bulky groups take, the lower are the observed moduli. compared to the neat copolyimides, a decrease in mechanical properties was observed when silica was introduced into the copolyimide matrix. this result may be due to the increased crosslinking density by the formation of an organic–inorganic network structure. table iv. physical and mechanical properties of the copolyimides and hybrid copolyimides samples modulus, gpa, at 40 °c tensile strength, mpa elongation at break, % contact angle θ / ° bappo–3,3′-dds/6fda 3.54 518 3.92 93 m-bappo–3,3′-dds/6fda 3.12 226 1.62 102 bappo–3,3′-dds/6fda/sio2 3.36 267 3.32 97 m-bappo–3,3′-dds/6fda/sio2 2.95 189 1.98 107 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1068 kizilkaya et al. contact angle measurements provide information about the hydrophobicity and hydrophilicity of a material surface. these measurements were performed with drops of distilled water at four different areas on the sample. the averages of these four measurements were calculated. the obtained contact angle values, for each material were given in table iv. the contact angles had a tendency to increase on addition of silica, demonstrating a more hydrophobic coating surface. the presence of –c(cf3)2– also provided for a more hydrophobic surface. sem analysis (fig. s-10a–d of the supplementary material) exhibited that the silica particles were dispersed in the copolyimide matrix. a microphase separation was observed in the m-bappo-containing copolyimide–silica hybrid. sem–energy-dispersive x-ray spectroscopy (sem–eds, fig. s-10e of the supplementary material) verified the presence of silica in the copolyimide–silica hybrids. in addition to the thermal, mechanical and morphological studies, the gas permeability coefficients and permselectivity data measured at 35 °c are reported in table v. the gas permeabilities and permselectivities can be calculated as follows: p ds= (1) αa/b = pa/pb (2) where p is the permeability coefficient, d is the diffusion coefficient and s is solubility coefficient. α represents the permselectivities of gases a and b.6 the order of the kinetic (å) diameters of the studied gases is co2, 3.3; o2, 3.46 and n2, 3.64. the gas permeabilities from this study were in the decreasing order: 2 2 2co o n .p p p> > thus, the larger is the kinetic diameter of a gas, the lower is its permeability. as seen in table v, the m-bappo-containing copolyimide without silica was better than the others in terms of the permeabilities of o2 and co2, whereas the bappo-containing copolyimide in the presence of silica was better than others for the permeability of n2. the introduction of silica increased the volume of the copolymer due to the effect of crosslinking so that the n2 gas, possessing the largest kinetic diameter, could easily diffuse throughout the table v. permeability coefficients and permselectivity of the copolyimides and hybrid copolyimides at 35 °c sample permeability coefficients permselectivities 2o p /barrier 2np /barrier 2cop /barrier 2 2o / na 2 2co / na bappo–3,3′-dds/6fda 0.310 0.046 0.365 6.74 7.93 m-bappo–3,3′-dds/6fda 0.405 0.084 1.137 4.82 13.54 bappo–3,3′-dds/6fda/ /sio2 0.229 0.104 0.232 2.20 2.23 m-bappo–3,3′-dds/ /6fda/sio2 0.165 0.093 0.366 1.78 3.94 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ properties of copolyimides and hybrids 1069 bappo-containing hybrid matrix. due to the increased volumes, the selectivities were adversely affected. the reason is that all gases can pass through without determining their diffusion rates in the presence of large volumes. since co2 is a condensable gas, it should have a higher permeability value than those of the other two gases. it is too difficult to obtain the highest permeability as well as the highest selectivity. as seen in table v, the permeability and permselectivity of co2 were the highest among the values for the copolyimides and copolyimide– silica hybrids in this study, when the m-bappo containing copolyimide in the absence of silica was used. conclusions in this study, copolyamic acid solutions (paas) were obtained by the reaction between m-bappo or bappo, dds and 6fda. copolyimide films were prepared by thermal (bulk) imidization. hybrid materials containing 5 % sio2 were synthesized by the sol–gel technique. the ftir, 31p-nmr and 1h-nmr spectra confirmed the expected structures. dma analysis showed that the m-bappo-based copolyimides had lower tg values than those of the bappobased copolyimides. tg of copolyimides increased when silica was introduced into the copolyimide matrix. thermal analysis showed that in the presence of silica, the weight loss was shifted to lower temperatures because of the removal of silica as sif4(g), compared to the neat copolyimide. however, the thermal stability was sufficiently high compared to that of branched-silica hybrid membranes.15 the loi results did not change significantly on addition of silica particles into the matrix. mechanical tests showed a decrease in mechanical properties on introduction of silica into the copolyimide matrix, compared to the neat copolyimides. this result may be due to the increased crosslinking density caused by the formation of organic–inorganic network structures. however, the moduli of the hybrids could be considered good.16 contact angle measurements confirmed the hydrophobic nature of the surface of the coating. sem analyses confirmed that silica particles are able to disperse in the polyimide matrix. these copolyimides with various properties may be used in the coating industry. in addition to the thermal, mechanical and morphological studies, gas permeability coefficients and permselectivity measurements were also performed. the condensable gas (i.e., co2) was observed to have higher permeability value than those of other two examined gases (i.e., n2 and o2). this could be explained by the plasticization effect. more soluble penetrants induce significant plasticization. the co2 permeability and the selectivity were the highest among the values of copolyimides and copolyimide–silica hybrids in this study when the m-bappo -containing copolyimide in the absence of silica was used. the gas permeabilities obtained from this work were in the decreasing order: 2 2 2co o n .p p p> > _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1070 kizilkaya et al. supplementary material ftir, 1h-nmr and 31p-nmr spectra, sem images, storage moduli and tan δ values, as well as the synthesis routes and structures of the samples, are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. и з в о д утицај структуре мономера на својства кополиимида и кополиимид/силицијум-диоксид хибридних материјала canan kizilkaya, merve bicen, sevim karatas и atilla gungor marmara university, faculty of science and letters, department of chemistry, 34722, istanbul, turkey у овом раду је приказан утицај структуре мономера, тј. два различита диамина на бази фосфорин-оксида, на термичка, механичка и морфолошка својства синтетисаних кополиимида и њихових хибридних материјала. поред тога, анализирана је способност синтетисаних кополиимида за раздвајање гасова. два различита мономера односно диамина на бази фосфор-оксида су коришћена: бис(3-аминофенил)фенилфосфин-оксид (bappo) и бис[4-(3-аминофенокси)фенил]фенилфосфорин-оксид (m-bappo). у синтезама кополиимида 3,3'-диаминодифенил-сулфон (3,3'-dds) је коришћен као диамин, а такође и 2,2-бис(3,4-дикарбоксифенил)хексафлуорпропан-дианхидрид (6fda). кополиимидни филмови су добијени термичком имидизацијом у другом ступњу реакције поликондензације. хибридни материјали са 5 % sio2 су синтетисани користећи сол–гел поступак. стуктура синтетисаних кополиимида је потврђена ftir и nmr спектроскопијом. динамичка механичка анализа (dma) је показала да кополиимиди на бази mbappo имају ниже температуре остакљивања (tg) у односу на bappo кополиимиде. m-bappo кополиимиди без sio2 испољавају бољу термичку стабилност. модули и затезна чврстоћа кополиимида на бази bappo диамина као и њихових хибридних материјала била је већа у односу на m-bappo аналогне материјале. мерења контактних углова су потврдила хидрофобност њихове површине. скенирајућа електронска микроскопија (sem) је показала диспергованост силицијум-диоксидних честица у кополиимидној матрици. синтетисани кополиимидни материјали могу се потенцијално користити у индустрији премаза. највећу пропустљивост према co2 и највећу селективну пропустљивост према гасовима је показао кополиимид на бази m-bappo без присуства честица sio2. пропустљивост према гасовима материјала, приказаних у овом раду, следила je опадајући тренд: 2 .> > 2 2co o n p p p (примљено 14. октобра 2014, ревидирано 26. фебруара, прихваћено 19. марта 2015) references 1. . y. k. yang, w. j. koros, h. b. hopfenberg, v. t. stannet, j. appl. polym. sci. 31 (1986) 1619 2. d. hofman, j. ulbrich, d. fritsch, d. paul, polymer 37 (1996) 4773 3. c. joly, s. goizet, j. c. schrotter, j. sanchez, m. escoubes, j. membr. sci. 130 (1997) 63 4. y. w. wang, c. t. yen, w. c. chen, polymer 46 (2005) 6959 5. k. u. jeong, j. j. kim, t. h. yoon, polymer 42 (2001) 6019 6. l. wang, y. cao, m. zhou, s. j. zhou, q. yuan, j. membr. sci. 305 (2007) 338 7. c. kızılkaya, s. karataş, n. k. apohan, a. güngör, j. appl. polym. sci. 115 (2009) 3256 8. a. gungor, c. smith, j. m. wescott, s. srinivasan, j. e. mcgrath, polym. prepr. (am. chem. soc., div. polym. chem.) 32 (1991) 172 9. t. t. moore, s. damle, p. williams, w. j. koros, j. membr. sci. 245 (2004) 227 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ properties of copolyimides and hybrids 1071 10. x. fang, z. wang, z. yang, l. gao, q. li, m. ding, polymer 44 (2003) 2641 11. s. xiao, x. feng, y. robert, m. huang, macromol. chem. phys. 208 (2007) 2665 12. a. y. ardashnikov, i. y. kardash, a. n. pravednikov, polym. sci. ussr 13 (1971) 2092 13. w. volksen, p. m. cotts, in polyimides: synthesis, characterization and applications, vol. 1, k. l. mittal, ed., plenum, new york, 1984, p. 163 14. c. hibshman, c. j. cornelius, e. marand, j. membr. sci. 211 (2003) 25 15. m. miki, t. suzuki, y. yamada, j. appl. polym. sci. 130 (2013) 54 16. a. c. lua, y. shen, chem. eng. j. 20 (2013) 441. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice hollow-fiber micro-extraction combined with hplc for the determination of sitagliptin in urine samples j. serb. chem. soc. 80 (10) 1311–1320 (2015) udc 543:615.2/.3:616.379–008.64:612.461: jscs–4799 43.544.5.068.7:543.544–14 original scientific paper 1311 hollow-fiber micro-extraction combined with hplc for the determination of sitagliptin in urine samples raheme rezaee, mahnaz qomi* and foroozan piroozi medicinal chemistry department, pharmaceutical sciences research center, pharmaceutical sciences branch, islamic azad university, tehran, iran (received 27 december 2014, revised 14 may, accepted 24 may 2015) abstract: in this study, a three-phase, hollow-fiber, liquid-phase micro-extraction (hf–lpme) method coupled with high performance liquid chromatography was successfully developed for the determination of trace levels of the anti-diabetic drug sitagliptin (stg) in urine samples. sitagliptin was extracted from 15 ml of the basic sample solution with a ph of 8.5 into an organic extracting solvent (n-octanol) impregnated in the pores of a hollow fiber and then back extracted into an acidified aqueous solution in the lumen of the hollow fiber with a ph of 3. after extraction, 20 µl of the acceptor phase was injected into hplc system. in order to obtain high extraction efficiency, the parameters affecting the hf–lpme, including ph of the source and receiving phases, type of organic phase, ionic strength, stirring rate, extraction time, the volume ratio of donor phase to acceptor phase and temperature, were studied and optimized. under the optimized conditions, enrichment factors up to 88 were achieved and the relative standard deviation of the method was in the range from 3 to 6 %. the results indicated that the hf–lpme method has an excellent clean-up capacity and a high pre-concentration factor and could serve as a simple and sensitive method for monitoring the drug in urine samples. keywords: anti-diabetic drug; drug analysis; chromatography; biological sample; pre-concentration. introduction type 2 diabetes mellitus is a common chronic condition that causes significant morbidity and mortality worldwide and is a growing global public health concern. sitagliptin (stg), (r)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8h)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine, is an orally active, potent and selective inhibitor of dipeptidylpeptidase-4 (dpp-4 inhibitor), for the treatment of type 2 diabetes.1,2 stg was the first dpp-4 inhibitor to gain * corresponding author. e-mail: qomi@iaups.ac.ir doi: 10.2298/jsc141227046r _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1312 rezaee, qomi and piroozi the approval of the food and drug administration (fda) in october 2006 for both monotherapy and in combination with other anti-diabetic agents for the treatment of type 2 diabetes mellitus.3 following oral administration of a 100-mg tablet, the absolute bioavailability is approximately 87 % and therapeutic concentration (cmax) is 950 nmol l–1 in healthy individuals. eighty seven percent of stg is eliminated via renal excretion (79 % unchanged) with a renal clearance in the region of 350 ml min–1.1,2,4 several liquid chromatographic methods with ultraviolet and mass spectrometric detection have been reported in the literature for the quantitative determination of stg in biological fluids. techniques utilizing hplc alone do not have an adequate lower limit of quantification (lloq) suitable for monitoring sitagliptin in low concentrations.5 recent lc/ms/ms methods showed improvement in sensitivity, however, a mass spectrometer is very expensive.6,7 therefore, due to the low concentration of sitagliptin in biological samples, a pretreatment and pre-concentration step is generally required prior to determination of trace amounts of the drug. various sample preparation procedures have been applied for stg in biological fluids, such as liquid–liquid extraction (lle) and solid phase extraction (spe).5,8 the liquid micro extraction method is a relatively new method for sample preparation. this method has some advantages over conventional extraction methods such as lle and spe.9–11 in 1999, pedersen -bjergaard and rasmussen suggested using porous hollow polypropylene fibers as a retainer to maintain the extraction phase, a method known as hollow fiber liquid phase micro extraction (hf–lpme).12,13 hf–lpme can be performed in two or three micro-extraction phase modes. the three phases involved in the extraction are the analyte solution (donor), an organic phase and second aqueous phase into which the extraction is conducted (acceptor phase). during the extraction, the desired analyte under proper conditions is first transferred into the organic phase and then into the acceptor phase. the rate of conducting the extraction depends on the rate of mass transfer between the two interfaces of the donor/organic phase and the organic/acceptor phase. the main reported limitation of this method is that it is only applicable for species which are capable of ionization.14,15 in the present study, a simple, inexpensive and sensitive three-phase hollow fiber micro-extraction technique combined with high performance liquid chromatography-ultraviolet detection (hplc– –uv) is described for the extraction and determination of stg in urine samples. experimental chemicals and materials sitagliptin reference standard was kindly donated by the food and drug organization (tehran, iran). a stock solution containing 200 µg ml-1 of sitagliptin was prepared in hplc grade methanol and stored at 4 °c. working solutions (from 2 to 300 µg l-1) were freshly prepared in hplc grade water by dilution of the stock solution and filtered using a millipore _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ determination of sitagliptin in urine 1313 filter (0.45 µm) each day prior to use. all chemicals were of analytical reagent grade unless otherwise stated. the urine samples were obtained from the taleghani clinic (tehran, iran). the samples were diluted using deionized water and were used for method development and calibration. the working standards for real sample analysis were prepared by spiking stg to 15 ml of urine samples, which were diluted 1:1 with ultra-pure water. apparatus and chromatographic conditions the chromatographic separations were realized on a youngling hplc instrument equipped with a yl 9110 quaternary hplc pump, a 20 µl sample loop and yl 9120 uv-vis detector. the chromatographic data were collected and analyzed using a younglin autochro3000. the separations were performed on a c18 column (150 mm×4.6 mm, with 5 µm particle size) from teknokroma (barcelona, spain). a mixture of methanol and 0.1 % perchloric acid (40:60) at a flow rate of 1.0 ml min-1 was used as the mobile phase. the injection volume was 20 µl for all the standards and samples, and the detection was performed at a wavelength of 268 nm. the ph measurements were performed using a gphr 1400a ph meter (berlin, germany). a magnetic stirrer model mr hei-standard (heidolph company, germany) and a 25 µl syringe model 702 nr from hamilton (bonaduz, switzerland) were also used. the ultrapure water was from a milliq ultrapure water purification system (millipore, bedford, ma, usa). all extractions were realized using a q3/2 accrual polypropylene hollow fiber membrane from membrana (wuppertal, germany) with a 0.2 µm pore size, 600 µm internal diameter and 200 µm wall thickness. extraction procedure initially, the polypropylene fibers were cut to the size of 8.5 cm (the volume of these fibers was approximately 22 µl). the fibers were completely washed with acetone in an ultrasonic bath for 10 min and then dried at room temperature before use for extraction. in order to avoid any possible memory effects, a new fiber was used for each extraction. in the extraction procedure, 15 ml of the sample solution with a known concentration and alkaline ph was transferred to a 20-ml sample vial containing a magnetic stirring bar. the sample vial was placed on a heidolph mr 3001 k magnetic stirrer. thirty µl of an acceptor phase with an acidic ph was withdrawn by a 25 µl hamilton micro-syringe and the syringe needle was inserted into the hollow fiber segment. the fiber was immersed in an organic solvent for 10 s (n-octanol) to impregnate the fiber pores with the organic solvent. the fiber was then placed in distilled water for 30 s in order to wash the extra solvent from its surface. the acceptor phase inside the syringe was injected into the lumen of the fiber. the end of the fiber was blocked by a piece of aluminum foil. the u-shaped fiber was placed in the sample vial. the sample solution was agitated at 1000 rpm. at the end of the extraction (after 50 min), the fiber was removed from the sample solution and the acceptor phase was then retracted back into the syringe. finally, the extraction phase (20 µl) was injected directly into the hplc system in order to analyze the drug. in 2006, pawliszyn and co-workers successfully automated the hf–lpme technique with a ctc combipal autosampler, and a new device was designed for the automation of hf–lpme in this study.16 results and discussion in this study, a three-phase hollow-fiber micro-extraction technique combined with hplc–uv was used for the extraction and determination of stg in biological samples. furthermore, the effect of different experimental parameters on the efficiency of extraction, such as the type of organic solvent, the ph of the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1314 rezaee, qomi and piroozi donor and acceptor phases, the stirring time, the ionic strength of the donor phase, temperature, the stirring rate and the volume ratio of donor phase to acceptor phase were all investigated and optimized to obtain the best analytical performance. type of organic solvent it is important to select a suitable solvent in order to achieve maximum extraction, good sensitivity, selectivity and high accuracy. the chosen solvent should enter easily into the fiber pores, be non-toxic, and ultimately have a high boiling point (be non-volatile) and a proper viscosity to be stable during the extraction process.10,17,18 in order to select a proper solvent, several solvents (n-octanol, n-octane, 1-decanol, 4-methyl-2-pentanone and benzyl alcohol) were examined. the highest extraction efficiency was obtained using n-octanol (fig. 1). fig. 1. the effect of organic solvent on hf–lpme efficiency in the determination of sitagliptin. ph of acceptor phase and donor phase the ph values of the donor and acceptor solutions are important factors in a three-phase lpme. for ionizable analytes, acid–base dissociation is the most common reaction utilized to facilitate the extraction of the analyte from the donor to the acceptor phase.18–20 the ph difference between the donor and acceptor phases can promote the transfer of analytes. for practical applications, the ph should differ from the pka value of the analyte (the pka of stg is 7.7) by at least 3 units.21 to promote extraction, the ph of the donor solution was adjusted in order to deionize the analyte. therefore, the effect of the donor phase ph on the extraction efficiency was studied by changing the sample ph from 8.0 to 12.5. the maximum efficiency of the analyte extraction was achieved at ph 10.5. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ determination of sitagliptin in urine 1315 the effect of the ph of the acceptor phase from 2.0 to 4.0 was studied. at ph of 3, the extraction rate increased because stg had an ionic structure at this ph and transferred easily into the aqueous phase. accordingly, ph values of 10.5 and 3 were selected as optimum values for the donor and acceptor phases respectively. stirring rate of analyte solution stirring the sample during extraction reduces the thermodynamic equilibrium time and increases the extraction efficiency as stirring provides a fresh donor solution for the organic phase to extract and enhances analyte transport from the donor phase to the organic solvent. to evaluate the effect of sample stirring, the aqueous donor sample was stirred for 50 min at different rates (from 250 to 1200 rpm). the efficiency of extraction increased with increasing stirring speed. however, at 1200 rpm there was a decrease in the extraction efficiency, probably due to the high speed creating air bubbles on the surface of the hollow fiber that ultimately leads to poorer precision and possible failure of the experiment.10,22,23 finally, a stirring speed of 1000 rpm was adopted for the subsequent experiments. ionic strength of the donor phase in this stage, the effect of adding salt to the donor phase on the extraction rate was investigated. generally, two phenomena occur simultaneously when salt is added to water. the first phenomenon is known as salting-out (this can increase the efficiency of the extraction) and the second phenomenon is electrostatic interactions between the analyte molecules and the salt,18,19 which reduces the mobility of the analyte molecules and increases the thickness of the emission layer between the sample solution and fibrated organic layer, that reduces the extraction efficiency. furthermore, the addition of salt could increase the viscosity of the sample solution and change the physical properties of the fiber wall, which could reduce the movement of analyte from the donor to solvent phase. overall, it is fair to comment that the addition of salt depends on the nature of the analyte and could increase, decrease or, in some cases, not change the efficiency of extractions.24,25 in this study, the effect of salt addition (nacl) from 0 to 350 g l–1 was investigated on the extraction rate. nacl addition significantly increased the drug extraction. the concentration of 350 g l–1 was selected as the optimum value for further studies. thus, according to the nature, structure and the pka value (7.7) of stg, the salting out phenomenon was dominant in this process and, therefore, increasing the salt concentration, increased the extraction rate of the drug. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1316 rezaee, qomi and piroozi extraction time the extraction and recovery of an analyte is affected by the stirring time of the sample solution, which facilitates the transfer of an analyte from the donor phase to the organic phase and finally to the acceptor phase. in this study, the effect of extraction time between 20 and 60 min was examined. normally, if the extraction time is too long, the loss of solvent and the formation of air bubbles may occur, both of which affect the extraction efficiency.17,10,26,27 the obtained results showed that with increasing time, the extraction rate increased. based on thee results, an extraction time of 50 min was selected as a suitable extraction time. temperature in this study, the effect of temperature on extraction rate was investigated in the range from 22 to 45 °c. the results demonstrated that the extraction efficiency was reduced on increasing the temperature. this could be due to solvent evaporation and bubble formation on the fiber wall.19 therefore, 25 °c was chosen as the optimum temperature for the extractions. volume ratio of donor phase to acceptor phase in the three phase hf–lpme, an analyte transfers from an aqueous donor phase to an organic solvent and finally to an acceptor phase existing in the fiber during an emission process. pre-concentration factor (pf) in this process is calculated based on the equation: d a 100 = v r pf v where vd and va are the volumes of the donor phase and acceptor phase, respectively, and r is the extraction efficiency.20,25 in this study, the volume of the acceptor phase was kept constant (20 µl), and the volume of donor phase was altered and the effects of these changes on the extraction rate were investigated. based on the obtained results, 15 ml was selected as the optimum volume of the donor phase. validation method the calibration curve for stg in aqueous solutions was plotted in the concentration range from 0.05 to 10 mg l–1. for each level, three replicate extractions were performed under the optimum pre-concentration conditions (table i). table i. optimum conditions for the pre-concentration of sitagliptin solvent ph of dp ph of ap stirring rate, rpmt / min[nacl] / g dl-1 t / °cvd / ml n-octanol 10.5 3 1000 50 35 25 15 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ determination of sitagliptin in urine 1317 the corresponding regression equation, coefficient of determination (r2), dynamic linear range (dlr), limit of detection (lod) and pre-concentration factor (pf) of stg were calculated and are summarized in table ii. the lod was calculated at a signal-to-noise ratio of 3 and was obtained by adding the standard stg solution to drug-free urine samples. the results indicated that the proposed method displayed good reproducibility to determine sitagliptin with intra-day rsd value of 3.4 % and inter-day rsd value of 5.25 % (for 3 consecutive days, the extraction was performed at an stg concentration of 1 mg l–1). under the optimized conditions, enrichment factors of up to 88 were achieved. table ii. the values of the corresponding regression equation and the analytical characteristics of the proposed hf–lpme method; pf, pre-concentration factor; lod, limit of detection; loq, limit of quantification; r2, coefficient of determination; dlr, dynamic linear range and rsd %: relative standard deviation; analyzed concentrations, mg l-1: 0.05, 0.2, 0.5, 3, 5, 7, 9 and 10 inter-day rsd / % intra-day rsd / % dlr range ng ml-1 r 2 intercept slope mv s l mg-1 loq ng ml-1 lod ng ml-1 pf 5.25 3.4 2.5–200 0.999 25.881 328.09 3.0 1.0 88 extraction of sitagliptin from urine samples under the optimized conditions, the developed hf–lpme–hplc technique was applied to the pre-concentration and determination of sitagliptin in urine samples. the ph of the real samples was adjusted to 10.5 by the addition of 0.1 mol l–1 naoh solution. prior to the spiking and extraction of the target drug, the urine samples were diluted 1:1 with water. typical chromatograms for spiked and non-spiked urine samples obtained by the hf–lpme–hplc method are shown in figs. 2 and 3, respectively. fig. 2. representative hf–lpme–hplc chromatograms of urine sample spiked with 10 ng/ml of sitagliptin under the optimum conditions (n-octanol as organic solvent; the donor phase ph of 10.5; the acceptor phase ph of 3; stirring speed of 1000 rpm; concentration of nacl 35 g dl-1; extraction time of 50 min; temperature of 25 °c; volume of donor phase of 15 ml). _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1318 rezaee, qomi and piroozi fig. 3. representative hf–lpme–hplc chromatograms of control urine sample. comparison of the present method with other reported methods the present method was compared with other studies in terms of the method of extraction, validation, and precision. the method compares well with those mentioned in table iii. as can be seen, the lod value of this method is comparable with those obtained in previous studies, and even lower than those reported in the literature. in addition, due to the simplicity and low cost of the extraction device, the hollow fiber, it can be discarded after each extraction to avoid carry-over and cross-contamination. table iii. comparison of the proposed method with other developed methods for determination of sitagliptin in biological samples ref. real sample rsd % loq µg ml-1 dlr µg ml-1 lod µg ml-1 extraction method determination method present work urine 3.4 0.003 0.0025–0.20.001 hf–lpme hplc–uv 28 plasma 10> 0.75 0.75–100 – lle hplc 6 plasma 2–6.1 0.001 0.001–1 – lle lc/ms/ms 29 urine 2.3–6.5 0.1 0.1–50 – tfoa htlc/ms/ms 30 plasma 0.93–1.620.075 0.25–200 0.02 – rp-lc 7 plasma <6 0.001 0.001–0.25– lle lc/ms/ms 8 urine 0.4 0.1 (µg l-1) 0.1–100 (µg l-1) 0.03 (µg l-1) mispeb lc atfo: turbulent flow online, bmispe: molecularly imprinted solid-phase extraction conclusions this work introduced a three-phase hf–lpme method combined with hplc–uv for the extraction, pre-concentration and determination of stg in urine samples. the lpme technique proved to have several advantages over the other extraction methods such as liquid–liquid extraction (lle) and solid phase extraction (spe). a simple, fast and inexpensive hf–lpme–hplc–uv with _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ determination of sitagliptin in urine 1319 high accuracy and good sensitivity for the extraction and determination of stg in human urine samples was developed and validated. acknowledgments. we would like to thank the food and drug organization (tehran, iran) for donating the sitagliptin reference standard for this study. we gratefully acknowledge the support and generosity of the pharmaceutical sciences research center, without which the present study could not have been completed. и з в о д екстракција шупљим влакнима комбинована са hplc за одређивање ситаглиптина у узорцима урина raheme rezaee, mahnaz qomi и foroozan piroozi medicinal chemistry department, pharmaceutical sciences research center, pharmaceutical sciences branch, islamic azad university, tehran, iran у овом раду је успешно развијена трофазна микроекстракција шупљим влакнима у течној фази (hf–lpme) спрегнута са високоефикасном течном хроматографијoм (hplc) за одређивање трагова лека, антидијабетика ситаглиптина (stg), у узорцима урина. ситаглиптин је екстрахован из 15 ml основног раствора, на ph 8,5 у органски растварач (n-oктанол) у порама шупљег влакна и ре-екстархован у закишењен раствор у лумену шупљег влакна на ph 3. после екстракције, 20 μl aкцепторске фазе је инјектирано у hplc систем. у циљу боље ефикасности екстракције, испитивани су и оптимизовани параметри који утичу на hf–lpme, као што су ph донорске и акцепторске фазе, тип органске фазе, јонска јачина, брзина мешања, време екстракције, запремински однос донорске и акцепторске фазе и температура. под оптималним условима је постигнут фактор преконцентрисања од 88 и добијена релативна стандардна девијација у опсегу 3–6 %. резултати указују да hf–lpme метода има одличан капацитет за пречишћавање и висок фактор преконцентрисања, па може бити примењена као једноставна и осетљива метода за праћење лекова у узорцима урина. (примљено 27. децембра 2014, ревидирано 14. маја, прихваћено 24. маја 2015) references 1. r. pathak, m. b. bridgeman, p&t 35 (2010) 509 2. d. k. badyal, j. kaur, jk science 10 (2008) 97 3. e. fleury-milfort, j. am. acad. nurse pract. 20 (2008) 295 4. a. j. scheen, diabetes obes. metab. 12 (2010) 648 5. s. terabe, anal. chem. 76 (2004) 241a 6. w. zeng, y. xu, m. constanzer, e. j. woolf, j. chromatogr., b 878 (2010) 1817 7. r. nirogi, v. kandikere, k. mudigonda, p. komarneni, r. aleti, r. boggavarapu, biomed. chromatogr. 22 (2008) 214 8. r. rao, p. maurya, s. khalid, talanta 85 (2011) 950 9. a. rodríguez, s. pedersen-bjergaard, k. e. rasmussen, c. nerín, j. chro matogr., a 38 (2008) 1198 10. k. m. al azzama, a. makahleaha, b. saada, s. mansorb, j. chromatogr., a 1217 (2010) 3654 11. y. tao, j. f. liu, x. l. hu, h. c. li, t. wang, g. b. jiang, j. c hromatogr., a 1216 (2009) 6259 12. s. pedersen-bjergaard, k. e. rasmussen, anal. chem. 71 (1999) 2650 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice ultrafast synthesis of isoquercitrin by enzymatic hydrolysis of rutin in a continuous-flow microreactor j. serb. chem. soc. 80 (7) 853–866 (2015) udc 547.9+544.473:577.15+547.918+ jscs–4764 542.913+66.094.941 original scientific paper 853 ultrafast synthesis of isoquercitrin by enzymatic hydrolysis of rutin in a continuous-flow microreactor jun wang1,2, an gong2, shuangshuang gu2, hongsheng cui2 and xiangyang wu1* 1school of environment and safety engineering, jiangsu university, zhenjiang 212013, p. r. china and 2school of biotechnology, jiangsu university of science and technology, zhenjiang 212018, p. r. china (received 15 september, revised 6 november, accepted 13 november 2014) abstract: isoquercitrin is a rare flavonol glycoside with a wide range of biological activities and is a key synthetic intermediate for the production of enzymatically modified isoquercitrin. in order to establish an ultrafast bioprocess for obtaining isoquercitrin, a novel continuous flow biosynthesis of isoquercitrin using the hesperidinase-catalyzed hydrolysis of rutin in a glass–polydimethylsiloxane (pdms) microreactor was first performed. using the developed microchannel reactor (200 μm width, 50 μm depth and 2 m length) with one t-shaped inlet and one outlet, the maximum yield of isoquercitrin (98.6 %) was achieved in a short time (40 min) under the following optimum conditions: rutin concentration at 1 g l-1, hesperidinase concentration at 0.1 g ml-1, reaction temperature 40 °c, and a flow rate of 2 μl min-1. the value of the activation energy, ea, of the enzymatic reaction was 4.61 kj mol -1, and the reaction rate and volume productivity were approximately 16.1-fold and 30 % higher, respectively, than those in a batch reactor were. thus, the use of a continuousflow microreactor for the enzymatic hydrolysis of rutin is an efficient and simple approach to achieve a relatively high yield of isoquercitrin. keywords: biocatalysis; continuous flow; hesperidinase; isoquercitrin; microreactor. introduction isoquercitrin is a rare flavonol glycoside with a wide range of biological activities and is a key synthetic intermediate for the production of enzymatically modified isoquercitrin (emiq), a new multiple food additive. due to its significant economic benefits and ecological acceptability when compared extraction from natural sources and chemical synthesis,1 there is a respectable number of reports indicating that isoquercitrin can be efficiently synthesized from rutin * corresponding author. e-mail: wuxy@ujs.edu.cn or jimwang_js@hotmail.com doi: 10.2298/jsc150914112w _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 854 wang et al. using an enzymatic hydrolysis process under suitable reaction conditions.2 recently, several methods for the transformation of rutin to isoquercitrin have been investigated, including acid hydrolysis,3 heating,4 microbial transformation,5 and enzymatic transformation techniques.6 among these methods, the biotransformation of rutin to isoquercitrin using selective hydrolysis would be a feasible procedure if the transformation could be performed at a reasonable cost. the application of commercial hesperidinase as a biocatalyst in the biosynthesis of isoquercitrin by the selective biotransformation of rutin has many advantages when compared with crude and recombinant enzymes. hesperidinase is more technically feasible for biological manufacturing approaches by controlling the ph instead of the temperature.3,7 consecutively, using an ionic liquid as a novel co-solvent in the [bmim][bf4]–glycine–sodium hydroxide buffer (ph 9; 10:90, v/v) to improve the isoquercitrin synthesis could allow for greater dissolution of the substrates and exercise a significant effect on the conversion, yield and selectivity of the enzymatic reaction system. these results indicated that ionic liquids could effectively enhance the selective synthesis of isoquercitrin and that the reaction process is simple and eco-friendly.4,8 in addition, the reaction time necessary to achieve the highest isoquercitrin yield of 91.41 % was reduced from 30 to 10 h, while the conversion of rutin and the yield of isoquercitrin were increased by 1.67-fold and 2.33-fold, respectively.9 however, the industrial production of isoquercitrin is hindered by a low reaction rate in a batch bioreactor. therefore, a longer reaction time would significantly increase the overall production cost of preparing isoquercitrin. for this reason, a better method for a high-efficiency synthesis of isoquercitrin over a short time needs to be developed. due to the rapid heat transfer and mixing in microreactors, reactions can be performed significantly faster than those in batch reactors, typically with increases in both yield and selectivity.10 recently, the use of microreactor technologies within the scope of bioprocesses as process intensification and production platforms is gaining momentum.11,12 compared to traditional batch reactors, this trend can be ascribed to a particular set of characteristics of microreactors, namely the enhanced mass and heat transfer, combined with easier handling and smaller volumes.13 haswell and co-workers demonstrated that the aldol reaction between aldehyde and silyl enol ether in the presence of tetrabutyl ammonium fluoride (tbaf) reaches completion in only 20 min when using a microreactor, versus 24 h in a typical reactor.14 herein, miniaturized devices are gaining widespread use in biocatalysis because this approach contributes to the rationalization of process development with a significant reduction in the work force, in the quantity of reagents required and in waste production, concomitantly contributing to a significant cost reduction.15 these microreactors have been shown to outperform conventional, large-scale vessels operating in the batch mode, given the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis of isoquercitrin in enzymatic microreactor 855 favorable mass and heat transfer characteristics due to a large area to volume ratio and the possibility of operating in a continuous mode.16 under the correct conditions, microreactors can also offer better selectivity, improved yields over shorter periods, increased process control, greater safety, and flexible production.17 kanno and co-workers showed that an enzyme-catalyzed reaction performed homogeneously in a flow could yield higher conversions than that in the batch counterpart.18 in this case, a solution of α-galactosidase in phosphate buffer at ph 8 was combined with a similarly buffered solution of p-nitrophenylα-d-galactopyranoside (pnpgal) in a 200 μm×200 μm microreactor. the authors were able to show that the hydrolysis in the microreactor was 5-fold faster than that in the analogous batch reaction performed in a micro-test tube.19 however, no report has hitherto been published detailing the use of a continuous-flow microreactor to significantly enhance the hesperidinase-catalyzed synthesis of isoquercitrin. the purpose of this study was to set up a continuous biocatalysis system for the selective and effective biotransformation of rutin to isoquercitrin in a glass– pdms microreactor. the effects of the channel length of the microreactor, reaction temperature, rutin concentration and enzyme concentration on isoquercitrin yield were investigated. in addition, the activation energy value ea of the enzymatic reaction was determined. materials and methods materials hesperidinase (contains both α-l-rhamnosidase and β-d-glucosidase activities, ≥1 units g-1 solid) produced by aspergillus niger and standard isoquercitrin were purchased from sigma (st. louis, mo, usa). the ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([bmim][bf4]) was purchased from shanghai cheng-jie chemical co. (shanghai, china) and had a residual chloride content of less than 50 ppm. all the reagents used were of analytical grade except methanol and acetonitrile, both of which were of hplc-grade (purchased from tedia co. (fairfield, oh, usa)). all other solvents and reagents were of analytical grade. water was purified using an elga purelab option-q purification system (elga labwater, high wycombe, uk) and had a resistivity of not less than 18.0 mω cm. this water was used for cleaning procedures and in the preparation of all buffer solutions. all aqueous solutions were prepared with ultrapure water and filtered through a 0.45 μm membrane filter. hesperidinase-catalyzed synthesis of isoquercitrin in a continuous-flow microreactor hesperidinase-catalyzed synthesis of isoquercitrin was realized in a glass–pdms microreactor with rectangular microchannels. the microreactor developed by the laser burn technology had a t-shaped inlet and an outlet channel. the main channel dimension was 200 μm wide, 50 μm deep and 2 m long (fig. 1). the reaction substrate (rutin at ph 9 in glycine– –sodium hydroxide buffer) and an enzyme solution containing 10 % [bmim][bf4] were both pumped into the microchannel by a two-channel syringe pump. the flow rates of the two phases were the same. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 856 wang et al. fig. 1. ultrafast synthesis of isoquercitrin through the enzymatic hydrolysis of rutin in a microreactor. a scheme of isoquercitrin synthesis using hesperidinase-catalyzed hydrolysis of rutin is presented in fig. 2, which shows the chemical structures of rutin, isoquercitrin and l-rhamnose. fig. 2. scheme of isoquercitrin synthesis using hesperidinase-catalyzed hydrolysis of rutin. hplc analysis and lc-ms analysis hplc quantitative analyses were performed using a constant flow pump (2pb0540, beijing satellite factory, beijing, china) with a uv–vis detector (l-7420, techcomp co., shanghai, china) and an n-2000 workstation (hangzhou mingtong s&t ltd., hangzhou, china). an alltima c18 column (250 mm×4.6 mm, i.d.; 5 μm; from w. r. grace & co., deerfield, il, usa) was used, and the column was maintained at 30 °c. the separation and determination of rutin and isoquercitrin using the hplc/uv method was performed on the alltima c18 column with a mobile phase consisting of acetonitrile:0.02 % phosphoric acid solution (20:80, v/v) at a flow rate of 1.0 ml min-1. rutin and isoquercitrin were detected at 360 nm. all solutions were filtered through a 0.45 μm filter before injection. all of the experiments were performed in triplicate. the isoquercitrin yield of the hesperidinase-catalyzed isoquercitrin synthesis was calculated using the following equation: moles of isoquercitrin isoquercitrin yield (%) = 100 initial moles of rutin × (1) lc-ms was performed on a thermo fisher system. the lc equipment comprised a finnigan mat spectra system p4000 pump, an autosampler with a 50 μl loop, a uv6000 lp diode array detector (dad) and a finnigan aqa mass spectrometer. lc separation was performed on the alltima c18 column (250 mm×4.6 mm, i.d.; 5 μm). the mobile phases consisted of 0.1 % formic acid in water (a) and 0.1 % formic acid in acetonitrile (b). separation was performed under the following conditions: 0–35 min, 6–100 % b; 35–40 min, back to 6 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis of isoquercitrin in enzymatic microreactor 857 % b. the column was equilibrated for 15 min prior to each analysis. the wavelength range of the dad was 200 to 400 nm. the flow rate was 1.0 ml min-1 for lc, and the column remained at 40 °c during da detection. electrospray ionization (esi) was performed using nitrogen to assist nebulization (1.0 ml min-1 flow rate). selected ion monitoring (sim) in the negative ion mode with 1.6 kv capillary voltage was used, and the temperature of the curved desolvation line (cdl) and heat block were both set at 200 °c. the data were processed using xcalibur 1.2 software. the intense peaks at m/z 463.17 in the esi-ms spectra under negative ion mode corresponded to the deprotonated [m–h]of isoquercitrin.9 kinetic analyses to study the kinetics of the enzymatic synthesis of isoquercitrin, the reactions were performed at different temperatures. the temperature ranged from 25 to 40 °c because the hesperidinase could exhibit its optimal activity at 40 °c and a temperature higher than 40 °c might deactivate the enzyme. the reaction rates were calculated according to: 1 ln ( ) a k a x t = − (2) where k is the reaction rate constant (min-1), a is the initial concentration of substrate (μmol l-1) and x is the concentration of isoquercitrin at time t, where t is the reaction time. statistical analyses triplicate experiments were performed for each investigated parameter. the standard deviation of the values was calculated to check the reliability of the results. the differences in mean values were evaluated using the analysis of variance (anova) method. significance was determined at the 95 % level of probability. results and discussion the effect of microchannel length the continuous-flow microreactor in this study consisted of channel diameters of 50 μm deep and 200 μm wide. the effects of the microreactor channel length on isoquercitrin yield using a continuous-flow microreactor at different flow rates are shown in fig. 3a. increasing the channel length from 0.5 to 2 m had a beneficial effect on isoquercitrin yield. when the microfluid flow rate was 2 μl min–1, the channel length was 2 m, and the residence time was 40 min, a maximum yield of 34.5 % was achieved. these results indicated that the shallow depth of the microreactor channels provided for very short diffusion lengths of reaction mixtures and induced the microfluid under laminar flow condition.20 this is one of the great advantages of using microscale reactors for rapid reactions. these results sufficiently confirmed that reducing the flow rate to induce a prolonged residence time would facilitate enzymatic reactions. however, when the liquid flow rate was too fast, the reaction droplets of hesperidinase increased friction with the inner wall of the channel. this phenomenon prompted the increased shear stress and local overheating and deactivated the enzyme.21 therefore, the yield of isoquercitrin was reduced. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 858 wang et al. fig. 3. using the continuous-flow microreactor developed to synthesize isoquercitrin by the enzymatic hydrolysis of rutin. a) effects of the channel length of the microreactor on the isoquercitrin yield at different flow rates; b) effects of residence time on the isoquercitrin yield. reaction temperature: 40 °c; rutin concentration: 1 g l-1; hesperidinase concentration: 0.01 g ml-1. the effects of residence time on the efficiency of isoquercitrin synthesis are shown in fig. 3b. using a 2 m long channel, the residence time (4–40 min) of the reaction mixture in the microreactor was tuned by varying the flow rates (20–2 μl min–1). as expected, the reaction appeared to be favored by an increase in the residence time. when the enzymatic reaction was conducted at 20 μl min–1, only a 29.0 % yield was obtained, while at 2 μl min–1, a maximum yield of 34.5 % was achieved. thus, a 2 m long channel was chosen for further experiments. the effect of reaction temperature the effects of temperature on isoquercitrin yield using a continuous-flow microreactor at different flow rates are shown in fig. 4a. the isoquercitrin yield initially increased with temperatures ranging from 25 to 40 °c, whereas higher temperatures led to a decline in isoquercitrin yield and enzyme denaturation, which lowered the efficiency of enzymatic hydrolysis. the yield had a similar _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis of isoquercitrin in enzymatic microreactor 859 tendency at different flow rates. an optimal temperature of 40 °c at 2 μl min–1 was chosen to promote the maximum yield of isoquercitrin. fig. 4. a) effects of temperature on the isoquercitrin yield and b) the arrhenius plot of ln k vs. t-1 using the developed continuous-flow microreactor at different flow rates at 40 °c. rutin concentration: 1 g l-1; hesperidinase concentration: 0.01 g ml-1. an analysis was performed to study the effects of temperature on reaction rate and energy of activation in the enzymatic synthesis of isoquercitrin. depending on the reaction rate constant values at various reaction temperatures, the value of energy of activation could be further estimated according to the arrhenius equation: aln ln+ e k = a rt (3) where k is the reaction rate constant, a is the frequency factor and ea is the energy of activation. an arrhenius plot made based on ln k vs. the reciprocal of temperature is shown in fig. 4b. the reaction rate increased with increasing temperature. in _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 860 wang et al. addition, the activation energy ea could be obtained from the slope of the straight line. thus, when the flow rate was 2 μl min–1, the ea of the reaction was calculated to be 4.61 kj mol–1. ea represents the ability of the enzymatic reaction to overcome the original “energy barrier”. in this continuous-flow microreactor, the ea value is relatively small, and the enzymatic reaction occurs more easily. these results suggest that heat transfer was compromised in the developed microreactor with rectangular microchannels and that local temperature changes were significant. heat transfer was enhanced as the flow rate increased; therefore, the heat dissipates faster from the active site of the enzyme,22 which subsequently decreased the yield of isoquercitrin when the temperature exceeded 40 °c. the effect of rutin concentration the effects of the rutin concentration on isoquercitrin yield using a microreactor at different flow rates are shown in fig. 5a. as expected, the yield of isoquercitrin increased with increasing inlet substrate concentration and lowering the flow rate. at inlet concentrations of rutin of 1 g l–1, a yield of isoquercitrin of fig. 5. a) effects of rutin concentration and b) enzyme concentration on the isoquercitrin yield using the developed continuous-flow microreactor at different flow rates. reaction temperature: 40 °c; a) hesperidinase concentration: 0.01 g ml-1; b) rutin concentration: 1.0 g l-1. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis of isoquercitrin in enzymatic microreactor 861 approximately 34.5 % was obtained after 40 min. however, further increasing the inlet concentration to 2 g l–1 decreased the yield of isoquercitrin significantly. the reasons mainly include two aspects: 1) the hesperidinase concentration in this experiment was very limited, resulting in an enzyme that could not fully contact with the substrate; 2) substrate inhibition was present and enzyme denaturation was detected at a high substrate concentrations. in addition, higher substrate concentrations caused clogging in continuous flow reactors.23 the inlet concentration of rutin used in the microreactor was in agreement with a previous study.9 however, some studies reported that the inlet substrate concentrations used in a microreactor were 10 times lower than those in a batch reactor.24 hence, the presented microreactor technology is economically feasible for the large-scale production of isoquercitrin without reducing the substrate concentration of rutin. usually, substrate inhibition and product degradation were two possible reasons for the decrease in isoquercitrin conversion when the concentration of rutin was higher. hypothetically, when the concentration of rutin is 1 g l–1, a higher isoquercitrin yield should be obtained. however, if isoquercitrin is abundantly produced and the substrate concentration is appropriately increased, the yield and efficiency should be greatly improved. thus, 1 g l–1 of rutin was chosen as a suitable substrate concentration for further study. the effect of hesperidinase concentration the effects of the hesperidinase concentration on isoquercitrin yield with different flow rates are shown in fig. 5b. under identical conditions of temperature and substrate concentration, the effect of the concentration of hesperidinase in the range of 0.01 to 0.12 g ml–1 on isoquercitrin yield at different flow rates was observed. an increase in the concentration of hesperidinase up to 0.1 g ml–1 had a positive effect on the isoquercitrin yield. when the concentration of hesperidinase was 0.1 g ml–1, the yield of isoquercitrin was near 98.6 %. further increases in the enzyme concentration up to 0.12 g ml–1 increased the yield of isoquercitrin only slightly. hence, from an economic perspective, the best hesperidinase concentration was 0.1 g ml–1. the enzyme concentration plays an important role in enzymatic syntheses,25 and the number of active sites in a particular enzyme can affect the reaction rate.26 in addition, the separation of the product can be easily attained using triacetin as an organic phase to extract isoquercitrin from the reaction mixture after completion of the enzymatic reaction.27 as expected, the yield under steady-state conditions increased with decreasing flow rate. a yield of approximately 98.6 % was achieved at an inlet rutin concentration of 1 g ml–1 and a flow rate of 2 μl min–1. the aqueous phase with the hesperidinase and unreacted rutin could be recycled, which makes this process much more economic. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 862 wang et al. the effect of flow rate and residence time to investigate the effect of flow rate on the hesperidinase-catalyzed synthesis of isoquercitrin by the enzymatic hydrolysis of rutin, five different flow rates, i.e., 2, 4, 8, 16 and 20 μl min–1, were selected for testing. as shown in figs. 3a, 4a, 5a and 5b, different isoquercitrin yields were obtained at various flow rates. in every figure, a flow rate of 2 μl min–1 resulted in a higher isoquercitrin yield than those obtained at the other rates. as the flow rate was further increased, the isoquercitrin yield was clearly reduced and reached the lowest yield at 20 μl min–1. in particular, fig. 5b shows that the isoquercitrin yield was the highest, up to 98.6 %, at a flow rate of 2 μl min–1. by using longer channels and reducing the flow rate, the residence time increases. longer residence times allow a more complete, full contact between substrates and enzymes. however, increasing the channel length from 2 m to 4 m did not obviously change the isoquercitrin yield, possibly because the stability of hesperidinase was lower at longer channel lengths. in addition, the pressure would increase with a further reduction in the channel dimension, and this is disadvantageous to the actual operation. under different flow rates in a 4 m long microreactor with rectangular microchannels, the faster the flow rate, the longer was the residence time of the hesperidinase-catalyzed synthesis of isoquercitrin by enzymatic hydrolysis of rutin. in this microreactor, flow rates ranging from 2 to 20 μl min–1 were employed, and the corresponding residence time was decreased from 2.5 h to 15 min. the isoquercitrin yield increased as the residence time was extended, which was most likely due to the increase in the mass transfer of the substrate towards hesperidinase molecules because of the increase in the reaction time.28,29 as the residence time increased, interactions between the substrate and enzyme increased, resulting in a higher yield of isoquercitrin. this result indicates that the flow rate is a vitally important factor for the operation of a microreactor with rectangular microchannels. at low flow rates, the increased residence time in the reaction system allowed for the completion of the enzymatic synthesis reaction.30 at high flow rates, the residence time was most likely not long enough for the completion of enzymatic synthesis because the synthesis of isoquercitrin was not fast enough to complete the enzymatic reaction in such a short time. comparing the technology between the microreactor and the batch reactor a maximum isoquercitrin yield of 98.6 % was obtained in the continuousflow microreactor after 40 min, which was 16.1-fold faster than the yield of 91.4 % in the batch reactor (10 h, table i). the calculated volume productivity at a rutin inlet concentration of 1 g l–1 was 3.23 µm min–1 at a residence time of 40 min, which was more than 1.2-fold higher than that reported within the batch reactor,9 where the highest volume productivity was 2.49 µm min–1. thus, the _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis of isoquercitrin in enzymatic microreactor 863 volume productivity in the microreactor was 30 % higher than that in the batch reactor. herein, although isoquercitrin production in a specific time was lower than that in a conventional reactor, the reaction time in the microreactor was 14 times lower than that in a batch reactor. considering these two factors, biocatalysis technology in the microreactor could be a feasible industrial tool in the future. table i. comparative results for the hesperidinase-catalyzed transformation of rutin to produce isoquercitrin in different reactors reactor substrate concentration, g l-1 enzyme (concentration) g ml-1 reaction time, h isoquercitrin yield, % volume productivity, µm min-1 ref. microreactora 1 hesperidinase (0.10) 0.67 98.60±3.25 3.23 this work batch reactorb 1 hesperidinase (0.05) 10 91.46±0.55 2.49 wang et al.3 batch reactorc 0.92 crude enzyme extract of a. niger (–) 4 – – you et al.1 batch reactord 0.46 naringinase (0.05) 6 61 – vila-real et al.31 areaction conditions: rutin concentration 1 g l-1, reaction temperature 40 °c, flow rate 2 μl min-1 for 40 min, [bmim][bf4]–buffer (ph 9.0), 10:90, v/v, as the reaction medium; breaction conditions: rutin concentration 1 g l-1, reaction temperature 40 °c, 120 rpm for 10 h, [bmim][bf4]–buffer (ph 9.0), 10:90, v/v, as the reaction medium; creaction conditions: rutin concentration 0.92 g l-1, reaction temperature 60 °c, 50 μl of crude enzyme extract of a. niger was resuspended in a reaction system; dreaction conditions: rutin concentration 0.46 g l-1, reaction temperature 60 °c for 6 h in a 20 mmol l-1 citrate buffer system as shown in table i, you et al.1 obtained isoquercitrin using a crude enzyme extract of aspergillus niger in 4 h. the reaction time and the substrate concentration were 7.97-fold longer and 0.92-fold lower, respectively, than those in the microreactor. in addition, the results of vila-real et al.31 indicated that isoquercitrin was obtained using the enzymatic hydrolysis of rutin with a production yield of 61 % under the conditions of 0.05 g ml–1 naringinase and 0.46 g l–1 rutin in 6 h. the isoquercitrin yield and rutin concentration were 37.6 % and 2.18-fold lower, respectively, than those in the microreactor, and the reaction time was much longer than that in the microreactor. this is due to the increased specific interfacial area and the reduced diffusion length in the microreactor, both improving the mass transfer rates in the reaction.32 these results imply that to determine the suitable performance of a continuous-flow microreactor on an industrial scale, a numbering up approach with parallel microreactors could be adopted. generally, the developed multi-channel microreactors and other similar multi-input microreactors are more inclined to the demands of industrial production.33,34 the higher the number of channels, the greater the injection volume _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 864 wang et al. of the substrates. the volume productivity of products would be significantly increased.35 thus, a shorter reaction time would significantly decrease the overall production costs of preparing isoquercitrin. due to the rapid heat transfer and mixing in microreactors, the reactions could be performed significantly faster than those in batch modes, typically with increases in both yield and selectivity.32 when examining rapid reactions that allowed for equivalent reaction times in flow and batch modes, the yield was improved.36 in addition to high yields, microreactors provide environments for highly selective chemistry, most likely due to the precise temperature control. when compared to a batch reactor, the microreactor had a higher efficiency.37 this improvement could be attributed to two factors:38 1) the surface-to-volume ratio in the microreactors is much higher than in batch reactors, which increases the enzyme active sites available to the reactants at any time; and 2) the volume of the microreactor is so limited that the reactants are forced to be in contact with enzyme active sites because the diffusion paths in microreactors are much smaller. another advantage of using the developed microreactor is that the biocatalytic reaction could be performed continuously in the preparation of isoquercitrin. in summary, the developed biocatalysis method using a continuous-flow microreactor in a shorter time (40 min) could produce a comparable amount of isoquercitrin as in a batch reactor (10 h). thus, the bioprocess is much more economical in the industrial preparation of isoquercitrin and other precious natural medicines.39 thus, the novel approach using a continuous-flow microreactor used to synthesize isoquercitrin through the enzymatic hydrolysis of rutin was both efficient and simple. conclusions an efficient and rapid process for isoquercitrin production using the hesperidinase-catalyzed hydrolysis of rutin was successfully developed in a continuous-flow microchannel reactor. the maximum isoquercitrin yield of 98.6 % and a volume productivity of 3.23 µmol l–1 min–1 were obtained under the following optimum conditions: a flow rate of 2 μl min–1 (residence time of 40 min), a rutin concentration of 1 g l–1, a hesperidinase concentration of 0.1 g ml–1, and a temperature of 40 °c. the value of the activation energy ea of the enzymatic reaction was 4.61 kj mol–1 at 2 μl min–1. the enzymatic reaction rate in the developed microreactor was approximately 26-fold higher than that in a batch reactor. thus, the novel approach using a continuous-flow microreactor for the synthesis of isoquercitrin through the enzymatic hydrolysis of rutin was both efficient and simple. moreover, this microtechnology could also be applied in the effective synthesis of other precious natural medicines. acknowledgements. this work was supported by the china postdoctoral science foundation funded projects (2013t60505, 2012m510124), the postdoctoral science foundation _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis of isoquercitrin in enzymatic microreactor 865 funded project of jiangsu university (1143002085), and the qing lan project of jiangsu province (2014). и з в о д брза синтеза изокверцитрина ензимском хидролизом рутина у микрореактору са непрекидним протоком jun wang 1,2 , an gong 2 , shuangshuang gu 2 , hongsheng cui 2 и xiangyang wu 1 1school of the environment and safety engineering, jiangsu university, zhenjiang 212013, p r china и 2school of biotechnology, jiangsu university of science and technology, zhenjiang 212018, p.r. china изокверцитрин је ретки флавонолни гликозид широког опсега биолошких активности и кључни је интермедијер у синтези ензимски модификованог изокверцитрина. да би се успоставио изузетно брз поступак за добијање изокверцитрина, коришћен је биореактор од стакла и полидиметилсилоксана са сталним протоком, а рутин је хидролизован хесперинидазом. применом новог микроканалног реактора (ширине 200 μm, дубине 50 μm и дужине 2 m), са једним улазом т облика и једним излазом, максимални принос изокверцитрина (98,6 %) је добијен у кратком времену (40 min), под следећим оптималним условима: концентрација рутина 1 g l-1, концентрација хесперинидазе 0,1 g ml-1, реакциона температура 40 °c и проток 2 μl min-1. енергија активације за ензимску реакцију је била 4,61 kj mol-1, а брзина реакције и запремински принос су биле 16,1 пута, односно за 30 % веће него у стандардном реактору. дакле, примена микрореактора са сталним протоком за синтезу изокверцитрина ензимском хидролизом рутина је ефикасан и једноставан поступак. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice poly(methyl methacrylate) denture base materials modified with ditetrahydrofurfuryl itaconate: significant applicative properties j. serb. chem. soc. 80 (9) 1177–1192 (2015) udc 547.462.3+678.744:66.081.2:661.742.2 jscs–4789 original scientific paper 1177 poly(methyl methacrylate) denture base materials modified with ditetrahydrofurfuryl itaconate: significant applicative properties pavle spasojević1*#, vesna panić1, sanja šešlija2#, vladimir nikolić3#, ivanka g. popović4# and sava veličković4 1innovation centre, faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11000 belgrade, serbia, 2institute of chemistry, technology and metallurgy, university of belgrade, njegoševa 12, 11000 belgrade, serbia, 3innovation centre, faculty of chemistry, university of belgrade, studentski trg 12–16, 11000 belgrade, serbia and 4faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11000 belgrade, serbia (received 23 january, revised 9 april, accepted 22 april 2015) abstract: the aim of this work was to examine the possibility of modification of commercial denture base materials with itaconic acid esters, in order to obtain materials with lower toxicity and higher biocompatibility. despite their relatively higher price compared to methacrylates, itaconic acid and itaconates are materials of choice for environmentally friendly applications, because they are not produced from petrochemical sources, but from plant products. a commercial system based on poly(methyl methacrylate) was modified using ditetrahydrofurfuryl itaconate (dthfi), whereby the ratio of dthfi was varied from 2.5 to 10 % by weight. copolymerization was confirmed using ftir spectroscopy, while sem analysis showed the absence of micro defects and pores in the structure. the effects of the itaconate content on the absorption of fluids, the residual monomer content, thermal, dynamic-mechanical and mechanical properties (hardness, toughness, stress and elongation at break) were investigated. it was found that the addition of dthfi significantly reduced the amount of residual methyl methacrylate, which made these materials less toxic. it was shown that increasing the dthfi content resulted in materials with decreased glass transition temperatures, as well as with decreased storage modulus, ultimate tensile strength and impact fracture resistance; however the mechanical properties were in the rang prescribed by ada standards, and the materials could be used in practice. the deterioration in mechanical properties was therefore worthwhile in order to gain lower toxicity of the leached monomer. keywords: dental; itaconic; methyl methacrylate; absorption, tensile. * corresponding author. e-mail: pspasojevic@tmf.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc150123034s _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1178 spasojević et al. introduction poly(methyl methacrylate) (pmma) is one of the most significant acrylic polymers. although it was discovered and commercialized many years ago, pmma is still the subject of intense scientific research. poly(methyl methacrylate) is used as a substitute for transparent glass and dielectric films,1 acrylic paints,2 micro-cell foam,3 etc. however, biomedicine represents the most attractive application area where this material is used to create denture bases,4 contact lenses, bone cement,5 inhalers,6 etc. materials based on pmma are often used as biomaterials due to their good biocompatibility, non-toxicity, stability of colour and shape,7 the absence of taste, smell and irritation of the surrounding tissue,8 good adhesion to teeth, insolubility in body fluids, the ease of handling and design, as well as good aesthetic properties.9 in spite of these advantages of the material, dental prosthesis based on pmma have several drawbacks. the most important of these drawbacks are toxicity of residual monomer,10–13 susceptibility to distortions as well as limitations in terms of mechanics.14 as a result of these deficiencies, residual monomer may leak out by diffusion from prosthesis and irritate the surrounding tissue. this leakage creates cracks and other structural damages to the dental prosthesis that could lead to mechanical fracture of the prosthesis and create an environment suitable for the development of different types of bacteria, moulds and fungi. in order to overcome the drawbacks and the limitations of pmma denture base materials, the possibility of modifying a commercial pmma-based formulation using itaconic acid derivatives was investigated. itaconic acid is structurally very similar to methacrylic acid, except that at the α-carbon atom a carboxyl group is attached instead of the h atom. despite the slightly higher market price compared to methacrylic acid, itaconic acid and itaconates are more acceptable in terms of ecology and sustainable development.15,16 the reason for this lies in the fact that the itaconic acid is obtained from plants (mostly by enzymatic transformations of molasses17), while methacrylic acid is derived from petrochemical sources. as a dibasic acid, itaconic acid provides more options when making its esters compared to methacrylic acid. due to the many similarities of itaconates with the corresponding methacrylates and the mentioned advantages, itaconates represent an interesting alternative to methacrylates in the synthesis of a variety of materials. in dentistry, itaconic acid and its esters are known. they have been widely used in the production of glass ionomer cements.18–20 itaconic acid and its derivatives are used as components in many systems for controlled drug release.21–26 it is important to note that itaconates have been extensively used in varies medical applications because of their very low toxicity.19 furthermore, itaconic acid and its derivatives are increasingly used in the preparation of paints and coatings,27–29 composite resins,30,31 contact lenses,32,33 and products for personal care.34 _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ properties of novel denture bases 1179 due to the structural similarities of the methacrylates and the itaconates, numerous studies concerning copolymers of methyl methacrylate and dialkyl itaconates have been published.11,12 fernandez-garcia and madruga13 examined the effect of copolymer composition on the glass transition temperature and came to the conclusion that the glass transition temperature of the copolymers decreased with increasing amount of itaconate, as well as with the increasing alkyl chain length of the ester group. investigation of the thermal stability of the copolymers of the methyl methacrylate and dialkyl itaconates showed that the relative thermal stability increased with increasing proportion of methyl methacrylate in the copolymer, following a similar trend as the change of glass transition temperature.14 in a previous study, the residual monomer content and water sorption for pmma denture base materials modified with dimethyl itaconate and dibutyl itaconate were investigated.35 it was shown that the addition of itaconate led to a reduction in the water uptake and greatly reduced the residual methyl methacrylate content. in this way, the applicative properties and biocompatibility of pmma denture base materials for the production of dental prostheses were greatly improved. in order to further investigate the effect of the substitution of methyl methacrylate in denture base materials with the esters of itaconic acid, in this study, a commercial denture base material was modified by ditetrahydrofurfuryl itaconate (dthfi). the applicative properties of significance of the novel materials were investigated. experimental materials a commercial system for denture base materials biokril® (galenika ad, serbia) was used as received. the system was delivered as two-components; one part was solid and the other was liquid. liquid component included monomer (mma) and ethylene glycol dimethacrylate (egdma) as crosslinker, while solid component included pmma powder and benzoyl peroxide (bpo) as initiator. itaconic acid (2-methylidenebutanedioic acid) was a commercial product (fluka), while ditetrahydrofurfuryl itaconate (dthfi) was synthesized as previously described in the literature.16 synthesis of pmma denture base materials modified with itaconates the liquid component was mixed with a precisely defined amount of ditetrahydrofurfuryl itaconate. the amount of dthfi that was added in liquid component was determined in a manner that mixture obtained by mixing modified liquid component with solid commercial component had satisfying characteristics (“working hours” and curing time). the mass percent of dthfi in the new procedures were 0, 2.5, 5.0, 7.5 and 10 (table i). polymerization under heating in a water bath polymerization was realised under pressure in metal mould that was placed in a water bath at 100 °c for 30 min. round moulds with diameter of 25 mm and thickness of 3 mm were used for the preparation of the samples for measurement of the material hardness, while _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1180 spasojević et al. moulds for the measurement of elongation, viscoelastic properties and toughness had a rectangular shape with the dimensions of 60 mm× 10 mm× 4 mm, 60 mm× 12 mm× 2 mm and 80 mm× 10 mm× 4 mm, respectively. table i. feed composition sample component biokril® pmma powder, % biokril® liquid component, % dthfi, % pmma 66.0 34.0 – pmma/2.5pdthfi 64.5 33.0 2.5 pmma/5pdthfi 63.0 32.0 5.0 pmma/7.5pdthfi 61.5 31.0 7.5 pmma/10pdthfi 59.5 30.5 10 determination of the amount of residual monomer in the obtained materials by high pressure liquid chromatography with a uv detector (hplc-uv) the amount of the residual monomer after polymerization was determined by high pressure liquid chromatography with a uv detector (hplc-uv). the residual monomers were extracted from samples with an average mass of 0.5 g. the samples were placed in cups and then immersed in 20 ml of methanol. to complete the extraction, the cups were closed, covered with parafilm® and stored in the dark for 10 days. a surveyor hplc system (thermo fisher scientific, waltham, ma, usa) was used for the determination of the amount of the residual monomers (mma and dthfi). the chromatographic separation of monomers was realised on the zorbax eclipse® xdb-c18 column that was preceded by a pre-column of the same producer. the mobile phase consisted of methanol (a) and deionised water (b). chromatographic separation of mma and dthfi was performed isocratically for a duration of 10 min and mobile phase consisted of 70 % a and 30 % b at a constant flow of 1.1 ml min-1. a 10-μl sample was injected into the hplc system. the uv spectra of monomers were obtained by a surveyor pda detector. only one absorption maximum for both monomers (λ = 210 nm) was observed in the obtained uv spectra. this absorption maximum was used for quantitative determination of the monomers. fourier transform infrared spectroscopy (ftir) ftir spectra were recorded by bomen mb 100 spectrophotometer (hartmann and braun, canada). the kbr pellet technique was used whereby the kbr:sample ratio was 10:1. the spectra were recorded with 10 repetitions at a resolution of 4 cm-1. win bomen software was used for the processing of the spectra. scanning electron microscopy (sem) prior to sem analysis, the samples were covered with platinum vapour in a leica scd005 nebulizer. the sem observations were performed using a jeol jsm 5800 scanning electron microscope at an acceleration voltage of 20 kv. water absorption water absorption of the commercial pmma and dthfi-modified materials for denture base was investigated gravimetrically at 37 °c. first, the masses of the dry samples were measured and then the samples were immersed in distilled water. in the first 3 days, the samples were measured in intervals of 3 h and then once per day until equilibrium were _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ properties of novel denture bases 1181 reached. before measuring, the excess water was removed by wiping the samples with filter paper. all of the calculations were realised using data from the second absorption cycle. the second absorption cycle lasted 28 days. water-soluble oligomers and other impurities were leached from the sample during the first adsorption cycle and hence, the results obtained from the second cycle were accepted as relevant for the water absorption–desorption process. equilibrium absorption (m∞ / %) was calculated using the equation: ( )0 0 100 m m m m ∞ ∞ − = (1) where m0 is mass of the dry sample and m∞ is the mass of the sample at equilibrium. water diffusion coefficient the diffusion of water into the synthesized materials was modelled by the higuchi equation: t n m kt m ∞ = (2) where mt is the % adsorption by the sample at time t, k is a kinetic constant and n is the diffusion exponent. in case of fickian diffusion, n is 0.5, but in literature values between 0.43 and 0.5 could be found.36 a value of n higher than 0.5 is indicative of an anomalous diffusion mechanism, while in case when n = 1, the diffusion kinetics is zero order, i.e., time-independent. according to the fickian second law, at the initial period of diffusion (mt/m∞), diffusion through a solid material could be calculated using the equation:37 1/ 2 2 2 π tm dt m l∞  =     (3) where d is the effective diffusion coefficient and 2l is the sample thickness. the dependence between mt/m∞ and t1/2 is linear and the diffusion coefficient can be calculated from the linear part using eq. (3). differential scanning calorimetry (dsc) the glass transition temperatures (tg) of the samples were determined by differential scanning calorimetry on a perkin elmer dsc-2 instrument under an inert nitrogen atmosphere. the heating and the cooling rates were 20 °c min-1 in the temperature range of 50 to 200 °c. the measurements were performed in two cycles in order to remove unreacted monomers in the first cycle. all calculations were performed using the data from the second cycle. dynamic mechanical analysis (dma) the dynamic mechanical properties of the samples were tested on a rheometrics mechanical spectrometer 605. the samples were exposed to a constant shear stress of 0.3 % at a frequency of 1 hz. the heating rate was 3 °c min-1 in the temperature range from 25 to 180 °c. the obtained experimental data were as follows: storage modulus (g' / gpa), loss modulus (g" / mpa) and damping factor (tan δ), while the tg was determined as the temperature at which tan δ had a maximum value. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1182 spasojević et al. tensile properties the analysis of the mechanical tensile properties was performed on an instron-1332 (fasttrack 8800 control system, high wycombe, uk) testing machine. the specimens (fig. s-1 of the supplementary material to this paper) were clamped by mechanical jaws. the testing speed was 0.5 mm min-1. for each sample five measurements were performed. the average values of the ultimate stress and deformation at break, as well as the standard deviations, were calculated. during the test, the deformations were continuously registered as a function of the stress. hardness the hardness of derived materials was measured on an instron d-xd durometer (instron, norwood, usa). five repetitions were performed for each sample. impact strength impact strength was measured on charpy pendulum using samples with a v notch (fig. s-2 of the supplementary material). length of pendulum was 60 mm with impact energy of 8.73 j. the impact edge of the cone-shaped pendulum with an angle of 30±1°, rounded with a radius r1= 2±0.5 mm, was made of hard steel. all tests were performed at 23 °c and repeated 5 times. for the calculation of the charpy impact strength for notched specimens, acn / kj m-2, the following equation was used: 3cn n 10 w a hb   =     (4) where w / j is the corrected energy absorbed by the fracture of the specimen, h / mm is the thickness and bn / mm is the remaining width of the specimen at the notch. results and discussion a commercial formulation of denture base material based on pmma was modified with dthfi. the modification was performed in order to obtain a material with better biocompatibility, minor risks for an immune response and preserved significant physicochemical properties. polymerization in moulds was used for the syntheses of the modified denture base materials in which a part of the mma in liquid phase was replaced with specified amounts of itaconate (table i). the samples prepared this way, including pmma and pdthfi homopolymers, were characterized by ftir spectroscopy. the ftir spectra of pmma and pdthfi homopolymers are shown in fig. 1, while the ftir spectra of pmma/pdthfi containing 2.5 and 10 wt. % itaconate are presented in fig. 2. from fig. 1, it could be seen that the ftir spectra of pmma and pdthfi were similar and the characteristic peaks were at the same wavenumbers (3000 and 2950 cm–1 for the c–h stretching vibrations of the methyl group, 1730 cm–1 for the c=o stretching vibrations of the ester group, 1450 and 1300 cm–1 for the asymmetric and symmetric c–h deformation vibrations, 1165 cm–1 for the stretching vibrations of the ester group and at 990, 850 and 750 cm–1 for the out _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ properties of novel denture bases 1183 of ring c–h vibrations). the ftir spectrum of pdthfi contained an absorption peak at 1266 cm–1 (c–o–c asymmetric stretching vibrations) but in the case of pmma, this peak was moved toward slightly a lower wavenumber. from fig. 2, it could be seen that if the amount of dthfi residues in monomer feed was increased, the absorption peak at 1266 cm–1 had a higher intensity; this led to the conclusion that polymerization between mma and dthfi had occurred. fig. 1. ftir spectra of the synthesized pmma and pdthfi homopolymers. fig. 2. ftir spectra of the synthesized copolymers pmma/pdthfi containing 2.5 and 10 wt. % of itaconate. the morphology of the samples of pmma denture base materials modified with dthfi is shown in fig. 3. serious micro defects in the structure could not be observed in the samples, as presented in micrographs of the representative samples modified with 2.5 and 10 wt. % of dthfi (fig. 3). macro pores (>200 μm) were not present in any of the prepared samples, indicating that during the polymerization process, no boiling of the residual monomer occurred and that the mechanical characteristics of the materials were preserved. the obtained results _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1184 spasojević et al. justified the continuation of the testing and detailed characterization of the pmma denture base materials modified with dthfi. fig. 3. sem micrographs of the denture base materials a) pmma/2.5dthfi and b) pmma/10dthfi; bar: 200 μm. the residual monomer contents of the synthesized pmma/pdthfi samples, as well as of the referent pmma sample, were analyzed using high-performance liquid chromatography with ultraviolet detection (table ii). table ii. residual mma and dthfi contents and total residual monomer (trm) content of the pmma and pmma/pdthfi denture base materials sample residual monomer, % trm / % mma dthfi pmma 1.27 – 1.27 pmma/2.5dthfi 0.79 0.95 1.74 pmma/5dthfi 0.62 1.66 2.28 pmma/7.5dthfi 0.27 2.37 2.64 pmma/10dthfi 0.33 2.97 3.30 the results presented in table ii showed that the highest amount of residual mma was found in case of the referent sample and its value was in accordance with literature data.27,38,39 it is clear from table ii that the addition of dthfi decreased the residual mma content in the synthesized samples. considering the lower toxicity of itaconates compared to methacrylates,40 the replacement of mma leads to the safer usage of denture base materials. at the same time, with increasing dthfi content in the copolymer, the amount of residual itaconate increased. this could be explained by the analysis of the kinetic parameters of the copolymerization reaction between mma and dthfi. it was found that the overall rate of copolymerization increased with increasing mma content in the feed composition. this also meant that a greater amount of itaconate in the system would result in a lower overall copolymerization rate and lead to a greater amount of total residual monomer for the same polymerization time. furthermore, the reactivity of mma monomer towards polymer radicals is greater compared to the reactivity of dthfi monomer and therefore, as the copolymerization _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ properties of novel denture bases 1185 reaction proceeded, the ratio of mma to dthfi decreased. from table ii it could easily seen be that even the minimum modification of denture base material with dthfi (2.5 wt. %) led to a decrease in the residual mma content by 37.8 %. modification with higher amounts of itaconate (> 7.5wt. %) led to the minimal residual mma content. the values of the residual mma and dthfi content were similar as in the case of the polymerization reaction between dmi and mma, as well as dbi and mma.35 the use of denture base materials implies absorption of water and others oral fluids. if the absorption is very pronounced, the dimensions of dental prosthesis could be significantly increased so they would not fit properly. the absorbed molecules act as plasticizers and hence they affect the mechanical properties of the material and the rate of aging of the material. furthermore, the fluids absorbed in micropores make the perfect environment for the propagation of many microorganisms. for these reasons, it is very important to determine the mechanism and rate of absorption. the adsorption–desorption characteristics of the denture base material modified with dthfi were investigated (fig. s-3 of the supplementary material). the water diffused from both the top and the bottom surfaces of the samples until equilibrium. many authors have used the weight gain as a parameter defining the capability of a material to absorb water, but this phenomenon needs a more detailed insight. namely, the weight of the samples after desorption were smaller than the initial weight, which indicates that a certain amount of impurities had leached from the sample during the first adsorption cycle. as already mentioned, the denture base materials produced by free radical polymerization have a small percentage of residual monomer as an undesirable part of the product. after the immersion of the samples, two processes occurred – entry of water into the sample, and desorption of residual monomer, water-soluble oligomers and other impurities.27 since the variation in weight is the cumulative result of both the increase in weight due to water penetration, and the decrease in weight due to elution of low-molecular weight components, it is impossible to conclude the exact amount of absorbed water only by measuring the increase in weight. several studies showed that the greatest amount of residual monomers leached during the first seven days of immersion, while the rest leached out over a longer period of time.28 according to this, in the present study, all results concerning the maximum water uptake and the diffusion coefficients were calculated using the data obtained from the second absorption cycle, as it was reasonable to assume that most of the impurities had diffused out from the samples during the first 28 days of absorption. the plots of water uptake in the second absorption cycle for the denture base materials modified with dthfi as a function of t1/2 / h1/2 are shown in figs. 4 and 5. the shape of the curves was the same for all the samples and hence, for _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1186 spasojević et al. better clarity, curves are shown for only three samples. the plots were initially linear with the respect to t1/2 (fig. 5), and thus, it could be concluded that the uptake was diffusion controlled. fig, 4. plot of water uptake in the second absorption cycle as a function of t1/2 for denture base material modified with ■ – 0, ● – 5 and ▲ – 10 % dthfi. fig. 5. plot of initial water uptake of the second absorption cycle as a function of t1/2 for denture base material modified with ◄ – 0, ■ – 2.5, ● – 5, ▲ – 7.5 and▼ – 10 % dthfi. the results for the diffusion exponent (n), the weight loss (δm / %), the maximum degree of absorption (m∞ / %) and desorption (m′∞ / %), as well as the calculated values of the diffusion coefficient for absorption (da / m2 s–1) and desorption (dd / m2 s–1) are summarised in table iii for the denture base materials modified with dthfi. the ratio dd/da is also presented, which is an index of the degree of the concentration dependence of the diffusion coefficient. dd/da > 1 indicates that the diffusion coefficient decreases with concentration. weight loss for the denture base materials (δm) involved a single absorption–desorption cycle. this loss was caused by the leaching of residual monomers, soluble oligomers and other impurities. after the polymerization was completed, residual monomer remained between the macromolecular chains: absorbed into the polymer network or trapped in micropores. furthermore, linear _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ properties of novel denture bases 1187 correlations of the total residual monomer content and weight loss as a function of dthfi content were found (fig, s-4 of the supplementary material). furthermore, the slopes of these linear correlations were almost the same indicating that the weight loss in this kind of materials depended mostly on the total residual monomer content. table iii. kinetic parameter (n), weight loss (δm), the maximum degree of absorption (m∞) and desorption (m′∞), and diffusion coefficients for absorption (da) and desorption (dd) for the studied denture base materials sample n m∞ / % m′∞ / % da×1012 m2 s-1 dd×1012 m2 s-1 dd / da δm / % pmma 0.49 1.87 2.04 3.00 7.27 2.42 0.34 pmma/2.5dthfi 0.47 1.94 2.15 3.27 4.44 1.36 0.75 pmma/5dthfi 0.49 1.93 2.11 3.83 5.32 1.39 1.21 pmma/7.5dthfi 0.53 1.96 2.20 3.76 4.54 1.21 1.49 pmma/10dthfi 0.51 1.98 2.20 4.06 5.32 1.31 1.87 the thermal properties of pmma denture base materials modified with dthfi were investigated via differential scanning calorimetry (fig. s-5 of the supplementary material). the commercial pmma denture base material had a single glass transition temperature (tg) at around 123 °c. the pmma materials modified with dthfi also showed a single tg indicating that copolymers were homogeneous in the range of 10–30 nm.41 substitution of a part of mma with dthfi lowered the values of the glass transition temperature of the polymer. the greater the amount of dthfi, the lower was the tg. however, the observed decrease in the tg was not as pronounced as in the case of modification of pmma denture base materials with monomers such as dimethyl itaconate (dmi) and dibutyl itaconate (dbi).42 this could be attributed to the presence of the tetrahydrofuran ring in the structure of dthfi, which is a not so movable side group. during the use of a denture, it undergoes diverse tensions that appear at different places and have enormously wide magnitude of intensities. the most common reason for denture deterioration is due to its breakage, and therefore, it is of a great importance to investigate the dynamic-mechanical properties of new materials. the dependences of the storage modulus (g′ / gpa) on temperature for all cured samples were of a similar shape. in the temperature range of 25–100 °c, the investigated denture base materials behaved like homogeneous rigid solids (fig. s-6 of the supplementary material). further increase in temperature led to a sharp decrease in the storage modulus, indicating transition from the glassy to the rubbery state. above 160 °c, storage moduli for all samples entered into a plateau. as the operating temperature for dentures is around 37 °c, special attention was paid on to values of the dma parameters precisely at this temperature. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1188 spasojević et al. the values of the storage modulus (g′ / gpa), loss modulus (g″ / mpa) and damping factor (tan δ37) at 37 °c, the damping factor (tan δmax) and glass transition temperatures calculated from dma (tg,dma / °c) and dsc (tg,dsc / °c) measurements for the pmma denture base materials modified with dthfi are listed in table iv. table iv. dynamic-mechanical parameters at 37 °c and glass transition temperatures calculated from dma (tg,dma) and dsc (tg,dsc) measurements for the pmma denture base materials modified with dthfi sample g′ / gpa g″ / mpa tan δ37 tan δmax tg,dsc / °c tg,dma / °c pmma 4.03 359 0.089 1.31 123.2 141.5 pmma/2.5dthfi 4.01 369 0.092 1.34 121.2 140.1 pmma/5dthfi 3.79 451 0.119 1.48 118.9 137.8 pmma/7.5dthfi 3.52 471 0.134 1.58 117.3 136.0 pmma/10dthfi 3.24 509 0.157 1.76 114.6 133.6 the “ideal” material for a denture base should exhibit great rigidity and strength (large g′), moderate deformation under the stress (g″) and the ability to return to the previous shape shortly after removal of a load (low tan δ). it could be noticed in table iv that increasing the dthfi content in the pmma denture base materials led to decreases in the values of the storage modulus and increases in the loss modulus and tan δ. these results indicated reduced stiffness and increased deformation under the load in the case of the materials modified with dthfi, which was due to the plasticizing effect of the side group of the employed itaconate. despite the noticed effect, the addition of small amounts of dthfi (see sample with 2.5 wt. % of dthfi) caused only negligible decreases in g′ (< 1 %) and ensured the same dynamic-mechanical properties as those of the commercial pmma denture base material. it could be noticed that the tg values determined from dma measurements, as temperature corresponding to the maximal value of damping factor (tan δmax), were somewhat higher than those obtained by dsc measurement, but the trend in the change in tg was the same for both methods. a similar observation was reported earlier.42 the differences in the tg values arise from the fact that the tg,dsc represents the temperature at which the material undergoes the maximum change in polymer chain mobility, which corresponds to the chemical definition of the tg, while tg,dma describes the damping characteristics of the material and has historical significance. as previously presented, the modified materials had better biocompatibility than the commercial pmma denture base material, but in order to prevent denture breakage during use, it was also necessary that they possess appropriate mechanical properties. for this reason, for all the modified and the commercial pmma denture base materials, the ultimate tensile strength (σult / mpa), elon_________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ properties of novel denture bases 1189 gation at break (ε / %), the shore d hardness and the charpy impact strength were determined and are listed in table v. table v. values of the ultimate tensile strength (σult), elongation at break (ε), shore d hardness and charpy impact strength of the investigated denture base materials sample σult / mpa ε / % shore d charpy, j cm-2 pmma 63.8±2.3 6.26±0.55 96.0±0.37 0.285±0.03 pmma/2.5pdthfi 63.7±2.5 6.3±0.31 96.3±0.17 0.313±0.02 pmma/5pdthfi 63.5±3.2 10.8±0.27 96.1±0.37 0.304±0.04 pmma/7.5pdthfi 64.2±1.7 9.5±0.35 94.8±0.28 0.295±0.03 pmma/10pdthfi 62.5±4.2 9.4±0.37 90.2±0.49 0.287±0.05 the tensile properties of modified denture base materials were investigated on an instron testing machine. modification of commercial pmma denture base material with dthfi gave materials with slightly lower values of the ultimate tensile strength. on the other hand, the modified materials elongated more at break. this behaviour could be explained by the fact that the dthfi molecule possesses an oxygen atom in its structure that might establish hydrogen bonds with hydrogen atoms. formed h-bonds were very weak and had no effect on the values of stress at break but affected the values of elongation at break. it should be noted that for all samples, the values of tensile strength were within the framework of the prescribed standards.43 the mean values of the shore d hardness for pmma denture base materials modified with dthfi ranged between 90.2 and 96.3 and thus fulfilled the required hardness values for denture base materials, as prescribed by the american dental standards institute.43 bearing in mind that many material properties, such as composition, surface porosity, residual monomer concentration, etc., affect its hardness, it could be concluded that investigated modifications of pmma denture base material did not have an effect on the shore d hardness. inclusion of dthfi in commercial pmma denture base formulation led to increase in the impact resistance of prepared samples (table v) except in the case of the sample with the highest dthfi content (10 wt. %). it was observed in all impact tests experiments that the specimens broke with a sharp fracture, exhibiting typical brittle fracture behaviour characterized by a lack of distortion of the broken parts. conclusions in this study, an itaconate (a sustainable, non-petrochemical raw material) was used as a co-monomer in the preparation of denture base materials. quantities of 2.5; 5.0; 7.5 and 10 wt. % of methyl methacrylate were replaced by ditetrahydrofurfuryl itaconate. copolymerization of mma and dthfi was confirmed by ftir, and dsc analysis. using liquid chromatography, it was found that the addition of itaconate led to a reduction in the amount of residual mma _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 1190 spasojević et al. and, therefore, made the base material significantly more biocompatible and drastically reduced the risk of a variety of immune responses. the glass transition temperatures of the synthesized samples were shifted to lower values, indicating that the side groups of dthfi acted as plasticizers. by dma, it was found that increasing the dthfi content led to a lowering the values of the storage modulus and stress at break. however, analysis of the mechanical properties showed that all the modified materials possessed characteristics prescribed by ada standards, and could be used in practice. the magnitude of the measured values indicated that the pmma denture base materials modified with dthfi could be developed into a less toxic, more environmentally and patient-friendly product than commercial pure pmma denture base material. the optimal mechanical properties were exhibited by the sample with the minimal dthfi modification (2.5 wt. %). supplementary material the geometry of the specimens and additional analysis are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the authors acknowledge funding from the ministry of education, science and technological development of the republic of serbia, project no. 172062 “synthesis and characterization of novel functional polymers and polymeric nanomaterials”. и з в о д поли(метилметакрилатни) материјали за базу протеза модификовани дитетрахидрофурфурил-итаконатом: својства важна за примену павле спасојевић1, весна панић1, сања шешлија2, владимир николић3, иванка г. поповић4 и сава величковић4 1иновациони центар, технолошко–металуршки факултет, универзитет у београду, карнегијева 4, 11000 београд, 2институт за хемију, технологију и металургију, универзитет у београду, његошева 12, 11000 београд, 3иновациони центар, хемијски факултет, универзитет у београду, студентски трг 12–16, 11000 београд и 4технолошко–металуршки факултет, универзитет у београду, карнегијева 4, 11000 београд циљ овог рада је испитивање могућности модификације комерцијалних система за добијање материјала за базу зубних протеза естрима итаконске киселине, ради добијања материјала смањене токсичности и већег степена биокомпатибилности. упркос већој цени итаконата у односу на метакрилате они се често користе у “зеленим” системима збoг чињенице да се добијају из биообновљивих, а не петрохемијских извора. комерцијални систем на бази поли(метил-метакрилата) (pmma) модификован је дитетрахидрофурфурил-итаконатом (dthfi), при чему је вариран удео dthfi од 2,5 до 10 мас. %. фтир спектроскопијом узорака утврђено је да је у систему дошло до кополимеризације, док је sem анализа потврдила одсуство озбиљнијих микродефеката и пора у структури. испитан је утицај итаконата на: апсорпцију течности (вода), количину заосталог мономера, као и термичке, динамичко-механичке и механичке карактеристике (тврдоћа, жилавост, напон и издужење при кидању) синтетисаних материјала. утврђено је да додатак итаконата у састав комерцијалних система значајно смањује количину заосталог метил-метакрилата што материјал чини биокомпатибилнијим. синтетисани узорци су _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ properties of novel denture bases 1191 имали задовољавајуће механичке карактеристике. нађено је да са повећањем удела dthfi долази до смањења вредности температуре остакљивања, модула сачуване енергије, напона кидања и ударне жилавости, међутим механичке карактеристике су и даље у границама прописаним аdа стандардима тако да се нови материјали могу користити у пракси. 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philadelphia, pa, 1994, p. 114 43. ada12-2002: ansi/ada specification no. 12 for denture base resins, 2002. _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {correction of the paper published in journal of the serbian chemical society 76 (2011) 987} j. serb. chem. soc. 82 (11) 1333 (2017) udc 582.293.378+634.22:543.613.3 jscs–5043 letters to the editor 1333 letter to the editor correction of the paper published in journal of the serbian chemical society 76 (2011) 987 gordana s. stojanović* department of chemistry, faculty of science and mathematics, university of niš, višegradska 33, 18000 niš, serbia (received 14 june, accepted 22 june 2017) dear editor, we would be very grateful if you could publish corrections considering the paper volatile constituents of selected parmeliaceae lichens, by i. ž. stojanović, n. s. radulović, t. lj. mitrović, s. m. stamenković, g. s. stojanović, published in j. serb. chem. soc. 76 (2011) 987. the original article was published with the incorrect names of the two identified compounds due to the authors’ oversight. although after a long time, the authors apologize to the editor and readers. the corrections are as follows: in the abstract, page 987 line 8: incorrect: olivetonide; correct: 3-hydroxy olivetonide in the table i, page 992: − incorrect: 6,8-dihydroxy-3-pentylisochromen-1-one; correct: 6,7,8-trihydroxy-3-pentylisochromen-1-one − incorrect: 2,4-dihydroxy-6-(2-oxoheptyl)benzoic acid; correct: 6,8-dihydroxy-3-pentylisochromen-1-one − incorrect: olivetonide; correct: 3-hydroxy olivetonide − incorrect: olivetonic acid; correct: olivetonide page 991, last line: − incorrect: olivetonide; correct: 3-hydroxy olivetonide − incorrect : olivetonic acid; correct: olivetonide page 993, in the conclusions, line 5: incorrect: olivetonide; correct: 3-hydroxy olivetonide page 994: in извод, line 5: incorrect: olivetonide; correct 3-hidroksiolivetonid once again, on behalf of all authors, my apologies. gordana stojanović * corresponding author. e-mail: stgocaus@yahoo.com https://doi.org/10.2298/jsc170614067s ________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {journal of the serbian chemical society} j. serb. chem. soc. 82 (4) 465–468 (2017) udc 543+061.3(4) euchems news division of analytical chemistry european association for chemical and molecular sciences 465 euchems news european analytical column no. 45 wolfgang buchberger1 and slavica ražić2*# 1analytical chemistry, university linz, altenbergerstrasse 69, 4040 linz, austria and 2department of analytical chemistry, faculty of pharmacy, university of belgrade, vojvode stepe 450, 11222 belgrade, serbia information from the euchems division of analytical chemistry (dac) the impact of analytical chemistry on progress in science is significant. vice versa, progress in analytical chemistry is essential to provide the necessary input to other research fields. the division of analytical chemistry (dac) can be seen as a common platform for activities initiated by the member societies of euchems. concerted actions will be much more efficient than a variety of isolated activities. dac has started the year 2017 with slavica ražić (university of belgrade) as the new chair. she will be supported by the members of the steering committee consisting of jiri barek (czech republic) as treasurer, wolfgang buchberger (austria) as secretary, sibel özkan (turkey), christian rolando (france), charlotta turner (sweden), and paul worsfold (united kingdom) as immediate past chair. at the annual meeting 2016 of dac in seville the delegates expressed their sincere thanks to paul worsfold for his highly efficient and most successful work as chair of dac from 2011 to 2016. this year the main dac event will be euroanalysis xix to be held in stockholm from 28 august to 1 september 2017 (http://euroanalysis2017.se). along the lines of the long established tradition of euroanalysis that started in heidelberg 1972, the meeting will cover all aspects where analytical chemistry plays a role, including fundamental and applied sciences. it has become a premier european meeting for the exchange of new ideas in analytical sciences. the organ * e-mails: wolfgang.buchberger@jku.at (w. buchberger); slavica.razic@pharmacy.bg.ac.rs (s. ražić) # serbian chemical society member. 466 buchberger and ražić izing committee chaired by charlotta turner and jonas bergquist invites the analytical community to participate and to present their most recent and innovative research work. various activities are initiated and carried out within the study groups of dac. in this european analytical column the current activities are summarized, which should stimulate input from analytical chemists potentially interested in cooperation with these study groups. study groups of dac the objectives of the study group “analytical chemistry in archaeology” (head: prof. miltiades karayannis, greece) are to demonstrate the importance of analytical chemistry for the collection of scientific information valuable to archaeologists and art historians in order to investigate, date and authenticate archaeological objects and works of fine art. the study group seeks to explore and propose methods for these purposes, and to recommend to archaeologists the proper analytical techniques for sampling, analysis, and evaluation of the data. collaboration and the exchange of ideas of analytical chemists between relevant european scientific laboratories will be strengthened and new trends in the scientific activities and publications in this area identified. the study group “bioanalytics” (head: prof. györgy horvai, hungary) aims to bring the analytical and the bioanalytical chemistry communities closer together. this is still a formidable task because a structured community of bioanalytical chemists does not appear to have formed yet. an indication of change is the fact that analytical journals publish more and more bioanalytical papers, including contributions from european authors. the programme of the 2016 euchems chemistry congress in seville included a session dedicated to bioanalytics. the main (invited) lecture in that session was presented by prof. jonas bergquist from sweden. in another session the invited lecture given by prof. romà tauler discussed chemometrics techniques applied to high throughput omics. members of the study group have closely followed bioanalytical events and the development of bioanalytical curricula at universities in several european countries. the euroanalysis conference 2017 will present an opportunity for establishing contact with colleagues involved in bioanalytics but not yet active in dac. the aims of the study group “chemometrics” (head: prof. romà tauler, spain) include the promotion of chemometrics in europe, the organization of chemometrics sessions at the euroanalysis conference and at other euchems -related conferences, and the realization of european schools and workshops on chemometrics. for the year 2017 two main activities are envisaged. first, the members of the study group are preparing a tutorial on chemometrics for the journal analytical and bioanalytical chemistry, consisting of two separate papers. the other activity is the preparation of the chemometrics session at the european analytical column no. 44 467 euroanalysis conference in stockholm where, in addition, a chemometrics course will be offered. the study group “education” (head: prof. reiner salzer, germany) investigates successful new approaches in teaching analytical chemistry. some approaches are based on didactic advances, some on reformed contents, and others introduce advanced digital tools. members of the study group also participate actively in establishing quality standards for the curricula in chemistry coursework. outstanding new approaches will be discussed at the symposium education during the euroanalysis conference in stockholm (scheduled for tuesday, 29th august 2017). everybody is invited to participate in the discussion or to get inspirations for her/his own teaching duties. the aim of the study group “history” (head: prof. d.t.burns, uk) is to study and publish accounts of the history of analytical chemistry in europe with reference to individual countries, specific locations, professional bodies and personalities. six recent publications can be found in the annual report 2016 on the dac website (http://www.euchems.eu/divisions/analytical-chemistry). meanwhile, the material on the history of the adulteration of coffee has been published (d. t. burns, l. tweed, m. j. walker, “ground roast coffee: review of analytical strategies to estimate the geographic origin, species authenticity and adulteration by dilution”, food anal. methods (2017), doi: 10.1007/s12161-016-0756 -3). other interesting publications to be mentioned include an article about paul coremans (1908–1965), a pioneer chemist in the application of scientific techniques to the visual arts (in press), and an account of the contributions of g. w. monier-williams to the improvement in quality and purity of foodstuffs (in preparation). monier-williams is best known for his method for the determination of sulphite in foods first published in 1927 but still in current use as a referee method. the study group “quality assurance” (head: prof. elin gjengedal, norway) follows the development of quality assurance and metrology in analytical chemistry and produces guidelines for assessing the quality of results, which ultimately can create a new basis for decision-making that may have an impact on the way research is carried out. the existing link to eurachem (https:// //www.eurachem.org) is particularly important, connecting the study group with a network of organisations in europe having the objective of establishing a system for the international traceability of chemical measurements and the promotion of good quality practices. among other things, eurachem is publishing guides that are reviewed five years after publication to assess the need for revision. recently, the eurachem education and training working group issued a new third edition of the well-known eurachem/citac “guide to quality in analytical chemistry an aid to accreditation”. in 2014, the second edition of “the fitness for purpose of analytical methods: a laboratory guide to method 468 buchberger and ražić validation and related topics” was published. this guide was first issued in 1998, and has over the years been one of the most popular of the eurachem guides. the working language of eurachem is english; however, thanks to the hard work of members of eurachem, the guides and leaflets are available in a number of other languages. activities planned for the dac study group “quality assurance” in 2017 include the continued input to eurachem, the publication of results of quality 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice j. serb. chem. soc. 81 (12) 1485–1490 (2016) 1485 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. author index abdelmalek, i., 1183 abdollahi, l., 91 abootalebi, m., 1069 acharjee, k., 1151 adebayo, a., 935 adekunle, o., 1251 agatonovic-kustrin, s., 1037 albayrak, e., 729 almeida, r. t., 1273 alosmanov, r., 907 anđelković, d., 255 anđelković, i., 403 anđelković, k., 1383 anđelković, t., 255 anders, b., 1 andoni, m., 177 andrejić, g., 447 andrić, d., 347 andrić, v., 813, 1415 aničić, n., 999 antić, m. p., 403, 1081, 1441 antić-stanković, j., 103 aonyas, m. m., 1097 ashok, d., 851 asli, m. a. n., 91 atbakar, m., 1121 azarin, k., 125 azizan, j., 29 bacchi, a., 1383 bagheri, f., 1111 bai, m., bajić, z., 947 bajuk-bogdanović, d., 777 bakare, o., 1251 bakirdere, e. g., 509 banjanac, k., 1371 bao, j., 1215 basić, z., 1141 baskaran, s., 859 batovska, d. i., 1231 battula, k., 233 bezbradica, d., 1371 bing, s., 209 bîrdeanu, a.-v., 163 birdeanu, m. i., 163 bogdanov, j., 947 bojić, a., 255 boljević, j., 999 bollepelli, p., 233 božić, a., 979 božić, d., 999 božinović, n., 1225 branković, g., 553 brkić, d., 979 brković, d. v., 589 brown, m. e., 1037 bubić-pajić, n. n., 1171 bučko, s., 35, 575 budimir, m., 777 budnyk, a., 751 bui, h. m., 959 bukhari, s. m., 1 bursać, m., 1239 bursić, v. p., 499, 707, 923 buruiana, e. c., 307 buruiana, t., 1055 butcher, r. j., 729, 1251 canpolat, e., 509 cao, y., 1215 capucci, d., 1383 carević, m., 1371 căta, a., 177 1486 journal of the serbian chemical society available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. cheng, h., 739 chiavelli, l. u., 1273 chibac, a. l., 1055 ciobanu, c. i., 133 ciobanu, c. s., 433 claus, t., 1273 costa, w. f., 1273 cvejić, j., 1239 cvetković, t., 255 čairović, a. d., 1307 čobeljić, b., 1383 dabić, d. č., 567 damjanov, n., 1127 damjanović, a., 103, 487 damjanović, lj., 897, 1415 danac, r., 133 das, a., 1151 das, k. c., 271 djokic, l., 871 dmitrović, s., 999 dobričić, v., 1171 dojčinović, b. p., 607, 829, 1097 dolić, s. d., 829, 1097 dondur, v., 897 drmanić, s. ž., 219, 589,979, 1199 dubrovina, v. a., 645 duteanu, n., 323 đorđević, n., 589 đorđević, t. m., 923 đorđević, t., 459 đuranović, d. 1441 đurić, s., 1239 đurović-pejčev, r. d., 923 el-asmy, a. a., 57 epurescu, g., 1055 erić, s., 103 eshaflu, h., 979 etinski, m., 1393 fagadar-cosma, e., 163 fagadar-cosma, g., 163 faraji, m., 1161 farajtabar, a., 1161 farmanzadeh, d., 277 fellah, m. f., 509 feuerherm, a. j., 1 figueiredo, l. c., 1273 filipović, j., 1013 filipović, v., 999 fincan, g. s. o., 729 florea, c. a., 475 fraj, j., 575 francuski, đ., 871 frem, d., 687 fuglset, e., 1 furtula, b., 271 gajica, g. đ. 459 galunsky, b., 1231 gašić, u. m., 567 ghasemnejad, h., 67 glass, b., 1037 gođevac, d., 883 golubchikov, o., 1025 gontier, e., 1183 gonzalez, s.v., 1 graf, h.-l., 799 grgur, b., 419 grgurić-šipka, s., 897 grozdanić, n., 789 grozescu, i., 323 grubin, j., 447 grujić letić, n., 333 gunduz, m. g., 729 gür, s., 509 gutman, i., 81, 271 haghdadi, m., 67 hansman, š., 707 he, h., 141 he, w., 187 heli, h., 395 holclajtner-antunović, i., 777 horvat, s., 717, 1455 hosny, n. m., 57 hrin, t. n., 717, 1455 hristov, n., 499 hu, x., 697 ibrahim, r., 57 ienaşcu, i., 177 ignjatović, lj., 403, 813, 1081, 1441 vol. 79: author index 1487 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. ilić, m., 1081 inđić, d. r., 829 ion, i., 1055 isaković, a., 1127 isaksen, s. m., 1 isli, f., 729 ito, t., 47 ivanković, n., 947 ivkov, m., 1013 jadranin, m., 487 janaćković, đ., 197,1295 jančić, d., 1141 janković častvan, i. j., 197, 1295 jevtić, v. v., 1383 jiao, l., 209 jitaru, f., 1055 johansen, b., 1 jokić, b. m., 291, 197 josić, lj., 459 jovančićević, b., 459 jovanović, d., 947 jovanović, s., 777 jovanović, u., 813 juranić, i., 487 juranić, z., 487 kalinić, m., 103 kaluđerović, m. r., 799 karkalić, r., 947 kašanin-grubin, m., 459 katona, j., 35, 575 katsikini, m., 1321 kavitha, r., 851 kaya, m., 509 kazemi-rad, r., 29 kefayati, h., 29 khaishbashev, g., 751 khorshidi, a., 1069 kijevčanin, m., 789, 1427 kiprovski, b., 499, 1239 koifman, o., 1025 kojić, m., 1393 kondić-špika, a., 499 kostić, i., 255 kostić-rajačić, s., 347 košutić, m., 1013 krstić, s., 219 kudryavtsev, e., 751 kuraica, m. m., 829 kuzmin, i., 1025 lashanizadegan, m., 153, 369 lastovina, t., 751 lazarević, a., 459 lazarević, s., 197, 1295 lazic, j., 871 lebete, m., 1037 li, h., 209 li, j., 521, 1263 li, y., 739 li, y.-w., 23 liang, l., 739 liese, a., 1231 lin, h., 141 liu, h., 13 liu, l., 1215 liu, q., 141 liu, x., 697 lu, x., 633 lv x., 13 ma, c.-l., 23 ma, k., 697 majlesi, k., 541 majstorović, d. m., 1427 maksimović, v. m., 1307 malenčić, đ., 499, 1239 manesh, a. a., 125 mangalagiu, i. i., 133 manojlović, d. d., 829, 1097 mao, h., 13 margetić, a., 1359 marić, i., 1335 marić-stojanović, m., 1415 marimuthu, d., 859 marinković, a. d., 219, 589, 979, 1199 marković, b., 1171 marković, m. d., 1097 marković, n., 883 marković, z., 777 maruyama, s. a., 1273 maslak, v., 871 matarneh, c. m. a., 133 matić, r., 459 matović, r., 1335 1488 journal of the serbian chemical society available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. mayzlish, v., 1025 medeleanu, m., 177 mehandzhiyski, a. y., 1231 menković, n., 607, 883 mesli, a., 1183 mihailović, m., 1371 mihailović, n., 447 mihajlović-lalić, lj. e., 897 milanović, m., 333 milenković, d. d., 717, 1455 milenković, m. r., 1383 milenković, m., 459 milić, n., 333 milić, s., 707 milinković, j., 575 milojković-opsenica, d. m., 567 milosavić, n., 1371 milosavljevic, m., 219 minato, m., 47 ming, j., 633 mioč, u. b., 1415 misaelides, p., 1321 mišić, d., 999 misirlić-denčić, s., 1127 mitrev, y. n., 1231 morton, d. w., 1037 mouffok, m., 1183 mousavi, s., 67 murariu, m., 307 mutić, j. j., 567 nagavelli, v., 233 najafi, m., 277 narsimha, s., 233 nastasović, a., 419 nataša bubić-pajić, n., 1171 natić, m. m., 567 nechifor, m., 673 nidhin, p. v., 763 nikodinović-runić, j., 871 nikolić, g., 255 nikolić, j. b., 219, 589, 979, 1199 nikolić, n. d., 291, 553 nikolić, r., 255 nikolić, v. d., 979 nikolić-đurović, m., 487 noli, f., 1321 novakovic, i., 347, 1345 obradović, b. m., 829, 1097 ocal, n., 1121 odunola, o., 1251 olyaei, a., 1111 onjia, a. e., 419, 661 opsenica, d., 999 opsenica, i. a., 1225 orhan, i. e., 357 oveisi, m., 91 pan, l., 623 pan, y., 935 pandit, b. k., 1283 pantelić, n., 487 peng, j.-q., 623 penjišević, j. z., 347 pergal, m. v., 829 perišić, n., 1415 petković, m., 1393 petride, h., 475 petronijević, n., 1127 petrović, l., 35, 575 petrović, r., 197, 1295 petrović, s., 219 pezo, l., 813, 1013 pinakidou, f., 1321 plavšić, d., 1013 pop, r. o., 177 popa, c. l., 433 popov, k. i., 291 popović, lj., 35 popović, z., 459 pourbahman, f., 395 predoi, d., 433 pržić, z., 883 pudar, g., 487 qi, y.-w., 623 radonić, j., 333 radovanović, f., 419 radović, i., 789 rajaković, lj. v., 403, 661 rakić, t., 447 ramaswamy, v., 859 rančić, m., 589, 1199 rao, m. s., 233 vol. 79: author index 1489 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. rao, v. h., 851 rašković, s., 487 razumov, m., 1025 ren, j.-g., 47 ren, x.-l., 623 rezvani, m. a., 91 roglić, g. m., 347, 1097 rušmirović, j., 1199 sabo, t., 897 sæther, m., 1 safak, c., 729 saha, s., 1151 saičić r. n., 1335 sarioglu, y., 729 sarkar, a., 1283 sarkheil, m., 153, 369 sarri, s., 1321 sattarahmady, n., 395 savić biserčić, m., 813 savić, a., 813 savić, a., 897 savić, s., 1171 savić, v., 103 schmid, r., 1 schreckenbach, j. p., 799 sebarchievici, i., 163 segedinac, m. d., 717, 1455 sekulić, m. t., 333 sekulić, p., 707 setamdideh, d., 971 shariati, s., 1069 shayegan, s., 153 sikora, v., 1239 simić, m. r., 103 simić, n., 1 simić-pašalić, k., 1127 simonova, t. n., 645 simsek, r., 729 singaram, k., 859 sinha, b., 1151 sinha, b., 1283 sinzar-sekulić, j., 447 skalicka-woźniak, k., 357 skorić, m., 999 sladić, d., 1345, 3764 soldatov, a., 751 soldatović, d., 789 spasic, j., 871 sredović ignjatović, i., 403, 813, 1081, 1441 srinivasan, p., 763 stanimirović, a. m., 1427 stanković, d. m., 567 stephen, a. d., 763 stevanović, b., 447 stevanović, j., 847 stevanović, s. i., 553 stoiljković, m. m., 1307 stojanović, d., 1295 stojković, d., 1383 stojković, t., 1127 striković, v., 947 subirade, m., 739 sudji, j., 333 sukalovic, v., 347 šavikin, k., 607, 883 šefik-bukilica, m., 1127 šobajić, s., 1141 šolaja, b. a., 1225 šumar-ristović, m., 897 šoškić, v., 347 šukalović, v., 347 ştefănuţ, m., 177 taleb, k., 1199 tan, x., 187 tanasie, c., 177 taranu, b., 163 taranu, i., 163 tasić, a., 403, 1081, 1441 tasić, g., 103 tešić, ž. lj., 567 thvedt, t. h., 1 todorović, v., 1141 todorović, ž. n., 661 todorović-marković, b., 777 tokic-vujosevic, z., 871 tomković, t., 419 tomović, g., 447 tong, j.-b., 383 topbaştekin, o., 1121 tosti, t. b., 829 tosun, f., 357 trbojević-ivić, j., 1371 1490 journal of the serbian chemical society available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. trifunović, s., 1383 trifunović-macedoljan, j., 487 tu, y., 13 tufegdžić, s., 1345 tunset, h. m., 1 uskoković, p. s., 589 vaida, m., 323 vashurin, a., 1025 vasiljević-radović, d., 419 vasin, j., 707 vaštag, ž. g., 35 veličković, d., 1371 veličković, z., 947, 1199 velikić, z., 1407 veselinović, d., 1407 vidoeski, b., 777 vilipić, j., 1345 višak, z., 789 vishnikin, a. b., 645 vladimirov, s., 1171 vojinović miloradov, m., 333 vojinović, j., 1127 vujčić, m., 1345 vujčić, z., 1359 vukić, m., 1383 vukićević, i., 219 vuković, g., 499 vuković, n., 1383 vuleta, g., 1171 vulović, b., 1335 wang x., 13 wang, f., 697 wang, j., 243 wang, l., 521, 1263 wang, n., 187 wang, s., 935 wang, x., 1215 wang, x., 1263 warchol, j., 1321 wei, w., 623 wu, w., 1263 xu, j., 697 yan, j., 141 yan, j., 697 yang, j., 141 yang, r., 141 yang, s., 935 yang, x., 697 yao, m. q., 1215 ye, j., 697 yildirim, s. o., 729 yildirim, s., 729 yin, x., 739 zamboulis, d., 1321 zdunić, g., 607, 883 zeighaminezhad, s., 541 zeremski, t., 707 zhang, h., 187 zhang, x., 209 zhang, y., 521 zhao x.-h., 243 zhao, x., zhong z., 13 zhu, b. x., 623 zlatković, b., 1 zlatović, m., 1345 živanović-radnić, t., 1127 živković, e. m., 1427 živković, j., 607, 883 živković, p. m., 291, 553, 1295 živković, p., 1295 subject index of vol. 81 and list of referees in 2016 are given in the electronic form at the internet address of the journal of the serbian chemical society: http://www.shd.org.rs/jscs/vol79/no12.html end of volume 81. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice j. serb. chem. soc. 82 (12) 1489–1494 (2017) 1489 author index abasaeed, a. e., 921 abdollahifar, m., 203 abedi, d., 841 abramović, b. f., 343 aftab, a., 905 ahmad, s., 151 ahmad, u., 151 ahrenkiel, p. s., 437 ajdačić, v., 641 ajduković, m., 449 akhtar, m. s., 943 akilandeswari, l., 1123 aksela, m. k., 1193 aleksić sabo, v., 651 aleksić, i., 1357 aleksić, n. m., 1461 aly, r., 509 amiri, s. s., 427 anačkov, g., 803 andrade, r. s., 1155 antić, m., 1395 antić, v., 1395 antonijević, m. d., 1461 apisarov, a. p., 51 ardakani, m. m., 1273 ashok, d., 117, 241, 357 asija, s., 13 babić, b., 1259 bahar, s., 317 bajić, z., 1175 banerjee, b., 755 banić, n. d., 343 banković, p., 449 baroud, a. a., 267 battula, k., 1 beara, i., 651 bechtel, a., 739 bedda, k., 107 bedilo, a. f., 523 behera, a. k., 985 benvidi, a., 1273 bjelogrlić, s., 495 bogdanović, g., 437 bogunović, m., 1445 božić, a., 495 božin, b., 803 branković, g., 539 buchberger, w., 465 budeanu, m. m., 891 budimirović, d., 1175 cakić, m., 1009 cao, y., 399 cardona-villada, w., 25 carević, m. b., 1223 carreras, a., 1155 chen, m., 579 chukhno, n., 791 coskun, d., 367 coskun, m. f., 367 cvejanov, j., 711 cvetković, d. j., 1009 cvetković, o., 739 cvijetić, i. n., 303 červenski, j. f., 1237 četojević-simin, d., 651 danojević, d., 1237 dapčević, a., 1433 darmawan, a., 921 davidović, s. z., 1223 dehestani, m., 289 dekanski, a., 411 del sol rúa, m., 417 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1490 journal of the serbian chemical society deniz, s., 215 despotović, v. n., 343 devetak, i., 1193 dimitrijević-branković, s. i., 1223 dimitrova pascova, r., 227 doroodi, z., 681 dragičević, v. d., 63 drakulić, b. j., 303 drašković, n. s., 289, 1357 drmanić, s., 1175, 495 dumitrescu, v., 891 dzunuzović, e. s., 437 đuran, m. i., 289 đokić-stojanović, d. r., 1039 đoković, n., 739 đorđević, a., 141 đorđević, b. d., 1379 đorđević, b. s., 1039 đorđević, i. s., 825 đorđević, j., 1259 đorđievski, s., 1147 đošić, m. s., 329 đukić, n. h., 379 elizalde, m. p., 417 elsamra, r. m. i., 851 emire, z., 99 eraković, s., 695 eren, b., 277 faiqa, n., 905 faraj, f. a. m., 1315 feng, d., 579 filipović, j. s., 253, 1097 filipović, n. r., 825, 495 filipović, v., 1097, 253 finčur, n. l., 343 florea, c. a., 627 gagnon, j., 509 gajica, g., 593, 1461 garg, s., 905 garrab, m., 509 gavarić, n., 803 gavrilović, m., 803 girija, k. k. n., 1087 gizir, a. m., 99 glišić, b. dj., 289, 1357 glossman-mitnik, d., 25 golovko, a., 1063 görmez, ö., 99 grgurić šipka, s. r., 267 grishenkova, o. v., 51 grozdanić, n., 1379 grubišić, s., 825 gruden, m., 1369 grujić, s. r., 329 gržetić, i., 723 gundu, s., 357 guo, y., 579 gur, s., 367 gurkan, y. y., 277 hafsa, m. b., 509 hamada, b., 107 hassan, s., 943 hemdan, s. s., 851 hidaryan, m., 203 hîrtopeanu, a., 627 huebra, m., 417 iglesias, m., 1155 ignjatović, n. l., 607 imanbayev, y., 1063 imran, m., 943 isaev, v. a., 51 isakov, a. v., 51 ismail, m. b., 509 ivančev-tumbas, i., 1445 ivanović, s., 1369 ivković pavlović, s., 1315 jafari, p., 203 jagličić, z., 1247 jahanbani, s., 1273 jang, g.-y., 1025 janjić, s. d., 83 jarić, s., 723 jin, y., 579 jindal, d. k., 995 jokić, b., 1259 jovančičević, b., 593, 1315, 1461 jovanov, p., 1343 jovanović, p. m., 1335 jovanović, v., 593 jović-jovičić, n., 449 ju, x.-h., 163 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. vol. 82: author index 1491 jugović, d., 607 juranić, i. o., 303 kahraman, m. v., 215 kajzerberger, m., 267 kalijadis, a., 1259, 1417 kalpana, a., 1123 karadžić, b., 723 karanović, lj., 1433 kašanin-grubin, m., 593 kaushik, c. p., 995 kavitha, r., 357 kermani, e. t., 483 khaled, e., 865 khan, k. s., 943 khatkar, p., 13 kijevčanin, m. lj., 1287, 1379 kiran, k., 241 kladar, n., 803 knezević, v. m., 253 knežević, d., 379 knežević, v., 1445 konststinović, s. s., 1039 kostić, m., 83, 1417 kostić, o., 723 košević, m., 411 kovač, t. s., 437 kovačević, n. n., 815 kralj, m., 411 kraljić roković, m., 411 kravić, n. b., 63 krivtsov, e., 1063 krogstad, t., 1303 krstić, v., 1147 kumar, a., 127, 995 kumar, d., 995 kumar, k., 995 kurmakyzy, r., 791 kuzichkin, n. v., 107 kuzmanović, m., 1135 lakshmi, b. v., 117 lakušić, b. s., 815 lakušić, d. v., 815 lal, b., 905 laušević, m. d., 1417 laušević, z. v., 1417 lazarević, m. j., 343 lazarević, n., 957 lazić, b. d., 83 lazić, j., 641 lazić, v. m., 1223 leghaei, v., 1211 leković, s. v., 379 lesjak, m., 651 li, j., 579 li, k.-n., 1025 li, x., 579 li, y.-y., 1025 liu, l., 39 lolić, a., 825 lončar, b. lj., 253 lv, j., 73 maheswari, p. r., 1087 maletić, s. p., 343 mandić, a., 1343 manojlović, d. d., 303, 933 mansurov, z., 1063 marčetić, m. d., 815 marinković, a., 1175, 495 marinović, d., 593 marinović, s., 449 marković, d., 1009 marković, m., 723 marković, s., 607 marković, s. m., 379 masoud, m. s., 851 matijašević, s. d., 329 mehmood, a., 943 mendoza-huizar, l. h., 25 menoyo, b., 417 mesarović, j. z., 63 metelka, r., 865 mihajlović-lalić, lj. e., 267 mihajlovski, k. r., 1223 mijin, d., 695 mikhailovskaya, t., 791 miladinović, j., 1433 milićević, z., 593 milonjić, s. k., 1075 milošević, d., 1175 milutinović-nikolić, a., 449 mimica-dukić, n., 651 mishakov, i. v., 523 mišan, a., 1343 mitić, v., 141 mitrović, d., 739 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1492 journal of the serbian chemical society mitrović, m., 723 mladenović drinić, s. d., 63 moeini, v., 681 mohil, r., 127 mojičević, m., 641 momčilović, m., 1135 mor, s., 127, 995 moradi, s. e., 551 moradi, s. m. j., 551 mrkonjić, z., 651 mudrinić, t., 449 nađpal, j., 651 nagavelli, v. r., 1 naghmouchi, k., 509 nagoria, s., 127 nallathambi, g., 567 naqvi, s. m. s., 943 narsimha, s., 1 nasrollahpour, a., 551 neamatollahi, d., 1053 nedeljković, j. m., 437 nedeljković, n., 1343 nedić-vasiljević, b., 449 nezam, f., 317 nićetin, m. r., 253 niketić, v., 955 nikodinović-runić, j., 289, 641, 1357 nikolić, j. b. 495, 1175 nikolić, j. d., 329 nikolić, n. d., 539 novaković, i., 495 nytoft, h. p., 1315 ongarbayev, y., 1063 opačić, m., 957 opsenica, i., 641 pacheco, a. g., 25 paczosa-bator, b., 879 padhy, g. k., 985 pajić-lijaković, i. s., 1417 panda, j., 985 panić, v., 695 papp, z., 1259 pavelkić, v., 695 pavlović, d., 723 pavlović, j. b., 1303 pavlović, m. g., 539 pavlović, m., 723 pavlović, p., 723 pejić, b. m., 1417 pergal, m., 1395 pestorić, m., 1343 petković, m. r., 1335 petride, h., 627 petrović, j., 1147 pezo, l. l., 253, 1343 piech, r., 879 poleti, d., 1247, 1433 portalone, g., 825 pouramiri, b., 483 pourestarabadi, s., 289 pourghobadi, z., 1053 pradhan, r., 189 putra, m. d., 921 radha, a. v., 1087 radovanović, l., 1247 radović, i. r., 1287, 1379 radulović, v. m., 933 rafajlovska, v., 1097 rajasekharan, k. n., 1087 rajić, n. z., 1303 rao, a. s. 241 rao, b. a., 241 rao, m. s., 1 rao, v. h., 357 rat, m., 803 ražić, s., 465 rezaeinasab, m., 1273 rijavec, t., 83 ristić, d. s., 63 rodić, m. v., 825,1247 rogan, j., 1247 roglić, g. m., 933 roglić-korica, v., 1075 rukh, s., 943 rumin, m., 879 saadati, f., 1211 sačer, d., 411 sakač, m., 1343 salgado-morán, g., 25 salimi, f., 203 sankarraj, n., 567 sarasija, m., 117, 241 sargolzaei, m., 665 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. vol. 82: author index 1493 satheesh, s. v., 1087 saurambaeva, l., 791 savec, v. f., 1193 savić, a., 267, 1237 savić, v. m., 1335 savović, j. j., 1135 semikin, k. v., 107 serebryanskaya, a., 791 shafiekhani, h., 317 shaikh, m. s., 905 shariff, a. m., 905 shirvani, m., 483 shoumkova, a. s., 227 shuvarakova, e. i., 523 si, s., 39 simeunović, j., 1445 simić, m. r., 1335 simonovska, j., 1097 singh, n., 13 sinha, b., 189 smajdor, j., 879 smiljanić, s. v., 329 soleimani, e., 665 song, h., 399 song, q., 1111 song, w., 1111 sopčić, s., 411 sovrlić, z., 1147 spasojević, v., 1259 stankov jovanović, v., 141 stanković, d. m., 267, 303 stanković, m., 141 stanojević, i. m., 1357 stanojević, j., 1009 stavarache, c., 627 stevanović, j., 695 stoiljković, m., 1135 stojanović, g., 141, 1333 stojanović, k., 739, 1315, 1461 stoyanova, v. b., 227 suručić, r. v., 815 sýs, m., 865 šajnović, a. m., 1461 šegan, s., 641 šekularac, g., 695 šerbanović, s. p., 1287, 1379 škrbić, b., 711 šojić merkulov, d. v., 343 šolević knudsen, t., 1315 špírková, m., 437 šuput, d. z., 253 taghizadeh, m., 427 taheri, s. a. n., 665 tang, d., 73 tang, p., 39 tang, x., 39 tang, y., 73 taqavian, m., 841 tasci, n., 215 tasić, a. m., 1395 tasić, g. d., 1335 teng, l., 73 teodorović, i., 1445 tezerjani, m. d., 1273 tileuberdi, y., 1063 todorović, n. m., 1287 todorović, t. r., 825 todorović, v. m., 1039 todorović, z. b., 1039 tong, j., 1025 tošić, m. b., 329 troter, d. z., 1039 trtica, m., 1135 trtić-petrović, t., 1259 urankar, b., 1193 urošević, t., 1147 uskoković, d. p., 607 van hecke, k., 267 vanaja, b., 117 vedyagin, a. a., 523 veličković, z., 1175 veljković, v. b., 1039 veljović, đ. n., 1223 verbić, t. ž., 303 vlahović, f. ž., 1369 vojnović, s., 389, 1357 vorobyev, p., 791 vukašinović, j., 825 vukčević, m. m., 1417 vuksanović, j. m., 1287 vytřas, k., 865 wahdini, i., 921 wang, x., 399 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1494 journal of the serbian chemical society wu, l., 1111 xu, s.-y., 163 yabalak, e., 99 yang, l., 1111 yao, s., 399 yao, t., 399 yardim, y., 175 yeganegi, s., 681 yetimoglu, e. k., 215 yousaf, r., 151 yu, j.-z., 163 yugay, o., 791 zaikov, y. p., 51 zamani, a., 1211 zdravković, s., 469 zeidabadinejad, l., 289, 841 zhang, j., 73 zhao, f.-q., 163 zhuk, s. i., 51 zigheimat, f., 841 zlatanović, i., 141 zlatar, m., 1369 zlatović, m., 825 zloh, m., 303 zrnzević, i., 141 živanović, v. d., 329 živković, p. m., 539 živković, s. m., 1135 životić, d., 739 subject index of vol. 82 and list of referees in 2017 are given in the electronic form at the internet address of the journal of the serbian chemical society: http://www.shd.org.rs/jscs/ end of volume 82. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. << /ascii85encodepages false 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {propene oxidation from air by atmospheric plasma-catalytic hybrid system} j. serb. chem. soc. 83 (5) 641–649 (2018) udc 547.313.3+66.094.3:533.9:544.478+546.98 jscs–5101 original scientific paper 641 oxidation of propene from air by atmospheric plasma-catalytic hybrid system thien huu pham1, ha manh bui2* and ahmed khacef3 1institute of applied material science, vietnam academy of science and technology, 01 thanh loc 29 st., district 12, ho chi minh city 700000, vietnam, 2department of environmental sciences, sai gon university, 273 an duong vuong st., district 5, ho chi minh city 700000, vietnam and 3research team in gremi laboratory, the national centre for scientific research, university of orleans, 14 issoudun st., 45067 orleans cedex 02, france (received 14 october, revised 19 november, accepted 27 december 2017) abstract: the pulsed dielectric barrier discharge (dbd) combined with the palladium supported on alumina beads, was investigated for propene (c3h6) removal from air. the effects of thermal-catalysis, plasma-catalysis (in-plasma catalysis and post-plasma catalysis), and plasma-alone on the propene removal were compared. results are presented in the terms of c3h6 removal efficiency, energy consumption, and by-products production. temperature dependence studies (20–250 °c) show that in all conditions of input plasma energy density explored (23–148 j l-1), the plasma-catalysis systems exhibit better propene conversion efficiencies than the thermal catalysis at low temperature (60% at 20 °c). plasma-alone treatment has a similar effectiveness compared to plasma-catalysis at room temperature, but it leads to the formation of high by-products concentrations. it appears that in the plasma-catalyst system, c3h6 removal was the most efficient, whatever was the configuration used, and it was helpful to minimize by-products formation. keywords: non-thermal plasma; c3h6 oxidation; palladium catalyst. introduction volatile organic compounds (vocs) are an important category of air pollutants and therefore, they have become a serious problem, damaging the human health and the environment in general. the well-established technologies for the removal of vocs, namely thermal and catalytic oxidation, require a substantial supply of thermal energy (200–800 °c) and are not well adapted and also are energetically expensive in the case of the moderate gas flow rates containing low vocs concentrations.1–3 * corresponding author. e-mail: manhhakg@sgu.edu.vn https://doi.org/10.2298/jsc171014012p 642 pham, bui and khacef as an alternative to the conventional vocs removal techniques, the atmospheric non-thermal plasma (ntp) technology received the increasing interest during the last decades for the removal of dilute vocs from many sources. the main advantage of these non-equilibrium plasmas consists of the ability to generate high energy electrons, while keeping the background gas close to room temperature. thus, a highly reactive environment is created without spending energy on the gas heating, as in thermal processes. however, ntp technology for indoor air treatment has the disadvantage, because it produces the undesirable by-products such as ozone, aldehyde, and nox.4–6 to overcome the by-products formation and to increase the energy efficiency, ntp could take the advantage of its synergetic effect through the combination with heterogeneous catalysts. this combination can be either single-stage (in-plasma catalysis) or two stages (postplasma catalysis). such a combination helps to bring down the disadvantages of both catalytic and plasma treatments.7,8 in this study, the dielectric-barrier discharge (dbd) reactor, combined to 1 wt.% of pd/al2o3 beads catalyst, were investigated for the removal of propene from air at the atmospheric pressure. reported results, as a function of the gas temperature and the input plasma energy density, consider the catalyst effect, the plasma effect and the plasma-catalyst effect on the efficiency of propene conversion. systematic investigations were carried out in order to select the optimal positioning of the catalyst regarding the plasma discharge. experimental catalyst preparation the catalysts based on palladium supported on alumina beads (1 wt.% of pd/al2o3) were prepared by the impregnation method as described in details elsewhere.8 the aqueous solution of tetraaminepalladium(ii) nitrate (5 wt.% of pd, strem chemicals) and alumina beads (1 mm diameter, sasol) were stirred in the rotary evaporator for 3 h at 50 °c, under atmospheric pressure. then, the sample catalyst was dried for 12 h at 120 °c and calcined for 4 h at 500 °c at a heating rate of about 3°c per min under air flow. catalysts characterization surface area/porosity measurements were conducted using a micromeritics asap 2010 apparatus with n2 as the sorbate at –196 °c. all the samples were outgassed prior to analysis at 300 °c under vacuum (5×10-3 torr) for 3 h. the total specific surface areas were determined by the multipoint bet (brunauer–emmett–teller) method. mesoporosity was evaluated by the barret–joyner–halenda (bjh) method. x-ray powder diffraction (xrd) analyses were conducted by a bruker d5005 powder diffractometer scanning, using cukα radiation. the samples were scanned at a rate of 0.02° per step in the 2θ range of 4–80° (scan time = 2s per step). the applied voltage and current were 50 kv and 35 ma, respectively. diffraction patterns were assigned using joint committee on powder diffraction standards (jcpds) cards supplied by the international centre for diffraction database (icdd). the average crystallite sizes of al2o3 support and pd-supported catalysts were estimated using the scherrer equation: plasma-catalytic oxidation of propene 643 i cos λ β θ = k d where di is the mean size of the ordered (crystalline) domains of (i) al2o3 or/and pdo and pd, which may be smaller or equal to the grain size, k (0.9) is the shape factor, λ (0.154 nm) is the x-ray wavelength, β is the line broadening at half maximum intensity (fwhm) in rad and θ is the bragg angle. chemical states of the atoms in the catalyst surface were investigated by the x-ray photoelectron spectroscopy (xps) on an axis ultra dld spectrometer produced by kratos analytical, operating with al (kα) radiation. xps data were calibrated using the binding energy of c 1s (284.6 ev) as the standard. the xps core level spectra were analyzed with a fitting routine, which decomposes each spectrum into individual, mixed gaussian–lorentzian peaks using a shirley background subtraction over the energy range of the fit. the surface composition was calculated from the integrated peaks, using empirical cross-section factors for xps (c 1s = 1, o 1s = 2.93, al 2p = 0.54, pd 3d (3d5/2+ 3d3/2) = 16.04). plasma-catalysis system the plasma reactor we used is a cylindrical dbd shown in fig. 1. that configuration gives the possibility to combine the catalyst with the plasma reactor in two different ways: by introducing the catalyst in the discharge zone (in-plasma catalysis, ipc) or by placing the catalyst downstream the discharge zone (post-plasma catalysis, ppc).the plasma reactor was powered by a pulsed sub-microsecond high voltage generator delivering hv amplitude (up to 20 kv) at frequency up to 200 hz. the electrical characterization of plasma (energy deposition) was performed by the current and voltage measurements. the discharge pulse energy was measured with a capacitive circuit. the energy deposition in the plasma reactor (j l-1) is given by ed = epq -1f, in which, ep is the discharge pulse energy, f the pulse repetition rate, and q is the gas flow rate at standard conditions (25 °c and 1 atm). the experiments were conducted maintaining the constant discharge pulse energy ep at about 80 mj and in varying the fig. 1. schematic view of the plasma-catalyst reactor: a) post -plasma catalysis; b) in-plasma catalysis. 644 pham, bui and khacef pulse repetition frequency in the range 30–190 hz, and as a consequence the desired energy deposition ranged from 23 to 148 j l-1. experimental conditions the propene oxidation was performed in a continuous flow fixed-bed reactor in the temperature range 20–250 °c. the total flow through the catalyst bed was kept at 1 l min-1, leading a weight hourly space velocity of about 15000 h-1. the initial propene concentration was fixed at about 1000 ppm. the reactant and reaction products were analyzed in-situ using the ftir spectrometer (nicolet 6700, thermo-scientific). results and discussion the n2 adsorption/desorption analysis show that both the bet surface area and the total pore volume increases with the alumina sphere diameter, corresponding to the mesoporous materials. the xrd patterns suggest the formation of the alumina phase with the presence of the characteristic peaks for γ-al2o3 phase. the xrd analysis also confirms a small pd metal peak to be present along with the major pdo peaks. however, the xps analyzes shows only the pd2+ peak, corresponding to the pdo phase, probably because the amount of the exposed pd metal is too small to be picked up by xps. thus, it can be expected that the exchange or equilibration should occur on the surface of pdo at lower temperatures, as pdo is quite stable and does not easily change the oxidation state of a metal.9 xps results also showed the formation of palladium species in a higher oxidation state, probably pdo2 (338 ev), inducing the formation of new interfacial sites for the oxidation reaction.10 fig. 2 shows the typical ftir spectra illustrating the plasma and plasma -catalysis processing of air-c3h6 mixture at 150 °c an energy deposition of 55 fig. 2. typical ftir spectra for plasma and plasma-catalytic processing of air–c3h6 mixture (150 °c, 55 j l -1). plasma-catalytic oxidation of propene 645 j l-1. in addition to co and co2, the other detected gaseous carbon-containing compounds are formaldehyde (hcho), formic acid (hcooh), and nitric acid (hno3). at higher temperature, the nitric acid decomposition leads to the formation of no and no2. before comparing the effect of the thermal catalysis, plasma-catalysis (ipc and ppc), and plasma alone on the propene removal, the preliminary studies with alumina beads were performed. in the absence of plasma, the alumina beads show a high activity at 450 °c and above. the propene conversion over alumina alone was about 60 % with co2, h2o and co which are the reaction products. for 1 wt.% of pd/al2o3 catalyst, the temperature of total propene oxidation was drastically reduced to 250 °c leading to co2 and h2o. the effect of the energy deposition on propene conversion was studied as function of temperature in the range 23–148 j l-1. the results indicated that propene could be converted by plasma at low temperature. however, the reaction by-products were hcho, hcooh, co and o3. in the plasma-catalyst system, the interaction of the catalyst active phase, with the reactive species produced by the plasma, changed the catalyst activity by the increasing of the conversion efficiency and the decrease of the concentration of by-products. in some cases, the plasma-catalyst system in ipc configuration is better than the plasma-catalyst system in ppc configuration. this could be explained by the interaction of the catalyst active phase with the reactive species produced by plasma (•o, •oh, •o2, etc.) in ipc configuration. in this study, only the data obtained at 148 j l–1 will be presented. fig. 3 shows the comparisons between the thermal and the plasma-catalysis for the removal of propene from air using both configurations: in-plasma cat fig. 3. thermal, plasma, and plasma-catalytic conversion efficiency of c3h6 in air as a function of temperature (ed = 148 j l -1). 646 pham, bui and khacef alysis and post-plasma catalysis. we can note that the removal of propene by the thermal catalysis has a threshold temperature of 150 °c and increases steadily with the temperature reaching 100 % removal at 250 °c. the processing of propene using plasma discharge (with and without catalyst) exhibits a lower threshold temperature and the reactions take place at room temperature. larger conversion efficiencies were observed with the plasma-catalysis systems at any temperature, over the range 20–150 °c as illustrated in fig. 3. at room temperature, the plasma-alone and the plasma-catalysis (ipc and ppc) exhibit 60 % of propene conversion, compared to 0 % for thermal-catalysis. while the conversion efficiencies are quite similar, the nature and the amounts of the end-products observed are different. the total propene conversion was achieved at 100°c (ipc and ppc) and 200 °c (plasma alone) leading to the production of the by-products such as co, hcho, hcooh, o3 and nox. fig. 4–7 show the amounts of co, co2, hcho and hcooh respectively, produced in the case of the plasma alone and the plasma-catalysis (ipc and ppc) processing of the air-propene mixture as a function of temperature. fig. 4. co selectivity according to temperature at input density energy 148 j l-1. at low temperature (<100 °c), co selectivity is quite similar for ipc and ppc configurations. at higher temperature, co concentration drastically increased in the case of plasma-alone and slightly decreased when plasma was combined to catalyst over two configurations. we observe that the addition of the catalyst to the plasma in both ipc and ppc configurations increased the co2 selectivity to about 60 %, when comparing to the thermal-catalysis at 150 °c. at a given temperature in the range of 20–250 °c, the concentrations of formaldehyde (hcho) and formic acid (hcooh), derived from the partial oxidation of propene, decrease when the catalyst is combined with plasma. we obs plasma-catalytic oxidation of propene 647 fig. 5. co2 selectivity according to temperature at input density energy 148 j l -1. fig. 6. hcho concentration according to temperature at input density energy 148 j l-1. fig. 7. hcooh concentration according to temperature at input density energy 148 j l-1. 648 pham, bui and khacef erve that the concentrations of these by-products could be drastically reduced by increasing the plasma energy density. conclusions in this research, the thermal catalysis, the plasma-catalysis (ipc and ppc), and the plasma-alone processing of the air–propene mixture were investigated as the function of temperature and of plasma energy deposition. alumina and palladium supported on alumina beads (1 wt.% of pd/al2o3) catalysts were used in combination with the sub-microsecond pulsed dielectric barrier discharge. the plasma-catalysis systems exhibit better propene conversion efficiencies than the thermal catalysis at low temperature. the plasma-alone treatment has a similar effectiveness to the plasma-catalysis at room temperature (up to 60 % propene conversion) but leads to the formation of the high concentration of by-products such as carbon monoxide, formaldehyde and formic acid. the total conversion of propene was achieved at 100 °c in plasma-catalysis case and 250 °c in catalysis alone case. it has been shown that at a given energy density, the plasma-catalyst was helpful in minimizing the by-products formation. the plasma-catalytic conversion processes could be explained by the specific plasma-induced interactions between plasma reactive species (o3, o, oh,…) and the catalyst active phase at low temperature, whereas at higher temperature the thermal activation of the catalyst becomes important, overtaking the contribution of the plasma-activated processes. acknowledgements. this research is funded by vietnam national foundation for science and technology development (nafosted) under grant number 103.99-2016.67. и з в о д оксидација пропана у ваздуху атмосферским плазма-каталитичким системом thien huu pham1, ha manh bui2 и ahmed khacef3 1 institute of applied material science, vietnam academy of science and technology, 01 thanh loc 29 st., district 12, ho chi minh city 700000, vietnam, 2 department of environmental sciences, sai gon university, 273 an duong vuong st., district 5, ho chi minh city 700000, vietnam и 3 research team in gremi laboratory, the national centre for scientific research, university of orleans, 14 issoudun st., 45067 orleans cedex 02, france метода пулсне диелектричне баријере (dbd), у комбинацији са паладијумом на носачу од алуминијумских перли, испитана је у циљу уклањања пропена из ваздуха. поређени су ефекти термалне катализе, катализе плазмом и саме плазме на уклањнање пропена. резултати су поређени у односу на ефикасност уклањања пропена, потрошњу енергије и стварање споредних проивода. проучавање зависности од температуре (20– –250 °c) показало је да је у било којим испитиваним условима уклањање пропена помоћу катализе плазмом ефикасније од термалне катализе. третман самом плазмом је на собној температури ефикасан слично као и катализа плазмом, али има превише споредних производа. катализа плазмом се показала као најефективнија. (примљено 14. октобра, ревидирано 19. новембра, прихваћено 27. децембра 2017) plasma-catalytic oxidation of propene 649 references 1. z. zhang, z. jiang, w. shangguan, catal. today 264 (2016) 270 2. x. zhu, x. gao, r. qin, y. zeng, r. qu, c. zheng, x. tu, appl. catal., b: environ. 170 (2015) 293 3. m. s. kamal, s. a. razzak, m. m. hossain, atmos. environ. 140 (2016) 117 4. r. zhu, y. mao, l. jiang, j. chen, chem. eng. j. 279 (2015) 463 5. x. xu, j. wu, w. xu, m. he, m. fu, l. chen, a. zhu, d. ye, catal. today 281 (2017) 527 6. c. norsic, j. m. tatibouët, c. batiot-dupeyrat, e. fourré, chem. eng. j. 304 (2016) 563 7. x. zhu, s. liu, y. cai, x. gao, j. zhou, c. zheng, x. tu, appl. catal., b: environ. 183 (2016) 124 8. t. h. pham, s. gil, p. da costa, a. giroir-fendler, a. khacef, catal. today 257 (2015) 86 9. h. lee, d. h. lee, y. h. song, w. c. choi, y. k. park, d. h. kim, chem. eng. j. 259 (2015) 761 10. t. h. pham, l. sivachandiran, p. da costa, a. khacef, topics catal. 60 (2017) 326. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {euchems news european analytical column no. 46} j. serb. chem. soc. 83 (5) 669–671 (2018) udc 543+061.3(4) euchems news division of analytical chemistry european association for chemical and molecular sciences 669 euchems news european analytical column no. 46 wolfgang buchberger1, sibel a. ozkan2 and slavica ražić3*# 1analytical chemistry, university linz, altenbergerstrasse 69, 4040 linz, austria, 2department of analytical chemistry, faculty of pharmacy, ankara university, turkey and 3department of analytical chemistry, faculty of pharmacy, university of belgrade, vojvode stepe 450, 11222 belgrade, serbia information from the euchems division of analytical chemistry (dac) regarding the activities of dac, the year 2018 may be called “the year between”, namely between a highly successful euroanalyis conference held in stockholm 2017, and the next one to be held in istanbul in 2019. the updated dac strategy for 2018-2020 approved at last year’s annual meeting of dac defines the promotion of the euroanalysis series of conferences as one of its main themes. already in 2015, the dac steering committee members voted for istanbul as the venue for euroanalysis xx in 2019. as the name of this conference indicates, the venues are rotated between all european countries, but it was believed that the neighbor countries of europe should be well integrated into the activities of dac. therefore, the turkish chemical society will organize this conference in istanbul (which is often called the bridge between europe and asia) from 1st to 5th september 2019 with two chairs, prof. dr. sibel a. özkan (ankara university) and prof. dr. mehmet mahramanlioglu (istanbul university). the venue will be located in the campus of istanbul university, which is in the heart of the city center and very close to road links and public transport. the detailed information about euroanalysis xx can be found at http://euroanalysis2019.com/. as a broad spectrum conference on analytical chemistry, it will cover many different topics, such as, “analytical methods for cultural heritage and art”, “analytical instrumentation”, “biomedical analysis”, * e-mails: wolfgang.buchberger@jku.at (w. buchberger); ozkan@pharmacy.ankara.edu.tr (s. a. ozkan); slavica.razic@pharmacy.bg.ac.rs (s. ražić) # serbian chemical society member. 670 buchberger, ozkan and ražić “biomolecular analysis”, “biosensors”, “chemical analysis”, “chemometrics”, “clinical chemistry”, “education in analytical chemistry”, “environmental analysis and monitoring”, “food analysis”, “forensic science”, “green analytical chemistry”, “laboratory automation”, “materials science”, “metabolomics”, “nano analysis”, “pesticide-residue analysis”, “pharmaceutical analysis”, “proteomics”, “surface science” and others. among the highlights of a euroanalysis conference, the robert-kellner-lecture, an award to distinguish a person who has made substantial contribution to the advancement of analytical chemistry research or education, can be found. another highlight will be the presentation of the “dac-euchems award” for sustained contributions to analytical chemistry in europe. this award distinguishes a european individual who has demonstrated significant and sustained achievements in analytical chemistry research or education throughout his/her career. nominations for the robert-kellner-lecture and for the dac -euchems award are due by 31st october 2018 and should be sent to the chair or the secretary of dac (addresses can be found at http://www.euchems.eu/ /divisions/analytical-chemistry/). euroanalysis in istanbul will be the 20th event within this series of conferences. over the years, it has become a well-established meeting for the presentation of recent advances in analytical chemistry at the highest possible scientific level. despite the digital revolution encountered during the last years, personal contacts within the scientific community are still vital. however, the landscape of conferences has recently changed a little, and it has not always changed for the better. suddenly, conferences have been announced with names similar to those of established conferences but with quite dubious practices regarding the scientific quality. it has become obvious that there are companies that take scientific conferences as an opportunity to make profits. you may check wikipedia for “predatory conferences” (“…meetings set up to appear like legitimate scientific conferences but which are exploitative as they do not provide proper editorial control over presentations and advertising…”). contrary to this, euroanalysis has always been and will always be embedded into scientific chemical societies and into dac as a non-profit organization. the promotion of euroanalysis defined by dac as one of its main themes guarantees the high scientific standards of this conference. other ongoing activities of dac are performed within study groups. these include “bioanalytics”, “chemometrics”, “education”, “history”, “quality assurance” and “nanoanalytics”. please check the dac website for their reports. in this context, it may be worth mentioning that sergei shtykov, head of the study group nanoanalytics, has recently edited a book “nanoanalytics – nanoobjects and nanotechnologies in analytical chemistry”, which contains various interesting contributions to this topic. european analytical column no. 46 671 dac also intends to intensify its cooperation with other divisions of euchems, especially with the division of chemistry and the environment (dce). at the 17th international conference on chemistry and the environment in thessaloniki next year, dac will have a full session dedicated to analytical chemistry. in 2018, one of the main events of euchems will be the 7th euchems chemistry congress (26-30 august, 2018) in liverpool with its theme “molecular frontiers & global challenges” (https://www.euchems2018.org/). there will be two special sessions dealing with specific aspects of analytical chemistry, namely “abc of analytics”, organized by günter gauglitz (a member of the study group bioanalytics), and “pattern recognition for chemometrics and metabolomics”, organized by richard brereton (a member of the study group chemometrics). last, but not least, the current team of the steering committee of dac will be happy to receive input for additional activities. feel free to contact one of the following persons: slavica ražić, university of belgrade, serbia (chair); marcela alves segundo, university of porto, portugal (secretary); jiri barek, charles university, czech republic (treasurer); charlotta turner, lund university, sweden; sibel a. özkan, ankara university, turkey; christian rolando, 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {preventing decomposition of 2-mercaptobenzothiazole during gas chromatography analysis using programmable temperature vaporization injection} j. serb. chem. soc. 82 (10) 1147–1153 (2017) udc 547.427.6’789.6:544.421.032.4+ jscs–5030 543.544.3 short communication 1147 short communication preventing decomposition of 2-mercaptobenzothiazole during gas chromatography analysis using programmable temperature vaporization injection stefan đorđievski*#, zorica sovrlić, tamara urošević, jelena petrović and vesna krstić mining and metallurgy institute bor, zeleni bulvar 35, 19210 bor, serbia (received 14 november 2016, revised 31 march, accepted 4 april 2017) abstract: 2-mercaptobenzothiazole (mbt) is a chemical compound which is widely used in various processes in chemical industry, and it was also detected in environmental samples. most of the researchers employed liquid chromatography (lc) or gas chromatography (gc) for determination of mbt. when gc was used, derivatization was necessary in order to prevent decomposition of mbt inside of the hot gc inlet. in this study, a new approach for preventing decomposition of mbt using programmable temperature vaporization (ptv) was presented. the sample was injected in a cold inlet (40 °c) and the temperature was raised gradually until the analyte was evaporated. contrary to hot splitless injection, no decomposition of mbt was observed using ptv. compared to derivatization, ptv requires no chemicals and the duration of analysis is reduced. keywords: solvent vent mode; inlet; gc; mbt; ptv. introduction 2-mercaptobenzothiazole (mbt) is an aromatic heterocyclic chemical compound consisted of 1,3-thiazole ring fused to benzene ring and substituted with mercapto functional group at methine position in thiazole ring.1 mbt may exist as thione and thiol tautomers (fig. 1),2 and crystallographic analysis showed that thione form is predominant.3 pk and kow of mbt are 7.2, and 2.41, respectively.4,5 some applications of mbt are as copper corrosion inhibitor,6 vulcanization accelerator in rubber production,7 in organic synthesis including deoxygenation of epoxides and in the preparation of alkynes.8 some derivatives of mbt have antimicrobial and antifungal activities.9 mbt has been extensively employed in * corresponding author. e-mail: stefan.djordjievski@irmbor.co.rs # serbian chemical society member. https://doi.org/10.2298/jsc161114041d ___________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1148 đorđievski et al. chemical industry before it was tried as a flotation collector;10 it has been used for flotation of copper, lead and zinc minerals.11–14 fig. 1. thione and thiol tautomer of mbt.2 mbt and related benzothiaziole derivates have been found in many environmental matrices. these compounds are released with treated municipal wastewater and have a considerable lifetime in surface waters.15 benzothiazoles enter the environment from a number of sources such as the leaching of rubber products, fine particles of automobile tires, and antifreeze.16 sorption and desorption processes of benzothiazoles onto sandy aquifer material is very important for the understanding of behaviour of these compounds in the environment.17 table i lists chromatographic methods applied for analysis of mbt, including matrix types, extraction techniques and limits of detection (lod). gas chromatography (gc) and liquid chromatography (lc) are both widely used for determination of mbt. sample preparation methods include solid phase extraction (spe), solid phase microextraction (spme), liquid–liquid extraction (lle), dispersive liquid–liquid microextraction (dllme) and direct injection. prior to lc determination, no derivatization of mbt is necessary. some researchers derivatized mbt to thioethers prior to gc analysis,18–20 while others carried out analysis without derivatization.21,22 main reason for derivatization of mbt, prior to gc analysis, is the reactivity of the thiol group inside of a gas chromatograph.19 other methods have also been reported for analys of mbt, including voltammetry and spectrophotometry.23,24 most of the researchers determined mbt simultaneously with other related chemical compounds. in our study, instead of derivatization, the programmed temperature vaporization (ptv) was used for the prevention of decomposition of mbt during gc determination. ptv was initially presented in 1979 by vogt et al.31,32 the three most important modes of ptv operation are cold split injection, cold splitless injection, and solvent elimination injection (also called solvent split injection or solvent vent mode).33–35 among these, the solvent elimination injection has been the most widely used ptv technique and it was also used in our study. in this mode, sample is introduced at temperature below the solvent boiling point and the solvent is eliminated via split exit while the higher-boiling analytes are retained in the liner. after the solvent elimination, ptv is rapidly heated and the retained analytes are transferred to the analytical column. when splitless transfer is completed, the split exit is reopened to remove the residual solvent vapour and low-volatile matrix compounds from the inlet.34 the advantages of ptv injection ___________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. preventing decomposition of 2-mercaptobenzothiazole 1149 over other techniques consist in decreased analyte discrimination during the injection step, better recoveries of thermodegradable compounds, less pronounced adverse effects of non-volatiles present in the sample on the injection process and the possibility to introduce large volumes of samples (up to hundreds µl) into gc system.36 table i. analytical techniques used for determination of mbt matrix extraction technique analytical technique lod for mbt reference tap water, surface water, urban effluent spme; polyacrylate gc–ms/ms 0.7–1.2 µg l-1 22 river water, urban and industrial wastewater spe lc–ms/ms 6 ng ml-1 25 wastewater spe gc–ms – 21 tap, river and industrial waters, wastewaters dllme lc–flu–uv 0.3 µg l-1 26 municipal wastewater spe lc–ms 50–120 ng l-1 27 tannery wastewater direct injection lc–ms/ms 20 ng l-1 28 ozonized water lle-derivatization gc–ms – 20 urine lle-derivatization gc–ms 0.12 µmol 1-1 19 industrial wastewater lle lc–uv, gc-fid 5 µg l-1 29 river water direct injection lc-ed 0.82 µg l-1 30 water and sediment lle-derivatization gc-fpd 0.04 ppb 18 this article provides useful information about the operating conditions of gc inlet that may be applied for the determination of mbt without the decomposition of this compound. it also provides information about conditions of gc inlets for determination of mbt developed by other authors, and a comparison of these conditions with those described in this study. experimental agilent gc/msd 7890b/5977a was employed for the qualitative determination of mbt. gc unit included g4513a autoinjector, multimode inlet and hp-5ms capillary column ((5 %-phenyl)-methylpolysiloxane packing, 30 m length, 0.25 mm id, 0.25 µm film). helium was used as a carrier gas. msd unit was consisted of a single quadrupole mass analyzer and ion source with electron ionization. technical flotation reagent skik (metoha technology) containing 50 % water solution of sodium-2-mercaptobenzothiazole was used for experiments in this study. 1 ml of the reagent was dissolved in 100 ml of deionized water. diluted hydrochloric acid was added to the solution to form the white precipitate composed of mbt. suspension was transferred to separatory funnel and the extraction was carried out using dichloromethane. after vigorous shaking of separatory funnel, the suspended particles from water layer were dissolved in dichloromethane. organic layer was separated, extract was dried using anhydrous sodium sulfate and diluted 100 times with hexane. in order to compare performances of ptv and hot splitless injection for the determination of mbt, two runs were performed using same injection volume of extract, temperature ___________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1150 đorđievski et al. program of oven and mass spectrometer parameters, but with different temperature programs of inlet. in both runs, injected volume of diluted extract was 1 µl, temperature program of oven was: 40 °c for 2 min, then 15 °c/min to 190 °c for 0 min, then 5 °c/min to 280 °c for 15 min; acquisition mode of mass spectrometer was scan; total run time was 45 min. in the first run, inlet mode was splitless, temperature was 280 °c and purge flow to split vent was set at 2 min. second run was carried out using solvent vent mode. temperature program of inlet was: 50 °c for 0.1 min, then 130 °c/min to 280 °c until the end of run. purge flow to split vent was set at 2 min, same as in the first run. results and discussion in the first run, when the hot splitless injection was used, two chemical compounds emerged on the chromatogram: benzothiazole (bt) and mbt. when the second run was performed, using the solvent vent mode, only mbt was identified on chromatogram. it was concluded that bt was the product of partial decomposition of mbt due to high inlet temperature during the first run. both compounds identified in first run exhibit the sharp peak shape, indicating that the degradation observed took place completely within the injector.37 retention times of bt and mbt were 9.024 and 16.559 min respectively. chromatograms acquired using the hot splitless and solvent vent mode are shown in fig. 2. fig. 2. chromatogram acquired by injecting sample solution using: a) hot splitless (280 °c) and b) solvent vent mode (40 °c). the mass spectrum of mbt, acquired by measurement of sample aliquot (fig. 3), matched the mass spectrum of mbt from database by 96.8 %. this indicates that the chemical compound was identified with high reliability. some researchers derivatized mbt prior to gc analysis in order to prevent decomposition, while the others used different analytical technique. prior to the gc-ms determination of mbt in ozonized samples, fiehn et al.20 carried out methylation using diazomethane in order to protect thiol group from decomposition. as a result, mbt was identified as s-methylated product. shinohara et al.18 methylated mbt prior to gc-fpd analysis using dimethyl formamide diacetal as methylation reagent, and manninen et al.19 derivatized samples with ___________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. preventing decomposition of 2-mercaptobenzothiazole 1151 pentafluorobenzyl bromide. rennie30 used hplc for determination of mbt instead of gas chromatography because of degradation problems. fig. 3. mass spectrum acquired by gc–ms analysis of mbt in hexane solution. some researchers preferred to determine mbt using gc without derivatization. domínguez et al.21 carried out gc–ms determination of benzothiazoles using six different ionic liquid stationary phases. in their study, samples were injected in splitless mode at injector temperature of 270 °c. six different columns were tested for mbt and related compounds. mbt have eluted from only one column, but with very poor response of mass spectrometric detector. among that, the response for bt was much higher than the responses of other compounds of the same concentration in standard mix solution. this may indicate that mbt was possibly partially degraded to bt using these gc conditions. naccarato et al.22 also performed analysis of mbt in splitless mode and by setting the injector temperature at 290 °c. the result of the present work suggests that hot splitless injection should be avoided for the determination of mbt. this suggestion is consistent with the results of researchers mentioned above who derivatized mbt prior to gc analysis. however, in some recent papers mbt was determined together with many other compounds in splitless mode and with high injector temperature. according to results obtained in our study and studies in which derivatization was performed, the response of mbt acquired using hot splitless injection might be lower than actual response due to decomposition. conclusion a new approach for preventing the decomposition of mbt was presented in this study. instead of derivatization, ptv injection was proven to be efficient in preventing the decomposition of mbt during gc determination. this approach requires no chemical reagents for derivatization and shortens the time of analysis. finally, it may be stated that the derivatization or the application of ptv injection is highly recommended when the analysis of mbt is performed using gc. hot splitless injection may cause partial decomposition of mbt and it may lead to biased results. ___________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1152 đorđievski et al. acknowledgment. the authors thank dr. kofi adomako-ansah, specially-appointed assistant professor at akita university, japan, for providing language assistance. и з в о д спречавање разградње 2-меркаптобензотиазола током гасно хроматографске анализе применом инјектора са могућношћу подешавања температуре испаравања стефан ђорђиевски, зорица соврлић, тамара урошевић, јелена петровић и весна крстић институт за рударство и металургију бор, зелени булевар 35, 19210 бор 2-меркаптобензотиазол (mbt) је хемијско једињење које се широко користи у многим гранама индустрије, а такође је детектовано и у узорцима из животне средине. највећи део истраживача користило је течну (lc) или гасну (gc) хроматографију за одређивање mbt. када је коришћена gc, дериватизација је била неопходна како би се спречила разградња mbt унутар загрејаног gc инлета. у овом раду представљен је нови приступ за спречавање разградње mbt коришћењем инјектора са могућношћу подешавања температуре испаравања (ptv инјектор). узорак је инјектован у охлађен инлет (40 °c) и температура је повишавана постепено док аналит није испарио. за разлику од splitless инјектовања, применом ptv инјектора није примећена разградња mbt. у поређењу са дериватизацијом, за ptv нису потребне хемикалије и време анализе је краће. 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//www.agilent.com/cs/library/usermanuals/public/5958-9468_041007.pdf (2016/09/30) 34. e. hoh, k. mastovska, j. chromatogr., a 1186 (2008) 2 35. j. á. gómez-ruiz, f. cordeiro, p. lópez, t. wenzl, talanta 80 (2009) 643 36. m. godula, j. hajšová, k. maštouska, j. křivánková, j. sep. sci. 24 (2001) 355 37. h. s. müller, h. j. stan, j. high resolut. chromatogr. 13 (1990) 759. ___________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis of 1,3-divalent glycoconjugates with diverse structure and functionalization} j. serb. chem. soc. 83 (5) 539–548 (2018) udc 547.424+547.455–124:542.913: jscs–5094 66.095.12:547.455.623’915.5 original scientific paper 539 synthesis of 1,3-divalent glycoconjugates with diverse structures and their functionalization laxminarayan sahoo1, anadi singhamahapatra2 and satyanarayan sahoo3* 1district forensic science laboratory, puri 752002, odisha, india, 2saraswati degree vidya mandir, neelakantha nagar, berhampur 760002, odisha, india and 3p. g. department of chemistry, berhampur university, berhampur 760007, odisha, india (received 5 september 2017, revised and accepted 14 february 2018) abstract: a series of novel 1,3-difunctionalized glycoconjugates were synthesized using a sequence of regioselective functionalization and stereoselective glycosidation of d-glucose and d-glcnac. regioselective c-3 functionalization of sugar molecules was achieved by chemical functionalization of isopropylidene or oxazoline protected sugar derivatives. the structural diversity at the anomeric carbon was explored by stereoselective chemical glycosidation. the oxazoline protected d-glcnac derivative gave either pyranose or furanose derivatives on glycosidation depending on the amount of lewis acid used. the diversely functionalized glycoconjugates with azide or alkyne groups are potentially useful for the synthesis of multifunctionalized complex glycoconjugates via click reactions. keywords: glycoconjugate; regioselective; stereoselective; chemical synthesis; click reaction, glycolipids. introduction chemical synthesis of diversely functionalized biomolecules with targeted medical applications is a rapidly growing area of research in synthetic organic chemistry and chemical biology. diversity oriented chemical synthesis of hybrid biomolecules not only generates a large number of compounds within a shorter period, but also produces a series of diversely functionalized molecules from the common starting material and by a common methodology. the concepts used regularly in the diversity-oriented synthesis are orthogonal protection and deprotection, functionalization of molecules by regioselective synthesis or cyclization of molecules to generate macrocycles with different scaffold structures.1–3 carbohydrates, being the most abundant biomolecule with unparalleled structural * corresponding author. e-mail: sns.chem@buodisha.edu.in https://doi.org/10.2298/jsc170905030s 540 sahoo, singhamahapatra and sahoo diversity, are regularly used as an instant source of chiral backbone in the area of diversity-oriented synthesis. in natural glycoconjugates, sugars are attached to other biomolecules like protein and lipid with different chemical linkages and with different stereochemistry.4–6 the glycan part controls the solubility, folding and other physical characteristics of the natural glycoconjugates modulating their biological functions.7 however, the potential of glycoconjugates in the area of medicinal chemistry are still not explored to the full extent due to their microheterogeneity and low bioavailability. glycoconjugates synthesized in pure form using an easy synthetic methodology are useful in this regard.8,9 the presence of a multiple number of chiral centres and the different relative orientation of the hydroxyl groups with respect to each other make carbohydrates a source of chiral backbones that can easily be diversely functionalized with the help of selective protection and deprotection methods.10–13 carbohydrates are frequently used for the synthesis of various structurally diverse biomolecules, such as multivalent compounds and glycoamino acid mimics. compared to monofunctionalized glycoconjugates, multifunctionalized glycoconjugates with different functional groups in the same sugar moiety have a better binding affinity with various bacteria, virus or lectins. in the literature, many diversely functionalized glycoconjugates are reported to be synthesized using a click reaction.14–16 recently, the synthesis of diversely functionalized “clickable” glycopeptoids17 and other glycoconjugates,18,19 such as triazole containing glycolipids, that are potentially useful in the area of chemical biology, were reported. in this present work, a series of novel difunctionalized glycoconjugate in which the sugar molecules were functionalized with azide or alkyne or both groups, which could be used for synthesis of complex glycoconjugates, was synthesized using cu(i) catalyzed click reactions. the diversity oriented synthesis of these complex glycoconjugates was achieved by selective protection of some of the hydroxyl groups of the sugar molecule followed by functionalization of the other groups. this synthetic methodology following the sequence of selective protection, chemical functionalization and glycosidation prior to selective deprotection resulted in the formation of novel 1,3-difunctionalized glycoconjugates not only with diverse functionalization, but also with different scaffolds with hexopyranose or hexofuranose rings of the sugar. 1,2:5,6-di-o-isopropylidene-α-d-glucofuranose is reported in the literature of carbohydrate synthesis for the chemical modification at the c-3 hydroxyl group of the sugar ring followed by modification of the other hydroxyl groups.20–23 similarly, 1,2-oxazoline protected sugar molecules can be used not only for selective functionalization, but also as acceptor in chemical glycosidation reactions.24–27 another advantage of 1,2-oxazoline protection is the β-selectivity in the chemical glycosidation reaction due to neighbouring group parti synthesis of 1,3-difunctionalized glycoconjugates 541 cipation of the c-2 acetamido group in addition to the scope for the formation of hexofuranose and hexopyranose rings as products (fig. 1). fig. 1. synthesis of 1,3-functionalized glycoconjugates. experimental general information all the solvents were used after distillation and dry solvents were prepared using standard methods. all reagents purchased from commercial sources were used without any purification. 1hand 13c-nmr spectra were recorded using 400 and 500 mhz nmr spectrometer. all mass spectra were recorded in q-tof electrospray ionization spectrometer. column chromatography was performed over 100–200 mesh silica with ethyl acetate and hexane as the eluent. analytical and spectral data of the synthesized compounds are given in supplementary material to this paper. syntheses synthesis of 1,2,4,6-tetra-o-acetyl-3-o-propargyl-d-glucopyranose (4). a solution of sodium hydride (240 mg, 10 mmol) in dry dmf (10 ml) in a 100-ml round bottom flask was cooled to 0 °c and a solution of diacetone glucose (1.3 g, 5.0 mmol in 10 ml dmf) was added. the reaction was continued at that temperature for 30 min and then propargyl bromide (10 mmol) was added under stirring. the reaction mixture was allowed to come to room temperature and the reaction continued for 24 h at room temperature. after completion of the reaction, as indicated by tlc, ethyl acetate (50 ml) was added followed by water (50 ml) and the two layers were separated. the organic layer was washed with distilled water (3×30 ml) followed by brine solution (30 ml), dried over anhydrous sodium sulphate and concentrated to dryness. the crude product was dissolved in a mixture of thf and water (9:1). to this solution, dowex h+ (30 wt. %) resin was added. the reaction mixture was stirred for 24 h at room temperature. after disappearance of the starting material, confirmed by tlc analysis, the reaction mixture was filtered through filter paper. after filtration of the resin, the filtrate was then concentrated and repeatedly washed with ethyl acetate to give a syrup. acetylation of the hydroxyl groups of the syrupy product was realised using acetyl chloride along with sodium acetate as base to obtain 1,2,4,6-tetra-o-acetyl-3-o-propargyl-dglucopyranose.28 the crude product was purified by column chromatography to give an overall yield of 80 %. synthesis of 2,4,6-tri-o-acetyl-3-o-propargyl-β-d-glucopyranosyl azide (5). 1,2,4,6 -tetra-o-acetyl-3-o-propargyl-d-glucopyranose (4, 386 mg, 1 mmol) was dissolved in dry dcm (10 ml). to this, tmsn3 (1.5 mmol) was added followed by a 1.0 m solution of sncl4 542 sahoo, singhamahapatra and sahoo in dry dcm (0.5 ml, 0.5 mmol). the reaction was continued at room temperature until the starting material was consumed. the reaction mixture was diluted with dcm (50 ml) and washed with distilled water (3×30 ml) followed by brine solution (30 ml). the crude product obtained after concentrating the organic layer was purified by column chromatography using a mixture of ethyl acetate and hexane (1:3) to afford 2,4,6-tri-o-acetyl-3-o-propargyl-β-d -glucopyranosyl azide (5) in 90 % yield. synthesis of 1,3-difunctionalized glycolipid 6. 1,2,4,6-tetra-o-acetyl-3-o-propargyl-d -glucopyranose (4, 386 mg, 1.0 mmol) was dissolved in dry dcm. to this, n-dodecanol (1.5 mmol) was added followed by a 1 m solution of sncl4 in dry dcm (0.5 ml, 0.5 mmol). the reaction was continued until the disappearance of compound 4. the reaction mixture was then diluted with dcm (50 ml) and washed with distilled water (3×30 ml). the organic layer was dried over anhydrous sodium sulphate and concentrated to dryness. the crude product was purified by column chromatography using a mixture of ethyl acetate and hexane (1:2) to give the corresponding n-dodecyl glucoside (6) in 80 % yield. synthesis of methyl 2,4,6-tri-o-acetyl-3-o-propargyl-α-d-glucopyranoside (7). 3-o-propargyl-d-glucopyranose (3, 1 mmol) was dissolved in dry methanol. to this, ir 120 h+ resin (0.5 g) was added and the reaction mixture was stirred at room temperature. after completion of the reaction, as indicated by tlc, the crude product was filtered and concentrated to dryness to give a syrup. the syrupy product was per-o-acetylated using acetyl chloride along with sodium acetate as base in acetonitrile at 60 °c to give the methyl 2,4,6-tri-o-acetyl-3-o -propargyl-α-d-glucopyranoside in 90 % yield after column purification using a mixture of ethyl acetate and hexane (1:2) as eluent. synthesis of 1,3-difunctionalized glcnac derivatives 10 and 11. 2-acetamido-2-deoxy -d-glucopyranose (5 mmol) was converted to the corresponding oxazoline derivative 8 following a literature procedure using dry acetone and ferric chloride.22 sodium hydride (96 mg, 4.0 mmol) dissolved in dry dmf (5 ml) was cooled to 0 °c. to this, oxazoline 8 (2 mmol) dissolved in dmf (5 ml) was added dropwise under stirring. after 30 min, propargyl bromide (2 mmol) was added to the reaction mixture and the reaction allowed to come to room temperature and continued until the starting material had been consumed. the reaction mixture was diluted with ethyl acetate (50 ml) and the organic layer was repeatedly washed with water, dried over sodium sulphate and concentrated to dryness to obtain the crude product 9, which was used for further reactions without any purification. the crude product 9 (1 mmol) obtained in the previous step was dissolved in anhydrous propargyl alcohol (1 ml). to this, p-tsa (0.5 equiv.) was added under nitrogen. the reaction was continued until disappearance of the starting material. the reaction mixture was dried under vacuum. acetylation of hydroxyl groups was realised using acetic anhydride and pyridine (1:1, 2 ml). the crude product was purified by column chromatography using a mixture of ethyl acetate and hexane (1:1) to afford compound 10. compound 11 was prepared from 9 using the same methodology as that for 10 except n-decanol (1 ml for 1 mmol) was used in place of propargyl alcohol. synthesis of 1,3-difunctionalized glcnac derivatives 13 and 14. a solution of oxazoline 8 (2 mmol) in dry dmf (5 ml) was added dropwise to a mixture of sodium hydride (96 mg, 4 mmol) in dry dmf (5 ml) under stirring at 0 °c. after 30 min, n-decyl bromide (2 mmol) was added to the reaction mixture and stirring was continued at room temperature until the starting material had disappeared. the reaction mixture was diluted with ethyl acetate (50 ml) and the organic layer was repeatedly washed with water, dried over sodium sulphate and concentrated to dryness to obtain the crude product 12. synthesis of 1,3-difunctionalized glycoconjugates 543 crude product 12 (1 mmol) was dissolved in anhydrous n-decanol (1 ml) and p-tsa (0.2 equiv.) was added under nitrogen. the reaction mixture was dried under vacuum after disappearance of the starting material. purification of the product using column chromatography with ethyl acetate and hexane (1:1) as the eluent resulted in compound 13. compound 14 was prepared from 12 using the similar methodology to that used for 13 except 1 equiv. of p-tsa (in place of 0.2 equiv. used in synthesis of compound 13) and propargyl alcohol (1 ml for 1 mmol) in place of n-decanol, followed by overnight reaction with a mixture of acetic anhydride and pyridine (1:1, 2 ml). results and discussion the synthesis of 1,3-difunctionalized glycoconjugates derived from d-glucose was initiated by the alkylation of the c-3 hydroxyl group of 1,2:5,6-di-o -isopropylidene-α-d-glucofuranose (1) with propargyl bromide using sodium hydride as the base in dry dmf to furnish the corresponding c-3 o-propargylated derivative 2. the two acetonide protecting groups were removed under acidic condition using dowex h+ resin in aqueous thf to obtain c-3 o-propargylated glucopyranose 3. per-o-acetylation of compound 3 was realised using acetyl chloride and sodium acetate in acetonitrile. the crude product was purified by column chromatography using a mixture of ethyl acetate and hexane as eluent to furnish 1,2,4,6-tetra-o-acetyl-3-o-propargyl-d-glucopyranose (4) in 80 % overall yield over three steps (scheme 1).27 scheme 1. synthesis of 1,2,4,6-tetra-o-acetyl-3-o-propargyl-d-glucopyranose (4). since compound 4 is suitably functionalised at the c-3 position and the anomeric acetate group is a good glycosyl donor, it was chosen as a versatile intermediate for the synthesis of various novel 1,3-difunctionalised glycoconjugates. 1,2,4,6-tetra-o-acetyl-3-o-propargyl-d-glucopyranose (4) was converted to the corresponding 2,4,6-tri-o-acetyl-3-o-propargyl-β-d-glucopyranosyl azide (5) in 90 % yield by reaction with tmsn3 and sncl4 as catalyst (scheme 2). in the 1h-nmr (500 mhz, cdcl3) spectrum of 5, the anomeric proton appeared as a doublet at 4.52 ppm with a coupling constant of 9.0 hz, confirming the β-linkage. the formation of the compound was further confirmed by 1h–1h cosy, 13c-nmr and esi-ms hrms spectral data. 544 sahoo, singhamahapatra and sahoo scheme 2. synthesis of 2,4,6-tri-o-acetyl-3-o-propargyl-β-d-glucopyranosyl azide 5. the methodology of synthesising 1,3-difunctionalized glycoconjugates was further extended to the preparation of other glycoconjugates, such as the α-linked c-3 o-propargylated glycolipid. for the synthesis of selectively functionalized glycolipids, 1,2,4,6-tetra-o-acetyl-3-o-propargyl-d-glucopyranose (4) was chosen as the glycosyl donor. glycosidation was realised by taking n-dodecanol and sncl4 in dry dcm as catalyst to furnish the 3-o-propargylated glycolipid 6 as the α-isomer (scheme 3). the formation of the α-isomer was confirmed by 1h-nmr where the anomeric proton appeared as a doublet at 5.06 ppm with a coupling constant of 3.6 hz. the formation of the compound was further confirmed by 1h–1h cosy, 13c-nmr and the presence of the molecular ion peak in the esi-ms hrms spectroscopic data. scheme 3. synthesis of c-3 propargylated glycolipid 6. in addition to the c-3 propargylated per-o-acetylated derivative 4, the 3-o -propargylated free sugar derivative 3 was also used in the glycosidation reaction under acidic conditions. the reaction of compound 3 with methanol in the presence of a catalytic amount of ir120 h+ resin followed by per-o-acetylation of the hydroxyl groups using acetyl chloride in presence of sodium acetate resulted in the formation of the 3-o-propargylated per-o-acetylated methyl glycoside 7 (scheme 4). the anomeric proton had a coupling constant of 2.8 hz with the h-2 proton in the 1h-nmr spectrum, which suggested the formation of the isomer as the only product. formation of the compound was further confirmed by other spectroscopic techniques, i.e., 1h–1h cosy, 13c-nmr and presence of the molecular ion peak in the esi-ms hrms spectroscopic data. scheme 4. synthesis of c-3 functionalized methyl glycoside 7. synthesis of 1,3-difunctionalized glycoconjugates 545 after synthesizing different 1,3-difunctionalized glycoconjugates derived from d-glucose, several 1,3-difuctionalized glycoconjugates were synthesized from 2-acetamido-2-deoxy-d-glucopyranose (d-glcnac). for the synthesis of d-glcnac derivatives, a novel oxazoline intermediate 8 was synthesized following a literature procedure, by reacting 2-acetamido-2-deoxy-d-glucopyranose with acetone catalyzed by ferric chloride. the 1,2-oxazoline ring of sugars has advantages over 1,2-isopropylidene protections, not only for its facile opening but also for selective formation of a β-glycosidic bond.22 oxazoline 8 was reacted with propargyl bromide using sodium hydride as base to synthesize the 3-o-propargylated oxazoline derivative 9. compound 9 was used for glycosidation using different alcohols, i.e., propargyl alcohol and n-decanol, using p-tsa (0.5 mol %) as catalyst followed by per-o-acetylation using acetic anhydride and pyridine to furnish the 1,3-dipropargylated glcnac derivative 10 and 3-o-propargylated n-decyl glucoside 11, respectively (scheme 5). formation of both the compounds was confirmed by nmr and esi-ms hrms spectroscopic data. scheme 5. synthesis of c-3 propargyl functionalized glcnac derivatives. the 1h-nmr spectrum of compound 10 revealed the formation of furanose glycoside where the anomeric proton appeared as a multiplet in the range 5.09– –5.03 ppm along with the h-4 proton. the exocyclic h-5 proton appeared as a multiplet in the 3.95–3.91 ppm range. two alkyne protons appeared as multiplets in the 2.51–2.49 ppm range. in case of compound 11, the anomeric proton appeared as a doublet at 4.85 ppm with a coupling constant of 2.4 hz, confirming the formation of the furanose ring. the lewis acid (0.5 mol % p-tsa) used in these reactions for glycosidation also catalyzed the deprotection of the 5,6-isopropylidene group. after synthesizing the 3-o-propargylated glycosides, attempts were made towards the synthesis of the corresponding c-3 decyl glycosides. for the synthesis of these derivatives, oxazoline 8 was used as the synthon and was reacted 546 sahoo, singhamahapatra and sahoo with n-decyl bromide using sodium hydride as the base to obtain the c-3 o-decyl derivative 12, which was used for the glycosidation reaction (scheme 6). compound 12 was reacted with two different alcohols in presence of p-tsa in different amounts. when it was reacted with n-decanol using 0.2 mol % of p-tsa, the resultant product, compound 13, was found to be a furanose glycoside without affecting the acetonide protection. in the 1h-nmr spectrum of compound 13, the anomeric proton appeared as a singlet at 4.88 ppm. two singlets appeared at 1.43 and 1.35 ppm, indicating the presence of acetonide protection. the formation of the compound was further confirmed by esi-ms hrms. when compound 12 was reacted with propargyl alcohol with 1 mol % of p-tsa followed by per-o-acetylation using acetic anhydride and pyridine, the resulting glycoconjugate 14 was found to be in the hexopyranose form, for which the anomeric proton was found to be a doublet at 4.85 ppm with a coupling constant of 8.0 hz. this experiment showed the scope of the reaction for the synthesis of glycosides with different conformations (hexopyranose or hexofuranose) using different amounts of the lewis acid. scheme 6. synthesis of c-3 decyl functionalized glcnac derivatives. conclusions in summary, seven 1,3-difunctionalized glycoconjugates were synthesized from oxazoline or isopropylidene protected carbohydrate derivatives that yielded either pyranose or furanose derivatives depending on the amount of lewis acid used in glycosidation. this is a unique example of substrate specific regio and stereoselectivity synthesis of furanose and pyranose ring containing glycolipids with one or two long chain alkyl groups or even propargyl groups. the propargyl functionalized glycolipids are useful synthetic biomolecules that can be utilized as clickable chemical ligating agent for the synthesis of hybrid glycolipids by a cu(i)-catalyzed click reaction with azide functionalized biomolecules. 2,4,6-trio-acetyl-3-o-propargyl-β-d-glucopyranosyl azide could be used for the synthesis of complex glycoconjugates or glycopolymers by a [3+2] cycloaddition reaction. the concept of selective protection and deprotection of the hydroxyl groups synthesis of 1,3-difunctionalized glycoconjugates 547 is potentially useful for the functionalization of the carbohydrate derivatives at different positions leading to biologically important glycoconjugates. this methodology for formation of hexopyranose or hexofuranose ring containing glycosides derived from d-glucose or d-glcnac could be explored for the synthesis of other diversely functionalized glycoconjugates. supplementary material analytical and spectral data of the synthesized compounds are available electronically at the pages of the journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. и з в о д синтеза 1,3-дивалентних гликоконјугата разноврсне структуре и функционализације laxminarayan sahoo,1 anadi singhamahapatra2 и satyanarayan sahoo3 1district forensic science laboratory, puri 752002, odisha, india, 2saraswati degree vidya mandir, neelakantha nagar, berhampur 760002, odisha, india и 3p. g. department of chemistry, berhampur university, berhampur 760007, odisha, india синтетисана је серија нових 1,3-дифункционализованих гликоконјугата, секвенцијама региоселективне функционализације и стереоселективне гликозидације d-глукозе и d-glcnac. региоселективна c-3 функционализација молекула шећера постигнута је на изопропилиденили оксазолин-заштићеним дериватима шећера. структурна разноврсност аномерних деривата је испитана стереоселективном гликозидацијом. оксазолин-заштићени дериват као производ реакције даје пиранозни или фуранозни дериват у зависности од количине примењене луисове киселине као катализатора. гликоконјугати са азидним или алкинским групама могу имати примену у синтези мултифункционализованих сложених гликоконјугата применом клик („click“) реакције. (примљено 5. септембра 2017, ревидирано и прихваћено 14. фебруара 2018) references 1. s. l. schreiber, m. d. burke, angew. chem. int. ed. 43 (2004) 46 2. s. l. schreiber, science 287 (2000) 1964 3. r. j. spandl, a. benderb, d. r. spring, org. biomol. chem. 6 (2008) 1149 4. c. manna, t. pathak, eur. j. org. chem. 27 (2013) 6084 5. a. cordeiro, e. quesada, m. c. bonache, s. velázquez, m. j. camarasa, a. san-félix, j. org. chem. 71 (2006) 7224 6. s. kashyap, s. hotha, tetrahedron lett. 47 (2006) 2021 7. a. varki, glycobiology 27 (2017) 3 8. v. wittmann, r. j. pictere, chem. soc. rev. 42 (2013) 4492 9. b. g. davis, j. chem. soc. perkin trans. 1 (1999) 3215 10. p. m. dandy, s. g. withers, acs chem. biol. 11 (2016) 1784 11. r. mahrwald, chem. commun. 51 (2015) 13868 12. k. villadsen, m. c. martos-maldonado, k. j. jensen, m. b. thygesen, chembiochem 18 (2017) 574 13. r. sangwan, p. k. mandal, rsc adv. 7 (2017) 26256 14. b. k. sharpless, angew. chem. int. ed. 41 (2002) 2596 548 sahoo, 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<feff04120438043a043e0440043804410442043e043204430439044204350020044604560020043f043004400430043c043504420440043800200434043b044f0020044104420432043e04400435043d043d044f00200434043e043a0443043c0435043d044204560432002000410064006f006200650020005000440046002c0020044f043a04560020043d04300439043a04400430044904350020043f045604340445043e0434044f0442044c00200434043b044f0020043204380441043e043a043e044f043a04560441043d043e0433043e0020043f0435044004350434043404400443043a043e0432043e0433043e0020043404400443043a0443002e00200020042104420432043e04400435043d045600200434043e043a0443043c0435043d0442043800200050004400460020043c043e0436043d04300020043204560434043a0440043804420438002004430020004100630072006f006200610074002004420430002000410064006f00620065002000520065006100640065007200200035002e0030002004300431043e0020043f04560437043d04560448043e04570020043204350440044104560457002e> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {electrochemical determination of 5-dodecylsalicylaldoxime and 2-hydroxy-5-nonylacetophenoneoxime in commercial extractants} j. serb. chem. soc. 82 (4) 417–425 (2017) udc 547.288.4–36:543.559+543.552:66.061 jscs–4977 original scientific paper 417 electrochemical determination of 5-dodecylsalicylaldoxime and 2′-hydroxy-5′-nonylacetophenone oxime in commercial extractants maría p. elizalde*, maría del sol rúa, marta huebra and begoña menoyo department of analytical chemistry, faculty of science and technology, university of the basque country (upv/ehu), p. o. box 644, 48080 bilbao, spain (received 7 july, revised 22 november, accepted 29 november 2016) abstract: a differential pulse voltammetric method using a hanging mercury drop electrode (hmde) was optimized for the determination of 5-dodecylsalicylaldoxime in hydro-alcoholic solutions using factorial and central composite designs. the taguchi design methodology was used to extend the formerly optimized method to the determination of 2′-hydroxy-5′-nonylacetophenone oxime. the method was successfully applied to quantify 5-dodecylsalicylaldoxime and 2′-hydroxy-5′-nonylacetophenone oxime in samples of commercial extractants over the concentration range 0.05–2.45 and 0.07–0.82 mg l-1, respectively, with detection limits of 0.034 and 0.019 mg l-1, respectively. keywords: voltammetry; hydroxyoxime-based extractants; factorial design. introduction 5-dodecylsalicylaldoxime (dsao) is the active component of the commercial extractants lix860, lix860-i and lix622. on the other hand, 2′-hydroxy-5′-nonylacetophenone oxime (hnapo) is the active component of lix84 and sme 529. these reagents, that are being mainly used for copper removal from industrial acidic effluents, are commercialized diluted in an organic solvent and they may contain small amounts of 4-nonylphenol (lix84), 4-dodecylphenol (lix860, lix860-i and lix622)1 or modifiers, such as tridecyl alcohol (lix 622).1,2 in the study of fundamental metal extraction processes directed towards the determination of the stoichiometry of the extracted metal complexes and the values of their equilibrium constants, the composition of the extractants must be known. consequently, it becomes necessary to develop methods for the quantification of the active component in industrial formulations since the manufac * corresponding author. e-mail: maria.elizalde@ehu.eus https://doi.org/10.2298/jsc160707005e 418 elizalde et al. turers usually do not provide precise information about the composition of commercial reagents. some electrochemical methods are reported in the literature for the analysis of the active components of several extractants. thus, the electrochemical behaviour3,4 and determination3 of 4-methyl-n-quinolin-8-ylbenzenesulfonamide, a compound closely related to the active component in lix 34 (4-alkaryl-n-quinolin-8-ylbenzenesulfonamide) were studied. the electrochemical quantification of 1-phenyldecane-1,3-dione5 and 7-(1-vinyl-3,3,5,5-tetramethylhexyl)-8-quinolinol,6 the active components of lix 54 and kelex 100, respectively, was also performed. as far as hydroxyoxime-based extractants are concerned, gc and hplc methods have been developed for either the identification or quantification of their active components.7–13 voltammetry has been used for both qualitative and quantitative determination of hnapo and 5-nonylsalicylaldoxime14 (the active component of lix 622n, lix 860n-i, lix 860n-ic, acorga p5100 and acorga pt5050). finally, the electrochemical behaviour of 2-hydroxy-5-methylbenzophenone oxime,15 a compound similar to the active component of the commercial extractant lix 65n (2-hydroxy-5-nonylbenzophenone oxime), was reported. in this work, a voltammetric method based on the electrochemical reduction of the oxime group of the hydroxyoximes dsao and hnapo to give the corresponding amines16 is proposed. the voltammetric behaviour of dsao in hydro-alcoholic solutions using differential pulse voltammetry (dpv) is reported. an experimental design methodology was used to establish the optimum values of the instrumental variables to determine dsao. the taguchi17 design methodology was used in order to extend the former optimized method to the determination of hnapo. the method was applied to quantify the dsao and hnapo contents in samples of commercial extractants. experimental reagents and chemicals standards of dsao were prepared from samples of lix 860-ic (90 % dsao), kindly supplied by cognis. a sample of pure hnapo (98.8 % purity) was obtained from the commercial extractant sme 529 (shell chemical co.) according to the method proposed by preston and whewell.18 hydro-alcoholic solutions of the standards and the commercial samples were prepared using methanol (carlo erba, p.a.) as diluent, kcl (panreac, p.a.) as ionic medium and britton–robinson buffer containing 0.04 m of each phosphoric acid (carlo erba, p.a.), acetic acid (carlo erba, p.a.) and boric acid (fluka, p.a.) to adjust the ph. the analyzed commercial extractants were lix 860, lix 860-i, lix 622 (cognis) and lix 84 (henkel). isotridecanol n (basf), 4-dodecylphenol (aldrich) and 4-nonylphenol (acros), that could be present in the commercial formulations,1,2 and the diluent shellsol d70 (shell) were also tested. electrochemical analysis of hydroxyoxime-based extractants 419 experimental procedure voltammetric measurements were performed with an autolab/general purpose electrochemical system (gpes) package, equipped with a potentiostat model iii connected to a metrohm 647 va stand. a three-electrode electroanalytical cell (50 ml) with a hanging mercury drop electrode (hmde) as the working electrode, a platinum auxiliary electrode and an ag/agcl reference electrode (3 m kcl) was used for all measurements, and all potentials in the paper refer to this electrode. the electrodes were washed with mixtures of ultra-pure water (milli-q water purification system) and methanol and tested prior to each experiment. the ph of the solutions was measured using a radiometer phm64 fitted with an ingold combined ag/agcl glass electrode. all the experiments were performed at 298.0±0.5 k. oxygen was removed from the solutions by purging with nitrogen gas for 4 min. results and discussion preliminary voltammetric experiments the differential pulse voltammograms of the dsao-based extractant lix 622 are shown in fig. 1. it could be observed that methanol percentage and ph affect both the peak potential and the peak intensity. the intensity of the reduction peak increased with increasing methanol percentage (fig. 1a). however, in order to avoid precipitation of the reagent and, simultaneously, maintain a minimum methanol percentage in the solutions, 40 % methanol was chosen. on the other hand, the intensity of the reduction peak of dsao decreased with ph (fig. 1b). taking into account that the solutions were not stable with time at ph values below 2.0, this ph value was selected. finally, an ionic medium of 0.1 m kcl was always used to keep the ionic strength constant. it could be observed that under these conditions (40 % methanol, ph 2.0), the reduction peak of the oxime group in dsao appeared at around –0.80 v. none of the other compounds that the extractants might contain gave rise to reduction peaks in the studied potential range. fig. 1. differential pulse voltammograms of the dsao based lix 622 extractant (5.84 mg l-1) as a function of: a) methanol content and b) ph; tm = 0.05 s, ti = 0.3 s, sp = 5 mv and am = 50 mv. 420 elizalde et al. optimization of the instrumental variables in the differential pulse voltammetric (dpv) determination of dsao to establish the optimum conditions for the determination of dsao by the dpv technique, a two-level factorial design was performed. the response variable was the peak intensity (ip), which could be influenced by a large number of instrumental and chemical variables. in this work, only the instrumental variables, modulation time (tm), interval time (ti), potential step (sp) and modulation amplitude (am) were optimized. a 24 factorial design was applied to estimate the residual error. prior to performing a design of experiments, the maximum and minimum values for each factor were established. all the experiments were realized in a random order and repeated. the values corresponding to the minimum (–) and maximum (+) levels for each factor were the following: 0.1 and 0.4 s (interval time), 2 and 15 mv (potential step), 0.002 and 0.060 s (modulation time) and 20.0 and 75.0 mv (modulation amplitude), respectively. by using the unscrambler program,19 the p factors for each variable and for the interactions between them were calculated, p being the probability of their coefficient being zero (null hypothesis). if the p value for a parameter is higher than 0.05, the parameter has no significant effect on the model (at a confidence level of 95 %) and it could be eliminated. for analysis, the results were presented as a pareto chart, shown in fig. 2. the bar lengths are proportional to the p value of each variable. as could be seen, the modulation time and modulation amplitude are the significant factors and there is an interaction between these factors. on the other hand, it was proven that the intermediate values of interval time and potential step, non-significant parameters, gave rise to good results and provided an acceptable scan rate (50 mv s–1). consequently, the interval time and potential step were fixed at 0.2 s and 10 mv, respectively. fig. 2. pareto chart showing the results of the experimental design applied to the dpv technique. electrochemical analysis of hydroxyoxime-based extractants 421 once the significant variables were found, a central composite design with three replications at the central point was performed. the values corresponding to the minimum (–), maximum (+) and centre point (0) levels were, respectively, 0.012 s (–), 0.060 s (+) and 0.036 s (0) for the modulation time, and 20.0 mv (–), 75.0 mv (+) and 47.5 mv (0) for the modulation amplitude. the data obtained were analyzed using anova. the proposed model was fitted to the following non-linear equation: 2 2p m m m m m m0.0229 0.0008 0.9680 0.0021 0.0095 0.0141i a t a t a t= + − + + − (1) where ip is the predicted response and the coefficients are the adjusted parameters. this second order function is adequate to model the data because the lack of fit was not significant at the 95 % confidence level. in addition, the value of the coefficient of determination, r2 = 0.986, indicates that most of the variability in the response is explained by the model. consequently, taking into account that outliers were not detected and the residuals showed no clear pattern, the experimental results are successfully interpreted. the anova test indicated that at a confidence level of 95 %, the modulation time and the modulation amplitude were the significant factors (p < 0.05), with an interaction between these variables. moreover, the values of the coefficients in eq. (1) indicate that the intensity of the reduction peak increases with increasing modulation amplitude and with decreasing modulation time. the optimum values for the variables modulation time (0.012 s) and modulation amplitude (75.0 mv) were obtained by graphical analysis of the response surface graph (fig. 3). fig. 3. response surface generated by the central composite design for the optimization of the variables modulation amplitude and modulation time in the dpv analysis of dsao. optimization of the instrumental variables in the dpv determination of hnapo the taguchi methodology17 was applied in order to check if the optimized design could also be applied to the determination of hnapo. an orthogonal l8 422 elizalde et al. matrix was used in order that the variables modulation amplitude, modulation time, potential step and interval time as well as the interaction between the two former factors could be evaluated. the values corresponding to the minimum and maximum levels were those previously selected for dsao. the results (fig. 4) showed that the modulation amplitude, modulation time and their interactions are the most influential factors. the anova tests indicated that the modulation amplitude had the greatest influence (57.3 %) followed by the modulation time (13.6 %). fig. 4. effect of the variables on the voltammetric peak intensity of hydro-alcoholic hnapo solutions (0.82 mg l-1) by applying the taguchi methodology. the results obtained showed that the optimum values for the significant factors, modulation amplitude and modulation time, are 75.0 mv and 0.012 s, respectively, whereas the maximum value of the potential step (10 mv) could be considered as the most appropriate, with the least influential factor, interval time, taking any value between 0.1 and 0.4 s. finally, confirmatory experiments were performed to verify that the predicted value for the response (ip) could be experimentally obtained. the theoretical response was obtained according to the following equation: p p2 m2 m1 m2 m1 m2 m1( ) ( ) ( ) ( ) ( ) ( )i t + s t a t t t a t t a t t t = − + − + − + − − − − −  (2) where t is the mean average of all the experimental results, and the values p2s , m2a and m1t are the average responses corresponding to the experiments in which the different factors had acquired the level indicated in the subscript. the theoretical current value obtained for a 0.82 mg l–1 hnapo solution using the optimum combination of variables ( p2 m2 m1s a t ) was –79.4 na, with –74.7 na being the experimental ip value obtained at interval time values between 0.1 and 0.4 s, thus confirming the independence of the response on the interval time factor, and evidencing that the optimum combination of variables electrochemical analysis of hydroxyoxime-based extractants 423 previously obtained for the analysis of dsao could also be used for the determination of hnapo. analysis of dsao and hnapo in commercial extractants for the determination of dsao and hnapo in hydro-alcoholic solutions, calibration curves of the standards (0.1 m kcl / 40 % methanol at ph 2.0) using the optimized method (interval time: 0.2 s, potential step: 10 mv, modulation time: 0.012 s and modulation amplitude: 75.0 mv) were constructed and are shown in figs. 5 and 6, respectively. fig. 5. dpv calibration curve of dsao in 0.1 m kcl / 40 % methanol at ph 2.0. fig. 6. dpv calibration curve of hnapo in 0.1 m kcl / 40 % methanol at ph 2.0. the statistical parameters of the calibration curves are summarized in table i. the limits of detection and quantification were taken as 3 and 10 times the standard deviation of the regression lines, respectively. the intraday repeatability was checked in each case by analyzing three to five samples independently prepared according to the established procedure. the results, expressed as rsd / %, are also given in table i. 424 elizalde et al. table i. statistical parameters of the calibration curves for the determination of dsao and hnapo by dpv parameter dsao hnapo linear range, mg l-1 0.05–2.45 0.07–0.82 slope ± s, na l mg-1 133.98±0.57 86.50±0.76 intercept ± s, na 2.27±0.71 0.55±0.32 r2 0.9999 0.9995 limit of detection, mg l-1 0.034 0.019 limit of quantification, mg l-1 0.113 0.064 intraday repeatability, rsd / % 0.63 (0.17 mg l-1, n = 3) 1.92 (2.14 mg l-1, n = 3) 2.47 (0.17 mg l-1, n = 5) 1.29 (0.57 mg l-1, n = 5) the developed dpv method was applied to determine dsao in lix 860, lix 860-i and lix 622 and hnapo in lix 84. all the solutions were prepared as described previously (40 % methanol, 0.1 m kcl at ph 2.0) and measured three times according to the optimized dpv conditions. the results are given in table ii. table ii. comparison of different methods for the determination of the percentages of dsao and hnapo in several commercial extractants analytical technique extractant compound mass % ref. hplc-uv lix 860 dsao 64.82±0.96 12 gc-ms lix860 dsao 66.6±2.0 13 dpv lix 860 dsao 65.7±1.9 this work dpv lix 860-i dsao 69.4±1.9 this work dpv lix 622 dsao 75.0±2.0 this work dpv (platinum electrode) lix 84 hnapo 49.8±3.4 14 dpv lix 84 hnapo 49.9±1.2 this work samples of lix 860 and lix 84 had been previously analyzed by chromatographic (hplc12 and gc-ms13) and voltammetric (dpv14) methods, respectively as seen in table ii, and the results acceptably agreed with those obtained using the optimized voltammetric method proposed in this work. conclusions the optimum combination of instrumental variables in the analysis of dsao by differential pulse voltammetry was obtained by means of an experimental design methodology. under these conditions, a well resolved reduction peak at – 0.80 v was recorded. the optimized method was extended by using the taguchi methodology to the analysis of hnapo under the same experimental conditions by the same technique. the differential pulse voltammetric method was successfully applied to the determination of dsao and hnapo in several commercial extractants. the results obtained from the developed method showed good agreement with pre electrochemical analysis of hydroxyoxime-based extractants 425 viously reported hplc-uv, gc-ms and electrochemical results. moreover, this is the first time that dsao was determined in lix 860-i and in lix622. и з в о д електрохемијско одређивање 5-додецилсалицилалдоксима и 2′-хидрокси-5′-нонилацетофенон-оксима у комерцијалним екстракционим средствима maría p. elizalde, maría del sol rúa, marta huebra и begoña menoyo department of analytical chemistry, faculty of science and technology, university of the basque country (upv/ehu), p. o. box 644, 48080 bilbao, spain оптимизована је метода диференцијалне пулсне волтаметрије уз коришћенње капљуће живине електроде (dme) за одређивање 5-додецилсалицилалдоксима у алкохолним растворима применом факторског и централног композитног дизајна. за одређивање 2′-хидрокси-5′-нонилацетофенон-оксима коришћена је методологија taguchi дизајна са циљем проширења претходно оптимизоване методе. метода је успешно примењена за одређивање 5-додецилсалицилалдоксима и 2′-хидрокси-5′-нонилацетофенон -оксима у узорцима комерцијалних екстркционих средстава, у концентрационим опсезима 0,05–2,45 и 0,07–0,82 mg l-1, редом, са границама детекције 0,034 и 0,019 mg l-1, редом. (примљено 7. јула, ревидирано 22. новембра, прихваћено 29. новембра 2016) references 1. b. menoyo, a. ocio, m. p. elizalde, chromatographia 65 (2007) 35 2. a. ocio, b. menoyo, m. p. elizalde, in proceedings of the international solvent extraction conference, ed. b. a. moyer, santiago, chile, 2011, pp. 5, 1 3. a. abelairas, m. j. puertollano, r. m. alonso, m. p. elizalde, r. m. jiménez, m. huebra, analyst 119 (1994) 323 4. m. huebra, m. p. elizalde, j. m. castresana, electroanalysis 6 (1994) 785 5. i. álava, m. p. elizalde, m. huebra, analyst 120 (1995) 1069 6. m. j. citores, r. m. alonso, l. a. fernández, electroanalysis 4 (1992) 733 7. w. sowa, b. h. light, i. j. itzkovitch, j. chromatogr., a 116 (1976) 197 8. j. w. hosking, n. m. rice, hydrometallurgy 3 (1978) 217 9. l. c. stone, t. n. andersen, j. c. parr, d. d. metcalf, anal. chem. 54 (1982) 1965 10. e. y. o. paatero, hydrometallurgy 13 (1984) 193 11. j. staniewski, j. szymanowski, j. chromatogr., a 503 (1990) 442 12. a. ocio, m. p. elizalde, j. a. prieto, j. chromatogr., a 1032 (2004) 129 13. a. ocio, b. menoyo, m. p. elizalde, can. j. anal. sci. spectrosc. 54 (2009) 63 14. m. p. elizalde, m. s. rúa, a. almela, m. huebra, in proceedings of the international solvent extraction conference, b. a. moyer, ed., tucson, ar, usa, 2008, pp. 2, 903 15. b. g. kumar, p. v. ramana, int. j. res. pharm. biomed. sci. 4 (2013) 1069 16. t. osterund, m. prytz, acta chem. scand. 10 (1956) 451 17. g. taguchi, s. chowdhury, y. hu, taguchi´s quality engineering handbook, wiley, hoboken, nj, 2005 18. j. s. preston, r. j. whewell, j. inorg. nucl. chem. 39 (1977) 1675 19. k. h. esbense, multivariate data analysis, camo process as, oslo, norway, 2001. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {ionic-interaction of aqueous and alcoholic poly(vinyl alcohol) in presence of protons} j. serb. chem. soc. 84 (3) 317–326 (2019) udc 539.196–71:678.744+546.212:532.13: jscs–5186 536.7 original scientific paper 317 ionic-interaction of aqueous and alcoholic poly(vinyl alcohol) in the presence of protons saima naz* and rehana saeed department of chemistry, university of karachi, karachi 75270, pakistan (received 1 april, revised 29 september, accepted 7 november 2018) abstract: the ionic-interactions of acetic acid in h2o–pvoh and h2o–alcohol–pvoh solvent systems were studied at different temperatures by the viscosity method. the viscosities of the poly(vinyl alcohol) (pvoh) were increased with increasing concentration of pvoh and decreased with the increasing concentration of acetic acid. the viscosity data were used to evaluate the ion–ion interactions and ion–solvent interactions in terms of the a and b coefficients of the jones–dole equation, respectively. the negative values of the b-coefficient increased with increasing temperature, showing that the acid behaves as a structure breaker in pvoh–solvent mixtures and consequently, the values of the a-coefficient were decreased with increasing temperature. thermodynamic parameters, such as energy of activation (ea*), gibbs energy change of activation (∆g*) and entropy change of activation (∆s*) were also calculated as a function of the acid (ch3cooh) concentration, pvoh composition, alcohols and temperature. keywords: viscosity; alcohols; ion–solvent interaction; thermodynamic parameters. introduction polymeric materials are widely used in many fields due to their vast applications.1,2 poly(vinyl alcohol) (pvoh) has unique characteristics of excellent filming ability, good water solubility and viscoelastic property.3–5 it is a highly hydrophilic, non-toxic and biocompatible polymer, which makes it industrially important and it is used in sizing agents and adhesives.6,7 these applications show the involvement of the polymer in the form of solution since the thermal degradation characteristics of pvoh limit its ability to be used as a conventional thermoplastic. viscosity is one of the simplest and reliable methods giving significant information about ion–ion, ion–solvent, and solvent–solvent interactions.8 the interaction between an ionic solute with non-ionic species in different solvent systems changes * corresponding author. e-mail: sam_cancer90@yahoo.com https://doi.org/10.2298/jsc180401097n 318 naz and saeed the physical and chemical properties of the system and the interactions between solute–solvent systems can be evaluated using the jones–dole equation.9 the viscosity of the polymer solution is influenced by the concentration of polymer in solution. at high concentrations of polymer, association of polymer chains starts, which results in inter-molecular interactions and decreased movement of the polymer.10 the extent of interand intra-hydrogen bonding between the polymer chain and water molecules decreases with increasing thermal energy. strong interand intra-molecular chain hydrogen bonding exists between the polar hydroxyl groups in pvoh molecules. the solubility and viscosity of pvoh solution is dependent on two forces, the interand intra-chain hydrogen bonding and solute-solvent hydrogen bonding.11,12 various factors directly influence the polymer–solvent associations such as temperature, nature of the solvent, polymer composition, acid concentration and electrolyte concentration. in the present study, viscometric and thermodynamic studies of pvoh in different aqueous alcoholic solvents in the presence of acetic acid were undertaken. ion–ion and ion–solvent interactions were analyzed by the jones–dole equation in terms of the a and b coefficients, respectively. the energy of activation (ea*), gibbs energy change of activation (∆g*) and change in entropy of activation (∆s*) were also evaluated as a function of the concentration of acetic acid, the concentration of polymer and temperature. the results are explained in terms of structural and electrostatic interactions. experimental materials and methods pyrex a-grade quality equipment was used throughout the experiment. methanol (analar, 99.8 %), ethanol (merck, 99.8 %), 2-propanol (merck, 99.7 %), 1-butanol (merck, 99.5 %), and acetic acid (merck) were used. poly(vinyl alcohol) (pvoh, merck) with a weight average molecular mass of 72000 g mol-1 and degree of hydrolysis of 98 % was used for the preparation of 0.5 % stock aqueous solutions of poly(vinyl alcohol) by dissolving 0.5 g of pvoh in 100 ml of solution at 80 °c under constant stirring using a magnetic stirrer with hot plate 78hw-1. acetic acid solutions with concentration ranging from 0.1 mol dm-3 to 0.5 mol dm-3 were prepared in 0.1, 0.35 and 0.5 % pvoh solutions in aqueous and 5 vol. % alcoholic medium (methanol, ethanol, 2-propanol or 1-butanol). a ph-meter model hi9813 was used to measure the ph values of the solutions. a relative density bottle having a capacity of 10.0 cm3 was used for the measurement of densities of solvent and solutions at various temperatures. the mass determination was made on a sartorius electronic balance model bl-150s (germany) with ±0.001 g uncertainty. the density of solutions and solvents was calculated using the relation: d = m/v (1) the viscosities of the solvents and solutions were measured using a constant volume ostwald viscometer (techniconominal constant 0.1 cs s-1 capillary astm d445). the flow time of a solution between two marks was recorded using a stopwatch (shanghai, china, having a least count of 0.2 s). three observations were made to ensure the reproducibility of the measurements. a thermostated water bath (model ycw-01, taiwan, r.o.c.) was used to ionic-interaction of pvoh 319 maintain a constant temperature, ranges from 305 to 323 k with the interval of 5 k. the viscosity of the solution was calculated using the following relation: ɳ = (dt/d0t0)ɳ0 (2) where, ɳ and ɳ0 are the viscosity of the solution and solvent, respectively, t and t0 are the time of flow of solution and solvent, respectively, d and d0 are the density of the solution and solvent, respectively. results and discussion the densities and viscosities of the aqueous and aqueous alcoholic poly(vinyl alcohol) (pvoh) solutions having concentrations 0.1, 0.35 and 0.5 % in acetic acid (0.1 mol dm–3 to 0.5 mol dm–3) were measured in the temperature range 305to 323 k at an interval of 5 k. the results tabulated in table s-i of the supplementary material to this paper show that densities of the pvoh–solvent system increased with increasing acetic acid concentration and increasing percent pvoh concentration whereas decreased with increasing temperature from 305 to 323 k. the densities of aqueous pvoh solutions were higher as compared to water–pvoh–alcohol systems and decreased in the order: 1-butanol > methanol > 2-propanol > ethanol. dipolar attraction, van der waals and hydrogen bonding make water denser as compared to alcohols. 1-butanol and methanol are tightly packed as compared to 2-propanol and ethanol due to more attractive forces that result in an increase in density. 2-propanol molecules, due to their branched structure, occupy more space and have fewer attractive forces and hence, there is more space between the molecules, hence density values are lower. molecules of ethanol are packed loosely and occupy more space, which results in a decrease in the density of the solution. the viscosity values of the solutions are tabulated in table s-ii of the supplementary material, which show that the values increased with increasing percent of pvoh but decreased with increasing concentration of acetic acid and with increasing temperature from 305 to 323 k. it was observed that the aqueous medium has the lowest values of viscosities, which increased in the following order: methanol < ethanol < 2-propanol < 1-butanol. with the addition of an alcohol, the interactions between pvoh and alcohols are increased that results in an increase in viscosity. viscosities of aqueous and 5 vol. % alcoholic pvoh systems decreased with increasing concentration of acetic acid, due to increasing degradation of pvoh. a representative graph showing the effect of the concentration of acetic acid on the viscosities of 0.1 and 0.5 % pvoh in 5 vol. % ethanol at 305 k is shown in fig. 1. the increase in percent composition of pvoh caused an increase in the viscosities of the solution due to the association of the polymer molecules by interand intra-molecular forces in the polymer solution. 320 naz and saeed � fig. 1. a plot of viscosity vs. concentration of acetic acid in pvoh–5 vol. % ethanol solvent systems at 305 k. electrostatic, hydrophobic interactions, van der waals forces and hydrogen bonding are the sources responsible for the formation of associations in polymer solutions leading to an increase in the viscosity. the results reported in table s-ii show that there is a regular decrease in the viscosities with increasing in temperature in aqueous pvoh and 5 vol. % alcoholic–pvoh solutions. as the temperature increased, due to a break down of the supramolecular structure, the intermolecular forces between solvent molecules decreased, resulting an increase in the molecular motion. therefore, the viscosities of a liquid were found to decrease. viscosity is affected by many factors as shown by the relation:13 η = η0 + η+ + ηa + ηe+ ηd (3) where, η0 is the viscosity for solvent, η+ is the positive increase in viscosity caused by long-range electrostatic interaction, ηa is the increase due to alignment or orientation of polar molecules by the ionic field, ηe is the positive increase in viscosity due to the shape and size of an ion, ηd is the decrease in viscosity arising due to distortion of the solvent structure by the ions.13 the results tabulated in table s-ii also showed that the viscosities of aqueous pvoh solutions at a fixed concentration of acetic acid are lower as compared to aqueous pvoh–alcohol systems and increased in the following order: methanol < ethanol < 2-propanol < 1-butanol. this is due to the change in the nature and extent of the interactions. in the case of aqueous, 5 vol. % methanol or 5 ethanol, the solvation power increased, respectively, and hence, the viscosities of the solutions increased. the viscosity was also influenced with the size of the molecule, and for this reason, the solutions containing 5 vol. % 1-butanol have higher values of viscosity as compared to those in 5 vol. % methanol. a representative graph showing the effect of dif ionic-interaction of pvoh 321 ferent solvents on the viscosity of pvoh in an acidic medium at 305 and 323 k is shown in fig. 2. � fig. 2. a plot of viscosity vs. different solvents in 0.1 mol dm-3 acetic acid–0.5 % pvoh systems at different temperatures. the increase in interaction of acid with pvoh degraded the supramolecular structure of water–pvoh system. in the case of an alcohol–pvoh system, the alcohol bound the pvoh molecules and increased the interaction between solvent and alcohol molecule, and as a result the fluidity of the solution decreased. it was observed that as the molecular weight of the alcohol increased from methanol to 1-butanol, the viscous nature of the solution increased leading to an increase in the viscosities of the solutions. the results tabulated in table s-ii showed that acid breaks the structure of resulting molecule and the viscosity of the system decreased with increasing concentration of acetic acid. the results tabulated in table s-iii of the supplementary material show that values of ph increased with increasing percentage composition of pvoh and decreased with increasing concentration of acetic acid. no significant change was observed on variation of the alcohol. the interaction between water and pvoh increased with the percentage composition of pvoh, therefore, h+ are less available in a free condition, which results in relatively high values of ph in 0.5 % pvoh solutions as compared to the 0.1 % pvoh solutions. on increasing the concentration of acid, the number of protons in the medium increased resulting in a decrease in the ph. a representative plot showing the effect of different solvents vs. the ph of the solutions containing 0.1 and 0.5 % pvoh in the presence of 0.1 mol dm–3 ch3cooh is shown in fig. 3. the ionic interactions of acetic acid in aqueous pvoh and aqueous alcoholic pvoh solvents were studied in terms of the aand b-coefficients of the jones–dole equation expressed by the relation:14,15 322 naz and saeed ηsp/√c = a + b√c (4) where ηsp is the specific viscosity, c is the concentration of acetic acid, and a and b are the jones–dole coefficients representing ion–ion and ion–solvent interactions. the linear regression method was employed to evaluate the values of the a and b coefficients from the intercepts and slopes of linear plots of ηsp/√c against the square root of acetic acid concentration. a representative plot of ηsp/√c vs. the square root concentration of acetic acid (ch3cooh) in 0.35 % pvoh–solvent systems at 308 k is shown in fig. 4. � fig. 3. a plot of ph vs. solvent solutions of pvoh in 0.1 mol dm-3 acetic acid. � fig. 4. a plot of ηsp/√c vs. √c for the 0.35 % pvoh–solvent system at 308 k. the values of the aand b-coefficients at different temperatures for solutions containing acetic acid and pvoh in aqueous and 5 vol. % alcoholic systems are tabulated in tables s-iv and s-v of the supplementary material, res ionic-interaction of pvoh 323 pectively. the results show that the ion–ion interactions decreased with rising temperature and increased with the increasing concentration of pvoh. the ion– –solvent interactions increased with increasing temperature and a decrease in the ion–solvent values was observed with increasing percent composition of pvoh. it was also observed that the values of the aand b-coefficients increased as the alkyl chain of the alcohol increased. higher values of ion–ion interactions and ion–solvent interactions were observed in water as compared to aqueous methanol and aqueous ethanol, but lower values were observed as compared to aqueous 2-propanol and aqueous 1-butanol. these results indicated that the interactions of acid are due to pvoh being less solvated in 2-propanol and 1-butanol. the negative values of the b-coefficient show the association between acetic acid and aqueous pvoh and 5 vol. % alcoholic–pvoh through ion–solvent interactions. the negative the b-coefficient values indicate the structure breaking behavior of the acid in aqueous pvoh and 5 vol. % alcoholic–pvoh systems. the negative values of the b-coefficient confirmed that acetic acid in the polymer solution decreased the viscosity by degrading the supramolecular structure of the polymer in different alcohol solvents. the thermodynamic parameters, such as energy of activation (ea*), gibbs energy change of activation (∆g*) and entropy change of activation (∆s*) of pvoh solutions were evaluated by the influence of temperature on the viscosities. the values of apparent activation energy (ea*) of various flows were obtained by the arrhenius relation:16 η = aexp (ea/rt) (5) ln η = ln (hna vm) + ea/rt (6) where a is the pre-exponential factor, r is gas constant, t is absolute temperature, h is plank’s constant, na is avogadro’s number, η is the viscosity of polymer solution and vm is the molar volume. the energy of activation (ea*) and molar volume (vm) were calculated from the slope and intercept of the plot of ln η vs. 1/t. a represented plot is shown in fig. 5. the results for the activation energy of pvoh in aqueous and 5 vol. % alcohol containing acetic acid are tabulated in table vi, from which it could be observed that the energy of activation of solutions containing acetic acid in aqueous and 5 vol. % alcoholic pvoh increased with increasing percent composition of pvoh. degradation of the structure and orientation of macromolecules occur during flow and hence, positive values of the activation energy were obtained. with increasing percent composition of pvoh, the increased in activation energy may be due to the fact that at higher concentrations, a greater number of ions are present, resulting in a decrease in the ionic mobility that make it difficult to produce vacant sites in the solvent matrix that led to the higher values of energy of activation. the aqueous pvoh system had the lowest value of energy of activation, which increased with 324 naz and saeed alcohol in the order: aqueous medium < methanol < ethanol << 2-propanol < 1-butanol. the gibbs energy change of activation (∆g*) and the change in entropy of activation (∆s*) are expressed by the relations:17–19 ∆g* = rtln (ηvm/hna) (7) ∆s* = (ea* – ∆g*)/t (8) � fig. 5. a plot of ln η vs. 1/t for different mediums in 0.5 % pvoh and 0.3 mol dm-3 acetic acid. the values of gibbs energy change of activation tabulated in table s-vi showed that an increase in the percent composition of pvoh resulted in higher values of the change in the gibbs energy of activation. whereas, increasing the concentration of acetic acid lowered the value of gibbs energy change of activation. this showed that in dilute solutions, the associations were weaker and could be easily overcome during flow while in high concentration of pvoh systems, these associations were stronger and were little affected during the flow process. therefore, the values of the gibbs energy change of activation were high. negative values of the entropy change were observed in the pvoh–solvent systems. the increase in the values of the entropy change with increasing concentration of acetic acid showed the randomness of the pvoh systems and explained the structure breaking ability of acetic acid in the pvoh–solvent systems. conclusions ionic interactions of acetic acid with poly(vinyl alcohol) (pvoh) in aqueous and different alcohols were studied by viscosity method. viscosities of solutions increased with increasing percentage composition of pvoh, and were influenced by the different alcohols in the order: methanol < ethanol < 2-propanol < 1-butanol. ionic-interaction of pvoh 325 decreased viscosities were observed with increasing concentration of acetic acid and with increasing temperature from 305 to 323 k. ionic interactions of acetic acid in the pvoh–solvent systems were evaluated in terms of the aand b-coefficients of the jones–dole equation. the negative values of the b-coefficient increased with increasing temperature showed that the acid behaves as a structure breaker in the h2o–pvoh and h2o–alcohol–pvoh solvent systems. it was observed that the water–pvoh systems have lower values of the activation energy as compare to water–alcohol–pvoh systems. the negative values of the entropy change also showed the structure breaking behavior of acetic acid in the pvoh–solvent systems. supplementary material additional data of the synthesized compounds are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. и з в о д јонске интеракције поли(винил-алкохола) у воденом и алкохолним растворима у присуству протона saima naz и rehana saeed department of chemistry, university of karachi, karachi 75270, pakistan јонске интеракције сирћетне киселине у h2o– поли(винил-алкохола) (pvoh) и h2o– –алкохол–pvoh системима растварача су испитане на различитим температурама применом вискозиметрије. вискозност раствора pvoh се повећавала са порастом концентрације полимера и смањивала са повећањем концентрације сирћетне киселине. подаци о вискозитету раствора су коришћени за одређивање коефицијента а и в у jones–dole једначини, на основу којих су процењиване јон–јон интеракције и јон–растварач интеракције. пораст негативне вредности коефицијента b са повећањем температуре је показао да присуство киселине нарушава надмолекулску структуру pvoh у смешама, а као последица тога и вредности коефицијента a су смањиване са порастом температуре. термодинамички параметри као што је енергија активације(ea*), промена слободне енергије активације (g*) и промена ентропије активације (s*) такође су израчунати као функција концентрације киселине(ch3cooh), садржаја pvoh, врсте алкохола и температуре. (примљено 1. априла, ревидирано 29. септембра, прихваћено 7. новембра 2018) references 1. e. a. el-hefian, m. m. nasef, a. h. yahaya, e.-j. chem. 7 (2010) s349 (http://dx.doi.org/10.1155/2010/275135) 2. p. shard, a. bhatia, d. sharma, int. j. drug del. 6 (2014) 58 3. l. r. shivakumara, b. p. dayananda, t. demappa, indian j. adv. chem. sci. s1 (2016) 13 4. s. mallakpour, m. dinari, e. azadi, int. j. polym. anal. charact. 20 (2015) 82 (https://doi.org/10.1080/1023666x.2015.976396) 5. a. f. sabirneeza, s. subhashini, j. adhes. sci. technol. 29 (2015) 1268 (https://doi.org/10.1080/01694243.2015.1025464) 6. z. abdeen, s. g. mohammad, m. s. mahmoud, environ. nanotechnol. monit. manage. 3 (2015) 1 (https://doi.org/10.1016/j.enmm.2014.10.001) 326 naz and saeed 7. y. dou, b. zhang, m. he, g. yin, y. cui, i. n. savina, polymer 7 (2015) 580 (https://doi.org/10.3390/polym7030580) 8. r. saeed, s. masood, n. h. siddiqui, pet. sci. technol. 32 (2014) 688 (https://doi.org/10.1080/10916466.2011.601509) 9. r. saeed, s. masood, s. m. s. nadeem, int. j. chem. 4 (2012) 28 (http://dx.doi.org/10.5539/ijc.v4n4p28) 10. p. hong, c. chou, c. he, polymer 42 (2001) 6105 (https://doi.org/10.1016/s00323861(01)00056-8) 11. b. briscoe, p. luckham, s. zhu, polymer 41 (2000) 3851 (https://doi.org/10.1016/s00323861(99)00550-9) 12. c. damas, t. leprince, t. huong, v. ngo, r. coudert, colloid polym. sci. 286 (2008) 999 (https://doi.org/10.1007/s00396-008-1862-6) 13. r. saeed, s. masood, s. khan, int. j. sci. 2 (2013) 83 14. r. saeed, f. uddin, a. fazal, j. chem. eng. data 47 (2002) 1359 (https://doi.org/10.1021/je020022w) 15. v. laxmi, m. singh, j. indian chem. soc. 85 (2008) 184 16. n. ahmed, s. akber, i. khan, a. saeed, j. chem. soc. pak. 10 (1988) 43 17. s. masood, r. saeed, m. ashfaq, eur. j. chem. 6 (2015) 37 (https://dx.doi.org/10.5155/eurjchem.6.1.37-43.1146) 18. n. d. shooto, c. w. dikio, d. wankasi, l. m. sikhwivhilu, f. m. mtunzi, e. d. dikio, nanoscale res. lett. 11 (2016) 426 (https://doi.org/10.1186/s11671-016-1631-2) 19. r. saeed, s. masood, f. uddin, phys. chem. liq. 46 (2008) 9 (https://doi.org/10.1080/00319100601188703). {extractive purification of hydrotreated gas oil with n-methylpyrrolidone} j. serb. chem. soc. 82 (1) 107–116 (2017) udc 665.75:54.05:66.061–911.4:66.094.522.8 jscs–4951 original scientific paper 107 extractive purification of hydro-treated gas oil with n-methylpyrrolidone kahina bedda1*, boudjema hamada1, nikolay v. kuzichkin2 and kirill v. semikin2 1laboratory of petrochemical synthesis, faculty of hydrocarbons and chemistry, university m’hamed bougara-boumerdes, algeria and 2st. petersburg state institute of technology (technical university), st. petersburg, russia (received 23 may, revised 21 november, accepted 28 november 2016) abstract: the purification of hydro-treated gas oil by liquid–liquid extraction with n-methylpyrrolidone as solvent was studied. the results showed that this method, under appropriate experimental conditions, reduced the sulphur content of the gas oil from 174 to 28 ppm, the nitrogen content was decreased from 58 to 15 ppm, the aromatics content was diminished from 27.1 to 13.8 % and the polycyclic aromatic hydrocarbons were totally extracted. the obtained refined gas oil could be used to produce clean diesel fuel, thus protecting the environment. keywords: diesel fuel; desulphurization; denitrification; de-aromatization. introduction diesel is extensively used as a fuel both in highway transportation vehicles (e.g., cars, buses and trucks), and non-highway transportation systems (e.g., locomotives, marine vessels, farm equipment, etc.).1–3 gas oil cuts contain a complex mixture of hydrocarbons having different molecular weights and boiling points, which are mainly composed of paraffins, naphthenes and aromatics together with small amounts of organic sulphur and nitrogen compounds.1,2,4 the sulphur present in the diesel fuel is a major source of air pollution. during combustion in the diesel engines, the sulphur compounds burn to form harmful sulphur oxides (sox) and sulphate particulates, and the nitrogen compounds are oxidized to nitrogen oxides (nox).4 aromatic hydrocarbons reduce the quality of diesel fuel and increase the emissions of particulate matter and polycyclic aromatic hydrocarbons (pah). many environmental problems and health hazards are caused by exhaust emissions from diesel engines, which contain sox, nox, co, co2, particulate * corresponding author. e-mail: bedda.kahina@gmail.com doi: 10.2298/jsc160523004b 108 bedda et al. matter and unburned hydrocarbons. for these main reasons, fuels specifications in many countries worldwide reduce the content of sulphur, nitrogen and aromatic hydrocarbons in diesel fuel to lower levels.5 hydro-treating is one of the most important processes in petroleum refining; it helps to improve the characteristics of diesel fuel to make it meet the required specifications and pollution standards. the gas oil hydro-treating process is a catalytic process in which gas oil obtained from either primary distillation of crude oil or conversion processes (visbreaking, coking and catalytic cracking) is treated under hydrogen pressure.6,7 this process helps obtain a quality diesel fuel with low contents of sulphur, nitrogen and aromatics together with a better stability and a high cetane number.1,5–7 the performance of gas oil hydro-treaters is affected by operating conditions, such as reactor temperature, liquid hourly space velocity (lhsv), hydrogen partial pressure and recycle gas to oil ratio.1,5 kinetics studies showed that the rates of hydrogenation of dibenzothiophene, benzothiophene, and thiophene homologues are low,1,5,8 but they can be increased by increasing the temperature and/or the hydrogen partial pressure and decreasing the space velocity, which leads to large capital investments and high specific power consumption.1,5 the use of higher operating severity for lowering sulphur levels to ultra low levels in conventional gas oil hydro-treaters have practical limitations related to cycle length, throughput, design pressure, hydrogen availability, thermodynamic equilibrium, etc. in view of this, alternative desulphurization methods that do not use hydrogen for catalytic decomposition of organosulphur compounds have been the subject of recent studies. oxidative desulphurization (ods) is a promising technology for the reduction of sulphur at low temperatures (≈50 °c) and atmospheric pressure.1,9 in ods, the organic sulphur compounds are oxidized by adding one or two oxygen atoms to the sulphur using appropriate oxidants without breaking any carbon–sulphur bonds, yielding sulphoxides and sulphone, respectively. these oxidized compounds are then extracted or adsorbed due to their increased relative polarity. thus, the ods is a two stage process; oxidation, followed by liquid extraction or adsorption. desulphurization by adsorption (ads) is an unconventional process of removing sulphur which is based on the ability of a solid sorbent to selectively adsorb organosulphur compounds from refinery streams.10 based on the mechanism of interaction the sulphur compound with the sorbent, ads could be divided into two groups: desulphurization by physical adsorption and reactive adsorption desulphurization. biodesulphurization (bds) has drawn wide attention recently because of its green processing of fossil fuels, which allows the removal of the sulphur present in organosulphur compounds by bacteria.1 the process operates in the presence extractive purification of gas oil 109 of water and oxygen under mild operating conditions (i.e., at room temperature and pressure) and requires no hydrogen.9 several bacterial species have now been identified for bds; these include arthrobacter, brevibacterium, pseudomonas, gordona and rhodococcus spp. extractive desulphurization is based on the fact that organosulphur compounds are more soluble than hydrocarbons in an appropriate solvent.10 the most attractive feature of the extractive desulphurization is the applicability at low temperature and low pressure. the process does not change the chemical structure of the fuel oil components. as the equipment used is rather conventional without special requirements, the process can be easily integrated into the refinery.10 to make the process efficient, the solvent must be carefully selected to satisfy a number of requirements. to be recycled, the solvent must have a boiling temperature different to that of the sulphur-containing compounds. it must be inexpensive to ensure economic feasibility of the process.1 the solvents used for this process can be divided into two categories: conventional organic solvents and ionic liquids. studies on extractive refining of non-hydro-treated gas oil cuts with various selective organic solvents and extraction systems have been performed.11–14 n,n-dimethylformamide (dmf), acetonitrile, dimethylacetamide (dma), methyl cellosolve, tetrahydrofurfuryl alcohol,13,14 furfuryl alcohol and phenol have been used for the extractive purification of atmospheric distillate gas oil fractions.15 in extractive refining of atmospheric gas oil, dibenzothiophene derivatives and polycyclic aromatic hydrocarbons, which are the most difficult to remove in hydro-treating, should be extracted most readily.15 aromatic components containing nitrogen (carbazole, indole, and quinoline homologous) are even more easily extracted with polar solvents. at the st. petersburg state institute of technology, n-methylpyrrolidone (nmp) was tested for the purification of different types of gas oil.16 in 2006, atmospheric gas oil was treated with nmp in the presence of undecane and the results showed that the raffinates obtained are promising as diesel fuel components.17 in 2011, refining of delayed-coking light gas oil using dmf, nmp and phenol with 8 % water as extractants was investigated; the quality of the raffinate obtained in a one stage extraction using water-free nmp was higher than for purification with phenol and dmf.18 in 2013, a study was conducted on a light visbreaker gas oil, phenol with 10 % water was used as a solvent for the light fraction, and nmp for the heavy fraction. the obtained raffinates could be used to produce ecological diesel fuel. the aim of this work was to study the capabilities of n-methylpyrrolidone for the desulphurization of hydro-treated diesel cut under different experimental conditions. this desulphurization should be accompanied by the de-aromatization and the denitrification of the diesel fraction. 110 bedda et al. experimental n-methylpyrrolidone (nmp) is a dipolar aprotic solvent having a normal boiling temperature of 202 °c and a density of 1.028 g ml-1 at 25 °c. in this study, n-methylpyrrolidone from sigma–aldrich (purity ≥ 99 %) was used as the extraction solvent. to increase the extraction selectivity and the raffinate yield, some water was added to the nmp. a hydrotreated gas oil fraction was supplied by the kstovo oil refinery (ooo lukoil-nizhegorodnefteorgsintez). the physicochemical properties of this feedstock are presented in table s-i of the supplementary material to this paper. the laboratory purification of the gas oil fraction with nmp was performed in a glass extractor in which a pre-set temperature was maintained with a thermostat and intense stirring was realized with a glass stirrer during 30 min (fig. 1). some experiments were performed in a single-stage while other in a five-stage process.19 the multistage extraction was performed in counterflow following the scheme simulating the operation of an extraction column (fig. 2). the experimental conditions of the extraction operations are listed in table i. fig. 1. glass extractor. fig. 2. five-stage extraction in counter flow. table i. experimental conditions of extractive purification of hydro-treated gas oil from the kstovo oil refinery with nmp exp. no. solvent: feed weight ratio water content in nmp, wt. % extraction stage t / °c 1 1:1 1 1 40 2 2:1 1 1 40 3 2:1 3 1 40 4 2:1 1 1 60 5 1:1 1 5 40 6 2:1 1 5 40 7 1:1 3 5 40 8 2:1 3 5 40 9 1:1 3 5 60 extractive purification of gas oil 111 in the extraction process with nmp, an oil-rich raffinate phase and a solvent-rich extract phase are formed. after decantation, the heavier extract phase collected at the bottom of the extractor was separated. the raffinate was washed with distilled water to remove traces of nmp and then dried with calcium chloride before being analyzed. results and discussion the yields of the raffinate and extract obtained in each experiment are given in table ii. the refractive index of these phases was determined by a classic abbe refractometer at room temperature and the obtained values were adjusted to the standard temperature of 20 °c using the eq. (1): nd20 = ndt + 0.00045 (t – 20 °c) (1) table ii. yields and characteristics of the raffinates and extracts obtained after extractive purification of the hydro-treated gas oil with nmp exp no. yielda, wt. % refractive index, nd20 density at 15 °c raffinate extract raffinate extract raffinate extract 1 95.8 4.2 1.4619 1.4902 0.8235 0.9892 2 77.0 23.0 1.4601 1.4760 0.8165 0.9792 3 88.3 11.7 1.4590 1.4823 0.8122 0.9834 4 60.0 40.0 1.4616 1.4734 0.8223 0.9763 5 83.0 17.0 1.4594 1.4791 0.8139 0.9811 6 59.0 41.0 1.4584 1.4746 0.8104 0.9785 7 95.2 4.8 1.4586 1.5027 0.8111 0.9987 8 81.6 18.4 1.4581 1.4798 0.8095 0.9817 9 87.4 12.6 1.4596 1.4809 0.8146 0.9825 afor 100 wt. units of feedstock the density of phases was measured using an electronic densimeter anton paar type dma 48 at 15 °c. in table ii, it could be seem that the refractive index and density for the raffinates and extracts vary with the experimental conditions, indicating the change in their composition. the density of the raffinate phase is governed by the density of the gas oil, whereas the density of the extract phase is governed not only by the density of the solvent, but also by the amount and the type of dissolved hydrocarbons in the solvent. the difference in density between these phases allowed their separation by decantation. the sulphur content in the raffinates was determined by x-ray fluorescence spectroscopy according to the standard test method astm d4294 and the sulphur concentration of the extracts was deduced by material balance. the results are presented in table iii. the degree of sulphur extraction is calculated as the ratio between the weight of sulphur extracted and the initial weight of sulphur in the feedstock. interesting results were obtained in experiment no. 8 performed in five stages at 40 °c. the solvent:feed ratio of this experiment was 2:1; the water con112 bedda et al. tent in nmp was 3 wt. %. the raffinate was collected with a yield of 81.6 % and its sulphur content was the lowest, 28 ppm. the degree of sulphur extraction in this experiment was 86.9 %.20 since the refractive index of aromatic compounds is higher than that of the paraffinic materials, the lower value of the refractive index for raffinate no. 8 indicates the lowest content of aromatic compounds. table iii. sulphur content of raffinates and extracts obtained after extractive purification of the hydro-treated gas oil with nmp exp no. sulphur content, wt. ppm degree of sulphur extraction, wt. % raffinate extract 1 102 1816 43.8 2 74 509 67.3 3 51 1102 74.1 4 98 288 66.2 5 63 716 69.9 6 32 378 89.1 7 35 2931 80.9 8 28 821 86.9 9 66 923 66.8 experiment no. 7 could be more economical from the point of view of the process than experiment no. 8. under the same conditions as extraction no. 8 and with a lower amount of solvent, extraction in experiment no. 7 allowed a raffinate to be obtain with only 7 ppm higher sulphur content than raffinate no. 8, but the yield of raffinate no. 7 (95.2 %) was significantly better than that obtained in experiment no. 8. the energy expended for regeneration of nmethylpyrrolidone by distillation in the case of experiment no. 8 would be greater than that used for experiment no. 7. in the extractive purification of gas oil realized according to experiment no. 7, the quantities of fuel and energy lost would be less important compared to those with experiment no. 8. the europe-wide regulation for diesel quality (en 590) limits the sulphur content of diesel fuel to 10 wt. ppm and the maximum content of polycyclic aromatic hydrocarbons tolerated is 8 wt. %. for these reasons, raffinate no. 8 having the lowest sulphur content and the lowest refractive index was selected for further analysis. effect of the solvent:feed weight ratio on the desulphurization the experiments nos. 1 and 2 are performed under the same conditions with ratios of solvent:feed equal to 1:1 and 2:1, respectively. this increase in the solvent ratio decreased the yield and the sulphur content of the raffinate. the same results were obtained by comparing experiments nos. 5 and 6, or experiments nos. 7 and 8. extractive purification of gas oil 113 effect of the water content in n-methylpyrrolidone on the desulphurization the water contents in nmp used as the extraction solvent in experiments nos. 5 and 7 were 1 and 3 %, respectively. the increase of this water content increased the yield of raffinate and decreased its sulphur content. the same results are obtained by comparing experiments nos. 2 and 3, or experiments nos. 6 and 8. kumar et al.21 studied the removal of sulphur compounds from straightrun gas oil (srgo), light cycle oil (lco) and coker gas oil (cgo) using nmp. the srgo was obtained from the atmospheric distillation of crude oil; the lco was produced in the fluidized catalytic cracking process (fcc) of heavy feed and the cgo from the coking process of heavy fractions. the obtained results showed that adding water increased both the yield and sulphur content of raffinates. this different result could be explained by the fact that these gas oils were produced from crude oils of different origins and they were treated by different processes. the composition of these cuts was certainly different from that of the hydro-treated gas oil used in the present study. the effect of water content in nmp on the efficiency of desulphurization depends on the nature of the hydrocarbons contained in the feedstock, in particular with regard to the organosulphur compounds. effect of extraction stage number on the desulphurization the experiments realized in five stages gave raffinates with lower yields and lower sulphur contents compared to the raffinates obtained under the same experimental conditions but with a single stage. effect of temperature on the desulphurization the raffinate of experiment no. 4 performed at 60 °c was collected in a lower yield and with higher sulphur content than the raffinate of experiment no. 2 performed at 40 °c. the same results were obtained by comparing the raffinates of experiments nos. 9 and 7 realized at 60 and 40 °c, respectively; the increase in temperature decreased the degree of sulphur extraction. mokhtar et al.22 found the same effect of temperature on the desulphurization of a synthetic gas oil with dmf as solvent. the selected model compounds were thiophene, dibenzothiophene and 4,6-dimethyldibenzothiophene, which are the most difficult sulphur compounds to hydrogenate, and these types of compounds represent the largest proportion of sulphur compounds contained in hydro-treated gas oils. adzamic et al.23 studied the extractive desulphurization of fcc gasoline with two solvents, sulfolane and furfural, the results showed that an increase of temperature from 50 °c reduced the desulphurization efficiency with sulfolane but had no significant effect on the extractive desulphurization with furfural. in their studies, kumar et al.20,21 reported that an increase in the extraction temperature increased the degree of sulphur removal and simultaneously decreased the raffin114 bedda et al. ate yield. this result was obtained for the extractive desulphurization of different types of gas oil using dmf, dma and nmp. the observed results indicate that the impact of temperature on the efficiency of the process is related to the nature of the sulphur compounds present in the feedstock, i.e., the composition and the origin of the petroleum fractions determine the choice of the solvent and the operating parameters of the process. denitrification and de-aromatization of the hydro-treated gas oil the nitrogen contents of the hydro-treated gas oil and the raffinate of experiment no. 8 were determined using a kjeldahl distillation apparatus according to the standard test method astm d3228 and the results are shown by the histogram in fig. 3. the extraction conditions of experiment no. 8 (table ii) reduced the nitrogen content of the gas oil from 58 to 15 ppm. fig. 3. sulphur and nitrogen contents in the gas oil and raffinate no. 8. the hydro-treated gas oil and the raffinate no. 8 were analyzed by high performance liquid chromatography with refractive index detection according to the standard test method astm d6591. this analysis determined the total aromatics content and the polycyclic aromatic hydrocarbons (pah) content of these samples and the results are shown in fig. 4. the experiment no. 8 reduced the fig. 4. aromatics and pah contents in gas oil and raffinate no. 8. extractive purification of gas oil 115 aromatics content of the gas oil from 27.1 to 13.8 wt. % and allowed the total amount of polycyclic aromatic hydrocarbons to be extracted. conclusions the extractive desulphurization of a hydro-treated gas oil fraction from the kstovo oil refinery was performed under different experimental conditions. the results showed that the desulphurization was improved by increasing the solvent ratio, increasing in water content of nmp and increasing the number of extraction stages. a higher temperature extraction was unfavourable for the desulphurization process. n-methylpyrrolidone had a high capacity for the removal of undesirable components from the hydro-treated gas oil cut. in the extractive purification performed in experiment no. 8, 86.9 % of the sulphur was removed, the nitrogen content was reduced by 3.8 times, the aromatics content was 2 times lower and the polycyclic aromatic hydrocarbons were totally extracted. the refined gas oil became cleaner for the environment met the european standard (en 590) in terms of the polycyclic aromatic hydrocarbons content, but not concerning the sulphur content. for these two required specifications, the purified gas oil respected the algerian (na 8110) and russian (gost r 52368-2005) standards. the combination of hydro-treatment and extractive purification is a promising method for producing ecological diesel fuels. supplementary material properties of the hydro-treated gas oil from the kstovo oil refinery are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding authors on request. acknowledgements. the authors express their deep gratitude to all the members of the st. petersburg state institute of technology for their assistance. и з в о д екстрактивно пречишћавање хидротретираног гасног уља помоћу n-метилпиролидона kahina bedda1, boudjema hamada1, nikolay v. kuzichkin2 и kirill v. semikin2 1laboratory of petrochemical synthesis, faculty of hydrocarbons and chemistry, university m’hamed bougara-boumerdes, algeria и 2st. petersburg state institute of technology (technical university), st. petersburg, russia проучавано је пречишћавање хидротретираног гасног уља екстракцијом течно– –течно помоћу n-метилпиролидона као растварача. резултати су показали да овај метод, под одређеним експерименталним условима, смањује садржај сумпора у гасном уљу са 174 на 28 ppm, садржај азота са 58 на 15 ppm, садржај аромата са 27,1 на 13,8 % и потпуно екстрахује полицикличне ароматичне угљоводонике. овако добијено рафинисано гасно уље може да се користи да би се добио за околину чист дизел за гориво моторних возила. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice stepwise or concerted? a dft study on the mechanism of ionic diels–alder reactions of chromanes j. serb. chem. soc. 81 (1) 67–80 (2016) udc 577.161.3+547.972.2:519.677:66.095.252+ jscs–4828 547.538:543.637 original scientific paper 67 stepwise or concerted? a dft study on the mechanism of ionic diels–alder reactions of chromanes mina haghdadi*, seyedeh soghra mousavi and hassan ghasemnejad department of chemistry, islamic azad university, p.o. box 755, babol branch, babol, iran (received 20 april, revised 26 september, accepted 5 october 2015) abstract: the stepwise and concerted ionic diels–alder reactions between phenyl(pyridin-2-ylmethylene)oxonium and styrene derivatives were explored theoretically. the results support the use of a computational method via persistent intermediates. the dft method was essential to reproduce a reasonable potential energy surface for these challenging systems. keywords: styrene; ionic diels–alder reaction; stepwise; concerted; dft study; reactivity indices. introduction the chromane skeleton appears in a number of natural products, such as tocopherols1 and flavans.2 they display a diverse array of biological activities, including antioxidant,3 antiestrogens,4 antiviral,5 antihypertensive6 and anticancer7 activity. common approaches to prepare the chromane skeleton8 are diels−alder reactions of o-quinone methides (1-oxadienes),9 additions of ohydroxy acetophenones10 and intramolecular nucleophilic substitution of phenols.11 alternative approaches to the chroman skeleton are of considerable interest for the formation of substituted chromanes. diels−alder (da) reactions and their formal equivalents provided a powerful means for the rapid construction of heterocyclic scaffolds. oxaand aza-da variants were developed in which the dienophile and/or dienes could incorporate the heterocomponents.12 one such aza-variant is the povarov reaction,13,14 originally developed 50 years ago, which has considerable utility. these da reactions, classified as ionic da reactions (i-da), in which positively or negatively charged ionic species can participate in these reactions. in i-da type of reactions, the reagents, transition states (tss), feasible intermediates and cycloadducts remain charged during the cycloaddition reaction.15 i-da reactions could be classified as anionic and cationic da reactions. however, while cationic da reac * corresponding author. e-mail: mhaghdadi2@gmail.com doi: 10.2298/jsc150420089h 68 haghdadi, soghra mousavi and ghasemnejad tions occur rapidly at very low temperatures,16 usually at –78 °c, due to the high electrophilic character of cationic species, there are few anionic da reactions because, in spite of the high nucleophilic character of anionic species, these reactions do not occur easily in the absence of strong electrophiles.17 the authors performed a series of experiments in order to establish the mechanism of these i-da reactions. several theoretical studies were devoted to i-da reactions.18–21 the theoretical studies of domingo et al. on i-da reactions of iminium cations indicated that both one-step and stepwise mechanisms could be found.18 the presence of the strong electron withdrawing pyridinium substituent in the iminium cation enabled the stabilization of a feasible intermediate once the first c–c single bond had completely formed, making corresponding process stepwise.19 recently, batey et al. reported the synthesis of chromanes via the i-da reaction of o-aryl oxonium species with some alkenes, including cyclopentene and styrene, yielding the formal [4+2] cycloadducts, which by one rapid loss of a proton afford chromanes.22a their results showed that such oxonium ion species are more reactive than the corresponding iminium ions and capable of undergoing either direct i-da reaction or the equivalent stepwise prins addition/intramolecular electrophilic aromatic substitution reaction to give chromans. moreover, in 2014, a theoretical study on the mechanism of oxa-povarov reactions was reported by domingo et al.22b they studied the stereoselectivity of the i-da reaction of o-aryl oxonium with cyclopentene and styrene at the b3lyp/6-31g* level in the gas phase and solvent, which was not in agreement with the experimental results. in the present study, the stereoselectivity, regioselectivity and conformational analysis on the stepwise and concerted mechanism of the oxa-povarov reaction leading to distereosynthesis of 2,4-substituted chromane22a were investigated by theoretical methods. in addition, the influence of substituents on styrene was analyzed in these reactions. herein, the i-da reactions between cationic aryl oxonium 1 and styrene derivatives 2a–c are analyzed (scheme 1) using the dft method. several pathways were analyzed in an attempt to elucidate the energetic difference between the stepwise mechanism and the concerted one. the calculations support the experimental finding of a two-step mechanism, as proposed by batey et al.22a in order to evaluate fully the possible reaction pathways, several tss and intermediates were optimized at the b3lyp/cc-pvdz and mpwb1k/aug-cc-pvtz level of theory. calculations the ubiquitous b3lyp23 hybrid functional has been the workhorse of quantum chemical studies on organic molecules for years.24 it is well-known that b3lyp could describe interactions in diels–alder reactions. recently, some functionals such as the mpwb1k,22b,25 were proposed to investigate the reaction energies, barrier heights and intermediates for diels– dft study on mechanism of diels–alder reaction of chromanes 69 –alder reactions. for a comprehensible comparison of geometries, we were prompted to the optimization of all species in the mentioned i-da reactions using both b3lyp and the mpwb1k exchange–correlation functional. the cc-pvdz basis set was used for full optimization using the b3lyp method and a single point with aug-cc-pvtz basis set of the mpwb1k method. energies were then recalculated at the mpwb1k/aug-cc-pvtz level, using the polarizable continuum model (pcm) as developed by the tomasi group26 within the framework at the self-consistent reaction field (scrf).27 dichloromethane (ɛ = 8.93) was selected as a moderately polar organic solvent. the electronic energies were corrected with zpe at the b3lyp/cc-pvdz level. all calculations were performed using the gaussian 09 program.28 the electronic structures of the stationary points were analyzed by the natural bond orbital (nbo) method.29 global reactivity indexes were estimated according to the equations recommended by parr and yang.30 the global electrophilicity index, ω, is given by the following expression:31 2 2     (1) where μ is the electronic chemical potential and η is the chemical hardness. both quantities may be approached in terms of the one-electron energies of the frontier molecular orbitals homo and lumo, ɛh and ɛl, respectively: 32 h l 2      (2) h l    (3) recently, domingo introduced an empirical (relative) nucleophilicity index,33 based on the homo energies obtained within the kohn sham scheme,34 that were defined as: homo homo(nu) (tce)  (4) nucleophilicity is referred to tetracyanoethylene (tce), because it presents the lowest homo energy in a large series of molecules already investigated in the context of polar cycloadditions. this choice allows a nucleophilicity scale of positive values to be conveniently handled. recently, domingo proposed two new electrophilic, +kp , and nucleophilic, kp , parr functions based on the atomic spin density distribution at the radical anion and cation of a neutral molecule.35 the electrophilic, +kp , and nucleophilic, kp , parr functions were obtained through the analysis of the mulliken atomic spin density of the radical anion and cation by single-point energy calculations over the optimized neutral geometries using the unrestricted ub3lyp formalism for radical species. the local electrophilicity indices, ωk, 36 and the local nucleophilicity indices, nk, 34 were calculated using the following expressions: +k k  p (5) -k kn np (6) where +kp and kp are the electrophilic and nucleophilic parr functions, 35 respectively. results and discussion experimentally, two concerted and a stepwise mechanism were suggested for these i-da reactions of which the stepwise mechanism may be preferred to 70 haghdadi, soghra mousavi and ghasemnejad the concerted,22a because the reaction is not stereospecific with respect to alkene geometry and the oxa-povarov reaction must proceed through stepwise pathways. therefore, two possible mechanisms for these reactions, concerted and stepwise mechanism were investigated herein to evaluate the energy differences between them by theoretical methods. then, the present study was divided into three parts: first, a mechanistic study of the i-da reactions of (e)-phenyl(pyridin -2-ylmethylene)oxonium (1) and styrene derivatives 2a–c, to yield the chromane derivatives 7–10, was performed along the concerted and stepwise mechanisms. thereafter, an analysis of the geometrical and electronic structure of the stationary points was undertaken and finally, an analysis of the dft reactivity indices of the reactants was performed. i) study of i-da reaction of (e)-phenyl (pyridin-2-ylmethylene)oxonium (1) and the styrene derivatives 2a–c along the concerted mechanism the reaction of aryl oxonium 1 species and styrene derivatives 2a–c comprises two consecutive steps (scheme 1): i) an i-da reaction between 1 and 2a–c to yield the corresponding intermediates 3–6 and ii) the elimination of hydrogen to give chromans 7–10. the relative energies and gibbs free energies for the stationary points are given in table i, and table s-i of the supplementary material to this paper. scheme 1. the calculated possible reaction pathways for the concerted mechanism of the i-da reaction between phenyl (pyridin-2-ylmethylene)oxonium (1) and styrene derivatives 2a–c. dft study on mechanism of diels–alder reaction of chromanes 71 table i. the calculated activation energies (δe#), activation free energies (∆g#) and reaction energies (∆er), all in kj mol -1, of the i-da reactions between phenyl(pyridin-2-ylmethylene)oxonium (1) and styrene derivatives 2a–c for the concerted mechanism (for a full comparison of energies see the supporting material) species ts ∆e#a ∆e#b ∆g#a ∆er a ∆er b 1+2a→3a-endo ts1a 15.12 –10.29 67.95 –87.26 –174.75 3a→7a-endo ts2a –75.23 –163.30 –12.55 –261.17 –328.57 1+2a→4a-exo ts3a 14.10 –9.13 66.00 –88.14 –179.91 4a→8a-exo ts4a –63.76 –146.07 –0.39 –245.93 –313.21 1+2b→3b-endo ts1b 21.21 –2.55 72.74 –78.60 –165.50 3b→7b-endo ts2b –74.93 –162.44 –10.71 –261.36 –337.05 1+2b→4b-exo ts3b 20.59 –1.95 72.31 –79.26 –170.15 4b→8b-exo ts4b –64.83 –147.09 –1.32 –246.50 –322.51 1+2c→3c-endo ts1c 5.53 –16.39 55.63 –90.62 –178.37 3c→7c-endo ts2c –74.66 –163.47 –10.23 –260.63 –337.13 1+2c→4c-exo ts3c 4.27 –17.67 61.47 –91.68 –182.74 4c→8c-exo ts4c –63.22 –145.42 0.30 –245.17 –321.63 aoptimization was performed at the b3lyp/cc-pvdz level of theory; b calculated at mpwb1k/aug-cc -pvtz//b3lyp/cc-pvdz level due to the asymmetry of the two reagents, four competitive pathways are feasible for the i-da reaction between aryl oxonium ion 1 and styrene derivatives 2a–c. they are related to the two stereoisomers corresponding to the endo and exo approach modes of the styrene aryl group relative to the phenyl group of the oxonium ion, and the two regioisomeric possibilities, ortho and meta (scheme 1). ts1, ts3, ts5 and ts7 were used to indicate the transition states (tss) of the first step, ts2, ts4, ts6 and ts8 are the tss for the second step, 3–6 are intermediates and 7–10 are cycloadducts of each pathway. the structures of the tss and intermediates are displayed in figs. s-1–s-3 of the supplementary material. as can be seen from the results of the calculations presented in tables i and s-i, the transition states of the first step (cycloaddition reaction), ts1, ts3, ts5 and ts7, are more energetic than the second step (elimination of a proton), ts2, ts4, ts6 and ts8, which suggests that the cycloaddition step is the rate-determining step. then in ortho-endo pathway, the energy barriers for two transition states, ts1a and ts2a are 15.12 and –75.23 kj mol–1 for the b3lyp geometries, and –10.29 and –163.30 kj mol–1 for the mpwb1k geometries, respectively, and the first step with ts1a could be the rate-controlling one. the energy barriers of the first step for ortho and meta pathways are 15.12, 14.10, 42.10 and 59.85 kj mol–1 for the b3lyp geometries, and –10.29, –9.13, 19.20 and 31.81 kj mol–1 for the mpwb1k geometries, respectively, which are higher than of the second steps and the intermediate 3a with –87.26 kj mol–1 could be a stable compound. in order to obtain a quantitative estimate of the conformational energies in such systems, conformational analyses of the chromane ring were performed for 72 haghdadi, soghra mousavi and ghasemnejad intermediates in all pathways at b3lyp/cc-pvdz level, which adopts two conformers, distorted half-chair and boat conformations. the conformational analysis showed that the distorted boat conformers are more stable than the chair conformers for the ortho-endo and meta-endo pathways by 16.73 and 27.28 kj mol–1, respectively, while in the ortho-exo and meta-exo pathways, the distorted chair conformers are the more stable ones by 15.21 and 11.33 kj mol–1, respectively. similar to the ortho-endo pathway, a common intermediate, 4a, is formed in the ortho-exo pathway, via ts3a with an energy barrier of 14.10 kj mol–1 at the b3lyp/cc-pvdz and –9.13 at the mpwb1k/aug-cc-pvtz level. then, 4a loses a proton (as acoh) to form the cycloadduct of 8a via ts4a, the barrier height of which is –63.76 kj mol–1 at the b3lyp method and –146.0 kj mol–1 at the mpwb1k method (in fig. s-1, aco– is not shown in all structures but it was considered in all of the calculations). as mentioned above, the energy barrier of the ortho-endo pathway is lower than those of the others are, and should be the most favorable pathway from the kinetic viewpoint. the processes are extremely exothermic and the corresponding cycloadducts, 7a and 8a, are stable because their energies are lower than their corresponding reactants by –261.17 and –245.93 kj mol–1 with b3lyp calculations, and –328.57 and –313.21 kj mol–1 with mpwb1k calculations, respectively. the most favorable product 7a was also confirmed, suggesting that the ortho -endo pathway is the favorable pathway from the thermodynamic viewpoint. the transition states of the meta pathways, ts5a, ts6a, ts7a and ts8a, have higher energy than those of the ortho ones, indicating that the ortho pathways are expected to be the dominant reaction pathways. therefore, the meta pathways were ignored due to their high potential energies and focus was directed to the ortho pathways, see more information about meta pathways in table s-i and fig. s-3 of the supplementary material. to understand the effects of electron donating (methyl) and electron withdrawing substituents (chloro) on the styrene ring on the i-da reaction, the reactions of the aryloxonium ion 1 with 4-chlorostyrene (2b) and 4-methylstyrene (2c) were investigated. these two i-da reactions also take place in the same way as mentioned earlier (i-da reaction of 1+2a). then, the two ortho pathways dominated the reaction pathways. as shown in table i, along the ortho pathway, the chloro group on styrene slightly increased the activation energies of ts1 and ts3, while the methyl group decreased the energy barriers (10 kj mol–1), relative to the parent (styrene). the activation energies associated to the regioisomeric pathways (ortho and meta) indicated a large regioselectivity in the i-da reaction of 1 with 2b and 2c. thus in this section, for the i-da reactions of 1 with 2b and 2c, the main focus dft study on mechanism of diels–alder reaction of chromanes 73 was on the two ortho pathways and meta ones were ignored due to the high potential energies (tables i and s-i). if the easy equilibrium between all stereoisomers is considered, the most favorable reaction pathway corresponds to the formation of 7c via ts1c, by 5.53 kj mol–1 at the b3lyp level and –16.39 kj mol–1 at the mpwb1k level. a comparison of the activation energies for all the i-da reactions indicated that the energy of the transition states associated with the ortho-endo pathway were slightly lower than those of the ortho-exo ones; which is in agreement with the experimental results.22 when the solvent effects of ch2cl2 were considered, the activation energies increased. for example, the activation barrier for the first step of the i-da reaction of 1 with 2a becomes 15.52 kj mol–1 with mpwb1k calculations, which is 25.81 kj mol–1 higher than that in gas phase (–10.29 kj mol–1). in addition, the relative energy of 3a is reduced (by about 30 kj mol–1) in gas phase, which shows that it becomes more stable than in the solvent. furthermore, the predicted stereoselectivity in the solvent remains almost the same as in the gas phase (the ortho-endo pathway is preferred). thus, it is apparent that in this work geometry optimization in the continuum solvent does not offer any direct advantage over the single-point calculations on the gas phase geometries. ii) study of i-da reaction of (e)-phenyl(pyridin-2-ylmethylene)oxonium (1) and the styrene derivatives 2a–c along the stepwise mechanism experimentally, these reactions were not stereospecific with respect to the alkene geometry and scrambling was observed, thus the oxa-povarov reaction must proceed through a stepwise path. hence, the second suggested mechanism is a stepwise one. the tss structures, suggested intermediates, products and related energies are provided in scheme 2, table ii and table s-ii of the supplementary material. the computational results indicated that these i-da reactions could be achieved by a stepwise mechanism along four competitive pathways, two stereoselective (endo and exo) and two regioselective pathways (ortho and meta). the initial stepwise prins-type addition of the styrene derivatives 2a–c to the oxonium 1 generates carbocation intermediates, 11, 14, 17 and 20 via ts9, ts13, ts17 and ts21. the relative stereochemistry of the c1–c5 or c1–c6 bond formation could be compared to the addition of styrene to the oxonium ion. the computational study revealed that i-da reactions between styrene derivatives and the oxonium ion occurred preferentially to give the syn carbocation intermediate. these observations were rationalized comparing the energy barriers of all tss, of which the lowest energy barrier as starting points were predicted for ts9 or ts13 in the ortho pathways. next, the newly attached bond c–c undergoes a rotation to form 12, 15, 18 and 21 via ts10, ts14, ts18 and ts22. then a rotation along the newly formed  bond in the intermediates yields new inter74 haghdadi, soghra mousavi and ghasemnejad mediates 13, 16, 19 and 22 via ts11, ts15, ts19 and ts23, respectively. now, the latter intermediates having the proper stereochemistry undergo friedel−crafts cyclization to form 3–6 via ts12, ts16, ts20 and ts24, respectively. scheme 2. the calculated possible reaction pathways for the stepwise mechanism of the i-da reaction between phenyl(pyridin-2-ylmethylene)oxonium (1) and the styrene derivatives 2a–c. a comparison among the activation energies and reaction energies for the i-da reaction of 1 with 2a in table ii and table s-ii of the supplementary material indicated that the meta pathways with extremely high potential energies should be ignored and thus attention was focused on the ortho pathways of these reactions. for more information about the meta pathways, see table s-ii of the supplementary material. in ortho-endo stepwise pathway, first, styrene 2a approaches to 1 through a synclinal orientation to generate the carbocation intermediate 11a via ts9a. the energy barrier for this process is –7.26 kj mol–1 at the b3lyp level and –29.93 kj mol–1 at the mpwb1k level with 11a being more stable than reactants by – 37.44 kj mol–1 according to the b3lyp method and –89.52 kj mol–1 according dft study on mechanism of diels–alder reaction of chromanes 75 to the mpwb1k method. then intermediate 11a must undergo c1–c5 bond rotation to generate intermediate 12a, which is antiperiplanar, via ts10a with an energy barrier of –35.41 and –79.85 kj mol–1 at the b3lyp and mpwb1k levels, respectively. next, 12a undergoes rotation around the same c1–c5 bond, with an energy cost of –16.45 kj mol–1 (–61.79 kj mol–1 at the mpwb1k level), to yield 13a via ts11a. subsequently, intermediate 13a undergoes friedel−crafts cyclization to produce 3a via ts12a. finally, 3a loses a proton to produce 7a, as discussed in the concerted section. table ii. the calculated activation energies (δe#), activation free energies (∆g#) and reaction energies (∆er), all in kj mol -1, of the i-da reactions between phenyl(pyridin-2-ylmethylene)oxonium (1) and styrene derivatives 2a–c for the stepwise mechanism (for a full comparison of the energies, see the supplementary material) species ts ∆e#a ∆e#b ∆g#a ∆er a ∆er b 1+2a→11a-endo ts9a –7.62 –29.93 42.44 –37.44 –89.52 11a→12a-endo ts10a –35.41 –79.85 30.39 –45.68 –91.18 12a→13a-endo ts11a –16.45 –61.79 43.93 –33.65 –79.52 13a→3a-endo ts12a –21.46 –75.72 40.28 –87.26 –174.75 1+2a→14a-exo ts13a –5.85 –28.31 45.58 –35.77 –88.96 14a→15a-exo ts14a –18.58 –68.66 42.02 –72.32 –118.12 15a→16a-exo ts15a –6.05 –56.07 53.42 –28.89 –75.91 16a→4a-exo ts16a –14.30 –63.82 51.15 –88.14 –179.91 1+2b→11b-endo ts9b –1.66 –23.56 47.84 –33.95 –84.80 11b→12b-endo ts10b –29.54 –72.84 36.64 –42.07 –86.83 12b→13b-endo ts11b –13.33 –57.43 47.45 –25.55 –71.62 13b→3b-endo ts12b –15.36 –69.28 46.97 –78.60 –165.50 1+2b→14b-exo ts13b 0.015 –21.41 51.45 –13.27 –85.59 14b→15b-exo ts14b –15.05 –64.25 46.18 –67.30 –112.17 15b→16b-exo ts15b 7.09 –32.38 70.46 –25.68 –71.94 16b→4b-exo ts16b –8.425 –56.87 29.67 –79.26 –170.15 1+2c→11c-endo ts9c –17.29 –38.26 32.19 –53.23 –106.06 11c→12c-endo ts10c –50.56 –95.89 15.68 –65.38 –112.65 12c→13c-endo ts11c –38.12 –84.02 22.37 –53.65 –101.37 13c→3c-endo ts12c –35.06 –90.43 27.30 –90.62 –178.37 1+2c→14c-exo ts13c –15.12 –35.15 35.51 –56.49 –110.39 14c→15c-exo ts14c –39.28 –91.24 22.13 –88.39 –135.34 15c→16c-exo ts15c –16.54 –59.82 46.21 –50.10 –101.07 16c→4c-exo ts16c –27.07 –79.41 37.93 –91.68 –182.74 aoptimization was performed at b3lyp/cc-pvdz level of theory; bcalculated at the mpwb1k/aug-cc-pvtz// b3lyp/cc-pvdz level from a comparison of the relative energies of the tss and intermediates in the ortho-endo stepwise pathway, some results could be concluded as follows: i) the formation of the c1–c5 bond (ts9a) is the rate-determining step, ii) as expected, the energy of intermediates are low compared to the surrounding barriers (ts9a–ts12a), iii) the low energy of the tss and intermediates (their ener76 haghdadi, soghra mousavi and ghasemnejad gies are lower than those of reactants) together with the rotation of the c1–c5 bond may suggest that these processes occur via an reversible stepwise mechanism, iv) furthermore, the lowest activation and relative energies were seen for ts10a and 3a, respectively. the optimized geometries of tss and intermediates involved in the domino pathway are given in figs. s-2 and s-3 of the supplementary material, respectively. similar results were obtained for the ortho-exo pathway, which are given in table ii. comparing the ortho pathways with regard to stereochemistry, the endo pathways are usually followed preferentially, as the activation energies in the more stable step (ts10a) are slightly lower than those in the exo addition and the resulting intermediates are more stable. these results confirmed that the ortho -endo pathway is the most energetically favorable one among the other proposed reaction pathways, in agreement with the experimental results. moreover, the stepwise pathways were investigated in i-da reaction of 4-chlorostyrene and 4-methylstyrene (2b and 2c, respectively) with aryl oxonium ion 1 (scheme 2) along the more favored pathways (ortho ones), and the results of their activation energies and reaction energies, given in table ii, indicated that the ortho-endo pathways are the more favorable ones. the theoretical results proved that the i-da reaction with the lowest activation barriers involved 4-methyl substituted styrene along the ortho-endo pathway, while 4-chloro substitution increased the activation and reaction energies. as can be seen in table ii, the activation energies varied within the series of dienophiles. of all the possible stepwise tss, ts10 is consequently favored over the others. furthermore, the energy of tss increased when going from 2a to 2b and became the lowest for 2c. a similar trend was observed for their intermediates, i.e., 11c, 12c, 13c and 3c are the most stable intermediates, while the intermediates of i-da reaction 1+2 appear to be less stable. iii) geometrical parameters selected geometry parameters of the tss on the concerted pathways at the b3lyp/cc-pvdz level are shown in fig. 1. as can be seen, the lengths of the c1–c5 and c4–c6 bonds (the atom numbering is given in scheme 1) for the concerted mechanism, at the ortho-endo pathway (ts1), are about 2.23 and 3.89 ǻ, and at the ortho-exo (ts3), the corresponding values are 2.25 and 3.89 ǻ, respectively. these bond lengths indicated that both tss structures are with highly asynchronous bond formation processes, where it seems only the c1–c5 bond is being formed. the extent of bond-formation along a reaction pathway is provided by the concept of bond order (bo).37 the bo values of the c4–c6 forming bonds for the concerted mechanism along the most favorable pathway is virtually zero, indicating a stepwise or at least highly asynchronous pathway for these reactions. dft study on mechanism of diels–alder reaction of chromanes 77 the polar nature of the two cyclization modes can be estimated by a charge transfer (ct) analysis at the tss.29 the ct descriptors (figs. s-1 and s-3) clearly show that these reactions are polar according to the domingo classification. moreover, the important dihedral angles and bond lengths for the tss and intermediates of stepwise mechanism are presented in table iii, and table s-iii of the supplementary material. table iii. selected geometrical parameters, bond lengths (r / ǻ) and dihedral angles (φ / °) for the stationary points of i-da reactions between phenyl(pyridin-2-ylmethylene)oxonium (1) and styrene (2a) for the ortho pathway of stepwise mechanism at the b3lyp/cc-pvdz level of theorya (for numbering of atoms, see scheme 2, and for full comparison of geometrical parameters, see the supporting information to this paper) species φo-c1-c5-c6 rc1-o rc3-c4 rc4-c6 rc5-c6 1 – 1.28 1.39 – – 2a – – – – 1.34 ts9a 69.15 1.31 1.39 4.93 1.37 11a 53.52 1.41 1.39 5.09 1.47 ts10a 116.23 1.43 1.39 5.74 1.48 12a 172.26 1.41 1.40 5.22 1.46 ts11a 115.01 1.44 1.39 4.93 1.45 13a 45.23 1.43 1.40 4.59 1.47 ts12a 65.20 1.45 1.44 4.51 1.48 3a 26.11 1.48 1.47 1.58 1.55 ts2a 49.10 1.46 1.48 1.58 1.55 7a 62.05 1.43 1.40 1.52 1.54 ts13a 67.86 1.30 1.39 4.55 1.37 14a 60.11 1.40 1.39 5.13 1.47 ts14a 120.32 1.42 1.39 5.52 1.47 15a 177.17 1.40 1.40 4.43 1.46 ts15a 119.75 1.42 1.40 4.89 1.46 16a 41.30 1.42 1.39 4.72 1.47 ts16a 45.52 1.45 1.44 2.49 1.49 4a 53.14 1.47 1.49 1.55 1.54 ts4a 44.33 1.46 1.49 1.57 1.55 8a 53.81 1.43 1.40 1.52 1.54 conclusion the molecular mechanism of i-da reactions between aryl oxonium 1 species and styrene derivatives 2a–c yielding chromanes 7–10 was studied using the dft method at the b3lyp/cc-pvdz level of theory. the formation of cycloadducts 7–10 occurs through two consecutive steps; first, a cycloaddition reaction between 1 and 2a–c occurred to yield the intermediates 3–6, then the elimination of hydrogen from these intermediates yielding 78 haghdadi, soghra mousavi and ghasemnejad chromanes 7–10. these i-da reactions are completely regioselective and slightly endo selective. the calculation results suggest that: i. the elimination of hydrogen is kinetically favored over the cycloaddition process (first step). ii. the concerted and stepwise mechanism of all the i-da reactions were investigated, the results of which showed that the stepwise mechanism is more favorable than the concerted ones. iii. the ortho-endo pathway with an energy barrier of 15.12 kj mol–1 for 4-cl styrene and 5.53 kj mol–1 for 4-me styrene, on the concerted mechanism is the most energetically favorable pathway; on the stepwise mechanism these energy barriers are reduced to –13.33 and –38.12 kj mol–1, respectively. iv. dft-based reactivity indices clearly predict the regiochemisty of the isolated cycloadducts. moreover, it is reasonable to conclude that the gas-phase geometry optimization at the b3lyp/cc-pvdz level can give quite good estimates of the mechanism and stereoselectivity in i-da reactions. supplementary material the calculated energies, geometrically optimized transition states and intermediates, as well as selected geometrical parameters are available electronically from http:// //www.shd.org.rs/jscs/, or from the corresponding author on request. и з в о д постепено или концертовано? dft студија механизма јонске дилс–алдерове реакције хромана mina haghdadi, seyedeh soghra mousavi и hassan ghasemnejad department of chemistry, islamic azad university, p.o. box 755, babol branch, babol, iran постепене и концертоване јонске дилс–алдерове рекције између фенил(пиридин -2-илметилен)оксонијумских и стиренских деривата изучаване су коришћењем теоријских метода. резултати дају подршку употреби рачунарких метода првенствено на стабилнијим интермедијерима. dft метод се показао битним за репродуковање реалистичне површине потенцијалне енергије за ове захтевне системе. 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press, new york, 1989, p. 16 31. r. g. parr, l. von szentpaly, s. liu, j. am. chem. soc. 121 (1999) 1922 32. r. g. parr, r. g. pearson, j. am. chem. soc. 105 (1983) 7512 33. l. r. domingo, p. pérez, j. org. chem. 73 (2008) 4615 34. p. pérez, l. r. domingo, m. duque-noreña, e. chamorro, j. mol. struct. theochem 895 (2009) 86 35. l. r. domingo, p. pérez, j. a. saez, rsc adv. 3 (2013) 1486 36. l. r. domingo, m. j. aurell, p. pérez, j. phys. chem., a 106 (2002) 6871 37. k. b. wiberg, tetrahedron 24 (1968) 1083 38. h. chemouri, s. m. mekelleche, int. j. quantum. chem. 112 (2012) 2294 39. w. benchouk, s. m. mekelleche, b. silivi, m. j. avrell, l. r. domingo, j. phys. org. chem. 24 (2011) 611. {determination of free phenolic acids from leaves within different colored maize} j. serb. chem. soc. 82 (1) 63–72 (2017) udc 633.15:635.074:547.562+543.544.5.068.7+ jscs–4947 621.383.5 original scientific paper determination of free phenolic acids from leaves within different colored maize jelena z. mesarović*, vesna d. dragičević, snežana d. mladenović drinić, danijela s. ristić and natalija b. kravić maize research institute zemun polje, slobodana bajića 1, 11185 belgrade, serbia (received 12 may, revised 22 november, accepted 29 november 2016) abstract: along with other plant parts, maize leaves are widely used for making fermented food for cattle, known as silage. since there have only been a few reports on studies concerning the extraction and determination of phenolic acids from maize leaves, the main goal of this investigation was to evaluate the content of free phenolic acids in the leaves of fifteen different maize inbred lines. reverse-phase, high performance liquid chromatography (rp -hplc), with a photodiode array detector (dad), was performed. under the optimized chromatographic conditions, referring to short time of sample preparation, small quantities of solvent and direct injection of the extract into hplc, phenolic acids (i.e., gallic, protocatechuic, caffeic, p-coumaric and ferulic acid) were successfully separated in less than 25 min, indicating that the method could be applied for routine analysis. the efficiency and validation of the method was evaluated by measuring the rate parameters: linearity, limit of detection and quantification, accuracy and precision. the obtained results showed that the most abundant free phenolic acid was p-coumaric acid (23.57 µg g-1 dry weight), followed by ferulic and caffeic acids (21.27 and 20.78 µg g-1 dry weight, respectively). principal component analysis (pca) revealed the existence of a link. keywords: hplc-dad; method validation; phenolic acids; corn. introduction maize (zea mays l.) is one of the oldest cultivated grain cereals and one of the most productive species worldwide, with an average yield of about 5.5 t ha–1.1 maize has food, feed, and industrial uses. namely, a high proportion of the produced maize is used in livestock feed as green chop, dry forage, silage or grain.2,3 in addition, differently processed and unprocessed maize grain is used in human diet.4 the rest of the plant is mostly used for the preparation of fermented * corresponding author. e-mail: jmesarovic@mrizp.rs doi: 10.2298/jsc160512104m 64 mesarović et al. high-moisture preserved food used in livestock feed (silage) or is discarded wherein a potentially valuable source of phytochemicals is lost. besides providing functional dietary micronutrients and fibers, plants are rich sources of phytochemicals, such as phenolic compounds. these are aromatic secondary plant metabolites that include a large number of naturally occurring compounds divided into several groups. due to their antioxidant and other biological properties that can promote human health, phenolic compounds in food have been constantly investigated over the past few years.5 in many studies the inhibitory effect of phenolics to oxidative damage, which could lead to atherosclerosis and cancer, were reported.6–8 the antioxidant activity of phenolic compounds can be explained by several possible mechanisms, such as their ability to chelate metals, inhibit lipoxygenase, modulate peroxide concentration, scavenge free radicals and stimulate the enzyme systems of antioxidative defense.9 due to harmful properties of synthetic antioxidants, such as butylated hydroxyanisole (bha) and hydroxytoluene (bht),10 demands of the food industry for antioxidants of natural origin, especially from industrial residues, have never been greater.11–13 phenolic acids are one of the main classes of phenolics and, according to their chemical structure, represent derivatives of benzoic and cinnamic acids. generally, they could be described as phenols with carboxylic acid and hydroxyl groups, the positions of which affects their antioxidative properties.14 phenolic acids in different concentrations are distributed in seeds, leaves, roots and stems.15 their functions in the plant are linked with photosynthesis, synthesis of proteins, enzyme activity and allelopathy.16 various factors affect the quantity and quality of the phenolic acids present in plant foods, including plant genetics, soil composition and growing conditions, maturity state and post-harvest conditions.17,18 many studies have reported the extraction of phenolic acids from the leaves of different plant species,19–21 but only a few from maize leaves.22 according to all the findings mentioned above, the aim of this study was to evaluate the content of free phenolic acids extracted from maize leaves. for this purpose, a new reverse phase hplc method with diode array detection was developed for identification and quantification of five phenolic acids. the method was performed on fifteen maize inbred lines. in addition, principal component analysis (pca) was performed to determine the relationship between compositions of the free phenolic acids in the leaves and the color of the maize kernel. experimental plant material and chemicals in this study, fifteen maize inbred lines (from il1 to il15) from the gene bank of the maize research institute “zemun polje”, belgrade, serbia, were used for the analysis. for each genotype (nine white, four orange and two red maize inbred lines), plant samples (i.e., leaf of the uppermost ear) were taken at flowering. leaves were dried to constant weight at 60 hplc/dad determination of phenolic acids from maize leaves 65 °c, milled (perten 120, sweden) into powder (particle size <500 μm) in order to obtain greater surface contact,23 and stored at –20 °c prior to analysis. methanol and formic acid, purchased from j.t. baker (netherlands), were of hplc grade. ethanol and standards of tested phenolic acids (i.e., gallic, protocatechuic, caffeic, p-coumaric and ferulic) were hplc grade and purchased from sigma–aldrich. for the analysis, ultrapure water (thermo fisher tka micro pure water purification system, 0.055 µs cm-1) was used. syringe filters (17 mm, ptfe membrane 0.45 µm) were purchased from thermo scientific (germany). samples preparation and extraction of free phenolic acids the method used for phenolic acids extraction was a slightly modified method proposed by sultana et al.24 approximately 0.3 g of the leaves powder was extracted (ika hs 501, germany) twice with 3 ml of 80 % methanol, for 30 min at 300 rpm, at room temperature. the collected extracts were centrifuged at 3000 rpm for 5 min, filtered through a 0.45-μm ptfe membrane filter and directly injected into the hplc. calibration curve and linearity working solutions were made by diluting the initial mixture containing all analyzed phenolic acids (100 µg ml-1) to the final concentration: 0.1, 0.3, 0.5, 1, 3, 5, 10 and 20 µg ml-1. calibration curves were obtained in ms excel, by plotting the peak areas (detector response) versus the concentration of the standard solutions. obtained correlation coefficients were used for determination of the linearity of the method. limit of detection and quantification limit of detection (lod) and limit of quantification (loq) were calculated from the following equations: 3 sd lod b = (1) 10 sd loq b = (2) where sd is the standard deviation of the response (standard error value for coefficient b) and b is the slope of the calibration curve obtained from the linear regression. precision and accuracy the repeatability of the method was determined by triplicate measurement of the relative standard deviation (rsd) of both the peak area for each phenolic compound (at a concentration of 1.0 µg ml-1), and two randomly chosen samples, on an intraand inter-daily basis. for the calculation, the following equation was used: / % 100 sd rsd x = (3) where sd is standard error value for the peak area and x is the average value of the peak area. the accuracy of the method was express as percentage of recovery. two samples were spiked with the working solution of the phenolic acids mixture (at a concentration of 1 µg ml-1), in three replicates. recovery (r / %) was determined from the equation: 0 / % 100 f r f a = + (4) 66 mesarović et al. where f represents the concentration of phenolic acid in the spiked sample, f0 is the concentration of phenolic acid in the unspiked sample and a is the added amount of phenolic acid (i.e., 1.0 µg ml-1). hplc method chromatographic separation of five phenolic acids was performed using a dionex ultimate 3000 liquid chromatography system (thermo scientific, germany), consisting of a quaternary pump (lpg-3400), autosampler (wps-300sl), column compartment (tcc -300sd) and a photodiode array detector (dad−3000). the analytical column used was acclaim polar advantage ii, c18 (150 mm× 4.6 mm, 3 μm) from thermo fisher scientific, operated at 25 °c. the mobile phase (flow rate 0.8 ml min-1) contained 0.1 vol. % aqueous formic acid solution (a) and pure methanol (b). the linear gradient program was as follows: 0.0−10.0 min, 15−45 % b; 10.0−20.0 min, 45−65 % b; 20.0−25.0 min, 65−15 % b. the injection volume was 5 μl. the uv detection wavelengths were set at 278, 280, 290 and 300 nm. phenolic acids were identified according to characteristic retention time and absorption spectra, whilst calibration curves of the corresponding standards were used for quantitative calculations. chromeleon software package (version 7.2) was used for instrument control, as well as for data acquisition and analysis. the contents of the phenolic acids are expressed as μg per g of dry weight (dw). data are reported as the mean value of three independent injections. statistical analysis all analyses were performed on triplicate measurements (n = 3) and the results are presented as mean values. the data were subjected to one-way analysis of variance (anova). the f-test was used for comparison of the means at the 0.05 probability level. principal component analysis (pca) was performed using the pls toolbox software package v.6.2.1, for matlab 7.12.0 (r2011a). to prevent the predominance of components existing in higher concentrations, compared to those present in lower concentrations, data were mean-centered and auto-scaled to unit variance before statistical processing. the singular value decomposition (svd) algorithm was used at the 0.95 confidence level for hotelling t2 limits. results and discussion extraction of free phenolic acids determination of extraction efficiency was performed using pure methanol, pure ethanol and mixtures of methanol–water and ethanol–water (both in volume ratio of 8:2, data not shown). the mixture of methanol–water exhibited the highest extraction efficiency, which is in agreement with the results of shabir et al.,25 and was used for further analysis. validation of the method the applied chromatographic method encompassed separation of all tested phenolic acids with good resolution, with a total separation time of 25 min. chromatograms of phenolic acids standards are shown in figs. 1a and b, recorded at 278 and 300 nm, respectively. the chromatograms obtained from maize leaves, also recorded at 278 and 300 nm, are presented in fig. 2a and b, respectively. hplc/dad determination of phenolic acids from maize leaves 67 fig. 1. chromatograms of the phenolic acid standards, recorded at 278 nm (a) and 300 nm (b). fig. 2. chromatograms for phenolic acids from maize leaves, recorded at 278 nm (a) and 300 nm (b). according to obtained maximum absorbance, the wavelength for protocatechuic acid (pa) and p-coumaric acid (p-couma) was set at 300 nm, for ferulic acid (fa) and caffeic acid (ca) at 290 nm and for gallic acid (ga) at 278 nm. these values of wavelengths are in line with those used in the study of nour et al.,26 but different to those used by lee et al.27 and kovacova et al.,28 when all phenolic acids were monitored at 280 nm. although recorded at 278 nm (fig. 2a), the peak of gallic acid had diminished absorbance at 300 nm, as is shown in fig. 2b. this was also confirmed in fig. 1a and b, obtained from phenolic standards chromatograms. retention time for ga, pa, ca, p-couma and fa were 6.78, 10.35, 14.51, 16.46 and 18.19 min, respectively. 68 mesarović et al. the parameters of linear regression (i.e., slope, intercept, coefficient of determination (r2), lod and loq), were obtained for the phenolic acids standards, based on their peak area, as presented in table i. the obtained linear correlation coefficients for all standards were not lower than 0.997, indicating good linearity of the method. the obtained lod values ranged from 16 to 52 ng ml–1 and those of loq from 54 to 173 ng ml–1. the obtained values were lower to those reported in method proposed by nour et al.,26 indicating higher sensitivity of the method used in this study. table i. parameters of linear regression, lod and loq for the phenolic acid standards phenolic acid intercept slope r2 lod / ng ml-1 loq / ng ml-1 ga –0.1180 0.3344 0.999 27 90 pa –0.0495 0.3426 0.999 32 107 ca –0.1424 0.7078 0.997 52 173 p-couma –0.0133 1.0225 0.999 29 97 fa –0.1181 2.0032 1.000 16 54 the repeatability of the method was investigated using intra-day and inter -day data obtained from the standards and two samples (table ii). for standards, the rsd of the intra-day (n = 5) and inter-day (n = 3) analysis were 0.17–1.19 and 0.81–2.42 %, respectively. for sample i, the rsd of the intra-day (n = 5) analysis was 0.21–1.41 %, being 1.13–3.05 % for the inter-day (n = 3) analysis. for sample ii, the rsd of the intra-day (n = 5) analysis was 0.32–1.18 %, being 1.69–2.93 % for the inter-day (n = 3) analysis. table ii. the obtained rsd (%) for the phenolic acids and the tested samples phenolic acid standard sample i sample ii intra-day inter-day intra-day inter-day intra-day inter-day ga 0.2 0.8 0.2 1.1 0.3 1.7 pa 1.2 2.4 0.7 1.9 0.6 2.1 ca 0.4 1.7 0.9 3.0 1.2 1.9 p-couma 0.9 2.0 1.0 2.5 0.8 2.9 fa 0.7 1.5 1.4 2.4 1.0 1.8 the standard additional method was used for recovery (r) determination, which represents the accuracy of the method. r for ga, pa, ca, p-couma and fa were 100.5, 97.8, 102.4, 99.7 and 98.9 %, respectively, indicating the good accuracy of the preformed method. free phenolic acids contents in relation to the maize kernel color the content of free phenolic acids in the leaves for each of the fifteen evaluated maize inbreds, as a well as total average values per parameter, are pre hplc/dad determination of phenolic acids from maize leaves 69 sented in table iii. the obtained results indicate that the most abundant evaluated free phenolic acid was p-coumaric acid, followed by ferulic and caffeic acids. table iii. obtained content of phenolic acids in the tested maize leaves sample maize kernel color ga µg g -1 pa µg g -1 ca µg g -1 p-couma µg g -1 fa µg g -1 il1 white 10.3014 7.1970 34.8993 21.8526 20.4395 il2 white 10.2993 5.5556 24.7301 21.0614 18.1406 il3 white 9.1026 7.0338 15.1407 18.0995 21.7998 il4 white 9.5499 7.0916 22.5808 16.2327 23.0608 il5 white 10.3803 11.4546 17.4689 23.5272 24.9282 il6 white 10.4429 9.8031 25.9565 21.3246 17.0967 il7 white 12.298 11.228 31.8369 17.2297 9.9062 il8 white 10.8847 6.3156 22.7485 19.0469 13.1095 il9 white 8.6345 8.5403 19.5884 15.6033 9.7336 averagea 10.1412 8.2466 23.8833 19.3309 17.5794 il10 orange 15.3776 22.0641 4.7345 18.2032 12.7739 il11 orange 14.1058 18.2494 27.705 28.665 28.9746 il12 orange 13.6257 19.7144 19.1517 35.3965 28.5013 il13 orange 13.2097 15.0719 17.8619 15.977 16.611 averagea 14.0797 18.775 17.3633 24.5604 21.7152 il14 red 9.0262 7.7717 20.9281 43.1607 29.5398 il15 red 9.1026 12.9652 6.4191 38.1813 44.4828 averagea 9.0644 10.3685 13.6736 40.671 37.0113 total average 11.0479 11.3371 20.7834 23.5708 21.2732 aaverage phenolic acids content of leaves total average values for ga, ca and fa contents obtained from all tested fifteen genotypes (i.e. 11.05, 20.78 and 21.27 µg g–1 dw, respectively), were shown to be higher than those in study on cob leaves (10.99, 6.0 and 1.87 µg g–1 dw, respectively), reported by pandey et al.22 average amounts of p-couma, fa and ca (i.e., 23.57, 21.27 and 20.78 µg g–1 dw, respectively), obtained from all analyzed genotypes, was higher than those in commercial red wine.29 mean values for pa and ga contents (i.e., 11.34 and 11.05 µg g–1 dw, respectively) obtained from leaves of all tested maize, was lower compared to leaf extracts of gold mohar (delonix regia (bojer ex hook.) raf.), having antimicrobial and antifungal properties and being widely used in folk medicine.25 on the contrary, the average contents of the other phenolic acids (i.e., p-couma, ga and fa) were higher.25 different kernel color in maize generally originates from the carotenoids and anthocyanins concentration, with positive correlations found between the antioxidant activity and the color of maize.30–32 similar studies showed that the color of the samples is also related to the content of phenolic acids.33,34 in this context, pca was performed in order to examine the possible relationship between the 70 mesarović et al. content of free phenolic acids in the leaves and the color of the maize kernel. pc analysis resulted in a four-component model that explains 98.93 % of the total variance. the first two principal components explain 42.80 (for pc1) and 36.23 % (for pc2) of the overall data variance. mutual projections of the factor scores and their loadings for these pcs are shown in fig. 3a and b, respectively. fig. 3. pca score (a) and loading plots (b). considering pc1 and pc2 score values (fig. 3a), three well-separated groups of samples (according to kernel color) were formed. this indicates that the leaves from different colored maize possibly have unique contents of phenolic acids. white kernel maize (il1–il9) formed a group in the plot center, while the group of orange kernel maize (il10–il13) was allocated in the upper right part of the plot. red kernel maize (il14 and il15) were separated in the lower right part of plot. the loading plot (fig. 3b) revealed that the most efficient parameters for distinguishing white kernel maize was ca, for orange maize ga and pa, while for red kernel maize fa and p-couma. these results are in agreement with the mean values for phenolic acids obtained in the leaves from different colored maize (table iii). leaves from white kernel maize had the highest average content of ca (i.e., 23.88 µg g–1 dw) compared to the leaves from orange and red maize (i.e., 17.36 and 13.67 µg g–1 dw, respectively). similarly, the leaves from the orange kernel maize showed the largest average content of ga and pa (i.e., 14.08 and 18.77 µg g–1 dw, respectively), compared to the leaves of white and red maize. moreover, leaves of the red kernel maize had the highest average concentration of p-couma and fa (40.67 and 37.01 µg g–1 dw, respectively), compared to the leaves from other colored maize. among the phenolic acids evaluated, the highest value for fa was found in red wheat, as was reported by ma et al.34 hplc/dad determination of phenolic acids from maize leaves 71 conclusions a new rp-hplc method with dad was developed for the quantification of five phenolic acids in maize leaves, due to the lack of information on this subject. the observed validation parameters confirmed that the performed method is of good accuracy and precision, with relatively low values for the lod and loq. this indicates that the method developed in this study could be usefully applied in further, more detailed analyses on phenolic acids content in maize leaves. the performed pca distinguished a relationship between the concentrations of phenolic acids in the leaves and the color of the maize kernel. in leaves, the most abundant phenolic acid was ca for white maize, and ga and pa for orange maize. moreover, the leaves from red maize showed the highest concentrations of fa and p-couma. acknowledgement. this research was supported by the ministry of education, science and technological development of the republic of serbia (projects tr31068 and tr31028). и з в о д одређивање слободних фенолних киселина у листу кукуруза различито обојеног зрна јелена з. месаровић, весна д. драгичевић, снежана д. младеновић дринић, данијела с. ристић и наталија б. кравић институт за кукуруз земун поље, слободана бајића 1, 11185 београд лист кукуруза, заједно са другим деловима биљака, доста се користи у производњи ферментисане хране за стоку, познате као силажа. с обзиром на то да је само неколико студија објављено на тему изоловања и квантификације фенолних киселина из листова кукуруза, главни циљ овог рада је одређивање садржаја слободних фенолних киселина у листовима петнаест различитих самооплодних линија кукуруза. коришћена је реверсно -фазна високо ефикасна течна хроматографија са dad детектором. под оптимизованим хроматографским условима, као што су кратко време припреме узорака, мале количине растварача и директно инјектовање екстракта узорка, фенолне киселине (тј. гална, протокатехинска, кафеинска, p-кумаринска и ферулинска киселина) успешно су раздвојене за мање од 25 min, што указује на могућу примену методе у рутинским анализама. ефикасност и валидација методе су процењенe мерењем параметара као што су: линеарност, граница детекције и квантификације, тачност и прецизност. добијени резултати указују да је најзаступљенија слободна фенолна киселина p-кумаринска киселина (23,57 μg g-1 суве масе), праћена ферулинском и кафеинскoм киселином (21,27 и 20,78 μg g-1 суве масе, редом). анализом главних компонената (pca) процењен је однос садржаја слободних фенолних киселина у листу и боје зрна кукуруза. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {microwave-assisted synthesis and antimicrobial evaluation of 6-(3'-(aryl)-1'-phenyl-3,4-dihydro-1'h,2h-[3,4'-bipyrazol]-5-yl)-2h-chromen-5-ols} j. serb. chem. soc. 84 (3) 237–244 (2019) udc 547.772.2+537.5–962:615.28 jscs–5179 original scientific paper 237 microwave-assisted synthesis and antimicrobial evaluation of 6-[3-aryl-1-phenyl-4′,5′-dihydro[4,5′-bi-1h-pyrazol]-3′-yl] -2h-chromen-5-ols dongamanti ashok1*, rangu kavitha1, srinivas gundu1 and madderla sarasija2 1green and medicinal chemistry laboratory, department of chemistry, osmania university, hyderabad500 007, india and 2department of chemistry, satavahana university, karimnagar505001, india (received 6 december 2017, revised 7 december, accepted 21 december 2018) abstract: a new series of 6-[3-aryl-1-phenyl-4′,5′-dihydro[4,5′-bi-1h-pyrazol]-3′ -yl]-2h-chromen-5-ol derivatives was synthesized by michael addition of chalcones 5a–j with hydrazine hydrate in presence of sodium acetate under conventional heating and microwave irradiation. structural assignment of the products was confirmed based on ir, 1h-nmr, 13c-nmr, ms and analytical data. all the synthesized compounds 6a–j were screened for their antimicrobial activity against various bacterial and fungal strains. most of the compounds exhibited variable range of antimicrobial activity and compounds 6c–f and 6i showed promising antimicrobial potency. keywords: pyrazole; pyrazoline; microwave irradiation; antimicrobial activity. introduction heterocyclic compounds containing nitrogen and oxygen play important roles in agrochemical and pharmaceuticals. heterocyclic compounds have great applicability in pharmaceutics because they have specific chemical reactivity and provide false synthons in biosynthetic processes or block the normal functioning of biological receptors. the interesting biological activities of heterocycles have stimulated considerable research work in recent years, including the synthetic utility. pyrazoles, an important member of heterocyclic compounds, are widely found as the core structure in a large variety of compounds that possess important agrochemical and pharmaceutical activities. many pyrazole derivatives are reported to have a broad spectrum of biological activities, such as anti-inflammatory,1,2 antifungal,3 herbicidal,4 insecticidal,5 anti-hiv,6 antiviral,7 anticonvulsant8 and anticancer9 activities. some of the drugs possessing a pyrazole scaf *corresponding author e-mail: ashokdou@gmail.com https://doi.org/10.2298/jsc171206113a 238 ashok et al. fold, such as celecoxib,10 rimonabant,11 deracoxib,12 and phenylbutazone, exhibiting anti-inflammatory, analgesic and antipyretic activities, are already on the market (fig. 1). fig. 1. commercially available pyrazole and pyrazoline drugs. pyrazolines are partially reduced form of pyrazoles that contain five-membered ring system with two adjacent nitrogens. pyrazolines are one of the emerging classes of compounds associated with a broad spectrum of biological activities. many compounds bearing pyrazoles and their reduced forms pyrazolines constitute an interesting class of heterocycles due to their synthetic versatility and effective biological activities, such as antiviral,13 anti-inflammatory,14,15 antitubercular,16 anti-amoebic,17 analgesic,18 antibacterial,19 analgesic,20 antifungal,21 anti-arthritic,22 cerebroprotective23 and antidepressant24 activities. they are also useful as biodegradable agrochemicals.25 a literature survey revealed several synthetic protocols for the synthesis of these compounds and the presence of this core in any molecule plays a key role in enhancing activity. prompted by the above-mentioned biological properties of pyrazole and pyrazoline incorporated heterocycles, it was contemplated to synthesize a novel series of pyrazole–chromene containing pyrazolines. in continuation to ongoing research, herein, the synthesis of 6-[3-aryl-1-phenyl-4′,5′ -dihydro[4,5′-bi-1h-pyrazol]-3′-yl]-2h-chromen-5-ol derivatives in excellent yields is reported. experimental materials all used materials were obtained commercially, mostly from sigma–aldrich, and were used without further purification. equipment all the microwave irradiation experiments were performed in a cem discover microwave system equipped with an ir sensor, with which the reaction temperatures were monitored. all the reactions were monitored on silica gel percolated tlc plates, 60 f254 from merck and the spots were visualized with uv light. melting points were determined by the open capillary method and are uncorrected. the 1h-nmr and 13c-nmr spectra were run on a bruker avance-400 spectrometer at 400 and 100 mhz, respectively, using tetramethylsilane (tms) as an internal reference. mass spectra were recorded on a shimadzu lcms 2020 mass spectrometer. elemental microanalysis was performed on a perkin elmer chn-2400 analyzer. synthesis and antibacterial activity of bipyrazolyl chromen-5-ols 239 spectral and analytical data of the synthesized compounds are given in supplementary material to this paper. general procedure for the synthesis of 6-[3-aryl-1-phenyl-4′,5′-dihydro[4,5′-bi-1h-pyrazol]-3′ -yl]-2h-chromen-5-ol derivatives (6a–j) conventional heating method. to a solution of 3-(3-aryl-1-phenyl-1h-pyrazol-4-yl)-1(5-hydroxy-2h-chromen-6-yl)prop-2-en-1-ones 5a–j (0.23 mmol) in dmf (5 ml) containing sodium acetate (0.23 mmol) and hydrazine hydrate (0.23 mmol), few drops of acetic acid was added and the reaction mixture heated at 80–90 °c for 1–3 h. the progress of the reaction was monitored by tlc. after completion of the reaction, ice-cold water was added. the solid product that separated was filtered, washed with water and dried. recrystallisation from meoh:chcl3 (1:1) afforded 6-[3-aryl-1-phenyl-4′,5′-dihydro[4,5′-bi-1h-pyrazol]-3′-yl]-2h -chromen-5-ols 6a–j. yield: 57–62 %. microwave irradiation method. a mixture of 3-(3-aryl-1-phenyl-1h-pyrazol-4-yl)-1-(5 -hydroxy-2h-chromen-6-yl)prop-2-en-1-ones 5a–j (0.23 mmol), hydrazine hydrate (0.23 mmol), sodium acetate (0.23 mmol) and few drops of acetic acid in dmf (2 ml) was taken in a glass vessel and then placed into a teflon vial with screw cap and the mixture was subjected to microwave irradiation at 100 w for 1–3 min. after completion of the reaction, indicated by tlc, the vial was cooled and the reaction mixture was poured into ice cold water. the solid product that separated was filtered, washed with water and dried. recrystallisation from meoh:chcl3 (1:1) furnished 6-[3-aryl-1-phenyl-4′,5′-dihydro[4,5′-bi-1h-pyrazol]-3′-yl]-2h-chromen-5-ol derivatives 6a–j. yield: 86–96 %. biological assays antibacterial activity. the synthesized novel compounds 6a–j were screened for their antibacterial activity against different types of bacterial strains, viz. gram-positive bacterial strains staphylococcus aureus and bacillus subtilis, and gram-negative bacterial strains escherichia coli and proteus vulgaris at concentrations of 10 and 20 μg ml-1. the cultures were diluted with 5 % saline, autoclaved, and the final volume was made with a concentration approximately 105–106 cfu ml-1. the synthesized compounds were diluted in acetone for the antibacterial biological assays. for agar disk diffusion method,26 the solution form of a test compound was allowed to air-dry, such that the disk became completely saturated with the test compound. the saturated chemical disks were introduced onto the upper layer of the medium evenly floated with the bacteria. the disks were dipped in different chemical samples and placed over the evenly spread bacterial nutrient media and incubated at 37 °c for 24–48 h for better inhibition of the bacteria. the zones of inhibition were measured after 24–48 h. all the experiments were performed in triplicate, and the results are expressed as zone of inhibition in mm. the zones of inhibition of synthesized compounds were compared with the zone of inhibition of the standard antibiotic gatifloxacin at concentrations of 10 and 20 μg ml-1. antifungal activity. the antifungal activities of the synthesized compounds 6a–j were tested against three pathogenic fungi, namely fusarium oxysporum, aspergillus niger and a. flavus by the poison plate technique at a concentration of 100 μg/ml. three kinds of fungi were incubated in potato dextrose agar (pda) at 25±1 °c for 5 days to obtain new mycelium for the antifungal assay; then the mycelia as disks of approximately 0.45 cm diameter cut from the culture medium were picked up with a sterilized inoculation needle and inoculated in the centre of a pda plate. the test compounds were dissolved in acetone (10 ml) and then added to the potato dextrose agar medium (pda, 90 ml). the final concentration of the compounds in the medium was adjusted to 100 μg ml-1. the inoculated plates were incubated at 25±1 °c for 5 240 ashok et al. days. acetone was diluted with sterilized distilled water and used as the control, while clotrimazole (100 μg ml-1) was used as the standard. for each treatment, three replicates of the experiments were performed. the radial growth of the fungal colonies was measured on the sixth day. results and discussion the synthetic route for 6-[3-aryl-1-phenyl-4′,5′-dihydro[4,5′-bi-1h-pyrazol]-3′-yl]-2h-chromen-5-ols is illustrated in schemes 1 and 2. the synthesis of title compounds involved the preliminary preparation of 1-(5-hydroxy-2h-chromen -6-yl)ethanone (3). starting from resacetophenone (1) upon treating with propargyl bromide in the presence of anhydrous k2co3 in dry acetone yielded 1-(2 -hydroxy-4-(prop-2-yn-1-yloxy)phenyl)ethanone (2), which was further refluxed in n,n-dimethylaniline at 180 °c for 3 h to give compound 327 (scheme 1). claisen–schmidt condensation between 1-(5-hydroxy-2h-chromen-6-yl)ethanone (3) and substituted pyrazole aldehydes (4a–j) in the presence of powdered koh under microwave irradiation for 4–7 min (scheme 2) gave 3-(3-aryl-1-phenyl-1h -pyrazol-4-yl)-1-(5-hydroxy-2h-chromen-6-yl)prop-2-en-1-ones27 5a–j. these chalcones were then cyclised by means of hydrazine hydrate and few drops of glacial acetic acid under conventional heating and microwave irradiation to furnish the title compounds 6a–j in excellent yields. scheme 1. synthesis of 1-(2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl)ethanone (3). scheme 2. synthesis of 6-[3-aryl-1-phenyl-4′,5′-dihydro[4,5′-bi-1h-pyrazol]-3′-yl]-2h -chromen-5-ols (6a–j). preliminarily, the synthesis of compounds 6a–j was carried out under conventional heating method, but this method suffered from poor yields (57–62 %). synthesis and antibacterial activity of bipyrazolyl chromen-5-ols 241 in order to improve the yields and reduce the reaction time, the synthesis approach was changed to the microwave irradiation method. microwave-assisted synthesis of title compounds 6a–j is advantageous over conventional method in terms of higher yields in shorter reaction times. a comparison of the yields of the title compounds prepared by the conventional and microwave irradiation methods is demonstrated in table i. table i. comparison of yields (isolated) of compounds 6a–j under different synthetic conditions compound melting point, °c conventional mwi time, h yield, %a time, min yield, %a 6 a 98–100 2 61 1 95 6 b 102–104 2 59 1 93 6 c 101–103 2 60 1 94 6 d 94–96 2 60 1 96 6 e 104–106 2 58 2 86 6 f 99–101 2 62 2 96 6 g 108–110 1 62 1 96 6 h 103–105 2 60 2 92 6 i 90–92 2 60 2 90 6 j 136–138 2 57 2 89 aisolated yields formation of the 6-[3-aryl-1-phenyl-4′,5′-dihydro[4,5′-bi-1h-pyrazol]-3′-yl]-2h-chromen-5-ols (6a–j) were confirmed by ir, 1h-nmr, 13c-nmr, ms and elemental analyses. the ir spectrum of compound 6h showed absorption bands at 3464, 3268 and 1596 cm–1 due to oh, nh and c=n groups, respectively. the 1h-nmr spectrum of 6h displayed two characteristic signals due to the diastereotopic protons (ha, hb). the ha proton, which is cis to hx resonated upfield at δ 3.09 ppm as a doublet of doublets (dd) with j values of 8.87 and 16.24 hz, while the hb proton which is trans to hx resonated downfield at δ 3.48 ppm (dd) with j values 10.19, 16.24 hz. the hx proton which is vicinal to two methylene protons (ha and hb) was observed as doublet of doublets (dd) at δ 5.08 ppm with j values of of 8.87 and 10.19 hz. a triplet appeared at δ 1.43 ppm was due to aliphatic ch3 proton and quartet at δ 4.09 ppm due to ar–o–ch2. the nh proton appeared at δ 5.90 ppm as a broad singlet. the pyrazole proton appeared as a singlet at δ 7.98 ppm and a broad singlet was observed at δ 11.38 ppm due to the oh proton. in the 13c-nmr spectrum of 6h, ch3, pyrazoline-ch2, pyrazoline ch and ar-och2 carbons appeared at δ 14.8, 40.9, 54.1 and 63.5 ppm, respectively. the mass spectra of 6h showed the molecular ion peak at m/z 479 [m+h]+. antimicrobial activity antibacterial activity. the synthesized compounds were screened in vitro for antibacterial activity against different types of bacterial strains viz. gram-positive 242 ashok et al. bacterial strains staphylococcus aureus (atcc 9144) and bacillus subtilis (atcc 6633), and gram-negative bacterial strains escherichia coli (atcc 25922) and proteus vulgaris at concentrations of 10 and 20 μg ml–1. the inhibitory efficiency of the synthesized compounds was measured through the zone of inhibition (in mm) compared with the standard drug gatifloxacin and the results are presented in table ii. the study of the antibacterial efficiency of the synthesized compounds revealed that most of the tested compounds displayed variable inhibitory effects on the growth of the tested gram-positive and gram -negative bacterial strains compared to the standard drug gatifloxacin at concentrations of 10 and 20 μg ml–1. the compounds 6d (ar = 4-methylphenyl), 6e (ar = 4-hydroxyphenyl), 6f (ar = 4-methoxyphenyl) and 6i (ar = 3,4-dimethoxyphenyl) showed the equipotent activity through the zone of inhibition (table ii) against s. aureus, b. subtilis, e. coli and p. vulgaris, respectively. compound 6e exhibited the most potent antibacterial activity against gram-positive and gram -negative bacterial strains. the remaining compounds showed moderate activity compared to the standard. an analysis of the antibacterial activity results indicated that compound with electron-donating groups, such as methyl, hydroxy and methoxy, on the phenyl ring were more potent as compared to the control drug gatifloxacin. table ii. antimicrobial activity of synthesized compounds (zone of inhibition, mm) compound gram-positive bacteria gram-negative bacteria fungal strains s. aureus b. subtilis e. coli p. vulgaris a. flavus a. niger f. oxysporum 6a 15a 26 12 22 12 19 09 12 13.3 13.6 13.9 6b 15 25 15 25 13 17 08 14 11.5 14.1 12.4 6c 13 23 14 21 10 16 09 11 17.2 17.5 18.1 6d 19 31 18 35 16 23 10 17 12.9 14.1 15.6 6e 21 34 19 36 18 25 11 18 08.7 13.4 15.3 6f 20 32 18 33 16 23 11 17 16.9 15.2 17.7 6g 17 28 16 30 16 20 10 16 15.1 14.7 16.1 6h 13 21 10 23 12 17 08 12 14.4 12.1 15.7 6i 21 33 19 34 17 25 11 16 17.0 17.3 18.0 6j 14 21 10 19 09 16 08 12 12.4 13.1 13.7 gatifloxacin 21 33 20 36 18 25 12 18 – – – clotrimazole – – – – – – – – 17.3 17.7 18.4 antifungal activity the antifungal activity of the synthesized compounds was tested against three pathogenic fungi, viz. aspergillus flavus, a. niger (atcc 9029) and fusarium oxysporum by the poison plate technique at a concentration of 100 μg ml–1. most of the synthesized compounds showed promising antifungal activity compared to standard drug clotrimazole (table ii). the compounds 6c, 6f and 6i synthesis and antibacterial activity of bipyrazolyl chromen-5-ols 243 showed equipotent inhibition compared to the standard drug against the tested fungi, whereas the remaining compounds showed moderate activity against the pathogenic fungi. conclusion in summary, a new series of compounds 6a–j was synthesized under conventional and microwave irradiation conditions. using the microwave irradiation method, the reactions were completed in short reaction times under mild reaction conditions, in high yields and convenient operation. all the titled compounds were screened for their in vitro antimicrobial activity. compound 6e was found to be the most potent and compounds 6d, 6f and 6i were found to be equipotent compared to the standard drug gatifloxacin against the pathogenic bacteria whereas compounds 6c, 6f and 6i exhibited potent activity against the pathogenic fungi compared to the standard drug clotrimazole at their respective concentrations. the antifungal screening results revealed that compounds 6f and 6i could be considered as promising antifungal drug candidates. supplementary material spectral and analytical data of the synthesized compounds are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. the authors are thankful to the head of department of chemistry, osmania university, for providing laboratory facilities and the director of central facilities for research and development (cfrd), osmania university, for providing ir and nmr spectral analysis. financial support from ugc, new delhi, india, for rk is gratefully acknowledged. и з в о д синтеза употребом микроталаса и испитивање антимикробне активности серије 6-[3-арил-1-фенил-[4,5′-би-1h-пиразол]-3′-ил]дихидро-2h-хромен-5-ола dongamanti ashok1, rangu kavitha1, srinivas gundu1 и madderla sarasija2 1green and medicinal chemistry laboratory, department of chemistry, osmania university, hyderabad 500 007, india и 2department of chemistry, satavahana university, karimnagar-505001, india синтетисана је серија деривата 6-[3-арил-1-фенил-[4,5′-би-1h-пиразол]-3′-ил]дихидро-2h-хромен-5-ола применом реакције мајклове адиције чалкона (5a–j) и хидразин-хидрата уз присуство натријум-ацетата, под условима традиционалног загревања и применом микроталасног зрачења. структура добијених једињења одређена је на основу ir, 1h-nmr, 13c-nmr, ms и аналитичких података. испитана је антимикробна активност свих синтетисаних једињења 6a–j према различитим сојевима бактерија и гљива. већина једињења показује антимикробну активност, а једињења 6c–f и 6i показују велики антимикробни потенцијал. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {physicochemical characterisation of pottery from vinca culture, serbia, regarding firing temperature and decoration technique} j. serb. chem. soc. 81 (12) 1415–1426 (2016) udc 544.2.004.12:738–035.67: jscs–4938 745(497.11vinča) original scientific paper 1415 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. physicochemical characterisation of pottery from the vinča culture, serbia, regarding the firing temperature and decoration techniques nebojša perišić1∀, milica marić-stojanović2, velibor andrić3, ubavka b. mioč1 and ljiljana damjanović1* 1university of belgrade, faculty of physical chemistry, studentski trg 12–16, 11000 belgrade, serbia, 2national museum belgrade, trg republike 1a, 11000 belgrade, serbia and 3university of belgrade, vinča institute of nuclear sciences, p. o. box 522, 11000 belgrade, serbia (received 23 august, revised 17 october, accepted 19 october 2016) abstract: a study of decorated neolithic pottery samples from the excavation site pločnik, serbia, was performed using x-ray powder diffraction (xrpd), fourier transform infrared (ftir) and x-ray fluorescence (xrf) spectroscopy. the investigated samples belong to the era of the vinča culture that existed in the central balkan region from the mid vi until the first half of the v millennium bce. the mineralogical composition of the pottery samples and comparison of the investigated pottery with thermally treated local clay indicated firing temperatures in the range from 600 to 800 °c. two different types of white pigments were identified in white incrusted decorations: calcium carbonate and bone white (composed of crushed bones). this is the first evidence of the use of bones for decorations in pottery of the vinča culture from the excavation site pločnik. in addition to this, it was revealed that the potters used the iron reduction technique for obtaining black decorations. keywords: ancient ceramics; bone; pigments; ftir spectroscopy; xrf spectroscopy. introduction ancient pottery is a significant source of information regarding the technological development. physicochemical analysis of pottery from the vinča culture excavated on the territory of serbia complements the visual characterisation and provides information on how the neolithic people produced and utilized their pottery (additional considerations are presented in the supplementary material to * corresponding author. e-mail: ljiljana@ffh.bg.ac.rs ∀ present address: vistin pharma as, stuttlidalen 4, fikkjebakke, 3766 sannidal, p. o. box 98, no-3791 kragerø, norway. doi: 10.2298/jsc160823100p 1416 perišić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. this paper). the detailed mineralogical composition provides information about the firing process, such as firing temperature, firing duration and atmosphere conditions.1 since the mineralogical composition of pottery highly depends on the temperature,2 not only the presence but also the absence of specific minerals and their phases can be conclusive for the firing temperature and conditions. when put in a wider context, these technological aspects provide primal and/or additional information on social developments, migrations and interactions between settlements, spread of religion, etc.3 for determination of the composition of crystalline phases in ancient pottery, x-ray powder diffraction (xrpd) has been extensively used.4 complementary to xrpd, fourier transform infrared (ftir) spectroscopy provides information about crystalline and non-crystalline minerals that are abundant in pottery, especially in pottery fired at relatively low firing temperatures.5 an additional advantage of ftir spectroscopy is the ability to follow specific changes in the mineral structures (e.g., aluminosilicates) caused by different firing temperatures.5–8 as a method of non-destructive elemental analysis of pottery body and the pigments, x-ray fluorescence (xrf) spectroscopy presents a powerful tool, which can provide information about the minerals and pigments discovered through the presence of key elements.9 several archaeometric studies have been conducted on the pottery of the vinča culture,10–14 but compositional data of vinča pottery are still scarcely available. moreover, to the best of authors’ knowledge, the characterisation of white decorations on vinča pottery has not hitherto been performed. the aim of this study was to determine important aspects of the technological developments of the vinča culture, such as firing conditions used for pottery manufacture and techniques adopted to produce particular decorations using the available raw materials. physicochemical methods such as xrpd, ftir and xrf spectroscopy were applied to assess the composition of the ceramic body and pigments used for decoration of pottery of the neolithic vinča culture. experimental pottery samples were provided by the national museum in belgrade and are part of a larger collection of serbian neolithic pottery. in this study, ten pottery samples collected at the pločnik excavation site in the toplica district near prokuplje, serbia, were investigated. description of investigated pottery samples is given in the supplementary material to this paper. raw clay was sampled approximately 2 km north of the pločnik excavation site from a locality that is currently in use as a source of clay material (fig. s-1 of the supplementary material). this clay sample was used as a potential raw material equivalent. pottery samples were used as powders for xrpd and ftir analyses. the surface layer of each pottery shard was removed by scraping; then about 50 mg of each sample was scraped and powdered in an agate mortar. physicochemical characterisation of vinča pottery, serbia 1417 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. x-ray powder diffraction (xrpd) patterns were recorded at room temperature on a philips pw-1710 diffractometer using cu kα radiation (λ = 1.54178 å) in the 2θ range 4–65° in 0.02° steps with 2.0 s per step. fourier-transform infrared (ftir) analysis was performed on the vinča pottery samples as well as on the raw clay sample. the raw clay material was heated at 7 different temperatures for six hours (100, 600, 700, 800, 900, 1000 and 1100 °c) in air. after heating and cooling to room temperature, ftir spectra were recorded. the ftir spectra of all the investigated samples were recorded on a nicolet 6700 spectrophotometer, using the kbr pellets technique in the wavenumber range from 4000 to 400 cm-1. the spectra were pre-processed by extended multiplicative signal correction (emsc) in matlab software (version 7.10.0.499 (r2010a), the mathworks, natick, ma, usa) in order to remove baseline shifts, and additive and multiplicative effects that could occur due to physical differences between the samples, e.g. kbr pellet thickness, scattering, etc. energy dispersive x-ray fluorescence (edxrf) spectroscopy measurements were performed on an edxrf spectrometer with a canberra si(li) semiconductor detector and mca analyser s35+. microsampo software was used for spectra acquisition and the measurement time was 600 s for all samples. for excitation, an annular radioisotope source 109cd (manufactured by isotope products) with a nominal activity of 740 mbq was used. qualitative edxrf measurements were performed on the decorated side of the pottery shards and the inner body for all investigated samples. in this way, the elemental composition of the pottery body and the decorations on the surface could be assessed and compared. results and discussion estimation of firing temperature in order to reconstruct the production techniques and better understand the knowledge and skills of the ancient potters, the firing temperature of the pottery was estimated. x-ray powder diffraction (xrpd) analysis. diffraction patterns of representative pottery samples and local clay sample are shown in fig. 1. as it can be seen, the powder diffraction patterns of all investigated samples were dominated by quartz reflections. besides quartz, characteristic reflections of feldspars were also detected in all samples. by comparing xrpd patterns of pottery shards with that of the raw clay sample, it can be concluded that these have highly similar mineralogical contents. the amount of material for analysis was limited due to the nature of the samples, which hindered further determination of more precise mineralogical compositions by other destructive methods. however, various archaeometric studies indicate that most likely local clay was used by neolithic potters from the balkan region for pottery production.12,15,16 the presence of illite/muscovite detected in all the investigated pottery samples indicated that the firing temperature did not exceed 900 °c, since phyllosilicates disappear at temperatures between 900–950 °c.6,17 even though an archaeometric study of pottery from the selevac excavation site showed that vinča potters routinely achieved high firing temperatures, about 1000 °c,11 the results obtained in this work indicated lower firing temperatures for pottery from 1418 perišić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. the pločnik excavation site. the firing temperature of pottery samples classified as vinča culture excavated on the territory of romania at the archaeological site limba, alba county, was estimated at about 700 °c, and for only a few samples at 850–900 °c.12 two samples from vinča culture uncovered on the territory of serbia were investigated as a part of a wider study of neolithic–bronze age pottery from central and south–east europe and the estimated firing temperatures were below 800 °c for the majority of the pottery made from non-calcareous clay and in particular, 750 °c for a sample from the gomolava excavation site and between 800–850 °c for a sample from the vinča excavation site.18 that finding is in excellent agreement with a study of comparable pottery from karanova in bulgaria, with an estimated firing temperature of less than about 750 °c.19 fig. 1. xrpd patterns of representative samples from pločnik excavation site and local raw clay, where q designates quartz, p – plagioclase, and il/ms – illite/muscovite. however, due to the poor mineralogical content of the vinča pottery and raw clay, xrpd could only provide a rough estimate of the firing temperature. therefore, an ftir spectroscopic analysis of investigated samples was performed. ftir analysis. ftir spectra were obtained for the ceramic body of all the investigated pottery samples and representative spectra are shown in fig. 2. in all ftir spectra, a doublet is present at 789 and 788 cm–1, which is assigned to quartz.20 the wide and intensive band at about 1040 cm–1, also present in all ftir spectra, originates from si–o stretching vibration of aluminosilicates.20 an additional vibrational band of aluminosilicates occurs at about 470 cm–1, which ori physicochemical characterisation of vinča pottery, serbia 1419 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. ginated from deformational si–o vibrations.20 the band that occurs at about 540 cm–1 originated from al–o deformational vibrations.21 various aluminosilicate minerals are common constituents of pottery (e.g., feldspars) and they can all contribute to these bands. therefore, signals overlap and broadening of the detected bands occurs. fig. 2. ftir spectra of representative pottery samples from pločnik excavation site; q designates quartz; the vertical line marks the position of the si–o stretching band at 1040 cm-1. it was shown that the positions of specific vibrations in the crystal lattice of aluminosilicates are sensitive to changes in temperature.5,22 according to this, determining the precise positions of specific si–o and al–o vibrations, can give an estimation of the temperature at which the raw clay was fired during pottery production. with this in mind, raw clay (sampled near the pločnik excavation site) was fired at different temperatures in air. as can be seen in fig. 3, a shift of the band at about 1000 cm–1 towards higher wavenumbers was evidenced as the firing temperature was increased from 100 to 1100 °c. the detected maxima were at 1031 cm–1 for 100 °c, 1040 cm–1 for 600 and 700 °c, 1040 and 1082 cm–1 (split band) for 800 °c and 1082 cm–1 for 900 °c. at higher temperatures, additional broadening of this band occurred and the maxima were estimated at 1081 cm–1 for 1000 °c and 1082 cm–1 for 1100 °c. the detected positions of the si–o stretching band for the investigated samples were as follows: 1033 cm–1 for p-7; 1035 cm–1 for p-4; 1040 cm–1 for p-2, p-3, p-5; 1047 cm–1 for p-9; 1050 cm–1 for p-6; 1035 and 1077 cm–1 (split band) for p-10; 1040 and 1080 cm–1 (split band) for p-3. the poor quality of 1420 perišić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. ftir spectra for samples p-1 and p-8 did not allow precise determination of the band position, but they were estimated to be in the same spectral region of about 1040 cm–1. hence, by comparing the positions of the si–o stretching band in the ftir spectra of the investigated pottery samples with the positions of the same bands obtained for thermally treated clay, it can be concluded that the firing temperature was in the range from 600 to 800 °c. this finding is in agreement with the conclusion obtained by xrpd analysis that the firing temperature did not exceed 900 °c. fig. 3. ftir spectra of raw clay from a locality near pločnik excavation site, heated for 6 h at different temperatures; the vertical line marks the position of the si–o stretching band at 100 °c. composition of pigments and decorations in addition to the ceramic body, the coloured areas on the pottery shards were analysed by ftir and xrf spectroscopy. white pigments. the ftir spectra of white pigments used for the white incrustations in the clay body of samples p-9 and p-10 are presented in fig. 4. as is apparent from the figure, these two white pigments have significantly different spectral features. the ftir spectrum of the white pigment from the sample p-9 (fig. 4a) is characterised by the presence of co32– bands (at 1459, 1415 and 872 cm–1) and po43– bands (at 1091, 1047, 961, 602 and 567 cm–1).23 this finding indicates presence of hydroxyapatite [ca5(po4)3(oh)] and carbonate–hydroxyapatite [ca10–x/2[(po4)6–x(co3)x][(oh)2–2y(co3)y]], which are mineral components of physicochemical characterisation of vinča pottery, serbia 1421 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. fig. 4. representative ftir spectra of white pigments from samples: a) p-9 and b) p-10 with the most relevant spectral features designated (q – quartz). bones.23,24 the detected po43– bands are sharp and well defined, which indicates that the bones were processed at a heightened temperature, most likely above 600 °c.25 furthermore, a presence of the characteristic band at 632 cm–1 in ftir spectrum, which is highly relevant for determining the temperature of firing, was also detected in the spectrum of this sample (p-9). this band originates from the vibrational mode of hydroxyl groups within hydroxyapatite and appears upon calcination at high temperatures in air.24 these findings are a strong indication that the bones used for producing the white decorations in sample p-9 were calcined at high temperatures between 700 and 900 °c.23,24 whether the bone material was fired separately or together with the clay cannot be determined with certainty. however, it is most likely that vinča potters calcined bones before crushing them in order to produce this white pigment, because it is much easier to produce very fine powder from burned bones than from crude ones.23,26 therefore, it can be concluded that ancient pigment bone white (or antler white) obtained from calcined animal bones, teeth and/or antlers was used for 1422 perišić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. the white decoration of sample p-9. production of white inlayed decorations on pottery is recognized as a widespread practice in prehistoric europe,23 but there are not many studies on the composition of the material used to fill white incrustations.23,24,26,27 this result shows that vinča culture potters were familiar with the technique of incrusting calcined bones for obtaining the white decorations on pottery. on the other hand, in the ftir spectrum of the white pigment obtained from sample p-10, an intensive and broad band at 1430 cm–1 together with two sharp and intensive bands at 874 and 713 cm–1 are apparent. these bands originate from the characteristic vibrations of caco3,20 which is the main constituent of this particular white decorative pigment. when caco3 is fired, the characteristic ftir bands shift towards lower wavenumbers and the ν4 band at 713 cm–1 broadens and decreases in intensity and almost completely disappears at temperatures above 600 °c.28,29 this is clearly not the case in the ftir spectrum shown in fig. 4b. therefore, it can be concluded that the vinča potters added the caco3 after the firing of the pottery as a consecutive production step. this is in line with visual observations of this sample, where it was postulated that additional decoration by the addition pigments was performed (see description of samples in supplementary material). in the edxrf spectra of white pigments from samples p-9 and p-10, a signal for ca was also observed (results not shown), which is in accordance with the obtained ftir spectroscopy results. detected characteristic doublet of quartz in both ftir spectra presented in fig. 4 and the si–o stretching band positioned at 1033 cm–1 with shoulder at about 1080 cm–1 in ftir spectrum of sample p-10 (fig. 4b) related to aluminosilicates,20 most probably originate from the underlying ceramic body collected during the scraping of the samples (the white layer of pigment was very thin and hence sampling was a difficult task) but could also be the result of impurities in the pigment mixture. red and black decorations. ftir spectra of the red and black decorations were obtained for all the investigated samples and representative ftir spectra are presented in fig. 5, from which it can be seen that these decorations contained highly comparable spectral features: si–o stretching vibrations of aluminosilicate minerals (at about 1000 cm–1) and quartz bands at 797, 779 and 693 cm–1.20 it can therefore be concluded that both the black and red decorations do not differ appreciably in chemical composition. the xrf spectra of red and black decorations were recorded for all the investigated pottery samples, and representative xrf spectra are presented in fig. 6. it is apparent that a strong signal of iron is present in the spectra of both the red and black decorations. moreover, the same trace elements were detected in the case of all red and black decorations. physicochemical characterisation of vinča pottery, serbia 1423 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. fig. 5. representative ftir spectra (samples p-3 and p-4) of red and black decorations on pottery shards (q – quartz). fig. 6. representative xrf spectra obtained directly on pottery shards: a) sample p-3, red decoration; b) sample p-4, black decoration. 1424 perišić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. this finding is relevant for the determination of the firing technique used by the vinča potters. more precisely, it is known that in the neolithic age on the territory of today’s south–east europe, two different techniques were used for obtaining black colour in the decorations of the pottery: i) manganese-reduction black and ii) iron-reduction black. the process in which black colours are produced during pottery firing by the reduction of ferruginous clays is widely distributed and very old, with certain indications that it is probably the oldest of all ceramic-decoration processes.30 in addition to this, the xrf spectra revealed that neither black decorations, nor any other part of the investigated pottery contained manganese. this finding underlines that the method for obtaining black colour on the pottery decorations was the technique of iron-reduction and not of manganese-reduction. the detected fe in red decoration indicates that this colour originated from hematite. the precision of the decorations, as well as the presence of both red and black colours on several of the analysed pottery shards, shows that the technology of pottery production was on a highly advanced level in the vinča culture. conclusion this multi-analytical study of decorated neolithic pottery found at excavation site pločnik was performed in order to determine the production technology, in particular the firing temperature and characterisation of materials used for decorations. for the analysed set of pottery shards, the firing temperature was estimated to be in the range from 600 to 800 °c, based on mineralogical composition of the pottery, determined by xrpd and ftir spectroscopy, and comparison of investigated pottery with thermally treated local clay with a similar mineralogical composition. in order to produce white decorations, the vinča potters used different white pigments, among which calcium carbonate and the ancient pigment bone white were confirmed by the herein presented ftir analysis. the analysed caco3 decoration was most likely added after the firing of the pottery. furthermore, the black decorations on the analysed pottery shards were obtained by the iron-reduction method, based on: i) identification of iron in the black pigments and ii) the lack of manganese in the analysed samples. the obtained results confirmed and further described that the ancient potters in the vinča culture had a high level of knowledge of raw materials and craftsmanship. supplementary material additional historical and archaeological information are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgments. the authors acknowledge the support from the ministry of education, science and technological development of the republic of serbia, projects nos. 177021 and physicochemical characterisation of vinča pottery, serbia 1425 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. 177012. the authors would like to thank archaeologist dr. dušan šljivar for useful discussions. и з в о д физичкохемијска карактеризација керамике која припада винчанској култури са циљем одређивања температуре печења и декоративне технике небојша перишић1, милица марић-стојановић2, велибор андрић3, убавка б. миоч1 и љиљана дамјановић1 1универзитет у београду, факултет за физичку хемију, студентски трг 12–16, 11000 београд, 2народни музеј, трг републике 1а, 11000 београд и 3универзитет у београду, институт за нуклеарне науке “винча”, п. пр. 522, 11000 београд карактеризација узорака украшене неолитске керамике са археолошког налазишта плочник је извршена применом дифракције x-зрачења на праху (xrpd), инфрацрвене спектроскопије са фуријеовом трансформацијом (ftir) и рендгенске флуоресцентне анализе (xrf). испитивани узорци припадају ери винчанске културе која је постојала у региону централног балкана од средине vi до прве половине v миленијума п.н.е. на основу минералошког састава керамике и поређења испитиваних узорака са термално третираном глином температура печења керамике процењена је на опсег од 600 до 800 °c. идентификоване су две врсте белих пигмената у белим урезаним декорацијама: калцијум-карбонат и коштано бело (који се састоји од смрвљених костију). ово је први доказ коришћења костију за украшавање винчанске керамике са археолошког налазишта плочник. такође, показано је да су древни грнчари користили технику редукције гвожђа за добијање црних украса. (примљено 23. августа, ревидирано 17.октобра, прихваћено 19. октобра 2016) references 1. l. maritan, c. mazzoli, l. nodari, u. russo, appl. clay sci. 29 (2005) 31 2. s. pavía, archaeometry 48 (2006) 201 3. b. sillar, m. s. tite, archaeometry 41 (2000) 2 4. l. maritan, p. holakooei, c. mazzoli, appl. clay sci. 114 (2015) 540 5. s. shoval, j. therm. anal. calorim. 42 (1994) 175 6. lj. damjanović, i. holclajtner-antunović, u. b. mioč, v. bikić, d. milovanović, i. r. evans, j. archaeol. sci. 38 (2011) 818 7. lj. damjanović, v. bikić, k. šarić, s. erić, i. holclajtner-antunović, j. archaeol. sci. 46 (2014) 156 8. i. holclajtner-antunović, d. bajuk-bogdanović, v. bikić, m. marić-stojanović, j. raman spectrosc. 43 (2012) 1101 9. a. j. sakalis, n. a. kazakis, n. merousis, n. c. tsirliganis, j. cult. herit. 14 (2013) 485 10. u. mioč, p. colomban, g. sagon, m. stojanović, a. rosić, j. raman spectrosc. 35 (2004) 843 11. t. kaiser, in selevac: a neolithic village in yugoslavia, r. tringham, d. krstić, eds., university of california press, los angeles, ca, 1990, p. 255 12. a. goleanu, a. marian, m. gligor, c. florescu, s. varvara, rev. roum. chim. 50 (2005) 939 13. m. gajić-kvaščev, m. marić-stojanović, r. jančić-heinemann, g. kvaščev, chem. cent. j. 6 (2012) 102 1426 perišić et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. 14. m. gajić-kvaščev, m. marić-stojanović, ž. šmit, v. kantarelou, a. g. karydas, d. šljivar, d. milovanović, v. andrić, j. archaeol. sci. 39 (2012) 1025 15. m. spataro, in a short walk through the balkans: the first farmers of the carpathian basin and adjacent regions, m. spataro, p. biagi, eds., società preistoria protostoria friuli-v.g., trieste, 2007, p. 149 16. m. spataro, j. archaeol. sci. 38 (2011) 255 17. g. e. de benedetto, r. laviano, l. sabbatini, p. g. zambonin, j. cult. herit. 3 (2002) 177 18. y. maniatis, m. s. tite, j. archaeol. sci. 8 (1981) 59 19. w. kingery, j. frierman, proc. prehist. soc. 40 (1974) 204 20. v. c. farmer, infrared spectra of minerals, mineralogical society, london, 1974 21. s. shoval, m. gaft, p. beck, y. kirsh, j. therm. anal. 40 (1993) 263 22. p. colomban, a. tournie, l. bellot-gurlet, j. raman spectrosc. 37 (2006) 841 23. r. giustetto, g. berruto, e. diana, e. costa, j. archaeol. sci. 40 (2013) 4249 24. c. p. odriozola, v. m. hurtado pérez, j. archaeol. sci. 34 (2007) 1794 25. m. figueiredo, a. fernando, g. martins, j. freitas, f. judas, h. figueiredo, ceram. int. 36 (2010) 2383 26. k. kos, h. posilović, a. durman, m. ristić, s. krehula, archaeometry 57 (2015) 636 27. d. všianský, j. kolář, j. petřík, j. archaeol. sci. 49 (2014) 414 28. j. balmain, b. hannoyer, e. lopez, j. biomed. mater. res., a 48 (1999) 749 29. b. xu, k. m. poduska, phys. chem. chem. phys. 16 (2014) 17634 30. w. noll, r. holm, l. born, angew. chem. int. ed. 14 (1975) 602. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {biological activities of umbilicaria crustulosa (ach.) frey acetone extract} j. serb. chem. soc. 82 (2) 141–150 (2017) udc 582umbilicaria crustulosa: 633.879.6: jscs–4954 547.284.3:577–188:615.279:612.112 original scientific paper 141 biological activities of umbilicaria crustulosa (ach.) frey acetone extract ivana zlatanović1*, miroslava stanković2, vesna stankov jovanović1, violeta mitić1, ivana zrnzević1, aleksandra đorđević1, and gordana stojanović1 1department of chemistry, faculty of science and mathematics, university of niš, višegradska 33, 18000 niš, serbia and 2 nuclear facilities of serbia, mike petrovića alasa 12–14, 11351 vinča, serbia (received 19 october, revised 16 december, accepted 21 december 2016) abstract: this paper reports for the first time the effect of an acetone extract of umbilicaria crustulosa on the micronucleus distribution of human lymphocytes, and on the cholinesterase activity and antioxidant activity by the cupric ion reducing antioxidant capacity (cuprac) method. additionally, the total phenolic compounds (tpc) and the antioxidant properties were estimated via dpph, abts and trp assays. moreover, the antibacterial activity against two gram-positive and three gram-negative bacteria were determined. acetone extract of u. crustulosa at concentration of 1 and 2 µg ml-1 decreased a frequency of micronuclei (mn) by 10.8 and 16.8 %, respectively, acting more or slightly less than the synthetic protector amifostine (amf, wr-2721, 11.4 %, at concentration of 1 µg ml-1). the tested extract did not inhibit cholinesterase activity nor did it exhibit activity toward the examined bacteria. the extract reduced the concentration of dpph and abts radicals by 88.7 and 96.2 %, respectively. values for total reducing power (trp) and cupric reducing capacity (cuprac) were 0.6197±0.0166 µg ascorbic acid equivalents (aae) per mg of dry extract, and 19.7641±1.6546 µg trolox equivalents (te) per mg of dry extract, respectively. the total phenol content was 350.4188 ±14.587 µg gallic acid equivalents (gae) per mg of dry extract. the results of the present study showed that u. crustulosa acetone extract is a promising candidate for in vivo experiments considering its antioxidant activity and protective effect on human lymphocytes. keywords: micronucleus test; antioxidant activity; cholinesterase inhibition; antimicrobial activity; chemical composition. * corresponding author. e-mail: ivana.z.988@gmail.com doi: 10.2298/jsc161019014z _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 142 zlatanović et al. introduction lichens of the genus umbilicaria are generally known as rock tripe. these species grow on siliceous rocks usually in the alpine vegetation zone and the northern hemisphere.1 some of these species are used as food and represent a part of folk medicine.2,3 umbilicaria crustulosa (ach.) frey is known as a crusty novel lichen. serina and arroyo found two u. crustulosa chemotypes in samples growing in spain: gyrophoric acid and crustinic acid chemotype. beside crustinic and gyrophoric acids, umbilicaria species are characterized by the presence of lecanoric, hiascic, ovoic, umbilicaric, salazinic and norstictic acids.3,4 the presence of phenolic groups in the structure of the constituents of u. crustulosa extracts results in their antioxidant activity.3,5 antioxidants could prevent oxidation of biomolecules in food as well as in human cells. industrial antioxidants are mostly synthetic components which could have negative impacts on human health.6 natural antioxidants are very important source of protection against free radicals, which could be related to many disease such as alzheimer's disease, atherosclerosis, emphysema and many forms of cancer.7,8 a decrease in the amount of acetylcholine in the brain is the hallmark of alzheimer's disease. some synthetic cholinesterase inhibitors are used for the treatment of disease but an alternative approach could be the utilization of natural anticholinesterases.9 according to published data, hypogimnia physodes extracts and lichens secondary metabolites have an impact on micronucleus (mn) expression in peripheral blood lymphocytes.10,11 micronuclei result from lesions/adducts at the level of dna or chromosomes, or at the level of proteins directly or indirectly involved in chromosome segregation. the cytokinesis-block micronucleus technique enables micronuclei to be specifically scored in cells that have completed nuclear division and is, therefore, not influenced by variations in cell division kinetics, and it has been shown to be a sensitive and reliable index of chromosome damage.12 taking into account the above-mentioned biological activities of the lichens, the aim of present study was to evaluate for the first time effect of u. crustulosa acetone extract on the micronucleus distribution in human lymphocytes, and the effect on cholinesterase and antioxidant activity by the cuprac method. additionally, total phenolic compounds (tpc), the antioxidant properties estimated via 2,2-diphenyl-1-picrylhydrazyl (dpph) scavenging radical capacity, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (abts) radical scavenging activity and trp (total reduction power) assays as well as the antibacterial activity against two gram-positive (bacillus subtilis subsp. spizizenii atcc 6633 and staphylococcus aureus atcc 6538) and three gram-negative bacteria (escherichia coli atcc 8739, pseudomonas aeruginosa atcc 9027 and salmonella abony nctc 6017) were determined. _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ biological activities of u. crustulosa 143 experimental lichen material the lichen umbilicaria crustulosa (ach.) frey (syn. gyrophora depressa (ach.) schaer, omphalodiscus crustulosus (ach.) schol, gyrophora crustulosa (ach.), gyrophora spodochroa var. crustulosa (ach.) arnold, gyrophoropsis crustulosa (ach.) räsänen) was collected in may 2015, from the peak of mount babin zub (1650 m above sea level, coordinates 43° 23′ n, 22° 40′ e; stara planina mountain, serbia) from the population growing on rock habitat (red sandstone, silicates). a voucher specimen has been deposited in the herbarium collection at the department of biology and ecology, faculty of science and mathematics, university of niš under the acquisition number 9373. the lichen material was air-dried without exposure to direct sunlight for 10 days and stored at ambient temperature (25±2 °c) prior to further treatment. extraction extracts were prepared in triplicate according to the following procedure: powdered lichen material (10 g) was first subjected to ultrasound-assisted extraction with acetone (50 ml; 30 min in an ultrasound bath, type uzk 8; manufactured by maget, bela palanka, serbia. subsequently, the extract was left in the dark (at room temperature) for an additional five days. dry residues of the extracts were obtained using a rotary evaporator with the water bath set at 40 °c. the extract yield was 7.4±0.5 %. hplc analysis hplc analysis was performed on an agilent, zorbax eclipse xdb-c18, 5 μm, 4.6 mm×150 mm column using an agilent 1200 series liquid chromatograph (equipped with a diode array detector (dad), chemstation software (agilent technologies), a quaternary pump, an online vacuum degasser, auto sampler and a thermostated column compartment. the mobile phase, methanol–water–formic acid (80:20:0.2 volume ratios), was pumped at a flow rate of 0.5 ml min-1, the injection volume was 5 μl (concentration 10 mg of the dry extract per 1 ml of acetone), at 25 °c. the spectra were acquired in the range 190–400 nm and the chromatogram plotted at 254 nm. identification was conducted using retention times and uv spectra. cytokinesis-block micronucleus assay (cbmn) the cytokinesis-block micronucleus assay was performed as previously described.10,12 cell culture lymphocytes were treated with 1.0, 2.0 and 3.0 μg ml-1 of the examined extracts. amifostine, wr-2721 (98 % s-[2-[(3-aminopropyl)amino]ethyl]phosphothioate; marligen biosciences) at a concentration of 1 μg ml-1 was used as a positive control. three experiments were performed for each sample. the results are expressed as the means ± standard deviation (sd). the statistical analysis was performed using origin software package version 7.0. the statistical significance of difference between the data pairs was evaluated by analysis of variance (one-way anova) followed by the tukey test. statistical difference was considered significant at p < 0.01 and p < 0.05. total phenolic content and antioxidant activity determination of the total phenolic content (tpc) and four antioxidant assays, i.e., dpph and abts scavenging radical capacity, cuprac (cupric reducing antioxidant capacity) and trp (total reducing power), were performed as previously described.13,14 all spectrophotometric assays were conducted on a perkin elmer lambda 15 double beam uv/vis spectrophotometer. for all the above-mentioned experiments, the concentration of the sample _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 144 zlatanović et al. solution was 15 mg of extract per ml of methanol. all analyses were performed in triplicate. the results are presented as mean ± standard deviation (sd). cholinesterase activity assessment of the effect of the extract on cholinesterase activity was performed as previously described.15 the activity was measured spectrophotometrically using a konelab 20 analyzer (thermo fisher scientific, helsinki, finland) with thermostated flow cells, length 7 mm (at a wavelength 405 nm). the sample concentration was 10 mg of dry extract per 1 ml of dmso. a solution of neostigmine bromide at a concentration of 200 µg ml-1 was used as the reference standard. antimicrobial activity antimicrobial activity was evaluated against two gram-positive (bacillus subtilis subsp. spizizenii atcc 6633 and staphylococcus aureus atcc 6538) and three gram-negative (escherichia coli atcc 8739, pseudomonas aeruginosa atcc 9027 and salmonella abony nctc 6017) bacteria. analysis was performed according to the national committee for clinical laboratory standards (nccls).16 each test was performed in triplicate. the sample concentration was 1 mg per disk (diameter 12 mm). streptomycin and chloramphenicol were used as positive controls, at a concentration of 10 mg and 30 mg per disk, respectively. results and discussion hplc analysis the hplc chromatogram of the acetone extract of u. crustulosa and the uv spectra of the identified constituents are presented in fig. 1. chemical composition and structures of the constituents are given in table i. the most abundant component of the acetone extract of u. crustulosa is the tridepside gyrophoric acid and this sample could be classified as a gyrophoric acid chemotype.4 the major components of the extract were the didepside lecanoric acid and the tridepside gyrophoric acid, comprised of two and three, respectively, orsellinic acid units joined with ester bonds at the para-positions. crustinic acid is an exception, as it has the ester bonded at the meta-position of the third ring. no direct evidence is available on the biosynthesis of meta-depsides. the co-occurrence and interconversion of the depsides metaand para-scrobiculin, which exist in dynamic equilibrium, in lobaria scrobiculata led to the hypothesis that meta-depsides are formed from para-depsides via an intramolecular rearrangement after hydroxylation of the carbon at the meta-position of the b-ring.17 cytokinesis-block micronucleus assay (cbmn) the extract was tested for in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using the cytochalasin-b blocked mn assay at concentrations of 1.0, 2.0 and 3.0 μg ml–1. the frequencies and distribution of mn in human lymphocytes were scored. the statistical significance of difference between the data pairs was evaluated by analysis of variance (oneway anova) followed by the tukey test. the results are presented in table ii. _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ biological activities of u. crustulosa 145 fi g. 1 . h pl c c hr om at og ra m o f u . c ru st ul os a ac et on e ex tr ac t a nd u v s pe ct ra o f i de nt if ie d co ns tit ue nt s. _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 146 zlatanović et al. table i. composition (as percentages of the total peak area of the hplc chromatograms recorded at 254 nm) and structures of the constituents of the u. crustulosa extract component number cmpd. structure rt / min hplc peak area, % 1 methyl orsellinate cooch3 ch3 ohho 1 2 3 4 5 6 7 8 h h 4.262 2.1 2 lecanoric acid cooho oh ch3 oh cooh ch3 1 23 4 5 6 7 1' 2'3' 4' 5' 6' 7' 8' 4.720 23.5 3 crustinic acid cooho oh ch3 oh coo ch3 1 23 4 5 6 7 8 1' 2'3' 4' 5' 6' 7' 8' h3c cooh oh ho 1" 2" 3"4" 5" 6" 7"8" 5.582 0.1 4 gyrophoric acid cooho oh ch3 oh coo ch3 oh cooh ch3 1 23 4 5 6 7 1' 2'3' 4' 5' 6' 7' 1'' 2''3'' 4'' 5'' 6'' 7'' 7.384 68.6 table ii. incidence of mn, cytokinesis-block proliferation index, distribution of mn per cells (mean ± sd) and frequency of mn in cell cultures of human lymphocytes treated with different concentration of umbilicaria crustulosa acetone extract (ae); mn/1000 bn cells – incidence of micronuclei in 1000 binucleated cells; % bn cells with micronuclei; mn/bn cells – incidence of micronuclei in binucleated cells; cbpi – cytokinesis-block proliferation index; frequency of mn – incidence of mn present as % from control groups in cell cultures of human lymphocytes treated with different concentration of extracts; a* – statistically significant difference from control at p < 0.05 concentrations mn/1000 bn cells bn cells with mn, % mn/bn cells cbpi frequency of mn rel. to control, % untreated cells 25.83±0.58 2.06±0.12 1.25±0.06 1.63±0.03 100.00 amifostin (1 µg ml-1) 22.89±0.68a* 1.87±0.06 1.22±0.06 1.62±0.04 88.62 ae (1 µg ml-1) 23.05±1.06 1.89±0.08 1.22±0.06 1.68±0.06 89.24 ae (2 µg ml-1) 21.60±1.68 1.62±0.13 1.34±0.13 1.63±0.01 83.62 ae (3 µg ml-1) 24.24±1.21 2.06±0.12 1.18±0.04 1.63±0.02 93.84 the cell cultures treated with amifostine (radioprotectant, previously known as wr-2721) at a concentration of 1 μg ml–1 resulted in a decrease of the mn frequency of 11.4 % compared to the control cell cultures (statistically significant, p < 0.05). the u. crustulosa extract at a concentration of 2 μg ml–1 gave a _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ biological activities of u. crustulosa 147 decrease in the mn frequency of 16.38 %, which was higher than that with amifostine, while at a concentration of 1 μg ml–1, the effect was 10.8 %, which is slightly less than the effect of amifostine. the u. crustulosa extract at a concentration of 3 µg ml–1 was less effective (6.2 %) than amifostine. since the number of micronuclei serves as an indicator of dna damage, these results indicate that the examined extract at a concentration of 2 μg ml–1 protects dna. previous results of h. physodes extracts suggested a similar conclusion. a diethyl ether extract of the lichen h. physodes at a concentration of 1 μg ml–1 caused a significant decrease in the frequency of micronuclei, 17.4 %, while it was less effective at a concentration of 2 μg ml–1.11 the effect of examined extracts on cell proliferation was investigated by determining the cytokinesis-block proliferation index (cbpi), calculated as [(mi + 2mii + 3(miii + miv)]/n, where mi–iv represent the number of cells with 1 to 4 nuclei, respectively, and n is the number of cells scored.18 the comparable cbpi values of extracts, amifostine wr-2721 and untreated cells have confirmed absence of the impact on the cell proliferation. this fact is important for validity of the results because mn expression is dependent on cell division. the total phenolic content and antioxidant activity tpc analysis showed a high content of phenolic components in the acetone extract of u. crustulosa, 350.4188±14.587 µg gae mg–1 due to the presence of the tridepside gyrophoric acid and the didepside lecanoric acid (total 92.0 % of the sample). it was not possible to compare the present results expressed as galic acid equivalents (gae) with those of kosanic et al. because they expressed the amount of total phenolic compounds as the pyrocatechol equivalent.19 the assessment of the dpph and abts scavenging activity showed that extract reduced the concentration of dpph and abts radicals by 88.7 and 96.2 %, respectively, which is consistent with previously published results. namely, kosanic et al. found that the scavenging activity of a methanol extract of u. crustulosa was 79.85 %, while buçukoglu et al. found that the values of the dpph scavenging activity of gyrophoric and leconaric acids were 50.96 and 32.48 %, respectively.19,20 the value of the total reducing power ability (trp, ability of antioxidants to reduce hexacyanidoferrate (iii) to hexacyanidoferrate (ii), which leads to an increase in the absorbance of the reaction mixtures) was 0.6197±0.0166 mg ascorbic acid equivalents (aae) per mg dry extract weight. again, it was not possible to compare the obtained results with those previously published due to the different expression of the results (absorbance vs. aae).19 the cuprac assay has some advantages compared to the other experiments since the reagent is stable, accessible, low-cost, and the reaction (reduction of a cupric neocuproine complex (cu(ii)–nc) by antioxidants to the cuprous _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ 148 zlatanović et al. form (cu(i)–nc)) is realized at nearly physiological ph. the result obtained for the u. crustulosa acetone extracts (19.7641±0.0166 mg trolox equivalents (te) per mg dry extract) is close to the highest cuprac capacity of mushrooms (21.738–4.164 mg te per mg dw).13 cholinesterase activity results obtained from the screening of the interaction of the extract with cholinesterase from pooled human serum showed that u. crustulosa possess a weak activation effect on the cholinesterase activity. in the conducted experiment, neostigmin bromide (commercial cholinesterase inhibitor) inhibited cholinesterase to extent of 96.6 % while u. crustulosa extract manifested weak activation effect on cholinesterase to an extent of 1.6 %. antibacterial activity the results of the antibacterial assays against two gram-positive (b. subtilis subsp. spizizenii atcc 6633 and s. aureus atcc 6538) and three gram -negative bacteria (e. coli atcc 8739, p. aeruginosa atcc 9027 and s. abony nctc 6017) showed that the acetone extract of u. crustulosa had no activity against the tested bacteria at a concentration of 1 mg per disk. these results were not entirely in accordance with those previously published. namely, rankovic et al. and kosanic et al. reported no activity toward escherichia coli, botrytis cinerea and candida albicans but moderate antimicrobial activity of methanol and acetone extracts of u. crustulosa to several bacteria and fungi, such as: bacillus mycoides, bacillus subtilis, enterobacter cloaceae, klebsiella pneumoniae, staphylococcus aureus, aspergillus flavus, aspergillus fumigatus, fusarium oxysporum, mucor mucedo, paecilomyces variotii , penicillium purpurescens, penicillium verrucosum, saccharomyces cerevisiae, trichoderma harzianum.2,19 bucukoglu et al. and candan et al. found that gyrophoric acid was not effective against b. subtilis, e. coli, k. pneumoniae, p. aeruginosa and salmonela typhimurium.20,21 since gyrophoric acid participated 68.5 % to the extract composition, the results obtained in the present study could be explained by its inactivity. conclusion the results of present study show that u. crustulosa acetone extract is a promising candidate for in vivo experiments considering antioxidant activity and protective effect on human lymphocytes, which could not be said for its antibacterial and anti-cholinesterase activity. acknowledgement. the authors acknowledge the ministry of education, science and technological development of the republic of serbia for the financial support (grant no. 172047). _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ biological activities of u. crustulosa 149 и з в о д биолошка активност ацетонског екстракта лишаја umbilicaria crustulosa (ach.) frey ивана златановић1, мирослава станковић2, весна станков јовановић1, виолета митић1, ивана зрнзевић1, александра ђорђевиђ1 и гордана стојановић1 1департман за хемију, природно–математички факултет, универзитет у нишу, вишеградска 33, 18 000 ниш и 2институт за нуклеарне науке винча, мике петровића аласа 12–14, 11351 винча у овом раду је по први пут испитан утицај ацетонског екстракта лишаја umbilicaria crustulosa (ach.) frey на дистрибуцију микронуклеуса у људским лимфоцитима, холинестеразну активност и антиоксидативну активност коришћењем cuprac методе. укупни садржај фенола, антиоксидативна својства (процењена коришћењем dpph, abts и trp тестова), као и антибактеријска активност према две грам-позитивне и три грам -негативне бактерије су такође одређени. ацетонски екстракт лишаја u. crustulosa у концентрацији од 1 и 2 μg ml-1 смањује фреквенцију микронуклеуса (мн) за 10,8 и 11,4 %, редом, делујући јаче или нешто слабије од комерцијалног заштитног агенса амифостина (amf, wr-2721), 11,4 % у концентрацији 1 μg ml-1). испитивани екстракт није показао инхибицију активности холинестеразе, као ни активност према тестираним бактеријама. екстракт смањује концентрацију dpph и abts радикала за 88,7 и 96,2 %, редом. вредности укупне редукционе моћи (trp) и купрак-редукционог капацитета (cuprac) су 0,6197±0,0166 mg еквивалента аскорбинске киселине (aae) по mg сувог екстракта и 19,7641±1,6546 mg тролокс еквивалента (te) по mg сувог екстракта, редом. укупни садржај фенола је 350,4188±14,587 mg еквивалената галне киселине по mg сувог екстракта. резултати испитивања су показали да је u. crustulosa обећавајући кандидат за in vivo испитивања у погледу антиоксидативне активности и заштитног ефекта на људске лимфоците. (примљено 19. октобра, ревидирано 16. децембра, прихваћено 21. децембра 2016) references 1. e. frey, ber. schweiz. bot. ges. 46 (1997) 412 2. b. rankovic, m. misic, s. sukdolak, biologia 64 (2009) 53 3. n. manojlovic, p. vasiljevic, p. z. maskovic, m. juskovic, g. bogdanovic-dusanovic, evidence based complementary altern. med. 2012 (2012), article id 452431 4. e. serina, r. arroyo, bryologist 99 (1996) 335 5. m. e. hidalgo, e. fernandez, w. quilhot, phytochemistry 37 (1994) 1585 6. f. shahidi, u. n. anasundra, r. amarowicz, food res. int. 27 (1994) 489 7. k. guddadarangavvanahally, r. jayaprakasha, z. naturforsch., c: j. biosci. 55 (2000) 1018 8. k. molnar, e. farakas, z. naturforsch., c: j. biosci. 65 (2010) 157 9. e. giacobini, drugs that target cholinesterase, in cognitive enhancing drugs, j. j. buccafusco, ed., springer, basel, 2004, p. 11 10. i. stojanović, m. stanković, o. jovanović, g. petrović, a. šmelcerović, g. stojanović, nat. prod. commun. 8 (2013) 109 11. g. stojanovic, m. 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xamena, a. creus, j. catalan, h. norppa, r. marcos, mutat. res. 341 (1995) 169 19. m. kosanic, s. šeklic, m. markovic, b. rankovic, dig. j. nanomater. biostruct. 9 (2014) 273 20. z. t. buçukoglu, s. albayrak, g. halici, y. tay, j. food process. preserv. 37 (2013) 1103 21. m. candan, m. yilmaz, t. tay, m. kivanç, h. turk, z. naturforsch., c: j. biosci. 61 (2006) 319. _________________________________________________________________________________________________________________________ (cc) 2017 scs. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice development and validation of an lc–ms/ms method for the determination of adapalene in pharmaceutical forms for skin application j. serb. chem. soc. 81 (10) 1171–1181 (2016) udc *adapalene:543.544+543.51:616.5:615 jscs–4617 original scientific paper 1171 development and validation of an lc–ms/ms method for the determination of adapalene in pharmaceutical forms for skin application vladimir dobričić1*, nataša bubić pajić2, bojan marković1, sote vladimirov1, snežana savić3 and gordana vuleta3 1department of pharmaceutical chemistry, university of belgrade, faculty of pharmacy, 11000 belgrade, serbia, 2department of pharmaceutical technology, university of banja luka, faculty of medicine, 78000 banja luka, bosnia and herzegovina and 3department of pharmaceutical technology, university of belgrade, faculty of pharmacy, 11000 belgrade, serbia (received 15 february, revised 11 may, accepted 27 june 2016) abstract. the development and validation of a liquid chromatography– tandem mass spectrometry (lc–ms/ms) method for the determination of adapalene in pharmaceutical forms for skin application were presented in this study. the ms/ms analysis of adapalene was performed by use of three mobile phases, consisted of acetonitrile and a) 0.1 % formic acid, b) 0.1 % trifluoroacetic acid and c) 20 mm ammonium acetate. the strongest signals of parent ion and dominant product ion were obtained in negative mode by use of the mobile phase c). the validation of this method was performed according to the ich guidelines. small variations of selected chromatographic parameters (concentration of ammonium acetate, mobile phase composition, column temperature and flow rate) did not affect significantly the qualitative and quantitative system responses, which proved the method’s robustness. the method is specific for the determination of adapalene. the linearity was proved in the concentration range of 6.7–700.0 ng ml-1 (r = 0.9990), with limits of detection and quantification of 2.0 and 6.7 ng ml-1, respectively. the accuracy was confirmed by calculated recoveries (98.4–101.5 %). the precision was tested at three levels: injection repeatability, analysis repeatability and intermediate precision. the calculated relative standard deviations were less than 1, 2 and 3 %, respectively. keywords: adapalene; ms/ms analysis; lc–ms/ms determination; pharmaceutical forms for skin application. * corresponding author. e-mail: vladimir@pharmacy.bg.ac.rs doi: 10.2298/jsc160215066d _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1172 dobričić et al. introduction adapalene (fig. s-1 of the supplementary material to this paper), chemically designated as 6-[3-(1-adamantyl)-4-methoxyphenyl]-2-naphthoic acid, is a third generation synthetic retinoid used in the topical treatment of acne vulgaris, psoriasis and photo aging.1 the adapalene was quantified by measuring the radioactivity in the plasma as well as in other biological samples (urine, faeces and skin strips). the measurements showed that its concentrations were below the limits of quantification.2 additionally, no evidence of teratogenicity has been reported.3 pharmaceutical forms of adapelene are creams, gels, emulsions, microemulsions, lotions, soy bean oil-ciclodextrin complexes and solid lipid microparticle dispersion.4–6 these forms contain complex matrices and specific and sensitive analytical methods are required for adapalene analysis. according to the european pharmacopoeia 7th edition, high performance liquid chromatography (hplc) determination of adapalene is performed by using a phenyl column (column length: 250 mm; column diameter: 4.6 mm; particle size: 5 µm) and mobile phase consisting of glacial acetic acid, water, acetonitrile and tetrahydrofuran.7 several hplc methods with uv detection for the determination of adapalene in gel topical formulations,8–11 suspensions of nanocapsules12 and in vitro release studies of adapalene from cream formulation13 have been reported so far. rühl and nau presented a method for the determination of adapalene and retinol in biological samples that combines solid-phase extraction and hplc with uv and fluorescence detection.14 brenna et al. used mass spectrometry (ms) and gas chromatography–mass spectrometry (gc–ms) for the characterization of three adapalene impurities, but these methods were not applied to the analysis of adapelene.15 ms has not been applied to the quantification of adapalene so far and there are no liquid chromatography–mass spectrometry (lc–ms) or liquid chromatography–tandem mass spectrometry (lc– –ms/ms) methods for the determination of this drug in pharmaceutical forms, biological samples, biopharmaceutical and formulation studies. lc–ms and lc– –ms/ms are usually used for the analysis of biological samples. however, in the last years these methods have been used more often for the analysis of pharmaceutical and cosmetic forms (e.g., tablets and creams).16–19 the goal of this study was to develop and validate a simple, specific and sensitive method for the determination of adapalene in pharmaceutical forms for skin application (cream, gel and microemulsion). for this purpose, tandem mass spectrometry (ms/ms) analysis of adapalene under different ionization conditions was performed and, on the basis of these results, an lc–ms/ms method was developed and validated. this method could be used as a starting point for the development of a bioanalytical method for the determination of adapalene in _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. lc–ms/ms determination of adapalene 1173 biopharmaceutical and pharmacokinetic studies (e.g., estimation of the adapalene potential to penetrate the skin using the tape stripping testing technique). experimental details related to chemicals and apparatus used and method optimization are given in supplementary material to this paper. method validation the validation of this method was performed according to the ich guidelines.20 a standard stock solution was prepared by dissolving 2 mg of adapalene in 100 ml of ethanol (20 μg ml-1). subsequently, this solution was diluted with ethanol to obtain working standard solutions. solutions of adapalene formulations (microemulsion apg-1, sona® 0.1 % gel and sona® 0.1 % cream) were prepared by dissolving these formulations in ethanol (the concentration of adapalene corresponding to 100 % accuracy and precision of the testing was 400 ng ml-1). solutions of placebos were prepared in accordance with the preparation of adapalene formulation solutions, which contain adapalene in concentration of 400 ng ml-1. the surfactants and solvents used in the formulation studies were diluted with ethanol prior to injecting. the robustness was tested by small variations of selected chromatographic parameters: column temperature (23 and 25 °c), flow rate (0.75 and 0.85 ml min-1), concentration of ammonium acetate solution (18 and 22 mm) and mobile phase composition (69 and 71 % acetonitrile). for this purpose, a mixture of adapalene (400 ng ml-1) and uracil (200 ng ml-1) was used. the uracil (lc–ms, negative mode, m/z = 111.0) was used as an unretained compound for the calculation of the retention factors (k). the changes in the adapalene peak shape, k and peak area (a) were monitored. the specificity was tested by injecting an adapalene standard solution (400 ng ml-1), placebos, as well as solvents and surfactants used in formulation studies. the chromatograms were examined at retention time corresponding to adapalene. the limit of detection (lod) and limit of quantification (loq) were concentrations of adapalene standard solutions whose signal-to-noise ratios were 3:1 and 10:1, respectively. the linearity of the method was tested in the range loq – 700 ng ml-1 (loq, 67, 250, 400, 500 and 700 ng ml-1). the influence of different weighting factors (1/x, 1/x2, 1/y and 1/y2) on the sum of percentage relative error was evaluated and the results were compared with an unweighted calibration curve. the accuracy was tested by spiking placebo or formulation with standard solution of the tested substance to obtain three concentration levels: 80, 100 and 120 % of the target concentration (in this study the target concentration of adapalene was 400 ng ml-1). the accuracy of this method was tested by spiking microemulsion apg-1 placebo, sona® 0.1% gel and sona® 0.1 % cream with adapalene standard stock solution (20 μg ml-1) to obtain three concentrations (around 320, 400 and 480 ng ml-1) and was evaluated on the basis of calculated recovery values. three levels of method precision (injection repeatability, analysis repeatability and intermediate precision) were tested by analysis of an adapalene microemulsion apg-1, sona® 0.1 % gel and sona® 0.1 % cream at concentration level 100 % (400 ng ml-1). the injection repeatability was determined after ten injections of the same sample solution of each formulation. the analysis repeatability was tested by preparing and injecting six sample solutions of each formulation, whereas the intermediate precision was tested in the same manner by another analyst, another day, with different set of reagents. the precision of the method was evaluated by calculating the relative standard deviations of adapalene determinations. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1174 dobričić et al. results and discussion the ms/ms spectra of adapalene, obtained by use of three mobile phases (in negative and positive mode) are presented in figs. 1–3. the applied collision energies were 25 (figs. 3a, 4a and 4b), 30 (figs. 1b and 2b) and 35 ev (fig. 1a). fig. 1. ms/ms spectra of adapalene, obtained by use of mobile phase acetonitrile/0.1 % trifluoroacetic acid (1:1 volume ratio), flow rate: 500 μl min-1, in: a) negative and b) positive mode. the most dominant fragment ion in negative mode is a product of the adapalene decarboxilation (fig. 4). an elimination of both carboxyl and methoxy group could be observed in negative mode (m/z 336.7 in fig. 2a and 337.1 in fig. 3a), whereas elimination of hydroxyl moiety from the carboxyl group was observed in positive mode (m/z 394.3, fig. 2b). in both negative and positive mode, the fragmentation of adapalene depends on the mobile phase used in ms/ms analysis. additionally, the increase in aceto_________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. lc–ms/ms determination of adapalene 1175 nitrile content and the decrease in flow rate improved the signal strength of parent ion and product ions. fig. 2. ms/ms spectra of adapalene obtained by use of mobile phase acetonitrile/0.1 % formic acid (1:1 volume ratio), flow rate: 500 μl min-1, in: a) negative and b) positive mode. the strongest signals of parent ion and product ions in positive mode were observed when a mobile phase with volume composition acetonitrile/0.1 % trifluoroacetic acid = 50:50 was used. this mobile phase has the lowest ph value (ph 2.1) and an efficient protonation of adapalene in positive mode could be expected, resulting in the strongest signals of parent ion and product ions. in comparison with the negative mode, the signal strength of parent ion and product ions in positive mode was significantly lower for all the tested mobile phases. the strongest signals of parent ion and product ions in negative mode were observed for a mobile phase with volume composition acetonitrile/20 mm ammonium acetate = 50:50. this mobile phase has the highest ph value (ph 7.2). therefore, it could be expected that the production of an adapalene anion in _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1176 dobričić et al. negative mode would be the most efficient with this mobile phase, thus resulting in the strongest signals of the parent ion and product ions. fig. 3. ms/ms spectra of adapalene obtained by use of mobile phase acetonitrile/20 mm ammonium acetate (1:1 volume ratio), flow rate: 500 μl min-1, in: a) negative and b) positive mode. among all the tested mobile phases and ionization modes, the strongest signals of parent ion and most dominant product ion were observed when a mobile phase with acetonitrile/20 mm ammonium acetate volume composition of 50:50 (negative mode, selected reaction monitoring srm transition: 410.8→367.0) was used. therefore, this mobile phase was selected for further development of the method. in the next step, the composition of the mobile phase was optimized. a simultaneous monitoring of ion 410.8 (lc–ms, negative mode) and srm transition 410.8→367.0 (lc–ms/ms, negative mode) was performed and the peak parameters (retention time, peak area and peak shape) were analyzed. the inc_________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. lc–ms/ms determination of adapalene 1177 rease in the percentage of acetonitrile above 70 % led to slight decrease in the adapalene retention time, whereas the use of a mobile phase with volume composition acetonitrile/20 mm ammonium acetate = 90:10 resulted in a peak shape deformation. the decrease in the acetonitrile content below 70 % resulted in adapalene retention times higher than 10 min, which significantly lengthened the analysis. therefore, the mobile phase with volume composition acetonitrile/20 mm ammonium acetate = 70:30 and flow rate 0.8 ml min–1 was selected as the optimal one. fig. 4. fragmentation of adapalene (negative mode; mobile phase: acetonitrile/20 mm ammonium acetate). the area of the adapalene peak in lc–ms/ms mode was approximately 20 % higher than the one obtained by lc-ms and there were no other peaks apart from those corresponding to adapalene (fig. 5). fig. 5. lc–ms and lc–ms/ms chromatograms of microemulsion apg-1. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1178 dobričić et al. this indicated the higher sensitivity and specificity of the lc–ms/ms method. finally, the lc–ms/ms method (negative mode, srm transition 410.8→ →367.0) with mobile phase volume composition acetonitrile/20 mm ammonium acetate = 70:30, mobile phase flow rate of 0.8 ml min–1 and column temperature 25 °c was selected as optimal and validated according to the ich guidelines.20 method validation robustness. small and deliberate changes in the column temperature, flow rate, concentration of ammonium acetate solution and mobile phase composition affected the retention factors and peak areas with less than 4.80 % (without any significant peak shape alterations), which proved the method’s robustness. specificity. the method is specific for the determination of adapalene as no interference was observed at retention time corresponding to this substance. overlaid sample chromatograms are presented in fig. s-2 of the supplementary material. since the specificity was proven by the use of different solvents and excipients, the application of this method to the analysis of adapalene in formulation studies (e.g., solubility experiments in tested solvents and excipients, optimization of formulation composition and stability evaluation of adapalene topical forms) could be considered. limits of detection and quantification. the limits of detection and quantification were 2.0 and 6.7 ng ml-1, respectively. the loq value was used as a first point in the linearity testing. linearity. due to the wide range of expected concentrations in formulation studies, the linearity of the method was tested and proved in the range of 6.7 (loq)–700.0 ng ml-1. the selected weighting factor was 1/y2, because its application resulted in the lowest sum of percentage relative error (17.63) in comparison to the other weighting factors (18.53–30.20) and unweighted calibration curve (115.44). the regression equation was y = 144236x + 526.31 and correlation coefficient (r) was 0.9990. accuracy. the recoveries of adapalene in tested formulations (microemulsion apg-1, sona® 0.1 % gel and sona® 0.1 % cream) at three concentration levels (80, 100 and 120 % of the target concentration) are presented in table i. the recovery at each level is presented as a mean value of three determinations. the recoveries were within the specified limits for active pharmaceutical ingredients (98–102 %), which proved the method to be accurate for the determination of adapalene in tested pharmaceutical forms.21 precision. the precision was tested at three levels: injection repeatability, analysis repeatability and intermediate precision. the results are expressed as relative standard deviations (rsd, table ii). _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. lc–ms/ms determination of adapalene 1179 table i. recovery values (n = 3) formulation level % total concentration, ng ml-1 found concentration, ng ml-1 recovery % rds % microemulsion apg-1 80 328.00 330.69 100.82 1.43 100 410.00 404.89 98.75 1.31 120 492.00 499.30 101.48 1.49 sona® 0.1% gel 80 320.00 324.67 101.46 1.81 100 400.00 405.92 101.48 0.78 120 480.00 484.61 100.96 1.32 sona® 0.1 % cream 80 340.00 341.81 100.53 1.11 100 412.00 411.32 99.83 1.86 120 484.00 476.16 98.38 1.64 table ii. precision of the method formulation level found concentration, mg g-1 rsd / % microemulsion apg-1 injection repeatability 1.04 0.86 analysis repeatability 1.19 1.83 intermediate precision 1.06 2.58 sona® 0.1% gel injection repeatability 0.99 0.81 analysis repeatability 1.02 1.19 intermediate precision 1.01 2.80 sona® 0.1% cream injection repeatability 1.04 0.91 analysis repeatability 0.98 1.83 intermediate precision 0.96 2.05 the relative standard deviations were within the specified limits for injection repeatability (<1 %), analysis repeatability (<2 %) and intermediate precision (<3 %), which proved the precision of the method.21 conclusions the development and validation of a lc–ms/ms method for the determination of adapalene in pharmaceutical forms for skin application were presented. the ms/ms analysis of adapalene was performed by use of three mobile phases (acetonitrile and 0.1 % formic acid; acetonitrile and 0.1 % trifluoroacetic acid; acetonitrile and 20 mm ammonium acetate). the strongest signals of parent ion and dominant product ion were obtained in negative mode, by use of the mobile phase consisting of acetonitrile and 20 mm ammonium acetate. subsequently, optimization of the mobile phase composition and flow rate was performed. the method was validated according to the ich guidelines, which confirmed its robustness, specificity, linearity, accuracy and precision. due to its specificity tested by using different solvents and excipients and low lod and loq values, application of this method to the analysis of adapalene in formulation studies (e.g., solubility experiments in tested solvents and excipients, optimization of formulation composition and stability evaluation of adapalene topical forms) could be considered. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1180 dobričić et al. supplementary material details about adapalene, chemicals and apparatus used and metod optimization and specificity are available electronically at the pages of journal website: http://www.shd.org.rs/ /jscs/, or from the corresponding author on request. acknowledgements. the authors would like to acknowledge the financial support from the ministry of education, science and technological development, republic of serbia, through projects oi172041 and tr34031. the authors are grateful to basf, kao chemicals and gattefosse for kindly supplying us with surfactants and solvents. и з в о д развој и валидација lc–ms/ms методе за одређивање адапалена у фармацеутским препаратима за примену на кожи владимир добричић1, наташа бубић пајић2, бојан марковић1, соте владимиров1, снежана савић3 и гордана вулета3 1катедра за фармацеутску хемију, универзитет у београду, фармацеутски факултет, 11000 београд, 2катедра за фармацеутску технологију, универзиет у бањој луци, медицински факултет, 78000 бања лука, босна и херцеговина и 3катедра за фармацеутску технологију, универзитет у београд, фармацеутски факултет, 11000 београд приказани су развој и валидација методе течне хроматографије са тандем масеном спектрометријом (lc–ms/ms) за одређивање адапалена у фармацеутским препаратима за примену на кожи. извршена је ms/ms анализа адапалена употребом три мобилне фазе, које су се састоје из ацетонитрила и: а) 0,1 % раствора мравље киселине, b) 0,1 % раствора трифлуоросирћетне киселине и c) 20 mm амонијум-ацетата. најјачи сигнал полазног јона и главног фрагмента је добијен у негативном моду употребом мобилне фазе c). валидација методе је извршена према смерницама ich. мале промене одабраних хроматографских параметара (концентрација амонијум-ацетата, састав мобилне фазе, температура колоне и проток) нису значајно утицале на квалитативне и квантитативне одговоре система, што доказује робусност методе. метода је специфична за одређивање адапалена. линеарност је доказана у опсегу 6,7–700,0 ng ml-1 (r = 0,9990), са границама детекције и квантификације од 2,0 и 6,7 ng ml-1, редом. тачност методе је потврђена израчунатим „recovery“ вредностима (98,4–101,5 %). прецизност је испитана на три нивоа: поновљивост инјектовања, поновљивост анализе и средња прецизност. израчунате релативне стандардне девијације су мање од 1, 2 и 3 %, редом. (примљено 15. фебруара, ревидирано 11. маја, прихваћено 27 јуна 2016) references 1. h. gollnick, w. cunliffe, d. berson, b. dreno, a. finlay, j. j. leyden, a. r. shalita, d. j. thiboutot, j. am. acad. dermatol. 49 (2003) s1 2. product information differin® adapalene 0.1 % topical gel, http://www.galderma.com.au/ /linkclick.aspx?fileticket=ko3vz2oz6ym%3d&portalid= 4 (january 10, 2016) 3. a. l. zaenglein, semin. cutan. med. surg. 27 (2008) 177 4. l. trichard, m. b. delgado-cherra, r. h. guy, e. fattal, a. bochot, pharm. res. 25 (2008) 435 5. g. bhatia, y. zhou, a. k. banga, j. pharm. sci. 8 (2013) 2622 6. a. lauterbach, c. mueller-goymann, int. j. pharm. 466 (2014) 122 7. european pharmacopoeia 7.0, council of europe, strasbourg, 2010, pp. 1324–1326 _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. lc–ms/ms determination of adapalene 1181 8. 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18. j. fiori, v. andrisano, j. pharm. biomed. sci. 91 (2014) 185 19. j. golubović, b. otašević, a. protić, a. stanković, m. zečević, rapid commun. mass spectrom. 29 (2015) 2319 20. international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use, ich harmonised tripartite guideline (2005), validation of analytical procedures: text and methodology q2(r1), complementary guideline on methodology, dated 06 november 1996, incorporated in november 2005, london, 2005 21. j. crowther, in handbook of modern pharmaceutical analysis, s. ahuja, s. scypinsky, eds., academic press, san diego, ca, 2001, p. 415. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice chemical composition and nutritive potential of cichorium intybus l. leaves from montenegro j. serb. chem. soc. 81 (10) 1141–1149 (2016) udc 633.78:641.1:577.164:577.118(497.16) jscs–4914 original scientific paper 1141 chemical composition and nutritive potential of cichorium intybus l. leaves from montenegro dejan jančić1*, vanja todorović2, zorica basić3 and slađana šobajić2 1llc center for ecotoxicological research podgorica, podgorica, montenegro, 2university of belgrade, faculty of pharmacy, department of bromatology, belgrade, serbia and 3the military medical academy, institute of hygiene, belgrade, serbia (received 13 march, revised 18 may, accepted 19 may 2016) abstract: the leaves of chicory (cichorium intybus l.) plant have been used for centuries in montenegro and other mediterranean countries as a vegetable in salads, sauces and other types of appetizers and meals. the wild and cultivated chicory leaves from different locations in montenegro were analysed regarding several nutrients, major and trace element as well as vitamin composition, using standard methods of analysis. the results of the study indicated that chicory leaves were rich in total dietary fiber and mineral content and had low energy value. also, they were potential sources of useful nutrients such as potassium, calcium, manganese, iron, and vitamin a, with the average content of 391.9, 164.7, 0.55, 2.33 and 0.47 mg/100 g in fresh leaves, respectively. wild plants were superior to the cultivated ones regarding carbohydrate, calcium and manganese content. origin of the chicory leaves significantly influenced most of the analyzed parameters. keywords: chicory; nutrients; minerals; vitamins. introduction indigenous plants play important role in human diet because they contain significant amounts of nutrients and trace elements. despite the wide-spread consumption of cultivated vegetables, some indigenous plants have been reported as more nutritious and less expensive. in montenegro many indigenous plants are traditionally collected from the nature, but precise and valid data for their nutritional value is not available. wild chicory (cichorium intybus l.) is an erect perennial herb, 80–90 cm in height usually with bright blue flowers, rarely white or pink. it has a fleshy taproot up to 75 cm in length. chicory is a member of asteraceae family. the genus cichorium consists of six species mainly distributed in the areas of europe * corresponding author. e-mail: dejan.jancic@ceti.co.me doi: 10.2298/jsc160313057s _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1142 jančić et al. and asia.1 several cichorium species have been used for centuries as part of the traditional diet in mediterranean countries.2 mostly used plant parts are leaves (as salads, cooked vegetable and meat dishes), chicons (blanched buds), or roots (var. sativum, which are baked, ground, and used as a coffee substitute and inulin source). most of the information on plant chemical composition refers to the roots and seeds, but leaves have been rarely investigated.2-4 chicory leaves are usually bitter. their bitterness is highly appreciated in certain cuisines, such as in italy and in spain, greece and turkey. the use of this herb is also common in the diet of people living in the montenegrin part of the adriatic coast, especially in the boka bay region. recently, due to the growing demand, this vegetable crop has been cultivated in certain montenegro areas. the goal of this study was to estimate nutritive potential of chicory leaves growing in montenegro and to compare selected nutritional parameters of wild and cultivated plants. in montenegro, this is the first study of chicory leaves composition. experimental details about sample collection and preparation are given in supplementary material to this paper. nutritional analyses the recommended methods by the association of official analytical chemists were used for the determination of moisture, ash, lipids, dietary fiber and nitrogen content.5-9 at least three replicate determinations were made for each sample. proteins were estimated by multiplying the sample percentage nitrogen content by factor 6.25. available carbohydrate was calculated by subtracting total sum of proteins, lipids, dietary fiber and ash from 100 % sample. total energy values were calculated in kcal* multiplying the amounts of protein and carbohydrate by the factor 4 and fat by the factor 9.10 determination of mineral content each fresh plant material (500 mg) was accurately weighted (±0.1 mg) and digested using closed microwave assisted digestion (berghof mws-4, germany) with 5 cm3 of nitric acid (≥69%, puriss. p.a., sigma aldrich) and 2 cm3 of hydrogen peroxide (≥30%, for trace analyses, sigma aldrich). digested samples were filtered and transferred into volumetric flasks by adding deionised water.11,12 all analyses were performed in triplicates. working standards for measurements of major and trace elements were prepared from sigma aldrich solutions of 1000 mg dm-3 each. atomic absorption measurements were carried out on shimadzu aa-6800 (japan). for analyses of iron, manganese, zinc and copper flame aas was used and for analyses of calcium, magnesium, sodium, potassium and phosphorus icp-oes thermo icap 6300 (uk) was used. the reliability of the analytical method was evaluated by using certified standard reference material ncs dc73348 (bush branches and leaves) from the china national analysis center for iron and steel, beijing. * 1 kcal = 4184 j _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. nutritive potential of c. intybus l. leaves 1143 vitamin analyses the vitamin content (b1, b2 and b6) was determined after acid hydrolysis of the samples in autoclave. after adjusting ph to 4.5 with ch3coona solution, the samples were filtered through membrane filter and were ready for the determination of vitamin b2 and b6. the determination of vitamin b1 was carried out with the pre-column oxidation of thiamine to tiochrom with oxidizing reagent (alkaline solution of potassium ferricyanide) and filtration through membrane filter. sample analysis was performed using hplc technique under the following conditions: column – lichrospher 100 rp-18 (5 µm) lichrocart 250-4; detector-rf-535 shimadzu fluorescence hplc monitor; eluent for vitamin b1 and b2: 450 ml ch3oh+620 ml 5 mm ch3coonh4; eluent for vitamin b6: 250 ml ch3oh+770 ml 5 mm hexansulfonic acid; flow rate 0.8 ml min-1; injected volume 20 μl; column temperature 20° c; wavelengths b1: λex = 370 nm and λem = 430 nm; b2: λex = 450 nm and λem = 530 nm; b6: λex = = 286 nm and λem = 392 nm. standard solutions of vitamins b1, b2 and b6 were purchased from sigma. methanol hplc grade, ammonium-acetate and hexansulphonic acid were all purchased from merck. β-carotene was determined using extraction with acetone and hexane in volume ratio of 70:30, followed by the separation of the other colouring matter on a chromatographic column. the absorbance of samples was measured at 430 nm.13 vitamin c was analyzed by volumetric method with 2,6-dichloro-phenol-indophenol.14 in this procedure, sample was first extracted with distilled water. for vitamins, at least three replicates for each sample were made. statistics statistical difference between averages of the two groups (wild and cultivated plants) was determined using student’s t-test – two sample assuming unequal variances, and p value <0.05 was considered statistically significant. results and discussion nutrient composition the results of the nutrients’ content and estimated energy values obtained for the nine samples of cichorium intybus l. leaves are presented in table i. determined average values for dry matter were 12.42 % for the leaves of wild plants and 8.85% for the cultivated plant leaves. the total mineral content expressed as ash content was 1.36 % for the wild plants and 1.16 % for the cultivated plants. the lipid contents varied between 0.22 and 0.49 % for the wild plant leaves, while the average content in cultivated plants was 0.44 %. the lipid content results indicated that the chicory leaves are poor sources of plant lipids, which is in agreement with general observation that leafy vegetables are low fat containing food.15 the dietary fiber content in the samples varied considerably between 2.90 and 6.16 % and represented about 35 % of dry matter in average. our results showed that plants grown on different locations had different carbohydrate content, but also different protein content. the protein content was the highest in those from pricelje (2.78 %) and the lowest in those from podgor (1.63 %). _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1144 jančić et al. table i. nutritive composition and energy value of cichorium intybus l. leaves; data are expressed per 100 g fresh weight and presented as mean±sd of three independent determinations; a: significant difference was stated at p < 0.05 location content, % energy value kcal/100 g dry matter ash lipids dietary fiber proteins available carbohydrate wild plants zoganje 11.94±0.40 1.54±0.03 0.45±0.09 4.99±0.14 2.19±0.10 2.77±0.25 24 risan 14.31±0.28 1.58±0.04 0.49±0.10 4.35±0.20 1.90±0.12 5.99±0.17 36 podgor 11.83±0.73 1.39±0.02 0.35±0.05 4.11±0.15 1.63±0.08 4.35±0.22 27 tivat 11.00±0.34 1.37±0.04 0.41±0.11 3.01±0.16 1.89±0.14 4.32±0.26 29 pricelje 14.47±0.19 1.42±0.04 0.45±0.10 3.39±0.14 2.78±0.06 6.43±0.38 41 plavnica 11.46±0.72 1.20±0.05 0.33±0.14 3.27±0.14 2.75±0.06 3.90±0.14 30 pljevlja 11.93±0.34 1.05±0.05 0.22±0.07 6.16±0.19 2.19±0.08 2.31±0.25 20 average 12.42±1.39 1.36±0.19 0.39±0.09 4.18±1.11 2.19±0.44 4.30±1.52 30±7 cultivated plants komani 9.48±0.18 1.23±0.02 0.48±0.06 4.29±0.15 2.16±0.11 1.32±0.19 18 susanj 8.21±0.09 1.08±0.04 0.39±0.10 2.90±0.11 2.28±0.11 1.56±0.16 19 average 8.85±0.90a 1.16±0.11 0.44±0.06 3.60±0.98 2.22±0.08 1.44±0.14a 19±1a compared to the literature data on composition of the most-commonly consumed leafy vegetable species in montenegro e.g. spinach (spinacia oleracea) and lettuce (lactuca sativa), an average value of dry matter in analysed chicory leaves was 1.4 and 2.5 times higher, respectively.16,17 the reason is the evident difference in dietary fiber and carbohydrate content between the analysed samples and the other leafy vegetables. also, the average protein content in chicory was about 25 % higher than in lettuce.18 on the basis of the estimated nutrient composition, energy value of one portion (100 g) of fresh chicory leaves was calculated. the average value was 27 kcal/100 g, which was 30% higher than in lettuce.18 the highest and lowest energy values were 41 kcal/100 g (pricelje) and 18 kcal/100 g (komani). the difference between dry matter and carbohydrate content in the leaves of wild and cultivated plants was significant (p < 0.05). cultivated plants had 30 % lower dry matter content than wild types. carbohydrate content in the wild plants was three times higher compared to the cultivated plants. our results for basic nutritive composition of fresh chicory leaves from montenegro are in good correlation with the data obtained for cultivated chicory leaves in holland.19 minerals the results of the major mineral content obtained for nine samples of chicory leaves are presented in table ii. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. nutritive potential of c. intybus l. leaves 1145 table ii. major mineral composition of cichorium intybus l. leaves; data are calculated based on the original weight and presented as mean±sd of three independent measurements; a: significant difference was stated at p < 0.05 location content of minerals, mg/100 g k na ca mg p wild plants zoganje 445.5±5.1 49.3±2.1 223.4±3.1 32.1±1.4 48.8±6.1 risan 439.1±4.2 52.1±2.0 275.4±2.4 41.0±2.0 28.8±7.0 podgor 511.2±4.0 15.3±2.2 146.0±2.1 25.8±1.9 35.1±7.4 tivat 383.4±4.1 60.4±3.4 158.8±3.9 28.1±1.9 38.4±6.0 pricelje 391.1±3.1 31.0±3.1 281.0±1.2 41.4±4.1 31.7±5.1 plavnica 335.9±3.2 45.0±2.4 149.2±1.4 24.8±2.2 23.2±4.1 pljevlja 311.4±4.2 10.8±1.3 88.8±0.9 20.1±1.9 32.8±5.1 average 402.5±68.6 37.7±19.1 188.9±72.5 30.5±8.2 34.1±8.1 cultivated plants komani 383.1±5.2 29.1±2.2 87.8±1.8 24.2±1.8 22.8±4.1 susanj 326.5±3.0 9.3±1.9 72.0±2.1 17.3±2.1 20.4±3.2 average 354.8±40.0 19.2±14.0 79.9±11.2a 20.8±4.9 21.6±1.7 all samples had low amount of na (average 33.6 mg/100 g), but relatively high concentration of k (average 391.9 mg/100 g). variations in ca content were in the range 72.0–281.0 mg/100 g. mg content in fresh cychorium leaves was low and in the range 17.3–41.4 mg/100 g, while p content was from 20.4 mg/100 g in the samples from susanj to 48.8 mg/100 g in the samples from zoganje. k content was about 40% higher than in lettuce, while the average value of ca (164.7 mg/100 g) was similar as in spinach, but five to ten times higher than in lettuce.18–20 these results proved that fresh cychorium leaves are poor p and mg dietary sources, while ca and k are present in higher amounts, which is in good correlation with the findings for organically grown forage chicory leaves from new zealand.22 the results of trace mineral content in fresh chicory leaves are presented in table iii. the values for cu content in fresh chicory leaves were low and in the range 0.02–0.12 mg/100 g. zn average content in chicory leaves was 0.68 mg/100 g. the lowest (0.26 mg/100 g) and the highest (1.07 mg/100 g) mn contents were recorded in the leaves from komani and pricelje, respectively. the average value for fe content was 2.33 mg/100 g. the obtained value for average zn content was about seven times higher than in lettuce. also, chicory leaves had 30 % more fe, but 50 % less cu than spinach.20,21 k, mg, p, cu and fe content did not show significant differences between wild and cultivated plants. difference in ca, zn and mn content in the leaves of wild and cultivated plants was significant (p < 0.05). the wild plants were about _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1146 jančić et al. 2.5 times richer in ca, 4 times richer in zn and 2 times richer in mn than the cultivated ones. although it is known that plant mineral content varies with the environmental and maturity conditions, these results indicate that wild growing plants can have more beneficial mineral profile than cultivated ones and these findings should be further investigated. table iii. content of cu, zn, mn and fe in cichorium intybus l. leaves; data are calculated based on the original weight and presented as mean±sd of three independent measurements; a: significant difference was stated at p < 0.05 location content of minerals, mg/100 g cu zn mn fe wild plants zoganje 0.12±0.02 1.14±0.02 0.56±0.01 1.64±0.07 risan 0.02±0.02 0.97±0.01 0.69±0.02 2.94±0.08 podgor 0.08±0.02 0.82±0.02 0.53±0.01 1.71±0.06 tivat 0.09±0.02 0.77±0.01 0.70±0.02 2.21±0.07 pricelje 0.08±0.01 0.96±0.02 1.07±0.03 4.51±0.09 plavnica 0.10±0.01 0.63±0.02 0.41±0.01 1.99±0.05 pljevlja 0.04±0.02 0.38±0.01 0.33±0.01 2.60±0.07 average 0.08±0.03 0.81±0.25 0.61±0.24 2.51±1.00 cultivated plants komani 0.05±0.02 0.22±0.01 0.26±0.01 1.60±0.06 susanj 0.03±0.02 0.18±0.01 0.36±0.01 1.80±0.07 average 0.04±0.01 0.20±0.03a 0.31±0.07a 1.70±0.14 vitamins the results for the vitamins b1, b2, b6, β-carotene and vitamin c content in fresh chicory leaves are given in table iv. the mean value of vitamin b1 content in fresh chicory leaves was 138.6 μg/100 g, while values for vitamin b2 content were in the range 85.1–181.6 μg/100 g, and for vitamin b6 137.0–252.6 μg/100 g. the values of β-carotene content in chicory leaves were 1.9-3.8 mg 100 g-1 and for vitamin c varied from 3.2 to 6.0 mg/100 g. our results for vitamin b6 content were about twenty times higher than results published for spinach and even 100 times higher than for lettuce.23 content of vitamin c in chicory leaves was about forty times higher than in lettuce, but compared to spinach four times lower.23 interestingly, the content of analyzed vitamins was not different between wild and greenhouse produced plants, while the total carbohydrate and mineral content (calcium, zinc and manganese) were significantly influenced by the production method. some health workers and consumers prefer wild plants than cultivated ones when given a choice, due to the notion that intensive plant production could _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. nutritive potential of c. intybus l. leaves 1147 result in lower content of certain valuable components. several investigations confirmed this hypothesis when polyphenols and essential oils were investigated.24,25 our results proved the hypothesis that wild chicory leaves have better nutritional profile of selected nutrients than cultivated ones. table iv. content of the vitamins b1, b2, b6 and β-carotene and vitamin c in chicory leaves; data are expressed based on the original weight and presented as mean±sd of three independent measurements location content of vitamins, μg / 100 g b1 b2 b6 β-carotene c wild plants zoganje 82.2±0.3 85.1±1.5 140.8±2.4 2.7±0.1 6.0±0.3 risan 137.8±1.2 118.5±0.7 161.6±1.4 3.6±0.2 4.6±0.4 podgor 126.7±1.6 99.4±2.2 161.6±1.3 2.7±0.1 3.5±0.3 tivat 215.8±1.7 115.7±0.9 148.9±1.3 3.8±0.1 3.5±0.3 pricelje 81.5±2.3 83.6±1.2 137.0±1.4 3.5±0.1 3.9±0.4 plavnica 160.3±2.9 181.6±1.1 252.6±1.1 1.9±0.1 3.2±0.1 pljevlja 129.4±1.4 129.1±1.5 178.7±2.5 2.6±0.2 3.2±0.2 average 133.4±46.4 116.1±33.6 168.7±39.6 3.0±0.7 4.0±1.0 cultivated plants komani 106.4±3.4 120.4±0.9 160.3±2.4 2.2±0.1 3.6±0.1 susanj 206.9±8.1 104.4±1.1 165.3±1.0 2.5±0.1 3.3±0.1 average 156.7±71.1 112.4±11.3 162.8±3.5 2.4±0.2 3.5±0.2 table v shows the results obtained for the content of major and trace minerals, as well as vitamins in one portion (100 g of fresh chicory leaves) compared to the reference daily intake values (rdi). table v. content of major and trace minerals, and vitamins in 100 g fresh leaves compared to reference daily intake (rdi10) major element rdi / % trace element rdi / % vitamin rdi / % k 19.2 zn 6.8 b1 12.5 ca 20.6 cu 8.0 b2 8.2 mg 7.5 mn 27.5 b6 11.9 p 4.4 fe 16.6 aa 58.8 c 4.8 acalculated from beta-carotene (1 μg of vitamin a = 6 μg of beta-carotene) considering the amount of analysed vitamins and minerals in one edible portion of fresh chicory leaves (100 g) and expressed as percentage of rdi values, it could be concluded that k, ca, mn, fe, and vitamin a (calculated from β-carotene) contents were >15% of rdi, which is the requirement for foods to carry a nutritional statement “source of”.26 _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. 1148 jančić et al. conclusion based on the results of our analyses it can be concluded that chicory leaves, which have been used as food in montenegro for centuries, are valuable sources of several nutrients, minerals and vitamins and therefore could be regarded as healthy foods in well-balanced diets. chicory leaves are especially rich in carbohydrates, ca, mn, fe and vitamins b1, b2 and b6. they could also be used in low caloric diets because of their low energy value. better knowledge of traditional foods’ nutritive potential can serve as a basis for their more intensive inclusion in modern dietary patterns. the case of chicory plant in montenegro also proves that revitalization of selected wild plants, either by harvesting them from nature or by cultivating process, is the best way for preserving traditional foods in certain geographical regions. supplementary material details about sample collection and preparation are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. и з в о д хемијски састав и нутритивни потенцијал лишћа cichorium intybus l. из црне горе дејан јанчић1, вања тодоровић2, зорица басић3 и слађана шобајић2 1доо центар за екотоксиколошка испитивања подгорица, црна гора, 2универзитет у београду, фармацеутски факултет, катедра за броматологију, београд и 3војно–медицинска академија, институт за хигијену, београд лишће биљке цикорије (cichorium intybus l.) вековима се користило као поврће у салатама, сосевима и другим врстама предјела и јела у црној гори и другим медитеранским земљама. лишће самоникле и гајене цикорије са различитих локација у црноj гори анализирано је у погледу састава нутријената, макрои микроелемената и витаминa коришћењем стандaрдних метода. резултати испитивања су показали да je лишће цикорије богато дијеталним влакнима и минералима и да има ниску енергетску вредност. такође, оно је значајан извор корисних нутријента као што су калијум, калцијум, манган, гвожђе и витамин а, са средњом вредности садржаја од 391,9, 164,7, 0,55, 2,33 и 0,47 mg/100 g свежег лишћа, редом. самоникле биљке су биле богатије од гајених у погледу садржаја угљених хидрата, калцијума и магнезијума. порекло лишћа цикорије имало је значајан утицај на већину анализираних параметара. (примљено 13. марта, ревидирано 18. маја, прихваћено 19. маја 2016) references 1. european medicines agency, assessment report on cichorium intybus l., radix, ema/ /hmpc/113041/2010, 2013 2. l. sinkovič, l. demšar, d. žnidarčič, r. vidrih, j. hribar, d. treutter, food chem. 166 (2015) 507 3. g. jan, m. kahan, m. ahmad, z. iqbal, a. afzal, m. afzal, g. m. shah, a. majid, m. fiaz, m. zafar, a. waheed, f. gul, j. med. plants res. 5 (2011) 2452 4. g. wen-ying, l. jin-gui, j. anim. plant sci. 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chem. soc. 18 (2007) 990 26. regulation (ec) no 1924/2006 of the european parliament and of the council of 20 december 2006 on nutrition and health claims made on foods, official journal of the european communities, l404, pp. 9–25. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2016 scs. all rights reserved. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice volume 81 (2016) 1 2016 list of referees editorial board of the journal is grateful to the following referees for reviewing the manuscripts during 2016: hebat-allah abbas, photochemistry department, national research centre, dokki, cairo, egypt syed nasir abbas bukhari, faculty of pharmacy, university kebangsaan malaysia, bangi, malaysia ameen abu-hashem, chemical industries division, national research centre, dokki giza, egypt borivoj adnađević, faculty of physical chemistry university of belgrade, serbia mara aleksić, faculty of pharmacy, university of belgrade, serbia davor antanasijević, faculty of technology and metallurgy, university of belgrade, serbia dušan antonović, faculty of technology and metallurgy, university of belgrade, serbia jelena arsеnijević, faculty of pharmacy, department of pharmacognosy university of belgrade, serbia ali reza ashrafi, university of kashan, iran mahmut aslani, institue of nuclear sciences, nuclear technology department, ege university, izmir, turkey elena azzini, crea-nut, food and nutrition research centre, rome, italy biljana babić, institute of nuclear sciences vinča, belgrade, serbia jelana bajat, faculty of technology and metallurgy, university of belgrade, serbia milica balaban, faculty of science, university of banja luka, bosnia and herzegovina rada baošić, department of analytical chemistry, faculty of chemistry, university of belgrade, serbia giancarlo barbieri, universita degli studi di napoli federico ii, department of agricultural sciences, naples, italy jiri barek, charles university, prague, czech republic zvezdana baščarević, institute for multidisciplinary research, university of belgrade, serbia fisseha andualem bezza, universiteit van pretoria, department of chemical engineering, pretoria, south africa filip bihelović, faculty of chemistry, university of belgrade, serbia polina blagojević, faculty of science and mathematics, department of chemistry, university of niš, serbia stevan blagojević, institute of general and physical chemistry, university of belgrade, serbia jovana bogojeski, department of chemistry, faculty of science, university of kragujevac, serbia tomislav bolanča, faculty of chemical engineering and technology, university of zagreb, croatia anita bosak, the institute for medical research and occupational health, zagreb, croatia nataša božić, institute of chemistry, technology and metallurgy, university of belgrade, serbia ilija brčeski, faculty of chemistry, university of belgrade, serbia 2 journal of the serbian chemical society sudhakar bhusare, department of chemistry, dnyanopasak college, parbhani, ms, india sulekh chandra, department of chemistry, zakir husain delhi college, university of delhi, jlnmarg, new delhi, india cheol-hong cheon, department of chemistry, korea university, anam-ro, seongbuk-gu, seoul, republic of korea manmohan chhibber, school of chemistry and biochemistry, thapar university, patiala, india otilia costisor, institute of chemistry of the romanian academy, timisoara, romania aurelien crochet, fribourg center for nanomaterials, frimat machine pool responsible, university of fribourg, germany dragana čičkarić-živojinović, department of analytical chemistry, faculty of technology and metallurgy, university of belgrade, serbia božidar čobeljić, faculty of chemistry, university of belgrade, serbia aleksandar ćosović, institute for technology of nuclear and other mineral raw materials, belgrade, serbia danica ćujić, institute for the application of nuclear energy, university of belgrade, serbia aleksandra daković, institute for technology of nuclear and other mineral raw materials, belgrade, serbia ivan damljanović, department of chemistry, faculty of science, university of kragujevac, serbia ljiljana damjanović, faculty of physical chemistry, university of belgrade, serbia graeme day, university of southampton, uk christopher davies, csiro agriculture flagship, brisbane, brisbane, australia jasmina dimitrić-marković, faculty for physical chemustry, university of belgrade, serbia biljana dojnov, institute of chemistry, technology and metallurgy, university of belgrade, serbia tomislav došlić, faculty of civil engineering, university of zagreb, croatia snežana dragović, institute for the application of nuclear energy, university of belgrade, belgrade, serbia milica drobac, faculty of pharmacy, university of belgrade, serbia som dubey, department of chemistry, gitam institute of technology, gitam university, india branko dunjić, faculty of technology and metallurgy, university of belgrade, serbia krystyna dzierzbicka, department of organic chemistry, politechnika gdanska, gdansk, poland dariusz dziki, university of life sciences in lublin, lublin, poland stojan đokić, department of chemical and materials engineering, university of alberta, edmonton, canada veljko r. đokić, ic of faculty of technology and metallurgy, university of belgrade, serbia svetlana đogo-mračević, department of analytical chemistry, faculty of pharmacy, university of belgrade, serbia jasna đonlagić, faculty of technology and metallurgy, university of belgrade, serbia miloš i. đuran, department of chemistry, faculty of science, university of kragujevac, serbia predrag đurđević, department of chemistry, faculty of science, university of kragujevac, serbia rada đurović-pejčev, institute of pesticides and environmental protection, belgrade, serbia enis džunuzović, faculty of technology and metallurgy, university of belgrade, serbia jasna džunuzović, institute of chemistry, technology and metallurgy, university of belgrade, serbia reda el-agmy, faculty of science, deparment of physics, cairo, egypt mihajlo etinski, faculty of physical chemistry, university of belgrade, serbia oleg farat, department of chemistry, m.v. lomonosov moscow state university, russian federation 2016 list of referees 3 nahed fathi, national research centre, cairo, egypt zorana ferjančić, faculty of chemistry, university of belgrade, serbia pedro fernandes, universidade de lisboa, lisbon, portugal zvjezdana findrik, faculty of chemical engineering and technology, university of zagreb, croatia melike firlak, lancaster university, uk boris furtula, department of chemistry, faculty of science, university of kragujevac, serbia gordana gajić, institute for biological research „sinisa stankovic”, university of belgrade, serbia subramaniapillai ganesan, school of chemical and biotechnology, sastra university, thanjavur, india ramesh gardas, department of chemistry, indian institute of technology madras, chennai, india sandra glišić, faculty of technology and metallurgy, university of belgrade, serbia gordana gojgić-cvijović, institute of chemistry, technology and metallurgy, university of belgrade, serbia snežana gojković, faculty of technology and metallurgy, university of belgrade, serbia mostafa ghorab, pharmacognosy department, college of pharmacy, king saud university, riyadh, saudi arabia william burgess grant, sunlight, nutrition and health research center, san francisco, united states sanja grgurić šipka, faculty of chemistry, university of belgrade. serbia maja gruden, faculty of chemistry, university of belgrade. serbia svetlana grujić, faculty of technology and metallurgy, university of belgrade, serbia ivan gutman, department of chemistry, faculty of science, university of kragujevac, serbia valéria guzsvány, faculty of sciences, department of chemistry, biochemistry and environmental protection, university of novi sad, geir martin haarberg, department of materials technology, norwegian university of science and technology, trondheim, norway abraha habtemariam, department of chemistry, university of warwick, gibbet hill, coventry, uk muhammad hanif, school of chemical sciences, university of auckland, new zealand ivanka holclajtner-antunović, faculty of physical chemistry, university of belgrade, serbia bob howell, central michigan university, mount pleasant, mi, usa zhang hui wen, institute of applied ecology, chinese academy of sciences, shenyang, china aziz ibragimov, institute of general and inorganic chemistry of uzbekistan academy of sciences, tashkent, uzbekistan ljubiša ignjatović, faculty of physical chemistry, university of belgrade, serbia nenad ignjatović, institute of technical sciences of the serbian academy of sciences and arts, belgrade, serbia aleksandar ilić, department of mathematics and informatics, faculty of science, university of niš, serbia žarko ilin, faculty of agriculture, university of novi sad, serbia salmah binti ismail, university of malaya, department of biohealth science, kuala lumpur, malaysia violeta ivanova-petropulos, faculty of agriculture, university goce delčev, štip, macedonia lidija jakobek, department of applied chemistry and ecology, josip juraj strossmayer university of osijek, croatia 4 journal of the serbian chemical society radmila jančić heinemann, faculty of technology and metallurgy, university of belgrade, serbia christoph janiak, institut für anorganische chemie und strukturchemie i, heinrich-heineuniversität düsseldorf, germany koen janssens, university of antwerp, belgium milan jokanović, faculty of medicine, university of niš, serbia milan joksović, department of chemistry, university of kragujevac, serbia ivan juranić, faculty of chemistry, university of belgrade, serbia zorica juranić, institute of oncology and radiology of serbia, belgrade, serbia melina kalagasidis-krušić, faculty of technology and metallurgy, university of belgrade, serbia ivanka karadžić, department of chemistry, school of medicine, university of belgrade, serbia i̇brahim karatas, faculty of science and art, chemistry department, university of selcuk, konya, turkey mirjana kijevčanin, faculty of technology and metallurgy, university of belgrade, serbia bijen kivçak, faculty of nursing, ege universitesi, izmir, turkey olivera klisurić, faculty of sciences, university of novi sad, serbia jakob kljun, faculty of chemistry and chemical technology, university of ljubljana, slovenia. ufuk kolak, bezmialem vakif universitesi, department of analytical chemistry, istanbul, turkey stoimir kolarević, faculty of biology, university of belgrade, serbia ju chang kim, department of chemistry, pukyong national university, pusan, south korea zorica knežević-jugović, faculty of technology and metallurgy, university of belgrade, serbia mirjana kostić, faculty of technology and metallurgy, university of belgrade, serbia milan kragović, institute for technology of nuclear and other raw materials, belgrade, serbia sandra kraljević-pavelić, department of biotechnology, university of rijeka, croatia marko krstić, faculty of pharmacy, university of belgrade, serbia tibor kurtán, university of debrecen, debrecen, hungary mila laušević, faculty of technology and metallurgy, university of belgrade, serbia hao-ran liu, college of chemistry and chemical engineering, hu’nan university, changsha, china jingyao liu, institute of theoretical chemistry, state key laboratory of theoretical and computational chemistry, jilin university, china magdalena maj-żurawska, faculty of chemistry,, university of warsaw, poland anđelija malenović, department of drug analysis, faculty of pharmacy, university of belgrade serbia ljuba mandić, department of biochemistry, faculty of chemistry, university of belgrade, serbia dragan manojlović, faculty of chemistry, university of belgrade, serbia nedeljko manojlović, department of pharmacy, faculty of science university of kragujevac, serbia huiming mao, department of central laboratory, shanghai east hospital, tongji university, shanghai, china xiangchao mao, ocean university of china, qingdao, shandong, china silvija markic, university og bremen, germany josé marco-contelles, laboratorio de química médica y computacional (iqog, csic), madrid, spain aleksandar marinković, faculty of technology and metallurgy, university of belgrade, serbia ana marjanović jeromela, institute of field and vegetable crops, novi sad, serbia smilja marković, institute of technical sciences of the serbian academy of sciences and arts, belgrade, serbia 2016 list of referees 5 zoran marković, state university of novi pazar, serbia violeta marković, department of chemistry, faculty of science, university of kragujevac, serbia joanna matysiak, department of chemistry, university of life sciences, lublin, poland sanja martinović, institute of chemistry, technology and metallurgy, university of belgrade, serbia mohamed néjib èjib marzouki, university of carthage, laboratory of protein engineering and bioactive molecules (lip-mb), tunis, tunisia zoran matović, department of chemistry, faculty of science, university of kragujevac, serbia nevena mihailović, institute for the application of nuclear energy, university of belgrade, serbia dušan mijin, faculty of technology and metallurgy, university of belgrade, serbia jelena miladinović, faculty of technology and metallurgy, university of belgrade, serbia nikola milašinović, department of forensics, academy of criminalistic and police studies, belgrade, serbia miloš milčić, faculty of chemistry, university of belgrade, serbia marina milenković, faculty of pharmacy, department of microbiology and immunology, university of belgrade, serbia slobodan milonjić, vinča institute of nuclear sciences, university of belgrade, serbia slobodan milosavljević, faculty of chemistry, university of belgrade, serbia tatjana mitrović, department of biology and ecology, faculty of sciences, university of niš, serbia hassan moghanian, department of organic chemistry, islamic azad university dezful branch, dezful, iran jelena molnar, faculty of sciences, university of novi sad, serbia shovan mondal, department of chemistry, visva-bharati university, santiniketan, india monica magureanu, national institute for lasers, plasma and radiation physics, magurele bucharest, romania ibrahim nassar, organic chemistry, ain-shams university, cairo, egypt olgica nedić, institute for the application of nuclear energy, university of belgrade, serbia nikola nikačević, faculty of technology and metallurgy, university of belgrade, serbia jasmina nikolić, faculty of technology and metallurgy, university of belgrade, serbia marija nikolić, faculty of technology and metallurgy, university of belgrade, serbia nagatoshi nishiwaki, school of environmental science and engineering, kochi university of technology, miyanokuchi, tosayamada, kami, kochi, japan jasmina novaković, apotex inc, toronto, ontario, canada slađana novaković, institute of nuclear sciences, laboratory of theoretical physics and condensed matter physics, university of belgrade, serbia rodica olar, department of inorganic chemistry, faculty of chemistry, university of bucharest, romania antonije onjia, vinča institute of nuclear sciences, university of belgrade, serbia feyyaz onur, faculty of pharmacy, ankara university, turkey dejan opsenica, faculty of chemistry, university of belgrade, serbia igor opsenica, faculty of chemistry, university of belgrade, serbia antonella pasqualone, universita degli studi di bari, dipartimento di scienze del suolo, bari, italy igor pašti, faculty of physical chemistry, university of belgrade, serbia manish patel, department of chemistry, sardar patel university, vallabh vidyanagar gujarat, india vladimir pavlović, faculty of agriculture, university of belgrade, serbia 6 journal of the serbian chemical society boris pejin, department of life science, institute for multidisciplinary research, university of belgrade, serbia ana penezić, institute for the application of nuclear energy, university of belgrade, serbia franc perdih, faculty of chemistry and chemical technology, ljubljana, slovenia josé luis pérez pavón, facultad de química, salamanca, spain marija pergal, institute of chemistry, technology and metallurgy, university of belgrade, serbia aleksandra perić-gujić, faculty of technology and metallurgy, university of belgrade, serbia vsevolod peshkov, department of chemistry, university of leuven (ku leuven), leuven, belgium milena petković, faculty of physical chemistry university of belgrade, serbia mirjana petronijević, department of chemistry, biochemistry and environment protection, faculty of sciences, university of novi sad, serbia slobodan petrović, faculty of technology and metallurgy, university of belgrade, serbia rada petrović, faculty of technology and metallurgy, university of belgrade, serbia vladimir petrović, faculty of science, university of kragujevac, serbia zdenka peršin, institute of engineering materials and design, faculty of mechanical engineering, university of maribor, slovenia andrej pevec, faculty of chemistry and chemical technology, university of ljubljana, slovenia vesna poleksić, faculty of agriculture, university of belgrade, serbia peter politzer, department of chemistry, university of new orleans, usa natalija polović, department of biochemistry, faculty of chemistry-university of belgrade, serbia jelena poljarević, faculty of chemistry, university of belgrade, serbia margarita popova, institute of organic chemistry, bulgarian academy of sciences, sofia, bulgaria daniela popović, faculty of technology and metallurgy, university of belgrade, serbia velimir popsavin, faculty of science, university of novi sad, serbia radivoje prodanović, department of biochemistry, faculty of chemistry, university of belgrade, serbia marion prudent, inra institut national de la recherche agronomique, paris, france surendra punganuru, biomedical sciences, school of pharmacy, texas tech university health sciences center, usa vladislav rac, faculty of agriculture, university of belgrade, zemun, serbia bojan radak, vinča institute of nuclear sciences, university of belgrade, serbia dušanka radanović, institute of chemistry, technology and metallurgy, university of belgrade, serbia mirjana radanović, faculty of sciences, university of novi sad, , serbia slavko radenković, department of chemistry, faculty of science, university of kragujevac, serbia dragan radivojević, faculty of agriculture, institute for horticulture, university of belgrade, serbia marija radoičić, vinča institute of nuclear sciences, university of belgrade, serbia serbia vesna radojević, faculty of technology and metallurgy, university of belgrade, serbia nataša radosavljević-stevanović, national criminalistic-technical centre, ministry of internal affairs of the republic of serbia, belgrade, serbia blaga radovanović, faculty of science and mathematics, university of niš, serbia ivona radović, faculty of technology and metallurgy, university of belgrade, serbia niko radulović, department of chemistry, faculty of science and mathematics, university of niš, serbia 2016 list of referees 7 beatriz ramos-solano, la fundacion universitaria san pablo (ceu), facultad de farmacia, madrid, spain vladana rajaković-ognjanović, faculty of civil engineering, university of belgrade, serbia nevenka rajić, faculty of technology and metallurgy, university of belgrade, serbia miloš rajković, faculty of agriculture, university of belgrade, serbia dejan raković, faculty of electrical engineering , university of belgrade, serbia slavica ražić, faculty of pharmacy university of belgrade, serbia sreedhar reddy, institute of organic chemistry, westfälischen wilhelms-universität, münster, germany moamen refat, department of chemistry, faculty of science, port said university, port said, egypt dragana robajac, institute for the application of nuclear energy, university of belgrade, serbia marko rodić, faculty of sciences, university of novi sad, serbia goran roglić, faculty of chemistry, university of belgrade, serbia zbigniew rozwadowski, department of inorganic and analytical chemistry, west pomeranian university of technology, szczecin, poland sridhar sahu, department of applied physics, ndian school of mines, dhanbad, india rachel sammons, university of birmingham, school of dentistry, birmingham, uk vladimir savić, faculty of pharmacy university of belgrade, serbia nagaiyan sekar, tinctorial chemistry group, department of dyestuff technology, institute of chemical technology, matunga, mumbai, india branislav simonović, institute of general and physical chemistry, university of belgrade, serbia liming shao, shanghai center for drug discovery & development, pudong, shanghai, china saira shahzadi, department of chemistry, quaid-i-azam university, islamabad, pakistan abeer nasr shehata, national research centre, department of biochemistry, cairo, egypt devanand shinde, department of chemical technology, dr. babasaheb ambedkar marathwada university, aurangabad, india. ivan spasojević, institute for multidisciplinary research, university of belgrade, serbia pavle spasojević, innovation centre of faculty of technology and metallurgy, serbia vuk spasojević, vinča institute of nuclear sciences, university of belgrade, serbia vladimir v. srdić, faculty of technology university of novi sad, serbia miroslav stanković, institute of chemistry, technology and metallurgy, department of catalysis and chemical engineering, university of belgrade, serbia dragomir stanisavljev, faculty of physical chemistry, university of belgrade, serbia mirjana stojanović, institute for technology of nuclear and other mineral raw matherials, belgrade, serbia ksenija stojanović, faculty of chemistry, university of belgrade, serbia biljana škrbić, faculty of technology, university of novi sad, serbia biljana šmit, faculty of science, university of kragujevac, serbia tatjana šolević knudsen, institute of chemistry, technology and metallurgy, university of belgrade, serbia maja šumar ristović, faculty of chemistry, university of belgrade, serbia lubomir švorc, faculty of chemical and food technology, slovak university of technology, bratislava, slovak republic carmen terán, departamento de química organica and instituto de investigaci on biom edica (ibi), universidade de vigo, spain anja terzić, institute for testing of materials ims institute, belgrade, serbia 8 journal of the serbian chemical society biljana tomasević, faculty of chemistry, university of belgrade, serbia jelena tričković, department of chemistry, biochemistry and environmental science, faculty of sciences, university of novi sad, serbia đorđe veljović, faculty of technology and metallurgy, university of belgrade, serbia manuel eugenio vázquez, department of organic chemistry, universidad de santiago de compostela, spain vesna vitnik, department of chemistry, institute of chemistry, technology and metallurgy, university of belgrade, serbia željko vitnik, department of chemistry, institute of chemistry, technology and metallurgy, university of belgrade, serbia vesna vodnik, vinča institute of nuclear sciences, belgrade, serbia ljljana vojinović ješić, faculty of sciences, university of novi sad, serbia marija vukčević, faculty of technology and metallurgy, university of belgrade, serbia vesna vukčević, faculty of agriculture, university of belgrade, serbia ivan vučković, faculty of chemistry, university of belgrade, serbia jelena vuksanović, faculty of technology and metallurgy, university of belgrade, serbia benjamin ward, school of chemistry, cardiff university, cardiff, uk klaus peter wilbois, research institute of organic agriculture, frankfurt am main, germany christian whitman, university of texas at austin, division of medicinal chemistry, austin, united states erdal yabalak, faculty of arts and science, mersin university, mersin, turkey constatinos zacharis, department of chemistry, aristotle university of thessaloniki, greece george a. zachariadis, aristotle university of thessaloniki, greece jing jing zhang, department of chemistry, university of illinois at urbana, illinois, usa jinsong zhang, anhui agricultural university, state key laboratory of tea plant biology and utilization, hefei, china snežana zarić, faculty of chemistry, university of belgrade, serbia goran zdunić, institute for adriatic crops and karst reclamation, split, croatia zongping zheng, state key laboratory of food science and technology, jiangnan university, wuxi, china anastasia-stella zotou, department of chemistry, aristotle university of thessaloniki, greece matija zlatar, institute of chemistry, technology and metallurgy, university of belgrade, serbia branka žarković, faculty of agriculture, university of belgrade, serbia slađana žilić, maize research institute, department of food technology and biochemistry, belgrade, serbia ljiljana živković, vinča institute of nuclear sciences, belgrade, serbia marija živković, faculty of science, university of kragujevac, 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis and biological evaluation of (3-aryl-1,2-oxazol-5-yl)methyl 6-fluoro-4-oxo-4h-chromene-2-carboxylates as antioxidant and antimicrobial agents} j. serb. chem. soc. 82 (1) 1–12 (2017) udc 547.786/.787+547.814.1: jscs–4942 615.279–188:615.28–188 original scientific paper 1 synthesis and biological evaluation of (3-arylisoxazol-5-yl)methyl 6-fluoro-4-oxo-4h-chro mene-2-carboxylates as antioxidant and antimicrobial agents kumaraswamy battula1, sirassu narsimha1, vasudeva reddy nagavelli1* and mutheneni srinivasa rao2 1department of chemistry, kakatiya university, warangal-506009, telangana, india and 2chemical biology laboratory, indian institute of chemical technology, hyderabad-500007, india (received 22 december 2015, revised 6 september, accepted 27 september 2016) abstract: a series of novel (3-arylisoxazol-5-yl)methyl 6-fluoro-4-oxo-4h -chromene-2-carboxylate derivatives (c1–c12) were synthesized by the cu(i) -catalyzed reaction of in situ generated nitrile oxides with prop-2-ynyl 6-fluoro-4-oxo-4h-chromene-2-carboxylate in good yields and their antioxidant and antimicrobial activities were investigated. among all the synthesized compounds, c1 (ic50: 16.43±0.57 µm) and c12 (ic50:15.98±0.72 µm) registered good antioxidant activity as compared to the standard drug trolox. compounds c1, c3 and c6 registered very good inhibition against all the tested gram-positive and gram-negative bacterial strains with mic values ranging from 9.375 to 37.5 µg ml-1. compounds c7–c11 registered good inhibition against bacillus subtilis and staphylococcus aureus with mic values ranging from 18.75 to 37.5 µg ml-1. compounds c10 and c11 against pseudomonas aeroginosa showed more prominent activity than the standard drug penicillin (mic: 12.5 µg ml-1) with an mic value of 9.375 µg ml-1 (≈1.33-fold more potent than penicillin). compounds c7–c9 registered good to moderate antifungal activity against the four tested fungal strains with mic values ranging from 18.75 to 37.5 µg ml-1. keywords: isoxazole; chromene; antioxidant; antimicrobial activity. introduction isoxazole and its derivatives have attracted much awareness because of their unique structure and applications.1 the isoxazole ring system is a five-membered heterocyclic ring structure composed of nitrogen and oxygen atoms at the 1,2 positions and is used in the synthesis of pharmaceuticals.2,3 the isoxazole moiety is a versatile lead molecule in pharmaceutical development and has a wide range * corresponding author. e-mail: vasujac3@gmail.com doi: 10.2298/jsc151222088b 2 battula et al. of biological activities. in the past few years, the therapeutic interest of isoxazole derivatives in the pharmaceutical and medicinal fields has been given great attention by medicinal chemist.4,5 a literature survey revealed that isoxazole derivatives are well known to exhibit antibacterial,6 gabaa antagonist,7 anticancer,8 antidiabetic9 and anti-hiv activities.10 the synthesis of isoxazole derivatives is obviously an important assignment in modern medicinal chemistry research. isoxazole is the basic moiety for several drugs, such as zonisamide (z, an anti-convulsant), leflunomide (l, a disease-modifying antirheumatic drug, dmard) and valdecoxib (v, a cox-2 inhibitor), fig. 1. although a number of synthetic methods are available,11 the copper(i)-catalyzed union of terminal alkynes and oximes to give 3,5-disubstituted isoxazole exhibits a remarkably broad scope and exquisite selectivity.12 in recent years, extensive studies have been focused on isoxazole derivatives because of their diverse chemical reactivity, accessibility and wide range of biological activities. fig. 1. structures of isoxazole-congaing drugs. chromone and its derivatives are reported to be physiologically and pharmacologically active and find applications in the treatment of several diseases. chromone derivatives are a broad class of chemical compounds with many important pharmacological properties.13,14 substituted chromone derivatives play a significant role in the medical field with many pharmacological activities, such as anti -hiv,15 antimicrobial,16 anticancer,17,18 antiviral,19 antioxidant,20 cytotoxic activities21 and anti-inflammatory activity.22 based on the above considerations and in continuation of ongoing research on biologically potent azole derivatives,23–31 herein, the synthesis of (3-arylisoxazol-5-yl)methyl 6-fluoro-4-oxo-4h-chromene2-carboxylate hybrids and their antioxidant and antimicrobial activities are reported. experimental all the reagents and solvents were purchased from sigma–aldrich or s.d. fine chemicals and used without further purification. thin-layer chromatography (tlc) was performed using merck silica gel 60 f254 pre-coated plates (0.25 mm) and silica gel (particle size 60–120 mesh) was used for column chromatography. melting points were determined using a cintex apparatus and are uncorrected. ftir spectra were recorded using a bruker spectrometer and are reported on the frequency of absorption (cm-1). elemental analysis was performed using a chromene-derived isoxazoles as antioxidant and antimicrobial agents 3 perkin elmer 2400 chn elemental analyzer. the 1h-nmr spectra were recorded on a varian gemini 400 mhz spectrometer and the 13c-nmr spectra on a bruker 100 mhz spectrometer. cdcl3 was used as the solvent. the 1h-nmr spectra are reported relative to me4si (δ 0.0 ppm). coupling constants (j) values are presented in hz and spin multiples are given as s (singlet), d (doublet), t (triplet), dd (doublet of doublets) and m (multiplet). the mass spectral analysis was recorded on a bruker hct mass spectrometer using the electrospray ionization mass spectrometry (esi-ms) technique. analytical and spectral data of the synthesized compounds are given in supplementary material to this paper. synthesis of ethyl 6-fluoro-4-oxo-4h-chromene-2-carboxylate (2) to a stirred solution of 1-(5-fluoro-2-hydroxyphenyl)ethanone (10 g, 64.93 mmol, 1 eq) and diethyl oxalate (194.79 mmol, 3 eq) in ethanol (200 ml) was added na metal (389.58 mmol, 6 eq) and the reaction mixture was refluxed under a nitrogen atmosphere for 4 h. after completion of the reaction (tlc), the reaction mixture was cooled, 6 m hci (100 ml) was added and the product was extracted with ch2ci2 (3×200 ml). the combined organic layer was washed with brine, dried over anhydrous na2so4 and evaporated under reduced pressure to afford the crude compound. recrystallization of the crude compound from ethyl acetate and diethyl ether afforded compound 2 (13.5 g, 88 %) as a light yellow solid. synthesis of 6-fluoro-4-oxo-4h-chromene-2-carboxylic acid (3) ethyl 6-fluoro-4-oxo-4h-chromene-2-carboxylate (10 g, 42.37 mmol) was dissolved in 50 % aqueous naoh solution and stirred at room temperature for 5 h. after completion of the reaction (tlc) the reaction mixture was neutralized with dilute hcl and extracted with ethyl acetate (3×50 ml). the combined organic layer was washed with brine, dried over anhydrous na2so4 and evaporated under reduced pressure to afford compound 3 (6.34 g, 72 %) as a light yellow powder. synthesis of prop-2-ynyl 6-fluoro-4-oxo-4h-chromene-2-carboxylate (4) to a stirred solution of 6-fluoro-4-oxo-4h-chromene-2-carboxylic acid (3) (5 g, 24.03 mmol) in dmf (50 ml) was added t-buok (48.06 mmol, 2 eq) portion-wise over a 10 min period. then, propargyl bromide (31.25 mmol, 1.3 eq) was added to the reaction mixture and stirred at room temperature for 4 h. the progress of the reaction was monitored by tlc. after the completion of the reaction, ice-cold water (100 ml) was added to the reaction mixture and extracted with ethyl acetate (3×50 ml). the combined organic layer was washed with brine, dried over na2so4 and evaporated under reduced pressure to afford compound 4 (4.73 g, 80 % yield) as a yellow solid. typical experimental procedure for synthesis of 3,5-disubstituted isoxazoles (c1–c12) as exemplified by the reaction of prop-2-ynyl 6-fluoro-4-oxo-4h-chromene-2-carboxylate and nicotinaldehyde nicotinaldehyde (500 mg, 4.67 mmol) was added to a solution of hydroxylamine hydrochloride (322 mg, 5.14 mmol) in 10 ml of 1:1 t-buoh:h2o. to this was added naoh (205 mg, 5.14 mmol), and after stirring for 30 min at ambient temperature, tlc analysis indicated that the oxime formation was complete. chloramine-t trihydrate (1.47 g, 5.14 mmol) was added in small portions over 10 min, followed by cui (44 mg, 0.233 mmol). compound 4 (1.26 g, 5.14 mmol) was added, the ph was adjusted to 6 by the addition of a few drops of 1 m naoh, and stirring was continued for a further 8 h. the reaction mixture was poured into cold water (50 ml), and 5 ml of dilute nh4oh was added to remove all copper salts. isox4 battula et al. azole c2 was collected by filtration, redissolved, and passed through a short plug of silica gel (ethyl acetate: hexanes 1:6, rf = 0.5) affording 3.6 g (72 %) of [3-(pyridin-3-yl)-isoxazol -5-yl]methyl 6-fluoro-4-oxo-4h-chromene-2-carboxylate as an off-white solid. antioxidant activity assay all the synthesized compounds c1–c12 were investigated for their in vitro antioxidant activity in terms of hydrogen donating or radical scavenging ability by the rapid and convenient 1,1-diphenyl-2-picrylhydrazyl (dpph) assay technique32 using trolox and ascorbic acid as standard drugs. methanol (95 %), dpph solution and standard drugs were used as the blank, control and references, respectively. the absorbance was measured at 517 nm (at an absorption maximum of dpph) after keeping a mixture of 100 ml of the synthesized compounds at a concentration 10 µg ml-1 (dissolved in dmso) and 900 ml of dpph radical solution (0.004 % solution of dpph in methanol) in the dark for 30 min incubation. the antioxidant activity was evaluated as the ic50 value in µm (the effective concentration at which 50 % of the radicals were scavenged). in vitro antimicrobial activity assay all the synthesized compounds (c1–c12) were examined for their in vitro antibacterial activity against gram-positive organisms, i.e., bacillus subtilis (mtcc 441), staphylococcus aureus (mtcc 96) and staphylococcus epidermidis (mtcc 2639), and gram-negative organisms, i.e., escherichia coli (mtcc 443), pseudomonas aeruginosa (mtcc 741) and klebsiella pneumoniae (mtcc 618), using the broth dilution method.33-37 in vitro antifungal activity of synthesized compounds was evaluated against the fungal strains candida albicans (mtcc 227), saccharomyces cerevisiae (mtcc 36), aspergillus niger (mtcc 282) and aspergillus flavus (mtcc 92) by the agar well diffusion method.38 the standard pathogenic microbial cultures were procured from the microbial type culture collection (mtcc), chandigarh, india. the antimicrobial activity was evaluated in terms of the minimum inhibitory concentration (mic) value (which corresponds to the lowest concentration that inhibits visible microbial growth) by the broth dilution method recommended by the national committee for clinical laboratory (nccl), standard protocol in liquid medium (nutrient agar) distributed in 96-well plates. the test compounds were dissolved in dimethylformamide (dmf) and further dilutions were made at the required concentrations of 300, 150, 75, 37.5, 18.75, 9.75, 6.25, 3.125 and 1.56 g ml-1. streptomycin and penicillin were used as reference standards for the antibacterial activity and amphotericin b was the reference standard for antifungal activity. results and discussion chemistry in this work, a series of 3,5-disubstituted isoxazoles (c1–c12) were synthesized by employing cu(i)-catalyzed cyclization between in situ generated nitrile oxide and the terminal alkyne as shown in scheme 1. 1-(5-fluoro-2-hydroxyphenyl)ethanone was treated with diethyl oxalate in the presence of naoet in etoh under refluxing condition to afford compound 2. the latter, on treated with 50 % naoh solution at room temperature, afforded 6-fluoro-4-oxo-4h-chromene-2-carboxylic acid in good yield. in initial experiments, the outlined reaction of 6-fluoro-4-oxo-4h-chromene-2-carboxylic acid with propargyl bromide chromene-derived isoxazoles as antioxidant and antimicrobial agents 5 using k2co3 in dmf at room temperature was investigated.39 this reaction afforded prop-2-ynyl 6-fluoro-4-oxo-4h-chromene-2-carboxylate in low yield (23 %). then, propargylation was performed using t-buok in dmf at room temperature for 4 h, which afforded the corresponding prop-2-ynyl 6-fluoro-4-oxo -4h-chromene-2-carboxylate in good yield (80 %). further, other aldehydes were converted to the corresponding aldoximes using hydroxylammonium chloride and 1 m naoh in t-buoh:h2o at room temperature. these aldoximes were converted to the corresponding nitrile oxide using chloramine-t trihydrate.40 the in situ generated nitrile oxide and alkyne in the presence of copper(i) catalyst at room temperature yielded 3,5-disubstitued isoxazoles (c1–c12) in good yields, table i. scheme 1. reagents and reaction conditions: i) diethyl oxalate / naoet, etoh, reflux, 4 h; ii) 50 % naoh, r.t., 5h; iii) propargyl bromide / t-buok, dmf, r.t., 4h; iv) a) ar-cho, nh2oh⋅hcl, naoh, t-buoh:h2o, r.t., 30 min. b) chloramine-t trihydrate, cu(i), r.t., 8–10 h. table i. synthesized 3,5-disubstituted isoxazoles c1–c12 from various aldehydes entry ar-cho time, h product yield, % c1 8.5 68 c2 9 70 c3 10 68 6 battula et al. table i. continued entry ar-cho time, h product yield, % c4 10 65 c5 8.5 70 c6 9 65 c7 cl o 8.5 66 c8 8.5 68 c9 8.5 66 c10 9 65 c11 10 62 c12 o 9 68 spectral analysis all the synthesized compounds were well characterized by spectral and analytical studies, such as 1h-nmr, 13c-nmr, ftir, esi-ms and elemental chromene-derived isoxazoles as antioxidant and antimicrobial agents 7 analysis. for convenience, compound c1 is discussed for spectral analysis. the presence of absorption bands at 3061 (c–h, ar), 1740 (c=o, ester), 1657 (c=o, chromene), 1608 (c=n, isoxazole) and 1220, 1130 cm–1 (ar-c–o–ch3) in the ftir spectrum confirmed the required functional groups present in compound c1. from its 1h-nmr spectrum, the presence of three multiplet signals (ppm) in the region δ 7.46–7.86 (3h, ar-h), two doublet signals at δ 7.37 (2h, ar-h) and δ 7.12 (2h, ar-h), two singlet signals at δ 7.15 (1h, chromene-h) and δ 6.82 (1h, isoxazole ch), and two singlet signals at δ 5.62 (2h, o–ch2) and 3.82 (3h, o–ch3) confirmed the formation of compound c1. similarly, from the 13c -nmr spectrum, the presence of characteristic carbon peaks at (ppm) 178.6 (c=o, ester), 168.7, (c=n, isoxazole), 162.3 (o–c=c), 162.1 (c=o, chromene), 100.1 (c–h, isoxazole), 61.9 (o–ch3) and 58.8 (ch2, ester) confirmed the presence of the characteristic carbon peaks in compound c1. the presence of the fluorine atom was confirmed by the additional carbon splitting pattern in the c-nmr spectra of c1 with a doublet coupling constant, j, values 245.6, 29.3, 10.5, and 9.2 hz, respectively. in addition, the molecular ion peak [396.0 (m+h)] from the esi-ms spectrum and elemental analysis (chn) data (c, 63.88 %; h, 3.50 %; n, 3.57 %) were further evidence for the formation of compound c1. antioxidant activity the evaluation of antioxidant activity results (table ii) revealed that some of the tested compounds exhibited good to moderate antioxidant activity as compared with the positive controls trolox and ascorbic acid. among them, compounds possessing the 4-methoxyphenyl and 1-napthyl group on the isoxazole ring (c1 and c12, respectively) registered very good antioxidant activity with ic50 values of 16.43±0.57 and 15.98±0.72 µm, respectively. compounds bearing 4-butylphenyl, 2,3-dimethylphenyl and 3,5-dimethylphenyl groups on the isoxazole ring (c5, c10 and c11, respectively) exhibited a moderate scavenging ability with ic50 values of 23.78±1.42, 27.15±1.47 and 18.87±0.82 µm, respectively. the remaining compounds exhibited moderate to poor antioxidant activity with ic50 values ranging from 34.66±2.10 to 82.31±3.02 µm. the potential scavenging ability may be attributed to the presence of pharmacologically active groups, such as 4-methoxyphenyl and 1-napthyl groups on the isoxazole ring. in vitro antibacterial activity the antibacterial screening results (table iii) revealed that some of the synthesized compounds exhibited excellent to moderate inhibition against the tested bacterial strains. compounds bearing 4-methoxyphenyl (c1), 2-(trifluoromethyl)phenyl (c3) and 2-hydroxyphenyl (c6) groups on the isoxazole core registered pro 8 battula et al. table ii. antioxidant activity of (3-arylisoxazol-5-yl)methyl 6-fluoro-4-oxo-4h-chromene2-carboxylates (c1–c12) determined by the dpph method product ic50 / µm c1 16.43±0.57 c2 82.31±3.02 c3 54.62±1.37 c4 66.17±2.21 c5 23.78±1.42 c6 45.30±1.31 c7 34.66±2.10 c8 77.40±1.88 c9 68.02±1.54 c10 27.15±1.47 c11 18.87±0.82 c12 15.98±0.72 trolox 13.24±0.80 ascorbic acid 3.54±0.40 table iii. in vitro antibacterial activity (mic / µg ml-1) of (3-arylisoxazol-5-yl)methyl 6 -fluoro-4-oxo-4h-chromene-2-carboxylates (c1–c12) against various bacterial strains compound bacteria b. subtilis s. aureus s. epidermidis e. coli p. aeruginosa k. pneumoniae c1 9.375 18.75 18.75 37.5 18.75 37.5 c2 75 75 150 75 75 150 c3 9.375 37.5 18.75 37.5 18.75 18.75 c4 75 75 75 150 75 150 c5 37.5 37.5 75 75 18.75 18.75 c6 9.375 18.75 18.75 37.5 18.75 37.5 c7 37.5 37.5 75 75 75 75 c8 18.75 37.5 75 75 75 75 c9 37.5 18.75 150 75 75 75 c10 37.5 37.5 75 75 9.375 37.5 c11 18.75 37.5 75 75 9.375 18.75 c12 37.5 75 75 75 18.75 75 penicillin 1.562 1.562 3.125 12.5 12.5 6.25 streptomycin 6.25 6.25 3.125 6.25 1.562 3.125 minent inhibition against all the tested gram-positive and gram-negative microorganisms with mic values ranging from 9.375 to 37.5 µg ml–1, as compared with the standard drugs penicillin and streptomycin. compounds possessing 4-butylphenyl (c5), 3-chlorophenyl (c7), 4-chlorophenyl (c8), 4-bromophenyl (c9), 2,3-dimethylphenyl (c10) and 3,5-dimethylphenyl (c11) groups on the isoxazole core registered good inhibition against b. subtilis and s. aureus bacterial strains, with mic values ranging from 18.75 to 37.5 µg ml–1. compounds c10 and c11 showed prominent activity against p. aeruginosa, greater than the stan chromene-derived isoxazoles as antioxidant and antimicrobial agents 9 dard drug penicillin (mic: 12.5 µg ml–1) with an mic value of 9.375 µg ml–1 (≈1.33 fold more potent than penicillin). compounds c10 and c11 also showed moderate inhibition against k. pneumoniae, with mic values of 37.5 and 18.75 µg ml–1, respectively. similarly, compound c5 also registered moderate inhibition against p. aeruginosa and k. pneumoniae, with an mic value 18.75 µg ml–1. compound c12 registered moderate inhibition against b. subtilis and p. aeruginosa, with mic values of 37.5 and 18.75 µg ml–1, respectively. the remaining compounds (c2 and c4) showed poor activity against all the bacterial strains. from the above observations, it is obvious that the presence of pharmacologically active moieties, such as 4-methoxyphenyl, 2-(trifluoromethyl)phenyl, 2-hydroxyphenyl, 2,3-dimethylphenyl and 3,5-dimethylphenyl groups, on the isoxazole core increased the antibacterial activity. in vitro antifungal activity the antifungal activity screening results (table iv) revealed that some of the synthesized compounds registered good to moderate activity against the tested microorganisms. compound c7, c8, and c9 showed better antifungal activity than the other synthesized compounds against the four fungal strains with mic values ranging from 18.75 and 37.5 µg ml–1. compounds c2 and c5 showed moderate activity against c. albicans and s. cerevisiae, with an mic value of 37.5 µg ml–1. compound c10 and c11 showed moderate antifungal activity against c. albicans and a. flavus, with mic values ranging from 18.75 to 37.5 µg ml–1. the remaining compounds (c1, c3, c4, c6 and c12) exhibited poor inhibition against all the tested fungal strains. from the above observations, it is obvious that the presence table iv. in vitro anti-fungal activity (mic / µg ml-1) of (3-arylisoxazol-5-yl)methyl 6-fluoro-4-oxo-4h-chromene-2-carboxylates (c1–c12) against various fungal strains compound fungi c. albicans s. cerevisiae a. niger a. flavus c1 150 75 150 75 c2 37.5 37.5 75 75 c3 75 150 75 75 c4 75 75 150 150 c5 37.5 37.5 75 75 c6 75 75 150 150 c7 18.75 37.5 18.75 37.5 c8 37.5 18.75 37.5 37.5 c9 37.5 37.5 37.5 37.5 c10 37.5 75 75 37.5 c11 18.75 75 75 37.5 c12 150 150 150 150 amphotericin b 6.25 6.25 1.562 6.25 10 battula et al. of 3-chlorophenyl, 4-chlorophenyl, and 4-bromophenyl groups on the isoxazole core influenced the antifungal activity of the synthesized compounds. conclusions in conclusion, a series of novel (3-arylisoxazol-5-yl)methyl 6-fluoro-4 -oxo-4h-chromene-2-carboxylates was synthesized by the cu(i)-catalyzed reaction between in situ generated nitrile oxides and prop-2-ynyl 6-fluoro-4-oxo -4h-chromene-2-carboxylate in good yields. all the synthesized compounds were investigated for their antioxidant and antimicrobial activities. compounds c1 and c12 exhibited very good antioxidant activity. compounds c1, c3 and c6 registered marked antibacterial activity against all bacterial strains, and c10 and c11 against p. aeroginosa. similarly, compounds c7, c8 and c9 showed better antifungal activity than remaining synthesized compounds. the biological activity of these compounds suggests that the synthesized compounds could be good candidates for future investigations. supplementary material analytical and spectral data for the synthesized compounds are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding authors on request. acknowledgements. the authors are thankful to the head of department of chemistry, kakatiya university, warangal, india, for providing the facilities and s. n. thanks csir-ugc new delhi, for the award of a senior research fellowship. и з в о д синтеза (3-арилизоксазол-5-ил)метил-6-флуор-4-оксо-4h-хромен-2 -карбоксилата и испитивање њихове антиоксидативне и антимикробне активности kumaraswamy battula1, sirassu narsimha1, vasudeva reddy nagavelli1 и mutheneni srinivasa rao2 1 department of chemistry, kakatiya university, warangal-506009, telangana, india и 2 chemical biology laboratory, indian institute of chemical technology, hyderabad-500007, india синтетисана је серија (3-арилизоксазол-5-ил)метил-6-флуор-4-оксо-4h-хромен -2-карбоксилата (c1–c12) у добром приносу, у реакцији in situ формираног нитрил-оксида са проп-2-инил-6-флуор-4-оксо-4h-хромен-2-карбоксилатом у присуству cu(i) као катализатора. испитана је антiиоксидативна и антимикробна активност синтетисаних једињења. од свих синтетисаних једињења, c1 (ic50: 16,43±0,57 μm) и c12 (ic50: 15,98±0,72 μm) имају добру антиоксидативну активност у поређењу са тролоксом, стандардним леком. једињења c1, c3 и c6 имају добру инхибиторну активност према свим грам-позитивним и грам-негативним бактеријама, са mic вредностима у опсегу од 9,375 до 37,5 μg ml-1. једињења c7–c11 имају добру инхибиторну активност према bacillus subtilis и staphylococus. aureus са mic вредностима у опсегу од 18,75 до 37,5 μg ml-1. једињења c10 и c11 показују истакнуту активност према pseudomonas aeruginosa у поређењу са пеницилином, као стандардним леком: mic, 12,5 μg ml-1 према 9,375 μg ml-1 (~1,33 активније од пеницилина). једињења c7–c9 chromene-derived isoxazoles as antioxidant and antimicrobial agents 11 имају добре до умерене антифунгалне активности према четири испитивана соја гљива са mic вредностима у опсегу од 18,75 до 37,5 μg ml-1. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis, spectral studies and in vitro antimicrobial activity of some new di/triorganotin (iv) complexes of schiff bases derived from 2-benzoyl pyridine} j. serb. chem. soc. 82 (1) 13–23 (2017) udc 542.9+547.571’551:546.657:546.811’13+ jscs–4943 547.814+66.095.3:615.281–188 original scientific paper 13 synthesis, spectral studies and in vitro antimicrobial activity of some new di-/tri-organotin(iv) complexes of schiff bases derived from 2-benzoylpyridine priyanka khatkar1, sonika asija1* and namita singh2 1department of chemistry, guru jambheshwar university of science & technology, hisar-125001, haryana, india and 2department of bio & nano technology, guru jambheshwar university of science & technology, hisar-125001, haryana, india (received 29 april, revised 12 september, accepted 28 september 2016) abstract: in the present work, a series of twenty-four organotin(iv) complexes of the type [r2sncll, r3snl] were synthesized by the condensation of 2-benzoylpyridine schiff bases with r2sncl2, r3sncl (r = me, n-bu or ph) in 1:1 mole ratio. these complexes were well characterized by ir, 1h-, and 13c-, 119sn-nmr, xrd and mass spectral techniques. in the search for biologically more effective antimicrobial agents, all the synthesized ligands and organotin complexes were evaluated for their in vitro antimicrobial activities against two gram-positive and two gram-negative bacteria, and two fungal strains by the serial dilution method. the results of spectral data revealed that the formed complexes were hexacoordinated with tridentate ligands coordinated through azomethine n, pyridine n and carboxylate o ligation sites. the ligands on coordination with tin metal showed a discernible augmentation in biocidal activity; however, the ph and bu complexes were found to be more intoxicating. the results revealed that the synthesized complexes were more noxious towards gram-positive strains as compared to gram-negative strains, which may be attributed to the presence of the outer lipid membrane of lipopolysaccharides. keywords: schiff bases; di-/tri-organotin complexes; antibacterial activity; antifungal activity. introduction there has been a tremendous growth in the synthesis of new safer antimicrobial drugs because of the resistance developed by microorganisms towards conventional drugs.1,2 therefore, extensive studies have been made on nitrogencontaining chelating schiff bases in recent years owing to their pronounced pharmacological applications.3,4 for instance, heterocyclic chelated schiff bases * corresponding author. e-mail: sc_ic2001@yahoo.co.in doi: 10.2298/jsc160429089k 14 khatkar, asija and singh with their metal complexes have shown remarkably enhanced biological activity.5,6 even lethal cancer-like disease was cured by the chelating complexes. therefore, active and well predictable schiff base ligands are considered ‘‘privileged’’ ligands. schiff bases and their complexes have been widely used in catalysis,3,7 organic light emitting diodes,8 wood preservatives and pesticides,9 non -linear optics,10,11 etc. consequently, they are regarded as potential scaffolds for chelating, which lead to the formation of organometallic complexes. schiff bases derived hydrazones and their metal complexes are promising compounds that have numerous pharmacological applications, such as antimicrobial,12,13 anticonvulsant,14 anti-inflammatory,15,16 anticancer,17,18 and antituberculosis19 agents. the bioactivity associated with metal complexes was increased as compared to their respective/parent ligands, while side effects may be decreased on complexation.20 the thriving utilization of organometallic complexes is a dynamically escalating area in biomedical and inorganic chemistry for the treatment of several human diseases. in particular, some unique characteristics of organotin complexes, such as geometries, variation in coordination number, thermodynamic and kinetic characteristics, accessible redox states and the inherent properties of the tin metal ion recommend chemistry researchers to develop varied approaches for their utilization in different fields, such as biomedical, industrial21,22 and agriculture.21 organotin complexes are potent motifs of organometallic compounds owing to their broad range of biocidal activities, in dependence on the nature and number of donor atoms of the ligands attached to the tin moiety. they are persistent moieties in a number of biocidal formulations in diverse areas, such as antifouling paints, fungicides, molluscicides, and they are found to be more effective against a large range of tumor lines than conventional metal anticancer drugs.23,24 in order to search for new antimicrobial agents with better molecular diversity and increased biological potency, some organotin complexes with 2-benzoylpyridine schiff bases were prepared that resulted in the formation of new drugs in which the ligand and metal might act synergistically. of meticulous exigency, the exploration for new antimicrobial agent should be more biospecific and less lethal to the environment and to the host. encouraged by these facts, herein, the synthesis of triand di-organotin complexes with different hydrazones of 2-benzoylpyridine is reported, which may lead to the formation of new pharmacophores with enhanced biological profiles. experimental materials and methods all the chemicals were obtained from sigma–aldrich and the solvents used were dried by conventional methods. the reactions were performed under an inert atmosphere. dimethyltin dichloride, di-n-butyltin dichloride, diphenyltin dichloride, tributyltin chloride, triphenyltin chloride, trimethyltin chloride and 2-benzoylpyridine were used as received without further purification. elements (c, h and n) were analyzed on a perkin–elmer 2400 instrument and synthesis, spectral studies and antimicrobial activity of di/triorganotin complexes 15 the measured data corresponded to the calculated data. the fourier transform infrared (ftir) spectra (4000–400 cm-1) were obtained in kbr pellets on a perkin–elmer spectrum rx1 instrument. the nmr spectra were recorded on a bruker avance ii 400 mhz nmr spectrometer in cdcl3 or dmso-d6 using tetramethylsilane (tms) as an internal standard. the mass spectra were recorded on an lcms ms 6410 agilent technologies spectrometer with an electron impact quadropole analyzer. the x-ray powder diffraction measurements were obtained using a rigaku table top x-ray diffractometer at a scan rate of 2° min-1 in the 2θ range 20–80°. tin was estimated gravimetrically as sno2.25 characterization data for the synthesized ligands are given in supplementary material to this paper. synthesis of di-/tri-organotin complexes with schiff base hydrazones derived from 2and 4-substituted benzoic acid hydrazide with 2-benzoylpyridine. 2-benzoylpyridine-derived hydrazones (m-no2, p-cl, p-no2, p-ch3) were prepared by reported methods.19,26 the schiff base ligands were prepared by dissolving (m-no2, p-cl, p-no2, p-ch3) benzoic acid hydrazides derivatives (5 mmol) in a methanolic solution of 2-benzoylpyridine (5 mmol, 1:1 mole ratio) and the reaction mixture was refluxed for 5–6 h. the mixture was left overnight and the obtained solid was separated, dried and washed with methanol. the schiff base ligands were recrystallized from methanol and chloroform. initially, the sodium salts of the ligands were prepared by dissolving and refluxing a weighed amount (5 mmol) of sodium in a methanolic solution of the schiff base ligands for 3 h. to this sodium salt solution, the starting material dialkyltin dichloride, diphenyltin dichloride (r2sncl2)/trialkyltin chloride, triphenyltin chloride (r3sncl), (r = ph, bu, me) were added dropwise in a 1:1 mole ratio. the mixture was refluxed for 9–10 h and filtered to remove the white-colored solid. the excess solvent was evaporated in vacuum to obtain the solid yellow complexes. the obtained complexes were washed with dry n-hexane. all the complexes were recrystallized from methanol and diethyl ether. pharmacology antimicrobial activity of all the synthesized ligands and organotin complexes were evaluated against four bacterial strains, i.e., gram-positive bacillus cereus (mtcc 10072), staphylococcus aureus (nicm 2901) and gram-negative escherichia coli (mtcc 732), pseudomonas aeruginosa (mtcc 424), and two fungal strains, i.e., aspergillus flavus (itcc 76801), aspergillus niger (mtcc 9933) by serial dilution method27,28 and their mic values were calculated. the bacteria and fungi were subcultured on nutrient agar and potato dextrose broth (pdb) from himedia, mumbai, india, respectively. the stock solution was diluted to make concentrations of 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.75 μg ml-1. the bacteria and the fungi were inoculated to each solution and the solutions were then kept in incubator at 37 °c for 24 h in the case of the bacteria and for 7 days in the case of the fungi. then, the minimum inhibitory concentration (mic) was determined. the experimental values were compared with standard drugs, i.e., ciprofloxacin for the antibacterial activity and fluconazole for the antifungal activity. results and discussion organotin(iv) complexes were synthesized by reacting benzoylpyridinederived hydrazones with dialkyltin dichloride, diphenyltin dichloride (r2sncl2)/ /trialkyltin chloride, triphenyltin chloride (r3sncl, r= ph, bu, me) in 1:1 mole ratio, scheme 1. the purity of synthesized compounds was checked by thin layer 16 khatkar, asija and singh chromatography (tlc). all the metal complexes were colored solids, and were stable on wide exposure to air. the spectroscopic techniques (ir, nmr and mass) were used to determine the geometry of complexes, which was found to be distorted octahedral. the ligands were found to be tridentate (onn) and chelated to the central tin atom with the replacement of one hydrogen atom through enolization and due to the chelation, the biocidal activities of the complexes were enhanced. scheme 1. synthetic route for the synthesis of schiff base ligands and their organotin complexes. ir spectra in the ligands, the sharp peaks that appeared due to ν(c=n) at 1603–1611 cm–1, ν(c=o) at 1671–1686 cm–1 and ν(nh) at 3190–3310 cm–1 disappeared on complexation. in the ir spectra of the complexes, the appearance of 2 new bands in the region 1345 and 1210 cm–1 due to ν(nco) and ν(c–o), respectively, indicated coordination through oxygen and nitrogen to the tin atom after deprotonation. the peak due to c=n shifted to lower frequency by 15–20 cm–1, showing the involvement of the electrons present on nitrogen of the azomethine group in bond formation to the central tin atom. the peak in the region 676–628 cm–1 in the spectra of the hydrazones is due to the in-plane vibration mode of the pyridine ring, which is shifted to a higher frequency in the complexes, confirming the mode of coordination of the pyridine nitrogen.12,29 new bands in appeared in the spectra of the complexes at 431–459 cm–1, 523–566 cm–1 and 608–731 cm–1, corresponding to (sn–n), (sn–o) and (sn–c) frequencies, respectively, which support the formation of complexes.30 based on these results, the coordination sites of the ligands with the tin atom were ascertained by comparing the fre synthesis, spectral studies and antimicrobial activity of di/triorganotin complexes 17 quency shifts in the spectra of ligand and complexes. the disappearance of the bands assigned to the carbonyl group in ir spectra of complexes divulges that the ligands coordinate with tin metal in the enolic form. electronic spectra electronic spectra of the ligands and their metal complexes were recorded in dmf. in the electronic spectra of the ligands, a band due to the phenyl ring was observed at 218 nm. this band shifts nearly 20–30 nm to higher wavelengths on complexation. in addition, the band at 293 nm, ascribed to the c=n chromophore in the ligands, was shifted to a longer wavelength and was observed at 296 nm in the complexes. an absorption band found at 351 nm for the ligands is due to n→π* transitions, and was decreased in the complexes. it is known that ligands containing nitrogen and oxygen as donor atoms are capable of forming dπ–pπ bonds with the metal, due to which charge transfer and intense intraligand bonds were observed.23 nmr spectral analysis the 1hand 13c-nmr spectra of ligands and their organotin complexes were recorded in cdcl3 or dmso. by comparing the spectra of the ligands with their organotin complexes, the coordination sites (onn) of the ligands were proposed. in the 1h-nmr spectra of the ligands, the main characteristic peak was a doublet obtained in the region at δ 8.63–8.82 ppm due to the proton present at the carbon adjacent to the n of the pyridine ring, which was further shifted downfield in the complexes as compared to the free ligands. this confirmed the coordination of pyridine n to the tin atom. the singlet due to azomethine proton observed in the region of δ 13.7–15.2 ppm was absent in the 1h-nmr spectra of the complexes,12 which substantiates the coordination mode of the carbonyl oxygen through enolization. the peaks due to remaining aromatic and aliphatic protons appeared in the expected regions. in the complexes, new signals appeared in the spectra at δ 1.07–1.67 ppm, 0.70–1.75 ppm and 7.18–8.78 ppm due to the methyl, butyl and phenyl protons attached to the tin atom. the 13c-nmr spectra also confirmed the proposed structures. in the spectra of the ligands, signals due to the carbonyl carbon and azomethine carbon appeared at δ 162.29–163.98 ppm and δ 153.11–158.66 ppm, respectively. on complexation, the signals due to the carbonyl carbon, azomethine carbon and the carbon adjacent to the coordinating atoms shifted downfield, which supported the coordination modes through the azomethine nitrogen and the carbonyl carbon. the aromatic carbons appeared in the δ range 153.94–122.50 ppm. in the complexes, new signals due to methyl and butyl groups appeared at δ 10.49–31.81 ppm and 13.57–35.43 ppm and the phenyl carbons appeared in their normal range. 18 khatkar, asija and singh the 119sn-nmr spectra of all the synthesized organotin complexes showed one sharp singlet depending upon the coordination number and the r group attached to the centre atom, which inferred the formation of single tin species. a large upfield shift was observed in the spectra of complexes with increasing coordination number. the occurrence of chemical shift in 119sn-nmr spectra in the ranges δ –208.18 to –228.23 ppm, –270.75 to –297.11 ppm and –331.45 to –376.12 ppm for the methyl, butyl and phenyl complexes, respectively, were in accordance with a hexacoordinated environment around the tin metal in the complexes.31,32 mass spectra the esi-ms spectra of the diorganotin complexes exhibited different fragmentation patterns as expected and results were found to be in good agreement with their molecular formulae. in some cases, the fragments were observed as groups of peaks due to different isotopes of chlorine and tin.24 the mass spectra of the compounds 1–28, in each case displayed the [m+h]+ peak, for instance, in the mass spectrum of bu2sncll2 (molecular mass 583.14), the peak due to [m+h]+ was observed at m/z 584.50. the other peaks due to [m–cl]+, [l]+ and [sn]+ fragments were observed at m/z 548.50 316.30 and 119.00, respectively. all these peaks are depicted in fig. 1. x-ray diffraction analysis of the synthesized complexes by x-ray powder diffraction was in accordance with the crystalline nature of the complexes.33 the patterns were recorded over the 2θ range 20–80° and the average crystallite sizes dxrd were calculated to obtain information about the dynamics of the complexes. the x-ray diffraction pattern of the bu2sncll2 complex displayed a clear crystalline peak with maxima at 2θ 31.680° and d = 2.822ǻ, fwhm = 0.286 rad (fig. 2). the particle size of the complexes was calculated with the debye–scherrer formula and it was approximately found to be in the range 54–65 nm. antimicrobial activities a detailed structure–activity relationship of hexacoordinated diorganotin(iv) complexes was made from the results of the antimicrobial evaluation presented in table i, which revealed that the organotin complexes have comparable activity to those of standard drugs against gram-positive bacteria and fungi rather than their respective ligands. these results show that chelation of a ligand to tin leads to augmentation in activity due to delocalization of electrons, which increases the lipophilic character of the complexes and proficient dissemination of the metal complexes into bacterial cell walls. furthermore, hexacoordinated complexes in which a chlorine atom is directly coordinated to the tin atom were found to be more active as com synthesis, spectral studies and antimicrobial activity of di/triorganotin complexes 19 fi g. 1 . m as s sp ec tr um o f b u 2 sn c ll 3. fi g. 2 . x r d p at te rn fo r b u 2 sn c ll 2. 20 khatkar, asija and singh table i. the in vitro antibacterial activity (mic / µmol ml-1) of 2-benzoylpyridine-derived hydrazone schiff base ligands, and their organotin(iv) complexes. compound bacteria e. coli p. aeruginosa b. cereus s. aureus a. niger a. flavus hl1 0.0361 0.0361 0.0361 0.0180 0.0090 0.0180 hl2 0.0361 0.0361 0.0361 0.0180 0.0090 0.0180 hl3 0.0396 0.0396 0.0396 0.0198 0.0198 0.0198 hl4 0.0372 0.0186 0.0186 0.0186 0.0186 0.0093 ph2sncll1 0.0096 0.0096 0.0191 0.0096 0.0048 0.0024 bu2sncll1 0.0102 0.0051 0.0051 0.0025 0.0051 0.0025 me2sncll1 0.0236 0.0059 0.0118 0.0118 0.0059 0.0059 ph3snl1 0.0090 0.0045 0.0090 0.0090 0.0090 0.0045 bu3snl1 0.0197 0.0098 0.0098 0.0098 0.0098 0.0049 me3snl1 0.0246 0.0123 0.0246 0.0123 0.0123 0.0061 ph2sncll2 0.0096 0.0048 0.0048 0.0024 0.0024 0.0048 bu2sncll2 0.0204 0.0051 0.0102 0.0051 0.0025 0.0051 me2sncll2 0.0236 0.0059 0.0118 0.0029 0.0059 0.0059 ph3snl2 0.0090 0.0090 0.0090 0.0045 0.0045 0.0045 bu3snl2 0.0197 0.0098 0.0098 0.0098 0.0025 0.0049 me3snl2 0.0246 0.0123 0.0123 0.0123 0.0061 0.0061 ph2sncll3 0.0100 0.0050 0.0100 0.0025 0.0025 0.0025 bu2sncll3 0.0215 0.0054 0.0107 0.0107 0.0107 0.0054 me2sncll3 0.0251 0.0125 0.0125 0.0031 0.0031 0.0031 ph3snl3 0.0094 0.0094 0.0094 0.0049 0.0049 0.0049 bu3snl3 0.0207 0.0103 0.0207 0.0103 0.0103 0.0052 me3snl3 0.0261 0.0131 0.0131 0.0131 0.0131 0.0065 ph2sncll4 0.0097 0.0097 0.0097 0.0024 0.0024 0.0024 bu2sncll4 0.0104 0.0052 0.0104 0.0052 0.0104 0.0052 me2sncll4 0.0241 0.0120 0.0120 0.0060 0.0120 0.006 ph3snl4 0.0091 0.0091 0.0046 0.0046 0.0046 0.0046 bu3snl4 0.0200 0.0100 0.0100 0.0100 0.0100 0.0100 me3snl4 0.0251 0.0125 0.0125 0.0125 0.0125 0.0063 ciprofloxacin 0.0047 0.0047 0.0047 0.0047 – – flucanazole – – – – 0.0051 0.0051 pared to the complexes which lack a chlorine atom in the complexes directly attached to the tin atom. therefore, chelation to the tin metal and the presence of a chlorine atom was assumed to impart a modification reaction in the biological system and it plays a significant role in the enhancement of biocidal activity, which may be due to a bacteriostatic or bactericidal effect. the order of biocidal activity for the complexes was ph > bu > me.34 this indicates that an r group directly attached to the tin atom also plays a significant role in the enhancement of activity, which is directly related to their electron donor ability. hence, a phenyl group binds with biological molecules by π–π interactions and increases the electron density above azomethine nitrogen, which leads to the stronger interactions with the active centers of cell constituents. synthesis, spectral studies and antimicrobial activity of di/triorganotin complexes 21 conclusion diorganotin and triorganotin complexes were obtained by reacting sodium salts of benzoylpyridine-derived hydrazones with organotin(iv) chloride. the synthesized complexes were characterized by different spectroscopic (1h-, 13c-, 119sn-nmr, ir and mass) and other physical techniques. the schiff base ligands were found to coordinate with tin metal in a tridentate manner (nno) producing hexacoordinated tin(iv) complexes with distorted octahedral geometry. the compounds were further evaluated for their in vitro antimicrobial activity against different pathogenic bacteria and fungi. the tested complexes exhibited greater biocidal activity as compared to the free ligands due to the coordination with tin metal. in addition, the activity varied with the substitution on the tin atom, which increased in the order me < n-bu < ph. a greater antimicrobial effect was observed in triphenyltin(iv) and diphenyltin(iv) complexes and the presence of a chlorine atom enhanced the biocidal activity against gram-positive bacteria as compared to the gram-negative bacteria, which may be due to difference in the nature of their cell wall and proficient diffusion of the complexes through the cell walls. supplementary material analytical and spectral data of the compounds, as well as copies of the corresponding 1h-nmr and 13c-nmr of the ligands hil2 and hil4 and 1h-nmr, 13c-nmr and 119sn spectra of the complexes bu2sncll2 and bu3snl4 and mass spectra of all the complexes are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding authors on request. acknowledgement. the authors are grateful to the guru jambheshwar university of science and technology, hisar, for providing financial support. и з в о д синтеза, спектрална испитивања и in vitro антимикробна активност неких нових комплекса ди/три-органокалаја(iv) са шифовим базама добијеним из 2-бензоилпиридина priyanka khatkar1, sonika asija1 и namita singh2 1 department of chemistry, guru jambheshwar university of science & technology, hisar-125001, haryana, india и 2 department of bio & nano technology, guru jambheshwar university of science & technology, hisar-125001, haryana, india у овом раду су у реакцији кондензације шифових база, које садрже 2-бензоилпиридин, са r2sncl2 и r3sncl (r = me, n-bu или ph) у молском односу 1:1 синтетисана двадесет четири комплекса калаја(iv), опште формуле [r2snlcl, r3snl]. комплекси су окарактерисани ir, 1h-, 13cи 119sn-nmr спектроскопијама, и техникама xrd и масене спектрометрије. у циљу проналажења најефекаснијег микробиолошког агенса, сви синтетисани лиганди и одговарајући комплекси калаја(iv) су испитивани на in vitro антимикробину активност на две грам-позитивне и две грам-негативне врсте бактерија, као и две врсте гљивица. спектроскопским методама је нађено да је координациони број калаја(iv) у испитиваним комплексима шест и да су у овим комплексима тридентатни 22 khatkar, asija and singh лиганди координовани за калај(iv) преко азометинског и пиридинског атома азота, као и атома кисеоника. лиганди координовани за калај(iv) су показали значајно већу биолошку активност, док су ph и bu комплекси показали већу токсичност. резултати антимикробних испитивања су показали да синтетисани комплекси калаја(iv) имају веће токсично дејство према грам-позитивним сојевима, што се може приписати присуству спољашње липидне мембране липополисахарида. 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(2005) 147 29. b. gleeson, j. claffey, d. ertler, m. hogan, h. m. bunz, f. paradisi, d. wallis, m. tacke, polyhedron 27 (2008) 3619 30. a. a. r. despaigne, j. g. da silva, a. c. m. do carmo, o. e. piro, e. e. castellano, h. beraldo, inorg. chim. acta 362 (2009) 2117 31. r. malhotra, j. mehta, k. bala, a. k. sharma, indian j. chem., a 47 (2008) 58 32. m. nath, p. k. saini, a. kumar, j. organomet. chem. 695 (2010) 1353 33. j. devi, s. devi, a. kumar, med. chem. commun. 7 (2016) 932 34. l. c. dias, g. m. de lima, j. a. takahashi, j. d. ardisson, appl. organomet. chem. 29 (2015) 305. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {discussion on the comments of slobodan k. milonji{\'{c}} on the article entitled “adsorption of strontium on different sodium-enriched bentonites” by sanja r. marinovi{\'{c}}, marija j. ajdukovi{\'{c}}, nata{\v{s}}a p. jovi{\'{c}}-jovi{\v{c}}i{\'{c}}, tihana m. mudrini{\'{c}}, bojana n. nedi{\'{c}}-vasiljevi{\'{c}}, predrag t. bankovi{\'{c}} and aleksandra d. milutinovi{\'{c}}-nikoli{\'{c}}, published in the journal of the serbian chemical society, vol. 82, issue 4, 2017, pp. 449–463} j. serb. chem. soc. 83 (3) 395–399 (2018) udc 546.42’33+666.32:544.723: jscs–5083 628.316.2(0.49.2) letters to the editor 395 letter to the editor discussion on the comments of slobodan k. milonjić on the article entitled “adsorption of strontium on different sodiumenriched bentonites” by sanja r. marinović, marija j. ajduković, nataša p. jović-jovičić, tihana m. mudrinić, bojana n. nedić-vasiljević, predrag t. banković and aleksandra d. milutinović-nikolić, published in the journal of the serbian chemical society, vol. 82, issue 4, 2017, pp. 449–463 sanja r. marinović1*, marija j. ajduković1, nataša p. jović-jovičić1**, tihana m. mudrinić1, bojana n. nedić-vasiljević2, predrag t. banković1 and aleksandra d. milutinović-nikolić1 1university of belgrade, institute of chemistry, technology and metallurgy, njegoševa 12, belgrade, serbia and 2university of belgrade, faculty of physical chemistry, studentski trg, 12–16, belgrade, serbia (received 23 january, accepted 30 january 2018) abstract: a thorough discussion on the comments on the article “adsorption of strontium on different sodium-enriched bentonites”, by sanja r. marinović, marija j. ajduković, nataša p. jović-jovičić, tihana m. mudrinić, bojana n. nedić-vasiljević, predrag t. banković and aleksandra d. milutinović-nikolić published in the journal of the serbian chemical society, volume 82, issue 4, pages: 449-463 (2017), given as letter to the editor by s. k. milonjić, is provided in this letter. the authors of the commented paper have considered all the comments, and responded to each of them, point by point. keywords: adsorption; bentonites; analysis of problematic interpretation. after the scientific paper entitled “adsorption of strontium on different sodium-enriched bentonites”, by marinović et al.1 had been published, it was commented on by milonjić.2 the authors of the paper1 have thoroughly considered all the comments.2 there are two typing errors that were, unfortunately, overlooked during the reviewing process and the proof reading. these accidental mistakes were, however, discussed by the critic as fundamental. beside them, *,** corresponding authors. e-mails: (*) sanja@nanosys.ihtm.bg.ac.rs; (**)natasha@nanosys.ihtm.bg.ac.rs https://doi.org/10.2298/jsc180123026м ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 396 marinović et al. other comments are questionable and imply superficial approach of the critic to the evaluation. this may be due to the specificity of the scientific field of clays and clay minerals, that perhaps has not been taken into consideration. the authors of the paper hereby address the editorial office of the journal of the serbian chemical society, with the detailed discussion that provides the relevant scientific data, and offers the references of the renowned authors and from the competitive scientific journals, aimed to prove the comments2 wrong, and to support their position. the authors have responded to each of the comments,2 point by point. in the eq. (1)1 the authors accidentally omitted one factor, namely volume (v), from the formula. however, and it is important to be stressed, they used the correct formula for the calculation of the amount of sr2+ adsorbed after time t (qt). the values of qt in tables ii and iii1 are calculated using the correct formula and the units are appropriate (mg g–1). therefore, the recalculating is redundant because the calculated data are certainly valid. consequently, the drawn conclusions can be based on the data, and should not be regarded as questionable. moreover, it should be noted that the authors are well aware of the correct form of the formula and have reported it previously in several scientific publications.3–6 the comment: “from fig. 4 (ref. 1), it is evident that the adsorbed amounts of sr2+ on na-t, naw and na-b bentonites in the initial phi range ~4 to ~9 are constant because the final phf values are constant” is unnecessary, because the authors commented the obtained results in the similar manner. in fig. 4, in the published paper, two aspects of the influence of the initial ph (phi) are presented. the first set of curves represents the influence of phi on sr2+ uptake. the other one represents the influence of phi on the final ph (phf) with the goal of determining the phi range for which phf is almost constant, exhibited as the plateau in the diagram. this plateau results from the constant ratio of the quantities of negative and positive charges on the surface of adsorbent. the direct consequence of this effect is the constant value of phf, which leads to the constant sr2+ uptake within the corresponding phi range. one of the goals of this part of investigation was the determination of the acidity range within which phi for further adsorption experiments should be chosen. accordingly, it is not important whether the constant ratio of the quantities of negative and positive charges on the surface of adsorbent was elaborated, as the relevant phenomenon (as the authors did in the published paper) or its direct consequence was evidenced as constant phf value. the remark that refers to the lack of the experimental proof of the ion exchange mechanism, as the dominant one, can be addressed as follows. the main property of smectites – a group of clay minerals – is the cation exchange ability, expressed through the high values of the cation exchange capacity (cec). this property is well described in literature7 as well as the fact that the ion ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. discussion on the comments of s. k. milonjić 397 exchange mechanism is considered to be the governing mechanism for the adsorption of cations. therefore, in the research area of clays and clay minerals, additional experimental proofs for the ion exchange mechanism are not a common practice. it was legitimate, though, for the reviewers of the submitted text, to request additional experimental proofs, which did not happen. otherwise, the authors would have readily provided additional data. beside the ion exchange mechanism, the mechanism of adsorption on clays is the surface adsorption. surface adsorption should be favoured by the developed specific surface area, which was not the case, and in the criticized paper1 a discussion on this topic was originally provided. further comments2 refer to the adsorption kinetics data analysis. according to the reference,8 recommended by the critic, results for longer adsorption times should be taken into account for porous materials (such as the investigated clays), which is opposed to the suggestions of the critic. tran et al.8 propose for this type of materials the extended adsorption times, even longer than 24 h. however, after receiving the remarks,2 the authors performed an additional calculation using fewer points, only for shorter times, than the one presented in the published paper. the equation for the pseudo-second order kinetics: 2 e2 e 1 = + t t t q qk q (2) was again used for the calculation. the newly obtained results for qe, differed for up to 5 % in comparison with the published ones. therefore, the conclusions in the published manuscript, based on the calculated data, are not erroneous. in addition, the distinguished experts in the field of clays and clay minerals published the kinetic analysis for the adsorption of cs+ onto the porous clay mineral sepiolite, using times that exceed the equilibrium time.9 the dimension of the freundlich adsorption constant, given in table ii as kf in dm3 mg–1, is a typing error. the authors are well aware of the correct unit, and the proof for this are the publications that preceded the commented one.4,5 the correct form of the unit is presented in corrections on the paper1 at the end of this text; however it differs from the one suggested by milonjić.2 here, the authors provide the dimensional analysis that explains how the unit is derived when the correct analytic form of the freundlich equation10–12 is used, as presented in eq. (3): 1 e f e= nq k c (3) the unit for kf is derived in eq. (4): ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 398 marinović et al. ( ) 1 1 3 1 f )mg g dm mg( nk − −= (4) the langmuir, freundlich and dubinin–raduskevich isotherm models were applied on the experimentally obtained data for the adsorption of sr2+ onto different bentonites. low values of the coefficient of determination (r2) obtained for the freundlich model suggested that the freundlich model is not appropriate for the investigated system. therefore, the freundlich adsorption constant (n) was not taken into consideration in the paper by marinović et al.1 for the determination of the nature of the investigated process. hence, the conclusions drawn from the isotherm analysis were not in disagreement, as stated by the critic. however, the corrections and the reformulated discussion on the mechanism of sr2+ uptake is given at the end of this text. the authors believe that the discussion given in the text above have completely addressed all the remarks given in the comments by milonjić.2 it is worth emphasizing that the original paper was already revised in the manner suggested by the reviewers and the editor of the journal of the serbian chemical society. therefore, the authors will avoid potential further polemics with dr milonjić regarding the marinović et al.1 paper that might become excessive. corrections on the paper published in the journal of the serbian chemical society, volume 82, issue 4, pages: 449-463 (2017) • the correct equation on page 452 is: ( )0 ads t t c c v q m − = (1) where c0 and ct are the concentrations (mg dm–3) of sr2+ in a solution initially and after adsorption time t, respectively, v is the volume of solution (dm3) and mads is the adsorbent mass. the correct unit for kf in table ii is mg g–1 (dm3 mg–1)1/n. the discussion in the paragraph following the table iii, page 460, is reformulated as follows: the squared correlation coefficients (r2) showed that the experimental data were in good agreement with the dubinin–radushkevich isotherm model (table iii). the calculated e values were similar for all the investigated adsorbents and slightly higher than 8 kj mol–1, within the range of the adsorption energy characteristic for the systems in which ion exchange is the dominant mechanism (8–16 kj mol–1).13 consequently, the sentence in the conclusion, page 461, where this result is emphasized, should be: the value obtained for the adsorption energy was slightly above 8 kj mol–1. this indicated the ion exchange as the dominant adsorption mechanism. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. discussion on the comments of s. k. milonjić 399 и з в о д дискусија коментара слободана к. милоњића на рад под насловом адсорпција стронцијума на различитим натријумом измењеним бентонитима, аутори: сања мариновић, марија ајдуковић, наташа јовић-јовичић, предраг банковић, тихана мудринић, бојана недић -васиљевић и александра милутиновић-николић, објављеног у journal of the serbian chemical society, волумен 82, број 4, 2017, стр. 449–463 сања р. мариновић1, марија ј. ајдуковић1, наташа п. јовић-јовичић1, предраг т. банковић1, тихана м. мудринић1, бојана м. недић васиљевић2 и александра д. милутиновић-николић1 1 универзитет у београду – институт за хемију, технологију и металургију, центар за катализу и хемијско инжењерство, његошева 12, београд и 2 универзитет у београду – факултет за физичку хемију, студентски трг 12–16, београд у овом тексту дата је детаљна дискусија која се односи на коментаре у писму с. к. милоњића уреднику часописа j. serb. chem. soc.: comments on the article entitled “adsorption of strontium on different sodium-enriched bentonites”, by sanja r. marinović, marija j. ajduković, nataša p. jović-jovičić, tihana m. mudrinić, bojana n. nedić-vasiljević, predrag t. banković and aleksandra d. milutinović-nikolić, published in the journal of the serbian chemical society, volume 82, issue 4, 2017, pp. 449–463. аутори коментарисаног рада су размотрили све коментаре и на сваки од њих одговорили. (примљено 23. јануара, прихваћено 30. јануара 2018) references 1. s. marinović, m. ajduković, n. jović-jovičić, t. mudrinić, b. nedić-vasiljević, p. banković, a. milutinović-nikolić, j. serb. chem. soc. 82 (2017) 449 2. s. milonjić 83 (2018) 391 3. n. jović-jovičić, a. milutinović-nikolić, m. žunić, z. mojović, p. banković, i. gržetić, d. jovanović, j. contam. hydrol. 150 (2013) 1 4. a. milutinović-nikolić, d. maksin, n. jović-jovičić, m. mirković, d. stanković, z. mojović, p. banković, appl. clay sci. 95 (2014) 294 5. s. marinović, m. ajduković, n. jović-jovičić, p. banković, z. mojović, a. milutinovićnikolić, d. jovanović, sci. sinter. 48 (2016)167 6. z. mojović, n. jović-jovičić, a. milutinović -nikolić, p. banković, a. abu rabistanković, d. jovanović, environ. prog. sustain. energy 32 (2013) 1124 7. f. bergaya, g. lagaly, developments in clay science, in handbook of clay science, vol.5a, 2nd ed., elsevier, amsterdam, 2013 8. h. n. tran, s.-j. you, a. hoseseini-bandegharaei, h.-p. chao, water res. 120 (2017) 88 9. m. darder, y. gonzalez-alfaro, p. aranda, e. ruiz-hitzky, rsc adv. 4 (2014) 35415 10. h. freundlich, z. phys. chem. 57 (1907) 385 11. n. viswanathan, c. s. sundaram, s. meenakshi, j. hazard. mater. 161 (2009) 423 12. s. abdi, m. nasiri, a. mesbahi, m. h. khani, j. hazard. mater. 332 (2017) 132 13. c. y. abasi, a. a. abia, j. c. igwe, envron. res. j. 5 (2011) 104. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {arsenate and arsenite adsorption in relation with chemical properties of alluvial and loess soils} j. serb. chem. soc. 82 (7–8) 943–954 (2017) udc 549.73’755+544.723.2:504.53.054+ jscs–5014 504.4.054 original scientific paper 943 arsenate and arsenite adsorption in relation with chemical properties of alluvial and loess soils shah rukh1*, mohammad saleem akhtar1, ayaz mehmood2, sayed hassan3, khalid s. khan1, syed m. s. naqvi4 and muhammad imran1 1department of soil science & swc, pmas-arid agriculture university rawalpindi-46000, pakistan, 2department of agricultural sciences, university of haripur, haripur-22620, pakistan, 3department of crop and soil sciences, university of georgia, athens-30602, ga, usa and 4institute of biochemistry and biotechnology, pmas-arid agriculture university rawalpindi-46000, pakistan (received 9 february, revised 18 march, accepted 20 march 2017) abstract: arsenic is one of the most toxic elements in the soil environment. understanding of the arsenic adsorption chemistry is essential for evolving the extent of soil and groundwater contaminations. this research was conducted to determine the variation in adsorption behaviour of arsenite and arsenate with depth in different lithology soils. we sampled two parent materials at genetic horizons, and within a parent material, we selected two soils. besides basic soil characterizations, a laboratory batch experiments were carried out to study the adsorption of arsenate and arsenite. freundlich adsorption approaches were employed to investigate the adsorption of arsenate and arsenite in the soils. freundlich isotherms fit arsenate and arsenite sorption data well with r2 values of 0.88–0.98 in most soils. arsenate and arsenite adsorption varied with the soil properties, especially in clay composition and in the oxides of iron and aluminum. arsenic adsorption parameters also varied with depth in parent materials, and loess derived soils had greater adsorption capacity as compared to alluvial soils in most of the adsorption parameters. this research concludes that the loess soils had higher arsenic adsorption capacity than the alluvial soils. keywords: arsenic species; parent material; freundlich isotherm. introduction arsenic is one of the most toxic trace element present in soil and water. naturally as occurs in rocks and mineral weathering and it can contaminate soil and groundwater.1 anthropogenic activities, including the application of agricultural pesticides, industrial waste and land filling of sewage sludge add as to soil and cause as mineral weathering. mineralization of organic matter and cyclic oxid * corresponding author. e-mail: shahrukhshah86@hotmail.com https://doi.org/10.2298/jsc170209042r (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 944 rukh et al. ation–reduction release as in the soluble form into the soil immediately after addition.2 arsenic forms a variety of inorganic and organic compounds in soils.3 inorganic as occurs dominantly as pentavalent arsenate (as(v)) and trivalent arsenite (as(iii)). arsenate species is predominant over as(iii) and organic as and it may be up to 40 percent of total as content in contaminated soils.4 the inorganic as phases are hundreds times more toxic than the organic phases; and among the inorganic as forms, as(iii) are twenty five to sixty times more toxic than as(v).5,6 therefore, the correct estimation of as form in soil is important for understanding bioavailability of as and its effects on biota.7 the adsorption of as is probably initial on metal oxides especially iron oxides, i.e., goethite, lepidocrocite and less crystalline ferrihydrite. a large portion of as becomes adsorbed on metal oxides and only a small portion remains in soil water.8 the arsenic adsorption process controls the toxicity, fate, mobility and bioavailability of as in soils. soil solution of as can be significantly different although the same amount of as exists in different soils due to its adsorption with the soil matrix. therefore the understanding of the adsorption of as is important for predicting the as behaviour in the soil environments.9 arsenic adsorption on clay, metal oxides of iron and aluminum and organic matter is the most common process for as solid phase formation generally.10,11 it is reported that oxides and hydroxides of iron and aluminum are major constituents which controls as adsorption in soils.12,13 calcite possibly has a role in the retention and the solubility of arsenic in carbonate rich environment. phosphate, organic matter and clay contents are the two important soil factors which significantly affect the adsorption of as in soils.14,15 arsenate and as(iii) adsorption studies used variety of adsorbents such as metal oxides, clays and soils. studies was conducted on as(v) and as(iii) adsorption by amorphous iron and aluminum oxides, as well as on clay minerals, i.e., kaolinite and montmorillonite.16 for the modeling of as(v) and as(iii) adsorption the most widely used analytical isotherms are the langmuir isotherm17 and the freundlich isotherm.18 the studies on the arsenic adsorption in relation with lithology and development are scarce. we assumed that the adsorption behaviour of as(v) and as(iii) may vary with the soil lithology and within a same lithology parent material, the soil development controls the arsenic forms adsorption. we sampled the two parent materials at genetic horizon level, and we selected two soils in each parent material. the objectives of the study were to evaluate the adsorption behaviour of as(v) and as(iii) of different parent materials soils and the effect of soil properties on as(v) and as(iii) adsorption. (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ arsenate and arsenite adsorption parameters 945 experimental soil sampling and characterization soils were taken from the pothwar plateau, having different parent material, and we selected alluvium and loess parent material for the present study. the gps location and usda taxonomy of selected soils is given in table i. triplicate profile for each soil was dug and soil samples were taken from each of the genetic horizon. samples were air dried and crushed to pass through a 2-mm sieve. table i. selected soils loess and alluvium parent materials soil location usda classification parent material rawal 33o38’46.28’’n and 73o04’57.82’’e fine silty, mixed, hyperthermic typic hapludalfs alluvium kotli 33o37’11.13’’n and 73o42’53.92’’e fine, mixed, hyperthermic entic chromosterts guliana 33°33’22.12’’n and 72°38’30.82’’e silty, mixed, thermic udic haplustalfs loess mansehra 34o24’56’’n and 73o14’06’’e fine loamy, mixed hyperthermic typic hapludalfs each sample was analyzed for texture, ph, calcium carbonate, cation exchange capacity (cec), na2s2o4-extractable fe and al and dissolved organic carbon (doc). the soil particle size distribution was determined by the dispersion in (napo3)6 solution and the soil ph of saturated soil paste was measured. soil caco3 was determined by the acetic acid consumption.19 the doc was extracted with k2so4 and determined by consumption of k2cr2o7. 20 soil iron and aluminum oxides were dissolved in na2s2o4 and c6h5na3o7 and nahco3 buffer solution. the concentrations of fe and al in the extracts were measured by icp-ms.21 cation exchange capacity was determined by saturating soil with an index cation.22 amorphous iron and aluminum were determined by extracting soil in acidified ammonium oxalate solution in dark.23 batch experiment for arsenate and arsenite sorption sorption isotherms for as(v) and arsenite were constructed to estimate fruendlich model parameters employing the batch sorption experiments.24,25 in triplicate, 3.0 g of soil was equilibrated with 30 ml 0.01 m kno3 solutions containing graded concentration of as(v) 0, 0.1, 2.5, 5, 8, 10, 15, 20, 25, 40 and 100 mg l-1 (na2haso4). the suspension was shaken for 48 h at room temperature and centrifuged for 20 min at 3000 rpm. the supernatant was filtered through 0.45 µm cellulose membrane and analyzed for total arsenic. separately, three g soil was equilibrated with 30 ml of 0.01 m kno3 solution containing graded concentration of as(iii) 0, 0.1, 0.5, 1, 3, 5, 7 and 8 mg l-1 from naaso2. the suspension was shaken for 48 h at room temperature and centrifuged for 20 min at 3000 rpm. the cellulose membrane of 0.45 µm was used to filter the supernatant and total arsenic was analyzed in the extract. sorbed amount of as(v) and as(iii) was calculated from the change in the solution phase concentration. the adsorption isotherm was fitted to the freundlich equation:26 1/f w/x m k c β= (1) (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 946 rukh et al. where x/m is the equilibrium concentration adsorbed by the soil (mg kg-1), cw is the equilibrium concentration in solution (mg l-1), β is an adsorption exponent related to adsorption intensity and kf is the fruendlich adsorption coefficient (l kg -1). chemical analysis soil ph and ph change for caco3 were determined by using cole parmer ph meter. for cec analysis, nh4 + determination was carried out using shimadzu uv–vis spectrophotometer. elemental analysis of fe, al, and total arsenic were determined by using perkin elmer’s icp-ms. statistical analysis the variety of the adsorption parameters were ascribed to the soil parent material at different depths. the multivariate analysis was implemented using proc general linear model in sas version 9.4 (sas institute inc., 2014).27 the parent material and soil (parent material) were class variable and the soil depths were multiple dependent variables. stepwise multiple regression analysis was applied to correlate the adsorption parameters and the soil properties that determine adsorption of as(v) and as(iii) in soils derived from loess and alluvium. results and discussion soil characteristics soils varied in chemical and physical characteristics which are important for as(v) and as(iii) adsorption (table ii). the soils were dominantly silt loam. overall, the alluvium derived kotli soil had larger clay content, followed by the guliana, mansehra and rawal soils. clay leaching and accumulation caused by the soil development processes was observed in bt horizon of all the soils except for the kotli, developed in clayey parent material. most soils were non-calcareous with lower doc. rawal soil under forest conditions showed higher doc content. dissolved organic carbon varied in most soils with higher contents in surface horizons and decreased with depth, in all the soils due to the accumulation of organic material at the surface horizons. most of the soils were non-calcareous except for the rawal soil which had caco3 in the range of 35 to 100 g kg–1 and increased with depth. the similar values for caco3 were reported earlier.28 the cec was in the range of 10 to 30 cmol+/kg. higher cec was observed in the guliana soil with the increasing trend with depth and seemed to increase at bt horizon level in all the soils. amount of citrate bicarbonate dithionate extractable iron seems increased with depth in all the soils. overall, the guliana soil showed greater amount of fe due to soil development process which results in the iron release from primary mineral. in the rawal, guliana and mansehra soils high fe content at bt horizon was due to the translocation and the accumulation at bt horizon level. amount of citrate bicarbonate dithionate aluminum was greater in the loess derived soils, as compared to alluvium derived soils indicating the significant change in al with the change in parent material.29 amount of citrate bicarbonate dithionate aluminum seems to be increased with the soil depth in all the soils. loess derived guliana and mansehra were highly weathered soils, as supported by greater content of fe and al, compared to allu(cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ arsenate and arsenite adsorption parameters 947 vium derived rawal and kotli soils. oxalate-extractable iron also varied with parent material. the loess derived guliana and mansehra soils had higher concentration of fe compared to the alluvial rawal and the kotli soils. high fe content was observed in soils at relatively higher stage of development and accumulated in bt horizon, whereas in the rawal and kotli soils higher fe was observed in the surface horizons due to less leaching.30 high amorphous iron contents in mansehra soils may attribute to high rainfall which reduces the crystallinity while dry climate promote crystallinity.31 oxalate-extractable aluminum was higher in soils at higher stage of development since the weathering processes result in an increase in hydrolytic breakdown and release of iron and aluminum from the primary minerals. oxalate-extractable aluminum content also increased with an increase in the depth. overall iron and aluminum oxides were present in greater amount as a result of weathering in guliana and mansehra soils. total as content varied between 3.4 to 6.92 mg kg–1 in most soil samples. in almost all the soils total as content increase with depth, which may indicate subsurface accumulation. table ii. chemical properties of studied soils; cec, cation exchange capacity; doc, dissolved organic carbon; fed, ald, dithionate extractable fe and al; feo, alo, oxalate-extractable fe and al; mean of n = 3; standard deviation in the parentheses horizon depth, cm ph clay, g kg-1 caco3, g kg-1 cec, cmol+/kg doc, mg kg-1 fed ald feo alo total as, mg kg-1 g kg-1 rawal: fine silty, mixed, hyperthermic, typic hapludalfs a 0–10 7.05 190 34(5.27) 9(0.60) 300(1.23) 5.0(0.33) 1.3(0.08) 0.10(0.02) 0.62(0.02) 3.52(0.05) bw 10–18 7.35 215 35(3.50) 12(2.37) 170(0.40) 6.6(0.40) 1.7(0.10) 0.09(0.04) 0.82(0.07) 3.40(0.10) bt 18–30 7.57 265 53(6.57) 14(1.19) 180(0.55) 9.4(0.33) 2.5(0.10) 0.09(0.00) 1.06(0.01) 3.57(0.13) bk 30–46 7.63 215 102(24.7) 10(1.45) 160(0.41) 8.8(0.38) 2.3(0.06) 0.03(0.02) 0.83(0.04) 3.97(0.07) kotli: fine, mixed, hyperthermic entic chromostert ap 0–10 7.93 345 5.9(0.30) 17(0.24) 150(0.78) 4.9(0.05) 1.4(0.02) 0.40(0.12) 0.69(0.04) 4.17(0.09) bw 10–18 8.40 345 7.3(0.32) 16(2.46) 113(0.10) 5.8(0.29) 1.6(0.06) 0.51(0.02) 0.67(0.04) 3.75(0.20) c 18+ 8.20 445 5.6(0.24) 18(0.10) 46(0.36) 8.7(0.10) 2.6(0.02) 0.24(0.04) 0.79(0.05) 4.43(0.35) guliana: silty, mixed, thermic udic haplustalfs ap 0–10 7.70 210 7.8(0.45) 26(0.65) 75(0.24) 8.0(0.41) 2.4(0.07) 0.21(0.02) 0.91(0.02) 5.21(0.04) bw 1–20 7.37 210 7.8(0.70) 18(0.85) 70(0.48) 7.7(0.40) 2.4(0.10) 0.22(0.05) 0.96(0.05) 5.48(0.18) bt1 20–30 7.48 295 6.4(1.05) 30(0.14) 60(0.08) 9.7(0.23) 3.3(0.06) 0.31(0.01) 1.25(0.04) 6.92(0.07) bt2 30–50 7.50 320 6.1(1.35) 26(1.46) 60(0.67) 9.3(0.38) 3.5(0.15) 0.27(0.01) 1.39(0.10) 6.66(0.08) mansehra: fine loamy, mixed hyperthermic typic hapludalfs ap 0–20 7.00 200 5.6(1.18) 17(0.60) 110 (0.40) 7.2(0.80) 2.0(0.62) 0.53(0.03) 0.91(0.03) 3.87(0.10) bw 20–40 7.10 200 5.2(0.74) 13(0.60) 88(0.21) 5.8(0.29) 2.5(0.07) 0.56(0.04) 0.43(0.03) 3.50(0.05) bt 40–70 7.08 310 6.2(0.17) 22(0.08) 66(0.75) 8.7(0.10) 4.0(0.10) 0.69(0.04) 1.48(0.06) 4.73(0.21) arsenate and arsenite sorption isotherm the isotherms for as(v) and as(iii) sorption depict variation in the shape of isotherms with the diversity in soil characteristics (fig. 1). overall, as(v) ads(cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 948 rukh et al. orption was greater than as(iii). both as(v) and as(iii) had fast rise in sorption, with a small increase in concentration in equilibrium solution. several scientists reported a fast sorption initially and a moderate increase at the later part of the isotherm.9,18 maximum sorption increase for as(v) was less than 150 mg kg–1. the bt horizons, especially in the case of the mansehra and the guliana soils, had faster adsorption rise compared to ap and bw horizons. in case of as(iii), where the concentration maxima was ≈ 30 mg kg–1 generally, had greater rise in the adsorbed concentration in case of bt horizons of all the soils. it appears that the clay content had a stronger role in the adsorption of both as(v) and as(iii). several studies indicated a strong correlation of clay content with the adsorption of as(v) and as(iii).32,33 fig. 1. adsorption isotherms for arsenate and arsenite constructed between adsorbed concentrations at ordinate and solution concentration at abscissa in, indicating a fast initial rise especially in case of bt horizon and the bk horizon of rawal had noteable low in adsorption. freundlich equation sorption parameters freundlich equation (eq. (1)) fit the isotherms with most of the r2 > 0.95 (fig. 2). adsorption of as(v) and as(iii) in these soils was well fitted by the freundlich isotherm model.34 the maximum average adsorption intensity of as(iii) (β) was lower than the adsorption intensity of as(v) in most soils, while the adsorption capacity (kf) was greater for as(v) than as(iii). it appears that as(v) had higher adsorption capacity than as(iii), but with lesser binding intensity than most soils. the distribution of β and kf for as(v) and as(iii) remained similar to the soil depth in both of the parent materials (fig. 3), as the hypothesis of nonsignificant depth–pm interaction was accepted through manova test criteria. fruendlich adsorption parameters increased with soil depth in loess and alluvium. the loess derived soils had higher adsorption parameters (β and kf) than alluvial soils at all soil depths. variation in β and kf of as(v) and as(iii) with the soil depth in each parent material was also remained similar (table iii). (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ arsenate and arsenite adsorption parameters 949 fig. 2. freundlich equation (eq. (1)) fit for arsenate and arsenite isotherms in the selected soil horizons. arsenate and arsenite adsorption parameters were calculated from the trendline. fig. 3. distribution of adsorption parameters (β and kf) of arsenate and arsenite in each parent material (mean = 6, and bars show standard error). (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 950 rukh et al. table iii. fitted freundlich sorption model parameters for arsenate and arsenite of each soil; adsorption intensity, β, and maximum adsorption capacity, kf, both in l kg -1, were calculated from the respective regression equation. values in parentheses are standard deviations horizon arsenate arsenite β kf β kf rawal: fine silty, mixed, hyperthermic, typic hapludalfs a 1.43(0.06) 19(0.34) 2.20(0.02) 13(0.36) bw 1.88(0.01) 35(0.55) 2.93(0.14) 24(0.70) bt 1.82(0.02) 40(0.62) 2.77(0.35) 34(0.80) bk 1.91(0.08) 38(0.76) 2.82(0.25) 29(0.10) kotli: fine, mixed, hyperthermic entic chromosterts ap 1.80(0.06) 35(1.40) 2.07(0.04) 32(0.18) bw 1.81(0.01) 36(0.45) 2.65(0.14) 32(0.29) c 2.18(0.05) 47(1.20) 3.43(0.21) 50(1.68) guliana: silty, mixed, thermic udic haplustalfs ap 1.73(0.03) 34(0.59) 2.74(0.18) 27(2.29) bw 1.90(0.11) 37(0.85) 2.98(0.04) 27(0.42) bt1 2.03(0.11) 53(0.50) 3.43(0.10) 57(0.58) bt2 2.22(0.02) 61(1.61) 3.31(0.13) 66(0.61) mansehra: fine loamy, mixed hyperthermic typic hapludalfs ap 1.74(0.013) 36(0.24) 2.40(0.02) 27(0.20) bw 2.01(0.031) 42(0.38) 3.11(0.12) 37(0.85) bt 2.50(0.05) 75(1.96) 2.61(0.14) 76(1.80) the adsorption intensity ranged from 1.45 to 2.50 and kf of as(v) ranged from 19 to 75 l kg-1 and. these results are in line with the findings of roy et al.,35 payne and abdel-fattah36 and zeng et al.37 arsenate β increased with depth in all the soils with the increase in clay content (r 0.48). as β is the intensity of adsorption, it is related to the abundance of metal oxides. it was evidenced that as(v) had high affinity for metal oxides from positive correlation of β with fed (r 0.77), ald (r 0.80), feo (r 0.42) and alo (r 0.71). similarly, kf, increased with depth with increase in clay content as evidenced by the positive correlation with clay (r 0.46). the cec is largely related to clay content38 because there are more exchange sites for adsorption, so that as(v) kf values positively correlated with cec (r 0.59). the kf had showed strong relation with iron (fed (r 0.83) and feo (r 0.50)) and aluminum (ald (r 0.89) and alo (r 0.87)) oxides as the increase in as(v) kf and in the metal oxides with increase in soil depth. several studies reported the role or iron and aluminum oxides in the as(v) adsorption.13,18 overall, as(v) β and kf were increased linearly with the increase in metal oxide contents. loess derived guliana and mansehra soils, which are more weathered soils, had greater amount of clay in bt horizons and ultimately higher iron and aluminum oxides, resulted in greater adsorption of as(v) when compared to alluvium derived rawal and kotli soils. the adsorption intensity, β, ranged from (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ arsenate and arsenite adsorption parameters 951 2.07 to 3.43 and kf ranged from 13 to 76 l kg–1. the adsorption parameters vary widely with the change in soil properties especially due to the differences in iron oxides.39 the β values showed non-significant relation with the metal oxides indicating the weak binding of as(iii) on the metal oxide surfaces. however, as(iii) β was positively related with clay content (r 0.38) and cec (r 0.36). the adsorption capacity of as(iii) was higher in weathered horizons of the guliana and the mansehra soils while β values are greater in bw/bt horizons of all the soils compared to remaining horizons. the kf for as(iii) increased with depth in all the soils except for the kotli soil. similar values for kf of as(iii) were observed by elkhatib et al.39 in loamy soils. arsenite kf showed similar behaviour as as(v) in the positive correlation with fed (r 0.77), ald (r 0.87), feo (r 0.46) and alo (r 0.82). the bt horizons of the guliana and the mansehra soil had higher kf values due to the higher clay and metal oxides content. prediction of adsorption parameters we selected the soil properties (independent parameters) by the stepwise regression maximizing r2 and predicted the adsorption parameters of as(v) and as(iii) obtained from the fitting of the isotherm to the fruendlich equation. soil was included as an independent parameter. intercept differed with the soil type in case r2 improves significantly, due to the addition of a soil type as a variable. the multiple regression equations have more significance in the understanding of processes rather than as predictive tools. the regression equations for as(v) and as(iii) adsorption parameters are given in the table iv. it is apparent that β for as(v) is positively related with ald while negatively correlated with cec, caco3, which confirms the observation of raven et al.18 that the adsorption intensity was more related to metal oxides. freundlich coefficient related to the adsorption capacity kf in all soils was more related to feo and alo as compared to fed and ald.40 however, β for as(iii) is suppressed by aluminum oxides (ald and alo) whereas it is increased in the presence of iron oxides and cec related to clay content.38 the regression equation for as(iii) kf showed that the adsorption capacity of as(iii) increases with the increase in ald, whereas it was suppressed by the increase in caco3 and cec. table iv. regression equations for different properties estimate for each adsorption parameter arsenic form regression equations r2 arsenate β = 3.63 + 12.24ald – 89.92cec – 7.76caco3 – 0.26clay 0.82 kf = 236.33 + 17.80feo – 72.44alo 0.15 arsenite β = 1.10 + 0.0055feo – 0.052alo – 0.021ald + 0.23cec 0.57 kf = 21.77 + 8.66ald – 66.50cec – 5.21caco3 – 0.22clay 0.82 (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 952 rukh et al. conclusions from our results, it can be concluded that as(v) adsorption was greater than as(iii) in all the soils and varied with the soil properties, among which clay content and iron and aluminum oxides exhibited the most important influence on as(v) and as(iii) adsorption. freundlich isotherms fit as(v) and as(iii) sorption data well with r2 values of 0.88–0.98 in most soils. loess soils had higher adsorption capacity than alluvial soils. metal oxides and clay contents are the major predictor for as(v) and as(iii) adsorption parameters. this study may help to understand the soil and groundwater contamination phenomena. acknowledgment. the authors thank the higher education commission of pakistan for financial assistance. и з в о д утицај хемијских својстава лесних и алувијалних земљишних наслага на адсорпцију арсената и арсенита shah rukh1, mohammad saleem akhtar1, ayaz mehmood2, sayed hassan3, khalid s. khan1, syed m. s. naqvi4 и muhammad imran1 1department of soil science & swc, pmas-arid agriculture university rawalpindi-46000, pakistan, 2department of agricultural sciences, university of haripur, haripur-22620, pakistan, 3department of crop and soil sciences, university of georgia, athens-30602, ga, usa и 4institute of biochemistry and biotechnology, pmas-arid agriculture university rawalpindi-46000, pakistan арсен је један од најтоксичнијих загађивача земљишта. познавање адсорпционе хемије арсена је суштински важно за одређивање степена загађења земљишта и подземних вода. представљена су истраживања помоћу којих је одређена промена адсорпционог понашања арсената и арсенита са литосферном дубином. узоркована су два матична материјала кроз различите типове земљишних наслага, а у оквиру та два одабрана су по два узорка земљишта. уз серијско лабораторијско испитивање адсорпције арсената и арсенита, урађена је и основна карактеризација земљишта. примењен је фројндлихов приступ анализи адсорпционих процеса. фројндлихова изотерма задовољавајуће описује сорпционе податке за арсенат и арсенит, са r2 вредностима у опсегу 0,88– –0,98 за већину узорака земљишта. адсорпциони параметри зависе од својстава земљишта, посебно од садржаја глине и оксида гвожђа и алуминијума. ови параметри су се мењали и у зависности од дубине слоја у оквиру матичног материјала, и закључено је да лесно-заснована земљишта поседују већи адсорпциони капацитет од алувијалних. (примљено 9. фебруара, ревидирано 18. марта, прихваћено 20. марта 2017) references 1. h. garelick, h. jones, a. dybowska, e. valsami-jones, rev. environ. contam. toxicol. 197 (2008) 17 2. e. moreno-jiménez, j. m. peñalosa, e. esteban, m. p. bernal, j. environ. monit. 11 (2009) 1375 3. g. t. vaughan, the environmental chemistry and fate of arsenical pesticides in cattle dip sites and banana plantations, csiro division of coal industry, center for advanced analytical chemistry, nsw, melbourne, 1993, p. 123 (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ arsenate and arsenite adsorption parameters 953 4. a. giacomino, m. malandrino, o. abollino, m. velayutham, t. chinnathangavel, e. mentasti, environ. pollut. 158 (2009) 416 5. x. c. le, x. lu, m. ma, w. r. cullen, v. aposhian, b. zheng, anal. chem. 72 (2000) 5172 6. c. k. jain, i. ali, wat. res. 34 (2000) 4304 7. c. j. ng, n. barry, s. bruce, h. crawley, m. r. moore, in proceedings of the fifth national workshop on the assessment of site contamination, australia, ephc, adelaide, 2003, p. 163 8. p. o’neill, in heavy metals in soil, b. j. alloway, ed., 2nd ed., blackie academic and professional, london, 1995, p. 105 9. h. zhang, h. m. selim, environ. sci. technol. 39 (2005) 6101 10. m. sadiq. water, air soil pollut. 93 (1997) 117 11. v. matera, i. l. hecho, a. laboudigue, p. thomas, s. tellier, m. a. astruc, environ. pollut. 12 (2003) 51 12. b. j. lafferty, r. h. loeppert, environ. sci. technol. 39 (2005) 2120 13. h. j. shipley, s. j. yean, a.t. kan, m. b. tomson, environ. toxicol. chem. 28 (2009) 509 14. a. mehmood, r. hayat, m. wasim, m. s. akhter, j. agri. biol. sci. 1 (2009) 59 15. j. alvarez-benedi, s. bolado, i. cancillo, c. calvo, d. garcia-sinovas, vadose zone j. 4 (2005) 282 16. s. goldberg, soil sci. soc. am. j. 66 (2002) 413 17. s. kundu, a. k. gupta, chem. eng. j. 122 (2006) 93 18. k. raven, a. jain, r. loeppert, environ. sci. technol. 32 (1998) 344 19. r. h. leoppert, c. t. hallmark, m. m. koshy, soil sci. soc. am. j. 48 (1984) 1030 20. d. w. nelson, l. e. sommers, in methods of soil analysis. part 2. chemical and microbiological properties, a. l. page, r. h. miller, d. r. keeney, eds., asa, madison, wi, 1982, p. 574 21. o. p. mehra, m. l. jackson, clay mineral 7 (1960) 317 22. h. d. chapmen, in methods of soil analysis. part 2. chemical and microbiological properties, c. a. black,ed., asa, madison, wi, 1965, p. 891 23. m. l. jackson, c. h. lim, l. w. zelazny, in methods of soil analysis. part 1, a. klute, ed., asa, madison, usa, 1986, p. 101 24. c. l. castro, d. l. rolston, soil sci. soc. am. j. 41 (1977) 1085 25. j. k. syers, r. f. harris, d. e. armstrong, j. environ. qual. 2 (1973) 1 26. h. freundlich, kapillarchemie. eine darstellung der chemie der kolloide und verwandter gebiete, akademische verlagsgellschaft, leipzig, 1909 27. sas institute inc. 2003. sas version 9. cary (nc): sas institute inc. 28. r. amin, m. ikram, keys to soils of punjab and sindh. soil survey of pakistan, multan road, lahore, 2007 29. a. mehmood, phd thesis, pmas-arid agriculture university, rawalpindi, 2014 30. h. jenny, factors of soil formation, mcgraw-hill, new york, 1941 31. a. mehmood, m. s. akhtar, r. hayat, m. memon, j. chem. soc. pak. 32 (2010) 129 32. n. ghorbanzadeh, w. jung, a. halajnia, a. lakzian, a. n. kabra, b. jeon, geosystem eng., doi: 10.1080/12269328.2015.1062436 33. wu, p., w. wu, s. li, n. xing, n. zhu, p. li and z. dang, j. haz. mat. 169 (2009) 824 34. q. feng, z. zhang, y. chen, l. liu, z. zhengjie, c. chen, procedia env. sci. 18 (2013) 26 35. w. r. roy, j. j. hassett, r. a. griffin, soil sci. soc. am. j. 50 (1986) 1176 36. k. b. payne, t. m. abdel-fattah. j. environ. sci. health 40 (2005) 723 37. x. zeng, p. wu, s. su, l. bai, q. feng, plant soil environ. 58 (2012) 405 (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 954 rukh et al. 38. h. l. bohn, b. l. mcneal, g. a. o'connor, soil chemistry, 2nd ed., john wiley & sons, new york, 1985 39. e. a. elkhatib, o. l. bennett, r. j. wright, soil sci. soc. am. j. 48 (1984) 1025 40. w. jiang, s. zhang, x. shan, m. feng, y. zhu, r. mclaren, environ. pollut. 138 (2005) 278. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {immobilization of maltase from saccharomyces cerevisiae on thiosulfonate supports} j. serb. chem. soc. 81 (12) 1371–1382 (2016) udc 633.6+547.543.05:547.458.2: jscs–4934 547.455.623 original scientific paper 1371 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. immobilization of maltase from saccharomyces cerevisiae on thiosulfonate supports mladen mihailović1, jovana trbojević-ivić2, katarina banjanac1, nenad milosavić2, dušan veličković2, milica carević1* and dejan bezbradica1 1department of biochemical engineering and biotechnology, faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11000 belgrade, serbia and 2department of biochemistry, faculty of chemistry, university of belgrade, studentski trg 12, 11000 belgrade, serbia (received 30 july, revised 14 october, accepted 19 october 2016) abstract: in this study, two commercial supports (eupergit® c and purolite® a109) were chemically modified in order to introduce thiosulfonate groups, which could subsequently exclusively react with the cysteine residues on the surface of enzymes. thereafter, the maltase from saccharomyces cerevisiae was immobilized onto the obtained thiosulfonate-activated supports, resulting in high expressed enzymatic activities (around 50 %), while on the other hand, immobilization on unmodified supports yielded expressed activities less than 5 %. moreover, protein loadings up to 12.3 mg g-1 and immobilized activities up to 3580 iu g-1 were achieved by employment of these thiosulfonate supports. desorption experiments, performed on samples taken during immobilization, proved that immobilization on the thiosulfonate supports was the first step of fast adsorption onto the supports and the formation of covalent bonds between the thiosulfonate groups and the thiol groups of cysteine represented a second slower step. more importantly, although enzyme coupling occurred via covalent bond formation, the performed immobilization proved to be reversible, since it was shown that 95 % of the immobilized activity could be detached from the support after treatment with a thiol reagent (β-mercaptoethanol). thus, the support could be reused after enzyme inactivation. keywords: α-glucosidase; covalent disulfide bridges; eupergit® c; purolite® a109. introduction maltase (α-glucosidase, e.c 3.2.1.20) catalyses hydrolysis of α-(1→4) glycosydic bonds between two glucose moieties, with a primary biological role to * corresponding author. e-mail: mcarevic@tmf.bg.ac.rs doi: 10.2298/jsc160730099m 1372 mihailović et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. hydrolyse maltose to glucose, thus enabling its prosperous employment in food processing, brewing, distilling and pharmaceutical industries. nowadays, maltase becomes even more attractive since it can catalyse the reaction of transglucosylation, provided efficient glucosyl acceptor molecules (various hydroxylated compounds) are present in reaction mixture. this corresponds to the contemporary trend of the utilization of the potential of transglycosylation of various glycosidases in the synthesis of bioactive glycosides.1–4 in previous studies, it was proven that maltase from saccharomyces cerevisiae is a very powerful catalyst since it can be used for the synthesis of glucosides with physiological activity using various aglycons (hydroquinone, pand m-hydroxybenzyl alcohol and vanillyl alcohol), and it exhibits high stability in organic solvents.5–8 rising interest for the application of this enzyme invokes the development of adequate immobilization protocols and the opportunity for multiple use and application in continuous immobilized enzyme reactors. hitherto, maltase from s. cerevisiae has been immobilized by encapsulation in polygalacturonic beads coated with chitosan,9 covalent immobilization via the oxirane groups on poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [poly(gma-co-edgma)],10,11 via the carbonyl groups on glutaraldehyde-activated sepabead® ec-ea support12 or on cyanogen bromide activated poly(2-hydroxyethylmethacrylate).13 the goal of this study was to evaluate supports bearing the thiosulfonate group in the immobilization of maltase from s. cerevisiae. thiosulfonate moieties react only with the cysteine residues on the enzyme surface and form stable covalent disulfide bridges.14–16 such a support seems to be a good candidate for immobilization of maltase, since the enzyme activity rapidly diminishes unless a thiol-containing compound is present in solution, plausibly because it prevents unwanted conformational rearrangements.17 hence, it seems that unpaired cysteine residues are susceptible to reactions that cause loss of the catalytic conformation of the enzyme and this could be avoided by immobilization on thiosulfonate supports. in addition, it provides the additional advantage of more site-directed immobilization than supports previously applied in maltase immobilization since oxirane or carbonyl groups react with a significantly wider range of amino acid residues.18 thiosulfonate supports were obtained by chemical modification of two commercial supports: eupergit® c, a copolymer of methacrylamide, n,nʹ -methylenebis(acrylamide) and a monomer carrying oxirane groups, and purolite® a109, a styrene–divinylbenzene copolymer with primary amine functional groups (table i). table i. the properties of eupergit® c and purolite® a10919,20 support functional group particle size, μm pore size, nm surface area, m2 g-1 eupergit® c epoxy 150–300 25–250 180 purolite® a109 primary amine 425–1000 4.5 38 maltase immobilization on thiosulfonate supports 1373 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. chemistry of the supports and their activation are depicted in fig. 1. the first step of purolite® a109 modification was the introduction of the epoxy group with epichlorohydrin, while further steps are mutual for the activation of both supports and result in thiosulfonate groups that react with cysteine residues on the enzyme surface. scheme 1. the chemistry of thiosulfonate modification of eupergit® c and purolite® a109. 1374 mihailović et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. different supports were applied in order to evaluate the eventual effects of the support surface and particularly two supports (eupergit® c and purolite® a109) were chosen bearing in mind their exceptional thermal and chemical stability and favorable mechanical and hydrodynamical features for application in different bioreactor configurations. moreover, their non-toxicity and biocompatibility ensures their safe application in the food and pharmaceutical industry. high activities and stabilities have already been achieved with these supports in the immobilization of various enzymes,2,20–25 including maltase.10 experimental the supports used in this study, purolite® a109 and eupergit® c were purchased from purolite (philadelphia, pa, usa) and sigma–aldrich, respectively. maltase used throughout the study was isolated from saccharomyces cerevisiae by a previously described procedure.5 reagents for activity and protein concentration assays: p-nitrophenyl-α-d-glucopiranosyde (p -npg), coomassie brilliant blue g-250 and bovine serum albumin (bsa) were purchased from sigma–aldrich, and all the other chemicals were of analytical grade and obtained from centrohem (stara pazova, serbia), unless specified otherwise. all experiments were performed in duplicate and the average values are presented in the figures throughout the paper. preparation of epoxy-activated supports (epoxy-purolite) in order to introduce epoxy groups on the surface of purolite® a109, a previously described modification method was applied.20 the obtained epoxy-activated purolite® a109 (epoxy-purolite) was used directly for enzyme immobilization, but it was also further functionalized with purpose of introducing thiosulfonate groups on its surface. preparation of thiosulfonate-activated supports (ts-purolite and ts-eupergit) for the introduction of thiosulfonate reactive groups on the surface of the epoxy-purolite and eupergit® c, a two-step procedure was performed. the first step comprised the introduction of thiol groups on the surface of the supports, using a slightly modified method previously described by axen et al.26 briefly, 1 g of support (epoxy-purolite or eupergit® c) was equilibrated with sodium phosphate buffer (0.5 m; ph 6.3), and then filtered through a sintered glass filter. subsequently, the suction dried support was suspended in 1 ml of the same buffer and 1 ml of 2 m sodium thiosulfate solution was added. the suspension was placed on an orbital shaker at 70 rpm and 22 °c for 6 h, and then the suspension was filtered and washed with an excessive amount of distilled water. then 1 g of suction dried support was resuspended in 1 ml of sodium bicarbonate buffer (0.2 m; ph 8.5) and dithiothreitol (dtt) solution (prepared by dissolving 200 mg of dtt in 1 ml of 1 mm ethylenediaminetetraacetic acid (edta)) was added. the reaction mixture was placed on an orbital shaker at 100 rpm and 22 °c for 1 h. when reaction was completed, the obtained thiol-support (thiol -purolite or thiol-eupergit) was filtered through a sintered glass filter, and washed with sodium bicarbonate buffer (0.2 m; ph 8.5), distilled water and 0.1 m acetic acid to eliminate any residual dtt. in the second step, the thiol groups of the supports (thiol-purolite or thiol-eupergit) were converted to thiosulfonate groups.27,28 thus, 1 g of suction dried thiol-support was suspended in 3 ml of sodium acetate (0.2 m at ph 5.0) and then 120 μl of 30 % hydrogen peroxide was added. the reaction mixture was placed on an orbital shaker at 100 rpm and 22 °c. during the maltase immobilization on thiosulfonate supports 1375 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. course of reaction, aliquots of 150 μl of 30 % hydrogen peroxide were added to the reaction mixture after 0.5 h, 1.5 h and 2.5 h. total reaction time should be no longer than 30 h. when the reaction was completed, the obtained thiosulfonate-supports (ts-purolite or ts-eupergit) were filtered and washed with 0.1 m acetic acid until the absence of peroxide. the ts-supports could be used immediately or stored in sodium acetate (0.2 m; ph 5.0) at 4 °c until use. immobilization of maltase immobilization of maltase was performed onto several unmodified and modified supports (purolite® a109, eupergit® c, epoxy-purolite, ts-eupergit and ts-purolite). the support (0.1 g) was equilibrated with 10 mm phosphate buffer (ph 6.8) and resuspended in 2 ml of maltase solution that was prepared in the same buffer so that 5 mg of proteins per g of support was offered. these suspensions were incubated on an orbital shaker at 25 °c and 100 rpm for 48 h. at different intervals, aliquots of the suspensions were removed and centrifuged for 90 s at 10,000 rpm. the separated immobilized preparations were washed three times with phosphate buffer (10 mm; ph 6.8), centrifuged at 10,000 rpm and then used for the determination of the maltase activity. the supernatants were sequestered for protein and maltase activity determination. for two selected supports (ts-purolite and ts-eupergit), which exhibited the best characteristics during preliminary screening, optimization of immobilization process was performed as follows: 0.05 g of equilibrated support was resuspended in 1 ml of maltase solutions prepared by dissolving different amounts of protein in 10 mm phosphate buffer (ph 6.8) so that 1–100 mg of proteins per g of support was offered. the obtained immobilized preparations were washed, centrifuged and further used as described in the previous paragraph. protein assay protein concentration was determined according to the bradford method using bovine serum albumin as the standard protein.29 the amounts of bound enzyme were determined indirectly from the difference between the amount of enzyme introduced into the immobilization process and the residual amount of the enzyme in the supernatant after the immobilization. protein immobilization yield (piy) was calculated using the equation: proteins in blank sample proteins in supernatan t after immobilization proteins in blank sample − =piy (1) blank sample represents the control sample of enzyme solution without an immobilized support, exposed to the same conditions as the immobilized samples during the immobilization procedure in order to eliminate external factors for eventual activity loss. maltase activity assay the activity of free and immobilized maltase was determined using p-nitrophenyl-α-d -glucopiranosyde (p-npg) as substrate.30 the substrate solution was prepared by dissolving 0.3 mg ml–1 p-npg in phosphate buffer (50 mm; ph 6.8) and 1 mm β-mercaptoethanol (2-sulfanylethanol). for the soluble enzyme, 0.2 ml of substrate solution was mixed with 0.005 ml of free enzyme sample. the enzyme activity was calculated from the slope of the p-nitrophenol (p-np) calibration curve. in the assay with immobilized enzyme, β-mercaptoethanol was omitted from the substrate solution. the assay mixture for immobilized maltase consisted of 0.005 g of immobilized preparation suspended in 0.4 ml of substrate solution. liberation of p-np was monitored at 405 nm in 30 s intervals for 2 min. 1 iu of enzyme activity was defined as the amount of enzyme that liberates 1 µmol of p-np at 25 °c in 1 min 1376 mihailović et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. from p-npg. the immobilization yield (iy), the expressed activity (ea) and the specific activity (sa) were calculated using the following equations: activity in blank sample activity in supernatant after immobilization 100 activity in blank sample − = ×iy (2) activity of immobilized preparation 100 ( /100) activity in blank sample ea iy = × × (3) activity of immobilized preparation bound proteins =sa (4) elution of maltase from ts-purolite nacl treatment. samples of the immobilized enzyme were taken after 3, 9 and 15 h of immobilization under optimum conditions. the wet immobilized preparations were resuspended in 1 m nacl and incubated on a thermoshaker at 600 rpm and 25 °c for 2 h. the activities of immobilized maltase and activities in the supernatants prior and after the nacl treatment were determined by the maltase activity assay. in addition, the protein contents in supernatants before and after treatment were determined by the protein assay. β-mercaptoethanol treatment. maltase immobilized on ts-purolite was treated with 1 m β-mercaptoethanol on a thermoshaker at 600 rpm and 25 °c for 10 h. the maltase activity and protein content in the supernatant and the activity of the immobilized preparations were determined by the activity and protein assays prior and after the β-mercaptoethanol treatment. results and discussion selection of immobilization support in a preliminary experiment, maltase from s. cerevisiae was immobilized on five supports differing with respect to the polymeric scaffolds and functional groups (primary amine, epoxy or thiosulfonate) in order to provide an adequate evaluation of the efficiency of the immobilization via cysteine residues. the obtained results of the protein immobilization yield indicated that the supports with epoxy groups enabled higher protein loadings than their thiosulfonate derivatives (table ii). the differences were more prominent with purolite as the support, since the second modification step resulted in a decrease of the protein immobilization yield from 88.8 to 62.1 %. unmodified purolite, the only support with amino functional groups, showed the lowest piy. with respect to iy, the differences between the results obtained with epoxyand thiosulfonate-supports were insignificant, since yields above 90 % were achieved. as the immobilization yield expresses the decrease of maltase activity in the supernatant, such a trend indicates that both functionalities have high affinity towards maltase immobilization. enzyme attachment is not always useful because its orientation and activity is influenced by the type of functional group on the enzyme surface, and hence for future use, immobilized enzymes need to be evaluated through the expressed activity.31–34 hugely higher expressed activities (54.6 % for eupergit and 48.9 % for purolite) were achieved with thiosulfonate supports than with epoxy supports maltase immobilization on thiosulfonate supports 1377 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. (only 4.6 % for eupergit and 1.5 % for purolite). such a trend indicates that maltase retains a higher portion of its initial activity if attached exclusively via cysteine residues on thiosulfonate supports, than in case of immobilization on epoxy supports, which can occur via both thiol and amino groups on the protein surface.18 table ii. the efficiency of maltase immobilization on different supports support piy / % iy / % ea / % sa / iu g-1 protein purolite® a109 55.2 75.1 3.1 0.97 eupergit® c 96.4 100 4.6 1.10 purolite-epoxy 88.8 91.2 1.5 0.35 ts-eupergit 89.3 98.3 52.3 8.16 ts-purolite 62.1 97.1 44.6 8.83 since the immobilization yields were similar in all experiments, provided that almost complete activity disappeared from the supernatant, lower activity yields measured on support indicate that attachment on epoxy supports occurred predominantly through basic amino acid residues (mostly lysine residues), which resulted in a less active enzyme conformation. furthermore, in terms of evaluating maltase immobilization selectivity towards the examined supports, the specific activities of different immobilized preparations were compared (table ii). the highest specific activities were obtained for the thiosulfonate supports (8.16 iu g–1 proteins for eupergit and 8.83 iu g–1 proteins for purolite). therefore, it could be concluded that maltase showed particular affinity towards the thiosulfonate supports. optimization of the immobilization on the thiosulfonate supports therefore, further experiments were focused on optimization of the immobilization on the thiosulfonate supports. the kinetics of the immobilization is depicted in fig. 1. immobilization on the thiosulfonate supports is very slow process, since a constant linear increase in the loaded protein occurred throughout 48 h. however, it is obvious that only initial 24–30 h of immobilization are productive with respect to activity of maltase, since maximum iy was reached and it seems that subsequently, only futile attachment of proteins in an inactive conformation occurred. hence, the optimum immobilization times were 24 h for ts-purolite and 30 h for ts-eupergit and all further experiments were performed at these immobilization times. subsequent experiments were performed to determine the capacity of the thiosulfonate supports by varying the concentrations of offered protein in the range 10–100 mg per g of dry support. the maximum was reached at offered protein concentration of 60 mg g–1 and further increases did not result in any improvements (fig. 2). similar protein loadings (12.1 and 12.3 mg g–1) and immo 1378 mihailović et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. fig. 1. immobilization kinetics on ts-purolite (triangles) and ts-eupergit (squares). immobilization yield is presented with filled symbols and protein immobilization yield with hollow symbols. bilized activities (3520 and 3580 iu g–1) were achieved with ts-eupergit and ts-purolite, respectively. when the obtained capacities are compared with those achieved using the other immobilization supports, namely purolite (35 mg g–1 support), purolite-epoxy (20 mg g–1 support) and eupergit c (90 mg g–1 support), it is evident that the immobilizations predominantly occurred via thiosulfonate fig. 2. the effect of the offered protein concentration on maltase immobilization on ts-purolite (triangles) and ts-eupergit (squares). activities are presented with filled symbols and protein loadings with hollow symbols. maltase immobilization on thiosulfonate supports 1379 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. moieties, resulting in highly active and favorable enzyme conformations. moreover, bearing in mind that both thiosulfonate supports exhibited similar results, it could be concluded that the surface of the supports did not influence the immobilization process. study of the immobilization mechanism the results of immobilization onto the unmodified supports (table ii) indicated that significant fractions of the proteins (approximately 50 %) were attached onto the supports, although with low activity yield and hence adsorptive forces between the enzyme and the support scaffolds could not be disregarded. covalent immobilization via thiosulfonate groups is a very slow process and hence fast enzyme adsorption inevitably occurred during the immobilization. however, the adsorption is also a reversible process and the formation of covalent bonds is irreversible under the examined circumstances, and overall immobilization is the sum of these competitive processes. it is plausible that during immobilization, adsorbed molecules gradually form covalent bonds with thiosulfonate moieties when in an appropriate conformation and covalent immobilization becomes predominant in the later stages. in order to test this hypothesis, desorption of immobilized enzymes obtained at different stages of immobilization (3, 9 and 15 h) with 1 m nacl was performed. obviously (fig. 3), in the initial stage of immobilization (3 h), a significant drop in the retained activity (38 %) was obtained by desorption, but during the course of immobilization, it steadily decreased to only 6 % of the desorbed activity. therefore, it was confirmed that the adsorbed enzyme molecules gradually formed covalent bonds with thiosulfonate groups and a stable covalently immobilized catalyst was obtained. finally, in order to confirm that immobilization occurred via cysteine residues, the immobilized enzyme was treated with the competitive thiol reagent, fig. 3. enzyme desorption with 1 m nacl. desorbed maltase activity is depicted with black bars and desorbed proteins with gray bars. 1380 mihailović et al. available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. β-mercaptoethanol. after treatment, 98 % of the proteins and 95 % of the immobilized maltase activity had been released into the supernatant, which is confirmation that immobilization occurred almost exclusively through the formation of covalent bonds between thiol groups of cysteine. more importantly, it also indicates that immobilization is reversible, hence enzyme could be liberated and the support used repeatedly, which significantly reduces the costs of immobilization and enables the use of more expensive immobilization supports. thermal stability major drawback concerning enzyme employment in industrial bioprocesses is the possibility of thermal inactivation, due to the partial unfolding of the enzyme structure. therefore, in terms of assessing the potential application of the obtained immobilized preparations (ts-purolite and ts-eupergit) on the industrial scale, the thermal stability of free and immobilized maltase was determined at 35 and 45 °c. the results (fig. 4) clearly indicated that the immobilized enzymes showed moderately higher stability under the examined conditions in comparison to free maltase. as expected, the benefits of immobilization were more pronounced at the higher temperature, confirming that immobilization enables improvement of the rigidity of the tertiary structure of the enzyme and thus prevents its unfolding during heat treatment. conclusions in this study, it was undoubtedly shown that thiosulfonate-supports provide favorable chemistry for the immobilization of maltase from s. cerevisiae. the fact that similar activities and protein loadings were achieved with two supports fig. 4. thermal stability of free maltase (circles) and maltase immobilized on ts-purolite (triangles) and ts-eupergit (squares) at 35 (filled symbols) and 45 °c (hollow symbols). maltase immobilization on thiosulfonate supports 1381 available on-line at: www.shd.org.rs/jscs (cc) 2016 scs. with significantly different polymer scaffolds indicates that this approach could be successfully applied to a wide range of immobilization supports with hydroxyl, amino and epoxy groups. finally, a simple method for the regeneration of the support using a competitive thiol agent indicates that even expensive immobilization supports could be used for maltase immobilization, since they could be reused after inactivation of the immobilized enzyme. acknowledgement. this research is performed within project iii 46010 funded by the ministry of education, science and technological development of the republic of serbia. и з в о д имобилизација малтазе из sаccharomyces cerevisiae на тиосулфонатне носаче младен михаиловић1, јована трбојевић-ивић2, катарина бањанац1, ненад милосавић2, душан величковић2, милица царевић1 и дејан безбрадица1 1катедра за биохемијско инжењерство и биотехнологију, технолошко–металуршки факултет, универзитет у београду, карнегијева 4, 11000 београд и 2катедра за биохемију, хемијски факултет, универзитет у београду, студентски трг 12, 11000 београд у овој студији извршена је хемијска модификација површина два комерцијална носача за имобилизацију ензима (purolite® a109 и eupergit® c) са циљем увођења тиосулфонатних група, које би потом искључиво реаговале са остацима цистеина на површини ензима. на модификоване носаче имобилисана је малтаза из saccharomyces cerevisiae. том приликом је утврђено да је модификацијом површине носача омогућено постизање високих приноса ензимске активности (око 50 %), док су са друге стране, приноси активности у случају немодификованих носача биле мање од 5 %. концентрација везаних протеина и добијене активности ензима имобилисаног на тиосулфонатне носаче износиле су 12,3 mg g-1 и 3580 iu g-1, редом. у експериментима који су подразумевали десорпцију ензима са носача, рађеним на узорцима у различитим фазама имобилизације, доказано је да се имобилизација на тиосулфонатне носаче одиграва у два корака. први корак подразумева фазу брзе адсорпције ензима на носач са тиосулфонатним групама, док је други корак спорији и подразумева формирање ковалентне везе између тиосулфонатних група носача и тиолних група остатака цистеина на површини ензима. такође, важно је истаћи да поред формирања стабилних ковалентних веза, ова имобилизација поседује значајне предности, имајући у виду да је експериментално показано да је реверзибилна, односно да се значајан део везане ензимске активности (95%) може десорбовати са носача након третмана са тиолним реагенсом (β-меркаптоетанол) што омогућава поновну употребу носача након инактивације ензима. 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fernandez-lafuente, process biochem. 47 (2012) 1220 32. d. i. bezbradica, c. mateo, j. m. guisan, j. mol. catal., b: enzym. 102 (2014) 218 33. k. banjanac, m. mihailović, n. prlainović, m. stojanović, m. carević, a. marinković, d. bezbradica, j. chem. technol. biotechnol. 91 (2016) 439. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {continuous flow synthesis of some 6and 1,6-substituted-4-methyl-3-cyano-2-pyridones} j. serb. chem. soc. 84 (6) 531–538 (2019) udc 66.017–033.5:539.24:547.491’824+621.039 jscs–5206 original scientific paper 531 continuous flow synthesis of some 6and 1,6-substituted 3-cyano-4-methyl-2-pyridones julijana tadić1#, marina mihajlović1, mića jovanović2 and dušan mijin2*# 1innovation center, faculty of technology and metallurgy, karnegijeva 4, 11120 belgrade, serbia and 2faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11120 belgrade, serbia (received 3 july, revised 24 october, accepted 29 october 2018) abstract: in this study, six 6and 1,6-substituted-3-cyano-4-methyl-2-pyridones were synthesized in a continuous flow microreactor system. the syntheses were realized at room temperature and the obtained results were compared to those achieved within classical syntheses. in order to optimize the continuous flow syntheses and increase the yield of the products, the retention time in the microreactor was varied by changing the flow rates of the reactant solutions. furthermore, the reaction was optimized for 3-cyano-4,6-dimethyl-2 -pyridone and 3-cyano-6-hydroxy-4-methyl-2-pyridone, which are comercially important in the pharmaceutical and dye industries. both 2-pyridones were obtained in satisfactory yield of circa 60 % in less than 10 min. the resulting compounds were characterized by their melting points, ft-ir, 1h-nmr and uv–vis spectra. the efficiency of the presented method for the synthesis of 2-pyridone-based molecules has promising potential for industrial production. keywords: microreactor; 2-pyridone; batch; flow synthesis; process intensification. introduction the continuous flow method has gained much attention in the field of organic synthesis.1,2 on the laboratory scale, a reaction in continuous flow is typically run in a capillary microreactor, which represents the mixing and reacting components of the continuous flow assembly.3 the specific characteristics of a microreactor are more efficient mass and heat transfers because of the large surface to volume ratio, which allows improved productivity with respect to a batch system.4,5 furthermore, automated process control and facilitated * corresponding author. e-mail: kavur@tmf.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc180703092т ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 532 tadić et al. scale-up reduce the costs and time needed to transfer the process from the laboratory to the industrial level.6 in the pharmaceutical industry, 3-cyano-2-pyridone and its derivatives are significant final products and intermediates in the synthesis of complex biologically active compounds. more specifically, the 2-pyridone structure can be found in various pharmaceutical products mentioned hereafter.7 commercially important cardiotonics used for the therapy of heart failure, milrinone and amrinone, possess a 2-pyridone core.8 antibiotics based on the 2-pyridone moiety (e.g., pilicides) could be applied in the treatment of bacterial infections caused by gram-negative bacteria.9 the 2-pyridone core has a significant role in the industrial production of disperse azo dyes.10,11 moreover, recent studies have shown that azo dyes could be synthesized in a continuous flow microreactor system, in high yields, indicating that this technology has great potential in organic synthesis.12 due to strong biological activity and wide commercial application in industry, the synthesis of 2-pyridone and its derivatives have been widely investigated.7 the general procedure for obtaining substituted 3-cyano-2-pyridones is in a condensation reaction of 1,3-dicarbonyl compounds with cyanoacetamide in the presence of various catalysts, typically in polar solvents, and external heating.7 the syntheses of these molecules were mostly studied in a classical batch system and under a microwave assisted method under different reaction conditions.13,14 microwave -assisted synthesis provides outstanding results in terms of yield and purity of the desired product,13 but the deficiencies of this method are difficulty in scale-up and transition into an industrial level of production. furthermore, traditional and microwave-assisted syntheses of 2-pyridone-based molecules include heating of the reaction mixture, either by conventional heating (e.g., oil or water bath) or microwave heating. altogether, the disadvantages of the classical synthetic method, such as the application of high temperature, prolonged reaction time, complicated process control and additional solvent consumption for purification of the final product, could be overcome by using a microreactor assembly where the synthesis is performed at the room temperature and the products are obtained in a relatively short period in satisfactory yield. in this paper, a novel approach to the synthesis of six 6and 1,6-substituted 3-cyano-4-methyl-2-pyridones was realized in a continuous flow system at room temperature. the obtained results were compared in terms of yields and reaction times to those achieved in a batch system. more specifically, the synthesis was optimized for derivatives 1 and 2, which were obtained in good yields of circa 60 % in less than 10 min. the chemical structure and purity of the resulting products were confirmed by their melting points, and ft-ir, 1h-nmr and uv–vis data. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. continuous flow synthesis 533 experimental general all starting materials were obtained from merck or fluka, and were used without further purification. the synthesis of six 6and 1,6-substituted 3-cyano-4-methyl-2-pyridones was performed in a continuous flow system, at the room temperature, and in a batch system (using heating and stirring). in the continuous flow, 6and 1,6-substituted 3-cyano-4-methyl-2-pyridones were obtained from the corresponding 1,3-dicarbonyl reagents and n-substituted cyanoacetamides, using naoh as a catalyst, in a methanol/water mixture (scheme 1). in the batch system, the same six 2-pyridones were synthesized according to modified literature procedures;13 details are given in the supplementary material to this paper. the efficiency of the continuous flow assembly was investigated by comparing reaction times and yields of the resultant products to those of the classical method of synthesis. compound r1 r2 r3 1 ch3 h ch3 2 och2ch3 h oh 3 ch3 ch2ch2oh ch3 4 och2ch3 ch2ch2oh oh 5 ch3 ch2ch2ch3 ch3 6 och2ch3 ch2ch2ch3 oh scheme 1. the synthetic path for the preparation of 6and 1,6-substituted 3-cyano-4-methyl-2-pyridones in the continuous flow system. the chemical structure and the purity of the obtained compounds were confirmed by melting points, and ft-ir, 1h-nmr and uv–vis spectral data. the melting points were determined in capillary tubes on an automated melting point system stuart smp30. the ft-ir spectra were recorded on a bomem mb-series ft-ir spectrophotometer, in the form of kbr pellets. the 1h-nmr spectral measurements were performed on a bruker ascend 400 instrument at 400 mhz. the spectra were recorded at room temperature in dmso-d6. the chemical shifts are expressed in ppm values referenced to tms. the uv–vis absorption spectra were recorded on a shimadzu uv–vis 1700 spectrophotometer in the region 200–600 nm at a concentration 5×10-5 mol∙l-1. the resulting data are given in the supplementary material and are in accordance with those known from the literature. the set-up of the continuous flow microreactor assembly the continuous flow microreactor assembly consisted of three pumps (lc-20ad xr, shimadzu manufacturing inc., usa), two t-shaped mixers and a peek (polyether ether ketone) capillary microreactor (inner diameter 0.5 mm, volume 5 ml, length 25 m), shown in fig. 1. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 534 tadić et al. fig. 1. the microreactor assembly. water solutions of the corresponding n-substituted cyanoacetamide and naoh were introduced into the first mixer. the resulting mixture was passed to the second mixer where a solution of a 1,3-dicarbonyl compound in methanol was added. the final mixture was delivered to the peek capillary microreactor to conduct the 2-pyridone condensation reaction (scheme 1). the following reaction was inhibited by concentrated hcl in the receiving test tube at the output of the capillary microreactor. in order to obtain higher yields, the residence time in microreactor was adjusted by changing the flow ratios. the reaction was performed at room temperature. the preparation of the reactant solutions for synthesis in the continuous flow microreactor system and the work-up of reaction mixture are given in the supplementary material. two sets of experiments were performed. the first set of experiments was conducted using equimolar concentrations of n-substituted cyanoacetamide and 1,3-dicarbonyl compound solutions. in the second part of the experiment, the reaction of condensation was further optimized for compounds 1 and 2 by changing mole ratios and concentrations of the starting reactants. results and discussion in the first set of experiments, six 6and 1,6-substituted 3-cyano-4-methyl-2 -pyridones were synthesized from 1,3-dicarbonyl reagent (dcr) and the corresponding n-substituted cyanoacetamide (caa, equimolar concentration, 0.6 mol·l–1) in the continuous flow microreactor system. sodium hydroxide solution was used as a catalyst (0.7 mol·l–1), and the synthesis was performed at the room temperature, as previously described. in order to improve the yield of the products, the flow rates of the starting reactants were varied. comparatively, the same 2-pyridones were synthesized in the batch system (using reflux and stirring) and the obtained results are given in table i. as indicated in table i, all the products were prepared starting from the total flow rate of the reaction mixture of 0.06 ml·min–1 except in the synthesis of compound 1. the use of this flow rate to produce compound 1, caused clogging of the capillary microreactor, induced by long residence time of the reaction ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. continuous flow synthesis 535 mixture in the continuous flow system. thus, the starting flow rate in the synthesis of compound 1 was 0.3 ml·min–1. furthermore, the increase of the flow rate generally led to a decrease of the yield, except in the case of compound 1 where increasing of the flow rate from 0.3 ml to 0.6 ml·min–1 led to increased yield. increasing the flow rate decreased the residence time in the microreactor and gives lower yields. table i. the optimization of the flow rate in first set of experiments and yields obtained in the batch system compound continuous flow system batch system f (dcr)a ml·min-1 c = 0.6 mol·l-1 f (caa)b ml·min-1 c = 0.6 mol·l-1 f (naoh)c ml·min-1 c = 0.7 mol·l-1 ftd ml·min-1 te min yf % tg min yh % 1 0.1 0.1 0.1 0.3 16.3 50 60 60 1 0.2 0.2 0.2 0.6 8.2 59 1 0.3 0.3 0.3 0.9 5.4 42 1 0.4 0.4 0.4 1.2 4.1 42 2 0.02 0.02 0.02 0.06 81.7 22 480 61 2 0.03 0.03 0.03 0.09 54.4 10 3 0.02 0.02 0.02 0.06 81.7 30 240 60 3 0.03 0.03 0.03 0.09 54.4 30 4 0.02 0.02 0.02 0.06 81.7 6 480 59 5 0.02 0.02 0.02 0.06 81.7 25.5 240 40 5 0.03 0.03 0.03 0.09 54.4 20 6 0.02 0.02 0.02 0.06 81.7 2 480 31 aflow rate of 1,3-dicarbonyl compound solution; b flow rate of n-substituted cyanoacetamide solution; c flow rate of sodium hydroxide solution; d total flow rate of the reacting mixture; eresidence time in the microreactor; fobtained yield in the continuous flow system; greaction time in the batch system; hobtained yield in the batch system however, the results obtained from the synthesis of n-substituted 3-cyano-4 -methyl-2-pyridones under continuous flow synthesis (table i) indicated that this method is not convenient for the synthesis of these molecules. mijin et al.13 reported the synthesis of different n-substituted 3-cyano-4,6-dimethyl-2-pyridones using a microwave-assisted method. the results obtained under microwave method have shown that is possible to obtain these products in very short reaction time (7 min) and high yield. therefore, it could be concluded that the microwave-assisted synthesis of n-substituted 2-pyridones is more efficient than the flow method. the synthesis in the continuous flow system was slower because it was performed at the room temperature, in comparison to the microwave method where the higher applied temperatures (and higher consumption of energy) increase the reaction rate, thus leading to good yields in a shorter reaction time. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 536 tadić et al. on the other hand, the flow synthesis acquired compound 1 in the good yield in a very short reaction time (60 %, 8.2 min, room temperature) in comparison to the classical method (60 %, 60 min, reflux) and thus, the synthesis of compound 1 was further optimized. furthermore, the synthesis of compound 2 did not result in the satisfactory level of conversion in the first set of experiments, but taking into account the commercial significance of this molecule in the production of various dyes,7 further investigation of its synthesis in the continuous flow system was also undertaken. the concentration of the starting materials for the synthesis of compounds 1 and 2 was increased, and the mole ratios were changed at the same flow rates of each starting compound. the mole ratios of acetylacetone or ethyl acetoacetate (1.0 mol·l–1), cyanoacetamide (1.5 mol·l–1) and naoh (2.0 mol·l–1) were set at 1:1.5:2, respectively. table ii. the optimization of the flow rate in the synthesis of 3-cyano-4,6-dimethyl-2-pyridone (1) and 3-cyano-6-hydroxy-4-methyl-2-pyridone (2) f (acetylacetone/ethyl acetoacetate)a / ml∙min-1 c = 1.0 mol∙l-1 f (cyanoacetamide)b ml∙min-1 c = 1.5 mol∙l-1 f (naoh)c ml∙min-1 c = 2.0 mol∙l-1 ftd ml∙min-1 te min y (1/2)f % 0.1 0.1 0.1 0.3 16.3 60/59 0.2 0.2 0.2 0.6 8.2 61/59 0.3 0.3 0.3 0.9 5.4 61/52 0.4 0.4 0.4 1.2 4.1 59/45 aflow rate of acetylacetone or ethyl acetoacetate solution; bflow rate of cyanoacetamide solution; cflow rate of sodium hydroxide solution; dtotal flow rate of reaction mixture; eresidence time in the microreactor; fobtained yield of compound 1 or 2 by comparing the results shown in tables i and ii, it could be concluded that the increase in concentration in the synthesis of compound 1 did not significantly affect an increase in the yield. on the contrary, when the optimization of compound 2 was performed, the change in the molar concentration and the ratios of ethyl acetoacetate, cyanoacetamide and naoh led to an increase of the yield, as shown in table ii. moreover, by comparing the yields of compound 2 obtained in the continuous flow system with those derived under conventional conditions (reflux, 480 minutes), it is evident that the former method acquired higher yields in a shorter reaction time, 59 % in 8.2 min in comparison to 61 % in 480 minutes, respectively. conclusions six different 2-pyridones were obtained under continuous flow synthesis. compared to the conventional method, the use of the particularly controlled continuous flow assembly allowed fast and effective preparation of compounds 1 and 2. the main advantage of this system is that the reaction was realized at ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. continuous flow synthesis 537 room temperature within a relatively short reaction time. optimization of the novel method for compounds 1 and 2 resulted in good yields (61 % yield in 5.4 min for compound 1 and 59 % yield in 8.2 min for compound 2). improved process control was enabled by managing the automated flow, which allows for changes in the flow ratio of the starting solutions in the system, during the synthesis. another advantage of the microreactor system compared to the batch method is the energy efficiency of the former, since the reaction was performed at room temperature. the continuous flow synthesis is a safe method, because the reaction occurs in a closed system and contact with potentially dangerous substances is minimized. additionally, scale up and the transition from a laboratory to an industrial level are facilitated, unlike in batch systems. thus, the presented method for the synthesis of 2-pyridone-based compounds has promising potential in the process industry. however, the level of conversion of n-substituted 4-methyl-3-cyano-2-pyridones was low, which led to the conclusion that this method is not suitable for the synthesis of these compounds. acknowledgement. the work was supported by the ministry of education, science and technological development of the republic of serbia under the research projects: oi172013 and tr34009. и з в о д синтеза 6и 1,6-супституисаних 3-цијано-4-метил-2-пиридона методом континуалног протока јулијана тадић1, марина михајловић1, мића јовановић2 и душан мијин2 1иновациони центар технолошко-металуршког факултета, карнегијева 4, 11120 београд и 2технолошко-металуршки факултет, универзитет у београду, карнегијева 4, 11120 београд у овом раду приказана је синтеза шест 6и 1,6-супституисаних 3-цијано-4-метил-2 -пиридона употребом микрореактора за континуалну синтезу. синтеза је извршена на собној температури и добијени резултати су упоређени са резултатима добијеним класичним поступком. у циљу оптимизације поступка синтезе континуалним протоком и повећања приноса производа, мењано је ретенционо време у микрореактору променом протока раствора реактаната. такође, оптимизована је реакција добијања 3-цијано -4,6-диметил-2-пиридона и 3-цијано-6-хидрокси-4-метил-2-пиридона који су комерцијално важни за фармацеутску индустрију и производњу боја. оба 2-пиридона су добијена у задовољавајућем приносу од око 60 % за мање од 10 min. синтетисана једињења су окарактерисана тачком топљења, ft-ir, 1h-nmr и uv–vis спектрима. ефикасност развијеног поступка има добар потенцијал за индустријску примену. (примљено 3. јула, ревидирано 24. октобра, прихваћено 29. октобра 2018) references 1. t. wirth, microreactors in organic synthesis and catalysis, 2nd ed., wiley–vch, weinheim, 2013, p. 6 2. c. wiles, p. watts, eur. j. org. chem. 2008 (2008) 1643 (https://dx.doi.org/10.1002/ejoc.200701041) 3. r. porta, m. benaglia, a. puglisi, org. process res. dev. 20 (2016) 2 (https://dx.doi.org/10.1021/acs.oprd.5b00325) ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 538 tadić et al. 4. s. lomel, l. falk, j. m. commenge, j. l. houzelot, k. ramdani, chem. eng. res. des. 84 (2006) 363 (https://dx.doi.org/10.1205/cherd05027) 5. p. plouffe, a. macchi, d. m. roberge, org. process res. dev. 18 (2014) 1286 (https://dx.doi.org/10.1021/op5001918) 6. n. g. anderson, org. process res. dev. 16 (2012) 852 (https://dx.doi.org/10.1021/op200347k) 7. d. ž. mijin, j. m. marković, d. v. 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mišić-vuković, j. serb. chem. soc. 59 (1994) 959. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {phenolic profiling and bioactivities of fresh fruits and jam of sorbus species} j. serb. chem. soc. 82 (6) 651–664 (2017) udc 634.18:576+615.9:615.279: jscs–4994 615.281:577.164.2 original scientific paper 651 phenolic profiling and bioactivities of fresh fruits and jam of sorbus species zorica o. mrkonjić1*, jelena d. nađpal1#, ivana n. beara1#, verica s. aleksić sabo2, dragana d. četojević-simin3, neda m. mimica-dukić1# and marija m. lesjak1# 1department of chemistry, biochemistry and environmental protection, faculty of sciences university of novi sad, trg dositeja obradovića 3, 21000 novi sad, serbia, 2department of biology and ecology, faculty of sciences university of novi sad, trg dositeja obradovića 2, 21000 novi sad, serbia and 3oncology institute of vojvodina, faculty of medicine, university of novi sad, dr goldmana 4, 21204 sremska kamenica, serbia (received 2 february, revised 6 march, accepted 10 april 2017) abstract: the purpose of this study was a comprehensive examination of the phenolic profile, the vitamin c content, and the antioxidant, anti-acetylcholinesterase, cytotoxic and antimicrobial activities of extracts and jams of fruits of two edible sorbus species: well characterized s. aucuparia and two insufficiently explored forms of s. torminalis (torminalis and semitorminalis). characterisation of 44 phenolics was realized using lc–ms/ms and 15 compounds were confirmed, with chlorogenic acid being the most dominant in s. aucuparia and ferulic acid in both s. torminalis forms. s. aucuparia demonstrated potent antioxidant activity, while that of both s. torminalis forms was moderate. jam extracts had the highest content of vitamin c. s. aucuparia exhibited some anti-acetylcholinesterase activity, while s. torminalis f. torminalis showed the best antimicrobial activity against staphylococcus aureus and both forms (torminalis and semitorminalis) possessed the highest activity against escherichia. coli. the results obtained herein are a great base for further research of edible sorbus species with the aim of promoting their better usage as nutraceuticals. keywords: phenolics; antioxidant; anti-acetylcholinesterase activity; cytotoxic activity; antimicrobial activity. introduction in spite of their widespread use in diet, preservatives preparation, beverage manufacture and traditional medicine (the details are presented in the supple * corresponding author. e-mail: zoricamrkonjic@gmail.com # serbian chemical society member. https://doi.org/10.2298/jsc170202049m 652 mrkonjić et al. mentary material to this paper), there are still very few reports concerning the detailed chemical composition and biological activities of fruits of different sorbus species. bearing this in mind, in this paper, the fruits of the well-known s. aucuparia and the unexplored s. torminalis f. torminalis and s. torminalis f. semitorminalis were the subjects of extensive study. namely, within the sorbus genus, the most renowned species is s. aucuparia (rowan), which is edible for humans and domestic animals. it was confirmed that rowan fruits contain a high content of natural products, such as phenolics,1–6 vitamins c, b1, b2, e and k,5,7 carotenoids,8 carboxylic acids9 and sugars,7 which surely contribute to their salubrity. s. aucuparia is a very variable species and occurs in nature as five subspecies: s. aucuparia subsp. aucuparia, s. aucuparia subsp. fenenkiana, s. aucuparia subsp. glabrata, s. aucuparia subsp. praemorsa and s. aucuparia subsp. sibirica. in this study, s. aucuparia subsp. aucuparia var. aucuparia fruits were the subject of in-depth investigation whereby their detailed phenolic profile was elucidated for the first time. herein, fresh fruit extracts and jam were investigated, as those are the forms in which the fruits of sorbus species are usually consumed. on the other hand, other species from sorbus genus, such as s. torminalis (wild service or chequer tree), have been poorly studied to date. as the fruits of s. aucuparia, the fruits of s. torminalis are commonly present in human and animal diet. this species occur in wide variety of forms, mostly differing in leaf shape. in this paper, two forms of s. torminalis, f. torminalis and f. semitorminalis, were studied. the morphological differences between leaves of these two forms are barely noticeable, while their colour and fruits are identical. the only variations between leaves are hairs on underside, which are hard to observe. specifically, leaves of f. torminalis are hairy only during a short period when young, while those of f. semitorminalis are permanently covered with hairs.10 thus, due to their strong morphological similarities, these two forms could be easily mistaken for each other if they were not harvested by professional plant collectors. consequently, it was considered worthwhile to determine whether their fruits have a similar chemical composition and biological potency, and conclude if substituting one of these species with the other in functional foods could result in quality differences. while the antioxidant and anti-acetylcholinesterase (ache) activities, as well as total phenolic and flavonoid contents of s. torminalis fruits have only been poorly investigated,11,12 no literature data related to the detailed phenolic profile, vitamin c content and other biological activities of these fruits has hitherto been reported. for this reason, the present research was focused on the examination and comparison of the phenolic profile, vitamin c content, and antioxidant, anti-ache, cytotoxic and antimicrobial activities of water and methanol extracts of the edible fresh fruits, and traditionally prepared sorbus – chemical and bioactivities profiling 653 jams of s. aucuparia and of the uninvestigated s. torminalis f. torminalis and s. torminalis f. semitorminalis plant species. experimental experimental details related to the employed chemicals and reagents, plant material collection and extracts preparation, are given in the supplementary material to this paper. lc–ms/ms analysis of single phenolic compounds determination of selected phenolic compounds in the extracts of s. aucuparia and both s. torminalis forms, torminalis and semitorminalis, was performed according to a previously reported procedure.13 additional brief details are given in the supplementary material. vitamin c content the vitamin c content was determined by a method given in the literature14 adapted for 96-well microplates.15 briefly, each sample was evaporated in vacuo at 40 °c and mixed with metaphosphoric acid (0.1 g ml-1) to obtain final concentrations of 60, 90 and 120 mg ml-1 for all extracts, except for the extracts of jam that were prepared in concentrations of 40, 60 and 80 mg ml-1. the mixtures were stirred for 45 min at room temperature. the prepared extracts in metaphosphoric acid (30 µl) were mixed with 270 µl of 2,6-dichlorophenolindophenol (72 mg ml-1) and the absorbance at 515 nm was measured within 5 min. the vitamin c content was determined using a standard calibration curve of vitamin c (ranging 0‒320 μg ml-1) and the results are presented as the mean value of three measurements. antioxidant and anti-ache activities the scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (dpph), superoxide anion (o2•-), nitric oxide (•no) and hydroxyl (ho•) radical, reducing power (frap assay) and inhibition of lipid peroxidation (lp) were tested according to previously published methods.16 to evaluate the inhibitory activity of the extracts, the ache ellman method17 with numerous modifications was used. details of these experiments are given in the supplementary material. cell growth activity preparation of the samples and standards, experimental conditions for maintenance of the cell lines, and the sulforhodamine b (srb) assay procedure were performed according to previously published procedures.16 each experimental method is briefly explained in the text below. preparation of samples and standards. for the evaluation of the cell growth activity, aqueous stock solutions of the extracts and podophyllotoxin (10 mg ml-1 in dimethyl sulfoxide (dmso)) were diluted in nacl (9 mg ml-1) to obtain ranges of concentrations 0.1‒1000 and 0.00001‒1 μg ml-1, respectively. maintenance of cell lines. cell growth activity was evaluated in vitro using human cell lines: hela (cervix epitheloid carcinoma, european collection of authenticated cell cultures (ecacc), no. 93021013), mcf7 (breast adenocarcinoma, ecacc no. 86012803), ht-29 (colon adenocarcinoma, ecacc no. 91072201) and mrc-5 (human foetal lung, ecacc 84101801). the cell lines were grown in dulbecco-modified eagle's medium with 4.5 % glucose, supplemented with 10 % heat-inactivated foetal calf serum (fcs), 100 iu ml-1 penicillin and 100 μg ml-1 streptomycin. the cells were cultured in 25-cm2 flasks at 37 °c, in a 5 % co2 atmosphere of high humidity, and sub-cultured twice a week. a single cell suspension was obtained using 0.1 % trypsin with 0.04 % edta. 654 mrkonjić et al. srb assay. the cell lines were harvested and plated into 96-well microtitre plates at seeding density of (3–5)×103 cells well-1 in a volume of 199 or 180 μl, and pre-incubated in complete medium supplemented with 5 % fcs at 37 °c for 24 h. serial dilutions of the extracts (1 μl) were added into 199 μl of medium, while serial dilutions of podophyllotoxin (20 μl) were added into 180 μl of medium, in order to achieve the required final concentrations. all samples were filtered through 0.22 μm microfilters to obtain sterility. equal volumes of water and dmso were added in the control wells. the concentration of dmso in the cell cultures was ≤ 5 μl ml-1. after adding the dilutions, the microplates were incubated at 37 °c for 48 h. the cell growth was evaluated by the colorimetric srb assay.18 colour development was measured using photometer at 540 nm against 620 nm as the background. antimicrobial activity to evaluate minimum inhibitory concentration (mic) of the extracts, the method for the determination of the antimicrobial activity of antimicrobial agents was used.19 first, two-fold dilutions of the tested plant extracts were prepared in microtitre plates. the final concentration of each extract ranged 1‒128 mg ml-1. subsequently, a bacterial suspension, previously adjusted to match a turbidity of a 0.5 mcfarland nephelometer standard, was diluted in double-strength mueller–hinton medium (1:100 volume ratio). into each well, inoculated double-strength medium were added to the same volume as each extract (1:1 volume ratio). the number of the bacteria in the assay was about 1×106 colony forming units (cfu) ml-1. antimicrobial activity of the extracts was tested against gram negative bacteria, escherichia coli american type culture collection (atcc) 25922 and gram-positive bacteria, staphylococcus aureus atcc 25923. the microtitre plates were incubated overnight at 37 °c, without shaking, amended with a 10 μl of a 1 % solution of 2,3,5-triphenyltetrazolium chloride and incubated additionally for 2 h until development of the red colour. controls for plate sterility and bacterial growth without extracts were also included. the lowest concentration of extracts that inhibited bacterial growth, which was identified by the absence of red formazane, was considered as the mic. the mic determination was performed in three replicates and three independent experiments. results and discussion phenolic profile qualitative and quantitative analyses of 44 phenolics in all extracts were performed using the lc–ms/ms technique. the content of the 12 determined phenolics are presented in table i and differences between the species and two s. torminalis forms are evident. concerning phenolic acids, chlorogenic acid was the most abundant compound in s. aucuparia extracts, while ferulic acid was the most dominant in extracts of both s. torminalis forms. analyses of the selected flavonoids showed that all extracts contain amentoflavone, which was dominant flavonoid in both s. torminalis forms, especially in semitorminalis form. additionally, rutin, quercetin-3-o-glucoside and hyperoside were characteristic for extracts of s. aucuparia. furthermore, coumarins and lignans were not detected, with the exception of aesculetin, which was present only in s. torminalis f. semitorminalis. sorbus – chemical and bioactivities profiling 655 it is possible to see a common trend among the extracts concerning determined phenolic amounts was recognizable. namely, aqueous methanol was more efficient than the extraction in water, while jam extracts contained significantly lower amounts of most phenolics, than did the fresh fruits. table i. contents of detected phenolics (µg g-1 dw) in s. aucuparia and s. torminalis (forms torminalis and semitorminalis) fruit extracts; means within each row with different letters (a‒i) differ significantly (p ≤ 0.05); compounds analyzed, but the peak was below the limit of quantification (loq):13 p-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, vanillic acid, cinnamic acid, caffeic acid, syringic acid, o-coumaric acid, p-coumaric acid, 3,4-dimethoxycinnamic acid, sinapic acid (phenolic acids); apigenin, apigenin-7-o-glucoside, baicalin, baicalein, apiin, daidzein, naringenin, vitexin, genistein, isorhamnetin, luteolin, luteolin-7-o-glucoside, myricetin, kaempferol, epigallocatechin gallate, chrysoeriol, quercetin, epicatechin (flavonoids); umbelliferone, scopoletin (coumarins); matairesinol, secoisolariciresinol (lignans) w – water extract, m – methanol extract, j – jam compound species s. aucuparia s. torminalis f. torminalis s. torminalis f. semitorminalis w m j w m j w m j phenolic acids gallic acid <loq <loq <loq <loq <loq <loq 5.69± 0.51a <loq <loq protocatechuic acid <loq <loq 12.5± 0.01a 13.7± 0.24b 23.2± 0.13c 5.92± 0.41d 4.61± 0.04e 3.44± 0.03f 2.11± 0.19g chlorogenic acid (5.69± 0.30)×103c (5.80± 0.30)×103c (2.60± 0.10)×103b < loq <loq <loq <loq <loq <loq ferulic acid 7.80± 0.46h 9.59± 0.80i 11.4± 0.93g 27.8± 0.97d 62.6± 3.18c 13.3± 0.67f 43.3± 1.44b 38.3± 1.46a 18.4± 0.55e flavonoids amentoflavone 10.7± 0.91h 11.9± 0.33g 8.40± 0.56i 15.8± 0.98f 19.3± 1.17d 16.8± 1.31e 362± 8.67b 974± 13.2c 195± 3.76a kaempferol -3-o-glucoside 9.00± 0.45c 8.56± 0.33c 3.99± 0.27b <loq <loq <loq 2.34± 0.02a 2.43± 0.01a <loq quercitrin <loq <loq <loq <loq <loq <loq 6.53± 0.51b 11.0± 0.97a 3.76± 0.27c quercetin-3 -o-glucoside 49.3± 1.34b 55.8± 2.16c 17.9± 1.24a <loq 13.6± 0.64d 2.53± 0.01f 3.33± 0.02e 2.06± 0.02f 1.60± 0.01g hyperoside 36.6± 1.47c 39.6± 1.57c 9.68± 0.33b <loq 10.4± 1.02b 1.61± 0.21c <loq <loq <loq rutin 82.3± 1.25c 80.4± 2.76c <loq <loq <loq <loq <loq <loq <loq catechin <loq <loq <loq <loq <loq <loq 10.6± 1.24c <loq <loq coumarin aesculetin <loq <loq <loq <loq <loq <loq <loq 3.07± 0.14c <loq 656 mrkonjić et al. furthermore, previously published data also highlighted fruits of sorbus species as a rich source of phenolic compounds. specifically, chlorogenic and neochlorogenic acid, as well as quercetin glycosides (rutin, hyperoside and isoquercitrin), were identified in fruit samples of nineteen different sorbus species, hybrids and varieties, including s. aucuparia.20 similarly, the same compounds were detected by others in ten fruit samples, including nine varieties of s. aucuparia.21 when compared to the results of these authors,21 the content of rutin was the same, while the content of chlorogenic acid was a few times higher than that found in the present study. on the other hand, others detected hyperoside in the fruits of s. aucuparia20–22 in higher amounts than in the presented data. according to a few previous studies, hydroxycinnamic acids, mainly chlorogenic and neochlorogenic acids, were identified in s. aucuparia fruits.1–3 these authors reported hydroxycinnamic acids to be the most abundant phenolics and obtained amounts of chlorogenic acid in s. aucuparia fruits that are in agreement with those found in the present research. moreover, another study pointed out that fruits of s. aucuparia present a valuable source of ferulic acid, which was higher than in the present extracts.23 significant contents of both quercetin and kaempferol and their glucosides were previously confirmed in the fruits of s. aucuparia.5,23 however, quercetin was absent in the present extracts, while the content of kaempferol reported previously was in agreement with data presented herein. similarly, s. aucuparia fruits were shown previously to be a rich source of rutin, quercetin-3-o-glucoside and quercetin-3-o-galactoside,22 whereby the determined contents of rutin and quercetin-3-o-glucoside were higher than in data obtained in the present study. in addition, another study indicates that the fruits of s. torminalis contain a high amount of quercetin as well as isorhamnetin.24 besides, amentoflavone was detected in the present s. aucuparia fruit extracts, while no prior study specifically mentioned this compound. generally, the obtained results were in a good accordance with a few previously published studies regarding the phenolic profile of s. aucuparia fruits. however, to the best of our knowledge, this is the first detailed report concerning the phenolic composition of fruits of either s. torminalis forms (torminalis and semitorminalis), as well as some phenolics in s. aucuparia fruits. interestingly, the dominant compounds found in the investigated sorbus species are proven to express valuable health benefits, which additionally support their usage as nutraceuticals. namely, it was previously reported that the consumption of fruits rich in chlorogenic acid and its derivates decrease the risk of type 2 diabetes. in addition, most of chlorogenic acids convert to caffeic acid, which reduces glucose absorption and oxidative stress in vitro and inhibits glucose-6-phosphate translocase, thereby decreasing glucose output in the liver.2 in addition, amentoflavone is recognized as a potent anti-inflammatory agent by sorbus – chemical and bioactivities profiling 657 inhibiting prostaglandin e2 biosynthesis via downregulation of cyclooxygenase-2 expression.25 vitamin c content as shown in table ii, the amount of vitamin c ranged from 40 to 420 μg g–1 dw in the extracts of fresh fruits and jam of the examined species. interestingly, the jam extracts were shown to be the richest sources of vitamin c. this could be explained by the fact that the preparation of the jam (cooking) lasted a short time (a few minutes) and it was stored in the cold (4 °c) until analysed. namely, a group of authors26 showed that long-time cooking, as well as storage at room temperature, causes an evident reduction in the vitamin c content. in addition, a study indicated that the stability of vitamin c is higher in a fruit matrix than in extracts.27 to the best of our knowledge, there are no prior investigations on vitamin c content in s. torminalis species. on comparing the two investigated forms of s. torminalis, it could be noted that the form semitorminalis was slightly richer in vitamin c than the form torminalis. table ii. total ascorbic acid contents (means within each column with different letters (a‒f) differ significantly (p ≤ 0.05)) in s. aucuparia and s. torminalis (forms torminalis and semitorminalis) fruit extracts; w – water extract, m – methanol extract, j – jam extract vitamin c content, µg g-1 dw s. aucuparia w 100±10.0a m 40.0±1.10b j 420±10.0c s. torminalis f. torminalis w 40.0±1.20ab m 220±20.3d j 300±10.1de s. torminalis f. semitorminalis w 80.0±7.90a m 320±20.1e j 360±10.7f generally, to date, there is a lack of information on the vitamin c content in sorbus species.5,28 one study indicated a higher amount of vitamin c in s. aucuparia fruits5 compared with the results obtained in the present study. this disagreement is probably due to differences in the maturity stage of the investigated fruits. namely, the others investigated unripe fruits, while in this study, the fruits where overripe. vitamin c is one of the most important antioxidants and everyday food supplement, which is purported to prevent respiratory tract infections and accelerate recovery from various conditions, such as fever, cold and flu.29 compared with 658 mrkonjić et al. some other fruits that are valuable sources of vitamin c, the content of vitamin c in sorbus fruits is about six times lower than in orange and eight times lower than in lemon and kiwi fruit.30 however, fresh sorbus fruits and jam are valuable sources of micronutritients and could serve in a healthy seasonable diet. antioxidant activity the reason to examine the antioxidant potency of s. aucuparia, s torminalis f. torminalis and s. torminalis f. semitorminalis derives from the fact that the antioxidant supplements in food, particularly those rich in phenolic compounds, could markedly diminish amount of free radicals, leading to the prevention of some severe diseases, such as metabolic syndrome, and cardiovascular and neurodegenerative disorders.31 in the light of this, antioxidant therapy by functional food is considered to be promising approach to postpone or retard such diseases.32 in order to extensively characterize the antioxidant activity of the fruits and jam, of s. aucuparia and two s. torminalis forms, several in vitro assays were applied: the dpph•, •no, ho• and o2•– scavenger capacity tests, frap assay and fe2+/ascorbate induced lp inhibition. the determined antioxidant activities are given in table iii. in comparison with well-known synthetic antioxidants butylated hydroxytoluene (bht) and propyl gallate (pg), all extracts exhibited considerable antioxidant effects. generally, s. aucuparia extracts were the most active in all the performed tests, except toward o2•– scavenging activity, where s. torminalis f. torminalis was the most powerful. namely, its fruit extracts (water and methanol) showed better or the same activity as pg, respectively, toward the neutralisation of o2•–. the water extract of s. aucuparia fruits demonstrated a significant ho• scavenger activity similar to that of the standard antioxidant bht. these facts are particularly important because o2•– reacts rapidly with other free radicals, such as no•, and can cause the biological damage occurring in many human diseases.33 additionally, ho• is the most potent radical in initiating lp in the cell and is capable of damaging almost every molecule found in living cells.34 overall, the extracts of the fruits of s. aucuparia had the highest antioxidant activity in the present study. by comparing two forms of s. torminalis, it could generally be noted that they express similar antioxidant potential, because the extracts of the form torminalis had a slightly better antiradical capacity towards •no, o2•– and ho•, while the extracts of the form semitorminalis were more effective in scavenging of dpph• and inhibition of lp and had a better reducing power. according to the results presented, it could be noted that the fresh fruits exhibited some antioxidant activity while the jam was shown to be a moderate source of antioxidants in the diet. sorbus – chemical and bioactivities profiling 659 anti-ache activity it has been suggested that ache may participate in the development of neurodegenerative disorders, such as alzheimer’s disease and tumorigenesis.35 thus, inhibition of ache serves as a promising strategy for the treatment of neurodegenerative disorders and the search for new sources of effective anti -ache compounds, such as natural products, is worthwile.36 table iii. antioxidant activities of s. aucuparia and s. torminalis (forms torminalis and semitorminalis) fruit extracts; means within each column with different letters (a–h) differ significantly (p ≤ 0.05); aae – ascorbic acid equivalents; na – 50 % inhibition not achieved; pg – propyl gallate; bht – butylated hydroxytoluene; w – water extract, m – methanol extract, j – jam extract ic50 / mg ml-1 frap mg of aae g-1 dw dpph· ·no o2·‾ (×103) ho· lp s. aucuparia w 0.07± 0.00a 1.43± 0.03a 20.16± 0.90b 0.16± 0.00c 6.40± 0.14c 10.6± 0.20c m 0.08± 0.01a 0.43± 0.03c 20.5± 0.50b 0.24± 0.00a 7.38± 0.08d 11.2± 0.43c j 0.13± 0.01b 2.26± 0.04d 67.8± 2.06e 0.61± 0.02e 4.08± 0.05a 4.22± 0.08b s. torminalis f. torminalis w 1.38± 0.01f naa 7.09± 0.50a 0.30± 0.00d na 1.11± 0.13f m 0.57± 0.02e 2.82± 0.19g 12.2± 1.21ca 0.26± 0.01ab na 2.12± 0.15e j 0.44± 0.02f 1.64± 0.03b 36.9± 2.19d 1.11± 0.01f na 3.10± 0.18g s. torminalis f. semitorminalis w 1.27± 0.02f na 12.8± 0.53a 0.43± 0.01g na 2.12± 0.27e m 0.42± 0.01g 3.12± 0.06f 12.5± 1.29cc 0.27± 0.01b na 3.81± 0.04d j 0.18± 0.00d 2.45± 0.01dg 50.3± 0.36g 0.29± 0.02bd 3.02± 0.02c 6.41± 0.11a references pg (0.38± 0.00)×10-3h (6.58± 0.43)×10-3h 9.68± 0.29c (20.0± 0.40)×10-3h na na bht (9.36± 0.05)×10-3c na na (160± 5.00)×10-3c (14.0± 3.00)×10-3h 124± 12.4h in this study, as shown in table iv, only the extracts of s. aucuparia possessed inhibitory activity toward ache. interestingly, the methanol extracts of fresh fruits as well as traditionally made jam exhibited the highest anti-ache activity, but were not as good as galanthamine. inhibitory potential against ache 660 mrkonjić et al. of s. aucuparia was also reported previously, but in the present study, the activity was weaker.37 even though some authors12 reported anti-ache activity of s. torminalis water extract, in the present study, neither form torminalis nor form semitorminalis were found to be active. the obtained results suggest that s. aucuparia fruits could potentially be used as sources of natural anti-ache agents. table iv. cytotoxic and anti-ache activities (ic50 / µg ml-1) of s. aucuparia fruit extracts; means within each column with different letters (a–c) differ significantly (p ≤ 0.05); na – 50 % inhibition not achieved; na – non-applicable; w – water extract, m – methanol extract, j – jam extract cytotoxic activity anti-ache activity mrc-5 hela mcf7 ht-29 w 532±7.79a na 526±7.21a 592±70.6a (3.81±0.21)×103a m 517±28.5b 965±27.9b 414±12.5b 432±4.15b (2.02±0.02)×103b j na na na na (2.08±0.04)×103b standard podophyllotoxin (4.70± 0.80)×10-3c (4.10± 0.30)×10-3c (1.30± 0.20)×10-3a (3.00± 0.50)×10-3c na galanthamine na na na na 0.39±0.01c cytotoxic activity the cytotoxity of the sorbus extracts was evaluated in vitro using tumour hela, mcf7 and ht-29 and healthy mrc-5 cell lines by the srb assay. in addition, the activity of podophyllotoxin, a potent cytotoxin, was investigated (table iv). overall, only extracts of s. aucuparia demonstrated cytotoxic activity, while the s. torminalis forms expressed no activity in the applied concentration range. unfortunately, s. aucuparia was not selective towards tumour cell lines, as it was also cytotoxic to the healthy mrc-5 cell line. furthermore, the growth of examined cell lines was not affected by jam. in comparison to podophyllotoxin, the extracts exhibited much lower activity. nevertheless, since there is only one literature reference on the antiproliferative activity of s. aucuparia,38 the obtained results could contribute to the overall knowledge of the bioactivity of sorbus species. antimicrobial activity the antimicrobial activity of extracts was evaluated against clinically relevant gram-positive and gram-negative bacterial strains (table v). one of the most important human and animal pathogens, e. coli, is responsible for a broad spectrum of diseases, including urinary tract infections and other clinical infections. on the other hand, s. aureus are causative agents of food poisoning in humans.39 it was previously reported that multiple mechanisms of organic and phenolic acids, tannins and anthocyanins are responsible for the antimicrobial activity of berries.39,40 in presented study all investigated extracts were sorbus – chemical and bioactivities profiling 661 more effective in growth inhibition of s. aureus compared to e. coli. extracts of s. torminalis f. torminalis possessed the best activity against s. aureus, while e. coli was more sensitive to both s. torminalis forms compared with s. aucuparia. it is noteworthy that among the jam extracts only the jam from s. torminalis f. semitorminalis exhibited inhibition of bacterial growth. the antibacterial activity of s. aucuparia was previously poorly argued,4,39 while there are no previous reports on either s. torminalis forms. taking into account that the investigated extracts showed moderate effects against bacterial growth, further studies are recommended in order to verify which constituents and mechanisms are responsible for the observable activity. table v. antimicrobial activities (mic / mg ml-1) of s. aucuparia and s. torminalis (forms torminalis and semitorminalis) fruit extracts; na – inhibition not achieved; w – water extract, m – methanol extract, j – jam extract test microorganism e. coli s. aureus s. aucuparia w 64 32 m 64 32 j na na s. torminalis f. torminalis w 32 16 m 32 16 j na na s. torminalis f. semitorminalis w 32 32 m 32 32 j 64 32 standard amikacin 4 4 overall, no clear correlation between the content of the examined phenolic compounds and results of antioxidant assays and analyzed activities were found. this indicates a strong synergistic effect of the phenolics, flavonoids and vitamin c, as well as other uninvestigated bioactive compounds, such as tannins and anthocyanins, which could be responsible for the determined bioactivities. conclusions in the present study, the edible fruits of s. aucuparia, and two forms of s. torminalis (torminalis and semitorminalis) were extensively investigated for their phenolic profile, vitamin c content and their antioxidant, anti-ache, cytotoxic and antimicrobial activities. the contents of 44 phenolics were investigated in the fresh fruits and jam using lc–ms/ms and 12 compounds were detected, with the most dominant being chlorogenic acid (s. aucuparia) and amentoflavone (s. 662 mrkonjić et al. torminalis f. semitorminalis). regarding the vitamin c content, the fruit jam was the richest source. although the fruits of both s. torminalis forms showed moderate antioxidant potency, those of s. aucuparia were the most powerful antioxidant agents. only s. aucuparia fruits were effective in the inhibition of ache and the cytotoxic assay, but regrettably, they showed no selectivity towards tumour cell lines. regarding antimicrobial activity, the fruits of s. torminalis f. torminalis were the most active in the inhibition of the growth of s. aureus, while both forms (torminalis and semitorminalis) exerted the best activity on e. coli. to sum up, the results obtained in this study indicated that the fruits of s. aucuparia had higher biopotential than those of the s. torminalis forms. furthermore, the fruits of the examined s. torminalis forms have somewhat different secondary metabolites profile and bioactivities and thus should be clearly distinguished from one another. the fruits of sorbus species should be consumed mainly fresh, even though the jams should not be disregarded from the diet, as they showed greater biopotential. the presented results firmly support further studies with the aim of promoting everyday consumption of edible sorbus fruits as functional food with valuable health benefits. supplementary material more details about studied species and additional experimental details are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. we wish to thank goran anačkov, phd, for determination of plant material and the voucher specimens. this research was supported by the ministry of education, science and technological development of the republic of serbia (grant no. 172058). и з в о д фенолни профил и биоактивност свежих плодова и пекмеза врста рода sorbus зорица о. мркоњић1, јелена д. нађпал1, ивана н. беара1, верица с. алексић caбо2, драгана д. четојевић-симин3, неда м. мимица-дукић1 и марија м. лесјак1 1департман за хемију, биохемију и заштиту животне средине, природно–математички факултет, универзитет у новом саду, трг доситеја обрадовића 3, 21000 нови сад, 2департман за биологију и екологију, природно–математички факултет, универзитет у новом саду, трг доситеја обрадовића 2, 21000 нови сад и 3институт за онкологију војводине, др голдмана 4, 21204 сремска каменица циљ приказаног рада представљао је детаљно испитивање фенолног профила, садржаја витамина це, антиоксидантне, анти-ацетилхолинестеразне, цитотоксичне и антимикробне активности екстраката и пекмеза плодова две јестиве врсте рода sorbus: добро окарактерисане врсте s. aucuparia и две недовољно проучене форме врсте s. torminalis (torminalis и semitorminalis). карактеризација 44 фенолна једињења урађена је помоћу lc–ms/ms технике и потрвђено је присуство 15 једињења. у екстрактима плодова s. aucuparia доминантно једињење била је хлорогенска киселина, док је у обе форме врсте s. torminalis доминантна била ферулна киселина. s. aucuparia је испољила снажну антиоксидантну активност, док су обе форме врсте s. torminalis показале умерен антиоксидантни потенцијал. највећи садржај витамина це детектован је у пекмезима. s. aucuparia sorbus – chemical and bioactivities profiling 663 је показала извесну анти-ацетилхолинестеразну активност, док је s. torminalis f. torminalis испољила најбољи антимикробни потенцијал. резултати овог рада представљају добру базу за даља исптраживања плодова јестивих врста рода sorbus у циљу њихове боље искоришћености као нутрацеутика. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice jscs :: 2015 list of referees volume 80 (2015) 1 2015 list of referees editorial board of the journal is grateful to the following referees for reviewing the manuscripts during 2015: shawkat a. abdelmohsen, department of chemistry, faculty of science, assiut university, assiut, egypt hatem a. abdel-aziz, department of applied organic chemistry, national research center, dokki, cairo, egypt snezana agatonovic-kustrin, latrobe university, school of pharmacy, bendigo, victoria, australia mohamed jawed ahsan, maharishi arvind college of pharmacy jaipur, rajasthan, india senem akkoç, erciyes university, faculty of science, department of chemistry, kayseri, turkey mariya al-rashida, department of chemistry, forman christian college, chartered university, lahore, pakistan joan albert, departament de química inorganica, facultat de química, universitat de barcelona, spain andrea aliboni, ente per le nuove tecnologie, l'energia e l'ambiente, energy and sustainable economic development, rome, italy mara aleksić, faculty of pharmacy, university of belgrade, serbia omar m. aly, medicinal chemistry department, faculty of pharmacy, minia university, egypt jelena arsеnijević, faculty of pharmacy, department of pharmacognosy university of belgrade, serbia athar ata, department of chemistry, the university of winnipeg; winnipeg, mb, canada tarek aysha, institute of organic chemistry and technology, faculty of chemical technology, university of pardubice, czech republic najmedin azizi, chemistry & chemical engineering research center of iran, tehran, iran darko babić, institut ruđer boskovć, zagreb milica balaban, faculty of science, university of banja luka, bosnia and herzegovina igor balać, faculty of mechanical engineering, university of belgrade, serbia taibi ben hadda, laboratoire chimie matériaux, faculté sciences, université med premier, oujda, morocco vladimir beškoski, faculty of chemistry, university of belgrade, serbia p. j. bindu, department of studies and research in chemistry, kuvempu university, shankaraghatt, india irani biswas, university of burdwan, department of botany, bardhaman, india jelena bobić, institute for multidisciplinary research, university of belgrade, serbia gordana bogoeva-gaceva, ss. cyril and methodius university, faculty of technology and metallurgy, skopje, fyr macedonia _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 2 journal of the serbian chemical society goran bošković, faculty of technology, university of novi sad, serbia stefano brenna, università dell'insubria, dipartimento di scienza e alta tecnologia, como, italy josip bronić, institute ruđer bošković, zagreb, croatia jaroslava budinski simendić, faculty of technology, university of novi sad, serbia antonio carta, department of chemistry and pharmacy, university of sassari, italy stamatis charalambos, university of ioannina, greece ping cheng, school of environmental and chemical engineering, shanghai university, p. r. china aleksandr n. chumakov, institute of physics, national academy of sciences of belorus, minsk, belarus valerio cristofori, department of agriculture, forests nature and energy, tuscia university, italy peter a. crooks, college of pharmacy, department of pharmaceutical sciences, little rock, ar, usa. jános csanádi, faculty of science, university of novi sad, serbia nikola cvjetićanin, faculty of physical chemistry, university of belgrade, serbia ana čučulović, institute for application of nuclear energy (inep), university of belgrade, serbia tanja ćirković veličković, faculty of chemistry, university of belgrade, serbia vladan ćosović, institute of chemistry, technology and metallurgy, university of belgrade, serbia danica ćujić, institute for the application of nuclear energy, university of belgrade, serbia aleksandra daković, institute for technology of nuclear and other mineral raw materials, belgrade, serbia ljiljana damjanović, faculty of physical chemistry, university of belgrade, serbia sara darakhshan, kermanshah university of medical sciences, department of pharmaceutics, kermanshah, iran claude daul, department of chemistry, university of fribourg, switzerland priyadarsi de, polymer research centre, indian institute of science education and research kolkata, mohanpur, nadia, west bengal, india ana de santiago-martín, dept. de génie civil et génie des eaux, faculté de sciences et de génie, univ. laval , qc, canada aleksandar dekanski, institute of chemistry, technology and metallurgy, university of belgrade, serbia dragana dekanski, biomedical research, r&d institute, galenika a.d., belgrade, serbia biljana dojnov, institute of chemistry, technology and metallurgy, university of belgrade, serbia andrey o. doroshenko, institute for chemistry, kharkov v.n. karazin national university, kharkov, ukraine branko drakulić, institute of chemistry, technology and metallurgy, university of belgrade, serbia gordana dražić, faculty for applied ecology futura, singidunum university, belgrade, serbia saša drmanić, faculty of technology and metallurgy, university of belgrade, serbia branko dunjić, faculty of technology and metallurgy, university of belgrade, serbia ljiljana đekić, faculty of pharmacy, university of belgrade, serbia veljko r. đokić, ic of faculty of technology and metallurgy, university of belgrade, serbia jasna đonlagić, faculty of technology and metallurgy, university of belgrade, serbia _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 2014 list of referees 3 dragana đorđević, , institute of chemistry, technology and metallurgy, university of belgrade, serbia milos i. đuran, department of chemistry, faculty of science, university of kragujevac, serbia enis džunuzović, faculty of technology and metallurgy, university of belgrade, serbia michail n. elinson, n. d. zelinsky institute of organic chemistry, moscow, russia oleg farat, department of chemistry, m.v. lomonosov moscow state university, russian federation daniela farinelli, dipartimento di scienze agrarie, alimentari e ambientali, perugia, italy m. h. fatemi, mazandaran university of babolsar, iran abel gomes martins ferreira, departamento de engenharia química, faculdade de ciências e tecnologia, universidade de coimbra, portugal sacramento ferrer, departament de química inorgànica, universitat de valència, burjassot, valencia, spain boris furtula, department of chemistry, faculty of science, university of kragujevac, serbia slobodan gadžurić, faculty of sciences, dep. of chemistry, university of novi sad dejan gođevac, institute of chemistry, technology and metallurgy, university of belgrade, serbia gordana gojgić-cvijović, institute of chemistry, technology and metallurgy, university of belgrade, serbia snežana gojković, faculty of technology and metallurgy, university of belgrade, serbia encarna gomez plaza, university of murcia, faculty of veterinary, spain juozas vidas grazulevicius, department of organic technology, kaunas university of technology, lithuania ludwig gruber, fraunhofer ivv, freising, germany sonja grubišić, faculty of chemistry, university of belgrade. serbia maja gruden, faculty of chemistry, university of belgrade. serbia mikhail goubko, russian academy of sciences, moscow, russia ivan gutman, department of chemistry, faculty of science, university of kragujevac, serbia valéria guzsvány, faculty of sciences, department of chemistry, biochemistry and environmental protection, university of novi sad, milica gvozdenovic, faculty of technology and metallurgy, university of belgrade, serbia ladislav habala, faculty of pharmacy, comenius university bratislava, slovakia sotiris k. hadjikakou, department of chemistry, university of ioannina, greece linda hall, faculty of agricultural, life & environmental sciences, university of alberta, edmonton, ab canada richard haynes, centre for infection, division of cellular and molecular medicine, st george’s, university of london, uk hooshang hamidian, department of chemistry, payame noor university (pnu), tehran, iran berta barta holló, department of chemistry, biochemistry and environmental protection faculty of sciences, university of novi sad, serbia tingjun hou, institute of functional nano & soft materials; soochow university, china nenad ignjatović, institute of technical sciences of the serbian academy of sciences and arts, belgrade, serbia mirela iličić, mirela iličić, university of novi sad, serbia moustafa issa , faculty of science, cairo university, giza, egypt ivana ivančev-tumbas, university of novi sad, serbia radmila jančić heinemann, faculty of technology and metallurgy, university of belgrade, serbia _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 4 journal of the serbian chemical society goran janjić, institute of chemistry, technology and metallurgy, university of belgrade, serbi j. jayabharathi, department of chemistry, annamalai university, annamalainagar, tamil nadu, india stanka jerosimić, faculty of physical chemistry, university of belgrade, serbia vladislava jovanović, institute of chemistry, technology and metallurgy, university of belgrade, serbia vladimir jović, institute for multidisciplinary research, university of belgrade, serbia ivan juranić, faculty of chemistry, university of belgrade, serbia ivanka karadžić, department of chemistry, school of medicine, university of belgrade, serbia bharti khungar, department of chemistry, birla institute of technology and science, pilani, rajasthan, india katarzyna kieć-kononowicz, department of technology and biotechnology of drugs, jagiellonian university medical college, kraków, poland mirjana kijevčanin, faculty of technology and metallurgy, university of belgrade, serbia nedeljko krstajić, faculty of technology and metallurgy, university of belgrade, serbia honnaiah kumar, department of organic chemistry, indian institute of science, bangalore, karnataka, india moslem mansour lakouraj, department of chemistry, mazandaran university, babolsa, iran isabel lara, universitat de lleida, spain gregory lefevre, cnrs-chimie paris tech / ircp, france wolfgang linert, technical university of vienna, vienna, austria iwona maciejowska, zakład dydaktyki chemii, wydziału chemii, kraków, poland kamran t. mahmudov, centro de química estrutural, instituto superior técnico, tu lisbon, portugal magdalena malecka, department of theoretical and structural chemistry, faculty of chemistry, university of lodz, poland roger mallion, university of kent, canterbury, uk miodar maksimović, faculty of technology and metallurgy, university of belgrade, serbia sven mangelinckx, department of sustainable organic chemistry and technology, faculty of bioscience engineering, ghent university, belgium dragan manojlović, faculty of chemistry, university of belgrade, serbia silvija markic, university og bremen, germany zoran marković, state university of novi pazar, serbia branko matović, department of materials science, vinča institute of nuclear sciences, belgrade, serbia violeta marković, faculty of science, university of kragujevac, serbia veselin maslak, faculty of chemistry, university of belgrade, serbia mihai medeleanu, politechnica university, faculty of chemical and environmental engineering, timisoara, romania mohammad mehdi ghanbari, department of chemistry, sarvestan branch, islamic azad university, sarvestan, iran slavko mentus, faculty of physical chemistry, university of belgrade, serbia nevena mihailović, institute for the application of nuclear energy, university of belgrade, serbia dušan mijin, faculty of technology and metallurgy, university of belgrade, serbia miloš milčić, faculty of chemistry, university of belgrade, serbia slobodan milonjić, vinča institute of nuclear sciences, university of belgrade, serbia _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 2014 list of referees 5 ingrid milošev, jozef stefan institute, ljubljana, slovenia ahmed mkadmh, chemsitry department, faculty of science, al aqsa university gaza palestine keith murray, school of chemistry, monash university, vic, autralia shanmugam muthusubramanian, department of organic chemistry, school of chemistry, madurai kamaraj university, india muhammad naseer, department of chemistry, quaid-i-azam university, islamabad, pakistan bojana nedić vasiljević, faculty of physical chemistry, university of belgrade, serbia olgica nedić, institute for the application of nuclear energy, university of belgrade, serbia aleksandra nešić, faculty of technology and metallurgy, university of belgrade, serbia biljana nigovic, department of pharmaceutical, analysis, faculty of pharmacy and biochemistry, university of zagreb, croatia katarina nikolić, faculty of chemistry, university of belgrade, serbia milan nikolić, faculty of chemistry, university of belgrade, serbia paolino ninfali, universita degli studi di urbino, dipartimento di scienze biomolecolari, urbino, italy emiliya v. nosova, chemical technology institute, urals federal university, ekaterinburg, russia miroslav novaković, institute of chemistry, tehnology and metallurgy, university of belgrade, serbia tatjana novaković, institute of chemistry, tehnology and metallurgy, university of belgrade, serbia jean ondo, aix-marseille université, cnrs, lce, marseille, france dejan opsenica, faculty of chemistry, university of belgrade, serbia igor opsenica, faculty of chemistry, university of belgrade, serbia adil a. othman, faculty of sciences, university of sciences and technology of oranmohamed boudiaf-usto-mb, oran, algeria magdalena owczarek, faculty of chemistry, university of wroclaw, poland sibel a. ozkan, faculty of pharmacy, ankara university, turkey jose palacios, the university of new mexico, albuquerque, usa vladimir panić, institute of chemistry, tehnology and metallurgy, university of belgrade, serbia andrea penoni, dipartimento di scienza e alta tecnologia università degli studi dell'insubria, como, italy marija pergal, institute of chemistry, technology and metallurgy, university of belgrade, serbia miljenko perić, faculty of physical chemistry, university of belgrade, serbia milena petković, faculty of physical chemistry university of belgrade, serbia menka petkovska, faculty of technology and metallurgy, university of belgrade, serbia srđan petrović, institute of chemistry, technology and metallurgy, university of belgrade, serbia rada petrović, faculty of technology and metallurgy, university of belgrade, serbia josé luis pérez pavón, facultad de química, salamanca, spain andrej perdih, national institute of chemistry, ljubljana, slovenia slobodan d. petrović, faculty of technology and metallurgy, university of belgrade, serbia velimir popsavin, faculty of science, university of novi sad, serbia radivoje prodanović, department of biochemistry, faculty of chemistry, university of belgrade, serbia _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 6 journal of the serbian chemical society hanna pruchnik, department of physics and biophysics, wrocław university of environmental and life sciences, wrocław, poland vladislav rac, faculty of agriculture, university of belgrade, zemun, serbia bojan radak, vinča institute of nuclear sciences, university of belgrade, serbia serbia slavko radenković, department of chemistry, faculty of science, university of kragujevac, serbia marija radoičić, vinča institute of nuclear sciences, university of belgrade, serbia serbia nevenka rajić, faculty of technology and metallurgy, university of belgrade, serbia vesna rakić, faculty of agriculture, university of belgrade, serbia jelena randjelović, faculty of pharmacy, university of belgrade, serbia slavica ražić, faculty of pharmacy university of belgrade, serbia tamás réti, obuda university, budapest, hungary enrico sanjust, department of biomedical sciences, university of cagliari, italy vladimir savić, faculty of pharmacy university of belgrade, serbia martin schlummer, fraunhofer ivv, freising, germany dr feng shi, school of chemistry and chemical engineering, jiangsu normal university, xuzhou, p. r. china samia shouman, cairo university, department of tumor biology, cairo, egypt zeba siddiqui, department of chemistry, aligarh muslim university, aligarh, india inder pal singh, department of natural products, national institute of pharmaceutical education and research (niper), nagar, punjab, india vishnu l. sharma, medicinal & process chemistry division, csir-central drug research institute, lucknow, india morteza shiri, department of chemistry, faculty of science, alzahra university, vanak, tehran, iran tesfaye r. soreta, department of chemistry, college of natural sciences, jimma university, jimma, ethiopia vladimir v. srdić, faculty of technology university of novi sad, serbia dragana stanić-vučinić, faculty of chemistry, university of belgrade, serbia miroslav stanković, institute of chemistry, technology and metallurgy, department of catalysis and chemical engineering, university of belgrade, serbia dragomir stanisavljev, faculty of physical chemistry, university of belgrade, serbia dragoslav stoiljković, faculty of technology, university of novi sad, serbia dušica b. stojanović, faculty of technology and metallurgy, university of belgrade, serbia gordana stojanović, department of chemistry, faculty of science and mathematics, university of niš, serbia biljana stojanović, department of drug analysis, faculty of pharmacy, university of belgrade, serbia ming-der su, national chiayi university, deptartment of applied chemical, chiayi, taiwan xuewu sui, department of pharmacology, case western reserve university, usa terézia szabό-plánka, department of physical chemistry, university of szeged, hungary zoran šaponjić, vinča institute of nuclear sciences, belgrade, serbia biljana šljukić paunković, faculty of physical chemistry, university of belgrade, serbia tatjana šolević knudsen, institute of chemistry, technology and metallurgy, university of belgrade, serbia _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 2014 list of referees 7 vladimir šukalović, institute of chemistry, technology and metallurgy department of chemistry, university of belgrade, serbia aziz tekin, ankara university, turkey vele tešević, faculty of chemistry, university of belgrade, serbia shiliang tian, university of illinois at urbana-champaign, urbana, il, usa manisha tiwari, dr. b.r. ambedkar centre for biomedical research, university of delhi, india theivasanthi thirugnanasambandan, international research centre, kalasalingam university, tamil nadu, india tamara todorović, faculty of chemistry, university of belgrade, serbia simonida tomić, faculty of technology and metallurgy, university of belgrade, serbia dragica trivić, faculty of chemistry, university of belgrade, serbia hakki türker akcay, recep tayyip erdoan university, rize, turkey vlatka vajs, institute of chemistry, technology and metallurgy department of chemistry, university of belgrade, serbia s. venkata mohan, bioengineering and environmental sciences (bees), csir-indian institute of chemical technology (csir-iict), hyderabad, india vesna vasić, vinča institute of nuclear sciences, belgrade, serbia cristina vázquez, universidad de buenos aires, facultad de ingeniería, buenos aires, argentina h. venkatachalam, department of chemistry, kasturba medical college international center, manipal university, india zhivko velkov, south-west university ‘‘neofit rilski’’, bulgaria tatjana ž. verbić, faculty of chemistry, university of belgrade, serbia miguel vilas vilas-boas, escola superior agraria braganca, braganca, portugal zoran višak, centro de química estrutural, instituto superior tecnico, universidade de lisboa, portugal vesna vitnik, department of chemistry, institute of chemistry, technology and metallurgy, university of belgrade, serbia željko vitnik, department of chemistry, institute of chemistry, technology and metallurgy, university of belgrade, serbia maja vukašinović-sekulić, faculty of technology and metallurgy, university of belgrade, serbia tatjana vulić, faculty of technology, university of novi sad, serbia agnieszka ewa wiącek, department of interfacial phenomena, faculty of chemistry, umcs lublin, poland cheli wang, changzhou university, school of petrochemical engineering, changzhou, pr china limin wang, key laboratory of advanced materials and institute of fine chemicals, east china university of science and technology, shanghai, pr china julian weghuber, university of applied sciences upper austria, school of engineering and environmental sciences, steyr, austria hong wei xi, technological university, singapore yumei xiao, department of applied chemistry, china agricultural university, beijing, p. r. china takehiko yamato, department of applied chemistry, faculty of science and engineering, saga university, japan okan zafer yeşilel, eskisehir osmangazi univ., eskisehir, turkey george a. zachariadis, aristotle university of thessaloniki, greece _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 8 journal of the serbian chemical society safi zaki, al-azhar university gaza, gaza · department of chemistry, palestine territory, occupied nilo zanatta, núcleo de química de heterociclos, departamento de química, universidade federal de santa maria, brazil snežana zarić, faculty of chemistry, university of belgrade, serbia gang zhao, shanghai institute of organic chemistry, chinese academy of sciences, 354 fenglin lu, p. r. china yimin zhao, beijing institute of pharmacology and toxicology, beijing, pr china matija zlatar, institute of chemistry, technology and metallurgy, university of belgrade, serbia mire zloh, university of hertfordshire, hatfield, uk jelena zvezdanović, faculty of technology, university of niš, leskovac, serbia branka žarković, faculty of agriculture, university of belgrade, serbia majda žigon, national institute of chemistry, ljubljana, slovenia irena žižović, faculty of technology and metallurgy, university of belgrade, serbia _________________________________________________________________________________________________________________________ (cc) 2015 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice investigation of different extraction procedures for the determination of major and trace elements in coal by icp-aes and ion chromatography j. serb. chem. soc. 81 (4) 403–417 (2016) udc 546.3+661.183.2:66.061+54– jscs–4856 732:543.421:543.544–71 original scientific paper 403 investigation of different extraction procedures for the determination of major and trace elements in coal by icp-aes and ion chromatography aleksandra m. tasić1#, ivana d. sredović ignjatović2*#, ljubiša m. ignjatović1#, ivan b. anđelković3#, mališa p. antić2# and ljubinka v. rajaković4 1university of belgrade, faculty of physical chemistry, studentski trg 12–16, 11000 belgrade, serbia, 2university of belgrade, faculty of agriculture, nemanjina 6, 11080 belgrade, serbia, 3university of belgrade, innovation center of the faculty of chemistry, studentski trg 12–16, 11000 belgrade, serbia and 4university of belgrade, faculty of technology and metallurgy, karnegijeva 4, 11000 belgrade, serbia (received 29 april, revised 2 august, accepted 14 september 2015) abstract: this paper presents the extraction of major and trace elements from a coal sample, in deionized water, by using three different extraction techniques. rotary mixing and ultrasonic extraction were examined for different extraction times, while the microwave-assisted extraction was performed at different temperatures. metal concentrations (na, k, ca, mg, al, fe, mn, be, cd, co, cr, hg, as, ni, se, sb and pb) in solution were determined employing inductively coupled plasma atomic emission spectrometry; whereas the results obtained for na, k, ca and mg were compared employing ion chromatography. comparing the rotaryand ultrasonic-assisted extractions, it was shown that the former technique was more efficient for the determination of fe, na and pb, whereas the latter one proved more efficient for the determination of co and cr ions. microwave-assisted extraction was shown to be the most efficient method for all the tested elements in coal. in addition, sequential extraction of the elements was realized using microwave digestion. the results of the sequential extraction experiments indicated associations of investigated elements with a mineral phase and organic matrix. sequential extraction provided information on possible leaching of as, cd, co, cr, ni, pb, fe and mn under environmental conditions. keywords: coal, extraction; microwave; ultrasonic; icp-aes; ion chromatography. * corresponding author. e-mail: isredovic@agrif.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc150429078t _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 404 tasić et al. introduction numerous studies conducted in the course of the 20th century clearly indicated that there was a direct connection between the contents of trace elements in coal and environmental pollution caused by the products of coal combustion.1–3 some of potentially toxic elements (as, be, cd, co, cr, hg, mn, ni, pb, sb, se, th and u) achieve multiple concentrations both in ash and fly ash and may cause land, air, and water pollution and affect vegetation.4 most methods of decomposition used in elemental analysis are based on digestion with strong acids, such as hno3, hcl, h2so4 and hf, at elevated temperatures, which could lead to loss of easily volatile elements, such as as, sb, se and sn.5 determination of cation concentrations can be realized in coal and fly ash extracts obtained by single or sequential procedures of extractions. many single or sequential procedures, mainly based on the community bureau of reference (bcr) procedure or different versions thereof, have been applied to coal and fly ash to fractionate elements by using different extractants or reagents to obtain more useful information about the bioavailability and mobility of elements.6–9 a useful sample preparation method for solid samples, such as coal, fly ash, ash, soil, sediment, biological and environmental samples, is ultrasound-assisted digestion.10–15 the main benefits of ultrasonic-assisted sample pre-treatment over other methods are the speed of digestion, high sample treatment capacity and low reagent usage. since a substantially lower pressure and temperature are used in ultrasonic-assisted digestion, it is safer than microwave digestion.11 microwave-assisted digestion can be performed in open or closed digestion systems and nowadays, the trend towards closed systems is the more dominant one.16–18 the main benefits of closed systems include the possibilities for controlling both temperature and pressure, and for pre-treating the sample without a significant loss of analyte elements.18 this paper presents a comparison of three techniques for the simultaneous extraction of elements using a rotary mixer, a microwave-digestion system and an ultrasonic bath. the metal concentrations in the water extracts were measured by ion chromatography (ic) and inductively coupled plasma-atomic emission spectrometry (icp-aes). deionized water was the agent used for all extractions. in the present study, the partitioning of the elements (na, k, ca, mg, al, fe, mn, be, cd, co, cr, hg, as, ni, se, sb and pb) was investigated using a microwave sequential extraction procedure. the bcr microwave extraction protocol was modified and the fractionations of the elements found in coal were examined and discussed. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ major and trace elements extraction from coal 405 experimental sampling and sample preparation a composite coal sample was taken from the kostolac coalmine, which is located approximately 90 km east of belgrade, serbia. prior to use, the sample was ground, homogenized, sieved through a 250 μm mesh and oven-dried overnight at 105 °c. reagents deionized water (18.2 mω cm) produced by a milli-q reagent grade system (millipore, usa) was used for the extractions and preparation of all solutions. acetic acid, hydrogen peroxide, nitric acid and hydrochloric acid were of analytical grade and obtained from merck (germany). hydroxylammonium chloride, oxalic acid, ammonium oxalate and ammonium acetate were of analar grade and procured from fluka (germany). instrumentation a 3540 conductivity/ph-meter (jenway, uk) was used to measure ph values of the extracts. a 761 compact ic ion chromatography system with a conductometric detector was used, as well as a metrosep c 2-150 separation column (all from metrohm, switzerland). the eluent was a 4 mmol dm-3 tartaric and a 0.75 mmol dm-3 dipicolinic acid solution, at a flow rate of 1 ml min-1. the eluent was made on a daily basis, and then filtered through a 0.2 μm membrane filter (phenomenex, usa). the injected sample volume was 20 μl for each test. for ic calibration, the certified standard solution of na, k, ca and mg (primary multiion standard solution, fluka, switzerland) was used to prepare working solutions by serial dilutions of standard solution. the icp-aes measurements were performed using an icap-6500 duo icp spectrometer (thermo fisher scientific, uk), with continuous wavelength coverage ranging from 166 to 847 nm, equipped with a racid86 charge injector detector, a pneumatic cross-flow type nebulizer, a quartz torch and a quartz detector. the instrumental conditions were set to an input power of 1150 w, an auxiliary gas flow of 0.5 dm3 min-1, a coolant gas flow of 12 dm3 min-1 and a nebulizer flow 0.5 dm3 min-1. all the concentration measurements were realized using a four-point calibration. for ic-aes calibration, multi-element plasma standard solution 4 (specpure, alfa aesar, germany) was used to prepare working solutions. analytes were determined at the following wavelengths (nm): 308.2 (al); 193.7 (as); 234.8 (be); 315.8 (ca); 226.5 (cd); 228.6 (co); 267.7 (cr); 240.4 (fe); 253.6 (hg); 769.8 (k); 279.0 (mg); 259.3 (mn); 818.3 (na); 231.6 (ni); 217.6 (sb); 206.3 (se) and 220.3 (pb). an overhead mixer reax 20/8 rotary mixer (carl roth, germany) and a transsonic t 760 dh ultrasonic bath (elma, germany), with an ultrasonic frequency of 40 khz and an effective ultrasound power of 170 w, were used. microwave digestion for coal sample preparation was accomplished using an ethos 1, advanced microwave digestion system (milestone, italy), equipped with 10 ptfe containers for microwave digestion. the capacity of the containers was 100 cm3 each, and the maximum pressure and temperature that could be achieved were 10 mpa and 240 °c, respectively. extraction procedures single extraction. the extraction suspension was prepared by mixing a coal sample with deionized water in the ratio 1 g:50 cm3. the extractions were realized using a rotary mixer (re) in which the suspension was processed for the following extraction times: 30, 60, 90, 120 and 180 min by mixing at 10 rpm at room temperature (20 °c). the second extraction technique involved the use of an ultrasonic bath (ue) for the following extraction times: 10, _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 406 tasić et al. 20, 30, 40 and 50 min. the third technique was microwave digestion (me) at the following temperatures: 50 (m50), 100 (m100) and 150 °c (m150). both temperature and pressure were controlled during the process; working temperature was achieved in 15 min for each cycle of extraction, while the extraction itself lasted for 15 min. after extraction, the samples were cooled to room temperature and phases were separated by centrifugation. blank extractions were performed for each of the employed extraction techniques. the coal extracts were acidified by the addition of a nitric acid solution (2.0 mol dm-3) and stored at 4 °c in a laboratory refrigerator. microwave sequential extraction. the coal samples were subjected to the extraction scheme, suggested by the standards, measurements and testing programme (bcr, formerly) of the european commission. in order to make a distinction between an easily reducible fraction bound to manganese oxides and a moderately and poorly reducible fraction bound to amorphous and crystalline iron oxides, the procedure was modified by adding a third extraction step. aqua regia was used in the fifth stage of the extraction according to the epa 3050b digestion method for determining the residual metal content of the analytes. the sequential extraction was investigated at three different temperatures and it was performed in triplicate. the complete scheme of the modified and applied procedure is given in table i. table i. the composition of extracting solutions and conditions of the sequential extraction procedures fraction no. elements extracted extraction time, min agitation method extractant volume, cm3 extractant i water-exchangeable, weakly adsorbed 15 me 40 0.11 mol dm-3 hoac ii easily reducible 15 me 40 0.1 mol dm-3 nh2oh·hcl / hno3, ph 2 iii moderately reducible 15 me 40 0.2 mol dm-3 (nh4)2c2o4 / 0.2 mol dm-3 h2c2o4 iv oxidizable 180 occasional agitation, 85 °c 2×10 30 % / 8.8 mol dm-3 h2o2 / hno3, ph 2 v residual 15 me 40 1 mol dm-3 nh4oac, ph 2 15 me 10 aqua regia results and discussion single extraction the measured ph value of the coal suspension in deionized water was 6.40. the extraction of potassium and sodium by means of re and ue are shown in fig. 1a and b, respectively, while ion concentrations were measured using ic and icp-aes. it could be seen from fig. 1a that the amount of extracted potassium increased as the time of shaking increased. a significant increase in the concentration of extracted potassium was observed when the time of shaking was inc _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ major and trace elements extraction from coal 407 (a) (b) fig. 1. the contents of na and k obtained by means of: a) re and b) ue at different extraction times, measured by ic and icp-aes. reased from 30 to 60 min. a further increase in the shaking time did not lead to any substantial increase in the quantity of extracted potassium ions and after 90 min, the concentration of potassium in the water was almost constant. the efficiency of ultrasonic-assisted extraction of potassium improved when the exposure to ultrasound was increased from 10 to 30 minutes (fig. 1b). the quantity of extracted potassium decreased with further increase in the duration of the ultrasonic-assisted extraction. potassium ion extraction using a rotary mixer gave almost the same results as the extraction using the ultrasonic bath. the quantity of sodium extracted by means of re increased with the time of shaking up to 60 min. a further increase in the time of shaking led to a decrease in the quantity sodium, after which a moderate increase was observed. the quantity of sodium obtained by means of ue increased as the duration of extraction increased up to _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 408 tasić et al. 30 min; thereafter a further increase in the time of extraction did not lead to any changes in the quantity of sodium. the quantity of sodium extracted after 30 min using an ultrasonic bath was almost the same as that obtained after 180 min using a rotary mixer. the extracted quantity of potassium obtained by microwave-assisted extraction at 100 °c was 2.2 times higher, while at 150 °c it was even 3.2 times higher than the one obtained using re and ue (fig. s-1 of the supplementary material to this paper). the quantity of sodium ions extracted employing me (fig. s-1) showed a tendency for a mild increase with increasing extraction temperature. according to results shown in fig. 2a and b, it is clear that there were no differences for ca and mg extraction using re and ue. the maximum extracted quantities of both mg and ca were obtained after 20 min of ue. from fig. s-2 of the supplementary material, it could be seen that with temperature increase, me (a) (b) fig. 2. the contents of ca and mg obtained by means of: a) re and b) ue at different extraction times, measured by ic and icp-aes. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ major and trace elements extraction from coal 409 became more efficient for ca than for mg extraction. when the me temperature was changed from 50 to 100 °c, the extraction of ca increased 5 times. this was expected since ca in coal occurs as various insoluble inorganic compounds. the similarity between the results obtained in the processes of ca and mg extraction could be associated with the fact that the similarity of elements in terms of their chemical characteristics leads to their similar behavior in coal and similarity in the process of their association and mobility. every parallel measurement of the concentrations k, na, mg and ca using the icp-aes and ic methods showed an adequate level of consistency, as well as great precision and accuracy in the measurement process. in a large number of measurements, the good agreement between the results obtained by ic and icp -aes confirmed that a readily available measurement method, such as ic, could be used effectively. the concentrations of be, sb, se and hg, obtained using the three different coal extraction procedures were below the detection limits of icp-aes (be < 2; se < 2; sb < 5 and hg < 10 μg dm–3). therefore, the investigated extraction methods cannot be used for the determination of these elements in coal. one of the reasons is their low concentrations, high volatility and forms of occurrence in coal.19,20 the amounts of cadmium extracted using the re and ue methods were below the detection limit (2 μg dm–3). microwave-assisted extraction at 100 °c resulted in an eleven times lower extraction amount when compared with the amount extracted at 150 °c (table s-i of the supplementary material). the amounts of extracted cobalt obtained by re, ue and m50 were quite low, close to or even below the detection limit (table s-i). the quantity of cobalt extracted using microwaves increased with increasing extraction temperature. the microwave-assisted extraction at 150 °c resulted in the highest extracted amounts of aluminum, manganese and, especially, iron compared to the other employed extraction techniques (table s-i). the results presented in the same table showed that re was more efficient for extraction of al and fe than ue. in the case of manganese, the efficiencies of the ue and re methods were comparable. the me at 50 °c resulted in a quantity of manganese ions that was twice as high, whereas an increase in temperature to 100 °c resulted in a ten times higher quantity of manganese and the quantity extracted at 150 °c was 30 times higher than those obtained by re and ue. in the case of arsenic, nickel and lead, the extraction using an ultrasonic bath or a rotary mixer proved equally efficient (table s-i). the quantities of all extracted elements increased with increasing me temperature. the influence of temperature on nickel extraction was more prominent than on arsenic extraction. considering the ue technique, the extracted amount of cations varied with the extraction time. this could be explained by the effect of ultrasonic energy on _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 410 tasić et al. the coal suspension that, alternately, influences the processes of ion adsorption and desorption. studies have shown that trace elements are most commonly bound by carboxyl groups of organic macromolecules and/or fulvic acids, when hydrogen ions are exchanged by metal ions, which is consistent with previous results.21 in the case of as, mn, ni and pb, the extracted quantities were almost constant at all re and ue extraction times. the efficiency of re and ue were the same for the above-mentioned elements. ue was more efficient in the case of co and cr, giving significantly higher extracted amounts than re. the use of re proved more efficient than ue for fe and al extraction. considering the me technique, there was an obvious positive correlation between the amount of extracted elements and extraction temperature, as expected. the microwave-assisted procedures were faster than the other two techniques. sequential microwave extraction of major and minor elements sequential extraction provides useful information for risk assessment since the amount of elements mobilizable under different changes in environmental conditions can be estimated.17 the time (and hence cost) required for extraction is considerable, with the bcr and tessier procedures requiring up to 4 and 5 days respectively.22 microwave technology provides an opportunity to reduce extraction time and offers an attractive alternative to conventional procedures. the average concentrations of major elements (for three measurements) in the coal sample extracted using sequential microwave extraction (sme) at three different temperatures are listed in table ii. as expected, an increase in the temperature of the sme resulted in an increase in the total quantity of extracted elements. the highest influence of temperature increase during sme was on al and k extraction, moderate on fe and mn extraction, and the lowest on ca, mg and na extraction. such results may be explained by the fact that a rise in temperature leads to the breakdown of minerals such as kaolinite, montmorillonite, illite and aluminum oxides and hydroxides.23 in the course of the sme, calcium was extracted in fraction i and ii at every temperature (table ii), which could be attributed to the breakdown of carbonates, namely calcite (caco3), dolomite (ca,mg(co3)2) and siderite (feco3).24 likewise, the occurrence of magnesium could be attributed to carbonates, to dolomite mainly. in addition, the fact that the highest quantities of magnesium were extracted in fraction i by means of acetic acid and in fraction ii by means of a hydroxylamine solution supports the view that it is bound to carbonates. the largest quantities of sodium, which can be found in a number of minerals, were extracted at all temperatures in fractions i and iv of the sme, an occurrence that is attributed to the breakdown of sulfide ores and the organically associated metal fraction. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ major and trace elements extraction from coal 411 table ii. the concentrations of major metals – al, ca, fe, k, mg, mn and na (μg g-1), obtained by sequential microwave extraction at 50, 100 and 150 °c fraction t / °c al ca fe k mg mn na i 50 26.74 6207 51.55 50.66 1236 38.54 48.21 ii 127.0 5507 2005 31.80 589.3 104.3 9.70 iii 521.7 26.30 3535 11.95 72.01 51.89 17.91 iv 1462 1238 5487 38.73 192.9 22.78 77.65 v 124.8 78.34 86.05 6.62 14.94 0.52 9.13 sum 2260 13060 11160 139.8 2105 218.0 162.6 i 100 22.99 8311 237.5 69.56 1651 111.3 51.80 ii 178.9 4531 4494 26.31 407.6 132.7 10.17 iii 1056 27.43 2049 11.57 72.53 15.34 12.06 iv 1253 596.6 4763 34.70 132.1 10.19 65.31 v 2213 29.95 622.0 49.03 85.05 1.69 13.57 sum 4724 13500 12170 191.2 2348 271.2 152.9 i 150 554.3 8920 1997 86.72 1754 156.7 58.74 ii 816.7 4289 4379 39.31 439.3 93.01 12.28 iii 4581 36.41 2166 63.78 178.3 12.44 17.12 iv 1086 395.9 2599 27.45 71.07 6.02 70.65 v 7138 55.93 2692 240.5 271.3 5.94 24.57 sum 14180 13700 13830 457.8 2714 274.1 183.4 astm d6349-13 45600 17400 31900 1878 4727 374.2 709.5 the largest quantities of potassium were extracted in the first fraction of sequential extraction at 50 and 100 °c and in the fifth fraction at 150 °c. this indicates that potassium was bound to hydrated aluminosilicate minerals. the main components of mineral matter from many coal and clay minerals which contain aluminum include: kaolinite (al2si2o5(oh)4, illite (kal2(alsi3o10)(oh)2, muscovite kal2(oh,f)2(alsi3o10) and montmorillonite (al2si4o10(oh)2·h2o). an oxalate reagent is an efficient agent to be used for the extraction of amorphous aluminum hydroxides, for which reason, a significant quantity of aluminum during was obtained in fraction iii. the reason the greatest quantity of al was extracted into fraction v is that with increasing temperature, the silicate material was decomposed. the presence of fe in the second extraction fraction indicates the presence of amorphous, highly mobile hydroxides of this element. it was expected that fe would be present during fraction iii due to the extraction of amorphous and crystalline oxides. in addition, a considerable amount of fe was found in fraction iv, whereas after an increase in the temperature of the sme up to 150 °c, it became the dominant component in fraction v as well. it is known that bentonite clays found at the kostolac basin contain high concentrations of fe2o3 (6.5–8.5 %).21 manganese quantities extracted in fraction i and fraction ii were considerable, due to decomposition of manganese oxides by the hydroxylamine solution. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 412 tasić et al. a significantly high concentration of manganese was evident and it was most probably bound to carbonates (calcite and siderite).21 the average concentrations of trace elements determined by sme are presented in table s-ii of the supplementary material. cobalt was released in all steps of the sequential extraction, but the easily mobilized form (easily reducible) and oxidizable were predominant at all extraction temperatures. this is in accordance with the fact that cobalt in coal is mainly associated with sulfide minerals, mostly in pyrite, or in organic associations in low-rank coals.25 the highest quantities of be extracted at 50 and 100 °c were obtained in fractions ii–iv of the sequential extraction, whereas those extracted at 150 °c were the highest in fraction i and ii (table s-ii). cadmium was released in all steps of the sequential extraction except for the residual fraction at 50 °c and presented similar percentages in the second and fourth fraction. cadmium usually occurs in sphalerite (zns).25 substantial quantities of arsenic was extracted in fractions iii and iv at 50 °c, in fractions ii–iv at 100 °c and in fractions i and ii at 150 °c. on the other hand, arsenic was almost not present in fraction v owing to its volatility. the greatest extracted quantities chromium were found in fraction iv of sequential extraction at 50 and 100 °c, but in fractions iii–v when the extraction was performed at 150 °c. chromium is found in association with oxides, most commonly, iron oxides.26 chromium in coal is organically associated, but coal with high concentrations of chromium (> 500 μg g–1) was found to contain chromite (fecro4).26 considerable quantities of nickel were extracted at all temperatures in fractions ii–iv. in many coals, substantial amounts of ni may be organically bound. the inorganically bound ni appears to be largely associated with sulfides.25 the largest amounts of lead were extracted in fractions iii and iv. lead occurs predominantly as sulfides or associated with sulfide minerals. galena (pbs) is the most common form of lead in coal, but galena can have several significantly different associations. galena can occur as large, epigenic crystals in cleat and fractures.27 the total contents of analyzed elements were determined by the standard astm method and the results are presented in tables ii and s-ii.28 the results of applied standard method were compared with results obtained for sme at 150 °c. the highest investigated temperature was selected, since, for most of the investigated elements, the extracted concentrations increased with temperature increase. according to the results listed in tables ii and s-ii, it could be seen that for all analyzed elements, less than 80 % of total content was determined by sme. considering the major elements (table ii), the lowest recovery (according to total content) was for al, k and na, moderate recovery was achieved for fe _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ major and trace elements extraction from coal 413 and mg, while the highest recovery was obtained for ca and mn. the reason for this is that in applied sme, aggressive reagents, such as concentrated mineral acids, were not used. in addition, al and k in coal are usually bonded to silica matter, and in this study hf, as proven reagent for silica decomposition, was not used. on the other hand, coal from the kostolac mine has a high silica content (up to 9 %). the results listed in table s-ii indicated that for the trace elements, lowest recoveries were for as and co, while moderate recoveries were achieved for cr, ni and pb. total contents for be and cd were below the detection limit, contrary to sme when these elements were detected. although, aqua regia was used in the fifth step of sme to determine the residual metal content, it is evident that some further investigations should be conducted in order to improve the precision and accuracy of the method. from a practical point of view, it is very important to evaluate the migration of the investigated major and, especially, trace elements to the environment. the mobility of elements from coal under environmental conditions depends on the ph of the rain and/or ground water, the ph values of which are lower than 7. the analyzed elements that are mobile under environmental conditions are bound to the water-exchangeable and weakly adsorbed fractions (the first step of the sme). the sme experiments of were conducted at raised temperatures, but from results presented in tables ii and s-ii , it is obviously that elements such as as, cd, co, cr, ni, pb, fe and mn from coal could influence environmental pollution. the element found in the other fractions could migrate to the environment under specific conditions, i.e. aggressive (fractions ii and iv) and/or in the presence of microorganisms (fractions ii–v).29 validation of extraction methods calibration curves were established for all investigated elements using aqueous standards. for this purpose, the certified reference solution multi-element standard solution 4 (specpure, alfa aesar) and diluted solutions were used. the parameters of the calibration lines, i.e., slopes, intercepts and correlation coefficients (r), as well as limits of detection (lod) and limits of quantifications (loq) are presented in table iii. the lod and loq values were calculated as 3 and 10 times the ratio of the relative standard deviation to the slope, respectively.30 the lod and loq values for investigated elements in coal sample (µg kg–1) are 10-times the corresponding values presented in table iii. it was found that the linear range is at least up to 3 orders of magnitude in relation to the loqs. the accuracy of the complete method, starting from the extraction (rotary, ultrasonic-assisted and microwave-assisted) to icp-aes measurements was checked using the standard addition method.30 different small amounts of aqueous crm were added to a real coal sample, which was air dried, at room tem_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 414 tasić et al. perature, before the extraction procedure. the obtained extracts were analyzed in triplicate, the standard deviations and the recoveries (r) were calculated for each element. the results are given in table s-iii of the supplementary material. the accuracy of the extraction methods were investigated under the optimal analytical conditions, as can be seen from figs. 1, 2 and s-1 and table s-i: re for 120 min, ue for 30 min, and me at 150 °c. table iii. calibration data, lod and loq for icp-aes measurement of the investigated elements element intercept, cps slope, cps dm3 µg-1 r lod / µg dm-3 loq / µg dm-3 al 298.6 5.01 0.9970 2.51 8.36 as –0.87 1.11 0.9999 1.24 4.14 ca 150.53 20.05 0.9993 0.72 2.4 cd 15.39 37.84 0.9999 0.06 0.22 co 2.31 15.88 0.9999 0.13 0.45 cr 10.07 28.07 0.9999 0.33 1.09 fe 20.58 10.15 0.9988 0.67 2.24 k 736.3 68.09 0.9993 0.50 1.66 mg 34.72 4.52 0.9998 2.22 7.39 mn 87.89 139.47 0.9997 0.06 0.20 na –144.85 4.82 0.9999 7.62 25.39 ni 10.64 12.22 0.9998 0.20 0.65 pb –1.04 2.73 0.9892 0.81 2.71 as can be seen from table s-iii, the used extraction methods provided good recoveries of all elements, ranging from 91 to 105 %. this means that the proposed methods are of good accuracy. the relative standard deviations (rsd) of the measured elements were in all cases, except na and k, less than 10 %, leading to the conclusion that the precision of the methods is satisfactory. the rsd values for na and k determination in spiked coal sample (table s-iii) were between 15 and 20 %. the rsd values for na and k were 12.24 and 11.30 %, respectively, for the calibration measurements with icp-aes. in these cases, the use of ic is more appropriate because the rsd for na and k were 0.35 and 0.29 %, respectively, and the contents of these metals in coal were in the range of the sensitivity of ic. conclusions three different extraction methods for the extraction and determination of elements in coal were investigated. the results of ion measurements in an extraction solution by ic and icp-aes indicated that ic could by successfully applied for the determination of the investigated alkali and earth alkali elements. the icp-aes method is very applicable in the study of a multi-element extraction process and metal partitioning in coal. the metal contents extracted using a rotary mixer and an ultrasonic bath were very similar and much lower than _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ major and trace elements extraction from coal 415 quantities extracted by a single microwave-assisted extraction. with respect to the extractions in which a rotary mixer or an ultrasonic bath was used, there were no positive correlations between the amount of extracted elements and the extraction time. in addition, the most efficient extraction technique for fe, na and pb ions was the re, while the ue was the most efficient method for co and cr extraction. the proposed microwave-assisted methods significantly reduced the time required for a conventional sequential extraction. with dependence on the analyte and temperature, the proposed microwave-assisted sequential procedure for coal digestion was considered suitable for the determination of the elements by icp-aes. the extracted amounts for each of the elements analyzed using the microwave-assisted digestion system correlated positively with increasing extraction temperature. additional research on microwave extraction at a constant temperature should be conducted in order to optimize the conditions for the total determination of the elements. results of sequential microwave extraction indicated possible leaching of as, cd, co, cr, ni, pb, fe and mn from coal under environmental conditions. among these elements, as, cd, cr, ni and pb are toxicologically the most important and they were present in the first phase fractions (the water-exchangeable and weakly adsorbed fractions). supplementary material the contents of na, k, ca and mg obtained by microwave assisted extraction at different temperatures, the concentrations of trace elements and the data of the determination of elements in a spiked coal sample are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. this research was supported by the ministry of education, science and technologic development of the republic of serbia, project no. 172030. и з в о д испитивање различитих процедура екстракције за одређивање макро и микро-елемената у угљу помоћу icp-aes и јонске хроматографије александра m. taсић1, ивана д. средовић игњатовић2, љубиша m. игњатовић1, иван б. aнђелковић3, maлишa п. aнtић2 и љубинка в. рајаковић4 1универзитет у београду, факултет за физичку хемију, студентски трг 12–16, 11000 београд, 2универзитет у београду, пољопривредни факултет, немањина 6, 11 080 београд, 3универзитет у београду, иновациони центар хемијског факултета, студентски трг 12–16, 11000 београд и 4универзитет у београду, технолошко–металуршки факултет, карнегијева 4, 11000 београд у раду је представљен поступак екстракције метала и неметала из угља дејонизованом водом применом три екстракционе технике: помоћу ротационе мућкалице, ултразвучне екстракције уз различито време трајања процеса, и микроталасну дигестију на различитим температурама. концентрације елемената (na, k, ca, mg, al, fe, mn, be, cd, co, cr, hg, as, ni, se, sb и pb) у раствору одређиване су применом атомске емисионе спектрометрије са индуковано спрегнутом плазмом, док су вредности за na, k, ca _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 416 tasić et al. и mg утврђене и применом јонске хроматографије. поређење ротационе и ултразвучне екстракције показује већу ефикасност ротационе екстракције за одређивање fe, na и pb, док је ултразвучна екстракција ефикаснија за одређивање co и cr. утврђено је да је микроталасна екстракција најефикаснија техника за екстракцију и одређивање садржаја испитиваних елемената у угљу. будући да се микроталасна екстракција показала бржом и ефикаснијом од друге две технике, спроведене су додатне секвенцијалне екстракције применом микроталаса: резултати указују да су испитивани елементи везани делом за минералну и делом за органску фазу. резултати секвенцијалне екстракције омогућавају процену излуживања as, cd, co, cr, ni, pb, fe и mn из угља у животну средину. 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r. b. finkelman, fuel process. technol. 63 (2000) 79 27. m. mastalerz, a. drobniak, int. j. coal geol. 71 (2007) 37 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ major and trace elements extraction from coal 417 28. astm d6349-13, standard test method for determination of major and minor elements in coal, coke, and solid residues from combustion of coal and coke by inductively coupled plasma-atomic emission spectrometry, 2013 29. e. sočo, j. kalembkiewicz, j. hazard. mater. 145 (2007) 482 30. d. c. harris, quantitative chemical analysis, 7th ed., w. h. freeman & co., new york, 2007, pp. 82–90. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {brown beer vinegar: potentially functional product based on its phenolic profile and antioxidant activity} j. serb. chem. soc. 83 (1) 19–30 (2018) udc 663.47:547.562+663.14.039.3:615.279 jscs–5055 original scientific paper 19 brown beer vinegar: a potentially functional product based on its phenolic profile and antioxidant activity elena mudura1, teodora emilia coldea1*, carmen socaciu2, floricuța ranga2, carmen rodica pop2, ancuța mihaela rotar2 and antonella pasqualone3 1department of food engineering, university of agricultural sciences and veterinary medicine cluj-napoca, romania, 2department of food science, university of agricultural sciences and veterinary medicine cluj-napoca, romania and 3food science and technology unit, department of soil, plant, and food science (di.s.s.p.a.), university of bari “aldo moro”, bari, italy (received 3 august, revised 5 october, accepted 9 october 2017) abstract: the aim of the present study was to create a functional, enriched in polyphenols and free of alcohol product obtained by acetic fermentation of beer. beer and vinegar were tested first for their phenolic content and antioxidant activity, by the folin ciocalteu and the free radical scavenging activity by the 1,1-diphenyl-2-picrylhydrazyl free-radical scavenging assay, respectively. then, the separation and identification of the 30 phenolic compounds was realized by high-performance liquid chromatography coupled with positive electrospray ionisation and diode array detection (hplc-dad–esi(+)-ms) analysis. identification of the phenolic compounds data was realized based on the uv spectra of each compound. based on a calibration curve (r2 = 0.9985), the amounts of the phenolic compounds, expressed as mg cathechin equivalents (ce)/l, were calculated. the total phenolic content of the beer and vinegar samples determined using folin–ciocalteu reagent were of 428.9±1.58 and 661.5±7.69 mg gae l-1, respectively, which contributed to the high antioxidant activity in the vinegar sample of 82.18 %. statistically significant differences were observed after acetic fermentation between each parameter (p < 0.05). brown beer vinegar represents a rich source of polyphenols and phenolic derivatives, compared to beer. by its increased phenolic content and antioxidant activity, brown beer vinegar could be considered another source of valuable compounds to beer, which could also be of interest in special diets. keywords: brewing; acetic fermentation; polyphenols quantification. * corresponding author. e-mail: teodora.coldea@usamvcluj.ro https://doi.org/10.2298/jsc170803107m ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 20 mudura et al. introduction vinegar is one of the fermented beverages used by consumers in their daily diet as a flavouring agent, as a preservative and as a healthy drink. beer vinegar is made in two steps involving yeast for alcoholic fermentation,1 followed by acetic acid bacteria for acetic fermentation.2 as a perspective, food industry manufactures support the capitalizing of bioactive compounds from beer, such as polyphenols, vitamins, minerals, nitrogenous compounds, fibre and carbohydrates.3 the profile of phenolic compounds found in beer is very complex due to their provenience and their transformations during the production process. polyphenols are considered the main natural antioxidants in brewing raw materials and beer, of which 70 % come from malt, and 30 % come from hop (humulus lupulus l.).4 important classes of phenolic compounds found in beer are hydroxybenzoic acids, cinnamic acids and flavonols.5 the total phenolic content in beer ranges from 250 to 500 mg gae l–1, with dark beer containing the highest amount (489 mg gae l–1).6 significant contributions to the in vitro antioxidant activity of beer was attributed to (+)-catechin, (–)-epicatechin, and ferulic, syringic, caffeic and protocatechuic acids7 extracted mostly from malt and in small amounts from hops.8 dietary phenolic compounds play a significant role as antioxidants in vivo.9 beer increases plasma antioxidant capacity in humans, thereby reducing chronic disease risk.10 the flavonoids in hops are considered derivatives of 2′,4,4′,6′-tetrahydroxy -3′-prenylated chalcone and the most important are xanthohumol, desmethylxanthohumol, dehydrocycloxanthohumol, and the flavanones isoxanthohumol, 8-prenylnaringenin and 6-prenylnaringenin.11 these hops prenylflavonoids have a positive effect on the human health due to their antioxidant, anticancer effect, antimicrobial and anti-inflammatory properties. hops have been considered to be an important source of substances having estrogenic effects. 8-prenylnaringenin has been shown to be one of the most potent phytoestrogens identified so far.12 beer is the major dietary source of prenylflavonoides. dark beers present higher levels of prenylflavonoids because dark malts contain maillard reaction compounds that inhibit the isomerisation of prenylflavonoids compounds with less bioactivity. a good yield of prenylflavonoids from hops to the final beer can be achieved by the usage of dark malt in the brewing process.12 beer as a natural drink, moderately consumed, may become a source of many health-promoting compounds.4,12,13 however, the beneficial health effects of moderate beer consumption are strongly related to damages to health that could occur due to the alcohol, such as pancreatitis, diabetes and pancreatic carcinoma.14 the brewing industry generates large amounts of by-products and recent research has been directed towards the reuse of spent grains, brewers spent hops and brewers spent yeasts, from nutritional, economic and environmental points of ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. beer vinegar: potentially functional product 21 view.15 less attention has been given to waste or unsold beers that could be valorised through acetic fermentation. beer vinegar is made from finished beers of all types, and the character of the beers carries through to the finished vinegar. vinegar is an important source of bioactive compounds and could be obtained by traditional (surface processes) or submerged methods, with the former contributing to an enhancement of the level of flavour compounds, increasing both the quality and acceptance of the products, due to the prolonged duration required for the acetic fermentation.16 considering the interest in hop-based products related to health benefits, every year new products are launched on the market. to the best of our knowledge, no paper has focused on an investigation of the phenolic profile of beer vinegar. therefore, the aim of the present study was to characterize the most important functional properties of this product, namely the phenolic profile and antioxidant activity. experimental reagents the reference catechin (min. 98 % purity) was provided by sigma–aldrich (steinheim, germany), gallic acid (min. 98 % purity) from carl roth (karlsruhe, germany), dpph (2,2 -diphenyl-1-picrylhydrazyl) from sigma–aldrich (steinheim, switzerland), methanol (min. 99.8 % purity) from chempur, piekary śląskie, poland, na2co3 of ≥ 99.9 % purity from sigma–aldrich and folin–ciocalteu phenol reagent from merck. beer and vinegar processing according to usual operative conditions developed in the brewery pilot plant of the uasvm cluj-napoca, romania, the following experimental plan was replicated three times. dark lager beer from 100 % all grain malted barley, i.e., pilsen, caramel and roasted (weyermann specialty malting company, bamberg, germany) was produced on the pilot scale in the microbrewery of the faculty of food science and technology uasvm cluj-napoca, romania. hop pellets 90 type (magnum and perle cultivars) crop 2016, were supplied from moragroind hops farm (targu mures, romania). two types of hops were used as pellets, magnum cultivar for bitter taste, and perle cultivar for flavour, which were added at the beginning and at the end of the boiling process, respectively, each in specific amounts (data not shown). a starter culture of fermentation yeast (saccharomyces carlsbergensis) was received from a local brewery and was added at a dose of 0.5 l yeast per 102 l wort (1.5×107 yeast cell per l wort). malt was ground in a motorized roller mill mav3 (tehnofavorit, bontida, romania) and grist was transferred to a saccharification kettle. the saccarification process was monitored by a controlled system (centra, cluj-napoca, romania) and the mash was transferred to a lauter tun. after separation, the wort was boiled for 90 min. the hopped wort was settled for clarification, refrigerated to 8 °c, pitched with slurry yeast and sent to the primary fermentation tank (pierre guerin, france). the primary fermentation lasted 7 days at 10 °c, then the process was set to secondary fermentation, for 14 days at 3 °c. the fermentation tank had 3 independent cooling areas, 3 temperature sensors, 1 ph sensor and tandem gas analyzer for dissolved o2/co2 (key instruments, usa). the fermentation was monitored daily (data not shown) with an automatic fermentostar analyzer type 3572 (funke–gerber, germany) for ethanol and real extract. the original gravity and ethanol content of the finished beer were of 12.8 °p, and 6.5 vol. %, respectively. when the degree of fermentation reached 85 %, the beer ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 22 mudura et al. was filtered and transferred to the next stage of fermentation. acetic fermentation was conducted by orleans-type surface fermentation, in 1 l vessel capacity, with magnetic stirrer bar daily homogenisation for 10 min, and lasted 60 days at 25 °c, in the presence of naturally occurring acetobacter aceti (at a count of 106 cfu ml-1). final chemical parameters of vinegar were 45 g/l acetic acid and 21.8 g/l dry extract. no thermal treatments were performed for beer or for vinegar. these experiments were performed in triplicate. prior to storage, the beer was freed of co2 in an erlenmeyer shaking flask. both beer and vinegar samples were stored in a freezer at –20 °c immediately after completion of the fermentation process (alcoholic and acetic, respectively) until analysis. determination of total phenolic content (tpc) before each analysis, the samples were filtered. measurement of the total phenolics content was performed using the folin–ciocalteu method,17 with some modifications. each 0.025 ml sample of brown lager beer or brown beer vinegar was mixed with 1.8 ml of distilled water and 0.12 ml of folin–ciocalteu reagent and immediately vortexed. after 5 min at room temperature, 0.34 ml of an aqueous na2co3 solution (7.5 %) were added and the mixture stood at room temperature for 2 h. the absorbance was measured at 750 nm on a uv– –vis spectrophotometer (pharmaspec uv-1700, shimadzu, kyoto, japan). a calibration curve was performed using different concentrations (0.0–1.0 mg ml-1) of standard gallic acid solutions (r2 = 0.9997). the concentration of tpc is expressed as mg gae l-1 of sample. determination of the free radical scavenging activity by the 1,1-diphenyl-2-picrylhydrazyl free-radical scavenging assay the scavenging activities of the beers and vinegars on the stable free radical dpph were assayed using the standard method,17 with some modifications. a beer or vinegar sample (0.01 ml) was transferred into a glass test tube with a screw cap, mixed with 0.09 ml distilled water and 3.9 ml methanolic dpph solution (0.025 g l-1). after 30 min incubation in the dark, the absorbance of the sample was measured at 515 nm against a methanol blank (a0) using a double-beam uv–vis spectrophotometer (pharmaspec uv-1700, shimadzu, kyoto, japan). three readings per sample were taken. positive and negative controls (0.5 mg ml-1 gallic acid and methanol, respectively) were treated in the same manner as the samples. the following equation was used to determine the dpph free radical scavenging activity (rsa) expressed in %: 0 1 0 100 − = a a rsa a where a1 is the absorbance of dpph free radicals in the sample. high-performance liquid chromatography coupled with positive electrospray ionisation and diode array detection (hplc-dad–esi(+)-ms) analysis the samples were filtered through a 0.45 µm nylon filter and injected (10 µl) into the hplc system. for the separation of the phenolic compounds, an agilent 1200 hplc system equipped with a diode array detector (dad) coupled with a mass detector (md) single quadruple agilent 6110 (agilent technologies, ca, usa) was used. the separation of the phenolic compounds was realised on an eclipse xdb c18 column (150 mm×4.6 mm, 5 μm) (agilent technologies, ca, usa). the mobile phases were water: 0.1 % acetic acid in acetonitrile (99:1, solvent a) and 0.1 % acetic acid in acetonitrile (solvent b), at a flow rate of 0.5 ml min-1.18 all runs were monitored at 280 nm. the following gradient was applied: % b: 0–2 min, 5 %; 2–18 min, 5–40 %; 18–20 min, 40–90 %; 20–24 min, isocratic, 90 %; 24–25 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. beer vinegar: potentially functional product 23 min 90–5 %. for ms fragmentation, the esi (+) ionization model was used with the following conditions: 3000 v capillary voltage, at 300 °c, an 8 l min-1 nitrogen flow and m/z: 100–1000 full-scan. two levels of energy were used to obtain 50 or 100 fragments in the range m/z: 100–1000 da. the phenolic compounds data identification was realised based on the uv spectrum of each compound. data acquisition and interpretation of the results were performed utilizing chemstation, agilent technologies, ca, usa. based on calibration curve (r2 = = 0.9985), the amounts of phenolic compounds, expressed as mg catechin equivalents (ce) l-1, were calculated. statistical analysis all the experiments were performed in triplicate. the data are reported as means ± standard deviation (sd). anova analysis of variance was used to compare the mean values, using spss 19.0 statistical analysis (ibm, new york, usa) and the tukey hsd test with a confidence interval of 95 or 99 %. a p-value below 0.05 was considered statistically significant. results and discussion total phenolic content and antioxidant activity the total phenolic content of the beer and vinegar samples determined using folin–ciocalteu reagent were 428.9±1.58 and 661.5±7.69 mg gae l–1, respectively, which contributed to the antioxidant activity in the vinegar sample of 82.18 %. similar values for the phenolic content were registered in other beers.7,19,20 both the content of phenolics and the antioxidant activity of the vinegar were in the range reported by other studies where balsamic vinegars were analyzed.21,22 the brown beer had a 63.03 % antioxidant activity, which is also in accordance with previous studies.20 after acetic fermentation of the beer, both tpc and antioxidant activity registered significant increases (p < 0.001). previously, the antioxidant activity of beer was attributed especially to xanthohumol, cinnamic, caffeic and ferulic acids and gallocatechin.15 even though the folin–ciocalteu method is widely applied for quantification of total phenolics in different matrices, vegetable or beverages, some of the beer contents may interfere, such as maillard reaction compounds or sulphites, for this reason, a complete evaluation of the phenolic profile via the identification and quantification of the individual phenolic compounds is recommended.7 individual phenolic compounds after the wort boiling phase, a large amount of polyphenols is lost in the cold break or later in the cold conditioning, and a little is lost during the alcoholic fermentation process of beer.23 due to the oxidative processes during acetic fermentation, the concentration of some of the hop-derived phenolic compounds decreased, mainly prenylflavonoids (table i). in the present study, the sum of individual phenolic contents revealed an increase of 40 % after the acetic fermentation of brown beer, because of the increasing concentration of solid pro________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 24 mudura et al. ducts due to water evaporation. statistically significant differences were observed after acetic fermentation between each parameter (p < 0.05). table i. individual phenolic contents in beer and vinegar expressed as mg ce l-1; the values are expressed as the mean of three replicates. n – number of samples. p ≥ 0.05, not significant compound rt / min [m–h]+ beer (n = 3) vinegar (n = 3) p protocatechuic acid o-glucoside 2.61 317, 155 7.57±0.05 7.42±0.03 <0.05 3-caffeoylquinic acid 2.89 355, 181,163 26.44±0.13 40.01±1.13 <0.001 (4-hydroxyphenyl)acetic acid 3.26 153, 136 7.53±0.27 11.84±0.02 <0.001 4-vinylguaiacol 3.42 151 2.25±0.07 10.22±0.04 <0.001 catechin 7 o-glucoside 3.65 453, 291 9.75±0.06 8.84±0.02 <0.001 4-hydroxybenzoic acid 3.90 139 9.47±0.02 38.23±0.05 <0.001 (3-hydroxyphenyl)acetic acid 4.24 153, 136 11.56±0.03 18.95±0.04 <0.001 catechin 5 o-glucoside 4.61 453, 291 23.81±1.73 7.24±0.06 <0.001 coumaric acid o-glucoside 5.19 327, 165 3.43±0.02 4.90±0.05 <0.01 ferulic acid o-glucoside 5.58 357, 195 3.93±0.10 4.33±0.02 <0.001 gallic acid 5.93 171, 153 2.87±0.16 5.72±0.04 < 0.01 vanilic acid o-glucoside 6.46 331, 169 12.07±0.38 10.25±0.03 < 0.01 gallocatechin 7.17 307 5.57±0.56 7.66±0.10 <0.001 sinapic acid o-glucoside 8.56 387, 255 3.17±0.15 14.03±0.12 <0.001 catechin o-diglucoside 9.05 615, 453, 291 4.08±0.09 8.41±0.04 <0.001 kaempferol o-glucoside 9.83 449, 297 4.16±0.05 6.28±0.04 <0.001 feruloylquinic acid 10.42 369, 195 10.81±0.11 6.60±0.15 <0.001 chlorogenic acid 10.74 355, 181, 163 10.06±0.01 18.30±0.02c <0.001 (+)-catechin 11.17 291 4.64±0.23 7.89±0.04 <0.001 (–)-epicatechin 12.30 291 4.08±0.05 7.78±0.12 <0.001 caffeic acid 12.70 181, 163 8.76±0.16 10.58±0.08 <0.001 sinapic acid 13.11 225 11.30±0.51 15.5±0.06 <0.001 apigenin o-glucoside 13.71 433, 271 7.08±0.08 6.15±0.02 < 0.01 quercetin o-glucoside 14.18 465, 303 7.77±0.11 7.05±0.06 < 0.01 cohumulone i 14.64 349, 237 4.87±0.10 4.44±0.02 <0.001 cohumulone ii 15.73 349, 280 3.62±0.04 6.58±0.10 <0.001 8-prenylnaringenin 16.74 341, 273 4.55±0.23 2.33±0.02 <0.001 6-prenylnaringenin 17.81 341, 273 2.43±0.05 1.86±0.02 <0.001 humulone 19.85 363 1.55±0.10 5.62±0.08 <0.001 isohumulone 20.92 363 1.67±0.19 4.14±0.03 <0.001 sum of individual phenolic compounds, mg ce l-1 220.85 309.15 – phenolic compounds play their role in beer not only as health benefits, but also they participate in the colloidal and sensory stability of beer.8 the bitter taste and astringency of beer is strongly related to the level of hydroxybenzoic and hydroxycinnamic acids.24 despite all the reports indicating the overall protective action of polyphenols, gallic acid shows the greatest pro-oxidant potential with negative implications for the physical stability of beer.25 the amount of gallic acid found in brown ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. beer vinegar: potentially functional product 25 beer (2.87 mg ce l–1) was higher compared to that found in other previously described beers,19 but similar to ones determined in other studies.26,27 4-hydroxybenzoic acid was also previously reported in beer.5,24 hydroxycinnamic acids are present in all plant matrices. many health effects are related to a diet rich in these compounds, such as reducing the risk of cardiovascular disease, strong antioxidant activity, anticarcinogen.28 a previous study reported 0.13–0.38 mg l–1 sinapic acid in commercial beer samples.19 still, the present results were higher for sinapic acid, 11.30 mg ce l–1 in brown beer, and the protective role of melanoidins from the coloured malt used could be an explanation. sinapic acid became a compound of interest to researchers for its antioxidant, antimicrobial, anti-inflammatory, anticancer, anti-anxiety activities, being considered as superior to ferulic or caffeic acids.29 it comes from malted barley, being formed during malting process, and its value is in the range 0.6–2.0 mg kg–1 dry weight.30 the concentration of sinapic acid can increase during beer maturing, while it is sensitive to thermal treatment.31 during acetic fermentation, the concentration of sinapic acid significantly increased to 15.5 mg ce l–1. the same sensitivity was determined for caffeic acid. the fact that the concentrations of caffeic and sinapic acids increased during acetic fermentation proved that they are not oxygen sensitive; on the contrary, their concentrations increased by 20, and 40 %, respectively. caffeic acid induces apoptosis in human cancer cells starting at a dose of 18 mg 100 ml–1.32 moreover, caffeic acid is a metabolite of chlorogenic acid. in the intestine, chlorogenic acid, which was found in the present samples of 10.06 mg ce l–1 beer, and almost doubled in vinegar, is hydrolyzed into caffeic acid, which has a stronger antioxidant activity than that of its precursor.33 the content of ferulic acid, found in its free form in beer and as feruloylquinic acid,5 was determined to be 10.81 mg ce l–1 in the beer sample, but, due to its oxygen sensitiveness, was lower in the vinegar sample, 6.60 mg ce l–1. other hydroxycinnamic acid derivatives identified and quantified in beer and vinegar were coumaric acid o-glucoside, ferulic acid o-glucoside and sinapic acid o-glucoside. hydroxycinnamic acid derivatives play an important role in the treatment of diabetes, obesity and metabolic syndrome.34 beer is a rich source of hydroxyphenylacetic acids, along with cider, olive oil and wine,5 and the present results proved their increasing content after acetic fermentation. phenolic compounds play an important role in beer and to its haze stability, catechin and epicatechin being involved in both haze formation23 and beer colloidal stability.35 the catechins in beer come from malt, 80 %, and the rest from hop (humulus lupulus l.). due to their affinity for proteins, the catechins from barley (hordeum vulgare l.) are known to be involved in the formation of beer haze, their role in beer stability becoming essential.36 (+)-catechin and (–)-epicatechin, known as contributors to beer bitterness, were previously identified in ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 26 mudura et al. beer samples.4,5,8,27,37 hop is a valuable ingredient in beer as it contains 14 % phenolic compounds and important amounts of catechin.4 the contents of (+) -catechin and (–)-epicatechin ranged between 0.57–1.27 mg l–1 and 0.08–0.39 mg l–1, respectively, in other beer samples.19 in brown beer vinegar, these compounds were almost doubled, due to the concentration process. derivatives of flavanols, catechin 7-o-diglucoside and gallocatechin, were also identified in beer.8,36 flavonol derivatives, quercetin o-glucoside and kaempferol o-glucoside, were recently reported in beer.5 quercetin o-glucoside can be metabolized in the small intestine.38 through its anti-inflammatory and antioxidant properties, it also plays a role in preventing cardiovascular diseases. as chemopreventive agents, by their demonstrated biological activities, i.e., antioxidant,39 anti-inflammatory, anti-proliferative, pro-apoptotic and anti-antigenic, they positively interfere in all stages of carcinogenesis.40 apigenin and its derivatives were previously reported in artichoke (cynara cardunculus var. scolymus l.), the same family as hop (humulus lupulus l.), cannabaceae,41 vegetables and other condimentary plants.5 their chemoprotective activity in unstabilised beer was demonstrated,42 and in hop products, their cytotoxic activities.43 frequently, hop is used in pellets form in the brewing process, as was the case in the present study, but can also be used as whole cones or extracts. the lupulin gland inside the cones contains hop bitter resins, essential oils, tannins and polyphenols, responsible for the flavour and bitterness of beers. the beneficial effects of bitter acids (α and β acids) can be summarized as potential anticancer activity, apoptotic effect, anti-inflammatory or protection against metabolic disorders (diabetes, cardiovascular diseases and metabolic syndrome). in addition, these valuable compounds are known for their antifungal and antibacterial activities, which impact beers preservation,44 suggesting their further medical and food industry applications.45 the content in α-acids is a very important attribute of hop because it determines their economical value, with implications in the brewing process when the constant quality of beers is imperative or simply when developing a new type of beer.13 unfortunately, many breweries tend to use lower hopping dosage rates (a reduction of approximately 30 % in the last decade) for economical reasons.13 additionally, bitter acids are recognized for their chemo-protective activities. for instance, humulone is known for its antioxidant, anti-inflammatory and antitumor promoting effects. the contents of α acids reported previously13 ranged between 2.20–13.09 % of the total contents of bitter acids (8.52–20.35 %), of which between 18.48–21.00 % were represented by cohumulone in the magnum variety hop pellets. in the brown beer vinegar sample, both humulone and isohumulone increased significantly to 5.62 and 4.14 mg ce l–1, respectively. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. beer vinegar: potentially functional product 27 during the brewing process, the content of bioactive compounds decreases due to process losses. the technological stages in beer making, i.e., boiling, must filtration, alcoholic fermentation, beer filtration and pasteurization, contribute to important losses of nutrients and aroma compounds. for example, prenylflavonoids are lost during wort boiling, hot wort separation, and during the fermentation, conditioning, and beer filtration. bitter resins, represented by iso-α acids, are compounds that give the specific bitter taste to beer during wort boiling with hops. iso-α acids are more soluble and bitter than α acids. unfortunately, the isomerisation yield of α acids to iso-α acids is quite low, only 20–35 % of the initial content could be retrieved in beer, depending on the beer type, because the remaining did not isomerise or was lost during boiling, fermentation or beer filtration. hops are a rich source of prenylflavonoids, 8-prenylnaringenin being the most active phytoestrogen currently known. prenylflavonoids found in beer have different biological activities, i.e., antioxidant, anticarcinogenic, anti-inflammatory, estrogenic and antiviral. still their amount in beer is strongly related to the applied brewing process, mainly by their insufficient extraction during wort boiling. it was demonstrated that the more intense the malt colour is, the higher are the yields of prenylflavonoids in beer, due to the protective role of maillard compounds.12 the group of alkylmethoxyphenols was represented in the present study by 4-vinylguaiacol, which was reported to be involved in the characteristic aroma of beer.5,46 it is formed during wort boiling, when ferulic acid releases 4-vinylguaiacol through decarboxylation by thermal decomposition, and during fermentation by an enzymatic reaction.5 previous studies recommended an amount of 2.2–3.5 mg l–1 was necessary for the pleasant aroma of beers, while an amount higher than 4 mg l–1 could contribute negatively to the sensorial quality of beers. in the present study, a value of 2.25 mg l–1 was found in the beer, which is in accordance with amounts found previously before in beer (0.02–2.7 mg l–1). conclusions finally, it is to consider that brown beer vinegar represents a richer source of polyphenols and phenolic derivatives, compared to beer. the complex composition of brown beer vinegar could recommend its use in the treatment or prevention of several diseases affecting elderly or young people, where special nutrition and dietetics are required. by its increased content of phenolic and antioxidant activity, and being free of alcohol, this potentially functional product could be considered a better source of valuable compounds than beer, which could also be used in special diets. the strategy proposed could also be useful to add value to the liquid waste from the brewing industry. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 28 mudura et al. acknowledgement. this work was supported by a grant from the romanian national authority for scientific research and innovation, cncs/cccdi – uefiscdi, project no. pn-iii-p2-2.1-ci-20170380, within pncdi iii. и з в о д сирће из тамног пива: потенцијално функционални производ захваљујући свом фенолном саставу и антиоксидативној активности elena mudura1, teodora emilia coldea1, carmen socaciu2, floricuța ranga2, carmen rodica pop2, ancuța mihaela rotar2 и antonella pasqualone3 1 department of food engineering, university of agricultural sciences and veterinary medicine cluj-napoca, romania, 2 department of food science, university of agricultural sciences and veterinary medicine cluj-napoca, romania и 3food science and technology unit, department of soil, plant, and food science (di.s.s.p.a.), university of bari ‘aldo moro’, bari, italy циљ рада је био добијање производа ферментације тамног пива који је функционалан, обогаћен полифенолима и који не садржи алкохол. у пиву и сирћету је одређен фенолни садржај и антиоксидативна активност, користећи folin–ciocalteu реагенс, као и способност хватања слободних радикала, користећи 1,1-дифенил-2-пикрилхидразил тест. раздвајање и идентификација 30 фенолних једињења је изведена користећи hplc -dad–esi(+)-ms. фенолна једињења су идентификована преко uv спектара. користећи калибрациону криву (r2 = 0,9985), израчунате су количине фенолних једињења и изражене као еквиваленти катехина (ce)/l. укупни садржај фенола у пиву и сирћету, одређен folin–ciocalteu реагенсом, је био 428,9±1,58 mg gae/l, односно 661,5±7,69 mg gae/l, доприносећи великој антиоксидативној активности сирћета од 82,18 %. статистички значајне разлике су нађене за сваки параметар након сирћетне ферментације (p < < 0,05). сирће тамног пива је бољи извор полифенола и фенолних деривата од пива, што га, заједно са већом антиоксидативном активношћу, чини бољим извором корисних састојака и треба га узети у обзир у специфичним дијетама. 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(voj. zdrav. listy) 85 (2016) 19 45. s. bedini, g. flamini, j. girardi, f. cosci, b. conti, j. pest. sci. 88 (2015) 583 46. y. cui, a. wang, z. zhang, r. a. speers, j. inst. brew. 121 (2015) 129. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {the physico-chemical and thermodynamic properties of the choline chloride-based deep eutectic solvents} j. serb. chem. soc. 82 (9) 1039–1052 (2017) udc 541.12.017:547.421–327:544.35: jscs–5021 544.2.004.12 original scientific paper 1039 the physicochemical and thermodynamic properties of the choline chloride-based deep eutectic solvents dragan z. troter1, zoran b. todorović1, dušica r. đokić-stojanović2, biljana s. đordević1, vanja m. todorović3, sandra s. konstantinović1 and vlada b. veljković1* 1faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia, 2zdravlje actavis, vlajkova 199, 16000 leskovac, serbia and 3faculty of pharmacy, university of belgrade, vojvode stepe 450, 11221 belgrade, serbia (received 25 february, revised 22 may, accepted 23 may 2017) abstract: this paper reports the physicochemical (density, dynamic viscosity, electrical conductivity and refractive index) and the thermodynamic (thermal expansion coefficient, molecular volume, lattice energy and heat capacity) properties of several choline chloride (chcl) based deep eutectic solvents (dess), with 1:2 mole ratio, respectively: chcl:propylene glycol, chcl:1,3-dimethylurea and chcl:thiourea, at atmospheric pressure as a function of temperature over the range of 293.15–363.15 k. their properties were also compared with those of some already characterized chcl-based dess, namely chcl:ethylene glycol, chcl:glycerol and chcl:urea (1:2 mole ratio). density, viscosity and refractive index of all dess decrease with the increasing temperature while the electrical conductivity increases. viscosity and conductivity of the tested dess were fitted by both arrhenius-type and vogel–tamman–fulcher equations. the changes of molar enthalpy, entropy and gibbs energy of activation, determined using the eyring theory, demonstrated the interactional factor as predominant over the structural factor for all des systems. the fractional walden rule, used to correlate molar conductivity and viscosity, showed an excellent linear behaviour. it was shown that chcl:propylene glycol des had properties similar to chcl:ethylene glycol and chcl:glycerol dess. however, the properties (density, viscosity and electrical conductivity) of chcl:1,3-dimethylurea and chcl: :thiourea dess were inferior to those of the chcl:urea des. keywords: physicochemical properties; eutectic; glycols; amides. introduction having highly low volatility and being less toxic, ionic liquids (ils) are very convenient to replace organic solvents. containing only ions, ils can easily be * corresponding author. e-mail: veljkovicvb@yahoo.com https://doi.org/10.2298/jsc170225065t _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs 1040 troter et al. designed to be in the liquid state at or even below room temperature by choosing adequate starting chemicals.1 their favorable properties make ils environmentally acceptable.2,3 many ils applications have been described, such as in electrochemistry and extraction4, catalysis and enzyme-catalyzed reactions.5 however, since some ils are still toxic,6,7 their use in drugand food-related products is unacceptable. deep eutectic solvents (dess) have been referred as promising alternative to the conventional ils. in comparison with ils, dess are cheaper, easier for preparation and biodegradable,8–10 referring them as green solvents. dess are usually made by combining a substituted quaternary ammonium salt and a complexing agent (usually a hydrogen bond donor, hbd), resulting in the compound with lower melting point.5,8,11 designing new green solvents requires using safe and environmentally friendly compounds. the most used substituted quaternary ammonium salt is choline chloride (2-hydroxy-ethyltrimethyl-amonium chloride, chcl), which is an essential micronutrient and human nutrient,12 used as an additive in animal feeds. as hbds, polyols are often used, and among them, glycerol and ethylene glycol are the most dominant. glycerol has a lot of advantages, such as being non-toxic, biodegradable and recyclable, and is produced on a large scale from renewable sources.13 on the other hand, ethylene glycol, although used in various industrial applications, is moderately toxic liquid. safer option is propylene glycol, a non-toxic polyol used in food processing, as a solvent in many pharmaceutical formulations, and for the production of polymers. it is also one of the major ingredients (together with glycerol) of the so -called “e-liquids” used for electronic cigarettes. besides polyols, urea and its derivates can also be chosen as safe hbds. urea is mostly used as a nitrogen -release fertilizer, while 1,3-dimethylurea and thiourea have been used in different chemical syntheses. for the general understanding, the optimization of the performance and the increase of the potential future application of dess, it is important to know or be able to predict their thermodynamic and physicochemical properties. therefore, further investigations of the most important properties of dess, such as density, viscosity, heat capacity, electrical conductivity or refractive index, are needed for expanding their use. both physicochemical and thermodynamic properties of dess must be known for their uses in different industrial and laboratory processes. knowing the density (ρ) of the dess is very important for many different industries, because the density measurement helps to determine the characteristics of the dess. besides being measured in a laboratory, the density data of different dess were also predicted by different ways, such as by rackett models,14 the parachor parameter and surface tension data,15 as well as the lorentz–lorentz equation.16 mjalli et al. modified the rackett model17 and recently mjalli used the molecular structure explained by the mass connectivity index for predicting the density of the dess.18 dynamic viscosity (η) is a very important _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs physicochemical properties of deep eutectic solvents 1041 transport property, especially for equipment design and fluid flow calculations. viscosity data of dess are useful in selecting the optimum salt:hbd ratio, which results in saving material and energy for the preparation of dess. knowing the electrical conductivity (κ) of a des is of great value, if it is used as a supporting electrolyte in electrochemical experiments. the higher is the electrical conductivity exhibited, the lower is the ohmic drop during electrolysis and the cell voltage, resulting in higher energy efficiency. the refractive index (nd) has a unique value for every material and has a very important role in material engineering. unlike other properties, the measurements of refractive index of dess are rarely done, so there is a lack of such data in the literature. once physical properties of dess have been fully characterized, they can be used to calculate thermodynamic properties of dess’ systems (such as entropy, enthalpy, etc). since these properties establish the criteria for the determination of the feasibility or spontaneity of a given transformation, the energy exchanges that occur in the dess’ systems can be predicted, which is crucial for the chemical industry. the present paper focuses onto the chcl-based dess with different hbds (propylene glycol, ethylene glycol, glycerol, urea and 1,3-dimethylurea and thiourea) prepared in the mole composition of 1:2. it is worth to emphasize, as far as the authors are familiar with, most physicochemical and thermodynamic properties of the chcl:propylene glycol, chcl:1,3-dimethylurea and chcl:thiourea dess have not been studied yet. both physical (density, dynamic viscosity, electrical conductivity and refractive index) and thermodynamic (thermal expansion coefficient, molecular volume, lattice energy and heat capacity) properties were studied at the atmospheric pressure in the temperature range of 293.15–363.15 k. while density and viscosity of chcl:ethylene glycol, chcl:glycerol and chcl: :urea have already been investigated,14,19–24 the data on their other properties are very scarce.19,22–24 the arrhenius exponential type dependence and vogel–tamman–fulcher equation were employed for the understanding of the transport properties of the studied dess. the thermodynamic characteristics of viscous flow were also determined. in addition, the fractional walden rule was employed for describing the relationship between molar conductivity and viscosity for the dess. experimental chemicals chcl, ethylene glycol and 1,3-dimethylurea (all ≥ 98.0 %) were obtained from sigma aldrich (st. louis, usa), while propylene glycol and glycerol (both ph eur grade) were purchased from meilab (belgrade, serbia). urea and thiourea (both 99.5 %) were obtained from zorka (šabac, serbia). all chemicals were used as purchased, without any purification. preparation of dess chcl was combined with the selected hbd, namely propylene glycol, ethylene glycol, glycerol, urea, 1,3-dimethylurea and thiourea, at the 1:2 mole ratio in a round-bottomed flask placed on a rotary evaporator and held at 348.15 k within 2 h (until the homogeneous, trans_________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs 1042 troter et al. parent liquid was formed). the dess were stored in the well-closed glass bottles in a desiccator containing cacl2. all dess are viscous, homogeneous and colorless liquids. however, chcl:urea des turns into white semi-solid with time, while chcl:1,3-dimethylurea and chcl:thiourea dess turn into white solid upon cooling. the water content of the dried dess, determined by the karl–fischer method (metrohm 73kf coulometer) were less than 350 ppm. fig. s-1 of the supplementary material to this paper shows the preparation of the chcl-based dess schematically and ftir spectra of the dess and their components are presented in fig. s-2 of the supplementary material. physicochemical properties of dess all physicochemical properties were measured in the temperature range between 293.15 and 363.15 k at the atmospheric pressure. the readings were taken after 20 min at each temperature to provide a good temperature equilibration. density, dynamic viscosity and electrical conductivity were measured using a dma 4500 anton paar densitometer, a rotational viscometer (visco basic plus, ver. 0.8, fungilab s.a., barcelona, spain) and a proline conductivity meter b250, respectively. refractive index values were obtained by an automatic atago refractometer a100. all measurements were performed in triplicates. results and discussions effect of temperature on density of the chcl-based dess fig. 1 shows the dependence of the density of the studied dess on temperature. the present results showed that the density decreases with the increase of the temperature, as usual for liquids. for the purpose of comparison, the literature data for the chcl:ethylene glycol, chcl:glycerol and chcl:urea systems are also presented. as in the case of pure ils,25,26 as well as chcl:ethylene glycol, chcl: :glycerol and chcl:urea,27–29 a very good linear correlation (see supplementary material) between density and temperature was observed for all dess systems studied. based on the coefficient of volume expansion, among dess with polyols, chcl:glycerol is the most thermally sensitive, followed by chcl:ethylene glycol and chcl:propylene glycol, while among dess with amides, the most thermally sensitive is chcl:1,3-dimethylurea, then chcl:thiourea and lastly chcl:urea. at the room temperature, the density of the tested dess is in the following order: chcl:glycerol ˃ chcl:propylene glycol ˃ chcl:urea ˃ chcl:ethylene glycol while chcl:1,3-dimethylurea and chcl:thiourea are solid and become liquid after heating. at 313.15 k, the densities of the tested dess follow the order: chcl:1,3-dimethylurea ˃ chcl:thiourea ˃ chcl:propylene glycol ˃ chcl:glycerol ˃ chcl:urea ˃ chcl:ethylene glycol. the difference in density can be explained by the different molecular arrangement, or packing of the des. chcl: :propylene glycol des has a higher density than other two polyol-based dess because of the higher intermolecular packing of the compound structure. the density of chcl:ethylene is lower than those of other polyol-based counterparts, showing that higher-density liquids have a more compact structure. in the case of _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs physicochemical properties of deep eutectic solvents 1043 chcl:1,3-dimethylurea and chcl:urea, the electron rich oxygen part easily interacts with chcl, but chcl:1,3-dimethylurea is denser, which can be attributed to the tight intermolecular packing between chcl and hbd. the presence of two methyl groups in this des influences its density greatly, since it is higher than the density of chcl:thiourea. fig. 1. temperature dependence of the density of the studied eutectic mixtures: a) chcl: :propylene glycol – ■ (our values); b) chcl:ethylene glycol – ■ (our values), 14, 19, 20 and 21; c) chcl:glycerol – ■ (our values), 19, 20, 21 and 22; d) chcl:urea – ■ (our values), 21, 23 and 24; e) chcl:1,3-dimethylurea – ■ (our values), f) chcl:thiourea – ■ (our values). the comparison with the literature data (fig. 1) leads to different conclusions, depending on the type of des. it should be noted that the slopes of the linear correlations for chcl:ethylene glycol and chcl:glycerol, corresponding to the coefficient of volume expansion (b), are almost the same with those found in the literature.14,19–22 the density of chcl:ethylene glycol is slightly lower than the already published ones in the same range of temperature,14,19–21 while the density _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs 1044 troter et al. of chcl:glycerol is slightly higher in the range of 293.15–303.15 k and lower in the range of 313.15–363.15 k than the literature values.19,20,22 on the other side, the slope of the linear correlation for chcl:urea differs from the reported one.21,23,24 the chcl:urea des had a moderately lower density in the range of 293.15–343.15 k and a higher density in the range of 353.15–363.15 k than those reported in the literature.23,24 also, compared to the values reported by mjalli and abdel-jabbar,21 the density of chcl:glycerol and chcl:urea determined in the present study in the temperature range of 293.15–353.15 k are slightly lower. since the complete physicochemical and thermodynamic properties of chcl:propylene glycol, chcl:1,3-dimethylurea and chcl:thiourea dess have not been studied yet, there are no literature data that can be compared with the present ones. density is commonly used for the calculation of thermal expansion coefficient, α, the molecular volume, vm, and the lattice energy, upot (see supplementary material).27,30 the calculated values of vm, upot and the heat capacity (cp) from equations from the literature27,30 at 303.15 k are presented in table s -iii of the supplementary material. the values for lattice energy are similar to those of molten salts; for instance, the fused csi31 has the lowest lattice energy among the alkaline halides (613 kj mol–1). this is the primary reason for the liquid state of the studied dess at the room temperature. effect of temperature on dynamic viscosity of the chcl-based dess fig. 2 shows the dynamic viscosity (η) of the prepared chcl-based dess as a function of temperature as both the arrhenius-type and vtf plots along with the literature data for the chcl:glycerol and chcl:urea systems (additional considerations are given in supplementary material). it can be noticed that the dess have higher viscosity at lower temperatures, which drops rapidly during heating due to the higher mobility of constituents. at 313.15 k, the viscosity of the tested dess is in following order: chcl: :1,3-dimethylurea ˃ chcl:thiourea ˃ chcl:urea ˃ chcl:glycerol ˃ chcl:propylene glycol ˃ chcl:ethylene glycol. the larger viscosity of the dess with urea -based compounds indicates the formation of stronger hydrogen bonds in these systems than in the dess with polyols. two methyl groups in 1,3-dimethylurea also have the major effect on the viscosity of its des. the highest viscosity among the polyol-based dess possesses chcl:glycerol, as expected, because glycerol has one more –oh group, thus forming stronger h-bonds. eη-values for the tested dess are larger than those for the conventional liquids or high temperature molten salts,32,33 which can be explained by a large ion-to-hole radius ratio in these eutectic mixtures. the differences among the studied dess in both density and viscosity values can be explained by a better ability of urea to form hydrogen bonds. ashworth et al.34 indicated that, on average, the individual h-bonds _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs physicochemical properties of deep eutectic solvents 1045 between urea and choline or even urea with another urea are stronger than those with the chloride. however, urea forms a h-bonded urea[choline]+ complexed cation that is energetically competitive with [cl(urea)2]–. fig. 2. temperature dependence of the viscosity (η) of the studied eutectic mixtures: a) arrhenius-type plot of viscosity for the dess; b) vtf plot of viscosity for dess (chcl:propylene glycol – , chcl:ethylene glycol –  , chcl:glycerol – , chcl:urea – , chcl:1,3-dimethylurea –  and chcl:thiourea –  (literature data: chcl:glycerol – 22, chcl:urea –  23. it was previously reported35 that alkyl chain lengthening, or fluorination of organic components causes an increase in van der waals interactions and hydrogen bonds, which makes ils more viscous. since high viscosity at low temperatures can negatively affect any technological process, it is recommended to use the studied dess at temperatures higher than 313.15 k. the comparison of the calculated eη values for chcl:ethylene glycol, chcl:glycerol and chcl:urea with the literature shows that they are lower for the first des22,23,28,29 but larger for the other two dess. for better understanding of the viscous flow, the eyring’s transition state theory is commonly used for calculating the thermodynamic functions of activation from the dynamic viscosity. usually, the following equation is used to calculate the activation enthalpy change (δh*) and entropy change (δs*) of viscous flow:27 a δ δ ln * *ηv h s hn rt r = − (6) for each dess system, ln (ηv/hna) as a function of t-1 (eq. (6)) is shown in fig. 3. the activation enthalpy (δh*) and entropy changes (δs*) of viscous flow were calculated from the slope and the intercept of the straight lines. the _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs 1046 troter et al. obtained values of the thermodynamic functions of activation at 313.15 k (since all tested dess are liquid at this temperature) are listed in table s-vi of the supplementary material. the molar enthalpy change of activation for viscous flow is higher than the tδs* values. thus, the energetic contribution which corresponds to the molar enthalpy change of activation for viscous flow is more important than the entropic contribution to the molar gibbs energy change of activation. fig. 3. plots of ln (ηv/hna) against inverse temperature of the studied dess: chcl:propylene glycol – ; chcl:ethylene glycol – ; chcl: :glycerol – ; chcl:urea – , chcl: :1,3-dimethylurea –  and chcl: :thiourea – . based on these results, it can be seen that the chcl:propylene glycol des has properties that are similar to those of the chcl:ethylene glycol and chcl: :glycerol dess, so it can replace them in the future possible industrial applications or as media for chemical reactions. on the other hand, since the density and viscosity of chcl:1,3-dimethylurea and chcl:thiourea dess are higher than those of the chcl:urea des at low temperatures, they can be used in the technological processes at temperatures above 313.15 k. effect of temperature on the electrical conductivity of the chcl-based dess the plots of electrical conductivity as a function of temperature in the temperature range of 303.15–363.15 k for the studied dess are shown in fig. 4 (additional considerations are given in supplementary material). at 313.15 k, the conductivity of the tested dess is in following order: chcl:ethylene glycol ˃ chcl:propylene glycol ˃ chcl:glycerol ˃ chcl:urea ˃ chcl:thiourea ˃ chcl:1,3-dimethylurea. the calculated values of both the pre -exponential coefficient (aκ) and the activation energy for electrical conductivity (eκ) are summarized in table s-vii of the supplementary material. similar values for some ils were described earlier.36 the obvious conclusion is that these dess are conductive, proving the fact that the ionic species are dissociated in the liquid and are able to move independently. however, the temperature dependence _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs physicochemical properties of deep eutectic solvents 1047 of the measured conductivities of dess is better correlated with the vogel–tamman–fulcher equation (see supplementary material).37 fig. 4. temperature dependence of the conductivity (κ) of the studied eutectic mixtures: a) arrhenius-type plot of conductivity for the dess; b) vtf plot of conductivity for dess (chcl:propylene glycol (), chcl:ethylene glycol (), chcl:glycerol (), chcl:urea (), chcl:1,3-dimethylurea –  and chcl:thiourea – ). molar conductivity and viscosity relationship the log λ–log η–1 relationship is shown in fig. 5 while the walden equation coefficients (additional considerations are given in supplementary material) for the dess are listed in table s-x of the supplementary material. fig. 5. application of the fractional walden rule for the studied dess (chcl:propylene glycol – ; chcl:ethylene glycol – ; chcl:glycerol – ; chcl:urea – ; chcl:1,3-dimethylurea – ; chcl: :thiourea – ). the α’ values are smaller than 1, as predicted by the ideal walden rule, implying a progressive raise in the quantity of less conductive ion pairs with heating.38 when no strong ion-ion interaction is present, the slope is unity. if the _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs 1048 troter et al. liquid is represented as an ensemble of the independent ions, the walden plot will be similar to the ideal line. for the ideal dotted walden line, the data for the dilute aqueous 0.01 m kcl solution,39 comprising of equal mobility of fully dissociated ions,40 were used. some authors used the δw-value, the vertical deviation to this ideal line, to characterize dess.41 “good” ils are fully dissociated and have a δw < 1 while ils with δw = 1 show 10 % of the ionic conductivity as expected at the ideal (kcl) line. the dess prepared in the present study have δw > 3, belonging to the category of “subionic liquids”. from the temperature dependence curves of log λ vs. log μ–1 shown in fig. 5, it can be seen that the polyol-based dess are closer to the ideal kcl line than the other dess. the deviation of the walden plot from the ideal line for these dess demonstrates the decreased electrostatic interaction between chcl and hbd. chcl:ethylene glycol is the closest to the ideal line, followed by chcl:glycerol and chcl:propylene glycol. among the amide-based dess, the closest to the ideal line is chcl:urea, followed by chcl:thiourea and chcl:1,3-dimethylurea. the fractional walden rule, which states that the activation energy for viscous flow is higher than activation energy for conductivity, finds an interpretation in terms of one of the ionic species being smaller than the other, making them capable of squeezing through smaller gaps in the condensed phase structure. all the studied dess obey the fractional walden rule very well and the values of the walden slope are all smaller than 1 (see table s-x), indicating the presence of the obvious ion association in the investigated dess. from fig. 5 it is clear that the curves of the studied dess are below the “ideal” walden line. the deviation of the walden plot from the ideal line for these dess clearly suggests an increased electrostatic interaction between chcl and the hbd. the second way for calculating the α’ value is the ratio of the temperature-dependent activation energies for viscosity and molar conductivity (α’ = = ea,λ/ea,η < 1).38 from these results, it can be concluded that there are not only charge carrying species simply formed by choline and chloride ions in the investigated dess because all slopes are different. in this case, it is presumed that the conduction involves some degree of cooperation of the ions. here, it can be seen a resemblance to the process of the viscous flow from molten salts, since in their case the translation of ions of both signs is involved due to the imposed condition of electro-neutrality. in addition, since all the investigated dess are “subionic”, probably their special accumulation structure also has a role in this phenomenon. effect of temperature on the refractive index of the chcl-based dess for all dess, the refractive index–temperature relationship is also a linear equation.19 refractive index of the tested dess as a function of temperature is shown in fig. 6 while the parameters of the linear equations are listed in table s-xi _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs physicochemical properties of deep eutectic solvents 1049 of the supplementary material. for the pure dess, the refractive index decreases linearly with the increase of temperature, as expected. with the increase of temperature, molecules move more vigorously, so the mixture becomes less dense due to thermal expansion, resulting in the decrease of the refractive index. at 303.15 k, the refractive index of the tested dess is in following order: chcl:thiourea ˃ chcl:urea ˃ chcl:ethylene glycol ˃ chcl:glycerol ˃ chcl:1,3-dimethylurea ˃ chcl:propylene glycol. for the temperature range of 293.15–363.15 k, the nd values for the tested dess lie within the range of 1.4384–1.5258. by comparing the obtained refractive index data for the chcl:ethylene glycol and chcl:glycerol dess with the literature data, it can be noticed that, at the range of 303.15– –333.15 k, higher values were found for the former des, and slightly lower values for the latter des.19 the ranges of the measured refractive index of the prepared dess are similar to the ranges for the other dess.42 at 303.15 k, the phase velocity of the tested dess is in following order: chcl:propylene glycol ˃ chcl:1,3-dimethylurea ˃ chcl:glycerol ˃ chcl:ethylene glycol ˃ chcl:urea ˃ chcl:thiourea. ranges for both phase velocity (υ) and molar refractivity (a)43 for the tested dess are listed in table s-xii of the supplementary material. fig. 6. temperature dependence of the refractive index of the studied dess: chcl:propylene glycol –  (our values); chcl:ethylene glycol –  (our values), 19; chcl:glycerol –  (our values), 19; chcl:urea –  (our values); chcl:1,3-dimethylurea –  (our values) and chcl: :thiourea –  (our values). conclusions the chcl-based dess with propylene glycol, ethylene glycol, glycerol, urea, 1,3-dimethylurea and thiourea (at 1:2 mole ratios) were prepared. density, viscosity, electrical conductivity and refractive index of these dess were measured in the range of 293.15–363.15 k at atmospheric pressure. it was found that density, viscosity and refractive index of the systems decrease with temperature, while electrical conductivity increases. the density and viscosity values were useful in calculating the thermal expansion coefficient, the molecular volume, the lattice energy and the heat capacity of these dess. after applying the absolute _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs 1050 troter et al. rate approach of the eyring theory concerning the viscosity flow, it was noted that the interactional factor was predominant over the structural factor for all the dess. the viscosity and conductivity values of the tested dess were fitted by both arrhenius-type and vogel–tamman–fulcher equation. also, according to the fractional walden rule, the density, viscosity and conductivity relationships were set up. the results of this study revealed that the chcl:propylene glycol des has the properties similar to the chcl:ethylene glycol and chcl:glycerol dess, thus suggesting them for several possible industrial applications and as media for chemical reactions. however, the properties (density, viscosity and electrical conductivity) of the chcl:1,3-dimethylurea and chcl:thiourea dess are inferior, compared to those of the chcl:urea des, but they can be used in processes at temperatures above 313.15 k. supplementary material additional data and considerations are available electronically at the pages of the journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgments. this paper has been funded by the ministry of education, science and technological development of the republic of serbia (project iii 45001). the authors would like to thank dr jasna janković from automotive fuel cell cooperation (daimler and ford), national research council canada, and university of british columbia, canada. also, the authors thank zdravlje actavis (leskovac, serbia) for technical support. и з в о д физичко–хемијскa и термодинамичка својства еутектичких растварача на бази холин-хлорида драган з. тротер1, зоран б. тодоровић1, душица р. ђокић-стојановић2, биљана с. ђорђевић1, вања м. тодоровић3, сандра с. константиновић1 и влада б. вељковић1 1технолошки факултет, универзитет у нишу, булевар ослобођења 124, 16000 лесковац, 2здравље актавис, влајкова 199, 16000 лесковац и 3фармацеутски факултет, универзитет у београду, војводе степе 450, 11221 београд у овом раду су описане физичко–хемијска (густина, динамички вискозитет, електрична проводљивост и индекс рефракције) и термодинамичка (коефицијент топлотне експанзије, молекулска запремина, енергија решетке и топлотни капацитет) својства еутектичких растварача холин-хлорид:пропилен гликол, холин-хлорид:1,3-диметилуреа и холин-хлорид:тиоуреа (у молском односу 1:2) на атмосферском притиску у функцији температуре у опсегу 293,15–363,15 k. ова својства су поређена са својствима неких већ окарактерисаних еутектичких растварача на бази холин-хлорида, и то са холин-хлорид:етилен-гликолом, холин-хлорид:глицеролом и холин-хлорид:уреом (у молском односу 1:2). густина, вискозитет и индекс рефракције свих еутектичких растварача опадају са порастом температуре, док електрична проводљивост расте. вискозитет и електрична проводљивост свих припремљених еутектичких растварача су фитоване помоћу једначине аренијусовог типа и vogel–tamman–fulcher једначине. промене моларне енталпије, ентропије и гибсове енергије активације, одређене путем eyring теорије, показују да је интеракциони фактор доминантнији у односу на структурни фактор за све еутектичке раствараче. фракционо walden правило, коришћенo je за одређење корелације између моларне проводљивости и вискозитета и резултати показују одличну лине_________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs physicochemical properties of deep eutectic solvents 1051 арност. доказано је да еутектички растварач холин-хлорид:пропилен-гликол има својства слична еутектичким растварачима холин-хлорид:етилен гликол и холин-хлорид:глицерол. међутим, својства еутектичких растварача холин-хлорид:1,3-диметилуреа и холин-хлорид:тиоуреа (густина, динамички вискозитет и електрична проводљивост) су инфериорнија у поређењу са својствима еутектичког растварача холин-хлорид:уреа. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {possibilities of assessing trace metal pollution using betula pendula roth. leaf and bark experience in serbia} j. serb. chem. soc. 82 (6) 723–737 (2017) udc 504.054:546.27’42’47:581.43/.45: jscs–4999 582.632.1:581.132(497.11) original scientific paper 723 possibilities of assessing trace metal pollution using betula pendula roth. leaf and bark – experience in serbia dragana pavlović1*, marija pavlović1, milica marković1, branko karadžić1, olga kostić1, snežana jarić1, miroslava mitrović1, ivan gržetić2 and pavle pavlović1 1department of ecology, institute for biological research “siniša stanković”, university of belgrade, bulevar despota stefana 142, 11000 belgrade, serbia and 2faculty of chemistry, university of belgrade, studentski trg 12–16, 11000 belgrade, serbia (received 13 january, revised 14 february, accepted 17 february 2017) abstract: in this study, both seasonal and spatial variations in trace metal uptake, as well as concentration of photosynthetic pigments in silver birch (betula pendula roth.) trees in five urban parks in pančevo, smederevo, obrenovac and belgrade (serbia) affected by different anthropogenic activities were studied. the characteristics of soil were assessed in terms of texture, ph and trace element content. concentrations of boron, strontium and zinc in both leaves and bark showed an increasing temporal trend, however, copper showed an opposite seasonal trend. a higher accumulation of trace elements was noticed in leaves compared to bark. the obtained results for photosynthetic pigments showed low sensitivity of birch to b, cu, sr and zn contamination, indicating that birch tolerates pollution and climate stress by increasing the amount of pigments. analysis of the effects on soil chemistry of trace element accumulation in plant tissues proved that soil chemistry poorly explains the variability of elements in bark (27.6 %) compared to leaves (82.99 %). discriminant analysis showed that belgrade and smederevo are clearly separated from the other three sites. keywords: urban pollution; biomonitoring; photosynthetic pigments; plant tissue; soil. introduction increasing industrialization and urbanization coupled with increased vehicular traffic intensifies the emission of various pollutants, such as trace elements, into the atmosphere.1 the urban environment is exposed to anthropogenic contaminants released from both stationary (power plants, industries and waste disposal) and mobile (road traffic) sources. trace elements are persistent and widely * corresponding author. e-mail: dragana.pavlovic@ibiss.bg.ac.rs https://doi.org/10.2298/jsc170113024p 724 pavlović et al. dispersed in the environment and can interact with different natural components, which results in toxic effects on the biosphere.2 heavy metals induce morphological, biochemical and physiological changes in plants.3,4 trees, as one of the most abundant plant types together with grass, found in urban green areas, intercept atmospheric pollutants from the air (through wet and dry deposition) and accumulate metals from the soil by their root system that later translocate to other plant parts.2,5 trace element absorption from soil is governed by soil characteristics such as the total concentration of elements present in the soil, ph, clay and hydrous oxide content, organic matter and redox conditions.6 many studies have shown that higher plants, especially different parts of trees (roots, leaves and bark), can be successfully used as biomonitors due to their accumulation properties, availability of biological material, simplicity of species identification, sampling and treatment, as well as their good tolerance to environmental changes.5–10 leaves are a often used for monitoring elemental deposition from the atmosphere. the deposition of pollutants on the leaf surface can be affected by the morphological and structural properties of the leaf, such as orientation and size, cuticle thickness, roughness, existence of surface wax layer.8 likewise, tree bark as a non-living plant material is also considered to be a promising indicator in air pollution monitoring and its surface has a very important influence on the accumulation of pollutants due to its structural porosity. furthermore, factors such as the concentrations of pollutants in the air, physiological and chemical properties of the bark, through fall, soil factors, contamination from other plants, climatic factors, etc., can also affect element accumulation.7,8 high accumulation of metals and other pollutants in plant photosynthesizing tissues can cause changes in the concentration of photosynthetic pigments (chlorophyll and carotenoids), which directly affects the plant productivity. therefore, their levels are an important tool used for the evaluation of the effects of air pollutants on plants.1,11 silver birch (betula pendula roth.) was studied in five urban parks in the localities exposed to different sources of pollution: pančevo, smederevo, obrenovac, belgrade and the arboretum of the faculty of forestry in belgrade (which is unexposed to a direct source of pollution). the elements b, cu, sr and zn were selected to be analyzed because b is one of the essential micronutrients for plants, cu is involved in many physiological processes in plants, sr is toxic and shares some chemical and physical characteristics with ca, therefore can enter the food chain and alter physiological process and zn is an essential element with a fundamental role in plant metabolism. seasonal variations in trace element concentrations (b, cu, sr and zn) were analysed in leaves and bark, as well as the content of photosynthetic pigments (chlorophill a and b, chl a and chl b, and total carotenoids, tot carrot) in leaves, in order to assess the impact of urban trace metal pollution assessment using b. pendula 725 environment pollution on the health status of b. pendula. in addition, the texture, ph and trace element concentration in soil was determined in order to distinguish the elements’ mobility and availability. silver birch has shallow roots spreading around the tree in all directions, so the sampled soil was always tightly bound to birch roots. the bioconcentration factor was determined to further clarify whether birch is a potential accumulator, indicator or excluder of trace elements.12,13 previous studies provided evidence that b. pendula is good bioaccumulator of trace elements in leaves, especially for zn, and is suitable as a biomonitor of trace element contamination due to its resistance to different types of industrial pollution.9,14-16 the aim of this study was to: 1) discern between significant variations throughout vegetation season in b, cu, sr, and zn content and their potentially toxic concentrations in leaf, bark and soil of b. pendula; 2) to investigate the effects of industrial and traffic pollution on the chlorophyll level in birch trees; 3) to compare the accumulation levels between bark and leaf samples of b. pendula. experimental soil and plant samples analyses plant and soil samples were collected from five urban parks in the localities exposed to different sources of pollution: pančevo, smederevo, obrenovac and belgrade. detailed information on study area, species description and sampling are given in supplementary material to this paper. soil ph values were measured using a glass electrode (1:2.5 soil-water ratio) after shaking the samples to equilibrium for approximately 30 min.17 the ph was measured directly in the suspension. leaves, bark and soil were dried to constant weight at 105 °c (binder, tuttlingen, germany). dried samples were ground using a stainless-steel mill and sieved through a 1.5 mm stainless-steel sieve. for trace element analysis, the leaf, bark and soil samples (0.3 g) were digested in a microwave (cem, 39 mds-2000), using the method usepa 3052. the final extracts were filtered into 50 ml polyethylene volumetric flasks, and then diluted to the mark with deionized water. the concentrations of b, cu, sr and zn in the samples were measured by inductively coupled plasma optical emission spectrometry (icp-oes) (spectro genesis fee, spectroanalytical instruments gmbh, kleve, germany). quality control was performed using the certified reference material for leaves–beech leaves–bcr–100, and soil–loam soil–erm– cc141 obtained from the irmm (institute for reference materials and measurements, geel, belgium) and is certified by the ec-jrc, european commission−joint research center. the concentrations found were within 95-110 % of the certified values for all measured elements. element concentrations were expressed as mg per kilogram of the dry leaf weight (mg kg-1 d.w.). chlorophyll concentration in the leaf tissue was determined spectrophotometrically based on the light absorption of a solution obtained after extraction with dimethyl sulfoxide (dmso).18 one disk (1 cm in diameter) per leaf was harvested from 5 leaves. chlorophylls and total carotenoids were extracted with 1 ml of dmso. after incubation at 65 °c until full extraction of chlorophyll was reached, the absorbance of extracts was measured at 663, 645 726 pavlović et al. and 480 nm with uv–visible spectrophotometry (shimdazy uv–160). the equations of arnon19 were used to calculate chl a and chl b. total carotenoids were calculated according to wellburn.20 chlorophyll and carotenoid concentrations in leaves were expressed also in mg per gram of the dry leaf weight (mg g-1 d.w.). calculation of bioconcentration factor (bcf) plant-to-soil bioconcentration (bcf) ratio estimates for element accumulation ability of the plants from the soil21 was calculated as a ratio of metal content in plant part (leaf and bark) and substrate expressed on a dry-weight basis. statistical analyses the results were analyzed using statistical analysis (anova) by computing the statistical significance of the mean differences of photopigments (chl a, chl b, tot carot) and trace element concentrations with respect to their locations. linear discriminant analysis22 was performed to assess the differentiation among the analyzed sites based on the variations of chl a, chl b, tot carot and elements. relationships among concentrations of all elements in the soil and tissues of b. pendula were assessed using both canonical correlation analysis (cca)23 and mantel’s test24-26 in order to assess the effects of soil chemistry on the concentrations of elements in birch tissues, redundancy analysis17 was performed. statistical analyses were performed using the “flora” software package.28 results and discussion trace element content in soil and plant material in the examined soil samples, the ph in aqueous solutions ranged from 8.30 at the control site to 8.51 in smederevo (table s-i of the supplementary material), which puts these soils into a group of alkaline soils.29 based on a specifically constructed triangle for soil classification by texture in parks in pančevo, smederevo, belgrade and at the control site, the dominant soil type was sandy clay loam, while in the park in obrenovac clay loam was ascertained.29 the concentrations of the examined elements in the soil were analyzed in order to characterize potential soil pollution and to determine the transfer efficiency from the soil. the seasonal variation in b, cu, sr and zn in soil, leaves and bark of b. pendula from each sampling site are provided in tables i and s-i. boron is one of the essential micronutrients for plants. it is unique among other essential elements in that a narrow range in its concentration can mean the difference between plant deficiency and plant toxicity.30 in our study, the average concentration of b in the soil from the five examined sites ranged from 128.63 mg kg-1 in pančevo to 166.28 mg kg-1 at the control site, and all obtained values were above the normal range for soil, as described by kabata-pendias and pendias,31 table s-ii. seasonal fluctuations of b concentrations in leaves were noted. the lowest concentrations of b were measured in june in obrenovac (30.96 mg kg–1), and the highest in october at all sites; toxic concentrations (table s-ii), as defined by kabata-pendias and pendias,31 were measured in belgrade (276.88 mg kg–1). toxic trace metal pollution assessment using b. pendula 727 b concentrations in soil can influence the b bioavailability for plants, which is linked to soil ph.32 namely, the availability of b to plants is highly dependent on ph, with a maximum at ph > 7.31 also, under conditions of low rainfall, as was the case throughout the entire year of the study (2012), b was not being sufficiently leached from soil surface and could therefore accumulate to toxic concentrations.33 the apparent accumulation of b to a toxic concentration resulted in the appearance of toxicity symptoms on older leaves in the form of marginal or tip chlorosis, or both, and necrotic patches. table i. average trace element concentrations (mg kg-1 d.w.) in leaves and bark of b. pendula sampled from urban sites pančevo-p, smederevo-s, obrenovac-o, belgrade-b, control -c. all values are mean with the standard deviation in parentheses (n = 5) site june august october b cu sr zn b cu sr zn b cu sr zn leaf p 36.59 (0.12) 4.89 (0.15) 34.42 (0.29) 29.80 (0.68) 44.53 (0.51) 4.08 (0.26) 36.28 (0.28) 24.88 (0.63) 59.20 (0.65) 3.63 (0.13) 21.61 (0.17) 39.39 (0.30) s 106.29 (0.65) 3.67 (0.18) 36.19 (0.45) 20.92 (0.51) 134.57 (1.22) 3.75 (0.14) 44.04 (0.41) 51.29 (0.73) 191.39 (1.53) 4.22 (0.21) 34.93 (0.41) 63.67 (0.67) o 30.96 (0.62) 5.81 (0.49) 35.55 (0.27) 36.68 (0.68) 79.00 (0.75) 3.72 (0.27) 41.99 (0.51) 38.29 (0.68) 86.11 (0.85) 4.10 (0.21) 29.73 (0.59) 34.36 (0.29) b 168.82 (2.74) 5.86 (0.23) 51.57 (0.19) 199.54 (1.06) 269.14 (2.94) 3.94 (0.10) 48.50 (0.28) 332.07 (4.60) 276.88 (1.65) 4.77 (0.08) 46.15 (0.37) 344.96 (2.10) c 45.89 (1.52) 5.62 (0.14) 28.98 (0.64) 30.02 (0.65) 67.80 (0.47) 4.64 (0.09) 31.44 (0.21) 40.41 (0.52) 105.56 (0.72) 4.03 (0.12) 27.14 (0.74) 35.52 (0.27) bark p 1.20 (0.08) 5.55 (0.19) 10.03 (0.18) 72.04 (0.68) 4.64 (0.15) 5.65 (0.30) 5.58 (0.16) 135.37 (0.71) 2.98 (0.24) 5.00 (0.08) 3.26 (0.15) 162.77 (1.02) s 5.01 (0.28) 5.58 (0.13) 13.17 (0.62) 73.34 (1.12) 7.81 (0.30) 5.19 (0.41) 7.93 (0.35) 145.41 (3.45) 16.63 (0.55) 5.80 (0.04) 9.12 (0.36) 162.69 (2.27) o 4.11 (0.12) 4.15 (0.08) 13.16 (0.12) 53.69 (0.64) 9.42 (0.34) 4.50 (0.18) 10.43 (0.33) 85.88 (1.25) 5.60 (0.20) 3.83 (0.11) 3.53 (0.37) 127.02 (12.79) b 9.02 (0.20) 9.25 (0.37) 21.26 (0.43) 74.41 (0.54) 14.53 (0.14) 12.49 (0.29) 18.24 (0.45) 105.06 (0.37) 11.96 (0.34) 4.72 (0.16) 9.89 (0.26) 160.15 (4.50) c 4.55 (0.23) 5.95 (0.18) 12.27 (0.35) 45.78 (0.82) 5.64 (0.24) 5.95 (0.17) 6.94 (0.18) 116.03 (0.02) 3.50 (0.34) 4.24 (0.44) 4.29 (0.28) 120.59 (0.75) unlike leaves, b concentrations in tree bark were very low during the growing season (table i). the lowest concentration was measured in pančevo in june (1.20 mg kg–1) and the highest at the end of season in smederevo (16.63 mg kg–1). copper is an essential transition metal that is involved in many physiological processes in plants.35 the concentrations of cu in the soil at all five investigated sites were in the normal range for soils (table s-ii). the lowest concentration was measured in pančevo (11.93 mg kg–1). the highest concentration was determined in smederevo (30.66 mg kg–1). 728 pavlović et al. the highest cu content at all examined sites was observed at the beginning of the vegetation season, with the highest (5.86 mg kg–1) measured in belgrade. this spike was followed by a decrease in the second part of the season. these results indicate a higher rate of plant metabolic activity in the first part of the season, resulting in a stronger uptake rate and metal accumulation. a similar seasonal trend was previously observed by kim and ferguson36 and to some extent by piczak et al.,15 who noted that cu concentrations were highest in new leaves and gradually decreased with time. in our study, in the second part of the season cu levels were below values for normal plant growth, and symptoms of deficiency occurred in the form of leaf-tip chlorosis or necrosis in young leaves, which later spread downward along the leaf margins in older leaves. in general, the deficiency effects of cu on plants are usually noted at concentrations lower than 5 mg kg–1. copper-deficient plants show morphological changes in leaf architecture and this represents the first symptom of cu deficiency.37 notwithstanding the normal concentration of copper in the soil (table s-ii), it is likely that the alkaline conditions (ph > 8.0) and cu low transfer coefficients cause lower element solubility and lower availability for plants, which explains its deficit in leaves.38,39 the concentration of cu in bark remained relatively constant from june to october, which is consistent with the findings of reimann et al.40 on the seasonal dynamics of different elements in birch leaves and bark under different anthropogenic conditions, i.e., cu values in the leaves and bark remained almost the same during the examined season. the only exception was belgrade with a maximum in august (12.49 mg kg–1) and a minimum in october (4.72 mg kg–1), which could be explained by the abundance of roads and traffic emissions, since most of the cu in urban environment originates from traffic and use of brakes.41 strontium is a toxic, nonessential element to plants which shares some chemical and physical characteristics with calcium, which is an essential element required for plant growth. the content of strontium in plants is highly variable.42 in our study, the concentration of sr in the soil exhibited considerable variability, ranging from a minimum of 18.32 mg kg–1 measured in pančevo, to a maximum of 85.53 mg kg–1 in obrenovac. however, all the measured values were below the average concentration for soils on a world scale (table s-ii). the concentration of sr in plants is very variable, although the highest concentrations are usually observed in plant tops.31 in this study, toxic concentrations of sr in birch leaves (>30 mg kg–1)34 were measured throughout the season at all the examined localities. the maximum concentration were reached during august, except in belgrade where it was reached in june (51.57 mg kg–1), while the lowest during october at all sites. given the fact that concentrations in range31 of 1–10 mg kg–1 are considered as normal sr values for plant tissues, concentrations in range of 21.61–29.73 mg kg–1 obtained for pančevo, obrenovac and control trace metal pollution assessment using b. pendula 729 site during october could be interpreted as excessive or even toxic. piczak et al.15 observed an increase in sr content in birch leaves with time. in general, leaves have the strongest accumulation capability among plant tissues.42 compared to leaves, the concentrations in the bark were much lower (in the range of 5–21 mg kg–1, table i), which is in accordance with previously published results regarding sr content in birch leaf and bark.40 the highest concentration of sr in the bark, at all five analyzed sites, was measured in june (maximum in belgrade, 21.26 mg kg–1), followed by a decrease through the season, with lowest values measured in october (minimum in pančevo, 3.26 mg kg–1). also, sr follows a similar distribution in plant tissues as observed for b; however, the relative accumulation of sr in leaves is not as extreme. after comparing the annual average concentration of sr in leaves and soil, it was observed that the concentration of sr in soil is slightly higher or equal to its concentration in leaves. bearing in mind that the uptake of sr by plant roots depends on the plant species and soil properties, such as organic matter content, ph and ionic composition,6 and that the root uptake of sr is much greater than leaf uptake,42 it can be assumed that a part of sr contents is airborne and deposited on leaf surfaces. in our study, concentrations of zn in soils were within the normal level for soils worldwide (table s-ii). the highest zn content was measured in belgrade (134.53 mg kg–1). for plants, zn is an essential element with a fundamental role in metabolism.31 unlike copper, the zn content in leaves was the lowest at the beginning of season, with the lowest concentration (20.92 mg kg–1) recorded in smederevo, reaching the maximum in late season. these results are in accordance with the findings of piczak et al.15 likewise, kabata-pendias and mukherjee31 found that zn is predominantly concentrated in mature leaves or during the phase of intensive growth of plant. in our study, the zn content was within the normal range during the entire season at all examined sites, except belgrade. toxic effects of zn are observed at concentrations higher than 100 mg kg–1 (table iii). the concentrations recorded in belgrade that ranged from 199.54 in june to 344.96 mg kg–1 in october were all within the toxic range and well over the values considered as optimal for plant development. such high concentrations of zn could be related to traffic. abrasion of tires is a source of zn contaminants in the environment, since zn is a catalyst in the manufacture of tires and additives containing zn are often used in tires and motor oil.44 the phytotoxicity of zn mostly depends on the species of plant, age, environmental conditions and the combination of other heavy metals.45 copper and zinc show antagonistic interactions, i.e., the uptake of one element is inhibited by the other due to competition for the same sites for absorption into the plant root.46 an excess of zn can also give rise to cu deficiencies in plant shoots.47 730 pavlović et al. zinc content in bark also showed an increasing tendency during the vegetation season with the lowest concentrations measured in june at the control site (45.78 mg kg–1), and highest in october at all the sites, the highest levels being measured in pančevo (162.77 mg kg–1). bark contained much higher zn concentration than leaves, with the exception of the belgrade urban park. these results agree with the findings of reimann et al.39 who found that bark was susceptible to a greater impact of urban pollution than leaves due to prolonged exposure time. high bioaccumulation of zn in tree bark could be the consequence of its high transfer coefficient, which is a reflection of its relatively poor sorption in the soil.37 bioconcentration factor after determining the concentrations of b, cu, sr and zn in soil, leaf and bark, their bioconcentration factor (bcf) was calculated to assess the possible role of b. pendula as a potential accumulator, indicator or excluder of these elements. bcf in b. pendula expresses the ratio of element concentrations (mg kg–1) in plant parts (leaves and bark) to its concentrations in soil. a bcf > 1 indicates that the plant is enriched with metals (accumulator); bcf = 1 indicates relatively indifferent response of the plant to metals (indicator); bcf < 1 shows that the plant excludes metals from uptake (excluder).12,13 the results in table ii shows that the bcf values for cu ranged from 0.1–0.3, for b from 0.3–1.5 (bcf > 1 was obtained at belgrade and smederevo sites), for sr from 0.4–1.6 (bcf > 1 was obtained for pančevo and the control site), and for zn they ranged from 0.3– –2.1 (bcf > 1 was obtained for the belgrade site). considering that the uptake of trace metals from the soil affected by several factors (ph, organic matter content and soil type), it can be assumed that the characteristics of the soil, e.g., clay loam in obrenovac, is the main reason for the low accumulation of elements in birch leaves at this site. the bcf values for b in bark (table ii) were in the table ii. bioconcentration factor for b. pendula leaves and bark sampled from from urban sites. all values are mean with the standard deviation in parentheses (n = 5) site leaf bark b cu sr zn b cu sr zn pančevo 0.36 (0.01) 0.35 (0.01) 1.68 (0.04) 0.51 (0.03) 0.06 (0.03) 0.45 (0.01) 0.34 (0.01) 1.99 (0.11) smederevo 1.04 (0.02) 0.13 (0.01) 0.81 (0.02) 0.38 (0.01) 0.71 (0.01) 0.18 (0.01) 0.21 (0.01) 1.02 (0.01) obrenovac 0.42 (0.01) 0.30 (0.01) 0.42 (0.02) 0.81 (0.07) 0.40 (0.01) 0.28 (0.01) 0.11 (0.01) 1.96 (0.12) belgrade 1.57 (0.02) 0.21 (0.01) 0.97 (0.02) 2.17 (0.01) 0.08 (0.01) 0.38 (0.01) 0.33 (0.01) 0.84 (0.01) control 0.44 (0.01) 0.38 (0.01) 1.04 (0.05) 0.64 (0.01) 0.03 (0.01) 0.43 (0.01) 0.28 (0.02) 1.72 (0.01) trace metal pollution assessment using b. pendula 731 range from 0.03–0.7, for cu from 0.2–0.4, for sr from 0.1–0.3 and for zn from 0.8–2.0 (bcf > 1 was obtained at all the examined sites except of belgrade site). effects of soil chemistry on the concentrations of the analyzed elements in birch tissues nutrient-cycling processes mutually connect plants and soil. plants absorb essential elements from soil. the absorption process ultimately depends on the availability of anions and cations in the soil. on the other hand, plants also control the nutrient-cycling process by nutrient-use efficiency, transpiration rate, root exudates, and by the tissue chemistry of falling leaves and twigs that are incorporated in decomposition processes. despite the strong mutual relationship among plants and soil, the correspondence between soil and plant chemistry is relatively poor because the absorption of elements is a process controlled by the several factors, such as ph, organic matter content, redox conditions, etc.6 in order to detect mutual relations among the concentrations of all elements in the soil and tissues of b. pendula, we used canonical correlation analysis, cca. a higher correlation coefficient was found between soil chemistry and the concentrations of b, cu, sr and zn in leaves in comparison to bark (table iii). this indicates that bark chemistry is determined not only by soil chemistry, but also by other factors (e.g., atmospheric deposits). table iii. hotelling’s correlation among the concentrations of b, cu, sr and zn in the soil and tissues of b. pendula principal axes soil and leaves of birch soil and bark of birch 1 0.853 0.243 2 0.311 0.044 3 0.194 0.034 4 0 0 the main disadvantage of canonical correlation analysis is the fact that the method calculates the correlation between two sets of variables for each principal axis. in order to detect the overall correlation between soil chemistry and the chemistry of birch tissues, mantel’s test was performed,24,25 (fig. 1). mantel’s test proved that the soil chemistry is significantly correlated with the concentrations of b, cu, sr and zn in the leaves of b. pendula. biotic indicators require a causal relationship between biotic components and the environment. if the causal relation is perfect, then an ideal bioindicator unambiguously points to the environmental conditions of a site.48 in order to detect causal effects of soil chemistry on the concentrations of the analyzed elements in birch tissues, we performed the redundancy analysis (ra). by the analogy with the univariate regression analysis, the redundancy analysis performs a variance partitioning to detect the effects of a set of independent variables on a 732 pavlović et al. set of dependent or “response” variables. redundancy analysis suggests that soil chemistry poorly explains the variability of the elements in birch bark. namely, soil chemistry explains only 27.6 % of variability of the analysed elements in the bark. however, the percentage of total variability of the elements in the leaves explained by soil chemistry is much greater (82.99 %), table iv. fig. 1. overall correlation between the chemistry of soil and birch bark (a) and soil and birch leaves (b). table iv. causal effects of soil chemistry on concentrations of elements in leaf and bark of b. pendula variance of concentrations of elements in leaves of b. pendula 17418.3 variance of concentrations of elements, predicted by regression model 14455.4 variance explained by soil chemistry 0.8299 variance of concentrations of elements in bark of b. pendula 1541.67 variance of concentrations of elements, predicted by regression model 425.21 variance explained by soil chemistry 0.276 photosynthetic pigments chlorophylls and carotenoids are the main photosynthetic pigments in plants. photosynthetic pigments have been used as sensitive markers of stress caused by heavy metals and other pollutants since they are very sensitive to changes in the environment. therefore, their quantification provides useful insight into the physiological performance of plants. carotenoids are accessory pigments and essential structural components of the photosynthetic antenna and reaction centers in higher plants. as non-enzymatic antioxidants, their main function is to protect the photosynthetic apparatus, dissipating energy to avoid harmful photooxidative processes.11 in our study, the concentration of chl a ranged from 2.26 in smederevo, to 6.82 mg g–1 in obrenovac, and increased significantly in the second part of season (table v). the lowest content of chl a was measured in belgrade during the entire vegetative season, which could be attributed to the toxic concentrations trace metal pollution assessment using b. pendula 733 table v. average chl a, chl b and tot carot (mg g-1 d.w.) in b.pendula sampled from urban sites in june, august and october 2012. the values are mean, with standard deviation in parentheses (n = 5) site june august october chl a chl b tot carot chl a chl b tot carot chl a chl b tot carot pančevo 3.63 (0.24) 1.81 (0.20) 1.03 (0.09) 4.62 (0.27) 1.37 (0.22) 1.41 (0.07) 5.48 (0.30) 2.04 (0.21) 1.72 (0.21) smederevo 2.26 (0.19) 2.50 (0.25) 0.93 (0.12) 3.44 (0.29) 1.27 (0.18) 1.27 (0.16) 5.09 (0.34) 1.52 (0.35) 1.22 (0.52) obrenovac 2.80 (0.06) 2.16 (0.14) 1.05 (0.02) 5.93 (0.19) 1.81 (0.08) 1.78 (0.18) 6.82 (0.09) 2.03 (0.14) 1.05 (0.51) belgrade 2.57 (0.05) 1.46 (0.14) 0.86 (0.09) 3.62 (0.23) 1.03 (0.16) 1.12 (0.12) 2.86 (0.20) 0.71 (0.08) 0.86 (0.06) control 2.75 (0.12) 2.57 (0.32) 0.87 (0.11) 5.56 (0.21) 1.63 (0.18) 1.58 (0.12) 4.72 (0.30) 1.21 (0.04) 1.16 (0.14) of zn and sr in birch leaves. moyen and roblin6 showed that sr probably blocks ion channels in the chloroplast envelope and decreases the content of mg, which is a main component of chlorophyll. in contrast, the highest chl a content was measured in leaves samples from obrenovac. our present results do not suggest clear reasons for such birch behaviour, so we can only assume that sensitivity of species to any element depends on concentration, exposure period, age and growth conditions. although, many authors have shown that an increased content in polluting agent leads to chlorophyll inhibition11,49–51 either through direct inhibition of several enzymatic steps or as a result of substitution of the central mg ion,52 several studies have shown that the exposure to heavy metals induces oxidative stress which is accompanied by an increase in the chlorophyll content.52–54 for example, kalaikandhan et al.55 examined the effects of different zn concentrations on the photosynthetic pigment content in sesuvium portulacastrum leaves during 120 days and established that an increase in chl a accompanied the increase in the concentration of zn in the substrate from 100 to 300 mg kg–1, with which our results are in good agreement. likewise, petrova56 tested the pigment response to urban air pollution in birch leaves and found that the highest concentrations of chlorophyll were present in plants growing in environments with a medium level of urbanization and moderate exposure to road traffic pressure. chl b content in birch leaves was in range from 0.71 in belgrade to 2.57 mg g–1 at the control site (table v); chl b exhibited an opposite trend and decreased during the season. a similar trend was found by gajić et al.4 at polluted sites (urban parks of belgrade), where higher content of chl b in ligustrum ovalifolium hassk. was measured in june than in october. the observations presented in this study are also consistent with the findings of fargašova49 who examined the aboveground parts of sinapis alba l. seedlings, however, our results are not 734 pavlović et al. in agreement with the findings of keser et al.54 in lepidium sativum l. different effects of pollution on chl b in relation to chl a have been reported in many studies.52,54 however, the different responses of chl a and chl b are not unexpected, since different types of pollution exert different effects on the pigment content, hence the response of a plant can be attributed to the interaction between various types of pollutants as well as abiotic factors (high temperature, drought, intense insolation, etc.). the minimum carotenoid concentration was measured in samples from belgrade (0.86 mg g–1) and the maximum in samples from obrenovac (1.78 mg g–1). their content was the highest in august when the climate conditions were the most unfavourable (table v). namely, the studied year was the warmest and the least rainy year (related period 1981–2010), with extremely high number of hot days and nights, since the beginning of their measurement in serbia. mean summer air temperature in serbia was categorized as extremely warm, with 62 days of maximum daily temperatures higher than 30 °c, and 52 nights with minimum temperatures higher than 20 °c.57 an increase in carotenoid concentration could be seen as a defence strategy to suppress above mentioned conditions, which has been shown with earlier studies.58 differentiation among examined sites the difference in the total content of b, cu, sr and zn and photosynthetic pigments in leaves of b. pendula between the examined sites was determined by discriminant analysis (da). based on the first discriminant function (dc1) which is responsible for 97.36 % of differences, and the second discriminant function (dc2) which explains 2.64 % of differences, it can be noted that the belgrade and smederevo sites are clearly separated from the other three sites. overlap exists in the case of pančevo, obrenovac and the control where they partially overlap (fig. s-2a of the supplementary material). the parameters that most contribute to the separation are b, sr and chl b on dc1 and b and zn on dc2 (fig. s-2b). conclusion the result of this research provides an insight of the pollution state of four urban localities in serbia, that are exposed to different sources of pollution using betula pendula leaf and bark. the concentrations of b, sr and zn in leaves reached maximal and sometimes toxic levels during the second part of the season, in august and october, unlike cu whose concentration was deficient or close to being deficient for normal plant development. a toxic concentration of b was measured in smederevo and belgrade, possibly as the result of high concentrations of b in the soil and conditions favouring its transfer from the soil to the plant. most of the sr was probably airborne as it was deposited on the leaf surface. toxic concentrations of trace metal pollution assessment using b. pendula 735 zn were measured in belgrade, which might relate to its high mobility and relatively poor sorption in the soil, and pollution resulting from the high traffic flow. despite the normal concentration of copper in the soil, it is likely that the alkalinity (ph > 8.0), low transfer coefficients and high concentration of zn in the rooting zone (zn and cu compete for the same sites for absorption) are responsible for the lower element solubility and its lower availability for plants. analysis of trace elements in leaves and bark showed that leaves have higher accumulation capability compared to bark. based on ra analysis of the effects of soil chemistry on the accumulation of trace elements in plant tissues it could be concluded that soil chemistry cannot explain the variability of the elements found in the bark (27.6 %), while the percentage of the total variability of elements in the leaves can be explained by the soil chemistry (82.99 %) which suggests that birch leaves are suitable for biomonitoring of pollution from different sources. the obtained results for photosynthetic pigments revealed a low sensitivity of b. pendula to b, cu, sr and zn contamination. it seems that birch, because of the increasing of the amount of pigments, is tolerant to pollution and climate stress and does not suffer damage and metabolic disorder. supplementary material detailed information on study area, species description and sampling are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. this work was supported by the ministry of education, science and technological development of the republic of serbia (grant no. 173018). и з в о д могућност коришћења листова и коре betula pendula roth. у процени загађености елементима у траговима –искуства у србији драгана павловић1, марија павловић1, мирослава митровић1, бранко караџић1, олга костић1, снежана јарић1, милица марковић1, иван гржетић2 и павле павловић1 1одељење за екологију, институт за биолошка истраживања „синиша станковић“, универзитет у београду, булевар деспота стефана 142, 11000 београд и 2хемијски факултет, универзитет у београду, студентски трг 12–16, 11000 београд у овом истраживању je проучаванa сезонска и просторна динамика акумулације елеменaта у траговима, као и концентрација фотосинтетичких пигмената у брезама (betula pendula roth.) урбаних паркова у панчеву, смедереву, обреновцу и београду (србија) који су под утицајем различитих антропогених активности. карактеристике земљишта су одређене у погледу текстуре, ph и садржаја елемената у траговима. концентрација b, sr и zn у оба биљна ткива је показала растући тренд током сезоне, за разлику од cu који је максималну концентрацију достигао почетком вегетацијске сезоне. већа акумулација елемената је забележена у листовима у односу на кору са изузетком садржаја zn. добијени резултати за фотосинтетичке пигменте су показали слабу осетљивост брезе на загађење b, cu, sr и zn указујући на то да бреза на одабраним локалитетима толерише стресне услове загађења повећавајући количину пигмената. анализа 736 pavlović et al. ефеката коју садржај елемената у земљишту има на њихову акумулацију је показала да садржај елемената у земљишту слабо потврђује варијабилност елемената у кори (27,6 %) у односу на листове (82,99 %). линеарна дискриминантна анализа је утврдила да су паркови у обреновцу и смедереву јасно одвојени од остала три парка. 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republic hydrometeorological service of serbia, belgrade, 2012 (in serbian) 58. e. brugnoli, a. cona, m. lauteri, photosynth. res. 41 (1994) 451. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams false /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {chemical composition and antiproliferative potential of dried wild apple and pear tea before and after in vitro simulated digestion} j. serb. chem. soc. 83 (12) 1315–1326 (2018) udc 634.12/.13:664.854+547.56:612.3 jscs–5153 original scientific paper 1315 chemical composition and antiproliferative potential of dried wild apple and pear tea before and after in vitro simulated digestion jelena živković1*, katarina šavikin1, nemanja stanisavljević2, gordana zdunić1, tatjana stanojković3 and jelena samardžić2 1institute for medicinal plants research “dr. josif pančić”, tadeuša košćuška 1, 11000 belgrade, serbia, 2institute of molecular genetics and genetic engineering, university of belgrade, vojvode stepe 444a, 11000 belgrade, serbia and 3institute of oncology and radiology of serbia, pasterova 14, 11000 belgrade, serbia (received 4 june, revised 29 august, accepted 30 august 2018) abstract: decoctions obtained from dried apple and pear fruits were subjected to in vitro digestion in the presence of a food matrix in order to determine changes in the polyphenol content and antiproliferative activity. the total phenolic and total proanthocyanidins contents were determined using spectrophotometrical methods, and the phenolic compounds were analyzed by rp-hplc/dad before and after digestion. chlorogenic acid and phlorizin dihydrate were the major identified compounds. the addition of a food matrix immediately decreased the contents of individual and total phenolics. after digestion, they were slightly elevated but still lower than in the initial samples. antiproliferative activity was investigated on human epithelial carcinoma cell line (hela), human colon carcinoma (ls174) and human foetal lung fibroblast (mrc-5) cells. the exhibited growth inhibition was not correlated with the content of phenolics in the tested samples, indicating that it could not be explained solely by their content. with the exception of the apple decoction effect on hela cells, the digestion process resulted in significant increases in the antiproliferative activity. keywords: bioaccessibility; phenolics; food matrix; fruit. introduction epidemiological studies demonstrated that diets rich in fruits could be linked with decreased mortality caused by certain types of cancer, mostly due to the additive and synergistic effects of bioactive compounds such as polyphenols, vitamins, minerals and fibre.1 apples (malus sp., rosaceae) and pears (pyrus sp., rosaceae) are widely consumed fruits in europe. there are various reports on the health-promoting * corresponding author. e-mail: jzivkovic@mocbilja.rs https://doi.org/10.2298/jsc180604073z ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1316 živković et al. effect of phenolic compounds present in these fruits,2,3 and as a result of a recent study,4 there is a new claim “an apple or pear a day helps keep strokes at bay”. thanks to recent advances in food processing technologies, the application of different drying techniques in order to reduce the moisture content have made achievable the prolongation of fresh fruit shelf life.5 dried fruits serve as important healthy snacks since they are available during the whole year. in addition, compared to fresh fruits, the dried ones are much easier for storage and transport. these products can be freely incorporated into other foods and recipes, and they present a healthy substitute for salty or sugary snacks. dried apples and pears are ingredients for tea infusions, popular due to their fruity fragrance, flavour, absence of caffeine and low adstringent and bitter taste.6 generally, six major polyphenolic groups are found in various apple varieties, i.e., hydroxycinnamic acids, flavan-3-ols, procyanidins, anthocyanins, flavonols and dihydrochalcones.7 similarly, four classes of polyphenolic compounds are reported to be present in pears, i.e., phenolic acids, flavonols, flavan-3-ols and anthocyanins.7 there are numerous literature data regarding antiproliferative potential of apples8 and pears,9 as well as compounds present in them.10 one of the limiting factors for the valuable effects of polyphenolics is their bioavailability, which depends on their release from the food matrix, as well as their stability during digestion and the efficiency of their transepithelial passage. prerequisite for their health effects is the quantitative liberation during the digestion process. although there are a large number of papers reporting the chemical composition and biological activity of apples and pears, there is a scarcity of reports dealing with apple and pear fruit teas. moreover, literature data concerning changes in the polyphenolic compounds of apple and pear during the digestion process are limited. the aim of this research was to assess the stability and functional properties of polyphenols present in decoctions of dried wild apple and pear fruits using an in vitro model of simulated digestion in the presence of the food matrix. experimental plant material and extraction procedure wild pear and apple fruits were collected on the mountain suvobor in central serbia. after rinsing with water, they were manually cut into pieces and dried in a laboratory oven at 40 °c overnight. preparation of the teas was realized in two traditional ways (as a decoction or infusion) according to ph. jug iv,11 with slight modifications. for decoction, plant material (10 g) was added to 100 ml of distilled water, heated in a steel kettle, and allowed to boil for 30 min. afterwards, the tea was filtered through filter paper and filled with distilled water up to 100 ml. for infusion, 10 g of plant material was added to 100 ml of boiling distilled water in a steel kettle and allowed to cool down to room temperature. after 30 min, the tea was filtered through filter paper and filled with distilled water up to 100 ml. all extraction procedures were performed in triplicate. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ digestion effect on fruit phenolics 1317 in vitro gastrointestinal digestion to examine the effects of digestion in the presence of a food matrix on the phenolic composition, a previously reported in vitro digestion method was applied.12 in the present experiments, an infant formula (juvitana, swisslion product d.o.o. inđija, serbia) of defined composition was used to simulate a food matrix. according to the manufacture’s specification, it contained 20 % turkey meat, 25 % boiled corn paste, 10 % boiled potato paste, 5 % rice flour, 0.1 % nacl and 39.9 % water. the digestive juices used during the in vitro digestion were prepared according to a previously reported method.12 the composition of the synthetic digestive juices is presented in table s-i of the supplementary material to this paper. all the enzymes used for in vitro digestion were purchased from sigma–aldrich (usa). the digestion started with the oral phase by mixing 6 ml of artificial saliva with 4.5 g of infant formula (food matrix) and 4.5 ml of prepared decoctions followed by gentle stirring for 5 min at 37 °c. the mixture was then transferred to 50 ml conical flasks and 3 ml of gastric juice was added. flasks were then agitated in a rotary shaker at 37 °c (1 h, 55 rpm). after one hour, the mixture was supplemented with 9 ml of gastric juice, the ph was adjusted to 2.0 and mixture was incubated under the same conditions for 1 h. in the next step, feeding conditions in the upper part of the duodenum were imitated and 12 ml of intestinal juice and 6 ml of bile juice were added. the ph was adjusted to 5.4 and the incubation was then continued for a further hour at 37 °c and 55 rpm. after completed digestion, the chyme was centrifuged for 5 min at 2750 g and the obtained supernatant was filtered through a 22-μm hydrophilic polyethersulfone membrane. samples were kept at –80 °c until further analyses. the whole procedure was repeated for 4.5 g of infant formula mixed with 4.5 ml of distilled water (instead of decoction) and subjected to digestion under the same conditions as digested sample in order to obtain digested matrix. for the preparation a control for decoction digestion or control for matrix digestion, decoctions or infant formula were immediately mixed with all components at 0 °c and ph was adjusted to a final value of 5.4, centrifuged at 2750 g for 5 min at 0 °c. the obtained supernatants were filtered through 22-μm filter and kept at –80 °c until further analysis. decoction dilution was prepared by mixing 4.5 ml with 40.5 ml of distilled water (instead of digestive enzyme cocktail) to reach the final volume after digestion and the ph was adjusted to 5.4. the diluted sample was then filtered through 22-μm filter and kept at –80 °c until further analysis. total phenolic content determination of total phenolic content (tpc) was conducted in agreement with a previously reported method.13 samples (20 μl) were combined with distilled water (1580 μl), folin–ciocalteu reagent (100 μl) and 20 % na2co3 (300 μl) and incubated at room temperature. after 2 h, the absorbance at 765 nm was measured. the content of total phenolics was calculated using a standard gallic acid curve and are expressed as milligrams of gallic acid equivalents per 1 ml of sample (mg gae 100 ml-1). all determinations were conducted in triplicate. total proanthocyanidin content the content of total proanthocyanidins (tpr) in samples was determined spectrophotometrically using the p-(dimethylamino)cinnamaldehyde (p-dmaca) method with slight modifications.14 the fruit teas and digested fractions (100 and 500 μl, respectively) were mixed with 80 μl of p-dmaca reagent and a drop of glycerol. the total volume was made up to 2 ml with methanol and after 7 min, the absorbance at 640 nm was read against methanol as the blank. the contents of proanthocyanidins in the samples are expressed as mg ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1318 živković et al. of catechin equivalents per 100 ml of sample (mg ce 100 ml-1). all the determinations were conducted in triplicate. identification of the phenolic compounds by hplc-dad analysis the obtained decoctions, digested fractions and controls were centrifuged for 10 min at 4000 g. the supernatants were subjected to solid phase extraction15 on c18 columns (bakerbond spe, 1000 mg capacity), obtained from j.t. baker, deventer, the netherlands. columns conditioning was achieved with 5 ml of water, followed by 5 ml of 0.5 % formic acid (v/v). after that step, 5 ml of supernatant solutions of the digested samples and controls were applied to the column and the bound material was eluted with 2 volumes of 0.5 % formic acid in water and finally with 5 ml of 0.5 % formic acid in acetonitrile (v/v). the fractions were collected and concentrated for further hplc analysis. the chemical analyses of the obtained decoctions, digested fractions and controls were performed on an agilent series 1200 rr hplc instrument with a dad detector, on a reverse phase lichrospher rp-18 (agilent) analytical column (250 mm×4 mm i.d., 5 µm particle size). the mobile phase consisted of solvent a (1 % of formic acid in water) and solvent b (acetonitrile). the fractions were separated using gradient elution according to the following scheme: 5–15 % b 0–5 min; 15–20 % b 5–8 min; 20 % b 8–12 min; 20–30 % b 12–15 min; 30 % b 15–17 min; 30–35 % b 17–20 min; 35 % b 20–22 min; 35–100 % b 22–25 min. the flow was adjusted to 1 ml min-1, and the detection wavelengths were set at 290, 350 and 520 nm. the standards, chlorogenic acid, phlorizine dihydrate, hyperoside, quercitrin and isoquercitrine were purchased from extrasynthese (cedex, france). all experiments were repeated in triplicate and the results are expressed as mean value ± standard deviation in µg ml-1 of sample. cell lines cervix adenocarcinoma cell line (hela), human colon carcinoma (ls174), and a noncancerous cell line, mrc-5 (human embryonic lung fibroblast) were grown in rpmi-1640 medium (sigma). all lines were from the atcc (american type culture collection) catalogue of human cell lines. the media were supplemented with 10% foetal bovine serum, 3 mmol/l l-glutamine and 1% penicillin-streptomycin (sigma). treatment of the cell lines the cell lines were treated according to a previously reported method.16 the target cells hela (2000 cells per well), ls174 (7000 cells per well) and non-cancerous mrc-5 (5000 cells per well) were seeded into wells of a 96-well flat-bottomed microtitre plate. twenty-four hours later, after cell adherence, different concentrations of the investigated teas and digested samples were added to the wells. exceptions were the control cells to which only a nutrient medium was added. the chosen concentrations range was 1–200 µg ml-1. the final concentration of dmso solvent did not exceeded 0.5 %, which is non-toxic to the cells. all concentrations were set up in triplicate. nutrient medium with the corresponding concentrations of the investigated samples, but without cells, was used as a blank, also in triplicate. the cultures were incubated for 72 h. determination of cell survival the effect of the prepared samples on the survival of the cancer cell was determined using the microculture tetrazolium test (mtt) according to mosmann17 with modification by ohno and abe18 72 h after addition of the compounds. briefly, 20 ml of mtt solution (5 mg ml-1 phosphate-buffered saline) was added to each well. the samples were incubated for a further 4 h at 37 °c in a humidified atmosphere of 95 vol.% air/5 vol.% co2. then 100 µl of ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ digestion effect on fruit phenolics 1319 100 g l-1 sodium dodecyl sulphate was added to the extract of the insoluble product formazan, resulting from mtt dye conversion by viable cells. the number of viable cells in each well was proportional to the intensity of the light absorbance, which was read in an enzyme-linked immunosorbent assay (elisa) plate reader at 570 nm. the absorbance (a) was measured at 570 nm 24 h later. cell survival rate (%) was calculated using the following equation: cell survival (%) = 100(as–ab)/(ac–ab) where ac is absorbance of the control cells grown only in nutrient medium, as – apsorbance of the tested sample and ab apsorbance of the blank. the ic50 value is defined as the concentration of an agent inhibiting cell survival by 50 % compared with a vehicle-treated control. as positive control, cis-diamminedichloroplatinum (cis-ddp) was used. all experiments were performed in triplicate. statistical analysis all results were calculated from triplicate measurements and expressed as the mean value ± standard deviation. differences between the group means and their significance were verified using one-way anova. statistically significant effects were further analyzed and means were compared using bonferroni test. a level of p < 0.05 was taken as statistically significant. results and discussion total phenolic and proanthocyanidin content the chemical composition of fruit teas is closely related to their biological activity. the first part of the present investigation was focused on the determination of the effect of the food matrix on the stability of polyphenolic compounds during in vitro digestion. in order to perform this quantification, the content of total phenolic compounds (tpc) and total proanthocyanidins (tpr) in fruit teas, digested fruit teas as well as the control of teas digestion were studied (table i). both groups of compounds were significantly higher in the apple samples compared to the pear samples. in addition, the decoctions contained higher tp and tpr contents compared to the infusions. due to this, decoctions were chosen for further study. table i. tp and tpr content in apple and pear samples and the food matrix subjected to in vitro digestion; n.d. – not detected. means with different letters are significantly different (p < 0.05) sample tpc / mg gae 100 ml-1 tpr / mg ce 100 ml-1 apple decoction 190.61±6.92a 71.82±3.75a pear decoction 84.18±4.21b 63.43±2.91a apple digested sample 38.93±1.89c n.d. pear digested sample 37.28±1.58c n.d. apple digested sample control 26.91±0.83d n.d. pear digested sample control 25.88±0.71d n.d. apple infusion 112.76±4.8e 28.55±1.36c pear infusion 45.28±2.12c 10.62±0.39d matrix 21.19±0.75d n.d. matrix control 18.54±0.51d n.d. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1320 živković et al. the total phenolics existing in food do not always reflect the amount that can be absorbed and metabolized in the human body. it is well known that bioavailability of phenolic compounds is influenced by numerous factors, such as the interaction with food matrix.19 in the present investigation, in control samples immediately after addition of food matrix and the digestive enzyme cocktail, the amounts of tpc were reduced by 85.88 and 69.26 % in case of apple and pear decoctions, respectively. this indicates that the constituents of the food matrix rapidly bound a high percent of soluble phenolic compounds from the investigated samples through hydrogen bonding and hydrophobic interactions.12 after simulated in vitro digestion, the tpc in the digested samples was slightly increased compared to the control samples. still it was 80 and 56 % lower in the case of apple and pear decoctions, respectively. this could be explained by the release of polyphenolic compounds from the complex with the food matrix components during time. this is in accordance with results of a previous in vitro study,20 in which a decrease of tpc in extracts of four commercial apple cultivars (jonaprinz, jonagold, golden and mutzu) was observed after simulated in vitro digestion. similarly, cilla et al.21 showed that the tpc decreased by at least 47 % in digested fruit beverages compared to undigested ones. a change in tpc during digestion was also observed for the food matrix. namely, the amount of total phenolics increased in the digested matrix (21.19 mg gae 100 ml–1) compared to the digestion of the control matrix (18.54 mg gae 100 ml–1). thus, it could be concluded that some of the phenolic compounds present in the digested samples originated from the food matrix. the amount of tpr was higher in the apple samples compared to the pear samples. the tpr content significantly decreased after digestion and it could not be quantified in the digested samples or in the control of the digested samples. similarly, fernandez and labra22 investigated the effect of in vitro simulated digestion on the stability and content of proanthocyanidins from grape skin and seeds. they showed that up to 80 % of the total proanthocyanidins were significantly degraded during the pancreatic digestion. in addition, the results of the present study compared well with those obtained in previous research with chokeberry juice,12 where the amount of tpr immediately decreased 10-fold after addition of a food matrix and a digestive enzyme cocktail. in the matrix and matrix control samples, proanthocyanidins were not detected, which is in accordance with the declaration for the used infant formula. chemical characterization expansion of knowledge on the bioavailability of plants metabolites is essential for an understanding of their health effect. the impact of a food matrix on the bioavailability of polyphenols has not been examined in detail. interact________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ digestion effect on fruit phenolics 1321 ions between food components and polyphenols (such as binding to proteins and polysaccharides) can exist and these interactions may modify their absorption.23 the quantification of phenolic compounds in apple and pear decoctions and their digested fractions was performed using an hplc-dad method, and results are presented in table ii. the decoction profiles encompassed hydroxycinnamic acid, flavonols and dihydrochalcones. the addition of the food matrix affected the content of individual phenolics in a similar way as was the case with tpc and tpr. after simulated in vitro digestion, the concentration of phenolic compounds was slightly (but not significantly) increased compared to the control samples, although their contents were still much lower than in the apple and pear decoctions. previous results24 indicated that during the digestion process, the amount of phenolic compounds may be metabolized into structurally different forms with different chemical and biological properties. in the present study, after examination of the hplc-dad chromatogram for the digested samples at 290, 350 and 520 nm, no new peak that might suggest the formation of new derivatives was detected. table ii. content of individual phenolics (in mg g-1) in the samples and food matrix subjected to in vitro digestion; nd – not detected, tr – in traces. means with different letters are significantly different (p < 0.05); lod – limit of detection, loq – limit of quantification sample chlorogenic acid phlorizine dihydrate hyperoside quercitrin isoquercitrin apple decoction 133.97±4.31a 43.65±1.09a 1.57±0.04a 13.82±0.42a 1.71±0.06a pear decoction 42.23±1.12b nd tr 4.97±0.11b 0.83±0.01b apple digested sample 12.39±0.33c 2.91±0.16b tr 3.52±0.09c tr pear digested sample 6.07±0.18d nd nd tr nd apple digested sample control 13.00±0.41b 2.06±0.10b tr 3.39±0.08c tr pear digested sample control 6.23±0.16d nd tr tr nd matrix digestion nd nd nd nd nd matrix digestion control nd nd nd nd nd lod 0.02 0.10 0.03 0.05 0.05 loq 0.10 0.50 0.20 0.30 0.40 according to previous results,25 chlorogenic acid is one of the dominant polyphenol compounds in apple fruit with an average concentration 9.02 mg in 100 g of fresh apple. in addition, it is one of the predominant compounds in pear fruit.26 during the drying process, the content of chlorogenic acid was significantly reduced compared to that in fresh fruit.27 in the present study, chlorogenic acid was also the main phenolic compound in amounts of 133.97 and 42.23 µg ml–1 for apple and pear decoctions, respectively. after gastric and pancreatic digestion, the ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1322 živković et al. content of chlorogenic acid was 90.75 and 85.63 % lower in the case of apple and pear decoctions, respectively. the obtained results are in accordance with previous literature data. according to boaventura et al.,20 the content of chlorogenic acid in yerba mate tea was decreased by 67 % after in vitro digestion, while in the case of sea fennel, it was approximately 82 % lower after gastric digestion.28 dupas et al.29 showed that chlorogenic acid in its native form is poorly absorbed in the body when ingested in a realistic food matrix. previous reports showed isomerisation of chlorogenic acid, an ester of caffeic acid with quinic acid, and even its hydrolysis into its free acid in both simulated and human duodenal juices.30 among the phenolic compounds in the apple decoction, phlorizine dihydrate was the most affected by the addition of a food matrix and digestion. on the other hand, in previous studies,30,31 the digestion process did not induce any significant change in the content of phloretin derivatives. the obtained differences might be explained by the different experimental conditions used in these studies. common to all previous assays is the complete gastric digestion in the absence of a food matrix and, therefore, they do not allow appropriate comparison with the results obtained in the present study. bearing in mind quercetin and its derivatives, previous results showed that aglycones and glucosides can be absorbed in the small intestine, while phenolics with a rhamnose, arabinose or xylose moiety must reach the colon and be hydrolyzed before absorption.23 the content of quercetin derivatives in investigated apple and pear decoctions ranged from 0.83 to 13.82 μg ml–1, while they were detected only in traces in the digested samples and controls. hollman and katan32 reported that the absorption of quercetin is more efficient after ingestion of onions rich in quercetin glucosides than after ingestion of apples (only 30 %) containing both glucosides and other glycosides. antiproliferative activity increased consumption of fruit and vegetables is recommended as a key component in a healthy diet for the prevention of certain types of cancer.34 owing to the high availability of apples and pears and their consumption among european nations, they could be considered as an important source of chemopreventive compounds. it was stated that regular consumption of one or more apples per day may reduce the risk for lung and colon cancer.34 previously, cetkovic et al.35 showed strong cytotoxic effect of apple pomace extracts on hela cell line with ic50 values below 10 mg ml–1. moreover, apple peel extract obtained from organic gala apples exhibited a significant decrease in the growth and clonogenic survival of human prostate carcinoma (cwr22rnu1 and du145) and breast carcinoma (mcf-7 and mcf-7:her18) cells.8 in a study conducted by sun et al.,9 an apple extract showed relatively potent antiproliferative activity on hepg2 human liver cancer cells with median effective dose (ec50) of 49.37 mg/ml, while a ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ digestion effect on fruit phenolics 1323 pear extract showed no antiproliferative activity under the employed experimental conditions. recent studies indicated that polyphenols are the main phytochemicals with antiproliferative property in higher plants.36 however, the intake of apple and pear fruits and their products does not automatically mean increased phenolics concentration in plasma. to exert pharmacological activity, they should be bioavailable, and their bioavailability is determined on the release from the food matrix during the digestion process and subsequent intestinal absorption.37 in this study, wild apple and pear decoctions were evaluated before and after in vitro digestion for their antiproliferative activity on human epithelial carcinoma cell line (hela), human colon cancer (ls174) cells and human foetal lung fibroblast (mrc-5) cells. the inhibition of cells proliferation and the cytotoxic effects of the investigated samples are presented in table iii. the results indicated that the exhibited growth inhibition was not correlated with the content of phenolic compounds in the tested samples, indicating that it could not be explained solely by their content. table iii. concentrations of samples that induced a 50 % decrease in hela and ls174, malignant cells, and mrc-5 normal cell survival (expressed as ic50 / µg ml-1); ic50 values are expressed as the mean±sd determined from the results of the mtt assay in three independent experiments. means with different letters are significantly different (p < 0.05) compound cell line hela ls174 mrc5 apple decoction 26.91±0.22a 41.01±0.55a >200 pear decoction 37.94±1.04b 69.94±1.82b >200 apple digested sample 56.21±2.02c 25.71±0.12c >200 pear digested sample 26.19±0.07a 25.97±0.32c >200 apple digested sample control 57.54±1.26c 27.25±0.21c >200 pear digested sample control 28.16±0.99a 25.33±0.31c >200 matrix >200 >200 >200 matrix digestion control >200 >200 >200 cisplatin 0.72±0.13d 2.64±0.26d 14.87±0.11 generally, with the exception of the effect of the apple decoction on hela cells, digestion process resulted in significant increases in antiproliferative activity. namely, inhibition of the proliferation of hela cells in the presence of apple decoction after in vitro digestion decreased approximately 2-fold in comparison to the non-digested sample, with the ic50 value decreasing from 56.21 to 26.91 μg ml–1. the decline in the content of bioactive compounds was probably the cause of the decrease in the inhibition of the proliferation of the tested cancer cell lines. in the cases of the effect of the apple decoction on ls174 cells, and of the pear decoction on hela and ls174 cells, the situation was different. lower ic50 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1324 živković et al. values were recorded with the digested samples in comparison to the non-digested ones. the decrease in total phenolic content did not result in a lowering of the exhibited antiproliferative potentials. there are numerous reports on increasing bioactivity of plant extracts after simulated in vitro digestion. the authors mainly reported increasing free radical scavenging capacites.38 chen et al.39 showed that wild raspberry extract produced after digestion had a pronounced protective effect against acrylamide induced cytotoxicity compared with that produced before digestion. it could be speculated that during digestion, a high proportion of apple and pear decoctions are transformed into other unknown and/or undetected compounds with different chemical and pharmacological properties. furthermore, numerous data suggest that phenolic compounds exert a pro-oxidative activity at higher concentrations. for example, for caffeic acid there are results that it can be carcinogenic at 2 %, tumour promoter at 0.5 to 1 % and anticancerogenic at 0.05 to 0.5 %.40 in both cases, the difference in the activity of the digested samples and control digestion samples was small, indicating that phenolic compounds entrapped in the food matrix showed small contributions to the exerted activity. since the food matrix is also a source of phenolic compounds, its effect on the proliferation of hela, ls174 and mrc5 cell lines was also investigated. no effect was observed for the food matrix or for its digested sample. the investigated samples showed no cytotoxic effects on normal mrc-5 cell line at concentration lower than 200 μg ml–1. conclusions in vitro simulated digestion in the presence of a food matrix induced significant decrease in the content of phenolic compounds in apple and pear decoctions. on the other hand, the antiproliferative effect was not correlated with the content of phenolic compounds in the tested samples, indicating that it could not be explained solely by their content. however, further research is needed to investigate the bioavailability and biological effects of these bioactive compounds in humans. supplementary material the additional data are available electronically at the pages of journal website: http:// //www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the authors are grateful to the serbian ministry of education, science and technology of the republic of serbia for financial support (grant nos. 46013, 175011 and 173005). ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ digestion effect on fruit phenolics 1325 и з в о д хемијски састав и антипролиферативно деловање чаја од дивље јабуке и крушке пре и након симулиране дигестије in vitro јелена живковић1, катарина шавикин1, немања станисављевић2, гордана здунић1, татјана станојковић3 и јелена самарџић2 1 институт за проучавање лековитог биља „др јосиф панчић“, тадеуша кошћушка 1, 11000, београд, 2 институт за молекуларну генетику и генетски инжињеринг, универзитет у београду, војводе степе 444а, 11000, београд и 3 институт за онкологију и радиологију србије, пастерова 14, 11000, београд у овом раду анализиране су измене у садржају полифенола и антипролиферативној активности декокта добијених од сувих плодова јабуке и крушке. ове промене испитиване су након in vitro дигестије у присуству компоненти матрикса хране. садржај укупних фенола и проантоцијанидина одређен је применом спектрофотометријских метода, док је састав појединачних фенола анализиран применом rp-hplc/dad технике пре и након дигестије. хлорогенска киселина и флоризин дихидрат била су доминантна идентификована једињења. додатак матрикса хране аутоматски је довео до смањења садржаја укупних и појединачних полифенола. након дигестије њихов садржај је благо порастао, али је и даље био нижи у односу на садржај у почетним узорцима. антипролиферативна активност испитиваних узорака анализирана је на две туморске ћелијске линије (hela и ls174). показана инхибиција раста туморских ћелија није била у корелацији са садржајем фенолних једињења у испитиваним узорцима, указујући на то да се она не може објаснити само њиховим садржајем. са изузетком ефекта декокта плода јабуке на раст hela ћелија, након дигестије узорака дошло је до значајног повећања њихове антипролиферативне активности. 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40. l. silva, f. shahidi, m. a. coimbra, in dried fruits. phytochemicals and health effects, c. alasalvar, f. shahidi, eds., wiley, oxford, 2013, pp. 325–356. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {in memoriam: professor bojana gruji{\'{c}}-injac} j. serb. chem. soc. 82 (7–8) 955–956 (2017) udc 929 bojana grujić-injac”1925/2017” jscs–5015 letters to the editor 955 in memoriam professor bojana grujić-injac (1925–2017) professor (retired) bojana grujić-injac, who elevated both the research and teaching of chemistry of natural products and biochemistry at the faculty of chemistry, university of belgrade, died on march 18, at the age of 92 after a short illness. bojana grujić was born in bašaid (vojvodina, serbia) on january 8, 1925. she spent her school years in kikinda and in vršac, after which she studied chemistry at the department of chemistry, faculty of natural sciences and mathematics, university of belgrade, where she obtained her diploma in 1950, followed by a doctorate in 1955 with professor djordje stefanović, who introduced her into the field of natural products chemistry. after graduation, bojana grujić was appointed by the ministry of education as an assistant for chemistry at the faculty of veterinarian medicine in belgrade, but after three years she returned to the department of chemistry and remained there (with short temporary stay at department of chemistry in priština, 1964– –1967) until her retirement in 1983. she was appointed assistant professor in 1956, associate professor in 1967, and full professor in 1971. the research interests of professor bojana grujić-injac in natural products chemistry comprised study of structure and biological activity of phospholipids, pheromones, terpenoids, alkaloids and plant pigments. by mid-1960 she also entered into the field of proteins, working on the isolation and basic characterization of a number of plant proteins and particularly on amino acid sequence determination of some haemoglobins. that work was done in close cooperation with the laboratory of professor gerhard braunitzer at the max-planck institute for biochemistry in munich of which she became (1972) an external co-worker. the research on proteins in general and on haemoglobin in particular that she initiated nearly 50 years ago did not cease; on the contrary, it extended and is progressing constantly in the department. professor bojana grujić-injac mentored 30 doctorates, co-authored 70 papers and delivered several plenary lectures at scientific meetings. (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 956 letters to the editor professor bojana grujić-injac made also great contributions to the teaching at the department. she taught chemistry of natural products and introduced (1969) and taught the undergraduate course of general biochemistry. she co -authored textbooks of the chemistry of natural products and wrote textbooks of biochemistry. as a head of the chair (“katedra”) for chemistry of natural products (later biochemistry) which she held from 1974 until 1983, she supported younger colleagues to develop a number of specialized biochemical courses. when opportunity arose in 1981 professor bojana grujić-injac initiated and took an active role in the formation of a distinct biochemistry group at the department. finally, it should be emphasized that research and teaching of biochemical disciplines within a chemical curriculum at our department was much ahead of the time, since this was not a common practice at chemistry departments worldwide until many years later. in parallel with her scientific and pedagogical work at the department, professor bojana grujić-injac was engaged in numerous administrative services to the department, university, and ministry of education. thanks to her, the department obtained earmarked equipment needed for modern biochemical research. through all her professional years professor bojana grujić-injac was engaged in activities of the serbian chemical society, particularly in its biochemistry section. she was elected for the merit member of the society (1982). she received a number of awards for her work, including the highest state medals. finally, it is to be noted with great regret and sadness that professor bojana grujić-injac was the last of our prominent professors coming from the first post -second world war generation of chemistry students, which greatly contributed to the development and expansion of this department. vesna niketić vniketic@chem.bg.ac.rs (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true 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<feff04120438043a043e0440043804410442043e043204430439044204350020044604560020043f043004400430043c043504420440043800200434043b044f0020044104420432043e04400435043d043d044f00200434043e043a0443043c0435043d044204560432002000410064006f006200650020005000440046002c0020044f043a04560020043d04300439043a04400430044904350020043f045604340445043e0434044f0442044c00200434043b044f0020043204380441043e043a043e044f043a04560441043d043e0433043e0020043f0435044004350434043404400443043a043e0432043e0433043e0020043404400443043a0443002e00200020042104420432043e04400435043d045600200434043e043a0443043c0435043d0442043800200050004400460020043c043e0436043d04300020043204560434043a0440043804420438002004430020004100630072006f006200610074002004420430002000410064006f00620065002000520065006100640065007200200035002e0030002004300431043e0020043f04560437043d04560448043e04570020043204350440044104560457002e> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice preparation and characterization of a new polyaniline salt with good conductivity and great solubility in dimethyl sulphoxide j. serb. chem. soc. 80 (11) 1435–1448 (2015) udc 678.652+541.48+547.462.3+ jscs–4809 547.212’544.2:544.351.3:66.094.522 original scientific paper 1435 preparation and characterization of a new polyaniline salt with good conductivity and great solubility in dimethyl sulphoxide hichem zeghioud1*, saad lamouri1, yasmine mahmoud1 and tarik hadj-ali2 1laboratoire de chimie macromoléculaire, ecole militaire polytechnique, bp 17, bordj el bahri, alger, algeria and 2laboratoire des systèmes électroniques et optroniques, ecole militaire polytechnique, bp 17, bordj el bahri, alger, algeria (received 5 march, revised 2 june, accepted 18 june 2015) abstract: in this study, a novel conducting and soluble polyaniline salt prepared by chemical polymerization in the presence of a new doping agent (ias) is proposed. this doping agent was prepared by the sulphonation of itaconic acid (ia) with concentrated sulphuric acid in tetrahydrofuran (thf). the obtained doped polyaniline (pani–ias) was extremely soluble in dimethyl sulphoxide (dmso) at room temperature, in which the solubility reached 44 mg ml-1. the conductivity measurement of doped polyaniline powder precipitated in thf as dispersing medium gave a value of 0.13 s cm-1 when the emeraldine base form of polyaniline was fully protonated. the polyaniline salt sample was characterized by thermogravimetry (tg), differential scanning calorimetry (dsc), x-ray diffraction and uv–vis and ftir spectroscopy. keywords: conducting polymer; pani; itaconic acid; sulphonation; solubility. introduction intrinsically conducting polymers (icps) have attracted important attention in many advanced applications. among icps, polyaniline (pani) is one of the essential conductive polymer for its wide use in many domains, such as organic lightweight batteries,1 electro-chromic display devices,2,3 photo-electronic devices,4 chemical sensors5,6 and hydrogen storage.7 polyaniline has been considered as one of the most promising electrode materials due to its simple synthesis, environmental stability, unique electronic properties, and simple acid– –base doping/dedoping chemistry.8,9 on the other hand, the intractable solubility of pani in common organic solvents has limited its practical applications in many fields. it is therefore a very important challenge to design doped polyaniline and its derivatives in forms that are easily processable and applicable. in * corresponding author. e-mail: hicheming@yahoo.fr doi: 10.2298/jsc150305064z 1436 zeghioud et al. recent years, a number of studies were focused on the improvement of pani solubility, a variety of methods were proposed that consisted in doping pani emeraldine base (pani–eb) by extended organic alkyls of sulphonic acids (dbsa and csa)10–12 or with polymeric acids.13–16 in 2009, peng studied an emulsion polymerization process that had been developed for the direct synthesis of organo-soluble polyaniline (pani) with oleic acid (oa) as a surfactant and dopant.17 gribkova et al, reported the chemical synthesis of polyaniline in the presence of polymeric acids (such as poly(amidosulphonic acids)), which contain sulphonic groups in the chemical structure. they found that the matrix polymerization of aniline resulted in the formation of water-soluble interpolymer complexes of pani with polyacid.18 in 2015, zeghioud et al prepared soluble polyaniline salts doped with poly (itaconic acid); they found that the solubility reaches 4 mg ml–1 in an optimized mixture of thf/water at room temperature.19 wang et al. prepared polyaniline nanorods by chemical oxidation polymerization using itaconic acid as dopant. the obtained polyaniline salt, compared to the undoped form, possesses high productivity, conductivity and excellent solubility in organic solvents (n-methylpyrrolidone, nmp, tetrahydrofuran, thf, and dimethyl formamide, dmf), which reached 19 mg ml–1.20 in this work, a new and low-cost sulphonic dopant was prepared from itaconic acid (a platform chemical produced from renewable resources) by a simple chemical reaction. therefore, the development of a new polyaniline salt with simultaneously very good solubility (in common solvents) and good electrical conductivity is a challenge and it was the aim of the present study. the structure, spectroscopic properties, thermal stability and conductivity of the obtained polyaniline were also investigated. experimental materials aniline (ani, 99b% pure) was purchased from fluka. the other employed materials were hydrochloric acid (35.37 %) from organics stinnes chemicals, methanol (99.5 %) and ammonium peroxodisulphate (aps, 99 %) from prolabo, acetone (99 %) from acros organics. while ammonia (25 % solution), n-methylpyrrolidone (nmp, 99.5 %) and tetrahydrofuran (99 %) were from merck. itaconic acid (ia, 99 %) was from aldrich, dimethyl sulphoxide (dmso, 100 %) and dimethyl formamide (dmf, 100 %) from sigma-aldrich. all these products were used without further purification. synthesis of pani polyaniline powder in the emeraldine salt form was synthesized as follows: 4.8 g of aniline (4.75 ml) and 15 ml of hcl were dispersed in 50 ml of deionised water under vigorous stirring at room temperature for 2 h, to obtain a uniform solution. an aqueous solution of aps (11.8 g of aps + 50 ml of deionised water) was slowly added to the above mixture with mole ratio equal to one (aps/ani = 1). the resulting solution was stirred for 30 min to ensure complete mixing. then, the reaction was followed by continuous stirring at 2 °c polyaniline salt with good conductivity 1437 for about 4 h. finally, the formed precipitate was filtered, washed successively with distilled water followed by methanol (to remove oligomers and other by-products) until the filtrate was colourless, and then dried at 60 °c for 48 h. the polyaniline salt (pani–hcl) was converted to base form (pani–eb) by treatment with aqueous nh4oh solution for 72 h, followed by washing with distilled water and methanol; the base form of pani was obtained as a blue powder. the polymerization yield was 80 %. synthesis of the dopant itaconic acid sulphonated (ias) was prepared by chemical reaction between itaconic acid (ia) and a concentrated sulfuric acid as follows: 3 g of ia was dissolved in 10 ml of thf under continuous stirring in round-bottom flask (triple-neck type); the system was placed in a thermostatic bath at 40 °c. a small quantity of sulfuric acid was added drop by drop to the above solution with the help of dropping funnel to a mole ratio equal to one. the solution was left under constant stirring for 3 days. subsequently, the product (ias) was separated by drying for 5 days at 70 °c. the possible reaction mechanism is presented in scheme 1. scheme 1. plausible mechanism of the sulphonation reaction. doping for the preparation of sulphonated acid doped polyaniline, undoped pani solution in thf (or another organic solvent) was mixed with ias in mole ratio equal to two between ias and tetrameric repeat unit of pani–eb. the mixture was transferred to an ultrasound bath for 2 h, followed by constant agitation for 6 h. a dark green colour was obtained. the pani–ias dispersion (in thf or nmp) was filtered using polytetrafluorothylene (ptfe) filters, washed and dried at 60 °c for 24 h. the reaction mechanism is shown in scheme 2. the obtained apparent viscometric average molar mass of the sulphonated itaconic acid-doped polyaniline (pani–ias) was m̅v = 4430 g mol-1. characterization fourier transform infrared (ftir) spectra were recorded between 400 and 4000 cm-1 from kbr pellets on an infrared fourier transform spectrometer (shimadzu type 8400 s). uv– vis spectra of all samples dissolved in different solvents were recorded using a shimadzu uv-2401pc uv–vis spectrometer with a resolution of 2 nm, in the wavelength range of 300– –900 nm. x-ray powder diffraction patterns were recorded on a panalytical x'pert pro diffractometer fitted with cukα radiation (λ = 1.5404 nm) at 40 kv and 40 ma in the 4–60° 2θ region. thermogravimetry (tg) was performed using setaram an mtb thermogravimetric instrument at a sensitivity of “10-8”, operating at a heating rate of 10 °c min-1, from room 1438 zeghioud et al. temperature up to 450 °c under an air atmosphere. the sample mass ranged between 3 and 6 mg. the differential scanning calorimetry (dsc) measurements were realised on a netzsch dsc 204 f1 at a scanning rate of 10 °c min-1 from room temperature up to 400 °c under an inert atmosphere. scheme 2. mechanism of pani doping with ias. results and discussion conductivity measurements the conductivity measurements on compressed pellets of various forms of pani powder prepared by chemical polymerization were made by the conventional four-point probe technique at room temperature. for ias-doped polyaniline, the powder was obtained by filtration and drying of the precipitate formed in the dispersion of thf and nmp. the band gap in ev was calculated by the equation:21 g 1237.5 e λ = (1) where λ represents the maximum wavelength (nm) in the band attributed to π–π* (band 1) transitions. the measured values of conductivity are given in table i; pani–hcl has better conductivity (4.6 s cm–1) than pani–ias. this was confirmed by the smaller band gap of pani–hcl, around 3.2 ev, compared with that of pani–ias (3.62 ev). however, the conductivity value of the latter depended polyaniline salt with good conductivity 1439 strongly on the dispersion medium during synthesis, whereby the highest value (0.13 s cm–1) was obtained in thf. table i. conductivities and energy band gap of different forms of pani sample (dispersion medium) uv–vis band, nm conductivity s cm-1 band gap energy ev 1 2 pani–hcl 387 467 4.60 3.20 pani–eb 331 626 8.14×10–10 – pani–ias (thf) – – 0.13 3.62 pani–ias (nmp) – – 0.01 pani–ias (dmso) 342 417 – uv–vis spectroscopy the uv–vis spectrum of undoped polyaniline included two absorption bands at 331 and 626 nm due to the excitation of the benzoid and quinoid segments in the polyaniline chain, respectively,22 (fig. 1, curve (a)). fig. 1. uv–vis spectra of: a) pani–eb and b) pani–ias recorded in dmso solvent. curve (b) in fig. 1 illustrates the uv–vis spectrum after ias had been added to the dmso containing pani–eb. thus, the absorption band at 626 nm disappeared. however, the characteristic polaron band would appear at 807 nm. the characteristic absorption bands in the spectrum of pani–ias are clearly observed at about 807 and 417 nm, which are attributed to the transitions from the π band to the polaron band and from the polaron band to the π* band of the alternating benzoid–quinoid structures, respectively.23 the band around 342 nm is referred to the transition from the π band to the π* band centred on the benzoid rings associated with the extended π orbitals on polymer backbone (band gap excitation).24 1440 zeghioud et al. fourier transform infrared spectroscopy (ftir) the ftir spectra of ia and ias samples are shown in fig. 2. the spectrum of ia and ias presented a broad band centred at 2926 cm–1, which is attributed to o–h stretching vibrations. the spectrum of ia (curve a) showed peaks around 1718, 1430, and 1190 cm–1, indicating the stretching c=o (carboxylic acid), c–o–h in the plane and c–o, respectively.25,26 fig. 2. ftir spectra of: a) ia and b) ias. the spectrum of ia obtained after sulphonation (curve b) showed the characteristic absorption band for the acid carbonylic groups at 1703 cm–1.27 the peaks around 1400 and 1170 cm–1 were assigned to the stretching vibrations of c–o–h of carboxylic acid and asymmetrical stretching of so2, respectively. new peaks appeared at 1070 and 855 cm–1, which are attributed to the stretching vibration of symmetrical so2 and the bending of o–s, respectively.27–29 the non-disappearance of the c=c peak at 1629 cm–1 confirmed the sulphonation of ia by sulphuric acid according to the mechanism proposed in the scheme 1. figure 3 shows the ftir spectra of pani–eb and pani–ias. in the spectrum of pani–eb (curve a), the characteristic absorption peaks at 158330 and 149431 cm–1 could be assigned to the c=c stretching mode for the quinoid rings and the c=c stretching mode for the benzoid rings, respectively. the absorption peak at 1303 cm–1 corresponds to the c–n stretching mode. the in-plane bending of c–h is reflected in the 1160 cm–1 peak, and the out-of-plane bending vibration of c–h occurred at 508 cm–1.31–33 in the spectrum of pani–ias (curve c), the characteristic peaks at 156227 and 147028 cm–1 could be assigned to the c=c stretching mode for the quinoid rings and the c=c stretching mode for the benzoid rings, respectively. two absorption peaks at 1303 and 1240 cm–1 corres polyaniline salt with good conductivity 1441 pond to the c–n stretching33 and c–n stretching of protonated mode,30 respectively. the in-plane bending of c–h was reflected in the 1121 cm–1 peak, and the out-of-plane bending vibration of c–h occurred at 508 cm–1.27 the shift of the characteristic peaks of pani indicated strong interaction between pani–eb and the dopant. fig. 3. ftir spectra of: a) pani–eb, b) ias and c) pani–ias. solubility determination the pani–ias was synthesized in different polar solvents, such as nmp, dmso, thf and dmf (fig. 4). the obtained dispersions (in thf and nmp) were filtered using 0.45-µm ptfe filters, washed and dried to recover the pani– –ias powder. fig. 4. pani–ias dispersions in different solvents. figure 4 shows the very good solubility in nmp and dmso just after doping. after 2 h, strong interactions between the polymers chains were clearly detected in all dispersion media except for the pani–ias/dmso solution. after filtration and uv–vis analysis of the resulting solutions, dmso was found to be 1442 zeghioud et al. the only solvent capable of dissolving the pani–ias chains at room temperature. the maximum solubility was determined by calculation of the difference between the densities of pani–ias/dmso solution and pure dmso; it was found that the solubility could reach 44 mg ml–1. from this, it was concluded that dmso is a very good solvent for pani–ias, which is confirmed by fig. 5. fig. 5. digital photos of solutions of: a) concentrated and b) dilute solution of pani–ias in dmso solvent. polyaniline is soluble only in dmso because the interactions between the pani chains and dmso are more important than the interactions between the polyaniline chains that increases the distance between the molecular chains and gives a relatively extended conformation to these chains. thus, the solubility of polyaniline is primarily controlled by the molecular structures of the polymer chain (including the doping agent) and the solvent properties (polarity, number of carboxylic groups and molecular size). x-ray diffraction pattern (xrd) the x-ray diffraction pattern of the pani–eb and pani–ias powder are presented in fig. 6. the crystaline region presented by the peak and the amorphous region presented by the remainder of diffractogramme were both integ fig. 6. xrd patterns of pani–eb and pani–ias. polyaniline salt with good conductivity 1443 rated in 2θ space. from these integrated peaks areas (ic, ia), the ratio xc/xa can be calculated from xc/xa = 1.8(ic/ia). a ryland factor of 1.8 is commonly used for semi-crystalline polymers.34 the percentage crystallinity xc (%) was obtained from eq. (2):34 ( ) ( )c c a 100 % 100 1 / x x x = − + (2) the xrd profile of pani–eb showed a number of narrow sharp reflection peaks at 2θ 9.54, 15.22, 20.75 and 24.29°, representing the (001), (011), (100) and (110) crystal planes, respectively (table ii). the peak centred at 2θ ∼20° may be ascribed to periodicity parallel to the polymer chain, while the peak at 2θ ∼25° may be caused by periodicity perpendicular to the polymer chain.35 table ii. the 2θ values, intensity and indexation (hkl) of pani–eb and pani–ias sample 2θ / ° d / å hkl ref. xc / % pani–eb 09.57 09.23 001 36 9.64 15.22 05.82 011 27 20.75 04.28 100 37 24.29 03.66 110 38 pani–ias 08.63 10.23 001 37 8.70 14.20 06.23 011 39 19.73 04.49 100 36 25.19 03.54 110 38 after conversion of pani–eb into pani–ias, broad diffuse peaks centred at 2θ 8.63 (001) and 14.2° (011) appeared, and two small peaks at 2θ 19.73 (100) and 25.19° (110) were observed. compared to pani–eb, the intensities of the two last peaks (2θ 19.73 and 25.19°) were strongly reduced.40 this was proved by the decrease in the degree of crystallinity (xc) from 9.64 % for pani–eb to 8.70 % for pani–ias. thermogravimetry (tg) the thermal stability of the pani–ias was evaluated using tg. polyaniline salt powder (pani–ias) showed poor thermal stability. as seen in fig. 7, the degradation occurred in three stages; the first mass loss was assigned to the evaporation of water and residual solvents,41,42 in the second temperature region (200–270 °c), the degradation was due to the start of loss of the sulphonic and carboxylic groups of itaconic acid.43 this was followed by a broad loss of mass starting from 270 °c. this mass loss was probably due to the dehydration and the decomposition of the chemical structure of the short pani chains.44 it is important to notice that more than 50% of the initial mass was conserved for temperatures up to 500 °c. this residual mass was related to the existence of reticule polymer, which is formed at high temperatures.45 1444 zeghioud et al. dsc thermal analysis the dsc curve for pani–ias (fig. 8) showed two endothermic peaks around 35–184 °c and 200–350 °c. the first endothermic transition is related to the excess water existing in the pani lattice that influences the conductivity and crystal structure of pani.46 the second thermal transition, which was centred at 290 °c, involved the beginning of pani–ias degradation (decarboxylation) and the detachment of doping agent. in fig. 9, there was an absence of dopant carboxylic groups after dsc analysis, which confirmed that complete decarboxylation had occurred. fig. 7. thermogravimetric analysis of pani–ias. fig. 8. dsc thermal analysis curves of the pani–ias powder under a nitrogen atmosphere: a) first run and b) second run (heating rate: 10 °c min-1). the curve b shown in fig. 8 represents the second scan dsc thermogram of the same pani–ias sample. there are no significant endothermic or exothermic polyaniline salt with good conductivity 1445 peaks on this curve. this is because almost all the moisture had evaporated from the pani–ias sample during the first thermal treatment run. moreover, a crosslinking reaction may have occurred during the first thermal analysis run and may have resulted in a 3d chemical structure. this crosslinking reaction, illustrated in scheme 3,47 was the result of the coupling of two neighbouring –n=q=n– groups. fig. 9. ftir spectra of pani–ias: a) before and b) after dsc analysis. scheme 3. thermal crosslinking reaction between the pani molecular chains. 1446 zeghioud et al. conclusions the sulphonation of itaconic acid was successfully performed in a simple chemical reaction. the doping of pani–eb by the synthesised dopant (ias) was effectively realised. the results of this study provide further that the use of ias as a novel sulphonic dopant for polyaniline delivers a significant improvement in the solubility of the polyaniline salt compared to those reported previously. the maximum solubility reached 44 mg ml–1 in pure dmso as polar organic solvent at room temperature. it was shown that a conducting form of pani that is thermally stable up to 200 °c could be prepared. the characterization of the pani–ias confirmed the success in obtaining good conductivity that reaches 0.13 s cm–1 for fully doped pani–eb. a low degree of crystallinity was found for the conducting form of pani compared to the non-doped one. и з в о д синтеза и карактеризација нове проводне и у диметилсулфоксиду веома растворне соли полианилина hichem zeghioud1, saad lamouri1, yasmine mahmoud1 и tarik hadj-ali2 1laboratoire de chimie macromoléculaire, ecole militaire polytechnique, bp 17, bordj el bahri, alger, algeria и 2laboratoire des systèmes électroniques et optroniques, ecole militaire polytechnique, bp 17, bordj el bahri, alger, algeria у раду је приказана синтеза нове растворне и проводне соли полианилина оксидативном полимеризацијом у присуству новог агенса за допирање. агенс за допирање (ias) је синтетисан сулфоновањем итаконске киселине (ia) концентрованом сумпорном киселином у тетрахидрофурану (thf). со допираног полианилина (pani–ias) је веома растворна у диметилсулфоксиду (dmso) на собној температури, при чему се достиже растворљивост од 44 mg ml-1. мерења проводљивости допираног полианилина, добијеног таложењем у thf, показала су да се максимална проводљивост од 0,13 s cm-1 достиже у при потпуном протоновању полианилина у облику емералдинске базе. поред тога, узорци соли полианилина су карактерисани термогравиметријском анализом (tga), диференцијалном сканирајућом спектроскопијом (dsc), дифракцијом x-зрака (xrd), као и uv–vis и ftir спектроскопијом. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {low-temperature-synthesized ruo2 from acidic chloride solution for the electrode coating applications} j. serb. chem. soc. 82 (6) 695–709 (2017) udc 546.96–31+542.913:544.35+546.135– jscs–4997 32+544.6.076.32 original scientific paper 695 low-temperature-synthesized ruo2 from acidic chloride solution for the electrode coating applications gavrilo šekularac1•#, sanja eraković1#, dušan mijin2#, vesna pavelkić1,3, jasmina stevanović1 and vladimir panić1*# 1institute of chemistry, technology and metallurgy, department of electrochemistry, university of belgrade, belgrade, serbia, 2faculty of technology and metallurgy, university of belgrade, belgrade, serbia and 3the railway college of vocational studies, zdravka čelara 14, belgrade, serbia (received 29 december 2016, revised 20 march, accepted 22 march 2017) abstract: for the preparation of ruo2 coatings on ti substrate, the ruo2 was synthesized in acidic aqueous medium by simple one-step low temperature-controlled microwave (mw) irradiation. the physical composition of synthesized solid phase was analysed through particle size distribution (psd), whereas the coating was investigated for its capacitive response and activity in oxygen evolution reaction (oer). the oxide phase was found highly polydisperse, with overlapped fractions within rather narrow particle size range and clear tendency toward agglomeration. the smallest particles and their best resolved fractions were synthesized at the temperature just above the boiling point of the reaction medium, and quite below the chloride-to-oxide conversion temperature. consequently, the highest oer activity was registered for ruo2/ti anodes prepared from this sample, with strong indication of different oxide structure, with respect to the electrodes prepared from samples synthesized at higher temperatures. however, the coatings from high temperature samples have considerably higher capacitance than those synthesized at lower temperatures. these findings can be rather correlated to the mw temperature-dependent oxide structure than to different morphology analysed through psd. keywords: electrocatalytic oxide materials; hydrothermal synthesis; microwave synthesis; electrochemical impedance spectroscopy; pseudocapacitance. introduction electrochemically active noble metal oxides found their application as coatings for industrial electrodes,1–5 due to the good electrocatalytic activity for chlor * corresponding author. e-mail: panic@ihtm.bg.ac.rs •present address: jožef stefan institute, department of physical and organic chemistry, jamova cesta 39, ljubljana, slovenia. # serbian chemical society member. doi: 10.2298/jsc161229040s 696 šekularac et al. ine evolution reaction (cer) and oxygen evolution reaction (oer),6–9 but also for many other electrochemical reactions.10–12 these oxides show also the excellent supercapacitive performances.15–19 the main electrocatalytic coating component is ruo2.6,20–27 typical procedure for the preparation of electrode oxide coatings on ti substrate is by the thermal decomposition of the chloride salts.6,27–30 in order to increase the oxide electrocatalytic activity, selectivity and operational stability, the variety of methods, based on the separation of the oxide synthesis and coating formation, have been applied. the aim was to establish the defined coating structure, which should be as much as possible independent from coating formation procedure itself.31–39 different types of hydrothermal oxide synthesis have been studied intensively, which promoted the sol–gel process due to improved coating stability in comparison to typical thermally prepared coatings.35 however, the hydrothermal procedures have appeared rather complex and long -lasting for the application in practice. the time required for the complete conversions of chlorides to oxides of defined structure is rather long, since the temperature/pressure conditions are far from those required for controlled conversion.35 microwave (mw)-assisted synthesis was proven to enhance many kinds of different processes, from organic synthesis40 to extraction procedures.41 it was shown39 that suitable conditions for the extremely short hydrothermal synthesis of ruo2 can be reached by highly controllable mw-assisted synthesis of the oxide from aqueous chloride solution. the corresponding coatings, prepared from the oxide synthesized at high pressures and mw temperatures above 200 °c, were found to be of high pseudocapacitance and activity for chlorine and oxygen evolution reaction. however, the applied mw conditions were rather extreme. the aim of this work is to investigate the physical properties of ruo2 synthesized at moderate mw conditions (low temperatures/pressure around boiling point of reaction medium) and consequently their influence on the electrochemical behaviour of corresponding ruo2/ti electrodes. the data were compared to those obtained in the investigation of mw-synthesized ruo2 at higher temperatures.39 experimental low temperature mw synthesis of ruo2 the precursor for the preparation of ruo2 coatings on ti, the ruo2 colloidal dispersion, was synthesized by the simple one-step temperature-controlled microwave (mw) irradiation, starting from aqueous rucl3 solution as described in previous work. 39 the reaction mixture (6 ml) was continuously mechanically stirred at 600 rpm inside a closed reactor (10 ml) and irradiated isothermally to 80, 120 and 150 °c in a mw oven (monowave 300, anton paar, ashland, va, usa). the initial heating speed was set to the most rapid possible and the temperature was maintained constant for 5 min. the reaction mixture was cooled to 60 °c in the reactor and afterwards to ambient temperature. the obtained dispersions are denoted as low-temperature-synthesized ruo2 as electrode coating 697 mwt80, mwt120 and mwt150. as a result of mw irradiation, initially brown precursor solution turned black with visible appearance of a solid phase. physical characterization the reaction medium was subjected to dynamic light scattering (dls) after the synthesis in order to analyse the solid phase presence. laser-based particle size analyzer zetasizer nano zs (malvern instruments, uk), operating at λ = 633 nm (produced by a he–ne laser at scattering angle 173°) at 25±0.1 °c was employed. all samples were diluted with di-water in volume ratio 1:100 and ultra-sonicated for 10 min before measurements. coating preparation the reaction mediums, after mw irradiation at different temperatures, were used for the formation of a ruo2 coating on ti. the sand-blasted titanium rods (commercially pure, krupp ag, essen, germany), 3 mm in diameter, were thoroughly etched in hot aqueous 18 % hcl solution for 20 min, rinsed by water, and dried at 100 °c. before the application of the reaction medium onto ti rods up to the height of 1 cm, the medium has been ultra-sonicated for 15 min. after deposition, the rod was dried at 120 °c for 30 min. these two steps were repeated and finally the coating was formed during the thermal treatment at 300 °c for 2 h. in the order to avoid possible detachment, the coating was deposited as rather thin layer in an amount of 0.11 mg cm-2. thus prepared ruo2/ti served as working electrodes in electrochemical testing. electrochemical measurements the electrochemical experiments were carried out with 0.5 cm2 working surface area in a three-electrode cell, with pt gauze electrode and saturated calomel electrode (sce; all potentials are expressed vs. sce) as counter and reference electrodes, respectively. cyclic voltammetry (cv) at 50 mv/s, quasi-steady-state polarization at 5 mv/s and electrochemical impedance spectroscopy (eis) measurements (at open circuit potential) in 1 m h2so4 were performed with a biologic sp-200 potentiostat/galvanostat (bio-logic sas, claix, france) at room temperature. polarization measurements were performed in the potential range 0.90– –1.32 v with preceding anode potentiostatic conditioning at 0.90 v for 10 min. eis data were recorded with ac potential amplitude of 10 mv (root mean square, rms) around open circuit potential in a multi-sine mode, within frequency range of 100 khz–5 mhz with 10 points per decade. the fitting of eis data was performed in zview® software (scribner associates inc., southern pine, nc, usa) with data-modulus type of data weighting in maximum 100 iterations. the fitting is assumed acceptable if the chi-squared and weighted sum of squares were of the orders of 10-5 and 10-3, respectively. with these fitting criteria, the relative error of the all equivalent circuit parameter values were below 10 % (the only exceptions was the value of r2 resistor for mwt150 sample, for which the fitting returned the error of 13 %). results and discussion the changes in parameters, temperature (tmw), the mw power (p) and heating rate ((dt/dτ)τ), of the mw reactor in the early stages of synthesis are shown in fig. 1. the data were analyzed during the time required to reach the desired isothermal condition, i.e., during the early stages of mw irradiation. the time of about 1 min was required to reach the temperature plateau under characteristic heating conditions, which depend on the pre-set isothermal requirements. the 698 šekularac et al. mw power was delivered continuously during ca. 6 s with no significant changes of temperature. when the power maxima, which considerably depend on required temperature (200, 400 and 650 w for 80, 120 and 150 °c, respectively), were reached after additional 5–6 s, the temperature started to increase with the different rate at the different pre-set temperatures. as in the case of power maxima, the peak heating rate can be reached faster if the required temperature is higher. in addition, the lower reaction temperature required finer power and heating rate tuning: there were two heating rate plateaus at 80 °c around 30 and 45 s, while the single one was seen at 120 °c around 30 s. no plateaus after heating rate peak could be observed at 150 °c. as a consequence, there was a minute overthrow of the pre-set temperature of 80 °c, as an indication of a hardly controllable heating conditions, at temperatures below boiling point of the reaction medium. it should be noted that the reactor pressure above standard conditions was registered only at the heating to 150 °c, which reached the steady-state value of 5.5 bar. fig. 1. the changes in mw reactor parameters: temperature (tmw), mw power (p) and heating rate ((dt/dτ)τ), in the early stages of synthesis. the considered changes in heating parameters which precede the conditions required to maintain the steady-state heating indicated that the synthesized solid phase should be of different physical properties, since the heating was expected to affect the particle nuclei formation and subsequent particle growth. this was low-temperature-synthesized ruo2 as electrode coating 699 checked by investigation of the particle size distribution (psd) by the dynamic light scattering (dls) as presented in following section. psd of synthesized ruo2 solid phase the psds by dls in reaction media for ruo2 synthesized by mw irradiation at 80, 120 and 150 °c are shown in fig. 2. the distribution was analysed during the ten consecutive runs of light irradiation in order to check the psd stability. all samples appeared highly polydisperse, with distinct appearance of the grains within the diameter fractions between 20 nm and 1 µm. the agglomerates of diameter above 3 µm were present in all samples, but their size distribution was becoming more pronounced upon increase in the pre-set temperature. the grains of the smallest diameter were obtained at 120 °c, grouped within well resolved fractions around 30 and 150 nm (fig. 2b). the separation of the fractions was considerably less pronounced at lower and higher temperature (fig. 2a and c). the initially registered peaks at higher mw temperatures (run 1, fig. 2b and c) were becoming better distinguished during the consecutive runs, which indicated the merging of the grains into larger ones. consequently, in steady state run 10 there were well resolved peaks around 50 and 300 nm for the sample synthesized at 120 °c (fig. 2b) and around 100 and 500 nm if the synthesis temperature is increased to 150 °c (fig. 2c). this effect was very weak, or even opposite at 80 °c (fig. 2a). the sample at 80 °c contained the µm-sized agglomerates, stable during the runs, of extremely narrow psd in comparison to other two samples. fig. 2. particle size distribution by intensity for ruo2 suspension synthetized at: a) 80; b) 120 and c) 150 °c in an mw reactor. the run relates to the number of successively repeated dls measurement. these considerations of dls data indicate that the pre-set temperature below the boiling point favours the particle growth formation and generates the grains of low tendency to join into agglomerates of different size. on the other hand, the intensification of the heating condition induces the nucleation and the subsequent generation of the grains, tending to form the defined grouped structures and the agglomerates of different sizes between 3 and 5 µm. this tendency of grains is reflected into the fraction of agglomerates themselves too, since they appeared 700 šekularac et al. joined between first and tenth run (fig. 2b and c) into distinct size similar to those present in sample synthesised at 80 °c (fig. 2a). further increase in temperature up to 220 °c did not affect the main grain fracture around 300 nm,39 but the fractions of smaller grains (below 100 nm) was found transferred to the regions of lower diameter. these findings are a strong confirmation of nuclei formation, favoured over particle growth. exclusively, the psd of the sample synthesized at 200 °c did not show any presence of the agglomerates.39 however, the particles appear to be of high surface energy, since they form stable grains and agglomerates. the electrochemical properties of the mw-synthesized ruo2/ti electrodes electrochemical properties of ruo2/ti electrodes prepared from mwt80, mwt120 and mwt150 samples were investigated by the cyclic voltammetry (cv), the polarization measurement and the electrochemical impedance spectroscopy (eis). the cv curves (as specific currents per mass of the coating) are shown in fig. 3. fig. 3. the cyclic voltammograms of ruo2/ti electrodes prepared from the oxide synthesized at the different temperatures. sweep rate: 50 mv/s; electrolyte: 1 m h2so4. cyclic voltammograms had usual shape for ruo2/ti electrodes obtained by the different synthesis procedures.35 in the most of cv potential region of cv anodic branch, the highest currents were registered for ruo2(mwt80)/ti and the lowest for ruo2(mwt120)/ti electrode, although the currents were quite similar for all samples. this indicated their almost equal capacitive ability. it should be noted that the order of currents was opposite right at the beginning of charging (–0.2 to 0.1 v). this part of the anodic charging branch follows the completion of the proton insertion into the hydrous structure of the oxide in the same potential region of the cathodic branch. it appeared that the oxide synthesized at low-temperature-synthesized ruo2 as electrode coating 701 higher temperatures was able to respond more efficiently to the redox transitions involving the lower oxidation states of ru.42 similarly, the transitions related to higher ru oxidation states were improved when the synthesis temperature was below the boiling point of the reaction (ruo2(mwt80)/ti). these findings indicate that the increasing temperature of mw synthesis to 120 and 150 °c produce more polycrystalline particles that create more accessible coatings with more defects sites (kink sites, defect sites, edges, etc.), which can be readily oxidized to higher oxidation states. total capacitance of anodes was calculated by integration of cv curves. a bit higher value was obtained for ruo2(mwt80)/ti, 108 f g–1, in comparison to ruo2(mwt120)/ti and ruo2(mwt150)/ti, 101 and 107 f g–1, respectively. in addition to small differences commented in previous paragraph, these small differences in cv capacitance could also originate from a bit more compact structure of the ruo2(mwt120)/ti and ruo2(mwt150)/ti coatings, for which more pronounced presence of a fraction of smaller particles was registered (fig. 2). the synthesis temperatures above 150 °c, which is assumed as optimal for good capacitive response of ruo2,43 caused considerable increase in cv currents.39 in order to investigate further coating structure-related pseudo-capacitive characteristics of synthesized ruo2 coatings, electrochemical impedance spectroscopy (eis) measurements at open circuit potential (ocp) were carried out. if it is assumed that ocp value corresponds to the fastest and the most convenient redox transition, the eis analysis is expected to give more details, related to the differences in various potential regions indicated by cv curves (fig. 3). experimental and fitted eis data, presented in form of complex plane and bode plots, along with the model of applied equivalent electrical circuit (eec), are shown in fig. 4. the registered eis response and the eec structures are typical for dsas with a well-developed tio2-rich interlayer.44 the semicircle-like dependence can be seen in the high frequency range down to 400 hz (fig. 4b), which precedes capacitive-like response in the intermediate frequency range, down to 0.5 hz. the semicircle is assignable to tio2-rich in the coating/ti substrate interphase, created during annealing of the electrode as the product of the substrate oxidation. its parameters found the equivalence in cpe1 and r2 eec elements in parallel (fig. 4a). the capacitive-like response is distributed through a transmission line generated by the elements c1-c4 and r3-r5 and is well developed as a capacitive loop, fig. 4c. in the low frequency range, there is an indication of finite diffusion limitations to the pseudo-capacitive response, which is represented by the element cpe2. the mean values (± abs. error) of eec parameters are shown in fig. 5. eis response which describes the properties of tio2-rich interlayer appears similar for three samples (fig. 4a), with corresponding resistance and capacitance around 0.6 ω and 2,5 f g–1, respectively. these findings indicate that the corresponding tio2-rich interlayer is of similar structure since the coat 702 šekularac et al. fig. 4. the results of the fitting of measured eis data to equivalent electrical circuit (eec, a) presented as impedance (b) and capacitance (c) complex plane and bode (d) plots of the ruo2/ti electrodes synthetized at: 80; 120 and 150 °c in an mw reactor at ocp; symbols: measured data, lines: eec data. ings should be of similar texture39 created from the particles of similar size distribution around 200 nm (fig. 2). owing to more narrower and more stable particle distribution in case of mwt80, the least compact coating structure was expected, and hence the most compact tio2 interlayer could be formed. this appears indicated by the larger interlayer capacitance for mwt80 in comparison to mwt120 and mwt150 (fig. 4a, branch no. 1, cpe1). however, the interlayer thickness is similar for all samples and seems to be considerably thinner than the coating, since the corresponding resistance (0.6 ω) is negligible in comparison to coating pore resistance around 100 ω. the capacitance complex plane plots show the difference in eis behaviour between investigated ruo2 coatings (fig. 4c). capacitive loop for ruo2(mwt80)/ti is larger than those obtained for ruo2(mwt120)/ti and ruo2(mwt150)/ti coatings. low-temperature-synthesized ruo2 as electrode coating 703 the fitting of eis response with eec model (fig. 4a) showed that ruo2(mwt80)/ti is of higher total capacitance of around 96,6 f g–1 in comparison to ruo2(mwt120)/ti and ruo2(mwt150)/ti coatings, which showed similar total capacitance of around 71.3 f g-1. this presents more pronounced difference between ruo2(mwt80)/ti and other two samples, while the values are lower, with respect to cv data. this means that mwt80 has better pseudo-capacitive characteristics in comparison with ruo2(mwt120)/ti and ruo2(mwt150)/ti. results obtained by fitting of experimental data are presented as capacitance and resistance through the branches of eec, fig. 5a and b, respectively. the more pronounced difference between the samples appears to be much higher pore resistances of ruo2(mwt120)/ti and ruo2(mwt150)/ti due to the more compact coating structure, which consequently showed the lower capacitance value in the eec branches 3–5. as it can be seen, the capacitance values in the second branch, associated to the most outer part of the coating, are quite similar with (a) (b) fig. 5. a) capacitance and b) resistance through the branches of the equivalent electrical circuit used for the fitting the impedance spectra of the ruo2/ti electrodes prepared from mwt80, mwt120 and mwt150 samples. 704 šekularac et al. even small opposite trend with respect to branches 3–5. these branches, according to the transmission line model,45 relates to coating inner structure. hence, higher pore resistance values were obtained for the branches 3–5. on the other hand, branch 2 presented the negligible resistance difference between samples, apparently related to the interspace of a fraction of large particles of similar size of around 200 nm for all samples. bearing in mind that the cv data where registered with rather high potential sweep rate in comparison to eis measurements, it could be noticed that registered cv data reflect fairly well the findings related to second eec branches of the lower order. it appears that the pseudo-capacitive response is sensitive to charging/discharging potential limits, with wider limits improving the capacitance performance (1.3 v in cv and only 10 mv rms amplitude in eis), since the cv conditions double the values of those obtained by eis. this sensitivity is less pronounced for ruo2(mwt80)/ti (55 % of cv capacitance is seen by eis) than for other two samples (45 %). this indicates that more defined structure is formed at higher synthesis temperatures, with the ability to promote electrocatalytic activity (fig. 3). figure 6 represents the total capacitance and total pore resistance for the samples synthesized at different mw temperatures. ruo2(mwt120)/ti is of largest resistance because of the compact structure created by the particles belonging to psd fractions of the smallest diameter in comparison to other samples (fig. 2b). consequently the total capacitance was small, as indicated by fig. 6. similarly, ruo2(mwt80)/ti and ruo2(mwt150)/ti were of lower resistance with corresponding higher values of total capacitance, owing to the structure created by larger particles (figs. 2a and c). the increase in mw temperature to 200 °c brought fig. 6. total capacitance and resistance of the ruo2/ti electrodes as a function of mw synthesis temperature. low-temperature-synthesized ruo2 as electrode coating 705 about the compromise between pore resistance and coating capacitance, which resulted in highest capacitance for moderate pore resistance, but also to more defined crystalline structure as shown in previous work.39 psd of ruo2(mwt200)/ /ti39 was consisted of similar small particle from fractions as psds for ruo2(mwt150)/ti and ruo2(mwt80)/ti, but with the appearance of huge agglomerates for these two samples. the intrinsic values of capacitance and resistance of ruo2(mwt200)/ti can also be assigned to the more uniform coating texture due to absence of the agglomerates. electrocatalytic properties of prepared ruo2/ti electrodes in oxygen evolution reaction (oer) are demonstrated by polarization curves shown in fig. 7. recorded polarization curves showed typical oer behaviour of ruo2/ti;39,45 all of the curves show regions of the two tafel slopes: around 40 mv below 1.23 v, and larger ones above this potential value. the region of lower tafel slopes is typical for polycrystalline, thermally prepared ruo2/ti, assignable to the mechanism with indirect water oxidation over simultaneous oh/o transition as rate -determining step, mostly on (110) ruo2. the data from previous work39 show that the synthesis temperature just above 150 °c makes the oxide less active in this potential region. the data for 200 °c show the increase of a slope to around 55 mv and the consequent decrease in currents, fig. 8a and b, respectively. fig. 7. quasi-steady-state polarization curves (5 mv s-1) for o2 evolution in 1 m h2so4 at room temperature on the ruo2/ti electrodes prepared from mwt80, mwt120 and mwt150 samples. 706 šekularac et al. (a) (b) fig. 8. a) tafel slopes in the regions of lower and higher potentials and b) corresponding current densities at 1.26 and 1.17 v (fig. 7) for the ruo2/ti electrodes as a function of ruo2 synthesis temperature. further increase in temperature to 220 °c recovers the slope (the electrocatalytic features of surface active sites) and the currents only partially, with respect to low-temperature samples. this indicates that considerable changes of oxide structure take place at and above 150 °c, as reported in literature.43 similarly, the discontinuity is registered for large tafel slopes, which could correspond to oer mechanism with a slowest step of direct water splitting on oxygen terminals at the oxide sites.44 however, the temperature of discontinuity is 120 °c, which relates to considerable promotion of the electroctalytic activity. the slope is considerably lower (fig. 8a) and corresponding currents are the highest (fig. 8b). it follows that substantial changes in the oxide structure during the nuclei growth are initiated at the synthesis temperature above the boiling low-temperature-synthesized ruo2 as electrode coating 707 point of the reaction mixture. it appears that mw temperature of 120 °c is able to generate a plenty of active sites at the surface of oxide particles, which is well preserved during the coating preparation and maintained as ruo2(mwt120)/ti unique electrocatalytic feature. it even appears that mechanism is not changed in the whole investigated potential region for ruo2(mwt120)/ti, and corresponds significantly to consecutive oer over oh/o transition of surface active sites. conclusions ruo2 coatings on ti were prepared by thermal treatment of the oxide solid phase synthesized from rucl3 aqueous solution by the low temperature-controlled microwave (mw) heating. the minor difference in capacitance between coatings prepared from oxides synthesized in temperature range of 80–150 °c was registered from voltammetric responses. the difference can be associated with more compact structure of the oxide synthesized at the temperature of 120 °c in comparison to the other samples. the more compact structure was correlated to the presence of the fractions of the smallest particles. the distribution of capacitance and pore resistance, as a result of impedance measurements, showed more pronounced difference between samples due to the same cause related to coating structure. the considerable differences in capacitance and pore resistance were found in inner parts of a coating, while similar behaviour was restricted only to the outer parts, as found by voltammetric measurements. the electrocatalytic activity of the coatings in oxygen evolution reaction (oer) was found considerably different and promoted the coatings prepared from the oxide synthesized at lower temperatures as more active. these differences were correlated to different oxide structure with characteristic structure transitions at the intrinsic temperatures just above the boiling point of reaction mixture and above the temperature recognized as crucial for optimum proton/ /electron conductivity of the oxide. although low synthesis temperatures were found beneficial for oer, the synthesis temperatures above 150 °c promoted the coating capacitance. this opposite finding was supposed to be due to the differences in the structure of oxide surface active sites, which appears to be generated during the mw synthesis and preserved during the formation of the coating. acknowledgments. financial support for the reported investigation from the ministry of education, science and technological development of the republic of serbia is acknowledged. the authors thank to dr. ivana drvenica for dls measurements. 708 šekularac et al. и з в о д ниско-температурна синтеза ruo2 из киселог хлоридног раствора за припрему eлектродне превлаке гаврило шекуларац1, сања ераковић1, душан мијин2, весна павелкић3, јасмина стевановић1 и владимир панић1 1институт за хемију, технологију и металургију, центар за електрохемију, универзитет у београду, београд, 2 технолошко–металуршки факултет, универзитет у београду, београд и 3висока железничка школа струковних студија, здравка челара 14, београд ruo2 је синтетисан у киселом воденом раствору једноставним микроталасним (mw) поступком у једном кораку на контролисаним ниским температурама и затим коришћен за припрему ruo2 превлаке на подлози од титана. синтетисана чврста фаза је окарактерисана анализом расподеле величине честица (psd), док су капацитивност превлаке и њена активност у реакцији издвајања кисеоника (oer) испитане електрохемијским техникама. нађено је да је оксидна фаза изражено полидисперзна, са фракцијама величине честица у уском опсегу пречника и тенденцијом ка укрупњавању. најситније честице и изражено раздвојене фракције синтетисане су на температурама блиским температури кључања реакционе смеше, знатно испод температуре конверзије хлорида у оксид. на овим температурама је регистрована и највећа активност ruo2/ti аноде за oer, вероватно због различите структуре оксида у односу на узорке припремљене на вишим температурама. међутим, нађено је да превлаке формиране од оксида синтетисаног на вишим температурама имају већу кaпацитивност од оних синтетисаних на нижим темпераутрама. овакви резултати су пре последица температурно-зависне структуре оксида, него промена у његовој морфологији које настају мw синтезом. 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice volume 82 (2017) 1 subject index 1,2,3-triazole, 241, 357 1,3-dicarbonyl compounds, 483 1,4-benzenedicarboxylate, 1247 2,4-diaminothiazolyl, 1087 3,5-di-t-butyl-4-hydroxyphenyl, 1087 11h-benzo[a]xanthenes, 483 aas, 215 acid dissociation constant, 851 acid sites, 523 acidity constants, 304 adsorption mechanism, 1395 adsorption, 39, 215, 227, 1175, 1303 aerogels, 523 agitation, 921 agricultural by-product, 1223 aldehydes, 1343 algae, 509 alkali treatment, 83 alpha, 1379 alprazolam, 933 amalgam film electrodes, 879 amides, 1039 anions in bottled water, 711 anova, 905 anti/pro-oxidants, 1009 anti-acetylcholinesterase activity, 651 antibacterial activity, 13, 641, 985, 995 antibacterial, 127, 241, 495 anti-cholinesterase activity, 803 antifungal activity, 13, 641, 985, 995 antifungal, 127, 495 antimicrobial activity, 1, 117, 141, 357, 367, 389, 509, 651, 1357 antimicrobial studies, 665 antioxidant activity, 141, 495, 825 antioxidant capacity, 253 antioxidant effect, 803 antioxidant, 1, 651 approximation model, 99 aqueous medium, 755 arsenic species, 943 arsenic, 1175 artificial neural network, 399 aryldiketo acids (adks), 304 asphaltene, 1063 atr-ftir, 567 attributes of a chemistry teacher, 1193 avena sativa, 379 azo dyes, 851 back propagation, 399 bast fibers, 83 bi2o3, 1417 bibliometric analysis, 1075 binary mixture, 399, 1155, 1433 biocarbon, 1395 biological study, 267 biomarkers, 593, 739 biomonitoring, 723 biophosphate glass, 329 biopolishing, 567 bipyrazoles, 127 bisbenzofuran, 367 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 2 journal of the serbian chemical society bisphenol a, 39 bn-yne, 289 boehmite, 203 bond lengths, 163 boron-doped diamond electrode, 175 cadmium, 1175 candida, 389, 641 carbohydrazones, 495 carbon ink, 865 carbon paste, 865 cast iron, 1135 catechol, 1053 cation complexed (η6-benzene)–tricarbonylchromium system, 1123 cation exchange capacity, 449 cations in bottled water, 711 cation–π-interaction energy, 1123 celery root, 253 cellulose, 567 chalcone, 241, 357 characterization, 267 charge transfer, 1123 charges, 163 chemical composition, 83, 141, 1395 chemical property, 289 chemistry teacher profession, 1193 chemometrics, 711 chlacone, 985 chlorite treatment, 83 chlorophyll, 921 choline chloride, 1287 cholinesterase inhibition, 141 chromene, 1, 117 cimetidine, 25 clay, 1303 click chemistry, 241 click reaction, 995 clinoptilolite, 1303 cluster analysis, 815 cobalt(iii) , 825 cold vapour atomic absorption spectrometry, 551 concordance correlation coefficient, 1316 contaminated water, 73 cookies, 1343 copper, 1211 corn, 63 cosmo, 277 cotton fabric, 567 coumarin, 117 cracking process, 1063 crystal structure, 1247 cu(ii) , 1247 cultivated plants, 815 cyclic voltammetry, 51, 411, 865, 1053 cyclohexane, 189 cytotoxic activity, 509, 651 cytotoxicity, 389, 1357 de-aromatization, 107 deep eutectic solvents, 1287 degradation, 99, 1223 dendrimer graphs, 151 denitrification, 107 density of state, 289 density, 905 derived properties, 1155 design of new inhibitors, 1025 destructive sorption, 523 desulphurization, 107 dft calculation, 665 dft, 25, 277, 289, 825, 841, 1369 di-/tri-organotin complexes, 13 diamines, 389 diastereotopic protons, 985 diazepam, 933 diesel fuel, 107 differential pulse voltammetry, 1273 differential scanning calorimetry, 437 diketoxime, 367 dimensionless, 921 dispersive solid phase micro-extraction, 551 disubstituted 1,2,3-triazoles, 995 dl-malic acid, 1287 docking simulations, 127 dopamine, 1053 dpph, 509 drugs, 879 dye removal, 227 eggs, 1097 electrocatalytic oxide materials, 695 electrochemical determination, 1259 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. volume 82: subject index 3 electrochemical impedance spectroscopy, 695, 1111 electrochemical sensor, 73, 1053 electrocrystallization, 51 electrodeposition, 539 electrografted glassy carbon electrode, 1053 electronic properties, 163 energy, 957 environment, 593 environmental remediation, 215 enzyme inhibition, 1087 enzymes, 1223 equilibrium phase diagram, 579 eras, 1433 esters, 189 ethanol, 891 eutectic, 1039 excess molar volume, 1433 excess properties, 189 exchange current density, 539 extracting mechanism, 579 ex-yugoslav republics, 1075 factorial design, 417 fiber properties, 83 flavonoids, 803 flory theory, 891 freon, 12, 523 freundlich isotherm, 943 fukui, 25, 825 functional materials, 607 fungicide, 1259 gaussian 09, 277 gc, 1147 gc/fid, 815 gc-ms, 509, 815, 1316 gc–ms-ms, 1316 glycols, 1039 goethite, 1175 graph automorphism, 151 graphene, 411 graphene nano-sheets, 1273 graphene oxide, 39, 411 graphyne, 289 green chemistry, 483 green pigments, 921 guanylhydrazones, 641 heavy metal ions, 215, 1395 heavy metal, 317 hemp fibers, 1395 herbicide, 343 heterocycles , 1335 heterocyclic chemistry, 755 heterometallic complex, 1247 hexadecyl trimethyl ammonium bromide, 175 hexa-hydro-1h-xanthene, 483 hindered phenol, 1087 hordeum sativum, 379 hplc-dad, 63 hsab, 25 hydrolysis products, 1223 hydrothermal synthesis, 695 hydroxyl radical, 277 hydroxyoxime-based extractants, 417 ifn-α-2a, 841 ifn-α-2b, 841 ifn-β-1a, 841 iiminoguanidines, 641 imines, 627 immobilization, 567 infrared spectroscopy, 1063 inlet, 1147 interbacterial communication, 1357 internal pressure, 681 ionic liquid, 551 iron oxide, 1303 isomers, 163 isomers, 495 isoxazole,1 kamlet–taft parameters, 851 kinetics, 329 kovin deposit, 739 laser-induced breakdown spectroscopy, 1135 leaching, 329, 1303 lead, 539, 1175 lignite, 739 lincomycin, 277 linewidth, 957 lipophilicity, 1369 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 4 journal of the serbian chemical society liposomes, 1009 liquid–liquid equilibria, 1287 lithotype, 739 lowest-energy, 163 lutein, 1009 macerals, 739 macrocycle ligand, 665 magnetic nanoparticles, 39 magnetic properties, 1247 magnetic reduced graphene oxide, 551 malondialdehyde, 1343 mathematical modelling, 1343 matrix effect, 343 mbt, 1147 md simulation, 681 mean-difference plot, 1316 mechanical measurements, 437 mechanism, 329 mefenamic acid (mfa), 1273 mercury, 317, 551 metal complex, 267, 1357 metal–non-metal transition, 681 method development, 1259 method validation, 63 metronidazole, 879 micronucleus test,141 microwave irradiation, 117, 357 microwave synthesis, 695 mineral content, 1097 mixing extractant, 579 mn(ii), 1247 modeling, 1287 molecular designing, 607 molecular modeling, 665 molecular motors, 469 molecular structure, 1063 molten salts, 51 morphology, 411 multi linear regression, 399 multi wall carbon nanotubes, 1273 multi-component reactions (mcrs), 483 mwcnt, 1175 na-enriched clays, 449 nanocomposites, 437, 1175 nanoparticles, 607, 1211 nanosecond transversely excited atmospheric carbon dioxide laser (tea co2), 1135 nanostructures, 203 natural bitumen, 1063 natural habitat, 815 n-heptane, 891 nickel(ii) complexes, 389 nitrogen-donor ligands, 1357 non-linear differential equations, 469 non-linear dynamics, 469 nucleus-independent chemical shifts (nics), 1123 nutritional composition, 1237 oils, 1316 omega-telechelic pdms, 1379 optimization, 253 optode, 317 orbits, 151 organic synthesis, 755 organophosphate pesticides, 1369 osmotic treatment, 253 oxidation, 627, 791, 933 p. chitinolyticus cks1, 1223 palladium, 1335 p-aminobenzene sulfonic acid, 73 paracetamol, 99 parent material, 943 particle representations, 1193 partition coefficient, 1369 pbo, 1417 pegylation, 841 peng–robinson equation of state, 189 petroleum pollutants, 593 pfp, 1433 ph, 203 phase diagram, 1417 phenolic acids, 63, 803 phenolics, 651 phonons, 957 photocatalytic degradation, 343 photosynthetic pigments, 723 physicochemical properties, 1039 plant tissue, 723 pollutants, 933 poly(vinyl chloride) , 317 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. volume 82: subject index 5 poly(vinyl pyrrolidone), 427 polygonum cuspidatum, 803 polysac-charides, 509 pore volume, 203 ppcps mixture, 1445 prediction model, 1433 prigogine–flory–patterson theory, 189, 891 process optimization, 1223 promoting effect, 791 propan-1-ol, 891 propan-2-ol, 891 propar-gylamine, 1211 proteasome inhibitors, 1025 pseudocapacitance, 695 ptv, 1147 pyrazole, 357 pyrazoline,117 , 241, 985 pyridinedicarboxylic acid, 665 qsar/qspr, 151 quantitative 13c-nmr spectroscopy, 1287 quantitative analysis, 1135 quantitative structure–activity relationship (qsar), 1025 quantum-chemical method, 791 rdf, 681 reactivity, 25 real samples, 1273 reciprocating plate column, 579 redox couples, 865 redox properties, 267, 304 refractive index, 905 resveratrol, 175 reused material, 1211 rheology quality, 1097 ruthenium tetraoxide, 627 scanning electron microscope (sem), 539, 1111 schiff base, 13, 825 scientific productivity, 1075 screen printed electrodes, 865 secondary amines, 627 selection, 379 sesame flour, 1097 seseli gracile, 815 shelf-life, 1343 silicon, 51 siloxane equilibration, 1379 single-wall carbon nanotubes (swcnts), 73 sno2, 411, 791 soil, 723 solitons, 469 solubility, 99 solvatochromism, 851 solvent activity, 427 solvent extraction, 921 solvent vent mode, 1147 solvent-free conditions, 483 sonochemistry, 755 spectrometry, 317 spin probe, 523 square-wave adsorptive stripping voltammetry, 175 sr2+, 449 stripping voltammetry, 1259 structure–property relationship, 304 subcritical water medium, 99 sugar beet molasses, 253 supercapacitor, 411 superconductors, 957 surface tension, 427 sustainable chemistry, 755 symmetry, 163 synthesis, 1335 tar, 1063 technological quality, 1097 technologically mature seed, 1237 temperature, 921 terpanes, 1316 textural properties, 449 theoretical models, 1155 thermal analysis, 665, 1111 thermal and thermo-oxidative degradation, 1379 thermal expansion coefficient, 905 thermos-dynamic model, 427 thermosetting resin, 437 thiophene, 367 time-dependent density functional theory (tddft), 1123 tio2 degussa p25, 343 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 6 journal of the serbian chemical society tolerance to low temperatures, 1237 topological indices, 151 topomer comfa, 1025 topomer search, 1025 total dissolved solids, 711 toxicity, 1369, 1445 transition state, 841 trans-resveratrol, 803 triazenes, 317 tricresyl phosphate, 1259 triple nature of chemical concepts, 1193 ultrasonic irradiation, 755 urban pollution, 593, 723 uv-irradiation, 1009 v2o5, 791 vanadium, 523 variability, 379 vilsmeier reagent, 117 viscosity, 905 voltammetry, 417, 879 walnut shell, 1211 waste recycling, 227 water purification, 449 winter varieties, 1237 wreath product, 151 x-ray, 267 х-ray crystal structure, 825 x-ray diffraction, 1111 zeolite linde f(na), 227 zeolite x, 227 zinc, 539 zno, 343 zro2, 791 α-aminonitriles, 627 β-carotene, 1009 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice spin state relaxation of iron complexes: the case for opbe and s12g j. serb. chem. soc. 80 (11) 1399–1410 (2015) udc 546.722/.723+533.6.013.7: jscs–4806 532.14:537.872 original scientific paper 1399 spin state relaxation of iron complexes: the case for opbe and s12g maja gruden1, stepan stepanović2# and marcel swart3,4* 1faculty of chemistry, university of belgrade, studentski trg 12–16, 11001 belgrade, serbia, 2center for chemistry, ictm, university of belgrade, njegoševa 12, 11001 belgrade, serbia, 3institut de química computacional i catàlisi (iqcc) and departament de química, universitat de girona, campus montilivi, facultat de ciències, 17071 girona, spain and 4institució catalana de recerca i estudis avançats (icrea), pg. lluís companys 23, 08010 barcelona, spain (received 11 june, revised 14 july, accepted 15 july 2015) abstract: the structures of nine iron complexes that show a diversity of experimentally observed spin ground states were optimized and analyzed using the density functional theory (dft). an extensive validation study of the new s12g functional was performed, with a discussion concerning the influence of the environment, geometry and its overall performance based on a comparison with the well-proven opbe functional. the opbe and s12g functionals gave the correct spin ground state for all investigated iron complexes. since s12g performs remarkably well, it could be considered a reliable tool for studying the energetics of the spin state in complicated transition metal systems. keywords: density functional theory; fe(ii) and fe(iii) coordination compounds; validation study; spin states. introduction spin is an intrinsic and inherent property of atoms and molecules.1 most transition metal ions with partially filled d-shells can exhibit different kinds of spin multiplicity in the ground state, i.e., can lead to different spin states. depending on the oxidation number, iron complexes usually have either 5 or 6 delectrons that can be distributed in an octahedral environment in at least two different ways: with a maximum number of unpaired electrons, leading to the high spin (hs) state, or with maximally paired electrons – giving the low spin (ls) state. other possibilities of the distribution of electrons represent an intermediate (is) spin state. since hs, is and ls complexes usually display quite different * corresponding author. e-mail: marcel.swart@icrea.cat # serbian chemical society member. doi: 10.2298/jsc150611068g 1400 gruden, stepanović and swart structural, spectral and magnetic properties, and often reactivity, it is of the utmost importance to have both experimental and theoretical methods to determine correctly the spin ground state of a system. however, both experiment and theory have difficulties and problems, and many studies have been devoted to this issue in the last decade.2–5 from a broad palette of quantum mechanical methods, the density functional theory (dft)6–8 has emerged into the mainstream, mainly because it gives a good compromise between accuracy of the results and computational efficiency.9–11 however, although the dft, in principle, gives an exact energy, a universal functional is still unknown, leading to density functional approximations (dfas). these dfas are parameterized for different properties and, noteworthy, spin-state energies were not included in the development for most of nowadays available dfas.12 it has been shown that the accuracy of the results not only strongly depends on the choice of the dfas, but also on the basis set that is used.1,3,13,14 early pure functionals, such as lda,15–17 bp86,18,19 blyp,19,20 and pw91,21,22 have a tendency to favor ls states,14 while hybrid functionals, such as b3lyp,23,24 pbe025 and m06,26,27 systematically favor hs states.14 for the reliable prediction of the correct spin ground state from a number of close lying states, opbe14 has emerged to be one of the best functionals for the task.28 recently, swart constructed a new density functional that combines the best of opbe (spin states, reaction barriers) with the best of pbe (weak interactions) into the s12g5 dfa. previously, the relative spin state energies of seven iron complexes (1–7, fig. 1) on olyp20,29 optimized geometries (1–3) and on crystal structures (4–7) with a variety of dfas were reported, which already showed the good performance of opbe for vertical spin state splittings.14 herein, a detailed dft study on opbe optimized geometries of iron complexes (1–7) with experimentally established spin ground states, ranging from singlet to sextet, is reported together with an extension to include two iron porphyrinato complexes (8 and 9, fig. 1) that were reported to have different electronic ground states in spite of their similarity.30–32 furthermore, a comprehensive validation study of the s12g dfa,5 together with an examination of the influence of the chemical environment, was performed on all the investigated complexes. methodology all dft calculations were performed with the amsterdam density functional (adf) suite of program.33,34 mos were expanded in an uncontracted set of slater type orbitals (stos) of triple-ζ quality containing diffuse functions (tzp)35 and one set of polarization functions. core electrons (1s for 2nd period and 1s2s2p for 3rd–4th period) were not treated explicitly during the geometry optimizations (frozen core approximation), as the core was shown to have a negligible effect on the obtained geometries.36 an auxiliary set of s, p, d, f, spin state energetics of iron complexes 1401 and g stos was used to fit the molecular density and to represent the coulomb and exchange potentials accurately for each self-consistent field (scf) cycle. fig. 1. fe(pypeps)2 1 (pypepsh2 = n-(2-mercaptophenyl)-2′-pyridinecarboxamide); fe(tsalen)cl 2 (tsalen = 2,2′-[1,2-ethanediylbis(nitrilomethylidyne)]bis[benzenethiolato]; fe(n(ch2-o-c6h4s)3)(1-me-imidazole) 3; (fe(nh)s4)l 4 (l=co), 5 (pme3), 6 (nh3), 7 (n2h4)((nh)s4 = bis(2-((2-mercaptophenyl)thio)ethyl)amine); iron porphyrin chloride (8, fepcl) and iron porphyrazine chloride (9, fepzcl). energies and gradients were calculated using the opbe and s12g functionals in the gas phase and with cosmo (methanol as a solvent)37-39 in the dielectric continuum model for a solvent environment. the geometries were optimized with the quild program40 using adapted delocalized coordinates41 until the maximum gradient component was less than 10-4 a.u. subsequent single point calculations that utilize all electron basis set were performed on all optimized geometries, with opbe and s12g. results and discussion the total set of molecules consists of both fe(iii) (1–3, 8 and 9) and fe(ii) (4–7) complexes, and show a diversity of experimentally observed spin ground states. a thorough examination with the opbe and s12g functionals in the gas phase and the cosmo solvent environment was performed. the discussion is 1402 gruden, stepanović and swart commenced by focusing on the influence of structure relaxation on the spin states of fe(iii)-complexes 1–3.42 experimentally, fe-(pypeps)2 (1, pypepsh2 = n-(2 -mercaptophenyl)-2-pyridinecarboxamide) has a ls doublet ground state,43 fe(tsalen)cl (2, tsalen = 2,2′-[1,2-ethanediylbis(nitrilomethylidyne)]bis[benzenethiolato] 2,2′-[1,2-ethanediylbis(nitrilomethylidyne)]bis[benzenethiolato]) an intermediate spin (is), quartet ground state44 and fe(n(ch2-o-c6h4s)3)(1-meimidazole, 3) a hs sextet ground state.45 then, the fe(ii)-complexes ((fe(nh)s4)l, (nh)s4 = bis(2-((2-mercaptophenyl)thio)ethyl)amine, l = co (4), pme3 (5), nh3 (6) and n2h4 (7)) are discussed. compounds 4 and 5 have a ls (singlet) state and compounds 6 and 7, reportedly, a hs (quintet) ground state.46–48 furthermore, focus is placed on feiii(porphyrinato)cl, fepcl (8) and, feiii(porphyrazinato)cl, fepzcl (9), which have a sextet and a quartet ground state, respectively. structure relaxation and spin state energies of fe(iii) compounds 1–3 the optimization of the three fe(iii) molecules (1–3) led in all cases to the expected structural changes for the different spin states (tables s-i–s-iii of the supplementary material to this paper). comparison of the optimized structures of 1–3 indicated the existence of an expansion of the ligand sphere. going from the doublet to the quartet state, first the equatorial ligands moved away from iron while the axial ligands stayed almost at the same position. in the sextet state, the equatorial ligands remained virtually at the same position, but the axial ligands (had to) moved out. comparing the vertical spin state energies, calculated on the experimental structure,14 with results from the optimized (“relaxed”) geometries, table i, it is evident that the energy gap between different spin states decreased. in the case of compound 1, the doublet state remained the spin ground state with the quartet state (from 22.5 kcal*·mol–1 “vertical” to 17.5 kcal·mol–1 “relaxed”) and the sextet state (from 33.9 kcal·mol–1 “vertical” to 10.2 kcal·mol–1 “relaxed”) in closer energetic proximity after geometry optimization. molecule 2 has the quartet ground state, and here, the relative energies of the doublet and sextet states were reduced after structure relaxation. the same trends apply for the sextet ground state of complex 3. for all complexes, after spin state relaxation, both opbe and its recently developed successor s12g gave the correct spin ground state. spin contamination was small for these complexes, and therefore, will not be discussed further. the choice of exchange-correlation functional had an obvious influence on the geometry, with a tendency of s12g to give somewhat longer bond lengths than opbe (tables s-i–s-iii). it should be noted that s12g gave structural parameters that were in excellent agreement with experimental values. unlike the * 1 kcal = 4184 j spin state energetics of iron complexes 1403 choice of functional, the influence of solvation on the geometrical parameters during the structural relaxation was not very significant, and it depended slightly on the system under consideration. in most cases, optimizations with cosmo gave slightly longer bonds, but without significant consequences for the spinstate splittings, table i. table i. spin state energies (kcal mol-1) for fe(iii) molecules 1–3 using tzp basis set, with opbe and s12g functionals, in vacuum and cosmo geo.a spb fe-(pypeps)2 1 fe(tsalen)cl 2 fe(n(ch2-o-c6h4s)3)(1mimb) 3 doublet quartet sextet doublet quartet sextet doublet quartet sextet opbe opbe 0 17.1 10.2 6.5 0 3.9 6.6 7.9 0 opbe cosmo 0 19.4 13.0 9.3 0 6.9 7.9 7.4 0 s12g 0 15.8 8.7 7.6 0 3.8 6.8 7.2 0 s12g cosmo 0 18.2 11.6 10.2 0 6.4 8.2 6.8 0 opbe cosmo opbe 0 18.8 13.1 5.2 0 2.9 6.2 7.5 0 opbe cosmo 0 17.4 10.2 9.7 0 7.7 8.0 7.2 0 s12g 0 18.4 13.3 6.0 0 3.0 6.5 6.8 0 s12g cosmo 0 17.1 10.6 10.2 0 7.4 8.3 6.5 0 s12g opbe 0 18.3 10.5 7.4 0 6.2 7.6 8.1 0 opbe cosmo 0 22.7 14.7 10 0 9.2 8.6 7.1 0 s12g 0 15.4 8.7 7.5 0 6.6 6.5 7.0 0 s12g cosmo 0 19.9 13.1 9.9 0 9.3 7.7 6.1 0 s12g cosmo opbe 0 17.5 10.6 7.0 0 4.7 7.5 8.4 0 opbe cosmo 0 20.5 14.9 11.2 0 6.7 8.8 7.2 0 s12g 0 15.7 9.2 6.6 0 4.7 6.1 6.8 0 s12g cosmo 0 18.8 13.7 10.4 0 6.5 7.6 5.9 0 ageometry optimization with frozen core electrons; bsubsequent single point calculations with all-electron basis sets structure relaxation and spin state energies of compounds 4–7 the spin state dependent structure relaxation for the fe(ii) compounds results in similar differences in fe–ligand distances as for the fe(iii) compounds (tables s-iv–s-vii). in the case of compounds 4–7, the fe–n, fe–s and fe–c distances were slightly elongated in comparison to the distances in the fe(iii) complexes due to the additional d-electron in the fe(ii) systems. the spin ground states of the fe(ii) complexes 4 and 5 were correctly predicted using both the opbe and s12g levels of theory (see table ii): the singlet state was the lowest in energy for both molecules, in agreement with expe 1404 gruden, stepanović and swart table ii. spin state energies (kcal mol-1) for labile (trans) complexes 4 and 5 using tzp basis, with opbe and s12g functionals, in vacuum and cosmo geo.a spb trans-(fe(nh)s4)co 4 trans-(fe(nh)s4)pme3 5 singlet triplet quintet singlet triplet quintet opbe opbe 0 23.4 34.8 0 16.3 20.1 opbe cosmo 0 24.5 36.6 0 17.3 18.6 s12g 0 19.1 28.0 0 14.5 17.7 s12g cosmo 0 20.2 29.7 0 15.4 16.3 opbe cosmo opbe 0 23.5 35.3 0 16.4 20.4 opbe cosmo 0 24.5 36.5 0 17.3 18.3 s12g 0 19.4 29.1 0 15.1 19.1 s12g cosmo 0 20.3 30.3 0 15.9 17.1 s12g opbe 0 23.4 34.2 0 19.6 19.4 opbe cosmo 0 24.3 36.4 0 20.3 19.2 s12g 0 18.7 29.3 0 15.6 16.8 s12g cosmo 0 19.6 31.4 0 16.3 16.6 s12g cosmo opbe 0 24.6 35.0 0 19.9 19.2 opbe cosmo 0 24.8 36.5 0 20.6 18.2 s12g 0 20.4 30.8 0 15.8 17.1 s12g cosmo 0 20.5 32.2 0 16.4 16.2 ageometry optimization with frozen core electrons; bsubsequent single point calculations with all-electron basis sets rimental data. for compound 4, the triplet and quintet states were significantly higher in energy. the energy differences between the different states were smaller for compound 5. similar to the fe(iii) complexes, spin contamination was small and will not be discussed any further. similar to compounds 4 and 5, after spin state structure relaxation, an ls ground state for iron complexes 6 and 7 was found, with is and hs higher in energy. unfortunately, the experimental determination of the spin states of compounds 6 and 7 were inconclusive, since anomalous high μeff values of 10–13 μb were measured that may indicate impurities, e.g., by metallic iron, or oligomer formation. for compound 7 in solution, an hs state was observed,48 but a compound similar to 7 showed a diamagnetic ls fe center.49 moreover, indications of dimer formation of the ligand-free [(fe(nh)s4)] complex were observed.47,48 since different forms of the (fe(nh)s4)l complex in these studies were obtained, both forms for compounds 4–7, i.e., with the “trans” and “meso” form (see fig. 2) had to be checked. for both forms of each of compounds 4–7, an ls ground state was found, albeit with smaller spin-state splitting for compounds 6 and 7. these findings could be traced back to the strength of the iron–ligand bond, which seems to be much weaker for compounds 6/7 than for compounds 4/5. the weakly bound nh3 and n2h4 ligands are easily exchanged with ch3oh, solvent (thf) or co.48 these experimental data corroborate the present computed ligand-binding energies, which indicate strong and favorable binding of co and p(me)3 to form the singlet ground state, but less favorable binding of the other ligands or spin states spin state energetics of iron complexes 1405 (see table s-viii of the supplementary material). interestingly, the monomeric fe(nh)s4 complex without a ligand was predicted to have a triplet spin ground state in the trans form, with the other spin states or the meso form lying higher in energy by at least 7 kcal·mol–1. the ligand-free complex may dimerize to give the experimentally observed hs state through ferromagnetic coupling. the latter process was not studied due to the complexity involved with ferromagnetic versus anti-ferromagnetic coupling of the many spin states that need to be considered. this was confirmed by a recent study using high-level ab initio methods that indeed found a singlet ground-state for these molecules.50 in another recent study, “accurate” spin ground states for molecules 6 and 7 were found with the double hybrid b2plyp functional, where an hs ground-state was obtained for molecule 6 with opbe.51 since the last result is in disagreement with the results of the present study, molecules 6 and 7 were re-optimized using the opbe functional with the geometries from their paper51 as the starting point. the re-optimized structures resulted in spin state splittings that were in accordance with the previous study,51 however, the structures were highly distorted representing only a local minimum on the potential energy surface (and ca. 5–20 kcal·mol–1 above the structures obtained here in table iii). fig. 2. different forms of compounds 4–7. table iii. spin state energies (kcal mol-1) for labile (meso) complexes 6 and 7 using tzp basis, with opbe and s12g functionals, in vacuum and cosmo geo.a spb meso-(fe(nh)s4)nh3 (6) meso-(fe(nh)s4)n2h4 (7) singlet triplet quintet singlet triplet quintet opbe opbe 0 10.3 6.6 0 11.3 6.6 opbe cosmo 0 10.1 3.9 0 10.7 4.4 s12g 0 7.7 2.6 0 8.5 2.5 s12g cosmo 0 7.4 –0.1 0 7.9 0.3 opbe cosmo opbe 0 10.6 7.2 0 11.5 7.1 opbe cosmo 0 9.9 3.5 0 10.1 3.8 s12g 0 7.9 3.5 0 9.7 3.6 s12g cosmo 0 7.0 –0.2 0 8.2 0.5 1406 gruden, stepanović and swart table iii. continued geo.a spb meso-(fe(nh)s4)nh3 (6) meso-(fe(nh)s4)n2h4 (7) singlet triplet quintet singlet triplet quintet s12g opbe 0 10.1 7.5 0 11.1 7.6 opbe cosmo 0 10.7 5.7 0 11.0 6.8 s12g 0 8.4 5.3 0 9.3 5.3 s12g cosmo 0 8.7 3.4 0 9.2 4.6 s12g cosmo opbe 0 10.1 6.8 0 10.8 6.6 opbe cosmo 0 10.5 4.4 0 10.4 5.2 s12g 0 8.7 4.9 0 9.4 4.6 s12g cosmo 0 8.8 2.4 0 8.9 3.2 ageometry optimization with frozen core electrons; bsubsequent single point calculations with all-electron basis sets as in the case of the previous investigated molecules 1–3, after optimization with s12g, somewhat longer bond lengths were obtained in comparison with the opbe geometries. nevertheless, both of them are again in good agreement with the experimental data (tables s-iv–s-vii). in contrast to the fe(ii) complexes 1–3, the fe(iii) p450 model systems 4–7 are prone to the influence of solvent (cosmo calculations) on the spin state ordering, tables ii and iii. calculations with the cosmo solvation model revealed a tendency to favor the hs state for complexes 5–7, and the ls state for complex 4. iron porphyrin chloride and the porphyrazine analogue the structures of fepcl (8) and fepzcl (9) were separately optimized in c4v symmetry for each spin state. similarly to previous results,30,52,53 it was found that the porphyrin core size increased when going from the ls to the hs state, while the fe–cl distance increased from the ls to the is state, and then was slightly decreased in the hs state (tables s-ix and s-x of the supplementary material). opbe and s12g predicted the correct sextet spin ground state for both, fepcl and fepzcl (see table iv). in the case of fepcl, a sextet ground state was predicted with the quartet higher in energy and vice versa for fepzcl, the quartet state was lower in energy. in both cases, the ls state was considerably higher in energy. cosmo calculations revealed a clear and unambiguous solvent effect on the electronic structure, table iv. introduction of the solvent favored the ls state, and as such had small effects on the spin ground state of molecule 9 that has a quartet ground state and a sextet quartet state that is similar in energy. in contrast, for molecule 8, that is in an hs state experimentally and has a low-lying quartet state, the quartet state is stabilized to the extent that it becomes the ground state within all cosmo calculations. of course, it should be added that the spin-state splittings were investigated here through looking at the electronic energy and spin state energetics of iron complexes 1407 hence, enthalpy and entropy effects were ignored. both of these favor the high -spin states. finally, s12g once again showed excellent agreement with the spin state energetics obtained at the opbe level of theory. table iv. spin state energy differences (kcal mol-1, tzp basis) for fepcl (8) and fepzcl (9), with opbe and s12g functionals, in vacuum and cosmo geo.a spb fepcl fepzcl doublet quartet sextet doublet quartet sextet opbe opbe 18.4 3.9 0 12.5 0 3.7 opbe cosmo 16.3 –1.0 0 15.6 0 7.6 s12g 15.7 1.5 0 12.8 0 4.9 s12g cosmo 13.8 –2.9 0 15.8 0 8.6 opbe cosmo opbe 18.0 4.8 0 11.6 0 2.9 opbe cosmo 16.9 –1.7 0 16.6 0 8.2 s12g 15.0 2.3 0 11.8 0 4.2 s12g cosmo 14.1 –3.6 0 16.5 0 9.2 s12g opbe 18.6 4.0 0 12.7 0 3.7 opbe cosmo 16.3 –0.7 0 15.7 0 7.5 s12g 15.4 1.5 0 12.6 0 5.0 s12g cosmo 13.4 –2.8 0 15.4 0 8.6 s12g cosmo opbe 18.3 4.5 0 12.0 0 3.2 opbe cosmo 17.1 –1.6 0 16.8 0 8.1 s12g 15.0 2.0 0 11.8 0 4.6 s12g cosmo 14.0 –3.5 0 16.3 0 9.2 ageometry optimization with frozen core electrons; bsubsequent single point calculations with all-electron basis sets conclusions within this paper, an extension of previous validation14 of dfas for a correct description of the spin states of fe(ii) and fe(iii) complexes is presented. in the present contribution, structure relaxation of the ls, is and hs states of the iron compounds was allowed separately at the opbe and s12g levels of theory and thereby, a more stringent test on the reliability of functionals for providing spin ground states of iron complexes was performed. a detailed comparison with the already proven opbe dfa for spin state energetics, and experimental findings, revealed that s12g performed remarkably well and thus, represents a very promising tool for studying spin states in complicated transition metal systems. moreover, for all iron complexes under investigation, s12g gave a good match with experimental geometries and thus, could be considered as a good starting point for the investigation of transition metal compounds. supplementary material selected bond lengths, opbe/tzp ligand binding energies, as well as coordinates of optimized structures (as additional supplementary file), are available electronically from http://www.shd.org.rs/jscs/ or from the corresponding author on request. 1408 gruden, stepanović and swart acknowledgments. the following organizations are thanked for financial support: the ministerio de ciencia e innovación (micinn, project ctq2011-25086/bqu), the ministerio de economia y competitividad (mineco, project ctq2014-59212/bqu) and the diue of the generalitat de catalunya (project 2014sgr1202, and xarxa de referència en química teòrica i computacional). financial support was provided by micinn and the feder fund (european fund for regional development) under grant ungi10-4e-801, and the serbian ministry of education, science and technological development (grant no. 172035). this work was performed within the framework of the cost action cm1305 ‘‘explicit control over spin-states in technology and biochemistry (ecostbio)’’ (stsm reference: ecost -stsm-cm1305-27360). we would like to thank dr. a. w. ehlers and prof. k. lammertsma (vu amsterdam, netherlands) for help and fruitful discussions in the initial stages of this study. и з в о д релаксација спинских стања код комплекса гвожђа: случај за opbe и s12g функционале маја груден1, степан степановић2 и marcel swart3,4 1хемијски факултет, универзитет у београду, студентски трг 16, 11001 београд, 2центар за хемију, ихтм, универзитет у београду, његошева 12, 11001 београд, 3institut de química computacional i catàlisi (iqcc) and departament de química, universitat de girona, campus montilivi, facultat de ciències, 17071 girona, spain и 4institució catalana de recerca i estudis avançats (icrea), pg. lluís companys 23, 08010 barcelona, spain структуре девет комплекса гвожђа који показују разноврсност експериментално одређених основних спинских стања оптимизоване су теоријом функционала густине (dft), а затим анализиране коришћењем различитих функционала. извршена је обимна валидациона студија новог s12g функционала, са дискусијом о утицају окружења, геометрије, као и његових перформанси у односу на opbe функционал који се већ показао као добар. opbe и s12g тачно предвиђају основно спинско стање код свих испитиваних комплекса гвожђа. како се s12g показао изузетно добро, он се може сматрати поузданим за проучавање енергетике спинских стања у компликованим системима прелазних метала. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {influence of various cosolvents on the calcium oxide-catalyzed sunflower oil ethanolysis} j. serb. chem. soc. 84 (3) 253–265 (2019) udc 546.41–31+544.4+665.334.9:66.095.13: jscs–5181 547.466.3 original scientific paper 253 influence of various cosolvents on the calcium oxide-catalyzed ethanolysis of sunflower oil dušica r. đokić-stojanović1, zoran b. todorović2, dragan z. troter2*, olivera s. stamenković2, ljiljana m. veselinović3, miodrag v. zdujić3#, dragan d. manojlović4# and vlada b. veljković2,5 1zdravlje actavis, vlajkova 199, 16000 leskovac, serbia, 2faculty of technology, university of niš, bulevar oslobođenja 124, 16000 leskovac, serbia, 3institute of technical sciences of the serbian academy of sciences and arts, knez mihailova 35, 11000 belgrade, serbia, 4faculty of chemistry, university of belgrade, studentski trg 12-16, 11000 belgrade, serbia and 5serbian academy of sciences and arts, knez mihailova 35, 11000 belgrade, serbia (received 27 august 2018, revised 28 december 2018, accepted 24 january 2019) abstract: ten organic solvents (triethanolamine, diethanolamine, ethylene glycol, methyl ethyl ketone, n-hexane, triethylamine, ethylene glycol dimethyl ether, glycerol, tetrahydrofuran and dioxane) were applied as cosolvents in the cao-catalyzed ethanolysis of sunflower oil performed in a batch stirred reactor under the following reaction conditions: temperature 70 °c, ethanol-to-oil mole ratio 12:1, initial catalyst concentration 1.374 mol·l-1 and amount of cosolvent 20 % based on the oil amount. the main goals were to assess the effect of the used cosolvents on the synthesis of fatty acid ethyl esters (faee) and to select the most efficient one with respect to the final faee content, reaction duration and safety profile. in the absence of any cosolvent, the reaction was rather slow, providing a faee content of only 89.7±1.7 % after 4 h. of the tested cosolvents, diethanolamine, triethanolamine and ethylene glycol significantly accelerated the ethanolysis reaction, whereby the last two provided a final faee content of 93.1±2.1 and 94.1±1.5 %, respectively, within 0.5 h. however, because of its safety profile, triethanolamine was selected as the best cosolvent for the ethanolysis of sunflower oil catalyzed by calcined cao. keywords: cosolvents; fatty acid ethyl esters; heterogeneous catalysis; transesterification. introduction biodiesel is usually produced by transesterification (alcoholysis) of vegetable oils or animal fats with methanol or ethanol in the presence of a catalyst. it is a promising alternative to mineral diesel due to its favorable properties, such as *corresponding author e-mail: drtroter@gmail.com # serbian chemical society member. https://doi.org/10.2298/jsc180827007d 254 đokić-stojanović et al. biodegradability, lower toxicity, and lower co2 and sulfur emission, can be used on its own or mixed with diesel for diesel-engine vehicles, etc. besides their numerous advantages, the ethanolysis reactions have been studied much less than the methanolysis reactions. ethanol is less toxic than methanol, can be obtained from agrarian waste and is more soluble in oils than methanol. by using ethanol obtained from biomaterials for biodiesel production, a completely agrarian fuel is obtained. compared to fatty acid methyl esters (fames), fatty acid ethyl esters (faees) have higher heat capacity, cetane number, cloud point and pour point, as well as better lubricity properties.1 from the environmental point of view, faees are characterized by less exhaust gas emissions and higher biodegradability in water than fames. in addition, higher esters’ yields can be obtained with ethanol than with methanol. ethanolysis has several drawbacks too, such as a higher cost and lower transesterification reactivity (caused by steric hindrance of the longer carbon chain) of ethanol compared to methanol, formation of azeotrope with water (making its separation more difficult), higher energy consumption, greater influence of water in the reaction mixture on the faees yield, formation of more stable emulsions, as well as higher viscosity and the acid value of faees than those of fames. the production of biodiesel is currently based on utilizing homogeneous catalysts, but it is expected that the importance of heterogeneous (solid) catalysts will increase in the near future. unlike homogeneous catalysts, solid catalysts can easily be separated from the final product and used repeatedly, thus reducing the production costs. the other benefits of solid catalysts are low corrosion risk and low environmental threats. on the other hand, their drawbacks are lower reaction rates, possible deactivation and leaching. one of the most attractive heterogeneous catalysts for transesterification is cao, since it is cheap, highly alkaline and catalytically active, easy for preparation from natural sources and plant or animal wastes, environmentally friendly and easily recovered from the reaction mixture.2,3 moreover, cao can be used repeatedly in batch processes without significant loss of catalytic activity and for long-term continuous biodiesel production.4 by using nano cao-based catalysts, both the specific surface and catalytic activity can be improved, resulting in high biodiesel yields.5 therefore, cao catalyst has a potential to be applied in industrial biodiesel production.6 after being exposed to the air, cao adsorbs co2 and moisture, forming caco3 and ca(oh)2 at the surface of catalyst particles, which lowers its catalytic activity. therefore, activation of cao prior to use, usually thermally, is necessary. vegetable oils and animal fats are not completely miscible with methanol and ethanol, which slows down the transesterification rate. the reaction rate in multiphase batch stirred reactors can be increased by enhancing the mass transfer rate, either by intensive agitation that increases the liquid–liquid contact area or by the addition of a cosolvent (usually an organic solvent) to the reaction mixture cosolvents in sunflower oil ethanolysis 255 that improves the mutual miscibility of the reactants. the preferable cosolvent should have the boiling point similar or close to that of the employed alcohol, allowing their simultaneous recovery by distillation. some of the commonly used cosolvents in transesterification reaction are organic solvents, such as tetrahydrofuran, acetone, dioxane, n-hexane, methyl esters and various ethers. specifically, ethyl acetate is used as either a reactant or cosolvent.7 recently, some ionic liquids and deep eutectic solvents have been used as cosolvents.8 cosolvents can be used in both homogeneously9,10 and heterogeneously9,11 catalyzed transesterification reactions, as well as in supercritical transesterifications.12 some cosolvents (diethyl ether, dioxane and methyl ethyl ketone), which promote the homogeneous koh-catalyzed methanolysis of sunflower oil, inhibit heterogeneous cao-catalyzed methanolysis.9 it could be expected that different cosolvents affect cao-based catalysts differently, as observed in the methanolysis of palm oil over a river snail shells-derived cao catalyst.13 in the present work, the ethanolysis of sunflower oil catalyzed by calcined cao in the presence of various cosolvents (diethanolamine, triethanolamine, ethylene glycol, methyl ethyl ketone, n-hexane, triethylamine, ethylene glycol dimethyl ether, glycerol, tetrahydrofuran and dioxane) was investigated. the main goals were to test the efficacy of these cosolvents in promoting cao activity and to select the cosolvent providing the highest final faee content in the shortest reaction time and having a good safety profile. according to the best of our knowledge, there are no available data on the comparison of the influence of different cosolvents in cao-catalyzed ethanolysis of vegetable oils. experimental materials commercial edible sunflower oil (dijamant, zrenjanin, serbia) was used. physico-chemical characteristics of oil were determined by the standard methods for oils.14 acid, saponification and iodine values were 0.29 and 190 mg koh·g-1 and 1.39 g i2·g -1 oil, respectively. the density (918.4 kg·m-3) and the dynamic viscosity (77.1 mpa∙s) were measured at 20 °c using a pycnometer and a rotational viscometer (visco basic plus v. 0.8, fungilab s.a., barcelona, spain), respectively. cao (extra pure) was provided by sigma-aldrich (st. louis, usa) and was activated by calcination at 550 °c for 2 h.15 the activated cao was cooled and then stored in well closed, glass bottles in a desiccator containing cacl2 and koh pellets. the cosolvents used were: diethanolamine, triethanolamine, ethylene glycol, triethylamine, tetrahydrofuran (all 99.0 %, sigma-aldrich, st. louis, usa), methyl ethyl ketone (hplc grade 99.5 %, jt baker, center valley, pennsylvania, usa), n-hexane (99 %, lgc promochem, wesel, germany), ethylene glycol dimethyl ether (99.0 %, acros organic, geel, belgium), glycerol (ph eur grade, meilab, belgrade, serbia) and dioxane (p.a., merck-millipore, darmstadt, germany). some properties of the used cosolvents and ethanol are listed in table s-i of the supplementary material to this paper. ethyl acetate (99.5 %, merck-millipore, darmstadt, germany) and glacial acetic acid (zorka, šabac, serbia) were employed as solvents. hplc grade methanol, 2-propanol and n-hexane were provided from lab-scan (dublin, ireland). hydrochloric acid (36.0 %) was purchased from centrohem (stara pazova, serbia). the standards 256 đokić-stojanović et al. containing ethyl esters of palmitic, stearic, oleic, linolenic and linoleic acids (20.0 % of each ester), as well as the standards of triolein, diolein and monoolein, were purchased from sigma-aldrich (st. louis, ca, usa). equipment and experimental procedure the reaction was performed in a 500 ml, three-necked glass flask, equipped with a reflux condenser and a magnetic stirrer, at the atmospheric pressure. the flask was placed in a glass vessel, through which heating water circulated from a thermostated bath. the desired amounts of ethanol, cosolvent and calcined cao were added to the flask and stirred at 70 °c for 30 min. the cao concentration was 1.374 mol·l-1 in all experiments. the stirrer was turned off and the corresponding amount of sunflower oil, heated separately to the same temperature, was added to the reaction flask. then, the stirrer was switched on again and the reaction was timed. during the reaction, samples were taken from the reaction mixture and immediately quenched by adding the required amount of aqueous hydrochloric acid solution (5 mol·l-1) to neutralize the catalyst. after centrifugation (sigma 2-6e, germany) at 3500 rpm for 15 min, the upper layer (ester/oil fraction) was withdrawn, dissolved in a 2-propanol/n -hexane (5:4 volume ratio) mixture in the appropriate ratio (1:10 or 1:200 for qualitative thin layer or quantitative liquid chromatography analysis, tlc and hplc, respectively) and filtered through a 0.45 μm millipore filter. the resulting filtrate was used for tlc and hplc analyses. at the end of the reaction, the cao catalyst was separated from the reaction mixture by centrifugation, washed with ethanol, filtered, dried for 2 h at 110 °c and analyzed by the x-ray powder diffraction (xrd) method. analytical methods the chemical composition of each sample from the reaction mixture was first estimated qualitatively by tlc and then quantitatively by the hplc method described elsewhere.15,16 calibration curves were prepared using standard mixtures of faee and acylglycerols, which were used for the quantification of the faee and acylglycerols present in samples of the reaction mixtures. calcium was determined in the upper (faee) and middle (alcohol) layers by icp/aes (inductively coupled plasma atomic emission spectrometry; icap-6500 duo, thermo scientific, uk) analysis. the xrd measurements were performed on a philips pw 1050 x-ray powder diffractometer using ni-filtered cu kα1,2 (λ = 1.54178 å) radiation and the bragg–brentano focusing geometry. the measurements were realized at room temperature over a 2θ range of 7–70° with a scanning step width of 0.05° and a counting time of 3 s per step. results and discussion impact of different cosolvents on the cao-catalyzed ethanolysis of sunflower oil the influence of the tested cosolvents on the cao-catalyzed ethanolysis of sunflower oil was investigated under the same reaction conditions (ethanol-to-oil molar ratio of 12:1, catalyst concentration of 1.374 mol·l-1 and cosolvent amount of 20 % of the oil mass) applied in a previous study of the calcined cao-catalyzed methanolysis of sunflower oil.9 the faee contents during the calcined cao-catalyzed ethanolysis of sunflower oil, compared to the control reaction in the absence of any cosolvent, are shown in fig. 1. the control reaction was slow and hence, quite time-consuming, as was previously observed.17 under the control reaction conditions, an faee content of cosolvents in sunflower oil ethanolysis 257 89.7±1.7 % was achieved in 4 h, while further prolongation of the reaction negligibly increased the faee content. another observance characterizing the control reaction system was slow separation of the two liquid phases of the final reaction mixture (more than 24 h). no reaction between the oil and ethanol occurred in the presence of cosolvents in the absence of cao, implying that the tested cosolvents had no catalytic activity. after 6 h, the faee contents were less than 0.5 % in these reactions (not shown in fig. 1). fig. 1. faee synthesis by the calcined cao-catalyzed ethanolysis of sunflower oil in the presence of different cosolvents: diethanolamine (•), triethanolamine (▲), ethylene glycol (■), dioxane (×), glycerol (○), methyl ethyl ketone (), n-hexane (□), triethylamine (), ethylene glycol dimethyl ether () and tetrahydrofuran (), compared to the control reaction with no cosolvent (, dotted line). reaction conditions: temperature 70 °c, ethanol-to-oil mole ratio 12:1, cao concentration of 1.374 mol·l-1 and amount of cosolvent 20 % based on the oil amount. based on their effects on the reaction performance, the cosolvents were classified as stimulative and inhibitory (represented in fig. 1 by the black and open symbols, respectively). the first group included diethanolamine, triethanolamine and ethylene glycol, while the second group consisted of methyl ethyl ketone, n-hexane, triethylamine, tetrahydrofuran, ethylene glycol dimethyl ether and glycerol. all curves were more or less sigmoidal, indicating a slow reaction in the initial induction stage of the reaction, an accelerated stage in the middle of the reaction and a deaccelerated stage approaching the final equilibrium. these effects were ascribed to mass transfer limitations (both liquid–liquid and liquid– –solid) and the kinetics of the reactions occurring on the surface of the solid catalyst particles,15 as well as the improved miscibility of the reactants that influenced the equilibrium of partition.18 the formed esters could also act as a cosolvent,19 thus contributing further to the mutual miscibility of the reactants. the slow reaction in the final stage was due to the reduced concentrations of the reactants. the curves corresponding to the stimulative cosolvents (diethanolamine, triethanolamine and ethylene glycol) are located above the curve corresponding to the control reaction (dotted curve, fig. 1). obviously, these cosolvents positively 258 đokić-stojanović et al. affected the ethanolysis reaction from its start by improving the mutual miscibility of the reactants (equilibrium of partition), as they shortened the initial induction period, accelerated the reaction and provided a higher final faee content in a shorter time, compared to the control reaction. these results highlighted the kinetic effects, leading to the highest yields for a significantly reduced operating time. the efficiency of these cosolvents was in the following order: triethanolamine > ethylene glycol > diethanolamine. moreover, deep eutectic solvents (dess) might be formed from these cosolvents and hydrogen bond donors, such as glycerol and water,20 shifting the reaction to the right, thus increasing the faee content. due to their strong water affinity, these cosolvents limited the availability of water molecules for triacylglycerols’ hydrolysis and soap formation. since ethylene glycol is moderately toxic for humans and animals, triethanolamine may be recommended as an optimal cosolvent for the cao-catalyzed ethanolysis sunflower oil. with this cosolvent, an faee content of 79.3±6.54 % was achieved in 20 min, which was much higher compared to the control reaction (2.3±1.6 %). in addition, an faee content of 93.1±2.07 % was achieved after 0.5 h, while the same content in the control reaction system was obtained after 6 h. some cosolvents from the second group, such as triethylamine and ethylene glycol dimethyl ether, inhibited the ethanolysis reaction from its beginning, while the others (glycerol, methyl ethyl ketone, n-hexane, triethylamine and tetrahydrofuran) did not affect the ethanolysis reaction up to its middle stage, compared to the control reaction system (no cosolvent present), as indicated by the gathering of their curves around the control curve (fig. 1). thereafter, the reaction rate deaccelerated and the faee content decreased, so the curves corresponding to these hydrophobic cosolvents were, more or less, far from the control curve. dioxane, as an exception, seemed not to affect the catalyst activity, compared to the control reaction, as its curve overlapped with the control curve (dotted line). for the cao-catalyzed methanolysis, todorović et al.9 reported that triethanolamine and ethyl acetate had no effect while dioxane, methyl ethyl ketone and diethyl ether had a negative influence on both the reaction rate and fame content. glycerol accelerated the reaction until 2 h; after which, the faee content was lower, compared to the content achieved in the control reaction. this means that the excess glycerol (amount not used for ca-diglyceroxide synthesis plus amount produced during the ethanolysis) retarded the reaction. this glycerol-induced loss of the ca-diglyceroxide catalytic activity was also observed in the methanolysis of soybean oil.21 the excess glycerol also favored the reverse reaction, hence reducing the faee content. unlike the hydrophilic cosolvents, the hydrophobic cosolvents did not react intensively with water and glycerol in the presence of a solid catalyst, thus having a low influence on the initial ethanolysis reaction rate. a possible inhibitory effect of the hydrophobic cosolvents might be ascribed to their multiple actions in the ethanolysis reaction. the hydrophobic cosolvents in sunflower oil ethanolysis 259 cosolvents did not effectively homogenize the alcohol and oil phases, thus not influencing the rates of monoand diacylglycerol formation, as already reported for thf and n-hexane.22 the blockage of the active sites on the catalyst surface by these cosolvents was also possible, which resulted in prevention of the formation of the catalytically active calcium alkoxide.9 the positive action of the hydrophilic cosolvents was related to the dramatic reduction in the viscosity of the reaction mixture. diethanolamine and triethanolamine could be also transesterified with sunflower oil using the cao catalysts to produce esteramines, known as cationic surfactants, which increased the homogenization of the alcohol and oil phases.23 moreover, because of their polarity, the hydrophilic cosolvents were well soluble in the alcohol, but not in sunflower oil, which aggravated the ethanolysis, thus leading to a decreased faee content.13 in an attempt to explain the impact of different cosolvents on the cao-catalyzed ethanolysis of sunflower oil, the faee content achieved after 1 h was correlated with the hydrophobicity of the cosolvents measured by logp (fig. 2), where p stands for the partition coefficient (log p values are listed in table s-i of the supplementary material). it was indicative that a slight change in the cosolvent hydrophobicity had a major effect on the ethanolysis reaction rate. the characteristic examples were the reactions with triethanolamine and ethylene glycol (improvers), as well as triethylamine and ethylene glycol dimethyl ether (inhibitors). generally, the activity of the cosolvents increased gradually with decreasing log p value, thus suggesting that the organic solvents with a negative logp value might improve the mass transfer rate and increase the mutual miscibility of methanol and oil. the group of hydrophilic cosolvents with a positive effect on the ethanolysis reaction, such as diethanolamine, triethanolamine and ethylene glycol, gather at lower log p values (<–1.5). the others (hydrophobic) fig. 2. correlation of faee content achieved after 1 h with the logp of the cosolvents (et – no cosolvent present, teoa – triethanolamine, deoa – diethanolamine, eg – ethylene glycol, mek – methyl ethyl ketone, hex – n-hexane, tea – triethylamine, glyme – ethylene glycol dimethyl ether, glyc – glycerol, thf – tetrahydrofuran and diox – dioxane). 260 đokić-stojanović et al. cosolvents that inhibited the ethanolysis reaction had a log p value higher than –0.5. although dioxane and ethylene glycol dimethyl ether have similar log p values to ethanol (table s-i), they affected the ethanolysis reaction differently, indicating that specific functional groups might also affect the reaction. however, it appeared that the polarity of the cosolvents was not the only factor with a decisive influence on the ethanolysis reaction rate and faee content. characterization of the used cao in order to gain more insight into the catalytically active phase(s), the used cao was separated from the reaction mixtures and analyzed by xrd. for comparison, commercial calcined cao was also analyzed. the xrd patterns of the samples of freshly calcined cao, calcined cao taken after 2 and 6 h from the control ethanolysis reaction, cao taken after 2 h from the ethanolysis reaction performed in the presence of different cosolvents, as well as cao taken after 6 h from the ethanolysis reaction realized in the presence of teoa are shown in fig. 3. the xrd analysis of the fresh calcined cao sample revealed almost a single well-crystallized cao phase. its characteristic peaks at 2θ 32.4, 37.55, 54.05, 64.35 and 67.55° were in a good agreement with the reported values (jcpds card 43-1001). in addition, the weak peaks at 2θ 18.15, 34.25 and 47.3° and the small peak at 2θ 29.5° indicated the presence of small amounts of ca(oh)2 (jcpds card 84-1263) and caco3 (jcpds card 81-2027), respectively. this verified the successful activation of cao by calcination. substantial changes of the fresh cao were observed during the first 6 h of the ethanolysis conducted in the presence and absence of cosolvents. in all the samples, ca(oh)2 was a dominant phase (the peaks at 2θ about 18.2, 28.8, 34.2, 47.2, 50.8 and 54.5°), along with an amorphous phase characterized by a broad peak (amorphous hump) with a maximum at 2θ about 20°. a small peak at 2θ about 29.5° indicated the presence of a caco3 phase. no xrd peak of a cao phase could be identified in these samples because of cao hydration during the collection step, which resulted in the formation of ca(oh)2.20,24 the amorphous phase probably corresponded to ca-ethoxide, produced in the catalyst preparation step when the calcined cao/ethanol mixture was stirred with or without the chosen cosolvent at 70 °c for 30 min. rodriguez-navarro et al.25 reported that after transformation of the ca(oh)2 particles into ca-ethoxide, the newly formed ca-ethoxide was amorphous and no xrd peaks assignable to crystalline alkoxide were noticed. amorphization occurred because of desolvation during ovendrying of a ca-ethoxide sample. the appearance of ca-diglyceroxide in the samples of calcined cao with glycerol (after 2 h), ethylene glycol (after 2 h) and triethanolamine (after 6 h) might only be speculated based on the weak peaks at 2θ 8.05 and 10.15° (jcpds card 21-1544). the presence of ca-diglyceroxide was explained by the reaction between glycerol and cao that produced ca-digly cosolvents in sunflower oil ethanolysis 261 fig. 3. the xrd patterns of the samples of freshly calcined cao, calcined cao taken after 2 and 6 h from the control ethanolysis reaction, calcined cao taken after 2 h from the ethanolysis reaction carried out in the presence of different cosolvents, as well as calcined cao taken after 6 h from the ethanolysis reaction carried out in the presence of teoa (teoa – triethanolamine, deoa – diethanolamine, eg – ethylene glycol, mek – methyl ethyl ketone, hex – n-hexane, tea – triethylamine, glyme – ethylene glycol dimethyl ether, glyc – glycerol, thf – tetrahydrofuran and diox – dioxane). ceroxide before the start of ethanolysis. moreover, when triethanolamine, diethanolamine or ethylene glycol were present in the reaction medium, they could interact with the produced glycerol, forming dess (first in 1:1 and later in 1:2 mole ratio). these dess could have a positive influence on the reaction.20 it 262 đokić-stojanović et al. should be mentioned that, in the methanolysis of castor oil, only ca(oh)2 and caco3 were identified by xrd analysis of the catalyst after the first use, ascribable to the easy dissolution of ca-diglyceroxide crystalline phase.26 this was confirmed by granados et al.27, who explained it by the lixiviation of the active phase, which deactivated the catalyst. during the ethanolysis, a partial solubilization of ca-diglyceroxide in ethanol could occur, resulting in a soluble precursor, which was transformed into the final solid base catalyst, as seen in the methanolysis of oil performed at 60 °c.21 finally, the amount of ca-diglyceroxide crystals could be below the detection limit of the xrd apparatus, but were active in ethanolysis.26 calcium leaching the well-known drawback of cao as a catalyst is its leaching during the reaction, leading to contamination of the reaction products and the hampering of commercialization. calcium contents in the faee and alcohol phases for the reactions performed with or without different cosolvents are summarized in table i. as the used sunflower oil had a calcium content of only 0.24±0.03 ppm, the increased calcium amount in both the faee and alcohol phases was ascribed to cao leaching. table i. calcium contents (ppm) in the faee and alcohol phases after 2 h for reactions performed under the following reaction conditions: temperature 70 °c, ethanol-to-oil mole ratio 12:1, cao concentration 1.374 mol·l-1 and amount of cosolvent 20 % based on the oil amount cosolvent phase faee alcohol none 50.3±0.5 470±7 nonea 17.1±0.2 – triethanolamine 35.4±0.3 34.2±0.2 triethanolaminea 47.7±0.1 – diethanolamine 236±3 250±2 ethylene glycol 44.4±0.1 – methyl ethyl ketone 13.9±0.1 23.2±0.1 n-hexane 124±1 354±4 triethylamine 442±5 – ethylene glycol dimethyl ether 13.17±0.03 – glycerol 36.9±0.1 33.0±0.3 tetrahydrofuran 56.9±0.2 130.4±2.0 dioxane 106.8±0.1 335±3 aafter 6 h the liquid fraction of the reaction system in the presence of triethanolamine, diethanolamine, methyl ethyl ketone, n-hexane, glycerol, tetrahydrofuran and dioxane after 2 h of reaction consisted of two-phases while it was single-phased cosolvents in sunflower oil ethanolysis 263 in the presence of ethylene glycol, trimethylamine and ethylene glycol dimethyl ether after 2 h of reaction, as well as in the case of triethanolamine after 6 h of reaction. a higher calcium leaching, compared to the reaction without the cosolvents, was observed for the reactions performed in the presence of diethanolamine, n-hexane, triethylamine, tetrahydrofuran or dioxane. the majority of the leached calcium in the two-phase liquid systems was present in the alcohol phase, which was attributed to the change in polarity of the reaction mixture. cao was more soluble in the glycerol/ethanol mixtures than in faee/glycerol/ethanol mixtures, and hence, the alcohol phase had a higher calcium content than did the ester phase, as already observed for methanolysis reactions.19,27,28 it was interesting to notice that, after 2 h of the reaction in the presence of glycerol, an insufficient amount of ca-diglyceroxide was produced, leading to a lower amount of leached calcium species, despite the higher solubility of ca-diglyceroxide compared to that of cao.27 conclusion among different cosolvents applied in the cao-catalyzed ethanolysis of sunflower oil performed in a batch stirred reactor, triethanolamine was selected as the best one for a temperature of 70 °c, ethanol-to-oil mole ratio 12:1, cao concentration of 1.374 mol·l-1 and amount of cosolvent 20 % based on the oil amount. triethanolamine is recommended as the optimal cosolvent for the cao -catalyzed ethanolysis of sunflower oil, as this reaction was fast and significantly improved at atmospheric pressure and a moderate temperature. nomenclature diox – dioxane eg – ethylene glycol et – ethanol faee – fatty acid ethyl ester glyc – glycerol glyme – ethylene glycol dimethyl ether hex – n-hexane hplc – high performance liquid chromatography mek – methyl ethyl ketone tea – triethylamine teoa – triethanolamine tag – triacylglycerol thf – tetrahydrofuran tlc – thin layer chromatography supplementary material additional data are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. this work was funded by the ministry of education, science and technological development of the republic of serbia (project iii 45001). it is also a part of the project 0-14-18 of the sasa branch in niš, serbia. 264 đokić-stojanović et al. и з в о д утицај различитих косолвената на етанолизу сунцокретовог уља катализовану калцијум-оксидом душица р. ђокић-стојановић1, зоран б. тодоровић2, драган з. тротер2, оливера с. стаменковић2, љиљана м. веселиновић3, миодраг в. здујић3, драган д. манојловић4 и влада б. вељковић2,5 1здравље актавис, влајкова 199, 16000 лесковац, 2технолошки факултет, универзитет у нишу, булевар ослобођења 124, 16000 лесковац, 3институт техничких наука српске академије наука и уметности, кнез михаилова 35, 11000 београд, 4хемијски факултет, универзитет у београду, студентски трг 12-16, 11000 београд и 5српска академија наука и уметности, кнез михаилова 35, 11000 београд анализиран је утицај десет органских растварача (триетаноламина, диетаноламина, етилен гликола, метил етил кетона, n-хексана, триетиламина, етиленгликол-диметилетра, глицерола, тетрахидрофурана и диоксана) на принос етил-естара масних киселина (faee) у етанолизи сунцокретовог уља у присуству cao као катализатора изведеној у шаржном реактору. услови реакције су били: температура 70 °c, молски однос етанол:уље 12:1, концентрација катализатора 1,374 mol·l-1 и количина косолвента 20 % у односу на количину уља. без косолвента, реакција је релативно спора, јер се добија садржај faee од 89,7±1,73 % тек после 4 h. од коришћених косолвената само су диетаноламин, триетаноламин и етиленгликол побољшали реакцију, при чему су последња два значајно убрзала реакцију уз садржаје faee од 93,1±2,1 и 94,1±1,5 %, редом, после 0,5 h. на основу експерименталних резултата и безбедносног профила, триетаноламин је одабран као најбољи косолвент за реакцију етанолизе са cao катализатором. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {mechanical characterization of copper coatings electrodeposited onto different substrates with and without ultrasound assistance} j. serb. chem. soc. 84 (7) 729–741 (2019) udc 546.56:531.004.12+544.654.2: jscs–5222 621.793+534–8 original scientific paper 729 mechanical characterization of copper coatings electrodeposited onto different substrates with and without ultrasound assistance ivana o. mladenović1, jelena s. lamovec1*, vesna b. jović1, marko obradov1, dana g. vasiljević radović1, nebojša d. nikolić2# and vesna j. radojević3 1institute of chemistry, technology and metallurgy – department of microelectronic technologies, university of belgrade, njegoševa 12, 11000 belgrade, serbia, 2institute of chemistry, technology and metallurgy – department of electrochemistry, university of belgrade, njegoševa 12, 11000 belgrade, serbia and 3faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11000 belgrade, serbia (received 3 october 2018, revised 7 march, accepted 11 march 2019) abstract: the mechanical properties of systems consisting of copper coatings electrodeposited on both brass sheet (bs) and thick electrodeposited nickel coating (ed ni) substrates have been investigated. the electrodeposition of copper coatings was performed with and without the ultrasound assistance. the ultrasound application decreases root mean square (rms) roughness of deposited cu coating on both applied substrates, as obtained from non-contact afm measurement. the coating roughness is highly dependent on the substrate roughness, being the smallest for the cu coatings deposited on ed ni substrate with the ultrasound mixing. the hardness and adhesion properties were characterized using the vickers microindentation test. model of korsunsky was applied to the experimental data for determination the film hardness and the model of chen-gao was used for the adhesion evaluation. the introduction of ultrasonic agitation caused the changes in the film microstructure, and consequently in the mechanical properties. the copper coatings on both substrates, have higher hardness when deposited from electrolyte with ultrasound agitation. although the type of the substrate has the major influence on the adhesion strength, it can be said that cu electrodeposition with ultrasonic mixing contributes to an increase in adhesion. keywords: cu electrodeposition; ultrasonic agitation; composite hardness; coating adhesion. * corresponding author. e-mail: jejal@nanosys.ihtm.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc181003023m 730 mladenović et al. introduction thin copper coatings are widely used material in electronic industry for the fabrication of contacts in integrated circuits, realization of har (high aspect ratio) channels or fabrication of different structures with copper as a sacrificial layer material.1,2 they are widely utilized in filling and covering flat substrates with regular holes of micro and nano-dimension (damascene and through silicon via (tsv) technologies).3,4,5 electrodeposited copper films have found their use in the fabrication of microelectromechanical (mems) devices for a wide range of applications.6 the copper electrochemical deposition (ed) is a low-temperature and easy -controlled technique with relatively high deposition rate. electrolytes that are commonly used for the copper deposition are sulphate based, with the possibility of adding different additives. the suppressor additives like polyethylene glycol (peg) and chloride ions inhibit the copper deposition, while the accelerator additives like 3-mercapto-1-propanesulfonate acid (mpsa) enhances the rate of the copper deposition.7,8 the introduction of ultrasound (us) into electrochemical deposition is a known way to improve the microstructural and mechanical properties of metal coatings of chromium, cobalt, silver, nickel, iron etc. the ultrasonic mixing of an electrolyte leads to changes in the film microstructure in terms of changing the direction of grain growth. the grains grow preferentially in the manner parallel to the substrate surface. the ultrasound-assisted electrodeposition is a method that can contribute to the improved surface morphology, adhesion and fatigue strength, tensile stress and hardness of the coatings.9,10 the two important mechanical properties of thin metallic coatings are hardness and adhesiveness. the adhesion strength of metallic coatings on various substrates is a serious problem in realization of mems devices due to the delamination of the coatings under stress. therefore, a new ways to achieve improved mechanical properties of electrodeposited metallic coatings are actively being researched. a coating and a substrate can be considered together as a composite system, the properties of which depend not only on particular material properties of the coating and the substrate, but also on the composite parameters such as good adhesion, controlled residual stresses, good corrosion resistance, etc. hardness testing is a widely used technique for assessing the structural and mechanical properties of the composite systems. as the thickness of the coating is very small, the influence of the substrate must be considered during the hardness determination. the measured hardness of composite systems is influenced by a number of factors such as coating thickness, indentation depth, coating and substrate hardness and hardness ratio as well as adhesion. it has been shown that the micro mechanical characterization of electrodeposited copper coatings 731 hardness testing can be a useful technique in assessing the adhesion of thin films to the substrate.11–15 the aim of the study was to analyze the hardness response of the selected composite systems and analyze the results of the quantitative assessment of coating adhesion based on the measured composite hardness. the versatility of composite systems was achieved by combining various substrates and copper coatings. the change of coating microstructure and hardness was performed using the electrodeposition with and without the ultrasonic assistance. the selected thickness of the coatings allowed the analysis of the composite hardness in a large load range, from low loads when the hardness of the film in the measured composite hardness is dominant, to higher loads when the influence of the substrate hardness is primary. the adhesion estimate, quantitatively expressed over a critical reduced depth (the ratio of the plastic zone radius to the indentation depth), was made based on the measurement of the composite hardness for all the composite systems. theory of composite hardness and adhesion models there is a problem of determining the coating hardness separately from the measured composite hardness. the composite and the coating hardness values depend on the applied loads. the change of the composite and the coating hardness with the load depends on the composite system structure. the composite hardness model of korsunsky was found to be appropriate for the experimental data analysis and film hardness determination.11 according to this descriptive model, the correlation between composite hardness, hc, coating hardness, hf, and substrate hardness, hs, is given as: ( )c s f s2 1 1 ' h h h h d k t       = + −    +        ; ' 49 k k t = (1) where t is the thickness of the film, d is the indent diagonal and k' is a dimensionless material parameter related to the composite response mode. for the evaluation the adhesion properties of thin coatings, chen-gao (c-g) method was chosen.12–15 this method introduces the composite hardness as a function of the critical reduced depth, b, beyond which the material will have no effect on the measured hardness. the critical reduced depth b represents the ratio between the radius of the plastic zone beneath the indentation and the indentation depth. a large value of the critical reduced depth corresponds to the good adhesion, while low values indicate poor adhesion of the coatings, as shown in fig. 1. 732 mladenović et al. according to c-g model, the correlation between composite, coating and substrate hardness values and the critical reduced depth b is given by: ( ) ( )c s f s 1m t h h h h mbd  + = + −    (2) where d is the indentation depth and m is the power index. critical reduced depth b has different values for various coating-substrate systems. fig. 1. schematic representation of deformation associated with indentation in a coated substrate.12 the appropriate value for the power index m is found to be 1.8 for a system of soft film on a hard substrate. this value is the intermediate between the value predicted by assuming an area law of mixtures (m = 1) and the mixtures of low volume of (m = 2).16,17 then, introducing the diagonal d of the indentation with d = 7d, for a vickers indentation test and δh = hs – hc, eq. (2), it becomes: ( ) ( )s f7 1m h h th mb d  + + δ =      (3) the critical reduced depth b can be calculated by using eq. (3) with experimental values of hc, hf, t and d. experimental two types of substrates were employed for experimental work. the first substrate was 125 µm-thick brass foil (2601/2 hard, astm b36, k&s engineering) and this substrate is denoted with bs in the further text. the second one was 50 µm-thick ni coating electrodeposited on brass foil, and this substrate is further denoted with ed ni. ed ni substrate was prepared by ni electrodeposition from sulphamate electrolyte consisting of 300 g l-1 ni(nh2so3)2⋅4h2o, 30 g l -1 nicl2⋅6h2o, 30 g l -1 h3bo3, 1 g l -1 saccharine on brass foil. prior to deposition, the brass foil was degreased and chemically polished in acid mixture of hno3:h3po4:ch3cooh of 4:11:5 volume ratio. electrochemical deposition was carried out using direct current (dc) galvanostatic mode with the current density value maintained at 50 ma cm-2. the temperature and ph-value were maintained at 50 °c and 4.20, respectively. copper coatings were electrodeposited on the both substrates from the sulfate electrolyte consisting of 240 g l-1 cuso4⋅5h2o, 60 g l -1 h2so, 0.124 g l -1 nacl, 1 mg l-1 polyethylene glycol (peg), 1.5 g l-1 3-mercapto-1-propanesulfonic acid (mpsa) and deionized water. this mechanical characterization of electrodeposited copper coatings 733 electrolyte was used because it enables electrodeposition of cu in the form of mirror bright coatings.18,19 dc-galvanostatic mode was used for the electrochemical deposition, with the current density value maintained at 50 ma cm-2. the process temperature and ph-value were maintained at 25 °c and 0.30, respectively. the deposition rates of the cu coatings were determined for the deposition performed under different mixing conditions: without stirring and with the assistance of agitation in ultrasonic bath (40 khz, bransonic 220 ultrasonic cleaner). then, the time of the deposition was determined according to the plating surface, current density value and projected film thickness of 20 μm. the thickness of the coatings was controlled by measuring the mass of the samples before and after the deposition process. the cross-sections of several samples were prepared and the thickness of the coatings was measured and checked by optical microscopy. the results of the measurement showed good agreement. the roughness and topographic details analysis of the two used substrates and electrodeposited copper coatings on them without and with ultrasound assistance was done by atomic force microscopy (afm, tm microscopes-vecco in non-contact mode). the root mean square (rms) roughness parameter, that represents the standard deviation of the distribution of surface heights and which is sensitive to large deviation from the mean line, was taken to express the roughness of the substrates and electrodeposited coatings. the mechanical properties of the composite systems were characterized using vickers microhardness tester “leitz, kleinharteprufer durimet i” with loads ranging from 1.96 down to 0.049 n. three indentations were made at each load from which the average value of composite hardness could be calculated. results and discussion surface morphology and roughness analysis the surface morphology and the roughness of used substrates and copper coatings electrodeposited on them, without and with ultrasound assistance, obtained by the afm technique are shown in figs. 2–4, respectively. a) b) fig. 2. substrates in the processes of electrodeposition: a) brass (bs); b) ed ni. surface roughness of the substrates and the coatings was expressed by their root mean square (rms) roughness derived from the afm images for a scanned area of 100 µm2. results given in table i show the influence of ultrasound mix734 mladenović et al. ing of electrolyte on rms roughness. from table i, it can be noticed that the rms roughness for ed ni substrate were about two times smaller than the same values for the bs substrate. a) b) fig. 3. afm images of copper coatings electrodeposited on bs substrate: a) cu coating deposited from silent bath, b) cu coating deposited from ultrasonically mixed electrolyte. a) b) fig. 4. afm images of copper coatings electrodeposited on ed ni substrate: a) cu coating deposited from silent bath; b) cu coating deposited from ultrasonically mixed electrolyte. table i. surface roughness values of substrates and cu coatings electrodeposited with and without the ultrasound agitation substrate coating ultrasound rms roughness, nm bs – – 34.1 bs cu – 126.3 bs cu + 119.5 ed-ni – – 18.1 ed-ni cu – 66.6 ed-ni cu + 52.8 at the first sight, it can be mentioned the considerable increase of rms roughness for cu coatings in relation to the same values for the substrates. in the case of cu electrodeposition on bs substrate without application of ultrasound, the values of rms roughness were 3.70 times larger than the corresponding values for the bs substrate. with ultrasound assisted electrodeposition, these mechanical characterization of electrodeposited copper coatings 735 values were 3.50 times larger than the rms roughness for the brass substrate. the similar changes are also observed with use of ed ni substrate. without the ultrasound assisted electrodeposition, the values of rms roughness were 3.7 times larger than the values for ed ni substrate. however, when cu electrodeposition was performed in the presence of ultrasound on the ed ni substrate, the rms roughness were 3.0 times larger than the values for this substrate. although the values obtained in the presence of ultrasound were smaller than those obtained without the ultrasound agitation, it is necessary to note that there is no any significant difference between the values obtained with and without application of ultrasound. according to expectations, the finest morphology of the electrodeposited cu film was achieved on the fine-grained 50 µm-thick ed ni film, as the substrate in the presence of ultrasound, what is a result of useful effects of both the addition of additives and the application of electrolyte stirring on the metal electrodeposition process.20 absolute hardness of the substrates the indentation tests were performed on brass foils and 50-µm thick ed ni coatings as the substrates in order to observe their response to indentation, due to their different microstructure. the load-independent microhardness values of the substrates were calculated according to the proportional specimen resistance (psr) model:17,20 2c1 2 0 p p a d d d   = +       (4) parameter pc is the critical applied load above which microhardness becomes load independent and d0 is the corresponding diagonal length of the indent. the measured values and linear fit of p/d against d are shown in fig. 5. fig. 5. psr plot of applied load trough indent diagonal, pd-1, vs. indent diagonal, d, for bs substrate and for ed ni substrate. 736 mladenović et al. the value of pc/d0 for the brass substrate was calculated as 7.6×10–4 gpa/µm and for the thick ed ni substrate was 2.4×10–3 gpa /µm. three independent measurements of indent diagonal size for each applied load were performed and the average values were calculated. the absolute substrate hardness and composite hardness values, h (in gpa), were calculated using the equation: 20.01854h p d −= (5) where 0.01854 is geometrical factor for the vickers indenter. variation in composite and coating hardness it is supposed that the systems of electrodeposited copper coatings on brass and thick ed ni coatings as the substrates belong to the “soft film on hard substrate” composite system type. the thickness of the electrodeposited nickel coatings of 50 µm is sufficient in terms of the hardness value to allow the coating to be chosen as the substrate.20 dependence of the composite hardness, hc, on the relative indentation depth (rid – the ratio between indent depth and coating thickness) for the mentioned systems is given in figs. 6. and 7. fig. 6. composite hardness, hc, variation with relative indentation depth, rid, for 20 µm thick cu coating on bs substrate with and without ultrasound assistance. fig.7. composite hardness, hc, variation with relative indentation depth, rid, for 20 µm thick cu coating on ed ni substrate with and without ultrasound assistance. mechanical characterization of electrodeposited copper coatings 737 as shown on fig. 6, the hardness of thin copper coatings electrodeposited with 50 ma cm–2 current density on bs substrate increases with introducing the ultrasound agitation into an electrolyte. the relative indentation depth between 0.1 and 1 corresponds to the hardness response of the whole composite system. the increase of the composite hardness values for the system of electrodeposited copper on ed ni substrate in the presence of ultrasound or without it, is also recorded. it is shown on fig. 7. the tendency of composite hardness hc with rid, as shown in figs. 6 and 7 is characteristic for the “soft film on hard substrate” type of composite systems. with the increase of the relative indentation depth above 1, the hardness values of the system will approach the hardness of the substrate for both systems.20 korsunsky model was applied to experimental data in order to determine the absolute hardness of copper coatings, hf. the fitting results are presented in table ii. table ii. absolute hardness of 20 µm thick ed copper coatings, according to korsunsky model substrate ultrasound hs / gpa hf / gpa k'×10 6 bs + 1.41 0.7355 47.41 bs – 1.41 0.6333 58.71 ed ni + 4.63 1.0700 0.984 ed ni – 4.63 0.9786 1.811 the dimensionless material parameter k' from korsunsky model, is related to the response mode of the composites and defined in eq. (1). as shown in table ii, the ultrasonic agitation contributes to the increase of the electrodeposited copper coatings hardness for coatings, which have been deposited on the same substrates. coatings deposited on ed ni have higher hardness in general, but the tendency of the hardness increase for coatings deposited under ultrasound agitation is preserved. higher absolute hardness for cu coatings, deposited on ed ni substrates, in comparison with cu coatings deposited on bs substrates under the same deposition and mixing conditions, can be explained by higher adhesion energy for cu coatings on ed ni than for cu coatings on bs, as discussed in next section. composite hardness and adhesion the evaluation of the interlayer adhesion strength of 20 µm-thick copper coatings electrodeposited on different substrates was performed according to the composite hardness model of chen-gao.12 the composite hardness of the coating/substrate system is expressed by eq. (2) and in the form of eq. (3) was used to calculate the critical reduced depth b (the ratio between the radius of the plastic zone beneath the indenter and the indentation depth). substrate (hs) and com738 mladenović et al. posite (hc) hardness were calculated using directly measured indent diagonals on substrate and coating surfaces, respectively. the hardness of the ed cu coatings, hf, was obtained as the result of the applied model of korsunsky (table ii). in fig. 8, the measured values of δh = hs – hc are plotted vs. td-1 (ratio between the coating thickness and the indentation diagonal). a linear fit of experimental data was performed, based on eq. (3), and the values of the fitted curve slope k are reported in the same figure. fig. 8. the micro-hardness difference, δh = hc – hs, vs. the ratio of coating thickness to indentation diagonal, td-1, for electrolytically obtained cu coatings on bs and ed ni substrates with and without ultrasound. the slope values (k) are indicated. by using m = 1.8 as the appropriate value of the power index, values of b were calculated and given in table iii. table iii. critical reduced parameter, b, for 20 μm thick ed copper coatings on different substrates substrate ultrasound m k b bs + 1.8 0.2976 24.679 bs – 1.8 0.4704 17.986 ed ni + 1.8 0.1390 278.88 ed ni – 1.8 0.4268 93.158 the good adhesion properties correspond to the increasing values of the plastic deformation zone radius around the indentation and the critical reduced depth, b. high values of the critical reduced depth correspond to better adhesion properties. it is obvious that the values of b are significantly higher for the ed cu coating on ed ni substrate than for the cu coating electrodeposited under same conditions on bs substrate. for both systems, the adhesion increased with the use of ultrasound agitation, which is more noticeable for the ed cu coating system on ed ni substrate, due to more similar microstructures between substrate and coating. mechanical characterization of electrodeposited copper coatings 739 according to fig. 8, it can be concluded that the quality of adhesion can be assessed based on the microhardness measurements. the difference of the substrate hardness and composite hardness, δh = hs – hc, decreases more rapidly with the increase of the indentation load, for poor adhesion. conclusion copper was electrodeposited from sulfate electrolyte with addition of additives for leveling and brightness on brass (bs) and thick electrodeposited nickel coatings (ed ni) substrates. dc-galvanostatic electrodeposition was performed with and without ultrasonic agitation of sulfate electrolyte. the analysis of the influence of the substrate type and ultrasonic mixing on microstructure and composite hardness properties was performed. the tests of microindentation were performed on bs and ed ni substrates to observe their hardness response. the thickness of nickel coating (50 µm) electrodeposited on bs was sufficient to allow the coating to be considered as the substrate. the bs substrate hardness was calculated as 1.41 gpa, and 4.63 gpa for the ed ni substrate. considering experimental results, the composite hardness model of korsunsky was applied to calculate the coating hardness. it is shown that cu coatings electrodeposited on the ed ni substrates have higher values of the hardness, than cu coatings electrodeposited on the brass substrates. higher values of the hardness were obtained for the ultrasound-assisted electrodeposition in comparison with those obtained without application of ultrasound. the composite hardness model of chen-gao was used for the adhesion assessment of cu coatings on different substrates through the values of microhardness. the system obtained by cu electrodeposition on ed ni substrate had significantly better adhesion strength than the system obtained by electrodeposition of cu on bs (brass) as the substrate, with high values of critical reduced depth, b, as the adhesion parameter. an increase in the adhesion strength was observed for the coatings electrodeposited under ultrasound mixing. the quality of adhesion can be assessed based on microhardness measurements. the microhardness difference δh = hs – hc decreases more rapidly with the increase of the indentation load, for poor adhesion. the coating roughness values depend on the substrate type and agitation conditions. the best morphology of the cu coatings was achieved with ultrasonic-assisted electrodeposition on the fine-grained ed ni substrate, with the average roughness value of 43 nm. acknowledgement. this work was funded by ministry of education, science and technological development of republic of serbia through the projects tr 32008, tr 34011 and iii 45019. 740 mladenović et al. и з в о д карактеризација механичких својстава превлака бакра електрохемијски исталожених на различитим подлогама уз примену и без примене ултразвучног мешања ивана о. младеновић1, јелена с. ламовец1, весна б. јовић1, марко обрадов1, дана васиљевић-радовић1, небојша д. николић2 и весна ј. радојевић3 1ихтм – центар за микроелектронске технологије, универзитет у београду, његошева 12, 11000 београд, 2ихтм – центар за електрохемију, универзитет у београду, његошева 12, 11000 београд и 3технолошко–металуршки факултет, универзитет у београду, карнегијева 4, 11000 београд испитиванa су механичкa својствa композитних система који се састоје од електрохемијски исталожених превлака бакра на месингу (bs) и дебелим електрохемијски исталоженим превлакама никла на месинганој фолији (ed ni) као супстратима. превлаке бакра на наведеним супстратима су исталожене из електролита без мешања или са ултразвучним мешањем. неконтактна микроскопија атомских сила (afm) је показала да храпавост, изражена средњом вредношћу квадратног одступања (rms), исталожених превлака бакра на обе врсте супстрата опада са применом ултразвучног мешања. храпавост превлака у највећој мери зависи од храпавости супстрата, при чему су електрохемијски исталожене превлаке бакра са најмањом храпавошћу реализоване на супстратима ni електрохемијским таложењем из електролита мешаног применом ултразвука. механичка својства тврдоће и адхезије превлака су анализирана викерсовим тестом утискивања са малим оптерећењима. за израчунавање апсолутне тврдоће превлака коришћен је модел korsunsky, док је за процену адхезије коришћен модел chen -gao. примена ултразвучног мешања током процеса електрохемијског таложења бакра довела је до промена у микроструктури превлака, па самим тим и промена у механичким својствима превлака. превлаке бакра на оба супстрата имају већу тврдоћу када се таложе из електролита уз ултразвучно мешање. на адхезију превлаке на подлогама највише утиче тип супстрата, али се може рећи да примена ултразвучног мешања доприноси побољшању адхезије електрохемијски исталоженог бакра на наведеним супстратима. (примљено 3. октобра 2018, ревидирано 7. марта, прихваћено 11. марта 2019) references 1. w. h. teh, l. t. koh, s. m. chen, j. hie, c. y. li, p. d. foo, microelectron. j. 32 (2001) 579 (https://doi.org/10.1016/s0026-2692(01)00035-0) 2. a. maciossek, b. lochel, h. j. quenzer, b. wagner, s. schulze, j. noetzel, microelectron. eng. 27 (1995) 503 (https://doi.org/10.1016/0167-9317(94)00154-m) 3. f. wang, p. zeng, y. wang, x. ren, h. xiao,w. zhu, microelectron. eng. 180 (2017) 30 (http://dx.doi.org/10.1016/j.mee.2017.05.052) 4. w. p. dow, m. y. yen, c. w. liu, c. c. huang, electrochim. acta 53 (2008) 3610 (https://doi.org/10.1016/j.electacta.2007.12.048) 5. k. kondo, n. yamakawa, z. tanaka, k. hayashi, j. electroanal.chem. 559 (2003) 137 (https://doi.org/10.1016/s0022-0728(03)00110-4) 6. j. lamovec, v. jović, i. mladenović, m. sarajlić, v. radojević, in proc. 57th etran conference, 3–6 june, 2013, zlatibor, serbia, pp. mo3.3 7. l. bonou, m. eyraud, y. massiani, electrochim. acta 47 (2002) 4139 (https://doi.org/10.1016/s0013-4686(02)00356-0) 8. m. datta, d. landolt, electrochim. acta 45 (2000) 2535 (https://doi.org/10.1016/s00134686(00)00350-9) mechanical characterization of electrodeposited copper coatings 741 9. c. t. walker, r. walker, electrodepos. surface treat. 1 (1973) 457 (https://doi.org/10.1016/0300-9416(73)90029-1) 10. r. cui, y. he, z. yu, w. shu, j. du, in proc. of 11th international conference on electronic packaging technology & high density packaging, 16-19.8, 2010, china, p.847 11. a. m. korsunsky, m. r. mcgurk, s. j. bull, t. f. page, surface coat. technol. 99 (1998) 171 (https://doi.org/10.1016/s0257-8972(97)00522-7) 12. m. chen, j. gao, modern phys. lett., b 14 (2000) 103 (https://doi.org/10.1142/s0217984900000161) 13. j. l. he, w. z. li, h. d. li, appl. phys. lett. 69 (1998) 1402 (https://doi.org/10.1063/1.117595) 14. h. qingrun, j. gao, modern phys. lett., b 11 (1997) 757 (https://doi.org/10.1142/s0217984997000931) 15. l. magagnin, r. maboudian, c. carraro, thin solid films 434 (2003) 100 (https://doi.org/10.1016/s0040-6090(03)00469-3) 16. q. r. hou, j. gao, s. j. li, eur. phys. j., b – cond. matter complex sys. 8 (1999) 493 (https://doi.org/10.1007/s100510050716) 17. h. li, r. c. bradt, j. mater. sci. 28 (1993) 917 (https://doi.org/10.1007/bf00400874) 18. n. d. nikolić, z. rakočević, k. i. popov, j. electroanal. chem. 514 (2001) 56 (https://doi.org/10.1016/s0022-0728(01)00626-x) 19. n. d. nikolić, z. rakočević, k. i. popov, j. solid state electrochem. 8 (2004) 526, (https://doi.org/10.1007/s10008-003-0467-8) 20. j. lamovec, v. jović, d. randjelovic, r. aleksic, v. radojevic, thin solid films 516 (2008) 8646 (http://dx.doi.org/10.1016/j.tsf.2008.06.035). {synthesis, fluorescent studies, antioxidative and $\alpha$-amylase inhibitory activity evaluation of some lanthanide(iii) complexes} j. serb. chem. soc. 83 (5) 561–574 (2018) udc 546.654+542.913: 535.371: 542.9+ jscs–5096 547.571’551 original scientific paper 561 synthesis, fluorescent studies, antioxidative and α-amylase inhibitory activity evaluation of some lanthanide(iii) complexes surya philip, preethy soosan thomas and kochukittan mohanan* department of chemistry, university of kerala, trivandrum, kerala-695581, india (received 18 september, revised 5 december, accepted 25 december 2017) abstract: a novel heterocyclic ligand, viz. 1,2-dihydro-1,5-dimethyl-4-[[1-2 -oxo-2h-1-benzopyran-3-yl)ethylidene]amino]-2-phenyl-3h-pyrazol-3-one, was prepared by condensing 3-acetylcoumarin with 4-aminoantipyrine. this ligand is versatile in forming complexes with lanthanum(iii), praseodymium(iii), samarium(iii), gadolinium(iii) and dysprosium(iii) ions. the ligand and the metal complexes were characterized through various physicochemical and spectral studies. the spectral studies revealed that the ligand is coordinated to the metal ion in a bidentate fashion, through the azomethine nitrogen and the oxygen atom of the pyrazolone ring. the powder xrd patterns of ligand and the dysprosium(iii) complex were studied. the photoluminescent properties of ligand and metal complexes were evaluated and the relative quantum yields were determined. it was observed that in all cases the metal ions enhanced the luminescence intensity. the α-amylase inhibitory activity of the ligand and the metal complexes was evaluated using the method of apostolidis. the metal complexes exhibited increased activity compared to the ligand. the antioxidant property was also examined using the dpph assay and the metal complexes were found to be more potent antioxidants than the ligand. keywords: 3-acetylcoumarin; lanthanides; luminescence; dpph assay; schiff base; rare earths. introduction the importance of lanthanide(iii) complexes in modern technological applications is mainly associated with their spectroscopic, magnetic, photophysical and biological properties.1 the coordination chemistry of lanthanide(iii) complexes with schiff base ligands has been investigated mainly due to their unique structures and potential pharmacological properties.2 in recent years lanthanide(iii) complexes also have attracted much attention, because of their use in various fields, such as organic light emitting diodes, fluorescent probes, sensors, * corresponding author. e-mail: drkmohanan@rediffmail.com https://doi.org/10.2298/jsc180918010p 562 philip, thomas and mohanan mri agents, laser materials, molecular optoelectronic devices, etc.3–6 in view of their unique electronic and coordination properties, schiff bases represent a group of organic ligands used for the preparation of coordination compounds showing interesting physical, chemical and biological properties. based on the sharp f–f transitions and high quantum yields, it is often thought that lanthanide(iii) complexes are the most important components in pursuit of new luminescence materials. these properties of lanthanide complexes and their potential applications were the encouragement to continue studies on some new lanthanide complexes with a schiff base ligand. the schiff base was prepared by condensing 3-acetylcoumarin with 4-aminoantipyrine possessing a wide spectrum of biological activity, such as antibacterial, antifungal, anti-inflammatory, anticoagulant, anti-hiv and antitumor properties.7,8 the resulting schiff base was expected to surpass either of the parent reagents in its effectiveness of complex formation and biological activities. apart from this, coumarin derivatives have been used as antenna ligands to harvest the absorption of photon energy and to enhance the efficiency of emission. considering these aspects, the particular ligand was selected and characterization of the ligand and the metal complexes were achieved on the basis of elemental analysis, molar conductance measurements, and ir, nmr and uv–vis spectral studies. the luminescence property of ligand and its effect on metallation was also examined. in view of the biological significance of the parent compounds, the antioxidant property and α-amylase inhibitory activity of the ligand and the metal complexes were also investigated. experimental materials all chemicals used in this work were of analytical reagent grade, purchased from aldrich, merck, fischer, sisco (india), etc. solvents used for physicochemical measurements, were purified by standard methods. elemental analysis for c, h and n were realised using microanalysis. the metal content was estimated by the oxalate-oxide method.9 molar conductance measurements were realised using 10-3 m solutions of the complexes in a suitable solvent at room temperatures using a systronic direct reading conductivity meter type 305. the infrared spectra were recorded using kbr discs on a shimadzu ftir 8000 spectrophotometer and electronic spectra were recorded on a hitachi 320 uv–vis spectrophometer. powder x-ray diffraction studies were conducted using a siemens d 5005 model x-ray spectrometer and indexing was performed using mcmaille software. proton nmr spectra were recorded using on a jeol gsx 400 nb 400 mhz spectrometer in dmso-d6. the fluorescence property was measured using jasco 8300 spectrofluorometer. synthesis of lanthanide(iii) chlorides lanthanide(iii) chlorides were prepared by dissolving the corresponding lanthanum oxide in 50 % hydrochloric acid and subsequently the salt formed was crystallised after concentrating the solution on a water-bath. synthesis, fluorescence, biological studies of lanthanide(iii) complexes 563 synthesis of ligand 3-acetylcoumarin, the starting material for the synthesis of the ligand (acap) was prepared by the knoevenagel method. the ligand was prepared by condensing 3-acetylcoumarin with 4-aminoantipyrane in 1:1 mole ratio in methanolic medium. a hot methanolic solution (30 ml) of 3-acetylcoumarin (0.01 mol) was mixed with a hot methanolic solution (20 ml) of 4-aminoantipyrine (0.01 mol). the mixture was refluxed on a water-bath for 3 h. the resulting solution was concentrated to half of its initial volume and allowed to crystallise. the crystals formed were collected on a filter paper, washed with methanol and dried and further purified by recrystallization from methanol. the melting point was found to be 194 °c. more details are given in the supplementary material to this paper. synthesis of metal complexes all the lanthanide(iii) complexes were prepared according to the following general procedure. to a hot methanolic solution (20 ml) of the ligand (0.01 mol), a methanolic solution (20 ml) of lanthanide(iii) chloride (0.01 mol) was added and refluxed for 1h on a water bath. the ph of the solution was adjusted to 6.5–7.0 and the refluxing process was continued for 8– –10 h. the resulting solution was concentrated to half of its initial volume. the solid mass separated was filtered, washed successively with small portions of water, methanol and ether and finally dried in vacuum over p4o10. α-amylase inhibitory assay the in vitro α-amylase inhibitory activities of the ligand and metal complexes were determined. different concentrations of sample were taken and made up to 100 µl using 25 mm phosphate buffer containing 25 µl of porcine α-amylase at a concentration of 0.5 mg ml-1 and were incubated at 25 °c for 10 min. after pre-incubation, 25 µl of 0.5 % starch solution in 25 mm phosphate buffer (ph 6.9) was added. the reaction mixtures were then incubated at 25 °c for 10 min. the reaction was stopped with 50 µl of 96 mm 3,5-dinitrosalicylic acid colour reagent. the microplate was then incubated in a boiling water bath for 5 min and cooled to room temperature. the solution thus prepared was taken as the test solution. absorbance was measured at 540 nm using a microplate reader. a control was prepared using the same procedure replacing the sample with distilled water: control test inhibition 100 control − = (1) antioxidant assay the radical scavenging activity of the ligand and the metal complexes were determined using dpph assay. the decrease in the absorption of the dpph solution after the addition of an antioxidant was measured at 517 nm. ascorbic acid (10 mg ml-1 dmso) was used as reference. 1,1-diphenyl-2-picrylhydrazyl is a stable free radical with a red colour that turns yellow when scavenged. this property of dpph is used in assessing free radical scavenging activity. the scavenging reaction between dpph and an antioxidant (h–a) could be written as: [ ]dpph h a dpph h a+ − → − + (2) antioxidants react with dpph and reduce it to dpph–h and as a consequence the absorbance decreases. the degree of discoloration indicates the scavenging potential of the antioxidant compounds or extracts in terms of hydrogen donating ability. different volumes (2.5–40 µl) of sample solutions (1.25, 2.5, 5.0, 10 and 20 mg/ml) were made up to a final volume of 40 µl with dmso and 2.96 ml dpph (0.1 mm) solution was added. the reaction 564 philip, thomas and mohanan mixture was incubated in the dark at room temperature for 20 min, after which, the absorbance of the mixture was read at 517 nm. the dpph was taken as the control. percentage of inhibition was measured using eq. (1). results and discussion analytical data indicated that condensation of 3-acetylcoumarin with 4-aminoantipyrine occurred in 1:1 mole ratio. the ligand is soluble in common organic solvents, such as methanol, ethanol, benzene, toluene, etc. analytical data of the complexes, given in table s-i of the supplementary material to this paper, are in good agreement with their formulation. all the complexes possess 1:1 metal–ligand stoichiometry and possess good keeping qualities. they are non-hygroscopic solids, soluble in dmso and dmf. the molar conductance values of the complexes, given in table s-i, were in the range of 6.4–13.1 s cm–2 mol–1 in dmso solution at room temperature. these values indicated that the complexes were non-electrolytes in nature.10 ir spectra the relevant ir spectral data provide important information of coordination of ligand to the metal ion and are presented in table s-ii of the supplementary material. the ir spectra of the ligand and its lanthanum(iii) complex are given in the figs. s-1 and s-2 of the supplementary material. the ligand shows a medium intensity band at 1590 cm–1, characteristic of ν(c=n) stretching vibration. this band in the complex is shifted to a lower frequency by ≈25 cm–1 indicating the involvement of the azomethine nitrogen in coordination to the metal ion.11 apart from this, the stretching frequency of the carbonyl group on the antipyrine moiety showed ν(c=o) at 1654 cm–1 in the free ligand that was shifted in the ir spectra of complexes (≈15 cm–1) to lower wave numbers. this indicated that the c=o was bonded to the metal ion.12 a medium intensity band in the ligand at 1721 cm–1 was attributed to the carbonyl group of the coumarin moiety. the lactone carbonyl frequency of the complexes remain unchanged in the position and intensity, indicating the non-coordination of the lactonic oxygen.13 the ir spectra of complexes showed a broad band in the region 3400–3460 cm–1 that was assigned to νo–h of co-ordinated water. this is further substantiated by the additional band occurring in the 850–840 cm−1 region.14 the complexes showed non-ligand bands that occurred in the region of 432–441, 420–427 and 338–354 cm–1, which were assigned to ν(m–o), ν(m–n) and ν(m–cl) vibrations, respectively.15 the ir spectral data indicated that the ligand behaves as a bidentate ligand with no donor sites coordinated to the metal ions via the azomethine nitrogen and the pyrazolone oxygen. synthesis, fluorescence, biological studies of lanthanide(iii) complexes 565 nmr spectra the 1h-nmr and 13c-nmr spectra of both the ligand and the lanthanum(iii) complex were recorded in dmso-d6 (figs. s-3 and s-4 of the supplementary material). the 1h-nmr spectrum of the ligand showed a multiplet at 7.25–7.57 ppm due to aromatic protons. the complex showed a slight upfield shift of the aromatic protons found in the range 7.23–7.54 ppm, which confirmed the bond formation. two singlets at 2.55 and 3.21 ppm were attributed to pyrazolone ring methyl protons of c–ch3 and n–ch3, respectively.16 a singlet was observed at 2.41 ppm in the spectrum of the ligand due to the c–ch3 methyl proton of the coumarin moiety.17 the 13c-nmr spectra of the ligand and the corresponding metal complexes were recorded in dmso-d6 using tms as the internal standard. in the case of the 13c-nmr spectra, the aromatic carbons in the ligand showed signals in the range of 113–134 ppm that were slightly shifted to 109– –129 ppm in the complexes. the considerable shifts in the position of the carbon atoms indicated the bonding of the azomethine nitrogen to the metal atom. the signal due to the lactone (c=o) carbon atom remained almost unchanged in the 13c-nmr spectra of the metal complexes.18 this is a clear indication of the non -coordination of the lactone (c=o). electronic spectra the electronic absorption spectra of the ligand and its complexes have been recorded in dmso solution in the range of 200–800 nm and the results are presented in table i. the electronic spectrum of ligand exhibited characteristic bands at 279 and 355 nm assigned to π→π* and n→π* transitions, respectively. upon complexation, the absorption bands of the complexes were slightly blue shifted compared to those of the free ligand (fig. 1). this is indirect evidence for coordination of the ligand with the lanthanide ion.19 it also gives conclusive support that the same structural form of the ligand also exists in the metal complexes. the visible spectral bands of the lanthanide complexes are hypersensitive to stereochemistry.20 an enhancement of the intensity of certain hypersensitive bands of the praseodymium(iii) complex, compared to respective aquated ion was observed.15 these variations could be attributed to the action of an inhomogeneous electromagnetic field and by changes in the symmetry of the field around the lanthanide ion. the spectra of the complexes exhibited alterations in intensity and shifted the positions of the absorption bands relative to the ligand. this slight shift was attributed to the effects of ligand field upon the inter-electronic repulsion between the 4f electrons.21 various spectral parameters, such the nephelauxetic ratio (β), covalency factor (b1/2), sinha parameter (δ), covalency angular overlap parameter (η) were calculated (table ii) from the hypersensitive transitions of the spectrum of the 566 philip, thomas and mohanan praseodymium(iii) complex.22 the β values being less than unity and the positive values of b1/2 and δ indicate covalent metal–ligand bonding. in the case of the praseodymium(iii) complex, an enhancement in the intensity of certain hypersensitive bands compared to the aquated ions was noted, table ii. this could be ascribed to quadrupolar effects owing to an inhomogeneous electrostatic field and changes in symmetry around the lanthanide(iii) ion. fig. 1. electronic absorption spectra of ligand and the complexes in 10-3m dmso. table i. electronic spectral data of ligand and the metal complexes complex λabs / nm π-π* n-π* apac 279 355 [la(acap)(h2o)3cl3] 276 348 [pr(acap)(h2o)3cl3] 277 336 [gd(acap)(h2o)3cl3] 258 350 [sm(acap)(h2o)3cl3] 279 352 [dy(acap)(h2o)3cl3] 271 351 complex aqua average value of β   =       b b (3) ( )1/ 21/ 2 1 2 b β− = (4) synthesis, fluorescence, biological studies of lanthanide(iii) complexes 567 ( )1 (%) = 100 β δ β − (5) 1/2 1/2 1 β η β − = (6) table ii. hypersensitive parameters of the praseodymium(iii) complex complex wavelength, nm hypersensitive parameters β b1/2 δ / % η (pr3+)aqua 340 0.9882 0.054 1.194 0.0595 [pr(acap)(h2o)3cl3] 277, 336 – – – – the nephelauxetic parameter is close to unity evidencing only a slight covalent character of the metal–ligand bond interaction. the low b1/2 value suggested a small participation of the 4f orbitals in the bonding and along with the δ value indicated the relatively low covalent character of the bond.23 the lanthanide(iii) ions possess unique spectral properties resulting from electronic f–f transitions within the 4f shells, which is shielded by the filled 5s and 5p orbitals. the shielding leads to minimal interactions with the field of surrounding molecules, so there were only weak perturbations of the electronic transition between the energy levels of the f electrons.24 luminescence properties the excitation and emission spectra of all the lanthanide(iii) complexes and the ligand in dmso at room temperature were recorded. the emission spectra were recorded in the range of 320–600 nm with an excitation wavelength of 300 nm. the excitation spectra of the complexes were obtained by observing characteristic emission of the lanthanide(iii) ion. the maximum of the excitation spectra showed similar features to those of the absorption spectra of the related complexes. the luminescent properties of lanthanide complexes are due to the efficient intramolecular energy transfer from the lowest energy level of the triplet state of the organic ligand to the resonance energy level of the lanthanide ions. the corresponding emission spectra of all the complexes of lanthanum(iii), praseodymium(iii), gadolinium(iii), dysprosium(iii) and samarium(iii) ions were recorded (fig. 2). in the case of ligand, two peaks were observed at 402 and 334 nm due to the presence of ligand chromophore moieties. all the lanthanide(iii) complexes showed an enhancement in the emission intensity compared to that of the ligand. the enhancement of luminescence intensity depends on the nature of the lanthanide ion that was in the order sm(iii)>dy(iii)>pr(iii)>la(iii)>gd(iii). the enhancement of the emission intensity of all metal complexes was clear evidence of metal–ligand complexation. enhancement of luminescence intensity might be 568 philip, thomas and mohanan attributed to the coordination of the ligand to the lanthanide(iii) ion, which effectively increases the rigidity of the complexes and reduces the loss of energy by radiationless decay.25 due to intraligand π→π* transitions, the emission spectra of la(iii), pr(iii), sm(iii), dy(iii) and gd(iii) complexes exhibited ligand emission bands. the luminescence emission bands were slightly blue shifted in the complexes compared to that of the free ligand. the blue-shift emission originated fig. 2. fluorescence emission spectra of the ligand acap and its lanthanide complexes. from the emission of a ligand-to-metal charge transfer.26 the lanthanide complexes do not exhibit typical emission in the visible region, which could be attributed to the energy gap between the triplet level of the organic ligand and the lowest resonance energy level for lanthanide ions. this gap may not favour the energy transfer process from organic ligand to the lanthanide ion. another possible reason is the fluorescence quenching effect of coordinated water molecules in these complexes. the samarium(iii) complex exhibited an increase in intensity compared to the ligand and a marginal blue shift in wavelength (376 nm) and the peak around 334 nm disappeared. the enhanced intensity of the samarium(iii) complex suggested a more efficient energy transfer from the lowest triplet state energy level (t1) of the ligand to the lowest resonance energy level of samarium(iii). the lanthanum(iii) complex showed a decrease in fluorescence intensity as the la(iii) ion has a completely empty 4f shell and no suitable level that could receive energy from the triplet state energy level of the ligand. for gd3+, the lowest lying resonance level lies far above triplet level of the ligand and energy transfer from the triplet to the rare earth ion does not take place, resulting in ligand fluorescence. the efficiency of triplet to ion energy transfer is con synthesis, fluorescence, biological studies of lanthanide(iii) complexes 569 trolled by the covalent nature of ligand-metal bonding. the enhancement of fluorescence through complexation opens up the opportunity for photochemical applications of these complexes. the fluorescence of the ligand was quenched by the photo-induced electron transfer process due to the presence of lone pair of electrons of the donor atoms in the ligand. this process was prevented by complexation of ligand with the metal ion. the ligand-to-lanthanide energy transfer was confirmed by the quantum yield values. the relative quantum yield of the ligand and the metal complexes were determined with reference to 2-aminopyridine, as given in table iii. table iii. photoluminescence data for the ligand and its metal complexes compound λem / nm max emission intensity, nm quantum yield, φ acap 402 374 0.22 [la(acap)(h2o)3cl3] 376 590 0.24 [pr(acap)(h2o)3cl3] 385 622 0.31 [sm(acap)(h2o)3cl3] 387 733 0.35 [gd(acap)(h2o)3cl3] 382 595 0.28 [dy(acap)(h2o)3cl3] 422 676 0.34 the relative quantum yields were determined using the equation: 2 r x x x r r a e a e η φ η     =         (7) in this case, the quantum yield of the unknown was compared with that of a reference sample: where φ is the luminescence quantum yield, a is the absorbance at the excitation wavenumber, e is the area under the corrected emission curve (expressed in number of photons), and η is the refractive index of the solvents used. the subscripts r and x refer to the reference and the unknown samples, respectively. samarium(iii) complex showed a higher quantum yield than that of the ligand. the stronger emission intensity was obtained by the photoexcitation of the coumarin-based ligand at a wavelength of 325 nm rather than by the direct photoexcitation of the lanthanide ions. for the lanthanide complexes, the quantum yield depends on the excited state of the ligand or metal ion because sensitization of the lanthanide ion can go through several energy migration paths, the efficiency of which depends on the particular levels involved. x-ray diffraction study the x-ray diffraction pattern of the ligand was recorded (fig. s-5), and the details are presented in table s-iii of the supplementary material. the bragg angles and the set of interplanar spacing obtained were related to the unit cell parameters and miller indices and these values were assigned to the individual 570 philip, thomas and mohanan reflections with the aid of expressions involving sin 2θ. the diffractogram of ligand recorded 20 reflections between 2θ ranging from 6 to 54° with a maxima at 2θ = 25°, which corresponds to an interplanar spacing d = 3.53 å.27 however the x-ray diffraction pattern of the corresponding dysprosium(iii) complex did not exhibit any prominent reflections and hence it could not be indexed to any crystalline form. hence it could be concluded that the dysprosium(iii) complex is amorphous in nature. the grain size was estimated using the scherer formula, dxrd = 0.9λ/(βcos θ) and average particle size of the ligand was found to be 30 nm.28 structure of the ligand and the complexes based on all the above physicochemical and spectral data, the structure of ligand and metal complexes were assigned and are given in figs. 3 and 4, respectively. fig. 3. structure of the ligand. fig. 4. structure of [la(acap)(h2o)3cl3]. α-amylase inhibitory activity an α-amylase inhibition assay was performed for the ligand and metal complexes using the method of apostolidis.29 pyrazole derivatives were used as synthetic hypoglycemic agents that reduced the formation and absorption of glucose in the intestine. as a result, the development of postprandial hyperglycemia was prevented. the α-amylase inhibitory activity of the ligand and the metal complexes are given in table iv. the ligand and the complexes inhibited the α-amylase activity in a dose dependent manner. synthesis, fluorescence, biological studies of lanthanide(iii) complexes 571 the ic50 values of the ligand and metal complexes are given in table iv. the ic50 value for ligand was found to be 2.32 mg ml–1. among the complexes, gadolinium(iii) showed an ic50 value of 0.29 mg ml–1 which is close to that of the standard acarbose. it was observed that the α-amylase inhibitory activity of the ligand was enhanced on complexation with metal ions. the gadolinium(iii) complex had a significantly higher percentage -amylase inhibition, i.e., 62.16 % at 0.5 mg ml–1. the observed reduction in enzyme activity was presumably due to enzyme deactivation through chelation. the insulin-like capacity of a metal complex is generally related to its ability to lower the blood glucose level by activating glucose transport into the cells of peripheral tissues.30 table iv. ic50 values for the α-amylase inhibitory activity of the ligand and metal complexes compound ic50 / mg ml-1 acap 2.32 [la(acap)cl3(h2o)3] 0.31 [pr(acap)cl3(h2o)3] 0.78 [sm(acap)cl3(h2o)3] 0.36 [gd(acap)cl3(h2o)3] 0.29 dy(acap)cl3(h2o)3] 0.68 acarbose 0.112 an increased α-amylase inhibition activity would decrease the rate of glucose absorption and concentration of postprandial serum glucose.31,32 this effect would delay the degradation of starch and oligosaccharides, which would cause a decrease in the absorption of glucose and consequently inhibit an increase in postprandial blood glucose. antioxidant activity oxidative stress is the basis of many serious diseases and one of its primary characteristics is the cellular imbalance between endogenous antioxidant defences and ros generation inside the cells. the antiradical properties of chromones have attracted increased interest and consequently, have been extensively investigated. the schiff base ligand and its lanthanide(iii) complexes were screened for free radical scavenging activity using the dpph method. dpph is a relatively stable nitrogen-centred free radical that easily accepts an electron or hydrogen radical to become a stable diamagnetic molecule. dpph radicals react with suitable reducing agents as a result of which the electrons become paired off and the solutions lose colour stoichiometrically depending on the incoming electron. the scavenging activity of the ligand and its metal complexes were compared with that of ascorbic acid. the results showed that the complexes exhibited scavenging effects with increasing concentration in the range of 1.25–20 μg ml–1. the ic50 values of the ligand and metal complexes were determined (table v). 572 philip, thomas and mohanan the complexes showed enhanced antioxidant activity at high concentrations than the ligand.33 the synthesized compounds scavenged dpph radicals in a concentration-dependent manner. the ic50 values of the complexes were found to be close to that of the standard ascorbic acid (table v). the increased activity of the complexes might be attributed to chelation of the organic molecules to the lanthanide ions that exert different and selective effects on the radicals of biological systems. electron-donating substituents to an aromatic ring can increase the radical scavenging activity as a result of increased electron density at the carbon atoms in the ring. in addition to this, the metal binding is also attributed to the antioxidant activities. generally, chelation prevents metal-catalyzed free radical generation and their subsequent reactions and accordingly protects very important biologically active molecules from oxidative stress.34 table v. ic50 values for the antioxidant activity of the ligand and metal complexes compound ic50 / mg ml-1 acap 8 [la(acap)cl3(h2o)3] 4.9 [pr(acap)cl3(h2o)3] 5.7 [sm(acap)cl3(h2o)3] 4.7 [gd(acap)cl3(h2o)3] 4.2 dy(acap)cl3(h2o)3] 5.9 ascorbic acid 2.5 thus, lanthanide (iii)complexes were found to be more effective free radical scavengers than their corresponding ligand. this study may be useful for the synthesis of lanthanide(iii) complexes that prevent oxidative damage because in the body, many free radicals are generated that produce diseases such as aging, cancer, and cardiovascular and neurodegenerative diseases. conclusions a novel heterocyclic schiff base, 1,2-dihydro-1,5-dimethyl-4-[[1-2-oxo-2h -1-benzopyran-3-yl)ethylidene]amino]-2-phenyl-3h-pyrazol-3-one, and its corresponding lanthanide(iii) complexes were synthesized and characterized based on various physicochemical spectral studies. elemental analysis, and 1h-nmr and 13c-nmr spectroscopy confirmed the compositions of the compounds. ir and uv–visible spectroscopy confirmed the suggested coordination of the ligand through the azomethine nitrogen and oxygen of the pyrozoline ring. the xrd patterns of the ligand and the dysprosium(iii) complex were recorded. the luminescence studies showed enhancement of the luminescence intensities for the complexes when compared to that of the ligand. from the α-amylase inhibitory assay, it was observed that the metal complexes, especially the gadolinium(iii) complex, showed greater inhibitory activity than that of the ligand. the antioxi synthesis, fluorescence, biological studies of lanthanide(iii) complexes 573 dant activity assay also evidenced the better antioxidant activity of the lanthanide complexes than that of the ligand. supplementary material the spectral data of the synthesized compounds are available electronically at the pages of the journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. we express our sincere gratitude to dr. s. m. a. shibli, head of the department of chemistry, university of kerala, kariavattom campus, trivandrum, for providing the necessary facilities for the realisation of this work. the fellowship given to surya philip by the university of kerala, trivandrum and the financial support given to prof. (dr.) k. mohanan by the kerala state council for science technology and environment (kscste) are gratefully acknowledged. we are also thankful to the sophisticated analytical instrument facility, cochin for providing the instrumental facilities. и з в о д синтеза, флуоросцентна и антиоксидативна својства неких комплекса лантаноида(iii) и испитивање њиховог утицаја на инхибиторску активност α-амилазе surya philip, preethy soosan thomas и k. mohanan department of chemistry, university of kerala, trivandrum, kerala-695581, india кондензационом методом, полазећи од 3-ацетилкумарина и 4-аминоантипирина, синтетисан је нови хетероциклични лиганд, 1,2-дихидро-1,5-диметил-4-[[1-2-оксо-2h-1 -бензопиран-3-ил)етилиден]амино]-2-фенил-3h-пиразол-3-он. овај лиганд се показао као веома погодан за комплексирање лантана(iii), празеодијума(iii), самаријума(iii), гадолинијума(iii) и диспросијума(iii). лиганд и одговарајући комплекси су окарактерисани помоћу различитих физичко–хемијских и спектроскопских метода. на основу спектроскопских мерења нађено је да се лиганд бидентатно координује преко азометинског атома азота и кисеониковог атома из пиразоловог прстена. структура лиганда и одговарајућег диспросијум(iii) комплекса одређена је дифракцијом x-зрака са прахова. испитиване су фотолуминисцентне особине лиганда и комплекса метала и одређени су релативни квантни приноси. нађено је да у свим случајевима јони метала повећавају луминесцентни интензитет. применом методе апостолидиса (apostolidis), испитиван је утицај лиганда и одговарајућих комплекса на активност α-амилазе, при чему је нађено да комплекси метала показују већу активност у односу на лиганд. на основу dpph тестова нађено је да су антиоксидативна својства комплекса израженија у односу на одговарајућа својства некоординованог лиганда. 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {oxidative ammonolysis of 3,4-dimethylpyridine on the vanadium oxide catalysts} j. serb. chem. soc. 85 (4) 427–437 (2020) jscs–5312 original scientific paper 427 oxidative ammonolysis of 3,4-dimethylpyridine on the vanadium oxide catalysts pavel vorobyev, anna serebryanskaya, olga yugay* and tatyana mikhailovskaya a. b. bekturov institute of chemical sciences jsc, almaty 050010, kazakhstan (received 7 august 2018, revised 4 october 2019, accepted 8 october 2019) abstract: oxidative ammonolysis of 3,4-dimethylpyridine on an individual vanadium oxide (v2o5) catalyst and binary vanadium oxide catalysts, modified by additions of sno2 and zro2, has been studied. a connection between снacidity of the methyl groups of the substrate in the gaseous phase and in the chemosorbed state and the sequence of their transformation into a cyano group has been established. it has been shown that nucleophilicity of vanadyl oxygen, calculated by the density functional theory method, increases with v2o5 modification by sno2 and zro2 additions. herewith, an increasing yield of 3methyl-4-cyanopyridine and imide of pyridine-3,4-dicarboxylic acid was observed. a proposed mechanism of the imide of pyridine-3,4-dicarboxylic acid formation has been discussed keywords: quantum-chemical method; imide of pyridine-3,4-dicarboxylic acid; mechanism; v2o5; sno2; zro2. introduction monoand dicyanopyridines have become very important in the recent years since they form the starting materials for a number of valuable intermediates of medicines, animal feed additives, food additives and other commodities.1–3 oxidative ammonolysis of monoand dimethylpyridines is the most rational method for obtaining monoand dicyanopyridines.4,5 oxidative ammonolysis of dialkylpyridines of the unsymmetrical structure showed that alkyl groups at positions 2 and 4 are more reactive than in positions 3 and 5.6 therefore, the primary product of the conversion of 2,3-dimethylpyridine on the v–sn–fe oxide catalyst is 2-cyano-3-methylpyridine. in the oxidative ammonolysis of 2,5-dimethyland 2-methyl-5-ethylpyridines, the methyl group in position 2 reacts first to form 2-cyano-5-methyland 2-cyano-5-ethylpyridine, respectively. it was established7 that the interaction of * corresponding author. e-mail: yu.ok@mail.ru https://doi.org/10.2298/jsc180807107v ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 428 vorobyev et al. the pyridine base and the acid site on the catalyst surface causes increase in the activity at the 2-position in all stages of the oxidative transformation of 2-methyl -5-ethylpyridine. oxidative ammonolysis of 2,5-lutidine was studied on a series of vanadium oxide catalysts (v2o5–al2o3, v2o5–k2o–al2o3, v2o5-pumice stone, v2o5– –moo3-pumice stone, v2o5–cr2o3-pumice stone). it was established that a methyl group in position 2 is oxidized into 2-cyano-5-methylpyridine, which then transforms into 2,5-dicyanopyridine.8 okada et al.9 have studied oxidative ammonolysis of isomeric dimethylpyridines on the catalyst 5 % cr2o3–9 % γ-al2o3 at 360 °с. it was established that the activity of methyl groups in positions 2-(6-) or 4is higher than that in position 3-(5-). in the case of 3,4-dimethylpyridine a methyl group in 4-position is oxidized by formation of monocyanomethylpyridine. selectivity of the formation of 3-methyl-4-cyanopyridine makes up 92 %. methyl groups of 2,4-dimethylpyridine showed a close reactivity: 2-methyl-4-cyanopyridine and 4-methyl-2-cyanopyridine were obtained with the same selectivity (42 %). the reasons of the observed regularities are not discussed in the article, but they are undoubtedly related to the structural features of the initial compounds and the nature of their interaction with the catalyst. earlier we had carried out modeling of chemosorption of dialkylpyridines on the surface of vanadium oxide catalyst with the participation of active centers of different nature. it was shown that the sequence of transformation of methyl substituents of dialkylpyridines into cyano group was determined by the enthalpy value of their deprotonation.6 the main goal of this work was an experimental and quantum chemical study of the reactivity of methyl groups of 3,4-dimethylpyridine in oxidative ammonolysis. the tasks of the work included studying the effect of oxide-promoters, sno2 and zro2, on the activity of binary vanadium oxide catalysts. experimental materials initial 3,4-dimethylpyridine was purchased from sigma-aldrich (583-58-4) and used without further purification. an individual vanadium oxide (v2o5) catalyst and binary catalysts v2o5·4meo2 (me: sn or zr) were obtained by mixing the initial oxides of analytical grade in the required mole ratio. further, the obtained mixture was pressed into tablets and calcined at 650 °с in the air flow for 3 h. the catalysts were ground into grains with the size of 1–2 mm. synthesis experiments catalytic testing was carried out on a flow-type microreactor with the length of 200 and diameter of 20 mm with the fixed layer of catalyst (0.01 l) at atmospheric pressure. 3,4 -dimethylpyridine and a vaporization zone at 250 °с, mixed with air oxygen and ammonia and finally fed into the microreactor. the feeding rate of 3,4-dimethylpyridine made up 40 g per 1 l of catalyst per hour. the mole ratio of the reagents was 3,4-dimethylpyridine: о2:nh3:н2о = 1:16:(6–22.5):32. oxi________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. ammoxidation of 3,4-dimethylpyridine 429 dative ammonolysis was carried out at the temperature interval from 270 to 370 °с. the reaction products were caught up in the air-lift type scrubbers irrigated by water. analysis of 3,4-dimethylpyridine ammoxidation products 3-methyl-4-cyanopyridine after recrystallization from chloroform melted at 46 °с. imide of pyridine-3,4-dicarboxylic acid after recrystallization from ethanol melted at 224 °с (with subliming). elemental analysis was performed by means of ce440 elemental analyzer. the ir spectra were recorded on a nicolet 5700 (thermo elecrtron corporation) spectrophotometer and the wavenumbers are given in cm-1. the nmr spectra were recorded on a jnm-eca jeol 400 in dimethyl sulfoxide-d6 and the chemical shifts (δ) are expressed in ppm values downfield from tms. the characterization data for 3-methyl-4-cyanopyridine and imide of pyridine-3,4-dicarboxylic acid are given in the supplementary material to this paper. the reaction products were determined by a gas chromatographic method with a flame -ionization detector. the products of deep oxidation were analyzed by a gas chromatographic method with a thermal conductivity detector.10 computing details of quantum-chemical modeling the description of quantum chemical calculations is given in the previous article.10 optimization of the geometry of the organic molecules and carbanions in the gaseous phase was carried out by the ab initio method (hf/6-31g*). modeling of chemisorption was carried by the density functional theory method,11 using a two-exponent basic set lanl2dz with an effective core potential. at non-empirical calculations of energy and optimization of geometry of molecules born–oppenheimer approximation that considers the movement of the nucleus, and just assumes that they are not strongly coupled with the changes in the electronic states, was used. for the elimination of possible errors in a definition of the total energy of molecule and taking account of the effect of molecular translation, rotation and vibration, the frequency calculations for initial chemical compounds and their complexes with the optimized geometry’s clusters were carried out (ab initio method with a different basis sets and dft method (b3lyp/lanl2dz freq test, temperature = 603.15 k), respectively. for obtaining the exact value of the total energy of a system at zero degrees kelvin, zero-point energy (zpe, eq. (1)) must be added to the received etot. similarly, for obtaining the exact value of the total energy of the complex at the reaction temperature thermal energy (etherm) of complexes (eq. (2)) must be added to the received etot:12 de = 2625.5[(eanion + zpeanion) – (emolecule + zpemolecule)] (1) de = 2625.5[(eanion/cluster + etherm.anion/cluster) – (emolecule/cluster + etherm.molecule/cluster)] (2) the energy of heterolytic cleavage of с−н bond (ec-h) and the enthalpy of deprotonation of the methyl group with proton transfer to vanadyl oxygen (de) and value of proton affinity (pav=o) of vanadyl oxygen was calculated by formulas (3) and (4), respectively: ec-h, de = 2625.5(еads.carbanion − еads.mol) (3) pav=o = 2625.5(еinit. − еfinal) (4) the calculations carried out by the gaussian 09w program, version d.01.13 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 430 vorobyev et al. results and discussion the experimental data of oxidative ammonolysis of 3,4-dimethylpyridine on the v2o5, v2o5·4sno2 and v2o5·4zro2 catalyst are presented in fig. 1. as seen, at 250–330 °с the main product is 3-methyl-4-cyanopyridine. with a further rise of temperature an increase of initial substance conversion (a), a decrease in the yield of 3-methyl-4-cyanopyridine (b) and the formation of imide of pyridine-3,4-dicarboxylic acid, the product of intramolecular cyclization (c), is observed. among the reaction products 3-cyanoand 4-cyanopyridines, pyridine and со2 are found, the yield of which depends on the reaction conditions and the catalyst used. it has been noted that 3,4-dicyanopyridine is not formed. fig. 1. the effect of temperature and composition of the catalyst on the conversion of 3,4-dimethylpyridine (a), yield of 3-methyl-4-cyanopyridine (b) and imide of pyridine-3,4-dicarboxylic acid (c): 1 – v2o5, 2 – v2o5·4sno2, 3 – v2o5·4zro2. the yield of 3-methyl-4-cyanopyridine and catalytic activity, estimated by the conversion of 3,4-dimethylpyridine, increases in the order: v2o5 < v2o5·4sno2 < v2o5·4zro2 it was found that an increase of the mole ratio of nh3 to 3,4-dimethylpyridine promotes a rise in the yield and selectivity of the 3-methyl-4-cyanopyridine formation on v2o5·4sno2 at 300 °с (fig. 2). the competing adsorption of 3,4-dimethylpyridine and ammonia on the same acid sites on the catalyst surface leads to decrease the conversion of the initial 3,4-dimethylpyridine. fig. 2. the effect of nh3/3,4-dimethylpyridine mole ratio on the conversion (1) of 3,4-dimethylpyridine, and yield (2) and selectivity (3) of 3-methyl-4-cyanopyridine. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. ammoxidation of 3,4-dimethylpyridine 431 upon interpretation of the experimental data on a relative reactivity of the methyl groups of 3,4-dimethylpyridine it is necessary to take into account that methylpyridines represent weak сн-acids. under the effect of a strong base they can lose a proton from the methyl group and form a carbanion.14 in a catalytic reaction a role of a proton acceptor can be played by nucleophilic oxygen of the surface. it is evident that reactivity of the methyl groups is influenced by their сн-acidity. the experimental data of сн-acidity in the gaseous phase are known only for monomethylpyridines, and at present, these characteristics are not available for dimethylpyridines. that is why with the help of an ab-initio quantum-chemical method (hf/6-31g*) we have calculated deprotonation enthalpy (de), which is an analogue of сн-acidity of the methyl groups of the molecule of the initial compound, coming from the values of total energy of the initial molecule and the corresponding carbanions: pych3  pych2− + h+−de de = еtot(pych2−) − еtot(pych3) (5) where еtot – total energy of the corresponding compound. an non-bonding electron pair of carbanion is situated at the highest occupied molecular orbital (homo).15 stabilization of the intermediate carbanions is a consequence of an interaction of an non-bonding electron pair of carbanion with π-electron system of the pyridine ring. this is favored by the aromatic nature of the analyzed position and the possibility of extensive anion delocalization. stability of carbanions may be characterized by an energy value of the highest occupied molecular orbitals (еhomo). a methyl substituent in position 4 has a lower value of de due to the conjugation effect, a higher extent of delocalization of an nonbonding electron pair of the formed carbanion, and the total energy (etot) of the latter is lower than that of the carbanion in position 3. the results of the ab initio quantum-chemical calculations, presented in table 1, are in accordance with the notions of classic organic chemistry and experimental data on the sequence of entering of the substituents of 3,4-dimethylpyridine into the reaction (fig. 1). table i shows the results of the quantum-chemical calculations for the isolated molecules of 3,4-dimethylpyridine and its aromatic analogue with the ortho-located methyl substituents – о-xylene. the data on the primary products of their transformation, 3-methyl-4-cyanopyridine and о-tolunitrile (o-methylbenzonitrile, o-cyanotoluene), are also provided here. the non-empirical method correctly describes the experimental regularity according to which the methyl group in the 4-position of 3,4-dimethylpyridine has a more mobile proton. so, oxidative transformations of the substrate under mild conditions proceed only with this substituent. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 432 vorobyev et al. table i. total energies of molecules of 3,4-dimethylpyridine, о-xylene, mononitriles and corresponding anions (etot), enthalpy of proton detachment from the methyl groups in the gaseous phase (de), energies of the highest occupied molecular orbitals of anions (еhomo), calculated by the ab initio method (hf/6-311+g**) initial molecule –etot(molecule)u –etot(anion) u de kj.mol-1 –еhomo kj.mol-1 3,4-dimethylpyridine 324.84460 324.21140 (3) 324.22261 (4) 1662.4 (3) 1633.0 (4) 90.3 (3) 144.5 (4) о-xylene 308.84755 308.20785 1679.5 77.8 3-methyl-4-cyanopyridine 377.54978 376.95097 (3) 1572.2 (3) 186.8 (3) о-tolunitrile 361.55825 360.94967 1597.8 166.0 similar results were obtained in modeling the chemisorption of 3,4-dimethylpyridine on the surface of a vanadium oxide catalyst (fig. 3). energy of heterolytic cleavage of c−h (ec–h) bond of the methyl groups of the substrate in positions 3 and 4 is rather high and is less for the substituent in position 4 (1178.2 and 1070.6 kj·mol–1, respectively, table ii). upon transfer of the proton, detached from the methyl group, to vanadyl oxygen a new o−h bond is formed. herewith, fig. 3. heterolytic cleavage of c−h bond without proton transfer to vanadyl oxygen (a) and with it (b). ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. ammoxidation of 3,4-dimethylpyridine 433 energy is released which compensates the expenditures for deprotonation of the methyl groups. as a result, deprotonation enthalpy (de) of the methyl groups in positions 3 and 4 has considerable lesser values (238.2 and 157.1 kj·mol–1, respectively). the substituent in position 4, possessing the least de value, should enter into the reaction first. thus, the results of the calculations for a molecule of 3,4-dimethylpyridine in the gaseous phase and in the adsorbed state correlate with the experimental data on the sequence of transformation of the methyl substituents of 3,4-dimethylpyridine into a cyano group. table ii. total energies of the connected with the lewis acidic center molecule of 3,4-dimethylpyridine and carbanions (еtot), energy of heterolytic cleavage of с−н bond of the methyl groups of the adsorbed substrate (ec-h), deprotonation enthalpy (de) of the methyl groups, calculated by the dft method (b3lyp/lanl2dz) structure −еtot / u ec-h / kj.mol-1 adsorbed molecule adsorbed carbanion cleavage of c−h bond (fig. 3a) 922.81280 922.40501 (4-position) 922.36405 (3-position) 1070.6 (4-position) 1178.2 (3-position) heterolytic cleavage of c−h bond with carry of a proton on vanadyl oxygen (fig. 3b) 922.81280 922.75297 (4-position) 922.72206 (3-position) 157.1 (4-position) 238.2 (3-position) the energies of the initial compounds and their complexes with clusters with optimized geometry were determined (tables iii and iv), using the born–oppenheimer approximation and taking into account the effects of molecular translation, rotation and vibration. table iii. total energies (еtot), zero-point energy (zpe) of the 3,4-dimethylpyridine and carbanions in gaseous phase and enthalpy deprotonation of substitutions, calculated by ab initio method (hf/6-311+g** freq test) structure −еtot / u zpe / u de / kj∙mol-1 3,4-dimethylpyridine 3-carbanion 4-carbanion 324.84460 324.21140 324.22261 0.15266 0.13647 0.13709 – 1620 1592.1 an effect of activated adsorption of the substrate upon a relative reactivity of the methyl substituents in the pyridine ring has been studied. we have carried out modeling of the adsorption of 3,4-dimethylpyridine by nitrogen’s heteroatom of the pyridine ring on the acidic lewis center of vanadium oxide catalyst (vanadium cation) at a temperature of reaction 603.15 k (table iv). thus, we have established that calculations using the born–oppenheimer approximation correctly describe the relative reactivity of methyl groups in the 3,4-dimethylpyridine molecule both in the gaseous phase and in the cluster– –bound state. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 434 vorobyev et al. table iv. total energies (еtot) and thermal energy (etherm) of the 3,4-dimethylpyridine and carbanions on cluster, enthalpy deprotonation of substitutions (de), calculated by dft method (b3lyp/lanl2dz) structure −еtot / u etherm / u de / kj∙mol-1 3,4-dimethyl-pyridine/cluster 3-carbanion/cluster 4-carbanion/cluster 922.81280 922.72206 922.75297 0.26333 0.26141 0.26192 – 233.2 153.4 the difference in the behavior of 3,4-dimethylpyridine and о-xylene in oxidative ammonolysis on v–o-catalysts was shown through experiments with 3,4 -dimethylpyridine in which we have not detected a corresponding dinitrile (3,4 -dicyanopyridine). the cause of this might be for the following reasons. the methyl group in the molecule of 3-methyl-4-cyanopyridine, more reactive than that in the molecule of o-tolunitrile, enters into reaction of intramolecular cyclization earlier, than a second cyano group is formed thereof. our quantum-chemical calculations (table i) testify to a greater mobility of the protons of the methyl substituent of 3-methyl-4-cyanopyridine in comparison with the protons of the methyl group of о-tolunitrile. we have established that upon oxidative ammonolysis of 3,4-dimethylpyridine the promotion of vanadium pentoxide by the additions of sno2 and zro2 results in a marked increase in the yield of 3-methyl-4-cyanopyridine and shifting of its maximum to the field of lower temperatures (fig. 1b). it is considered,16 that activity and selectivity of vanadium oxide catalysts of hydrocarbons oxidation are connected with the existence of different forms of active oxygen at the catalyst surface. an important role is played here by a double bond oxygen of the lattice (v=o). an intermediate action of sno2 and zro2 upon the activity of the vanadium oxide catalysts, studied by us, may be stipulated by their effect upon the reactivity of v=o bond. indeed, the calculations have shown (table v), that upon the transfer from v2o5 to binary catalysts a value of proton affinity of vanadyl oxygen increases. these results confirm that the mechanism of the promoting effect of sno2 and zro2 additives to v2o5 is to activate both the chemisorbed substrate (table ii) and the vanadyl oxygen responsible for the deprotonation of the oxidizing methyl group at the initial stages of the process. it has been established that additions of the above-mentioned oxides to v2o5 increase nucleophilicity of vanadium oxygen (pa, table v), the conversion of substrate and the yield of the primary reaction product, 3-methyl-4-cyanopyridine (fig. 1a and b). the mechanism of formation of an imide cycle in oxidative ammonolysis of o-xylene and 3,4-dimethylpyridine is of the interest. by its ability to intramolecular cyclization 3,4-dimethylpyridine is similar to о-xylene. the authors17 have assumed that the transformation of o-xylene into phthalimide proceeds through the stages of formation of о-tolunitrile, oxidation of a methyl substituent of the ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. ammoxidation of 3,4-dimethylpyridine 435 latter and interaction of its oxidized forms with a triple bond of a nitrile group, as a result of which isoimide of phthalic acid is formed. the unstable isoimide is easily transformed into phthalimide at a high temperature. table v. the total energies of the vanadium-containing clusters and their protonated forms (etot), proton affinity of oxygen bound to the vanadium ion (pa), calculated by the dft method (b3lyp/lanl2dz) reaction − еtot / u pa / kj·mol-1 initial state kj·mol-1 a 1038.17019 1038.45849 756.9 b 672.90766 673.20234 773.7 c 716.26601 716.58135 827.9 accounting for the above-stated and the analogous structure of o-xylene and 3,4-dimethylpyridine, the following scheme 1 of the formation of imide of pyridine-3,4-dicarboxylic acid from 3,4-dimethylpyridine can be proposed. scheme 1. formation of imide of pyridine-3,4-dicarboxylic acid from 3,4-dimethylpyridine. the scheme includes 4-cyanonicotinic acid and isoimide of pyridine-3,4 -dicarboxylic acid as hypothetic intermediate products. one must suppose that ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 436 vorobyev et al. the transformation of 3-methyl-4-cyanopyridine into imide includes the superficial stages of oxidation of the methyl group in position 3 and an electrophilic attack of a proton at a nitrogen atom of the nitrile group. in the scheme the proton belongs to the carboxylic group, however, protons as potential hydrogen donors, present in the reaction mixture (ammonia, water vapors), may induce heterocyclic cleavage of n−h or o−h bonds under the effect of a catalyst. the results of our ab initio quantum-chemical calculations (hf/6-31g*) testify in favor of this hypothesis of the imide formation mechanism. an isolated molecule of 3-methyl-4-cyanopyridine is characterized by the pronounced polarization of a triple bond in the nitrile group, as a result of which a carbon atom carries a positive charge (δc = +0.274), and a nitrogen atom carries a negative charge (δn = -0.447). this favors the electrophilic attack of the proton at the nitrogen atom with the formation of isoimide. the total energy level of isoimide (etot = -526.039 u) is higher than that of imide (etot = –526.067 u) by 73.5 kj·mol–1. this indicates a possibility of transformation of the thermodynamically less stable isoimide into imide of pyridine-3,4-dicarboxylic acid. conclusion the correlation between the position of methyl substituents in pyridine cycle and their reactivity in oxidative ammonolysis of 3,4-dimethylpyridine on modified vanadium oxide catalysts has been found. it was found that due to the high ch-acidity in the gas phase and in conditions modelling chemisorption on the lewis acid sites of the catalyst surface, methyl substituent at 4-position has higher reactivity and the first reacts obtaining 3-methyl-4-cyanopyridine. the temperature increase favors intramolecular cyclization with the formation of imide of pyridine-3,4-dicarboxylic acid. a possible mechanism of 3,4-dimethylpyridine transformation into imide of pyridine-3,4 -dicarboxylic acid has been proposed. the main stages of the mechanism were confirmed by physicochemical methods and quantum chemical calculations. it was established that the modification of v2o5 by additions of sno2 and zro2 increases the nucleophilicity of vanadyl oxygen and promotes the yield of 3-methyl-4-cyanopyridine, the primary reaction product. supplementary material analytical and spectral data of the synthesized compounds are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. ammoxidation of 3,4-dimethylpyridine 437 и з в о д оксидативна аминолиза 3,4-диметилпиридина на ванадијум-оксидним катализаторима pavel vorobyev, anna serebryanskaya, olga yugay и tatyana mikhailovskaya а. b. bekturov institute of chemical sciences jsc, almaty 050010, kazakhstan испитивана је оксидативна аминолиза 3,4-диметилпиридина на v2o5 и бинарним ванадијум-оксидним катализаторима, модификованим додавањем sno2 и zro2. утврђена је повезаност сн-киселости метил-група супстрата у гасовитој фази, стања хемисорпције и корака њихове трансформације у циано-групу. показано је да нуклеофилност ванадил-кисеоника, израчуната методом теорије функционала густине (density functional theory, dft), расте приликом модификације услед додавања sno2 и zro2 и утиче на повећавање приноса у добијању 3-метил-4-цијанопиридина и имида пиридин3,4-дикарбоксилне киселине. размотрен је предложени механизам реакције формирања имида пиридин-3,4-дикарбоксилне киселине. (примљено 7 августа 2018, ревидирано 4 октобра 2019, прихваћено 8. октобра 2019) references 1. т. delaine, v. bernandes-genisson, a. quemard, p. constant, b. meunier, j. bernadou, eur. j. med. chem. 45 (2010) 4554 (https://doi.org/10.1016/j.ejmech.2010.07.016) 2. t. r. k. reddy, r. mutter, w. heal, k. guo, v.j. gillet, s. pratt, b. chen, j. med. chem. 49 (2006) 607 (https://doi.org/10.1021/jm050610f) 3. e. e. ergozhin, b. a. mukhitdinova, v. v. shaligina, b. zh. shekeeva, k. m. sartbaeva, react. funct. polym. 65 (2005) 93 (https://doi.org/10.1016/j.reactfunctpolym.2004.12.003) 4. b. v. suvorov, oxidative ammonolysis of organic compounds, nauka, alma-ata, 1971, p. 210 5. r. chuck, appl. catal., a 280 (2005) 75 (https://doi.org/10.1016/j.apcata.2004.08.029) 6. p. b. vorob’ev, d. kh. sembaev, russ. j. gen. chem. 75 (2005) 147 (https://doi.org/10.1007/s11176-005-0186-1) 7. d. kh. sembaev, o. k. yugay, s. g. klepikova, eurasian chem.-tech. j. 6 (2004) 127 (https://doi.org/10.18321/ectj601) 8. j. okada, s. morita, y. miwa, t. tashima, yakugaku zasshi 98 (1978) 1391 9. j. okada, s. morita, y. miwa, chem. pharm. bull. 22 (1974) 2402 (https://doi.org/10.1248/cpb.22.2402) 10. p. vorobyev, t. mikhailovskaya, o. yugay, a. serebryanskaya, n. chukhno, r. kurmakizy, j. serb.chem.soc. 82 (2017) 791 (http://doi.org/10.2298/jsc161220023z) 11. w. koсh, m. c. holthausen, chemist’s guide to density functional theory. 2nd ed., wiley-vch, weinheim, 2001, p. 293 12. j. b. foresman, a. frish, exploring chemistry with electronic structures methods, 2nd ed., gaussian inc., pittsburgh, pa, 1996, p. 335 13. gaussian09, revision d.01., gaussian inc., wallingford, ct, 2009 14. d. j. cram, fundamentals of carbanion chemistry, academic press, new york, 1965, p. 290 15. k. higasi, h. baba, a. rembaum, quantum organic chemistry. interscience publishers, division of john wiley & sons, inc., new york, 1965, p. 380 16. l. ya. margolis, v. n. korchak, russ. chem. rev. 67 (1998) 1073 (https://doi.org/10.1070/rc1998v067n12abeh000428) 17. n. i. chukhno, i. s. kolodina, d. kh. sembaev, izv. akad. nauk kazssr. ser.chim. 4 (1989) 31. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {electrochemical deposition and characterization of agpd alloy layers} j. serb. chem. soc. 83 (5) 593–609 (2018) udc 544.654.2:546.57’98–034+543.552+ jscs–5098 543.42 original scientific paper 593 electrochemical deposition and characterization of agpd alloy layers nevenka r. elezović1#, piotr zabinski2, mila n. krstajić pajić3#, tomasz tokarski4, borka m. jović1# and vladimir d. jović1* 1institute for multidisciplinary research university of belgrade, kneza višeslava 1, 11030 belgrade, serbia, 2agh university of science and technology, faculty of non-ferrous metals, al. mickiewicza 30, 30-059 krakow, poland, 3faculty of technology and metallurgy university of belgrade, karnegijeva 4, 11000 belgrade, serbia and 4agh university of science and technology, academic centre for materials and nanotechnology, al. mickiewicza 30,30-059 krakow, poland (received 3 november, revised and accepted 25 december 2017) abstract: the agpd alloys were electrodeposited onto au and glassy carbon disc electrodes from the solution containing 0.001 mol dm-3 pdcl2 + 0.04 mol dm-3 agcl + 0.1 mol dm-3 hcl + 12 mol dm-3 licl under the non-stationary diffusion (quiescent electrolyte) and convective diffusion (ω = 1000 rpm) to the different amounts of charge and at different current densities. electrodeposited alloy layers were characterized by the anodic linear sweep voltammetry (alsv), scanning electron microscopy, energy dispersive x-ray spectroscopy (eds) and x-ray photoelectron spectroscopy (xps). the compositions of the agpd alloys determined by the eds were almost identical to the theoretically predicted ones, while the compositions obtained by xps and alsv analysis were similar to each other, but different from those obtained by eds. deviation from the theoretically predicted values (determined by the ratio jl(pd)/j(ag)) was more pronounced at lower current densities and lower charges of agpd alloys electrodeposition, due to the lower current efficiencies for alloys electrodeposition. the alsv analysis indicated the presence of ag and pd, expressed by two alsv peaks, and in some cases the presence of the additional peak, which was found to correspond to the dissolution of large agpd crystals, formed at thicker electrodeposits (higher electrodeposition charge), indicating, for the first time, that besides the phase structure, the morphology of alloy electrodeposit could also influence the shape of the alsv response. in addition to ag and pd, the xps analysis confirmed the presence of agcl at the surface of samples electrodeposited to low thicknesses (amounts of charge). keywords: agpd alloys electrodeposition; alsv; xps; eds; sem. * corresponding author. e-mail: vladajovic@imsi.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc171103011e 594 elezović et al. introduction the possibility of agpd alloys electrodeposition from the solution containing high concentration of chloride ions (12 mol dm–3 licl) was first mentioned by brenner,1 considering the results obtained by graham et al.2,3 for the electrodeposition of agpt alloys. it was shown later4–7 that in the excess of chloride ions agcl could dissolve to the concentrations sufficient for the electrodeposition of agpd alloys, with pd electrodeposition starting at more positive potentials than ag. in our previous work,7 agpd alloys were characterized by the anodic linear sweep voltammetery (alsv) technique, indicating the possibility of the formation of an additional phase in the electrodeposited alloys, as a consequence of the appearance of additional peak on the alsv responses. these investigations were in accordance with some other work,8 where the measurement of microhardness and the specific electric resistivity indicated the possibility of the formation of ordered structures (or intermetallic compounds) with the approximate stoichiometric compositions of ag2pd3 and agpd in the agpd alloys below 1200 °c, while all of the data found in the binary alloys literature9 claimed that the agpd alloy consists of a homogeneous solid solution phase over the entire composition range. müller et al.10 conducted an unbiased search of fcc-based ag1–xpdx structures consisting of up to many thousand atoms by using a mixed-space cluster expansion (msce). they found an unsuspected ground state at 50–50 % composition – the l11 structure, currently known in binary metallurgy only for the cu0.5pt0.5 alloy system. they also provided predicted short-range-order profiles and mixing enthalpies for the high temperature, disordered alloy. using msce they predicted the existence of following ordered structures in the system agpd: ag3pd, ag2pd, agpd, ag2pd2, ag3pd3, agpd2 and agpd3. in this work, an attempt was made to characterize agpd alloy layers, electrodeposited from high concentration chloride solutions, by different techniques and compare their results, with the intention to define the appearance of the additional peak on the alsv responses. experimental all experiments of the agpd alloys electrodeposition and alsv analysis were performed in standard electrochemical cells at the temperature of 333±1 k using rotating au and glassy carbon (gc) disc electrodes (d = 5 mm) and ctv 101 speed control unit (radiometer analytical, s.a.). the agpd alloys were electrodeposited onto au and gc disc electrodes from the solution containing 0.001 mol dm-3 pdcl2 + 0.04 mol dm -3 agcl + 0.1 mol dm-3 hcl + 12 mol dm-3 licl under the conditions of non-stationary diffusion (quiescent electrolyte) and the convective diffusion (ω = 1000 rpm) to the different amounts of charge (0.05 to 3.0 c cm-2), at a constant current density and at various current densities (–0.178 to –0.415 ma cm-2) to the constant amount of charge (0.2 c cm-2). pure pd and ag samples were electrodeposited onto the au disc electrode from the solutions of 0.04 mol dm-3 pdcl2 + 0.1 mol dm -3 hcl + 12 mol dm-3 licl and 0.04 mol dm-3 agcl + 0.1 mol dm-3 hcl + 12 mol dm-3 licl, respectively, electrodeposition and characterization of agpd alloy layers 595 under the conditions of non-stationary and convective diffusion. the dissolution of all coatings by the alsv technique was performed in another cell containing 0.1 mol dm-3 hcl + 12 mol dm-3 licl under the conditions of convective diffusion (ω = 1000 rpm) at the same temperature of 333±1 k. the alloy samples for the alsv analysis were electrodeposited onto the au disc electrode, while those for eds and xps analysis were electrodeposited onto gc electrode. all solutions were made from the extra pure uv water (18.2 mω cm), smart2pureuv, tka, and p.a. chemicals (pdcl2, agcl, licl, hcl), sigma–aldrich. the saturated calomel electrode (sce), radiometer analytical, was used as the reference electrode in all measurements, while the platinum mesh (pt mesh), placed parallel to the working electrode, was used as a counter electrode in both cells. all potentials in the paper are referred at sce scale. the j–e curves for ag, pd and agpd alloys electrodeposition and the alsv responses for their dissolution were recorded using the potentiostat reference 600 and the software phe 200 and dc 105 (gamry instruments). before the each experiment all solutions were purged with n2 (5.0 purity) for 30 min. the rotating disc electrodes were polished with the emery papers 1200–4000 and the alumina polishing slurries (1, 0.3 and 0.05 µm) before the electrodeposition of each sample. the sem–eds characterization of all coatings was performed with fei versa 3d field emission gun (feg)-sem equipped with the energy dispersive spectrometer (eds). the xps analysis of samples was carried out in the ultrahigh vacuum system (3×10-10 mbar) equipped with the hemispherical analyzer (ses r4000, gammadata scienta). the mgkα source of the incident energy of 1256.6 ev was applied to generate the core excitation. the spectrometer was calibrated according to iso 15472:2001. the energy resolution of the system, measured at full width at half maximum for ag 3d5/2 excitation line, was 0.9 ev. the analysis area of the prepared sample was about 3 mm2. no gas release and no changes in the sample composition were observed during the measurements. the casaxps 2.3.12 software was applied for the analysis of the xps spectra. no charging was observed for the studied sample, therefore no additional calibration of the spectra energy scale was applied. in the spectra, the background was approximated by a shirley profile. the spectra deconvolution into a minimum number of components was done by the application of the voigt-type line shapes (70:30 gaussian/lorentzian product). the analytic depth of the xps method was estimated as 10.2 nm. the calculations were performed with the quases-imfp-tpp2m, ver. 2.2, software according to an algorithm proposed by tanuma et al.11 this estimation takes into account 95 % of photoelectrons escaping from the surface. the experimental error of the xps analysis was ±3 %. results and discussion electrodeposition of samples for the alsv, xps and eds analysis agpd alloys electrodeposited under the conditions of non-stationary diffusion. the polarization, j–e curves, for the electrodeposition of pure ag, pure pd and agpd alloys, onto the au disc electrode, recorded at a sweep rate of 1 mv s–1 and ω = 0 rpm, are presented in fig. 1. the electrodeposition of pd is characterized with the well-defined diffusion limiting current density (jl(pd)) in the solution containing only pdcl2 (pdcl42–), as well as in the solution containing pdcl2 and agcl (agcl43–), with the one recorded in the presence of agcl being 596 elezović et al. slightly higher (jl(pd) = –59.18 µa cm–2). in the absence of agcl, the sharp peak corresponding to the formation of pd–h6,12,13 is detected at –0.10 v, while in the presence of agcl such peak does not exist, indicating that the pd–h cannot be formed during the agpd alloy electrodeposition. electrodeposition of ag is seen to commence at about –0.11 v, being characterized with the sharp increase of the current density until the peak at about –0.15 v has been reached. the j–e curve for agpd alloy electrodeposition practically represents the sum of those for pd and ag. the current density values for agpd alloy electrodeposition are marked in the figure as j(1) = 3jl(pd), j(2) = 5jl(pd) and j(3) = 7jl(pd). the composition of the agpd alloy could be calculated from the parameters of each metal electrodeposition. masses of the electrodeposited pd (gpd) and ag (gag) are given as: ( )l pd pd pd = j tm s g zf (1) ( ) ag ag ag = j tm s g zf (2) where: jl(pd) is the diffusion limiting current density for pd electrodeposition; t is the time of electrodeposition; mpd is the molecular weight of pd; s is the electrode surface area; z is the number of exchanged electrons; f is the faraday’s constant; jd is the current density for the agpd alloy electrodeposition; j(ag) is the current density for the ag electrodeposition (j(ag) = jd – jl(pd)); mag is the molecular weight of ag. -0.3 -0.2 -0.1 0.0 0.1 0.2 -0.9 -0.8 -0.7 -0.6 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 -0.3 -0.2 -0.1 0.0 0.1 0.2 -0.8 -0.6 -0.4 -0.2 0.0 pd ag-pd ag j d (3)=7j l (pd) j d (2)=5j l (pd) j d (1)=3j l (pd) j l (pd) j / m a c m -2 e / v vs. sce fig. 1. the j–e curves for electrodeposition of pure ag (dashed green line), pure pd (dotted red line) and agpd alloy (solid black line), recorded at a sweep rate of 1 mv s-1 from the following solutions: (ag) – 0.04 mol dm-3 agcl + 0.1 mol dm-3 hcl + 12 mol dm-3 licl; (pd) – 0.001 mol dm-3 pdcl2 + 0.1 mol dm -3 hcl + 12 mol dm-3 licl; (agpd) – 0.001 mol dm-3 pdcl2 + 0.04 mol dm -3 agcl + 0.1 mol dm-3 hcl + 12 mol dm-3 licl. electrodeposition and characterization of agpd alloy layers 597 the composition of the agpd alloy expressed as a mass share of each metal is obtained from the following equations: pd pd ag (pd) = + g w g g (3) ag pd ag (ag) = + g w g g (4) these equations, presenting the mass share of metals, are practically identical to the atomic share of metals, since the molecular weights of both metals are very close. using eqs. (1)–(4) the agpd alloy compositions for the different current densities and the constant charge (q = 0.2 c cm–2) were calculated and presented in fig. 2 as a function of the current density for the alloy electrodeposition, jd (, ). it can be seen that the content of both metals (at.%) changes exponentially with the increase of the current density, starting from jl(pd). the amount of pd decreases, while the amount of ag increases and already at jd = 1.5jl(pd) the equal amounts of ag and pd should be electrodeposited. 0 100 200 300 400 500 600 0 20 40 60 80 100 0 100 200 300 400 500 600 0 20 40 60 80 100 0 100 200 300 400 500 600 0 20 40 60 80 100 0 100 200 300 400 500 600 0 20 40 60 80 100 j l( p d) / μa c m -2 c on te nt o f p d, a g / % j d / μa cm-2 fig. 2. composition of the agpd alloys (at.%) calculated from eqs. (1)–(4): () pd; () ag. composition of agpd alloys obtained by different techniques: () pd and () ag obtained from the alsv; () pd and () ag obtained from the xps; () pd and () ag obtained from the eds (see tables s-i–s-iii of the supplementary material to this paper). agpd alloys electrodeposited under the conditions of convective diffusion. in the case of pure pd electrodeposition from the solution containing 0.001 mol dm–3 pdcl2 + 0.1 mol dm-3 hcl + 12 mol dm-3 licl under the conditions of the convective diffusion, the diffusion limiting current density for pd cannot be precisely defined, as it is shown in fig. 3a. although the commencement of pd electro598 elezović et al. deposition is moved to slightly more cathodic potentials in comparison with that at non-stationary diffusion (fig. 1), a well-defined plateau of the jl(pd) does not exist at any applied rotation rate. a sharp peak of the pd–h formation appears at the same potential (about –0.10 v), as in the case of the non-stationary diffusion. the reason for such behaviour might be that the sweep rate of 1 mv s–1 is faster than the one which is necessary for the pd electrodeposition. an attempt was made to apply the sweep rate of 0.1 mv s–1, but with that sweep rate the plateau of the diffusion limiting the current density could be obtained only at 400 rpm, while at higher ω values such plateau could not be detected. taking into account that at ω = 900 rpm the diffusion limiting current density for pd electrodeposition is 10 times higher than that for the non-stationary diffusion (see figs. 1 and 3a) the amount of the electrodeposited pd is 10 times higher and in the region of the plateau of the diffusion limiting current density the electrodeposit becomes rougher with the increasing cathodic potential producing the increase of the jl(pd), due to the increase of the real surface area. this increase is more pronounced than in the case of the non-stationary diffusion and jl(pd) cannot be well-defined. -0.2 -0.1 0.0 0.1 0.2 -8 -6 -4 -2 0 -0.2 -0.1 0.0 0.1 0.2 -8 -6 -4 -2 0 -0.1 0.0 0.1 0.2 0.3 -2.0 -1.5 -1.0 -0.5 0.0 -0.1 0.0 0.1 0.2 0.3 -2.0 -1.5 -1.0 -0.5 0.0 -0.1 0.0 0.1 0.2 0.3 -2.0 -1.5 -1.0 -0.5 0.0 (b) rpm=1000 agpd pdag j / m a c m -2 e / v vs. sce j / m a c m -2 e / v vs. sce (a) rpm=400 rpm=900 rpm=1600 rpm=2500 j / m a c m -2 e / v vs. sce fig. 3. a) the j–e curves for electrodeposition of pure pd recorded at a sweep rate of 1 mv s-1 and different ω (given in the figure) form the solution 0.001 mol dm-3 pdcl2 + 0.1 mol dm -3 hcl + 12 mol dm-3 licl. b) the j–e curves for electrodeposition of pure ag (green dashed line), pure pd (red dotted line) and agpd alloy (black solid line), recorded at the sweep rate of 1 mv s-1 and ω = 1000 rpm, form the following solutions: (ag) – 0.04 mol dm-3 agcl + 0.1 mol dm-3 hcl + 12 mol dm-3 licl; (pd) – 0.001 mol dm-3 pdcl2 + 0.1 mol dm -3 hcl + 12 mol dm-3 licl; (agpd) – 0.001 mol dm-3 pdcl2 + 0.04 mol dm -3 agcl + 0.1 mol dm-3 hcl + 12 mol dm-3 licl. for the agpd alloy electrodeposition in the solution 0.001 mol dm–3 pdcl2 + 0.04 mol dm–3 agcl + 0.1 mol dm–3 hcl + 12 mol dm–3 licl the situation is even electrodeposition and characterization of agpd alloy layers 599 worse. the current density of the pd electrodeposition increases in the potential range where a plateau is expected, as seen in fig. 3b (black solid line). as in the case of non-stationary diffusion, the electrodeposition of ag commences at the same potential, characterized with the sharp increase of the current density. hence, the calculation of the alloy composition using eqs. (1)–(4) is impossible and the composition must be determined by other techniques, such as alsv, xps or eds. characterization of electrodeposited agpd alloys by the alsv technique the samples for the alsv analysis were electrodeposited onto the au disc electrode, due to the reasons explained in our previous work.7 characterization of agpd alloys electrodeposited under the conditions of non-stationary diffusion. the typical alsv responses14 for the dissolution of agpd alloys electrodeposited under the conditions of non-stationary diffusion (fig. 1) are shown in fig. 4a, for the samples electrodeposited at different jd (qd = 0.2 c cm–2) and in fig. 4b, for the samples electrodeposited to different qd (jd = –178 µa cm–2). -0.2 -0.1 0.0 0.1 0.2 0 1 2 3 -0.2 -0.1 0.0 0.1 0.2 0 1 2 3 4 5 6 7 q(pd) alsv q(ag) alsv (b) qd / c cm-2 0.05 0.1 0.2 0.4 0.6 j / m a c m -2 e / v vs. sce q(pd) alsv q(ag) alsv (a) 4 23 1 j / m a c m -2 e / v vs. sce fig. 4. alsv responses for dissolution of agpd alloys recorded at ω = 1000 rpm and a sweep rate of 1 mv s-1 in the solution containing 0.1 mol dm-3 hcl + 12 mol dm-3 licl. a) alloys electrodeposited to the charge qd = 0.2 c cm -2 at different current densities: (1) jd(1) = 3jl(pd) = –178 µa cm-2; (2) jd(2) = 5jl(pd) = –296 µa cm -2; (3) jd(3) = 7jl(pd) = –415 µa cm -2; (4) jd(4) = 5jl(pd) = –296 µa cm -2; qd = 1.0 c cm -2. b) alloys electrodeposited to different charges (given in the figure) at a constant current density jd = –178 µa cm -2. considering these alsv responses, it seems that all of them are characterized with two dissolution peaks, one corresponding to the dissolution of ag and one corresponding to the dissolution of pd. with the increase of the amount 600 elezović et al. of charge for agpd alloy electrodeposition (thickness of the alloy layer), the peak current densities on the alsv responses become higher. in order to determine approximate composition of the agpd alloys (taking into account high values of current efficiencies for alloy electrodeposition, see tables s-i–s-iii), the alsv responses were analyzed as schematically presented in fig. 4a for the sample 4, as well as in fig. 4b for the sample corresponding to the dissolution of agpd alloy electrodeposited to qd = 0.6 c cm–2 (see also fig. 5b). the alsv responses were divided in two parts, the one corresponding to the dissolution of ag (q(ag)alsv) and the another one corresponding to the dissolution of pd (q(pd)alsv). the at.% of pd and ag, calculated from their charges on the alsv responses, are presented in fig. 2 ( and , respectively), while the calculated percentages are presented with  for pd and  for ag. the percentages of pd and ag obtained from the xps analysis are presented with  for pd and  for ag, while those obtained from the eds analysis are presented with  for pd and  for ag, respectively. it should be stated here that only two samples presented in the fig. 2 were analyzed by the xps and eds (see table s-i). the results of the composition analysis with all three techniques were different (tables s-i–s-iii). relatively good agreement was obtained for those obtained from the xps and alsv analysis, where the at.% pd was the lowest for sample agpd2. the difference between the calculated percentages of pd () and ag () and those determined from their charges on the alsv responses (pd (), ag ()), as well as those determined by xps (pd(), ag()), have increased with the decrease of the electrodeposition current density. actually, for the alsv and xps analysis, the increase in the at.% pd is equal to the decrease in the at.% ag. taking into account that the results of xps and alsv analysis are in relatively good agreement, the difference between the theoretically predicted and the experimentally detected alloy compositions could be the consequence of lower current efficiency for the alloy electrodeposition at lower current densities (jd) and lower amounts of charge for alloy electrodeposition (qd) (see tables s-i and s-ii). at higher current densities (< –400 µa cm–2) and higher values of qd the current efficiency ηj for alloy electrodeposition is very high (> 97 %), causing the identical theoretical and experimentally determined compositions of agpd alloys (see fig. 2 and tables s-i and s-ii). due to the low current densities for agpd alloys electrodeposition, the highest amount of charge was qd = 1.5 c cm–2 (see table s-i, sample (5)). characterization of agpd alloys electrodeposited under the conditions of convective diffusion. alsv responses for the dissolution of agpd alloys electrodeposited under the conditions of the convective diffusion are shown in fig. 5a for samples electrodeposited at different jd (qd = 0.2 c cm–2) and in fig. 5b for the sample electrodeposited at jd = –7.0 ma cm–2 to the qd = 3.0 c cm–2. for the dis electrodeposition and characterization of agpd alloy layers 601 solution of these samples the same comments and conclusions as those for the samples electrodeposited under the conditions of non-stationary diffusion are valid. -0.2 -0.1 0.0 0.1 0.2 0 1 2 3 4 5 -0.1 0.0 0.1 0.2 0 2 4 6 8 10 12 14 16 (a) rpm = 1000 q d = -0.2 c cm-2 j d / ma cm-2 -0.5 -4.0 -6.0 -8.0 j / m a c m -2 e / v vs. sce rpm = 1000 j d = -7.0 ma cm-2 q d = -3.0 c cm-2 q (p d) a ls v q(ag) alsv (b) j / m a c m -2 e / v vs. sce fig. 5. alsv responses for dissolution of agpd alloys recorded at ω = 1000 rpm and a sweep rate of 1 mv s-1 in the solution 0.1 mol dm-3 hcl + 12 mol dm-3 licl. a) alloys electrodeposited to the charge qd = 0.2 c cm -2 at different current densities (marked in the figure). b) alloy electrodeposited to the highest amount of charge. with the increase of jd the peak of the ag dissolution increases, while the peak of the pd dissolution decreases (fig. 5a), as in the case of the samples electrodeposited under the conditions of non-stationary diffusion. for the samples electrodeposited under the conditions of the convective diffusion the peak potential of pd dissolution has become more negative with the increase of jd (fig. 5a), while the one for the samples electrodeposited under the conditions of the non-stationary diffusion remains practically the same (fig. 4a). such behaviour could be the result of the different morphology of agpd samples electrodeposited by these two procedures (see figs. 9–11). for the sample electrodeposited to the highest charge (qd = 3.0 c cm–2), the separation of ag and pd peaks is not as well defined as for samples electrodeposited to the lower charges. only one sample (alsv presented in fig. 5b) was analyzed by the xps and eds. the results of conditions for agpd alloy electrodeposition and their compositions are shown in table iii. procedure of fitting alsv responses. according to the theoretical predictions for the alsv technique,14 alsv responses of the solid solution type alloys should be characterized with the two separate peaks only (corresponding to the dissolution of each metal), since in the case of alloys with the intermediate phases (intermetallic compounds) each phase should be characterized with a sep602 elezović et al. arate peak on the alsv. the shape of the alsv responses of agpd alloys is seen to change from alsv responses with two peaks only to the alsv responses with more than two peaks, depending on the value of jd and qd (see figs. 4 and 5). the procedure of fitting alsv responses with two peaks (using origin multi-peaks lorentzian function – origin 8.0, originlab, usa) was possible only for the samples electrodeposited at smaller current densities (< 200 µa cm–2) and the samples electrodeposited to the cathodic charges lower than 0.4 c cm–2. in fig. 6 are presented results of fitting the alsv responses of the agpd alloys dissolution electrodeposited at different values of jd to a different qd under the conditions of the non-stationary diffusion. the fitting lines (black solid lines) are in a good agreement with the experimental ones (the magenta dash-dot-dot lines), with the ag peak being labelled with the red dash lines, pd peak with the green dotted lines and a third, unknown peak, up, whose origin was undefined, was labelled with a blue dash-dot lines. it should be emphasized that the fit of the most positive peak, pd peak, cannot follow the shape of the lorentzian peak after the peak maximum has been reached, since the peak current density sharply decreases due to the removal of the traces of the electrodeposit and to the certain discrepancy between the fitting and the experimental lines must exist, as it could be seen in figs. 6 and 7. as it was already stated, the only sample electrodeposited at jd = 3jl(pd) could be fitted with two peaks, fig. 6a and b, while the appearance of a third peak (up) could already be observed for the sample electrodeposited at jd = 7jl(pd). -0.2 -0.1 0.0 0.1 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -0.1 0.0 0.1 0.0 0.5 1.0 1.5 2.0 -0.1 0.0 0.1 0.0 0.2 0.4 0.6 0.8 exp. j d = 7j l (pd) q d = -0.2 c cm-2 pd ag up (c) e / v vs. sce (b) jd = 3jl(pd) q d = -0.2 c cm-2 e / v vs. sce ag pd exp. pd ag exp. (a) jd = 3jl(pd) q d = -0.1 c cm-2 j / m a c m -2 e / v vs. sce fig. 6. results of fitting alsv responses of the agpd alloys electrodeposited at jd = 3jl(pd), qd = 0.1 c cm -2 (a), jd = 3jl(pd), qd = 0.2 c cm -2 (b) and jd = 7jl(pd), qd = 0.2 c cm -2 (c). fitting (black solid lines), experimental (magenta dash-dot-dot lines), ag (red dashed lines), pd (green dotted lines), unknown peak (up) (blue dash-dot lines). electrodeposition and characterization of agpd alloy layers 603 for the agpd alloys electrodeposited at a constant current density jd = –178 µa cm–2 (jd = 3jl(pd)) and ω = 0 rpm to the different amounts of charge (fig. 4b, table ii), the appearance of a third peak (up) was detected at the cathodic charges higher than 0.2 c cm–2. 0.0 0.1 0.2 0 2 4 6 8 10 12 14 -0.1 0.0 0.1 0.2 0 2 4 6 8 10 12 14 -0.1 0.0 0.1 0.2 0 2 4 6 8 10 12 14 16 (a)qd = -1 c cm -2 j / m a c m -2 e / v vs. sce (b)qd = -2 c cm -2 e / v vs. sce pd upag (c)qd = -3 c cm -2 e / v vs. sce fit exp. fig. 7. results of fitting alsv responses of the agpd alloys electrodeposited at ω = 1000 rpm: a) jd = –5 ma cm -2, qd = 1.0 c cm -2; b) jd = –5 ma cm -2, qd = 2.0 c cm -2; c) jd = –7 ma cm-2, qd = 3.0 c cm -2. fitting (black solid lines), experimental (magenta dash-dot-dot lines), ag (red dashed lines), pd (green dotted lines), unknown phase (up) (blue dash-dot lines). in addition to the q(pd)alsv and q(ag)alsv in tables i–iii are presented the results obtained by the analysis of the charge under the third peak, q(up)alsv, and the whole alsv responses (qalsv). third peak appears to be more pronounced since its charge, expressed in the percentages of the total charge under the alsv (qalsv), increases with the rise of the cathodic current density and the cathodic charge. it should be stated that such approach is a rough approximation, since all three peaks obtained by the fitting procedure overlap and it is practically not possible to determine the exact amount of charge for the up peak. in fig. 7 are presented the alsv responses of agpd alloy layers electrodeposited at various current densities and the various amounts of charge under the conditions of convective diffusion (ω = 1000 rpm). since the cathodic charges are higher than 0.4 c cm–2 all alsv responses had to be fitted with three peaks. the up peak is seen to increase with the increase of qd. the results presented in table iii are obtained by the analysis of alsv responses shown in fig. 7. as it can be seen, the charge for up reaches high values in the samples electrodeposited to higher cathodic charges (qd = 2.0 and 3.0 c 604 elezović et al. cm–2). the composition of only one sample (agpd3, qd = 3.0 c cm–2) was analyzed by the xps and eds techniques. hence, considering the results of the alsv analysis it appears that all electrodeposited agpd alloy layers do not represent solid the solution type alloy, being characterized with the presence of two peaks only (corresponding to the dissolution of ag and pd). the appearance of a up peak could be either the consequence of the formation of an additional phase, or the properties of the agpd electrodeposit. theoretically, there is a possibility for the formation of the different ordered structures in the agpd system,10 but the question arises, is it possible to detect these ordered structures by the application of the alsv technique. in order to define the appearance of up peak on the alsv responses, the additional analysis, using xps and sem–eds techniques was performed. characterization of the agpd alloys by the xps analysis five peaks, namely o1s, ag3d, pd3d, c1s and cl2p were detected on the surface of agpd1, agpd2 and agpd3 samples. for the composition analysis the most important peaks are ag3d and pd3d and their percentages were taken from the complete xps spectra. the xps results for these two elements are shown in fig. 8. by their deconvolution it was discovered that a certain amount of agcl could be detected on the ag3d spectra. the amount of agcl was seen to decrease from agpd1 to agpd3 samples, being 19.3 % for agpd1, 16.4 % for agpd2 and 5.3 % for agpd3. a certain amount (smaller in percentages than agcl) of pdo was detected under the pd3d spectra, was also decreasing from agpd1 to agpd3. characterization of the agpd alloys by the eds analysis the sem micrographs for the sample agpd1 are shown in fig. 9a and b, while the eds spectra recorded at the marked surface positions in fig.9a are presented in the supplementary material. the surface is heterogeneous and rough, with the presence of white and gray parts and the substrate is not completely covered with the electrodeposit (a,b). eds analysis of the white parts on the fig.9a contains approximately 12.6 at.% cl, while the eds of gray parts shows only 0.6 at.% cl, indicating that white parts of the electrodeposit could be agcl with the excess of chloride since the ratio ag/cl is higher than 1. the agcl was detected on much larger surface area (3 mm2) and on much lower depth (10.2 nm) by the xps analysis. hence, for the eds analysis only grey parts of the electrodeposit were considered and the corresponding results are given in tables s-i–s-iii. as can be seen in fig. 9a, agpd crystals are characterized by the rounded shape with the dimensions of about 200 nm. the sem micrographs for sample agpd2 are presented in fig. 9c and d, while the eds spectra recorded at the marked surface positions in fig.9c are shown in supplementary material. again, the surface is heterogeneous and rough, electrodeposition and characterization of agpd alloy layers 605 366 368 370 372 374 376 378 2x105 3x105 4x105 5x10 ag3d5/2 (a) in te ns ity , a .u . binding energy, ev ag3d3/2 measured agcl;ag-o agcl;ag-o ag metallic ag metallic background fitted 366 368 370 372 374 376 378 1x105 2x105 3x105 4x105 ag3d5/2 (c) in te ns ity , a .u . binding energy, ev measured agcl;ag-o agcl;ag-o ag metallic ag metallic background fitted ag3d3/2 366 368 370 372 374 376 378 0x105 0x105 0x105 0x106 2x106 4x106 ag3d5/2 (b) in te ns ity , a .u . binding energy, ev ag3d3/2 measured agcl;ag-o agcl;ag-o ag metallic ag metallic background fitted 334 336 338 340 342 344 2.0x105 2.5x105 3.0x105 3.5x105 4.0x105 pd3d5/2 (e) in te ns ity , a .u . binding energy, ev pd3d3/2 measured pd metallic pd metallic pdo pdo background fitted 334 336 338 340 342 344 1.25x105 1.50x105 1.75x105 2.00x105 2.25x105 pd3d5/2 (d) in te ns ity , a .u . binding energy, ev pd3d3/2 measured pd metallic pd metallic pdo pdo background fitted 332 334 336 338 340 342 344 6x104 7x104 8x104 9x104 1x105 1x105 pd3d5/2 (f) in te ns ity , a .u . binding energy, ev measured pd metallic pd metallic pdo pdo background fitted pd3d3/2 fig. 8. results of the xps analysis for ag3d (a,b and c) and pd3d (d,e and f) in samples agpd1, agpd2 and agpd3, respectively. with the presence of white and grey parts and the substrate is not completely covered with electrodeposit (c,d), but the non-covered substrate surface is smaller 606 elezović et al. in comparison with that for the sample agpd1. the white parts on the fig. 9c contain approximately 13.1 at.% cl, while the eds of the grey parts shows only 0.9 at.% cl. concerning the presence of agcl (xps) on the surface of sample agpd2, the same conclusion, as that for the sample agpd1, is valid. the agpd crystals (fig. 9c) are characterized by the rounded shape with the dimensions of about 300 nm. fig. 9. sem micrographs of the surface of agpd layers: a) and b) agpd1 sample, c) and d) agpd2 sample and e) and f) agpd3 sample. on microphotographs a), c) and e) the positions for the eds analysis are marked (eds spectra are given in supplementary material, the average composition given in table s-i). the sem micrographs for the sample agpd3 are presented in fig. 9e and f, while the eds spectra recorded at the marked surface positions in fig. 9e are shown in supplementary material. the surface is rough, composed of much larger crystals than those of samples agpd1 and agpd2. no presence of white electrodeposition and characterization of agpd alloy layers 607 parts was detected on the sample surface, indicating the absence of agcl. since this electrodeposit is much thicker, the substrate is completely covered with the electrodeposit (e,f). at some positions the large agpd crystals, of about 10–20 µm high (marked with arrows in fig. 9f), growing normally to the electrode surface, are detected (f), which is typical for the metals with fast nucleation rate, as are ag and pd.15–17 considering the results of alsv, xps and eds analysis for the samples electrodeposited under the conditions of the non-stationary diffusion (fig. 2), it could be stated that the similar compositions obtained by the alsv and xps are the result of the analysis of the large sample area (in the case of alsv the whole sample, in the case of xps 3 mm2), taking into account the presence of agcl on their surfaces. for the eds analysis only grey parts of the surfaces were analyzed, giving higher percentages of ag in the electrodeposit (tables s-i–s-iii). it should be mentioned that the average composition is analyzed by alsv and xps, while eds usually presents the local composition – at the specific, chosen surface locations. the appearance of up on the alsv responses, recorded for samples obtained at higher electrodeposition current densities and higher charges, is most probably the consequence of different morphologies. actually, with the increase of jd and qd the morphology becomes similar to that presented in fig. 9f and the ag from large agpd crystals, particularly those of higher heights (marked with arrows in fig. 9f), dissolves easier and faster than from the rest of the surface (the dissolution is a pure inversion of deposition).18 when the whole amount of ag from the large agpd crystals is dissolved, the current density on the alsvs starts to decrease until the ag begins to dissolve from the rest of the surface, causing the appearance of an additional peak on the alsv responses. hence, the appearance of an additional peak could be the consequence of this phenomenon.15–18 the alsv response for the thin agpd alloy layer (fig. 6a and b – low jd and qd) shows no additional peaks and could be fitted with two peaks using origin multi-peaks lorentzian function. hence, it appears that the additional peak on the alsv responses is the result of faster dissolution of large crystals of higher heights. this means that q(up)alsv, given in tables s-i–s-iii, roughly represents the percentage of the total surface area containing large agpd crystals of higher heights. conclusions the agpd alloys electrodeposited from the solution containing 0.001 mol dm–3 pdcl2 + 0.04 mol dm–3 agcl + 0.1 mol dm–3 hcl + 12 mol dm–3 licl under the conditions of non-stationary (ω = 0 rpm) and the convective diffusion (ω = = 1000 rpm), to the different amounts of charge and at different current densities, were characterized by the alsv, sem, eds and xps techniques. the compo608 elezović et al. sitions of the agpd alloys determined by the eds were almost identical to the theoretically predicted ones, while the compositions obtained by the xps and the alsv analysis (they were almost identical) were different. deviation from the theoretically predicted values (determined by the ratio jl(pd)/j(ag)) was more pronounced at lower current densities and at lower charges of agpd alloys electrodeposition, due to lower current efficiencies for alloys electrodeposition. the alsv analysis indicated the presence of ag, pd, expressed by the two alsv peaks, and the additional peak, which was found to correspond to the dissolution of large agpd crystals formed at electrodeposits with higher charge and larger cathodic current densities. the xps analysis, among the ag and pd, confirmed the presence of agcl at the surface of samples electrodeposited to low amounts of charge. supplementary material additional calculation and sem–eds data are available electronically at the pages of the journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. the authors are indebted to the ministry of education, science and technological development of the republic of serbia (project no. 172054) for the financial support of this work. the authors would like to acknowledge the cost mp 1407 action for networking support. и з в о д карактеризација електрохемијски исталожених слојева сребропаладијум легуpa различитим техникама невенка р. елезoвић1, piotr zabinski2, мила н. крстајић пајић3, tomasz tokarski4, борка м. јовић1 и владимир д. јовић1 1 институт за мултидисциплинарна истраживања универзитета у београду, кнеза вишеслава 1, 11030 београд, 2 agh university of science and technology, faculty of non-ferrous metals, al. mickiewicza 30,30-059 krakow, poland, 3 технолошко–металуршки факултет универзитета у београду, карнегијева 4, 11000 београд и 4 agh university of science and technology, academic centre for materials and nanotechnology, al. mickiewicza 30,30-059 krakow, poland танки слојеви сребро–паладијум легура исталожени су електрохемијским поступком на дисковима од злата и стакластог угљеника из раствора 0,001 mol dm -3 pdcl2 + 0,04 mol dm -3 agcl + 0,1 mol dm -3 hcl + 12 mol dm -3 licl под условима нестационарне (ω = 0 o min-1) и конвективне (ω = 1000 o min-1) дифузије, при различитим вредностима густине струје таложења и количине наелектрисања. електрохемијски исталожени слојеви окарактерисани су анодном линеарном волтаметријом (alsv), скенирајућом електронском микроскопијом (sем), енергетски диспергованом рендгенском спектроскопијом (eds) и рендгенском фотоелектронском спектроскопијом (xps). састав сребро– паладијум легура одређен eds техником био је скоро идентичан теоријски предвиђеном, док су састави одређени xps и alsv техникама (који су били скори идентични) одступали од теоријски предвиђеног, при чему је то одступање било израженије код узорака исталожених мањим густинама струје и узорака исталожених до мањих количина наелектрисања. alsv анализа је показала присуство сребра и паладијума, изражено као два струјна врха, као и присуство још једног струјног врха, за који је установљено sем и eds анализама да је последица растварања великих сребро–паладијум electrodeposition and characterization of agpd alloy layers 609 кристала који се формирају код дебљих превлака, исталожених до већих количина наелектрисања. на тај начин је по први пут показано да поред фазног састава и морфологија талога легура може утицати на облик alsv одговора. xps анализом је на површини тањих узорака (исталожених до мањих количина наелектрисања у условима нестационарне дифузије) потврђено присуство сребро-хлорида, поред сребра и паладијума. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {comments on the article entitled adsorption of strontium on different sodium-enriched bentonites by sonja r. marinovic, marija j. ajdukovic, natasa p. jovic-jovicic, tihana m. mudrinic, bojana n. nedic-vasiljevic, predrag t. bankovic and aleksandra d. milutinovic-nikolic, published in the journal of the serbian chemical society, volume 82, issue 4, 2017, pp. 449-463} j. serb. chem. soc. 83 (3) 391–393 (2018) udc 546.42’33+666.32:544.723: jscs–5082 628.316.2(0.072) letters to the editor 391 letter to the editor comments on the article entitled adsorption of strontium on different sodium-enriched bentonites by sonja r. marinović, marija j. ajduković, nataša p. jović-jovičić, tihana m. mudrinić, bojana n. nedić-vasiljević, predrag t. banković and aleksandra d. milutinović-nikolić, published in the journal of the serbian chemical society, volume 82, issue 4, 2017, pp. 449–463 slobodan k. milonjić*# vinča institute of nuclear sciences, university of belgrade, belgrade, serbia (received 16 december 2017, accepted 15 january 2018) abstract. recently, marinović et al.1 have presented a study on the adsorption of strontium on different sodium-enriched bentonites. however, some experimental procedures and methods used, as well as presentation and interpretation of some data, are questionable, i.e., erroneous. keywords: adsorption; bentonites; problematic interpretation. recently, i have read the article of marinović et al.1 on the adsorption of strontium on different sodium-enriched bentonites with great interest. but, it seems to me that a few scientific questions about the presentation and interpretation of some experimental data have to be raised. in experimental, adsorption experiments, the authors wrote that the amount of sr2+ adsorbed, qt (mg g–1), after time t was calculated from the following mass balance relationship (eq. (1)):1 0 ads t t c c q m − = (1) where c0 and ct are the concentration (mg dm–3) of sr2+ in the initial solution and after adsorption time t, respectively. * e-mail: smiloni@vinca.rs # serbian chemical society member. https://doi.org/10.2298/jsc171216017m ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 392 milonjić the expression of eq. (1), however, is incorrect. according to this equation, the obtained/resulting qt values have the unit mg g–1 dm–3. equation (1) should be correctly expressed as eq .(2): 0 ads ( )− = tt c c v q m (2) where v is the volume of solution (dm3) in authors’ case, 0.050 dm3. therefore, the qt values calculated according to eq. (1) are incorrect. the q values plotted on the y-axis of figs. 3–7 and given in tables ii and iii1 are also incorrect. therefore, the values obtained from these data, as well as the discussions related to the adsorption of sr2+ on different sodium-enriched bentonites are invalid, and consequently re-calculation of all parameters is strongly recommended. from fig. 4,1 it is evident that the adsorbed amounts of sr 2+ on na-t, na-w and na-b bentonites in the initial phi range ~4 to ~9 are constant, because the final phf values are constant. please note that the adsorbed amount depends on the equilibrium solution ph, i.e., phf. on pages 457 and 459,1 the authors wrote: “this indicates that the ion exchange mechanism was the dominant adsorption mechanism” and “as previously stated, the ion exchange mechanism could be regarded as the dominant adsorption mechanism”, respectively, giving however no experimental proof for such a statement. in the case of ion exchange, the amount of sorbed cation(s) from a solution (in this case sr2+) should be equivalent to the amount of released cation(s) from the sorbent (in this case na+, and other cations present in the investigated bentonites). in fig. 5,1 the authors illustrate the effect of the contact time on the adsorption of sr2+ on three studied bentonites. it is evident that the adsorption equilibrium for na-b and na-t bentonites was reached within 20–25 min. however, in fig. 6,1 they show linear plots for the pseudo-second-order kinetics. the use of data recorded after the reached equilibrium (in this case after 25 min), or very close to the equilibrium, for a significant interval of time is likely to lead to erroneous conclusions in the adsorption kinetics analysis. the measurements of the adsorption of sr2+ on the studied bentonites should be started at an initial time shorter than 10 min. the dimension of the freundlich adsorption constant given in table ii, as kf in dm3 mg–1, is incorrect. the correct dimension is (mg g–1)/(mg dm–3)n. the values for the energy of adsorption (e) according to the dubinin–raduskevich isotherm model are given in table iii. on the basis of the calculated e values the authors wrote: “the calculated e values were similar for all investigated adsorbents and slightly higher than 8 kj mol–1, indicating that the type of adsorption was chemisorption.” in the same table, the authors gave the freundlich adsorption constant (n) data, from 5.78 to 9.558, for all investigated bentonites. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. comments on an article published in jscs 393 based on this (n > 1), it means that the adsorption process is physical. it is evident that the given data, and the conclusion drawn from them, are in disagreement. finally, all the results and discussions related to the adsorption of sr2+ on studied bentonites, as well as drawn conclusions are highly questionable. i hope that these comments, intended to prevent future mistakes, will be of assistance not only to the authors of commented paper1 but also to others working in this field. to conclude, i suggest the authors of the paper,1 as well as other researchers and reviewers working in the field of adsorption/sorption from solutions, to read the paper (a critical review) of tran et al.2 devoted to mistakes and inconsistencies regarding the adsorption of contaminants from aqueous solutions. и з в о д коментари на рад под насловом адсорпција стронцијума на различитим натријумом измењеним бентонитима, аутори: сања мариновић, марија ајдуковић, наташа јовић-јовичић, предраг бранковић, тихана мудринић, бојана недић-васиљевић и александра милутиновић-николић, објављеног у journal of the serbian chemical society, volume 82, issue 4, 2017, стр. 449–463 слободан к. милоњић институт за нуклеарне науке “винча”, универзитет у београду, београд недавно је у journal of the serbian chemical society објављен рад (jscs 82(2017)449– –463) под насловом “адсорпција стронцијума на различитим натријумом измењеним бентонитима”, чији су аутори сања мариновић, марија ајдуковић, наташа јовић-јовичић, предраг банковић, тихана мудринић, бојана недић васиљевић и александра милутиновић-николић. циљ ових добронамерних коментара је био да се укаже на: погрешно израчунавање појединих величина, грешке у интерпретацији добијених/израчунатих резултата као и грешке изнете у закључцима рада. аутор ових коментара верује да ће изнети коментари бити од користи не само ауторима предметног рада већ и осталим ауторима и рецензентима који раде у области адсорпције/сорпције из раствора. (примљено 16. децембра 2017, прихваћено 15. јануара 2018) references 1. s. r. marinović, m. j. ajduković, n. p. jović-jovičić, t. m. mudrinić, b. m. nedić -vasiljević, p. t. branković, a. d. milutinović-nikolić, j. serb. chem soc. 82 (2017) 449 2. h. n. tran, s.-j. you, a. hosseini-bandegharaei, h.-p. chao, water res. 120 (2017) 88. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {discussion on the comments on dft calculation, biological activity, anion sensing studies and crystal structure of (e)-4-chloro-2-((pyridin-2-ylimino)-methyl)phenol by nuray yıldırım, neslihan demir, g{\"{o}}khan alpaslan, bahadir boyacıoğlu, mustafa yıldız, and huseyin {\"{u}}nver, published in the journal of the serbian chemical society, volume 83, issue 6, 2018, pp. 707–721} j. serb. chem. soc. 84 (1) 117 (2019) letters to the editor jscs–5169 117 letter to editor discussion on the comments on dft calculation, biological activity, anion sensing studies and crystal structure of (e)-4-chloro-2-((pyridin-2-ylimino)-methyl)phenol by nuray yıldırım, neslihan demir, gökhan alpaslan, bahadir boyacıoğlu, mustafa yıldız and huseyin ünver, published in the journal of the serbian chemical society, volume 83, issue 6, 2018, pp. 707–721 mustafa yildiz* çanakkale onsekiz mart university, department of chemistry, 17100 çanakkale, turkey (received 1 october, accepted 3 october 2018) we re-evaluated the x-ray data carefully. since we worked on the schiff bases of 2-aminopyridine and 3-aminopyridine, the possibility that the crystals have been confused cannot be ruled out. therefore, we agree with marko v. rodić on b structure and would like to thank for his careful consideration. the data and studies in the paper https://doi.org/10.2298/jsc180925082r are correct. we really regret this undesirable confusion. * e-mail: myildiz@comu.edu.tr ________________________________________________________________________________________________________________________ (cc) 2019 scs. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {application of the eco-friendly subcritical water oxidation method in the degradation of epichlorohydrin} j. serb. chem. soc. 84 (7) 757–767 (2019) udc 547.583.1’422+628.16.094.3:66.092:574 jscs–5224 original scientific paper 757 application of the eco-friendly subcritical water oxidation method in the degradation of epichlorohydrin erdal yabalak*, i̇pek topaloğlu and ahmet murat gi̇zi̇r department of chemistry, faculty of arts and science, mersin university, çiftlikköy campus, tr-33343, mersin, turkey (received 8 december 2018; revised and accepted 4 april 2019) abstract: degradation of epichlorohydrin was investigated using subcritical water oxidation method in the presence of hydrogen peroxide. degradation rate was monitored by means of total organic carbon (toc) analysis. the central composite design was used to determine optimal toc removal conditions and modelling experimental process. the effects of all experimental variables (temperature, oxidant concentration of hydrogen peroxide and treatment time) on the toc removal rates were evaluated and the theoretical prediction model was proposed. reliability of the employed model was evaluated using anova. f value and the p-value of the model were found to be 84.60 and <0.0001, respectively. 93.78 % of toc removal was achieved in the degradation of epichlorohydrin at 373 k of temperature and 75 min of treatment time using 90 mm of h2o2. keywords: epichlorohydrin; degradation, anova; eco-friendly method; subcritical water. introduction epichlorohydrin (1-chloro-2-epoxypropane, epc) is a raw compound used in the production of epoxy resins.1,2 it is widely used in the paper and pharmaceutical industries, production of drinking water pipes and synthesis of cationic polyelectrolytes.3,4 industrial wastewater and other contaminants are released during the epc production process.5 epc is listed by the epa as being toxic to the aquatic environment.1 central nervous system destruction, inflammation of lungs, local irrigation and nausea are some of the known harmful effects of epc to human health.6,7 moreover, epc has been listed in group 2a by the international agency for research on cancer as having the potential carcinogenic effect.5,6 therefore, some effective methods for treating water containing epc are needed. however, conventional methods are far from being a solution due to the * corresponding author. e-mail: yabalakerdal@gmail.com https://doi.org/10.2298/jsc181208027y 758 yabalak, topaloğlu and gi̇zi̇r hard-to-degrade structured contaminants. further, these methods may cause the formation of even more harmful intermediates than the main pollutant.8 subcritical water has been widely used, especially in oxidation, solubility, extraction and synthesis processes.8–11 in this study, subcritical water oxidation method (swo), which is known as an environmentally friendly and effective method, was performed in the degradation of epc. subcritical water oxidation depends on the oxidation of organic compounds in the aqueous phase at a high temperature (373–647 k) and high enough pressure to keep water in the liquid state. hazardous organic compounds and micro-pollutants can be degraded to harmless organic compounds such as co2 and h2o by using this method. the oxidation process in which h2o2 is used as an oxidizer is effective in the application of wastewater containing medium and high concentration levels of organic carbon. in addition, h2o2 is a non-toxic and an ecological oxidant that does not cause any harmful by-product formation.11 the degradation of epc was investigated using subcritical water oxidation and h2o2. degradation rate was monitored by toc analysis, which is the best -known method for determining the organic content of an aqueous sample. the experimental parameters (temperature, time, oxidant concentration) and the optimum degradation percentage were assessed by the response surface method (rsm). rsm consists of a set of mathematical and statistical techniques and it is used to define the relationship between the response and the independent variables of a system. though several design methods of the response surface method have been studied, central composite design (ccd) arises as the most preferred one.8,11,12 experimental reagents and apparatus the analytical grade epc and h2o2 were purchased from sigma–aldrich (st. louis, mo, usa). n2 gas was provided from linde gas (turkey). ultra-pure water (18 mω cm, 25 °c) was obtained using millipore milli-q advantage a10 apparatus (darmstadt, germany). experiments were performed in the home-made stainless steel reactor which was given in the previous work in detail.8 toc analyses of the stock solution (100 ppm) and treated samples were performed using toc-l analyzer with an asi-l autosampler (shimadzu). degradation method the experimental design of the independent variables was determined using ccd. the five levels of the three independent variables such as temperature, concentration of h2o2 and treatment time were assigned after preliminary experiments and established design model was applied in the experimental process (table i). the degradation experiments were carried out according to the previously published methods which were briefly given below.8,11 150 ml of the stock solution of epc was placed in the reactor followed by the specific amount of h2o2. the inner pressure of the reactor was fixed at 30 bar using n2 gas for providing the subcritical water medium. the reactor was heated to a certain temperature during a specific treatment degradation of epichlorohydrin using subcritical water oxidation 759 time. the mentioned amounts of h2o2, temperature and treatment time are given in table ii. 20 ml of treated sample was kept at 281 k after each run for further analyses. table i. ccd model of the experimental variables along with their coded levels factor independent variable coded levels –1.682 –1 0 1 1.682 x1 temperature, k 352.55 373 403 433 453.45 x2 concentration of h2o2, mm 7.96 25 50 75 92.05 x3 treatment time, min 9.55 30 60 90 110.45 table ii. experimental and predicted results of the toc removal efficiency of epc run x1 x2 x3 toc removal, % exp. ccd pred. 1 403 (0) 50 (0) 60 (0) 88.05 87.56 2 403 (0) 50 (0) 60 (0) 87.29 87.56 3 373 (–1) 25 (–1) 90 (+1) 65.49 64.45 4 373 (–1) 75 (+1) 90 (+1) 93.78 92.43 5 403 (0) 50 (0) 60 (0) 85.94 87.56 6 433 (+1) 75 (+1) 30 (–1) 89.52 88.36 7 433(+1) 25 (–1) 30 (–1) 64.72 63.88 8 433(+1) 25 (–1) 90 (+1) 69.13 69.04 9 403 (0) 50 (0) 60 (0) 88.20 87.56 10 352.55 (–1.682) 50 (0) 60 (0) 56.39 60.25 11 373 (–1) 75 (+1) 30 (–1) 69.43 67.33 12 403 (0) 7.96 (–1.682) 60 (0) 47.60 49.96 13 403 (0) 50 (0) 60 (0) 88.38 87.56 14 403 (0) 50 (0) 60 (0) 88.05 87.56 15 433 (0) 75 (+1) 90 (+1) 89.68 91.26 16 403 (0) 50 (0) 110.45 (+1.682) 88.12 87.60 17 403 (0) 92.05 (+1.682) 60 (0) 93.32 94.07 18 403 (0) 50 (0) 9.55 (–1.682) 58.53 62.16 19 453.45 (+1.682) 50 (0) 60 (0) 82.54 81.79 20 373 (–1) 25 (–1) 30 (–1) 40.87 37.10 toc method the toc analysis was known as being a safe and practical method to measure the organic content of an aqueous sample.11,13 the toc content of the stock solution and the treated samples was measured by toc-l analyzer with an asi-l autosampler (shimadzu). the toc removal percentages of the stock solution and the treated samples were calculated according to the equation given in the previous work. 8,11 ccd modeling rsm provides several advantages such as saving time, reagent, labour and etc. through representing efficient experimental designs and requiring a limited number of experiments.14 not only does rsm reduce the number of experiments, but it also allows to determine the relationship between variables and the effect of the variables on the response.11 ccd, as one of the rsm models, provides an evaluation of interaction effects between the independent 760 yabalak, topaloğlu and gi̇zi̇r variables and the response and enables establishing the approximation equations for the prediction of the response.15 the ccd model was employed to establish the experimental design and the experimental parameters of each run, and they were demonstrated along with the experimental and the predicted toc removal percentages in table ii. x1, x2 and x3 represent the temperature, the concentration of h2o2 and the treatment time, respectively. results and discussion the experimental and the predicted results of the toc removal efficiency of epc were given in table ii along with the running parameters. the highest and the lowest experimental toc removal rates were obtained to be 93.78 and 40.87 %, respectively at runs 4 and 20. moreover, the predicted toc removal of 92.43 and 37.10 %, which were obtained at run 4 and 20, respectively, show the accordance between the experimental and the predicted results. also, these results proved the applicability of the employed ccd model. statistical analysis of ccd modeling the significance of the model can be proved by means of statistical analysis.8,11 tables iii and iv demonstrate the anova results and the regression coefficients of the ccd model of the degradation of epc, respectively. p-value and f value were obtained as 84.60 and <0.0001, respectively for the model. both of the p-value and f value are at a satisfactory level.8,11 thus, the employed ccd model can be used to navigate design, determine the combined effects of experimental factors on the response and achieve the approximation model. moreover, x1, x2, x3, x1x3, x12, x22 and x32 are the other significant terms of the model. table iii. anova results of the ccd model of the degradation of epc source sum of squares df mean square f value p-value prob > f model 4976.32 9 552.92 84.60 < 0.0001 x1 560.09 1 560.09 85.60 < 0.0001 x2 2348.55 1 2348.55 359.34 < 0.0001 x3 781.42 1 781.42 119.56 < 0.0001 x1x2 16.53 1 16.53 2.53 0.1428 x1x3 246.42 1 246.42 37.70 0.0001 x2x3 2.55 1 2.55 0.39 0.5459 x1 2 493.16 1 493.16 75.46 < 0.0001 x2 2 435.63 1 435.63 66.65 < 0.0001 x3 2 289.90 1 289.90 44.36 < 0.0001 residual 65.36 10 6.54 – – lack of fit 61.15 5 12.23 14.53 0.0053 pure error 4.21 5 0.84 cor total 5041.68 19 – the reliability of the ccd model was also supported by regression and correlation analysis (table iv). the 472.50 value of the predicted residual sum of squares (press) indicates that the model fits each point in the design and differ degradation of epichlorohydrin using subcritical water oxidation 761 ences between the actual and the predicted results are at an acceptable level.11 the r2 value of 0.9870 supports the above-mentioned findings. in addition, the obtained adjusted r2 (0.9754) and predicted r2 (0.9063) values are quite close to each other. this closeness demonstrates the high correlation level between the experimental and the predicted results of the employed model. table iv. regression coefficients of the ccd model standard deviation 2.56 r2 0.9870 mean 76.75 adjusted r2 0.9754 coefficient of variation value (c.v.), % 3.33 predicted r2 0.9063 press 472.50 adequate precision 31.515 the accordance between the actual and the predicted values of the toc removal of epc was demonstrated in fig. 1. this figure clearly shows the compatibility within the points which represent the actual and predicted values. almost all points (each point represent one run) are aligned on the line. also, the closeness of adjusted r2 and predicted r2, which was mentioned above, supports this accordance: 1 2 3 1 2 1 3 2 2 2 2 3 1 2 3 7.56 1.44 5.55 0.56 5.50 4.49 6.40 13.11 5.858 87.56 y x x x x x x x x x x x x + − − − +− −−− = + (1) fig. 1. correlation between actual and predicted values. the predicted values of epc degradation percentages were obtained by second-order equation (eq. (1)). the degradation rates can be predicted in the working range of each system variables and the interaction of these variables and their contribution to the efficiency of the process can be analysed by this equation. thus, the concentration of h2o2 was found to be the most effective variable on the toc removal of epc, following by the treatment time and the temperature. 762 yabalak, topaloğlu and gi̇zi̇r evaluation of the combined effects of the experimental variables on the toc removal of epc experimental toc removal percentages of epc were evaluated using three -dimensional (3d) plots. these plots are useful to demonstrate the interactive effects of experimental variables on the response. also, they allow easy evaluation of the optimum conditions for the maximum theoretical toc removal rate.8,11 the combined effects of the concentration of h2o2 and the temperature on the toc removal of epc at the fixed treatment time of 60 min were displayed in fig. 2. the increase in the temperature favours the formation of hydroxyl and other radicalic species, thus allowing an increase in the toc removal of target pollutant.11 it is clearly seen from fig. 2 that the temperature values above 383 k and the concentration of h2o2 above 45 mm are adequate for the obtaining of the elevated toc removal rates. thus, the higher temperature and concentration of h2o2 should be seen as redundant. the broad red area of fig. 2, which demonstrates the high yielded region, also supports this results. for instance, the toc removal of epc can be increased from 55.25 to 75.31% through doubling the 25 mm of concentration of h2o2 at 373 k and the fixed treatment time of 60 min. however, an extra 25 mm increase in the concentration of h2o2 at the same treatment time can only contribute 9% in the toc removal. moreover, increasing both of the temperature and the concentration of h2o2 to their highest levels (433 k and 75 mm, respectively) provides 94.30 % of the toc removal at the fixed treatment time of 60 min. fig. 2. combined effects of the concentration of h2o2 and temperature on the toc removal of epc at the fixed treatment time of 60 min. fig. 3 demonstrates the combined effects of the treatment time and the concentration of h2o2 on the toc removal of epc at the fixed temperature of 400 k. it is clearly seen from this figure that high toc removal rates can be achieved at degradation of epichlorohydrin using subcritical water oxidation 763 fig. 3. combined effects of treatment time and concentration of h2o2 on the toc removal of epc at the fixed temperature of 400 k. high concentration levels of h2o2 and in the short treatment time or moderate concentration level of h2o2 and in the moderate-long treatment time at the fixed temperature of 400 k. 87.59 % of toc removal can be increased to 92.18 % by decreasing the concentration of h2o2 from 75 to 55 mm and increasing the treatment time from of and 40 to 75 min, at the fixed temperature of 400 k. however, it is hardly possible to increase the toc removal from 97.28 to 97.69 % by prolonging the treatment time from 75 to 90 min and adding 75 mm of the concentration of h2o2 at the fixed temperature of 400 k. besides, the co-effect of the concentration of h2o2 and the treatment time was found to be crucial on the toc removal of epc. though the concentration of h2o2 has a significant effect on the degradation of epc, a specific time is required for efficient formation of free radicals from h2o2. fig. 4 demonstrates the combined effects of the treatment time and the temperature on the toc removal of epc at the fixed concentration of h2o2 of 60 fig. 4. combined effects of treatment time and temperature on the toc removal of epc at the fixed concentration of h2o2 of 60 mm. 764 yabalak, topaloğlu and gi̇zi̇r mm. as can be seen from this figure, the red area is relatively wide and scattered. this means that 60 mm of the concentration of h2o2 offers an appropriate medium for obtaining the elevated toc removal. nevertheless, the low levels of the temperature and the treatment time are not enough to achieve high efficiency at the fixed concentration of h2o2 of 60 mm. thus, the temperature and the treatment time should be increased. the toc of 62.88 % removal can be achieved at 373 k and 30 min of the treatment time at the fixed concentration of h2o2 of 60 mm. only 10 units of the raise of temperature and the treatment time provide 13.21 % of the increase in the toc removal. moreover, an extra 10 units of the increase of temperature and the treatment time provides an extra 9.69 % increase in toc removal. fig. 5 demonstrates the cube plot of the ccd model for toc removal rates of epc. this figure facilitates the evaluation of response over changing all experimental variables. the possible toc removal percentages can be predicted by adjusting the experimental variables from –1 and +1 levels. furthermore, this plot enables further predictions and demonstrates the required conditions. for instance, the treatment time and the concentration of h2o2 must be adjusted to their highest level and the temperature must be adjusted to its lowest level to achieve 92.43 % of toc removal. fig. 5. cube plot of the employed ccd model. fig. 6 displays the perturbation plot of the toc removal of epc. the optimal conditions for the efficient toc removal percentages of epc can be seen in this figure. the slope and the direction of curves for each variable provide the determination of the toc removal rates. fig. 6 shows that a reasonable toc removal percentage can be obtained at 422 k of the temperature, 53 mm of the concentration of h2o2 and 61 min of the treatment time. degradation of epichlorohydrin using subcritical water oxidation 765 fig. 6. perturbation plot of toc removal of epc validation of the ccd model a series of experiments were performed to validate the precision of the employed method. the table v demonstrates the validation experiments and the obtained results with standard deviation values. the experimental conditions of each variable were selected in the working range, but not the same as the ones given in table ii. it is clearly seen from table v that the experimental and the theoretical results are in agreement with each other. the differences between the experimental and the theoretical results are at a reasonable level, considering the standard deviation value of the model (2.56). thus, the reliability of the employed model was proved by the validation experiments beside being statistically evaluated. table v. validation of the ccd model run x1 x2 x3 toc removal, % exp. pred. 1 383 60 40 75.19±0.87 76.09 2 420 40 60 85.01±1.23 83.51 3 400 70 80 95.84±0.96 97.07 conclusion the degradation of epc was extensively investigated using the eco-friendly subcritical water degradation method and a green oxidising agent, h2o2. the rate of the degradation of epc was monitored by the measuring of toc removal. the maximum toc removal was achieved as 93.78 %, at the temperature of 373 k, 75 min of the treatment time, using 90 mm of h2o2. the co-effects of the main parameters such as the temperature, the concentration of h2o2, and the treatment time on the toc removal rates were determined using the ccd modeling. the reliability of the employed ccd model was evaluated by anova. the approxi766 yabalak, topaloğlu and gi̇zi̇r mation model for the toc removal percentage of epc was proposed and the optimal conditions for efficient toc removal were evaluated. it was obtained that the concentration of h2o2 is the most effective factor on the toc removal rates of epc. и з в о д примена еколошки прихватљиве разградње епихлорохидрина методом оксидације у подкритичној води erdal yabalak i̇pek topaloğlu и ahmet murat gi̇zi̇r department of chemistry, faculty of arts and science, mersin university, çiftlikköy campus, tr-33343, mersin, turkey проучавана је разградња епихлорохидрина методом оксидације у подкритичној води у присуству водоник-пероксида. брзина разградње праћена је анализирањем toc. за одређивање оптималних услова за смањење toc коришћено је моделовање експерименталног процеса конструисањем централног композита (central composite design). оцењен је утицај свих експерименталних променљивих (температура, концентрација оксиданса водоник-пероксида и време третирања) на брзину смањивања toc, те је предложен теоријски модел. поузданост овог модела је оцењена методом anova. по моделу, вредности за f и p биле су 84,60 односно <0,0001. постигнуто је 93,78 % смањења toc у разградњи епихлорохидрина на температури од 373 k, 75 min третирања коришћењем 90 mm h2o2. (примљено 8. децембра 2018, ревидирано и прихваћено 4. априла 2019) references 1. c. sarzanini, m.c. bruzzoniti, e. mentasti, j. chromatogr., a 884 (2000) 251 (https://doi.org/10.1016/s0021-9673(00)00252-1) 2. h. hindsø landin, t. grummt, c. laurent, a. tates, mutat. res. 381 (1997) 217 (https://doi.org/10.1016/s0027-5107(97)00171-1) 3. l. lucentini, e. ferretti, e. veschetti, v. sibio, g. citti and m. ottaviani, microchem. j. 80 (2005) 89 (https://doi.org/10.1016/j.microc.2004.12.003) 4. m.c. bruzzoniti, s. andrensek, m. novic, d. perrachon, c. sarzanini, j. chromatogr., a 1034 (2004) 243 (https://doi.org/10.1016/j.chroma.2004.02.033) 5. j. gaca and w.grażyna, talanta 70 (2006) 1044 (https://doi.org/10.1016/j.talanta.2006.02.017) 6. m. lasa, r. garcia, e. millán, j. chromatogr. sci. 44 (2006) 438 (https://doi.org/10.1093/chromsci/44.7.438) 7. k. chandrasekara pillai, g. muthuraman, i.-s. moon, electrochim. acta 232 (2017) 570 (https://doi.org/10.1016/j.electacta.2017.02.147) 8. e. yabalak, j. environ. chem. eng. 6 (2018) 7132 (https://doi.org/10.1016/j.jece.2018.10.010) 9. e. yabalak, a.m. gizir, j. serb. chem. soc. 78 (2013) 1013 (https://doi.org/10.2298/jsc120321123y) 10. y. nural, m. gemili, e. yabalak, l. de coen, m. ulger, arkivoc 2018 (5) (2018) 51 (https://doi.org/10.24820/ark.5550190.p010.573) 11. e. yabalak, j. environ. sci. health, a 53 (2018) 975 (https://doi.org/10.1080/10934529.2018.1471023) 12. j.l. pilkington, c. preston, r.l. gomes, ind. crop. prod. 58 (2014) 15 (https://doi.org/10.1016/j.indcrop.2014.03.016) degradation of epichlorohydrin using subcritical water oxidation 767 13. a. llop, e. pocurull, f. borrull, water air soil pollut. 197 (2009) 349 (https://doi.org/10.1007/s11270-008-9816-7) 14. s. karimifard, m.r.a. moghaddam, sci. total environ. 640–641 (2018) 772 (https://doi.org/10.1016/j.scitotenv.2018.05.355) 15. c. r. t. tarley, g. silveira, w. n. l. dos santos, g. d. matos, e. g. p. da silva, m. a. bezerra, m. miró, s. l. c. ferreira, microchem. j. 92 (2009) 58 (https://doi.org/10.1016/j.microc.2009.02.002). {oxidized humic acids from the soil of heat power plant} j. serb. chem. soc. 85 (3) 421–426 (2020) udc 504.53.05+620.4:547.992.2+579.6 jscs–5311 short communication 421 short communication oxidized humic acids from the soil of heat power plant srđan b. miletić1#, jelena avdalović1#, jelena milić1#, mila ilić1, aleksandra žerađanin1#, kristina joksimović2# and snežana spasić1*# 1university of belgrade, national institute for chemistry, technology and metallurgy, serbia and 2university of belgrade, innovation center of the faculty of chemistry, serbia (received 27 july, revised and accepted 9 september 2019) abstract: humic acids isolated from the soil of a heat power plant (ha-e) contaminated with oil were analyzed by fourier transform infrared spectroscopy (ftir). in comparison with a humic acids standard (ha-s), a lack of an intense broad band of the stretching vibrations of hydrogen-bonded hydroxyl groups (3600–3200 cm-1) is evident. the ha-e spectra have a peak at 1649 cm-1, which could belong to carbonyl groups. ha-e are heavily oxidized and among the isolated microorganisms, achromobacter denitrificans may be responsible for such intensive oxidation of ha-e. to the phylogenetically diverse nitrate-reducing microorganisms that have the capacity to utilize reduced ha as electron donors in soils, a. denitrificans can be added. keywords: humic acid; microorganism; ftir; contaminated soil. introduction structurally diverse, humic acids (ha) contain numerous functional moieties, including carboxylic acid, ketone, quinone, and phenolic/alcoholic hydroxyl groups. ha may be of particular relevance to inherent soil denitrification.1 ha are recalcitrant and degrade relatively slowly. however, reduced redox-active functional groups within ha can be readily oxidized as electron donors for bacterial respiration.1,2 the hydroquinone content of reduced ha are considered important humus-borne electron donors of this type, and microbial oxidation of hydroquinones to the corresponding quinones has been demonstrated to support nitrate, perchlorate, arsenate, and selenate reduction.1–7 although nitrate-dependent humic acid-oxidizing bacteria (nhox) are common in the environment,2 their prevalence and metabolic activity in soils are still not well known. * corresponding author. e-mail: svujin@chem.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc190726099m ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 422 miletić et al. it was decided to isolate ha-e to determine the number of presented microorgansims by the serial dilutions method on agar plates, to identify microorganisms and to compare the ftir spectra of humic acid standard with the ftir spectra of the humic acid isolated from samples of soil contaminated with oil from a heat power plant. experimental the number of microorganisms the number of microorganisms present was determined by the serial dilutions method on agar plates at 28 °c. several types of media were used: • nutrient agar for total chemoorganoheterotrophs (na), • malt agar for yeasts and molds (sa), • mineral base medium for hydrocarbon degraders (ug)8 with d2 diesel fuel9 and • nutrient agar for anaerobes (hag). identification of microorganisms using api tests analytical profile index (api) tests were realized using the “biomerieux industry” manual. the api 20 ne, rapid 20e and api coryne systems were used for the detection of the microorganisms. isolation of humic acids following the standard procedure, the humic acids were isolated from soil contaminated with oil from the heat power plant with an alkaline sodium pyrophosphate solution (iso 5073:1999). the ha-e was precipitated with hydrochloric acid. ftir ftir spectra were obtained in solid state using the attenuated total reflectance (atr) sampling technique on thermo-nicolet 6700 spectrophotometer (thermo fisher scientific, usa) at wave numbers between 400 and 4000 cm-1 with a spectral resolution of 4.0 cm-1 in the transmission mode. the spectra were analyzed using omnic 7.3 software. results and discussion the number of microorganisms the obtained results (table i) show that the number of ug microorganisms was high in comparison with other types of microorganisms, which could indicate a high level of oil pollution of the soil. table i. number of microorganisms media number of microorganisms, cfu g-1 na 4.3×106 sa 2.6×103 ug 3.5×104 hag 1.8×102 ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ humic acids from the soil 423 identification of microorganisms using api tests several microorganisms, such as pseudomonas putida, pseudomonas aeruginosa, achromobacter denitrificans, pseudomonas sp., bacillus cereus, rhodococcus sp., aeromonas hydrophila, etc., were identified. ftir spectra in ftir spectrum of ha-s, intense broad band of stretching vibrations of hydrogen bonded hydroxyl groups (3600–3200 cm–1) could be observed (fig. 1). the standard has a very strong band at 1737 cm–1 that is assigned to c=o stretching of cooh group and a band at 1229 cm–1 that corresponds to in-plane bending vibrations of oh groups from cooh (fig. 1). fig. 1. ftir of ha-s. extracted humic acids (ha-e) lack oh groups and aliphatic chains compared to the standard ha, because peaks is absent in the 3000–3700 cm–1 and 2920–2850 cm–1 regions are absent. the peak at 1649 cm–1 could belong to carbonyl group (fig. 2). it was previously reported that ha promote zymogenous microbial consortium growth.10 ha influences microbial consortium growth via protection from oxidative stress, and this mechanism is highly significant in oxidative conditions (i.e., in the presence of free iron). a. denitrificans was identified from samples of ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 424 miletić et al. soil contaminated with oil from the heat power plant. it was shown that a. denitrificans can induce biodegradation of sulfamethoxazole in a bacterial consortium and leucobacter sp. gp.11 a. denitrificans efficiently utilizes 16 phthalate diesters and their downstream products through the protocatechuate 3,4-cleavage pathway, which makes a. denitrificans sp1 a very attractive candidate to be employed as an efficient biofactory in waste water treatment processes.12 a. denitrificans strain sp1 efficiently remediates di(2-ethylhexyl) phthalate.13 fig. 2. ftir spectrum of ha-e. nitrate-dependent ha oxidizing organisms isolated from agricultural soils were phylogenetically diverse and included members of the alphaproteobacteria, betaproteobacteria and gammaproteobacteria.14 conclusions the present results indicate that ha from samples of soil contaminated with oil from a heat power plant are oxidatively degraded mainly by achromobacter denitrificans. when the isolated ha was compared with aldrich humic acid sodium salt (h16752) as standard, there were differences that may offer a new promising improvement in cleaning technologies in all environments, especially for soil (bioremediation) and high levels of pollution in water and air. acknowledgment. this research is a part of project iii 43004 funded by the ministry of education, science and technological development of the republic of serbia. the authors have declared no conflict of interest. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ humic acids from the soil 425 и з в о д оксидоване хуминске киселине из топлане срђан б. милетић1, јелена авдаловић1, јелена милић1, мила илић1, александра жерађанин1, кристина јоксимовић2 и снежана спасић1 универзитет у београду, институт за хемију, технологију и металургију, институт од националног значаја, београд и 1иновациони центар хемијског факултета, универзитет у београду, београд хуминске киселине, изоловане из земљишта контаминираног нафтом из топлане (ha-e) је анализирано уз помоћ инфрацрвеног спектрометра (ftir). у поређењу са стандардом хуминских киселина (ha-s) уочљив је недостатак интензивног пика вибрација водоничне везе из хидоксилне групе (3600–3200 cm-1). ha-e има пик на 1649 cm-1 који вероватно припада карбонилној групи. ha-e су веома оксидоване, а претпоставља се да су микроорганизми achromobacter denitrificans за ову интензивну оксидацију ha-e. они су филогенетски различити нитрат-редукујући микроорганизми који имају капацитет да редукују хуминске киселине где се понашају као електрон донори у земљишту. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {microwave-assisted synthesis of azepines via nucleophilic aromatic substitution} j. serb. chem. soc. 81 (11) 1225–1230 (2016) udc 547.235.2’7+547.963.3:547.5– jscs–4921 962:61:54 original scientific paper 1225 microwave-assisted synthesis of azepines via nucleophilic aromatic substitution nina božinović#, bogdan a. šolaja#* and igor m. opsenica#** faculty of chemistry, university of belgrade, studentski trg 16, p. o. box 51, 11158 belgrade, serbia (received 24 august, revised 30 august, accepted 30 august 2016) abstract: a novel and efficient route has been developed to afford dipyridoazepine derivatives from primary amines and 3,3′-(z)-ethene-1,2-diylbis(4-chloropyridine). the procedure based on a double nucleophilic aromatic substitution provides a valuable synthetic tool for the synthesis of dipyridoazepines. the reaction proceeds without catalyst, under microwave irradiation conditions. keywords: azepines; heterocycles; nucleophilic aromatic substitution; transition metal-free conditions; microwave irradiation. introduction iminostilbene (5h-dibenz[b,f]azepine, 1) is tricyclic heterocycle (fig. 1). derivatives of this scaffold have been reported to exhibit various biological activities, including antioxidant1 and anticancer activity.2 5h-dibenz[b,f]azepine is important structural element for the registered anticonvulsant drug carbamazepine (2)3 and the tricyclic antidepressant (tca) opipramol (3).4 strong interest in the synthesis of 5h-dibenz[b,f]azepine derivatives still exists as well as their applications in medicinal chemistry.5 methods based on the double n-arylation reaction of primary amines are of great significance since they provide a route to a variety of n-substituted iminostilbenes.6 fig. 1. iminostilbene and tricyclic 5h-dibenz[b,f]azepine drugs. * corresponding authors. e-mail: (*)bsolaja@chem.bg.ac.rs; (**)igorop@chem.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc160824074b _________________________________________________________________________________________________________________________ (cc) 2016 scs. available on line at www.shd.org.rs/jscs/ 1226 božinović, šolaja and opsenica recently, an efficient synthetic procedure for the synthesis of 5h-dipyrido[4,3-b:3′,4′-f]azepine compounds via a double palladium-catalyzed amination– cyclization reaction (scheme 1) was disclosed.7 the synthesized compounds were found to exhibit interesting in vitro antibacterial activities.8 scheme 1. pd-catalyzed synthesis of 5h-dipyrido[4,3-b:3′,4′-f]azepine compounds. the electron-deficient nature of γ-halopyridines enables the reaction with different nucleophiles in a nucleophilic aromatic substitution reaction (snar) via an addition–elimination mechanism. the displacement of chloride from 4-chloropyridine by primary amines has been well studied and shown to be an efficient approach for the synthesis of 4-(alkylamino)pyridine derivatives. however, high temperatures and prolonged reaction times are usually required.9 in continuation of ongoing studies of biologically active tricyclic heterocycles,7,8,10 herein the synthesis of 5h-dipyrido[4,3-b:3′,4′-f]azepines using the double snar reaction of 3,3′-(z)-ethene-1,2-diylbis(4-chloropyridine) with primary aliphatic amines under microwave irradiation is reported. experimental instrumentation microwave reactions were performed in a biotage initiator 2.5 microwave reactor. the ir spectra were recorded on a perkin–elmer ftir 1725x spectrophotometer. the nmr spectra were recorded on a bruker ultrashield avance iii spectrometer (500 mhz) using tms as the internal standard. the chemical shifts are expressed in ppm (δ) values and coupling constants (j) in hz. the esi–ms (hrms) spectra were acquired on an agilent technologies 1200 series instrument equipped with a zorbax eclipse plus c18 column and a dad detector in combination with a 6210 time-of-flight lc/ms instrument operated in the positive ion mode. the samples were dissolved in meoh. thin-layer chromatography was performed on pre-coated merck silica gel 60 f254 and merck rp-18 f254 plates. the solution meoh (nh3) stands for a combination meoh/nh3 (aq.) in 9:1 volume ratio. chemistry general procedure for azepine synthesis. a reaction tube, containing a stirring bar, was charged with 3,3′-(z)-ethene-1,2-diylbis(4-chloropyridine), n-methylpyrrolidone (nmp) and amine (10 equiv.), and capped. after pre-stirring at room temperature for 2 min, the reaction mixture was heated to 150 °c and stirred at same temperature for 3 h under microwave conditions. the products were purified by preparative column chromatography: sio2, ch2cl2/meoh(nh3) = 9:1. _________________________________________________________________________________________________________________________ (cc) 2016 scs. available on line at www.shd.org.rs/jscs/ microwave-assisted synthesis of azepines 1227 n,n-dimethyl-5h-dipyrido[4,3-b:3′,4′-f]azepine-5-propanamine (5).7 following the general procedure, a mixture of 3,3′-(z)-ethene-1,2-diylbis(4-chloropyridine) (20 mg, 0.080 mmol), n,n-dimethylpropane-1,3-diamine (100 μl, 0.80 mmol), and n-methyl-2-pyrrolidone (0.5 ml) was stirred at 150 °c for 24 h or for 3 h under microwave conditions. yield: 14 mg, 61 %. 5-[3-(morpholin-4-yl)propyl]-5h-dipyrido[4,3-b:3′,4′-f]azepine (6).7 following the general procedure, a mixture of 3,3′-(z)-ethene-1,2-diylbis(4-chloropyridine) (20 mg, 0.080 mmol), n-(3-aminopropyl)morpholine (90 μl, 0.80 mmol), and n-methyl-2-pyrrolidone (0.5 ml) was stirred at 150 °c for 24 h or for 3 h under microwave conditions. yield: 16 mg, 62 %. n,n-dimethyl-5h-dipyrido[4,3-b:3′,4′-f]azepine-5-ethanamine (7). following the general procedure, a mixture of 3,3′-(z)-ethene-1,2-diylbis(4-chloropyridine) (20 mg, 0.080 mmol), n,n-dimethylethane-1,2-diamine (75 μl, 0.80 mmol), and n-methyl-2-pyrrolidone (0.5 ml) was stirred at 150 °c for 24 h or for 3 h under microwave conditions. yield: 12 mg, 56 %. 5h-dipyrido[4,3-b:3′,4′-f]azepine-5-propanol (8). following the general procedure, a mixture of 3,3′-(z)-ethene-1,2-diylbis(4-chloropyridine) (20 mg, 0.080 mmol), 3-(dimethylamino)propan-1-ol (60 μl, 0.80 mmol), and n-methyl-2-pyrrolidone (0.5 ml) was stirred at 150 °c for 24 h or for 3 h under microwave conditions. yield: 9 mg, 45 %. the characterization data for compounds 5–8 are given in the supplementary material to this paper. results and discussion chemistry the previously reported method7 for the synthesis of n,n-dimethyl-5h -dipyrido[4,3-b:3′,4′-f]azepine-5-propanamine (5) (scheme 1, table i, entry 1) is simple and efficient. nevertheless, the use of palladium as a catalyst makes it an expensive procedure. in order to simplify and to re-examine the reaction mechanism, an experiment was performed that focused on pd-free conditions. in the absence of pd(oac)2, ligand and base, the reaction of stilbene 4 and n,n -dimethylpropane-1,3-diamine in phme did not occur at 100 °c (table i, entry 2), which strongly supported the earlier conclusion7 that palladium really is needed for the formation of the product. next, an attempt was made to optimize the reaction of stilbene 4 with n,n-dimethylpropane-1,3-diamine under snar reaction conditions in order to define the reaction parameters that could possibly result in comparable yield to that given by the pd-catalyzed method. since the snar reaction of 4-chloropyridine and primary amines is often performed at elevated temperatures, toluene was replaced with n-methylpyrrolidone (nmp), a solvent with a higher boiling point. when the reaction temperature was elevated to 150 °c, the azepine 5 was obtained in moderate yield (table i, entry 4). the same reaction in nmp at 100 °c did not occur thus confirming that elevated temperatures are necessary for the formation of the desired product (table i, entry 3). finally, microwave irradiation was applied in order to shorten the reaction time. performing the reaction in a microwave reactor for 3 h at 150 °c, resulted in azepine 5 in 61 % yield (table i, entry 5). _________________________________________________________________________________________________________________________ (cc) 2016 scs. available on line at www.shd.org.rs/jscs/ 1228 božinović, šolaja and opsenica table i. synthesis of 5 under various conditions entry solvent t / °c pd-catalyzed mw time, h yield, % 1a phme 100 + – 24 87 2b phme 100 – – 24 0 3b nmp 100 – – 24 0 4b nmp 150 – – 24 58 5b nmp 150 – + 3 61 apd(oac)2 (5 mol %), johnphos (10 mol %), amine (3 equiv), naot-bu (2.8 equiv); b10 equiv. of amine the same reaction conditions were then applied for the synthesis of one known and two new azepine derivatives (6–8, respectively; scheme 2). scheme 2. synthesis of azepine derivatives. conclusions in conclusion, the first microwave-assisted synthetic procedure for the formation of 5h-dipyrido[4,3-b:3′,4′-f]azepines based on the double snar reaction of 3,3′-(z)-ethene-1,2-diylbis(4-chloropyridine) with primary aliphatic amines has been reported. this novel methodology was successfully applied for the synthesis of new azepine derivatives. although the yields were lower than in the previously reported pd-catalyzed method, this procedure has the great advantage of being transition metal-free, and the time of the reaction was significantly shortened by microwave heating. the next step in the development of methodology will be the replacement of nmp with less toxic solvent, towards a green procedure for the synthesis of azepines. supplementary material the characterization data for compounds 5–8 are available electronically electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. _________________________________________________________________________________________________________________________ (cc) 2016 scs. available on line at www.shd.org.rs/jscs/ microwave-assisted synthesis of azepines 1229 acknowledgements. this research was financially supported by the ministry of education, science and technological development of the republic of serbia (grant no. 172008) and the serbian academy of sciences and arts. и з в о д синтеза азепина нуклеофилном ароматичном супституцијом у микроталасним реакционим условима нина божиновић, богдан а. шолаја и игор м. опсеница хемијски факултет, универзитет у београду, студентски трг 16, п. пр. 51, 11158 београд развијена је нова метода за синтезу дипиридоазепинских једињења. азепински прстен формиран је реакцијом двоструке нуклеофилне ароматичне супституције, реакцијом примарног амина и одговарајућег стилбена. предност нове методе, у односу на раније описане синтезе азепина, јесте ефикасна синтеза без присуствa катализатора на бази прелазних метала. додатно, реакционо време значајно је смањено применом микроталасних реакционих услова. (примљено 24. августа, ревидирано 30. августа, прихваћено 30. августа 2016) references 1. h. v. kumar, n. naik, eur. j. med. chem. 45 (2010) 2 2. m. p. sadashiva, basappa, s. nanjunda swamy, f. li, k. a. manu, m. sengottuvelan, d. s. prasanna, n. c. anilkumar, g. sethi, k. sugahara, k. s. rangappa, bmc chem. biol. 12 (2012) 5 3. b. leduc, in foye's principles of medicinal chemistry, 6th ed., t. l. lemke, d. a. williams, eds., lippincott williams & wilkins, philadelphia, pa, 2007, p. 521 4. k. c. miles, in emergency medicine: a comprehensive study guide, 6th ed., j. e. tintinalli, g. d. kelen, j. s. stapczynski, eds., mcgraw–hill, new york, 2004, p. 1025 5. a) l. j. kricka, a. ledwith, chem. rev. 74 (1974) 101, and references cited therein; b) a. knell, d. monti, m maciejewski, a. baiker, appl. catal., a 121 (1995) 139; c) g. p. tokmakov, i. i. grandberg, tetrahedron 51 (1995) 2091; d) e.-c. elliott, e. r. bowkett, j. l. maggs, j. bacsa, b. k. park, s. l. regan, p. m. o’neill, a. v. 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309; b) s. hashimoto, s. otani, t. okamoto, k. matsumoto, heterocycles 27 (1988) 319; c) y.-k. lee, d. j. parks, t. lu, t. v. thieu, t. markotan, w. pan, d. f. mccomsey, k. l. milkiewicz, c. s. crysler, n. ninan, m. c. abad, e. c. giardino, b. e. maryanoff, b. p. damiano, m. r. player, j. med. chem. 51 (2008) 282; d) b. c. pérez, c. teixeira, i. s. _________________________________________________________________________________________________________________________ (cc) 2016 scs. available on line at www.shd.org.rs/jscs/ 1230 božinović, šolaja and opsenica albuquerque, j. gut, p. j. rosenthal, j. r. b. gomes, m. prudêncio, p. gomes, j. med. chem. 56 (2013) 556 10. n. božinović, s. šegan, s. vojnovic, a. pavic, b. a. šolaja, j. nikodinovic-runic, i. m. opsenica, chem. biol. drug des. 88 (2016) 795. _________________________________________________________________________________________________________________________ (cc) 2016 scs. available on line at www.shd.org.rs/jscs/ << 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice j. serb. chem. soc. 83 (12) 1417–1422 (2018) 1417 author index abadi, z. a., 941 abbasi, k., 745 abbasoglu, r., 837 abdul, a. b. h., 575 abdullah, r., 575 ačanski, m., 317 ahmad, z. a., 75 ahmad, z., 745 ahmed, w., 745 ajaj, i., 139 ajduković, m. j., 395 akhtar, m., 745 al-abdullah, e. s., 1 al-shakban, m., 425 alan, y., 795 alavi, s. m., 473 alie, h. m., 425 aljohar, h. i., 1 alpaslan, g., 707 anđelković, d., 1157 anđelković, k., 1327 anđelković, lj., 699 anđelković, lj. n., 625 anđelković, t., 1157 anjum, u., 911 antić, m., 1167 arar, o., 1059 armaković, s., 1 armaković, s. j., 1 ashok, d., 1305 atalay, a., 837 attri, p., 213 ausloos, m., 1391 avili, m. g., 1047 ayaz mehmood, a., 745 azhari, c. h., 75 bai, y., 265 balaban, m., 1167 banerjee, b., 1071 bao, j., 549 banković, p. t., 395 benkhaled, m., 31 banu, i., 759 bechtel, a., 923 beitollahi, h., 863 beškoski, v., 745 bercaru, g., 759 beyramabadi, s. a., 167, 305 bildirici, i̇., 795, 953 biparva, p., 863 branković, g., 1351 brborić, j. s., 875 bogojeski, j., 1229 bourezzane, s., 31 boyacioglu, b., 707 bozga, g., 759 brčeski, i., 699 brothers, e. n., 899 brusko, v., 51 bučko, s. đ., 847 bui, h. m., 641 cai, x., 969 carrillo-romo, f. j., 181 călinescu, m., 271 cetin, a., 795 chao, l., 1031 chen, w., 995 çiçek, a., 1059 cinarli, a., 1099 coldea, t. e., 19 corciova, a., 515 coşkun, m., 1017 cui, y., 969 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1418 journal of the serbian chemical society cvetković, t., 1157 cvetković, v. s., 1351 ćetković, g., 1223 čanadanović-brunet, j., 1223 čanović, p. p., 1229 čanović, p. s., 1229 čobeljić, b., 1327 čomić, lj. r., 1229 dahbi, a., 1243 darroudi, m., 821 dasireddy, v. d. b. c., 39 de la rosa cruz, e., 181 dekanski, a. b., 1391 demir, n., 707 dharmadhikari, s., 611 dilber, s. p., 875 din, i., 425 dinić, j., 1193 doğan, ö. m., 1005 dorraji., p. s., 449 drăghici, c., 271 drmanić, s. ž., 139 drvenica, i., 1391 dumitraşcu, f., 271 đorđević, a., 1209 đorđević, m., 699 đuričić, i. d., 685 džunuzović, e. s., 899 džunuzović, j. v., 899 el-emam, a. a., 1 elezović, n. r., 593 esmaeili. h., 199, 237 fatima, a., 575 fraj, j. l., 847 furtula, b., 1339 galaguz, v. a., 1123 gavrilović, m., 1273 ghoreishi, s. m., 1047 ghosh, p., 611 gilani, s. r., 425 gligorijević, a., 1339 gochev, v., 107 gojgić-cvijović, g., 745 görmez, ö., 489 gozmen, b., 93 gržetić, i., 773 guemra, k., 1243 gungor, a., 885 gurbuz, d., 1099 haba, h., 31 haghdadi, m., 285 hakimi, m., 167 han, j., 969 hao, z., 265 hamzehloueian, m., 821 hasan, a., 745 hassan, a., 367 hassel, e. p., 61, 1005 hernandez, a. g., 181 hongwei, x., 1031 hussain, r., 733 hussain, s., 911 iftikhar, s. h., 425 ijaz, s. s., 367 ilić pajić, j. m., 331 ilijević, k., 773 imran, m., 745 ivanescu, b., 515 ivaniš, g. r., 331 ivanković, n., 463 jagličić, z., 1327 jakovljević, d., 745 janković, d., 463 janković, d., 463 janković-častvan, i., 1143 jeremić, d. a., 699 jeremić, m., 1193 jeremić, s., 1273 jeremić, v., 1285 jerosimić, s., 439 jevtić, a. s., 1273 jiang, x., 995 jin, c., 379 johra, f. t., 503 jovančićević, b., 1167 jovanović, đ., 317 jović, b. m., 593 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ vol. 82: author index 1419 jović, v. d., 593 jović-jovičić, n. p., 395 jovićević, j., 1351 jung, w.-g., 503 karim-nezhad, g., 449 karimi, g., 411 karjiban, r. a., 575 karkalić, r., 463 kasmaei, l. s., 941 katona, j. m., 847 kaur, g., 1071 kayaman-apohan, n., 885 kazeri-shandiz, s., 305 khacef, a., 641 khaligh, a., 651 khan, a., 745 khan, j., 733 khanaliloo, s., 449 khoo, s. f., 237 khorablou, z., 449 kijevčanin, m. lj., 331 kim, i. t., 213 kim, s. h., 213 kirova, g. k., 107 kittinger, f., 923 kojić, i., 923 koljančić, n., 1167 kostić, i., 1157 kovač, t. s., 899 krstajić pajić, m. n., 593 kundie, f., 75 lakshmi, b. v., 1305 lal, r., 367 larbi, o. c., 1243 lashanizadegan, m., 221 lashkarbolooki, m., 199 lashkari, m., 673 lazarova, h., 39 lazarević, n., 1209 lee, v. s., 575 leovac, v. m., 157 li, b., 1261 li, p., 379 li, p., 1261 li, s., 995 li, w., 995 li. x. z., 549 li, y., 265 likozar, b., 39 lin, l., 995 liu, b., 379 liu, h., 345 long, c., 31 lukić, i., 345 mahmood, m. h., 733 mahmood, t., 745 mahmoud, y., 1363 makrlík, e., 51 malovanyi, s. m., 1123 mandić, a., 1223 manta, c. m., 759 maofa, j., 1031 marinković, a. d., 139 marinović, s. r., 395 marković, d., 1379 marković, s., 1339 mary, s. y., 1 matović, b., 745 medina velasquez, d. y., 181 mehmood, a., 367 mendoza-huizar, l. h., 981 menges, n., 795, 953 merine, h., 1243 mihalache, m., 271 mihályi, m. r., 39 milanović, v. d., 1177 milenković, i., 745 milenković, m. r., 1327 milenković, m., 1327 miletić, s., 699 miller, r., 847 milinković, j. r., 847 milićević, b., 899 milićević, d. b., 625 miljković, d., 1297 milonjić, s., 391 milovanović, m., 439 milovanović, s., 1379 milutinović-nikolić, a. d., 395 miranda, j. r., 181 mirković, s., 1273 mirzazadeh, h., 221 misaelides, p., 251 mishra, n. k., 401 mitić, m., 439 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1420 journal of the serbian chemical society mitić, m. p., 625 mitić, v., 1209 mladenović, k. g., 1229 mohammad, m., 733 mohamadpour, f., 673 mohanan, k., 561 morales ramirez, a. j., 181 morsali, a., 167, 305 mousavi, h. z., 651 mowla, d., 411 mudrinić, t. m., 395 mudura, e., 19 murillo, a. g., 181 nab, n., 285 nagaraju, n., 1305 namazova, a. t., 61 narayana, b., 1 naseriazar, a., 121 nayak, p. s., 1 nedeljković, j. m., 899 nedić, o., 1391 nedić-vasiljević, b. n., 395 negreanu-pirjol, t., 271 nguyen, h. t., 1113 nguyen, x. s., 1113 ni, z. j., 549 nikolić, i., 1143 nikolić, j. b., 139 nikolić, n. d., 1351 novaković, i., 1193 novaković, s. b., 157 obradović, m., 923 okoro, u. c., 401 oktay, b., 885 özmen, h., 121 panov, e. v., 1123 pasqualone, a., 19 pastor, k., 317 pavlićević, m. ž., 723 pavlović, d., 1157 pekdemir, m. e., 1017 perić, m., 439 pešić, m., 1193 petrović, l. b., 847 petrović, s., 1229 pevec, a., 1327 pezo, l., 317 pham, t. h., 641 philip, s., 561 pilčević, i., 1285 poleksić, d. t., 723 pop, c. r., 19 popova, m., 39 popovska jovičić, b. d., 1229 popsavin, m., 1297 popsavin, v., 1297 prekajski, m., 745 prlainović, n. ž., 139 purenović, j. m., 625 purenović, m. m., 625 qayyum, a., 745 qu, s., 265 qui, m., 265 rac, v. a., 723 radanović, d., 1327 radanović, m. m., 157 radetić, m., 1379 radmilović, v. r., 1143 radmilović, v. v., 1143 radoičić, m., 1379 radovanović, ž., 463, 1379 radojević, i. d., 1229 radotić, k., 745 radović, i. r., 331 ragna, f., 19 raheel, a., 425 raissi, h., 167 rajić, d., 463 rajković, k. m., 1273 rakić, v. m., 723 raković, i. r., 1229 raković-simić, j. m., 723 ramachandran, m., 611 ramli, y., 1243 rančić, m. p., 139 rangus, m., 39 ranković, r., 439 rashidi, a., 651 raziq, a., 733 redžepović, i., 1339 ristić-medić, d. k., 685 rodić, m. v., 157 rosevenis, a., 1131 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ vol. 82: author index 1421 roshanaei, a., 473 rotar, a. m., 19 rukh, s., 745 sabet, j. k., 1047 safarov, j. t., 61, 1005 safarzadeh, s., 941 safidine, z., 1363 sahoo, l., 539 sahoo, s., 539 samardžić, j., 1315 samelak, i., 1167 sandoval, c. f., 181 sarasija, m., 1305 saropjini, b. k., 1 sarrafi, y., 821 sarri, s., 251 savić, j. s., 875 savikin, k., 1315 selucký, p., 51 shafeeq, a., 911 shahverdyiev, a. n., 61 shahzad, k., 745 shaki, h., 167 shariati, a., 199 sharma, a., 1071 shen, x., 345 shirkhanloo, h., 651 simić, v., 1273 singhamahapatra, a., 539 skala, d., 345 sladić, d., 699, 1193 sobola, a. o., 809 socaciu, c., 19 sonmez, o., 93 sozutek, a., 93 sönmez, b. g., 489 spasojević, lj. m., 847 sredojević, d. n., 899 stajčić, s., 1223 stajić-trošić, j. t., 331 stanisavljević, lj., 773 stanisavljević, n., 1315 stanković, m., 1209 stanojković, t., 1315 stepanović, m., 1193 stevanović, n., 923 stijepović, m. z., 331 stoica, i., 759 stoiljković, i., 139 stojanović, g., 1209 stojanović, k., 923 stoytcheva, m. s., 107 stupar, s., 463 sun, g., 969 sun, s. y., 549 sundararajan, v., 1131 szegedi, a., 28 šaponjić, z., 1379 šegan, d., 1193 šeregelj, v., 1223 šobajić, s. s., 685 šolević knudsen, t., 1167 tadić, m., 1143 taj, m. b., 425 tajik, s., 863 tan, m., 953 tariq, m., 733 tavman, a., 1099 thomas, p. s., 561 timić, j. b., 685 tirmizi, s. a., 425 tokarski, t., 593 toumi, f. b., 1243 trivić, d. d., 1177 tumbas šaponjac, v., 1223 turel, i., 1327 ugur, m. h., 885 ugwu, d. i., 401 ullah, i., 733 umasivakami, k., 1131 ünver, h., 707 uysal, b. z., 1005 vaferi, b., 199 van alsenoy, c. k., 1 van brecht, b., 809 vaňura, p., 51 velkova, z. y., 107 vidović, n., 1167 vladimirov, s. m., 875 vojinović-ješić, lj. s., 157 vucelić-radović, b. v., 723 vujić, đ., 317 vujić, z. b., 875 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1422 journal of the serbian chemical society vujošević, d., 1285 vukićević, n., 1351 vulić, j., 1223 wan, j., 379 wang, x., 549 warchoł, j., 251 watkins, g. m., 809 wu, w., 995 wu, x., 995 xiao, h., 995 xu, h.-y., 1261 yabalak, e., 489 yan, h., 379 yildiz, m., 707 yildirim, n., 707 yılmaz, o., 1059 yilmaz, s., 1017 yue, k., 1031 yue, l., 1031 yuzhu, z., 1031 zabihi, r., 411 zabinski, p., 593 zamboulis, d., 251 zarić, n. m., 773 zeghioud, h., 1363 zdunić, g., 1315 zdujić, m., 345 zhang, l., 995 zhang, y., 345 zhang, z., 995 zhu, x., 345 ziraman, d. u., 1005 zlatanović, i., 1209 zlatković, b., 1209 živković, j., 1315 živković, lj., 745 žižovic, i., 1379 subject index of vol. 83 and list of referees in 2018 are given in the electronic form at the internet address of the journal of the serbian chemical society: http://www.shd.org.rs/jscs/vol83/no12.html end of volume 83. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {quantitative structure-property relationship studies for prediction vapor pressure of volatile organic compounds} j. serb. chem. soc. 84 (12) 1405–1414 (2019) udc 547.1+532.11+621.5.02:544.112:539.12 jscs–5273 original scientific paper 1405 quantitative structure–property relationship studies for the prediction of the vapor pressure of volatile organic compounds mounia zine1, amel bouakkadia1,2, leila lourici3 and djelloul messadi1* 1environmental and food safety laboratory, badji mokhtar-annaba university, bp. 12, 23000 annaba, algeria, 2abbes laghrour university, faculty of sciences and technology, khenchela, algeria and 3chadeli ben djedid university, bp. 73, 3600 el taref, algeria (received 6 march, accepted 13 june 2019) abstract: a theoretical model (qspr) using multiple linear regression analysis for predicting the vapor pressure (pv) of volatile organic compounds (vocs) has been developed. a series of 51 compounds were analyzed by multiple linear regression analysis. first, the data set was separated arbitrarily into a training set (39 chemicals) and a test set (12 chemicals) for statistical external validation. a four-dimensional model was developed using as independent variables theoretical descriptors derived from dragon software when applying the ga (genetic algorithm)–vss (variable subset selection) procedure. the obtained model was used to predict the vapor pressure of the test set compounds, and an agreement between experimental and predicted values was verified. this model, with high statistical significance (r2 = 0.9090, q2loo = 0.8748, q2ext = 0.8307, s = 0.24), could be used adequately for the prediction and description of the log pv value of other vocs. the applicability domain of mlr model was investigated using a william‘s plot to detect outliers and outsides compounds. keywords: molecular descriptors; vocs; log pv; multiple linear regression. introduction volatile organic compounds (vocs) are molecules which can contain h and c atoms but also other elements such as o, n, cl, f, p, s,... and metals and/or metalloids, and which are almost entirely in the vapor state under normal conditions of temperature and pressure. they include 210 species and 23 large families. these compounds can be of natural origin (terpenes) but very often they are contaminants mainly from human activity.1 the sectors of activities that are more strongly transmitters of vocs are road transport, industry, agriculture, and the tertiary sector. other air pollutants that could be cited are biological contaminants (bacteria, pollen, fungi), physical con * corresponding author. e-mail: d_messadi@yahoo.fr https://doi.org/10.2298/jsc190306059z 1406 zine et al. taminants (metals, particles, dust, radioactivity), and chemical contaminants that are part of the vocs represented by gases (co, o3, nox, so2, fluorocarbons), dioxins and furans.1 experience is a direct way to obtain activity data for organic compounds, which have many shortcomings, such as the need for large test organisms, high costs, long time duration, and value differences measured between different researchers. consequently, it would be impossible to test experimentally the activity values for all organic compounds. as new compounds are emerging, other difficulties will also arise. therefore, it is necessary to use theoretical methods to waiver the disadvantages of the experiment and to predict the data of compounds exactly. with the rapid development of computer science and theoretical quantum chemical studies, quantum chemical parameters of compounds can speedily and precisely obtained by computation. these structural parameters along with the introduction of the quantitative structure–activity relationship (qsar) models can increase the interpretability and predict the activity of new organic compounds.2 quantitative structure–property relationships (qspr) have gained wide attention in the area of separation science recently. these models are based on the relationship between structures and property of compounds.3 the vapor pressure of different compounds can be predicted from their formula and even unknown compounds can be identified using this method. in general, qspr models attempt to predict the vapor pressure of a molecule by characterizing it with a series of molecular descriptors. these models can effectively be used for the prediction of molecular structures and determination of vapor pressure. the aim of this study was to find a statistical model for the prediction of vapor pressures of some volatiles organic compounds. the model was validated by dividing the data set arbitrarily into training (39 compounds) and test (12 compounds) sets. different statistical techniques were used to develop the model to highlight the structural requirements for the ideal vapor pressure. the three objectives of the present paper were first, to explore the structure–activity relationships of vapor pressure of diverse volatiles organic compounds, second, to select the best predictive model from among all comparable chemometric models for the property and third, verification of the performance and stability of the obtained model by two approaches (mlr). the model obtained shows which descriptors play a significant role in the variation of log pv value of these compounds. qspr stadies of vapor pressure 1407 experimental data set the pv experimental values of 51 selected, structurally heterogeneous vocs were collected from previous works,4 and converted to log pv values. molecular descriptors generation the structures of the molecules were drawn using hyperchem 6.03 software.5 the final geometries were obtained with the semi empirical pm3method. all calculations were realized at the rhf (restricted hartree–fock) level with non configuration interaction. the molecular structures were optimized using the polak–ribiere algorithm and a gradient norm limit of 0.01 kcal ǻ-1 mol-1. the resulting geometry was transferred into the software dragon version 5.5 to calculate 1600 descriptors of the type geometrical and getaway (geometry, topology and atoms weighted assembly).6 descriptors with constant or near constant values inside each group were discarded. for each pair of correlated descriptors (with correlation coefficient r ≥ 0.95), the one showing the highest pair correlation with the other descriptors was excluded. the genetic algorithm (ga)7 was considered superior to other methods of the variable selection techniques. thus, variable selection was performed on the training set using ga in the mobydigs version of todeschini8 by maximizing the cross-validated explained variance q2loo. model development and validation models with four variables were performed by the software mobydygs.8 the goodness of fit of the calculated models were assessed by means of the multiple determination coefficients, r2, and the standard deviation error in calculation (sdec): ( )2i i 1 1 ˆ = = − n i sdec y y n (1) cross validation techniques allow the assessment of the internal predictivity (q2lmo cross validation; bootstrap) in addition to the robustness of the model (q2loo cross validation). cross validation methods consist in leaving out a given number of compounds from the training set and rebuilding the model, which is then used to predict the compounds left out. this procedure is repeated for all compounds of the training set, obtaining a prediction for everyone. if each compound is taken away one at a time, the cross validation procedure is called the leave-one-out technique (loo), otherwise the leave-more-out technique (lmo). the loo or lmo correlation coefficient, generally indicated with q2, was computed by evaluating the accuracy of the prediction of these “test” compounds: 2 / 12 2 1 ˆ( ) 1 1 ( ) = = − = − = − −   n i i i i n i i y y press q tss y y (2) the “hat” of the variable y, as is the usual statistical notation, indicates that it is a predicted value of the studied property, and the sub index “i/i” indicates that the predicted values come from models built without the predicted compound. tss is the total sum of squares. the predictive residual sum of squares (press) measures the dispersion of the predicted values. it is used to define q2 and the standard deviation error in prediction (sdep). 1408 zine et al. =sdep press n (3) a value q2 > 0.5 is generally regarded as a good result and q2 > 0.9 as excellent.9,10 however, studies11,12 have indicated that while q2 is a necessary condition for high predictive power a model, is not sufficient. to avoid overestimating the predictive power of the model the lmo procedure (repeated 5000 times, with 4 objects left out at each step) was also performed (q2l(4)o). in the bootstrap validation technique k n-dimensional groups are generated by a randomly repeated selection of n-objects from the original data set. the model obtained on the first selected objects is used to predict the values for the excluded sample, and then q2 is calculated for each model. the bootstrapping was repeated 8000 times for each validated model. by using the selected model, the values of the response for the test objects are calculated and the quality of these predictions is defined in terms of q2ext, which is defined as: ext tr / ext ext12 ext tr2 tr tr 1 ˆ( )² / / 1 1 / ( ) / = = − = − = − −   n i i i i n i i y y n press n q tss n y y n (4) here next and ntr are the number of objects in the external set (or left out by bootstrap) and the number of training set objects, respectively. the data set was divided arbitrary into a training set (39 objects) used to develop the qsar models and a validation set (12 objects), used only for statistical external validation. other useful parameters are r2, calculated for the validation chemicals by applying the model developed from the training set, and external standard deviation error of the prediction (sdepext), which is defined as: ( ) ext 2 ext ext 1 1 = = − n i i sdep y y n (5) where the sum runs over the test set objects (next). according to golbraikh and tropsha,12 a qspr model is successful if it satisfies several criteria as follows: 2cvext 0.5>r (6) r2 > 0.6 (7) ( ) ( )0 0² ² / ² 0.1 or ² ²’ / ² 0.1− < − <r r r r r r (8) 0.85 < k < 1.15 or 0.85 <k’ < 1.15 (9) here: ( )( ) ( ) ( )22 = − − − −          i i i i i y y y y r y y y y (10) 0 2 2 ( ) ² 1 ( ) − = − −   r i i i y y r y y (11) ( ) ( ) 0 2 i2 0 2' = 1 y − − −       r i i y y r y (12) qspr stadies of vapor pressure 1409 ( ) ( ) ' 2=     i i i y y k y (13) ( ) ( )2 =   i i i y y k y (14) where r is the correlation coefficient between the calculated and experimental values in the test set; r²0 (calculated versus observed values) and r02 (observed versus calculated values) are the coefficients of determination; k and k’ are the slopes of regression lines through the origin of the calculated versus observed and observed versus calculated, respectively yr0i, y r0i; are defined as yr0i = k y i and y r0i = k’yi and the summations runs over the test set. the applicability domain (ad) was discussed by the williams plot8,9 of jacknified residuals versus leverages (hat diagonal values (hi). the jacknifed residuals (or studentized residuals) are the standardized cross-validated residuals. each residual is divided by its standard deviation, which is calculated without the ith observation. the leverage (hi) value of a chemical in the original variable space is defined as: hi = xi(xtx)-1xit (i = 1,….,n) (15) where xi is the descriptor row-vector of the query compound and x is the n(p+1) matrix of p model parameter values for n training set compounds. the superscript t refers to the transpose of the matrix/vector. the warning leverage value (h*) is defined as 3(m+1)/n. when h value of a compound is lower than h*, the probability of accordance between predicted and actual values is as high as that for the compounds in the training set. a chemical with hi > h* will reinforce the model if the chemical is in the training set. but such a chemical in the validation set and its predicted data may be unreliable. however, this chemical may not appear to be an outlier because its residual may be low. thus the leverage and the jacknified residual should be combined for the characterization of the ad. in this stage, linear qspr model was developed and evaluated to predict the log pv of the compounds. the study we conducted consists of the multiple linear regressions (mlr) available in the mobydygs software. results and discussion in order to predict log pv, application of the ga–vss lead to several good models for the prediction based on different sets of molecular descriptors. the best model obtained using 39 compounds is a four-dimensional model (x0sol, spposa_h2, gats2e and hy) with a high predictive power. the multiple linear regression model (mlr) is given by: log pv = 11.0490 – 0.4602x0sol – 12.3322spposa_h2 + + 1.1372gats2e – 1.2333hy (16) statistical parameters for the model with: ntr = 39 next = 12 are: r² = 0.9009q²loo = 0.8748q²boot = 0.8555f = 77. 25q2ext = 0.8307 sdep = 0.256sdec = 0. 227sdepext =0.297s = 0.24kxx = 38.51 kxy = 45.57 1410 zine et al. the reported fitting and validation parameters have, as expected, high values indicating that the model has a very good predictive performance and the descriptors involved in it well describe the vapor pressure. the high absolute t-values shown in table i express that the regression coefficients of the descriptors involved in the mlr model are significantly larger than the standard deviation. the t-probability of a descriptor can describe the statistical significance when combined together within an overall collective qspr model (descriptors interactions). table i. characteristics of the selected descriptors in the best ga/mlr model descriptor coefficient regressed standard error coefficient t t-probability vif constant 11.0490 0.6288 17.57 0.000 – x0sol –0.4602 0.0460 –9.81 0.000 1.720 spposa_h2 –12.3320 1.2100 –10.19 0.000 1.528 gats2e 1.1372 0.1423 7.99 0.000 1.074 hy –1.2333 0.1275 –9.68 0.000 1.944 descriptors with t-probability values below 0.05 (95 % confidence) are usually considered statistically significant in a particular model, which means that their influence on the response variable is not merely by chance.13 a smaller t-probability suggests a more significant descriptor. the t-probability values of the four descriptors are very small, indicating that all of them are highly significant descriptors. the vif is uniform and equal to 1.00 if there is no linear correlation between a given variable and rest of the variables in the regression equations. higher values of vif in table i indicate a more serious multi-co linearity problem. models would not be accepted if they contain descriptors with vifs above a value of five.14 2 1 1 = − j vif r (17) where rj2 is the squared correlation coefficient between the jth coefficient regressed against all the other descriptors in the model.15 applicability domain on analyzing the domain of the applicability of the model from a williams plot, all residuals were located within the range of three standard deviations, and there was no influential compound both for the training or the prediction set, fig. 1, which means that the model has a good external predictivity. a diagram of the statistical coefficients q2 and r2 is presented fig. 2 to compare the results obtained for the randomized models (circle) with the starting model (square). it is clear that the statistics obtained for the modified vectors of qspr stadies of vapor pressure 1411 the log pv are smaller than those of the real models; the q2 values are lower (<10 %), as for the major part are the r2 values (<30 %). this ensures that the established model has a real base, and is not due arbitrarity. fig. 1. williams plot: jackknifed residuals and leverages. fig. 2. randomization test associated to the previous qspr model. this technique, plot of cross-validation log pv versus experimental log pv values, shown in fig. 3, ensures the robustness of a qspr model (see also tables s-i and s-ii of the supplementary material to this paper).16 descriptor contributions analysis based on a previously described procedure,17,18 the relative contributions of the four descriptors to the model were determined. it should be noted that the difference in contribution between two descriptors used in the model indicates that all the descriptors are essential to generate the predictive model (fig. 4). descriptors decrease according to the following order: spposa_h2 (27.7811 %) > x0sol (26.6809 %) > hy (25.7967 %) > gats2e (19.7413 %). 1412 zine et al. fig. 3. cross-validation versus experimental log pv for the entire data set. fig. 4. relative contributions of the selected descriptors to the mlr model. conclusions multiple linear regressions were used to construct a quantitative structure property relation model of 51 vocs for their vapor pressures binding propriety. the model was developed by a genetic algorithm selection of theoretical molecular descriptors from among a wide set obtained with several softwares. the data set were separated randomly into two subsets of 39 elements for training and 12 for external validation. the mlr model has good stability, robustness and predictivity. the chemical applicability domain of the studied mlr model and the reliability of the predictions were verified by the leverage approach. the equation obtained could be used successfully to estimate the vapor pressures for new compounds or for other compounds the experimental values of which are unknown. supplementary material additional data are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. qspr stadies of vapor pressure 1413 и з в о д проучавања квантитативних релација структуре и реактивности за предвиђање напона паре испарљивих органских једињења mounia zine1, amel bouakkadia1,2, leila lourici3 и djelloul messadi1 1environmental and food safety laboratory, badji mokhtar-annaba university, bp. 12, 23000 annaba, algeria, 2abbes laghrour university, faculty of sciences and technology, khenchela, algeria и 3chadeli ben djedid university, bp.73, 3600 el taref, algeria развијен је теоријски модел (qspr) коришћењем вишеструке линеарне регресионе анализе за предвиђање напона паре (pv/pa) испарљивих органских једињења (voc), за серију од 51 једињења. на почетку је скуп података произвољно подељен у скуп за проучавање (39 једињења) и скуп за проверу (12 једињења) за екстерну статистичку валидацију. развијен је четвородимензионални модел користећи као независне варијабле теоријске описнике изведене из софтвера dragon применом ga (генетски алгоритам)–vss (variable subset selection) процедуре. добијени модел је употребљен за предвиђање напона паре једињења скупа за проверу, и потврђена је сагласност између експерименталних и претсказаних вредности. овај модел, са високом статистичком значајношћу (r2 = 0,9090, q2loo = 0,8748, q 2 ext = 0,8307, s = 0,24), може бити адекватно употребљен за предвиђање и описивање log (pv/pa) вредности других voc. област применљивости mlr модела испитивана је коришћењем виљемсовог графа (william‘s plot) да се детектују једињења која се не уклапају у модел. 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {supercapacitors based on graphene/pseudocapacitive materials} j. serb. chem. soc. 82 (4) 411–416 (2017) udc 620.1:66.017.004.12+546.812–31: jscs–4976 544.032.72 extended abstract 411 extended abstract supercapacitors based on graphene/pseudocapacitive materials• denis sačer1*, magdalena kralj1, suzana sopčić1, milica košević2#, aleksandar dekanski2# and marijana kraljić roković1 1faculty of chemical engineering and technology, university of zagreb, marulićev trg 19, 10000 zagreb, croatia and 2institute of chemistry, technology and metallurgy, department of electrochemistry, university of belgrade, njegoševa 12, 11000 belgrade, serbia (received 7 february, accepted 20 february 2017) abstract: composites of graphene and sno2 were successfully prepared by a single step simultaneous synthesis of sno2 and reduction of graphene oxide (go). three different compositions of precursor solution resulted in different composite materials containing graphene and sno2. the reaction was realized by microwave-assisted hydrothermal synthesis. scanning electron microscopy (sem) and energy-dispersive x-ray spectroscopy (edx) gave insight into the morphology and composition of the obtained materials. good capacitive/ /pseudocapacitive properties of the obtained material suitable for supercapacitor application were registered by cyclic voltammetry, from where specific capacitance values up to 93 f g-1 were determined. keywords: graphene oxide, graphene, sno2, supercapacitor, morphology, cyclic voltammetry. materials having pseudocapacitive redox reactions are promising candidates for applications demanding high energy/high power. their specific capacitance value, cs, goes up to 750 f g–1, while for double layer charging/discharging, it goes up to 150 f g–1. the most important metal oxides with pseudocapacitive redox reactions are ruo2, mno2, iro2, moo2, v2o5 and sno2.1–3 in order to improve their properties and increase their specific capacitance, a great deal of research effort has been directed at the optimization of electron and ion conductivities.1 this can be accomplished by application of 2d graphene sheets structures as supports for pseudocapacitive materials. due to its properties, such as * corresponding author. e-mail: dsacer@fkit.hr • lecture at the meeting of electrochemical division of the serbian chemical society held on 15 december, 2016. # serbian chemical society member. https://doi.org/10.2298/jsc170207027s 412 sačer et al. high electrical conductivity, high surface area and flexibility, graphene fulfils the basic requirements for improving the properties of pseudocapacitive materials.4 sno2 has unique electrical and optical properties that enable its application in catalysis, gas sensors, solar cells and transparent electrode materials. among the various metal oxides, tin-based materials have attracted considerable attention as an electrode material for energy storage due to its low cost, abundance and environmental aspects.5–9 it has been used as the anode material for li secondary batteries,5,6 whereby its pseudocapacitive properties were studied.7,8 in this work, a simple simultaneous synthesis of sno2 and reduction of graphene oxide (go) was realised using microwave-assisted hydrothermal synthesis. this method was selected due to its effects, such as rapid volumetric heating, increased reaction rates and shortened reaction time. hydrothermal synthesis and microwave assisted hydrothermal synthesis are useful for accelerating the de-protonation reaction of sn(h2o)x(oh–)y(4-y)+ to sno2·nh2o offering good control of both particle size and particle size distribution.10 a hydrothermal reaction was mostly used for the preparation graphene/sno2 composite materials,5,6,11,12 while lim et al.11 successfully prepared a graphene/sno2 composite in a microwave process. lim et al. also suggested electrostatic interaction between the negatively charged go functional groups and sn2+, thereby providing nucleation centres for growth of sno2 nanoparticles on the graphene sheets. in this work, three different approaches for the synthesis of the composite materials were studied. good interaction of sn2+ and go molecule could be achieved by retarding the hydrolysis rate of sn2+ and for this reason, sncl2 dissolved in hcl solution or an ethanol/water mixture was used during the synthesis. in the first procedure, sncl2 was dissolved directly in a go solution (1.85 g dm–3) containing water and ethanol, to obtain a 15 or 25 mmol dm–3 solution of sn2+. the mixture was heated in a microwave assisted hydrothermal reactor at temperatures ranging from 60–70 °c for 5 min (composite 1). in the second procedure, the same amount of sncl2 was dissolved in ethanol and subsequently mixed with a go water solution and heated to the same temperatures as in the first approach (composite 2). in the third procedure, sncl2 was dissolved in 25 % hcl (5 ml) with addition of ethanol (4 ml) to obtain 0.24 mol dm–3 solution12 and subsequently, it was mixed with the go water solution and heated to the same temperatures as previously reported (composite 3). the go used in this work was obtained by the hummers method.13 in all experiments after the heat treatment, the brown colour of go had become black, indicating successful reduction of go. the temperature used in this work was limited to 60–70 °c due to the large pressure increase that was obtained during the heating process. usually, higher temperatures were used for both graphene reduction14 and sno2 synthesis, approaching 90 °c for graphene oxide reduction and approaching 160– –180 °c for sno2 synthesis.5,6,11,12 it is believed that the pressure increase is the graphene/pseudocapacitive supercapacitors 413 result of the go reduction process during which oxygen functionalities are removed and co2 released. after the reduction process, the obtained solid product suspended in the reaction mixture was washed intensively with water and finally it was left with an appropriate amount of water in order to obtain a paste or a suspension. in addition, some of it was dried in order to perform characterisation by scanning electron microscopy (sem) and energy-dispersive x-ray spectroscopy (edx) (fig. 1). the typical microstructure of the graphene and graphene oxide surface shows wrinkled sheets interconnecting with each other in a continuous structure.15 the sem images of the composite materials revealed that the obtained morphology was different from those of graphene or graphene oxide and from the edx analysis it was evident that the resulting composite materials contained tin and carbon. therefore, it could be concluded that the hydrothermal synthesis of graphene/sno2 was successfully performed. the most significant impact of sno2 on the go microstructure was registered for 2, where the microstructural characteristic of go had almost disappeared. it is also evident from edx analysis that 2 contained the highest amount of sno2. on the other hand, 1 showed the presence of sno2 but a go-like microstructure was also evident. since the amount of sno2 was similar to that in 2, this indicates that the layer was less homogenous with respect to that of 2. in the case of 3, cl was included within the structure of the material, go microstructural properties were present and the lowest amount of sno2 was obtained. from the reported results, it follows that sno2 particles in situ decorated the surface of the graphene nanosheets. however, it is important to stress that the composites had different microstructures and contained different amounts of sno2 (fig. 1). fig. 1. sem–edx analysis of composites 1(a), 2 (b) and 3(c). the pseudocapacitive properties of the obtained sno2/graphene composite materials were examined in a three-electrode cell. the working electrode was 414 sačer et al. prepared by applying a water suspension or paste of the composite material on a carbon support (aluminium coated with carbon, gelon lib group, china, a = = 0.48 cm2). the amount of active material varied from 0.03–0.30 mg. the counter electrode was a pt-foil and the reference electrode was a saturated calomel electrode. cyclic voltammetry was performed using a potentiostat/galvanostat princeton applied research 236a in the potential range from –0.8 to 0.8 v at a scan rate of 50 mv s–1. high currents and high reversibility were achieved over a wide potential range during electrode polarisation in 0.5 mol dm–3 na2so4 solution (fig 2a). it is well known that during polarisation graphene shows a constant current over a wide potential range,14 while sno2 shows non-ideal pseudocapacitive behaviour with continuously increasing current at potentials more negative than 0 v. in this potential range, the sn4+ to sn2+ transition occurs (fig 2b). a) b) -0.8 -0.4 0.0 0.4 0.8 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 i s / a g -1 e / v fig. 2. cyclic voltammograms of: a) go and different composite materials and b) sno2 deposited at a glassy carbon support. for the composite materials investigated in this work, almost constant currents, originating from charging/discharging of the graphene layer, were registered within the potential window of investigation. the good capacitive response is evidence that the go was reduced to the more conductive graphene form. moreover, in the potential range from –0.4 to –0.8 v, a current response characteristic for sno2 is also evident, indicating that sno2 contributes to the overall capacitive/pseudocapacitive response. the continuous increase in the cathodic current and the anodic current peak representing the sno2 redox reaction did not develop the same for each layer. they were the most prominent in the case of 2 and the least prominent in the case of 3. these observations are in accordance with sem results that revealed almost complete coverage of graphene with sno2 for 2 and a go-like microstructure for 1 and 3. it is possible to determine the cs values of the obtained layers by integration of the cyclic voltammogram curves (fig. 2) and such calculations were reported graphene/pseudocapacitive supercapacitors 415 previously.15 it was found that the cs values of the prepared composites depend significantly on the amount of material deposited on the carbon support. thus, in the case when 0.25 or 0.03 mg of 2 was applied to the carbon support, cs values of 26 and 91 f g–1 were obtained, respectively. in the case of 3 for 0.25 or 0.03 mg, the cs values were 14 and 93 f g–1, respectively. these observations are a consequence of the better utilization of the composite material in the case of thinner layers. furthermore, it was found out that electrodes containing from 0.10 to 0.30 mg of active material showed specific capacitance values, in f g–1, ranging from 5–17, 4–26 and 3–14 for 1, 2 and 3, respectively. the obtained results indicated that the capacitive properties depended on the method of preparations and the highest cs values were registered for 2, when the sncl2 was dissolved in absolute ethanol and afterwards mixed with the go solution. the lowest cs value was registered for 3. evidently, although the acid environment used during the synthesis of 3 retarded the hydrolysis process and although the reduction was realised at increased sn2+ concentration, these experimental conditions did not improve the degree of go reduction and, consequently, in this case lower cs values were registered. such behaviour could be explained by the fact that go is more easily reduced at increased ph values.16 the amount of sno2 was also lower, compared to the amount in the other composites, which additionally influenced the cs value. from the results obtained in this work, it can be concluded that composite 2 exhibited the best capacitive properties with a cs value up to 91 f g–1, which is comparable or even higher compared to the values obtained previously for graphene or sno2.2,6,17 it is obvious that the synergistic effect of these two constituents resulted in enhanced capacitive properties suitable for supercapacitor application. acknowledgements. this work was fully supported by the bilateral croatian–serbian project “high-power graphene/redox pseudocapacitive materials-based electrochemical supercapacitors”, the croatian science foundation under project no. ip-2013-11-8825 and the ministry of education, science and technological development of the republic of serbia under project no. 172060. и з в о д суперкондензатори засновани на графен/псеудокапацитивним материјалима denis sačer1, magdalena kralj1, suzana sopčić1, милица кошевић2, александар декански2 и marijana kraljić roković1 1 faculty of chemical engineering and technology, university of zagreb, marulićev trg 19, 10000 zagreb, croatia и 2 институт за хемију, технологију и металургију, центар за електрохемију, универзитет у београду, његошева 12, 11000 београд коришћењем микроталасног реактора у поступку хидротермалне синтезе успешно су припремљени композити графена и sno2. током реакције извршена је симултана синтеза sno2 и редукција графен-оксида. три различита састава раствора прекурсора 416 sačer et al. резултирала су ситнтезом три различита композитна материјала гафена и sno2. скенирајућа електронска микроскопија (sem) и спектроскопија дифракције x-зрака (edx) омогућили су увид у морфологију и састав добијених композитних материјала. добра капацитивна/псеудокапацитивна својства композита, погодна за примену у суперкондензаторима, регистрована су цикличном волтаметијом. вредности специфичне капацитивности испитаних материјала износиле су до 93 f g-1 . 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice volume 84 (2019) 1 subject index 1,3,4-oxadiazole, 649 1,3-thiazole, 649 16s rna, 225 1-butanol, 599 1h-indole-2,3-dione, 1 2,4-d, 187 2-pyridone, 531 2πu electronic state, 801 3d-qsar, 303 4,7-phenanthroline, 689 aas, 267 ab initio, 391 abietates, 1073 absorption, 1183 abundance, 1249 acetylcholinesterase, 649 acidity constants, 391 activated chlorine, 99 activity descriptor, 417 additives, 41 adhesion, 1305 adsorption capacity, 69 adsorption, 327 advanced oxidation process (aop), 517 advanced oxidation processes, 1041 afm, 1415 ageing, 663 alkaloids, 935 alcohols, 317 aldehydes, 267 alginate beads, 679 alkylation, 1 alpha-2-macroglobulin, 943 alpha-amylase, 1093 alstonia scholaris, 935 amides, 477 amino-functionalized copolymer, 1391 amlodipine determination, 1005 analgesics, 639 androgen receptor, 153 anodization, 915 anova, 757 anthocyanins, 141 antibacterial coating, 1287 antibiofilm, 1355 anticancer studies, 277 antigenotoxic property, 539 antimicrobial activity, 119, 237, 355, 1355 antimicrobial, 277 antioxidant activity, 877 antioxidant property, 119 antioxidants, 891 anti-proliferative activity, 991 antiquorum potential, 1355 antitumor agents, 1119, 1345 antoine equation, 599 aquatic sediment, 1439 aqueous solubility, 575 artificial neural network, 575, 713 arylpiperazines, 925 atom economy, 119 au electrode, 1319 authenticity of oils, 1017 azabenzenes, 819 b3lyp/cc-pvdz, 591 2 journal of the serbian chemical society bacillus paralicheniformis, 1093 bare tio2 nanoparticle, 1455 batch, 531 bathochromic shift, 343 batteries, 1261 benzohydrazides, 555 bicyclodicarboxylate, 961 binding free energy, 837 biodegradation, 679, 743 biofilm dispersion, 99 biological activity, 267 biological warfare agents, 99 biopolymers, 1427 bischromanone, 355 bischromene, 355 bismuth (iii) oxide, 483 bone implants, 1287 botanical origin, 1017 bpa migration, 377 brine shrimps, 649 broccoli, 445 building material, 55 butyrylcholinesterase, 649 cadmium, 11 caffeic acid, 391 calcined condition, 69 calcium ionophore i, 199 can damage, 377 candida albicans, 1 canned food, 377 captopril, 175 carbon electrocatalyst, 901 carbon paste electrode, 175 carbonate/bicarbonate, 1041 catalase, 455 catalyst selectivity, 901 catalyst, 609, 1055 characterization, 1119 chemical deposition, 1199 chemical kinetics, 975 chemical nuclease activity, 563 chemical reactions, 1055 chitosan membrane, 83 chitosan, 327 chloride, 1041 chloroaluminate melt, 1329 choline, 991 choronoamperometry, 41 chronoamperometry, 1155 citrates, 1261 clausius–clapeyron type equation, 599 cmcase and avicelase activity, 129 coacervation, 1427 coal ash, 527 coal slag, 527 coating adhesion, 729 coatings, 1221 combination therapy, 951 competitive ions, 327 composite hardness, 729 composite membrane, 211 composite, 1287 constant current density, 1271 contaminant pattern, 1439 control, 483 coordination isomers, 1391 copper(ii), 489 copper, 1209, 1415 corrosion, 1221 cosolvents, 253 cotton, 743 csd, 1391 cu electrodeposition, 729 cu(ii)-phenanthroline, 563 cultivars, 141 current density, 483 cyclic voltammetry, 41, 1155, 1271, 1319 cytotoxic lactones, 1345 cytotoxicity, 1119, 1455 decontaminant, 99 deep eutectic solvents, 1221 degradation, 757 dendrite, 1209 denitration, 1183 density functional theory, 27 , 779, 819, 891 derivative of vouacapane, 153 derivatization, 555 destoning, 1381 dft calculations, 539, 1129 dft modelling, 1391 dft, 277, 877, 975 dft/6-31+g (d,p), 153 d-glucose, 1345 dibenzaldehydes, 119 diesel fuel, 599 dihydrotestosterone, 153 volume 84: subject index 3 dimeric and polymeric cu(ii) complexes, 467 dioxolanes, 1 dipolar cycloaddition, 1073 dipole moment, 861 direct anodic oxidation, 187 direct bioautography, 1355 distillate, 663 distortion, 779 double emulsions, 1427 dmfc, 1155 dna interaction, 277 dna-binding affinity, 563 doc, 623 dopa-minergic, 639 double layer capacitance, 901 drug design, 153 dye photodegradation, 743 dye removal, 701 dynamic light scattering, 1105 eco-friendly method, 757 eco-friendly, 435 electric field, 1235 electrocatalysis, 417 electrochemical activity, 1155 electrochemical metal deposition, 1329 electrochemical oxidation, 713 electrode, 483 electrodeposition, 41, 1221 electrolysis, 1209 electronic structure methods, 417 electrophoresis, 943 electrophoretic deposition, 1287 electrostatic surface potential, 27 emulsion, 99 energetic compounds, 27 enthalpy, 1143 enthalpy of combustion, 477 entropy, 1143 environmental pollutants, 861 environmental waters, 1455 epichlorohydrin, 757 equations of state, 1169 equilibrium, 1143 essential oils, 405 europium and americium, 199 evacuation, 83 exothermic, 1143 extinction coefficient, 343 extraction and stability constants, 199 fatty acid ethyl esters, 253 fe-based phase, 503 fenton, 517 flame atomic absorption spectrometry, 489 flow synthesis, 531 fluorescence, 343 flux agents, 1055 fly ash, 211 food additive, 991 fotodegradation, 623 four-atomic molecules, 801 four-membered ring stannylene, 293 fresnel equations, 1235 freundlich, 1143 fuel cells, 1155 fuller clay, 211 fullerene, 837 functional assay, 943 gamma al2o3, 915 gc electrode, 1319 gc/ms, 1017 genotoxic property, 539 gfaas, 435 gfn2-xtb, 837 gibbs energy change, 391 glucosidase, 649 glucosinolates, 445 glutamic acid, 1083 glutathione-s-transferase, 455 glycerol carbonate, 609 glycerol, 609 goniofufurone mimics, 1345 graphene/zno nanocomposite, 175 graphite, 423 green and biodegradable α-amino acid catalyst, 1083 greenhouse gas, 69 groundwater, 327 gum, 55 h2c=sn:, 293 heavy metal ions, 435 hedgehog inhibitors, 951 heme-iron protein, 1105 hemolysate, 455 herbicide, 455 herzberg–teller coupling, 819 4 journal of the serbian chemical society heterocycles, 639 heterogeneous catalysis, 253 heterotopic ossification, 951 htra pdz domain, 1367 hybrid material, 1027 hybrid nanomaterials, 539 hydrogen evolution, 1221, 1271 hydrolysis, 1199 hydroxyapatite coating, 1305 hydroxyapatite, 1287 (hyper)polarizability, 861 immobilized system, 679 in situ anaphoretic deposition, 1305 indigenous bacteria, 225 inorganic ion, 623 instantaneous nucleation, 1415 interacting quantum atoms, 891 intermetallics, 1329 intrinsic distortion path, 779 ionic liquids, 991 ion–solvent interaction, 317 irox electrode, 1319 irras, 1235 isomerization, 975 isotherm, 1143 isotopic variants of hnco, 845 ketoprofen, 1027 kretschmann configuration, 1235 lactobacillus fermentum, 365 lemon balm, 11 lignocellulosic waste, 129 linear regression, 591 liquid–liquid extraction, 199 log pv, 1405 magnetic core shell nanoparticles, 1005 malachite green, 701 materials design, 417 mechanism, 327, 1199 meconium, 365 melatonin, 11 mesenchymal stem cells, 951 metal complexes, 277 metal(ii) complexes, 267 metals in human use, 1249 methanesulfonate electrolyte, 187 micro-arc oxidation, 915 microbiological and antiproliferative activity, 961 microextraction, 435 micropollutant, 623 microreactor, 531 microstructure, 503 microwave irradiation, 237, 355 microwave-assisted extraction, 129 mineralizers, 1055 minor groove binding, 563 mixing rules, 1169 modified multiwall carbon nanotubes, 701 molecular descriptors, 1405 molecular docking, 303, 649, 925 molecular dynamics, 925 molten salts, 1129 monte carlo, 55 mrci-f12 calculations, 801 multiple linear regression, 405, 575 multiple linear regression, 1405 multireference configuration interaction, 819 multivariate cluster analysis, 1017 mycobacterium tuberculosis, 555 n-(5-chlorosalicylidene)-2-aminopyridine, 111 n-(5-chlorosalicylidene)-3-aminopyridine, 111 n-(ferrocenylmethylidene)fluorene-2amine, 175 naclo3, 1183 n-alkylation, 639 nanocluster, 1415 nanoporous sorbent, 489 naphthalene, 225 nbo, 975 ndo gene, 225 net analyte signal (nas), 391 neuronal stem cells, 935 new drug design, 303 n-hydroxy, 343 ni(ii) and zn(ii) complexes, 961 nics, 861 nir spectroscopy, 663 nitro explosives, 27 nitro polycyclic aromatic hydrocarbons, 861 nitrogen oxides, 1183 nmr spectroscopy, 1073 nnrtis, 303 volume 84: subject index 5 one-pot procedure, 1083 optical emission spectroscopy, 915 optimization, 1093 organic ions and radicals, 779 organotin(iv) compounds, 1119 orr activity measurements, 901 orthogonal π-cation-π stacking, 1367 otto configuration, 1235 oxidation, 1183 oxygen evolution, 187 palm oil mill effluent (pome), 517 palomar–pardavé model, 41 particle swarm optimization, 713 peptide ligand, 1367 peroxidases,11 pesticides, 1455 phenol removal, 211 phenol, 575, 679 photo-electro-fenton hybrid system, 517 phylogenetic tree, 225 piperazine, 639 piperidine, 639 plasma temperature, 915 plum cultivars, 1381 plum wine, 141 polyaniline, 1261 polycyclic aromatic hydrocarbons, 1439 poly-functionalized dihydro-2-oxypyrroles, 1083 polynuclear complexes, 689 polyphenols, 141, 891 potential energy profile, 293 potential, 483 potentiostatic, 1415 powder, 1209 pr, 1169 principal component analysis, 663 probiotic, 365 process intensification, 531 protease, 1367 proteins, 1427 pseudocapacitors, 1261 pva, 211 pyrazole, 119, 237 pyrazoline, 237 pyrometallurgical method, 423 qspr, 405, 575 quantum yield, 343 quasilinear molecules, 845 quercetin 3-o-β-d-glucopyranoside, 245 radical, hole, and electron scavengers, 1455 radicals, 877, 891 raman spectra, 861 rate constant, 975 raw materials, 1249 reactive blue 19, 1041 receptor binding site, 925 remediation, 225 reserves, 1249 response surface methodology, 1093 retention indices, 405 revision, 111 rigid chain linkers, 119 roughness, 1305 safety, 365 saxs, 1027 scanning electron microscope (sem), 1209 schiff base, 467 screen-printed carbon electrode, 1005 sensory characteristics, 1381 sharifker–hills model, 41 silicon complexes, 1129 silver(i) complexes, 689 simple work-up, 1083 singlet–triplet transitions, 819 site selectivity, 1073 slag, 69 slaughterhouse blood, 1105 sod1, 455 sol–gel process, 1027 solid pharmaceutical compounds, 1169 solid state interdiffusion, 1329 solid state transformations, 1055 solid-state fermentation, 129 sorbifolin, 245 sorption of metal ions, 1391 spectrophotometry, 391 spectroscopic, 267 spectroscopy, 689 spinel zinc ferrite nanoparticle, 701 spirits, 1381 spiro-sn-heterocyclic ring compound, 293 spontaneous fermentation, 1381 square wave voltammetry, 1319 6 journal of the serbian chemical society srk, 1169 stability, 1249 static method, 599 statistical analysis, 591 storage temperature, 377 structural identification, 245 structure, 1129 structure-activity relationships, 1345 subcritical water, 757 sulfamethoxazole, 713 sulforaphane, 445 superoxide dismutase, 11 survey, 1 synergistic effect, 1367 synergistic effect, 991 synthesis, 445, 483 tafel curves, 503 taurine, 991 tetradentate coordination mode, 467 thermochemistry, 477 thermodynamic parameters, 317 thiocarbohydrazide, 489 thp-1, 445 thymus vulgaris (lamiaceae), 405 tin chloride, 41 tin sulfide, 423 tio2 nanoparticles, 743 tio2/polyaniline nanocomposite, 1455 titanium anodization, 1305 titanium oxide, 1305 tnf-α, 445 topomer comfa, 303 topomer search, 303 total organic carbon analysis, 1319 tpmc, 961 trace metals, 83 transesterification, 253, 609 transition metal complexes, 779 tribology, 423 ultrasonic agitation, 729 urease, 649 uv protection, 743 uv–vis spectroscopy, 343, 877, 1105 valerian, 11 vapor pressure, 599 vibronic coupling, 779 vibronic levels, 801 vilsmeier–haack reagent, 355 virtual screening, 153 viscosity, 317 vocs, 1405 volatile compounds, 1381 water glass, 609 water quality, 527 water–nitrobenzene system, 199 well plate method, 555 x-ray crystallography, 111 , 467, 689 zeolite, 83 zinc, 11, 1261 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {empirical method for predicting the enthalpies of combustion of amides} j. serb. chem. soc. 84 (5) 477–481 (2019) udc 536.722:662.611/.612+547.1–327 jscs–5200 short communication 477 short communication empirical method for predicting the enthalpy changes of combustion of amides alma k. ryskaliyeva*, murat e. baltabayev and kurmankul t. abaeva department of plant protection and quarantine, kazakh national agrarian university, 8 abay av., almaty 050010, kazakhstan (received 9 august, revised 17 october, accepted 6 november 2018) abstract: the enthalpy change of combustion is one of the basic thermochemical characteristics of an organic compound, indicating the amount of heat produced in the complete combustion of the substance. the enthalpy changes of combustion of many organic compounds have been experimentally determined. hitherto, the existing empirical approaches have aimed at calculating the combustion enthalpy change of individual classes only, which do not always satisfactorily agree with the experimental results. therefore, the method of correlation–regression analysis is proposed herein to establish the combustion enthalpy changes of previously unexplored compounds. as is known, a linear relationship between two characteristics is possible only if both of them obey the principle of additivity, i.e., in a homologous series of organic compounds with increasing number of ch2-groups, the value of additive characteristic (for example, the enthalpy change of vaporization) should increase by a certain constant amount. keywords: enthalpy change of combustion; amides; thermochemistry. introduction correlation–regression analysis of experimental data is widely applied in all sections of chemistry to quantitatively describe chemical phenomena, systematize facts and perform calculations. in particular, correlation–regression analysis of thermochemical data enables convenient and reliable formulas for practical calculations to be obtained.1,2 herein, a correlation analysis of the results of experimental thermochemical studies on the combustion of amides was conducted, which would allow regularity in the values of the obtained combustion enthalpy changes to be revealed. * corresponding author. e-mail: aryskalieva@mail.ru https://doi.org/10.2298/jsc180809094r ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 478 ryskaliyeva et al experimental an industrial calorimeter b-08-ma with an isothermal shell and a stationary self-packing calorimetric bomb (vint. = 325 cm3) equipped with two valves (for input and output of gases) was used to determine the enthalpy changes of combustion of the studied compounds. the error in the measuring of the calorimeter b-08-ma is ±0.1 %, which is clearly insufficient for precision measurements. therefore, in order to improve the accuracy of the determination of the energy of combustion of the substances, some parts of the calorimeter were refined. in particular, a system for thermostating the shell, an oxygen purification system, a system for sample ignition, a calorimetric vessel, and a calorimetric bomb were modified. the changes to the calorimeter enabled the accuracy of the obtained thermochemical values to be improved to ±0.01 %. the test samples of amides, anilides and amidic acid were burned as tablets with or without an auxiliary substance in the form of a special polyethylene film. the masses of the combustible samples and polyethylene were determined on a microbalance with an accuracy of ±2×10-5 g. moreover, the amount of substance was chosen as recommended in the reference literature.3 after the completion of the experiment, the combustion gases were analyzed for their co2 content by the rossini method (accuracy ±0.05 %).4 the analysis for the co content was realized with the help of indicator tubes (sensitivity 6×10-6 kg l-1). the experimental values of the enthalpies of combustion of the studied amides are presented in table i. table i. the experimental and calculated values of the combustion enthalpy changes of the studied amides compound –δснexp./ kj mol-1 n –δснcalc./ kj mol-1 ε / % n,n-methylacetylcarbamide 2154.32 10 2180 1.2 oxamide 851.70 4 872 2.3 α-cyanacetamide 1565.43 7 1526 2.5 α-phenylacetamide 4210.40 19.5 4251 0.9 valeramid 3140.62 14.5 3161 0.6 salicylamide 3352.32 15.5 3379 0.8 nicotinamide 3083.78 14 3052 1.0 n,n-dimethylbenzamide 4959.91 22.5 4905 1.1 results and discussion inside the calorimeter, the process of complete combustion of amides occurs according to the general equation: cahbocnd + (n/2)o2 = aco2 + (b/2)h2o + (d/2)n2 (1) the enthalpy changes of combustion of the studied amides and the number of oxygen atoms, n = 2a+b/2–c, required for the complete combustion of one mole of the studied amide are presented in table i and a simple regularity that relates these quantities to each other was found: –δсн= 218n (2) ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. predicting the enthalpy changes of combustion of amides 479 the linear nature of this relationship was confirmed by the value of the correlation coefficient, which was 0.992. the observed dependence enables the clarification of the physicochemical meaning of the found correlation (2) and an examination of the accuracy of the predictive calculations based on it (table i). it is possible to assess qualitatively the degree of reliability of the proposed calculation for enthalpy change of combustion of amides from the relation (2) from table i, which presents the values established experimentally and the calculated values. the greater the number of ch groups in the amides, the greater will be the quantity of carbon dioxide and water formed because of combustion, and the greater will be the number of oxygen atoms n necessary for their formation. if the process of complete combustion of amides would be accompanied by the formation of nitrogen oxides, it could similarly be assumed that the greater the number of nitro groups in the amide formula, the greater would be the amount of oxygen required for the combustion of the amide. as can be seen from the general eq. (1) for the combustion of amides, this was not the case. therefore, according to table i, it is easy to determine that with increasing number of nitro groups in the amide formula, the relative deviation of the predicted values from the experimental values of the enthalpy change of combustion increases. however, on the other hand, it can be seen from table i that the relative deviations of the calculated values of the enthalpy changes of combustion of amides from the experimental values do not exceed 2.5 %, which indicates a sufficient reliability of the prediction method. the logic of the reasoning and the good agreement between the predicted and experimental values of combustion enthalpy changes (table i) allows the assumption that the mole fraction of nitrogen atoms in the amide formula might serve as a certain quantitative limiting factor, which would indicate the upper margins of the relative deviation of the predicted and experimental values of the enthalpy change of combustion. in this regard, the correlation coefficient between the relative deviation and xn, the mole fraction of nitrogen atoms in the amide, was found to be rather high and equal to 0.929. thus, based on the regression analysis, the following relationship was obtained: |ε| = 12xn (3) based on this relationship, it is possible to estimate the expected maximum errors of the predicted values of the enthalpy change of combustion of amides calculated according to formula (2). in accordance with the calculated formulas (2) and (3), the enthalpy changes of combustion and their maximum expected errors of amides were calculated and compared with the thermochemical properties experimentally studied by other ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 480 ryskaliyeva et al authors. a comparison of the calculated and experimental values is given in table ii. table ii. comparison of the literature and calculated values of δch and |ε | of some amides compound –δснexp./ kj mol-1 n –δснcalc./ kj mol-1 εexp/εtheor 1-methylcarbamide 1311.74 6 1308 –0.3/2.1 ethylcarbamide 1966.10 9 1962 –0.2/2.0 isopropylcarbamide 2613.70 12 2616 0.09/1.4 tert-butylcarbamide 3267.80 15 3270 0.07/1.2 acetamide 1186.03 5.5 1199 1.1/1.3 as can be seen from table ii, the values given in the literature and the calculated values are in a good agreement with each other. the table also shows that the relative deviations of the predicted values from the experimental values do not exceed the upper margin of the relative deviations calculated by formula (3). conclusions the thermochemical properties of amides should be known when studying the mechanism of chemical reactions and thermodynamic equilibrium for the purposeful synthesis of amides with given properties and when developing optimal technological processes for the industrial production of these compounds. the good agreement between the predicted values of the enthalpy change of combustion of amides calculated by correlation (2) and the experimental values suggests that researchers can use the methods proposed above to predict the enthalpy changes of combustion of amides for which there is no reliable experimental data, and to predict the expected marginal error in calculating these quantities. technologists can also use these methods for performing engineering calculations. the effectiveness of correlations (2) and (3), despite their simplicity, suggests that the next stage of study can find out a more general relationship that would reflect a correlational dependency of the enthalpy changes of combustion on two variables – the number of oxygen atoms and the mole fraction of nitrogen atoms in an amide. thus, the potential relation would combine formulas (2) and (3). и з в о д емпиријски модел за предвиђање промена енталпија сагоревања амида alma k. ryskaliyeva, murat e. baltabayev и kurmankul t. abaeva department of plant protection and quarantine, kazakh national agrarian university, 8 abay av., almaty 050010, kazakhstan промена енталпије сагоревања је једна од основих термодинамичких карактеристика органских једињења која представља количину топлоте произведене при комплетном сагоревању супстанције. промена енталпије сагоревања великог броја органских једињења су одређене експериментално. постојећи емпријски приступи су имали за циљ ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. predicting the enthalpy changes of combustion of amides 481 израчунавање промене енталпије сагоревања само за индивидуалне класе, што се не слаже увек на задовољавајући начин са експерименталним подацима. због тога је у овом раду предложен метод корелационо–регресионе анализе за одређивање промене енталпије сагоревања једињења за која та вредност није одређена. као што је познато, линеарна веза између две величине је могућа само онда када обе величине задовољавају услов адитивности, тј. у хомологим серијама органских једињења код којих расте број ch2 група вредност адитивних карактеристика (на пример, промена енталпије испаравања) треба да се повећава за констатан износ. (примљено 9. августа, ревидирано 17 октобра, прихваћено 6. новембра 2018) references 1. e. v. sagadeev, v. v. sagadeev, high temp. 4 (2002) 581 (https://doi.org/10.1023/a:1019663013424) 2. a. k. ryskaliyeva, m. e. baltabayev, a. m. zhubatova, acta chim. slov. 65 (2018) 127 (https://doi.org/10.17344/acsi.2017.3683) 3. v. p. kolesov, osnovy termokhimii (fundamentals of thermochemistry), msu, moscow, 1996, p. 205 (isbn 5211033795) 4. f. d. rossini, experimental thermochemistry: measurement of heats of reaction, interscience publishers, new york, 1956, p. 326 (isbn 9110141278). ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {antibacterial and antifungal properties of guanylhydrazones} j. serb. chem. soc. 82 (6) 641–649 (2017) udc 547.495.9–304.2+547.556.9: jscs–4993 615.281/.282.004.12 original scientific paper 641 antibacterial and antifungal properties of guanylhydrazones vladimir ajdačić1#, jelena lazić1#, marija mojićević2, sandra šegan3, jasmina nikodinovic-runic2*# and igor m. opsenica1**# 1faculty of chemistry, university of belgrade, studentski trg 16, p. o. box 51, 11158, belgrade, serbia, 2institute of molecular genetics and genetic engineering, university of belgrade, vojvode stepe 444a, 11000 belgrade, serbia and 3institute of chemistry, technology, and metallurgy, university of belgrade, njegoševa 12, 11000 belgrade, serbia (received 13 february, revised 3 march, accepted 7 march 2017) abstract: a series of novel guanylhydrazones were designed, synthesized and characterized. all the compounds were screened for their antibacterial and antifungal activity. compounds 26 and 27 showed excellent antibacterial activities against staphylococcus aureus atcc 25923 and micrococcus luteus atcc 379 with minimal inhibitory concentrations of 4 µg ml-1, and good antifungal activity against candida parapsilosis atcc 22019. these results suggested that the selected guanylhydrazones could serve as promising leads for improved antimicrobial development. keywords: guanylhydrazones; iminoguanidines; antibacterial activity; antifungal activity; candida spp. introduction infectious diseases caused by human pathogens, both bacteria and fungi, result in significant morbidity and mortality worldwide. treatment of these diseases is often hampered by limited therapeutic options and the development of resistance. bacteremia is a major cause of life-threatening complications in patients in intensive care units, neonates, or cancer patients, who are at extremely high risk for infections caused by antibiotic resistant bacteria.1 invasive candidiasis is the fourth most common bloodstream infection with mortality rates remaining disturbingly high at 40 %.2 this makes the quest for new molecules that are effective against the threat of drug resistance a significant issue in modern medicine. *,** corresponding authors. e-mail: (*)jasmina.nikodinovic@imgge.bg.ac.rs; (**)igorop@chem.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc170213033a 642 ajdačić et al. guanylhydrazones have been a long-standing point of interest in medicinal chemistry.3 recently, a two-step procedure was demonstrated for the preparation of simple guanylhydrazones 1 (fig. 1).4 the synthesized compounds were evaluated for their in vitro antifungal activities against a wide range of medically important fungal strains. among the series, compound 2 proved to be an effective, broad-spectrum antifungal compound (fig. 1). in particular, compound 2 exhibited excellent activity against the voriconazole-resistant candida albicans ca5 strain. given the good antifungal properties shown by selected guanylhydrazones and as a continuation of research on the development of new antimicrobial agents, in the present work, the synthesis, characterization and evaluation of the antibacterial and antifungal properties of new guanylhydrazone derivatives are reported. fig. 1. guanylhydrazones. results and discussion chemistry the suzuki–miyaura reaction enabled access to a range of aldehydes 4–7 from easily obtainable starting compounds (scheme 1). the low yield of aldehyde 5 is associated with problems during purification and isolation of the desired product from the crude reaction mixture. the reported yield is also non -optimized and certainly could be further improved through variation of the reaction conditions. scheme 1. synthesis of aldehydes 4–7. bromination of aldehydes 85 and 9 with bromine and n-bromosuccinimide afforded the corresponding monobromo derivatives 10 and 11, respectively (scheme 2). aldehydes 12 and 13 were prepared by a suzuki–miyaura reaction starting from bromide 10 (scheme 2). access to 4-fluoro-5-phenyl-2-furaldehyde (15) was accomplished by a halogen/metal exchange reaction. the lithiated intermediate formed in the reaction medium was trapped with the electrophilic fluor guanylhydrazones as antimicrobials 643 inating reagent n-fluorobenzenesulfonimide (nfsi, scheme 2). the thiophene aldehyde 8 underwent nitration with a mixture of nitric acid and acetic anhydride affording the corresponding 4-nitro derivative 16 in 71 % yield (scheme 2). subsequent acid hydrolysis of 16 gave the nitroaldehyde 17 (scheme 2). scheme 2. synthesis of aldehydes 10–13, 15 and 17. the guanylhydrazones 18–27 were synthesized using a one-step condensation reaction of aminoguanidine hydrochloride and the corresponding aldehyde in good to excellent yields (scheme 3). all the guanylhydrazones were obtained as hydrochlorides. scheme 3. synthesis of guanylhydrazones 18–27. antimicrobial activity all synthesized guanylhydrazones were assessed for their antimicrobial activity against one gram-negative strain (pseudomonas aeruginosa pao1, nctc 10332), and three gram-positive strains (staphylococcus aureus atcc 25923, micrococcus luteus atcc 379 and listeria monocytogenes nctc 11994) and 644 ajdačić et al. three fungal strains (candida albicans atcc 10231, issatchenkia orientalis atcc 6258 and candida parapsilosis atcc 22019). the minimum inhibitory concentration (mic) values obtained by the standard broth dilution method were compared to those of clinically used antibiotics (kanamycin and nystatin). most of the tested compounds were found to display poor to moderate activities against the tested bacterial strains, with the exception of compound 22, which displayed excellent antibacterial activity against p. aeruginosa pao1, and compounds 26 and 27, which exhibited excellent activities against s. aureus atcc 25923 and m. luteus atcc 379 (table i). it is noteworthy that compounds 22, 26 and 27 showed better antibacterial activities against these three bacterial strains in comparison to the control drug, kanamycin (table i). table i. minimal inhibitory concentrations, mic / µg ml-1 compound p. aera s. aurb m. lutc l. mond c. albe i. orif c. parg 18 150 62.5 62.5 125 50 62.5 31.2 19 200 125 31.2 500 250 125 62.5 20 125 62.5 125 250 200 125 62.5 21 >500 15.6 125 >500 31.2 62.5 31.2 22 4 62.5 62.5 125 125 100 62.5 23 >500 125 15.6 15.6 31.2 31.2 15.6 24 250 62.5 62.5 62.5 62.5 18.8 31.2 25 >500 >500 >500 >500 62.5 62.5 31.2 26 >500 4 4 >500 15.6 15.6 6 27 >500 4 4 >500 31.2 31.2 6 kanamycinh 50 10 12.5 12.5 – – – nystatinh – – – – 1 7.8 2 apseudomonas aeruginosa pao1 nctc 10332; bstaphylococcus aureus atcc 25923; cmicrococcus luteus atcc 379; dlisteria monocytogenes nctc 11994; ecandida albicans atcc 10231; fissatchenkia orientalis atcc 6258; gcandida parapsilosis atcc 22019; hcontrol drug from a perusal of the data, it could be seen that all the tested compounds showed moderate antifungal activity against all the tested fungal strains, while two compounds 26 and 27 exhibited the most promising activity against c. parapsilosis atcc 22019 (table i). overall, the minimal inhibitory concentrations (mic) values lead to the conclusion that the additional aromatic ring on thiophene was beneficial to the antibacterial and antifungal activity of compounds 26 and 27. experimental instrumentation dry-flash chromatography was performed on sio2 (0.018–0.032 mm). melting points were determined on a boetius pmhk apparatus and were not corrected. ir spectra were recorded on a thermo-scientific nicolet 6700 ft-ir diamond crystal instrument. 1hand 13c -nmr spectra were recorded on a bruker ultrashield advance iii spectrometer (at 500 and 125 mhz, respectively) using tetramethylsilane (tms) as the internal standard. chemical guanylhydrazones as antimicrobials 645 shifts are expressed in ppm (δ) values and coupling constants (j) in hz. esi ms spectra of the synthesized compounds were recorded on an agilent technologies 6210 time-of-flight lc– –ms instrument in the positive ion mode using meoh/h2o = 1/1 with 0.2 % hcooh as the carrying solvent solution. the samples were dissolved in pure meoh (hplc grade). the selected settings were as follows: capillary voltage, 4 kv; gas temperature, 350 °c; drying gas, n2, 12 l·min-1; nebulizer pressure, 45 psig*; fragmentator voltage, 70–200 v. the gc–ms spectra of the synthesized compounds were acquired on an agilent technologies 7890a apparatus equipped with a db-5 ms column (30 m×0.25 mm×0.25 μm), a 5975c msd and fid detector. selected settings were as follows: carrier gas he (1.0 ml min-1), temperature linearly increased from 40–315 °c (10 °c min-1), injection volume, 1 μl, temperature, 250 °c, temperature (fid detector), 300 °c, and ei mass spectra range: m/z 40–550. thin-layer chromatography was performed on pre-coated merck silica gel 60 f254 and merck rp-18 f254 plates. all the reported yields refer to isolated yields. the compounds were analyzed for purity (hplc) using a agilent 1200 hplc system equipped with quat pump (g1311b), injector (g1329b) 1260 als, tcc 1260 (g1316a) and detector 1260 dad vl + (g1315c) (other details are presented in the supplementary material to this paper). all compounds were >95 % pure. the analytical and spectral data of the synthesized compounds are given in the supplementary material to this paper. chemistry general procedure. 5-(4-methylphenyl)furan-2-carbaldehyde (4).6 in a dry glass flask purged with argon, pd(oac)2 (3.4 mg, 0.015 mmol) was dissolved in dry dimethoxyethane (dme) (2 ml) and pph3 (16.2 mg, 0.060 mmol) was added. the resultant solution was stirred at room temperature for 10 min and 3 (113.8 mg, 0.650 mmol) and na2co3 (aq.) (2m, 0.65 ml, 1.3 mmol) were added. after 5 min stirring at room temperature, a solution of (4-methylphenyl)boronic acid (111.5 mg, 0.820 mmol) was added and the reaction mixture was purged with argon and refluxed at 90 °c overnight under argon. the solution was cooled to room temperature and filtered through a celite pad, washed with ch2cl2 and dried with anh. na2so4. the organic solvent was removed under reduced pressure and the crude product was purified by dry-flash chromatography (sio2: hexane/etoac = 95/5 to 9/1) to afford the title compound 4 (113.6 mg, 94 %). 5-(4-bromophenyl)furan-2-carbaldehyde (5).7 to a glass flask, 3 (113.8 mg, 0.650 mmol), (4-bromophenyl)boronic acid (143.6 mg, 0.715 mmol), tetrabutylammonium bromide (209.6 mg, 0.650 mmol), pd(oac)2 (2.9 mg, 0.013 mmol) and k2co3 (224.6 mg, 1.63 mmol) were added and then dissolved in deionized water (3 ml). the reaction mixture was stirred vigorously for 5 h at room temperature. after the white reaction mixture had become yellow and non-homogeneous, the mixture was diluted with water (10 ml), and the product was extracted with etoac. the organics were separated, filtered through a celite pad, and dried with mgso4. the organic solvent was removed under reduced pressure and the crude product was purified by dry-flash chromatography (sio2: hexane/etoac = 9/1 to 7/3) to afford the title compound 5 (11 mg, 7 %). 5-(4-fluorophenyl)furan-2-carbaldehyde (6).6 the general suzuki coupling procedure was followed, except 4-fluorophenylboronic acid (114.7 mg) was used. the reaction mixture was refluxed for 12 h. the crude product was purified by dry-flash chromatography (sio2: hexane/etoac = 95/5 to 8/2) to afford the title compound 6 (108.3 mg, 88 %). * 45 psig = 310.3 kpa 646 ajdačić et al. 5-(4-methoxyphenyl)furan-2-carbaldehyde (7).6 the general suzuki coupling procedure was followed, except 4-methoxyphenylboronic acid (124.6 mg) was used. the reaction mixture was refluxed for 12 h. the crude product was purified by dry-flash chromatography (sio2: hexane/etoac = 9/1 to 6/4) to afford the title compound 7 (118 mg, 90%). 4-bromo-5-phenylthiophene-2-carbaldehyde (10).5 to a solution of aldehyde 8 (76 mg, 0.40 mmol) in dry chcl3 (700 μl) was added a solution of bromine (33 μl, 0.64 mmol) in dry chcl3 (330 μl). the resulting solution was stirred at r.t. for 3 h. to the reaction mixture was added sat. na2s2o3 solution and the reaction mixture extracted with ch2cl2 (2×10 ml). the organic layer was washed with sat. nahco3 solution and brine and dried over anhydrous na2so4. the organic solvent was removed under reduced pressure and the crude residue was purified by dry-flash chromatography (sio2: hexane/etoac = 9/1) to yield 10 (81 mg, 75 %). 4-bromo-5-phenyl-2-furaldehyde (11).8 to a solution of aldehyde 9 (100 mg, 0.58 mmol) in mecn (10 ml) was added nbs (114 mg, 0.64 mmol). the resulting solution was stirred at r.t. for 24 h. to the reaction mixture was added h2o (10 ml) and the reaction mixture was extracted with ch2cl2 (2×10 ml). the organic layer was washed with brine and dried over anhydrous na2so4. the organic solvent was removed under reduced pressure and the crude residue was purified by dry-flash chromatography (sio2: hexane/etoac = 95/5) to yield 11 (72.9 mg, 50 %). 4,5-diphenylthiophene-2-carbaldehyde (12).6 the general suzuki coupling procedure was followed, except phenylboronic acid (38.3 mg) was used. the reaction mixture was refluxed for 12 h. the crude product was purified by dry-flash chromatography (sio2: hexane/etoac = 9/1) to afford the title compound 12 (53 mg, 89 %). 4-(4-fluorophenyl)-5-phenylthiophene-2-carbaldehyde (13). the general suzuki coupling procedure was followed, except 4-fluorophenylboronic acid (36.6 mg) was used. the reaction mixture was refluxed for 12 h. the crude product was purified by dry-flash chromatography (sio2: hexane/etoac = 9/1) to afford the title compound 13 (42 mg, 77 %). 2-(4-bromo-5-phenyl-2-furyl)-1,3-dioxolane (14). aldehyde 11 (117 mg, 0.46 mmol), ethylene glycol (130 μl, 2.33 mmol), and p-toluenesulfonic acid monohydrate (1.8 mg, 9.32×10-3 mmol) were dissolved in phme (4 ml). under dean–stark conditions, the reaction mixture was refluxed for 3h, and then washed sequentially three times with 3 m naoh and water. the phme layer was dried, filtered, evaporated under vacuum and the crude residue was purified by dry-flash chromatography (sio2: hexane/etoac = 9/1) to yield acetal 14 (109 mg, 80 %). 4-fluoro-5-phenyl-2-furaldehyde (15). to a solution of acetal 14 (49 mg, 0.167 mmol) in dry thf (2.5 ml), nbuli (1.6 m in hexane, 125 μl, 0.2 mmol) was added dropwise at a temperature below –60 °c under ar. after stirring the mixture for 2 h, n-fluorobenzenesulfonimide (58 mg, 0.18 mmol) in thf (1 ml) was added dropwise. the reaction mixture was allowed to warm to room temperature. h2o was added to the reaction mixture, which was then extracted with ch2cl2, washed with brine, dried with mgso4 and concentrated. to a stirred solution of crude residue in thf (2 ml) at 25 °c was added 2m hcl (0.6 ml), and the reaction mixture was stirred at 25 °c for 1 h. the reaction mixture was slowly quenched with sat. aq. nahco3 (2 ml), the biphasic mixture was extracted with ch2cl2 (2×10 ml), and the combined organic layers were dried over mgso4 and concentrated. the crude residue was purified by dry-flash chromatography (sio2: hexane/etoac = 9/1) to yield 15 (9.4 mg, 30 % for 2 steps). (4-nitro-5-phenyl-2-thienyl)methylene diacetate (16). to a cold solution of aldehyde 8 (35 mg, 0.186 mmol) in ac2o (500 μl) was added a solution of hno3 (9.5 μl, 0.223 mmol) guanylhydrazones as antimicrobials 647 in acoh (190 μl). the resulting solution was stirred at r.t. for 2 h, and then ice was added and the reaction mixture was extracted with ch2cl2 (2×10 ml). the organic layer was washed with sat. nahco3 solution, brine and dried over anhydrous na2so4. the organic solvent was removed under reduced pressure and the crude residue was purified by dry-flash chromatography (sio2: hexane/etoac = 8/2) to yield 14 (44 mg, 71 %). 4-nitro-5-phenylthiophene-2-carbaldehyde (17). to a solution of diacetate 16 (17 mg, 0.05 mmol) in meoh/h2o (1/1volume ratio, 1 ml), was added h2so4 (100 µl). the resulting solution was stirred at r.t. for 2 h, and then h2o was added and the reaction mixture extracted with ch2cl2 (2×10 ml). the organic layer was washed with brine and dried over anhydrous na2so4. the organic solvent was removed under reduced pressure and the crude product was used in the next step. general procedure for the preparation of guanylhydrazones.4 (2e)-2-{[5-(4-methylphenyl)furan-2-yl]methylidene}hydrazinecarboximidamide hydrochloride (18). to a solution of aldehyde 4 (86.2 mg, 0.463 mmol) in absolute ethanol (10 ml), aminoguanidine hydrochloride (51.2 mg, 0.463 mmol) was added. the resultant solution was stirred at room temperature for 5 min, and a solution of concentrated hcl (5 mol %) in absolute etoh (50 µl, 1/25, v/v) was added. the reaction mixture was heated to 90 °c, refluxed for 18 h and allowed to cool to room temperature. the solvent was removed under reduced pressure, the crude product was washed with ch2cl2 (1 ml) and then crystallized from etoh/hexane (9/1) to provide the title compound 18 (127.8 mg, 99 %). (2e)-2-{[5-(4-methoxyphenyl)furan-2-yl]methylidene}hydrazinecarboximidamide hydrochloride (19). following the general procedure for guanylhydrazone formation, 19 (144 mg, 100 %) was obtained from 7. (2e)-2-{[5-(4-fluorophenyl)furan-2-yl]methylidene}hydrazinecarboximidamide hydrochloride (20). following the general procedure for guanylhydrazone formation, 20 (93.2 mg, 100 %) was obtained from 6. (2e)-2-{[5-(4-bromophenyl)furan-2-yl]methylidene}hydrazinecarboximidamide hydrochloride (21). following the general procedure for guanylhydrazone formation, 21 (17.2 mg, 100 %) was obtained from 5. (2e)-2-[(4-fluoro-5-phenyl-furan-2-yl)methylidene]hydrazinecarboximidamide hydrochloride (22). following the general procedure for guanylhydrazone formation, 22 (7.1 mg, 79 %) was obtained from 15. (2e)-2-[(4-bromo-5-phenyl-2-thienyl)methylidene]hydrazinecarboximidamide hydrochloride (23). following the general procedure for guanylhydrazone formation, 23 (41 mg, 92 %) was obtained from 10. (2e)-2-[(4-bromo-5-phenyl-furan-2-yl)methylidene]hydrazinecarboximidamide hydrochloride (24). following the general procedure for guanylhydrazone formation, 24 (44 mg, 78 %) was obtained from 11. (2e)-2-[(4-nitro-5-phenyl-2-thienyl)methylidene]hydrazinecarboximidamide hydrochloride (25). following the general procedure for guanylhydrazone formation, 25 (19.5 mg, 75 %) was obtained from 17. (2e)-2-[(4,5-diphenyl-2-thienyl)methylidene]hydrazinecarboximidamide hydrochloride (26). following the general procedure for guanylhydrazone formation, 26 (51.9 mg, 99%) was obtained from 12. (2e)-2-{[4-(4-fluorophenyl)-5-phenyl-2-thienyl]methylidene}hydrazinecarboximidamide hydrochloride (27). following the general procedure for guanylhydrazone formation, 27 (36.5 mg, 92%) was obtained from 13. 648 ajdačić et al. antimicrobial activity guanylhydrazones were dissolved in dmso in stock concentrations of 50 mg ml-1 and used immediately for antimicrobial activity assessments. mic concentrations (concentration value corresponding to the lowest concentration that inhibited the growth after 24 h at 37 °c) were determined according to the standard broth microdilution assays, recommended by the national committee for clinical laboratory standards (m07-a8) for bacteria in lb (luria -bertani) broth and standards of european committee on antimicrobial susceptibility testing (edef7.1.) in sab (sabouraud dextrose) broth. the highest concentration used was 500 µg ml-1. the test organisms included pseudomonas aeruginosa pao1 (nctc 10332), staphylococcus aureus (atcc 25923), micrococcus luteus (atcc 379), listeria monocytogenes (nctc 11994) candida albicans (atcc 10231), issatchenkia orientalis (atcc 6258) and candida parapsilosis (atcc 22019). the inoculums were 105 colony forming units, cfu ml-1, for the bacteria and 104 cfu ml-1 for the candida strains. conclusions in the present work, an efficient synthesis of novel guanylhydrazones was designed. the activity of these compounds against the panel of human pathogens consisting of one gram-negative strain, three gram-positive strains, and three fungal strains, was assessed. noticeably, compounds 26 and 27 showed excellent antibacterial activities against gram-positive s. aureus atcc 25923 and m. luteus atcc 379, even better than the control drug, kanamycin. furthermore, compounds 26 and 27 displayed good antifungal activity against c. parapsilosis atcc 22019. altogether, the reported results indicate that the selected guanylhydrazones could form the basis for further development of new and effective antimicrobial agents. supplementary material analytical and spectral data of the synthesized compounds are available at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. this research was financially supported by the ministry of education, science and technological development of the republic of serbia (grant no. 172008 and 173048) and the serbian academy of sciences and arts. и з в о д антибактеријска и антифунгална активност гванилхидразонских деривата владимир ајдачић1, јелена лазић1, марија мојићевић2, сандра шеган3, јасмина никодиновић-рунић2 и игор м. опсеница1 1хемијски факултет, универзитет у београду, студентски трг 16, п. пр. 51, 11158, београд, 2институт за молекуларну генетику и генетичко инжењерство, универзитет у београду, војводе степе 444а, 11000 београд и 3институт за хемију, технологију и металургију, универзитет у београду, његошева 12, 11000 београд у овом раду приказана је синтеза серије гуванилхидразонских деривата. добијеним дериватима испитана је антибактеријска и антифунгална активност. утврђено је да једињења 26 и 27 показују изражену антибактеријску активност према staphylococcus aureus guanylhydrazones as antimicrobials 649 atcc 25923 и micrococcus luteus atcc 379 сојевима. истовремено, ова једињења показала су и изражену антифунгалну активност према candida parapsilosis atcc 22019 соју. (примљено 13. фебруара, ревидирано 3. марта, прихваћено 7. марта 2017) references 1. m. oliveira, i. serrano, frontiers in antimicrobial agents: the challenging of antibiotic resistance in the development of new therapeutics, bentham science publ., sharjah, 2015 2. m. a. pfaller, s. a. messer, g. j. moet, r. n. jones, m. castanheira, int. j. antimicrob. agents 38 (2011) 65 3. a) p. ulrich, a. cerami, j. med. chem. 27 (1984) 35; b) m. n. borges, j. c. messeder, j. d. figueroa-villar, eur. j. med. chem. 39 (2004) 925; c) z. li, m. khaliq, z. zhou, c. b. post, r. j. kuhn, m. cushman, j. med. chem. 51 (2008) 4660; d) a. s. mayhoub, m. khaliq, c. botting, z. li, r. j. kuhn, m. cushman, bioorg. med. chem. 19 (2011) 3845; e) h. mohammad, a. s. mayhoub, a. ghafoor, m. soofi, r. a. alajlouni, m. cushman, m. n. seleem, j. med. chem. 57 (2014) 1609; f) h. mohammad, p. v. n. reddy, d. monteleone, a. s. mayhoub, m. cushman, m. n. seleem, eur. j. med. chem. 94 (2015) 306 4. v. ajdačić, l. senerovic, m. vranić, m. pekmezovic, v. arsic-arsnijevic, a. veselinovic, j. veselinovic, b. a. šolaja, j. nikodinovic-runic, i. m. opsenica, bioorg. med. chem. 24 (2016) 1277 5. v. ajdačić, s. stepanović, m. zlatović, m. gruden, i. m. opsenica, synthesis 48 (2016) 4423 6. t. hosoya, h. aoyama,t. ikemoto,y. kihara,t. hiramatsu, m. endo, m. suzuki, bioorg. med. chem. 11 (2003) 663 7. j. c. bussolari, d. c. rehborn, org. lett. 1 (1991) 965 8. k. l. milkiewicz, d. j. parks, t. lu, tetrahedron lett. 44 (2013) 4257. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {in vitro antimicrobial activity and cytotoxicity of nickel(ii) complexes with different diamine ligands} j. serb. chem. soc. 82 (4) 389–398 (2017) udc 546.742+547.415.1:615.28–188:576+615.9 jscs–4974 original scientific paper 389 in vitro antimicrobial activity and cytotoxicity of nickel(ii) complexes with different diamine ligands nenad s. drašković1#, biljana đ. glišić2*#, sandra vojnovic3, jasmina nikodinovic-runic3** and miloš i. djuran2# 1university of priština, faculty of agriculture, kopaonička bb, 38228 lešak, serbia, 2department of chemistry, faculty of science, university of kragujevac, r. domanovića 12, 34000 kragujevac, serbia and 3institute of molecular genetics and genetic engineering, university of belgrade, vojvode stepe 444a, 11000 belgrade, serbia (received 13 january, revised 30 january, accepted 13 february 2017) abstract: three diamines, 1,3-propanediamine (1,3-pd), 2,2-dimethyl-1,3-propanediamine (2,2-dime-1,3-pd) and (±)-1,3-pentanediamine (1,3-pnd), were used for the synthesis of nickel(ii) complexes 1–3, respectively, of the general formula [ni(l)2(h2o)2]cl2. the stoichiometries of the complexes were confirmed by elemental microanalysis, and their structures were elucidated by spectroscopic (uv–vis and ir) and molar conductivity measurements. the complexes 1–3, along with nicl2·6h2o and the diamine ligands, were evaluated against a panel of microbial strains that are associated with skin, wound, urinary tract and nosocomial infections. the obtained results revealed no significant activity of 1–3 against the investigated bacterial strains. on the other hand, they showed good antifungal activity against pathogenic candida strains, with minimum inhibitory concentration (mic) values in the range from 15.6 to 62.5 µg ml-1. the best anti-candida activity was observed for complex 2 against c. parapsilosis, while the least susceptible to the effect of the complexes was c. krusei. the antiproliferative effect on normal human lung fibroblast cell line mrc-5 was also evaluated in order to determine the therapeutic potential of nickel(ii) complexes 1–3. these complexes showed lower negative effects on the viability of the mrc-5 cell line than the clinically used nystatin and comparable selectivity indexes to that of this antifungal drug. keywords: nickel(ii) complexes; diamines; antimicrobial activity; candida; cytotoxicity. introduction the field of medicinal application of metal-based compounds has attracted widespread attention of researchers over the decades.1 this broad interest is due *,** corresponding authors. e-mail: (*)bglisic@kg.ac.rs; (**)jasmina.nikodinovic@gmail.com # serbian chemical society member. https://doi.org/10.2298/jsc170113026d 390 drašković et al. to the constant demand of medicinal chemistry for innovation and input of novel metal-based compounds to cope with very important challenges, such as broader spectrum of activity, selectivity, reduced toxicity and emerging resistance. the development of a new therapeutic agent is a multi-stage process involving synthesis, characterization, evaluation of biological activity, and pre-clinical and clinical testing. considering the fact that this process becomes very expensive in the later stage, it is important to use the best possible models in the identification of those compounds that have the desired biological activity. furthermore, it was found previously that microorganisms and infections play major roles in carcinogenesis, as well as in antitumor response.2 of the 12.7 million new cancer cases that occurred in 2008, around 2 million could be attributed to infections. bacteremia is a major cause of life-threatening complications in patients with cancer, who are at extremely high risk for infections caused by antibiotic-resistant gram -negative bacteria. invasive candidiasis is the fourth most common bloodstream infection (surpassing many bacterial pathogens) with mortality rates remaining disturbingly high at 40 %.3 more than 17 different candida species are known to be etiological agents of human infection, however, more than 90 % of invasive infections are caused by c. albicans, c. glabrata, c. parapsilosis and c. krusei.4 considering this and in the view of the global problem of multi-drug resistant microbial strains, the search for new antibacterial and antifungal therapeutics is of paramount importance. nickel was considered for many years as an element without important biological significance, until its existence in the active center of the enzyme urease was established in 1975.5,6 since then, ni2+ was confirmed to be competitive antagonists with both mg2+ and ca2+ and the presence of nickel was established in the active sites of different metallo-enzymes, and hence, interest for the evaluation of its biological properties has rapidly expanded.7,8 it was found that chronic exposure to nickel could be connected with increased risk of lung cancer, cardiovascular disease, neurological deficits, developmental deficits in childhood and high blood pressure.9 nickel is also considered as a potential allergen, which may cause contact dermatitis.10 nevertheless, a broad spectrum of beneficial biological activities of various nickel(ii) complexes has hitherto been reported. nickel(ii) complexes were reported to act as anticonvulsant,11 antiepileptic,12 antibacterial,13 antifungal,14 antileishmanial,15 antioxidant16,17 and antiproliferative agents.18 considering this, in the present study, three diamines, 1,3-propanediamine (1,3-pd), 2,2-dimethyl-1,3-propanediamine (2,2-dime-1,3-pd) and (±)-1,3-pentanediamine (1,3-pnd) were used for the synthesis of nickel(ii) complexes of the general formula [ni(l)2(h2o)2]cl2 (scheme 1). although the synthesis of these complexes was reported previously,19–22 their antimicrobial effects have not been investigated. in order to determine the therapeutic potential biological evaluation of bis(diamine)nickel(ii) complexes 391 of these complexes, their antiproliferative effect on the normal human lung fibroblast cell line mrc-5 was also evaluated. scheme 1. structural representation of [ni(l)2(h2o)2]cl2 complexes 1–3 (l = 1,3-pd (1), 2,2-dime-1,3-pd (2) and 1,3-pnd (3)). experimental reagents distilled water was demineralized and purified to a resistance of greater than 10 mω cm. nickel(ii) chloride hexahydrate, 1,3-propanediamine (1,3-pd), 2,2-dimethyl-1,3-propanediamine (2,2-dime-1,3-pd) and (±)-1,3-pentanediamine (1,3-pnd) were purchased from sigma– –aldrich. all the employed chemicals were of analytical reagent grade. synthesis of the nickel(ii) complexes 1–3 the nickel(ii) complexes with the above-mentioned diamine ligands were synthesized by modification of a previously described method.19 the corresponding diamine (0.02 mol; 1.7 ml of 97 % 1,3-pd, ρ = 0.887 g ml-1; 2.4 ml of 99 % 2,2-dime-1,3-pd, ρ = 0.851 g ml-1 and 2.4 ml of 98 % 1,3-pnd, ρ = 0.855 g ml-1) was added slowly under stirring to a solution containing 0.01 mol of nicl2·6h2o (2.38 g) in 10.0 ml of water. the formed nickel(ii) hydroxide was removed by filtration and the filtrate was stirred at 40 °c for 15 min, and then left standing at ambient temperature to evaporate slowly to a volume of 3.0 ml. the concentrated solution was then stored in refrigerator and purple crystals of the nickel(ii) complexes had formed after two days. these crystals were filtered off and dried at ambient temperature. the yield was 83 % for [ni(1,3-pd)2(h2o)2]cl2 (1; 2.61 g), 77 % for [ni(2,2-dime-1,3 -pd)2(h2o)2]cl2 (2; 2.85 g) and 79 % for [ni(1,3-pnd)2(h2o)2]cl2 (3; 2.92 g). measurements elemental microanalyses of the nickel(ii) complexes for carbon, hydrogen and nitrogen were performed by the microanalytical laboratory, faculty of chemistry, university of belgrade. the ir spectra were recorded as kbr pellets on a perkin elmer spectrum one spectrometer over the wavenumber range 4000–450 cm-1. the uv–vis spectra were recorded over the wavelength range of 1100–190 nm on a shimadzu uv-1800 spectrophotometer after dissolving the corresponding nickel(ii) complex in water. the concentration of the nickel(ii) 392 drašković et al. complexes was 5×10-2 m. the molar conductivities were measured at room temperature on a crison multimeter mm 41 digital conductivity-meter. the concentration of the aqueous solutions of nickel(ii) complexes used for conductivity measurements was 1×10-3 m. analytical and spectral data of the synthesized compounds are given in the supplementary material to this paper. determination of the biological activity nickel(ii) complexes 1–3, nicl2·6h2o and the diamine ligands were dissolved in distilled water to give stock solutions of 50 mg ml-1, which were used immediately for biological assessment of their activities. the mic concentrations (concentration value corresponding to the lowest concentration that inhibited the growth after 24 h at 37 °c) were determined according to the standard broth microdilution assays, recommended by the national committee for clinical laboratory standards (m07-a8) for bacteria and standards of the european committee on antimicrobial susceptibility testing (edef 7.1.). the highest concentration used was 500 µg ml-1. the test organisms included pseudomonas aeruginosa pao1 (nctc 10332), staphylococcus aureus (nctc 6571), candida albicans (atcc 10231), c. glabrata (atcc 2001), c. parapsilosis (atcc 22019) and c. krusei (atcc 14243). the inoculums were 105 colony-forming units, cfu, per ml for the bacteria and 104 cfu ml-1 for the candida strains. cell viability was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay.23 the assay was performed using human lung fibroblasts (mrc-5) after 48 h of cell incubation in the media containing the compounds at concentrations ranging from 0.1–500 µg ml-1. the mrc-5 cell line was maintained in rpmi-1640 medium supplemented with 100 μg ml-1 streptomycin, 100 u ml-1 penicillin and 10 vol.% fetal bovine serum (fbs), all from sigma, as a monolayer (1×104 cells per well) and grown in a humidified atmosphere of 95 % air and 5 % co2 at 37 °c. the extent of mtt reduction was measured spectrophotometrically at 540 nm using a tecan infinite 200 pro multiplate reader (tecan group, männedorf, switzerland), and the cell survival was expressed as percentage of the control (untreated cells). results and discussion synthesis and structural features of the nickel(ii) complexes 1–3 three nickel(ii) complexes of the general formula [ni(l)2(h2o)2]cl2, where l stands for bidentately coordinated 1,3-pd (1), 2,2-dime-1,3-pd (2) or 1,3-pnd (3), were prepared in high yields (≈80 %) by reacting nicl2·6h2o with the corresponding diamine in 1:2 mole ratio in water using a modified procedure reported previously.19 the crystal structures of the [ni(l)2(h2o)2]2+ complexes were previously determined by single-crystal x-ray diffraction analysis.19–22 in this study, spectroscopic (uv–vis and ir) and conductivity measurements were used for structural characterization of the [ni(l)2(h2o)2]cl2 complexes. the uv–vis spectra of the investigated complexes 1–3 are presented in fig. 1, while the wavelengths of the maximum absorption (λmax / nm) and molar extinction coefficients (ε / m–1 cm–1), determined immediately after dissolution of the complexes, are listed in table i. the shape of the uv–vis spectra for the investigated complexes was similar to that of the octahedral [ni(en)2(h2o)2]co3 biological evaluation of bis(diamine)nickel(ii) complexes 393 complex (en is bidentately coordinated ethylenediamine) with the same n4o2 coordination environment.24 in accordance with the previously established results for the [ni(en)2(h2o)2]co3 complex,24 the interpretation of uv–vis spectra of the presently investigated complexes 1–3 was realized using an octahedral model (oh): 3a2g → 3t2g(f) (band i), 3a2g → 3t1g(f) (band iii) and 3a2g → 3t1g(p) (band iv). in addition, in each spectrum, there was a shoulder at approximately 735 nm (band ii, table i), which occurs on the higher-energy side of the spin allowed band i. as was previously found, this shoulder arises from a spin forbidden triplet-to-singlet transition, 3a2g → 1eg(d).24,25 as could be seen from fig. 1, the absorption maxima of the bands i and iii for the investigated nickel(ii) complexes were slightly shifted to higher energy in the following order 1 > 3 > 2. moreover, the molar absorptivity of the absorption maxima for these bands increased in the same order. these differences in the spectra could be attributed to the presence of the substituent in the six-membered 1,3-propanediamine ring of the corresponding nickel(ii) complex, i.e., two methyls for 2 and an ethyl for 3. it could be assumed that these substituents affect some changes in the strain of the six-membered 1,3-propandiamine ring. moreover, all absorption maxima of the investigated complexes 1–3 were shifted to lower energies with respect to those for the [ni(en)2(h2o)2]2+ complex.24 this shifting results from the presence of a six-membered 1,3-propanediamine ring in 1–3, which is less strained than the five-membered ethylenediamine ring in the [ni(en)2(h2o)2]2+ complex. fig. 1. electronic absorption spectra of the investigated nickel(ii) complexes 1–3 measured in water (c = 5×10-2 m). the ir spectroscopic data for the nickel(ii) complexes are listed in the supplementary material to this paper and are consistent with the structural formula presented in scheme 1. the ir spectra of these complexes recorded in the range 394 drašković et al. of 4000–450 cm–1 showed the expected peaks attributable to the coordinated diamine and water ligands. thus, a broad absorption in the 3400–3300 cm–1 region attributed to the stretching vibration of oh confirmed the presence of a coordinated water molecule.26 moreover, the complexes exhibited two very strong and sharp bands at approximately 3300 and 3200 cm–1, which were assigned to the asymmetric and symmetric stretching vibration of the coordinated amino group, respectively.22 table i. electronic absorption data for the nickel(ii) complexes 1–3. for comparison the corresponding data for the previously reported [ni(en)2(h2o)2]co3 complex is given24 complex absorption assignments λ / nm ε / m-1 cm-1 [ni(en)2(h2o)2]co3 i 905 – 3a2g → 3t2g(f) ii 690 – → 1eg(d) iii 555 – → 3t1g(f) iv 349 – → 3t1g(p) [ni(1,3-pd)2(h2o)2]cl2 (1) i 946 5.1 ii 738 2.0 iii 581 9.7 iv 356 20.7 [ni(2,2-dime-1,3-pd)2(h2o)2]cl2 (2) i 931 7.5 ii 734 4.3 iii 575 13.9 iv 358 22.7 [ni(1,3-pnd)2(h2o)2]cl2 (3) i 941 5.8 ii 734 2.6 iii 580 11.2 iv 361 16.8 molar conductivity values for the nickel(ii) complexes 1–3, being approximately 250 ω–1 cm2 mol–1 (see supplementary material), are sufficiently high to assess the non-coordinated nature of the two chloride anions, i.e., these values in water solvent are in agreement with 1:2 electrolytic nature of the synthesized complexes.27 biological activity of the nickel(ii) complexes 1–3 in vitro antimicrobial activity assays of nickel(ii) complexes 1–3, nicl2·6h2o and the corresponding diamine ligands revealed no significant activity against two bacterial strains (p. aeruginosa pao1 and s. aureus) even at 500 µg ml–1 (data not shown), while mic values against the pathogenic candida strains were between 15.6–62.5 µg ml–1 for complexes 1–3 and 250 µg ml–1 for the inorganic salt (table ii). therefore, a certain level of selectivity of 1–3 towards fungal strains could be concluded. the best anti-candida activity was that of complex 2 against c. parapsilosis, while c. krusei was the least susceptible to the effects of biological evaluation of bis(diamine)nickel(ii) complexes 395 the complexes. in contrast, nickel(ii) thiohydrazide and thiodiamine complexes exhibited significant activity towards p. aeruginosa and escherichia coli, and comparable activity against a selection of fungal aspergillus strains.28 antifungal activity of nicl2·6h2o and nickel(ii) complexes derived from amino sugars against c. albicans was reported by yano et al. with mic values in the 200–250 µm range, which is 2.5–5-fold higher in comparison to the mic values of 1–3.29 furthermore, the complexes from the present study showed better anti-candida activities in comparison to nickel(ii) complexes with pyrazoline-based ligand, which had mic values ranging from 100 – 1000 µg ml–1.30 it was shown that certain selectivity against candida strains by nickel(ii) compounds was due to the competitive inhibition of fungal chitinase (chitin-degradation enzyme).29 table ii. minimal inhibitory concentrations (mic / µg ml-1) against candida strains and ic50 values against mrc-5 cells (concentration that inhibits 50 % of cell growth after treatment with the tested compounds, µg ml-1); the results are from three independent experiments, each performed in triplicate. standard deviations were within 1–3 % compound c. albicans atcc 10231 c. glabrata atcc 2001 c. parapsilosis atcc 22019 c. krusei atcc 14243 mrc-5 1 31.2 31.2 31.2 62.5 500 2 31.2 31.2 15.6 62.5 80 3 31.2 31.2 31.2 62.5 500 nicl2·6h2o 250 250 250 250 100 1,3-pd >500 >500 >500 >500 >500 2,2-dime-1,3-pd >500 >500 >500 >500 100 1,3-pnd >500 >500 >500 >500 50 nystatin 4 2 2 8 40 in parallel, to determine the applicability of complexes 1–3 as potential antifungals, their in vitro cytotoxicity against healthy human lung fibroblasts was examined (table ii, fig. 2). while the ligands exerted no activity against candida strains at 500 µg ml–1, 2,2-dime-1,3-pd and 1,3-pnd had ic50 values of 100 and 50 µg ml–1, respectively. 1,3-propanediamine was not cytotoxic even at 500 µg ml–1 (table ii). accordingly, 2 was the most cytotoxic of the nickel(ii) complexes, while 1 and 3 had ic50 values of 500 µg ml–1. the inorganic salt nicl2·6h2o had a toxic effect on the cells in a dose-dependent manner (ic50 value of 100 µg ml–1), while the cytotoxicity of 1–3 did not follow this trend (fig. 2). this may be due to differing dissociation dynamics of the complexes and different toxicity of the ligands. thus, the selectivity index for the complexes was between 1.3 and 16, while the antiproliferative effect of the inorganic salt was higher than its antifungal effect (table ii). although the mic values of 1–3 were 8–16-fold higher in comparison to that of the clinically used nystatin, the selectivity indexes were comparable. this finding is encouraging for further development of nickel(ii)-based complexes for antifungal treatment. 396 drašković et al. fig. 2. in vitro cytotoxic effect on healthy human fibroblasts (mrc-5) of various concentrations of nickel(ii) compounds upon 48 h treatment. conclusions this work presents a modified procedure for the preparation of [ni(l)2(h2o)2]cl2 complexes 1–3 in high yields. the octahedral geometry of these complexes was confirmed by spectroscopic and conductivity measurements. in vitro antimicrobial activity assays of these complexes showed their good selectivity towards the investigated candida strains. the best anti-candida activity was observed for complex 2 against c. parapsilosis, while the least susceptible to the effect of complexes was c. krusei. moreover, an in vitro cytotoxicity study showed that complex 2 was the most cytotoxic against healthy human lung fibroblasts. this arises from the presence of two methyl substituents in the six-membered 1,3-propanediamine ring of 2, indicating that better antimicrobial and cytotoxic activities of bis(diamine)nickel(ii) complexes could be achieved by structural modification of the chelated diamine ligand. the obtained results are encouraging for further development of nickel(ii) complexes with diamine ligands as antifungal agents. a study in this sense is in progress. supplementary material analytical and spectral data of the synthesized compounds are available electronically at the pages of the journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. this work was funded in part by the ministry of education, science and technological development of the republic of serbia (project nos. 172036 and 173048). biological evaluation of bis(diamine)nickel(ii) complexes 397 и з в о д in vitro антимикробна активност и цитотоксичност комплекса никла(ii) са различитим диаминским лигандима ненад с. драшковић1, биљана ђ. глишић2, сандра војновић3, јасмина никодиновић-рунић3 и милош и. ђуран2 1 пољопривредни факултет, универзитет у приштини, копаоничка бб, 38228 лешак, 2 институт за хемију, природно–математички факултет, универзитет у крагујевцу, р. домановића 12, 34000 крагујевац и 3 институт за молекуларну генетику и генетичко инжењерство, универзитет у београду, војводе степе 444а, 11000 београд три диамина, 1,3-пропандиамин (1,3-pd), 2,2-диметил-1,3-пропандиамин (2,2 -dime-1,3-pd) и (±)-1,3-пентандиамин (1,3-pnd), коришћена су за синтезу никaл(ii) комплекса 1–3 опште формуле [ni(l)2(h2o)2]cl2. комплекси су окарактерисани применом елементалне микроанализе, uv–vis и ir спектроскопије и мерењем моларне проводљивости. никал(ii) комплекси 1–3, nicl2·6h2o и одговарајући диамини су испитивани као потенцијални антимикробни агенси према различитим сојевима бактерија и гљива, који могу узроковати инфекције коже и рана, као и уринарне и интрахоспиталне инфекције. добијени резултати су показали да комплекси 1–3 немају значајну активност према испитиваним сојевима бактерија. насупрот томе, ови комплекси показују добру активност према испитиваним патогеним сојевима гљива, при чему су вредности минималне инхибиторне концентрације (mic) у опсегу од 15,6 до 62,5 μg ml-1. највећу антифунгалну активност према c. parapsilosis показује комплекс 2, док је активност комплекса најмања према c. krusei. у циљу одређивања терапеутског потенцијала ових комплекса, испитивана је њихова антипролиферативна активност према нормалној ћелијској линији фибробласта плућа. добијени резултати су показали да су комплекси никла(ii) мање токсични на mrc-5 ћелијској линији у односу на нистатин и да имају индексе селективности сличне овом антифунгалном агенсу. (примљено 13. јануара, ревидирано 30. јануара, прихваћено 13. фебруара 2017) references 1. e. alessio, bioinorganic medicinal chemistry, wiley–vch, weinheim, germany, 2011 2. n. d. savić, d. r. milivojevic, b. đ. glišić, t. ilic-tomic, j. veselinovic, a. pavic, b. vasiljevic, j. nikodinovic-runic, m. i. djuran, rsc adv. 6 (2016) 13193 3. m. a. pfaller, s. a. messer, g. j. moet, r. n. jones, m. castanheira, int. j. antimicrob. agents 38 (2011) 65 4. j. c. sardi, l. scorzoni, t. bernardi, a. m. fusco-almeida, m. j. mendes giannini, j. med. microbiol. 62 (2013) 10 5. n. e. dixon, c. gazzola, r. l. blakeley, b. zerner, j. am. chem. soc. 97 (1975) 4131 6. c. tserkezidou, a. g. hatzidimitriou, g. psomas, polyhedron 117 (2016) 184 7. r. r. crichton, biological inorganic chemistry: an introduction, elsevier, amsterdam, 2003 8. d. m. di toro, h. e. allen, h. l. bergman, j. s. meyer, p. r. paquin, r. c. santore, environ. toxicol. chem. 20 (2001) 2383 9. y. chervona, a. arita, m. costa, 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {structure-activity relationship and in silico study of unique bi-heterocycles: 5-[(2-amino-1,3-thiazol-4-yl)methyl]-1,3,4-oxadiazole-2-thiol derivatives} j. serb. chem. soc. 84 (7) 649–661 (2019) udc 547.7/8+547.245:548.022–188 jscs–5216 original scientific paper 649 structure–activity relationship and in silico study of unique bi-heterocycles: 5-[(2-amino-1,3-thiazol-4-yl)methyl]-1,3,4 -oxadiazole-2-thiol derivatives muhammad athar abbasi1,2*, muhammad shahid ramzan1, aziz-ur-rehman1, sabahat zahra siddiqui1, mubashir hassan2, hussain raza2, syed adnan ali shah3, bushra mirza4 and sung-yum seo2** 1department of chemistry, government college university, lahore-54000, pakistan, 2college of natural sciences, department of biological sciences, kongju national university, gongju, 32588, south korea, 3faculty of pharmacy and atta-ur-rahman institute for natural products discovery (aurins), level 9, ff3, universiti teknologi mara, puncak alam campus, 42300 bandar puncak alam, selangor darul ehsan, malaysia and 4department of biochemistry, quaid-i-azam university, islamabad, 45320, pakistan (received 2 february, revised 29 october 2018, accepted 4 march 2019) abstract: this paper presents the synthesis of some unique bi-heterocyclic hybrid molecules with a thiazole and an oxadiazole ring. the synthesis was initiated by the conversion of ethyl 2-(2-amino-1,3-thiazol-4-yl)acetate (1) to the corresponding 2-(2-amino-1,3-thiazol-4-yl)acetohydrazide (2) by the reaction with hydrazine hydrate in methanol. the treatment of the acid hydrazide, 2, with carbon disulfide gave the bi-heterocyclic nucleophile, 5-[(2-amino-1,3 -thiazol-4-yl)methyl]-1,3,4-oxadiazole-2-thiol (3). finally, the target compounds, 5a–o, were synthesized by stirring the nucleophile 3 with different electrophiles, 4a–o, in dmf using lih as a base and an activator. the structures of the newly synthesized molecules were confirmed through spectroscopic techniques, such as ir, ei-ms, 1h-nmr and 13c-nmr. the structure– –activity relationship of all these bi-heterocycles was established by evaluating them against four enzymes, namely, acetylcholinesterase, butyrylcholinesterase, urease and α-glucosidase, followed by their in silico study. moreover, their cytotoxicity was also profiled by killing data of brine shrimps at various concentrations. keywords: 1,3-thiazole; 1,3,4-oxadiazole; acetylcholinesterase; butyrylcholinesterase; urease; glucosidase; molecular docking; brine shrimps. *,** corresponding authors. e-mail: (*)abbasi@gcu.edu.pk; (**)dnalove@kongju.ac.kr https://doi.org/10.2298/jsc180203019a 650 abbasi et al. introduction heterocyclic compounds are major division of organic chemistry and are of immense use biologically and industrially. the thiazole nucleus imparts an important function in medicinal chemistry and serves as a key template for the development of various therapeutic agents. a large number of thiazole-containing derivatives have been reported to have a wide variety of biological activities, such as antibacterial, antifungal, antitubercular, anti-mycobacterial, anticancer and antiviral.1–6 2-amino-thiazol acetate esters are present in most organic compounds of interest in biology, pharmacology, and material sciences.7 similarly, 1,3,4-oxadiazoles have had appealing attention for the last two decades due to their extensive range of biological activities, such as anti-inflammatory, antifungal, antiparasitic and antimicrobial effects. such compounds have also demonstrated a very remarkable anti-tumor activity against leukemia, colon and breast cancer.8,9 cholinesterase enzymes, i.e., acetylcholinesterase (ache) and butyrylcholinesterase (bche) belong to serine hydrolases. acetylcholine is terminated at cholinergic synapses by these enzymes and these enzymes are also part of neuromuscular junctions and cholinergic brain synapses.10 alzheimer’s plaque is known to possess bche in notably elevated quantities. the inhibitors of these enzymes are involved in the treatment of alzheimer’s disease.11 urease is known to be involved in different pathogenic processes. it is known to be involved in pyelonephritis, peptic ulceration, kidney stone, hepatic encephalopathy, urolithiasis and urinary catheter incrustation.12,13 α-glucosidase inhibitors (agis) are drugs that inhibit the absorption of carbohydrates from the gut and may be used in the treatment of patients with type 2 diabetes or impaired glucose tolerance. inhibitors such as acarbose and miglitol have been approved for clinical use in the management of type 2 diabetes, as well as in the treatment of diabetic complications.14–16 molecular docking analysis approximates the ligands regarding their orientation and conformation at binding site of a target protein. the precise forecast of activity and precise structural modeling can be achieved by docking studies. furthermore, the elaborate interactions the target protein active site and inhibitors.17 a literature survey revealed that minor modification in the structure of heterocycles could lead to quantitative as well as qualitative changes in the biological activity. this prompted the present synthesize the various bi-heterocyclic molecules bearing thiazole and oxadiazole rings with the goal of lowering toxicity and improving activity. experimental general all the chemicals, together with analytical grade solvents, were purchased from sigma– –aldrich, alfa aesar (germany) or merck through local suppliers. pre-coated silica gel al structure-activity relationship and in silico study of unique bi-heterocycles 651 -plates were used for tlc with ethyl acetate and n-hexane as the solvent system. spots were detected by uv254. a gallonkamp apparatus was used to detect melting points (uncorrected) in capillary tubes. ir spectra (ν / cm-1) were recorded by kbr pellet method in a jasco-320-a spectrophotometer. elemental analyses were realized on a foss heraeus chn-o-rapid instrument and were within±0.4 % of the theoretical values. ei-ms spectra were measured on a jeol jms-600h instrument with data processing system. the 1h-nmr spectra (δ / ppm) were recorded at 600 mhz (13c-nmr spectra, at 150 mhz) in dmso-d6 using the bruker advance iii 600 as-cend spectrometer using a bbo probe. the coupling constant (j) is given in hz and chemical shift (δ / ppm). the abbreviations used in interpretation of 1h-nmr spectra are as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; brt, broad triplet; q, quartet; quint, quintet; sex, sextet; sep, septet; m, multiplet; dist, distorted. analytical and spectral data of the investigated compounds are given in supplementary material to this paper. procedure for the preparation of 2-(2-amino-1,3-thiazol-4-yl)acetohydrazide (2) ethyl 2-(2-amino-1,3-thiazol-4-yl)acetate (1; 10 g) and methanol (200 ml) were taken in a 500 ml rb flask, then, hydrazine hydrate (2.5 ml, 0.050 mol) was added drop wise and the mixture was allowed to reflux for 30 min. the reaction progress was observed by tlc using n-hexane and ethyl acetate as the solvent system (40:60). after completion, the reaction mixture was allowed to cool to room temperature to obtain a white colored precipitate of hydrazide 2. this product was filtered and washed with methanol. 5-[(2-amino-1,3-thiazol-4-yl)methyl]-1,3,4-oxadiazole-2-thiol (3) 2-(2-amino-1,3-thiazol-4-yl)acetohydrazide (2; 4 g, 0.024 mol) was dissolved in c2h5oh (20 ml) in a 250 ml rb flask at 28 °c and then solid koh (1.34 g, 0.024 mol) was added. the mixture was refluxed to dissolve the koh. then, carbon disulphide (3.70 ml, 0.048 mol) was added drop wise into the reaction mixture at 28 °c and was allowed to reflux again for 10 h. the reaction completion was checked by tlc using n-hexane and ethyl acetate as the solvent system (70:30). the excess of ethanol was evaporated. an excess of ice-cold distilled water was added followed by addition of dilute hcl until a ph of 4–5. the light peach colored precipitate of 3 was filtered and washed with distilled water. general procedure for the synthesis of 4-{[5-(substituted-sulfanyl)-1,3,4-oxadiazol-2-yl]methyl}-1,3-thiazol-2-amines (5a–o) the parent nucleophile (3; 0.1 g; 0.00047 mol) was dissolved in n,n-dimethyl formamide (dmf, 5–10 ml) in a 100 ml rb flask. solid lih (0.005 g) was added and mixture was stirred for 30 min. then, different electrophiles, alkyl/aralkyl halides (4a–o, one in each reaction) were added in equimolar ratios and further stirred for 3–5 h. the reaction was monitored by tlc using n-hexane and ethyl acetate solvent system (80:20). after reaction completion, ice-cold distilled water was added and the respective products, 5a–o, were collected by filtration or solvent extraction. cholinesterases assays the ache and bche inhibition activities were obtained according to the ellman method with slight modifications.20 the total volume of the reaction mixture was 100 µl. it contained 60 µl na2hpo4 buffer with a concentration of 50 mm and a ph of 7.7. 10 µl test compound (0.5 mm well-1) was added, followed by the addition of 10 µl (0.005 unit well-1 of ache or 0.5 unit well-1 bche) enzyme. the contents were mixed and pre-red at 405 nm. then the contents were pre-incubated for 10 min at 37 °c. the reaction was initiated by the addition of 10 652 abbasi et al. µl of 0.5 mm well-1 substrate (acetylthiocholine iodide for ache or butyrylthiocholine chloride for bche), followed by the addition of 10 µl dtnb (0.5 mm well-1). after 15 min of incubation at 37 °c, the absorbance was measured at 405 nm using 96-well plate reader synergy ht, biotek, usa. all experiments were performed with their respective controls in triplicate. eserine (0.5 mm well-1) was used as the positive control. the percent inhibition was calculated using the following formula: control testinhibition, % = 100 control − where control = total enzyme activity without inhibitor and test = activity in the presence of a test compound ic50 values were calculated using ez–fit enzyme kinetics software (perrella scientific inc. amherst, usa). urease inhibition assay this enzyme assay was a customized form of the commonly known berthelot assay.21 the assay mixture of 85 µl was prepared containing 10 µl of phosphate buffer of ph 7.0 (in each well in a 96-well plate), 10 µl of sample solution and 25 µl of enzyme solution (0.135 units). the contents were pre-incubated at 37 °c for 5 min. 40 µl of urea stock solution (20 mm) was added to each well with incubation for 10 min at 37 °c. this was followed by the addition of 115 µl phenol–hypochlorite reagent (freshly prepared by mixing 45 µl phenol with 70 µl of alkali) per well. for color development, the mixture was incubated for a further 10 min at 37 °c. the absorbance was measured at 625 nm. the percentage enzyme inhibition and ic50 values were calculated by the same procedure as mentioned above. α-glucosidase inhibition assay the α-glucosidase inhibition activity was performed according to a slightly modified literature method.22 the total volume of the reaction mixture was 100 µl containing 70 µl phosphate buffer saline (50 mm) with a ph of 6.8, 10 µl of test compound (0.5 mm) and 10 µl enzyme (0.057 units). the contents were mixed, pre-incubated for 10 min at 37 °c and pre-red at 400 nm. the reaction was initiated by the addition of 10 µl of 0.5 mm substrate (p-nitrophenylglucopyranoside). acarbose was used as the positive control. after 30 min of incubation at 37°c, the absorbance was measured at 400 nm using synergy ht microplate reader. all experiments were performed in duplicate. the % inhibition and ic50 values were calculated by the same equation as discussed for cholinesterase enzymes. cytotoxicity assay the cytotoxicity was studied by the brine–shrimp cytotoxic assay method.23,24 artificial sea water was prepared using sea salt 34 g l-1. brine shrimp (artemia salina) eggs (sera, heidelberg, germany) were hatched in shallow rectangular dish (22 cm×32 cm) under constant aeration for 48 h at room temperature. after hatching, the active shrimps free from eggs were collected from the brighter portion of the hatching chamber and used for the assay. ten shrimps were transferred to each vial using a pasteur pipette vial containing 5 ml of artificial sea water with 200, 20, 2 and 0.2 µg ml-1 final concentration of test compound from their stock solution. the vials were maintained under illumination at room temperature 25 to 28 °c. after 24 h, the number of surviving shrimps was counted. experiment was performed in triplicate. data was analyzed with finney computer program to determine ld50 (lethal dose that killed 50 % of shrimps) values. structure-activity relationship and in silico study of unique bi-heterocycles 653 statistical analysis statistical analysis was performed by microsoft excel 2010 for all the thrice measured values and the results are presented as mean ± sem. grid generation and molecular docking the ache, bche, urease and α-glucosidase structures were retrieved from protein data bank (pdb, www.rcsb.org) with pdbids 4pqe, 4bds, 4h9m and 4j5t in protein preparation wizard. the selected crystal structures of the proteins were preprocessed and minimized using default parameters in maestro interface. bond orders were assigned, and hydrogen atoms were added to the protein. all four structures were minimized separately to reach a converged root mean square deviation (rmsd) of 0.30 å with the opls_2005 force field. the active site of the enzyme was defined from the co-crystallized ligands from protein data bank and literature data.25-28 furthermore, docking experiment was performed against all synthesized ligands and target protein using the glide docking protocol.29 the predicted binding energies (docking scores) and conformational positions of ligands within active region of protein were also defined using glide experiment. throughout the docking simulations, both partial flexibility and full flexibility around the active site residues were considered using the glide/sp/xp and induced fit docking (ifd) approaches.29 results and discussion chemistry the targeted bi-heterocycles, 5a–o, were acquired in excellent yields by starting the synthesis from ethyl 2-(2-amino-1,3-thiazol-4-yl)acetate (1), which was transformed into 2-(2-amino-1,3-thiazol-4-yl)acetohydrazide (2) by reaction with hydrazine hydrate in methanol. the cyclization of 2 was realized with carbon disulfide in basic medium to afford the bi-heterocyclic core molecule, 5-[(2 -amino-1,3-thiazol-4-yl)methyl]-1,3,4-oxadiazole-2-thiol (3) having a light peach color. finally, its s-substituted derivatives, 5a–o, were prepared by reacting the nucleophilic 3 with different aralkyl/alkyl halides, 4a–o, as electrophiles. this synthesis is outlined in scheme i and the varying substituents are listed in table i. the structures of these synthesized derivatives were corroborated by their ir ei-ms, 1h-nmr and 13c-nmr spectral data and chn analysis data. the structural assignment of one of the compounds is discussed here in detail for the expediency of the readers. the molecular formula, c13h11brn4os2, of 5g was established through its chn analysis data together with its ei-ms data showing [m+2]+ peak at m/z 384 and a molecular ion peak [m]+ of almost equal intensity at m/z 382. this assignment was supported by counting the number of protons in its 1h-nmr spectrum (figs. s-1 and s-2 of the supplementary material). the number of the carbon atoms resonating in its 13c-nmr spectrum (fig. s-3 of the supplementary material) was also in agreement. the prominent absorption bands in the ir spectrum appeared at 3350 (–nh2 str.), 3173 (c–h str. of the aromatic ring), 2923 (–ch2– str.), 1672 (c=c str. of aromatic ring) and 1590 cm–1 (c=n str.). an a2b2 spin system for a 4-bromobenzyl moiety in the molecule was clearly indicated by two ortho-coupled doub 654 abbasi et al. scheme 1. outline for the synthesis of 4-{[5-(substituted-sulfanyl)-1,3,4-oxadiazol-2-yl]methyl}-1,3-thiazol-2-amines. reagents and conditions: i) meoh/n2h4•h2o/reflux for 30 min; ii) etoh/cs2/koh/refluxing for 10–11 h; iii) dmf/lih/stirring for 3–5 h. table i. the different –r (aralkyl/alkyl) groups in scheme i n sh2n n n o s r 12 3 4 5 6 1' 2' 5' 3' 4' compd. –r compd. –r 4a, 5a h2c 7'' 1'' 3'' 5'' 4h, 5h h2c f 7'' 1'' 3'' 5'' 4b, 5b h2c cl 7'' 1'' 3'' 5'' 4i, 5i h2c 7'' 1'' 3'' 5'' 8''h3c 4c, 5c h2c 7'' 1'' 3'' 5'' cl 4j, 5j ch2 ch3 1'' 2'' 4d, 5d h2c cl 7'' 1'' 3'' 5'' 4k, 5k ch2 ch2 1'' 2'' ch3 3'' 4e, 5e h2c br 7'' 1'' 3'' 5'' 4l, 5l ch2 ch2 1'' 2'' ch2 3'' ch3 4'' structure-activity relationship and in silico study of unique bi-heterocycles 655 table i. continued compd. –r compd. –r 4f, 5f h2c 7'' 1'' 3'' 5'' br 4m, 5m ch2 ch2 1'' 2'' ch2 3'' ch2 ch3 4'' 5'' 4g, 5g h2c br 7'' 1'' 3'' 5'' 4n, 5n ch2 ch2 1'' 2'' ch2 3'' ch2 ch2 4'' 5'' ch2 ch3 6'' 7'' 4o, 5o h2ch2ch2c 8'' 7'' 1'' 3'' 5''9'' lets at δ 7.51 (2h, brd, j = 8.2 hz, h-2′′ & h-6′′) and 7.35 ppm (2h, brd, j = 8.2 hz, h-3′′ & h-5′′), along with a benzylic methylene signal at δ 4.42 (2h, s, ch2-7′′). the 2-amino-1,3-thiazol-4-yl heterocycle was characterized by two singlets at δ 7.02 (2h, –nh2) and 6.40 ppm (1h, h-5), while a singlet at δ 4.03 ppm (2h, ch2-6) was assignable to a methylene group connecting the two heterocycles in the molecule. all these assignments are also substantiated by its 13c -nmr spectrum that exhibited overall eleven carbon resonances due to some equivalent carbons in the molecule. the 2-amino-1,3-thiazol-4-yl heterocycle was undoubtedly ascribed by two quaternary signals at δ 162.68 (c-2) and 143.79 ppm (c-4), along with a methine signal at δ 103.27 ppm (c-5). similarly, the other heterocycle, i.e., (5-substituted-1,3,4-oxadiazol-2-yl)sulfanyl was also signified by two quaternary signals at δ 168.78 (c-2′) and 165.85 ppm (c-5′)30 while a methylene connecting the two heterocycle was obvious at δ 27.52 ppm (c-6).18 the 4-bromobenzyl moiety was also apparent with two symmetric doublet methine signals 131.39 (c-3′′ & c-5′′), and 131.28 ppm (c-2′′ & c-6′′) along with two quaternary signals at δ 136.30 (c-1′′) and 120.87 ppm (c-4′′). while a methylene signal at δ 34.95 ppm (c-7′′) was attributed to the benzylic methylene of this moiety.18 these structural units of the molecule were also fully coherent with various fragment ion peaks observed in its ei-ms spectrum. based on the complete consolidated discussion, the deduced structure of 5g was named as 4-({5-[(4-bromobenzyl)sulfanyl]-1,3,4-oxadiazol-2-yl}methyl)-1,3-thiazol-2-amine. a similar exercise was implemented for the structural analysis of all the other synthesized bi-heterocycles. enzyme inhibition and structure–activity relationship the screening of these synthesized compounds against acetylcholinesterase (ache) and butyrylcholinesterase (bche) enzymes (cholinesterases) revealed that these molecules exhibited variable inhibitory potential as shown by their ic50 values (table s-i of the supplementary material). 656 abbasi et al. the ic50 results against the ache enzyme demonstrated that 5-({5-[(3 -chlorobenzyl)sulfanyl]-1,3,4-oxadiazol-2-yl}methyl)-1,3-thiazol-2-amine (5c) was a better inhibitor having an ic50 value of 14.27±0.19 µm relative to the other derivatives in the series. eserine was used as a reference standard, with an ic50 value of 0.04±0.01 µm, against this enzyme. the better activity of 5c might be attributed to the substitution of 3-chlorobenzyl group in the molecule. the synthesized compounds could be arranged in the following order according to their inhibitory activity: 5c > 5n > 5g > 5o > 5i > 5l > 5d > 5b > 5a > 5k > 5h > 5j > 5f > 5m > 5e. against the bche enzyme, only 5-({5-[(4-bromobenzyl)sulfanyl]-1,3,4-oxadiazol-2-yl}methyl)-1,3-thiazol-2-amine (5g) exhibited considerable inhibitory potential with an ic50 value of 63.51±0.12 µm, as compared to the other derivatives in the series. here also, the very potent eserine was used the reference standard, having an ic50 value of 0.85±0.01 µm. the notable inhibitory potential of 5g against this enzyme might be an attribute of the substitution of 4-bromobenzyl group in this molecule. the overall order of the active compounds against this enzyme was established as: 5g > 5d > 5b > 5c > 5o > 5i > 5m > 5a > 5f > 5l > 5j > 5e > 5h. similarly, against the urease enzyme, most of the molecules also exhibited a very moderate inhibitory potential, relative to the reference thiourea, having an ic50 value of 21.11±0.12 μm. in comparison among the synthesized derivatives, 5c possessed relatively better inhibitory activity, indicating that the incorporation of 3-chlorobenzyl moiety was also adequate for the inhibition of this enzyme. the overall order for the active compound was inferred as: 5c > 5b > 5e > 5g > 5n > 5k > 5i > 5d > 5a > 5f > 5j > 5o > 5c > 5h. their ic50 results were also in agreement with molecular docking data. against the α-glucosidase enzyme, the reference used was acarbose with an ic50 value of 37.38±0.12 µm. here, 5f was identified as the most active compound among the series, having an ic50 value of 46.17±0.14 µm. the better activity of 5f against this enzyme might be the outcome of the substitution of 3-bromobenzyl group into the molecule. the inhibitory potential order of the active compounds was observed to be: 5f > 5l > 5e > 5j > 5b > 5c > 5d > 5h > 5a > 5g > 5i > 5m > 5n > 5k > 5o. all these derivatives were docked into the active pocket of the urease enzyme in a similar way. docking energy and binding interaction pattern molecular docking experiments are the best approach to study the binding conformation of ligands within the active region of target proteins.31–34 to predict the best-fitted conformational position of synthesized compounds 5a–o, they were docked against ache, bche, jack bean urease and α-glucosidase. the generated docked complexes were examined based on the glide docking energy structure-activity relationship and in silico study of unique bi-heterocycles 657 values (kcal* mol–1) and the bonding interaction (hydrogen/hydrophobic) pattern. the binding energy values depict the conformational positions of ligands within the active region of target proteins. the docking energy values of ligands against different targeted proteins, ache, bche, jack bean urease and α-glucosidase, are given in table ii. table ii. docking energy values of all ligands against selected target proteins docking ache bche urease α-glucosidase 5a –7.12 –4.00 –4.57 –7.24 5b –8.09 –4.00 –5.18 –7.22 5c –8.38 –4.72 –4.37 –7.26 5d –8.08 –5.13 –4.94 –7.27 5e –7.87 –4.28 –4.33 –6.82 5f –8.11 –4.56 –4.76 –7.12 5g –7.45 –4.27 –4.62 –6.92 5h –7.31 –4.73 –4.55 –6.05 5i –7.84 –3.53 –4.59 –7.02 5j –6.83 –4.14 –4.88 –5.81 5k –6.63 –3.45 –4.04 –5.61 5l –5.43 –3.30 –4.40 –6.85 5m –5.66 –2.60 –3.62 –5.62 5n –6.85 –3.74 –4.84 –6.39 5o –8.13 –4.27 –4.94 –5.56 docking analysis showed that all compounds were confined in the active binding region of the receptor molecules with different conformational poses. in the ache docking results, three bonds were observed between the most active compound (5f) and the target protein. the 2-aminothiazole group of 5f forms good interaction with ser293, while the oxadiazole and 3-cholorobenzyl rings were involved in π–π interactions with aromatic residues tyr341 and trp86, respectively (figs. 1a and 2a). the present docking results showed good correlation with already published data.35 the synthesized derivatives were computationally docked against bche to explore their binding modes. among them, 5g made a couple of interactions with asn57 and trp56 (figs. 1b and 2b). the amino group of thiazole ring was involved in hydrogen bonding with asn57, while, the thiazole ring forms π–π interaction with trp56. the docking depiction of 5c against jack bean urease is shown in figs. 1c and 2c, where it was surrounded by residues in the active binding pocket of the target enzyme. no direct interaction was seen however. already reported data confirmed its good conformational position within the active region of the target protein.26,27 from the α-glucosidase docking results, it was recognized that com * 1 kcal = 4184 j 658 abbasi et al. fig. 1. the 3d interactions depiction of: a) 5c–ache, b) 5g–bche, c) 5c–urease and d) 5f–α-glucosidase. fig. 2. the depiction of the 2d interactions: a) 5c–ache, b) 5g–bche, c) 5c–urease and d) 5f–α-glucosidase. structure-activity relationship and in silico study of unique bi-heterocycles 659 pound 5f was bound very strongly in the active pocket of enzyme by making two strong interactions. the 2-aminothiazole group forms interactions with val152 and gln145 (figs. 1d and 2d). all other graphical docking results are mentioned in the supplementary material to this paper (figs. s-4–s-63). cytotoxicity the cytotoxicity of the synthesized compounds was evaluated through the brine shrimp lethality assay. higher ed50 values of brine shrimp lethality demonstrated the lower toxicity of the compounds (table iii). compounds 5i, and 5k exhibited higher ed50 values, 6.98 and 11.43 mm, respectively, and were less cytotoxic as compared to the standard doxorubicin having an ed50 value of 5.21 mm. table iii. brine shrimp activity; doxorubicin was used as a standard compd. ed50 μg ml-1 compd. ed50 μg ml-1 compd. ed50 μg ml-1 compd. ed50 μg ml-1 5a 1.21 5e 2.04 5i 6.98 5m 1.25 5b 1.45 5f 2.43 5j 2.32 5n 2.00 5c 1.15 5g 4.53 5k 11.43 5o 1.01 5d 3.46 5h 2.15 5l 2.76 doxorubicin 5.21 conclusions the targeted series of bi-heterocyclic compounds was synthesized in good yields by a facile strategy in multi-steps and some of the compounds exhibited considerable enzyme inhibitory potential. therefore, these molecules could be utilized as possible therapeutic agents in drug discovery and design programs. supplementary material analytical and spectral data, as well as other additional data, are available electronically at the pages of journal website: http:// //shd.org.rs/jscs, or from the corresponding author on request. acknowledgment. this work was supported by a research grant of the kongju national university in 2018. 660 abbasi et al. и з в о д однос структуре и активности и in silico анализа би-хетероцикличних једињења: деривати 5-[(2-амино-1,3-тиазол-4-ил)метил]-1,3,4 -оксадиазол-2-тиолa muhammad athar abbasi1,2, muhammad shahid ramzan1, aziz-ur-rehman1, sabahat zahra siddiqui1, mubashir hassan2, syed adnan ali shah3, bushra mirza4 и sung-yum seo2 1 department of chemistry, government college university, lahore-54000, pakistan; 2 college of natural sciences, department of biological science, kongju national university, gongju, 32588, south korea; 3 faculty of pharmacy and atta-ur-rahman institute for natural products discovery (aurins), level 9, ff3, universiti teknologi mara, puncak alam campus, 42300 bandar puncak alam, selangor darul ehsan, malaysia и 4 department of biochemistry, quaid-i-azam university, islamabad, 45320, pakistan у овом истраживању приказана је синтеза неких би-хетероцикличних хибридних једињења тиазола и оксадиазолског прстена. синтеза почиње трансформацијом етил 2-(2-амино-1,3-тиазол-4-ил)ацетата (1) до одговарајућег 2-(2-амино-1,3-тиазол-4 -ил)ацетохидразида (2), реакцијом са хидразин-хидратом у метанолу. реакцијом деривата 2, са угљен-дисулфидом у киселој средини, добијен је би-хетероциклични производ 5-[(2-амино-1,3-тиазол-4-ил)метил]-1,3,4-оксадиазол-2-тиол (3). на крају, циљана једињења, 5а–о, синтетисана су полазећи од једињења 3 са различитим електрофилима, 4a–o, у дмф, употребом lih као базе и активатора. структуре нових једињења су потврђене ir, ei-ms, 1h-нмр и 13c-нмр спектрима. однос структуре и активности је анализиран испитивањем активности добијених једињења према ензимима, ацетилхолинестерази, бутирилхолинестерази, уреази α-глукозидази, као и in silico испитивању. осим тога, испитана је и цитотоксичност добијених једињења према рачићима, при различитим концентрацијама. 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oxidation of olefins j. serb. chem. soc. 81 (2) 153–162 (2016) udc 546.562+547.592’415.1+547.576: jscs–4835 542.9+547.571+547.551:66.094.3.097 original scientific paper 153 copper(ii) complex of (±)trans-1,2-cyclohexanediamine azo-linked schiff base ligand encapsulated in the nanocavity of zeolite-y for the catalytic oxidation of olefins maryam lashanizadegan*, sahar shayegan and marzieh sarkheil department of chemistry, faculty of physics and chemistry, al-zahra university, p. o. box 1993893973, tehran, iran (received 8 july, revised 2 october, accepted 7 october 2015) abstract: a schiff base ligand derived from 2-hydroxy-5-(phenylazo)-benzaldehyde and (±)trans-1,2-cyclohexanediamine (h2l) and its corresponding cu(ii) complex (cul) were synthesized and characterized by ft-ir, uv–vis and 1h-nmr spectroscopy. the copper schiff base complex was encapsulated in the nanopores of zeolite-y (cul-y) by the flexible ligand method and its encapsulation was confirmed by different studies. the homogeneous and the corresponding heterogeneous catalysts were used for the oxidation of different alkenes with tert-butyl hydroperoxide. under the optimized reaction conditions, the oxidation of cyclooctene, cyclohexene, styrene and norbornene catalyzed by cul gave 89, 63, 46 and 13 % conversion, respectively. these olefins were oxidized efficiently with 50, 96, 95 and 92 % conversion, respectively, in the presence of cul-y. comparison of the catalytic behavior of cul and cul-y showed the higher catalytic activity and selectivity of the heterogeneous catalyst with respect to the homogenous one. keywords: catalyst; 2-hydroxy-5-(phenylazo)-benzaldehyde; styrene; zeolite; encapsulated. introduction various transition metal complexes have been used in the catalytic oxidation of organic substrates.1 in particular, the catalysis of alkene oxidation by transition metal complexes is an area of current interest.2,3 schiff base ligands are easily synthesized by the condensation of amines and aldehydes.4 the development of the salen transition metal complexes has provided a useful catalyst for epoxidation reactions. in 1990, the jacobsen5 and katsuki6 groups first reported that mn(iii) salen complexes were applied in the epoxidation of non-functionalized alkenes. they extensively studied the steric7 and electronic effects8 of sub * corresponding author. e-mail: m_lashani@alzahra.ac.ir doi: 10.2298/jsc150708085l _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 154 lashanizadegan, shayegan and sarkheil stituent groups on the salen ligand that could affect the enantioselectivity and rate of this reaction.9 schiff bases having both azo and azomethine groups are called azo schiff bases. these compounds are used in the textile, leather and plastic industries10–12 and have the potential for use in electronic and drug delivery applications.13,14 azo compounds are useful in analytical applications, e.g., as complexometric and ph indicators.15 moreover, these derivatives show biological activities.16,17 a few azo schiff base complexes were reported to be catalytically active towards oxidation.18 therefore, it is of interest to study azo -linked compounds as catalysts. immobilization of homogeneous catalysts onto or into an insoluble solid, which can be either an inorganic solid or an organic polymer, provides higher activity, selectivity and reusability of catalysts.19–22 several examples of heterogenization of homogeneous catalysts onto some inorganic supports, such as mcm-41,23,24 sba-1525,26 and zeolites27–29 have been reported. among the different metal complexes, cu(ii) schiff base complexes are known for their oxygenation reactions. various copper complexes catalyzed olefin oxidation reactions in homogeneous30,31 and heterogeneous32,33 media and the mechanisms of these reactions are well established. in this study, the copper(ii) complex of an azo-linked schiff base ligand was encapsulated in the nanocavity of zeolite-y and the catalytic activity of the homogeneous and heterogeneous catalysts in the oxidation of various olefins with tert-butyl hydroperoxide (tbhp) was investigated. experimental materials all starting materials and solvents were purchased from merck and were used without further purification. physical measurements ir spectra (kbr discs, 500–4000 cm-1) were recorded using a bruker ftir model tensor 27 spectrometer. uv–vis absorption spectra were recorded on a perkin–elmer lambda 35 spectrophotometer. 1h-nmr spectra were collected on a bruker ft-nmr 250 mhz spectrometer in cdcl3 with tms as the internal reference. x-ray diffractograms were recorded using a seifect 3003 pts diffractometer with a cu-kα target. the reaction products of oxidation were determined and analyzed by gc–ms quadrupole agilent 5973 msd spectrometer. preparation of 2-hydroxy-5-(phenylazo)-benzaldehyde 4-(benzeneazo)salicylaldehyde was prepared using a standard procedure.34 preparation of the ligand (h2l) (±)trans-1,2-cyclohexanediamine (0.5 mmol, 0.06 g) in 5 ml ethanol was added to a solution of 2-hydroxy-5-(phenylazo)-benzaldehyde (1 mmol, 0.23 g) in 10 ml ethanol and chloroform at a ratio of 2 to 1. the resulting mixture was refluxed for 4 h. finally, the precipitate of ligand (h2l) was recovered by filtration, washed with ethanol and dried. the ligand was recrystallized from ethanol to give the pure product (fig. 1). yield: 57 %. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ copper(ii) complex as catalyst in olefin oxidation 155 fig. 1. structure of the ligand h2l. preparation of cul the cul complex was prepared by adding a chloroform solution of the ligand (h2l) (1.5 mmol, 0.29 g) to an ethanolic solution of cu(ch3coo)2⋅h2o (1.5 mmol, 0.79 g). the resulting mixture was refluxed for about 2 h and a brown precipitate was obtained. finally, the precipitate of the complex was recovered by filtration, washed with ethanol and dried at room temperature. yield: 85 %. some physical and spectral data for h2l and cul are given in supplementary material to this paper. incorporation of the copper(ii) in na-y (metal exchanged zeolite-y) the cu-y was prepared using the standard procedure.35 na zeolite-y (4 g) was suspended in 100 ml distilled water that contained copper(ii) nitrate (4 mmol). the mixture was then stirred for 24 h. the solid was filtered and washed with deionized water and dried at room temperature to give a light blue powder of cu-y. immobilization of h2l in cu-y cu-y (0.6 g) and ligand h2l (0.1 g) were mixed in 50 ml of acetonitrile and the reaction mixture was refluxed for 7 h in an oil bath under constant stirring. the resulting material was removed and extracted with acetonitrile using a soxhlet extractor to remove the unreacted ligand from the cavities of the zeolite and the surface of the zeolite along with neat complex, if any. the non-complexed metal ions present in the zeolite were removed by exchanging with an aqueous 0.01 m nacl solution. the resulting solid was finally washed with hot distilled water until no precipitation of agcl was observed in filtrate reacted with agno3 solution. the final solid was then dried at 150 °c for several hours until a constant weight was achieved (fig. 2). fig. 2. preparation of encapsulated complex in the supercages of zeolite-y. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 156 lashanizadegan, shayegan and sarkheil homogeneous oxidation to a solution of cyclooctene (10 mmol) and cul (10 mg, 0.02 mmol) in ch3cn (5 ml), tbhp (25 mmol) was added. the resulting mixture was refluxed and the products were collected at different time intervals, identified and quantified by gc and verified by gc–ms. heterogeneous oxidation catalyst (20 mg), cyclooctene (10 mmol) and tbhp (25 mmol) were mixed in 5 ml of ch3cn and the reaction mixture was refluxed under continuous stirring in an oil bath for 8 h. the products were collected at different time intervals, identified, quantified by gc, and verified by gc–ms. results and discussion spectral studies in the ir spectrum of the ligand (h2l), a sharp band due to the azomethine ν(c=n) appeared at 1630 cm–1. the bands at 2853 and 2919 cm–1 are indicative of the presence of 1,2-cyclohexanediamine.36,37 the band due to ν(c=n) in h2l was shifted to a lower wave number and appeared at 1608 cm–1 in cul. this indicates the involvement of azomethine nitrogen in the coordination to the copper center. moreover, ν(c=n) of cul-y appeared at 1633 cm–1. this observation suggested that the structure of cul in the zeolite is not identical to that of the neat complex due to some host–guest interactions. the ir spectrum of the hybrid material showed an intense band at 1022 cm–1 attributable to the asymmetric stretching of al–o–si chain of the zeolite. the symmetric stretching and bending frequency bands of al–o–si framework of zeolite appeared at 789 and 458 cm–1, respectively.38 the x-ray powder diffraction patterns of cul-y and na-y were essentially similar except the intensities were weaker in the immobilized complex (fig. 3). this observation indicates that the framework of the zeolite had not structurally changed during the immobilization. the 1h-nmr spectrum of the ligand (h2l) was recorded using cdcl3 solvent. hydrogen atoms of the azomethine groups of h2l appeared at δ 8.23– –8.41 ppm. the hydrogen atoms of the ch2/ch groups in the cyclohexane ring were observed in the δ 1.49–3.46 ppm range. the phenolic oh protons were found at 10.05 and 11.35 ppm. the aromatic protons appeared in the range δ 7.00–7.94 ppm as a multiplet.36,37,39 the electronic spectrum of cul consisted of one broad d–d transition band at 524 nm, as is usual for square planar geometry cu(ii). the higher energy bands were due to π–π* or n–π* transition.40 the catalytic oxidation of alkenes the catalytic performance of cul in the oxidation of cyclooctene, as a model substrate, with h2o2 and tbhp as oxidizing agents was investigated. the results of a series of blank experiments are shown in table i, which confirmed the presence of the catalyst is essential for the oxidation of alkenes. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ copper(ii) complex as catalyst in olefin oxidation 157 fig. 3. xrd patterns of na-y, cu-y and cul-y. table i. blank experiments; reaction conditions: substrate, 10 mmol, tbhp, 25 mmol, acetonitrile, 5 ml; the reactions were run for 8 h under reflux entry alkene conversion, % selectivity, % 1 cycloctene 5 50a 2 cyclohexene 7 64b 3 styrene 5 72c 4 norbornene 9 58d acyclooctene oxide; b2-cyclohexen-1-ol; cbenzoic acid; dnorbornene epoxide different reaction parameters, such as reaction time, reaction solvent, catalyst concentration, the nature and the concentration of the oxidant that may affect the conversion and selectivity of the reaction were optimized. the influence of reaction time in the catalytic oxidation of cyclooctene by cul is illustrated in fig. 4. it was observed that oxidation of cyclooctene required 8 h for maximum conversion. the effect of the nature of the solvent in the catalytic activity of cul for the oxidation of cyclooctene was studied. thus, acetonitrile, ethanol, dichloromethane and chloroform were used and the highest conversion was obtained in acetonitrile (table ii). the higher conversions in acetonitrile (69 %) relative to the others possibly may be due to the polarity, hydrophilicity, size of the solvent molecule and higher boiling point of acetonitrile.41 the effect of amount of catalyst was investigated in the oxidation of cyclooctene. as seen in table iii, the highest conversion was obtained with 0.04 mmol (0.02 g) of catalyst. different amounts of oxidant (tbhp) were studied in _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 158 lashanizadegan, shayegan and sarkheil the oxidation of cyclooctene (table iv). the results indicate that the highest conversion (89 %) was obtained at 1:2.5 mole ratio of cyclooctene to tbhp. fig. 4. effect of the reaction time on the oxidation of cyclooctene. reaction conditions: cyclooctene, 10 mmol, cul, 0.02 mmol, tbhp, 25 mmol, solvent, ch3cn, 5 ml, under reflux. table ii. the influence of various solvents on the oxidation of cyclooctene in the presence of cul; reaction conditions: cyclooctene, 10 mmol, cul, 0.02 mmol, tbhp, 25 mmol and solvent, 5 ml; the reactions were run for 8 h under reflux entry solvent conversion% selectivity, % cyclooctene oxide 2-cycloocten-1-one other products 1 acetonitrile 69 63 34 3 2 ethanol 28 60 40 0 3 chloroform 18 57 32 11 4 dichloromethane 16 49 43 8 table iii. the effect of the amount of cul on the oxidation of cyclooctene; reaction conditions: cyclooctene (10 mmol), ch3cn (5 ml) and tbhp (25 mmol); the reactions were run for 8 h under reflux entry cul mmol conversion % selectivity, % cyclooctene oxide 2-cycloocten-1-one other products 1 0.02 69 63 34 3 2 0.04 89 53 38 9 3 0.06 80 59 37 4 4 0.08 66 65 31 4 table iv. the effect of amount of oxidant (tbhp) on the oxidation of cyclooctene in the presence of cul; reaction conditions: cyclooctene (10 mmol), cul (0.04 mmol) and ch3cn (5 ml), the reactions were run for 8 h under reflux entry tbhp mmol conversion % selectivity, % cyclooctene oxide 2-cycloocten-1-one other products 1 20 59 63 35 9 2 25 89 53 38 9 3 30 81 59 37 5 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ copper(ii) complex as catalyst in olefin oxidation 159 in order to investigate the effect of the oxidizing agent in the oxidation reaction, h2o2 and tbhp were used (table v). in the presence of tbhp, a higher conversion (89 %) was obtained. table v. the influence of the kind of oxidant on the oxidation of cyclooctene in the presence of cul; reaction conditions: cyclooctene (10 mmol), cul (0.04mmol), ch3cn (5 ml), oxidant (25 mmol); the reactions were run for 8 h under reflux entry oxidant conversion % selectivity, % cyclooctene oxide 2-cycloocten-1-one other products 1 tbhp 89 53 38 9 2 h2o2 23 100 0 0 to establish the general applicability of the method, under the optimized conditions, different olefins were subjected to oxidation in the presence of a catalytic amount of cul and cul-y, the results are given in tables vi and vii respectively. table vi. oxidation of olefins using tbhp catalyzed by cul; reaction conditions: cul (0.04 mmol), substrate (10 mmol), tbhp (25 mmol), acetonitrile (5 ml); the reactions were run for 8 h under reflux alkene conversion, % selectivity, % main product others 89 53a 38b 9 63 60c 40d 0 46 60e 25f 9 13 100g 0 0 acyclooctene oxide; b2-cycloocten-1-one; ccyclohexene oxide; d2-cyclohexen-1-ol; ebenzaldehyde; fbenzoic acid; gnorbornene epoxide comparison of the catalytic behavior of the copper schiff base complex encapsulated in the super cages of zeolite-y and free cul showed the higher catalytic activity and selectivity of the heterogeneous catalyst with respect to the homogenous one. when the reaction occurred in the cavity of zeolite, the imp_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 160 lashanizadegan, shayegan and sarkheil rovement of catalysis event was probably due to diffusional selectivity of the reaction and potentially increased stability via site isolation. table vii. oxidation of olefins using tbhp catalyzed by cul-y; reaction conditions: culy (20 mg), substrate (10 mmol), tbhp (25 mmol), acetonitrile (5 ml); the reactions were run for 8 h under reflux alkene conversion, % selectivity, % main product other 50 68a 32b 96 80c 20d 95 80e 20f 92 100g 0 acyclooctene oxide; b2-cycloocten-1-one; ccyclohexene oxide; d2-cyclohexen-1-ol; ebenzaldehyde; fbenzoic acid; gnorbornene epoxide conclusions in summary, the azo schiff base ligand (h2l) derivative of (±)trans-1,2-cyclohexanediamine and 2-hydroxy-5-(phenylazo)-benzaldehyde was prepared. the copper schiff base complex (cul) was encapsulated in the nanopores of zeolitey (cul-y). furthermore, these heterogeneous and homogeneous catalysts were used for the oxidation of different alkenes with tert-butyl hydroperoxide. various reaction parameters were investigated and optimized in the oxidation reaction. the oxidation of cyclooctene, cyclohexene, styrene and norbornene catalyzed by cul gave 89, 63, 46 and 13 % conversion, respectively. under the heterogeneous conditions, the oxidation of these olefins with 50, 96, 95 and 92 % conversion, respectively, was obtained. it was observed that cul-y has higher catalytic activity and selectivity than cul. this change was specially seen for norbornene. supplementary material some physical and spectral data for h2l and cul are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. financial assistance from alzahra university is acknowledged. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ copper(ii) complex as catalyst in olefin oxidation 161 извод комплекси бакра(ii) са (±)trans-1,2-циклохександиамин шифовом базом са азо групом, као лигандом, капсулирани у нанопорама зеoлита-y као катализатори оксидације олефина maryam lashanizadegan, sahar shayegan и marzieh sarkheil department of chemistry, faculty of physics and chemistry, al-zahra university p. o. box 1993893973, tehran, iran полазећи од 5-(фенилазо)-2-хидроксибензалдехида и (±)trans-1,2-циклохександиамина синтетисани су шифова база као лиганд (h2l) и одговарајући cu(ii) комплекс (cul). за карактеризацију ових једињења коришћене су ft-ir, uv–vis и 1h-nmr спектроскопске методе. бакар(ii) комплекс је методом флексибилног лиганда капсулиран у нанопoре зеолита-y (cul-y) и ово капсулирање је испитивано различитим методама. хомогено и хетерогено каталитичко својство овог капсулираног бакар(ii) комплекса је коришћено за оксидацију различитих алкена у присуству tert-бутил-хидропероксида. под одговарајућим експерименталним условима, оксидација циклооктена, циклохексена, стирена и норборнена уз каталитичко дејство cul комплекса се одиграва са 89, 63, 46, односно 13 % конверзијом. ови олефини су у присуству cul-y комплекса веома ефикасно оксидовани са 50, 96, 95, односно 92 % конверзијом. поређена су каталитичка својства cul и cul-y, при чему је нађено да хетерогена катализа показује веће каталитичко дејство и селективност у односу на хомогену катализу. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<feff04120438043a043e0440043804410442043e043204430439044204350020044604560020043f043004400430043c043504420440043800200434043b044f0020044104420432043e04400435043d043d044f00200434043e043a0443043c0435043d044204560432002000410064006f006200650020005000440046002c0020044f043a04560020043d04300439043a04400430044904350020043f045604340445043e0434044f0442044c00200434043b044f0020043204380441043e043a043e044f043a04560441043d043e0433043e0020043f0435044004350434043404400443043a043e0432043e0433043e0020043404400443043a0443002e00200020042104420432043e04400435043d045600200434043e043a0443043c0435043d0442043800200050004400460020043c043e0436043d04300020043204560434043a0440043804420438002004430020004100630072006f006200610074002004420430002000410064006f00620065002000520065006100640065007200200035002e0030002004300431043e0020043f04560437043d04560448043e04570020043204350440044104560457002e> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {tuning the copper cluster´s size on hopg by electrodeposition from perchlorate aqueous solutions. an afm study} j. serb. chem. soc. 84 (12) 1415–1426 (2019) udc 546.56+546.137–145.2:544.654.2: jscs–5274 544.653.1 original scientific paper 1415 tuning the copper cluster’s size on hopg by electrodeposition from perchlorate aqueous solutions. an afm study diana elizabeth garcia-rodriguez1, clara hilda rios reyes2 and luis humberto mendoza huizar3* 1comunidad educativa entorno. eugenio garza sada no. 72. los pocitos. aguascalientes, méxico, 2artes. san felipe 215. providence. mineral de la reforma hidalgo, hgo. méxico and 3universidad autónoma del estado de hidalgo. academic area of chemistry. carretera pachuca-tulancingo km. 4.5 mineral de la reforma, hgo, méxico (received 23 january, revised 2 april, accepted 29 may 2019) abstract: in this work, we report the electrochemical synthesis of nanometric copper clusters smaller than 3 nm of height and 14.3 nm of diameter from perchlorate solutions. from the number of copper clusters counted directly from the afm (atomic force microscopy) images, it was possible to derive an equation, which is able to predict the number of clusters formed in function on the applied potential. also, qualitative afm images were simulated employing a spreadsheet and the freeware imagej, the results obtained show concordance with the experimental afm images. keywords: copper; nanocluster; instantaneous nucleation; potentiostatic; afm. introduction copper nanoparticles (cun) have been widely used as cooling fluids for electronic systems,1 conductive inks,2 biosensors,3 electrochemical sensors,4 antimicrobial agents,5 among others. also, cun exhibit great catalytic activities, and nonlinear optical properties, which could result in many applications in optical devices and nonlinear optical materials, such as optical switches or photochromic glasses.6,7 moreover, recently, it was reported that cun are able to convert co2 in methanol, which represents an important advance in diminishing some environmental issues in big cities.8 thus, it is not so strange that many researchers have been developing new methods to synthesize cun. currently the main methods to synthesize them include chemical reduction,9–12 thermal decomposition,13 polyol,14,15 laser ablation,16 in situ chemical synthetic route,17 low energy cluster beam deposition technique18 and electrodeposition.4,19–22 specifically, electrodeposition is commonly used to synthesize nanoparticles * corresponding author. e-mail: hhuizar@uaeh.edu.mx https://doi.org/10.2298/jsc190123054g 1416 garcia-rodriguez, rios reyes and mendoza huizar because it is a relatively simple and low cost procedure in comparison with other techniques.22 however, it is important to highlight that the physical and chemical properties exhibited by cun are highly dependent on their size and morphology by which, one of the main difficulty during their synthesis is tailoring and fine -tuning the cluster’s size and the properties of these for a given application.23 in the case of the electrodeposition process, there are two main approaches to control the size of metallic nanoparticles. the first one is to vary the experimental conditions associated with the plating bath, while the second one is to modify the electrode surface roughness.22 in the first approach, several parameters including the nature of additives, concentration of nanoparticle precursors, temperature, deposition potential, and the ph value of the plating bath, can be varied to ensure shape and size control of the nanoparticles,24–28 while in the second approach, the electrode surface roughness is modified using either physical methods29 or depositing conducting films27. employing the first approach, huang et al. were able to synthesize cun of 51 nm on hopg applying a potential step to nucleate copper particles, followed of a strip peak potential to strip smaller metal nuclei partly, at last, a growth potential.21 also, cun have been synthesized modifying the composition of the plating bath by pectin scaffold4 or using sulfate solutions.30 on the other hand, the roughness surface may be modified depositing a polypyrrole conducting film on a substrate, and the size distribution of the cun is highly dependent on the thickness of the polypyrrole film, due to the increased surface roughness and the decreased uniformity of the surface potential.27 thus, employing any of the two procedures above mentioned it has been possible to synthesize cun with different sizes and distributions. from these results, it is clear that the modulation of the cluster’s size depends on an extensive number of parameters, which have to be modified to get nanoparticles with a defined size and specific properties. therefore, the development of simple methodologies is desirable to modulate the cun’s size electrodeposited on different substrates. up to the best of our knowledge, the influence of the applied potential on the electrode surface to control the size of the copper nanoclusters from a plating bath based on perchlorate ions has not been reported in the literature. this kind of electrolyte shows advantages because clo4– are weakly adsorbed on the electrode surface, which allows to study the nucleation process without a strong interference of anions adsorbed on the substrate.19 thus, in the present work we employed the potentiostatic technique to synthesize the copper clusters with different size, maintaining constant the composition of the plating bath and modifying exclusively the applied potential on the electrode surface. we carry out the morphological characterization of the cun employing atomic force microscopy at different applied overpotentials. also, we performed the prediction of qualitative afm images of the cun synthesized employing a spreadsheet and the freeware imagej.31 tuning the copper cluster´s size 1417 experimental the copper electrodeposition onto hopg electrode was carried out from a plating bath containing 0.01 m cu(clo4)2 + 0.02 m naclo4 at ph 5. all plating baths were prepared using analytic grade reagents and ultra pure water (millipore-q system) and they were deoxygenated by bubbling n2 for 15 min before each experiment. for these experiments, freshly cleaved hopg surfaces were employed in each experiment. in all cases a graphite bar was used as counter electrode, while an ag/agcl electrode (in saturated kcl), with a luggin capillary was used as reference electrode. all potentials are expressed on ag/agcl scale. all experiments were carried out at 25oc in unstirred solutions. an epsilon potentiostat with the basiepsilonec software was employed to control the parameters involved during the electrochemical experiments. cyclic voltammetry was carried out in the 0.600 v to –0.800 v potential range. the characterization of cun was performed with an atomic force microscopy (afm) a jeol jspm 4210 microscope in the lift mode. results and discussion a cyclic voltammogram was obtained from the system 0.01 m cu(clo4)2 + 0.02 m naclo4 at ph 5, see fig. 1. this voltammogram is similar to those reported previously by our research group.19 note that at the direct scan, the copper reduction process starts at 0.020 v approximately. copper reduction process involves two-steps, the first one is a slow step, related to the reduction of cu2+ to cu1+ (peak a), while the second one is a fast step related to cu1+ to cu (peak b).32 the peak a at –0.050 v has been associated with the slow step, while peak b at –0.2 v, is related to the fast step. thus, from this voltammogram it can be seen that metallic copper can be electrodeposited at lower potentials than –0.050 v. fig. 1. a typical cyclic voltammetric curve obtained onto a hopg electrode from an aqueous solution containing 0.01m cu(clo4)2 + 0.02m naclo4 (ph 5.0). the potential scan rate was started at 0.600 v toward the negative direction with a potential scan rate of 20 mv s-1. it is possible to synthesize metallic clusters employing the chronoamperemetric technique, however, it is necessary to know the potential value, and the 1418 garcia-rodriguez, rios reyes and mendoza huizar duration of the potential pulse applied on the electrode surface. here, it is important to mention that from j vs. t plots is possible to infer the experimental potential values where is possible to form disperse clusters. in this work, the transients were obtained by applying an initial potential of 0.600 v on the electrode surface. after the application of this initial potential, a step of negative potential was varied on the electrode surface in the range -0.075 to -0.175 v. in this sense, the formation of a current density maximum in a transient may serve to identify the regions in where it is possible synthesize, either disperse or overlapped clusters. thus, before this maximum it is possible to synthesize clusters because the nucleation process is separated from the overlapping process.35 in fig. 2, it is reported a chronoamperogram obtained from the hopg/0.01m cu(clo4)2 + 0.02 m naclo4 (ph 5) system at –0.175 v vs. ag/agcl for 30 s. note that this potential value corresponds to the extreme of the potential range analyzed in the present work. this transient is similar to those reported previously for the copper electrodeposition of copper onto hopg electrodes by our research group.19 fig. 2. chronoamperogram obtained with the potentiostatic technique from the hopg/0.01 m cu(clo4)2 + 0.02 m naclo4 (ph 5) system at –0.175 v vs. ag/agcl. moreover, in the transients reported in the previous work, is clear that the transients obtained at more negative potentials than –0.175 v, show diffusion limitations which become significant and may alter the getting of disperse clusters. thus, we selected the potential range in the present work as –0.075 to –0.175 v to carry out the present study, because in this range it is possible to adequately control the influence of the applied potential involved in the formation of copper clusters. also, from fig. 2 it is possible to note that the current density maximum is obtained at 5 s, therefore, at this applied potential value the formation of copper clusters should be obtained at t ≤ 5 s, and at t > 5 s is expected the overlapping of the copper clusters. thus, in the present work, in all cases, the potential pulse was finalized at 5 s to avoid the overlapping of nuclei (clusters) on the electrode surface, see fig. 3. tuning the copper cluster´s size 1419 the transients obtained at the potentials values reported in fig. 3 but at t > 30 s, have already been reported previously by our research group.19 in the transients reported in fig. 3, note that at shorter times than 0.5 s, there is a falling current transient, which may be associated with a process where the adsorption and the step cu2+ to cu1+ occurs simultaneously at the hopg electrode surface.33,34 also, note that after this falling current, in each case, the j vs. t plot increase the density current value without reaches a maximum, this fact ensures the formation of disperse copper clusters on the hopg surface. fig. 3. a set of current transients obtained from aqueous solution 0.01 m cu(clo4)2 + 0.02 m naclo4 (ph 5) onto a hopg by means of the potential step technique for different potential step values indicated in the figure. in all the cases, the initial potential was 0.600 v. the general shape of the transients reported in fig. 3 is very similar to the reported for a three dimensional nucleation process with a diffusion control (3d-c).36,37 a classification of the nucleation as instantaneous or progressive from transients showed in fig. 3 is possible by following the criteria established by sharifker et al. where the experimental transients in a non-dimensional form by plotting j2/jm2 vs. t/tm are compared with those theoretically generated from eqs. (1) and (2) for instantaneous and progressive nucleation, respectively:36 212 2 m mm 1.9254 1 exp 1.2564 −       = − −              j t t t tj (1) 2 1 22 2 m mm 1.2254 1 exp 2.3367 −        = − −             j t t t tj (2) where jm is the current density on the maximum reached at the time tm. however, it is important to mention that these equations are valid only at the initial stages 1420 garcia-rodriguez, rios reyes and mendoza huizar of the electrocrystallization process, when the nuclei are sufficiently small and growth to independently from each other.38 fig. 4 shows a comparison of the theoretical dimensionless transients, generated by eqs. (1) and (2) with the experimental dimensionless current transients reported in fig. 3. this analysis was carried out for the transients obtained at –0.175, –0.150 and –0.125 v during a pulse potential of 5 s, to ensure the formation of disperse copper clusters on the hopg surface. note that the nucleation process may be classified as instantaneous. in an instantaneous nucleation the saturation number density of nuclei is achieved immediately after applying the potential step, by which is possible to control the number of cluster formed on the electrode. moreover, in an instantaneous nucleation is expected that all nuclei have similar sizes. fig. 4. comparison of experimental transients obtained at different potential values normalized through the coordinates of its respective local maximum (tm, jm), with the theoretical non-dimensional curves corresponding to 3d instantaneous nucleation (eq. (1)) and 3d progressive nucleation (eq. (2)). in order to verify the formation of cun on the hopg surface through the potentiostatic technique, we analyzed the copper deposits electrochemically synthesized when the potentials applied on the electrode surface were –0.075, –0.100, –0.125, –0.150 and –0.175 v. for the case of the transients obtained at –0.075 v (see fig s-1a of the supplementary material to this paper) and –0.100 v (see fig. 5); it is possible to observe the formation of disperse copper clusters on the hopg surface. the afm images reported in these figures were visualized with the software wsxm.39 note that when the applied potential was –0.125 v (fig. s-1b) the number of copper clusters is major in comparison with the nuclei population observed at –0.075 and –0.100 v. a similar behaviour is observed at the –0.150 (fig. s-1c) and –0.175 v (fig. s-1d) applied potentials. thus, at lower potentials the population of nuclei on the electrode surface is bigger. moreover, note that in all cases the copper clusters synthesized are homogeneous and they tuning the copper cluster´s size 1421 are disperse on the electrode surface, which agrees with the concluded from the non-dimensional plots, see fig. 4. last results suggest that it is possible to control the population of copper clusters synthesized on the hopg surface with a simple change on the applied potential on the electrode surface. fig. 5. afm image of the copper clusters electrodeposited on hopg electrode at –0.100 v, from a plating bath containing 0.01 m cu(clo4)2 + 0.02 m naclo4 at ph 5; a) 2d; b) 3d. in order to determine the average cluster’s size of the copper clusters obtained at each potential; we plotted the distribution of cluster’s size as function of their height employing the wsxm software.39 also, the roughness of the hopg substrate in an area of 3.0×3.0 μm2, covered with copper clusters, was measured; resulting 0.35, 0.39, 0.51, 0.83 and 0.89 nm for the deposits obtained at -0.075, -0.100, –0.125, –0.150 and –0.175 v, respectively. note that the roughness becomes higher as the number of nuclei or clusters increases due to the changes on the surface morphology. the average cluster’s size at each case was evaluated considering a gaussian distribution. in fig. 6, it is reported the histo fig. 6. histogram of the height distribution of copper clusters deposited at –0.100 v potential value in an area of 3.0×3.0 μm2, and its corresponding gaussian distribution. also, height cluster distribution is depicted according to the three-sigma rule. 1422 garcia-rodriguez, rios reyes and mendoza huizar gram for the number of copper clusters deposited at –0.100 v in an area of 3.0×3.0 μm2; and its corresponding gaussian distribution, for this potential value, the average cluster’s size is 3.3 nm. a similar analysis was carried out for the other cases, and the average cluster’s size is 3.6, 3.1, 2.4, and 2.3 nm for -0.075, –0.125, –0.150 and –0.175 v, respectively. also, note in fig. 6 that, according to the three-sigma rule, 68 % of the cluster’s size is in the range of 2.06–4.63 nm, 95 % in the range 0.8–5.91 nm and 99.7 % in the range 0–7.20 nm. thus, copper clusters bigger than 7.2 nm are few and their influence is negligible in the gaussian distribution. considering the above mentioned we selected different zones of the afm images reported in figs. 5 and s-1 (see supplementary material), where the copper cluster’s size is into the 95 % of the gaussian distribution, to avoid the influence of the clusters, which are not significant in the analysis. in fig. s-2 of the supplementary material, these zones can be seen, on each image reported in this figure; we carried out a similar analysis to that shown in fig. 6. the results obtained indicates that the average cluster’s size for –0.075, –0.100, –0.125, –0.150 and –0.175 v applied potentials values are 2.84, 2.77, 2.69, 2.65, and 1.98 nm, respectively. the rms (root mean square) associated with these electrodeposits were 0.35, 0.31, 0.27, 0.35 and 0.45 nm. also, to determine the diameter of the copper clusters we used the otsu thresholding method as implemented in the software gwyddion.40 thus, for –0.075, –0.100, –0.125, –0.150 and –0.175 v the average diameters are 14.3, 13.71, 11.41, 11.05 and 11.02 nm, respectively. these results are indicating that the cluster’s size increases as the applied potential is augmented. these results suggest that small copper clusters may be obtained when lower potential values are applied on the electrode surface, while bigger clusters were obtained at more positive potentials than –0.075 v. from fig. s-2, the number of clusters obtained for –0.075, –0.100, –0.125, –0.150, and –0.175 v were 62, 84, 135, 167, and 248 per μm2, respectively. it is clear that the population of nuclei is increased when the applied potential is more negative than –0.075 v. also, observe that this increment follows an exponential tendency with the applied potential, see fig. 7, which may be represented by the following equation: n = 21.956e–0.0138e (3) where n is the number of nuclei per μm2, and e is the applied potential in mv. also, in this work we propose to simulate the qualitative afm images of the copper clusters synthesized, through the number of nuclei and their respective xyz coordinates. thus, it is possible to consider that all nuclei should have similar heigths because under our conditions the copper electrodeposition is instantaneous, see figs. 5, s-1 and s-2. in order to perform the simulation of the afm images, the number of nuclei at each potential were determined employing the eq. tuning the copper cluster´s size 1423 fig. 7. copper cluster’s population in function of the applied potential derived from afm images reported in fig. s-2. (3), and their respective xy coordinates were determined through the “random function” implemented in any spreadsheet, such as exceltm, multiplied by the long and wide of the modelled surface. for example the “x” coordinate is obtained as (“random function”)×(wide), while the “y” coordinate is calculated as (“random function”)×long, where “wide” and “long” correspond to the surface’s dimensions. this approximation is valid because at the initial stages of the electrodeposition process the nuclei formation on the electrode surface follows a random distribution.41 this procedure is repeated as many times as nuclei have been formed or predicted by eq. (3) at a specific potential value. once the xy coordinates of each nuclei are obtained they are plotted in a bidimensional arrengment, then this figure is visualized employing the freeware imagej,31 and the plugin “interactive 3d surface plot” as implemented in this software. fig. 8 shows the simulation and distribution of nuclei on hopg according to procedure above mentioned, on an area of 1 μm2 (wide = 1 μm and long = 1 μm) for the experimental applied potential value of –0.075 v, the simulation for the other applied potentials are reported in fig. s-3. also, a theoretical additional applied potential value (–0.200 v) was analyzed employing the methodology above mentioned. fig. 8. simulation of the distribution of the copper cluster nuclei per μm2, according to eq. (3) at the potential values of –0.075 v considering a random distribution of the nuclei on the hopg surface; a) 2d; b) 3d. 1424 garcia-rodriguez, rios reyes and mendoza huizar note that, according to this simulation, the formation of disperse clusters may be synthesized at higher potential than –0.175 v. from these simulations note that the agglomeration of nuclei is major at –0.175 and –0.200 v, which suggests that the synthesis of disperse copper clusters with nanometric dimensions may be carried out at higher potentials than –0.175 v. these results show a similar tendency to that observed from the experimental afm study, see fig. s-2, suggesting that this methodogy may be employed to simulate the population and distribution of cun on the hopg surface at different applied potential values. conclusion in this work, we employed the potentiostatic technique to synthesize nanometric copper clusters from a plating bath based on perchlorate solutions on a hopg electrode. our results indicate that it is possible to synthesize copper clusters smaller than 3 nm of height and 14.3 nm of diameter from perchlorate solutions, modifying exclusively the applied potential on the electrode surface. from the afm study, it was observed that the population of copper clusters is increased when the applied potential is more negative than –0.075 v, however higher clusters are obtained at more positive potentials. supplementary material additional data are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. lhmh gratefully acknowledges financial support from conacyt (project cb2015-257823) and to the universidad autónoma del estado de hidalgo. guanajuato national laboratory (conacyt 123732) is acknowledged for supercomputing resources. lhmh acknowledges to the sni for the distinction of their membership and the stipend received. и з в о д подешавање величине кластера бакра електрохемијским таложењем на високо оријентисаном пиролитичком графиту. исптивање микроскопијом атомских сила diana elizabeth garcia-rodriguez1, clara hilda rios reyes2 и luis humberto mendoza huizar3 1 comunidad educativa entorno. eugenio garza sada no. 72. los pocitos. aguascalientes, méxicо, 2artes. san felipe 215. providence. mineral de la reforma hidalgo, hgo. méxico и 3universidad autónoma del estado de hidalgo. academic area of chemistry. carretera pachuca-tulancingo km. 4.5 mineral de la reforma, hgo, méxico у раду је приказана синтеза нанометарских кластера бакра висине испод 3 nm и пречника мањег од 14,3 nm електрохемијским таложењем из перхлоратног раствора. на основу броја кластера бакра одређеног микроскопијом атомских сила (afm) изведена је једначина која предвиђа број формираних кластера у зависности од примењеног потенцијала таложења. такође, извршена је квалитативна симулација afm слика коришћењем слободно доступног програма imagej и показано је да су оне у сагласности са експерименталним afm сликама. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice j. serb. chem. soc. 83 (10) 1167–1175 (2018) udc 686.862.6.000.57:665.008.68:631.4: jscs–5141 627.8.034(497.6) original scientific paper 1167 application of alkane biological markers in the assessment of the origin of oil pollutants in the soil and recent river sediments (river vrbas, bosnia and herzegovina) ivan samelak1, milica balaban1*#, nada vidović1#, nemanja koljančić1, mališa antić2, tatjana šolević-knudsen3 and branimir jovančićević4 1university of banja luka, faculty of natural sciences and mathematics, mladena stojanovića 2, 78000 banja luka, bosnia and herzegovina, 2university in belgrade, faculty of agriculture, nemanjina 6, 11080 zemun, serbia, 3university of belgrade, center of chemistry, institute of chemistry, technology and metallurgy, njegoševa 12, 11001 belgrade, serbia and 4university of belgrade, faculty of chemistry, studentski trg 12–16, 11001 belgrade, serbia (received 1 may, revised 11 june, accepted 12 june 2018) abstract: in this paper, an attempt was made to contribute to the use of biological markers, n-alkanes, and polycyclic alkanes of the sterane and terpane type, in the assessment of the source of oil pollutants in the environment using the example of the correlation between recent river and coastal sediments,. four samples of recent river sediments of the vrbas river and four samples of adjacent bank sediments (soils), in the part of the river that belongs to the city of banja luka (bosnia and herzegovina) were analyzed. in the alkane fractions of isolated extracts, a bimodal distribution of the n-alkanes was observed. lower homologues dominated in the recent river sediments with maximum at c15, but higher n-alkanes dominated in the soil samples, with a maximum at n-c29 or at n-c31. the higher concentration of steranes and terpanes with oil type distributions in the recent/fresh river sediments (compared to the bank sediments) represents evidence that lower n-alkanes originate from oil sources. the higher content of total hydrocarbons in the recent river sediments than in the bank sediments further confirms this conclusion. at the same time, these fundamental considerations provide evidence that oil pollution primarily occurred in the water flow and then the oil pollutants propagated towards the riverbank. keywords: oil type pollutant; recent river sediments; riverbank sediments correlation; native and anthropogenic organic substances; biomarkers. * corresponding author. e-mail: milica.balaban@pmf.unibl.org # serbian chemical society member. https://doi.org/10.2298/jsc180501061s ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1168 samelak et al. introduction the amount of native bitumen/oil type organic substances (soluble in organic solvents) in soil and recent sediments is not defined and, theoretically, can be in the range from 0.00 (recent sediments without any organic substance) to up to 100 % (sediment formations containing so-called “surface oil”). within this wide range are the quantities found in different sediments, or, for example, in surface deposits of oil shales or coals of a different degree of carbonification.1,2 for this reason, it is not possible to determine “maximal allowable quantity” for this type of organic pollutant in the soil and in recent sediments. in other words, the amount of organic extract in a particular sample is not a sufficient parameter to determine the presence of an oil type pollutant, because it is not possible to estimate the origin (native or anthropogenic) of the isolated organic substance based only on the amount of extract in a soil or in a recent sediment. previous studies of the origin and fate of oil pollutants in the environment have shown how native and the anthropogenic organic substance can be distinguished. the approach is based on the fact that oil represents a mature organic substance in the geosphere. compared to the organic substance of recent sediments, oil contains a larger amount of carbon isotope 13c. furthermore, the most thermodynamically stabile structural and stereo isomers dominate in the biomarker distributions of alkane and aromatic fractions. in this sense, n-alkanes and isoprenoid aliphatic alkanes, as well as polycyclic alkanes of sterane and terpane type could be advantageous. the use of these biological markers and δ13cpdb for “forensic” purposes, i.e., the distinction of native and anthropogenic organic substance, was demonstrated in previous papers.3–9 moreover, the study of biological markers in oil-type pollution has proved beneficial in terms of assessing its fate in various recent sediment formations, including groundwater and surface waters. by monitoring the change in the abundance and distribution of the alkane and aromatic oil biomarkers under natural conditions, as well as under laboratory-simulated conditions (n-alkanes, isoprenoids, steranes, terpanes, phenanthrenes and their methyl derivatives), the intensity and kinetics of oil biodegradation in the environment,10–13 and the mechanism of its migration through “water wet” sediments were estimated.14 it is well known that processes in the environment are very complex. previous empirical studies set the transformation of oil pollutants into theoretical frameworks well. however, the specific features of each particular environment lead to specific phenomena and specific transformations of the oil, i.e., to the transformation of its constituent compounds. this provides opportunities for establishing new theoretical regularities. in this study, four samples of recent river sediments of the vrbas river, as well as four samples of adjacent bank sediments (soils) in the part of the river flowing through the city of banja luka (bosnia and herzegovina) were analyzed. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ petroleum biological markers in river sediments 1169 based on organic–geochemical knowledge of oil as a form of organic substance in the geosphere, and up-to-date findings of methods for the identification of anthropogenic organic matter of an oil type and its fate in the environment, on the example of the interaction of recent river and bank sediments, an attempt was made to contribute to the use of biological markers, n-alkanes and polycyclic alkanes of the sterane and terpane type, in assessing the source and fate of oil pollutants in the environment. experimental samples the sampling details are given in supplementary material to this paper. separation and instrumental techniques the sediment samples were cleaned from plant and animal tissues and debris, air-dried and then homogenized. the soluble organic matter (extract) was obtained by the soxhlet method during 36 h using chloroform. the content of soluble organic matter in the samples was determined from the ratio of the extract mass (dried to constant mass) and the initial mass of the sample. the following method was employed to separate the extracts into fractions (fig. 1). the extract was first saponified using a potassium hydroxide solution in methanol (5 %). the solution was then neutralized by the addition of hydrochloric acid (10 %). after that, the sample was dissolved in n-hexane and transferred to the top of the adsorption layer placed in the glass column. aluminum oxide and silica gel were used as adsorbents. the saturated hydrocarbon fraction was eluted using hexane, the aromatic fraction using dichloromethane, while the alcohols were eluted using a mixture of dichloromethane and methanol (1:1). the fourth fraction, i.e., fatty acid (in the form of their methyl esters) was eluted with a sulfuric acid solution in methanol (5 %). the solution of the fourth fraction was further treated with aqueous sodium chloride solution (20 %) and n-hexane. fig. 1. schematic representation of the extract fractionation. the saturated hydrocarbons from the extracts were analyzed by gas chromatography– –mass spectrometry (gc–ms). gc–ms was performed using an agilent 7890a gas chroma________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1170 samelak et al. tograph (hp5-ms column, 30 m×0.25 mm, 0.25 μm film thickness, he carrier gas, 1.5 cm3 min-1) coupled to an agilent 5975c mass selective detector (70 ev). the column was heated from 80 to 310 °c, at a rate of 2 °c min-1, and the final temperature of 310 °c was maintained for additional 25 min. detailed analyses of the target compounds were conducted using the following ion chromatograms: m/z 71 (n-alkanes), 217 (steranes) and 191 (terpanes). the individual peaks were identified by comparison with literature data1,15 and based on their mass spectra (library: nist11). results and discussion group parameters the extractable amounts from the examined river sediments and soils, as well as the contents of the total hydrocarbons (i.e., the sum of the saturated and aromatic hydrocarbons) and polar nso compounds in the extracts are given in table i. table i. the content of soluble organic matter (extract) in the investigated samples and content of total hydrocarbons and polar compounds in the extracts sample content of extract, % total hydrocarbons content in extract, %a content of polar compounds in extract, %b rs1 0.31 29.13 46.32 rs2 0.39 11.05 40.23 rs3 0.04 36.48 59.73 rs4 0.06 42.36 54.41 s1 0.09 12.54 83.45 s2 0.05 14.28 65.09 s3 0.19 6.61 59.02 s4 0.06 10.89 85.10 atotal content of saturated and aromatic hydrocarbons; bpolar compounds represent the sum of 3rd and 4th fractions (see fig. 1) the amount of the extracts in the samples rs1 and rs2 was slightly increased (0.31 and 0.39 %, respectively, table i) in comparison to the other samples, which contained extractable amounts in a relatively narrow range of 0.04 to 0.19 % (table i). according to group composition, the extracts of the fresh river sediments were characterized by a slightly higher percentage of hydrocarbons, and a lower content of polar compounds compared to the soil samples (11.05–42.36 % vs. 6.61–14.28 % and 40.23–59.73 % vs. 59.02–83.45 %, respectively, table i). such a ratio of hydrocarbons and polar compounds might be indicative of a higher proportion of oil pollutants in the fresh river sediment samples (rs) compared to the soil samples (s).1,2,15 in the fresh river sediments and in the soil samples, highly polar molecular compounds were present, which were not eluted in the described chromatographic procedure. their percentage content represents ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ petroleum biological markers in river sediments 1171 the difference in the total of hydrocarbon and polar components (table i) up to 100 %. specific parameters the fragmentograms of n-alkanes (m/z 71), steranes (m/z 217) and terpanes (m/z 191) for the samples rs1 and s1, which are typical for all the investigated extracts of the river sediments and soils, are given in fig. s-2 of the supplementary material. in addition, the fragmentograms of the other samples are given in fig. s-3 of the supplementary material. the values of the parameters calculated from n-alkane distributions in the river sediments and soils are given in table ii. table ii. parameters of n-alkane distributions sample n-alkane range the most abundant n-alkane n-c15/n-c29 cpic22-c36 a rs1 c14–c35 c15; c29 5.31 1.74 rs2 c14–c34 c15; c29 2.15 4.13 rs3 c14–c35 c15; c29 3.62 1.52 rs4 c14–c36 c15; c29 8.80 1.21 s1 c15–c36 c16; c31 0.20 4.08 s2 c15–c36 c16; c29 0.25 1.45 s3 c15–c36 c15; c29 0.24 3.48 s4 c14–c38 c16; c31 0.38 1.22 acarbon preference index determined for the distribution of n-alkanes c22–c36, cpic22-c36 = 1/2 [σodd(n-c23 – – n-c35) / σeven(n-c22 – n-c34) + σodd(n-c23 – n-c35) / σeven(n-c24 – n-c36)] the n-alkane distributions of the river sediment extracts are characterized by bimodal distribution with maximums at c15 and c29 (fig. s-2a, table ii). the second part of bimodal distribution of n-alkanes is characterized by the prevalence of homologues with an odd number of c-atoms, whereby the carbon preference index values, cpic24-c36, are in the range of 1.21–4.13. based on previous organic geochemical findings,1,2,15 it could be concluded, with a great degree of reliability, that these n-alkanes belong to the organic substances of terrestrial origin. on the other hand, the first part of chromatogram with maximum at n-c15 could be indicative for native aquatic organic matter. however, these lower normal alkanes could also originate from oil.1,2 the latter suggests that the studied extracts of the river sediments may contain a mixture of native organic substances and anthropogenic organic substances of an oil type. in all samples, n-c15 was considerably more abundant than n-c29 (n-c15/n-c29 is in the range of 2.15–8.80, table ii), and this indicates that the proportion of native aquatic organic matter or anthropogenic organic substances of an oil type is higher than that of a naturally occurring terrestrial organic substances. the identification of representative peaks from sterane and terpane fragmentograms is given in table s-i of the supplementary material. the distribution ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1172 samelak et al. of steranes and terpanes, which is shown in fig. s-2b and c, supports the assumption of the presence of anthropogenic organic matter of the oil type in the investigated samples of fresh river sediments rs1–rs4. these polycyclic hydrocarbons are characterized by structural and stereochemical isomers that are typical for the oil as a mature form of the organic substances in the geosphere.1,2,15 for example, in the fragmentogram of steranes, in addition to the biolipid isomers 14α(h), 17α(h), 20r c27–c29 (peaks 3, 5 and 7; fig. s-2b, table s-i), diasteranes, typical geoisomers, and thermodynamically more stable geolipid sterane isomers having hydrogen atoms in the β-position at c14 and c17 and s-configuration at c20 (for example, peaks 1, 2, 4 and 6; fig. s-2b, table s-i) are present. terpanes were also present in all the investigated samples of fresh river sediments, rs1–rs4, with distributions that are characteristic for oil (fig. s-2c). in the fragmentograms of these samples, the peaks of thermodynamically stable isomers c29 17α(h)21β(h)-hopane and c30 17α(h)21β(h)-hopane (peaks 8 and 9, table s-i) are dominant. among the c31–c35 homohopanes, the more stable 22s isomers dominate in comparison to 22r isomers (peaks 11–14). it could be concluded that the identified steranes and terpanes in the recent river sediments, together with a part of lower n-alkanes (fig. s-2a), originate from anthropogenic organic substances. higher n-alkanes characterized by domination of odd homologues (cpi in the range 1.21–4.13) with a maximum at c29 (the second part of the bimodal distribution; table ii) are native and originate from higher terrestrial plants, n-alkanes in coastal sediments, i.e., soils (s1–s4) also have bimodal distributions with maximums at c15 or c16 and c29 or c31 (m/z 71, fig. s-2d, table ii). the second part of bimodal distribution of nalkanes, similar to the fresh river sediments rs1–rs4, is characterized by distinct domination of homologues with an odd number of c-atoms (cpic24-c36 in the range 1.22–4.08). as has already been mentioned, it could be reliably claimed that these n-alkanes belong to the native organic substances of terrestrial origin. on the other hand, the lower n-alkanes with a maximum at c15 or c16 could also originate from anthropogenic oil sources. thus, similarly to the rs1–rs4 river sediment samples, in the four investigated soil samples from nearby localities, s1–s4, there is a mixture of native organic matter and anthropogenic organic substance of an oil type. however, compared to the recent river sediments, the ratio of lower and higher n-alkanes is different. in the soil samples, higher n-alkanes dominate in comparison to the lower ones. in all four samples, the abundance of n-c29 is considerably higher than that of n-c15 (n-c15/n-c29 is in the range of 0.20–0.38, table ii). therefore, it could be concluded that in this case, the share of native organic matter is significantly greater than the proportion of anthropogenic organic substance. the steranes and terpanes in the fragmentograms corresponding to the s1–s4 samples provide “fingerprints” that are characteristic for the oils (fragmento________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ petroleum biological markers in river sediments 1173 grams of steranes, m/z 217 and terpanes m/z 191 of the s1 sample, typical for all the studied soil samples, are given in fig. s-2e and f).1,2,15 however, the intensity of the peaks is at the level of the electronic “noise” of the instrument, which indicates their extremely low concentrations. these low concentrations of the steraneand terpane-type polycyclic alkanes correspond to the low concentrations of lower n-alkanes in the soil samples s1–s4 (fig. s-2d). the aforementioned values of the parameters, distributions and the relative proportions of n-alkanes, steranes and terpanes in the extracts of the fresh river sediments and the riverbank sediments (i.e., soils) at the site could be indicative of pollution, as well as of the path of the dispersion of oil pollutants in the river system. the higher content of anthropogenic organic substance of an oil type in the recent river sediments compared to the soils (only a few meters away from the water flow) unambiguously suggests that the oil pollution occurred in the water flow. a small quantity of oil was then dispersed from the fresh river to the coastal sediments, i.e., soil. this conclusion does not exclude factories and utility systems located near the sampling sites as potential sources of pollution. conclusions the use of biological markers, n-alkanes and polycyclic alkanes of the sterane and terpane type in the assessment of the source and fate of oil pollutants in the environment has been demonstrated in the study of the fresh river and riverbank sediments of the vrbas river in the part belonging to the city of banja luka (bosnia and herzegovina). based on the slightly higher proportion of hydrocarbons and the smaller content of polar compounds in the extracts of the fresh river sediments, rs1– –rs4, in comparison with the soil samples, s1–s4, it was assumed that a higher amount of oil pollutant was present in the recent river sediments. the n-alkanes in the extracts of the fresh river sediments are characterized by a bimodal distribution with the maximums at c15 and c29. among the higher n-alkanes, the apparent dominance of homologues with an odd number of c-atoms was evident. these n-alkanes belong to the native organic substance of terrestrial origin. the lower homologues with the peak maximum at n-c15, and the identified steranes and terpanes confirm the presence of oil-type pollutants. therefore, the extracts of the recent river sediments contain a mixture of native organic matter and anthropogenic organic matter of an oil type. considering the fact that in these river sediments the lower n-alkanes are more abundant than the higher ones, it was concluded that the proportion of anthropogenic organic substance was greater than the proportion of native organic matter. n-alkanes of the banks sediments, i.e., soils (s1–s4), also had bimodal distributions with maximums at c15 or c16 and c29 or c31. the second part of bimodal distribution of n-alkanes was characterized by a distinct domination of ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1174 samelak et al. homologues with an odd number of c-atoms, presenting strong evidence for native organic substances of terrestrial origin. however, based on the distributions and abundances of n-alkanes, steranes and terpanes, it was concluded, contrary to the fresh river sediments, that native organic matter notably prevailed over the anthropogenic organic substance in these samples. the higher content of anthropogenic organic substance of an oil type in recent river sediments compared to adjacent land (which is a few meters away from the water flow) unambiguously suggested that oil pollution occurred in the water flow. after pollution, the small quantity of oil was probably dispersed from the river to the coastal sediments, i.e., soil. however, this conclusion does not exclude factories and utility systems, located nearby sampling sites, as potential sources of pollution. this study undoubtedly gives a fundamental contribution to the definition of the use of n-alkanes and polycyclic alkanes (steranes and terpanes) in the assessment of the origin and fate of oil type pollutants in river systems. supplementary material additional information and data are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement: this study was supported by the ministry of education, science and technological development of the republic of serbia (project no. 176006). и з в о д нови приступи у примени алканских биомаркера у процени порекла и судбине нафтних полутаната у речним седиментима (река врбас, босна и херцеговина) иван самелак1, милица балабан1, нада видовић1, немања кољанчић1, mалиша антић2, tатјана шолевић кнудсен3 и бранимир јованчићевић4 1универзитет у бањој луци, природно–математички факултет, , младена стојановића 2, 78000 бања лука, босна и херцеговина, 2универзитет у београду, пољопривредни факултет, немањина 6, 11080 београд, 3универзитет у београду, центар за хемију, институт за хемију, технологију и металургију, његошева 12, 11000 београд и 4универзитет у београду, хемијски факултет, студентски трг 12–16, 11001 београд у овом раду је учињен покушај да се на примеру интеракције рецентни речни седимент–приобални седимент да допринос употреби биолошких маркера типа нормалних алкана, и полицикличних алкана типа стерана и терпана у процени извора нафтног полутанта у животној средини. испитивани су свежи речни талози и приобални седименти блиских локалитета реке врбас (по 4 узорка), у делу тока који припада граду бања лука (босна и херецеговина). у алканским фракцијама изолованих екстракта идентификована је бимодална расподела нормалних алкана, с тим што су у свежим речним седиментима доминирали нижи хомолози са максимумом на n-c15, а код приобалних седимената, виши са максимумом на n-c29, односно n-c31. виша концентрација стерана и терпана са нафтним расподелама у рецентним речним седиментима (у поређењу са приобалним седиментима) доказ су да нижи нормални алкани у њима потичу од нафте. већа количина укупних угљоводоника у свежим речним седиментима у односу на приобалне, иде у прилог изнешеном закључку. у исто време ова фундаментална разматрања ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ petroleum biological markers in river sediments 1175 пружају доказ да се нафтно загађење реке одиграло у самом току и да је нафтни полутант расејан у приобаље. (примљено 1. маја, ревидирано 11. јуна, прихваћено 12. јуна 2018) references 1. k. e. peters, c. c. walters, j. m. moldowan, the biomarker guide, vol. 2: biomarkers and isotopes in the petroleum exploration and earth history, cambridge university press, cambridge, 2005, pp. 483, 488, 499, 608, 612, 643, 645, 964 2. j. schwarzbauer, b. jovančićević, fundamentals in organic geochemistry – fossil matter in the geosphere, springer, heidelberg, 2015, pp. 1–158 3. b. jovančićević, lj. tasić, h. wehner, e. faber, n. šušić, p. polić, fresenius envir. bull. 6 (1997) 67 4. m. mićić, b. jovančićević, p. polić, n. šušić, d. marković, fresenius envir. bull. 7 (1998) 648 5. b. jovančićević, p. polić, b. mikašinović, g. scheeder, m. teschner, h. wehner, fresenius envir. bull. 10 (2001) 527 6. d. djurka, p. pfendt, b. jovančićević, o. cvetković, h. wehner, environ. chem. lett. 3 (2005) 39 7. b. jovančićević, m. vrvić, j. schwarzbauer, h. wehner, g. scheeder, d. vitorović, water air soil pollut. 183 (2007) 225 8. s. štrbac, g. gajica, a. šajnović, n. vasić, k. stojanović, b. jovančićević, j. serb. chem. soc. 79 (2014) 597 9. z. milićević, d. marinović, g. gajica, m. kašanin-grubin, v. jovanović, b. jovančićević, j. serb. chem. soc. 82 (2017) 593 10. b. jovančićević, p. polić, d. vitorović, g. scheeder, m. teschner, h. wehner, fresenius envir. bull. 10 (2001) 178 11. m. antić, b. jovančićević, m. ilić, m.m. vrvić, j. schwarzbauer, environ. sci. pollut. res. 13 (2006) 320 12. b. jovančićević, m. antić, i. pavlović, m. vrvić, v. beškoski, a. kronimus, j. schwarzbauer, water air soil pollut. 190 (2008) 299 13. v. beškoski, s. miletić, m. ilić, g. gojgić-cvijović, p. papić, m. marić, t. šolevićknudsen, b. jovančićević, t. nakano, m. vrvić, clean (weinh.) 45 (2017) 1600023 14. b. jovančićević, lj. tasić, s. vujasinović, i. matić, d. malović, p. pfendt, j. serb. chem. soc. 61 (1996) 1023 15. b. p. tissot, d. h. welte, petroleum formation and occurrence, 2nd ed., springerverlag, 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {gas chromatography mass spectrometry system applied to determine botanical origin of various types of edible vegetable oils} j. serb. chem. soc. 84 (9) 1017–1025 (2019) udc 665.32/.33+543.51+543.544.3:633.8:581.6 jscs–5243 original scientific paper 1017 gas chromatography–mass spectrometry system applied to determine botanical origin of various types of edible vegetable oils kristian pastor1*#, vesna vujasinović2, ana marjanović jeromela3, djura vujić1, djordje jovanović4 and marijana ačanski1# 1faculty of technology, university of novi sad, bul. cara lazara 1, 21000 novi sad, serbia, 2the college of hotel management, kneza višeslava 70, 11030 belgrade, serbia, 3institute of field and vegetable crops, maksima gorkog 30, 21000 novi sad, serbia and 4university union nikola tesla, faculty of engineering management, bulevar vojvode mišića 43, belgrade, serbia (received 19 july, revised 10 december, accepted 11 december 2018) abstract: this study represents a new strategy for discrimination of 59 samples of various cold-pressed, virgin and refined edible vegetable oils according to the corresponding botanical origin. samples were produced from 17 plant species: olive, sunflower, safflower, flax, pumpkin, sesame, hemp, walnut, hazelnut, almond, grape, black cumin, apricot, plum, soybean, wheat and rapeseed. a gc/ms device performing in a ion current (ic) mode, combined with multivariate clustering, was employed in the analysis. derivatization reaction occurred in the injector of a gas chromatograph. the discriminations between species were based on marker-peaks of 9 molecular ions of dominant fatty acid methyl esters (fames), which were chosen as descriptors: m/z 268, 270, 292, 294, 296, 298, 324, 326 and 354. dendrogram obtained after performing cluster analysis shows clear discriminations of the analyzed samples, based on the belonging botanical origin. these results demonstrate that ic-gc/ms approach with cluster analysis could be a useful tool in rapid screening for botanical origin of commercial samples of various edible vegetable oils. keywords: authenticity of oils; botanical origin; gc/ms; multivariate cluster analysis. introduction vegetable oils of different types and quality are important from nutritional and economical point of view. they are widely used in homemade cooking and food industry.1,2 virgin olive oil is a big success on the market, but over the last years other vegetable oils that are produced by mechanical extraction without the * corresponding author. e-mail: pastor@tf.uns.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc180719109p ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 1018 pastor et al. use of any solvent emerged and are now available for the consumer, resulting in increased competition for market share.3,4 these oils are a natural source of bioactive compunds, which are proposed to have a wide spectrum of biological and health beneficial effects including anti-inflammatory, anti-oxidative, anticarcinogenic and cholesterol-lowering.5 a report of a joint who/fao expert consultation demonstrates a decreased risk of cardiovascular disease for unsaturated fatty acids, such as linoleic acid, α-linolenic acid, oleic acid, but also plant sterols and stanols.6 higher quality products demand higher market prices, therefore unscrupulous traders may attempt to increase profits by deliberately mislabeling foods, or by increasing the volume of a good quality batch through adulteration with low value ingredients. the substitution or adulteration of food products with a cheap ingredient is not only an economic fraud, but may also have severe health implications to consumers.7–9 edible vegetable oils can be also purposely modified using different methods to enhance their commercial applications, to improve their nutritional quality and to create new specific products at affordable prices. these products are characterized by desired textural, oxidative and nutritional properties, such as changed fatty acid composition and increased bioactive components and natural antioxidants. therefore, in the future, there are many economical and health reasons for the production of new oil blends using new and conventional oil sources to be introduced to the market.10 verifying the description of food in terms of its composition, processing or origin is challenging, but important in protecting consumers and enforcing food law. consumers need clear and accurate information so that they can make informed choices about their diet and the foods they buy.11 the assessment of quality needs to be based on the exploitation of high-tech analytical methodologies. at the present, chromatographic methods are the most popular ones used in routine measurements in monitoring authenticity, adulteration, and in traceability studies of fats and oils. the analysis of fatty acid methyl esters by gc-fid or gc/ms is the simplest and most commonly used technique.7,12 the analysis performed by the new and sophisticated analytical instruments provide a vast amount of data, which is difficult to process. however, developments in computer science have allowed the extensive use of multivariate procedures, in order to efficiently extract the maximum useful information from obtained data.13,14 therefore, the information content of the total fatty acid profile, obtained after gc/ms analysis, can be more efficiently exploited by multivariate data analysis in order to classify oils or determine their authenticity.15 various analytical methods utilizing gc, hplc, ms, nmr, ftir, raman spectroscopy etc., associated with different multivariate techniques, have been extensively used for the characterization and authentication of different types of edible vegetable oils.16–25 the aim of this paper was to demonstrate the potential ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. vegetable oils discrimination with gc/ms 1019 of a new and rapid analytical approach, utilizing a gc/ms device in ic mode combined with a multivariate clustering tool, that will discriminate 59 samples of various edible oils according to belonging botanical origin. the aim was not to discover new or identify known eluting components, but to establish a rapid approach for differentiation based on comparing the influences of selected factors present in and specific for an edible oil sample of each botanical origin. experimental analyzed samples the number of 29 samples of certified cold-pressed (21), virgin (7) and refined (1) edible vegetable oils were purchased from the department of food preservation engineering, faculty of technology novi sad, republic of serbia. the other 30 samples of non-certified cold-pressed oils were obtained from the oil crops department at the institute of field and vegetable crops, novi sad, republic of serbia. these samples were extracted mechanically, by pressing the oilseeds under 200 bar and kept in the nitrogen atmosphere until the analysis. analyzed certified and non-certified edible oil samples, their origin, source and labeling are given in table i. table i. certified and non-certified samples of cold-pressed, virgin and refined edible vegetable oils used in the analysis, their origin, source and labels botanical origin and source oil sample labels certified non-certified olive o1-o5 (virgin) – sunflower (seed) su1 (refined), su2, su3 – safflower (seed) – sf1-sf8 flax (seed) fx1 fx2 fx7 pumpkin (seed) pu1, pu2 pu3, pu4 sesame (seed) se1 (virgin), se2 se3 hemp (seed) he1, he2 – walnut wn1, wn2 – hazelnut hn1 (virgin), hn2 – almond al – grape (seed) gs1, gs2 – black cumin cu1, cu2 – apricot (seed) ap1, ap2 – plum (seed) pl – soybean so – wheat germ wh – rape (seed) – rs1 rs13 sample preparation the volume of 10 µl of each sample was pipetted into a glass vial with a micropipette, and further dissolved by the addition of 1 ml of methylene chloride. the volume of 50 µl of a 0.2 m tmsh derivatization solution (trimethylsulfonium hydroxide, macherey-nagel) in methanol, was added, and each sample was transferred to a gc/ms device. transesterification derivatization procedure occurred in the injection port of a gc/ms device itself, thus con________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 1020 pastor et al. verting the components of oil saponifiable fraction into corresponding volatile fatty acid methyl esters (fames). gc/ms analysis a gas chromatography device (agilent technologies 7890) with mass spectrometric detection (agilent technologies 5975 msd), was employed in the analysis. electron ionization with the energy of 70 ev was applied for the purpose of fragmentation. a db-5 ms column (30 m×0.25 mm×25 µm) was used for separation. the temperature of the injection port was 250 °c, the flow of the carrier gas (helium) 0.8 ml min-1, and the applied temperature program was: 50–130 °c, 30 °c min-1 and 130–300 °c, 10 °c min-1. the volume of 1 µl of each sample was injected with a split ratio of 1:50. samples were analyzed in duplicate. data matrix construction chromatograms were processed using a chemstation program (agilent technologies, palo alto, usa). the retention times/the time position of peaks of the eluting fames were established in total ion current (tic) mode using both nist14 and wiley7 mass spectra libraries, giving the match quality of over 90 %. molecular ions of methyl esters of 9 dominant fatty acids were extracted in ic mode from tic chromatograms of all investigated oil samples. peaks of the selected molecular ions on ic chromatograms with the following m/z ratios were integrated: 268 (cis-9-hexadecenoic acid, methyl ester), 270 (hexadecanoic acid, methyl ester), 292 (cis,cis,cis-9,12,15-octadecatrienoic acid, methyl ester), 294 (cis,cis-9,12 -octadecadienoic acid, methyl ester), 296 (cis-9-octadecenoic acid, methyl ester), 298 (octadecanoic acid, methyl ester), 324 (cis-11-eicosenoic acid, methyl ester), 326 (eicosanoic acid, methyl ester) and 354 (docosanoic acid, methyl ester). mean numerical values of ion peak surface areas were used in further data processing. data processing collected raw numerical data matrix of the ic-gc/ms analysis was firstly variance -scaled, and then subjected to multivariate data analysis. cluster analysis (ca) comprises classification algorithms designed to understand the information of data matrices, to describe similarities and dissimilarities among objects and to single out categories grouping similar objects.26,27 the performances of ca with correlation algorithm were employed in order to classify investigated edible oil samples according to the corresponding botanical origin of plant species used in the oils production. multivariate statistical procedures were carried out using a freely available past 3.21 program (palaeontological association).28 results and discussion marker ions of all lipid compounds eluting on chromatograms of every analyzed oil sample were taken into consideration for discrimination purposes. marker ions present only in oil samples of certain plant species represent a good discriminating factor of these samples. overlaid chromatograms of 9 marker ions of edible oil samples, each thereby representing one specific vegetable oil group, are given in supplementary material to this paper. one oil sample of each plant species was chosen for visualization purposes. the abundances of selected ions are shown on vertical axes and their specific retention times on horizontal axes. chromatograms of plum oils were not presented because they belong to the same ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. vegetable oils discrimination with gc/ms 1021 group like apricot oil, and are therefore grouped together in a dendrogram shown on fig. 1. fig. 1. multivariate clustering of investigated edible vegetable oil samples, discriminated according to botanical origin. labels of cold-pressed, virgin and refined oil samples are indicated in table i. selected marker ions show the similar qualitative pattern, based on the observed retention times, but different amounts of eluting fames in oil samples belonging to different botanical group. however, differences in ionic functions are scarcely visually observed. therefore, a multivariate statistical tool has to be applied in order to visualize the differences between analyzed edible oil samples, taking into account 9 fames, as discriminating variables. the obtained dendrogram of the ratios of 9 discriminating marker ions which are present in 59 investigated edible vegetable oil samples is presented in fig. 1. according to the literature, the number of discriminating variables, i.e. chemical compounds, should be lower than objects, i.e., samples.26 obtained dendrogram suggested that 9 observed fames contributed sufficiently to the separation of 59 edible oil samples into 16 groups: 1) cold-pressed flax seed oils fx1– –fx7; 2) cold-pressed wheat germ oil; 3) cold-pressed rapeseed oils rs1–rs13; 4) virgin olive oils o1–o5; 5) cold-pressed apricot and plum seed oils; 6) refined su1 and cold-pressed sunflower seed oils su2 and su3; 7) cold-pressed safflower seed oils sf1–sf8; 8) cold-pressed soybean oil; 9) cold-pressed hemp seed oils he1 and he2; 10) cold-pressed black cumin oils cu1, cu2; 11) cold-pressed grape seed oils gs1 and gs2; 12) cold-pressed walnut oils wn1 and wn2; 13) cold-pressed almond oil; 14) virgin hn1 and cold-pressed hn2 hazelnut oils; 15) cold-pressed pumpkin seed oils pu1–pu4; 16) virgin se1 and cold-pressed se2 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 1022 pastor et al. and se3 sesame seed oils. the overlap between some edible oil samples, specifically samples of cold-pressed apricot and plum seed oils, might be related to either location, growing season or, even more probable, due to a high level of genetic similarity between varieties of these plants used for cold-pressing in oil extraction. they express a high degree of mutual similarities of fames ratios. the group of oil samples of flaxseed (group 1) is mostly differentiated from all the other samples. the samples of olive oil, apricot seed oil and plum seed oil (groups 4 and 5) express high levels of similarities, as well as the samples of safflower and soybean oil (groups 7 and 8), samples of cumine, grapeseed and walnut oil (groups 10, 11 and 12), samples of almond and hazelnut oil (groups 13 and 14), and samples of pumpkin seed and sesame seed oils (groups 15 and 16). rapeseed oils (group 3) and sunflower seed oils (group 6) show strong mutual groupings and inter-group separations in the obtained dendrogram. there was an established opinion that the ability of an analytical method to characterize a vegetable oil is based on the identification and quantification of minor constituents, such as fatty alcohols, waxes, hydrocarbons, tocopherols and tocotrienols, phenolic compounds, volatiles, pigments and triterpenic acids, that are expected to be in connection with the oil’s origin. this is, however, a difficult task because these groups contain numerous compounds with a wide range of polarities, concentrations and chemical structures. therefore, such approach would require the isolation and analysis of minor constituents by means of several procedures of separation, identification and quantification.30 furthermore, it is possible to “de-sterolize” oils and remove sterols and related minor compounds characteristic to a certain botanical oilseed species. also, levels of tocopherols and tocotrienols can be affected by oil age and refining process.30 this study delivers a rapid, qualitative approach without the analysis of minor constituents, which are present in higher quantities in samples of cold-pressed oils. in contrast, this approach is focused on the characteristic ratios of fatty acid profiles between the analyzed edible vegetable oil samples, as major and mandatory constituents of such products. conclusions an approach which combines the utilization of a ic-gc/ms device with multivariate clustering has been employed to discriminate a wide spectrum of edible vegetable oils, based on relative proportions of 9 dominant fatty acids, as discrimination factors. oil samples were clustered into 16 groups based on corresponding botanical origin, independently of the production procedure. it is important to emphasize that the proposed qualitative approach does not require exact qualitative nor quantitative determinations of eluting fames. in addition, a simple sample processing is required and derivatization occurs directly in an injector of a gas chromatography device. also, using this approach determin________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. vegetable oils discrimination with gc/ms 1023 ations of various minor constituents using other instrumental techniques or/and methodologies is thus avoided. therefore, the proposed approach displays a high potential to rapidly confirm the correctness of the label on expensive and valuable vegetable oil products, and determine possible adulterations in their production process and sale on the market. supplementary material additional data are available electronically from the journal web site: http:// //www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. this research was funded by the ministry of education, science and technological development of the republic of serbia. authors would also like to acknowledge the support from cost action ca16233 and the project of matica srpska „environment and perspectives of quality development and health safety of food in ap vojvodina“. и з в о д примена система гасне хроматографије–масене спектрометрије у одређивању ботаничког порекла различитих јестивих биљних уља кристиан пастор1, весна вујасиновић2, ана марјановић јеромела3, ђура вујић1, ђорђе јовановић4 и маријана ачански1 1технолошки факултет, универзитет у новом саду, булевар цара лазара 1, 21000 нови сад, 2висока хотелијерска школа, кнеза вишеслава 7, 11030 београд, 3институт за ратарство и повртарство нови сад, максима горког 30, 21000 нови сад и 4унион – универзитет никола тесла, факултет за инжењерски менаџмент, булевар војводе мишића 43, 11000 београд ова студија представља нову стратегију разликовања 59 узорака различитих хладно пресованих, девичанских и рафинисаних јестивих биљних уља према одговарајућем ботаничком пореклу. узорци су произведени од 17 биљних врста: маслине, сунцокрета, шафрана, лана, бундеве, сусама, конопље, ораха, лешника, бадема, коштице грожђа, црног кима, коштице кајсије, коштице шљиве, соје, пшеничних клица и уљане репице. у анализи је коришћен gc/ms уређај у режиму јонске струје (ic), уз мултиваријантну кластер анализу добијених података. реакција дериватизације се одиграва директно у инјектору гасног хроматографа. разлике између биљних врста базирају се на маркер-пиковима молекулских јона метил-естара 9 доминантних масних киселина (fames), који су изабрани као дескриптори. ови резултати показују да би ic-gc/ms приступ у комбинацији са кластер анализом могао бити корисно средство у брзом разликовању комерцијалних узорака различитих врста јестивих биљних уља према ботаничком пореклу. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice short communication design, synthesis and antimycobacterial evaluation of some new azaheterocycles with the 4,7-phenanthroline skeleton. part vi j. serb. chem. soc. 81 (2) 133–140 (2016) udc 547.235.2’7+547.677.5:547.639.1’583.1: jscs–4833 615.281/.282 short communication 133 short communication design, synthesis and antimycobacterial evaluation of some new azaheterocycles with the 4,7-phenanthroline skeleton. part vi cristina m. al matarneh1, catalina i. ciobanu2, ionel i. mangalagiu1 and ramona danac1* 1alexandru ioan cuza university of iasi, faculty of chemistry, chemistry department, 11 carol i, iasi 700506, romania and 2alexandru ioan cuza university of iasi, faculty of chemistry, research department, 11 carol i, iasi 700506, romania (received 14 may, revised 31 august, accepted 5 october 2015) abstract: a feasibility study concerning the synthesis, structure and in vitro antimycobacterial evaluation of new 4,7-phenanthroline derivatives is reported. the preparation is straightforward and efficient, involving an n-alkylation reaction of 4,7-phenanthroline. the structure of the new compounds were verified by elemental and spectral (ir, 1hand 13c-nmr) analysis. the in vitro antimycobacterial evaluation of the five synthesized compounds was investigated against mycobacterium tuberculosis h37rv under aerobic conditions. a certain influence of substituents on the para position of the benzoyl moiety was observed; the 4,7-phenanthrolin-4-ium salt substituted with p-chlorobenzoyl group showing the most pronounced antimycobacterial activity. keywords: p-halogenobenzoyl; cycloimmonium salts; antimycobacterial; 4,7-phenanthroline; n-alkylation. introduction phenanthroline derivatives have attracted attention especially due to their biological effects,1–6 crystal engineering,7–11 unique π-electrons delocalization12,13 and complexation properties, especially in the case of 1,10-phenanthroline.14,15 while 1,10-phenathroline derivatives have been widely studied both for synthesis and applications, much less interest has been shown for the other phenanthrolines because of difficulties in their synthesis. however, there are several reports regarding biological properties of 4,7 -phenanthroline and its derivatives, such as inhibition of several enzymes,16–18 microbicide activity especially as an amoebicide19 and antiviral activity.20 more * corresponding author. e-mail: rdanac@uaic.ro doi: 10.2298/jsc150514084a _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 134 al matarneh et al. recently, 4,7-phenanthroline derivatives were found to stabilize triple-helix dna21 and to possess in vitro and in silico antiviral activity against singlestranded positive-sense rna genome viruses.22 as part of ongoing research in the field of heterocyclic compounds, especially in the synthesis of (aza)indolizine and poly(aza)indolizine derivatives via (3+2) cycloaddition of cycloimmonium ylides,23–25 and encouraged by previous promising results in the field of anti-tb derivatives with a nitrogen heterocycle skeleton,26–30 it was decided to study the synthesis, structure and in vitro antimycobacterial activity of new 4,7-phenanthrolin-4-ium salts. experimental chemistry melting points were recorded on an a. krüss optronic melting point meter kspi and are uncorrected. proton and carbon nuclear magnetic resonance (δh, δc) spectra were recorded on a drx-500 bruker (500 mhz) instrument. all chemical shifts are quoted on the δ-scale in ppm. coupling constants are given in hz. ir spectra were recorded on a shimadzu prestige 8400s ftir spectrophotometer. all commercially available products were used without further purification unless otherwise specified. general procedure for the synthesis of 4,7-phenanthrolin-4-ium salts (1–8) 4,7-phenanthroline (28 mmol, 1 equiv.) was dissolved in 6 ml anhydrous acetonitrile. then a reactive halide 30.8 mmol (1.1 equiv.) was added and the resulting mixture was stirred at reflux for 24 h. the formed precipitate was filtered and washed with acetonitrile and diethyl ether to give the desired product. the following compounds were synthesized: 4-(2-oxo-2-p-tolylethyl)-4,7-phenanthrolin4-ium bromide (1), 4-(2-(4-methoxyphenyl)-2-oxoethyl)-4,7-phenanthrolin-4-ium bromide (2), 4-(2-(4-nitrophenyl)-2-oxoethyl)-4,7-phenanthrolin-4-ium bromide (3), 4-(2-(4-chlorophenyl)-2-oxoethyl)-4,7-phenanthrolin-4-ium bromide (4), 4-(2-(3-methoxyphenyl)-2-oxoethyl)-4,7-phenanthrolin-4-ium bromide (5), 4-(2-amino-2-oxoethyl)-4,7-phenanthrolin-4-ium iodide (6), 4-(cyanomethyl)-4,7-phenanthrolin-4-ium bromide (7) and 4-(2-methoxy-2-oxoethyl)-4,7-phenanthrolin-4-ium bromide (8). the physical and spectral data for compounds 1–8 are given in the supplementary material to this paper. microbiology the compounds were evaluated for their antimycobacterial activity against mycobacterium tuberculosis, as a part of the taacf tb screening program under direction of the us national institute of health, the niaid division. the evaluation of the antimycobacterial activities of the compounds were performed at the center of tuberculosis antimicrobial acquisition and coordinating facility (taacf) of the southern research institute.31-34 primary cycle high throughput screening (hts). determination of the 90 % inhibitory concentration (ic90), 50 % inhibitory concentration (ic50) and minimum inhibitory concentration (mic) the mic values of the compounds were determined by measuring the bacterial growth after 5 days in the presence of test compounds. compounds were prepared as 10-point two -fold serial dilutions in dmso and diluted into 7h9-tw-oadc medium in 96-well plates with a final dmso concentration of 2 %. the highest concentration of the compounds was _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis of new 4,7-phenanthroline derivatives 135 200 µm when the compounds were soluble in dmso at 10 mm. for compounds with limited solubility, the highest concentration was 50× less than the stock concentration, e.g., 100 µm for 5 mm dmso stock and 20 µm for 1 mm dmso stock. for potent compounds, the assays were repeated at lower starting concentrations. each plate included assay controls for background (medium/dmso only, no bacterial cells), zero growth (100 µm rifampicin) and maximum growth (dmso only), as well as a rifampicin dose response curve. the plates were inoculated with m. tuberculosis and incubated for 5 days: growth was measured by the optical density at 590 nm (od590) and fluorescence (ex. 560 nm/em. 590 nm) using a biotek™ synergy 4 plate reader. growth was calculated separately for od590 and relative fluorescence units (rfu). to calculate the mic, the 10-point dose response curve was plotted as % growth and fitted to the gompertz model using graphpad prism 5. the mic was defined as the minimum concentration at which growth was completely inhibited and was calculated the gompertz model from the inflection point of the fitted curve to the lower asymptote (zero growth, fig. 1a). in addition, dose response curves were generated using the levenberg– –marquardt algorithm and the concentrations that resulted in 50 and 90 % inhibition of growth were determined (ic50 and ic90, respectively, fig. 1b). fig. 1. dose response curves used to calculate the mic, ic50 and ic90. mic values were reported when the following quality control criteria were satisfied: – for each plate: no growth in the background (un-inoculated) control wells; od590 >0.3 in maximum growth wells; rifampicin mic within 3-fold of the expected value. – for each compound curve, the mics were reported if there were 2 points with growth >75 %. if only one point was >75 % inhibition, then the mic value was reported as the maximum concentration tested. if no point reached 75 % inhibition, the mic was reported as >maximum concentration tested. results and discussion chemistry according with the goal of this study, it was decided to synthesize new phenanthroline derivatives: 4,7-phenanthrolinium monoquaternary salts 1–8 and 4,7-phenanthrolinium diquaternary salts 9–16. the strategies adopted for the con_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 136 al matarneh et al. struction of the phenanthroline derivatives was straightforward and efficient, involving an n-alkylation reaction of 4,7-phenanthroline, scheme 1. scheme 1. reaction pathway to obtain the 4,7-phenanthrolin-4-ium salts. unfortunately, it was not possible to synthesize compounds 9–16, under any of the employed conditions (ambient temperature or refluxing, content of 4,7 -phenanthroline. thus, on varying the amount of the reactive halides from 1:2 to 1:7, and using different solvents, i.e,. acetone, acetonitrile and dimethylformamide, only 4,7-phenanthrolin-4-ium mono salts 1–8 were obtained. as in the case of 1,7-phenanthroline,26 a feasible explanation for this behavior could be related to the basicity of the n7-nitrogen atom, i.e., after the n4-alkylation of 4,7-phenanthroline, the basicity of the n7-nitrogen is decreased in the obtained phenanthrolin-4-ium mono salts so that the second alkylation could not occur. the salts 1–8 were prepared in moderate to good yields (58–94 %), using a minimum volume of acetonitrile by refluxing the reaction mixture for 24 h. the structure of the new compounds was assigned by elemental and spectroscopic analysis: ir, 1hand 13c-nmr. the ir spectra of compounds 1–6 and 8 are characterized by intense absorption bands in the region of 1672–1698 cm–1 specific to c=o stretching, whereas in the ir spectrum of compound 7 the cyano group furnishes an absorption band at 2201 cm–1. in the 1h-nmr spectra of the new monoquaternary salts 1–5, the signals for methylene protons h15 appear at low fields (7.07–7.20 ppm, singlet), according to the substituent on the para or meta position of the benzoyl ring. the same protons appear more shielded at 5.27, 6.58 and 6.35 ppm for compounds 6–8, respectively, due to the weaker withdrawing effect of the adjacent carbonyl amide, cyano and ester groups. in the aromatic region, the most unshielded protons are h3 (the signals of which appear at 10.22–10.33 ppm) situated in the proximity of the positive nitrogen atom n4. in the 1h-nmr spectra of compound 6, the two-amide protons (nh2) furnished two singlet signals at 7.89 and 8.21 ppm, whereas in the spectrum of compound 8 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis of new 4,7-phenanthroline derivatives 137 the methyl ester protons appear as a singlet at 3.81 ppm. in the 13c-nmr spectra of compounds 1–5, the signals for c16 from the c=o ketone groups appear at 190.6–188.8 ppm, while for compound 6, having an amide group, and for compound 8 with an ester group, it appears at 165.8 and 166.6 ppm, respectively. the methylene c15 atoms give signals at 63.7–64.3 ppm for compounds 1–5 and at 45.6–59.5 ppm for compounds 6–8. all the other signals from nmr spectra are in agreement with the proposed structures. design and biological activity in the continuing battle against m. tuberculosis, researchers have found that the 1,10-phenanthroline skeleton and a p-halogenobenzoyl moiety are useful pharmacophoric units for the antimycobacterial activity.35,36 moreover, recent results26 in the area of 1,7-phenanthroline salts indicate that derivatives containing a p-substituted benzoyl moiety exhibit activity against m. tuberculosis h37rv, the relative order of activity being p-cl> p-br> p-methyl-. encouraged by these promising results in the field of anti-tb derivatives with the phenanthroline skeleton and, especially by recent results in the area of 1,7-phenanthroline salts which contain a p-substituted-benzoyl moiety, it was decided to combine the biological potentials of 4,7-phenanthroline and the p-substituted benzoyl moiety, with the intention of obtaining compounds with better activity and better pharmacological properties, and to determine whether changing the nitrogen atoms position of phenanthroline from 1,7to 4,7would somehow affect the activity, scheme 2. also in view were other 4,7-phenanthrolinium salts with the alkoxycarbonyl, cyano and acetamide moiety, in order to allow structure–activity relationship (sar) comparisons with p-substituted benzoyl salts. scheme 2. design in the class of 4,7-phenanthrolin-4-ium derivatives with p-halogeno benzoyl moiety. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 138 al matarneh et al. a selection of compounds, salts 1–4 and 6, were evaluated for in vitro antimycobacterial activity against m. tuberculosis h37rv (grown under aerobic conditions), as a part of ongoing collaboration with the taacf tb screening program under direction of the us national institute of health, the niaid division. the mic value were determined by measuring bacterial growth after 5 days in the presence of the test compounds.30–34 the assay was based on measurement of the growth in liquid medium of a fluorescent reporter strain of h37rv where the readout was either optical density (od) or fluorescence. a linear relationship between the od and fluorescence readout was established, justifying the use of fluorescence as a measure of bacterial growth. the mic values generated from the od measurements are reported in the summary data. the mic was defined as the minimum concentration at which growth was completely inhibited and was calculated from the inflection point of the fitted curve to the lower asymptote (zero growth, fig. 1a). in addition, dose response curves were generated using the levenberg–marquardt algorithm and the concentrations that resulted in 50 and 90 % inhibition of growth were determined (ic50 and ic90, respectively, fig. 1b). the strain has been fully characterized and is equivalent to the parental strain in microbiological phenotypes and virulence. the obtained results are listed in table i. table i. antimycobacterial activity of phenanthrolinium salts 1–4 and 6 against m. tuberculosis h37rv under aerobic conditions tested compound ic50 / μm ic90 / μm mic / μm 1 (r=c6h4–mep) 110 >200 >200 2 (r=c6h4–omep) >200 >200 >200 3 (r=c6h4–no2p) >200 >200 >200 4 (r=c6h4–clp) 83 >200 >200 6 (r=nh2) >200 >200 >200 rifampicin 0.0036 0.0061 0.0055 although the results are not spectacular, the data from table i illustrate that two of the five tested compounds exhibited activity against m. tuberculosis h37rv: salt 4 (substituted with p-chlorobenzoyl moiety) and salt 1 (substituted with p-methylbenzoyl moiety), the first one having a more pronounced antimycobacterial activity. by comparison with similar 1,7-phenanthrolin-7-ium monoquaternary salts it could be seen that the heterocycle has only a minor influence of on antimycobacterial activity, the ic50 values of the derivatives (1,7 or 4,7 -phenanthroline) substituted with the same substituents being similar, e.g., for 7-(2-(4-chlorophenyl)-2-oxoethyl)-1,7-phenthrolin-7-ium bromide, ic50 = 88 μm26, while for 7-(2-oxo-2-p-tolylethyl)-1,7-phenanthrolin-7-ium bromide, ic50 = = 100 μm.26 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis of new 4,7-phenanthroline derivatives 139 conclusions the synthesis, structure and in vitro antimycobacterial activity of a new class of 4,7-phenanthrolin-4-ium monoquaternary halides are presented. the compounds were prepared by a straightforward and efficient method. the structure of the new compounds was assigned by elemental and spectroscopic analysis: ir, 1h-nmr and 13c-nmr. the in vitro antimycobacterial activity of the synthesized compounds was investigated against m. tuberculosis h37rv under aerobic conditions. two of the five tested compounds showed activity against m. tuberculosis h37rv, the 4,7-phenanthrolin-7-ium derivative substituted with p-chloro-benzoyl moiety being the most active. supplementary material the physical and spectral data for the prepared compounds are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. authors c.m. al matarneh and c.i. ciobanu are thankful to posdru/159/1.5/s/137750 for financial support. part of this work (biological tests) was supported by national institutes of health and the national institute of allergy and infectious diseases, contract no. hhsn272201100012i. we also thank the poscce-o 2.2.1, smis -csnr 13984-901, no. 257/28.09.2010 project, cernesim, for the nmr results. и з в о д дизајн, синтеза и испитивање антимикобактеријске активности нових азахетероцикла са 4,7-фенантролинским скелетом. 6. део cristina m. al matarneh1, catalina i. ciobanu2, ionel i. mangalagiu1 и ramona danac1 1alexandru ioan cuza university of iasi, faculty of chemistry, chemistry department, 11 carol i, iasi 700506, romania и 2 alexandru ioan cuza university of iasi, faculty of chemistry, research department, 11 carol i, iasi 700506, romania приказано је истраживање у ком је описана синтеза, одређивање структуре и in vitro испитивање антимикобактеријске активности нових деривата 4,7-фенантролина. синтеза деривата је директна и ефикасна и укључује реакције n-алкиловања 4,7-фенантролина. структура добијених деривата утврђена је елементалном анализом и спектроскопским методама (ir, 1hи 13c-nmr). антимикобактеријска in vitro активност пет синтетисаних једињења испитана је према mycobacterium tuberculosis h37rv под аеробним условима. утврђено је да постоји утицај супституената у пара положају бензоил језгра и да 4,7-фенантролин-4-ијум со са p-хлорбензоил групом показује веома изражену антимикобактеријску активност. 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"al.i.cuza" iasi 11 (2003) 367 [ca 142 (2004) 56249] 36. h. r. dholariya, k. s. patel, j. c. patel, k. d. patel, spectrochim. acta, a 108 (2013) 31. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {removal of lithium from water by aminomethylphosphonic acid containing resin} j. serb. chem. soc. 83 (9) 1059–1069 (2018) udc 628.316.12+546.34:665.941:544.142.3: jscs–5133 544.726 original scientific paper 1059 removal of lithium from water by aminomethylphosphonic acid-containing resin aydin çiçek, onur yilmaz and özgür arar* ege university, faculty of science, department of chemistry, 35100, izmir turkey (received 30 september 2017, revised 1 january, accepted 22 january 2018) abstract: this paper gives an overview of the ability of an aminomethylphosphonic acid-containing chelating resin for the removal of lithium from water. the studies were performed under various conditions, such as resin dose, initial li+ concentration, solution ph and solution temperature. the results showed that the sorption of li+ reached equilibrium within 15 min and the experimental data were well-fitted by the pseudo-second-order kinetic model. the li+ sorption was highly ph dependent, and the optimum ph for li+ removal was ≥3. isotherm sorption data displayed good correlation with the langmuir model, and the maximum monolayer sorption capacity of the resin found to be 13.65 mg g-1. thermodynamic studies suggested that li+ sorption onto the chelating resin was an exothermic and spontaneous process in nature. the resin could be regenerated by 0.1 m hcl, nacl or h2so4 with > 99 % efficiency. desorption of li+ with 0.1 m nacl resulted in no changes in the uptake capacity through four sequential sorption/desorption cycles. keywords: chelating resin; ion exchange; lithium; lewatit tp 260. introduction lithium is the 25th most abundant element (at 20 mg kg–1) in the earth’s crust.1 the major lithium minerals with commercial value are classified into three major groups, namely, silicates, micas, and phosphates.2 lithium finds application in rechargeable lithium ion batteries (libs) because of its very high energy density by weight and high electrochemical potential (3.045 v).1 besides batteries, lithium compounds are used in ceramics and special glass industries, in primary aluminum production, rocket propellants, nuclear and pharmaceutical industries, in the manufacture of lubricants and greases, synthesis of vitamin a, synthesis of organic compounds, silver solders, underwater buoyancy devices, and batteries. lithium is alloyed with aluminum and magnesium as light metals to form stronger and lightweight alloys.3 it is recovered from mines and salt * corresponding author. e-mails: ozgur.arar@ege.edu.tr, ozgurarar@gmail.com https://doi.org/10.2298/jsc170930020c ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 1060 çiçek, yilmaz and arar lakes, which contain about 17 million t of lithium in total, while seawater is also considered as a vast source of lithium (about 2.5×1014 kg), although the concentration of lithium is very low, i.e., 0.17 mg l–1.4 various methods have been studied for the recovery of lithium from seawater, brine, and geothermal water. these can be classified into three groups: adsorption (ion-exchange), solvent extraction and co-precipitation.5 the efficiency and limits of such methods have been reviewed elsewhere.3 problems in separation or in the concentration of trace constituents are sometimes encountered. ion exchange resins with different cation-exchange groups may possess different selectivity coefficients. among the factors that determine the ionic selectivity of resins, the most important ones are: a) the nature of the acidic functional group and b) the density of the structure, which is largely determined by the degree of cross linkage. regarding the first point, it is characteristic that the affinity of alkali ions toward strongly acidic groups (sulfonic acid groups, etc.) decreases in the order k >> na > li, whereas the order is precisely the opposite on resins with carboxylic, phosphonous, and phosphonic acids (especially the latter) as the functional groups.6,7 the purpose of this study was to evaluate an aminomethylphosphonic acid -containing chelating resin for li+ sorption. the sorption process was optimized by varying different parameters, such as resin dose, initial solution ph, concentration of li+ and temperature. elution of li+ from the resin was also examined. experimental materials lewatit tp 260 resin was used in the experiments. it is a weak acidic, macroporous resin contains chelating aminomethylphosphonic acid groups. the properties of resin are listed in table i.8 the resin was converted into the sodium form by treatment (100 ml wet resin) with 2 m nacl (250 ml) solution for 24 h and then washing thoroughly with water. it was dried in an oven and used in the experiments. table i. physicochemical properties of lewatit tp 260 matrix cross-linked polystyrene functional group aminomethylphosphonic acid ionic form na+ total capacity (h-form) 2.3 eq. l-1 water retention 59–61 % stable in ph range 0–14 reagents analytical grade reagents were used in the experimental studies. a stock solution of li+ was prepared by dissolving appropriate quantities of li2co3 (merck) salt. the weighed amount of li2co3 was transferred into a beaker containing pure water and then hcl was added for complete dissolution. the solution was heated and swirled to expel dissolved co2. the prepared solution was cooled to room temperature then transferred to a volumetric flask and diluted to volume with water. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. removal of lithium from water 1061 the ph of the solutions used in the batch test was adjusted to its optimum value by the addition of sufficient amounts of 0.1 m hcl and 0.1 m naoh solutions. lithium analysis the concentration of li+ was determined using a flame photometer (jenway pfp7). the concentration ranges of li+ standards for the calibration curve were in the range of 0.1 to 5 mg l-1. high concentration of li+ (which was used in the isotherm study) was measured after appropriate dilutions. batch adsorption tests experimental conditions for li+ sorption were summarized in table ii. table ii. experimental parameters for li+ removal investigated parameter li+ concentration mg l-1 amount of resin used, g volume of solution, ml ph of solution temperature °c resin dose 5.0 0.02–0.5 25 6 25 ph of solution 5.0 0.3 25 1–6 25 isotherm study from 25 to 300 0.3 25 6 25 temperature effect 5.0 0.3 25 6 30,40, 50, 55 and 60 kinetic study 5.0 12 1000 6 25 the removal efficiency (r) and capacity (q) of the resin were calculated according to eqs. (1) and (2), respectively: 0 e 0 100 c c r c − = (1) 0 e c c q v m − = (2) where, c0 and ce (mg l-1) are the initial and equilibrium li+ concentrations, respectively, v is the volume of solution (l) and m is the mass of the resin (g). results and discussions effect of resin dose the effect of resin dosage on the uptake of li+ was studied to understand the efficacy of the resin for li+ removal. the uptake of li+ plotted as percent removal vs. resin dose is illustrate in fig. 1, from which it could be seen that on increasing the resin dose from 0.02 to 0.3 g per 25 ml, the removal efficiency improved from 53 to 98 %. this could be attributed to an increase in the availability of more sorption sites as the dose of resin was increased.9 the optimum resin dose was found to be 0.3 g for 25 ml of solution and this amount was used in further experiments. effect of solution ph the solution ph is a one of the important factor in sorption studies, because it can influence the structure of resin (for weak acidic and weak base), the struc________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 1062 çiçek, yilmaz and arar ture of target molecule (especially if it has weak acidic or weak basic character) and h+ or oh– act as competing ions in the ion exchange process. the effect of initial solution ph values (1–6) on li+ ion sorption onto tp 260 resin was investigated, and the results are shown in fig. 2. li+ removal was unfavorable in acidic media (ph ≤ 1) but it increased with increasing ph value. there was only 4 % removal at ph 1 that increased to 99 % at ph ≥ 3. the pka values of aminomethylphosphonic acid (ampa) are 2.35 and 5.9.10 at ph 1, the functional group of the resin is in the molecular form, thus removal of li+ was not possible. on the other hand, when the ph of solution was increased, the functional group of the resin ionized and sorption of li+ was enabled. fig. 1. effect of resin dose on li+ removal. fig. 2. effect of initial solution ph on li+ removal. isotherm analysis of li+ removal the sorption isotherm is one of the important parameters for understanding sorption behavior and mechanism. the sorption capacity of the resin for li+ removal was studied at different initial li+ concentrations and the obtained results were applied to the langmuir and freundlich models. the langmuir isotherm model is used to describe a monolayer adsorption process, and the model can be described in linear form by eq. (3): ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. removal of lithium from water 1063 ce/qe = 1/bq0 + ce/q0 (3) in this equation, qe / mg g–1 is the amount of li+ sorbed per gram of dry resin at equilibrium, q0 / mg g–1, and b / l mg–1 are the langmuir constants related to the capacity and energy of sorption, respectively.11,12 the freundlich isotherm model shows that a multilayer of the adsorption process occurs on heterogeneous surfaces, and is expressed by eq. (4): log qe = log kf + (1/n)log ce (4) where kf / dm3 g–1 is the isotherm constant of the freundlich model and n is the exponent of the freundlich model. kf and n are characteristics of the system and are indicators of the sorbent capacity (or affinity for the solute) and sorption intensity, respectively.13 the related parameters of the two models were calculated and are summarized in table iii. from the values of the linear correlation coefficients (r2), the langmuir model was found more suitable for describing the li+ sorption than the freundlich model. the results suggest that monolayer sorption of li+ on such resin is the main mechanism. additionally, the values of q0 calculated from the langmuir model was 13.65 mg g–1. table iii. calculated isotherm constants of langmuir and freundlich models langmuir isotherm constants freundlich isotherm constants q0 /mg g-1 b / l g-1 r2 kf / dm3 g-1 n r2 13.6553 0.0370 0.9914 2.4526 3.4710 0.9577 dubinin–radushkevich model the dubinin–radushkevich (d–r) model is another model that is used for clarification of the mechanism of sorption (i.e., physical or chemical). the linear d–r equation is given by eq. (5): ln qe = ln xm – βε2 (5) where β is a constant related to the sorption energy (mol2 j–2), xm (mol g–1) is the d–r monolayer capacity, ε (mol l–1) is the polanyi potential that is calculated as shown in eq. (6): ε = rtln (ce+1/ce) (6) the mean free energy, e (kj mol–1), of sorption can be estimated by using β values as expressed in the following equations:14 e = 1/(β1/2) (7) for values of e < 8 kj mol–1, physical forces may have an effect on the sorption mechanism while e values between 8 and 16 kj mol–1 depict sorption controlled by ion exchange and e > 16 kj mol–1 signifies the process is chemisorption.15 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 1064 çiçek, yilmaz and arar in the present case, xm was found to be 0.0027 mol g–1, which is equal to 18.74 mg li+ per g-resin, β was found to be 0.0050 mol2 j–2, and mean free energy, e, was 14.1 kj mol–1, indicating that removal mechanism was ion exchange. the ion-exchange reaction of the resin with li+ can be expressed as:16 2 2 2 2r ch po(ona) 2li r ch po(oli) 2na+ +⎯⎯→− + − +←⎯⎯ (8) kinetics of li+ removal ion exchange time-dependent experiments were performed to evaluate the sorption kinetics. the kinetic data shown in fig. 3 indicate that the sorption of li+ increased rapidly within 5 min, followed by a relatively slow process, and then the sorption equilibrium was achieved within 15 min. in addition, no remarkable changes were observed from 15 to 45 min. fig. 3. removal of li+ versus time. reaction-based model the kinetic data were analyzed using pseudo-first-order, and pseudo-second -order models. the equations for these models are given as eqs. (9) and (10):17,18 log (qe–qt) = log qe – k1t/2.303 (9) t/qt = 1/k2qe2 + t/qe (10) experimental results were applied to the kinetic models and the results are summarized in table iv, from which it could be seen that the correlation coefficient (r2) obtained from the pseudo-second-order model was larger than that from the pseudo-first-order model. diffusion-based model the intraparticle diffusion model proposed by weber and morris in 1963 was also taken into account in the experiments. they concluded that the sorption is proportional to the square root of the contact time:19 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. removal of lithium from water 1065 qt =kidt1/2 (11) where kid is the intraparticle diffusion rate constant (mg g–1 min–0.5). when the intraparticle diffusion model controls the sorption, the graph of qt against t0.5 should be a straight line passing through the origin. the rate constant could be calculated from the slope of the line, the liquid film diffusion equation is given as: –ln (1–f) = kfdt (12) where f is the fractional attainment of equilibrium f  =  (qt/qe), kfd (1/min) is the rate constant. a linear plot of −ln (1 − f) vs. t with zero intercept would suggest that the adsorption process was controlled by liquid film diffusion.19 the experimental data were fitted in eqs. (9)–(12) and the obtained results are summarized in table iv. the r2 revealed that the retention process is the film diffusion controlled mechanism. table iv. the calculated parameters of pseudo first and pseudo second order kinetic model kinetic model parameter calculated value pseudo first order k1 / min-1 0.4443 qe / mg g-1 0.782 r2 0.2594 pseudo second order k2 / g mg-1 min-1 17.056 qe / mg g-1 0.6085 h / mg g-1min-1 0.6314 r2 0.9999 intraparticle diffusion model r2 0.9756 kid 0.1998 diffusion model r2 0.9890 kfd 0.4083 thermodynamic parameters the effect of the temperature on the removal of li+ from water was examined by changing the solution temperature from 30 to 60 °c under the optimized conditions of resin dose and solution ph. changes in the free energy (δg), entropy (δs) and enthalpy (δh) were estimated by the usual procedure.20 the calculated values are summarized in table v. table v. thermodynamic parameters for li+ sorption δs / j mol-1 k-1 δh / kj mol-1 δg / kj mol-1 (t / k) 10 –8.16 –12.1 (303) –12.2 (313) –12.4 (323) –12.4 (328) –12.5 (333) ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 1066 çiçek, yilmaz and arar the negative values of δg shows that ion exchange reaction is spontaneous. the negative value of δh suggests the exothermic nature of the sorption. the positive value of δs suggests increased randomness at the solid/solution interface during the sorption of li+ onto the resin. regeneration of the resin regeneration experiments were performed as explained in the literature.21 recovery of li+ from the resin was checked with hcl, h2so4 and nacl solutions at various concentrations. the regeneration efficiency (re / %) was calculated using eq. (13): desorbed amount of li from the resin (%) 100 sorbed amount of li onto the resin re + + = (13) the obtained results are summarized in table vi, from which it could be seen that 0.1 mol dm–3 hcl, h2so4 or nacl was enough for complete regeneration of the resin. table vi. effect of acid concentration on the desorption of li+ from the resin regenerant concentration, mol dm-3 desorbed amount of li+, % hcl 0.01 75 0.05 88 0.1 >99 0.5 >99 h2so4 0.01 85 0.05 96 0.1 >99 0.5 >99 nacl 0.01 75 0.05 95 0.1 >99 0.5 >99 multiple sorption/regeneration tests multiple sorption/regeneration tests were performed to determine the reusability of the ion exchange resin and the recovery of sorbed li+. the sorption test was realized by contacting 0.3 g resin with 25 ml of li+ solution (5 mg li l–1, ph 6) for 2 h. after decantation of the solution, the resin was washed with pure water until the conductivity of the eluting water reached that of pure water conductivity value. the elution step was performed by contacting 0.1 mol dm–3 25 ml of regenerant solution with the washed resin for 2 h. at the end of this time, the solution was decanted and washed with pure water as explained above. this sorption/regeneration cycle was repeated 4 times and the obtained results are summarized in table vii. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. removal of lithium from water 1067 table vii. evaluation of sorption/regeneration cycle studies for li+ removal, %, from water by lewatit tp 260 resin cycle number hcl nacl 1 99 99 2 54 99 3 53 99 4 53 99 as can be seen from table vii, the type of the regenerant influences the sorption capacity. when nacl was used for regeneration, the sorption capacity of the resin did not change but when hcl was used, sorption capacity decreased. after regeneration with hcl, the resin was converted to the h-form and used in the next sorption cycle. in the second sorption process, li+ replaces h+ and thus, the h+ concentration in the solution increased and the ph of the solution decreased. as explained in effect of solution ph section, when the ph of solution decreased and the sorption capacity of resin decreased. previously in the literature, various research studies were conducted for lithium removal/recovery. the results obtained in such research studies are summarized in table viii. the capacities of the sorbents vary from 4.07 to 62.5 mg g–1 sorbent. the removal performance of such materials strongly depended on the experimental conditions, particle size, and loaded metal. table viii. capacities of sorbents used for li+ removal/recovery resin/sorbent capacity, mg g-1 reference manganese oxide adsorbent (h1.6mn1.6o4) 40 22 magnetic lithium ion-imprinted polymer 4.07 23 mno2 ion sieve 62.5 24 nano-mno2 28.2 25 ion-sieve manganese oxide, hmno(mg) 8.5 5 spinel lithium manganese oxide 27.62 26 λ-mno2 31.55 27 aminomethylphosphonic acid containing chelating resin 13.65 this study conclusions in this study, the removal of lithium ions from aqueous solutions onto an ion-exchange resin was investigated under various experimental conditions, such as resin dose, initial solution ph, and temperature. the results clearly demonstrated that the phosphonic acid groups contributed to the sorption mechanism through electrostatic interactions between the phosphonic acid of the resin and the li+. the ion exchange process was quite fast and equilibrium was established within 15 min. the percentage removal of li+ was ph dependent, decreasing with decreasing initial ph of the solution. the equilibrium sorption behavior li+ onto lewatit tp 260 resin followed the langmuir adsorption isotherm with a ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 1068 çiçek, yilmaz and arar maximum theoretical sorption capacity of 13.65 mg g–1 resin. the sorption of li+ onto the resin was found to be mainly based on ion-exchange interactions, and these were confirmed by the dubinin–radushkevich model. regeneration and reuse of resin work well for nacl regenerant. regeneration of resin with acid solution decreased the removal efficiency of resin. temperature variations were used to evaluate enthalpy, entropy and free energy changes. the negative value of free energy change showed the spontaneous nature of the adsorption. in the temperature range 303–333 k, enthalpy change was negative, and the ion exchange reaction was exothermic. acknowledgments. we would like to acknowledge lanxess–lewatit and their distributor in turkey, ökotek environmental technologies and chemical industries, for providing the resin sample. we thank the mr. emre seyyal and mr. ekrem kandemir for their kind suggestions to improve this paper. и з в о д уклањање литијума из воде смолом која садржи аминометилфосфонску киселину aydin çiçek, onur yilmaz и özgür arar ege university, faculty of science, department of chemistry, 35100, izmir turkey ова студија даје увид у способност аминометилфосфонске киселине која садржи хелатну смолу да уклони литијум из воде. испитивања су изведена под различитим условима као што су количина смоле, почетна концентрација li+, ph раствора и његова температура. резултати су показали да сорпција литијума достиже равнотежу за 15 min, а експериментални подаци су добро корелисани кинетиком псеудо-другог реда. сорпција li+ је показала велику зависност од ph, а оптимална ph за уклањање li+ била је изнад 3. подаци за изотермну сорпцију показали су добру корелацију са лeнгмировим моделом, а максимални капацитет монослојне сорпције у смолу је био 13,65 mg/g. термодинамичка испитивања су указала на егзотермност односно спонтаност процеса ове сорпције. смола се може регенерисати из 0,1 m hcl, nacl или h2so4 са ефикасношћу преко 99 %. десорпција li+ у 0,1 m nacl није дала никакве промене у капацитету преузимања у четири узастопна сорпциона/десорпциона циклуса. (примљено 30. септембра 2017, ревидирано 1. јануара, прихваћено 22. јануара 2018) references 1. p. meshram, b. d. pandey, t. r. mankhand, hydrometallurgy 150 (2014) 192 2. h. aral, a. vecchio-sadus, ecotoxicol. environ. saf. 70 (2008) 349 3. b. swain, sep. purif. technol. 172 (2017) 388 4. l. wang, c. g. meng, w. ma, colloids surfaces, a 334 (2009) 34 5. y. miyai, k. ooi, s. katoh, sep. sci. technol. 23 (1988) 179 6. h. h. ussing, p. kruhoffer, h. j. thaysen, n. h. thorn, the alkali metal ions in biology: i. the alkali metal ions in isolated systems and tissues, springer, berlin, 2013 7. w. g. berl, physical methods in chemical analysis, elsevier, amsterdam, 1961 8. g. coşkun, i̇. şimşek, ö. arar, ü. yüksel, m. yüksel, desalin. water treat. 57 (2016) 25739 9. e. özbunar, s. kırca, ö. arar, ü. yüksel, anal. lett. 50 (2017) 1657 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {hydrogen conversion using gasification of tea factory wastes} j. serb. chem. soc. 85 (7) 967–977 (2020) udc 66.026+546.11+57.018.8: jscs–5352 661.566.097.3:502/504 original scientific paper 967 hydrogen conversion using gasification of tea factory wastes ali akyüz1, zuhal akyurek2, muhammad y. naz3, shaharin a. sulaiman4 and afsin gungor5,6* 1bucak emin gulmez v. s., burdur mehmet akif ersoy university, turkey, 2department of polymer engineering, faculty of engineering and architecture, burdur mehmet akif ersoy university, turkey, 3department of physics, university of agriculture, faisalabad, pakistan, 4department of mechanical engineering, universiti teknologi petronas, perak, malaysia and 5department of mechanical engineering, akdeniz university, turkey and 6bucak technology faculty, burdur mehmet akif ersoy university, turkey (received 15 february 2019, revised 7 january, accepted 10 february 2020) abstract: in this study, gasification performance and importance of hydrogen production using waste of a tea factory were evaluated. a mathematical model was developed for the gasification system, which includes a water gas shift reactor used for hydrogen purification. the gasifier temperature was 877 °c for the developed model. the model has been validated against experimental data from an 80 kw t h cylindrical downdraft gasifier, given in the literature for syngas composition for three different air-to-fuel ratios. with the developed model, hydrogen production from tea wastes was achieved to yield a higher level by additionally using a water gas shift reactor. tea waste (1000 kg) was gasified and after the hydrogen purification process, a total of 4.1 kmol hydrogen was achieved, whereas the amount would be 2.8 kmol gas hydrogen if a normal gasification method were used. the validity of the developed model was verified by comparing the experimental results obtained from the literature with the results of the model under the same conditions. after verification of the developed model, the effect of the moisture content of the biomass and the air/fuel ratio on the composition of the product gas were investigated. these investigations were also confirmed by experimental data. the results show that it is important to convert biomass waste into a clean energy source of hydrogen to minimize its environmental impact. keywords: biomass; environment; conversion; syngas; thermodynamic; equilibrium. introduction biomass will be one of the most significant energy sources in the near future owing to its immense accessibility and encouraging potential to decrease global * corresponding author. e-mail: afsingungor@hotmail.com https://doi.org/10.2298/jsc190215013a available on line at ________________________________________________________________________________________________________________________www.shd.org.rs/jscs/ (cc) 2020 scs. 968 akyüz et al. warming problems.1 many researchers believe that hydrogen is a clean and efficient gas and hence, hydrogen-powered systems could be an important solution to the global energy problem.2,3 biomass is an important source for hydrogen production and could be the primary energy-supply system used for implementing hydrogen economy and energy production as a major fuel of the future.3–6 thermo-chemical conversion processes have been of utmost importance in the field of scientific research and these processes have been widely and commercially employed all over the world. gasification is one of the conventional thermo-chemical conversion processes that enables partial oxidation of biomass at a high temperature in the range of 800–1000 °c, resulting in the conversion of biomass into a flammable gas mixture. the gasification process is realized using air, oxygen and/or steam like gasifying agents. product of gasification process is the producer gas that contains h2, co, co2, ch4 and n2.7,8 mathematical modeling of biomass gasification could improve both its design and operation, reduce associated problems and facilitate the implantation of this technology.9,10 the advantages of mathematical modeling are now widely acknowledged by researchers and have been performed by many researchers in the scientific studies.8,11–13 there are three well-known modeling ways of biomass gasification, the computational fluid dynamic model (cfd), kinetic models and equilibrium models.14 in the cfd model, the examination of fluid flow is conducted.15 in order to virtually generate a solution for a physical phenomenon associated with fluid flow, without compromise on accuracy, fluid properties and some major inputs, such as detailed geometry, well mesh structure and boundary conditions have to be considered simultaneously. kinetic modeling can give precise results, in particular concerning the time evolution of the process.16 nonetheless, this kind of modeling is rather multifaceted. an advantage these models have is that they could be appropriate for studies that focus on reactor design and parameters of different processes (reaction rate, residence time, etc.). equilibrium models are known as being simple and speedy. it is almost impossible to reach the equilibrium conditions within the gasifier but these models can define gasification processes with successful approximations. the thermodynamic equilibrium model (tem) is a simple and beneficial tool for the first estimate and initial assessment of the findings of the gasification process. through tem, the influence of fuel and process parameters are studied conveniently independent of gasifier design and thus it enables a reasonable prediction of the maximum achievable yield of a particular product useful for a designer.17 there are few advanced technologies to separate hydrogen from the product gas. to obtain high purity hydrogen, the product gas is treated through several steps. mainly, there are four types of additional operations such as: 1) water gas shift (wgs), 2) scrubber for drying and cleaning, 3) membrane separation and 4) ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. gasification of tea factory wastes 969 pressure swing adsorption. very recently, a wgs process with gasification has gained more interest in the production of hydrogen from biomass.18–20 turkey is one of the most important tea producers in the world, and comes immediately after china, india, sri lanka, kenya, indonesia and vietnam. the black sea region of turkey is where tea is planted and grown widely. approximately 70.000 ha are covered with tea plantations and 150.000 t of dry black tea is produced each year. the tea factories in and around the eastern black sea region produce about 30.000 t of waste every year.21 despite the fact that the production area remains almost the same, the amount of production increases. according to the production statistics for the year 2016, 1,350,000 t of wet tea were produced.21 especially in the eastern black sea region where tea cultivated, solid tea leaves of organic origin, fiber and dust result during the transformation of wet tea leaves to black tea. during tea production, overgrown tea plants are not used in the production process and are directly collected as factory waste.22 the increasing amount of tea waste creates environmental problems due to limited storage capacity. this massive amount of tea waste is not used for any purpose and hence, they have to be stored in depository areas.21,22 although interest is growing for the use of low cost materials and abundantly available lignocellulosic materials to produce liquid and gaseous products and activated carbon, there are very rare studies about tea factory waste treatments, such as pyrolysis, gasification, carbonization, etc. in the literature.23 uzun et al. pyrolysed tea waste and characterized the obtained bio-oil and bio-char fractions.21 the effect of temperature (400–700 °c), heating rate (5–700°c/min) and n2 flow rate (200–800 cm3 min–1) on the product yield was investigated. maximum yield of the liquid product was determined as 30.4 % at 500 °c, 500 °c min–1 heating rate and 200 cm3 min–1 n2 flow rate.21 mahmood et al. performed a three step process in order to produce biofuels from spent tea.24 in the first step, spent tea was gasified in the presence of co nanocatalyst at 300 °c and atmospheric pressure. catalytic gasification of spent tea waste yielded 60 % liquid extract, 28 % gaseous product and 12 % charcoal. gullu investigated the effect of na2co3 and k2co3 catalysts on the yield of the liquid product obtained from flash pyrolysis of tea waste.25 catalytic pyrolysis experiments were performed in order to increase the yield of methanol, which is one of the most valuable fractions in the liquid product. methanol yield was 8.65 % for non-catalytic run. the methanol yield increased from 8.65 to 10.93 % in the presence of na2co3.26 in this study, the potential of converting the tea factory wastes to renewable energy by gasification was examined. in this context, a mathematical modeling for a biomass gasifier was developed. in the model, the stoichiometric thermodynamic equilibrium biomass gasification model was used. simulations of models were performed with the optimal rates, which are assumed to be 100 % for carbon conversion efficiency in the gasification process. the most important ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 970 akyüz et al. difference of the developed model from the studies in the literature is that the gasifier has a wgs unit and the model includes both gasifier and wgs reactor operations. the validity of the developed model was verified by comparing the experimental data obtained from the literature with the model results under the same conditions. experimental model achieving the best synthesis gas composition for production of biomass chemicals and power generation is a challenging problem. the correct understanding of the gasification phenomenon, reliable performance prediction through modeling can greatly avoid expensive upsets. briefly, developing a model is very advantageous for the evaluation, design and analysis of the process. from this viewpoint, in this study, evaluation of tea factory residues gasification performance was simulated by a presently developed stoichiometric thermodynamic equilibrium model. for the gasification model, air and steam are utilized as the gasification agent, and model includes a wgs reactor for hydrogen purification. the concept of the thermodynamic equilibrium model is based on the second law of thermodynamics as applied to chemical reacting systems. a stoichiometric thermodynamic equilibrium model (stem) based on specific chemical reactions is used here for the estimation of the composition of the product gas. thermodynamic equilibrium models do not require any knowledge of the mechanisms of transformation. moreover, they are independent of the reactor and are not limited to a specified range of operating conditions. a stem was formulated to assess the hydrogen yield from different locally available biomasses at specified operating conditions to select the most appropriate one.27 in this study, the assumptions of the biomass gasification model are as follows. − the biomass is modeled considering the carbon, hydrogen and oxygen atoms only. − the gasifier is a steady state system with uniform pressure and temperature. − at equilibrium, the reaction system achieves the most stable composition. − the gases h2, co, co2, ch4, h2o and n2 considered in the reaction system behave ideally. − gasification reaction rate is fast enough and residence time is long enough for the equilibrium state. − no tar leaves the reaction system at the end of the process. − carbon conversion efficiency in the gasification process is assumed as 100 %. in this study, stoichiometric thermodynamic equilibrium model for biomass gasification is based on the work of basu.28 a denotes the air supply in kg dry air per kg dry fuel. f kg of dry fuel is required to obtain n m3 of the gas, and xc is the carbon content of the fuel (kg carbon per kg dry fuel). carbon is split between co, co2 and ch4. for 1 n m3 of gas produced, one can write the carbon molar balance between inflow and outflow streams as: fxc/12 = (vco + vco2 + vch4)/22.4 (1) where v represents the volumetric fraction of a constituent of the gas. the molar balance of h2 is as follows: f(s/18 + xh/2 + w/18) = (vh2 + vh2o + vch4)/22.4 (2) where xh is hydrogen content of the fuel (kg hydrogen/kg dry fuel). s represents the total steam supplied as humidifier associated with air and added steam (kg steam/kg of dry fuel), ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. gasification of tea factory wastes 971 and w represents the moisture content of fuel (kg water / kg dry fuel). if oa represents the mass fraction of oxygen in air and xo is the oxygen content of the fuel (kg oxygen/kg dry fuel), the molar balance of o2 is as follows: f(s/36 + xo/32 + w/36 + aoa/32) = (0.5vco + 0.5 vh2o + vco2)/22.4 (3) where a is kg air / kg dry fuel. if xn is the nitrogen content of the fuel (kg nitrogen/kg dry fuel) and na is the mass fraction of nitrogen in air, the molar balance of n2 gives: f(xn/28 + ana/28) = vn2/22.4 (4) the volume fractions of all constituents of the product gas are equal to 1.0. vco + vco2 + vh2 + vch4 + vh2o + vn2 = 1 (5) to estimate the values of the seven unknowns: vco, vco2, vh2, vh2o, vn2, vch4 and f, seven equations are needed. the remaining two equations can be formed by assuming that all the reactions occurring in the gasification zone are in thermodynamic equilibrium. for the boudouard reaction, the equilibrium constant is: kpb = pco2/pco2 = (vcop) 2/vco2p (6) where pco is the partial pressure of co, which is equal to volume fraction of co (vcop – the pressure of the reactor, p). similarly, for the water–gas reaction: kpw = (phpco)/ph2o = (vh2p)(vcop)/(vh2op) (7) volume fractions of the product gas constituents can be obtained by simultaneously solving these equations using the combined relaxation newton–raphson method in the visual basic net platform, which uses the results of ultimate and proximate analysis of biomass as input data. it is assumed that all of the reactions in biomass gasifiers are thermodynamically balanced with each other in the equilibrium model in this work. the reactions used in the model are given in table i. the equilibrium constants of the water–gas reaction (kpw), boudouard reaction (kpb), and the wgs reaction (kps) are given in table ii.28,29 table i. chemical reactions involved in the gasification system8 no. reaction equals δh / kj mol-1 1 oxidation i c + o2 = co2 –394.5 2 oxidation ii c + ½o2 = co –111.5 3 water–gas c + h2o = co + h2 131.4 4 boudouard c + co2 = 2co 172.6 5 methanation c + 2h2 = ch4 –74.9 6 steam reforming ch4 + h2o = co + 3h2 206.2 7 wgs co + h2o = co2 + h2 –41.2 table ii. equilibrium constants temperature, k kpw, eq. (3) kpb, eq. (4) kps, eq. (7) 400 7.7×10-11 5.2×10-14 4050.00 600 5.1×10-5 1.9×10-6 27.00 800 4.410-2 1.1×10-2 4.04 1000 2.62 1.90 1.38 1500 6.08×102 1.62×103 0.37 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 972 akyüz et al. when the volume fractions are determined, the lower heating value of the product gas (lhv) can be calculated from gas composition and this value is stated in volume basis as shown in eq. (8):30 lhv = 10.79vh2 + 12.26vco + 35.81vch4 (8) the input parameters for the model are biomass-feed rate, biomass properties, gasification temperature and pressure, air-to-fuel (a/f) ratio and steam-to-fuel (s/f) ratio. the simulation model calculates the synthesis gas composition, h2/co and lhv for the a/f and s/f under the given operating conditions. the gasifier temperature has to be conserved within the range of 700–850 °c. at lower temperatures, the efficiency of gasification is lower, and the tar content of the gas is excessive.8 the a/f rate and s/f rate are 0.2 due to the high content of o2 in biomass residues. the gasifier temperature is 877 °c for the developed model. in this study, the developed biomass gasification model also includes a wgs reactor for hydrogen purification. in the wgs process, co is converted to h2 via the reaction between steam and co at different conditions. in fig. 1, the flow diagram of the model calculations is given. the input parameters for the wgs reactor are syngas composition from the gasifier outlet, the wgs reactor temperature and pressure, and steam feed. in wgs reactor, the model assumptions are as follows: − the wgs reactor is a steady state system with uniform pressure (1 atm) and temperature (310 °c). − at equilibrium, the reaction system achieves the most stable composition. the wgs reaction rate is fast enough and residence time long enough for the equilibrium state. fig. 1. flowchart of the biomass gasification model. in this study, a high temperature wgs reactor was used. it is clear from the model results that using the wgs reactor is important for h2 production. however, results and discussion, the high temperature wgs reactor did not provide complete conversion of co. it should be noted that the wgs reaction is exothermic and the hydrogen product is thermodynamically favored at low temperature, 200 °c.31,32 however, at low temperatures, the reaction kinetics are slow, even using catalysts and excess steam needs to be recycled.33 typically, a high temperature shift, operating 300–350 °c maximizes the conversion34 and on an industrial scale, a higher temperature wgs is usually realized.35 therefore, in this study, a wgs reactor operating at 310 °c was considered in the model. model validation the predictions of the stem model developed in this study were validated with the experimental data obtained from the literature for an 80 kwth cylindrical downdraft gasifier (inner reactor diameter is 0.92 m and reactor height is 1.15 m) fed with rubber wood.36 the ultimate and proximate analysis of rubber wood is given in table iii. three experimental data from ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. gasification of tea factory wastes 973 jayah et al. for three different air-to-fuel ratios were used to compare with the model predictions in terms of the gas composition on the gasifier exit.36 the comparison of the gas composition at the gasifier exit between experimental data and model predictions is given in table iv. the model results have a good consistence with experimental ones at air-to-fuel ratios of 1.86, 1.96 and 2.03. the model predictions fit very closely with the experimental data in table iv. the maximum error between the results of experiments and the model is less than 2.09 %. table iii. proximate and ultimate analysis of rubber wood36 component value content (proximate analysis, dry basis), wt.% volatile matter 80.10 ash 0.70 fixed carbon 19.20 content (ultimate analysis, dry basis), wt.% c 50.60 h 6.50 n 0.20 o 42.00 table iv. a comparison of model predictions with the experimental data from jayah et al.36 contenta wt.% air/fuel ratio gas composition, dry basis, vol.% data source co error h2 error co2 error ch4 error n2 error 18.5 2.03 19.6 2.061 17.2 1.872 9.9 1.909 1.4 2.071 51.9 2.025 model 20.8 15.0 9.0 1.5 53.2 experiment 16.0 1.96 18.4 2.032 17.0 1.864 10.6 1.924 1.3 2.076 52.7 2.024 model 19.0 14.7 9.8 1.4 54.0 experiment 14.7 1.86 19.1 1.952 15.5 1.864 11.4 1.929 1.1 2.090 52.9 2.039 model 18.2 13.4 10.6 1.2 55.0 experiment afuel moisture content results and discussion in this study, hydrogen production from tea wastes by the gasification method was evaluated with the developed mathematical model. the composition of the tea wastes used in the study is given in table v in detail. table v. proximate and ultimate analysis of tea waste37 component value content (proximate analysis, as received basis), wt.% moisture 4.00 ash 4.13 fixed carbon 31.90 content (ultimate analysis, dry basis), wt.% c 46.40 h 5.32 n 4.14 o 44.14 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 974 akyüz et al. an overview of the hydrogen production process and the results of the gasification and wgs reactor are given in mol and percentages in fig. 2. besides, the mathematical model results for tea waste gasification also resulted in the observation of the higher heating value of fuel and hydrogen content. fig. 2 illustrates the yield of h2 is 2.8 mol h2/kg tea waste and co yield is 6.2 mol co/kg tea waste at the gasifier outlet. however, co2 yield is relatively low under this condition. this can be associated with the boudouard reaction, which occurs in the gasification of tea waste. ergun reported that carbon has ability of retaining oxygen at sites on its surfaces by chemical bonding. co2 is reduced to co on the carbon surface at temperatures as low as 600 °c. oxygen retained on the carbon surface can be removed by co. at equilibrium, most of the co2 produced in the gasification was converted back to co by the boudouard reaction.37,38 these results are also in agreement with parametric study of ayas and esen in which tea waste was gasified at temperatures of 450, 650 and 850 °c and 1 atm in an updraft air gasifier.37 by the gasification of tea wastes, it was found that the amount of co is nearly two times higher than the amount h2 in the syngas at the gasifier outlet. the o2 and c ratios of tea wastes are quite high, as shown in the ultimate analysis in table iii. this causes the c in the tea wastes to turn into co with the air used as the agent during the gasification. therefore, a high amount of co emerges from the tea waste in the gasification process. the wgs reaction is exothermic and hence, at higher reaction temperatures (300–400 °c) the reaction shifts to the reagents and reduces co and favors h2 production, as can also be seen from fig. 2. fig. 2. production of h2 from tea waste. conclusions hydrogen plays a very important role in the relationship between sustainable environment and energy. hydrogen is seen as the fuel of the future because it is clean and environmentally friendly. in this study, the gasification performance of the waste of a tea factory was investigated by a developed stoichiometric thermodynamic equilibrium biomass gasification model. this study proves that a wgs reactor system plays a very important role in production of hydrogen from the ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. gasification of tea factory wastes 975 conversion of co. with the development of hydrogen production technology, biomass will play a vital role in the development of hydrogen usage. in this study, it was revealed that tea wastes have a serious hydrogen potential and they should be reused with gasification technology. in the scope of the study, it was shown that the addition of wgs reactors in biomass gasifiers increases the potential of converting biomass fuels to hydrogen. и з в о д конверзија водоника гасификацијом отпада фабрике чаја ali akyüz1, zuhal akyurek2, muhammad y. naz3, shaharin a. sulaiman4 и afsin gungor5,6 1 bucak emin gulmez v. s., burdur mehmet akif ersoy university, turkey, 2 department of polymer engineering, faculty of engineering and architecture, burdur mehmet akif ersoy university, turkey, 3 department of physics, university of agriculture, faisalabad, pakistan, 4 department of mechanical engineering, universiti teknologi petronas, perak, malaysia, 5 department of mechanical engineering, akdeniz university, turkey и 6 bucak technology faculty, burdur mehmet akif ersoy university, turkey у овој студији процењене су перформансе гасификације и значај производње водоника коришћењем отпада фабрике чаја. израђен је математички модел за гасификациони систем, који укључује реактор за измену воде и гаса који се користи за пречишћавање водоника. температура гасификатора је 877 °c за развијени модел. модел је валидиран на основу експерименталних података цилиндричног гасификатора снаге 80 kw t h датих у литератури за састав синтетског гаса за три различита односа ваздуха и горива. са развијеним моделом, постигнута је већа производња водоника из отпадног чаја уз употребу реактора за измену вода–гас. укупно 1000 kg чајног отпада је гасификвано и после процеса пречишћавања водоника добијено је укупно 4,1 kmol водоника. употребом уобичајене методе гасификације, резултат би био 2,8 kmol водоника. валидност развијеног модела проверена је упоређивањем експерименталних резултата из доступне литературе и резултата модела под истим условима. након верификације развијеног модела, испитиван је утицај садржаја влаге биомасе и ваздух/гориво однос на састав произведеног гаса. ова истраживања су потврђена и експерименталним подацима. резултати указују да је важно претворити отпад из биомасе у чист извор енергије попут водоника како би се смањио утицај отпада на животну средину. 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hydrogen energy 41 (2016) 8067 (https://doi.org/10.1016/j.ijhydene.2015.09.156) 38. s. ergun, j. phys. chem. 60 (1956) 480 (https://doi.org/10.1021/j150538a022). ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice volume 82 (2017) 1 2017 list of referees editorial board of the journal is grateful to the following referees for reviewing the manuscripts during 2017: mohsen abbaspour, hakim sabzevari university, sabzevar, iran ornella abollino, dipartimento di chimica, università di torino, italia biljana abramović, faculty of science, university of novi sad, serbia salah akkal, laboratoire de phytochimie et analyses physico-chimiques et biologiques, département de chimie, faculté des sciences, université mentouri, constantine, algérie ana alil, faculty of technology and metallurgy, university of belgrade, serbia mara aleksić, faculty of pharmacy, university of belgrade, serbia maría jesús almendral parra, department of analytical chemistry, nutrition and food sciences, universidad de salamanca, spain francisco alonso, departamento de química orgánica, facultad de ciencias and instituto de síntesis orgánica (iso), universidad de alicante, spain omar m. aly, medicinal chemistry department, faculty of pharmacy, minia university, egypt marius andruh, inorganic chemistry laboratory, faculty of chemistry, university of bucharest, romania katarina anđelković, faculty of chemistry, university of belgrade, serbia robert angelici, department of chemistry, iowa state university, ames, ia, us vesna antić, faculty of agriculture, university of belgrade, serbia mališa antić, faculty of agriculture, university of belgrade, serbia davor antanasijević, faculty of technology and metallurgy, university of belgrade, serbia zorana arsenijević, institute of chemistry, technology and metallurgy, university of belgrade, serbia muhammad arshad, university of veterinary and animal sciences lahore, jhang, pakistan mahmut aslani, institue of nuclear sciences, nuclear technology department, ege university, izmir, turkey jelana bajat, faculty of technology and metallurgy, university of belgrade, serbia divna bajić, faculty of technology and metallurgy, university of belgrade, serbia robert baker, school of chemistry, university of dublin, trinity college, dublin, ireland wojciech bal, institute of biochemistry and biophysics, polish academy of sciences, warsaw, poland milica balaban, faculty of science, university of banja luka, bosnia and herzegovina antun balaž, scientific computing laboratory, institute of physics university of belgrade, serbia jiří barek, charles university, prague, czech republic andreas baum, walther meissner institute, bavarian academy of scinces, munich, germany ivana n. beara, department of chemistry, biochemistry and environmental protection, faculty of sciences university of novi sad, serbia ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 2 journal of the serbian chemical society snežana belanović simić, faculty of forestry, university of belgrade, serbia vladimir beškoski, faculty of chemistry, university of belgrade, serbia filip bihelović, faculty of chemistry, university of belgrade, serbia mira bjelaković, institute of chemistry, technology and metallurgy department of chemistry, university of belgrade, serbia polina blagojević, faculty of science and mathematics, department of chemistry, university of niš, serbia jovana bogojeski, department of chemistry, faculty of science, university of kragujevac, serbia mohammad ali bodaghifard, department of chemistry, arak university, iran nevenka bošković-vragolović, faculty of technology and metallurgy, university of belgrade, serbia mohammad bozorgmehr, department of chemistry, islamic azad university, mashhad branch, mashhad, iran damien brady, athlone institute of technology, bioscience research institute, athlone, ireland ilija brčeski, faculty of chemistry, university of belgrade, serbia alan brown, department of chemistry, florida institute of technology, melbourne, usa ha bui, sai gon university, viet nam jorge cárdenas, instituto de química, universidad nacional autónoma de méxico circuito exterior,, méxico d.f., mexico ram chaudhary, sant longowal institute of engineering & technology, longowal (sangrur), punjab, india gui-xin chou, institute of chinese materia medica of shanghai university of traditional chinese medicine, zhangjiang, shanghai, pr china jános csanádi, faculty of science, university of novi sad, serbia ilija cvijetić, inovation center of faculty of chemistry, university of belgrade, serbia mirjana čomor, vinča institute of nuclear sciences, university of belgrade, serbia aleksandra daković, institute for technology of nuclear and other mineral raw materials, belgrade, serbia ljiljana damjanović vasilić, faculty of physical chemistry, university of belgrade, serbia aleksandar dekanski, institute of chemistry, technology and metallurgy, university of belgrade, serbia sabahattin deniz, marmara üniversitesi, kadıköy, i̇stanbul, turkey david dixon, department of chemistry, the university of alabama, tuscaloosa, al, usa konstantin v. domasevitch, inorganic chemistry department, national taras shevchenko university of kyiv, ukraine ivana drvenica, institut za medicinska istraživanja, univerzitet u beogradu, serbia branko dunjić, faculty of technology and metallurgy, university of belgrade, serbia dragana đorđević, institute of chemistry, technology and metallurgy, university of belgrade, serbia miloš i. đuran, department of chemistry, faculty of science, university of kragujevac, serbia predrag đurđević, department of chemistry, faculty of science, university of kragujevac, serbia rada đurović-pejčev, institute of pesticides and environmental protection, belgrade, serbia jasna džunuzović, institute of chemistry, technology and metallurgy, university of belgrade, serbia anastasios ecconomou, department of analytical chemistry, university of athens, greece mihajlo etinski, faculty of physical chemistry, university of belgrade, serbia m. h. fatemi, mazandaran university of babolsar, iran ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 2017 list of referees 3 marta figueredo, department of chemistry, autonomous university of barcelona, spain érico m. m. flores, federal university of santa maria, brazil jovana francuz, faculty of sciences, department of chemistry, biochemistry and environmental protection, university of novi sad, serbia gordana gajić, institute for biological research „sinisa stankovic”, university of belgrade, serbia marija gavrović-jankulović, faculty of chemistry, university of belgrade, serbia sandra glišić, faculty of technology and metallurgy, university of belgrade, serbia biljana dj. glišić, department of chemistry, faculty of science, university of kragujevac, serbia gordana gojgić-cvijović, institute of chemistry, technology and metallurgy, university of belgrade, serbia santiago gómez-ruiz, departamento de química inorgánica y analítica, e.s.c.e.t., universidad rey juan carlos, móstoles, madrid, spain branimir grgur, faculty of technology and metallurgy, university of belgrade, serbia sanja grgurić šipka, faculty of chemistry, university of belgrade. serbia dušan grozdanić, faculty of technology and metallurgy, university of belgrade, serbia nikola grozdanić, faculty of technology and metallurgy, university of belgrade, serbia maja gruden, faculty of chemistry, university of belgrade. serbia svetlana grujić, faculty of technology and metallurgy, university of belgrade, serbia snežana grujić, faculty of technology and metallurgy, university of belgrade, serbia raquel guiné, instituto politcnico de viseu, viseu, portugal özlem güngör, department of chemistry, faculty of science, gazi university, teknikokullar, ankara, turkey ivan gutman, department of chemistry, faculty of science, university of kragujevac, serbia valéria guzsvány, faculty of sciences, department of chemistry, biochemistry and environmental protection, university of novi sad, serbia milica gvozdenovic, faculty of technology and metallurgy, university of belgrade, serbia stylianos hamilakis, school of chemical engineering, national technicval university of athens, greece mohamed hassan, national research centre, department of chemistry of natural and microbial products, cairo, egypt radmila hercigonja, faculty of physical chemistry, university of belgrade. serbia berta barta holló, department of chemistry, biochemistry and environmental protection faculty of sciences, university of novi sad, serbia cemil ibis, istanbul university, department of chemistry, avcılar, i̇stanbul, turkey hamada ibrahim, chemistry department, faculty of science, fayoum university, egypt mila ilić, institute of chemistry, technology and metallurgy, university of belgrade, serbia esma isenović, vinča institute of nuclear sciences, university of belgrade, serbia gorica ivaniš, faculty of technology and metallurgy, university of belgrade, serbia romee jan, sant longowal institute of engineering and technology, department of food engineering and technology, sangrur, india radmila jančić heinemann, faculty of technology and metallurgy, university of belgrade, serbia ivona janković-častvan, faculty of technology and metallurgy, university of belgrade, serbia mirjana jankulovska, department of genetics and plant breeding, faculty of agricultural sciences and food, ss cyril and methodius university, skopje, macedonia verica jevtić, department of chemistry, university of kragujevac, serbia zhiqin ji, institute of pesticide science, northwest a & f university, shaanxi, pr china ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 4 journal of the serbian chemical society jovan jovanović, faculty of technology and metallurgy, university of belgrade, serbia ivan juranić, faculty of chemistry, university of belgrade, serbia melina kalagasidis-krušić, faculty of technology and metallurgy, university of belgrade, serbia goran kaluđerović, department of bioorganic chemistry, leibniz-institute of plant biochemistry, halle (saale), germany robert kargbo, medicinal chemistry department, albany molecular research, inc. albany,usa dejan kepić, vinča institute of nuclear sciences, belgrade, serbia dmytro khomenko, department of chemistry, kyiv national taras shevchenko university, kyiv, ukraine sezgin kiren, winston salem state university, winston salem, united states mirjana kijevčanin, faculty of technology and metallurgy, university of belgrade, serbia anton kokalj, jozef stefan institute, department of physical and organic chemistry, ljubljana, slovenia adam kokotkiewicz, department of pharmacognosy, gdanski uniwersytet medyczny, gdańsk, poland ljiljana kolar-anić, faculty of physical chemistry, university of belgrade, serbia beata kołodziej, faculty of chemical technology and engineering, department of inorganic and analytical chemistry, szczecin, poland sandra konstantinović, faculty of technology, university of niš, leskovac, serbia aleksandar kostić, faculty of mining and geology, university of belgrade, serbia mirjana kostić, faculty of technology and metallurgy, university of belgrade, serbia tatjana kop, institute of chemistry, technology and metallurgy department of chemistry, university of belgrade, serbia milan kragović, institute for technology of nuclear and other raw materials, belgrade, serbia marijana kraljić roković, faculty of chemical engineering and technology, university of zagreb, croatia natalija krstić, faculty of chemistry, university of belgrade, serbia ajaykumar kulkarn, department of applied sciences, mit academy of engineering, dehu phata, pune, india thymios liarokapis, national technical university of athens, greece rodolfo lavilla, laboratori de química orgànica, facultat de farmàcia, universitat de barcelona, spain slavica lazarević, faculty of technology and metallurgy, university of belgrade, serbia angélica lazarin, departamento de química, universidade estadual de maringá, brazil vera lazić, faculty of technology, university of novi sad, serbia marija lesjak, department of chemistry, biochemistry and environmental protection, faculty of sciences, university of novi sad, serbia xiaodong li, northeast agricultural university, key laboratory of dairy science, harbin, china rossitza mancheva, medical university, plovdiv, bulgaria dragan manojlović, faculty of chemistry, university of belgrade, serbia aleksandra margetić, institute of chemistry, technology and metallurgy, university of belgrade, serbia silvija markic, university og bremen, germany aleksandar marinković, faculty of technology and metallurgy, university of belgrade, serbia bratislav marinković, institute of physics, university of belgrade, zemun, serbia sanja marinović, institute of chemistry, technology and metallurgy, university of belgrade, serbia ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 2017 list of referees 5 ana marjanović jeromela, institute of field and vegetable crops, novi sad, serbia zoran marković, state university of novi pazar, serbia romana masnikosa, faculty of physical chemistry, university of belgrade, serbia branko matović, department of materials science, vinča institute of nuclear sciences, belgrade, serbia violeta marković, faculty of science, university of kragujevac, serbia veselin maslak, faculty of chemistry, university of belgrade, serbia christopher mcerlean, school of chemistry, the university of sydney, australia slavko mentus, faculty of physical chemistry, university of belgrade, serbia radovan metelka, fakulta chemicko-technologická, university of pardubice, czech republic vladimir mihailović, department of chemistry, faculty of science, university of kragujevac, serbia jelena miladinović, faculty of technology and metallurgy, university of belgrade, serbia miloš milčić, faculty of chemistry, university of belgrade, serbia dušica milenković, faculty of sciences, university of novi sad, serbia srđan miletić, faculty of chemistry, university of belgrade, serbia milan milivojević, faculty of technology and metallurgy, university of belgrade, serbia šćepan miljanić, faculty of physical chemistry, university of belgrade, serbia slobodan milonjić, vinča institute of nuclear sciences, university of belgrade, serbia nebojša milošević, institut za voćarstvo, univerzitet u kragujevcu, čačak, serbia francesca mocci, department of chemical sciencies, university of cagliari, monserrato, italy ljiljana mojović, faculty of technology and metallurgy, university of belgrade, serbia majid moosavi, department of chemistry, faculty of science, university of isfahan, iran subbiah nagarajan, school of chemical and biotechnology, sastra university, thanjavur tamil nadu, india vesna najdanović-višak, l engineering department , ancaster university, uk olgica nedić, institute for the application of nuclear energy, university of belgrade, serbia mikko nikinmaa, turun yliopisto, department of biology, abo (turku), finland nikola nikačević, faculty of technology and metallurgy, university of belgrade, serbia branislav ž nikolić, serbian chemical society, belgrade, serbia jasmina nikolić, faculty of technology and metallurgy, university of belgrade, serbia ljubiša nikolić, faculty of technology, university of nis, lescovac, serbia katarina nikolić, faculty of chemistry, university of belgrade, serbia marija nikolić, faculty of technology and metallurgy, university of belgrade, serbia milan nikolić, faculty of chemistry, university of belgrade, serbia rodica olar, department of inorganic chemistry, faculty of chemistry, university of bucharest, romania radovan omorjan, faculty of technology, university of novi sad, serbia antonije onjia, vinča institute of nuclear sciences, university of belgrade, serbia dejan opsenica, faculty of chemistry, university of belgrade, serbia igor opsenica, faculty of chemistry, university of belgrade, serbia ilkay erdoğan orhan, gazi universitesi, department of pharmacognosy, ankara, turkey sibel a. özkan, faculty of pharmacy, ankara university, turkey musa özil, department of chemistry, recep tayyip erdogan university, rize, turkey vesna panić, institute for medical research, university of belgrade, serbia selvarengan paranthaman, department of physics & irc, kalasalingam university, india antonella pasqualone, universita degli studi di bari, dipartimento di scienze del suolo, bari, italy ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 6 journal of the serbian chemical society vladimir pavićević, faculty of technology and metallurgy, university of belgrade, serbia srđan pejanović, faculty of technology and metallurgy, university of belgrade, serbia aleksandra perić-gujić, faculty of technology and metallurgy, university of belgrade, serbia menka petkovska, faculty of technology and metallurgy, university of belgrade, serbia miloš petković, faculty of pharmacy university of belgrade, serbia milena petković, faculty of physical chemistry university of belgrade, serbia dušan petrović, forschungszentrum jülich gmbh rada petrović, faculty of technology and metallurgy, university of belgrade, serbia marija pergal, institute of chemistry, technology and metallurgy, university of belgrade, serbia maría-jesús pérez-pérez, instituto de química m edica (iqm-csic), madrid, spain carmelo garcía pinto, department for analytical chemistry and bromatology, university of salamanca. spain mahtab pirouzmand, department of inorganic chemistry, faculty of chemistry, university of tabriz, iran aurelia magdalena pisoschi, university of agronomic sciences and veterinary of bucharest, faculty of veterinary medicine, bucharest, romania rada pjanović, faculty of technology and metallurgy, university of belgrade, serbia marija popović, faculty of pharmacy university of belgrade, serbia sedigheh pourestarabadi, department of chemistry, shahid bahonar university of kerman, kerman, iran bojan radak, vinča institute of nuclear sciences, university of belgrade, serbia dušanka radanović, institute of chemistry, technology and metallurgy, university of belgrade, serbia slavko radenković, department of chemistry, faculty of science, university of kragujevac, serbia marija radoičić, vinča institute of nuclear sciences, university of belgrade, serbia serbia ivona radović, faculty of technology and metallurgy, university of belgrade, serbia aleksandar radu, keele university, uk muhammad khalid rafiq, college of pastoral agriculture, science and technology agric, lanzhou university, china kadalipura p. rakesh, school of chemistry, chemical engineering and life science, wuhan university of technology, wuhan, pr china tamara rakić, institute of botany and botanical garden “jevremovac”, faculty of biology, university of belgrade, serbia vesna rakić, faculty of agriculture, university of belgrade, serbia marica rakin, faculty of technology and metallurgy, university of belgrade, serbia vladana rajaković-ognjanović, faculty of civil engineering, university of belgrade, serbia nevenka rajić, faculty of technology and metallurgy, university of belgrade, serbia harry ramanantoanina, university of fribourgh, switzerland bahman ramavandi, bushehr university of medical sciences, iran lina raudone, lithuanian university of health sciences, department of pharmacognosy, kaunas, lithuania slavica ražić, faculty of pharmacy university of belgrade, serbia damoder reddy motati, union university, jackson, tennessee, usa ivan ristić, faculty of technology, university of novi sad dragana robajac, institute for the application of nuclear energy, university of belgrade, serbia ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 2017 list of referees 7 marko rodić, faculty of sciences, university of novi sad, serbia isao sakurada, discovery research, mochida pharmaceutical co. ltd., jimba, gotemba, shizuoka, japan marija sakač, faculty of sciences, university of novi sad, serbia victoria samanidou, aristotle university of thessaloniki, greece diganta sarma, department of chemistry, dibrugarh university, dibrugarh, assam, india snežana savić, department of pharmaceutical technology and cosmetology, faculty of pharmacy, university of belgrade, serbia jan schwarzbauer, rwth aachen university, institute of geology and geochemistry of petroleum and coal, aachen, germany milan senćanski, vinča institute of nuclear sciences, university of belgrade, serbia dušan sladić, faculty of chemistry, university of belgrade, serbia ivana smičiklas, vinča institute of nuclear sciences, university of belgrade, serbia tesfaye r. soreta, department of chemistry, college of natural sciences, jimma university, jimma, ethiopia ivan spasojević, institute for multidisciplinary research, university of belgrade, serbia miroslav spasojević, faculty of agronomy, university of kragujevac, čačak, serbia pavle spasojević, innovation centre of faculty of technology and metallurgy, serbia dušan sredojević, vinča institute of nuclear sciences, university of belgrade, serbia marko stamenić, faculty of technology and metallurgy, university of belgrade, serbia miroslav stanković, institute of chemistry, technology and metallurgy, department of catalysis and chemical engineering, university of belgrade, serbia dragomir stanisavljev, faculty of physical chemistry, university of belgrade, serbia dalibor stanković, vinča institute of nuclear sciences, university of belgrade, serbia velizar stanković, department of metallurgy, technical faculty bor, university of belgrade; bor, serbia stepan stepanović, institute of chemistry, technology and metallurgy, department of chemistry, university of belgrade, serbia sanja stevanović, institute of chemistry, technology and metallurgy, department of electrochemistry, university of belgrade, serbia mirko stijepović, faculty of technology and metallurgy, university of belgrade, serbia gordana stojanović, department of chemistry, faculty of science and mathematics, university of niš, serbia ksenija stojanović, faculty of chemistry, university of belgrade, serbia ljiljana stanojević, faculty of technology, university of niš, leskovac, serbia marina stojanovska, institute of chemistry, faculty of natural science and mathematics ss cyril and methodius university, skopje, macedonia ivana stojković-simatović, faculty of physical chemistry, university of belgrade, serbia derek stewart, heriot-watt university, school of life sciences, edinburgh, united kingdom zoran šaponjić, vinča institute of nuclear sciences, belgrade, serbia radovan šebesta, department of organic chemistry, comenius university in bratislava, slovakia slavica šiler, faculty of technology and metallurgy, university of belgrade, serbia tatjana šolević knudsen, institute of chemistry, technology and metallurgy, university of belgrade, serbia maja šumar ristović, faculty of chemistry, university of belgrade, serbia lubomir švorc, faculty of chemical and food technology, slovak university of technology, bratislava, slovak republic ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 8 journal of the serbian chemical society yoshiaki takaya, faculty of pharmacy, meijo university, tempaku, nagoya, japan tan kok tat, faculty of engineering and green technology, university tunku abdul rahman, perak, malaysia javad tavakoli, chemical and biomolecular engineering, lafayette college, easton, pennsylvania, usa ibrahim tegin, department of chemistry, faculty of arts and science, siirt university, tyrkey anja terzić, institute for testing of materials ims institute, belgrade, serbia shiliang tian, university of illinois at urbana-champaign, urbana, il, usa dragana todorović, vinča institute of nuclear sciences, belgrade, serbia nina todorović, institute of chemistry, technology and metallurgy department of chemistry, university of belgrade, serbia tamara todorović, faculty of chemistry, university of belgrade, serbia nemanja trišović, faculty of technology and metallurgy, university of belgrade, serbia dragica trivić, faculty of chemistry, university of belgrade, serbia tanja trtić-petrović, vinča institute of nuclear sciences, belgrade, serbia aleksandra tubić, faculty of sciences, university of novi sad, serbia maris turks, faculty of materials science and applied chemistry, riga technical university, latvia najeeb ullah, the university of sydney, nsw, australia pothiappan vairaprakash, department of chemistry, school of chemical and biotechnology, sastra university, thanjavur, tamil nadu, india zoran višak, centro de química estrutural, instituto superior tecnico, universidade de lisboa, portugal željko vitnik, department of chemistry, institute of chemistry, technology and metallurgy, university of belgrade, serbia sandra vojnović, institute of molecular genetics and genetic engineering, university of belgrade, serbia milan vraneš, faculty of sciences, university of novi sad, serbia vesna vučić, institute for medical research, university of belgrade, serbia marija vukčević, faculty of technology and metallurgy, university of belgrade, serbia jelena vuksanović, faculty of technology and metallurgy, university of belgrade, serbia jelena vulić, department of biotechnology and pharmaceutical engineering,university of novi sad, serbia dong wang, college of biotechnology, tianjin university of science and technology, tianjin, pr china jingli xie, east china university of science and technology, shanghai, china erdal yabalak, faculty of arts and science, mersin university, mersin, turkey ilkay yildiz, faculty of pharmacy, ankara university, turkey peng yu, college of biotechnology, tianjin university of science and technology, tianjin, pr china mohamad yusuf, department of chemistry, punjabi university, patiala, punjab, india george a. zachariadis, aristotle university of thessaloniki, greece slavica zec, vinča institute of nuclear sciences, belgrade, serbia yan zhang, department of food science, nutrition and health promotion, mississippi state university, mississippi, united states guo-qing zhong, school of material science and engineering, southwest university of science and technology, mianyang, sichuan, china ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 2017 list of referees 9 peng zhou, school of life science and technology, university of electronic science and technology of china, chengdou, sichuan, pr china matija zlatar, institute of chemistry, technology and metallurgy, university of belgrade, serbia mario zlatović, faculty of chemistry, university of belgrade, serbia vladimir zyryanov, institute of solid state chemistry and mechanochemistry, siberian branch of the russian academy of sciences, novosibirsk, russia branka žarković, faculty of agriculture, university of belgrade, serbia majda žigon, national institute of chemistry, ljubljana, slovenia ljiljana živković, vinča institute of nuclear sciences, belgrade, serbia dragana životić, faculty of mining and geology, university of belgrade, serbia irena žižović, faculty of technology and metallurgy, university of belgrade, serbia ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice un co rre cte d p ro of j. serb. chem. soc. 80 (0) 1–11 (2015) udc jscs–72 letter to the editor 1 letter to the editor 1 a survey on publishing policies of the journal of the serbian 2 chemical society– on the occasion of the 80th volume 3 olgica nedić1*# and aleksandar dekanski2# 4 1institute for the application of nuclear energy (inep), university of belgrade, serbia and 5 2institute of chemistry, technology and metallurgy, department of electrochemistry, 6 university of belgrade, serbia 7 (received 6 march, accepted 6 april 2015) 8 abstract: journal of the serbian chemical society (jscs) is the scientific 9 journal of the serbian chemical society and this year is celebrating 85 years of 10 its publishing and the 80th volume. after so many years of publishing, the idea 11 of the editorial board of the jscs was to investigate the opinion of the authors, 12 reviewers and sub-editors concerning the journal and whether their evaluation 13 and suggestions could aid in its improvement. questionnaires were sent to the 14 three investigated groups as an e-mail link. the responses were analyzed and 15 only the most general and the most important data are presented in this article. 16 the grades, comments and suggestions showed that most of the contributors 17 are satisfied with the present handling and publishing policy of the jscs, but 18 certain technical aspects should be improved. after a thorough inspection of 19 the data, the editorial board decided to introduce a fully automatic on-line 20 system, to speed-up the peer review process, to improve the instructions to 21 authors and reviewer’s report form. all these novelties commenced from the 22 beginning of march 2015. 23 keywords: peer review; publishing; questionnaire; evaluation 24 introduction 25 evaluation of journals, especially the peer review process, has attracted great 26 attention in the last decade. there are number of scientometric methods, mostly 27 of the quantitative type, to express the “value” of the journal.1–3 the best known 28 is the one that measures journal’s impact via the citation index. an intensive 29 debate on the positive and negative aspects of the journal evaluation via the 30 impact factor (if) has been in progress for a long time, but officially the if has 31 been recognized as the measure of impact recognition and a tool for journal 32 * corresponding author. e-mail: olgica@inep.co.rs # serbian chemical society member. doi: 10.2298/jsc150306036n send back the proof corrections to jscs-proofs@shd.org.rs within next 48 hours! un co rre cte d p ro of 2 nedić and dekanski ranking.4 there are other ways to investigate the quality of a periodical and one 33 of which is to determine the opinion of associates of the journal. 34 after so many years of publishing, the idea of the editorial board of the 35 journal of the serbian chemical society (jscs) was to investigate the opinion of 36 authors, reviewers and sub-editors concerning the journal and to determine 37 whether their evaluation and suggestions could help in the improvement of the 38 quality of the journal and the managing process, and raise the prestige of the 39 jscs. prior to this study, an educational article recommending how to write a 40 good scientific paper was published.6 both scientific and technical aspect were 41 discussed, as it is equally important to obtain significant research results and to 42 know how to present them. 43 journal of the serbian chemical society, as its name says, is an official 44 journal of the society. the society was founded in 1897 and its first bulletin 45 appeared in 1899.6,7 the journal was first published in 1930 as the journal of the 46 chemical society of the kingdom of yugoslavia, the name was changed in 1947 47 to the journal of the chemical society belgrade and under the present name, it 48 exists since 1985.7 in this year, the 80th volume of the jscs is being published. 49 all papers are published only in english. there are 12 issues per volume, 10–13 50 articles per issue and 2000–2200 pages a year. the jscs is an open access 51 publication, without page charges and with on-line submission. it has been 52 indexed in the science citation index expanded since 1995, in the category 53 chemistry: multidisciplinary and its last impact factor (if 2014) is 0.871 (rank 54 114/157); 5-year if is 1.009 (rank 105/157)*. 55 the journal is managed as a non-profit making periodical by the members of 56 the serbian chemical society (scs), who work mostly voluntarily. it is sup-57 ported by membership fees, various institutions of the university of belgrade, the 58 ministry of education, science and technological development of serbia and 59 occasionally by sponsors. in the last five years, 2130 articles were submitted of 60 which 872 were accepted. during this period, 7 authors complained about reject-61 ion of their papers, 33 withdrew their manuscripts after reviewing and 3 62 manuscripts were recognized as plagiatorism. 63 a questionnaire is a widely recognized method to obtain relatively reliable 64 data on the posed questions and it is used by many publishers. in contrast to the 65 most publishers who interview the authors of accepted papers, it was decided to 66 interview all contributors to the publishing process. it was felt that by examining 67 the entire partnership network, a more reliable overview could be obtained. more-68 over, potential authors, a category of researchers whose manuscripts were not 69 accepted for publication, were also interviewed. in addition, a very important 70 decision was made by the editor-in-chief to publish openly the results of the 71 survey. 72 * data announced in june 2015. send back the proof corrections to jscs-proofs@shd.org.rs within next 48 hours! un co rre cte d p ro of jscs publishing policies survey 3 thus, three questionnaires adapted to suit the three investigated population 73 groups were composed and sent as an e-mail link to all participants in the 74 publishing activity of the journal in the last five years. there were cases when 75 individuals performed two or even all three roles, so they received the appro-76 priate number of questionnaires. the questions were composed to evaluate the 77 publishing process in quantitative and qualitative ways, both at the level of peer 78 review and managing, and the results obtained are presented accordingly. 79 method and approach 80 study population 81 there were three groups of persons involved in the study. in total, 2422 invitations were 82 sent: 13 for sub-editors, 980 for reviewers and 1429 for authors. certain number of invitations 83 returned undelivered (80 for reviewers and 170 for authors). the inclusion criterion for the 84 survey was at least one type of activity in relation to the jscs over a five-year period 85 (november 2009–october 2014). 86 questionnaires 87 the three questionnaires contained some questions that were the same for all participants 88 and others that were more specific, suitable for the role played by the surveyed persons (see 89 supplementary material to this letter). the participants were asked for their academic title, 90 research field, professional background, previous experience in the same kind of activity that 91 was being investigated in the survey, before being asked direct questions about the jscs. the 92 survey contained two types of questions: those to be answered by scaling (from 5, excellent to 93 1, poor) and those to be answered by choosing offered responses (in some cases more than 94 one response could be chosen). finally, in the last section of the survey, the participants were 95 given the opportunity to express their personal suggestions and remarks. 96 the surveys were sent time-shifted: the sub-editors first, the reviewers two weeks later 97 and the authors a month later. two weeks after the first invitation, a reminder was sent to 98 those who had not responded. the reports were collected and analyzed. the results of the 99 survey offered many more correlations and conclusions than presented in this paper, but in 100 order not to overload the article, only the most general and the most important data are shown. 101 thus, the three surveyed populations were analyzed as entire entities. 102 data analysis 103 data on questions that were answered by scaling (5–1) are given as an average grade. 104 data on questions that could be answered by multiple responses were grouped as the 105 frequency of each response and are reported as such (in % of the total number of quest-106 ionnaires). suggestions and remarks were grouped according to their similarity and are rep-107 orted as lists. 108 results and discussion 109 twelve sub-editors responded to the survey (92 % of the interviewed), 309 110 reviewers (43 %) and 511 authors (41 %). depending on the type of data, the 111 analyzed results are presented graphically (in the case of frequency distributions), 112 in tables (in the case of scaling) or in lists (suggestions and remarks). all surveys 113 were analyzed separately and the results reported by the sub-editors are given in 114 send back the proof corrections to jscs-proofs@shd.org.rs within next 48 hours! un co rre cte d p ro of 4 nedić and dekanski fig. 1, table i and frame 1, for the reviewers in fig. 2, table ii and frame 2, and 115 for the authors in fig. 3, table iii and frame 3. 116 117 fig. 1. the responses of sub-editors to particular questions, expressed as frequency 118 distributions (individual answers). 119 comment [l1]: the question d should read “do you” and not “you do” send back the proof corrections to jscs-proofs@shd.org.rs within next 48 hours! un co rre cte d p ro of jscs publishing policies survey 5 table i. sub-editors’ responses to questions that were recorded by scaling 120 question grade average grade 5 4 3 2 1 number of responses comprehensiveness of the guide for authors 4 6 2 0 0 4.2 choice of the questions for reviewers in the form 2 7 3 0 0 3.9 communication with the editorial office 9 2 1 0 0 4.7 frame 1. sub-editors’ major suggestions and remarks grouped by topics 121 1. introduction of fully automatic on-line system for submission and management of manuscripts 2. improvement of the instructions to authors (considerable number of inappropriate figures) 3. high tendency of potential reviewers to refuse the invitation to review 4. examination of manuscripts by use of software to detect plagiarism sub-editors were first asked about the procedure they conduct while search-122 ing for reviewers and their general opinion on this process. they responded that 123 they most often find reviewers by using scientific databases (scopus, pubmed) or 124 by asking colleagues who they know (fig. 1a). in the first round, some sub-edi-125 tors invited only one or two reviewers, whereas others invited more, even more 126 than four (fig. 1b). the responses to this question illustrate the different indi-127 vidual approaches of the sub-editors to peer review. six sub-editors conduct a 128 second round of search for reviewers in the case of less than 25 % of the 129 manuscripts, while another six reported a greater number of papers that could not 130 be finalized after the first call (fig. 1c). the number of unanswered calls for peer 131 review is rather high (expressed as the percentage of the total number of calls in 132 fig. 1d). sub-editors found between 1 and 15 % of reviewers’ reports inade-133 quate (fig. 1e), due to a complete absence of peer review (“publish as is” in con-134 trast to other reports that suggested major revision or even rejection) or due to 135 unprofessional conduct of reviewers (humiliating or malicious attitudes). as for 136 the quality of the reports in terms of their usefulness to authors to improve the 137 manuscripts, ten sub-editors evaluated the reports as mostly good, while two 138 sub-editors stated that they receive the same number of good and poor reports. 139 responses to this question probably illustrate the different individual criteria of 140 sub-editors. on the grading scale, sub-editors valued relatively highly the tech-141 nical aspects of the publishing process (table i), giving an overall average grade 142 of 4.3. to improve the work of the jscs, most sub-editors suggested a complete 143 on-line submission and management system (frame 1). 144 reviewers who responded to the survey were from serbia (43 % of the total 145 number) and from other countries (57 %, fig. 2a). slightly more than half 146 reviewers defined themselves as chemists (fig. 2b) and approximately 70 % 147 were experienced reviewers (fig. 2c). reviewers accept to review manuscripts 148 comment [n2]: is this term related to instructions to authors and is there any connection to the “instruction for authors” as it appears on jscs website? if it is, then please choose unique term throughout the paper, maybe the best choice is the term officially mentioned at the jscs website. send back the proof corrections to jscs-proofs@shd.org.rs within next 48 hours! un co rre cte d p ro of 6 nedić and dekanski for the jscs for many reasons, but the predominant one is the professional ethics 149 of an expert who feels that it is part of his scientific activity (fig. 2d). reviewers 150 graded technical aspects of the peer review with an average grade of 4.0 (table 151 ii). most reviewers (58 %) either had no additional remarks or expressed an 152 affirmative opinion in a free form of comments and the greatest number of sug-153 gestions were focused on the introduction of a complete on-line system that 154 would enable easier submission and communication (frame 2). 155 156 fig. 2. reviewers’ responses to particular questions expressed as frequency distributions. 157 table ii. reviewers’ responses to questions that were recorded by scaling 158 question grade average grade 5 4 3 2 1 share of the number of responses,% comprehensiveness of the reviewer’s report form 20 57 23 1 0 4.0 period given for reviewing 20 51 25 4 0 3.9 communication with sub-editors and editor 40 39 18 3 0 4.2 send back the proof corrections to jscs-proofs@shd.org.rs within next 48 hours! un co rre cte d p ro of jscs publishing policies survey 7 frame 2. reviewers’ major suggestions and remarks grouped by topics (number of 159 individual comments) 160 58 % of all comments were affirmative +: 1. introduction of fully automatic on-line system for submission and management of manuscripts 26 2. free access to scopus or other databases for the reviewers 11 3. prolongation of the period for reviewing 8 4. improvement of the instructions for reviewers 7 5. information to reviewers on the final decision on the manuscript 5 6. introduction of scaling in the reviewer’s report form 4 authors who responded to the survey were from serbia (30 % of the total 161 number), as well as from the other countries (70 %, fig. 3a). the affiliation of 162 the authors illustrates the international character of the jscs. the main research 163 fields of the authors who submit papers to the jscs were organic chemistry, 164 analytical chemistry, biochemistry and biotechnology, environmental and inorg-165 anic chemistry (fig. 3b). early-stage researchers made up 31 % of all authors 166 and the others were more experienced ones (fig. 3c). the main reasons for sub-167 mitting their manuscripts to the jscs, authors report as: quality and the impact of 168 the journal, no publishing fee, previous positive experience and the speed of 169 reviewing (fig. 3d). grading of the technical parameters related to jscs, inc-170 luding periods for reviewing and publishing, resulted in an average grade of 3.7. 171 as for the suggestions and remarks, besides no additional comments or affirm-172 ative opinion of 50 % of the interviewed authors, many contributors stated that 173 they would appreciate faster reviewing and publishing after manuscript accept-174 ance (approximately 30 % of suggestions). a significant number of authors sug-175 gested improvement of the instructions to authors and reduction of the technical 176 requirements for submission (especially for figures). additionally, a few authors 177 suggested a more careful choice of reviewers and a few recommended elevation 178 of the criteria for article acceptance. 179 after collection of the surveys, the responses were summarized and anal-180 yzed. a meeting of the editorial board was organized on this occasion and all 181 points were discussed individually. certain conclusions were drawn enabling a 182 detailed overview of the entire publishing process of the jscs, which further led 183 to decisions directed at improvement of the process. 184 all participants in the survey, in one way or another, strongly supported the 185 idea of a fully automatic on-line system: a) sub-editors in order not to have to 186 remind or thank reviewers by themselves and not to have to archive all reports 187 and letters to authors as their own database, b) reviewers in order to have the 188 ability to quickly review the abstract on-line, to have a direct choice to accept or 189 decline to review and to have access to an on-line report form and c) authors in 190 order to facilitate the submission step and to speed-up the reviewing process. 191 send back the proof corrections to jscs-proofs@shd.org.rs within next 48 hours! un co rre cte d p ro of 8 nedić and dekanski members of the editorial board agreed that a complete on-line system would 192 improve the management of the jscs and the decision was made to practice 193 exclusively on-line communication from march 2015. 194 195 fig. 3. authors’ responses to particular questions expressed as frequency distributions. 196 send back the proof corrections to jscs-proofs@shd.org.rs within next 48 hours! un co rre cte d p ro of jscs publishing policies survey 9 table iii. authors’ responses to questions that were recorded by scaling 197 question grade average grade 5 4 3 2 1 share of the number of responses,% comprehensiveness of the guide for authors 22 41 27 5 5 3.7 usefulness of the reviewers’ reports 19 46 27 5 3 3.7 period for reviewing 22 36 30 9 3 3.6 period for publishing 19 38 31 7 5 3.6 communication with sub-editors and editor 35 37 23 3 2 4.0 frame 3. major suggestions and remarks of authors, grouped by topics (number of indi-198 vidual comments) 199 50 % of all comments were affirmative +: 1. speeding-up reviewing and publishing process 150 2. improvement of the instructions for authors and reduction of technical requirements for manuscript submission 88 3. better choice of reviewers and “blind” review 30 4. promotion and advertising of the jscs 15 5. increasing the number of published articles per year 12 6. periodical special issues 5 7. invitation of respectable scientists to write review articles 5 8. addition of new research fields (chemical education, nano-chemistry, chemistry in agriculture) 5 it was noticed that similar number of reviewers suggested longer (expected) 200 and shorter (unexpected) period for reviewing which initiated deeper data 201 analysis. reviewers who were also authors could not separate these two roles and 202 the impression of the author dominated the impression of the reviewer. 203 authors who suggested faster publishing do not seem to differentiate clearly 204 between peer review and publishing. members of the editorial board agreed to 205 contribute personally to speed-up the reviewing part by faster communication 206 with (potential) reviewers and/or by increasing the number of initially invited 207 reviewers. it is, however, difficult to stimulate researchers to review for the 208 jscs. many invited persons do not respond at all, many refuse, some accept but 209 never send the report and a considerable number of reviewers send inappropriate 210 reports. the reasons for such a situation may be seen as a lack of profession-211 alism, not very good opinion on the jscs based on the journal’s ranking and/or 212 prejudices. potential reviewers from west europe and north america are among 213 those who most frequently do not respond or decline to review. a similar attitude 214 towards the journal is also valid for the (potential) authors. when nationalities of 215 the jscs authors were analyzed, it became obvious that contributors from west 216 europe and north america are rare. 217 the second part of the publishing process, which includes the actual print-218 ing, can hardly be faster, as there are many accepted papers and the jscs is 219 send back the proof corrections to jscs-proofs@shd.org.rs within next 48 hours! un co rre cte d p ro of 10 nedić and dekanski already publishing more articles per year than similar non-profitable journals run 220 by scientific societies. since accepted papers receive doi numbers and are avail-221 able in an on-line version of the journal few days after their acceptance, the print-222 ing step is not crucial from the point of article visibility by the scientific com-223 munity. steps will be taken to see how this part of the process could also be 224 improved. 225 it was interesting to notice that certain number of authors suggested the 226 application of more rigorous criteria for the evaluation of manuscripts. in their 227 opinion, some of the published articles should not have been accepted. thus, a 228 considerable number of our authors have the potential to produce high quality 229 papers and they will certainly be engaged as reviewers if they agree. 230 instructions to authors and reviewer’s report form were revised and new 231 versions up-loaded. moreover, the editorial board decided to reduce the technical 232 requirements for figures and to make small alterations if necessary without 233 asking authors to do so. 234 for the moment, the comments and suggestions that raised the greatest con-235 cerns were dealt with and some new approaches applied. other conclusions that 236 could be drawn from the responses in the questionnaires and members of the 237 editorial board will continue to analyze the data, both at the level of the entire 238 journal and at the level of sections run by particular sub-editors. 239 the editorial board thanks all participants of the survey who have helped to 240 evaluate the publishing process in the journal of the serbian chemical society 241 and to become aware of the imperfections. it is also hoped that the actions 242 undertaken after the survey will be welcomed by past, present and future contri-243 butors. 244 supplementary material 245 the questionnaires that the sub-editors, reviewers and authors were requested to fill out 246 online are available electronically from http://www.shd.org.rs/jscs/, or from the corres-247 ponding authors on request, as portable document format (pdf) files. 248 acknowledgment. this survey and article resulted as part of scientific activity in the 249 cost action td1306 “new frontiers of peer review (peere)”. 250 и з в о д 251 анкета о часопису „journal of the serbian chemical society“ – 252 обележавање штампања 80. годишта 253 олгица недић1 и александар декански2 254 1 институт за примену нукеларне енергије (инеп), универзитет у београду и 2 институт за хемију, 255 технологију и металургију, центар за електрохемију, универзитет у београду 256 часопис journal of the serbian chemical society (jscs) је научни часопис српског 257 хемијског друштва и ове године се обележава 85 година његовог излажења и излазак 80. 258 годишта. после толико година објављивања научних радова, жеља уредништва је била да 259 утврди шта аутори, рецензенти и подручни уредници мисле о часопису и уређивачкој 260 send back the proof corrections to jscs-proofs@shd.org.rs within next 48 hours! un co rre cte d p ro of jscs publishing policies survey 11 политици, и могу ли њихове процене и предлози помоћи да се делатност часописа по-261 бољша. упитници анкете су послати е-поштом испитаницима. добијени одговори су ана-262 лизирани и овде су приказани само најопштији и најзначајнији подаци. оцене, комен-263 тари и предлози су показали да је већина учесника у поступку објављивања задовољна 264 постојећим начином рада и уређивачком политиком, али мисле и да би се одређени 265 технички детаљи поступка могли побољшати. након сагледавања добијених одговора, 266 уредништво је донело одлуку да уведе високо-аутоматизовани кориснички сервис за 267 пријаву и обраду радова и тако убрза поступак рецензирања и објављивања, и да појед-268 ностави и додатно појасни упутство за ауторе и рецензентски формулар. све наведене 269 новине су уведене 1. марта 2015. 270 (примљено 6. марта, прихваћено 6. априла 2015) 271 references 272 1. k. iyengar, v. balijepally, scientometrics 102 (2015) 5 273 2. j. garner, a. l. porter, n. c. newman, scientometrics 100 (2014) 687 274 3. c. w. holsapple, j. am. soc. inform. sci. technol. 59 (2008) 166 275 4. p. o. seglen, br. med. j. 314 (1997) 498 276 5. a. dekanski, j. serb. chem. soc. 79 (2014) 1561 277 6. s. bojović, bull. soc. chim. 49 (1984) 751 278 7. a. r. despić, j. a. jovanović, b. ž. nikolić, d. vitorović, the serbian chemical society. 279 history, organization, activity, serbian chemical society, belgrade, 1996. 280 send back the proof corrections to jscs-proofs@shd.org.rs within next 48 hours! << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {prediction of excess molar volumes of binary mixtures by prigogine-flory-patterson (pfp) and extended real association solution (eras) models} j. serb. chem. soc. 82 (12) 1379–1390 (2017) udc 544.038.2+544.344.2’032:53.02:544.3 jscs–5048 original scientific paper 1379 prediction of excess molar volumes of binary mixtures by prigogine–flory–patterson (pfp) and extended real association solution (eras) models• ivona r. radović#, nikola d. grozdanić#, bojan d. djordjević#, slobodan p. šerbanović# and mirjana lj. kijevčanin*# faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11120 belgrade, serbia (received 17 august, revised 31 august, accepted 8 september 2017) abstract: in this paper, the prigogine–flory–patterson (pfp) theoretical model and the extended real association solution (eras) model for the prediction of the excess molar volumes of 17 binary sets of alcohols (methanol, ethanol, propan-1-ol, butan-1-ol, butan-2-ol and pentan-1-ol) with benzene, chlorobenzene, acetonitrile, hexan-1-amine and n-heptane were tested at 298.15 k and atmospheric pressure. the binary sets contained 333 experimental data points for 11 different compounds. the eras model demonstrated better correlative characteristics for binary mixtures comparing to pfp model. the eras model is successfully applied on all binary systems except ethanol + benzene and propan-1-ol + benzene. pfp model gave less satisfactory results for all systems except butan-1-ol + hexan-1-amine, for which obtained results were acceptable. keywords: binary mixtures; excess molar volume; prediction model; pfp; eras. introduction design of process and industrial equipment is based on the knowledge of the thermodynamic properties of fluids. the knowledge of thermodynamic and transport properties of pure components and their mixtures is very important for understanding intermolecular interactions in binary mixtures. solvent mixtures play an important role in chemical industries and their physiochemical properties often show large deviations from ideal behaviour. data of this properties are important for various engineering calculations and represent the starting points for the designing, analysis and optimization of technological processes. in addition to the experimental determination of thermodynamic parameters of pure sub * corresponding author. e-mail: mirjana@tmf.bg.ac.rs # serbian chemical society member. • this paper is dedicated to prof. bojan d. djordjević on the occasion of his 80th birthday. https://doi.org/10.2298/jsc170817103r ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1380 radović et al. stances and their mixtures, it is necessary to develop and test a reliable thermosdynamic model to replace, in some cases, very expensive experiments. prigogine–flory–patterson (pfp) model1–7 is the theoretical approach that can describe and predict the non-ideal behaviour of multicomponent systems. on the other hand, the original extended real associated solution (eras) model, introduced by heintz,8 combines the association effects arising from hydrogen bonding with free volume effects from the differences of van der waals interactions between unlike molecules in a mixture. the aim of this work was to test the applicability of these two models to correlate the excess molar volume data of 17 binary systems containing the widely applied organic compounds, alcohols (methanol, ethanol, propan-1-ol, butan-1-ol, butan-2-ol and pentan-1-ol) and benzene, chlorobenzene, acetonitrile, hexan-1-amine and n-heptane.9–13 the analyzed systems are divided into five groups: alcohols + aromates, alcohols + n-alkane, alcohols + amine, alcohols + acetonitrile and amine + n-alkane. selected systems show diverse deviations from ideal behaviour, e.g., highly negative, positive or s-shaped excess molar volumes vs. mixture composition curve. in this way, the applicability of the model for a specific type of non-ideal behaviour is confirmed. the modelling was performed at 298.15 k and atmospheric pressure. modelling in this paper predictive capabilities of pfp and eras models were tested for density calculations and excess molar volumes for large number of binary systems based on the previously measured experimental data. the selected systems are characterized by different forms of non-ideal behaviour and the results of this work can show the applicability of both models for different groups of compounds and mixtures. the pfp model is designed to analyze the thermodynamic functions of the binary systems. this theory is used for interpretation of the measurement results of some excess properties of the binary systems formed by molecular species that differ in size and shape. pure empirical application of the flory model4 can provide some interesting correlations of excess molar properties for complex mixtures. according to pfp model, the excess molar volume contains three contributions: interactional contribution, a free volume contribution and a pressure contribution. the expression for excess molar volume which separates the three contributions is given as: ( ) ( ) ( )( ) ( ) ( )( ) ( )( ) ( )( ) 2 1/3 2/3 1/3 1 2 12 1 2 1 2 * * 1/3 * 1/31 1 2 2 1 * * 1 2 1 2 1 2 * * 1 2 2 1 1 14 / 9 / 1 4 / 3 / 1 4 / 3 / 1 e v v v v vv x v x v v p v v v v p p p p ψ θ χ ψ ψ ψ ψ ψ ψ − − − = − + + − − − − + +           (1) where ve is excess molar volume, x mole fraction, v* characteristic volume, p* characteristic pressure, ψ molecular contact energy fraction, θ molecular surface fraction and χ12 parameter. reduced volume of binary mixture is defined as:14 1 1 2 2v v vψ ψ= +   (2) ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. pfp and eras models 1381 the characteristic parameters v* and p* are obtained from thermal expansion coefficient (1 / )( / )pv v tα = ∂ ∂ and isothermal compressibility, (1 / )( / )v v p τκ = ∂ ∂ : ( )( ) ( ) 3 1 4 / 3 , 1, 2 1 i i i t v i t α α  + = =  +   (3) * /i i iv v v= (4) 2 * , 1, 2i iii ii p tv p i p α κ = = =   (5) the molecular contact energy fraction is calculated by: ( ) * 1 1 1 2 * * 1 1 2 2 1 p p p ϕ ψ ψ ϕ ϕ = − = + (6) ( ) * 1 1 1 2 * * 1 1 2 2 1 x v x v x v ϕ ϕ= − = + (7) where ϕ is a hard-core volume fraction. the molecular surface fraction is calculated by: 1 1 11 2 1 1 2 2 1 2 1 2 1 ( / ) s s s s s ϕ ϕ θ θ ϕ ϕ ϕ ϕ = − = = + + (8) where si is the molecular fraction surface/volume ratio for the components determined by bondi’s method:15 1/3* 1 1 * 2 2 s v s v −   =     (9) , ln ρ α ∂ = − ∂ p xt (10) 2 21 / ( ) p t u c α κ ρ ρ = + (11) the values of the parameters for the pure liquid components and the mixture are obtained using the flory theory.4 the cross-interaction parameter required for the calculation of ve using pfp theory was derived from values of excess molar volumes at equimolar composition for each mixture. on the other hand, eras model combines real association solution model16–19 with flory equation of state. the excess function is written as: e e echem physv v v= + (12) where vechem is the chemical contribution, mainly due to the association reactions, and v e phys represents the physical contribution, which results from the physical interactions between molecules: ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1382 radović et al. ( ) ( ) ( ) ( ) e * 0 * 0 m a a a a1 b b b b11 1chem b1 a a1* a ab ab 1 b b b1 ab b1 1 1 a b a v v x k v x k v k x k v v v k k δ φ φ δ φ φ φ φ δ φ φ− = − + − + − +  − +   (13) ( )( )e * *a a b b m a a b bphys v x v x v v v vφ φ= + − −   (14) where ki are the association constants (i is a, b or ab) and it is assumed to be independent of the chain length. their temperature dependence is given by: * i i 0 0 1 1 exp h k k r t t δ    = − −        , i = a, b or ab (15) where k0 is the equilibrium constant at the atmospheric pressure and standard temperature t = = 298.15 k and ∆hi * the enthalpy change of reaction, which corresponds to the hydrogen bonding energy. these reactions are also characterized by the volume change ∆vi *, related to the formation of the linear chains. eras model is used to correlate the experimental values for the investigated mixtures and needs pure component properties to obtain the characteristic parameters v* and p* of each compound. it also needs data for density, ρ, isobaric thermal expansivity, α, and isothermal compressibility, κ, at atmospheric pressure and t = 298.15 k. results and discussion the goal of this research was to check if pfp and eras models are capable to correlate the excess molar volumes of different binary mixtures containing alcohols, aromates, n-alkanes, amines and nitriles. table i shows the literature density and the molar mass data for all constituents of investigated binary systems at atmospheric pressure and t = 298.15 k. table i. densities, ρ, and molar masses, m, of the pure compounds at 298.15 k and atmospheric pressure compound ρ / g cm-3 m / g mol-1 methanol9 0.78665 32.04 ethanol9 0.78525 46.07 propan-1-ol10 0.799692 60.09 butan-1-ol11 0.805762 74.12 butan-2-ol12 0.802528 74.12 pentan-1-ol13 0.810968 88.15 acetonitrile9 0.77669 41.05 hexan-1-amine11 0.760128 101.19 benzene10 0.873582 78.11 chlorobenzene13 1.101038 112.56 n-heptane11 0.679494 100.02 the values of parameters for the pure liquid components required for both pfp and eras models were obtained from the literature.20–26 table s-i of the supplementary material to this paper lists the literature for pure component data, ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. pfp and eras models 1383 self-association constant, k, thermal expansion coefficient, α, isothermal compressibility κ, reduced pressure p*, reduced volume v*, molar enthalpy change of association, ∆h*, molar volume change of association, ∆v*, and surface volume ratio s at atmospheric pressure and t = 298.15 k. the pfp parameter, χ12, in eq. (1), can be calculated at given temperature by fitting the experimental excess molar volumes of binary mixture over the entire composition range. the values of the interaction parameter χ12 for the studied mixture have been calculated using the equimolar ve values and they are listed in table s-ii (supplementary material) together with the equimolar values for all contributions to ve (interactional, free volume and p* effect contribution). the parameters kab, ∆vab*, and xab in the eras model were correlated and listed in table s-iii (supplementary material). the quality of the fit for the applied models was estimated by the standard deviation, σ and average percent deviation, d: ( ) 0.5 2 exp 1 n e e cal i v v n npar σ =   −    =  −       (16) e e exp cal e exp1 100 n i v v d n v= − =  (17) where npar is the number of parameters. the obtained results with pfp and eras models, for all systems, are listed in table ii. the results obtained for the selected binary systems, representing each group of analyzed binary mixtures, are presented in figs. 1–7. the systems selected for the analyses are divided into five groups: alcohols + aromates, alcohols + n-alkane, alcohols + amine, alcohols + acetonitrile and amine + n-alkane. selected systems show different deviations from ideal behaviour, e.g., negative, positive or s-shape (positive and negative) ve vs. x1 curves. in all cases the eras model was significantly superior comparing to pfp model. the systems of alcohols with aromates were the largest group in this research, i.e., five systems with benzene and six systems with chlorobenzene were analyzed. the systems of analyzed alcohols with chlorobenzene exhibit s-shape ve vs. x1 curve. as it was already discussed in the literature10 the addition of large amounts of chlorobenzene to a pure alcohol induces the de-polymerization of normal alcohols, resulting in volume expansion and positive ve values. the contraction in volume is characteristic for higher alcohol concentrations due to the ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1384 radović et al. formation of oh–cl atom hydrogen bonded complexes, between an alcohol and chlorobenzene. structural effects can also contribute to closer geometrical packing and volume contraction, which is the prevailing factor in the mixture with methanol, since methanol is the smallest molecule. the systems with benzene (figs. 1 and 2) also show sigmoidal ve vs. x1 curves, but the negative effects are less pronounced comparing to the equivalent systems with chlorobenzene. the possible interactions between alcohols and benzene aromatic ring are the oh-π type27, which are much weaker comparing to oh-cl interactions between alcohols and chlorobenzene. it is obvious that pfp model is not capable to correlate ve experimental data of those systems, with errors exceeding 100 % (fig. 2). on the other hand eras model was rather successful in some cases. for the alcohol + chlorobenzene the systems percentage deviations are ranged from about 7 to 15 %, while for the alcohol + benzene systems the deviations were higher especially for the systems containing ethanol or 1-propanol (see fig. 2). although the errors for the ethanol + benzene system are high (10.97 %), fig. 1 confirms that eras model fits experimental point rather well, except in the dilute regions. table ii. average relative deviation, d and the standard deviation, σ obtained with pfp and eras models mixture pfp eras d / % σ / cm3 mol-1 d / % σ / cm3 mol-1 methanol + benzene27 103.25 60.097 10.97 0.0017 ethanol + benzene28 203.20 51.452 217.45 0.0276 propan-1-ol + benzene9 1120.70 37.7 181.05 0.0203 butan-1-ol + benzene29 56.65 21.7 12.65 0.0096 butan-2-ol + benzene31 18.34 9.55 6.71 0.0194 methanol + chlorobenzene27 865.59 13.7 7.28 0.0030 ethanol + chlorobenzene27 202.61 17.6 14.02 0.0051 propan-1-ol + chlorobenzne12 156.60 26.1 8.53 0.0062 butan-1-ol + chlorobenzene12 249.09 32.8 15.18 0.0061 butan-2-ol + chlorobenzene12 229.09 36.5 6.72 0.0030 pentan-1-ol + chlorobenzene12 158.62 37.9 14.17 0.0063 butan-1-ol + n-heptane30 45.45 20.9 9.87 0.0078 butan-2-ol + n-heptane32 20.18 10.2 5.36 0.0160 butan-1-ol + hexan-1-amine11 2.66 1.41 1.95 0.0129 methanol + acetonitrile7 26.11 15.4 3.03 0.0026 ethanol + acetonitrile27 240.09 39.0 29.06 0.0037 hexan-1-amine + n-heptane9 20.90 8.95 3.16 0.0042 two systems containing an alcohol (butan-1-ol and butan-2-ol) and n-heptane and one system of an amine (hexan-1-amine) and n-heptane were also analyzed. all systems exhibit positive ve vs. x1 trend. in these mixtures the unlike molecules are not capable to make heteroassociates due to hydrogen bonding. con ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. pfp and eras models 1385 0.0 0.2 0.4 0.6 0.8 -0.02 -0.01 0.00 0.01 0.02 0.03 10 6 v e /m 3 m ol -1 x 1 0.0 0.2 0.4 0.6 0.8 -0.03 -0.02 -0.01 0.00 0.01 0.02 0.03 0.04 10 6 v e /m 3 m ol -1 x 1 a) b) fig. 1. the experimental excess molar volumes compared with the correlated values via pfp theory and eras model for binary mixture of methanol + benzene at 298.15k; a) pfp theory and b) eras model (-) physical contribution, (– ·–) chemical contribution. 0.0 0.2 0.4 0.6 0.8 1.0 -0.06 -0.04 -0.02 0.00 0.02 0.04 0.06 0.08 10 6 v e / m 3 m ol =1 x 1 0.0 0.2 0.4 0.6 0.8 1.0 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 10 6 v e /m 3 m ol -1 x 1 a) b) fig. 2. the experimental excess molar volumes compared with the correlated values via pfp theory and eras model for binary mixture of ethanol + benzene at 298.15k; a) pfp theory and b) eras model (-) physical contribution, (– ·–) chemical contribution. trary to that, both alcohol and amine molecules are making homoassociates. it is assumed that n-heptane added to a pure alcohol/amine causes disruption of alcohol or amine homoassociates and volume expansion, i.e., positive ve values. increase in the volume in butan-2-ol + n-heptane mixture is higher, compared to butan-1-ol + n-heptane system, while both ve curves are slightly skewed towards mixtures rich in n-heptane. the same trend, the ve–x1 shape, is also characteristic for the hexan-1-amine + n-heptane system. both models work better for the system butan-2-ol + n-heptane, since the ve curves are more symmetric. the average percentage deviation for the eras model and butan-1-ol + n-heptane system ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1386 radović et al. goes up to 10 %, though eras model curve follows experimental data very well (see figs. 3 and 4). 0.0 0.2 0.4 0.6 0.8 0.00 0.05 0.10 0.15 0.20 10 6 v e / m 3 m ol -1 x 1 0.0 0.2 0.4 0.6 0.8 -0.05 0.00 0.05 0.10 0.15 0.20 0.25 1 06 v e /m 3 m ol -1 x 1 a) b) fig. 3. the experimental excess molar volumes compared with the correlated values via pfp theory and eras model for binary mixture of butan-1-ol + n-heptane at 298.15k; a) pfp theory and b) eras model(-) physical contribution, (– ·–) chemical contribution. 0.0 0.2 0.4 0.6 0.8 0.0 0.1 0.2 0.3 0.4 0.5 10 6 v e /m 3 m ol -1 x 1 0.0 0.2 0.4 0.6 0.8 0.0 0.1 0.2 0.3 0.4 0.5 10 6 v e /m 3 m ol -1 x 1 a) b) fig. 4. the experimental excess molar volumes compared with the correlated values via pfp theory and eras model for binary mixture of butan-2-ol + n-heptane at 298.15k; a) pfp theory and b) eras model(-) physical contribution, (– ·–) chemical contribution. in figs. 5 and 6 two systems of methanol and ethanol with acetonitrile are shown. the excess molar volumes for the system with methanol show small negative values, while the ve–x1 curve for the system with ethanol is s-shaped, i.e., positive for the mixtures rich in acetonitrile and negative for higher ethanol concentrations. from shown figures it is obvious that eras model succeeds to follow the s-shaped ve curve, although with much higher average relative deviation, comparing to the system with ethanol. on the other hand pfp model com________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. pfp and eras models 1387 pletely fails to describe the complex ve behaviour of ethanol + acetronitrile system. for the system with methanol the situation is better, however the pfp model still does not follow the asymmetric ve vs. x1 trend. 0.0 0.2 0.4 0.6 0.8 -0.16 -0.14 -0.12 -0.10 -0.08 -0.06 -0.04 10 6 v e / m 3 m ol -1 x 1 0.0 0.2 0.4 0.6 0.8 -0.16 -0.14 -0.12 -0.10 -0.08 -0.06 -0.04 -0.02 0.00 10 6 v e /m 3 m ol -1 x 1 a) b) fig. 5. the experimental excess molar volumes compared with the correlated values via pfp theory and eras model for binary mixture of methanol + acetonitrile at 298.15k; a) pfp theory and b) eras model(-) physical contribution, (– ·–) chemical contribution. 0.0 0.2 0.4 0.6 0.8 1.0 -0.08 -0.06 -0.04 -0.02 0.00 0.02 10 6 v e /m 3 m ol -1 x 1 0.0 0.2 0.4 0.6 0.8 -0.10 -0.08 -0.06 -0.04 -0.02 0.00 0.02 0.04 10 6 v e /m 3 m ol -1 x 1 a) b) fig. 6. the experimental excess molar volumes compared with the correlated values via pfp theory and eras model for binary mixture of ethanol + acetonitrile at 298.15k; a) pfp theory and b) eras model (-) physical contribution, (– ·–) chemical contribution. the mixtures of alcohols and amines show highly negative excess molar volume data with the symmetric ve vs. x1 curve,9 due to the hydrogen bonding and hetero-association between alcohol and amine molecules. this type of nonideal behaviour can be successfully fitted by both pfp and eras models, yet the eras model fits minimal region slightly better than pfp model. from the all mentioned above it is obvious that except for the alcohol + amine mixtures, the ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1388 radović et al. pfp model was not able to describe the different ve–x1 curve shapes. the best results were achieved for the hexan-1-amine + n-heptane system (see fig. 7). 0.0 0.2 0.4 0.6 0.8 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 10 6 v e /m 3 m ol -1 x 1 0.0 0.2 0.4 0.6 0.8 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 10 6 v e /m 3 m ol -1 x 1 a) b) fig. 7. the experimental excess molar volumes compared with the correlated values via pfp theory and eras model for binary mixture of hexan-1-amine + n-heptane at 298.15k; a) pfp theory and b) eras model (-) physical contribution, (– ·–) chemical contribution. conclusions in this paper, two theoretical models, the prigogine–flory–patterson (pfp) and the extended real association solution (eras) for the excess molar volume correlation were tested on 17 binary mixture systems divided into five groups: alcohols + aromates, alcohols + n-alkane, alcohols + amine, alcohols + acetonitrile and amine + n-alkane. the excess molar volumes for binary mixtures are described qualitatively for all ranges of mole fractions by the pfp and the eras model. both models can be applied on the systems that have only positive or negative excess molar volume values over the whole range of mole fractions, where the eras model gives better results. also, the eras model was much better comparing to the pfp for systems that have both, positive and negative excess molar volume values, over the whole range of mole fractions. the pfp theory is able to describe the systems with only negative or only positive excess molar volume values over the whole range of mole fractions. however, if the ve– –x1 curve is asymmetric and tends to shifts towards higher or lower concentration than an equimolar point, the errors are getting higher. the comparison of calculated and experimental excess properties of binary systems shows that the eras model is able to describe all the excess functions ranging from positive values to strong negative values, as well as the s-shaped ve–x1 curves, which cannot be completed for the pfp model. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. pfp and eras models 1389 supplementary material tables s-i–s-iii are available electronically at the pages of journal website: http:// //www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. this work was financially supported by the research fund of ministry of education, science and environmental protection, serbia, the faculty of technology and metallurgy, university of belgrade, serbia (project no 172063). и з в о д одређивање допунске моларне запремине бинарних система помоћу prigogine–flory–patterson (pfp) и extended real association solution (eras) модела ивона р. радовић, никола д. грозданић, бојан д. ђорђевић, слободан п. шербановић и мирјана љ. кијевчанин технолошко–металуршки факултет, универзитет у београду, карнегијева 4, 11120 београд у овом раду је извршено израчунавање допунских моларних запремина 17 бинарних система алкохола (метанол, етанол, пропан-1-ол, бутан-1-ол, бутан-2-ол и пентан-1 -ол) са бензеном, хлорбензеном, ацетонитрилом, хексан-1-амином и n-хептаном на температури од 298,15 k и на атмосферском притиску, коришћењем теоријских модела: prigogine–flory–patterson (pfp) и extended real association solution (eras). бинарни системи садржали су 333 експерименталне тачке за 11 различитих једињења. eras модел показао је боље корелативне карактеристике за бинарне смеше у поређењу са pfp моделом. eras модел успешно је примењен на свим бинарним системима, осим на смеше етанол + бензен и пропан-1-ол + бензен. pfp модел генерално даје лошије резултате за све системе, осим за систем бутан-1-ол + хексан-1-амин, за који су добијени прихватљиви резултати. (примљено 17. августа, ревидирано 31. августа, прихваћено 8. септембра 2017) references 1. i. prigogine, a. bellemans, v. mathot, the molecular theory of solutions, north holland, amsterdam, 1957 2. p. j. flory, r. a. orwoll, a. vrij, j. am. chem. soc. 86 (1964) 3507 3. p. j. flory, r. a. orwoll, a. vrij,j. am. chem. soc. 86 (1964) 3515 4. p. j.flory, j. am. chem. soc. 87 (1965) 1833 5. a. abe, p. j.flory, j. am. chem. soc. 87 (1965) 1838 6. r. a. orwoll, p. j.flory, j. am. chem. soc. 89 (1967)6814 7. d. patterson, g. delmas, discuss. faraday soc. 49 (1970) 98 8. a. heintz, ber. bunsenges. phys. chem. 89 (1985) 172 9. i. r. grgurić, a. ž. tasić, b. d. djordjević, m. lj. kijevčanin, s. p. šerbanović, j. serb. chem. soc. 67 (2002) 581 10. m. lj. kijevčanin, i. m. purić, i. r. radović, b. d. djordjević, s. p. šerbanović, j. chem. eng. data 52 (2007) 2067 11. i. r. radović, m. lj. kijevčanin, s. p. šerbanović, b. d. djordjević, fluid phase equilib. 298 (2010) 117 12. i. r. radović, m. lj. kijevčanin, e. m. djordjević, b. d. djordjević, s. p. šerbanović, fluid phase equilib. 263 (2008) 205 13. i. r. radović, m. lj. kijevčanin, a. ž. tasić, b. d. djordjević, s. p. šerbanović, j. serb. chem. soc. 74 (2009) 1303 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1390 radović et al. 14. y. sun, l. su, h.wang, j. chem. thermodynamics 41 (2009) 1154 15. a. bondi, physical properties of molecular crystals, liquids and glasses, wiley, new york, 1968 16. c. b. kretschmer, r. wiebe, j. chem. phys. 22 (1954) 1697 17. h. renon, j.m. prausnitz, chem. eng. sci. 22 (1967) 299 18. h. v. kehiaian, bull. acad. pol. sci. 16 (1968) 165 19. h. v. kehiaian, a. j. treszczanowicz, bull. acad. pol. sci. 16 (1968) 171 20. m. bender, a. heintz, fluid phase equilib. 89 (1993) 197 21. e. n. rezanova, k. kammerer, r. n. lichtenthaler, j. chem. thermodynamics 32 (2000) 1569 22. a. heintz, p. k. naicker, s. p. verevkin, r. pfestorf, ber. bunsen – ges. phys. chem. 102 (1998) 953 23. r. b. torres, a. z. francesconi, p. l. volpe, fluid phase equilib. 210 (2003) 287 24. u. domanska, m. gloskowska, fluid phase equilib. 216 (2004) 135 25. b. orge, m. iglesias, g. marino, m. domınguez, m. m. pineiro, j. tojo, fluid phase equilib. 170 (2000) 151 26. s. p. ijardar, n. i. malek, s. l. oswal, ind. j. chem., a 50 (2011) 1709 27. s. p. šerbanović, m. lj. kijevčanin, i. r. radović, b. d. djordjević, fluid phase equilib. 239 (2006) 69 28. i. r. grgurić, s. p. šerbanović, m. lj. kijevčanin, a. ž. tasić, b. d. djordjević, thermochim. acta 412 (2004) 25 29. j. d. smiljanić, m. lj. kijevčanin, b. d. djordjević, d. k. grozdanić, s. p. šerbanović, int. j. thermophys. 29 (2008) 586 30. m. lj. kijevčanin, i. r. radović, s. p. šerbanović, a. ž. tasić, b. d. djordjević, thermochim. acta 496 (2009) 71 31. 31. j. d. smiljanić, m. lj. kijevčanin, b. d. djordjević, d. k. grozdanić, s. p. šerbanović, j. chem. eng. data 53 (2008) 1965 32. m. lj. kijevčanin, i. r. radović, s. p. šerbanović, e. m. živković, b. d. djordjević, j. chem. eng. data 55 (2010) 1739. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1391 a tribute to professor bojan djordjević in honour of his 80th birthday journal of the serbian chemical society wishes to draw attention to professor djordjević’s rich legacy and superior contribution to the field of thermodynamics in general and to jscs in particular. prof. dr. bojan d. djordjević was born in 1937 in belgrade where he graduated from the faculty of technology, university of belgrade in 1961 and commenced his career as research fellow in ihtm, belgrade. he stayed in ihtm for the period from 1961 to 1964. he received his phd in 1970 from the institute of nuclear sciences “vinča“ and the faculty of technology where in 1971 he was promoted to the position of the assistant professor and in 1976 to the position of the associate professor. since 1982 he had been full professor at the faculty of technology and metallurgy, where he had been extensively involved in curricula development and teaching of various subjects of thermodynamics, including but not limited to chemical engineering thermodynamics, energy of process industry, and molecular thermodynamics. during his fruitful carrier he also mentored hundreds of graduate students supporting them selflessly to commence their own academic paths. further, professor djordjević lectured different subjects of thermodynamics at many other university centers (novi sad, podgorica, priština, the military–technical academy, belgrade, higher technical school, kosovska mitrovica, etc.). he is the author of 27 text-books and monographs including thermodynamics and thermotechnics, thermodynamics of fluids, chemical engineering thermodynamics, and heat transfer. professor djordjević was a foundational member of the laboratory for chemical engineering parameters. in two terms he was the head of fundamental technical science department and, also, led a national research projects and subprojects. he contributed to over 50 technical studies and projects, many of which have been applied in chemical industry (mineloprema, pančevo; oil refinery, ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1392 beolgrade; oil refinery, pančevo; hip, pančevo; miloje zakić, kruševac, železara, smederevo, etc.) and he also established cooperation with imperial chemical industries, united kingdom and university of berkley, usa. his research has incorporated a wide range of fundamental areas, with a focus on equations of state, vapour–liquid equilibria, excess properties of solution and thermo-hydraulic design of heat equipment. professor djordjevic has also been very active in the yugoslav association of chemists and technologists. he is a member of serbian scientific society, serbian chemical society and american chemical society. he received a number of national and international acknowledgments, awards, plaques and certificates: men of achievement for distinguished achievement, cambridge, england, 1982.; 5.000 personalities of the word, american biographical institute; the international who’ s who of intellectuals, cambridge, england, 1983; certificate of merit for distinguished service to the community, cambridge, england, 1984.; who’s who in the world 1980/1981; who’s who in the world 1982/1983; medal for outstanding and long-lasting contribution to science (for development of chemical engineering thermodynamics), serbian chemical society, 2000; honorary diploma on the 25th anniversary of the faculty of technology, university of novi sad, for a significant contribution to the work and development of the faculty of technology, university of novi sad, 1984; certificate as a sign of acknowledgment and gratitude for the great contribution to the work and development of the serbian chemical society on the occasion of 100th anniversary 1997. professor djordjević authored and contributed to over 100 scientific articles, more than hounded scientific papers presented on international and national congresses and symposia and over 30 professional papers. his articles were published in international journals, such as: journal of chemical thermodynamic, fluid phase equilibria, journal of chemical engineering data, chemical engineering science, aiche journal, canadian journal of chemical engineering, industrial & engineering chemistry process design and development, industrial & engineering chemistry research, thermochimica acta, physics and chemistry of liquids, chemie ingenieur technik, korean journal of chemical engineering, journal of applied polymer science, hungarian journal of industrial chemistry, chemische technik, journal of chemical education, etc. being internationally recognized and respected, professor djordjević was cited over 500 times in the international journals, university textbooks and monographs. he was a referee in the leading international and national journals and congresses and was a member of editorial boards of the journal of the serbian chemical society and chemical industry. professor djordjević has made an exceptional contribution to the field of thermodynamics throughout his career, and has also been graciously involved in supporting the journal of the serbian chemical society. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1393 being a member of editorial board of the journal of the serbian chemical society (more than 30 years) and publishing more than 40 scientific papers in this journal*, professor djordjević provided an outstanding contribution to journal of the serbian chemical society development and reputation as well. it is therefore great pleasure to honour him in this tribute, on the celebration of his 80th birthday. we join in wishing him well in the future and thanking him for all the work and the high standards he set for all of us. mirjana kijevčanin ivona radović slobodan šerbanović * the paper jscs–5048 in this issue is dedicated to professor djordjević on the occasion of his jubilee. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {computational, antimicrobial, dna binding and anticancer activities of pyrimidine incorporated ligand and its copper(ii) and zinc(ii) complexes} j. serb. chem. soc. 84 (3) 277–291 (2019) udc 546.562’472+519.677:547.963.32+ jscs–5183 547.853:615.277 original scientific paper 277 computational, antimicrobial, dna binding and anticancer activities of pyrimidine incorporated ligand and its copper(ii) and zinc(ii) complexes murugesan sankarganesh1, nagaraj revathi2,3, jeyaraj dhaveethu raja1*, karunganathan sakthikumar1, gujuluva gangatharan vinoth kumar1, jegathalaprathaban rajesh1, manikkam rajalakshmi4 and liviu mitu5** 1chemistry research centre, mohamed sathak engineering college, kilakarai, ramanathapuram, tamil nadu 623 806 india, 2department of chemistry, ramco institute of technology, rajapalayam, virudhunagar, tamil nadu 626 117, india, 3department of chemistry, manonmanium sundaranar university, tirunelvi, tamil nadu 627 012, india, 4bioinformatics centre, pg & research centre department of biotechnology and bioinformatics, holy cross college (autonomous), tiruchirapalli, tamil nadu 620 002, india and 5department of nature sciences, university of pitesti, pitesti 110 040, romania (received 6 june, revised 7 august, accepted 5 september 2018) abstract: in this research article, the synthesis, structural characterization, and biological, dna binding and anticancer properties of pyrimidine incorporated schiff base ligand l and its [cul2](clo4)2 (1) and [znl2](clo4)2 (2) complexes are reported. the isolated complexes 1 and 2 have significant antibacterial and antifungal properties, greater than those of ligand l. the interaction between protein and l were analyzed by an in silico method. the intercalative binding of the prepared compounds was proved from electronic absorption, fluorometric, cyclic voltammetric and viscometric methods. the calculated binding parameters such as, kb (2.65×103, l; 7.74×103, 1 and 2.99×103, 2); ksv (3.30×103, l; 4.31×103, 1 and 3.89×103, 2), and kapp (2.15×105, l; 3.30×105, 1 and 2.82×105, 2) indicted that complex 1 has better interaction ability than l and complex 2. the in vitro anticancer properties of l, and complexes 1 and 2 against human cancer (mcf-7, hela and hep-2) and normal (nhdf) cell lines were determined by the mtt assay method. the obtained results designated that complexes 1 and 2 exhibited substantial anticancer activity against the cancer cell lines, better than that of l. keywords: metal complexes; dft; antimicrobial; dna interaction; anticancer studies. *,** corresponding authors. e-mail: (*)jdrajapriya@gmail.com; (**)ktm7ro@yahoo.com https://doi.org/10.2298/jsc180609080s 278 sankarganesh et al. introduction pyrimidine is a heterocyclic compound and it forms the backbone of deoxyribonucleic acid (dna) and ribonucleic acid (rna). pyrimidine derivative drugs reveal various pharmacological and biological activities, such as, antitumor,1,2 antiamoebic,3 antimalarial,4,5 antipneumocystis carinii pneumonia6 and antimicrobial.2,7,8 moreover, the attachment of cf3 groups results in compounds that are lipophilically and pharmacologically more active than the corresponding non -fluorinated compounds. these –cf3-substituted organic compounds have various biological competences, such as, herbicidal, fungicidal, analgesic, antihyperglycemic and antipyretic, etc.9–13 pyrimidine-containing drugs, such as gefitinib, erlotinib and afatinib were used in cancer-related treatments.14 therefore, schiff base ligands were prepared from biologically and pharmacologically active pyrimidine derivative compounds. these schiff base ligands possess analytical, biological and pharmacological applications.15 in earlier days, schiff bases were employed to develop metal-based complexes16,17 because metal complexes acquire diverse biological abilities, such as antidiabetic, antimicrobial, antioxidant, dna cleavage or binding and anticancer activities.18–20 especially, copper(ii) and zinc(ii) complexes were synthesized with much more interest due to their enhanced biological applications.21–23 the interest in the biological properties of copper(ii) and zinc(ii) complexes has been continued in the present study through incorporation of a bio-active pyrimidine ligand. the formations of l, and complexes 1 and 2 were confirmed from analytical, spectroscopic and theoretical techniques. moreover, their biological properties, such as antimicrobial, dna binding and in vitro anticancer activities were studied. experimental materials and methods 4-(4-morpholinyl)benzaldehyde, 2-hydrazino-4-(trifluoromethyl)pyrimidine, tris-hcl, sodium chloride and ethidium bromide were procured from sigma aldrich. cu(clo4)2·6h2o and zn(clo4)2·6h2o were received from alfa aesar company. calf thymus (ct) dna was purchased from genei bangalore, india. elemental analyses were realized on an elementar vario el iii analyzer. ftir and nmr spectra were recorded using an ftir affinity-1 shimadzu instrument and bruker (400 and 125 mhz) spectrometers. the mass spectra were obtained on an esi-ms spectrometer. the esr spectra were recorded at 300 and 77 k in an iit, mumbai. cyclic voltammetric studies were recorded on a chi650c instrument. the absorption and fluorescence spectra were recorded on a uv-1800 shimadzu spectrophotometer and fluoromax-4 spectrometer, horiba, respectively. analytical and spectral data are given in supplementary material to this paper. synthesis of ligand l ligand l was synthesized by refluxing 4-(4-morpholinyl)benzaldehyde (0.1912 g, 1 mmol) with 2-hydrazino-4-(trifluoromethyl)pyrimidine (0.1781 g, 1 mmol) in 25 ml of ethanol for 6 h. the resulting solution was slowly evaporated on a water bath. the yellow col pyrimidine incorporating metal(ii) complexes 279 oured product was collected by filtration and washed with 10 ml of cold methanol and recrystallized from hot chloroform. synthesis of complexes 1 and 2 an ethanolic solution of ligand l (2 mmol) was mixed with metal perchlorates (1 mmol) in 10 ml of ethanol maintaining a metal : ligand ratio of 1 : 2. the resulting solution was refluxed under stirring for 5 h. then the solution was evaporated on a water bath, the precipitated solid filtered and washed thoroughly with 10 ml of petroleum ether and dried in vacuo over cacl2. computational studies density functional theory (dft) calculations were performed with the hybrid exchange -correlation function using the 6-311g(d,p) and lanl2dz basis set by the gaussian 09 program to understand the mode of complexation between the ligand and its complexes.24 initially, the optimized geometries of both the ligand and its complexes were determined by the b3lyp functional but using 6-311g(d,p) and lanl2dz basis set, respectively. antimicrobial studies the antimicrobial activity of the prepared compounds were screened against the bacterial species, escherichia coli, klebsiella pneumoniae, pseudomonas fluorescens, shigella sonnei and staphylococcus aureus, and the fungal species, aspergillus niger, candida albicans, c. tropicalis, mucor indicus and rhizopus by the well diffusion method.21 the reference drugs streptomycin and amphotericin were used for the antibacterial and antifungal studies, respectively. anticancer studies the anticancer capabilities of ligand l, complexes 1 and 2 against various cancer cell lines (mcf-7, breast adenocarcinoma; hela, cervical, hep-2, laryngeal) and a single normal cell line (nhdf, normal human dermal fibroblasts) were performed by the mtt assay25: absorbance of sample inhibition, % 100 100 absorbamce of control = − (1) protein–ligand docking the docking of ligand l with protein was studied by a previously reported method.26 the prepared proteins and ligand l were docked using the libdock module of accelrys discovery studio software, version 2.1, to acquire the drug with protein interaction. dna interaction the ct-dna interaction of ligand l and complexes 1 and 2 in 5 mm and 50 mm trishcl buffer solution, respectively, under physiological conditions were appraised using absorption spectroscopy and fluorescence, cyclic voltammetric and viscometric measurements.27-29 results and discussion the synthetic route to ligand l, and complexes 1 and 2 are depicted in schemes 1 and 2, respectively. the ligand l (4-(4-morpholinyl)benzaldehyde, 2-[4-(trifluoromethyl)-2-pyrimidinyl]hydrazone) is lemon yellow in colour and well soluble in acetone, acetonitrile, dmso and dmf solvents. the complexes 1 and 2 are brown and dark brown colour and soluble in dmso and dmf solvents. the molar conductance at infinite dilution (λm) of complexes 1 and 2 in dmso 280 sankarganesh et al. was found at 104 ω–1 cm2 mol–1 for complex 1 and 109 ω–1 cm2 mol–1 for complex 2, which signified them to be electrolytes. moreover, the obtained conductance values confirmed that percholarate ions are present in the outside coordination sphere of complexes 1 and 2. scheme 1. synthesis of ligand l. scheme 2. synthesis of complexes 1 and 2. nmr spectra the 1h-nmr spectra of ligand l and complex 2 were recorded in dmso-d6 solution (supplementary material). in the 1h-nmr spectrum of ligand l, the azomethine (–ch=n–) proton signal appeared at 8.10 (1h, s) and the nh proton signal at 9.90 (1h, s) ppm. the pyrimidine (–ch=c–cf3 and =ch–n=) protons signals were observed at 7.19 (1h, d) and 8.77 (1h, d) ppm, respectively. the signals of the aromatic protons were found at 6.99 (2h, d) and 7.55 (2h, d) ppm. the morpholine moiety protons (mor-ch2–n–ch2– and mor-ch2–o–ch2–) signals appeared at 3.20 (4h, t) and 3.75 (4h, t) ppm, respectively. in the 1h-nmr spectra of complex 2, the signals of the azomethine (–ch=n–) and pyrimidine moiety protons (=ch–n=) appeared at 8.64 (1h, s) and 8.83 (1h, d), respectively. these spectral results reveal that the azomethine and pyrimidine nitrogen atoms participate in the complexation. as per comparison with the free ligand l, the signals of the pyrimidine (–ch=c–cf3 and –nh), aromatic and morpholine (mor-ch2–n–ch2– and mor-ch2–o–ch2–) protons remained more or less at the same positions in complex 2. the 13c-nmr spectra of ligand l and complex 2 were taken in dmso-d6 solution and the obtained data are given in the supplementary material. in the pyrimidine incorporating metal(ii) complexes 281 spectra of ligand l, the azomethine (–ch=n), pyrimidine c2 and c6 appeared at 138.60, 160.61 and 153.60 ppm, respectively. in complex 2, these peaks appeared at 139.45, 161.10 and 154.16 ppm, respectively. these results indicate that ligand l formed coordinate bonds with the central metal atom via azomethine and pyrimidine ring (c2) nitrogen atoms. ft-ir spectra the ft-ir spectrum of ligand l showed a characteristic band at 1544 cm–1 due to ν(–ch=n) stretching vibrations.30 the bands at 1591, 1465 and 1402 cm–1 are associated with ν(–nh) and ν(=ch–n=) aromatic ring nitrogen stretching vibrations, respectively. in the spectra of complexes 1 and 2, characteristic bands appeared at 1522 (1) and 1535 cm–1 (2), and 1388 (1) and 1384 cm–1 (2), which are due to ν(ch=n) and ν(aromatic, =ch–n=) group vibrations. this result indicates that nitrogen atoms of azomethine ν(ch=n) and the pyrimidine ring ν(aromatic, =ch–n=) participated in the complexation. bands are observed at 1087 and 1088 cm–1 in complexes 1 and 2, respectively, due to the presence of perchlorates in the outer coordination spheres. mass spectra in the esi-ms spectrum of ligand l, the molecular ion peak was observed at m/z 350.9 and daughter peaks were observed for c12h7n4f3+, c6h5n4f3+ and c5h4n3f3+. in complexes 1 and 2, molecular ion peaks were obtained at m/z, 765 (1) and 767 (2). these values evidence that one mole of complex 1 or 2 contained with two moles of ligand l. electronic absorption spectra in the uv–vis spectra of ligand l, and complexes 1 and 2, bands were observed at 266 and 336 nm owing to π→π* and n→n* transitions of ligand l.31 the ligand to metal charge transfer (lmct) spectra of complexes 1 and 2 showed bands at 272 and 329 nm for complex 1 and 270 and 338 nm for complex 2. in the uv–vis spectrum of complex 1, a broad band was displayed at 777 nm and the band assignments are 2b1g→2a1g, 2b1g→2b2g and 2b1g→2eg transitions in a square planar arrangement.32 complex 2 possess d10 electronic configuration, which authenticates the absence of a d–d transition, and therefore complex 2 showed inct bands at 270 and 338 nm, which confirms the complexation. esr spectra esr spectral data give knowledge of the environment of a metal ion. the esr spectrum data of complex 1 recorded in dmso at liquid nitrogen temperature gave gii and gi values of 2.35 and 2.07, respectively. the spectrum of complex 1 showed that g-tensor value are gii > gi > 2.0027, indicating the unpaired electrons lie predominantly in the dx2–dy2 orbital, which suggests square 282 sankarganesh et al. planar geometry around the central metal ion.33–36 the gii/aii value is useful for predicting the structure of the complex 1 and the value of 154 suggests square planar arrangement of complex 1 around the central metal ion.37 according to hathaway and billing,38–40 the g value greater than 4 suggested that the local tetragonal axes are aligned parallel or only slightly misaligned. computational studies the 3d structure of ligand l, and complexes 1 and 2 were optimized using density functional theory (dft) calculations accomplished through the (b3lyp/ /6-311g(d,p)/lanl2dz) basis set using the gaussian 09 program.24 the basis set 6-311g(d,p) was used for the n, o, c and h atoms and lanl2dz was exploited for complexes 1 and 2. in ligand l, the homo is spread over the whole π-moiety, while the lumo is spread over the pyrimidine and imine moieties. after the introduction of cu(ii), the homo is spread over the surrounding of the metal ion and its lumo is spread on the pyrimidine moiety with little contribution of the metal ion. similarly, in 2, the homo and lumo are more spread over the pyrimidine moiety and slightly in the morpholine moiety with a metal ion contribution (fig. 1). in addition, the calculated homo–lumo energy gap for complexes 1 and 2 was lower than that for the free ligand. the order of the energy band gap is l > 2 > 1. the obtained results obviously indicate that the complexation of cu(ii) and zn(ii) to the ligand l result in a disruption of internal charge transfer, which may be responsible for the appearance of new absorption band in the visible region. fig. 1. frontier molecular orbitals of ligand l, complexes 1 and 2 (from left to right). pyrimidine incorporating metal(ii) complexes 283 biological studies the pharmacological, dna binding and in vitro anticancer activities of ligand l, complexes 1 and 2 were studied as follows. antimicrobial studies. the antimicrobial activity of ligand l, and complexes 1 and 2 were screened against the bacterial species e. coli, k. pneumoniae, p. fluorescens, s. sonnei and s. aureus, and the fungal species a. niger, c. albicans, c. tropicalis, m. indicus and rhizopus. the reference drugs streptomycin and amphotericin were used for the antibacterial and antifungal studies, respectively. the zone of inhibition values of isolated compounds are presented in table i. the obtained results established that the synthesized complexes 1 and 2 acted as better antibacterial and antifungal agents than ligand l due to their chelation ability.41 additionally, ligand l, and complexes 1 and 2 show superior activity against e. coli bacteria and c. albicans fungi than against the other pathogens. table i. antimicrobial activities (zone of inhibition, mm) of ligand l, and complexes 1 and 2 compound bacterial strains e. coli k. pneumoniae p. fluorescens s. sonnei s. aureus 1 9 8 8 8 8 2 8 6 6 7 6 l 6 5 4 5 4 streptomycin 18 12 14 16 16 fungal strains a. niger c. albicans c. tropicalis m. indicus rhizopus 1 9 15 12 7 9 2 8 13 9 6 8 l 5 10 6 5 6 amphotericin 18 17 31 9 24 anticancer studies. the positive results received from the biological studies encouraged the investigation of the anticancer ability of the pyrimidine incorporated compounds. the in vitro anticancer activities of the ligand l, and complexes 1 and 2 against human cancer cell lines (mcf-7, breast adenocarcinoma; hela, cervical; hep-2, laryngeal) and nhdf (normal human dermal fibroblasts) cell line were examined by using the mtt assay (fig. s-1). the 50 % inhibitory concentration (ic50, µg ml–1) values of the prepared compounds against human normal and cancer cell lines are given in table ii. from the obtained data, cis platin can affect the cancer as well as normal cell lines at lower concentration (6.93±0.35 µg ml–1 mcf-7; 7.46±0.37 µg ml–1 hela; 10.28±0.51 µg ml–1 hep-2). when compared to the above results, l had no significant anticancer activity against the cancer cell lines or the normal cell lines (75.82±3.79 µg ml–1 mcf-7; 76.26±3.81 µg ml–1 hela; 81.03±4.05 µg ml–1 hep-2). moreover, the ic50 values of the complexes 1 and 2 against cancer cell lines revealed that 284 sankarganesh et al. complex 1 has moderate anticancer ability on mcf-7 (54.51±2.73 µg ml–1) and on hela (55.40±2.77 µg ml–1) cell lines which was not so obvious on the hep-2 cell line (77.57±3.88 µg ml–1). however, complex 2 expressed modest anticancer activities on all three selected cancer cell lines (58.89±2.94 µg ml–1, mcf-7; 59.98±2.99 µg ml–1, hela; 60.79±3.04 µg ml–1, hep2). from these observations, the pyrimidine incorporated complexes 1 and 2 could control the growth of cancer cells. table ii. anticancer activities of ligand l, and complexes 1 and 2 on cancer and normal cell lines compound ic50 / µg ml-1 mcf-7 hela hep-2 nhdf cisplatin 6.93±0.35 7.26±0.36 7.46±0.373 10.28±0.51 l 75.82±3.79 76.26±3.81 81.03±4.05 100.48±5.02 1 54.51±2.73 55.40±2.77 77.57±3.88 105.67±5.28 2 58.89±2.94 59.98±2.99 60.79±3.04 107.04±5.35 protein–ligand interaction the mode of interaction of ligand l with antioxidant enzymes and the estrogen receptor alpha was analyzed using molecular docking studies. from the various obtained docked poses, the best pose was chosen based on the higher absolute energy and the libdock score. the protein–ligand interaction was found to be stronger in the presence of hydrogen bond interactions, especially with bond length less than 0.3 nm (table iii).42,43 the present study also revealed the presence of h-bond interaction between ligand l and the proteins indicating a better protein–ligand interaction. binding of ligand l with the antioxidant enzymes and the estrogen receptor alpha predicts the mode of action through which the compound acts as an anticancer agent by regulating their involvement in cancer progression. table iii. in silico results for protein–ligand interactions interaction of ligand l with no. of poses absolute energy, kcala mol-1 libdock score, kcal mol-1 no. of h-bonds bond length, nm interacting residues 1spd_a 12 74.225 60.174 1 0.24 val 81 1qqw_a 25 74.426 112.491 1 0.23 tyr 358 2he3_a 19 74.426 101.122 1 0.25 ser 132 1a52_a 67 74.669 97.379 4 0.240 arg 394 a1 kcal = 4184 j dna binding absorption titration. the interaction between the compounds and ct-dna was ascertained by electronic absorption spectroscopy. the absorption spectra of complexes 1 and 2 (50 µm) in the presence and absence of ct-dna (0–50 µm) pyrimidine incorporating metal(ii) complexes 285 were assessed by typical absorption spectral titrations (fig. 2a and b). as shown in fig. 2a and 2b, the incremental addition of ct-dna to solutions of complex 1 and 2 led to hypochromism (6.27, l; 15.60, 1 and 13.57, 2) with bathochromic shifts of 3–6 nm. these observed spectral titration results clearly indicate that complexes 1 and 2 can bind with ct-dna through intercalative interaction due to the presence of the pyrimidine substituent. in order to determine the intrinsic binding constants (kb) of the prepared compounds, the following equation25 was used: ( ) 1dna dna b b f a f b f c c k ε ε ε ε ε ε − = +  −  − − (2) where cdna is the concentration of ct–dna. fig. 2. absorption spectra of complexes 1 (a) and 2 (b) in tris-hcl/nacl buffer at room temperature in the presence of ct-dna solutions. dotted lines refer to the free compound; the solid lines to absorption spectra of the compound in the presence of different concentrations of dna. the kb values were obtained from the ratio of slope to the intercept from plots of cdna/(εa–εf) vs. cdna. the obtained kb values of the synthesized compounds (2.65×103, l; 7.74×103, 1 and 2.99×103, 2) are given in table iv. the obtained kb values are low as compared to that for ethidium bromide (eb) (1.4×106). the lower kb values for synthesized compounds are due to the flexible, versatile morpholine moiety in the complexes that greatly facilitate table iv. absorption spectral properties of the synthesized compounds on interaction with ct-dna compound λmax / nm δλ / nm hypochromism, % kb / 103 free bound 1 329.0 335.0 6.0 15.60 7.74 2 338.0 343.0 5.0 13.57 2.99 l 336.0 339.0 3.0 6.27 2.65 286 sankarganesh et al. intercalation with the base pairs. the binding strengths of the studied compounds with dna show that complex 1 has a higher binding affinity as compared to ligand l and complex 2, in the following order: 1 > 2 > l. competitive binding. the fluorescence spectra of eb–dna in the presence of the prepared compounds are presented in fig. 3. as shown in fig. 3a and b, the concentration of complexes 1 and 2 enlarges to the eb–dna, the fluorescence intensity of the complexes 1 and 2 steadily decreases. these results confirm that competitive interactions occurred. the intercalative binding of ligand l, complexes 1 and 2 with eb–dna could be summarized by the stern–volmer equation:44 i0/i = 1 + ksvcq (3) where i and i0 are the emission intensities in the presence and absence of the quenchers (ligand l, complexes 1 and 2), respectively, ksv is the linear stern– –volmer quenching constant and cq is the concentration of the quencher. the ksv values were obtained from the slope of plots of i0 / i vs. cq (fig. 3). additionally, the binding affinity (kapp) of ligand l complexes 1 and 2 in contrast to that of eb was evaluated using the following equation:45 fig. 3. emission spectra of eb–ct-dna in the presence of increasing amounts of complex 1 (a) and 2 (b). pyrimidine incorporating metal(ii) complexes 287 kebceb = kappccomplex (4) where, ccomplex is the concentration of the newly prepared compounds necessary for a 50 % reduction in the fluorescence intensity of eb and keb = 1.0×107. the binding constant (ksv) and binding affinity (kapp) values of the present compounds are given in table v. the ksv and kapp values of the prepared compounds are in the following order: 1 (ksv 4.31×103 and kapp 3.30×105) > 2 (ksv 3.89×103 and kapp 2.82×105) > l (ksv 3.30×103 and kapp 2.15×105). table v. the quenching constants and binding affinity of ligand l, and complexes 1 and 2 with eb–dna compound ksv / 103 kapp / 105 1 4.31 3.30 2 3.89 2.82 l 3.30 2.15 electrochemical studies the results of cyclic voltammetric measurements of complexes 1 and 2 in the presence and absence of ct-dna (0–50 µm) are presented in fig. 4a and b. here, as the concentration of ct-dna rises, the cathodic and anodic peak currents of the prepared complexes 1 and 2 increase, and their peak potentials are shifted to negative values. these findings confirm that the newly prepared compounds can interact with ct-dna. viscosity measurements the viscometric studies were very useful in the clarification of the nature of the binding of the synthesized compounds to ct-dna. the graphs of the relative viscosity vs. ccomplex/cdna are presented in fig. 5. as the concentration of compounds increases, the viscous flow of ct-dna increases. these results correlate fig. 4. cyclic voltammograms of complexes 1 (a) and 2 (b) in tris-hcl buffer at 25 °c in the presence of increasing amounts of ct-dna. 288 sankarganesh et al. with the spectroscopic and cyclic voltammetric results that suggested the prepared compounds should interact with ct-dna through an intercalation mode. fig. 5. effect of increasing amount of complexes 1 and 2 on the relative viscosity of dna. 1/r = ccompound/cdna. conclusion the schiff base ligand l, and complexes 1 and 2 were isolated from pyrimidine backbones. the analytical data confirmed that the prepared complexes 1 and 2 possess 1:2 stoichiometry ratios. square planar geometry of complexes 1 and 2 were finalized from the results of spectroscopic techniques. the prepared compounds possess better antibacterial and antifungal activity against e. coli (bacteria) and c. albicans (fungi) than against the other tested pathogenic species. the molecular docking results revealed that ligand l can interact with proteins. the interacting ability of ligand l, and complexes 1 and 2 with ct-dna were explored by spectroscopic, cyclic voltammetric and viscometric techniques and the results suggested that the prepared complexes can bind with ct-dna by intercalatation. the in vitro antitumor results suggested that the pyrimidine incorporating complexes 1 and 2 have moderate anticancer activities. supplementary material spectral and analytical data of the synthesized compounds, as well as graphical representation of anticancer activities, are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the authors acknowledge the department of science and technology (dst)-science and engineering research board (serb-ref. no.: sr/ft/cs-117/2011 dated 29.06.2012), government of india, new delhi for the financial support. the authors express their sincere and heartfelt thanks to managing board, dean, principal, head and staff members, chemistry research centre, mohamed sathak engineering college, kilakarai for their constant encouragement and providing the research facilities. pyrimidine incorporating metal(ii) complexes 289 и з в о д компјутерска анализа, днк интеракције, антимикробна и антиканцерска активност бакар(ii) и цинк(ii) комплекса са лигандом који садржи пиримидински прстен murugesan sankarganesh1, nagaraj revathi2,3, jeyaraj dhaveethu raja1, karunganathan sakthikumar1, gujuluva gangatharan vinoth kumar1, jegathalaprathaban rajesh1, manikkam rajalakshmi4 и liviu mitu5 1chemistry research centre, mohamed sathak engineering college, kilakarai, ramanathapuram, tamil nadu 623 806 india, 2department of chemistry, ramco institute of technology, rajapalayam, virudhunagar, tamil nadu 626 117, india, 3department of chemistry, manonmanium sundaranar university, tirunelvi, tamil nadu 627 012, india, 4bioinformatics centre, pg & research centre department of biotechnology and bioinformatics, holy cross college (autonomous), tiruchirapalli, tamil nadu 620 002, india и 5department of nature sciences, university of pitesti, pitesti 110 040, romania у овом раду описана је синтеза, структурна карактеризација, антимикробна и антиканцерска својства, као и испитивање интеракција са молекулом днк, комплекса [cul2] .(clo4)2 (1) и [znl2] .(clo4)2 (2) који као лиганд (l) садрже шифову базу. у односу на лиганд, одговарајући комплекси (1 и 2) су показали значајну антибактеријску и антифунгалну активност. на основу електронских апсорпционих, флуорометријских и циклично-волтаметријских мерења, као и на основу мерења вискозитета, потврђено је интеркалативно везивање комплекса за хеликс днк. израчунати су следећи параметри везивања испитиваних комплекса за днк: kb (2.65×103 (l); 7.74×103 (1) и 2.99×10 3 (2)), ksv (3.30×103 (l); 4.31×103 (1) и 3.89×103 (2)) и kapp (2.15×105 (l); 3.30×105 (1) и 2.82×105 (2)). ове вредности показују да комплекс 1 има бољу интеркалациону способност везивања за днк у односу на лиганд l и комплекс 2. in vitro антиканцерска својства лиганда l и комплекса 1 и 2 према хуманим ћелијама тумора (mcf-7, hela и hep-2) и нормалним (nhdf) ћелијским линијама одређена су помоћу mtt методе. добијени резултати су показали да комплекси 1 и 2 показују значајно већу активност према ћелијама канцера у односу на лиганд l. 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(https://doi.org/10.1016/s1386-1425(00)00322-x) 41. n. raman, s. s. a. fathima, j. dhaveethu raja, j. serb. chem. soc. 73 (2008) 1063 (https://doi.org/10.2298/jsc0811063r) 42. p. daisy, s. suveena, v. lilly sr., j. chem. pharm. res. 3 (3) (2011) 557 (http://www.jocpr.com/articles/molecular-docking-of-medicinal-compound-lupeol-withautolysin-and-potential-drug-target-of-uti.pdf) 43. p. daisy, p. vijayalakshmi, c. selvaraj s. k. singh, k. saipriya, indian j. pharm. sci. 74 (2012) 217 (https://doi.org/10.4103/0250-474x.106063) 44. p. adwin jose, j. dhaveethu raja, m. sankarganesh, j. rajesh, j. photochem. photobiol., b 178 (2018) 143 (https://doi.org/10.1016/j.jphotobiol.2017.11.005) 45. s. m. kumar, m. p. kesavan, g. g. vinoth kumar, m. sankarganesh, g. chakkaravarthi, g. rajagopal, j rajesh, j. mol. struct. 1153 (2018) 1 (https://doi.org/10.1016/j.molstruc.2017.09.070). << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis and sar studies of pyrazole-3-carboxamides and -thioureides including chiral moiety: novel candidates as antibacterial agents} j. serb. chem. soc. 83 (7–8) 795–807 (2018) udc 547.435+547.772:542.913:57– jscs–5113 188:615.281’272 original scientific paper 795 synthesis and sar studies of pyrazole-3-carboxamides and -3-carbonyl thioureides including chiral moiety: novel candidates as antibacterial agents ishak bildirici1, adnan cetin2*, nurettin menges1 and yusuf alan3 1van yuzuncu yil university, faculty of pharmacy, department of pharmaceutical chemistry, van, turkey, 2muş alparslan university, faculty of education, department of science, 49250, muş, turkey and 3muş alparslan university, faculty of science and arts, department of biology, 49250, muş, turkey (received 13 march, revised 6 december 2017, accepted 14 february 2018) abstract: a series of tetrasubstituted pyrazole-3-carboxamides (3a–c) and pyrazole-3-carbonyl thioureides (6a–c) were synthesized and their structures characterized by ir, nmr and elemental analysis. the antibacterial potential against specific gram-positive and gram-negative strains and the antifungal activities of all novel compounds were investigated. structure–activity relationships (sar) studies and some theoretical parameters (clogp, cmr, psa and esp) of the compounds were performed on these two pyrazole derivatives. pyrazole-3-carboxylate ester 2 was used for the synthesis of the carboxamide derivatives. the reactions of pyrazole-3-carbonyl isothiocyanate 5 with appropriate chiral amino alcohols were utilized for synthesizing the thioureide derivatives. both of these types of pyrazole derivatives including a chiral moiety exhibited pronounced antibacterial activities. according to the present in vitro study, some of the promising compounds might be new candidates for a new generation of antibacterial drugs. keywords: biological activity; chiral amino alcohols; pyrazole; heterocyclic compounds. introduction chiral structures are important target molecules in chemistry since they have significant properties, such as catalytic,1 biological,2 pharmaceutical,3 agricultural4 and industrial implications.5 chiral properties have brought a third dimension to the all these fields of science. on the other hand, carboxamide derivatives bearing a chiral stereocentre have also extensive workspace. for example, chiral carboxamides were used as a transporter for drug in pharmaceuticals.6 * corresponding author. e-mail: adnankimya@gmail.com https://doi.org/10.2298/jsc170313029b ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 796 bildirici et al. furthermore, compounds having thiourea are known to exhibit various biological properties, such as antibacterial,7 antifungal,8 antitubercular,9 antithyroid,10 anthelmintic,11 rodenticidal,12 insecticidal,13 herbicidal14 and plant growth regulator15 activities. however, some thiourea derivatives including chiral moiety not only can serve as catalysts16 for the synthesis of optically active compounds, but can also be employed as medicines.17–19 studies on the biological activities of chiral thioureas are relatively rare and the reported studies showed that they have a broad spectrum of biological activities, such as anti-hiv,20 anticancer21 and antiviral.22 some of the biologically active chiral thiourea and amide derivatives are showed in fig. 1. since the beginning of this century, pyrazoles, which are an important scaffold of heterocyclic compounds, have increasingly drawn the attention of researchers because of their wider range of properties, particularly for their biological activities. these compounds have been used in the development of agricultural products and in drug research since they have diverse biological activities.23,24 some known activities, such as pharmaceutical, agricultural and biological activities of these compounds containing a pyrazole ring system in the structure can be listed as high antihyperglycemic,25 analgesic,26 inflammatory,27 antipyretic,28,29 anti-bacterial30 and antidepressant.31 fig. 1. some of the important examples of pyrazole, carboxamide and thiourea derivatives. they provided protection against plant pests,32 as insecticides33 and against fungal organisms.34 they were also found to be hypotensive35 and anticancer.36,37 compounds including synthetic pyrazoles, such as the anti-rheumatic celecoxib, have been used in the treatment of inflammation38 and pain.39 rimonabant has been used for the treatment of obesity,40 difenzoquat exerted lethal ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. synthesis of novel pyrazole-based antibacterial agents 797 effects against plant pests41 and tartrazine lemon yellow dye is widely used as a colouring agent for food in the uk and the usa42 (fig. 1). studies on the implications of the stereochemistry of antibacterial agents have high significance in medicinal chemistry.43 however, the antimicrobial and antifungal activities of pyrazole-3-carboxamides and 3-carbonyl thioureides containing a chiral stereocentre have not hitherto been reported in the literature. recently the design, synthesis, and antimicrobial activity of a series of novel carboxamide and thioureide derivatives including the pyrazole scaffold were reported.44 encouraged by these successful efforts, we aimed both to expand this study by synthesis new pyrazole derivatives containing chiral moiety and to evaluate the antimicrobial potential against various gram-positive and gram-negative strains and antifungal activities of these novel compounds. moreover, structure– –activity relationships (sar) studies were undertaken and some theoretical parameters of these newly synthesized derivatives were investigated. experimental materials and equipment the 1hand 13c-nmr spectra were recorded at 400 and 100 mhz, respectively, on a bruker drx-400 high performance ft-nmr spectrometer. the nmr spectra were obtained in solutions of dmso-d6 and cdcl3. analytical tlc of all reactions was performed on merck prepared plates. the infrared spectra were recorded on a shimadzu ir-470 spectrophotometer. the elemental analyses were obtained with a carlo erba model 1108 apparatus. the optical rotations were taken on a perkinelmer 341 model polarimeter. refraction indices were measured using an atago abbe refractometer. the mass spectrum was measured on thermo scientific tsq-quantum access lc/ms spectrometers. analytical and spectral data of the synthesized compounds are given in supplementary material to this paper. compounds 1 (m.p.: 202–204 °c) and 2 (m.p.: 180 °c) were obtained according to a previous study.44 general procedure for the synthesis of chiral pyrazole-3-carboxamides (3a–c) to compound 2 (0.404 g, 1.0 mmol) dissolved in methanol (5 ml) in the reaction vessel was added dropwise a solution of a chiral amino alcohol (1 mmol, 2-amino-2-phenylethanol, 2-amino-3-methyl-1-butanol and 2-amino-1-butanol, respectively) in methanol (5 ml) at room temperature over 2 h. after the addition, solid product had precipitated that was filtered and washed with diethyl ether. the obtained product was purified by silica gel column chromatography (n-hexane/ethyl acetate in 7:1 volume ratio). general procedure for the synthesis of chiral pyrazole-3-carbonyl thioureides (6a–c) compound 1 (0.397 g, 1 mmol) was refluxed with an excess of socl2 at 80 °c for about 7 h. the excess socl2 was evaporated. the remaining oily product was purified in a dry ether/cyclohexane mixture. as a result, 4-benzoyl-1-(2,5-dimethylphenyl)-5-phenyl-1h-pyrazole-3-carbonyl chloride (4, m.p.: 145 °c) was obtained, according to the literature.44 the resulting acylchloride compound (4, 1.0 mmol) was dissolved in anhydrous acetone (15 ml) and a solution of 1 mmol ammonium thiocyanate in acetone (5 ml) was added to the reaction vessel. the reaction mixture was refluxed in a round-bottom flask equipped with a condenser ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 798 bildirici et al. for 4 h. the solvent was evaporated and the residue was washed with diethyl ether. the solid product was filtered and then the crude product was crystallized from n-hexane/diethyl ether. finally, 4-benzoyl-1-(2,5-dimethylphenyl)-5-phenyl-1h-pyrazole-3-carbonyl isothiocyanate (5, m.p.: 159–160.5 °c) was obtained.44 compound 5 (0.219 g, 0.50 mmol) dissolved in anhydrous acetone (5 ml) was added dropwise to the appropriate chiral amino alcohol (1 mmol, 2-amino-2-phenylethanol, 2-amino-3-methyl-1-butanol and 2-amino-1-butanol, respectively) in acetone (5 ml). this mixture was kept at room temperature for 4–6 h, after which the reaction mixture was poured onto ice-cold water. the formed precipitate was filtered, dried and the product was purified by crystallization in diethyl ether/n-hexane. microorganisms and antimicrobial assays although the antimicrobial activities of the starting compounds 1 and 2 were previously investigated,44 their antimicrobial activities were examined again in order to compare together. all samples (1, 2, 3a–c, 5 and 6a–c) were separately tested against enterobacter aerogenes atcc 13048, bacillus subtilis atcc 6633, staphylococcus aureus 6538, bacillus megaterium dsm 32, pseudomonas aeruginosa 9027, klebsiella pneumoniae rskk 574, escherichia coli, candida albicans, yarrovia lipolytica and saccaromyces cerevisiae atcc 10231 fungi. all the bacterial and fungal strains examined in the present study were supplied by the microbiology laboratory of muş alparslan university (turkey). penicillin (10 mg), amikacin (30 mg), erythromycin (15 mg), rifampicin (5 mg) and ampicillin (10 mg) antibiotics were used as the reference drugs. the antimicrobial activities of the samples were determined by the well diffusion method.45,46 for this purpose, the bacterial and fungal strains were cultured overnight at 37 °c in nutrient agar and 25 °c in sabouraud dextrose agar medium, respectively. 100 µl of suspensions of test microorganisms, containing 1×108 colony-forming units (cfu) per ml-1 of bacteria cells and 1×104 cfu ml-1 spores of fungal strains were spread on nutrient agar and sabouraud dextrose agar medium, respectively. subsequently, the medium was poured into a petri dish on a horizontally levelled surface. after the medium had solidified, 9 mm diameter wells per dish were made in the agar medium. then 0.005, 0.01 and 0.02 g ml-1 doses of 1, 2, 3a–c, 5 and 6a–c suspensions dispersed in dmso as 50 mg ml-1 were loaded into the wells separately. the petri dishes were incubated at 37 °c for 24 h for bacteria and at 25 °c for 48 h for fungal strains. the average diameters of the inhibition zones were measured by repeating the experiment at least three times. results and discussion chemistry in the present study, two series of new chiral derivatives, 3a–c and 6a–c, containing a 1,4,5-trisubstituted-pyrazole-3-carbonyl moiety attached to the chirality centre, were synthesized. initially, 4-benzoyl-1-(2,5-dimethylphenyl)-5-phenyl-1h-pyrazole-3-carboxylic acid (1) was synthesized according to a newly published manuscript and the corresponding methyl 4-benzoyl-1-(2,5-dimethylphenyl)-5-phenyl-1h-pyrazole-3-carboxylate (2) was obtained by heating the pyrazole-3-carboxylic acid 1 and methanol with a catalytic amount of sulphuric acid.44 then, these compounds were derivitized by amino alcohols, (r) -2-amino-2-phenylethanol, (r)-2-amino-3-methyl-1-butanol and (r)-2-amino-1 -butanol, respectively. the chiral pyrazole-3-carboxamides compounds were syn________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. synthesis of novel pyrazole-based antibacterial agents 799 thesized by means of the reaction between methylpyrazole-3-carboxylate 2 and chiral amino alcohols at room temperature (scheme 1). as a result of this reaction, different new chiral pyrazole-3-carboxamides 3a–c were obtained in overall yield 72–78 % (scheme 1). scheme 1. synthesis of pyrazole-3-carboxamides of chiral amines. all new synthesized compounds were confirmed by analytical and spectral data (see supplementary material). in the case of compounds 3a–c, the correct structures were established by ir and 1h-nmr spectroscopies in which characteristic –nh absorption bands were observed at 3325–3210 cm–1 in the ir spectra. protons of the hydroxyl signals and nh protons of amide group signals were observed at δ 3.53– –2.50 ppm and at δ 8.42–8.34 ppm in the 1h-nmr spectra, respectively. in order to incorporate potent active pharmacophores, such as pyrazole, thiourea and chiral amino alcohols, in a single molecule, a series of chiral pyrazolo -thiourea derivatives 6a–c were synthesized by the reactions of 4-benzoyl-1-(2,5 -dimethylphenyl)-5-phenyl-1h-pyrazole-3-carbonyl isothiocyanate (5) with the above-mentioned chiral amino alcohols (scheme 2). therefore, first 4-benzoyl-1 -(2,5-dimethylphenyl)-5-phenyl-1h-pyrazole-3-carbonyl chloride (4) was obtained by heating pyrazole-3-carboxylic acid (1) with excess socl2 and then compound 5 was synthesized by heating the pyrazole-3-carbonyl chloride with ammonium thiocyanate in acetone for about 5 h.44 the chiral thioureide compounds 6a–c were prepared by a basic chemical procedure. thus 6a–c were synthesized by heating pyrazole-3-carbonyl isothiocyanate (5) and the corresponding chiral amino alcohol derivative for 4–6 h as outlined in scheme 2 (overall yield 60–80 %). these types of heterocyclic compounds including chiral structures are not found in the literature to date. structure elucidations of the compounds 6a–c were based on 13c-nmr spectroscopy. benzoyl carbonyl (c=o) signals were ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 800 bildirici et al. observed at δ 196.8–194.3 ppm, thioamide (c=s) at δ 185.6–180.3 ppm and amide carbonyl (c=o) signals at δ 167.7–165.9 ppm. socl2 nh4scn n n ho o ph ch3 h3c o ph n n cl o ph ch3 h3c o ph n n n o ph ch3 h3c o phc s 1 4 5 nh2 oh r 6a-c n n o n h ph n h s r ho h3c ch3 ph o 6 r a -ph b -ch(ch3)2 c c2h5 scheme 2. synthesis of chiral substituted thiourea derivatives 6 via pyrazolecarbonyl isothiocyanate 5. biology antibacterial activity. the antimicrobial activities of 1, 2, 3a–c, 5 and 6a–c against gram-positive bacteria bacteria (bacillus subtilis atcc 6633, staphylococcus aureus 6538, bacillus megaterium dsm 32) and gram-negative bacteria (enterobacter aerogenes atcc 13048, pseudomonas aeruginosa 9027, klebsiella pneumoniae rskk 574 and escherichia coli atcc 25922) as expressed minimal inhibitory concentration (mic). representative inhibition zone images were showed in fig. 2. no antifungal activities of the compounds were detected against fungal strains (candida albicans, saccharomyces cerevisiae and yarrovia lipolytica atcc 10231, data not shown). table i. the zones of inhibition (mm) of the materials with antibiotics against bacterial strains (mic in µg/ml) mic: minimal inhibitory concentration values with sem = 0.02, –: totally inactive (no inhibition) cmpd. dose µg/ml bacteria gram-positive bacteria gram-negative bacteria b. subtilis s. aureus b. megaterium e. aerogenes e. coli p. aeruginosa k. pneumonia 1 5 12 – – – – – 15 10 13 – – – 12 14 15 20 14 – 13 13 16 17 16 2 5 14 12 12 12 14 14 12 10 10 – – 12 12 12 12 20 12 – 10 14 16 16 16 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. synthesis of novel pyrazole-based antibacterial agents 801 table i. continued cmpd. dose µg/ml bacteria gram–positive bacteria gram–negative bacteria b. subtilis s. aureus b. megaterium e. aerogenes e. coli p. aeruginosa k. pneumonia 3a 5 – – – – 10 – 10 10 14 14 15 12 16 12 15 20 19 20 16 19 20 21 19 3b 5 11 12 11 10 12 10 10 10 13 12 15 14 15 12 14 20 16 22 18 18 16 20 18 3c 5 12 – – 12 12 – – 10 15 11 11 16 15 12 11 20 16 17 17 18 18 19 15 5 5 – – – – 11 – 13 10 12 12 12 14 13 – 15 20 16 17 16 16 17 18 16 6a 5 14 – 13 – 13 13 12 10 16 15 16 12 15 18 16 20 18 18 19 19 18 20 18 6b 5 12 12 – 12 – – 12 10 15 14 15 15 17 13 14 20 18 18 18 20 18 17 19 6c 5 13 – 12 – 14 13 – 10 14 13 13 13 14 13 14 20 15 15 15 15 15 14 16 positive controls erythromycin 15 20 21 25 27 19 19 19 amikacin 30 14 10 – 10 13 – 16 penicillin 30 21 18 16 16 18 9 19 ampicillin 10 9 – 10 – – – 10 rifampicin – 9 – 10 – – – 10 the in vitro antimicrobial and antifungal activities of the compounds and positive controls were tested in a dose manner (5, 10 and 20 µg ml–1) against ten microorganisms (table i). the current results indicated that the synthesized compounds showed a broad spectrum of antibacterial activities producing 10–22 mm zones of inhibition. among the tested compounds, 3b and 6a exhibited the highest antimicrobial activities. all tested compounds showed higher antimicrobial activity compared to amikacin and rifampicin, except compound 2. compounds 3b and 6a had similar or higher antimicrobial activity compared to penicillin. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 802 bildirici et al. moreover, the obtained data indicated strong antibacterial activity of pyrazole-3-carbonyl derivatives, i.e., 3a–c and 6a–c exhibited pronounced activity against b. subtilis, 3a and 3b exhibited pronounced activity against s. aureus, 3b, 3c, 6a and 6b exhibited pronounced activity against b. megaterium, 3a–c, 6a and 6b exhibited pronounced activity against e. aerogenes, 3a exhibited strong activity against e. coli. 3a, 3b and 6a exhibited pronounced activity against p. aeruginosa. compound 3b exhibited the highest antimicrobial activity against gram -positive bacteria, followed by 3a and 3b. with regards to gram-negative bacteria, compound 3a showed the highest antibacterial activity followed by 3b and 3c. compound 6a–c showed similar antibacterial activities against gram-positive and gram-negative bacteria. the pattern of the antimicrobial activity potential was 6a > 6b > 6c for both of gram-positive and gram-negative bacteria. in general, all compounds had pronounced antimicrobial activities against the bacteria strains. however, s. aureus and b. megaterium seemed to be more resistant to the compounds and the tested positive controls than the other bacteria strains. there was a positive correlation between antimicrobial activity and the concentrations tested, as shown in table i. all tested compounds were pyrazole derivatives with certain modifications. among the tested compound groups, 3 and 6 had the highest antimicrobial activities. the pronounced antimicrobial activities of compounds 3 and 6 might be explained by the presence of chiral amino alcohol moiety. relatively higher antimicrobial activities of 3b and 6a might be due to the presence of isopropyl and phenyl groups. the structure–activity relationships (sar) studies the synthesized compounds were tested against many different types of bacteria and fungi. three gram-positive and four gram-negative bacteria were applied and some important activities on the bacteria were found. unfortunately, for all compounds, no inhibition was observed against any of the fungi. the main skeleton of synthesized molecule was 1-(2,5-dimethyl-phenyl)-4-benzoyl-5-phenylpyrazole. changeable units were selected as ester (2), carboxamide (3a–c), thiocyanate (5) and thioureide (6a–c). all synthesized molecules were prepared at a concentration of 10 mm in dmso. the molecules with chiral unit showed the best activity against all bacteria. chiral molecules which have stereogenic centre are important structures because bioactive molecules usually involve the activities of enantiomers. chiral molecules should play a dominant role in their interactions with bioactive substances. moreover, they should be applied only to molecules that contain the stereogenic centre in close proximity to the bioactive centre of the molecule, as was the case with the synthesised compounds. synthetic routes to chiral molecules can be properly turned from achiral structures. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. synthesis of novel pyrazole-based antibacterial agents 803 molecules having a chiral structure are synthesized by chiral transfer, using a chiral starting material such as chiral amino alcohol. the ester group should be a derivative with some more potent and suitable groups. for this reason, compound 2 was substituted by (r)-amino-alcohol derivatives to obtain compounds 3a–c. the most active derivative of them was found to be 3a, which bears a phenyl group, against all bacteria except for s. aureus and b. megaterium. it was found that a bulky group on the (r)-amino-alcohol affected the activity against bacteria. to clear this idea, some physicochemical parameters were calculated.47 hence, hydrophobic (clogp: calculated partition coefficient), steric (cmr: calculated molecular refractivity) and electronic (psa: polar surface area) parameters for all the synthesized compounds were calculated. the theoretical lipophilicity of 3a–c decreases from 3a to 3c and was calculated as 6.67, 6.14 and 5.77, respectively. calculation of cmr and clogp for 3a–c revealed that cmr and clogp affect the antibacterial activity and increasing them has a positive impact on the biological activity. the worst activity was detected for 3c, which bears an ethyl group, for which the values for cmr and clogp are 138.43 and 5.77, respectively (table ii). the polar surface areas for 3a–c were calculated and it was seen that there is no change for compounds 3a–c. table ii. some theoretical parameters for the synthesized molecules compound psa / å2 cmr / cm3 mol-1 clogp 1 69.97 116.47 5.03 2 49.74 120.33 6.31 3a 82 153.7 6.67 3b 82 142.90 6.14 3c 82 138.43 5.77 5 62 129.83 6.90 6a 94 170 6.83 6b 94 159 6.38 6c 94 154 6.02 thiocyanate group attached to the molecule 5 shown lower activities than 3a–c. on the other hand, activities of 5 on bacteria were moderate. compounds 6a–c which have thiourea and (r)-amino-alcohol chains were tested against same bacteria and it was calculated that reactivity against bacteria decreased and the most potent molecule of compounds 6a–c was 6a which bears phenyl ring. theoretical parameters of compound 6a–c were also shown that biological activity could be affected by increasing of cmr and clogp (table ii). moreover, although cmr and clogp of 6c was very close to 3a, which has the best potent molecule, biological activity of 6c is lower in comparison to 3a. decreasing of reactivity on bacteria have shown that (r)-amino-alcohol unit should be adjacent to the carbonyl group and there should be no any other chain between carbonyl and (r)-amino-alcohol groups. after sar study, it was worthy to say that (r) ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 804 bildirici et al. -amino-alcohol unit increase the biological activity against some gram-(–) and gram-(+) bacteria. the electrostatic potential (esp) of 3a was investigated to understand its hdonor and -acceptor units and its total electronic surface. according to the esp map, the oxygen atoms of the two carbonyl groups and of the alcohol group are h-acceptors and the alcohol group is an h-donor group (fig. 3). fig. 3. electrostatic potential (esp) of 3a. the proton of the amide group is less acidic than the others and due to this, this proton might not be a good candidate for proton donation. the rest of molecule showed the expected electronic potential and these surfaces are not worthy of discussion. conclusions within this study, a series of tetra-substituted pyrazole-3-carboxamides (3a– –c) and pyrazole-3-carbonyl thioureides (6a–c) binding a chiral amino alcohol were synthesized and their antibacterial and antifungal properties were investigated. the structure–activity relationships (sar) studies and some theoretical parameters (clogp, cmr, psa and esp) of these pyrazole-carboxamide and -thioureide derivatives were investigated. compound series 3 and 6 exhibited superior antimicrobial activities among the tested compounds. the pronounced antimicrobial activities of compound series of 3 and 6 can be explained by the presence of a chiral amino alcohol moiety. the relatively higher antimicrobial activities of 3b and 6a might be due to the presence of isopropyl and phenyl groups. the present results indicated that the synthesized compounds were active in a broad spectrum against important human pathogenic microorganisms. therefore, these compounds might be new candidates for efficient antibacterial agents. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. synthesis of novel pyrazole-based antibacterial agents 805 supplementary material analytical and spectral data of the synthesized compounds are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the authors thank to the management unit of scientific research projects of muş alparslan university (msübap) for financial support under project msü14 -emf-g05. the authors thank dr. abdullah dalar for his valuable comments and corrections in the antimicrobial section. и з в о д синтеза и sar испитивање пиразол-3-карбоксамида и 3-карбонил-тиоуреида и хиралних структура: нови кандидати за антибактеријске агенсе ishak bildirici1, adnan cetin2, nurettin menges1 и yusuf alan3 1 van yuzuncu yil university, faculty of pharmacy, department of pharmaceutical chemistry, van, turkey, 2 muş alparslan university, faculty of education, department of science, 49250, muş, turkey и 3muş alparslan university, faculty of science and arts, department of biology, 49250, muş, turkey синтетисана је серија пиразол-3-карбоксамида (3a–c) и пиразол-3-карбохил-тиоуреида (6a–c), а добијеним дериватима структура је одређена ir, nmr и елементалном анализом. испитана је антибактеријска активност према специфичним грам-позитивним и грам-негативним сојевима бактерија и антифунгална активност свих нових синтетисаних једињења. урађена је анализа утицаја структуре на активност (structure–activity relationships, sar) и неких теоријских параметара (clogp, cmr, psa и esp). за синтезу карбоксамидних деривата коришћен је пиразол-3-карбоксилатни естар 2. реакцијом пиразол-3-карбонил-изотиоцијаната 5 и одговарајућих хиралних алкохола добијени су деривати тиоурее. обе групе добијених једињења показују запажену антибактеријску активност. према приказаном in vitro испитивању, неки од деривата могу бити кандидати за даља испитивања антибактеријске активности. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis and efficacy of copper(ii) complexes bearing n(4)-substituted thiosemicarbazide and diimine co-ligands on plasmid dna and hela cell lines} j. serb. chem. soc. 85 (3) 321–334 (2020) udc 546.562:547.496.3–304.2:547.7:576+ jscs–5303 615.9:577.213.3 original scientific paper 321 synthesis and efficacy of copper(ii) complexes bearing n(4)-substituted thiosemicarbazide and diimine co-ligands on plasmid dna and hela cell lines neelaveni rajendran1, abirami periyasamy2, nithya kamatchi3 and vasantha solomon1* 1pg and research department of chemistry, lady doak college, madurai – 625002, tamil nadu, india; 2department of biotechnology, lady doak college, madurai – 625002, tamil nadu, india and 3pg and research department of zoology, lady doak college, madurai – 625002, tamil nadu, india (received 29 april, revised 19 august, accepted 29 august 2019) abstract: this present work deals with the synthesizes of nine novel thiosemicarbazone copper(ii) complexes {[cu(l)2]cl c3, [cu(l)(bpy)]cl c4–c6, [cu(l) (phen)]cl c7–c9 (where, l = h(l1)–h(l3), h(l1) = (e)-n-methyl-2 -(1-phenyl-2-((5-(pyridin-3-yl)-4h-1,2,4-triazol-3-yl)thio)ethylidene)hydrazinecarbothioamide, h(l2) = (e)-n-ethyl-2-(1-phenyl-2-((5-(pyridin-3-yl)-4h -1,2,4-triazol-3-yl)thio)ethylidene) hydrazinecarbothioamide, h(l3) = (e)-n -phenyl-2-(1-phenyl-2-((5-(pyridin-3-yl)-4h-1,2,4-triazol-3-yl)thio)ethylidene) hydrazinecarbothioamide, bpy = 2,2′-bipyridyl and phen = 1,10-phenanthroline) with improved pharmacological results. the synthesized complexes were characterized by various spectral-analytical techniques. the structure of the copper(ii) complexes c1–c9 was proposed by epr spectroscopy. it confirmed the square planar coordination around cu(ii) complexes. the antibacterial screening of the complexes revealed that complexes c7 and c8 demonstrated significant activity against gram-positive (b. thuringiensis) and gram-negative (e. coli) bacteria. the concentration-dependent dna cleavage activity of supercoiled (sc) puc18 dna exhibited complete dna degradation effect on complex c6 at a minimum concentration of 40 μm. in vitro cytotoxic results showed that the mixed ligand copper(ii) complexes c4, c5 and c7 exhibited higher effects on human cervical cancer cell lines, hela, when compared to cisplatin. hence, the results obtained from each biological screening indicated the superior biological efficacy of the mixed ligand copper(ii) complexes bearing diimine moieties. it could be considered as a promising alternative to an existing anticancer drug. keywords: thiosemicarbazone; heterocyclic compounds; copper(ii) complexes; diimine; cytotoxicity. * corresponding author. e-mail: vasantha@ldc.edu.in https://doi.org/10.2298/jsc190429093r ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 322 rajendran et al. introduction over a few decades, platinum-based metal compounds have had a tremendous starting point in the development of anticancer drugs. however, the clinical success of cisplatin, oxaliplatin and carboplatin exhibited limited action against various cancers due to severe side effects, immune efficiency, less pharmacological stability and toxicity.1,2 as a result, several efforts were made to prevail with the development of thiosemicarbazone copper(ii) complexes to reduce the toxicity and increase specificity. thiosemicarbazones metal complexes are an important group of compounds in the field of inorganic and bioinorganic chemistry. moreover, these compounds were synthesized by simple and cost-effective procedures with small changes in their structures. such changes in the thioamide nitrogen and its metal complexes provided interesting results due to their π-delocalization over the ring system.3,4among them, some developed biologically active thiosemicarbazone compounds, such as triapine (3 -aminopyridine-2-carboxaldehyde thiosemicarbazone), di-2-pyridylketone thiosemicarbazone, 2-benzoylpyridine thiosemicarbazone, di(2-pyridyl)ketone 4,4 -dimethyl-3-thiosemicarbazone, di(2-pyridyl)ketone 4-cyclohexyl-4-methyl-3 -thiosemicarbazone, 2-formyl thiosemicarbazone, 2-formyl-4-(m-amino)phenylpyridine thiosemicarbazone and 2-formyl(m-amino)phenylpyridine thiosemicarbazones, are currently under various stages of preclinical trials and have emerged for the development a new selective anticancer drug.5–7 recently, haribabu and co-workers synthesized in vitro anticancer compounds, i.e., water-soluble ruthenium metal complexes with substituted thiosemicarbazone derivatives. these mono and binuclear complexes showed significant toxicity against human lung carcinoma (a549) and human liver carcinoma cell lines (hepg-2).8 more recently, kallus (2019) et al demonstrated that iron(iii) and copper(ii) complexes of biotin-conjugated thiosemicarbazone were quite interesting owing to their improved in vivo anticancer activity against ct-26 colon cancer-bearing mice.9 presently, dna interaction and anticancer activities of copper(ii) complexes were extensively studied in association with both metabolism and oxidative dna damage. based on the assumption, investigations of copper-thiosemicarbazone-based complexes have been widely studied. in addition to this, metal complexes with diimine moieties have demonstrated potential in vitro anticancer activity with strong interaction and also capable to be exploited as dna-targeted anticancer drug.10,11 based on these observations, the present study was undertaken to synthesize and characterize new thiosemicarbazone-based copper(ii) complexes derived from (e)-n-methyl-2-(1-phenyl-2 -((5-(pyridin-3-yl)-4h-1,2,4-triazol-3-yl)thio)ethylidene) hydrazine carbothioamide h(l1), (e)-n-ethyl-2-(1-phenyl-2-((5-(pyridin-3-yl)-4h-1,2,4-triazol-3 -yl)thio)ethylidene)hydrazine carbothioamide h(l2) and (e)-n-phenyl-2-(1 -phenyl-2-((5-(pyridin-3-yl)-4h-1,2,4-triazol-3-yl)thio)ethylidene)hydrazinecarbo________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ copper(ii) complexes with n(4)-substituted thiosemicarbazide and diimine ligands 323 thioamide h(l3) with a varying suitable substitutions at the terminal nitrogen. thus, the present study was aimed at achieving the biological effect of these compounds for clinical application using the disc diffusion method, agarose gel electrophoresis and mtt assay. experimental materials and methods all the reagents and solvents used for the synthesis of ligands and copper(ii) complexes were of analytical or spectroscopic grade. the commercially available chemicals were purchased from sigma–aldrich, such as 4-methyl-3-thiosemicarbazide, 4-ethyl-3-thiosemicarbazide, 4-phenyl-3-thiosemicarbazide, phenacyl bromide, 5-(3-pyridyl)-4h-1,2,4-triazole-3 -thiol, 2,2′-bipyridyl, 1,10-phenanthroline and copper(ii) chloride. the chemicals tris–hcl, boric acid, tris-base, ethidium bromide, edta (ethylenediaminetetraacetic acid) disodium salt used for the dna cleavage studies were procured from merck. the chemicals used for biological studies, such as penicillin, streptomycin, chloramphenicol, 10 % fetal bovine serum (fbs) and mtt (3-(4,5-dimathylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were acquired from himedia, india. the puc18 dna was obtained from genei, bangalore, india, and stored at –20 °c. the cervical cancer cell lines (hela) were purchased from the national centre for cell science (nccs), pune, india. the molar conductance of the ligands as well as the complexes was analyzed using elico cm 183 ec-tds analyzer in 1×10-3 m solution in dmf at room temperature. the elemental analyses (c, h, n and s) were obtained from vario el-iii elemental analyzer. the 1hand 13c-nmr spectra of free thiosemicarbazone ligands were recorded on a 300 mhz spectrophotometer using cdcl3 or dmso-d6 as solvent and tms as an internal standard. the electronic spectra of the synthesized ligands and copper(ii) complexes were taken using a jasco v-630 uv–vis spectrophotometer within the range 200–1000 nm using 1×10-3 m solution in dmf. the ft-ir spectra were recorded on a shimadzu ir tracer 100 spectrophotometer as kbr discs in the range 4000–400 cm-1. the electrochemical performance of the copper(ii) complexes was realized using an electrochemical analyzer on 600 c version 5.01 in dmf using platinum and ag–agcl as a working and reference electrode, respectively. tetrabutylammonium perchlorate (tbap, 0.1 m) was used as supporting electrolyte. the esi-ms spectrum of the copper(ii) complex was identified using bruker daltonics. the x-band epr spectra of the copper(ii) complexes were taken at room temperature as well as frozen solutions in dmf by varian esr-112 sspectrometer and esr jeol jes-fa200, respectively. analytical and spectral data of the complexes are given in supplementary material to this paper. evaluation of the biological activity minimum inhibitory concentration (mic). the antibacterial activity of the synthesized thiosemicarbazone ligands h(l1)–h(l3) and copper(ii) complexes c1–c9 were tested using the method described earlier.12 the minimum inhibitory concentration of the synthesized compounds (as 5 and 50 μg ml-1) against b. thuringiensis (gram-positive) and e. coli (gramnegative) was evaluated in mm using the disc diffusion method in nutrient agar medium. chloramphenicol and dmf were used as the positive and negative control, respectively. filter paper of about 6 mm diameter was dipped into the compounds and placed in the nutrient agar plate inoculated with microorganisms under sterile conditions. then the plates were incubated at 37 °c for 24 h to observe the zone of inhibition. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 324 rajendran et al. dna cleavage activity. the dna cleavage efficiency of the thiosemicarbazone ligands and their copper(ii) complexes were assessed by agarose gel electrophoresis.13 the experiments were performed at 40 and 60 μm concentration of the complexes treated with sc puc18 dna under the experimental conditions in 5 mm tris–hcl/50 mm nacl buffer at ph 7.2 and incubated for 1 h at 37 °c in the presence of an activating agent (ascorbic acid). later, the sample was mixed with the gel loading buffer (25 % bromophenol blue, 0.25 % xylene cyanol, 30 % glycerol) and loaded on to 1.2 % agarose gel containing 0.5 mg/ml ethidium bromide for 1 h. after electrophoresis, the extent of dna cleavage of the copper(ii) complexes was visualized and photographed by spectroline ultraviolet transilluminator (uvitec geneitm fire reader, india) gel documentation system. a complete conversion of sc dna (form i) to nc dna (form ii) and linear (form iii) was monitored. in vitro cytotoxicity analysis maintenance of the cell lines. the human cervical carcinoma cell line (hela) was routinely maintained in eagle’s minimal essential medium (mem) containing penicillin (100 u ml-1) and streptomycin (100 µg ml-1) and supplemented with 10 % fetal bovine serum (fbs, himedia, india) in a humidified atmosphere of 5 % co2 incubator at 37 °c. cisplatin (platinex) was used as the positive control and the cells without treatment as the negative control. mtt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. the complex stimulated cell toxicity study was realized using human cervical cancer cell line (hela). it is mainly based on the metabolic reduction of soluble mtt to insoluble formazan crystal by mitochondrial enzyme activity of viable cells.14 the hela cell line was seeded onto 96 well plates with a seeding density of 2×105 cells/well and kept in a co2 incubator at 37 °c for attachment of the cells. after 16 h, the test sample at 5 μm concentration was added and incubated for 24 h. to the cells were added 20 μl of mtt (5 mg ml-1 in pbs buffer) and kept for 4 h. then the supernatant was completely removed and 50 μl of dmso was added into each well to dissolve the precipitants. the absorbance was measured at 570 nm using an elisa plate reader (read well touch, robonik, india) with a reference wavelength at 690 nm. the assay was performed in triplicate and the values were compared with that of the positive control (cisplatin). synthesis of thiosemicarbazone ligands preparation of 1-phenyl-2-((5-(pyridin-3-yl)-4h-1,2,4-triazol-3-yl)thio)ethanone (hl). the starting material hl was synthesized by stirring (2 h) in a 1: 1 molar ratio 5-(3-pyridyl) -4h-1,2,4-triazole-3-thiol (0.18 g, 1 mmol) and phenacyl bromide (0.20 g, 1 mmol) in dimethylformamide (2 ml) and ethanol (10 ml). then, the obtained colorless solid was set aside under cold conditions for a day. it was then filtered and washed with cold ethanol and dried in vacuo over anhydrous cacl2. preparation of h(l1)–h(l3). the compound h(l1) was synthesized using the following procedure: 1-phenyl-2-((5-(pyridin-3-yl)-4h-1,2,4-triazol-3-yl)thio)ethanone (0.30 g, 1 mmol) in ethanol was mixed with 4-methyl-3-thiosemicarbazide (0.12 g, 1 mmol) in water (20 ml). to this, a few drops of glacial acetic acid were added and the mixture was refluxed for 2 h at 90 °c in a water bath. the resulting clear yellow solution was set aside under cold condition for 24 h in a refrigerator. thereafter the obtained yellow solid was filtered, dried and recrystallized from chloroform. the similar method was applied for the synthesis of h(l2) and h(l3), but using 4-ethyl-3-thiosemicarbazide or 4-phenyl-3-thiosemicarbazide, respectively, instead of 4-methyl-3-thiosemicarbazide (scheme 1). ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ copper(ii) complexes with n(4)-substituted thiosemicarbazide and diimine ligands 325 scheme 1. schematic representation of synthesis of thiosemicarbazone ligands. synthesis of copper(ii) bis complexes (c1–c3) to a 2 mmol ethanolic solution of ligand h(l1), a solution of copper(ii) chloride dihydrate (1 mmol in ethanol) was added over 1 h under constant stirring to obtain a clear green solution. the resulting solution was set aside overnight at room temperature. subsequently, the obtained green precipitate was washed several times with cold ethanol and dried to afford complex ci (scheme 2). a similar method was applied for the synthesis of complexes c2 and c3 using h(l2) and h(l3), respectively, instead of h(l1). scheme 2. schematic representation of the preparation of copper(ii) bis complexes c1–c3. synthesis of mixed ligand copper(ii) complexes (c4–c9) to a 2 mmol ethanolic solution of ligand h(l1), a solution of 2,2′-bipyridyl (1 mmol in ethanol) was added over 1 h under constant stirring. to this solution, copper(ii) chloride dihydrate was added dropwise and again stirred for 30 min. subsequently, the obtained green precipitate was washed several times with cold ethanol and dried. a similar method was applied for the synthesis of complexes c5 and c6 using h(l2) and h(l3), respectively, instead of h(l1) (scheme 3). complexes c7–c9 were prepared in a similar manner to c4–c7, respectively, using 1,10-phenanthroline instead of 2,2′-bipyridyl (scheme 3). ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 326 rajendran et al. scheme 3. schematic representation of the preparation of the mixed ligand copper(ii) complexes c4–c9. results and discussion 1hand 13c-nmr spectra of the thiosemicarbazone ligands h(l1) – h(l3) the structures of the free thiosemicarbazone ligands were verified by their 1hand 13c-nmr spectra in dmso-d6. the 1h-nmr spectra of thiosemicarbazone ligand h(l1) is given in the supplementary material, figs. s-1 and s-2. all the synthesized thiosemicarbazone ligands h(l1)–h(l3) showed a singlet of the hydrazinic proton at δ 10.88 and 11.08 ppm for the methyl and ethyl substituted thiosemicarbazone ligands h(l1) and h(l2) except for h(l3) that appeared in δ 11.60 ppm as a result of the inductive effect of the electron withdrawing phenyl group.15,16 a sharp singlet of azomethine proton was observed at around δ 8.55 ppm. the signals at around δ 7.50–6.90 ppm were attributed to the aromatic protons of the ligands. the chemical shift of ch2 of the free ligand was found at about δ 4.50 ppm. the signals found near δ 6 ppm were assigned to methyl and ethyl protons. similarly, in the 13c-nmr spectrum, signals of c=n and c=s appeared within the region of δ 145 and 175 ppm, respectively. the signals due to aromatic carbons were observed in the expected region (δ 140–112 ppm). the signals corresponding to the ch2 carbon showed a signal near to 38.5 ppm.17 the rest of the methyl and ethyl carbon appeared in the downfield region. hence, it was clear that the data obtained from the 1h-nmr and 13c-nmr data were in good agreement with each other and confirmed the structures of the thiosemicarbazone ligands. the data of the other thiosemicarbazone derivatives, h(l2) and h(l3), are given in the supplementary material. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ copper(ii) complexes with n(4)-substituted thiosemicarbazide and diimine ligands 327 electronic spectra the electronic spectra of thiosemicarbazone ligands h(l1)–h(l3) and their respective copper(ii) complexes c1–c9 provided good evidence for the geometry of the complexes. the electronic spectra and the absorption spectral data of ligand h(l1) and complex c5 are given in the supplementary material, figs. s-3 and s-4, respectively. the electronic spectra of thiosemicarbazone ligands in dmf displayed two intense absorption bands near 240 and 350 nm that are assigned to π→π∗ and n→π∗ transitions, respectively.18 the band around 240 nm corresponding to the azomethine chromophore experienced a bathochromic shift as a result of the donation of the lone pair of electrons to the central metal ion. the absorption band of the thioamide chromophore near 350 nm experienced blue shift owing to thioenolization.11,18 in the electronic spectra of copper(ii) complexes displayed three to four bands. a significant low intensity d→d band was observed near 600–750 nm for all the copper(ii) complexes. this might be due to the structural differences of the complexes through the extended conjugation of the present aromatic ring. however, complex c9 exhibited a moderate intense band in the region 735 nm due to 2b1g→2a1g transitions of square planar copper(ii) complexes.19 noticeably, all the thiosemicarbazone copper(ii) complexes in dmf were in concurrence with the d9 electronic configuration of the cu(ii) ion. ft-ir spectroscopy the coordinating sites of the copper and free thiosemicarbazone ligands were attained from ft-ir spectroscopy. the characteristics ft-ir spectral data afforded suitable evidence for the coordination of ligand to the central metal ion through deprotonated sulfur, the azomethine nitrogen and n,n′-donor of heterocyclic bases. the free thiosemicarbazone ligands displayed ν(n(4)–h) band around 3055–3350 cm–1.19 in general, thiosemicarbazone can exist in thione– –thiol tautomerism. a strong band around 800–856 cm–1 in the ligand was attributed to the presence of –nh–c(=s)–n group that might be converted to c–s in the spectra of complexes as a result of enothiolization by loss of the n(2)h hydrogen and the formation of a new –c=n– group, found at around 1530–1560 cm–1.15 in addition, a characteristic high intensity c=n band of a ligand in the region of 1520–1540 cm–1 could be shifted to higher frequencies (1550–1580 cm–1) in the spectra of complexes, suggesting the coordination of azomethine to copper. the ν(n–n) band of thiosemicarbazone was found near to 970–990 cm–1 and this might be increased by 20–25 cm–1 in the complexes upon chelation that increases in the double bond character as well as a loss of electron density.20 the infrared spectra of complexes showed medium bands around 420 and 550 cm–1, which is consistent with ν(cu–n) and ν(cu–s), respectively.17 ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 328 rajendran et al. epr spectral study the epr spectra of the thiosemicarbazone copper(ii) complexes offer valuable information regarding the extent of delocalization of the unpaired electron in copper(ii) complexes, the geometry and metal ligand bonding. the epr spectra of the complexes were recorded at room temperature and at liquid nitrogen temperature (77 k) to facilitate the determination of epr parameters, such as g||, g⊥, a|| and g. the x-band epr spectra of mixed ligand thiosemicarbazone copper(ii) complex c7 in dmf are presented in the supplementary material, fig. s-5). the x-band epr spectra of the copper(ii) complexes displayed three wellresolved peaks at g|| region, corresponding to nuclear spin i = 3/2. the thiosemicarbazone copper(ii) complexes were found to be g|| (2.252–2.268) > g⊥ (2.056– –2.074) > 2.0023, which revealed that the unpaired electron of the copper(ii) complexes had a dx2–y2 ground state that is characteristic of square planar geometry.21 the exchange interaction parameter of the copper(ii) complexes could be calculated using the following expression: g = ((g|| – 2)/(g⊥ – 2)) and values within 3.55–4.54 were found, indicating negligible interaction in the copper(ii) complexes according to hathaway.22 meanwhile, g|| values less than 2.3 were found for these complexes, suggesting the covalent nature of the copper(ii) complexes. it is renowned that a cun4 chromophore with square planar geometry is expected to display g|| and a|| values within the range of 2.220 and (180–200)×10–4 cm–1, respectively.23,24 the replacement of any coordinated nitrogen by sulfur/ /nitrogen would increase the g|| value and decrease the a|| likewise. at this point, the observed values for copper(ii) bis complexes (cun2s2) c1–c3 and mixed ligand copper(ii) complexes (cun3s) c4–c9 were found to be g|| (2.252 to 2.268) and a|| (158 to 163×10–4 cm–1), revealing the presence of square planar geometry. furthermore, the geometrical distortions of the copper(ii) complexes were measured using the g||/a|| ratio. in general, the values for square planar complexes fall between 130–138×10–4 cm. the values for the synthesized copper(ii) complexes were in the range (138–142)×10–4 cm, suggesting that the complexes reliably have perfect square planar geometry with the absence of any significant distortion from planarity.25 this was further supported by molar conductivity measurement in dmf the obtained values of which fall in the neutral and 1:1 electrolyte range of copper(ii) bis complexes and mixed ligand copper(ii) complexes, respectively. 26 high resolution mass spectra the stoichiometric composition of the synthesized thiosemicarbazone copper(ii) complexes was identified using high-resolution mass spectroscopy and their molecular ion peaks was used to authenticate the proposed formulae of the complexes. the high-resolution mass spectra of complex c9 is given in the supplementary material, fig. s-6. it showed a molecular ion peak at m/z 765.16, ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ copper(ii) complexes with n(4)-substituted thiosemicarbazide and diimine ligands 329 which corresponds to the existence of 99 % of the relative abundance of [c34h26cun9s2]+cl– species with an ethanol adduct. thus, the m/z of c9 confirmed the stoichiometry of complex c9 as a [cu(l3)(phen)]cl type. electrochemistry the electrochemical actions of the thiosemicarbazone copper(ii) complexes were studied using cyclic voltammetry and differential pulse voltammetry (supplementary material, fig. s-7 and table s-i). the measurements were performed using a platinum working electrode and tbap as the supporting electrolyte in dmf. the cu(ii)/cu(i) redox potential (dpv) of the bis copper(ii) complexes and mixed ligand copper(ii) complexes of bpy/phen could be in the following order: 0.601 (c1) > 0.529 (c2) > 0.471 (c3); 0.637 (c4) > 0.598 (c5) > 0.446 (c6); 0.643 (c7) > 0.612 (c8) > 0.587 (c9). the cu(ii)/cu(i) redox potential values of the mixed ligand copper(ii) complexes c4, c5, c8 and c9 were comparatively more positive than the copper(ii) bis complexes, which might be attributed to the existence of electron donating –nme, –net groups and diimine co -ligands. it was shown that the incorporation of –nme, –net and bpy/phen in the coordination would predominantly raise the cu(ii)/cu(i) redox potential rather than –nph electron withdrawing substituents. thus, in the case of copper(ii) bis complexes c1 and c2, were slightly more positive upon –nme2 and –net2 substitution. similarly, c3 with two electron withdrawing groups (–nph2) shows lower redox potentials than c1 and c2. therefore, the mixed ligand copper(ii) complexes facilitating cu(ii)→cu(i) reduction were more feasible than the copper(ii) bis complexes.24,27 pharmacological results in vitro antibacterial activity. the comparative results of antibacterial activity of thiosemicarbazone ligands h(l1)–h(l3) and their copper(ii) complexes c1–c9 in association with a zone of inhibition with the standard positive control (chloramphenicol) are presented in the supplementary material, table s-ii. the tabulated antibacterial results suggested the following: the thiosemicarbazone derivatives displayed minimal activity against e. coli at 50 μg ml–1 concentration and showed no activity on the other tested gram-positive bacteria, specifically b. thuringiensis. only the copper(ii) complexes c7 and c8 showed higher antibacterial activity and act as a potent bacteriostatic agent against both bacterial strains in comparison with chloramphenicol. this was due to the existence of n(4)-substituted thiosemicarbazones and diimine co-ligands.12 on the other hand, all the other complexes were found to exhibit only moderate activity. this could be explained based on the chelation theory.11 on chelation, the polarity of the copper(ii) complexes would be reduced owing to the overlapping of the donor groups of a ligand with the orbital and partial positive charge of the copper. moreover, it enhances the delocalization of π-electrons over the chelating ring system as well ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 330 rajendran et al. as the lipophilicity of the complexes.28 this might enhance the penetration of lipid layer and therefore affects the growth of the microorganisms. cleavage of puc18dna by the copper(ii) complexes. the effect of concentration-dependent dna cleavage efficiency of the copper(ii) complexes on puc18 dna in the presence of the activating agent was studied by agarose gel electrophoresis (figs. 1 and 2). it is interesting to note that many approved anticancer agents can trigger cell death by damaging dna. for this reason, the extent of dna cleavage activity of thiosemicarbazone-based copper(ii) complexes has been considered for the development of a new novel anticancer drug.29 during electrophoresis, relatively fastest migration was observed in form i. while, form ii occurs on the strand by the nick produced. if both strands were cleaved, linearized form (form iii) is generated that migrates in the midst of forms i and ii. nc sc fig. 1. a) gel electrophoresis of copper(ii) complexes (40 µm) in buffer containing 50 mm tris/-–hcl/50 mm nacl (ph 7.2) in the presence of ascorbic acid at 37 °c. lane 1: dna control; lane 2: dna + ascorbic acid; lane 3: dna + ligand hl+ ascorbic acid; lane 4: dna + ligand h(l1)+ ascorbic acid; lane 5: dna + [cu(l1)2] + ascorbic acid; lane 6: dna + [cu(l1)(phen)]cl + ascorbic acid; lane 7: dna+[cu(l1)(bpy)]cl + ascorbic acid; lane 8: dna + ligand h(l3) + ascorbic acid; lane 9: dna + [cu(l3)2]cl + ascorbic acid; lane 10: dna + [cu(l3)(phen)]cl + ascorbic acid; lane 11: dna + [cu(l3)(bpy)]cl + ascorbic acid; lane 12: dna + ligand h(l2) + ascorbic acid; lane 13: dna + [cu(l2)2] + ascorbic acid; lane 14: dna + [cu(l2)(phen)]cl + ascorbic acid; lane 15: dna + [cu(l3)(bpy)]cl + ascorbic acid. nc sc fig. 2. b) gel electrophoresis of copper(ii) complexes (60 µm) in buffer containing 50 mm tris–hcl/50 mm nacl (ph 7.2) in the presence of ascorbic acid at 37 °c. lane 1: dna + ligand hl + ascorbic acid; lane 2: dna + ligand h(l1) + ascorbic acid; lane 3: dna + [cu(l1)2] + ascorbic acid; lane 4: dna + [cu(l1)(phen)]cl + ascorbic acid; lane 5: dna + [cu(l1)(bpy)]cl + ascorbic acid; lane 6: dna + ligand h(l3) + ascorbic acid; lane 7: dna + [cu(l3)2]cl + ascorbic acid; lane 8: dna + [cu(l3)(phen)]cl + ascorbic acid; lane 9: dna + [cu(l3)(bpy)]cl + ascorbic acid; lane 10: dna + ligand h(l2) + ascorbic acid; lane 11: dna + [cu(l2)2] + ascorbic acid; lane 12: dna + [cu(l2)(phen)]cl + ascorbic acid; lane 13: dna + [cu(l3)(bpy)]cl + ascorbic acid. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ copper(ii) complexes with n(4)-substituted thiosemicarbazide and diimine ligands 331 as illustrated in fig. s-7a, the cleavage activity of plasmid dna at 40 μm concentration involved the effect of complete dna degradation on c6 into unnoticeable minor fragments in the presence of ascorbic acid. the predominant dna cleavage efficiency of c6 could be strong intercalation of the diimine moiety (bpy) and the generation of a large amount of reactive oxygen species by stabilizing cu(i) species.24 however, other complexes showed partial and moderate activity. in addition, under the same experimental conditions at 60 μm concentration resulting in the complete conversion of form i to form ii by means of increasing concentration of copper(ii) complexes. however, no cleavage activity was observed in the control dna and dna with ascorbic acid. in vitro cytotoxic evaluation by the mtt assay encouraged by the positive results achieved from the antibacterial and oxidative dna cleavage activities, the in vitro cytotoxicity of the thiosemicarbazone ligands and copper(ii) complexes were tested against human cervical cancer cell line (hela). the percentage of cytotoxicity of each compound is displayed in table s-iii of the supplementary material. the thiosemicarbazone ligands alone showed less cytotoxicity towards hela cell lines. interestingly, complexation of thiosemicarbazone ligands with heterocyclic bases would predominantly enhance their cytotoxicity even at 5 μm concentration with 24 h exposure. in addition, it was clearly indicated that the cytotoxicity was time and dose-dependent. thus, the activity of complexes c4, c5 and c7 were significantly superior when compared to cisplatin. it was also confirmed that the complexes were very specific to the cervical cancer cell line. consequently, varying of heterocyclic bases with the existence of bulky n(4)-substituents at the thioamide nitrogen could improve their cell-killing activity through stronger and efficient oxidative cleavage.12,13 consequently the data obtained for each complex showed the quality of the results with minimum concentration (5 μm) and shorter incubation period (24 h) when compared to the results of similar observations.12,31,32 conclusions in this study, the coordinating ability of the nine thiosemicarbazone-based copper(ii) complexes c1–c9 was derived from three sulfur-containing ligands h(l1)–h(l3) with copper and diimine co-ligands. it was concluded that the copper(ii) bis complexes c1–c3 coordinated to the central metal copper through two molecules, deprotonated thiolic sulfur and azomethine nitrogen. while the mixed ligand copper(ii) complexes c4–c9 coordinated through deprotonated thiolic sulfur and the n,n′-donor of the heterocyclic bases. remarkably, sc puc18 dna efficiently cleaves the mixed ligand copper(ii) complexes and completely degrades complex c6 through intercalation of bpy/phen in the presence of an activating agent. the antibacterial studies of the compounds displayed superior activity of c7 and c8 at higher concentrations. however, the mixed ligand ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 332 rajendran et al. copper(ii) complexes c4, c5 and c7 exhibited an enhanced cytotoxic effect on cervical hela cell lines upon the existence of n(4)-substituted thiosemicarbazone ligands and diimine co-ligands. hence, the remarkable biological activity of the mixed ligand copper(ii) complexes c7 should be further studied with different cancer cells to elucidate its mode of action. supplementary material additional data are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgments. the authors express their honest thanks to the principal, lady doak college and the management for providing research facilities. the authors heartfelt thanks to professor dr. m. palaniandavar, frsc, fasc, fna, professor of eminence, department of chemistry, bharathidasan university, tiruchirappali, for helping to elucidate the epr spectra. the authors would like to thank dr. r. shenbagarathai, former coordinator, department of biotechnology, lady doak college for helping to complete the cytotoxicity study successfully. the authors thank saif, iit bombay for recording the epr spectra and also thank the science instrumentation centre, lady doak college. и з в о д синтеза комплекса бакра(ii) са n(4)-супституисаним тиосемикарбазидом и диимином као лигандима и испитивање њихове активности према плазмоидној dnk и hela ћелијским линијама neelaveni rajendran1, abirami periyasamy2, nithya kamatch3 и vasantha solomon1 1 pg and research department of chemistry, lady doak college, madurai – 625002, tamil nadu, india, 2 department of biotechnology, lady doak college, madurai – 625002, tamil nadu, india и 3 pg and 3 research department of zoology, lady doak college, madurai – 625002, tamil nadu, india у овом раду описане су синтезе девет нових комплекса бакра(ii) са тиосемикарбазонским типом лиганада, {[cu(l)2] c1–c3, [cu(l)(bpy)]cl c4–c6, [cu(l)(phen)]cl c7–c9 (l = = h(l1)–h(l3), h(l1) = (e)-n-метил-2-(1-фенил-2-((5-(пиридин-3-ил)-4h-1,2,4-триазол-3 -ил)тио)етилиден)хидразинкарботиоамид, h(l2) = (e)-n-етил-2-(1-фенил-2-((5-(пиридин-3 -ил)-4h-1,2,4-триазол-3-ил)тио)етилиденхидра-зинкарботиоамид, h(l3) = (e)-n-фенил-2 -(1-фенил-2-((5-(пирадин-3-ил)-4h-1,2,4-триазол-3-ил)тио)етилиден)хидразинкарботиоамид, bpy = 2,2′-бипиридил и phen =1,10-фенантролин). синтетисани комплекси су окарактерисани применом различитих спектроскопских метода. квадратно-планарна геометрија c1–c9 комплекса је претпостављена на бази epr спектроскопских мерења. на основу испитивања антибактеријске активности ових комплекса нађено је да c7 и c8 комплекси показују најбољу активност према грам-позитивним (b. thuringiensis) и грам-негативним (e. coli) бактеријама. испитивање степена деградације суперколоидне (sc) puc18 dnk у зависности од концентрације комплекса је показало да c6 комплекс показује комплетну деградацију dnk при минималној концентрацији (40 μm). in vitro испитивања цитотоксичне активности синтетисаних комплекса су показала да, у поређењу са цисплатином, комплекси c4, c5 и c7 имају највећу активност према хуманој ћелијској линији канцера грлића материце (hela). на основу добијених резултата биолошких испитивања закључено је да најбољу активност показују комплекси бакра(ii) који као лиганд имају диимински остатак. добијени резултати могу бити од значаја за синтезу нових алтернативних антиканцерних цитостатика у односу на оне који су до сада у медицинској примени. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {studies on the chemical compositions of hyptis suaveolens (l.) poit} j. serb. chem. soc. 84 (3) 245–252 (2019) udc 582.94:581.19:544.2.004.12:547(510) jscs–5180 original scientific paper 245 studies on the chemical compositions of hyptis suaveolens (l.) poit. gengqiu tang, xile liu, xue gong, xiaojing lin, xiudi lai, dong wang and shengguo ji* school of traditional chinese medicine, guangdong pharmaceutical university, p. r. china (received 8 december 2017, revised 21 may, accepted 20 september 2018) abstract: this study aimed at identifying the chemical compounds isolated from the aerial parts and roots of hyptis suaveolens (l.) poit. the compounds were isolated and purified by a combination of various chromatographic techniques including silica gel column chromatography, silica gel reverse phase column chromatography, sephadex lh-20 gel column chromatography, semi-preparative hplc and recrystallization. the chemical structures were analyzed and elucidated based on physiochemical properties and spectroscopic analysis. from the aerial parts of h. suaveolens, eight compounds were isolated and identified as quercetin 3-o-β-d-glucopyranoside (1), apigenin (2), sorbifolin (3), quercetin (4), kaempferol (5), genkwanin (6), rosmarinic acid (7) and methyl rosmarinate (8). two compounds were isolated from the roots of h. suaveolens and identified as podophyllotoxin (9) and picropodophyllotoxin (10). compounds 2–6 were isolated from h. suaveolens for the first time while compound 10 was isolated from the genus of hyptis for the first time. the results of this study provided a scientific basis for the development of the medicinal value of h. suaveolens and have important theoretical significance for the chemical utilization of h. suaveolens resources. keywords: structural identification; quercetin 3-o-β-d-glucopyranoside; sorbifolin. introduction hyptis suaveolens (l.) poit, locally known in china as maolaohu belongs to the family lamiaceae, covers important horticultural crops having economical value and is an important medicinal plant that is distributed in dense clumps along roadsides, in over-grazed pastures and around stockyards throughout the tropics and subtropics. h. suaveolens has a number of synonyms, such as ballota suaveolens l., bystropogon graveolens blume, bystropogon suaveolens (l.) l'hér., gnoteris cordata raf., gnoteris villosa raf., hyptis congesta leonard, hyptis * corresponding author: e-mail shengguo_ji@163.com https://doi.org/10.2298/jsc171208078t 246 tang et al. ebracteata r.br., hyptis graveolens schrank, hyptis plumieri poit., marrubium indicum blanco, mesosphaerum suaveolens (l.) kuntze, schaueria graveolens (blume) hassk. or schaueria suaveolens (l.) hassk. h. suaveolens is a fast-growing perennial and aromatic herb, 0.4–2 m tall with hairy stems and leaves, having branches and usually woody at the base. the leaves are weak, oval in outline, tip broadly pointed.1,2 the plant is native to the tropics of america and now considered as a weed worldwide. there are also many distributions on the southern coast of china. the medicinal use of the plant has been reported in 22 countries, which emphasizes its potential values of application in modern pharmacy. in traditional medicine, almost all parts of this plant are used to treat numerous ailments, such as respiratory and gastrointestinal infections, as antirheumatic and antisuporific baths, to treat indigestion, colic, stomachache, colds, fever, burns, wounds, cramps and various skin complaints.3–6 h. suaveolens has both medicinal individuality as well as insecticidal properties. in several countries, it is commonly used as an insect repellent by incinerating or placing it naturally.7–10 among these traditional medicinal uses, the leaves are used most frequently, followed by seeds and thirdly the whole plants. in addition, the decoction of the roots was reported to contain urosolic acid, a natural hiv integrase inhibitor, and was highlighted for the preparation of an appetizer.11 the chemical components of the essential oils obtained from the leaves of h. suaveolens were intensively investigated in previous studies. many new chemical compounds, such as monoterpenes and sesquiterpenoids, obtained from the essential oils of h. suaveolens leaves were isolated and their structures elucidated, and their antibacterial, antifungal and anti-aspergillus activity confirmed.12–16 in addition, it was reported that the leaves had anticancer and antifertility properties, while their aqueous extract showed an antinociceptive effect and acute toxicity, especially for the essential oil.17,18 however, little attention has been given to the extraction, separation and structural identification of chemical compositions from the entire plant, leading to a lack of a systematic study. therefore, information concerning the chemical composition of h. suaveolens is still incomplete. as the drug is endowed with huge exploitation and utilization value, it is medicinally important to know precisely and comprehensively its chemical composition. in view of the importance of this, the present investigation is concerned with the extraction, separation, purification and structural identification of the aerial parts and roots of h. suaveolens using physicochemical and chromatographic methods to help in the development of the utilization and pharmacological activity of this drug. materials and methods general procedures all reagents used for the analyses were of analytical grade and were not subjected to further purification. column chromatography was performed on silica gel (200–300 mesh, qingdao haiyang chemical works, china), ods (50–70 µm, tianjin jianhexiang chromato chemical compositions of hyptis suaveolens (l.) 247 graphic works, china) and sephadex lh-20 (beijing ruida henghui scientific and technical corporation, china). semi-preparative hplc was realized on a gilson 322 instrument, using a shim-pack gis c18 column (3.0 cm×250 mm, 5 µm, shimadzu, japan). the esi-ms spectra were recorded on a waters acquity uplc/q-tof micro lc-ms spectrometer (waters, usa). the nmr spectra were obtained on an avance 500 spectrometer (bruker, germany). collection, identification and preparation of the plant materials fresh whole plants of h. suaveolens growing on the grounds of guangdong pharmaceutical university, guangzhou higher education mega center were randomly collected, taxonomically identified and authenticated by prof. shengguo ji, school of traditional chinese medicine, guangdong pharmaceutical university. they were cleaned to remove physical impurities, air-dried in the shade, mechanically powdered, sieved using 10 meshes and stored in hermetic containers under dry air for further studies. this research was conducted in the key laboratory of the state administration of traditional chinese medicine for the production & development of cantonese medicinal materials, school of traditional chinese medicine, guangdong pharmaceutical university, china, during 2016–2017. extraction and purification of samples the coarse powder materials of aerial parts (8.67 kg) and roots (1 kg) of h. suaveolens were subjected to ultrasonic extraction three times with ethanol (90 % and 2×70 %, the ratio of solid (powder) to liquid (ethanol) was 1:10), and these extracts were concentrated in a rotary evaporator under reduced pressure and controlled temperature (< 45 °c). they were then extracted separately and successively with petroleum ether, ethyl acetate and n-butanol. (b.p. 60–90 °c, 10 l). the ethyl acetate extracts were placed in an airtight container and stored in a refrigerator. purification of the samples were performed using silica gel column chromatography, silica gel reverse phase column chromatography, sephadex lh-20 gel column chromatography, gilson semi-preparative chromatography and recrystallization. identification of constituents the constituents of the extracts were determined by a combination of physicochemical properties, uv spectrometry and nuclear magnetic resonance spectrometry (nmr), and identified by comparing their mass spectral patterns with the data for original samples and by matching those cited in the literature.19-28 results and discussion the chemical compounds of the aerial parts and roots of h. suaveolens were determined and are listed in table i. their structures were identified by comparison of physicochemical analysis and their spectral data (esi-ms, 1h-nmr and 13c-nmr data) with literature values. the characteristics, solubility and melting points (m.p.) of compounds are also given in table i, which were used to deduce the functional group. by comparing with the uv absorption and esi-ms data, given in table ii, the molecular formula of each compound could be obtained. the 13c-nmr and 1h-nmr spectra data are presented in tables s-i and s-ii, respectively, of the supplementary material to this paper. from these data, detailed structural information of compounds, shown in fig. 1, were obtained. the results revealed that the identified compounds from the aerial parts of h. suaveolens included quercetin 3-o-β-d-glucopyranoside (1), apigenin (2), sorbi248 tang et al. folin (3), quercetin (4), kaempferol (5), genkwanin (6), rosmarinic acid (7) and methyl rosmarinate (8), while the identified compounds from the roots of this plant included podophyllotoxin (9) and picropodophyllotoxin (10). compounds 2–6 were found in h. suaveolens for the first time, while compound 10 was isolated for the first time from the genus of hyptis. compound 1, also called isoquercetin, was found in this plant in a previous study,29 which was identified in course of lc-ms measurements, but it was not isolated. compounds 7–9 were also obtained from the lc-ms in this plant according to previous literature.30,31 table i. components detected in the extracts of h. suaveolens with solubility and melting point data compound characteristics solubility m.p. / °c functional group classes quercetin 3-o-β-d-glucopyranoside yellow powder methanol-soluble; waterand ethyl acetate-insoluble – flavonoid glycoside apigenin yellow powder methanol-soluble; water-insoluble 347–348 flavone sorbifolin yellowish white needle crystals methanol-soluble; water-insoluble n.a. flavone quercetin yellow powder methanol-microsoluble; chloroform and ethyl acetate-insoluble 313–314 flavone kaempferol yellow powder methanol-microsoluble; chloroformand ethyl acetate-insoluble 276–278 flavone genkwanin yellow powder methanoland water-insoluble 293–296 flavone rosmarinic acid white powder waterand methanol-soluble; acetone-insoluble 171–175 polyphenol methyl rosmarinate yellow syrup methanoland ethyl acetate-soluble; water-insoluble – polyphenol podophyllotoxin white powder methanol-soluble; water-insoluble 183–184 lignan picropodophyllotoxin white powder acetone-soluble; water-insoluble 225–227 lignan table ii. uv and esi-ms data for the isolated compounds compound max. absorption in uv, nm esi-ms exact mass molecular formula ref. quercetin 3-o-β-d-glucopyranoside 268, 364 487.0857[m+na]+, 463.0887[m–h]－ 465.391 c21h20o12 19 apigenin 268, 336 271.0635[m+h]+ 270.241 c15h10o5 20 sorbifolin 262, 325 323.0542[m+na]+, 299.0536[m–h]－ 300.268 c16h12o6 21 quercetin 283, 336 303.0445[m+h]+, 301.0344[m–h]－ 302.201 c15h10o7 22 kaempferol 222, 267, 366 285.0378[m–h]－ 286.241 c15h10o6 23 genkwanin – 283.0619[m–h]－ 284.268 c16h12o5 24 chemical compositions of hyptis suaveolens (l.) 249 table ii. continued compound max. absorption in uv, nm esi-ms exact mass molecular formula ref. rosmarinic acid 199, 261, 325 361.0931[m+h]+, 383.0746[m+na]+, 359.0785[m–h]－ 360.32 c18h16o8 25 methyl rosmarinate 205, 264, 331 374.0961[m+h]+, 397.0901[m+na]+, 373.0933[m–h]－ 374.347 c19h18o8 26 podophyllotoxin 203, 288 432.0534[m+nh4]+, 415.0847 [m+h]+ 414.412 c22h22o8 27 picropodophyllotoxin 264, 322 851.0301[2m+na]+, 438.0634[m+na+1], 415.0688[m+1]+ 414.412 c22h22o8 28 fig 1. chemical structures of 1–10. 1. quercetin 3-o-β-d-glucopyranoside; 2. apigenin; 3. sorbifolin; 4. quercetin; 5. kaempferol; 6. genkwanin; 7. rosmarinic acid; 8. methyl rosmarinate; 9. podophyllotoxin; 10. picropodophyllotoxin most previous studies focused on the identification of polyphenols, sesquiterpenes, diterpenes, triterpenes and steroids extracted from h. suaveolens, but little attention was given to the flavonoids extracted from this plant. it was indi250 tang et al. cated that flavonoids have antibacterial and antiviral, antitumor, antioxidant, antiinflammatory, analgesic and other biological activities. the results showed that compounds obtained from the aerial parts of h. suaveolens were flavonoid constituents mostly. generally, dissociative flavonoids were extracted by ethanol (90–95 %), flavonoid glycosides were extracted by ethanol (60 %), while both could be extracted by ethanol (70 %). in this study, the flavonoid constituents of the aerial parts of h. suaveolens were studied by the ultrasonic extraction method using ethanol (70 and 90 %), which could be used for the comprehensive extraction of flavonoids. however, in this experiment, tailing of peaks of preparation liquid chromatogram could easily occur when extracting flavonoids monomer. in this case, the hydrothermal re-crystallization purification method can be tried to remove water-soluble impurities and improve the purity of the product when the percentage of methanol in methanol-water is more than 30 %. the compounds podophyllotoxin and picropodophyllotoxin obtained in this research belong to the functional group of aryl lignans that are mainly derived from plants of the family berberidaceae, and are isomers to each other. it was reported that podophyllotoxin had potent inhibitory effect on tumors and virus, which reveal the huge exploitation and utilization value of the roots of h. suaveolens.31 this research provides essential information for further studies on the medicinal values of h. suaveolens. however, h. suaveolens has not been recorded in the chinese pharmacopoeia. in order to develop the curative value of it, a series of further scientific research, such as its pharmacological research, clinical tests and formulation of the quality standard have to be completed. conclusions the chemical compounds isolated from the aerial parts and roots of hyptis suaveolens (l.) poit. were identified by comparison of physico-chemical analysis and their spectral data. the components obtained from the aerial parts were identified as quercetin 3-o-β-d-glucopyranoside (1), apigenin (2), sorbifolin (3), quercetin (4), kaempferol (5), genkwanin (6), rosmarinic acid (7) and methyl rosmarinate (8), while the components obtained from the roots were identified as podophyllotoxin (9) and picropodophyllotoxin (10). in this regard, compounds 129 and 7–930,31 have been reported to be found in h. suaveolens, while compounds 2–6 are found in h. suaveolens for the first time and compound 10 is isolated from the genus of hyptis for the first time. supplementary material the 1h-nmr and 13c-nmr data, as well as all recorded spectra and preparation liquid chromatograms, are available electronically at the pages of journal website: http:// //www.shd.org.rs/jscs/, or from the corresponding author on request. chemical compositions of hyptis suaveolens (l.) 251 и з в о д испитивање хемијског састава hyptis suaveolens (l.) poit. gengqiu tang, xile liu, xue gong, xiaojing lin, xiudi lai, dong wang и shengguo ji school of traditional chinese medicine, guangdong pharmaceutical university, p. r. china циљ истраживања је био да се изврши идентификација хемијских једињења која су изолована из надземних делова и корена биљке hyptis suaveolens (l.) poit. једињења су изолована и пречишћена комбиновањем различитих хроматографских и препаративних техника, којe укључују хроматографију на колони силика-гела, хроматографију на реверсној фази модификованог силика-гела, хроматографију на колони sephadex lh-20, семи-препаративну hplc хроматографију и кристализацију. хемијске структуре једињења анализиране су и утврђене на основу физичко–хемијских особина и спектроскопске анализе. осам једињења је изоловано из надземних делова h. suaveolens и идентификована су као кверцетин-3-o-β-d-глукопиранозид (1), апигенин (2), сорбифолин (3), кверцетин (4), камферол (5), генкванин (6), рузмаринска киселина (7) и метил -розмаринат (8). два једињења су изолована из корена h. suaveolens и идентификована су као подофилотоксин (9) и пикроподофилотоксин (10). једињења 2–6 изолована су по први пут из h. suaveolens док је једињење 10 изоловано из рода hyptis по први пут. резултати истраживања пружају научну заснованост за резвијање примене h. suaveolens у медицини и имају важан теоретски значај за ширу употребу h. suaveolens. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice tetraoxanes as inhibitors of apicomplexan parasites plasmodium falciparum and toxoplasma gondii growth and anti-cancer molecules j. serb. chem. soc. 80 (11) 1339–1359 (2015) udc *plasmodium falciparum*toxoplasma jscs–4801 gondii+547.495.9+541.459:576+615.9:615.9: 615.285:615.277 original scientific paper 1339 tetraoxanes as inhibitors of apicomplexan parasites plasmodium falciparum and toxoplasma gondii growth and anti-cancer molecules dejan m. opsenica1*#, jelena radivojević2, ivana z. matić3, tijana štajner4, slavica knežević-ušaj5, olgica djurković-djaković4 and bogdan a. šolaja6# 1institute of chemistry, technology, and metallurgy, university of belgrade, njegoševa 12, 11000 belgrade, serbia, 2institute of molecular genetics and genetic engineering, university of belgrade, vojvode stepe 444a, p. o. box 23, 11010 belgrade, serbia, 3institute for oncology and radiology of serbia, pasterova 14, 11000 belgrade, serbia, 4national reference laboratory for toxoplasmosis, institute for medical research, university of belgrade, dr. subotića 4, p. o. box 102, 11129 belgrade, serbia, 5institute for pathology, medical faculty, university of novi sad, hajduk veljkova 3, 21000 novi sad, serbia and 6faculty of chemistry, university of belgrade, studentski trg 12–16, p. o. box 51, 11158, belgrade, serbia (received 30 april, revised and accepted 22 june 2015) abstract: new cyclohexylidene 1,2,4,5-tetraoxanes with polar guanidine and urea based groups were synthesized and evaluated for their antimalarial activity against chloroquine resistant and susceptible plasmodium falciparum strains. the derivatives showed moderate, nm range antimalarial activities and low cytotoxicity. the n-phenylurea derivative 24 exhibited the best resistance indices (riw2 = 0.44, ritm91c235 = 0.80) and was not toxic against human normal peripheral blood mononuclear cells (ic50 > 200 μm). seven derivatives were tested in vitro against four human cancer cell lines and they demonstrated high selectivity toward leukaemia k562 cells. one compound, derivative 21 with a primary amino group, was the first tetraoxane tested in vivo against toxoplasma gondii as another apicomplexan parasite. subcutaneous administration at a dose of 10 mg kg-1 day-1 for 8 days allowed the survival of 20 % of infected mice, thus demonstrating the high potential of tetraoxanes for the treatment of apicomplexan parasites. keywords: antimalarials; antiparasitic; peroxides; cancer; cytotoxicity. * corresponding author. e-mail: dopsen@chem.bg.ac.rs # serbian chemical society member. doi: 10.2298/jsc150430063o 1340 opsenica et al. introduction apicomplexan protozoa are single-celled parasites, with significant medical, veterinary and economic effects worldwide. from the aspect of human medicine, two apicomplexan infections are of major health concern – malaria, caused by plasmodium spp., and toxoplasmosis, caused by toxoplasma gondii. a potentially devastating disease, malaria represents an enormous public health problem in the majority of developing countries, further emphasized by the fact that nearly half of the world population is exposed to the infection.1 malaria may be caused by five plasmodium species, p. falciparum, p. ovale, p. vivax, p. malariae and p. knowlesi, of which p. falciparum that causes cerebral malaria is the major threat. on a cellular level, all plasmodium parasites contain acidic food vacuoles (fv), where digestion of haemoglobin occurs and it is generally accepted that the fv are the site of action for a number of quinoline-like drugs. the haem obtained from haemoglobin degradation is toxic to the parasite and is transformed into insoluble haemozoin pigment, while the globin is hydrolysed into individual amino acids. antimalarial drugs active within fv appear to kill the parasite either by producing toxic free radicals2 or by blocking haemozoin formation, as in the case of the 4-amino-7-chloroquinolines (acqs).3 the development of widespread drug-resistance to chloroquine (cq), the most successful antimalarial drug, has resulted in severe health issues in malaria endemic regions. although thorough investigation led to the realization that mutations in p. falciparum chloroquine resistant transporter (pfcrt), multidrug resistance protein 1 (pfmdr1) and multidrug resistance-associated protein (pfmrp) are responsible for the development of resistance of the malaria parasite against chloroquine and its analogues,4 additional analysis appears necessary.5 therefore, significant focus has been placed on the synthesis of peroxide antimalarials active in fv6 or interfering in nadph balance acting as leuco-methylene blue (lmb) and fadh2 oxidisers,7 as well as on the development of new 4-aminoquinolines,8 acridines9 and other molecules that prevent haem polymerisation.10 t. gondii is an obligatory intracellular apicomplexan protozoan with worldwide distribution. globally, it has been estimated that one in three persons is infected with t. gondii. in the united states, nearly 25 % of the adult population has been infected with this organism,11 while most studies of european populations report 20−35 % seropositivity.12 however, in south america, these rates can reach 50−75 %.13 individuals with a compromised immune system, such as hiv-infected persons and transplant recipients, are particularly prone to severe t. gondii induced disease, mainly as a result of reactivation of a previously latent infection.14 when primary t. gondii infection occurs during pregnancy, it can be vertically transmitted, which may result in intrauterine death, birth defects, or to late sequellae, such as ocular disease and mental retardation. tetraoxanes as inhibitors of apicomplexan parasites growth 1341 as intracellular pathogens, plasmodium spp. and t. gondii both rely on the invasion of host cells for survival and proliferation. since both parasites belong to the same apicomplexan phylum, they have common metabolic pathways and thus may be susceptible to same drugs for prevention or treatment.15–17 however, there are a number of differences between the two pathogens. during the asexual reproduction stage, p. falciparum exclusively targets human erythrocytes, whereas t. gondii can infect any cell in most vertebrates.17 and, while malaria is transmitted only via the mosquito vector or blood exchange, infection with t. gondii occurs via ingestion of sporulated oocysts shed into the environment by infected cats, or by consuming bradyzoites in the form of tissue cysts from infected animals. current therapies for t. gondii include drugs which inhibit folate metabolism, protein synthesis, or disrupt electron transport.18,19 for the inhibition of folate metabolism, a combination of diaminopyrimidine antimalarials, such as trimethoprim 1 or pyrimethamine 2 (fig. 1), and sulfonamides, such as sulfadiazine 3 (fig. 1) or sulfamethoxazole, is the recommended therapy. this combination acts synergistically not only against t. gondii, but also against various bacterial and other parasitic microorganisms. in patients sensitive to sulfonamides, protein synthesis inhibitors such as macrolide and lincosamide antibiotics are the second line of drugs. another antimalarial, atovaquone, an inhibitor of mitochondrial electron-transport processes, is the drug of choice as third line therapy.20 in all these treatments, drug resistance, high cost, limited efficacy, and side effects21 often result in discontinuation of therapy. therefore, new agents with better activity and safety profiles and that are less expensive are fig. 1. various inhibitors of the t. gondii parasite. 1342 opsenica et al. urgently needed.19 a variety of t. gondii inhibitors designed for different targets have been reported in the last few years, such as inhibitors of ca-dependent protein kinase-1 (tgcdpk1),22,23 compounds which cause the disappearance of apicoplasts and plastid-like organelles,16 phosphodiesterase,17 purine nucleoside phosphorylase,18 and biosynthesis of pantothenic acid.24 of these, outstanding activities were exhibited by tgcdpk1 inhibitors 4 and 5,22,23 and the inhibitor of the vitamin b5 biosynthetic pathway, derivative 6 (fig. 1).24 outstanding examples of drugs active against both plasmodium spp. and t. gondii parasites include artemisinin (7, art, fig. 2) and its semi-synthetic derivatives. it was established that their main mechanism of action is interfering in calcium homeostasis and triggering of micronema.25,26 art and artemether 8 exhibited 100 times better in vitro activity against t. gondii than trimethoprim 1, the current front-line drug.19 other semi-synthetic artemisinin derivatives, including 9,19 artemisone 10 or artemiside 11 have shown even better activities in the nm region (fig. 2).27 these derivatives inhibit multiple steps in the t. gondii cycle,19,28 and the absence of activity of deoxyartemisinine confirmed the peroxide group as the pharmacophore.25 in vivo results are rare and incomplete,23 and only few studies are reliable. thus, artemisinin showed complete clearance of parasitaemia after a 5-day treatment with 1.3 µg ml–1 day–1.29 an 8-day administration of artemiside at 10 mg kg–1 day–1 3 days after sulfadiazine treatment prolonged survival in 80 % of the mice, without toxic side-effects.24 fig. 2. structures of artemisinin derivatives. artemisinin and its derivatives,30 artemisone31 and other synthetic peroxides32,33 also exert potent in vitro and in vivo anticancer activities affecting diverse signalling pathways that regulate cell cycle, differentiation, apoptosis, invasion and angiogenesis in cancer cells.30 artemisinins can functionally be clearly distinguished from the synthetic peroxides, in spite of the fact that they all share a common peroxide pharmacophore, which suggests that these molecules have multiple modes of action.33 furthermore, it was found that synthetic and semi-synthetic peroxides exhibited specific activities against cancer cells. thus, steroidal mixed tetraoxanes show highly specific activity toward uo31 renal tetraoxanes as inhibitors of apicomplexan parasites growth 1343 cancer cells,2b and artesunate against non-small cell lung cancer and colorectal cancer.34 the high antimalarial activities of dicyclohexylidene tetraoxanes that possess polar end groups attached to one of the cyclohexane units35 encouraged us to explore the influence of modifications to the polar groups on their antimalarial and cytotoxic activities. for this, compound 21 was chosen since it showed the most promising in vivo activity against the malaria parasite.35c results and discussions synthesis synthesis of targeted derivatives is presented in schemes 1 and 2. the key intermediary tetraoxane amine 21 was prepared according to a previously described procedure (scheme 1).35c in brief, cyclohexane was transformed into gemdihydroperoxide 17 using 50 % h2o2 in the presence of re2o7 as catalyst,36 which was further coupled with benzyl 4-oxocyclohexanecarboxylate 1535a,37 producing tetraoxane benzyl ester 18. the presence of the benzyl-group enabled a more efficacious purification of the crude product using a biotage sp chromatography system. reduction with lialh4 produced alcohol 19, which was transformed via the corresponding azide 20 into amine 21. final derivatives 22– 25 (scheme 2) were obtained from 21 and corresponding coupling reactants, in high to excellent yield (72–98 %). all derivatives were fully characterized and their purity, as determined by hplc, was ≥95 %, with the exception of compound 23 that was 79–81 % pure. full details are given in the experimental and the supplementary material to this paper. scheme 1. reaction pathway for the synthesis of derivative 21. 1344 opsenica et al. scheme 2. reaction pathway for the synthesis of derivatives 22–25. in the 1h-nmr spectra, all derivatives showed a characteristic signal at around 3.0 ppm for the methylene groups bonded to the polar groups (imidazoline, guanidine, urea or thiourea), and characteristic signals for the introduced polar groups: singlet at 3.38 ppm for the ethylene group in 22 and multiplets in the region 7.50–6.80 ppm for the aromatic protons in 24 and 25. in the 13c-nmr spectra, all derivatives showed signals for peroxy-acetal carbons in 107–108 ppm region and signals in the 45–50 ppm region for the methylene groups bonded to the polar groups. additionally, a signal at 160 ppm for sp2 carbon in polar groups in 22, 23 and 24, and a signal at 180 ppm for 25 were present. furthermore, aromatic carbons from the phenyl-groups in 24 and 25 appeared in the 117–140 ppm region. antimalarial activity compounds were screened in vitro against four p. falciparum strains: d6 (cq susceptible (cqs) strain), w2 (cq resistant (cqr) strain), tm91c235 (cq and mfq resistant) and tm90c2b (atovaquone resistant), following wellestablished protocols.38 in brief, the malaria sybr green i based fluorescence (msf) assay is a microtiter plate drug sensitivity assay that uses the presence of malarial dna as a measure of parasitic proliferation in the presence of antimalarial drugs or experimental compounds. the intercalation of sybr green i dye, and its resulting fluorescence, is relative to parasite growth, and a compound that inhibits the growth of the parasite will result in lower fluorescence intensities. the antimalarial activities are presented in table i. tetraoxane 2135c was also screened here in order to enable a more reliable comparison to be drawn tetraoxanes as inhibitors of apicomplexan parasites growth 1345 with the activities of the new derivatives. all derivatives, with the exception of 23, showed low nanomolar activities against all four strains. table i. in vitro antimalarial activities against p. falciparum strains and cytotoxicity (pbmc) of tetraoxanes 21–25; n.t. – not tested compd. p. falciparum, ic50 / nm pbmc, ic50 / μm rih sii d6a w2b tm91c235c tm90c2bd (–)phae,f (+)phag 21 13.99 7.26 10.81 7.03 185.61 182.22 0.52/ 0.77 13265/25560/ 17165/26388 22 92.75 72.86 208.54 85.89 166.10 168.70 0.79/ 2.25 1791/2280/ 796/1934 23 243.84 494.37 1489.80 n.t. >200 >200 2.03/ 6.11 820/400/ 134/– 24 36.50 15.97 29.14 21.40 >200 >200 0.44/ 0.80 5480/12525/ 6863/9343 25 16.30 9.94 28.28 n.t. 32.74 32.01 0.61/ 1.73 2008/3295/ 1158/– artj 6.66 3.79 16.90 6.23 n.t. n.t. 0.57/ 2.54 cq 7.63 462.99 198.70 181.08 n.t. n.t. 60.70/ 26.05 mq 14.69 6.52 80.28 11.60 n.t. n.t. 0.44/ 5.47 ap. falciparum african d6 strain; bp. falciparum indochina w2 strain; cp. falciparum tm91c235 strain; dp. falciparum tm90c2b strain; enon-stimulated with pha; fpha = phytohaemagglutinin, gstimulated with pha; hresistance index (ri) is defined as the ratio of the ic50 values for the cqr versus cqs strain, w2/d6 and tm91c235/d6, respectively; iselectivity index (si) is defined as the ratio of the ic50 values for pbmc (–)pha/d6, pbmc (–)pha/w2, pbmc (–)pha/tm91c235 and pbmc (–)pha/tm90c2b, respectively; javerage of greater than eight replicates, cq = chloroquine, mq = mefloquine simultaneously, all derivatives except 23 were more active against the chloroquine-resistant (cqr) w2 strain than against the chloroquine-susceptible (cqs) d6 strain, which could be seen from the favourable ri values. the derivatives were somewhat less active against the multi-drug resistant tm91c235 (cqr and mqr strain) in comparison with the activities exhibited toward the w2 strain. in addition, the corresponding ri values were less favourable, with the exceptions 21 and 25. however, three of the five derivatives were 7.4–2.8 times more active than mq against the tm91c235 strain – 21, ic50 = 10.81 nm, 24, ic50 = 29.14 nm and 25, ic50 = 28.28 vs. mq, ic50 = 80.28 nm. the most active derivative was 21 that showed exceptional activities against all the resistant strains. even against the atovaquone resistant strain tm90c2b, this amine showed activities comparable with those of mq. removing the basic character of the terminal amino-group attenuates the anti-malarial activity, as seen in the case of compounds 21, 24 and 25. transformation of the primary amino-group into n-phenyl urea or thiourea reduced the antimalarial activity. however, 25 pre1346 opsenica et al. served high activity against the w2 strain, with an ic50 of 9.94 nm, which is comparable to those of mq and 21 with values of 6.52 nm and 7.26 nm, respectively. on the other hand, increasing basic character of the terminal group did not increase the activity unconditionally. introduction of more basic groups, such as imidazolidine or guanidine, led to a sharp decrease in activity. this was even more striking with the guanidine derivative 23, which was 17, 68 and 138 times less active than 21 against d6, w2 and tm91c235 strains, respectively. such sharp decreases in activity caused by more basic groups is in accordance to the behaviour observed with trioxolanes.39 derivative 23 was also less active than 22, which makes it the least active compound within this series. derivatives 22 and 23 have same pka values (table s-i of the supplementary material to this paper), which are two orders of magnitude higher than the pka for 21, and correlations between pka or log p values (table s-i) with antimalarial activity could not be established.40 the derivatives were in general less active than art against all four strains. tetraoxane 21 was the only one with activities comparable with those of art. however, two tetraoxanes 21 and 25 had better ri values compared to that of art against multi-drug resistant strain tm91c235 – 21, ri = 0.77 and 24, ri = 0.80 vs. art, ri = 2.54. the toxicity of the compounds was estimated using human normal peripheral blood mononuclear cells (pbmc), non-stimulated (pbmc (–)pha) and stimulated (pbmc (+) pha) with pha (table i). in general, the pbmc assay for cytotoxicity estimation revealed that all compounds are well tolerated by normal immunocompetent cells, possessing ic50 ≥ 32 µm. in addition, high selectivity indices (si) were obtained for all compounds toward all p. falciparum strains. derivative 21 had the best si indices, which were in the range 13000–26000, depending on the strain. the second best si was exhibited by the urea derivative 24, which had the lowest cytotoxicity >200 µm and moderate antimalarial activities in the range 16–36 nm. in vitro cytotoxic activity cytotoxic activities of the new tetraoxanes and the previous compounds 21 and 2035c were evaluated against four human cancer cell lines: cervix adenocarcinoma hela, melanoma fem-x, breast adenocarcinoma mda-mb-361 and chronic myelogenous leukaemia k562 cells. the intensities of the cytotoxic activities are given in table ii and fig. 3. first, it should be emphasized that the tested tetraoxanes showed the strongest cytotoxic activities against myelogenous leukaemia k562 cells. the highest cytotoxicity against k562 cells was exerted by derivatives 25 with an ic50 of 6.15 µm, 24 with 13.23 µm, and 18, 20 and 22 with an ic50 of ca. 18.5 µm. tetraoxanes as inhibitors of apicomplexan parasites growth 1347 table ii. in vitroa activities (ic50 / μm) of tetraoxanes against cancer cell lines (fem-x, human melanoma cell line; hela, human cervical adenocarcinoma cell line; k562, myelogenous leukaemia cell line; mda-mb-361, human breast adenocarcinoma cell line) and peripheral blood mononuclear cells (pbmc), non-stimulated or stimulated with pha (phytohaemagglutinin) compd. fem-x hela k562 mda-mb--361 pbmc (–)pha pbmc (+)pha sifem-xa sihela sik562 simda-mb-361 21 79.75 63.56 33.68 >100 185.61 182.22 2 3 6 2 18 31.86 27.01 18.84 73.92 >200 >200 6 7 11 3 20 98.82 37.67 18.43 94.7 >200 >200 2 5 11 2 22 43.84 34.56 18.38 80.29 166.10 168.70 4 5 9 2 23 >100 75.66 81.49 90.91 >200 >200 2 3 2 2 24 52.68 39.85 13.23 96.85 >200 >200 4 5 15 2 25 15.67 10.51 6.15 41.13 32.74 32.01 2 3 5 1 cis-pt 8.10 7.89 5.64 >33.34 >66.67 >66.67 8 8 12 2 athe selectivity index (si) is defined as the ratio of the ic50 for pbmc (–)pha / cancer cell line fig. 3. survival (s%) of: a) cervical adenocarcinoma hela, b) melanoma fem-x, c) myelogenous leukaemia k562 and d) breast adenocarcinoma mda-mb-361 cells grown for 72h in the presence of increasing concentrations of the investigated tetraoxanes, determined by mtt test. 1348 opsenica et al. against cervix adenocarcinoma hela cells, compounds 18, 20, 22, 24 and 25 exerted pronounced cytotoxic actions with ic50 values in the range of 10.51 and 39.85 µm. the highest cytotoxic effect was exhibited by derivative 25 with ic50 = 10.51 µm. against melanoma fem-x cells, compound 25 was the most active with an ic50 of 15.67 µm; derivative 18 exerted pronounced cytotoxic effects with ic50 value of 31.86 µm. compounds 22 and 24 exerted moderate cytotoxic action, while the cytotoxic effects of the other derivatives were lower, with derivative 23 being the least active, showing an ic50 value higher than 100 µm. all tested tetraoxanes exerted low cytotoxic actions against breast adenocarcinoma mda-mb-361 cells with ic50 values >73.92 µm, except derivative 25 which had moderate activity against this cell line, with an ic50 value of 41.13 µm. tetraoxane 23 with a guanidine group was the least active compound in this series, showing lowest activities towards all cell lines in comparison with other members of the series. all tetraoxanes exerted selective concentration-dependent cytotoxic activity against specific malignant cell lines, which was especially high against myelogenous leukaemia k562 cells. it is noteworthy that the cytotoxic actions of compounds 21, 18, 22, 24 and 25 were notably higher against k562, hela and fem-x cells than against mda-mb-361 breast cancer cells, indicating selectivity in their anticancer action against specific malignant cell types. to further evaluate the anticancer potential of the tested tetraoxanes, their cytotoxic activities were examined against normal healthy peripheral blood mononuclear cells (pbmc), both resting and stimulated to proliferate by mitogen phytohaemagglutinin (pha), table ii and fig. 4. each of the examined tetraoxanes exerted remarkably higher cytotoxic activities against k562, hela and fem-x malignant cell lines than against both non-stimulated and stimulated pbmc, indicating a significant specificity in their anticancer action. the highest fig. 4. survival of (a) resting pbmc and (b) pbmc stimulated to proliferate by pha grown for 72h in the presence of increasing concentrations of the investigated tetraoxanes, determined by the mtt test. tetraoxanes as inhibitors of apicomplexan parasites growth 1349 selectivity in antitumor action was observed against myelogenous leukaemia k562 cells in comparison with normal healthy pbmc with tetraoxanes 18, 20 and 24, which showed the highest si values, 11, 11 and 15 respectively. in addition, the tested tetraoxanes showed good selectivity as follows: 18 and 24 against cervix adenocarcinoma hela and melanoma fem-x cells, 20 against hela cells, and 22 against k562, hela and fem-x cells. low selectivity in the anticancer action of the tested tetraoxanes was observed against breast adenocarcinoma mda-mb-361 cells, which showed the lowest sensitivity to the cytotoxic action of these compounds. in contrast to antimalarial activity, it could not be concluded that activities depend on the presence of a specific functional group. rather, the activity was specific towards particular cell lines. however, in general, the obtained results showed that derivatives 21–23 with basic groups have lower activities. derivatives were less active than cisplatin with the exception of derivative 25. the thiourea derivative 25 was the most active tetraoxane in this series, with ic50 values in the range 41–6.0 µm, which are the same as for cisplatin. however, the low si of 25 for all tested cell lines makes this derivative less interesting, in spite of having good cytotoxic activities. conversely, derivatives 18, 20 and 24 had si values for the k562 cell line in the same range as that for cisplatin, which makes them very interesting candidates for further examinations. the mechanisms of cytotoxic action of 18, 20 and 24 were examined by cell cycle analysis and morphological assessment of cell death modalities induced by these tetraoxanes using fluorescence microscopy. the tested compounds at ic50 concentrations induced time-dependent percentage increases of the target k562 cells in the subg1 phase, as could be seen in fig. 5. it is noteworthy that compound 20 at the ic50 concentration caused the most remarkable increase in the percentage of k562 cells in the subg1 cell cycle phase after 48 h treatment. in addition, treatment with 2ic50 concentrations of three tetraoxanes resulted in an increase in the percentages of apoptotic subg1 k562 cells after 24 h exposure. after 48h, the percentages of subg1 k562 cells exposed to 2ic50 concentrations of 18, 20 and 24 were approximately 12 times higher compared to the control cell sample, pointing to a strong proapoptotic activity of these compounds. examination of changes in the morphological features confirmed that the tested tetraoxanes triggered apoptosis in k562 leukaemia cells after 24 h treatment. typical hallmarks of apoptotic k562 cells induced by tetraoxanes, such as shrinkage of the nucleus, chromatin condensation and fragmentation of the nucleus in addition to orange–red stained cells in the final stages of apoptosis are presented in fig. 6. prominent proapoptotic effects of tetraoxanes 18, 20 and 24 in addition to high selectivity in their anticancer activity against myelogenous leukaemia k562 cells in comparison to normal immunocompetent pbmc, suggest significant anticancer potential of these compounds. 1350 opsenica et al. fig. 5. changes in the cell cycle phase distribution of k562 cells induced by the investigated tetraoxanes 18, 20 and 24 (tested concentrations corresponded to ic50 and 2ic50 values). c: control k562 cell sample. fig. 6. photomicrographs of acridine orange/ethidium bromide-stained control k562 cells and k562 cells treated with 2×ic50 concentrations of 18, 20 and 24, for 24 h. tetraoxanes as inhibitors of apicomplexan parasites growth 1351 in vivo activity against t. gondii derivative 21 was chosen for examination of in vivo activity against t. gondii. the activity was examined in murine models of infection with tachyzoites of the highly virulent rh strain of t. gondii following a well-established protocol.41 the compound exhibited no visible toxicity as judged by the quality of fur, gait and general activity of the uninfected treated mice. the activity of 21 was compared to that of art. the results obtained in the treated animals are presented in fig. 7. in the infection model where infection was established with 102 per ml, all untreated mice died between day 6 and day 9 p.i. (mean 8 days). most importantly, treatment with both 21 and art in 10 mg kg–1 day–1 doses allowed survival of 20 % of infected treated mice after 15 days (fig. 7a). compared with the control mice, treatment with both drugs significantly prolonged survival, but the effect of 21 was more pronounced (p = 0.014) than that of art (p = 0.0413), whereas the effects of the two compounds were mutually comparable (p = 0.9086). fig. 7. survival of infected mice after treatment with 21 or art; parasite concentration: a) 102 and b) 106 ml-1. 1352 opsenica et al. in mice infected with 106 ml–1, all untreated mice died within 5 days. compared to these, treatment with either 21 or art significantly prolonged survival (p = 0.0092 and p = 0.0013, respectively) but did not afford complete protection. the antitoxoplasmatic activity of the two compounds was similar (p = 0.8358). pathohistological analysis was performed on all surviving infected treated mice (four treated with 21 and two treated with art), as well as on representative controls (non-infected treated mice), which were all sacrificed day 24 p.i. no pathological changes other than extramedullary haematopoiesis (characteristic of these mice) and mild lymphocyte infiltration of the liver were revealed in both 21 and art-treated mice, except for the finding of focal myocarditis in one art-treated infected mouse. these results indicate no significant macroscopic or microscopic toxicity of the tested compounds, and moreover, show the potential of derivative 21 to clear t. gondii infection. experimental chemistry melting points were determined on a boetius pmhk or a mel-temp apparatus and were not corrected. optical rotations were measured on a rudolph research analytical automatic polarimeter, autopol iv in dichloromethane (dcm) or methanol (meoh) as solvent. the ir spectra were recorded on a perkin-elmer ft-ir 1725x spectrophotometer. the 1hand 13c-nmr spectra were recorded on a varian gemini-200 spectrometer (at 200 and 50 mhz, respectively), and on a bruker ultrashield advance iii spectrometer (at 500 and 125 mhz, respectively) employing the indicated solvents (supplementary material) using tms as the internal standard. chemical shifts are expressed in ppm (δ) values and coupling constants (j) in hz. the esi-ms spectra were recorded on an agilent technologies 6210 time-of-flight lc-ms instrument in the positive ion mode with ch3cn/h2o 1/1 with 0.2 % hcooh as the carrying solvent solution. the samples were dissolved in ch3cn or meoh (hplc grade purity). the selected values were as follows: capillary voltage, 4 kv; gas temperature, 350 °c; drying gas, 12.l min-1; nebulizer pressure, 310 kpa and fragmentator voltage, 70 v. elemental analyses were realized on the vario el iiic,h,n,s/o elemental analyzer (elementar analysensysteme gmbh, hanau, germany). thin-layer chromatography (tlc) was performed on precoated merck silica gel 60 f254 and rp-18 f254 plates. column chromatography was performed on lobar lichroprep si 60 (40–63 µm), or rp-18 (40–63 µm) columns coupled to a waters ri 401 detector, and on biotage sp1 system with a uv detector and flash 12+, flash 25+ or flash 40+ columns charged with kp-sil (40–63 µm, pore diameter 60 å), or kp-c18-hs (40–63 µm, pore diameter 90 å) as adsorbent. compounds 18 and 20–25 were analyzed for purity (hplc) using an agilent 1200 hplc system equipped with quat pump (g1311b), injector (g1329b) 1260 als, tcc 1260 (g1316a) and a 1260 infinity refractive index detector (rid) was. hplc analyses were performed in two diverse systems. method a: zorbax eclipse plus c18, 4.6 mm×150 mm, 1.8 µm, s.n. uswky01594 was used as the stationary phase. the compounds were eluted using an isocratic protocol and eluent was made from water/meoh, 30/70 (v/v). the compounds were dissolved in methanol; the final concentrations were 0.5 mg ml-1. method b: poroshell 120 ec-c18, 4.6 mm×50 mm, 2.7 µm, s.n. uscfu07797 was used as the stationary phase. the compounds were tetraoxanes as inhibitors of apicomplexan parasites growth 1353 eluted using an isocratic protocol and the eluent was made from water/meoh, 30/70 (v/v). the compounds were dissolved in methanol; the final concentrations were 0.5 mg ml-1. synthetic procedures benzyl 7,8,15,16-tetraoxadispiro[5.2.5.2]hexadecane-3-carboxylate (18). into ice-cold (ice–water mixture) solution of gem-dihydroperoxide 17 (412.7 mg, 2.53 mmol) and ketone 15 (374.8 mg, 2.53 mmol) in ch2cl2 (5 ml), under intensive stirring, precooled freshly prepared solution of conc. h2so4/ch3cn (1/10, v/v, 1.82 ml) was added dropwise. after one hour stirring at the same temperature, the reaction was quenched with water and the layers were separated. water layer was extracted with ch2cl2 (3×50 ml). the combined organic layers were washed with sat. nahco3 (3×20 ml), brine (2×10 ml) and dried over anh. na2so4. the solvent was removed under reduce pressure and the product was isolated after column chromatography purification (dry-flash, sio2-column, eluent hexane/etoac, gradient 97/3 → 9/1). yield: 241.9 mg (26 %), as an amorphous powder. n-(7,8,15,16-tetraoxadispiro[5.2.5.2]hexadec-3-ylmethyl)-4,5-dihydro-1h-imidazol-2 -amine (22). a mixture of 21 (100.0 mg, 0.39 mmol), i (142.2 mg, 0.58 mmol) and et3n (0.5 ml) in methanol (3 ml) was stirred under an inert atmosphere (ar) for 3 days at 70 °c. the solvent was evaporated, the residue dissolved in ch2cl2, 10 % naoh solution added and the mixture intensively shaken. the layers were separated, the organic layer was washed once with water, once with brine and dried over anh. na2so4. the solvent was removed under reduce pressure and the product isolated after column chromatography purification (lobar, sio2-column a, eluent ch2cl2/ch3oh sat. nh3 = 1/1). yield: 91 mg (72 %). 1-(7,8,15,16-tetraoxadispiro[5.2.5.2]hexadec-3-ylmethyl)guanidine (23). a mixture of 21 (100.0 mg, 0.39 mmol), s-methylthiourea sulphate (162.0 mg, 0.58 mmol) and et3n (0.5 ml) in methanol (3 ml) was stirred at 70 °c under an inert atmosphere (ar) for 3 days. into the warm solution, methanol was added (10 ml) and the warm mixture was filtered, the solvent removed under reduce pressure and product isolated after column chromatography purification (dry-flash, sio2-column, gradient ch2cl2/meoh, = 9/1 → 1/1) as a pale yellow oil that became solid with time. yield: 108.6 mg (93 %). 1-phenyl-3-(7,8,15,16-tetraoxadispiro[5.2.5.2]hexadec-3-ylmethyl)urea (24). a mixture of 21 (150.0 mg, 0.58 mmol) and phenyl-isocyanate (63.4 μl, 0.58 ml) in ch2cl2 (10 ml), was stirred at r.t. under an inert atmosphere (ar) for 45 min. the solvent was evaporated and product isolated after column chromatography purification (dry-flash, sio2-column, gradient hexane/etoac = 1/1 → etoac → etoac/meoh = 7/3). yield: 215 mg (98 %). 1-phenyl-3-(7,8,15,16-tetraoxadispiro[5.2.5.2]hexadec-3-ylmethyl)thiourea (25). a mixture of 21 (78.2 mg, 0.30 mmol) and phenyl-isothiocyanate (35.57 μl, 0.30 ml) in ch2cl2 (15 ml) was stirred under an inert atmosphere at r.t. for 2 h. the solvent was removed under reduce pressure and the product isolated after column chromatography purification (dry-flash, sio2-column, gradient hexane/etoac = 9/1 → etoac). yield: 87.3 mg (73 %). in vitro antimalarial activity the in vitro antimalarial drug susceptibility screen is a modification of the procedures first published by desjardins et al.42 with modifications developed by milhous et al., the details of which are given in ref. 8b. all the synthesized aminoquinolines were screened in vitro against the p. falciparum strains cq and mfq, as well as the susceptible strain d6 (clone of the sierra i/unc isolate), the cq resistant but mfq susceptible strain w2 (clone of the indochina i isolate), and the cq and mfq resistant strain tm91c235 (clone of the south– east asian isolate), and tm90c2b (clone of the thailand isolate). 1354 opsenica et al. cytotoxic activity reagents. stock solutions of investigated tetraoxanes were prepared in dimethyl sulphoxide (dmso) at concentrations of 10 mm and subsequently diluted with complete nutrient medium (rpmi-1640 without phenol red) supplemented with 3 mmol l-1 l-glutamine, 100 µg ml-1 streptomycin, 100 iu ml-1 penicillin, 10 % heat inactivated foetal bovine serum (fbs), and 25 mm hepes, and adjusted to ph 7.2 with bicarbonate solution. rpmi-1640, fbs, hepes, and l-glutamine were purchased from sigma–aldrich, st. louis, mo, usa. cell cultures. human cervical adenocarcinoma hela, human melanoma fem-x and human breast adenocarcinoma mda-mb-361 cells were cultured as a monolayer, while human chronic myelogenous leukaemia k562 cells were grown in a suspension in the complete nutrient medium, at 37 °c in a humidified air atmosphere with 5 % co2, as previously described.43 preparation of peripheral blood mononuclear cells (pbmc). pbmc were separated from whole heparinised blood (obtained from two healthy volunteers) by histopaque®-1077 (sigma–aldrich) density gradient centrifugation. interface cells, washed three times with haemaccel (aqueous solution supplemented with 145 mm na+, 5.1 mm k+, 6.2 mm ca2+, 145 mm cland 35 g l-1 gelatine polymers, ph 7.4), were counted and suspended in nutrient medium with 10 % fbs.43 treatment of human malignant cell lines. hela (2,000 cells per well), fem-x (5,000 cells per well) and mda-mb-361 cells (10,000 cells per well) were seeded into 96-well microtiter plates. twenty hours later, after cell adherence, five different concentrations of the investigated compounds in complete nutrient medium were added to the cells (range 6.25–100 µm or 3.125–50 µm), except for the control cells to which nutrient medium only was added. k562 cells (5,000 cells per well) were seeded two hours before addition of the investigated compounds to obtain final concentrations within the above-mentioned range. all experiments were realised in triplicate. cisplatin was used as a positive control. treatment of pbmc from healthy donors. pbmc cells were seeded at a density of 150,000 cells per well in nutrient medium only, or in the nutrient medium enriched with 5 µg ml-1 of phytohaemagglutinin (pha – inep, belgrade, serbia) in 96-well microtiter plates. two hours later, five different concentrations of the investigated tetraoxanes were added to the wells with non-stimulated and pha-stimulated pbmc (in order to obtain five final concentrations within the range of 12.5–200 µm). all experiments were realised in triplicate. cisplatin was used as a positive control. determination of cell survival. cell survival 72 h after addition of the drug was determined by the mtt test according to the method of mosmann44 as modified by ohno and abe.45 briefly, 10 µl of mtt solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg ml-1 in phosphate buffered saline) was added to each well.43 the samples were incubated for a further four hours at 37 °c in a humidified atmosphere with 5 % co2. then, 100 µl of 10 % sds solution was added to the wells. the absorbance was measured at 570 nm the next day. to obtain cell survival (s / %), the absorbance at 570 nm of a sample with cells grown in the presence of various concentrations of agent was divided by the absorbance of the control sample (the absorbance of cells grown only in nutrient medium), implying that the absorbance of the blank was always subtracted from the absorbance of a corresponding sample with target cells. the ic50 concentration was defined as the concentration of an agent inhibiting cell survival by 50 %, compared to the corresponding control. cell cycle analysis. k562 cells were exposed to two different concentrations of the examined compounds 18, 20 and 24 (corresponding to the ic50 and 2ic50 values determined tetraoxanes as inhibitors of apicomplexan parasites growth 1355 after 72 h treatment) for 24 and 48 h. after incubation, the target cells were collected, washed and fixed in 70 % ethanol on ice. the cell samples were stored at −20 °c for at least one week before staining. the cells were collected by centrifugation, washed, resuspended in pbs containing rnase a at a final concentration of 200 μg ml-1 and incubated for 30 min at 37 °c. subsequently, the propidium iodide solution was added to the cells at a final concentration of 40 μg ml-1.43 the phase distribution of the cell cycle was determined using a facscalibur flow cytometer (bd biosciences, franklin lakes, nj, usa). the data (10,000 events collected for each sample) were analysed using cellquest software (bd biosciences). morphological evaluation of k562 cell death. to examine the mode of death of human chronic myelogenous leukaemia k562 cells induced by the investigated tetraoxanes, morphological analysis by microscopic examination of acridine orange/ethidium bromide-stained target cells was performed, as already described.43 the k562 cells were seeded in 6-well plates (200,000 cells per well) in complete nutrient medium. after 2 h, the cells were treated with the investigated compounds for 24 h at concentrations corresponding to double the ic50 values obtained after 72 h treatments. after this period, the target cells were collected by centrifugation and stained with 20 µl of a mixture of acridine orange and ethidium bromide (3 μg ml-1 ao and 10 μg ml-1 eb in pbs) dyes, and visualized under a fluorescence microscope – carl zeiss palm microbeam with axio observer.z1 using axiocam mrm (filters alexa 488 and alexa 568). anti-toxoplasmatic activity mice. female swiss webster mice (medical military academy animal research facility, belgrade, serbia) weighing 18 to 20 g at the beginning of each experiment were used. the mice were housed six to a cage and offered drinking water ad libitum. t. gondii. tachyzoites of the virulent rh strain maintained through serial intraperitoneal (i.p.) passages were used. for the experimental infections, tachyzoites were harvested from mouse peritoneal fluids 72 h post infection and purified by centrifugation, cotton wool filtration, and needle extraction. the parasites were counted in a haemocytometer, and their numbers were adjusted to 2×106 ml-1 with saline. the suspensions were also serially 10-fold diluted, and 0.5-ml aliquots of 2×102 and 2×106 ml-1 dilutions were inoculated i.p. into fresh mice. infected non-treated mice served as infection controls. mice were randomly assigned into experimental groups and treated with 21 or art. both drugs were administered at a dose of 0.2 mg per mouse per day (10 mg kg-1 day-1), subcutaneously (s.c.) for 8 consecutive days starting from the day of infection (day 0). survival of the mice was monitored for another week after the end of treatment, meaning a total follow-up period of 15 days post infection (p.i.). to control for drug side effects (toxicity), separate groups of non-infected animals were given 21 (10 mg kg-1 day-1) and art (10 mg kg-1 day-1) in the same manner and duration as in the experimental groups. statistical analysis the rates of survival in particular treatment groups were estimated by the kaplan–meier product limit method and compared by the log-rank test. the level of statistical significance was 0.05. 1356 opsenica et al. conclusions herein, the synthesis and biological activity of new cyclohexylidene mixed 1,2,4,5-tetraoxanes containing polar guanidine and urea based groups were reported. four new tetraoxanes were tested in vitro against p. falciparum cqr and cqs strains. the derivatives showed moderate nanomolar antimalarial activities, and differences in activities were clearly influenced by changes in the structures of the introduced polar groups. the n-phenylurea derivative 24 showed the best resistance indices (riw2 = = 0.44, ritm91c235 = 0.80), the highest si score against all four tested p. falciparum strains and the lowest toxicity against pbmc (ic50 > 200 μm). seven tetraoxanes were tested in vitro against four human cancer cell lines and five of them showed pronounced cytotoxic effects against myelogenous leukaemia k562 cells in 6.15–18.84 μm concentrations with high sik562 index. the amino-tetraoxane 21 was the first one evaluated for its anti t. gondii effect in vivo, in a murine model of acute toxoplasmosis. an 8-day treatment at a dose of 10 mg kg–1 day–1 allowed survival of 20 % of the infected mice. the obtained results clearly showed the activity of the investigated tetraoxanes against acute murine toxoplasmosis, suggesting the potential of synthetic organic peroxides in t. gondii treatment. supplementary material calculated pka and log p values for derivatives 21–23, synthetic procedures for derivatives 13–17 and 19–21 and analytical data for derivatives 13–25 and hplc chromatograms for determination of the purity of tested compounds are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. this work was supported by the ministry of education, science and technological development of the republic of serbia (grants 172008 and 175011). и з в о д тетраоксани као инхибитори апикомплексних паразита plasmodium falciparum и toxoplasma gondii и анти-канцерски молекули дејан м. опсеница1, јелена радивојевић2, ивана з. матић3, тијана штајнер4, славица кнежевић-ушај5, олгица ђурковић-ђаковић4 и богдан а. шолаја6 1институт за хемију, технолохгију и металургију, универзитет у београду, његошева 12, 11000 београд, 2институт за молекуларну генетику и генетски инжењеринг, универзитет у београду, војводе степе 444a, п. пр. 23, 11010 београд, 3институт за онкологију и радиологију србије, пастерова 14, 11000 београд, 4национална референтна лабораторија за токсоплазмозу, институт за медицинска истраживања, универзитет у београду, др суботића 4, п. пр. 102, 11129 београд, 5институт за патологију, медицински факултет, универзитет у новом саду, хajдук вељкова 3, 21000 нови сад и 6хемијски факултет, универзитет у београду, студентски трг 12–16, п. пр. 51, 11158 београд синтетисана је група нових циклохексилиденских 1,2,4,5-тетраоксана који имају гванидино и уреидо поларне групе и испитана је њихова антималаријска активност према хлорокивин-резистентним и осетљивим сојевима plasmodium falciparum. деривати tetraoxanes as inhibitors of apicomplexan parasites growth 1357 показују умерене активности у nm опсегу и ниску цитотоксичност. дериват n-фенил -урее 24 има најбољи индекс резистенције (riw2 = 0,44; ritm91c235 = 0,80) и није токсичан према хуманим нормалним мононуклеарним ћелијама периферне крви (ic50 > 200 μm). седам деривата је тестирано in vitro према четири хумане малигне ћелијске линије и показало је високу селективност према k562 ћелијама леукемије. дериват 21 који има примарну амино групу је први тетраоксан тестиран in vivo према још једном паразиту из филума apicomplexa, toxoplasma gondii. приликом супкутаног администрирања, дневна доза од 10 mg kg-1 омогућила је преживљавање 20 % инфицираних мишева, што показује висок потенцијал тетраоксана за терапију инфекција изазваних апикомплексним паразитима. 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(1985) 525 39. y. tang, y. dong, s. wittlin, s. a. charman, j. chollet, f. c. k. chiu, w. n. charman, h. matile, h. urwyler, a. dorn, s. bajpai, x. wang, m. padmanilayam, j. m. karle, r. brun, j. l. vennerstrom, bioorg. med. chem. lett. 17 (2007) 1260 40. for pka calculations, epik, version 2.9, schrödinger, llc, new york, ny, 2014 and for log p calculations, qikprop, version 4.1, schrödinger, llc, new york, ny, 2014 were used. 41. o. djurković-djaković, t. nikolić, f. robert-gangneux, b. bobić, a. nikolić, antimicrob. agents chemother. 43 (1999) 2240 42. r. e. desjardins, c. j. canfield, d. e. haynes, j. d. chulay, antimicrob. agents chemother. 16 (1979) 710 43. n. m. krstić, i. z. matić, z. d. juranić, i. t. novaković, d. m. sladić, j. steroid. biochem. mol. biol. 143 (2014) 365 44. t. mosmann, j. immunol. methods 65 (1983) 55 45. m. ohno, t abe, j. immunol. methods 145 (1991) 199. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {effects of 5es learning model on pre-service science teachers' conceptual understanding and science process skills: a case of gases and gas laws} j. serb. chem. soc. 85 (4) 559–573 (2020) jscs–5322 original scientific paper 559 effects of a 5es learning model on the conceptual understanding and science process skills of pre-service science teachers: the case of gases and gas laws fethiye karsli baydere1, alipaşa ayas2 and muammer çalik3* 1giresun university, faculty of education, department of science education, giresun, turkey, e-mail: fethiyekarsli28@gmail.com, 2bilkent university, graduate school of education, ankara, turkey, e-mail: alipasaayas@yahoo.com and 3trabzon university, fatih faculty of education, department of elementary teacher education, 61335 trabzon, turkey (received 29 march, revised 28 october, accepted 22 november 2019) abstract: the aim of this study was to investigate the effects of using a 5es learning model on the pre-service science teachers’ conceptual understanding and science process skills for “gases and gas laws”. the sample of the study consisted 49 pre-service science teachers enrolled for the “science laboratory practices-i” course within the department of science education of a turkish state university. through a quasi-experimental research method (preand post -test research design), an experimental group was exposed to the 5es learning model (engage-explore-explain-elaborate-evaluate) with different conceptual change methods/techniques (i.e., worksheets, computer animations, analogies and experiments). a control group was also taught through existing instruction (e.g., experiments, lecture and question–answer). data were collected through the gas laws test and the science process skills test. the results of partial eta squared (η2) revealed large-size effects for the control (0.61) and experimental groups (0.73). the 5es learning model was more effective than the existing instruction in overcoming the pre-service science teachers’ alternative conceptions of “gases and gas laws”, as well as in improving their science process skills. the current study recommends that the 5es learning model be tested with a larger sample throughout a long-term teaching intervention. keywords: chemistry education; conceptual change; science process skills. introduction the properties of gases are difficult to observe with the naked eye; furthermore, they are explained by abstract ideas and laws, making them difficult for students to understand.1,2 conventional teaching methods have shortcomings * corresponding author. e-mail: muammer38@hotmail.com  this study is a part of the first author’s phd dissertation, whose earlier version was presented at the 1st eurasian educational research congress, istanbul, turkey. https://doi.org/10.2298/jsc190329123d ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 560 karsli baydere, ayas and çalik with facilitating students’ learning of fundamental molecular behaviors (e.g., interlinks at macroscopic, sub-microscopic and symbolic levels) and developing graphical skills of “gases and gas laws”.3–4 similarly, chemistry textbooks have generally handled the subject of “gases and gas laws” within procedural learning instead of conceptual understanding.5 since everyday life experiences informally shape students’ conceptions of the subject of ”gases and gas laws”,6,7 several alternative conceptions (see table s-i of the supplementary material to this paper) have been developed by students from different grades (7th, 10th and 11th grades), pre-service and in-service teachers.2,5,7–13 identifying these alternative conceptions is critical for determining the steps needed to support conceptual change. unfortunately, teachers may be the inadvertent source of students’ alternative conceptions, because they either miscommunicate information or have their own alternative conceptions. it is important that teacher preparation programs are aware of this issue. given these arguments, conceptual change researches have employed varied pedagogical approaches related to the subject of “gases and gas laws”. these include problem-based learning,13 research-based approach,11,14 research-based computer simulations,15 conceptual change-oriented instruction,9,12 hands-on laboratory activities accompanied with computer modeling,16 argumentation-based pedagogy,17 augmented virtual science laboratories3,18 and case-based instruction.7 of these studies, only three concentrated on teacher preparation programs and studied with pre-service teachers.11,13,17 pabuccu and erduran19 called for more studies related to facilitating pre-service science teachers’ understanding of the gases and gas laws. the current study, therefore, was undertaken in response to this call. even though conceptual change methods used in the aforementioned studies were somewhat effective in overcoming the pre-service science teachers’ alternative conceptions related to “gases and gas laws”, some of their participants were still resistant to change even after the teaching intervention. hence, the implications for future research in these studies recommended enhancing pre-service science teachers’ conceptual change levels.20 furthermore, studies have shown that using only one type of conceptual change method may limit students’ learning motivation and satisfaction.20,21 previous studies used only one or two conceptual change method(s)/technique(s)3,7,12,14–17 instead of combining various conceptual change methods/techniques within the 5es learning model. by doing this, the current study illustrated an alternative pedagogical approach for gases and gas laws and enriched the learning environment with different conceptual change methods/techniques. science process skills (sps) school science courses explicitly focus on content knowledge, while sps implicitly includes scientific attitudes, scientific communication, scientific arg________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. effects of 5es learning model 561 umentation and so forth as well.22 however, science education literature continues to debate which is more important: sps or content knowledge.23 that is, sps drives the doing of science, whilst science content is the knowing of science.24 there are basically two ways to develop sps, either through teaching sps solely, and directly or by integrating sps into content knowledge.24 the first one designs specific science activities for each of the sps to help students learn how to do science, the nature of science and the origin of facts. the second one integrates sps into science content knowledge and employs the power of activity-based learning. the latter approach offers a context for both sps and content.24 that is, science experiments/activities deploy sps while teaching science content knowledge. scharmann25 supports that teaching science content knowledge and sps simultaneously helps them complement each other. this combined approach is more promising as it leverages regular content teaching time to develop sps. we, therefore, adhered to this view while designing the current study. to illustrate the interconnection between content knowledge and sps, experiments are used to teach sps development, while sps is the tools for learning content knowledge. for example, while students test how temperature affects the volume of a fixed quantity of gas (charles gas law), they observe, measure, identify variables, formulate hypotheses, interpret data, define operationally, do experiments and formulate models. students will learn by doing and experiencing personally how to access information about this process. hence, sps plays a key role in teaching and learning scientific knowledge.26,27 a review of the literature shows other studies that support the idea of integrating sps and content knowledge to: i) improve better understanding of subject/topic/content, ii) facilitate science learning and iii) encourage students to take their own responsibility for their learning through the scientific inquiry process.22,24,25,28,29 furthermore, developing sps helps to improve higher-order-thinking skills, such as problem solving, critical thinking, and decision making.29 along the same lines, sps and scientific literacy overlap with each other. sps promotes components of scientific literacy, such as the ability to think scientifically, the ability to use scientific knowledge in problem solving, appreciation of science, and the ability to think critically about science and scientific expertise.30 despite the advantages of developing individuals’ sps outlined above, pre-service teachers still have a poor understanding of such skills31,32 and also possessed pitfalls in performing the basic and integrated sps.32 hence, this necessitates an alternative pedagogical approach that more directly emphasizes sps. relationship between science curriculum and science process skills to achieve the targeted goals of science education (i.e., conceptual understanding, sps, critical thinking), science education studies of conceptual change ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 562 karsli baydere, ayas and çalik and constructivist learning theory have focused on the students’ pre-existing knowledge to enhance scientific understanding through active engagement and ensure ownership of the constructed ideas.21,28,29 for example, the most recent versions of the turkish science curricula emphasize student engagement, creative thinking, innovative thinking, constructivist learning theory, as well as varied teaching methods/strategies to stimulate students’ interests in science.29 these studies claim that the 5es learning model (engage-explore-explain-elaborateevaluate), which enriches the learning environment with different conceptual change methods, significantly increases the probability of achievement gains (i.e., conceptual understanding, long-term learning, developing sps, logical -creative-reasoning skills) in science classes.21,28,30,31 any educational change (e.g., constructivist learning theory) promotes national educational systems to update to the needs of new trends (i.e., economic development, work force). for example, the turkish science curricula have challenged traditional or teacher-centered instruction and fostered any innovative view for science learning and teaching. even though various learning models of constructivism have been launched, e.g., 3es (explore-explain-elaborate, called learning cycle), 4es (engage-explore-explain-evaluate), 5es and 7es (excite-explore-explain-expand-extend-exchange-examine),38 the most recent versions of the turkish science curricula have especially suggested the 5es learning model.20,33 since each “e” represents a particular part of the learning process and calls for varied conceptual change methods/techniques (i.e., computer animation, worksheet, analogy and experiment), the 5es learning model purposes to actively assist students in linking their prior knowledge with new concepts. due to the aforementioned issues,20,29,33 the turkish science teacher education programs have paid more attention to the 5es learning model. therefore, pre-service experiences of teachers (e.g., 5es learning model, sps and content knowledge) should be enhanced within teacher preparation programs since qualified teachers are only able to choose and use proper pedagogical approaches in their classrooms. in other words, teacher preparation programs should not only equip preservice teachers with these abilities but also pedagogically illustrate how to integrate these approaches into science classes. hence, science educators should look for any alternative pedagogical approach challenging any negative disposition towards deeper conceptual understanding and sps. given the interaction between content knowledge and sps, the current study explored the degree to which the 5es learning model makes concepts of abstract chemistry understandable and/or meaningful. in summary, the current study integrated content knowledge and sps within the 5es learning model to facilitate conceptual, procedural and multidimensional functional levels of scientific literacy.34 the 5es learning model guides teaching and learning processes in science classes21,33,34 by stimulating the pre-service science teachers’ learning capacities ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. effects of 5es learning model 563 of the subject of “gases and gas laws” drawn from the regular general chemistry curriculum in the science teacher preparation program. even though some studies investigated the effect(s) of the 5es learning model with varied conceptual change on the pre-service science teachers’ understanding of other science subjects (e.g., electrochemistry),20 none of the earlier studies tested how the 5es learning model facilitates the pre-service science teachers’ understanding of the subject of “gas and gas laws” and sps. on the other hand, previous studies have also highlighted that the 5es learning model directly influences their conceptual understanding and sps rather than the content/topic (e.g., gas and gas laws) or separate techniques (i.e., computer animation, worksheet, and analogy).20,21,28 to address the goal of the current study, the researchers utilized worksheets, computer animations, analogy/analogical reasoning, experiments, and sps within the 5es learning model. for example, while the pre-service science teachers actively discovered the charles gas law (content knowledge) in small groups of three or four, they employed such sps as identifying variables, formulating hypotheses, performing experiments, interpreting data and defining operationally. in this way, they wrote their observations down on the worksheet (see appendix 1 of the supplementary material) and discussed their results to reveal the charles gas law. hence, they linked content knowledge (i.e., charles gas law) with sps and constructivist learning (i.e., the 5es learning model). the researchers, in turn, hypothesized that the 5es learning model would result in better conceptual understanding of the subject of “gases and gas laws” and improvements in their sps. in addition, their combined pedagogical features (i.e., content knowledge, sps and constructive learning) may boost the pre-service science teachers’ learning capacities in practicum. therefore, the aim of this study was to investigate the effects of using the 5es learning model on the pre-service science teachers’ conceptual understanding and sps for gases and gas laws. the following research questions guided the current study: 1. how do the 5es learning model and existing instructions influence the pre-service science teachers’ conceptual understanding of “gases and gas laws” and sps? 2. are there any significant differences between the experimental and control groups’ preand post-test mean scores of the gas laws test and sps test? experimental through a quasi-experimental research design (with a non-equivalent pretest–post-test control group),7 the study investigated the effects of independent variables (e.g., the 5es learning model and existing instruction) on the dependent variables (e.g., the pre-service science teachers’ conceptions and development of their sps). the department of science education under investigation possessed two regular cohorts (classes a and b) for the third-year of the four-year teacher preparation program. hence, the experimental (15 females and 9 males) and control (15 females and 10 males) groups were randomly assigned to these two cohorts ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 564 karsli baydere, ayas and çalik instead of individually assigning the participants to the groups. each group was exposed to different teaching designs (i.e., the 5es learning model and existing instruction) for the same duration (i.e., 8 class-hours). after the teaching intervention, the post-tests were re-administered to elicit any improvement in conceptual change and sps. participants the sample of the study consisted of 49 pre-service science teachers (aged 20 to 22 years) from a middle-sized public university in turkey. the students, who were in the third year of their four-year program, were of average socioeconomic status and incomes. they had successfully passed a high-stakes nationwide examination and voluntarily chose this department. by this time, they had studied the subject of “gases and gas laws” in the 11th grade and then again in the first-year of their science education program. as the turkish science curriculum follows a top–down model in curriculum development, all students take compulsory science courses in lower and upper secondary schools. they were initially introduced to gases and the related underlying gas concepts (i.e., gas laws, kinetic theory, diffusion/ /effusion, ideal gases and gas mixtures) in 11th grade chemistry course. additionally, in the first-year of the science education program, they attended general chemistry i–ii including such topics as structure of matter, solution chemistry, gases and gas laws, chemical bonding, mole and chemical calculations, periodic table, and redox. a four-year-science teacher education program, which is an integrated framework of physics, chemistry, biology, earth science and astronomy, covers 240 european credit transfer system (ects)-180 ects for compulsory courses and 60 ects for elective courses. all turkish science teacher education programs have to track the same syllabus of any compulsory course offered by higher education council (yüksek öğretim kurumu). these courses are labeled under four categories: subject matter knowledge (i.e., general chemistry i–ii, science laboratory practices i–ii, special topics in chemistry), pedagogical content knowledge (e.g., science teaching methods i–ii), general pedagogy knowledge (i.e., philosophy of education, sociology, guidance and counseling) and general cultural knowledge (e.g., foreign language – english, german, french). all the participants were currently enrolled in the “science laboratory practices-i” course in which they actively implemented several tasks and experiments with gases. before the teaching intervention, all official permissions were completed. in addition, the pre-service science teachers were informed about the teaching intervention and invited to sign a consent form. the first author informed them that they were not obliged to participate in the study and would not get any extra points for their participation. furthermore, they could freely leave the study if they felt uncomfortable. consequently, the current study evaluated and only reported data from the pre-service science teachers who had signed the consent form and participated voluntarily. teaching intervention the subject of “gases and gas laws” covers the effects of temperature, volume, pressure and mole on gases. the researchers developed the 5es lesson plans and then sent them to a group of experts (four chemistry educators and one science educator) to ensure content validity. to assure further validity, the 5es lesson plans and related guide materials were pilot-tested with 28 pre-service science teachers, who were not participants for the actual study. the reviews resulted in some minor revisions (i.e., typographical errors and images). the first author firstly asked them to generate their own small groups of 3–4 and then carried out all teaching sessions in a science laboratory (a total of 8 class-hours–eight 50 min) for ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. effects of 5es learning model 565 both groups. overall, the experimental group pursued the 5es learning model, whilst the control one followed the existing instructions (see appendices 2 and 3 of the supplementary material). in the experimental group, the worksheets purposed to revise and reinforce their gained knowledge by directly challenging the alternative conceptions 1, 3, 5–8 of gases and gas laws (see table s-i). furthermore, these worksheets requested them to use explicitly such sps as observing, measuring, classifying, identifying variables, formulating hypotheses, interpreting data, defining operationally and doing experiments. moreover, the analogy used in the current study intended to familiarize unfamiliar concepts by dealing with the alternative conceptions 5, 11 and 15 (see table s-i). for example, a “sumo wrestler” analogy was used to make the relationship between the temperature and volume of a fixed quantity of gas familiar. later, the pre-service science teachers were asked to compare analogue features (i.e., sumo wrestling ring) with target ones (e.g., charles gas law, and the particulate nature of matter) via analogy mapping. furthermore, it directly asked them to exploit “interpreting data” as an sps. the computer animations helped them visualize sub-microscopic issues of movements of gas molecules and link macroscopic level with sub-microscopic one by handling the alternative conceptions 2, 4, 9–14 (see table s-i). hence, the computer animations required them to employ such sps as identifying variables, defining operationally and formulating models. the 5es learning model highlighted class/group discussion, therefore, the pre-service science teachers were encouraged to share their ideas/results with peers through real life examples and to negotiate the foregoing alternative conceptions. in addition, discussion processes engaged them in such sps as communicating, predicting and inferring. lessons for the control group were comprised of existing laboratory activities (e.g., experiments, lecture and question–answer, see appendix 3 of the supplementary material). instruments to collect data, the researchers developed the gas laws test with 12 two-tier items. in developing the instrument, they firstly decided relevant concepts to ensure content validity. later, they examined the literature to learn common alternative conceptions (see table s-i) and wrote multiple-choice questions by taking these alternative conceptions into account.20,21 all multiple-choice questions included one correct answer and four distracters including alternative conceptions. a multiple-choice question was the first-tier of the question, while the second-tier asked the pre-service science teachers to write down the reason for their selection. a group of experts (three chemistry educators and two chemistry teachers) ensured face validity, readability and content validity of the gas laws test. the instrument was further validated when the participants in the pilot of the 5es lesson plans (28 pre-service science teachers) were asked to read the gas laws test and depict any unclear point. thereafter, they suggested some minor revisions (i.e., typographical errors). finally, the test was pilot-tested with 115 pre-service science teachers in the third-year of their four-year science education program, who were not participants of the actual study. the reliability coefficient for the gas laws test was found to be 0.603 (see appendix 4 of the supplementary material). this sample question, which measured the relationship between the volume and temperature of a fixed quantity of gas (charles gas law), took the following alternative conceptions into account: “an increase in the temperature does not change the volume of gas”, “changes in the pressure and volume affect the temperature of a compressed gas” and “cold environment increases the volume of the balloon and decreases its pressure”. in other words, related alternative conceptions led the researchers to develop and administer the gas laws test. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 566 karsli baydere, ayas and çalik the researchers also used the science process skills test (spst, with 25 multiple-choice and 11 open-ended questions).35 multiple-choice questions focused on recalling the sps and could quickly be scored (time-efficient) by the participants. the open-ended questions, on the other hand, concentrated on their understanding of sps, and the use of scientific language (i.e., scientific terminology). hence, the researchers were able to combine the advantages of multiple-choice and open-ended questions within the same instrument (see appendix 5 at supplementary material). this sample question (question 19) measured “formulating hypothesis and identifying variables (e.g., independent, dependent and controlled variables)” of sps with which students and pre-service teachers had difficulties.28,29,32 that is, the spst paid more attention to the sps difficulties depicted by the relevant literature. the instruments were administered as pretests one week before the teaching intervention. after the teaching intervention, the same instruments were re-administered as post-tests. furthermore, the researchers did not give any information about the data collection tools and their possible responses to the participants. for this reason, they did not know that the data collection tools would be employed as post-tests after the treatment. however, they may have considered their responses and discussed them with their peers. this may be seen as the effect(s) of the teaching intervention on building their own knowledge and facilitating their science learning. data analysis in analyzing the gas laws test, the responses were ranked from the most scientifically accepted ones to scientifically incorrect ones. given the framework of the expected responses and context of the current study, the researchers had made changes in the scoring criteria provided in the related literature.20,21 that is, the first-tier of each item in the gas laws test was scored using the following criteria: “correct option (4 points), incorrect option (one point), and blank (zero point)”. the second-tier of each item in the gas laws test was assessed with the criteria: “sound understanding (8 points), partial understanding (6 points), partial understanding with alternative conception (2 points), and no understanding (zero point, see table s-ii for details)”.20,21 to highlight their explanations in the second-tier of each item, the researchers decided to give higher points to the open-ended questions and calculated total scores to run statistical and inferential analysis.20,21 the responses of pre-service science teachers to the multiple-choice questions in the spst were scored with either one point (correct response) or zero point (incorrect response). the open-ended questions in the spst were analyzed through rubrics adapted from related literature (see table s-iii for a sample rubric).36 since the one-sample kolmogorov smirnov test did not show a normal distribution of the data, this study employed non-parametric statistical methods (i.e., the mann whitney u test and the wilcoxon signed rank test). results and discussion results from the gas laws test as seen from table i, there was no statistically significant difference between the mean scores of the experimental and control groups in the pre-gas laws test (u = 289.000, p > 0.05, η2 = 0.13). their rank average values showed that the pre-gas laws test scores were similar. in the post-gas laws test, the mann–whitney u test indicated a statistically significant difference between the groups (u = 80.500, p < 0.05, η2 = 0.66) in favor of the experimental group. this ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. effects of 5es learning model 567 revealed that the 5es learning model assisted the pre-service science teachers in improving their conceptual understanding of gases and gas laws better than the existing method. the closer the η2 value is to 1, the more the experimental group out-performs the control group. the results of partial eta squared (η2) indicated a small-size effect for the pre-gas laws test (0.13) and a large-size effect for the post-gas laws test (0.66). table i. the results of the mann–whitney u test for the preand post-gas laws tests; cg: control group, eg: experimental group; note: u means a derived statistic to compare a specific value in a mann–whitney distribution table for statistical significance group type of test n rank average rank total u p η2 cg pre-test 25 18.15 614.00 289.000 0.82 0.13 eg 24 17.46 611.00 cg post-test 25 20.86 540.50 80.500 0.00 0.66 eg 24 34.52 828.50 as can be seen from table ii, both groups had statistically significant differences between their mean scores in the preand post-gas laws test in favor of the post-gas laws test (control group; z = 3.05, p < 0.05, η2 = 0.61; experimental group; z = 3.57, p < 0.05, η2 = 0.73). rank averages of the preand post-gas laws test scores showed positive ranks in favor of the post ones. the results of partial eta squared (η2) revealed large-size effects for both the control (0.61) and experimental groups (0.73). the partial eta squared (η2) values denote that the teaching interventions in the experimental and control groups were effective in facilitating the conceptual understanding of pre-service science teachers (e.g., a medium-size effect for the control group and, a large-size effect for the experimental group). however, as compared with the existing instruction, the 5es learning model was more efficient. this may stem from macroscopic and submicroscopic representations in the computer animations. furthermore, the experimental group’s engagements with the relational variables and graphics (i.e., the effects of temperature, volume, pressure and mole on gases) may have resulted in their improved performances. in addition, having yielded any mathematical equation between the relational variables seems to have evolved their conceptions of symbolic representation(s) of gases. although not as pronounced as the experimental group, the control group did show improvements after participating in regular (existing) teaching methods. the existing experiments and lecturing might have allowed them to imagine mentally complex/abstract dynamic processes at the sub-microscopic level and molecular behaviors of gases. furthermore, medium-size effects in the conceptual understanding of the control group may have come from the question–answer processes and feedbacks in their regular learning. moreover, the fact that the existing instruction somehow resulted in remedying some of the alternative con________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 568 karsli baydere, ayas and çalik ceptions in the pre-test may stem from the engagement of the control group with the regular experiments (see table s-iv). table ii. the results of wilcoxon signed rank test for the preand post-gas laws tests; cg: control group, eg: experimental group; the z value is compared with the critical z value to determine the null or research hypothesis group post-test–pre-test n rank average rank total za p η2 cg negative rank 4 8.42 26.50 3.05 0.00 0.61 positive rank 21 16.74 298.50 equal – – – eg negative rank – – – 3.57 0.00 0.73 positive rank 24 12.50 300.00 equal – – – abased on negative ranking percentages of the alternative conceptions of the pre-service science teachers of gases and gas laws were calculated through their responses to the preand post-gas laws test. as observed in table s-iv, the pre-gas laws test drew out 9 alternative conceptions for the control group and 6 alternative conceptions for the experimental group. after the treatments in both groups, the control and experimental groups accomplished positive conceptual change for 8 and 6 alternative conceptions, respectively. furthermore, the control group showed one negative conceptual change for the ninth alternative conception and one neutral conceptual change for the fourth alternative conception. this means that the experimental group demonstrated a better performance in achieving conceptual change than did the control one. for example, the percentages of the experimental and control groups holding the first alternative conception were 40 and 48 for the pre-gas laws test; and 13 and 32 for the post-one, respectively. decreases in the post-test percentages of the experimental and control groups showed that the students remedied their alternative conceptions after the teaching intervention. in this case, the positive conceptual change (cc) was indicated with (+). for instance, the conceptual change rates of the control and experimental groups for the first alternative conception (+8 and +27, respectively – see table s-iv) mean that the teaching intervention in the experimental group had a greater impact on remedying the first alternative conception than that in the control one. on the other hand, the control group’s percentages of the ninth alternative conception (4 % in the pre-test and 8 % in the post-test) showed an increase in their post-test percentage. in this case, this negative cc was signaled with (–). for the ninth alternative conception, the control group revealed a negative conceptual change (–4). the results from the post-gas laws test showed that the 5es learning model helped the pre-service science teachers to overcome their alternative conceptions and significantly improved their conceptual understanding of the subject of “gases and gas laws”. phrased differently, the 5es learning model, which took ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. effects of 5es learning model 569 common alternative conceptions of ‘gases and gas laws’ into consideration, seems to have enhanced the pre-service science teachers’ learning capabilities20,28 and to have resulted in a better understanding of scientific concepts.12 even through the experimental and control groups’ alternative conceptions of “gases and gas laws” decreased in the post-gas laws test, the experimental group performed better in remedying relevant alternative conceptions than did the control one. for instance, the computer animations and experimental activities, which afforded the pre-service science teachers to imagine gay–lussac gas law at the sub-microscopic level and its processes,16 overcame the third alternative conception (see table s-iv). similarly, the fact that the computer animations, experimental activities and analogy explicitly challenged such alternative conceptions as “students possess pitfalls at understanding relationships amongst pressure, temperature, volume, and mole” seems to have somewhat overcome the first four alternative conceptions (see table s-iv). the analogy and experimental activities handling the fifth alternative conception (see table s-iv) may have dealt with their misunderstanding of the particulate nature of matter (i.e., gas molecules expand or shrink when heated or cooled, respectively).37 similarly, the worksheets and computer animations clearly seem to have remedied the seventh alternative conception since the 5es learning model took common alternative conceptions into account. however, the teaching interventions in the experimental and control groups were unable to remedy completely the first alternative conception of “gases and gas laws”. results from the science process skills test (spst) the results of the preand post-spst are given in table iii. table iii. the results of mann–whitney u test for the preand postspst; u means a derived statistic to compare a specific value in a mann–whitney distribution table for statistical significance group type of test rank average rank total u p η2 control group pre-test 23.08 577 252 0.33 0.12 experimental group 27 648 control group post-test 13.82 345 20.5 0.00 0.79 experimental group 36.65 879 as seen from table iii, there was no statistically significant difference between the mean scores of the pre-spst of the experimental and control groups (p > 0.05). this means that the experimental and control groups were very similar to each other. furthermore, there was a statistically significant difference between the mean scores of the post-spst in favor of the experimental group (p < 0.05). this showed that the 5es learning model was more effective in improving their sps than the existing instruction. the results of partial eta squared ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 570 karsli baydere, ayas and çalik (η2) indicated a small-size effect for the pre-spst (0.12) and a large-size effect for the post-spst (0.79). the results of wilcoxon signed rank test for the pre and post-spst are presented in table iv. table iv. the results of wilcoxon signed rank test for the spst; the z value is compared with the critical z value to determine the null or research hypothesis group post-test–pre-test n rank average z* p η2 control group negative rank 2 3.25 3.19 0.00 0.53 positive rank 23 13.84 equal – – experimental group negative rank 0 .00 4.29 0.00 0.88 positive rank 24 12.50 equal – – abased on negative ranking as observed in table iv, the rank averages of the preand post-spst scores of the experimental and control groups revealed positive ranks in favor of the post-spst ones (p < 0.05). that is, both groups had changes in the sps after the treatment. the results of the partial eta squared (η2) indicated a medium-size effect for the control group (0.53) and a large-size effect for the experimental group (0.88). the results indicated that the 5es learning model was also more effective in improving the sps scores of pre-service science teachers. this result complies with related studies.28,29 the sps scores of the control group, which were significantly lower than those of the experimental group, supported the idea that concept-related experiments are insufficient in improving the sps levels of students. these findings provide further evidence that an interaction between content knowledge (e.g., gases and gas laws) and sps not only improve sps25,28,29 but also make abstract chemistry concepts more meaningful. the partial eta squared (η2) values revealed that the teaching interventions in the experimental and control groups were efficient in developing their sps (e.g., a medium-size effect for the control group and a large-size effect for the experimental group). however, as compared with the existing instruction, the 5es learning model was more effective. this may stem from the engagements of the experimental group with the relational variables, graphics (i.e., the effects of temperature, volume, pressure and mole on gases) and computer animations. furthermore, a medium-size effect in sps of the control group may arise from the question–answer processes and feedbacks in their regular learning (see table s-v for the descriptive parameters). conclusions the present study revealed that the 5es learning model resulted in better gains in the conceptions of pre-service science teachers of “gases and gas laws” ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. effects of 5es learning model 571 and improved their sps. this means that pedagogical interlinks amongst conceptual change methods (e.g., worksheets, computer animations, and analogies), content knowledge (e.g., gases and gas laws) and sps increase their learning capacities of conceptual understanding and sps. the study found, however, that there were alternative conceptions that were resistant to change in both groups. this implies that the 5es learning model and existing instruction seem to have mainly influenced soft-core alternative conceptions. fortunately, it appears that neither the 5es learning model nor the existing instruction resulted in any new alternative conception after the teaching intervention. this may come from the 5es learning model directly handling common alternative conceptions of “gases and gas laws”. although the existing instruction did not concentrate specifically on common alternative conceptions of “gases and gas laws”, the alternative conceptions of pre-service teachers were somewhat reduced and showed improvements. this may be because the lecturer might intuitively address these alternative conceptions during the question/answer sessions. phrased differently, the lecturer who taught the subject to both the experimental and control groups may have transferred her awareness of common alternative conceptions to the existing instruction (control group). this may be seen as an uncontrolled variable (limitation) of the current study. even though the pre-service science teachers held some common alternative conceptions reported by the related literature (see table s-i), the current study elicited several new alternative conceptions (i.e., the first, fourth, sixth and tenth alternative conceptions – see table si-v). this may stem from their pre-existing experiences or learning/class cultures or contextual differences in various countries. since the 5es learning model engaged the pre-service science teachers in actively building their own understanding, they should be confronted with possible alternative approaches/pedagogies suggested by the turkish science curriculum. future studies may investigate how student-generated-animations influence their imagination of macroscopic, submicroscopic and symbolic representations. the current study recommends that the 5es learning model be tested with a large sample size throughout a long-term teaching intervention. in addition, to challenge effectively alternative conceptions of students, future studies should exploit alternative conceptions elicited in the pre-test/treatment rather than common ones reported in the literature. supplementary material additional analytical and crystallographic data are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. we would like to thank dr. jennie farber lane from bilkent university, turkey for her kind help in language polishing. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 572 karsli baydere, ayas and çalik и з в о д ефекти 5es модела учења на концептуално разумевање и научне вештине будућих наставника науке: случај гасова и гасних закона fethiye karsli baydere1, alipaşa ayas2 и muammer çalik3 1 giresun university, faculty of education, department of science education, giresun, turkey, 2 bilkent university, graduate school of education, ankara, turkey и 3 trabzon university, fatih faculty of education, department of elementary teacher education, 61335 trabzon, turkey циљ овог истраживања био је испитивање ефеката примене 5es модела учења (енгл., engage-explore-explain-elaborate-evaluate) на концептуално разумевање и научне вештине будућих наставника науке у вези с гасовима и гасним законима. узорком истраживања обухваћено је 49 студената, будућих наставника науке, уписаних на курс научне лабораторијске праксе i, у оквиру катедре за научно образовање на турском државном универзитету. применом квази-експеримента (дизајн који је обухватио иницијално и финално тестирање), експериментална група била је изложена 5es моделу учења с различитим концептуалним променама метода/техника (на пример, радни листови, компјутерске анимације, аналогије и експерименти). контролна група била је подучавана кроз уобичајену наставу (на пример, експерименти, предавања и одговарање на питања). подаци су прикупљени применом тестова (тест који се односио на гасне законе и теcт у вези с научним вештинама). резултати парцијалне квадриране ете (η2) указали су на велике ефекте за контролну (0,61) и експерименталну групу (0,73). модел учења 5es показао се ефикаснијим од постојеће наставе у превазилажењу алтернативних концепата будућих наставника науке у вези с гасовима и гасним законима, као и у унапређивању нивоа њихових научних вештина. препорука изведеног истраживања је да се модел учења 5es тестира на већем узорку укључујући дужи временски период интервенције. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {use of honeybees (apis mellifera l.) as bioindicators of spatial variations and origin determination of metal pollution in serbia} j. serb. chem. soc. 83 (6) 773–784 (2018) udc 595.799+57.08:504.054–034(497.11) jscs–5111 original scientific paper 773 use of honeybees (apis mellifera l.) as bioindicators of spatial variations and origin determination of metal pollution in serbia nenad m. zarić1*, konstantin ilijević2, ljubiša stanisavljević3 and ivan gržetić2# 1university of belgrade, innovation center, faculty of technology and metallurgy, karnegijeva 4, 11120 belgarde, serbia, 2university of belgrade, faculty of chemistry, studentski trg 12–16, 11000 belgrade, serbia and 3university of belgrade, faculty of biology, studentski trg 16, 11000 belgrade, serbia (received 10 november 2017, revised 11 january, accepted 15 january 2018) abstract: honeybees have been proposed and used as bioindicators for the last few decades, because of their nature. until now they have mostly been used to determine the present pollution and to distinguish the differences between the sampling locations and the sampling periods. with the use of multivariate statistical methods honeybees can also be used to distinguish the origin of this pollution. in this study the concentrations of al, ba, ca, cd, co, cr, cu, fe, mg, mn, na, ni, sr and zn were measured in the bodies of adult honeybees collected from nine different apiaries in serbia. with the help of the statistical methods it was established that the least polluted area was the one that has no industrial activities or the intense traffic nearby. the most polluted was the urban region, followed by a region close to thermal power plants and ash disposal site. using pca and ca the origin of the analyzed metals were proposed. it was suggested that al, ba, ca, cd, co, cr, cu, fe, mn and ni have anthropogenic origin mainly from the intensive agriculture, traffic and coal combustion. key words: biomonitoring; honeybees; metal; pollution; pca; ca. introduction pollution can be of anthropogenic or natural origin. pollution that represents a bigger concern is the one emitted by mankind. the number of humans on the planet is rapidly increasing, which has led to the increase in the anthropogenic pollution as well. metals are one of the pollutants that have the negative effect on the environment, even more so since they are not bio-degradable and are accumulated in soil, water and air, and have a potentially negative effect on human health.1–3 * corresponding author. e-mail: nzaric@tmf.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc171110018z ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 774 zarić et al. the metal pollution can have different origin. the combustion of fossil fuels in the thermal power plants (tpp) causes the emission of fly ash and smoke containing particulate matter (pm) rich in toxic metals.4–8 the industrial areas can also have different industries that emit metal pollution: oil refineries, petrochemical factories, steel manufacturing companies, etc.9–11 the burning of fossil fuels for the purposes of heating, together with the intense traffic are major pollution sources in urban areas.12,13 in rural areas major pollution comes from the intensive agriculture. the use of different pesticides, fertilizers, insecticides, herbicides and fungicides can contribute to the accumulation of metals in soil and water.14 considering the negative effects that toxic metals have on human health and that the anthropogenic influence is expanding, with it the emissions of toxic metals too, it is vital to monitor their concentrations in the environment. this means that there is a need for more sampling stations, whose price and application are expensive, which is a drawback for larger areas that need to be monitored. this has led to the research and development of methods that are faster, cheaper and cover larger areas. consequently the use of bioindicators has been increasing, since they have all of these characteristics. many different animal and plant spices have been proposed and used as suitable bioindicators.15–21 toxic metals can be accumulated in the bodies of honeybees because they are in general not deadly for bees. these metals can originate from water the bees drink, from the suspended pm in the air, depositing directly on the body of the bee, or from soil that can either be transferred from roots through plants to flowers (pollen and nectar) or can be re-suspended in the air and deposited on the plant organs that honeybees visit.22 so, honeybees reflect pollution that is present in all aspects of the environment: soil, water and air. this is why in the last few decades they have been increasingly used as bioindicators of pollution, including the toxic metal pollution.22–29 the another important part of monitoring the toxic metal pollution is the method used for the detection of these metals. for the detection of metals in the biological samples, some different traditional methods are used, such as ion chromatography (ic), atomic absorption spectroscopy (aas), inductively coupled plasma optical emission spectroscopy (icp-oes), inductively coupled plasma mass spectrometry (icp-ms).16,30–33 these methods require the complicated sample preparation, that usually involves expensive equipment.34 this is why in the past few years different new methods for detection of metals in biological samples are being explored, amongst which are x-ray fluorescence (xrf), scanning electron microscopy (sem), which can be coupled with x-ray spectroscopy (sem-edx), and fluorescence nanoprobes.34–36 the aim of this study was to compare the concentration of al, ba, ca, cd, co, cr, cu, fe, mg, mn, na, ni, sr and zn found in bodies of adult honeybees ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ honeybees as bioindicators of metal pollution 775 collected during year 2014, from nine different apiaries in serbia and to assess the origin of these metals. experimental study area and samples the samples were adult honeybees obtained from nine different apiaries in serbia. two apiaries were located in belgrade (bg), one at the faculty of veterinary medicine (bgvt) and one at the faculty of agriculture (bgpo); two in the city of pančevo (pa), one in the eastern part of the city (paba), one in the western part of the city (pazv); one in the village of pavliš (pv); one in the village of mesić (ms); one in the village of drmno (dr); one in the town of kostolac (ko); one in the village of stari kostolac (stk). at each of the apiaries at least two colonies were sampled. detailed maps and explanations of sampling locations are provided in the supplementary material to this paper (figs. s1 – s4). between 5 and 10 g of the sample (50–100 bees) were collected from the outer frame of the hive that was occupied with bees but without brood.26 samples were transferred into sterile plastic containers and frozen in the laboratory and kept at –21±3 °c until analyzed. honeybee analysis samples were measured and dried in the oven at 60 °c until the constant mass was obtained (approximately 96 h). a test portion of dried samples between 0.5 and 0.6 g was taken and digested according to the us epa sw-846 method 3052, under high pressure in closed teflon vessels, with 7 ml of concentrated hno3 and 2 ml of concentrated h2o2. the mineralization was performed in a closed microwave digestion system (ethos 1, advanced microwave digestion system, milestone, italy) by heating the samples up to 200 °c (15 min), followed by another 15 min at the same temperature. each sample was cooled, transferred to a 25-ml volumetric flask and diluted to 25 ml with deionised water. the concentrations of metals were measured by the inductively coupled plasma-optical emission spectrometry, icp-oes (icap 6500duo, thermo scientific, cambridge, uk). the multi-element standard solutions were used (multi-element plasma standard solution 4, specpure®alfa aesar, karlsruhe, germany) for the determination of the elements of interest. the quality control was based on the analysis of blanks (containing 7 ml of concentrated hno3 and 2 ml of concentrated h2o2 but no analyte, prepared following the whole sample preparation procedure), duplicates and analysis of the standard solutions. statistical analysis the grubbs test for outliers was performed first, and the outliers were removed. the assessment of the data normality was done by the shapiro–wilk test. for some elements, the data were not normally distributed; therefore, they were log-transformed and the normality was reassessed. the results were processed by the one-way analysis of variance (anova), followed by tukey’s honestly significant difference (hsd) multiple comparisons test. differences in concentrations were considered significant if p values were ≤0.05. for the source appointment principal component analysis (pca) with varimax, the normalized rotation was used, as well as the hierarchical cluster analysis (ca) with the variables standardized by means of the z – score and ward’s method with euclidian distances as a measure of similarity. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 776 zarić et al. results and discussion metal concentrations the range and average concentrations found in honeybees are given in table i. the highest concentrations were observed for ca (1167 mg kg–1 d.m.) followed, in the declining order, by mg, na, fe, zn, mn, al, cu, sr, ba, ni, cr, cd, and the lowest concentrations was observed for co (0.104 mg kg–1 d.m.). table i. range and mean concentrations of analyzed elements detected in the adult bodies of honeybees; the range is presented from measurements of concentrations for each individual colony, and not from average concentrations per apiary (as displayed in table ii) element range of concentrations, mg kg-1 dry mass x̅ / mg kg-1 al 7–146 36 ba 0.54–3.97 1.69 ca 660–1838 1167 cd 0.03–0.260 0.125 co 0.022–0.221 0.104 cr 0.045–0.333 0.144 cu 11.8–29.2 19.1 fe 77–227 145 mg 590–1312 979 mn 21–78 48 na 216–630 415 ni 0.12–1.88 0.74 sr 0.79–3.71 1.99 zn 59–179 99 the mean concentrations and the standard deviations for the metals analyzed were calculated for each apiary, and are given in table ii. high standard deviations are present for some of the analyzed elements, which was to be expected. there were at least two colonies sampled at each location. the samples from each colony were prepared and analyzed separately and the mean concentrations were calculated from three repeated measurements of each sample. considering that the bees from different colonies do not necessarily fly in the same direction, even if they are in the same apiary, the detected metal concentrations can vary between colonies. this is the reason behind the high standard deviations for some of the elements. analysis of variance (anova) using one-way anova statistical differences between at least two of the analyzed locations were observed for al, ba, ca, cd, co, cr, cu, fe, mg, mn, na, ni and sr (s-5 of the supplementary material). only for the concentration of zn found in bodies of adult honeybees there were no statistically significant differences between any of the analyzed locations. this can suggest that the distribution of zn in the environment is even. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ honeybees as bioindicators of metal pollution 777 table ii. mean metal concentrations in bodies of honeybees (mg kg-1 dry mass) at different apiaries and standard deviations element location bgpo bgvt dr ko ms paba pazv pv stk al 24±7 25±13 52±47 48±45 48±50 26±11 25.8±6.0 16.8±5.4 74.5±8.1 ba 1.83± ±0.66 2.15± ±0.79 1.41± ±0.99 1.28± ±0.88 2.78± ±0.62 0.86± ±0.30 1.28± ±0.69 1.16± ±0.72 2.3± ±1.0 ca 1178± ±155 1173± ±34 955± ±176 915± ±174 1322± ±240 1147± ±249 1294± ±303 907± ±239 1653± ±164 cd 0.194± ±0.068 0.113± ±0.065 0.058± ±0.014 0.121± ±0.034 0.181± ±0.043 0.072± ±0.033 0.129± ±0.093 0.086± ±0.062 0.20± ±0.11 co 0.132± ±0.026 0.12± ±0.015 0.099± ±0.040 0.174± ±0.067 0.111± ±0.034 0.056± ±0.010 0.101± ±0.078 0.062± ±0.051 0.149± ±0.056 cr 0.132± ±0.045 0.169± ±0.047 0.158± ±0.095 0.084± ±0.012 0.149± ±0.068 0.124± ±0.018 0.212± ±0.098 0.090± ±0.042 0.169± ±0.006 cu 24.9±1.8 26.6± ±2.7 15.6±4.2 16.6± ±1.6 21.8±3.0 15.95± ±0.90 16.4± ±2.4 16.4±3.3 17.2±3.6 fe 151±31 186±37 114.3± ±7.6 196±45 139±43 113±22 158±54 114±49 203.5±3.0 mg 1117±123 985±30 899±224 916± ±195 1019±84 979± ±113 1107± ±150 751±193 1097±64 mn 54.8±9.2 50±10 47.5±2.6 63.4±4.2 61.1±7.7 37.2±4.6 57±21 30.2±5.8 41.7±6.9 na 432±99 379±34 311±111 399±102 454±101 406±138 531±74 357±84 501±105 ni 0.783± ±0.038 0.63± ±0.24 0.68± ±0.24 0.42± ±0.23 1.62± ±0.20 0.48± ±0.23 0.55± ±0.31 0.21± ±0.11 0.982± ±0.085 sr 2.17±0.40 2.10± ±0.73 1.80± ±0.59 1.88± ±0.27 2.18± ±0.14 1.18± ±0.45 2.0±1.2 1.88± ±0.77 3.20± ±0.34 zn 105±21 94.6±5.6 80±11 115±12 104±12 100±20 108±18 102±46 87.9±8.4 for al and fe it was observed that stk had significantly higher concentrations then pv (table ii, fig. s-5). higher concentrations of al and fe at stk sampling site was expected, due to its vicinity to an ash disposal site. popović et al.37 already established that the fly ash from thermal power plants “kostolac” is rich in this element. in our preliminary research the honeybees from this region have proven to have higher concentration of these two elements compared to urban and agricultural-woodland regions.27 ba and sr had significantly higher concentrations in ms compared to paba (table ii, fig. s-5). ba had also higher concentrations in ms compared to pv, while sr had higher concentrations in stk compared to paba. ms is surrounded by agricultural land and vineyards, which use different ba compounds, that can be the source of the elevated ba concentrations at this location.27,38 the higher, statistically significant concentrations, for ca were detected in the honeybees from stk apiary, than in those coming from dr, pv and ko. also higher concentrations can be seen for ms compared to pv (table ii, fig. s-5). again high concentrations of ca in stk can also be associated with its vicinity ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 778 zarić et al. to the ash dump site of the thermal power plants “kostolac”. after the production ash is suspended in water and after that it is transported to the dump site. considering that the ionic strength change in the aquatic environments will cause the exchange reactions on the ash particles, and that the fly ash is a significant source of abundant cations, including ca, it is expected for the concentration of this metal to be higher near the ash dump site.37,39,40 the concentrations of cd were statistically higher in bgpo and ms compared to dr (table ii, fig. s-5). cd is present in many artificial phosphate fertilizers and its presence in the agricultural region surrounding ms is not surprising.41 the elevated presence of cd in an urban area of bgpo can be due to traffic. it has already been concluded that the soil and the vegetation in urban areas are more burdened by this toxic metal.42 co concentrations were significantly higher at three locations, namely ko, stk and bgpo compared to pv (table ii, fig. s-5). coal combustion can have a negative effect on co concentrations in the environment due to its presence in the fly ash produced.43 therefore, ko and stk sampling locations are positioned in the vicinity of the thermal power plants “kostolac”, and životić et al.44 have concluded that coal from this basin has relatively high content of co, therefore this can explain the higher concentrations of this element at these sites. co is also present in higher concentration in urban environments due to intense traffic.42 at pazv sampling site concentrations of cr were higher than in pv (table ii, fig. s-5). pazv is located in an industrial area, and cr can come from the industrial processes happening at petrochemical industry and oil refinery in the region. levels of cu were higher at bgpo sampling site compared to dr, paba, pazv and pv sampling sites, as well as at bgvt in comparison to dr, ko, paba, pazv, pv and stk, and at ms sampling site compared to dr (table ii, fig. s-5). bgpo and bgvt are urban areas located in the capitol of serbia belgrade. it has been established earlier that cu is an constituent of the vehicle brake pads.45 in our earlier research we have already determined that honeybees from urban regions have higher cu content then those from other locations. also it has been proposed that honeybees from ms region have higher cu content because of the vineyard vicinity that is treated with bordeaux mixture containing copper sulfate.27 mg concentrations were higher in bgpo and pazv compared to pv (table ii, fig. s-5). considering that mg is one of the most abundant elements that is essential to honeybees it can be that these locations are naturally richer in this element. this can also be for na concentrations that are higher at pazv sampling site compared to dr. concentrations of mn were higher in ko and ms compared to pv and paba. also bgpo, pazv, bgvt, and dr had higher concentrations of this element compared to pv sampling site. it can be concluded that the concentration ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ honeybees as bioindicators of metal pollution 779 of mn are low at pv location (table ii, fig. s-5). in urban sampling locations (bgvt and bgpo) and sampling locations located near the thermal power plant “kostolac a” (ko) and “kostolac b” (dr) mn concentration are higher due to traffic and power plant emissions.37,46 coal burned in thermal power plants “kostolac a & b” comes from the kostolac basin. this coal has relatively high concentrations of mn, which is much higher than clarke values for brown coals, and this explains the higher mn content in honeybees sampled at locations near thermal power plants “kostolac a & b”.44 ni had statistically higher concentrations at the ms sampling site compared to pv, ko, paba, pazv, bgvt and dr sampling sites. statistically lower concentrations were found at pv in relation to stk, bgpo, dr, bgvt, pazv and paba (table ii, fig. s-5). higher concentrations of ni at ms can be explained because of the agricultural nature of the surrounding environment. some commercial phosphate fertilizers, as well as the animal manure that are used in this region can be rich in ni.47,48 honeybees in our preliminary research coming from this region also had high concentrations of ni.27 source appointment for the source appointment of trace metals the most commonly used multivariate statistical methods are principal the component analysis (pca) and the cluster analysis (ca).49 pca and ca are mostly used to identify pollution sources in street dust and soil.50,51 for the identification of sources of metal pollution in bioindicators these methods are rarely used.52 the source appointment of the metal pollution in honeybees using pca and ca was done only once.32 the factor loadings greater than 0.70 were considered excellent, while those under 0.30 were regarded as very poor.53 it can be observed in table iii that half of the elements, co, mn, cu, al, ca, cr, zn and na had loadings higher than 0.70. there were three principal components with eigenvalues larger than 1. these three components explain 70.92 % of total variance. the first component is mainly characterized by co, mn and cu. to a smaller extend it is also characterized by cd, ba and to a small extend fe and ni (table iii). this is confirmed by ca where cu, co, fe, ba, mn and cd form one large cluster (fig. 1). this component can be attributed to anthropogenic sources mainly related to agriculture and traffic. cu is known to be a constituent of the vehicle brake pads.45 it is also a part of bordeaux mixture used for treatment of crops.27 ba is used as an insecticide in the form of barium fluorosilicate and carbonate.38 cd, as well as co and mn concentrations are higher at locations with intense traffic.42,54 the second component is characterized by al, ca and cr, and to a lesser extent ba, ni and sr (table iii). the origin of these elements could be related to the ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 780 zarić et al. table iii. principal component analysis (pca) of metals measured in bodies of honeybees (pca loadings >0.70 are shown in bold) element component 1 2 3 co 0.818 0.335 0.129 mn 0.790 0.077 0.167 cu 0.715 0.100 0.224 cd 0.648 0.116 0.574 ba 0.618 0.606 0.173 fe 0.534 0.450 0.355 al 0.009 0.887 -0.085 ca 0.196 0.743 0.490 cr 0.228 0.737 0.234 ni 0.557 0.592 0.066 sr 0.487 0.571 0.262 zn 0.297 -0.066 0.821 na 0.090 0.372 0.780 mg 0.305 0.524 0.537 initial eigenvalues 7.173 1.586 1.170 variance, % 51.24 11.33 8.36 cumulative variance, % 51.24 62.56 70.92 fig. 1. dendrogram results from hierarchical cluster analysis for the analyzed elements in bodies of adult honeybees. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ honeybees as bioindicators of metal pollution 781 emissions and the fly ash produced by thermal power plants “kostolac”. at different places it was already concluded that emissions al and cr are coming from the fly ash of these power plants.27,40,44,32 this is also confirmed by their separate cluster in ca (fig. 1). earlier in the text it was also pointed out that ca is also an element that has higher concentrations around the thermal power plants, and its ash disposal sites.39,40 the third principal component is mainly characterized by zn and na, and to some extend mg (table iii). considering that these elements are abundant in the environment, and are essential for insects it can be concluded that these metals are from natural origin. zn and na form a distinctive cluster in ca as well as they are confirming their mutual source (fig. 1). some of the elements had similar loadings for different components. ba, fe, ni and sr had similar loading for the first and second component. this means that they have multiple sources. they can originate from agriculture, traffic or industrial processes. conclusions the data presented above about the concentrations of metals in the bodies of adult honeybees sampled from different apiaries can give us the invaluable data on the pollution of the environment surrounding these apiaries. the chosen method for the sample preparation and the analysis of metals gives good results. this method is widely used with good limits of detection for the metals analyzed in this study. the disadvantages of this method is the complicated sample preparation and costly equipment required. this study showed that the least polluted location is pv, considering that there is no industry or high intensity traffic nearby this which was expected. the most polluted area was in the urban region, followed by the region located around an ash disposal site of a thermal power plant. with the help of pca and ca origin of the metal pollution measured by the use of honeybees can be explained. it was suggested that co, mn, cu, cd, ba, fe, al, ca, cr and ni, although grouped into two different principal components, both have anthropogenic origin, either from the intense agriculture, traffic or the burning of coal and the disposing of ash from thermal power plants. zn, na and mg have natural origin. honeybees were again proven to be excellent bioindicators that can help track metal pollution in the environment and with the help of the multivariate statistics, can also be used to determine the origin of pollution. acknowledgement. this paper was realized as part of project no 176006, which was financed by the ministry of education, science and technological development of the republic of serbia. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 782 zarić et al. supplementary material aditional information and data are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. и з в о д употреба медоносне пчеле (apis mellifera) за одређивање просторних варијација и порекла загађења металима у србији ненад м. зарић1, константин илијевић2, љубиша станисављевић3 и иван гржетић2 1универзитет у београду, иновациони центар технолошко–металуршког факултета, карнегијева 4, 11120 београд, 2хемијски факултет универзитета у београду, студентски трг 16, 11000 београд и 2биолошки факултет универзитета у београду, студентски трг 16, 11000 београд због својих особина, медоносне пчеле се већ деценијама користе као биоиндикатори. до сада су се углавном користиле како би се одредиле просторне и временске разлике у присутном загађењу. коришћењем мултиваријантних статистичких метода могуће је користити пчеле и за одређивање извора загађења. у овом истраживању су измерене концентрације al, ba, ca, cd, co, cr, cu, fe, mg, mn, na, ni, sr и zn у телима одраслих медоносних пчела прикупљених из 9 пчелињака у србији. употребом статистичких метода утврђено је да је најмање загађено оно подручје на којем нема индустријске активности и у чијој близини нема саобраћајница са интензивним саобраћајем. најзагађеније подручје је урбани регион, па затим регион у близини термоелектрана и пепелишта. уз помоћ синтетичких метода (pca и ca) предложени су извори загађења. предложено је да al, ba, ca, cd, co, cr, cu, fe, mn и ni потичу из антропогених извора, превасходно пољопривреде, саобраћаја и од сагоревања угља. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {biodegradation of cotton fabric impregnated with tio2 nanoparticles} j. serb. chem. soc. 84 (7) 743–755 (2019) udc 677.21+667.2+546.824–31:66.092.000.57: jscs–5223 544.526.2 original scientific paper 743 biodegradation of cotton fabric impregnated with tio2 nanoparticles darka marković1#, jelena vasiljević2, barbara golja2, brigita tomšič2, barbara simončič2 and maja radetić3*# 1innovation center of the faculty of technology and metallurgy, university of belgrade, karnegijeva 4, belgrade, serbia, 2faculty of natural sciences and engineering, university of ljubljana, aškerčeva cesta 12, ljubljana, slovenia and 3faculty of technology and metallurgy, university of belgrade, karnegijeva 4, belgrade, serbia (received 13 december 2018, accepted 16 january 2019) abstract: commercial p25 tio2 nanoparticles are widely exploited as an efficient photocatalyst. in the textile domain, these nanoparticles are used for the production of self-cleaning, highly uv protective textiles, with an antimicrobial activity. the disposed textile products may end up in a landfill where they are subjected to the biodegradation process. considering the importance of the later, this study discusses the biodegradation behaviour of cotton fabric impregnated with commercial p25 tio2 nanoparticles. photocatalytic activity of tio2 nanoparticles immobilized on cotton fabric was proved by the photodegradation of dyes c.i. acid orange 7 and methylene blue in aqueous solution. biodegradation of fabrics was assessed by soil burial test in periods of 3, 9 and 18 days. chemical and morphological changes induced by biodegradation were analyzed by ftir, sem and eds. a colour of the samples gradually changed from white to yellow/brown due to rotting. sem analysis revealed a severe destruction of the control and impregnated cotton fibres after 18 days of soil burial which was in line with visual appearance of completely damaged fabrics. the results confirmed that biodegradation behaviour of both the control and impregnated sample was equivalent, indicating that p25 tio2 nanoparticles did not inhibit the biodegradation process of cellulose. keywords: cotton; tio2 nanoparticles; biodegradation; dye photodegradation; uv protection. introduction various goods with immobilized metal and metal oxide nanoparticles (nps) can be already found on the market. although ag nps are enormously exploited due to their antimicrobial action against a wide range of microorganisms,1–3 the * corresponding author. e-mail: maja@tmf.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc181213004m 744 marković et al. commercialization of products with tio2 nps should not be underestimated. excellent photocatalytic activity, uv protective properties, high stability and relatively low price made them attractive for numerous applications (photovoltaic cells, treatment of effluents, manufacturing of cosmetic products, self-cleaning ceramics and glass, etc). these features are also utilized for manufacturing of textiles with high uv protective, self-cleaning and antimicrobial efficiency.4–6 the commercially available p25 powdered tio2 nps (degussa and aeroxide) are the most commonly consumed tio2 nps and they can be considered as de-facto standard titania photocatalyst.7 generally, tio2 nps in anatase crystalline form exhibit extraordinary photocatalytic activity. however, it has been shown that p25 tio2 nps possess absolutely superior photocatalytic activity, which is attributed to their specific anatase/rutile crystalline structure.4 the addition of rutile significantly improves the photocatalytic activity of anatase phase.8 namely, when anatase and rutile phase are in the close contact, photo-excited electrons and holes are preferentially trapped in the anatase and rutile phases, inhibiting an unfavourable electron-hole recombination.8,9 although the mechanism of their action is well established, little is known on their environmental impact and health safety at this point. these aspects gained much attention recently, but the specific nature of nps that highly differ from the behaviour of equivalent bulk materials makes such research very complex and time consuming. the environmental impact of nps in general is strongly affected by their size, shape, chemical composition, surface structure and area, charge, solubility and aggregation state10 and hence, an extensive and systematic research on the interaction of the nps with various ecosystems should be encouraged.11 keeping in mind the significance of biodegradation of textile products in post-exploitation period, we made many efforts in the last several years to learn more on the possible influence of ag, tio2 and tio2/ag nps on biodegradation behaviour of cotton and cotton/pet fabrics in soil.12–16 since the biodegradation trends vary with a nature of the substrate and applied nps, it is very important to understand that some general conclusion will be drawn in the future only on the ground of the results acquired from the plenty of individual case studies. the reported studies so far indicated that the complexity is even larger than expected as the temperature, the humidity and the type of soil, including the type of enzymes involved, also play an important role in biodegradation processes of textiles in the soil.14,17,18 as anticipated, the results confirmed that ag nps in various forms inhibit the biodegradation of cotton and cotton/pet fabrics due to outstanding antimicrobial activity.12–15,17 recently, milošević et al. reported that ag/tio2 nps also suppress the biodegradation of cotton and cotton/pet fabrics in the soil.15 it is well established that antimicrobial activity of tio2 nps is initiated by uv irradiation. therefore, it was surprising that cotton fabrics loaded with colloidal tio2 nps synthesized by acidic hydrolysis of ticl4 hindered the biodegradation of cotton with tio2 nanoparticles 745 biodegradation process, which was carried out in the dark.14 the latest study pointed out that the biodegradation behaviour of the cotton fabric corona pretreated and impregnated with the same tio2 nps insignificantly differed from the control sample.16 to our knowledge, a biodegradation behaviour of cotton fabrics loaded with p25 tio2 nps, which are dominant in textile nanocomposites studied in literature and commercial textile products, has not been reported yet. therefore, the aim of this study was to consider their influence on biodegradation of cotton fabric in soil by the standard soil burial test. chemical and morphological changes were assessed by ftir and sem, respectively. as already mentioned, p25 tio2 nps impart photocatalytic and uv protective properties to textile materials. in order to demonstrate that tio2 nps in applied concentrations are truly efficient from that point of view, uv protection efficiency and discoloration of selected textile dyes (c.i. acid orange 7 and methylene blue) from aqueous solution were also evaluated. experimental materials and methods desized and bleached cotton (co) woven fabric (117.5 g/m2, 52 picks/cm, 27 ends/cm, thickness of 0.26 mm) was kindly supplied by the slovenian textile company tekstina d.d. ajdovščina (ajdovščina, slovenia). the samples were cleaned from impurities before the impregnation with aeroxide p25 tio2 nps in a manner described elsewhere. 19 co fabric was dipped into 0.1 m dispersion of tio2 nps (liquor to fabric ratio 20:1) for 5 min and after squeezing at a pressure of 2 kg/cm2, it was dried at room temperature. dry fabric was rinsed twice (5 min) with deionised water and dried again at room temperature. this fabric is denoted as a co+tio2. the uv protection factor (upf value) of the control and impregnated co fabrics was measured by uv/vis spectrophotometer cary 100 scan (varian). the uv protection factor (upf) was automatically calculated based on recorded data in accordance with australia/new zealand standard as/nzs 4399:1996 using a startek uv fabric protection application software, version 3.0 (startek technology). photocatalytic activity of tio2 nps deposited onto co fabric was studied by measuring the removal of dyes c.i. acid orange 7 (ao7) and methylene blue (mb) from aqueous solution under uv illumination (ultra-vitalux lamp, 300 w, osram). a 0.5 g of the co fabric was dipped into 25 ml of ao7 and mb solutions (10 mg/l) and illuminated for 30, 60, 90, 120, 180, 240, 300, 360 and 1440 min. the ao7 and mb concentrations were calculated based on spectrophotometric measurements (uv/vis spectrophotometer cary 100 scan, varian) at λmax of 664 and 484 nm, respectively. biodegradation of the co and the co+tio2 fabrics was accomplished by a soil burial test according to iso 11721-1:2001 and iso 11721: 2003 standards. in this standard procedure, a container was filled with commercial grade compost. the water content of the soil was 60±5 % of its maximum moisture retention capacity. it was held constant during the experiment by spraying with water. the ph of the soil was between 4.0 and 7.5. the samples were buried in the soil for 3, 9 and 18 days. after certain incubation times, the samples were rem746 marković et al. oved from the soil and rinsed with running tap water. afterwards, they were immersed in 70 % ethanol for 30 min and dried at room temperature. the presence of tio2 nps on the surface of the co fibres after impregnation was detected by field emission scanning electron microscopy (fesem, tescan mira3 feg). in addition, energy-dispersive x-ray spectroscopy (eds) of the fibres was performed using a jeol jsm 5800 sem with a sili x-ray detector (oxford link isis series 300, uk). morphological changes of co and co+tio2 fabrics induced by the biodegradation process after 3, 9 and 18 days of exposure to the soil microflora were analyzed by jeol jsm 5610 and 6060 lv scanning electron microscope equipped with eds. the samples were coated with a thin layer of au before the analysis. the wettability of co and co+tio2 fabrics was studied using a thin-layer wicking test of wettability (tlw) which was carried out in the horizontal direction according to chibowski and gonzales-caballero.20 fourier-transform infrared (ftir) spectra were obtained by a spectrum gx i spectrophotometer (perkin elmer, great britain) equipped with an attenuated total reflection (atr) cell and a diamond crystal (n = 2.0). the spectra were recorded over a range of 4000 to 600 cm-1 using 32 scans at a resolution of 4 cm-1. results and discussion a successful impregnation of co fabric with p25 tio2 nps was confirmed by fesem and eds analyses. large amounts of agglomerated tio2 nps across the surface of co fibre can be seen in fesem image (fig. 1a). peaks corresponding to ti are also visible in eds spectrum of the co+tio2 fabric which is presented in fig. 1b. fig. 1. fesem image (a) and eds spectrum (b) of the co+tio2 fiber. the influence of tio2 nps on the wickability of the impregnated co fabric was evaluated on the basis of the results obtained by tlw measurements. the time at which the water penetrated a certain distance was plotted as a function of the wetted surface and the results are presented in the fig. 2. apparently, the impregnation of co fabric with tio2 nps resulted in decreased wickability. the rate of thin-layer wicking of water into the co+tio2 fabric was slower compared to the control co fabric. taking into account that wickability and wettability are biodegradation of cotton with tio2 nanoparticles 747 closely related,21 the obtained results indicate that tio2 nps created the hydrophobic effect. such finding is in a good correlation with literature data.14,16,22 fig. 2. rate (x2/t) of thin-layer wicking of water into co and co+tio2 fabrics. growing depletion of ozone layer requires adequate uv protection which can be utilized with tio2 nps due to their ability to absorb uv irradiation. the uv protection of textiles is commonly expressed as uv protection factor (upf) or upf rating. it is recommended that upf rating for garments should not be less than 40 to 50+. in this study, the upf values of co and co+tio2 fabrics were 5.24±0.21 and 83.79±12.32, respectively. the upf rating of only 5 in the case of co fabric means that it does not provide uv protection. in contrast, the impregnated fabric ensured maximum upf rating of 50+. the photocatalytic activity of tio2 nps immobilized onto co fabric was evaluated by monitoring the discoloration of aqueous solutions of dyes ao7 and mb under the lamp which simulated the sunlight. the dependence of c/c0 vs. time of illumination for the co and the co+tio2 fabrics is shown in fig. 3. fig. 3a reveals that the control co fabric caused the removal of ao7 dye by approximately 10 % after 24 h of exposure to illumination. on the other hand, mb was almost completely removed from the solution in the same period. such results are attributed to the absorption of dyes which was more prominent in the case of mb as it is cationic dye. the photographs of the co and the co+tio2 fabric samples after the repeated illumination cycles (fig s-1, supplementary material to this paper) proved that photodegradation did not occur in the presence of co fabric. it is evident that both co samples were orange/blue coloured after 24 h of illumination. in contrast, co+tio2 fabric remained white, independently of investigated dye, which indicates the photodegradation of both ao7 and mb dyes. the ao7 dye was totally removed from the solution after 3.5 h, while in the case 748 marković et al. of the mb dye, the complete discoloration of the solution occurred after 3 h. the obtained results confirmed that the amount of deposited tio2 nps was sufficient for the efficient removal of ao7 and mb dyes from aqueous solutions. fig. 3. removal of dyes ao7 (a) and mb (b) from aqueous solutions in the presence of the co and the co+ +tio2 fabrics under sun-like illumination (inset: repeated illumination cycles with co+tio2 fabric). in order to examine the durability of photocatalytic activity of tio2 nps in the impregnated co+tio2 fabric, the discoloration procedure was repeated two more times under the same experimental conditions. the changes in relative ao7 and mb concentrations after repeated illumination cycles for the co+tio2 fabric are presented as insets in fig. 3. obviously, the co+tio2 fabric preserved excellent photocatalytic activity in repeated cycles, but its efficiency slightly decreased for the ao7 dye though it can be still considered as satisfactory. such results demonstrate that the co+tio2 fabric is stable and it could be exploited several times. biodegradation of the co and the co+tio2 fabrics was evaluated by the soil burial test. co fabrics consist of cellulosic fibers that are prone to action of bacteria and fungi during the biodegradation process. the photographs of the co biodegradation of cotton with tio2 nanoparticles 749 and the co+tio2 fabrics before and after 3, 9 and 18 days of soil burial are shown in fig. 4. apparently, there is no significant difference in appearance between the control and impregnated fabric. the yellowish/brown colour of both samples appeared already after 3 days spent in soil. the prolongation of incubation time in soil led to a further darkening of the samples due to severe rotting. it can be also noticed that both fabrics lost their form after 18 days of soil burial, i.e., they were disintegrated into pieces. such macroscopic, visually detectable changes are caused by the changes in fibre morphology, crystallinity and chemical structure. fig. 4. photographs of the co and the co+tio2 fabrics before and after 3, 9 and 18 days of soil burial. morphology of co and co+tio2 fibres before burial and after excavation from the soil after 3, 9 and 18 days were assessed by sem analysis (fig. 5). on the other hand, eds analysis provided the data on the surface elemental composition of the co+tio2 fibres before and after the soil burial test (fig. s-2 of the supplementary material). 750 marković et al. fig. 5. sem images of the co and the co+tio2 fabrics before and after 9 and 18 days of soil burial. the indications of fibres damage were apparent already after 3 days of burial. deep cracks can be observed on both co and co+tio2 fibres. the damage was slightly more pronounced on the co fibres indicating higher biodegradation rate. as expected, further prolongation of burial time brought about more severe fibre destruction which is in line with the photos shown in fig. 4 and previous results.16 the presence of considerable fibre defibrillation was also reported by milošević et al. and primc et al.15,23 the sem images of the co+tio2 fibres reveal considerable decrease in amounts of deposited tio2 nps with a burial time, which implies the leaching of tio2 nps into the soil environment. on the other hand, the eds results showed that ti was detected on the co+tio2 fibre surface even after 18 days of burial. it is clear that both the co and the co+tio2 fibres underwent the equivalent destruction. this indicates that tio2 nps did not affect the biodegradation process. biodegradation of cotton with tio2 nanoparticles 751 it is generally accepted that tio2 nps exhibit antimicrobial activity when they are exposed to uv light. however, some studies indicated that tio2 nps can interact with bacteria even in the absence of uv light.24,25 lazić et al. also demonstrated that co fabric impregnated with 0.1 m colloid of anatase tio2 nps (with significantly smaller dimensions than currently applied p25 tio2 nps) which were synthesized by acidic hydrolysis of ticl4, exhibited similar biodegradation behaviour as the control sample within 20 days of testing.14 however, unlike in the current study, the former samples were not destroyed within this time interval and impregnated sample in the following weeks showed a resistivity towards biodegradation. this collision in the results implies that the structure, size and shape of tio2 nps may highly influence their behaviour in soil. on the other hand, weaker degradation is attributed to lower temperature and humidity, as well as to different soil type in previous study. commonly, microorganisms in soil are more active at elevated temperature and humidity. so far reported results undoubtedly convinced us that these parameters are extremely important in biodegradation process.14–18 it is suggested that cuticle layer of the co fiber is digested first.26 the action of various microorganisms is supposed to be different. fungi penetrate through the second wall until they reach fibre lumen where they grow. in fact, mycelium grows towards the fibre wall secreting the enzymes which cause the degradation.26 in contrast, the action of bacteria is oriented in opposite direction: from the fibre surface towards fibre interior. tomšič et al. assumed that fungi play the main role in biodegradation of cellulose fibres since bacteria need medium with larger moisture.13 in other words, co fabric should be saturated throughout the whole biodegradation process. the chemical changes induced by biodegradation were examined by ftir analysis. the ftir spectra of co and co+tio2 samples before and after 3, 9 and 18 days of soil burial are presented in fig. 6. the characteristic absorption bands for cellulosic fibres can be clearly read from these spectra and they were discussed in detail in our previous work.15,16 the emergence of intensive absorption bands at 1641 and 1535 cm–1 corresponding to amide i and ii in excavated samples are attributed to secondary amides originating from the proteins that were produced during the growth of microorganisms and they remain irreversibly bound to the fibres.12,13 the intensity of these bands gradually increased with a prolongation of the burial time for both the co and the co+tio2 samples. the clear presence of these bands in the co+tio2 fabric revealed that tio2 nps did not significantly affect the biodegradation process in soil. since the existence of these bands was reported and discussed in many studies so far, they can be adopted as an indicator of microbial growth and thus, the degree of fibre deterioration.12,13,15,16,23 rough conclusions on the alterations in crystallinity of cellulose caused by biodegradation process can be drawn also on the basis of ftir measurements. 752 marković et al. the degree of crystallinity is commonly expressed as a crystallinity index (ik). in accordance with methods proposed by o’connor et al., nelson and o’connor, and hulleman et al. in the current study, ik was determined from the proportion of intensity of absorption bands at 1429 (ch2 bending) and 893 cm–1 (c–o stretching), at 1372 (vibration of c–h group of cellulose ring) and 2900 cm–1 (c–h stretching) as well as at 1280 (c–h bending) and 1200 cm–1 (–oh bending)27–29. the ik changes of cellulose in the co and the co+tio2 fabrics caused by biodegradation after 3, 9 and 18 days of soil burial are presented in fig. 7. evidently, the degree of crystallinity increased within 18 days of soil burial for both samples. such finding is attributed to initial attack of amorphous fibre regions by microorganisms.13,18 it is believed that further exposure to the action of microorganisms would result in deterioration of both amorphous and crystalline regions of the fibre.18 obtained results are in accordance with the reported data.16 fig. 6. ftir spectra of the co and the co+tio2 fabrics before and after 3, 9 and 18 days of soil burial. fig. 7. dependence of crystallinity index, ik, from burial time of the co and the co+tio2 samples. method of ik determination:  and  are proportions of intensity of the bands at 1429 and 898 cm-1,  and  are proportions of intensity of the bands at 1372 and 2900 cm-1. biodegradation of cotton with tio2 nanoparticles 753 conclusion the commercial p25 tio2 nanoparticles present on the fibres of the impregnated cotton fabric decreased the fabric wickability, provided maximum upf rating and excellent photocatalytic activity. after 18 days of soil burial, both the control cotton fabric and impregnated fabric were disintegrated into pieces. the observed defibrillation and degradation of the control and impregnated fibres during the biodegradation in the soil environment showed that presence of the p25 tio2 nanoparticles did not significantly influence the biodegradation process. furthermore, the detected amide groups from the generated proteins and increased crystallinity index with a prolonged burial time, for both the control and impregnated fabrics, suggested that tio2 nanoparticles did not obstruct the growth of the microorganisms. obtained results clearly demonstrated that p25 tio2 nanoparticles did not hinder the biodegradation of cotton fabric which is very beneficial from the environmental standpoint. supplementary material the additional data are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the financial support for this study was provided by the slovenian research agency (programme p2-0213 textiles and ecology) and the ministry of education, science and technological development of republic of serbia (projects no. 172056 and 45020). this research has been done under the umbrella of the bilateral cooperation between republic of slovenia and republic of serbia (project “biodegradability of textile materials impregnated with silver and titania nanoparticles”, 451-03-38/2016-09/41). и з в о д биодеградација памучне тканине импрегниране наночестицама tio2 дарка марковић1, јелена васиљевић2, brigita tomšič2, barbara golja2, barbara simončič2 и маја радетић3 1универзитет у београду, иновациони центар технолошко–металуршког факултета, карнегијева 4, 11120 београд, 2faculty of natural sciences and engineering, university of ljubljana; aškerčeva cesta 12, ljubljana, slovenia и 3универзитет у београду, технолошко–металуршки факултет, карнегијева 4, 11120 београд комерцијалне наночестице p25 tio2 се широко употребљавају као катализатор. у области текстила се ове честице често користе у производњи самочистећег текстила са ув заштитом и антимикробном активношћу. одбачени текстилни производи могу завршити на депонијама где долази до њихове биодеградације. узимајући ово у обзир, у раду је дискутовано понашање памучне тканине импрегниране комерцијалним наночестицама p25 tio2 приликом процеса биодеградације. фотокаталитичка активност наночестица tio2 имобилисаних на памучноj тканини је доказана праћењем фотодеградације воденог раствора боја c.i. acid orange 7 и метиленско плаво. биодеградација памучне тканине је испитана методом закопавања у земљу на период од 3, 9 или 18 дана. хемијске и морфолошке промене настале услед биодеградације материјала су испитане ftir, sем и eds анализом. боја узорака се променила из беле у жуто/браон услед процеса труљења. sем анализа је указала на изражено оштећење контролног узорка и импрегни754 marković et al. раних влакана након 18 дана стајања у земљи, што је у складу са изгледом распаднутих тканина. такође је утврђено да присуство наночестица p25 tio2 нема негативан утицај на способност биодеградације памучне тканине. 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(https://doi.org/10.1177%2f004051755902900411) 30. s. h. d. hulleman, j. m. van hazendonk, j. e. g. van dam, carbohyd. res. 261(1994) 163 (https://doi.org/10.1016/0008-6215(94)80015-4). novel use of biocl as an efficient and selective reagent for cleavage of 2,4-dinitrophenylhydrazones to carbonyl compounds j. serb. chem. soc. 81 (2) 125–131 (2016) udc 546.872’131:548.512:547.556.9:547.594 jscs–4832 original scientific paper 125 novel use of biocl as an efficient and selective reagent for cleavage of 2,4-dinitrophenylhydrazones to carbonyl compounds abbas amini manesh* and khadijeh azarin department of chemistry, payame noor university, 19395–4697 tehran, iran (received 4 may, revised 14 august, accepted 25 august 2015) abstract: a novel use of bismuth oxychloride (biocl) as an efficient and selective catalyst for the clean cleavage of 2,4-dinitrophenylhydrazones under mild conditions is reported. the reactions proceed very smoothly, and the yields are good to excellent. over oxidation of aldehydes to carboxylic acid and the formation of by-products were not observed. the catalyst could be recovered and reused for at least four reaction cycles without considerable loss of reactivity. keywords: bismuth oxychloride; aldehydes; hydrazones; ketones; selective. introduction hydrazones are well-known and synthetically useful derivatives of aldehydes and ketones. they are usually formed easily from carbonyl compounds and hydrazines in a reversible reaction.1 hydrazones are important derivatives in carbonyl chemistry and are used extensively as protecting, activating, and directing groups.2 regeneration of carbonyl compounds from stable and readily prepared oximes, hydrazones and semicabazones has received considerable attention in recent years.3–6 there are only a few reports available dealing with these reactions.7–11 although some are realized under mild conditions, most of these regenerations are often hazardous or use very toxic, expensive, or not readily available reagents, or reagents which need to be freshly prepared.12–14 thus, there is still a need to develop new and facile procedures for the regeneration of carbonyl compounds from 2,4-dinitrophenylhydrazones. in continuation of a systematic study and research on oxidation methods,15–19 herein, a new simple and selective method for the conversion of 2,4-dinitrophenylhydrazones to their corresponding aldehydes and ketones by the novel use of bismuth oxychloride is reported. * corresponding author. e-mail: a_aminima@yahoo.com doi: 10.2298/jsc150504071m _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 126 amini manesh and azarin according to a literature search, there is not a great deal of work on the use of biocl as an oxidant and hence, this is the first report on the use of biocl as a reagent for the regeneration of the carbonyl group from 2,4-dinitrophenylhydrazones that act as a catalyst. the simple reaction of different hydrazones by biocl in acetonitrile under reflux conditions gave the corresponding carbonyl compounds in good yields (scheme 1). scheme 1. cleavage of hydrazones by biocl. experimental chemicals and apparatus chemicals were purchased from merck, fluka and aldrich chemical companies. the solvents were used as received. all products were known and identified by comparison of their m.p., b.p., ir and nmr data with those reported for the authentic samples. all yields refer to the isolated products. the progress of the reaction was monitored by tlc. the ir and 1h-nmr spectra were recorded on a shimadzu infrared spectrophotometer ft-ir, model ir prestige 21 (kbr pellets), and a 90 mhz jeol ft-nmr spectrometer, respectively. the 1h -nmr chemical shifts were measured relative to tms. biocl was prepared according to previously reported procedure.20 the 2,4-dinitrophenylhydrazones were prepared by a standard procedure.21 general procedure for the cleavage of hydrazones by bismuth oxychloride: a solution of the hydrazone (1 mmol) in acetonitrile (10 ml) was refluxed for 10 min. then biocl (1 mmol) was added to the solution and the mixture was refluxed for the appropriate time, as indicated in table i. after completion of the reaction (tlc monitored), the reaction mixture was cooled to room temperature and h2o (1 mmol) was added and the mixture stirred for 10 min. then the solvent was evaporated and chcl3 or thf was added. after stirring the mixture for 10 min, the solid residue was filtered off and washed with chcl3 or thf (10 ml). evaporation of the solvent gave the pure carbonyl products in excellent yields. the products were characterized by their physical constants and ir and nmr spectra, which were compared with those of authentic samples.22-31 recovery of the biocl the residue on the filter paper was separated, hcl was added, the ph of the aqueous solution adjusted to 3 and the mixture stirred until the biocl was dissolved. the mixture was filtered (2,4-dinitrophenylhydrazine was separated as residue on the filter paper), na2co3 (5 % aqueous solution) was added to the filtrate and the ph of the aqueous solution was adjusted to 8–9. after stirring at room temperature for 30 min, the resulting precipitate was filtrated and washed with deionized water several times. finally, the white powders of biocl were obtained by drying the precipitate at 80 °c for 5 h. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ biocl: efficient reagent for cleavage of hydrazones 127 result and discussion the results illustrated in table i indicate that the reaction was successful for a variety of aliphatic and aromatic 2,4-dinitrophenylhydrazones (scheme 1). table i. conversion of 2,4-dinitrophenylhydrazones to the corresponding carbonyl compounds by biocl; reaction conditions: hydrazine, 1 mmol, biocl, 1 mmol, h2o, 1 mmol, ch3cn; under reflux yielda, % time, h product reactant no. 8522 2:20 no2 o2n nhnch 1 8222 2:15 2 7822 2 3 9222 3:15 4 8923 2:10 h3c o ch3 5 9022 1 6 9224,25 2:35 7 8126 1:15 8 8827 1:20 9 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 128 amini manesh and azarin table i. continued yielda, % time, h product reactant no. 7028 3:20 10 8529 4 11 8530,31 4:25 12 6027 2:50 13 aisolated yields all these carbonyl derivatives were converted back to their corresponding aldehydes and ketones in acetonitrile as the optimal solvent among the tested solvents, including: methanol, ethanol, chloroform, diethyl ether and acetonitrile, taking benzophenone 2,4-dinitrophenylhydrazone as a representative example (table ii). table ii. optimization of the solvent entry solvent time, h t / °c yield, % 1 meoh 1 64 65 2 etoh 1 78 70 3 chcl3 1 62 60 4 (c2h5)2o 1 40 10 5 ch3cn 1 80 90 moreover, the reactions were run with different amounts of biocl. on analyzing the different results, it was concluded that the best reaction conditions were 1 mmol of oxidant biocl in ch3cn media under reflux conditions. a sterically hindered ketone hydrazine was successfully cleaved to the corresponding ketone in good yield. the hydrazones of aldehyde were converted into the corresponding aldehydes and no acid, due to oxidation of the regenerated aldehyde, was formed (entries 1–3 and 10–13, scheme 2). based on the above results, a plausible reaction mechanism is shown in scheme 3. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ biocl: efficient reagent for cleavage of hydrazones 129 85 % 0 % scheme 2. selective cleavage of hydrazone. scheme 3. proposed mechanism for the cleavage of hydrazones. catalyst reuse and stability an important advantage of biocl in these reactions is its facile recovery from the reaction mixture and reusability. the catalyst reuse and stability was checked using benzophenone 2,4-dinitrophenylhydrazone as a model substrate. the recovery of catalyst was very easy. the products are soluble in chcl3 or thf, while the catalyst remains insoluble. the catalyst was separated from the reaction mixture after each experiment by simple filtration. as shown in table iii, the biocl catalyst was reusable four times and its activity did not show any significant decrease. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 130 amini manesh and azarin table iii. reusability of biocl catalyst in the hydrolysis of benzophenone 2,4-dinitrophenylhydrazone run no. time, h yielda, % 1 1 90 2 1:30 83 3 1:45 75 4 2 70 aisolated yield conclusions in this study, oxidation of the aldehydes to carboxylic acids and the formation of by products were not observed. moreover, the synthesis of the catalyst (bismuth oxychloride) is very simple and the required starting materials are commercially available and cheap. the stability and activity of the catalyst are other advantages of this reagent. the catalyst could be recovered and reused for at least four reaction cycles without considerable loss of reactivity. the striking features of the proposed method are availability, the cost of reagent, easy preparation of the catalyst, easy and clean work-up procedure (most do not require chromatography), absence of the formation of oxidation products due to the high selectivity and mild nature of the catalyst, high yields and recovery and reusability of the catalyst. overall, this simple, clean, selective and efficient procedure for the hydrolysis of various hydrazones to carbonyl compounds on a medium to large scale could be recommended. acknowledgement. the authors acknowledge the support of this work by the hamedan payame noor university. и з в о д нова примена biocl као ефикасног и селективног реагенса за раскидање 2,4-динитрофенилхидразона до карбонилних једињења abbas amini manesh и khadijeh azarin department of chemistry, payame noor university, 19395–4697 tehran, iran приказана је нова примена бизмут-оксихлорида као ефикасног и селективног реагенса за раскидање 2,4-динитрофенилхидразона под благим реакционим условима. реакције се одигравају без потешкоћа у добром до одличном приносу. није уочена оксидација алдехида до карбоксилних киселина или формирање других споредних производа. реагенс се може изоловати из реакционе смеше, и поновно користити у бар четири наредна реакциона циклуса без значајнијег губитка реактивности. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis of novel piperazino-alkyl-1h-benzo[d]imidazole derivates and assessment of their interactions with the d2 dopamine receptor} j. serb. chem. soc. 84 (9) 925–934 (2019) udc 547.861.3’53.024+547.233– jscs–5235 304.2:539.196:615 original scientific paper 925 synthesis of novel 2-(piperazino-1-yl-alkyl)-1h-benzimidazole derivates and assessment of their interactions with the d2 dopamine receptor jelena z. penjišević1, deana b. andrić2, vladimir b. šukalović1, goran m. roglić2#, vukić šoškić3 and slađana v. kostić-rajačić1* 1ictm-department of chemistry, university of belgrade, njegoševa 12, 11000 belgrade, serbia, 2faculty of chemistry, university of belgrade, studentski trg 12–16, 11000 belgrade, serbia and 3orgentec gmbh, carl-zeiss-str. 49, 55129 mainz, germany (received 29 october, revised 3 december, accepted 4 december 2018) abstract: a total of 14 novel arylpiperazines were synthesized, and pharmacologically evaluated by measuring their affinities towards the d2 dopamine receptor (drd2) in a [3h]spiperone competition assay. all the herein described compounds consist of a benzimidazole moiety connected to n-(2-methoxyphenyl)piperazine via linkers of various lengths. molecular docking analysis and molecular dynamics simulations were performed on the drd2–arylpiperazine complexes with the objective of exploring the receptor–ligand interactions and properties of the receptor binding site. the recently published crystal structure of drd2 was used throughout this study. the major finding is that high affinity arylpiperazines must interact with both the orthosteric binding site and the extended binding pocket of drd2 and therefore should contain a linker of 5 or 6 methylene groups long. keywords: arylpiperazines; molecular dynamics; molecular docking; receptor binding site. introduction dopamine receptors belong to the rhodopsin-like, aminergic g protein -coupled receptors (gpcrs) group. they are involved in many physiological processes and play important role in the central nervous system (cns).1–4 targeting the dopamine d2 receptors (drd2) is a common strategy for the treatment of neurodegenerative diseases, such as schizophrenia, parkinson’s disease, dementia and depression.5–8 it is a well-documented fact that n-substituted arylpiperazines are compounds with pronounced drd2 activity.9,10 since arylpiperazines have a wide * corresponding author. e-mail: srkostic@chem.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc181029104p ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 926 penjišević et al. spectrum of therapeutic potentials and the design, synthesis and characterization of new arylpiperazine like drugs is an ever growing field of research.11–14 in this paper, the synthesis of 14 new n-(2-methoxyphenyl)piperazines of the general structure 5 (scheme 1) is presented. their affinities towards drd2 were evaluated in the [3h]spiperone competition assay. recent discovery of drd2 crystal structure with bound risperidone15 defined the receptor binding site with greater accuracy than existing homology models. this finding prompted us to investigate drd2–arylpiperazine binding features, using molecular docking analysis and molecular dynamics simulations in order to define key receptor–ligand interactions and explain the dopaminergic properties of the herein described compounds. experimental the reagents and solvents used in this work were obtained from alfa–aesar or sigma– –aldrich and used without further purification. solvents were routinely dried over anhydrous na2so4 prior to evaporation. general a boetius phmk apparatus (veb analytic, dresden, germany) was used to determine the melting points, which are here presented uncorrected. the 1h-nmr and 13c-nmr spectra were recorded at 200 and 50 mhz, respectively, on a gemini 2000 (varian, oxford). the spectra were recorded in deuterochloroform with tetramethylsilane as the internal standard; the chemical shifts (δ) are reported in parts per million (ppm); all coupling constants (j values) are reported in hz. lc/ms was performed on a 6210 time-of-flight lc–ms system (agilent technologies, germany). for data analysis, masshunter workstation software was used. the infrared (ir) spectra were obtained on a thermo scientific spectrometer. for analytical thin-layer chromatography (tlc), polygram sil g/uv254 plastic-backed thin layer silica gel plates were used (macherey–nagel, germany). the chromatographic purifications were performed on merck-60 silica gel columns (230–400 mesh astm) under medium pressure (dry column flash chromatography). analytical and spectral data for the synthesized compounds are given in supplementary material to this paper. a microsynth milestone and a biotage initiator 2.5 exp were used for the microwave experiments. chemistry general procedure for the synthesis of compounds 3a–g. a suspension of 1-(2-methoxyphenyl)piperazine (1, 0.084 mol), triethylamine (0.0874 mol), k2co3 (0.175 mol) and bromoester 2a–g (0.084 mol) in 2-butanone (100 ml) was stirred for 24 h at 80 °c. after cooling, the mixture was poured into cold water and the organic layer was extracted with ch2cl2 and concentrated in vacuo. the resulting ester was purified by silica gel column chromatography using a gradient of methanol (0–5 %) in dichloromethane. general procedure for the synthesis of compounds 5a–n. compounds 3a–g (0.0035 mol) and diamines 4a–c (0.0035 mol) were suspended in 8 ml 50 % methanesulfonic acid in water, transferred into a sealed tube, and microwave irradiated at 180 °c for 45 min at 300 w. after cooling to room temperature, the reaction mixture was poured into ice-cold water and neutralized with a saturated solution of naoh. the product was extracted with ch2cl2 and concentrated in vacuo. the resulting 1h-benzimidazoles were purified by silica gel column chromatography using a gradient of methanol (0–5 %) in dichloromethane. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. investigation of synthesized dopaminergic ligands 927 biological assays membrane preparation. rat caudate nuclei synaptosomal membranes for the drd2 binding experiments were prepared as previously described.16 striatal tissue acquired from male wistar rats (150–200 g) was used as the source of drd2. the tissue was homogenized in 20 volumes of ice-cold 50 mm tris-hcl buffer containing 120 mm nacl, 5 mm kcl, 1 mm mgcl2 and 2 mm cacl2 using a potter–elvehjem homogenizer (6×800 rpm). the membrane fraction obtained after centrifugation at 20000 rpm for 15 min was used in the binding experiments. [3h]spiperone receptor binding assay. [3h]spiperone (73.36 ci mmol-1, perkin elmer las gmbh, rodgau, germany) binding was assayed in 1.0 mm edta, 4 mm mgcl2, 1.5 mm cacl2, 5 mm kcl, 120 mm nacl, 25 mm tris-hcl solution, ph 7.4, with rat caudate nuclei synaptosomal membranes (protein concentration 0.6 mg ml-1), at 37 °c for 10 min in a total volume of incubation mixture of 0.4 ml. the binding of the radioligand to 5-ht2 receptors was prevented by 50 mm ketanserin. the ki values of the tested compounds were determined by competition binding at 0.2 nm of the radioligand and eight different concentrations of each compound (10-4–10-10 m). nonspecific binding was determined in the presence of 10 µm spiperone. the reaction was terminated by rapid filtration through whatman gf/c filters, washed three times with 5.0 ml of ice-cold incubation buffer, and the retained radioactivity was measured in a 1219 rackbeta wallac scintillation counter (eg&g wallac, turku, finland). inhibition curve construction and statistical (student’s t-test) analysis were performed by graph-pad prism (graphpad software inc). hill slope coefficients were fixed to unity during the calculations. computational study docking simulations. the docking procedure was performed using forecaster software.17 the receptor model pdb code 6cm418 was used together with 2d structures of the ligands, prepared in chemdraw.19 all structures were prepared in the software using default procedures. rigid receptor, flexible ligand docking was carried out. the obtained docking structures were examined and structures with the maximum number of receptor–ligand interactions were selected for further analysis. binding poses metadynamics. the docking pose quality was assessed in terms of the fluctuations of the ligand rmsd (the root-mean-square deviation of atomic positions), and the persistence of important contacts between the ligand and the receptor (metadynamics binding posescore and metadynamics binding persistence) using desmond software and default parameters.20 one docking pose with the lowest rmsd and best overall score was selected for molecular dynamics (md) simulations. construction of a protein–membrane system for molecular dynamics. the protein protonation state was adjusted using the schrodinger protein preparation module, at physiological ph (ph 7.4). the prepared protein was embedded into a popc membrane bilayer using the desmond system builder module,20 and oriented according to data from the orientations of proteins in membranes (opm) server.21 the embedded protein was solvated with tip3p explicit water model, and the system was neutralized via counter ions and a salt solution of 0.15 m kcl. in this way, systems were obtained that were subjected to membrane relaxation protocol.20 md simulations. molecular dynamics (md) simulations of the system were performed using schrodinger desmond software packages.20 opls 2003 forcefield22 was used to calculate the interactions between all the atoms. for the calculation of long-range coulombic interactions, the particle–mesh ewald (pme) method was used, with a cut-off radius of 9 å for short-range van der waals (vdw) and electrostatic interactions. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 928 penjišević et al. during the course of the simulation, a constant temperature of 310 k and a pressure of 1.01235 bar were maintained, using a nose–hoover thermostat,23 and the martyna–tobias– –klein method.24 time increments of 2.0 fs were used in the simulations. finally, 100 ns md simulation for the each ligand– drd2 complex was performed and the collected trajectory frames used in the md analysis to quantify the protein–ligand interactions. results and discussion compounds 5a–n were synthesized according to scheme 1. the synthesis started with n-(2-methoxyphenyl)piperazine (1) that was alkylated with a series of homologous bromo-esters 2a–g, providing n-alkylated products 3a–g. counterpart diamines 4a–c were obtained by reduction of the corresponding 2-nitro precursors, using raney-ni and hydrazine hydrate under conditions described in earlier publications.25,26 microwave assisted condensation of piperazines 3a–g and diamines 4a–c, under forcing, strongly acidic conditions, secured the desired benzimidazoles 5a–n. scheme 1. synthesis of the compounds 5a–n n = 1–7 for compounds 2a–g and 3a–g; ethyl esters of the general structure 2 were used in the synthesis of 3b, 3c, 3e and 3f; 4a (r = h); 4b (r= ome); 4c (r= cl); structures 5a–n are presented in table i. drd2 binding affinities of compounds 5a–5n were evaluated in vitro using [3h]spiperone as a standard dopaminergic radioactive ligand (table i).27 molecular docking simulation of the herein described 2-{[4-(2-methoxyphenyl)piperazin-1-yl]alkyl}-1h-benzo[d]imidazoles on d2dr was performed on the d2dr crystal structure published recently by wang et al.15 they reported that the benzisoxazole moiety of risperidone interact with d2dr through cys1183.36, thr1193.37, ser1975.46, phe1985.47, phe3826.44, phe3906.52 and trp3866.48 in the orthosteric binding site (obs). obs of d2dr is defined by the amino acid side chains of helices iii, v and vi and also harbour asp1143.32. asp1143.32 forms an essential salt-bridge with protonated piperidine nitrogen of risperidone molecule. in addition d2dr has a secondary binding pocket, extended binding pocket (ebp), that encloses the tetrahydropyridopyrimidinone ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. investigation of synthesized dopaminergic ligands 929 moiety of risperidone. ebp is bordered by the extracellular part of tm vii consisting of an extracellular loop 1 (el1) and the junction of helices i, ii and vii.15 tаble i. chemical structures and drd2 binding constants of 2-{[4-(2-methoxyphenyl)piperazin-1-yl]alkyl}-1h-benzimidazoles (5a–n); drd2 binding constants (ki) were determined in a [3h]spiperone displacement assay. the values are the mean of three independent experiments realized in triplicate, performed at eight competing ligand concentrations ligand n r ki ± sem /nm 5a 1 h >1000 5b 2 h >1000 5c 3 h >1000 5d 4 h >1000 5e 5 h 24±1 5f 6 h 16±2 5g 7 h >1000 5h 4 och3 124±5 5i 5 och3 12±3 5j 6 och3 76±8 5k 7 och3 >1000 5l 4 cl 109±9 5m 5 cl 25±3 5n 6 cl 102±3 molecular docking simulations on the binding of 2-{[4-(2-methoxyphenyl)piperazin-1-yl]alkyl}-1h-benzimidazoles into the crystal structure of drd2 show that the (2-methoxyphenyl)piperazine moiety occupies drd2 obs, and interacts with asp1143.32, cys1183.36, trp3866.48 and phe3906.52, while the benzimidazole part interacts with leu942.64, ile184el2,trp100el1, phe3896.51, thr4127.39 and tyr4087.35 in the ebp (fig. 1). compounds with optimal linker length (five or six methylene groups in the linker) allow the benzimidazole moiety to reach ebp and to interact with leu942.64, trp100el1, phe3896.51, thr4127.39 and tyr4087.35 (fig. 2). compounds with shorter linker (5a–d) do not reach into the ebp, while ligands with seven methylene groups in the linker (5g and 5k) are too long to fit optimally into the d2dr binding cleft and protrude into the extracellular space. these results are in agreement with experimental data: compound 5d (with a 4 methylene groups linker) has affinity of over 1000 nm, while compounds 5e and 5f (with 5 and 6 methylene groups linker, respectively) have 24 and 16 nm, respectively. compound 5g shows a sharp drop in affinity because of the length of the linker, which cannot be accommodated in the drd2 bind cleft. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 930 penjišević et al. fig. 1. docking of ligand 5i to drd2 is presented. view of the interactions between the 3d model of the drd2 binding site and ligand 5i. the images show only the key amino acid residues of the receptor binding pocket. figures (side view–left and top view–right) show docking of 5i viewed from different angles. binding site ligand accessible surface is shown in the top view. in series of compounds substituted with methoxy and chloro groups, the highest drd2 affinity was obtained with compounds 5i and 5m. linker with 5 methylene groups facilitates optimal positioning of substituted benzimidazole part in the receptor ebp (fig. 1). shorter linkers, as it is obvious in series 5h–k and 5l–n, lead to decrease in receptor affinity due to sub-optimal placement of benzimidazole part in regard to the interacting residues trp100el1 and tyr4087.35. to test the stability of obtained docking poses, md simulations of the drd2 and selected ligands were performed on inactive receptor state for 100 ns for each ligand. obtained trajectories were analyzed with focus on the residues that form obs and ebp (table s-i of the supplementary material). most of the receptor–ligand interactions in obs, observed in molecular docking simulations, persisted for a significant portion of md run (>20 % total simulation time). compounds with significant drd2 affinity (5e–f, 5h–j and 5l–n) had a salt bridge between the protonated piperazine nitrogen of the ligand and asp1143.32 of drd2 preserved for more than 79–84 % of the simulation time. additional interactions in obs are aromatic interaction with cys1183.36 (32–75 % of the simulation time), and edge-to-face interactions with trp3866.48 (76–98 % of the simulation time) and phe3906.52 (20–49 % of the simulation time). in the ebp, significant interactions are aromatic interactions (edge-to-face type) with trp100el1, phe3896.51 and tyr4087.35. compounds 5e, 5f, 5i and 5m form an additional hydrogen bond with thr4127.39. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. investigation of synthesized dopaminergic ligands 931 fig. 2. results of docking simulations for ligand 5e (a), 5f (b), 5i (c) and 5m (d) are presented. schematic representation of the best docking pose for all ligands are provided. for clarity, only amino acid residues in close contact with ligands are shown. solid lines represent aromatic, while dotted lines represent electrostatic interactions. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 932 penjišević et al. conclusions molecular docking and md simulation provide important information that explains how the receptor–ligand complexes are formed. high affinity 2-{[4-(2 -methoxyphenyl)piperazin-1-yl]alkyl}-1h-benzimidazoles must simultaneously occupy both obs and ebp. to establish key interactions both in obs (salt bridge formation and aromatic interactions) and ebp (aromatic interactions and hydrogen bond formation), the ligands should have a linker of five or six methylene groups. linker flexibility and substituent size in the benzimidazole moiety determine ligand positioning inside the ebp and brings it in close contact with trp100el1 and tyr4087.35, which are key interacting residues. additionally, as can be concluded from the results of molecular dynamics, the affinity of the arylpiperazine ligands benefit greatly from possible formation of interactions of the arylpiperazine part of ligands with thr4127.39 in ebp. it is clear that both trp100el1 and tyr4087.35 can form aromatic interactions and/or hydrogen bonds. to establish the exact nature of interactions in ebp, modification of presented ligands, in terms of target synthesis of the compounds which can strictly form only one of these interactions, represent a guideline for further investigation. supplementary material analytical and spectral data for the synthesized compounds, as well as additional results, are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. this work was funded by the ministry of education, science, and technological development of the republic of serbia (grant 172032). и з в о д синтеза нових деривата 2-(пиперазино-1-ил-алкил)-1н-бензимидазола и проучавање интеракција са д2 допаминским рецептором јелена з. пењишевић1, деана б. андрић2, владимир б. шукаловић1, горан м. роглић2, вукић шошкић3 и слађана в. костић-рајачић1 1ихтм-центар за хемију, универзитет у београду, његошева 12, 11000 београд, 2хемијски факултет, универзитет у београду, студентски трг12–16, 11000 београд и 3orgentec gmbh, carl-zeiss-str. 49, 55129 mainz, germany у овом раду је презентована синтеза 14 нових арилпиперазина и одређен је њихов афинитет везивања за д2 допамински рецептор (drd2) тестовима компетиције са [3h]спипероном. по својој хемијској структури ова једињења представљају супституисане бензимидазоле повезане са n-(2-метоксифенил)пиперазинским делом, линкерима различитих дужина. у циљу испитивања лиганд-рецептор интеракција и особина везивног места drd2, урађена је докинг анализа новосинтетисаних једињења и симулација молекулске динамике, користећи кристалну структуру рецептора. резултати добијени у овом раду указују да арилпиперазини високог афинитета остварују интеракције у ортостерном везив________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. investigation of synthesized dopaminergic ligands 933 ном месту и у екстензији ортостерног места везивања drd2 и да стога треба да поседују линкер оптималне дужине, од 5 или 6 метиленских група. 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice volume 83 (2018) 1 subject index 2-aminopyridine, 707 2-arylidene-5-oxopyrazolidin-2-ium-1-ide, 285 3,4-dihydropyrimidin-2(1h)-ones/tihones derivatives, 673 3-acetylcoumarin, 561 4-(dimethylamino)pyridinum acetate, 265 4-nitroaniline, 221 4-(1h-tetrazol-5-yl) benzaldehyde, 1305 8-parametric approximative formula, 1339 ab initio calculations, 439 acetic fermentation, 19 acid black 2, 379 acidity, 1031 adapted strain, 411 adsorption, 237, 391, 395, 503, 1261 advanced electro-oxidation processes, 93 agitation, 911 agpd alloys electrodeposition, 593 alie, 1 alsv, 593 amantadine, 699 americium, 51 ammonium il, 213 analysis of problematic interpretation, 395 anatase, 181, 899 anthelmintics, 401 antibacterial assay, 1209 antibacterial, 515 anticancer activity, 1193 antiinflammatory compounds, 875 antimicrobial activity, 707, 1193, 1305 antimicrobial agents, 271 antimicrobial, 425 antioxidant assays, 1209 antioxidant, 425, 515 antioxidative activity, 31, 723 apoptosis, 1193 apparent molar volume, 1005 aqueous calcium acetate solution, 1005 artificial neural network, 199, 379 astragalus monspessulanus l., 31 asymmetric, 1099 aurone, 1305 azo dye, 93 azo dyes, 221 b3lyp, 1113 barbituric derivatives, 821 bentonites, 391, 395 bde, 1 bibliometric indicators, 1285 bicyclic pyrazolidinone, 285 biginelli reaction, 673 biguanide metal complexes, 271 binary gc–ms data, 317 bioaccessibility, 1315 biological activity, 699, 795, 1229 biomarkers, 1167 biomaterial, 745 biomonitoring, 773 bioproducts, 411 biosorption, 107 bis-benzimidazole, 1099 bisphenol a, 1017 bis-urea, 51 black walnut, 1273 blast furnace slag, 1031 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 2 journal of the serbian chemical society [bmim]pf6, 1071 boron removal, 251 brewing, 19 brucite, 1143 bubble pressure, 199 c use efficiency, 367 c3h6 oxidation, 641 ca, 773 caffeine, 673 cage-fused dienes, 837 calcareous soil, 941 caloric properties, 61 cao/fe3o4 composite, 237 carbon nanotubes, 305 carbon stocks, 785 carboxamides, 401 carboxylic acids, 875 carotenoids, 1223 catalysis, 401 cathode, 1123 cationic dyes, 651 ccsd(t), 1113 ceo2, 745 c-equivalence inputs, 367 c-equivalence outputs, 367 cereal and pseudocereal flour, 317 chalcone, 1305 charge transfer complex, 899 chelates, 809 chelating resin, 1059 chemical equation, 1177 chemical equilibrium, 121 chemical oxygen demand, 611 chemical reaction, 1177 chemical synthesis, 539 chemistry education, 121 chiral amino alcohols, 795 chitosan, 549 cholinesterase test, 1209 chronoamperometry, 733 ck-saft, 331 click reaction, 539 climate, 785 clostridium acetobutylicum, 411 cobalt(ii), 1099 cod, 489 collaboration patterns, 1285 complex, 1099 complexes, 271 composite, 1017 conceptual change, 121 contact time, 911 conversion of propane to aromatics, 473 copolymer, 1017 copper nanoparticles, 449 copper sulfide, 503 covalent and non-covalent functionalization, 167 crystal structure, 157, 809 crystallite size, 1261 crystallization, 1031 cu/tio2 nanocomposite, 863 cultivation, 785 current density, 611 cyclic voltammetry (cv) 733, 995, 1193 cycloaddition reaction, 285 cytotoxic, 515 deactivation of catalyst, 473 decolourization, 93 degradation, 221, 489 demonstration experiment, 1177 density functional theory, 167 density, 61, 331, 1005 deoxy quinocetone, 265 deposit and scale formation, 625 desorption, 941 dew pressure, 199 dft calculations, 837 dft study, 285, 821 dft, 1, 305, 981 dideoxy quinocetone, 265 diels–alder cycloadditions, 837 differential pulse voltammetry, 449 differentiation, 317 diffraction efficiency, 213 diglycolamide (dga), 969 dihedral angle, 953 dilatational elasticity, 847 diode-pumped solid-state, 213 dissociation constant, 875 distillation column, 1047 distribution ratio, 969 dixon ring, 1047 dna binding, 425, 707 dpph assay, 561 dpph test, 723 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ volume 83: subject index 3 dual descriptor, 981 dye photodegradation, 1379 dyes, 107 eco-safe procedure, 673 editorial experience, 1391 eds, 593 electrochemical oxidation, 379 electrolysis, 1351 electrospinning, 885 enol tautomerism, 953 enzymatic hydrolysis, 847 equation of state, 61 equilibrium and thermodynamics study, 237 ethanol, 1099 europium, 51 excess values, 1131 extraction and stability constants, 51 extraction, 1223 fast adsorption, 651 fabaceae, 31 fenton, 93 fiber properties, 463 filtration dynamics, 463 fixed bed reactor, 759 flame retardant, 1363 flavonoids, 31 fluorescence, 1099 folin–ciocalteu method, 723 food matrix, 1315 frizzled (fzd) protein, 575 fruit, 1315 fukui function, 981 gas chromatography, 685 gas–solid mass transfer, 759 gc–ms, 923 genetic algorithm, 379 glassy carbon electrode, 449 glucose, 745 gluten content, 317 glycerol esterification, 39 glycoconjugate, 539 glycolipids, 539 gpdc chart, 1047 granulation, 1031 graphite screen printed electrode, 863 green chemistry, 515 halofuginone, 305 hcl, 969 heavy metals, 237 heterocycles, 1071 heterocyclic compounds, 795 hexafluorophosphate, 1071 high density polyethylene, 923 hmbc correlation, 953 honeybees, 773 honeycomb-like structure, 1351 hybrid nanofibers, 885 hydrochemical and hydromechanical parameters, 625 hydrazone, 1327 hydrogen abstraction reactions, 1113 hydrogen evolution, 1351 ibuprofen, 875 imidazolium il, 213, 1071 immobilized laccase, 549 in vitro digestion, 723 in vitro release, 1243 in vitro, 809 index of sustainability, 367 interfacial dilatational rheology, 847 interpretation, 391 intra-institutional publication performance, 1285 ion exchange capacity, 611 ion exchange, 1059 ionic liquid, 61, 213 isobaric thermal expansibility, 1005 isotherm, 651 isothermal compressibility, 1005 kinetic model, 759 kinetic modelling, 473 kinetic study, 1243 kinetics, 251, 345, 503, 651, 941 la-doped ruo2–tio2/ti coating, 995 land use, 785 langmuir–hinshelwood, 759 lanthanides, 561 levan, 745 lewatit tp 260, 1059 light-off curve, 759 lignan, 31 lignite, 923 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 4 journal of the serbian chemical society lipid profile, 317, 685 liquid chromatography, 875 liquid–liquid extraction, 51, 969 lithium ion batteries, 1123 lithium, 1059 low-cost adsorbent, 651 lser, 139 luminescence, 561 lump, 473 manure, 941 mechanical properties, 75 mechanism, 821 mefenamic acid, 221 magnesium nitrate melt, 1351 magnetic properties, 1327 melamine, 449 mesoporous silica, 39 metal complexes, 157 metal, 773 methyldopa, 863 methylene blue, 503 micronucleus test, 1209 microparticles, 1243 microwave irradiation, 1305 mnco3/na-silicate, 345 modelling, 251, 331 modified adjacency matrix, 1339 molecular docking, 575 molecular interactions, 1131 molecular structure, 1339 morphology, 1261 multidrug resistant, 1193 mwcnt, 1017 nano-particles, 75 nanocomposite, 1363 narrow column, 1047 native and anthropogenic organic substances, 1167 natural and green biodegradable catalyst, 673 natural surfactants, 847 neighbouring group participation, 1297 nmr, 31 nmr spectroscopy, 699 non-associative compounds, 331 non-thermal plasma, 641 n-phosphorylated, 51 nylon net, 549 oil type pollutant, 1167 oil–water interface, 847 olive mill wastewater, 489 ommt, 1363 open circuit voltage, 611 open pyrolysis system, 923 optical properties, 899 organotin(iv) complexes, 425 packed bed, 1047 packing characterization, 1047 palladium catalyst, 641 palladium(ii) complexes, 1229 palladium(ii), 1099 parameterization, 1339 parsimonious model, 1391 particle size, 911 particle swarm optimization, 379 pattern recognition tool, 317 pca, 773 pcm, 305 pc-saft, 331 persulfate, 93 phase equilibria, 199 phenolics, 1315 photocatalysis, 1261 photoelectrodes, 181 photosensitive materials, 213 phthalate amount, 1157 phthalides, 139 physicochemical characterization, 157 p-ionic liquid, 1071 piperazine, 1017 plastic bags, 1157 plastic tubings, 1157 pmma denture base, 75 polarization measurements, 995 pollution, 773 poly(ε-caprolactone), 1379 poly(vinyl alcohol), 885 polycyclic aromatic hydrocarbons (pahs), 549 polyethylenimine–epichlorohydrin resin, 251 polymer carriers, 1243 polyol method, 733 polyol process, 1123 polyphenols, 1223, 1273 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ volume 83: subject index 5 polyphenols quantification, 19 power density, 611 praseodymium (pr), 969 primary school, 1177 problematic, 391 pseudohalides, 1327 pullulan, 745 pur rigid foam, 1363 pyrazole, 795 pyridoxine hydrochloride, 899 quantum chemical calculation, 139 quantum chemical calculations, 899 quantum molecular descriptors, 167 quinocetone, 265 quinones, 1193 radionuclides, 1209 rare earth, 969 rare earths, 561 rate constants, 1113 reaction mechanisms, 167 reactivity indices, 285 reactivity, 1229 recent river sediments, 1167 redlich–kister equation, 1131 reduced graphene oxide, 503 reducing power, 1223 refrigerant, 199 regioselective, 539 renner–teller effect, 439 response surface methodology, 1273 reusability, 345 rdf, 1 rice bran oil, 911 rigid and bulky ligand, 699 riverbank sediments correlation, 1167 saft, 331 salicylaldimine, 809 salinity, 411 saponins, 31 savory snacks, 685 sba-15, 39 sba-16, 39 scanning electronic microscopy (sem), 463, 1351 scavengers, 221 scavenging activity, 1223 schiff base, 157, 561 schiff bases, 809 seawater, 1143 sem, 593, 733 silver(i) complexes, 699 simulation, 1031 so3h, 39 soil genesis, 785 soil remediation, 549 sol–gel, 181, 885 solvatochromism, 139 solvent evaporation method, 1243 solvent extraction, 911 solvent-free conditions, 673 solvolysis, 1297 sonocatalysis, 221 sorption equilibrium, 251 sorptivity, 1143 spectral moments, 1339 spectroscopy, 425 speed of sound, 61 square planar, 809 steel slag, 1143 stereoselective, 539 structure porosity, 463 structure, 1229 students’ learning, 121 subcritical water medium, 489 substituent effect, 139 sugar beet molasses, 411 sulphonamides, 401 sumatriptan, 449 supercritical foaming, 1379 surfaces, 745 survey, 1391 synergetic effect, 923 synthesis, 265, 401, 515, 995 tautomerism, 305 temperature, 911 ternary mixture, 1131 tga–ftir, 923 thermal decomposition, 995 thermal properties, 61 thermodynamic properties, 1131 thermodynamics, 651 thio-ortoester, 1297 thiobencarb, 981 thiosugars, 1297 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 6 journal of the serbian chemical society tio2 nanoparticles, 1379 tosyloxy leaving group, 1297 total polyphenol, 489 toughening mechanisms, 75 transesterification, 345 transition metal, 271 transparent films, 181 triazoles, 821 trifluoroacetyl group, 285 ultrasonic waves, 221 uv–vis spectroscopy, 707 variational approach; c6–, 439 vermicompost, 941 voltammetry, 863 waste biomass, 107 water absorption, 75 water hardness, 625 water solubility, 75 water treatment, 107 water uptake, 611 water–nitrobenzene system, 51 wnt–β-catenin pathway, 575 xps, 593 x-ray crystallography, 707 x-ray structure, 1327 zerumbone, 575 zinc(ii), 1099 zn/hzsm-5 catalyst, 473 znfe2o4, 1261 β-catenin–transcription factor 4 complex, 575 γ-fe2o3 nanoparticles, 305 π-facial selectivity, 837 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {green and efficient synthesis of new $\beta$-amido-aroyl carbonyl derivatives catalyzed by choline chloride/urea as a deep eutectic solvent} j. serb. chem. soc. 86 (6) 547–553 (2021) original scientific paper jscs–5441 547 green and efficient synthesis of new β-amido-aroyl carbonyl derivatives catalyzed by choline chloride/urea as a deep eutectic solvent anita berjis1, behrooz mirza1* and hossein anaraki-ardakani2 1department of chemistry, islamic azad university, karaj branch, karaj, iran and 2department of chemistry, mahshahr branch, islamic azad university, mahshahr, iran (received 6 may 2020, revised 9 march, accepted 11 march 2021) abstract: a green and highly efficient synthesis of some new β-amido-aroyl carbonyl derivatives has been achieved through a one-pot, three-component reaction of dimedone/barbituric acid derivatives, arylglyoxals, and amides in choline chloride/urea as a deep eutectic solvent (des). the use of biodegradable materials, short reaction time and high yields of products introduced this protocol as an efficient environmentally friendly method. the des could be easily recovered and reused about four times with satisfied catalytic activity. keywords: deep eutectic solvents; green chemistry; multi-component reaction; β-amido-aroyl carbonyl compounds. introduction multicomponent reactions (mcrs) have been identified as one of the most efficient methods for the synthesis of heterocyclic compounds.1,2 mcrs provide a powerful synthetic method in which a wide range of raw materials could react through one-pot reactions to produce valuable compounds. in general, most of the atoms in the substrates are also found in the structure of the newly-formed products.3 green chemistry, as an essential and evolving research field, has focused on designing processes that reduce the consumption and production of environmentally harmful substances.4 recently, deep eutectic solvents (des) have been utilized as a new green solvent in research. dess have received more attention due to their exciting properties, such as high thermal stability, high purity, high solubility, no waterreactivity, low cost, and simple preparation methods. therefore, many valuable review articles examine the types and features of des.5 a deep eutectic solvent (des) is defined as a mixture of two or more components with effective hydro * corresponding author. e-mail: b_mirza@azad.ac.ir https://doi.org/10.2298/jsc200506019b ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 548 berjis, mirza and anaraki-ardakani gen-bond interactions that result in the observed melting point being much lower than either of the individual components.6–10 choline chloride (chcl) is one of the most common materials utilized in the synthesis of dess. this quaternary ammonium salt has received more attention due to its special properties, such as low cost, biodegradability, environmental friendliness, and reusability. in addition, choline chloride exhibits high thermal and chemical stability.11 chcl as hydrogen bond acceptor (hba) could produce des in reaction with hydrogen bond donors (hbd), such as acids,12 alcohols,13 amines14 or amides.15 β-acetamido carbonyl compounds have been identified as precursors of 1,3-amino alcohols and γ-lactams.16,17 they also exhibit biological and pharmaceutical activities18 and are used to prepare of antibiotic drugs, such as nikkomycin or neopolyoxins.19 the modified dakin–west condensation of aromatic aldehyde, acetophenone and acetonitrile is highly recommended in the synthesis of β-acetamido carbonyl compounds.20,21 moreover, several efficient methods have been reported for the synthesis of β-acetamido ketones through three-component reactions of acetophenone, an aryl aldehyde, and acetyl chloride in acetonitrile using cocl2,17 montmorillonite k-10 clay22 and heteropoly acids23 as useful catalysts. three-component reactions of 1,3-dicarbonyl compounds, arylglyoxals, and heteroaryl amines/2-aminobenzimidazoles were reported in order to synthesis of 6,7-dihydrobenzofuran-4(5h)-one24/12-aroylbenzimidazo[2,1-b]quinazolin-1(2h)-one25 derivatives. moreover, some reports have been found on the synthesis of β-amido-aryl carbonyl derivatives through three-component reaction of 1,3-dicarbonyl compounds, arylglyoxal, and benzamide.26–28 following previous works, herein an efficient approach is reported for the one-pot synthesis of β-amido-aroyl carbonyl derivatives (4) using three-component reactions of dimedone, barbituric/thiobarbituric acid derivatives (1), arylglyoxals (2) and amides (3), in the presence of choline chloride/urea as a deep eutectic solvent (des), scheme 1. ᴼc scheme 1. synthesis of β-amido-aroyl carbonyl derivatives in des as solvent and catalyst. aryglyoxals 2 were synthesized by the reported reaction of the corresponding acetophenone and seo229 (scheme 2). ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ synthesis of new β-amido-aroyl carbonyl derivatives 549 ar o o h ar o dioxane /h2o 2 ar = 4-nitrophenyl, 4-chlorophenyl, 4-bromophenyl,4-ch3-phenyl seo2 scheme 2. the synthesis of aryglyoxals. experimental all used chemicals and solvents were purchased from fluka (buchs, switzerland) and used without further purification. melting points of the synthesized products were determined with an electrothermal 9100 apparatus. the ir spectra were recorded on a shimadzu ir-470 spectrometer. 1hand 13c-nmr spectra were recorded on bruker drx-250 avance spectrometer in dmso-d6 or cdcl3 with tms as the internal standard. in addition, elemental analyses were performed using a heraeus chn-o-rapid analyzer. general procedure for the synthesis of β-amido-aroyl carbonyl derivatives a mixture of dimedone/barbituric acid derivatives (1 mmol), arylglyoxals (1 mmol), and amides (1 mmol) were added to choline chloride/urea (1:2, 4 ml). the resulting mixture was stirred and heated 90 °c for 145–165 min (scheme 1). after completion of the reaction (tlc, ethyl acetate/n-hexane, 2:1), the reaction mixture was washed with water (10 ml) and the solid residue recrystallized from ethanol to obtain the pure product. the spectral and analytical data for the new compounds are presented in the supplementary material to this paper. results and discussion first, the reaction of dimedone, 4-nitrophenylglyoxal, and benzamide was selected as a model reaction. then the model reaction was performed in various dess based on choline chloride (chcl), and the results are listed in table i. the mixture of choline chloride:urea (1:2) was identified as the best des. next, the reaction was tested at different temperatures to find the most suitable conditions, whereby the best reaction result was observed in the presence of choline chloride:urea (1:2) at 90 °c. performing the reaction at lower temperatures reduced the reaction yields (table i, entries 9–11). furthermore only 20 % yield of the product was detected in the absence of any des (table i, entry 8). in order to prove the efficiency of the method, several cyclic 1,3-diketones (1), various substituted arylglyoxal (2, including electron-donating and electronwithdrawing groups), and different amides (3) were employed in presence of choline chloride:urea (1:2) at 90 °c (table ii). excellent yields of products and short reaction times were found as the advantages of the method. novel synthesized products (4a–h) were characterized by ir, 1h-nmr and 13c-nmr spectral data, as well as elemental analyses. for example, the ir spectrum of 4a showed absorptions at 3417 and 3258 cm–1 for oh and nh groups and at 1710, 1610 cm–1 for carbonyl groups, indi________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 550 berjis, mirza and anaraki-ardakani cating the presence of these functional groups in the proposed structure. the 1h -nmr spectrum of 4a exhibited two singlet signals at δ 0.63 and 0.99 ppm for methyl groups, and the methylene group was observed at δ 2.01 and 2.67 ppm. additionally, a single signal was observed at δ 5.81 ppm for the methine group, and multiplet signals were observed at 7.50–8.27 ppm related to aromatic and nh hydrogens and finally a broad signals at δ 12.43 ppm related to the oh proton. the decoupled 13c-nmr spectrum of 4a showed 19 resonances that are consistent with the proposed structure. the elemental analysis confirmed the amounts of c, h and n in the final product. table i. optimization of reaction in various choline chloride-based dess; reaction conditions: dimedone (0.25 mmol), 4-nitrophenyl glyoxal (0.25 mmol) and benzamide (0.25 mmol) in des (1 ml) entry des t / °c time, min yielda, % 1 choline chloride 90 200 20 2 choline chloride:zncl2 (1:2) 90 200 40 3 choline chloride:ptsa (1:1) 90 200 45 4 choline chloride:malonic acid (1:1) 90 200 55 5 choline chloride:succinic acid (1:1) 90 200 55 6 choline chloride:citric acid (1:1) 90 200 50 7 choline chloride:oxalic acid (1:1) 90 200 55 8 – 100 220 20 9 choline chloride:urea (1:2) 70 150 70 10 choline chloride:urea (1:2) 80 150 82 11 choline chloride:urea (1:2) 90 150 90 12 choline chloride:urea (1:2) 100 150 90 aisolated yield table ii. three-component reaction of dimedone or barbituric acid derivatives, arylglyoxals, and amides in a deep eutectic solvent (des); mp – melting point entry substrate ar r2 time, min yielda, % mp / °c 4a dimedone 4-no2-c6h4 ph 150 90 200 4b dimedone 4-cl-c6h4 ph 160 89 205 4c dimedone 4-br-c6h4 ph 160 85 225 4d dimedone 4-br-c6h4 ch3 155 90 210 4e dimedone 4-no2-c6h4 ch3 145 88 215 4f dimedone 4-br-c6h4 c2h5 150 85 210 4g 1,3-dimethylbarbituric acid 4-ch3-c6h4 ch3 165 80 200–203 4h thiobarbituric acid 4-cl-c6h4 c2h5 165 78 215–217 aisolated yield a plausible mechanism for the synthesis of β-amido-aroyl carbonyl derivatives based on the previously reported30,31 is presented in scheme 3. first, a knoevenagel condensation of enolic form of dimedone 1 with more electrophilic formyl group of the arylglyoxal 2 (path a) in the presence of des is proposed to ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ synthesis of new β-amido-aroyl carbonyl derivatives 551 give intermediate 5.28,29 then michael addition of amide 3 to intermediate 5 forms the β-amido-aroyl carbonyl derivatives products. des activates all carbonyl groups via hydrogen bonding. (scheme 3). o oh n + ohnh nh2 o nh2 nh o hh h o o r 1 cl nh2 r 2 o n + oh cl nh2 nh o h nh nh2 o h o o o r 1 o o o r 1 path a path b 4a-h + -h2o 1 2 3 55 scheme 3. suggested pathway for the formation of compounds 4a–h. finally, the reusability of the catalyst for the synthesis of n-[1-(2-hydroxy -4,4-dimethyl-6-oxo-cyclohex-1-enyl)-2-(4-nitro-phenyl)-2-oxo-ethyl]-benzamide (4a) was studied (fig. 1). after completion of the reaction, the reaction mixture was washed with water and the solid residue recrystallized to obtain the pure product. the des was recovered from the aqueous phase by evaporation at 80 °c under vacuum and prepared for the next run. it was applied for four runs without noticeable decrease in the catalyst activity (fig. 1). fig. 1. reusability of the des. conclusions in conclusion, a simple and efficient methodology for the synthesis of β -amido-aroyl carbonyl derivatives was successfully developed by the one-pot three-component reaction of dimedone/barbituric acid derivatives, arylglyoxals, and amides in the presence of choline chloride/urea as a green and eco-friendly ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 552 berjis, mirza and anaraki-ardakani catalyst and solvent. several noticeable advantages such as simplicity of operation, safe method, high yields of products, and biodegradable, non-toxic, inexpensive materials were found. also, the deep eutectic solvent (des) could be easily recycled and reused in at least four consecutive runs without significant loss of catalytic activity. supplementary material additional data are available electronically at the pages of journal website: https:// //www.shd-pub.org.rs/index.php/jscs/index, or from the corresponding author on request. acknowledgement. we are thankful to the islamic azad university, karaj branch, for the support of this work. и з в о д зелена и ефикасна синтеза нових β-амидо-ароил карбонилних деривата у еутектичној смеши холин-хлорид/уреа anita berjis1, behrooz mirza1 и hossein anaraki-ardakani2 1department of chemistry, faculty of science, islamic azad university, karaj branch, alborz, iran и 2department of chemistry, mahshahr branch, islamic azad university, mahshahr, iran зелена и веома ефикасна синтеза нових β-амидо-ароил карбонилних деривата постигнута је у једном реакционом кораку у тро-компонентној реакционој смеши која садржи димедон/деривате барбитурне киселине, арил-глиоксале и амиде у смеши холин хлорида/ /урее као дубоком еутектичком растварачу (deep eutectic solvent, des). због употреба биодеградабилних материjала, кратког реакционог времена и високог приноса производа овај поступак припада ефикасним и еколошки прихватљивим методама синтезе. еутектички растварач се лако може рециклирати и поново користити четири пута без губитка каталитичке активности. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {biosynthesis, characterisation and therapeutic applications of plant mediated silver nanoparticles} j. serb. chem. soc. 83 (5) 515–538 (2018) udc 542.913.000.57+577.1:581.6.001.5: jscs–5093 546.57+620.3:615.279:576+615.3 survey 515 survey biosynthesis, characterization and therapeutic applications of plant-mediated silver nanoparticles andreia corciova1* and bianca ivanescu2 1department of drugs analysis, “grigore t. popa” university of medicine and pharmacy, 16 universitatii street, 700115, iasi, romania and 2department of pharmaceutical botany, “grigore t. popa” university of medicine and pharmacy, 16 universitatii street, 700115, iasi, romania (received 31 july, revised 20 december 2017, accepted 22 january 2018) abstract: nanotechnology is one of the most studied domains, and nanoparticle synthesis, especially of silver nanoparticles, has gained special importance due to their properties, biocompatibility and applications. today, the processes of nanoparticles synthesis tend toward the development of inexpensive, simple, non-toxic and environmentally friendly methods. thus, the use of plants in the synthesis of silver nanoparticles has attracted considerable interest because biomolecules can act as both reducing and stabilizing agents. this survey aims at discussing the conditions for obtaining silver nanoparticles using plants and their characterization by several methods, such as ftir and uv–vis spectroscopy, x-ray diffraction, and scanning and transmission electron microscopy. in addition, it examines some of the most common biological uses of silver nanoparticles: antibacterial, antioxidant and cytotoxic. keywords: green chemistry; synthesis; antibacterial; antioxidant; cytotoxic. contents 1. introduction 2. synthesis of silver nanoparticles 2.1. conditions for agnps synthesis 2.1.1. concentration and amount of plant extract 2.1.2. concentration of silver salt 2.1.3. ph 2.1.4. temperature 2.1.5. reaction time 2.2. methods for the analysis of agnps * corresponding author. e-mail: acorciova@yahoo.com https://doi.org/10.2298/jsc170731021c 516 corciova and ivanescu 2.2.1. visual inspection 2.2.2. uv-vis spectroscopy 2.2.3. dynamic light scattering and determination of zeta potential 2.2.4. ftir spectroscopy 2.2.5. x-ray diffraction (xrd) 2.2.6. scanning and transmission electron microscopy (sem and tem) 2.2.7. photoluminescence spectroscopy 3. therapeutic applications of agnps 3.1. antimicrobial activity 3.2. antioxidant activity 3.3. cytotoxic activity 4. conclusions 1. introduction nanotechnology, which refers to the manipulation of particles with sizes smaller than 100 nm, named nanoparticles, is a developing domain. special attention is attributed to zero-valent metal nanoparticles that can serve as the basis for various physical and biological systems. metallic nanoparticles have numerous biological applications and can be used as therapeutic and diagnostic agents, as carriers for targeted drug delivery, for biomolecules detection, and they can be employed in other fields too, such as food, agricultural or treatment of waste.1 in this survey, the focus is on the silver nanoparticles (agnps), considered among the most potent antimicrobial agents, as evidenced by the numerous studies that have explored their antibacterial1,2 and antifungal activity3 in the past decade. agnps can be prepared and stabilized by physical and chemical methods. besides the fact that these methods raise problems of toxicity and have a relatively inadequate cost–efficiency ratio, many experimental conditions affect their size, morphology, stability and properties.1,4 preparation of nanoparticles involves the reduction of metal ions by a reducing agent and their stabilization at the zero valence state through capture by a stabilizing agent. this process requires the use of chemicals that pose a potential risk to the environment and influence the size and shape of the particles.3 therefore, the current trend is the biological synthesis of agnps through the use of various biological agents, such as yeasts, enzymes, bacteria, polysaccharides, algae, oligosaccharides, fungi, dna and human cell lines.1,4–8 perhaps one of the most accessible and environmentally safe methods compared to others methods for nanoparticles production consists in the use of plant extracts and whole plants. the plants are easily accessible, the process is less laborious and shorter, the synthesis can be realized on a large scale and the method is environmentally friendly.3,9 biosynthesis, characterisation and therapeutic applications of agnps 517 thus, the paper presents an analysis of the conditions for obtaining agnps, with plants, followed by a presentation of the methods by which they could be characterized and their biological applications. 2. synthesis of silver nanoparticles an extract of unicellular green algae chlorella vulgaris was used for the first time to synthesize single-crystalline agnps at room temperature. the components that participated in the synthesis were proteins that had two functions: reduction of ag+ (via the carboxyl groups in aspartate or glutamate residues and hydroxyl groups in tyrosine residues) and control of the shape of nanoparticles (anisotropic growth of silver nanocrystals into nanoplates). this synthesis process gave a good yield (> 55 %) of low polydispersity agnps.10 obtaining agnps using plants was first reported by gardea-torresdey et al.11 they demonstrated that silver ions ag+ are reduced to ag in a solid agar medium and then adsorbed from alfalfa roots and transferred into shoots, where the silver atoms are rearranged to form nanoparticles that could form further various other arrangements. the majority of plants used for the synthesis of agnps belong to the following families: acanthaceae, amaranthaceae, apocynaceae, asteraceae, burseraceae, dioscoreaceae, euphorbiaceae, fabaceae, lamiaceae, moraceae, myrtaceae, poaceae, ranunculaceae, rutaceae, solanaceae and asphodelaceae.1 among the plants used for the synthesis of agnps, the most frequent were: punica granatum, cydonia oblonga, castanea sativa, ficus carica, juglans cinerea, morus nigra, morus alba,12 piper nigrum,13,14 morinda citrifolia,15 calendula officinalis,16 glycyrrhiza glabra,17 artemisia annua,18 sida acuta,18 emblica officinalis, terminalia catappa, eucalyptus hybrida,19 choerospondias axillaris, nyctanthes arbortristis, moringa oleifera, zanthoxylum armatum, areca catechu,20 argemone mexicana and ocimum tenuiflorum.21 biomolecules that participate in reduction and capping of the nanoparticles are distributed in the leaves, stems, fruit, seeds and roots, and belong to the following classes of compounds: alcoholic compounds, alkaloids, amino acids, phenols, flavones, enzymes, polysaccharides, proteins, terpenoids, some vitamins, chlorophyll and other metabolites.22 in the phytosynthesis of agnps, primary and secondary metabolites from plants act as both reducing and capping agents. primary metabolites, such as carbohydrates, proteins, peptides, amino acids and vitamins, are always present in plants and are involved in the reduction and stabilization of metallic silver in nanoparticles.23 li et al.24 showed that amine groups in proteins from the aqueous extract of capsicum anuum anuum played an important role in the formation of agnps. furthermore, the carbonyl group of amino acids and proteins has the 518 corciova and ivanescu ability to bind metal ions, to cap nanoparticles and to prevent agglomeration, thereby stabilizing the medium.25 some secondary metabolites with biological activities, such as terpenoids, alkaloids, flavonoids, phenolic acids and other polyphenols, have been reported to act as either reducing or stabilizing agents in the formation of agnps. sathishkumaret et al.26 reported that the terpenoids (linalool, eugenol, methyl chavicol) present in cinnamomum zeylanicum bark are responsible for the reduction of agnps. the alcohols, ketones, aldehydes, and lactones of terpenoids are involved in both the reduction and capping of silver nanoparticles. in adequate concentration, terpenoids could be adsorbed on the surface of agnps, possibly by interaction through π-electrons or carbonyl groups.27 secondary metabolites are active principles from plants and their inclusion in silver nanoparticles could potentially enhance their biological activities or help target the compounds. for example, numerous studies focused on the synthesis of agnps with extracts from artemisia species28,29 and evaluation of their antibacterial, antifungal, antioxidant and anticancer effects.18,30,31 artemisia is a widely spread genus including numerous medicinal and aromatic plants known to be a rich source of bioactive compounds (sesquiterpene lactones, flavonoids, phenolic acids, coumarins) with antimicrobial, anticancer and anti-inflammatory activities.32 in addition to having intrinsic therapeutic value, these compounds are also involved in the synthesis of nanoparticles. various functional groups of flavonoids can actively chelate and reduce metal ions into nanoparticles.33 zargar et al.34 showed that the hydroxyl groups in the structure of flavonoids present in an aqueous extract of vitex negundo are responsible for the bioreduction of metal ions during the synthesis of agnps. moreover, polyphenols and caffeine present in tea or coffee extracts play a crucial role in the synthesis of agnps.35 in the synthesis of agnps in a lycopersicon esculentum leaf extract, the hydroxyl and carbonyl groups of phenols reduced the silver ions to silver, while the amines present in the extract capped the agnps.36 polyols in a papaya fruit extract are the compounds implicated in the bioreduction of silver ions and formation of agnps.37 different antioxidants with varied structures present in ananas comosus juice act synergistically in reducing silver ions.38 for the extraction of biomolecules, solvents such as water,12,16 ethanol,39,40 70 % ethanol,41 etc. are used. among these, water is preferred, because it is the most polar solvent, easy to use for active principles extraction and further is a non-toxic solvent.42 the synthesis of nanoparticles has several stages: preparation of the plant extract and silver salt solution; preparation of agnps by mixing those two solutions in different proportions, at certain ph and temperature values and for different times. in the presence biosynthesis, characterisation and therapeutic applications of agnps 519 of plant compounds, ag+ is reduced to ag, and then oligomeric clusters are formed, which lead to silver nanoparticles; agnps purification is realized by centrifugation to remove the unreacted plant extract, followed by resuspension in distilled water and again centrifugation, repeatedly, to remove unwanted substances; confirmation of agnps formation through different analytical methods. 2.1. conditions for agnps synthesis for the agnps synthesis, various parameters need to be taken into account in order to achieve the maximum yield, nanoparticles with a certain shape and size and to achieve stability. parameters that require investigation: concentration and amount of plant extract; concentration of silver salt; ph; temperature; reaction time. 2.1.1. concentration and amount of plant extract to obtain agnps from aegle marmelos leaves, rao and paria43 used an initial 5 % concentration extract that was diluted 4–50 times. as or the 20and 50-fold dilutions, unreacted agno3 was observed in the medium, a dilution of 17 times (0.3 %) was considered appropriate for the production of agnps. concerning the concentration of agno3, concentrations of 1–4 mm were tested. a concentration of 1 mm was considered appropriate, since an increase in the particle size was observed when the concentration of agno3 was further increased. this situation could be explained on the one hand by the small concentration of active principles in the extract as capping agent, insufficient to prevent agglomeration. on the other hand, a high concentration of agno3 decreases the rate of reaction and nuclei formation. another example is the generation of agnps with tealeaf extract of different concentrations: 1, 5, 10, 25, 50 and 100 % and 10 mm solution agno3, by stirring at 700 rpm, at 25 °c for 120 min. it was observed that the yield of nanoparticles production was above 94 % in all cases. in the following studies, a 5 % plant extract concentration was used, as the zeta potential was found to increase with increasing extract concentration. example: from –20.7 mv (1 % extract), – 21.3 mv (5 % extract) to –12 mv (50 % extract) and –11.3 mv (100 % extract). with the increase of the zeta potential, agnps dispersion becomes unstable due to particles precipitation from the dispersion.44 520 corciova and ivanescu halawani42 showed that at a higher concentration of the plant extract, the biocompounds act both as reducing agents and as surface coatings of nanoparticles to protect them from aggregation. moreover, utilization of different amounts of extract can control certain properties of the resulting nanoparticles. in the case of agnps synthesis from malus domestica extract, by using a different ratio extract, agno3 (1–5 ml extract: 50 ml of 0.1 m agno3), it was observed that the formation of uniformly dispersed agnps was significantly influenced by this ratio. peak absorption increased with increasing amount of extract, which showed that as the amount of extract added was increased, the possibility of reduction of silver nitrate increased, which will lead to the formation of well defined and stable silver nanoparticles.45 the same was observed for agnps from artemisia absinthium, i.e., the maximum absorbance increased with increasing extract concentration (from 10 to 50 %) and decreased with decreasing of extract concentration (from 60 to 10 %). augmentation of the extract concentration raises the average hydrodynamic sizes of agnps.28 in the case of pinus eldarica bark, on raising the amount of extract, the absorbance increased, and production of nanoparticles grew, but no directly proportionally, and the agnps size decreased.46 the use of equal volumes of plant extract and silver salt led to comparatively smaller size and more stable nanoparticles.28 in some cases, stabilizing agents such as sodium dodecyl sulfate (sds) and sodium citrate were used. these were used to prepare agnps from artemisia nilagirica leaf extract. the authors/researchers also used silver nitrate as the precursor metal and hydrazine hydrate as a reducing agent, thereby obtaining particles with a diameter of 70–90 nm.29 2.1.2. concentration of silver salt for the preparation of agnps, silver acetate41 or silver nitrate may be used, the most common being silver nitrate (agno3), of various concentrations (0.1– –10 mm). for garcinia mangostana leaf extract, the optimal concentration to obtain agnps was found to be 1 mm.47 in the case of agnps obtained from angelica pubescens, the most effective conditions were: equal volumes of the two solutions, 5 % extract (obtained at 100 °c, for 30 min) and 5 mm agno3, temperature 80 °c and a reaction time of 50 min. the average crystallite size was 12.48 nm.48 generally, by augmenting the concentration of agno3, the production of agnps increases46 and the color of the solution intensifies, because aggregation of silver ions occurs and larger nanoparticles are obtained.14 in the case of agnps from solanum trilobatum using 1 mm agno3, monodisperse nanoparticles were formed without aggregation. in the 2–4 mm agno3 biosynthesis, characterisation and therapeutic applications of agnps 521 concentration range a band at 460 nm was observed with aggregation and at 5 mm agno3 concentration a broad band at 500 nm was observed, indicating that the particles were polydisperse. the plasmon resonance band broadened with increasing agno3 concentration.49 2.1.3. ph the ph plays an important role in the synthesis of agnps, particularly influencing the size and morphology of the nanoparticles. to demonstrate the effect of ph on the formation of agnps, iravani and zolfaghari46 used pinus eldarica bark extract (20 %, boiled for 15 min), agno3 of different concentrations and phosphate buffer with a ph in the range of 3–11. on increasing the ph, the reduction intensified, the absorbance was augmented and the production and stability of agnps enhanced. more4over, the ph influences the size of agnps, i.e., at low ph values, large agnps were obtained while small-sized agnps were obtained at high ph values. the same conclusion was reached in the case of agnps obtained from garcinia mangostana leaves extract. acid ph values suppress the formation of agnps, with aggregation of nanoparticles and formation of large nanoparticles (ph 4), while alkaline ph (ph 8) favors the formation of agnps, with nucleation and formation of small highly dispersed agnps. the optimal ph was found to be neutral.47 another example of the influence of ph is given by acalypha indica fresh leaves extract, at 2–13 ph range. in this case, at acid ph values, agnps were not obtained while at alkaline ph values, a rapid color formation was observed, which proves the formation of agnps with the absorption peak shifted to 500 nm, but at ph 13, agglomeration was immediate. at neutral ph the reaction started after the addition of agno3 and agnps formation was complete within 30 min of incubation.50 in the case of agnps production using crataegus douglasii fruit, for a period of 24 h, the absorption wavelength of the sol decreased from ph 2 to ph 6. at ph 2, agnps were not generated and flocculation observed, while in alkaline media an important decrease in the flocculation parameter occurred, which may have been the result of aggregation. the stability of the nanoparticles in the presence of additives depends on the ph of the solution because hydroxide ions may change the surface charge of the nanoparticles. cluster distribution stability was improved in the alkaline ph range due to the complete charging of the cluster, thus maximizing the electrostatic/electrosteric repulsive interactions. at ph values above 8, ag+ partially hydrolyzes in solution to form bioorganic–ag(oh)x or bioorganic–ag(nh3)2 complexes on the surface of the particles and agoh/ag2o colloid in the medium. the degree of hydrolysis and the formation of colloids are enhanced by an increase in the solution ph.51,52 522 corciova and ivanescu for agnps from solanum trilobatum, ph 5.8 suppressed the formation of the nanoparticles due to the limited availability of functional groups of the compounds in the extract. at ph 8.8, a broad absorption band peaking at 480 nm was obtained, while at ph 7.8, a narrow peak positioned at 440 nm was observed, with the maximum yield of agnps.49 2.1.4. temperature the reaction temperature influences the production and size of agnps. in order to obtain agnps, various temperatures could be used. room temperature was found to be optimal because small and spherical agnps are formed, having a single surface plasmon band, at lower wavelengths.1 thus, kumar et al.53 demonstrated the production of agnps with 35 ± 5 nm mean particle size and irregular spherical shape using a 10 % annona squamosa extract and 1 mm agno3 at room temperature for 4 h. ndikau et al.54 synthesized agnps from 0.001 m citrullus lanatus fruit peel extract, and 250 g l–1 agno3 in 4:5 ratios at 80 °c and ph 10. the agnps were stable, spherical, with diameters of 17.96±0.16 nm. das et al.55 prepared agnps from sesbania grandiflora leaf extract and 1.0 mm agno3 solution by incubation at 37 °c in the dark. the agnps were stable for 6 months at room temperature, had a spherical shape and a size in the 10–25 nm range. preparation of agnps from solanum trilobatum by varying the temperature led to the following conclusions: at 20 °c no plasmon resonance band was observed, at 35, 45 and 70 °c characteristic peaks of the formation of agnps were observed, and the maximum yield was obtained at the highest temperature. the plasmon resonance peaks of the formed agnps shifted significantly toward the blue region from 460 nm to 440 nm, suggesting that the same form of nanoparticles strongly influences the plasmon resonance bands at higher temperatures.49 at high temperatures (25–150 °c), a rise in agnps production, an increase of absorbance and a reduction of nanoparticles size, which resulted in an increase in the sharpness of their plasmon resonance band, were observed.46 in the case of agnps from garcinia mangostana leaf extract, by varying the temperature (37– –90 °c), the production of nanoparticles was enhanced with increasing temperature. the agnps initial size was reduced because of the decrease in the aggregation of the growing nanoparticles, while at over 75 °c, the crystal grew around the nucleus that led to decrease in the absorbance.47 2.1.5. reaction time reaction time influences the production and stability of agnps. thus, by testing some different reaction times (0–70 min), it was found that the production of agnps increased with increasing reaction time. however, agnps instability biosynthesis, characterisation and therapeutic applications of agnps 523 should be taken into account, because after an optimal period of 60 min, the agnps agglomerate and the particle size increases.47 logeswari et al.56 obtained agnps using ethanol extracts of solanum trilobatum, syzygium cumini, centella asiatica and citrus sinensis, 1 mm agno3, ammonium solution, at 37 °c using reaction times of 24–48 h. the silver ions were reduced to agnps, which could be seen from the change in the color of the solution from yellow to dark brown. the agnps had irregular shapes and the nanoparticle size was between 41–53 nm. in addition, vanaja et al.49 studied the generation of agnps from solanum trilobatum extract and observed, at 20 min, the appearance of a sharp, narrow peak at 420 nm indicating the formation of an isotropic shape and a uniform size of the nanoparticles. after 20 min, the peak had shifted to 440 nm. on increasing the reaction time, the synthesis of nanoparticles was enhanced and the maximum yield was confirmed by the absorption maximum. the reaction was completed in 4 h and was visually observed through the occurrence of a precipitate at the bottom of the vial. in case of agnps obtained from ocimum sanctum, the bioreduction of silver ions into nanoparticles was observed to begin at 3 min and reached an optimum level within 30 min (optimum absorption at 430 nm). the absorption band measured at 60, 90 and 120 min intervals had not achieved better values. the widening of the absorption peak with increased time indicated increased polydispersity of the nanoparticles.57 besides using the stirring method, ultrasonic cavitation technique could be used on a mixture of extract and silver salt to obtain agnps. thus, shimpi et al.3 prepared agnps from an extract of alstonia scholaris leaves (25 %) obtained by the same method and 0.01m agno3 for 40 min at an on–off pulse rate of 5 s at an intensity of 20 khz. the ultrasonic cavitation technique is based on cavitation, which consists in the formation, growth and collapse of bubbles due to the transmission of acoustic vibrations through a medium. according to shimpi et al.57 the silver ions diffuse into the cavitation bubbles and concentrate at the interface where they react with high-energy species, with the start of nucleation. during cavitation, the bubbles grow and when the maximum size is reached they collapse by implosion, and agnps are created. these nanoparticles have a large number of dangling bonds on their surface. in order to gain surface stability, it is necessary to establish connections, which leads to the development of nuclei. when growth is terminated prematurely, agnps stabilization occurs through the formation of bonds with the stabilizers present in the plant extract, which prevents aggregation of the nanoparticles. some examples of plants that have been used to obtain agnps are given in table i, together with the conditions used to obtain agnps. 524 corciova and ivanescu table i. plants and conditions used for agnps synthesis plant vegetal material extract concentration (in water) agno3 concentration method temperature°c, ph time of reaction agnps size (nm), shape distribution reference peumus boldus leaves 5 %, soxhlet extraction 2 mm stirring ambient 3 h 18 nm, spherical 58 heritiera fomes, sonneratia apetala leaves barks 10 %, boiled for 10 min 1 mm stirring, under direct sun light ambient 12 h 50, 400 nm 20–30, 70–100 nm 59 urtica dioica leaves 20 %, boiled for 15 min 10-3m incubati on in the dark 40 °c 60 min 20–30 nm, crystallized in facecentered cubic 60 acalypha indica leaves 10 %, heated at 65 °c for 5 min 1 mm incubati on in the dark, under static conditio ns 37 ºc – – 50 hedera helix leaves 20 %, boiled for 20 min 1 mm stirring, under sun light ambient 15 min more or less spherical, 10–30 ± 2 nm in diameter 61 ziziphus spinachristi leaves 25 %, boiled for 10 min 1 mm shaking, under dark conditio ns ambient 30 min 21.5–59.67 nm, hexagonal shape 42 sambucus nigra fruits 3 %, stirred for 1 h 1 mm mixed ambient, ph 8 10 min 8–33 nm spherical 62 artemisia annua, sida acuta leaves 10 %, boiled 15 min 0.1 m manual shaking, under sunlight ambient 10 min – 18 ocimum sanctum leaves 10 %, boiled for 10 min 10-2 m kept under direct sunlight ambient 30 min 7–11 nm, circular shape 57 ocimum tenuiflorum leaves 1.5 %, boiled 1 mm stirring 37 °c, in presence of ammonium solution 24–48 h 26 nm (xrd)a 63 biosynthesis, characterisation and therapeutic applications of agnps 525 table i. continued plant vegetal material extract concentration (in water) agno3 concentration method temperature°c, ph time of reaction agnps size (nm), shape distribution reference solanum trilobatum leaves 1.5 %, boiled 1 mm stirring 37 °c, in presence of ammonium solution 24–48 h 20–22.3 nm 63 syzygium cumini leaves 1.5 %, boiled 1 mm stirring 37 °c, in presence of ammonium solution 24–48 h 26–26.5 nm 63 centella asiatica leaves 1.5 %, boiled 1 mm stirring 37 °c, in presence of ammonium solution 24–48 h 24–28.4 nm 63 citrus sinensis peel 1.5 %, boiled 1 mm stirring 37 °c, in presence of ammonium solution 24–48 h 59–65 nm 63 crataegus douglasii fruit 10 %, boiled 15 min 0.01 m shaking 28 °c 24 h 29.28 nm, nearly spherical shape 51 carica papaya peel 10 %, refluxed for 1 h, at 80 °c 1 mm stirring ambient 1 h 3–5 nm, polydispersed spherical in shape 64 matricaria chamomilla plant 25 % boiled for 5 min 20 mm kept in a dark place – – 60–65 nm, different forms 65 ax-ray diffraction 2.2. methods for the analysis of agnps among the methods used to characterize the agnps encountered in the literature, the following will be mentioned: visual inspection; uv-vis spectroscopy; dynamic light scattering and determination of zeta potential; ftir spectroscopy; x-ray diffraction (xrd) scanning and transmission electron microscopy (sem and tem); photoluminescence spectroscopy. 526 corciova and ivanescu 2.2.1. visual inspection first, the demonstration of agnps formation is made visually by the color change from a colorless,47 pale yellow,66,67 yellow,49,67 yellow–brown46 or yellowish–brown45,67 solution to a brown,44,47,49 or dark brown sol45 due to excitation of the surface plasmon resonance in the agnps.46,47,66,67 the color is due to surface plasmon resonance that occurs due to the presence of collective oscillation of conducting free electrons induced by an electromagnetic field that interact,17 and electrons gathering around the surface of the metal particles.20 the frequency and width of the surface plasmon resonance depends on the size and shape of the metal nanoparticles, the dielectric constant of the metal and the environment.19 2.2.2. uv–vis spectroscopy uv–vis spectroscopy is used to observe the change in agnps size, based on the wavelength of the surface plasmon resonance band.49 allafchian et al.66 synthesized 25-nm spherical agnps using a phlomis sp. extract rich in flavonoids, phenylpropanoids and other phenolic compounds and a 0.01 m agno3 solution at room temperature. the uv–vis spectra showed the a peak corresponding to the surface plasmon resonance agnps at 440 nm, after 5 min of reaction and the absorbance increased up to 30 min of reaction. for agnps obtained from pinus eldarica bark, monitoring was performed at a wavelength of 430 nm, observing an increase in the intensity of the absorbance over time, which demonstrates the increase in the concentration of agnps. a broad peak, with maximum absorbance at 438 nm showed that agnps from garcinia mangostana leaf extract were polydisperse.47 the uv–vis spectra obtained from a 5 % extract of the fresh fruits of malus domestica and 0.001 m agno3 proved the production of nanoparticles with different sizes: a large absorption peak was registered after a reaction time of 1 h and sharp peaks after 6 and 24 h, with the wavelength of the absorption peak shifting to higher values with increasing reaction time.45 in the case of the agnps synthesized using olive leaves extract, it was observed that as the concentration of the extract increases, the absorption peak becomes more defined and sharper, and the wavelength shifts to blue, 458–441 nm. this behavior indicates a reduction of the average particle diameter and a spherical and homogeneous distribution of agnps.67 on the other hand, agnps from piper nigrum leaves extract, with increasing concentration of agno3 solution (1–5 mm) showed a blue shift of the absorption maximum, indicating an increase in the average diameter of nanoparticles in the range of 5–50 nm.14 the blue shift of the absorption peak located at 420 nm could be explained by the small particle size ≤ 25 nm.68,69 biosynthesis, characterisation and therapeutic applications of agnps 527 2.2.3. dynamic light scattering and determination of the zeta potential the particle size is determined by dynamic light scattering, a technique that characterizes the size of colloidal dispersion, by studying the illumination of a particle or molecule suspension in brownian motion with a laser beam. fluctuations in the intensity of scattered light in dependence on time are analyzed with the help of an autocorrelator that determines the autocorrelation depending on the signal. the zeta potential describes the degree of stability of a colloidal dispersion of nanoparticles.66 the zeta potential value may be positive or negative; a negative value may occur due to the possible incorporation of bioactive components present in the extract.45,70–72 in addition, a negative potential suggests that the surface of the nanoparticles is negatively charged, which causes a strong rejection force between the particles, thus preventing their aggregation.73 a strong negative potential may be due to a shielding effect determined by the bioactive compounds in plants.74 gengan et al.74 are of the opinion that a zeta potential higher than 30 mv or lower than –30 mv demonstrates a stable system. in the case of malus domestica, the agnps obtained were polydisperse mixtures with sizes in the range of 50–300 nm, and a mean size of about 150 nm in diameter. the zeta potential of the synthesized agnps was determined in water as dispersant and was –65.07 mv; the high value confirms a particle repulsion phenomenon and an increase in the stability of the formulation.45 2.2.4. ftir spectroscopy ftir is a useful method in the analysis of the chemical composition of the agnps surface and the local molecular environment of nanoparticle incorporation agents. thus, in order to demonstrate the production of agnps by ftir analysis, the ftir spectra of the extract and agnps are compared to observe the common peaks and the changes that had occurred. the ftir spectrum of agnps from malus domestica had a broad band at 3450 cm–1, attributed to oh stretching vibrations due to the presence of hydroxyl groups in the reducing agent, and intense peaks at 1379 and 1625 cm–1 corresponding to c–n stretching vibrations and to amide i band of proteins from the apple fruit extract. this confirms that malus domestica extracts have the ability to reduce and stabilize agnps.45 ftir spectra of agnps and calendula officinalis extract showed common but displaced bands: the bands at 3350 (extract) and 3323 cm–1 (agnps) are assigned to the oh groups, the bands at 2928 (extract) and 2943 cm–1 (agnps) are assigned to the alkyl and ch groups, the bands at 1622 (extract) and 1640 cm–1 (agnps) are assigned to type i amides, 1237 (extract) and 1217 cm–1 (agnps) are assigned to type iii amides. in addition, in agnps spectra, there are 528 corciova and ivanescu bands not found in the extract spectrum: at 1406, 1021 and 840 cm–1, bands specific for the presence of silver.40 shifted peaks were also observed in the ftir spectra of agnps synthesized from the tea leaf extract: from 3420 to 3371 cm–1 due to n–h stretching amides, from 2931 to 2925 cm–1 due to c–h stretching alkanes, from 1383 to 1371 cm–1 for hydroxyl groups and from 1051 to 1044 cm–1 for c-stretching from ether groups. in the agnps, new peaks were observed at 1695, 1452, 1241, and 926 cm–1 related to alkene groups (c–c stretching), tertiary ammonium ions, polyphenols, aliphatic amines (c–n stretching vibrations) and alkene groups (c–h stretching).44 in the case of agnps from garcinia mangostana leaf extract, in addition to the stretching absorption bands corresponding to c–c (aromatic ring), c–o–c (ethers) and c–o (c–oh), at 1160 cm–1, a band corresponding to c–o from the –oh group of hydroxyflavones and hydroxyxanthones was observed. this band was not present in the spectrum of the agnps because these compounds were responsible for reducing ag+, thereby becoming oxidized to an unsaturated carbonyl group, resulting in a broad peak at 1660 cm–1.47 the same explanation is given for the agnps from morinda pubescens, i.e., the band at 1226 cm–1 most likely corresponding to c–o group of hydroxyflavones and catechins disappeared after the bioreduction, and a peak at 1650 cm–1 was observed.75 the presence of secondary metabolites acting as reducing agents in the synthesis of agnps from solanum nigrum was demonstrated by a peak at 2357 cm–1, attributed to hydrogen-bonded oh stretching.69 the ftir spectra of agnps and glycyrrhiza glabra root extract demonstrate that the potential biomolecules responsible for the synthesis and stabilization of agnps are flavonoids, terpenoids, thiamine and reducing sugars.17 2.2.5. x-ray diffraction (xrd) the mean size of the crystallized particles of agnps can be calculated using the debye–scherrer formula.66,76 in order to apply xrd testing, the samples were prepared as follows: the dispersion resulting in the process of nanoparticles production was centrifuged at 10000 rpm for 30 min. the agnps residue was washed and resuspended in absolute ethanol that was evaporated to dryness at 80 °c to obtain agnps powder, which was used for the x-ray diffraction measurements.45 the xrd spectrum of agnps from malus domestica showed four distinct peaks at degrees (2θ) 38.28, 44.330, 64.33 and 77.53°, and from solanum trilobatum at 38.13, 46.2, 64.44 and 77.36°. these values correspond to the (111), (200), (220) and (311) planes of face-centered-cubic (fcc) silver, with a network parameter of 4.08 ǻ, which is consistent with the joint committee pow biosynthesis, characterisation and therapeutic applications of agnps 529 der diffraction standards (jcpds) card no-087-0720.44,45,49 another peak was also observed at 2θ 31.9°, possibly due to ag2o.44 the xrd spectrum of agnps synthesized from solanum nigrum showed 6 peaks at 2θ 10.00, 11.51, 12.00, 27.75, 32.15 and 45.94°, corresponding to six diffraction facets of silver.69 in the case of agnps obtained from the mussaenda erythrophylla leaf extract, the pattern showed characteristic bragg reflections. the xrd spectrum shows only one clear signal at 2θ 38.50°, corresponding to (111) plane of face -centered cubic silver, demonstrating that the synthesized nanoparticles were highly pure.70 2.2.6. scanning and transmission electron microscopy (sem and tem) sem was used to determine the surface morphology and topography of the synthesized silver nanoparticles. samples are prepared by placing a drop from the colloidal dispersion onto a support and drying at room temperature. the sem images for agnps obtained from malus domestica show flower structures45 and those from solanum trilobatum have spherical and monodisperse forms, well distributed with aggregation in the range 50–70 nm.49 agnps synthesized using phlomis sp. extract were spherical, well dispersed, and the size ranged from 19 to 30 nm, with an average size of 25 nm, confirmed by sem and tem.66 sem and tem analyzes of agnps obtained from musa balbisiana, azadirachta indica and ocimum tenuiflorum demonstrated the production of nanoparticles of different shapes due to the different availability, quantity and nature of the capture agents present in the extracts. the nanoparticles were predominantly spherical but also in the form of triangles, pentagons and hexagons with dimensions up to 200 nm.77 a spherical form of nanoparticles was observed for the agnps obtained from garcinia mangostana47 and tea leaf extract44. in the case of agnps obtained from alstonia scholaris leaf extract, tem analysis demonstrated the production of spherical and triangular pyramidal shaped nanoparticles with minimal aggregation and diameters of 15–38 nm.3 for the agnps synthesized from olive extract leaves, the tem images showed that the average particle size was influenced by the concentration of the plant extract: as the concentration of extract increases, the particle size decreases. in addition, small amounts of extract reduce silver ions, but do not protect most of the quasi-spherical agnps from aggregation, because there is a deficiency of biomolecules that act as protective agents. at high concentration, biomolecules act as reducing agents and cover the surfaces of nanoparticles, protecting them from aggregation.67 530 corciova and ivanescu high resolution tem images for the agnps obtained from a terminalia catappa extract, showed that each nanoparticle was surrounded by a material with a lower contrast, which could be a bio-organic extract component that acts as a stabilizer agent for the nanoparticles.19 for the agnps obtained from couroupita guianensis leaf extract, devaraj et al.78 consider that the same phenomenon in sem images were due to hydrogen bonding interactions and electrostatic interactions between organic biomolecules bound to the surface of the agnps, and hence, the nanoparticles were not in direct contact, even within aggregates, indicating stabilization of the nanoparticles by a coating agent. 2.2.7. photoluminescence spectroscopy the visible luminescence of agnps is due to the excitation of electrons from occupied bands into states above the fermi level.79,80 the agnps obtained from alstonia scholaris leaf extract were photoluminescent at a 370 nm excitation wavelength, the obtained peak having a bathochromic shift to 447 nm.3 3. therapeutic applications of agnps 3.1. antimicrobial activity agnps were shown to be active against both gram-positive and gramnegative bacteria, as well as fungi. for example, the antifungal activity of agnps synthesized from acalypha indica leaf extract intensified with increasing concentrations of agnps. the mechanism of action includes the destruction of membrane integrity of fungal spores, the interaction of agnps with compounds containing phosphorus and sulfur, leading to the destruction of dna and proteins of the microorganisms and ultimately to cell death.50 the agnps obtained from solanum trilobatum inhibited the growth of pathogenic bacteria, the highest percentage of inhibition being observed against escherichia coli, klebsiella planticola, klebsiella pneumoniae and streptococcus sp.49 the antibacterial action could be explained by several mechanisms. thus, the antibacterial effect of the agnps obtained from garcinia mangostana leaf extract against e. coli and staphylococcus aureus has a double mechanism of action, namely, the bactericidal effect of ag+ and the damaging effect on some polymeric subunits of the membrane.47 kvitek et al.81 and feng et al.82 suggested that agnps attach themselves to the surface of the bacterial cell membrane by interacting with sulfur-containing proteins, disrupting the membrane permeability and the respiratory functions of the cell leading to cell death. in this regard, li et al.83,84 demonstrated the biosynthesis, characterisation and therapeutic applications of agnps 531 interaction of agnps with compounds containing thiol from the respiratory enzymes of bacterial cells, thus inhibiting the respiration process in bacteria. it is also evident that the binding of the particles to the microorganisms depends on the surface available for interaction. generally, small nanoparticles have a larger surface area for bacterial interaction compared to larger particles.85–87 for example, the stronger bactericidal effect of agnps containing higher amounts of olea extract was justified by the extremely large surface area of small nanoparticles that provide for better contact and better interaction with bacterial cells than larger nanoparticles.67 morones et al.88 explained the antibacterial action of agnps not only by interaction with the membrane surface, but also by penetration into the bacteria. li et al.84 showed that agnps enter the bacterial cells and condense the dna so that dna replication and cell replication are prevented. since antimicrobial activity proved to be different for gram-positive bacteria and gram-negative bacteria, the difference in sensitivity was explained by the difference in the thickness of bacterial cell membrane and the constituents of the membrane. this fact was demonstrated by khalil et al.67 who tested the antimicrobial activity of agnps obtained from olea leaves extract against s. aureus, pseudomonas aeruginosa and e. coli. gram-negative bacteria were less sensitive to agnps than gram-positive bacteria. this effect was also observed for agnps obtained from piper nigrum leaves extract due to the difference between the cell wall of gram-positive and gram-negative bacteria.14 augustine et al.14 tested agnps obtained from piper nigrum leaves extract and agno3 with different concentrations. it was observed that by increasing the agno3 concentration, the antibacterial activity decreased, which was attributed to the larger size of the agnps, and thus, the antibacterial activity was dependent on the size of agnps. moreover, in the case of agnps obtained from the phlomis sp. leaf extract, it was observed that the antimicrobial action decreased in the order e. coli, salmonella typhimurium, s. aureus and bacillus cereus.67 this may be due to the gram-positive bacterial cell wall composed of a thick layer of peptidoglycan with a rigid structure made of linear polysaccharides chains cross-linked by short peptides, which prevents nanoparticles from entering the cell wall.89,90 variation in the chemical composition of the bacterial cell wall also explains the varied degree of antibacterial activity of agnps synthesized from avicennia alba leaves versus gram-positive and gram-negative bacteria. the best activity was observed for arthrobacter protophormia and proteus mirabilis, even better than streptomycin, as a positive control.91 agnps obtained from cassia roxburghii stem were tested against bacteria and fungi in combination with an antibiotic/antifungal agent and compared with antibiotic/antifungal agent alone. a synergistic action was observed. antibac532 corciova and ivanescu terial activity was higher in the case of gram-negative bacteria than gram-positive bacteria, and the antifungal activity was moderate.92 some examples of antifungal and antibacterial activity of agnps from plant extracts are presented in table ii. table ii. antifungal and antibacterial activity of agnps from plant extracts plant species and part of plant used for agnps synthesis sensitive fungi sensitive bacteria reference gram-positive bacteria gram-negative bacteria acalypha indica leaf alternaria alternata, sclerotinia sclerotiorum, macrophomina phaseolina, rhizoctonia solani, botrytis cinerea, curvularia lunata 50 solanum trilobatum leaf b. subtilis, streptococcus sp. serratia sp., e. coli, k. pneumoniae, k. planticola 49 olea europaea leaf s. aureus e. coli, p. aeruginosa 67 piper nigrum leaves s. aureus e. coli 14 avicennia alba leaves micrococcus luteus, arthrobacter protophormia, rhodococcus rhodochrous, enterococcus faecalis, streptococcus mutans, b. subtilis enterobacter aerogenes, alcaligenes faecalis, proteus vulgaris, proteus mirabilis, p. aeruginosa, salmonella enterica 91 cassia roxburghii stem candida glabrata, candida albicans, cryptococcus neoformans s. aureus, b. cereus e. coli, p. aeruginosa 92 3.2. antioxidant activity one of the methods used for testing the antioxidant activity of agnps is the dpph (2,2-diphenylamine-1-picryl hydrazyl) scavenging method. thus, agnps synthesized from heritiera fomes and sonneratia apetala and 1 mm agno3, by stirring, under exposure to sunlight, for up to 12 h showed potent antioxidant activity, exhibiting ic50 (the effective concentration that shows 50 % inhibition activity) values in the range 53.64–169.71 μg ml–1, due to the ability to donate electrons or hydrogen ions to neutralize dpph free radicals.59 in the case of biosynthesis, characterisation and therapeutic applications of agnps 533 agnps obtained from angelica pubescens, a dose-dependent activity was observed, the ic50 value was 1.01 mg ml–1. the free radical scavenging activity was due to biomolecules such as flavonoids, sesquiterpenes, and phenols.48 abdel-aziz et al.94 determined the antioxidant activity of agnps (30–50 nm) obtained from chenopodium murale extract and 5 mm agno3 by the dpph scavenging and β-carotene bleaching assays. the results showed significant differences between the antioxidant values of the plant aqueous extract and agnps due to the higher total phenolic content and total flavonoids content in the case of agnps. the ic50 values for the two methods increased in a dose-dependent manner. goodharzi et al.95 determined the antioxidant potential by the dpph method and the reducing capacity by the folin–ciocalteu method for some agnps synthesized from extracts of the following plants: rosmarinus sp., zataria multiflora, pelargonium graveolens, chamaemelum nobile, francoeuria undulata, achillea wilhelmsii and carthamus tinctorius. the highest antioxidant and reduction capacity were observed in the case of rosmarinus sp. and z. multiflora nanoparticles. the lowest antioxidant activity and reduction capacity were observed in the case of c. tinctorius nanoparticles. in addition, the relationship between antioxidant and reduction capacity demonstrates that plants with high antioxidant activity have high reducing capacity and high capacity for agnps synthesis. 3.3. cytotoxic activity currently, the development of molecules that could safely transport active principles inside the human body is a top priority and the synthesis of nanoparticles derived from plants could prove to be an efficient solution. however, the major concern is still the safety of these carriers. evaluation of cytotoxic activity can be achieved by the mtt (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) method. the 50 % cytotoxic concentration (cc50) is defined, as the concentration of a compound (μg ml–1) required to reduce cell viability by 50 %, which is calculated by regression analysis.96 agnps obtained from salvia officinalis and ricinus communis were tested for cytotoxicity. the mtt test indicated that treatment with different plant extracts and nanoparticles reduced the cell growth of the vero cell line in a concentration-dependent manner. cc50 values for the vero cell line were 22.7, 11.7, 16.5, 10.0, 14.0 and 15.0 mg ml–1 for salvia officinalis leaf extract, agnps obtained from salvia officinalis leaf extract, ricinus communis leaf extract, agnps obtained from ricinus communis leaf extract, ricinus communis fruit extract and agnps obtained from ricinus communis fruit extract. the results did not involve the activity of the extracts on other cell lines (primary or transformed), or 534 corciova and ivanescu the antiproliferative actions in vivo. however, the cytotoxic effect of these nanoparticles must be taken into account for future use.96 agnps synthesized from tanacetum vulgare leaf extract, at concentrations ranging from 0.5 to 35 μg l–1, were tested for their cytotoxic action compared to the aqueous leaf extract. it was observed that both samples show cytotoxicity against breast cancer (mcf-7) cell lines. the cytotoxicity of agnps was dose dependent while that of the plant extract was not because some of the bioconstituents may increase cell growth, while others may decrease it.97 the same dose-dependent cytotoxicity on the mcf-7 breast cancer cell line was observed for agnps synthesized from couroupita guianensis leaf extract.78 piao et al.98 explained the mechanisms of agnps cytotoxicity as follows: the destruction of cellular components by agnps is determined by the induction of generation of reactive oxygen species and depletion of intracellular glutathione. it also leads to c-jun n-terminal kinase activation, mitochondrial disruption, caspase activation and apoptosis. by testing the agnps obtained from podophyllum hexandrum leaf extract against human cervical carcinoma hela cells, it was shown that the synthesized nanoparticles act by decreasing cellular proliferation, induction of oxidative stress, dna damage and apoptosis by caspasemediated and mitochondria-dependent pathways.99 4. conclusions the emergence of agnps synthesis by biological methods has been a significant development in the field of nanoparticles. the interest for this type of synthesis has increased rapidly because these methods do not require the use of hazardous/toxic chemicals, as did previously employed methods. the use of plants, easily accessible factors, has led to the production of agnps through a simple, fast, cost-effective and eco-friendly process. the synthesis of nanoparticles was attributed to the abundance of biomolecules in plant extracts. studies have shown that the size, morphology, stability and biological properties of metallic nanoparticles are strongly influenced by the type of molecules found in plant extracts and by the experimental conditions, and hence, designing the synthesis of agnps with desired characteristics is a real challenge in the production of nanoparticles from plants. и з в о д биосинтеза потпомогнута биљкама, карактеризација и терапијска примена наночестица сребра andreia corciova1 и bianca ivanescu2 1 department of drugs analysis, “grigore t. popa” university of medicine and pharmacy, 16 universitatii street, 700115, iasi, romania and 2 department of pharmaceutical botany, “grigore t. popa” university of medicine and pharmacy, 16 universitatii street, 700115, iasi, romania нанотехнологија, поготову синтеза наночестица сребра, привлачи посебну пажњу због њихових својстава, биоподударности и примене. савремени процеси синтезе biosynthesis, characterisation and therapeutic applications of agnps 535 наночестица заснивају се на јефтиним, једноставним и нетоксичним методама, које не нарушавају животну средину. синтеза наночестица сребра коришћењем биљака привлачи велико интересовање зато што биомолекули могу да делују као редукујуће, али и стабилишуће врсте. услови за добијање наночестица сребра употребом биљака, као и карактеризација ових честица помоћу неколико метода, као што су инфрацрвена спектроскопија са фуријеовим трансформацијама, uv–vis спектроскопија, дифракција x-зрака и скенирајућа и трансмисиона електронска микроскопија, дискутовани су у овом прегледном раду. поред овога, разматрани су и неки од уобичајених примера биолошке примене наночестица сребра, као што су: антибактеријски, антиоксидативни и цитотоксични. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {improved synthesis of quinocetone and its two desoxymetabolites} j. serb. chem. soc. 83 (3) 265–270 (2018) udc 66.094.3:547.567.3+547.477.2+ jscs–5073 577.121:546.33’131 short communication 265 short communication improved synthesis of quinocetone and its two deoxy metabolites yuwen li, mei qiu, yubin bai, shaoqi qu and zhihui hao* agricultural bio-pharmaceutical laboratory, qingdao agricultural university, qingdao 266109, china and national-local joint engineering laboratory of agricultural bio-pharmaceutical technology, qingdao 266109, china (received 14 june, revised 9 november, accepted 13 november 2017) abstract: oxidation of o-nitroaniline with sodium hypochlorite afforded benzofurazan oxide in 96 % yield, and treatment of benzofurazan oxide with acetylacetone in the presence of triethylamine gave 2-acetyl-3-methyl-quinoxaline -1,4-dioxide in 94 % yield. finally, condensation of 2-acetyl-3-methyl-quinoxaline-1,4-dioxide with benzaldehyde using 4-(dimethylamino)pyridinium acetate as a catalyst led to quinocetone in 95 % yield. subsequently, reduction of the synthesized quinocetone with sodium dithionite resulted in two deoxy derivatives, 1-(3-methyl-4-oxido-2-quinoxalinyl)-3-phenyl-2-propen-1-one and 1-(3-methyl-2-quinoxalinyl)-3-phenyl-2-propen-1-one in 88.5 and 92 % yield, respectively. furthermore, the synthesized quinocetone, and its deoxy derivatives were characterized by 1h-nmr, 13c-nmr and elemental analysis. keywords: quinocetone; deoxy quinocetone; 4-(dimethylamino)pyridinum acetate; dideoxy quinocetone; synthesis. introduction chemically known as 1-(3-methyl-1,4-dioxide-2-quinoxalinyl)-3-phenyl-2 -propen-1-one, quinocetone (qct, scheme 1) is a quinoxaline-1,4-n-dioxide, the family members of which are bioactive compounds displaying antibacterial, antiviral, and antifungal activities.1 qct is widely used in veterinary medicine for swine, poultry, and aquatic animals due to its effectiveness and low toxicity. two other family members, carbadox and olaquindox, were banned in 1999 due to their toxicity and food safety concerns.2 in addition, qct is currently applied as an antibacterial feed additive and as a growth promoter.3 thus, a facile and efficient synthesis of qct would be agriculturally beneficial, particularly in livestock breeding and aquaculture industry. * corresponding author. e-mail: 81975048@qq.com https://doi.org/10.2298/jsc170614118l ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 266 li et al. currently, there are several methods for the synthesis of qct, but all are challenged by low yields, use of toxic reagents and unrecyclable catalysts, leading to environmental concerns.4,5 previous studies revealed that qct is metabolized in the liver and kidneys of pigs and at least 31 metabolites were identified in pig urine,6 including two deoxy metabolites 4 and 5 (scheme 1). research on the metabolites of a drug is beneficial to drug design and optimization, as well as guiding a reasonable clinical prescription, and hence, several syntheses of deoxy metabolites of quinocetone were developed.7,8 however, these methods are tedious due to the use of different starting materials, and other toxic and corrosive reagents. to obviate these drawbacks associated with the synthesis of quinocetone and its deoxy metabolites, an improved protocol for the chemical synthesis of quinocetone and its deoxy metabolites (scheme 1) was developed in the present study. scheme 1. improved synthesis of quinocetone and its two deoxy metabolites. experimental chemicals 4-(dimethylamino)pyridinium acetate was synthesized according to a published procedure.9 a sodium hypochlorite solution was freshly prepared prior to use according to a literature procedure.10 other chemicals of analytical reagent grade were purchased from commercial sources and used without further purification. apparatus melting points were determined on a digital melting point apparatus (wrs-1b) without correction. 1h-nmr and 13c-nmr spectra were recorded in cdcl3 solvent on a bruker avance iii400 spectrometer, operating at 400 and 500 mhz for protons and 100 and 125 mhz for carbons. the chemical shift values are expressed in δ values relative to the internal standard tetramethylsilane. elemental analysis was realised using an elementar vario el iii analyzer (hanau, germany). ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. synthesis of quinocetone and deoxy metabolites 267 synthesis of benzofurazan oxide (1) a mixture of sodium hydroxide (25 g, 0.625 mol) and water 100 ml was stirred until the solid had dissolved. the solution was cooled to 0 °c, and 50 g of crushed ice was added. the flask was then placed in an ice bath, and chlorine gas from a tank was bubbled through the solution until 0.29 mol chlorine had been absorbed. the solution of sodium hypochlorite was stored in the dark at 0 °c prior to use. a mixture of potassium hydroxide (8.96 g, 0.160 mol) and 95 % ethanol (125 ml) was heated at 80 °c on an oil bath to obtain a clear alkali solution. to the warm alkali solution, o-nitroaniline (20.0 g, 0.145 mol) was added to obtain a deep red solution. the deep red solution was then cooled to 0 °c, and a freshly prepared sodium hypochlorite solution was added slowly under good stirring within 10 min. the flocculent yellow precipitate was collected by filtration on a büchner funnel, and the cake was washed with 100 ml water and air -dried. recrystallization of the crude product from 95 % ethanol gave benzofurazan oxide (1). yield: 20.9 g (96 %); m.p.: 72.2–73.0 °c (lit:10 72–73 °c). synthesis of 2-acetyl-3-methyl-quinoxaline 1,4-dioxide (2) a mixture of benzofurazan oxide 1 (10.2 g, 0.075 mol) and acetylacetone (12 g, 0.12 mol) in 25 ml ethanol was stirred at 45 °c, then triethylamine (4.55 g, 0.045 mol) was added to the solution and the mixture stirred for 2 h at 45 °c. on cooling, a yellow precipitate formed, which was collected by filtration, washed with 10 ml 95 % ethanol and air-dried. recrystallization of the yellow precipitate from 95 % ethanol afforded compound 2. yield: 15.38 g (94 %); m.p.: 154.2–154.8 °c (lit:4 153–154 °c). synthesis of 1-(3-methyl-1,4-dioxide-2-quinoxalinyl)-3-phenyl-2-propen-1-one (3) a mixture of 2-acetyl-3-methyl-quinoxaline 1,4-dioxide (2) (8.09 g, 0.040 mol) and benzaldehyde (6.37 g, 0.060 mol) in 50 ml ethanol was heated at 70 °c for 30 min to obtain a clear solution, and then 4-(dimethylamino)pyridinium acetate (0.364 g, 2.0 mmol), readily prepared according to literature,9 was added to the solution. the solution was then stirred at 70 °c for 3 h. on cooling the solution to 0 °c, yellow crystals precipitated within 3 h. the yellow crystals were collected by filtration, washed with ethanol, and air-dried. the mother liquor was evaporated to recycle the catalyst 4-(dimethylamino)pyridinium acetate. recrystallization of the yellow crystals afforded compound 3. yield: 12.3 g (95 %). analytical and spectral data for 3 are presented in the supplementary material to this paper. synthesis of 1-(3-methyl-4-oxido-2-quinoxalinyl)-3-phenyl-2-propen-1-one (4) sodium dithionite (90 %, 12 mmol) was added portionwise to a mixture of 1-(3-methyl1,4-dioxide-2-quinoxalinyl)-3-phenyl-2-propen-1-one (3, 2.90 g, 10 mmol) in 50 ml 95 % ethanol under good stirring. the mixture was stirred under reflux for 3 h and then cooled in an ice-bath, whereby a yellow precipitate formed. the yellow precipitate was washed with water and air-dried to give 2.57 g yellow crystal of 4. yield: 88.5 %. analytical and spectral data for 4 are presented in the supplementary material to this paper. synthesis of 1-(3-methyl-2-quinoxalinyl)-3-phenyl-2-propen-1-one (5) 90 % sodium dithionite (30 mmol) was added portionwise to a mixture of 1-(3-methyl1,4-dioxide-2-quinoxalinyl)-3-phenyl-2-propen-1-one (3, 3.48 g, 12 mmol) in 120 ml 95 % ethanol with good stirring. the mixture was stirred under reflux for 4 h and then cooled in an ice-bath, and yellow precipitate produced. the yellow precipitate was washed with water, and air-dried to give 3.02 g yellow crystal 5. yield: 92 %. analytical and spectral data of 5 were presented in supplementary material to this paper. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 268 li et al. results and discussion as shown in scheme 1, benzofurazan oxide 1 was readily synthesized in 96 % yield as reported in the literature.10 according to a previous synthesis of intermediate 2,4 reaction of intermediate 1 with acetylacetone in the presence of triethylamine generated intermediate 2 in 94 % yield. conventionally, condensation of intermediate 2 with benzaldehyde using different catalysts produced quinocetone (3) in various yields, ranging from 65.2 to 83 %. both diethylamine4 and sodium carbonate5 are conventional catalysts in the synthesis of quinocetone (3) by reaction of intermediate 2 with benzaldehyde. however, these catalysts are difficult to recycle and are detrimental to the environment, especially for large -scale preparations. 4-(dimethylamino)pyridinium acetate (dmapa) was initially developed by nowrouzi, farahi and irajzadeh9 to catalyze synthesis of 5-substituted-1h-tetrazoles through reaction of nitriles with sodium azide. for the first time, we applied 4-(dimethylamino)pyridinium acetate as a recyclable catalyst to synthesize quinocetone (3) by reaction of intermediate 2 with benzaldehyde. details for the screening of the dosage of catalyst dmapa are summarized in table i. table i. optimization of the dosage of catalyst dmapa entry mole ratioa dmapab / mol % temperature, °c time, h yield, % 1 1:1.5 1 70 3 79.2 2 1:1.5 3 70 3 86.4 3 1:1.5 5 70 3 95.1 4 1:1.5 7 70 3 94.9 5 1:1.5 9 70 3 94.8 amole ratio of compound 2 to benzaldehyde; bcontent of compound 2 the data in table i revealed that the optimal dosage of dmapa was 5 mol % of compound 2. in terms of the yield of quinocetone (3), dmapa catalyst is superior to the existing catalysts for the synthesis of quinocetone. moreover, this catalyst is recyclable, making the procedure for synthesis of 3 environmentallyfriendly and commercially-viable. reduction of quinocetone (3) with sodium dithionite yielded compounds 4 and 5 by altering the mole ratio of sodium dithionite to quinocetone. experiments showed that a mole ratio of sodium dithionite to quinocetone (3) of 1.2:1 generated deoxy metabolite 4 in 88.5 % yield. however, further increasing the mole ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. synthesis of quinocetone and deoxy metabolites 269 ratio of sodium dithionite to quinocetone (3) decreased the yield of 4, while increasing the yield of dideoxy quinocetone 5. when the mole ratio of sodium dithionite to 3 was set at 3:1, the dideoxy quinocetone 5 was acquired in 92 % yield. conclusions in summary, an improved and practical protocol for the synthesis of quinocetone (3) in three sequential chemical steps was developed in 85.7 % total yield, which was superior to the existing protocol for the synthesis of quinocetone. moreover, deoxyquinocetone 4 and dideoxyquinocetone 5 were conveniently synthesized in 88.5 and 92 % yield, respectively, by reduction of the synthesized quinocetone 3 with sodium dithionite. in addition to the higher yields of quinocetone (3), deoxy quinocetone 4 and dideoxy quinocetone 5, this protocol has the advantages of operational simplicity, chromatography-free separation and purification by recrystallization throughout the whole procedure and the recyclability of the 4-(dimethylamino)pyridinium acetate catalyst. supplementary material analytical and spectral data of the synthesized compounds are available electronically at the pages of the journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the authors thank the financial support from national natural science foundation of china (grant 31402256), international advanced agricultural science and technology foundation of china (grant 2016-x28), and high-level talents start-up foundation of qingdao agricultural university of china (grant 631206). и з в о д унапређена синтеза хиноцетона и његова два деокси метаболита yuwen li, mei qiu, yubin bai, shaoqi qu и zhihui hao agricultural bio-pharmaceutical laboratory, qingdao agricultural university, qingdao 266109, china и national-local joint engineering laboratory of agricultural bio-pharmaceutical technology, qingdao 266109, china оксидацијом о-нитроанилина натријум-хипохлоритом добијен је у приносу од 96 % бензофуразан-оксид, који у реакцији са ацетилацетоном, у присуству триетиламина, даје 2-ацетил-3-метил-хиноксалин-1,4-диоксид у приносу од 94 %. у наредном реакционом кораку, у реакцији кондензације 2-ацетил-3-метил-хиноксалин-1,4-диоксида и бензалдехида, у присуству 4-(диметиламино)пиридинијум-ацетата као катализатора, добијен је хиноцетон у приносу од 95 %. реакцијом редукције у наредном реакционом кораку, употребом натријум-дитионита, добијени су деокси деривати 1-(3-метил-4-оксидо-2хиноксалинил)-3-фенил-2-пропен-1-он и 1-(3-метил-4-оксидо-2-хиноксалинил)-3-фенил-2пропен-1-он у приносима 88,5 и 93 %, редом. синтетисана једињења, хиноцетон и његови деокси деривати, окарактерисани су 1h-nmr и 13c-nmr спектроскопијом и елементалном анализом. (примљено 14. јуна, ревидирано 9. новембра, прихваћено 13. новембра 2017) ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 270 li et al. references 1. a. carta, m. corona, m. loriga, curr. med. chem. 12 (2005) 2259 2. commission regulation (ec) no. 2788/98, off. j. eur. commun. 3 (1998) l347 3. b. yang, l. l. huang, y. l. wang, y. liu, y. f. tao, d. m. chen, z. l. liu, k. fang, y. p. chen, z. h. yuan, j. agric. food chem. 58 (2010) 937 4. x. l. zhao, g. j. zheng, chin. vet. pharm. feed addit. 3 (2008) 13 5. s. h. tang, s. m. tang, s. h. sun, chem. bulletin 4 (2009) 381 (in chinese) 6. j. z. shen, c. y. yang, c. m. wu, p. s. feng, z. h .wang, y. li, y. s. li, s. x. zhang, rapid commun. mass spectrom. 24 (2010) 375 7. j. y. li, j. y. zhang, x. z. zhou, j. s. li, r. h. lu, heterocycl. commun. 13 (2007) 49 8. j. h. zhang, l. x. li, y. b. li, b. peng, s. q. li, z. q. zhaou, h. x. gao, s. x. zhang, j. mol. struct. 1022 (2012) 32 9. n. nowrouzi, s. farahi, m. irajzadeh, tetrahedron lett. 56 (2015) 739 10. f. b. mallory, org. synth. 37 (1957) 1. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {study of carbon dioxide and methane adsorption on carbon molecular sieves, raw and modified by waste engine oil} j. serb. chem. soc. 85 (8) 1083–1093 (2020) udc 546.264–31+547.211+544.723: jscs–5361 665.6/.7.002.68 original scientific paper 1083 study of carbon dioxide and methane adsorption on carbon molecular sieves, raw and modified by waste engine oil reza zahedi1, hossein ghafourian2*, yahya zamani3, shahrzad khoramnejhadian1 and reza dabbagh4 1department of environment, damavand branch, islamic azad university, damavand, iran, 2department of environment engineering, tehran north branch, islamic azad university, tehran, iran, 3gas research division research institute of petroleum industry (ripi), tehran, iran and 4materials & nuclear fuel research school, nuclear sciences & technology research institute (nstri), tehran, iran (received 4 september 2019, revised 14 january 2020, accepted 16 january 2020) abstract: in this study, a carbon molecular sieve (cms) was synthesized from walnut shells, followed by physical activation of the carbon content of the cms. adsorption of co2 and ch4 onto raw and acid treated adsorbents were investigated using two sizes, 300–600 and 600–1180 μm. the adsorbents were impregnated with two solvent mixtures of waste engine oil with kerosene and with thinner at a proportion of 1:1 at 25 °c. the highest adsorption for co2 and ch4 was obtained at the suitable size of a group of cms(a) adsorbents, with acid treated adsorbents being more efficient than the raw adsorbents. the acid treated cms(a-3) sample adsorbed 0.925 mol co2 g-1 and 0.353 mol ch4 g-1. the results indicated that by decreasing the granulation size of group cms(r) adsorbents, the adsorption capacities for co2 and ch4 were reduced while increasing the granulation size of group cms(a) adsorbents resulted in an enhancement in the adsorption capacity for co2 and ch4. moreover, acid treated adsorbents achieved enhanced adsorption capacity for co2 and ch4. further modifications reduced the adsorption capacity for co2 and ch4 in impregnated adsorbents, due to a decrease in surface area, pore volume and pore size of the adsorbent. keywords: adsorption capacity; carbon molecular sieve (cms); impregnated adsorbents; acid treated adsorbent. introduction nowadays, adsorption and separation processes play a major role in various industries, such as chemical, oil, gas, petrochemical, and biochemical plants. with the advancement of adsorbents and process cycles, adsorption is currently * corresponding author. e-mail: h_ghaforian@iau-tnb.ac.ir https://doi.org/10.2298/jsc190904012z ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1084 zahedi et al. used as an important and essential process in gas separation in various industries.1 carbon molecular sieves (cmss) are adsorbents that perform the separation based on molecular dimensions, which are interesting both theoretically and empirically. in their carbon structure, they have narrow channels the diameters of which are similar to those of the adsorbed particles. therefore, these channels allow the passage of such particles with diameter as large as the openings of the channels and thus, larger molecules would not be stopped from the passage. the use of cms in gas separation processes has been known for decades. chemical processes have become increasingly interested in the development of low-cost gas separation processes and gas separation technology is in quite close connection with cms.2 cmss are special classes of activated carbons that may exhibit several advantages, such as excellent and sharp selectivity for the adsorption of planar molecules, higher hydrophobicity, higher resistance to both alkaline and acidic media, and higher thermal stability at high temperatures under an inert atmosphere.3 they can be prepared from a variety of precursors, such as polymers, coconut shells, wood, bituminous carbon, and other lignin cellulosic materials.4 in this mechanism, smaller particles have high potential and selectivity. cmss have narrow pores with a diameter size near to that of particles in the gas mixture. in these narrow channels, the energy of the interaction between carbon and gas molecules involves repulsive and diffusive interactions. when the channels are much narrower than the size of the penetrating molecules, repulsive forces are dominant and the molecules need activation energy to pass through the channels.5 compared to other structures of molecular sieves, such as zeolites, cmss are among the most desirable candidates in the adsorption and separation of gases due to typical advantages such as high hydrophobicity, suitable thermal and chemical resistance against acids and bases, the stability of the carbon structure in neutral environments, and low fabrication costs.6,7 having the above features as well as being cheap and abundant, agricultural wastes are the most suitable raw materials for producing activated carbon, among which are the following raw materials: bagasse, rice bar, cardamom core, olive stone, almond shell, apricot stone, cherry stone, grape seed,8 hazelnut shell, walnut shell,9 coconut shell,10 pomegranate seed,11 etc. in addition to agriculture, other polymeric and mineral materials, such as worn tires,12 waste newspapers,13 coal,14 etc. might also be used as raw materials. cmss have not only been used for adsorbing methane, carbon dioxide,15 carbon monoxide, and nitrogen,16 but also for separating multiple gas mixtures, such as carbon monoxide–hydrogen–methane,17 methane–carbon dioxide,18 and methane–nitrogen.19 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. co2 and ch4 adsorption by cms 1085 in this study, the cms was prepared from walnut shell followed by physical activation. following the preparation of the cms, it was granulated, acid treated, and impregnated by waste oil mixed with various solvents in the proportion of 1:1. then, the adsorption capacity of prepared samples for carbon dioxide and methane was measured under the same conditions. experimental steps for preparing the cms using walnut shell walnut shells (5 kg) were used to prepare 300 g of cms. first, the walnut shells were wiped, crushed, and heated at 110 °c in order to remove moisture from the material. they were then heated at about 300 °c for 60 min to remove all organic compounds. in the final step, they were heated at 450–550 °c for 2–4 h to be converted into charcoal. all the above -mentioned procedures were performed in the absence of air. in this research, the physical method was selected and performed for carbon activation. carbon was activated in two steps in a rotary furnace at 700–900 °c in the presence of water vapor and carbon dioxide: 1. at 700–800 °c in the presence of water vapor; 2. at 850–900 °c in the presence of water vapor. all preparation steps are presented in fig. 1. fig. 1. an overview of the steps involved in the synthesis of activated carbon.20 by consuming some of the existing carbon and removing the bitumen-like materials remaining from the pyrolysis phase, these reactions cause the opening, better expansion, and formation of internal connection between the carbon pores. as a result, the internal area of the pores was increased significantly. the following reactions were used to convert carbonized materials into gases using water vapor and carbon dioxide: c + h2o  co2 + h2 (1) c + co2  2co (2) co + h2o  h2 + co2 (3) finally, 300 g of cms was obtained. division based on granulation the material was granulated using the following standard astm e001121 sieves to synthesize cmss with meshes of 300–600–1180 μm. division based on ph group cms – raw. following the preparation of the cms, its ph was measured according to astm d-383822 instructions. first, distilled water acidity was measured and then 100 ml of distilled water was added to the beaker containing 10 g of activated carbon. a gentle turbulence was created in the beaker using a magnetic stirrer and then the solution was passed through a filter. the acidity, which was neutral (ph 6.5), was measured immediately. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1086 zahedi et al. group cms – acid treated. acid treatment was performed to release the pores from sedimentary materials. cms (100 g) was mixed with 100 ml of 0.1 m nitric acid solution and then placed under a hood for 2 h at 90 °c. the mixture was then cooled and washed several times with distilled water. next, the mixture was filtered, and the acidity of the solution was measured immediately, which was acidic (ph 4.5). division based on modification preparation of the modifying solution. a mixture of waste engine oil with kerosene, thinner, ethanol and benzine solvents was considered for modification. however, the mixture of waste engine oil with ethanol and benzine was not selected due to the inability to process and reduce the dimensions of the pores of the adsorbent. the waste engine oil was first passed through a filter and chemical treatment (physical separation) was performed. the kerosene and thinner solvents were separately mixed with waste oil at a mixing proportion of 1:1 and the resulting mixture was thoroughly stirred. then, the mixture was kept at 25 °c for 30 min and then diluted and deposited. finally, the deposited sludge was separated. once the cmss were granulated, they were acid treated and impregnated. the cmss were dried at room temperature (25 °c) for 24 h. then, the samples were placed in an oven at 125 °c for 2 h. subsequently, the samples were heated at 10 °c min-1 to 500 °c and calcined at this temperature for 1 h. cms modifying method. the prepared cmss (1 g) were immersed in 50 ml of filtered solution for 1 h and then separated from the solution using filter paper. drying and calcination encoding. adsorbent samples were acid treated, impregnated and encoded based on the granulation (tables i and ii). table i. encoding of the acid treated samples sample code u.s. std sieve no. particle size, µm ph calcination temperature, °c time, h cms(r) – – 6.5 – – cms(r-2) 30–50 300–600 6.5 500 1 cms(r-3) 16–30 600–1180 6.5 500 1 cms(a-2) 30–50 300–600 4.5 500 1 cms(a-3) 16–30 600–1180 4.5 500 1 table ii. encoding of the impregnated samples; calcination temperature: 500 °c; calcination time: 1 h sample code used sample chemical treatment, proportion: 1:1 cms(r-2-k) cms(r-2) oil waste:kerosene cms(r-3-k) cms(r-3) oil waste:kerosene cms(r-2-t) cms(r-2) oil waste:thinner cms(r-3-t) cms(r-3) oil waste:thinner cms(a-2-k) cms(a-2) oil waste:kerosene cms(a-3-k) cms(a-3) oil waste:kerosene cms(a-2-t) cms(a-2) oil waste:thinner cms(a-3-t) cms(a-3) oil waste:thinner ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. co2 and ch4 adsorption by cms 1087 reactor tests description of gas adsorption method and the measurement device. weighted and volumetric methods are two common approaches for measuring the adsorption equilibrium. in the weighted method, the adsorption equilibrium is examined based on the measurement of weight changes in the adsorbent sample during adsorption; while in the volumetric method, the absorption equilibrium is measured through the difference in pre and post-absorption gas content. in the present study, the adsorption of carbon dioxide and methane by the synthesized adsorbents was investigated using a gas adsorption measurement device that was designed and made based on the volumetric method. a schematic view of the device is shown in fig. 2. in this device, a tank was used for the adsorbed gas, and another tank was used as an adsorption column. furthermore, a water–ice bath was used to adjust the temperature. by adjusting the regulator and opening the capsule valve, the gases enter the gas tank through a valve 1. by reading from the digital display and pressure gauge, the gas pressure in the tank is set to 2 bars through valves 1 and 4. all parts of the device, such as pipes, valves, fittings, gas tanks and absorption columns, are made of steel and have a maximum pressure tolerance of about 40 bars. fig. 2. schematic view of the device for measuring the gas adsorption capacity using a constant volume–variable pressure (volumetric) method. measurement of ch4 and co2 adsorption capacity after exposing the whole system to a vacuum for 5 min, 2 g of an adsorbent sample was placed in the adsorption column and its temperature was increased to 100 °c for 5 min. using a heating jacket. then the absorption column was again placed under vacuum to remove all the gases adsorbed by the adsorbent. next, the temperature of the absorption column was reduced to 25 °c using a water–ice bath container. by closing valves 2 and 4 and opening valve 1, the pressure of the gas tank was set to 2 bars by valves 1 and 4. after closing valve 1 and recording p0, the gas was allowed to occupy all of the available space between valves 1, 2 and 4 by opening valve 2. from this moment on, changes in the gas pressure with time inside the system were recorded in a computer and continued until reaching the equilibrium state (p1). in order to determine the adsorbed content, a control test was performed with helium and that part of the pressure drop that occurs after the expansion of the gas in the system (dead vol________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1088 zahedi et al. ume) that was not related to gas adsorption was deducted from the total observed pressure drop in each test. the adsorbent volume was also measured and deducted from the volume of the system. after all the above steps, examination of the adsorption for carbon dioxide and methane was commenced and the slope of the pressure variations with time were recorded until the pressure remained constant over time. characterization of the adsorbents analysis of bet specific surface area of the cms adsorbent selected in terms of adsorption capacity. the surface area, pore volume and pore size of the synthetic cmss were characterized by micromeritix3020 instrument using the bet method. first, a certain amount of the adsorbent sample was degassed at 200 °c for 2 h and then, desorption was performed in the presence of liquid nitrogen. results and discussion in order to determine the effects of granulation, the number of moles of carbon dioxide and methane adsorbed by the adsorbent samples at a pressure of 2 bar at 25 °c were investigated and calculated using eqs. (4) and (5) and excel software:18 pv = nrt (4) ( )0 1,2,3,...,nvp p p n rt − = (5) where, p0 is the equilibrium pressure, p1,2,3,…n is the pressure at different times (atm), v is the volume (l), t is the temperature (k), r is the universal gas constant (l·atm·k–1·mol–1), and n is the moles of adsorbed gas. granulation effects on the adsorption capacity of carbon dioxide and methane. the results indicated that in the group cms(a) adsorbents, the adsorption capacity increased with increasing size, while in the group cms(r) adsorbents, the adsorption capacity decreased with increasing size. the cms(a-3) adsorbent had the highest adsorption capacity for methane and carbon dioxide, while the cms(a-2-t) and cms(r-3-k) adsorbents had the lowest adsorption capacity for carbon dioxide and methane, tables iii and iv. table iii. effects of granulation on the adsorption capacity for carbon dioxide and methane (mmol g-1) by group cms(a) adsorbents cms(a-2) cms(a-3) cms(a-2-k) cms(a-3-k) cms(a-2-t) cms(a-3-t) co2 adsorption capacity 0.639 0.925 0.477 0.61 0.381 0.629 ch4 adsorption capacity 0.343 0.353 0.191 0.248 0.114 0.343 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. co2 and ch4 adsorption by cms 1089 effects of acid treatment of granulated cmss on the adsorption capacity of methane and carbon dioxide the results showed that acid treated samples, as compared to raw (non-acid treated) ones, have a higher adsorption capacity for carbon dioxide and methane. as compared to the cms(r-3) adsorbent, the adsorption capacity for carbon diox table iv. effects of granulation on the adsorption capacity for carbon dioxide and methane (mmol g-1) by group cms(r) adsorbents cms(r-2) cms(r-3) cms(r-2-k) cms(r-3-k) cms(r-2-t) cms(r-3-t) co2 adsorption capacity 0.620 0.600 0.532 0.41 0.534 0.524 ch4 adsorption capacity 0.315 0.276 0.114 0.095 0.165 0.162 ide and methane of the cms(a-3) adsorbent was increased by 54 and 28 %, respectively. however, the adsorption capacity for carbon dioxide and methane of the cms(a-2) adsorbent was only slightly increased compared to the cms(r-2) adsorbent. the reason for the significant increase in the adsorption capacity of cmss could be the granulation size in the acid treatment process. this is because coarser granules have larger channels that make it possible for the acidic solution to gain more access into the pores, which would result in more pores to be released from sediments (as shown in table v). table v. effects of acid treatment of granulated cmss on their adsorption capacity for methane and carbon dioxide (mmol g-1) cms(r-2) cms(a-2) cms(r-3) cms(a-3) co2 adsorption capacity 0.62 0.639 0.6 0.925 ch4 adsorption capacity 0.315 0.343 0.276 0.353 examination of the effects of impregnation on methane and carbon dioxide adsorption capacity the results suggested that the acid treated adsorbents have a higher adsorption capacity compared to the impregnated ones. in comparison to cms(a-2t), with a granulation size of 300–600 µm, which was acid treated and impregnated with waste engine oil and thinner, cms(a-2-k) with a granulation size of 300–600 µm, which was acid treated and impregnated with waste engine oil and kerosene, had a higher adsorption capacity for carbon dioxide and methane. in the other adsorbents, those that were impregnated with waste engine oil and thinner had a higher adsorption capacity for carbon dioxide and methane as com________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1090 zahedi et al. pared to the adsorbents which were impregnated with waste engine oil and kerosene (as shown in table vi). table vi. the results of the effects of modification of the adsorbent on its adsorption capacity for carbon dioxide and methane (mmol g-1) co2 adsorption capacity ch4 adsorption capacity cms(a-2-t) cms(a-2-k) cms(a-2) cms(a-2-t) cms(a-2-k) cms(a-2) 0.381 0.477 0.639 0.114 0.191 0.343 cms(a-3-k) cms(a-3-t) cms(a-3) cms(a-3-k) cms(a-3-t) cms(a-3) 0.61 0.629 0.925 0.248 0.343 0.353 cms(r-2-k) cms(r-2-t) cms(r-2) cms(r-2-k) cms(r-2-t) cms(r-2) 0.532 0.534 0.620 0.114 0.165 0.315 cms(r-3-k) cms(r-3-t) cms(r-3) cms(r-3-k) cms(r-3-t) cms(r-3) 0.410 0.524 0.600 0.095 0.162 0.276 bet special surface area analysis the results of bet test indicated that acid treated cms(a-3) sample with a granulation size of 600–1180 μm that had a higher adsorption capacity for carbon dioxide and methane compared to the other adsorbents had a larger bet special surface area compared to non-granulated, raw, acid treated cms(r) (as shown in table vii). additional data related to adsorption capacities are given as supplementary material to this paper. table vii. textural properties of cms(r) and cms(a-3) sample bet surface area, m2 g-1 micro pore area m2 g-1 micro pore volume cm3 g-1 external surface area m2 g-1 cms(r) 330 250 0.115 80 cms(a-3) 432 334 0.155 98 the performance of the prepared adsorbents are compared with those of other adsorbents with almost similar components and operating conditions are presented in table viii. the main purpose of the current investigation was to enhance the adsorption capacity. according to table viii, among all listed prepared adsorbents, cms(a-3) has the highest adsorption capacity. table viii. performance of the four adsorbents and some other in similar works adsorbent p / kpa t / °c adsorption capacity, mmol g-1 reference co2 ch4 cms 198.9 25 2.527 – 16 cms s-6 40 27 0.352 0.412 24 cms – 25 1.15 0.95 25 cms 206 25 – 1.41 26 cms-i 200 – 1.15 – 27 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. co2 and ch4 adsorption by cms 1091 table viii. continued adsorbent p / kpa t / °c adsorption capacity, mmol g-1 reference co2 ch4 cms-iii 200 – 0.85 – 27 cms-3k-12 292.5 – 2.364 – 28 c-102 100 25 0.003 – 29 cms(a-2) 200 25 0.639 0.343 current work cms(a-3) 200 25 0.925 0.353 current work cms(a-2-k) 200 25 0.477 0.191 current work cms(a-3-k) 200 25 0.610 0.248 current work conclusions the results of the present study showed that the cms that was synthesized from walnut shells was capable of adsorbing carbon dioxide and methane at a pressure of 2 bar and a temperature of 25 °c. this cms adsorbs more carbon dioxide (with a molecular diameter of 0.33 nm) than methane (with a molecular diameter of 0.38 nm). the results suggest that the acid treatment of an adsorbent increased its adsorption capacity for carbon dioxide and methane. the adsorption capacity for carbon dioxide and methane in the group cms(r) adsorbents (impregnated and acid treated cmss that are not acid treated) decreased with increasing granulation size; while the adsorption capacity for carbon dioxide and methane in the group cms(r) adsorbents (impregnated and acid treated cmss that were acid treated) increased with increasing the granulation size. the results showed that the modification reduced the adsorption capacity. in all samples, the adsorption capacity of the adsorbents was decreased after modification. the results of bet analysis indicated that the acid treated cms(a-3) with a granulation size of 600– 1180 μm had higher surface area, volume, and pore size as compared to non -granulated, non-acid treated and acid treated cms(r). the highest adsorption capacity for carbon dioxide and methane was obtained with the cms(a-3) sample (0.925 mmol g–1 for carbon dioxide and 0.353 mmol g–1 for methane). supplementary material details of the adsorption capacity for carbon dioxide and methane to the adsorbents that were impregnated with waste engine oil and kerosene are available electronically from http://www.shd.org.rs/jscs/. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1092 zahedi et al. и з в о д испитивање адсорпције угљен-диоксида и метана на сировим угљеничним молекулским ситима и модификованим помоћу истрошеног моторног уља reza zahedi1, hossein ghafourian2, yahya zamani3, shahrzad khoramnejhadian1 и reza dabbagh4 1 department of environment, damavand branch, islamic azad university, damavand, iran, 2 department of environment engineering, tehran north branch, islamic azad university, tehran, iran, 3 gas research division research institute of petroleum industry (ripi), tehran, iran и 4 materials & nuclear fuel research school, nuclear sciences & technology research institute (nstri), tehran, iran угљенично молекулско сито (cms) је синтетизовано из љуске ораха, уз накнадну физичку активацију угљеничне компоненте. испитана је адсорпција co2 и ch4 на сировом адсорбенсу, као и на адсорбенсу третираном киселином, коришћењем две величине, од 300–600 μm и 600–1180 μm. адсорбенси су импрегнирани истрошеним моторним уљем од керозена са разређивачем у односу 1:1 на 25 °c. највиша вредност адсорпције co2 и ch4 добијена је при погодној величини адсорбенса из групе (а), при чему су се боље показали они третирани киселином него сирови. киселином третирани узорак (а-3) адсорбовао је 0,925 mol co2 g -1 и 0,353 mol ch4 g -1. према резултатима, смањење величине гранулације групе (r), адсорпциони капацитети co2 и ch4 су се смањили, док је повећање гранулације код групе (а) увећало адсорпциони капацитет за co2 and ch4. штавише, киселином третирани адсорбенси увећали су адсорпциони капацитет за co2 and ch4. даља модификација умањила је адсорпциони капацитет код импрегнираних адсорбенаса, због умањивања расположиве површине, запремине и величине пора адсорбенса. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {chemical and sensory characterization of plum spirits obtained from cv cacanska rodna and its parent cultivars} j. serb. chem. soc. 84 (12) 1381–1390 (2019) udc 634.22+663.142/.143:663.51: jscs–5271 54–004.12(497.11) original scientific paper 1381 chemical and sensory characterization of plum spirits obtained from cultivar čačanska rodna and its parent cultivars branko t. popović1, olga v. mitrović1, aleksandar p. leposavić1, svetlana a. paunović1, darko r. jevremović1, ninoslav j. nikićević2 and vele v. tešević3*# 1fruit research institute čačak, kralja petra i 9, 32000 čačak, serbia, 2faculty of agriculture, university in belgrade, nemanjina 6, 11080 zemun, serbia and 3faculty of chemistry, university in belgrade, studentski trg 12–16, 11000 beograd, serbia (received 7 march, revised 3 june, accepted 19 june 2019) abstract: during the two-year research, a comparative analysis of the contents of 24 major volatile compounds and sensory characteristics of the plum spirits produced by spontaneous alcoholic fermentation of the plum mashes with or without stones from cultivar čačanska rodna and its parent cultivars – stanley and požegača were carried out. the plum spirits obtained from čačanska rodna cultivar contain lower amounts of methanol, 1-hexanol, ethyl acetate and acetaldehyde and higher amounts of 3-methyl-1-butanol and 2-phenylethanol than the spirits from its parent cultivars. contents of 1-propanol, 1-butanol and hexanoic acid in the spirits obtained from čačanska rodna cultivar were lower than the contents in the spirits from požegača and higher than those in the spirits obtained from stanley. the sensory quality of plum spirits, in addition to the cultivar, has been significantly influenced by the presence of stones in the mash during processing of plums into spirits. in processing plums with stones, the best-graded plum spirit was from požegača (17.88), whereas in processing without stones, the best was čačanska rodna spirit (17.78). the spirits obtained from stanley cultivar had the lowest sensory grades regardless of the processing method. keywords: plum cultivars; destoning; spontaneous fermentation; spirits; volatile compounds; sensory characteristics. introduction the old plum cultivar požegača have been traditionally used for the production of supreme quality plum spirits in serbia,1 and in europe as well2–4 (the characteristics of plum cultivars for plum spirit production are presented in the * corresponding author. e-mail: vtesevic@chem.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc190307061p 1382 popović et al. supplementary material to this paper). although with a small fruit, it was also an indispensable raw material for the production of prunes. nowadays, the area under this cultivar has been reduced in all plum growing european countries, as it is extremely susceptible to plum pox virus that causes premature fruit drop, decrease of total yield and fruit quality.5,6 in many plum-growing countries, the cultivars tolerant to plum pox virus, among which is the cultivar stanley, have been introduced in the production. plum cultivar stanley was created in 1912 in the usa and it has been grown in serbia since 1956. similar to požegača, it has been used for fresh market, for freezing, as well as for the production of prunes, jam and plum spirit. due to its large fruit size, it is particularly suitable for drying, although with no harmonic ratio between sugar and acid contents as in požegača.5 the cultivar čačanska rodna was developed from the cross of stanley and požegača cultivars at the fruit research institute in čačak. it was released and named in 1975, and protected in 1991. the main objective of breeding was the creation of a cultivar which would be tolerant to plum pox virus such as stanley, and enable the production of similar quality (in terms of colour and taste), though larger prunes compared to požegača.7 plum cultivar čačanska rodna has become significantly present in serbian plum orchards in the last quarter of the century, as well as in some european countries, united states and new zealand.5,6 due to the decreased production of prunes in serbia today, market surpluses of the fruits of both stanley and čačanska rodna cultivars have been increasingly used as a raw material for spirit production.5 in our earlier studies, the quality of plum spirit from the cultivar čačanska rodna was compared to the quality of plum spirits from other cultivars created in čačak, intended primarily for fresh consumption and drying.8 also, in our previous work we considered the compliance of the content of the most important components in the čačanska rodna plum spirits, obtained from the top-quality fruits originating from different localities, with legislation.9 however, there were no comparative studies of the characteristics of plum spirits obtained from plum cultivar čačanska rodna and its parent cultivars – stanley and požegača to this date. the aim of this work was to comparatively examine the influence of these cultivars on the content of the major volatile compounds and sensory characteristics of monovarietal plum spirits produced in the same manner (from the plum mashes with or without stones). experimental experimental details related to the plum fruits used, plum spirits production, the employed chemicals and statistical analysis are given in the supplementary material to this paper. gc analysis of volatile compounds gc analysis of volatile compounds in the plum spirits was performed according to previously reported procedures.10,11 details are briefly described in the supplementary material. monovarietal plum spirits – volatile profiles 1383 sensory analysis for sensory analysis of the plum spirits, the buxbaum method was used.3,12 additional brief details are given in the supplementary material. results and discussion contents of volatile compounds in plum spirits the contents of the analysed volatile compounds in g hl–1 of absolute alcohol (a. a.) and the ratios of a certain higher alcohols in the twelve plum spirits produced from three plum cultivars are shown in the supplementary material, tables s-i (processing fruits with stones) and s-ii (processing fruits without stones). zhang et al.13 and satora et al.14 found that methanol contents in plum spirits vary significantly depending on the cultivars used. the contents of toxic methanol were considerably lower (p ≤ 0.001) in the plum spirits from čačanska rodna (668.67 to 715.03 g hl–1 a. a.) than in spirits produced in the same manner and in the same year from its parent cultivars (709.30 to 939.37 g hl–1 a. a.). also, they were almost two times lower than the maximum content (1200 g hl–1 a. a.) prescribed by eu regulations.15 our earlier studies have shown that the plum spirits with low methanol content (275 to 715 g hl–1 a. a.) can be obtained from čačanska rodna.8,9 interestingly, the požegača plum spirits contained methanol in the similar range as the spirits from the same cultivar produced traditionally in poland3 and in ex-yugoslavia.8,16 higher alcohols are the most common aroma volatiles in the plum spirits, characterized by pleasant or unpleasant aroma depending on the type and their concentration.1 the contents of higher alcohols in plum spirits vary significantly depending on the plum cultivar.8 the contents of 3-methyl-1-butanol and 2-phenylethanol were significantly higher (p ≤ 0.001) in čačanska rodna spirits than in those produced in the same way (with or without stones) and in the same year from parent cultivars, while it was contrary for the content of 1-hexanol. the concentrations of 1-propanol and 1-butanol were significantly different (p ≤ 0.001) in monovarietal plum spirits produced in the same manner, within the same year. their concentrations in čačanska rodna plum spirits were always between the values found in spirits from parent cultivars (higher than in stanley plum spirit, and lower than in požegača plum spirit). for other higher alcohols (2-butanol, 2-methyl-1-propanol and 2-methyl-1-butanol) as well as for the total content of higher alcohols no such regularities have been observed. these results confirm our previous findings regarding differences in the content of certain higher alcohols in čačanska rodna and požegača plum spirits.8 yeasts form most of the analysed higher alcohols during fermentation from the corresponding amino acids and/or sugars.17 among higher alcohols having direct precursors in amino acids, 3-methyl-1-butanol was predominant in all plum spirits, followed by 2-methyl-1-propanol, 2-methyl-1-butanol and 2-phe1384 popović et al. nylethanol. fruits of the cultivar čačanska rodna had larger share of leucine and phenylalanine, in the total amino acids, than its parent cultivars,18 which could explain the significantly higher contents of 3-methyl-1-butanol and 2-phenylethanol in čačanska rodna plum spirits. because of 2-butanol dual (yeast and bacterial) origin, differences in 2-butanol contents in monovarietal plum spirits can probably be explained by the existence of subtle differences in the composition of the microflora during spontaneous alcoholic fermentation of plum mashes. among the higher alcohols analysed, only 1-hexanol does not derive from amino acids and/or sugars. in cognac production, content of 1-hexanol in wine distillates is considered a cultivar characteristic.19 based on the results obtained, the same goes for the monovarietal plum spirits. contents of 1-hexanol in plum spirits made from požegača are in accordance with the contents that are found in spirits of the same cultivar produced in ex-yugoslavia,8,16 and are considerably lower than the contents found in požegača spirits produced in poland.2,3 ratios of certain higher alcohols may be useful for the closer characterization of monovarietal plum spirits. by analysis of variance it was found that higher alcohols ratios vary considerably (p ≤ 0.001) in plum spirits depending on the plum cultivar used for processing. the highest regularity was observed for 2-phenylethanol/1-hexanol ratio, i.e., its value was always greater in čačanska rodna plum spirits (1.24–2.71) than in those from the parent cultivars (0.27– –1.28). ratios of other higher alcohols were similar in čačanska rodna and stanley plum spirits and most often higher than the same ratios in požegača plum spirits. their values in požegača plum spirits were in compliance with the values in the spirits of the same cultivar produced in poland, applying spontaneous mash fermentation as well.2,3 among the analysed esters, only ethyl acetate contents in čačanska rodna plum spirits were always significantly lower (p ≤ 0.001) than the contents in spirits of stanley and požegača. differences in the content of ethyl acetate in the experimentally produced spirits are likely the result of subtle differences in the compositions of bacterial and yeast microflora in spontaneously fermented mashes of čačanska rodna and its parent cultivars. in the produced plum spirits, ethyl acetate is followed by ethyl lactate. regardless of the significant differences in the contents of this ester in monovarietal spirits, there were no regularities in these differences that can be attributed to plum cultivar. in stanley plum spirits, it was observed that the content of ethyl lactate is several times greater in the second year than in the first. it may be caused by differences in the size of lactic acid bacteria population in fruits, which may arise due to different climatic conditions.20 depending on the concentration, both ethyl acetate and ethyl lactate have a positive or negative effect on the quality of plum spirit. monovarietal plum spirits – volatile profiles 1385 tables s-i and s-ii show that in some years there are no significant differences in the content of ethyl esters of fatty acids, diethyl succinate and isoamyl acetate in the monovarietal plum spirits produced. furthermore, in the cases where a statistically significant difference was observed, there is no regularity in differences of these esters contents between plum spirits from čačanska rodna and its parent cultivars. it is most likely that these differences occur due to the existence of subtle differences in the microflora, which may occur in the spontaneous mash fermentations of different plum cultivars.21 it should be noted that contents of these esters (characterized by pleasant fruity odours) in all produced monovarietal spirits were within the range for spirits obtained by spontaneous fermentation of požegača and čačanska rodna plums previously presented in our earlier study8 and studies of other authors.1,2,14,16 the contents of hexanoic acid in čačanska rodna spirits were in most cases significantly (p ≤ 0.01 and p ≤ 0.001) higher than in stanley spirits and lower than in požegača spirits. however, plum cultivar rarely affected the occurrence of significant differences in the contents of octanoic acid, decanoic acid and the total fatty acids in monovarietal spirits. compared with the data reported in the literature for traditional požegača plum spirits,16 the contents of volatile fatty acids were 2–3 times lower in experimental samples produced from the same cultivar. because volatile fatty acids are typical congeners of tail fraction, these differences are mainly due to earlier start of cutting between heart and tail fractions during re-distillation in experimental plum spirits production compared to traditional re-distillation. in the same year, plum cultivars significantly (p ≤ 0.01 and p ≤ 0.001) influenced the contents of acetaldehyde in plum spirits produced in the same manner. this finding is in agreement with our previously reported results,8 and are most likely the result of differences in the monovarietal plum mashes microflora.21 contents of acetaldehyde in the čačanska rodna spirits was most frequently lower than in the spirits from parent cultivars, except for the spirits obtained from plums without stones in year 2012 when the opposite was found. experimentally produced požegača plum spirits contained between 10.53 and 17.07 g hl–1 a. a. acetaldehyde, similar to požegača plum spirits from poland (between 9.2 and 17.5 g hl–1 a. a.).3 significant differences (p ≤ 0.001) in the benzaldehyde content were observed only in monovarietal plum spirits obtained from mashes with stones. regardless of the cultivar, higher the stone ratio in fruits (see table s-iii), higher is the content of benzaldehyde in spirits. this is understandable, since most of the benzaldehyde in spirits derives from the amygdalin, cyanogenic glycoside present in the fruit stones.22 in monovarietal spirits produced from mashes without stones, no statistically significant differences in the content of benzaldehyde have been observed. in this case, benzaldehyde is formed by degradation of the cyano1386 popović et al. genic glycoside – prunasin, present at a considerably smaller concentration in the plum fruits than amygdalin.23 the concentrations of 1-propanol, 2-butanol, 2/3-methyl-1-butanol, 1-hexanol, ethyl acetate, ethyl lactate, acetaldehyde and benzaldehyde in all monovarietal plum spirits were significantly lower than their limit concentrations above which they exhibit a negative impact on the sensory characteristics.3,24 sensory analysis the results of sensory evaluations of produced plum spirits are shown in fig. 1, both for the processing plums with stones (w) and without stones (wo). fig. 1. sensory analysis of monovarietal plum spirits obtained from plums with stones and without stones čr – čačanska rodna, st – stanley, po – požegača, w – processing with stones, wo – processing without stones, 1 – year 2011; 2 – year 2012. according to the levels of sensory ranking, which were in most cases > 17.00, the freshly distilled monovarietal spirits were considered to have no defects, forming a good basis for maturation and production of plum spirits of very good and superior quality.1 pecić et al.25 have found that plum cultivars strongly influence the sensory characteristics of old serbian plum spirits. our results, however, show that differences in sensory characteristics of freshly monovarietal plum spirits can be statistically significant in one year, and yet not significantly pronounced in another. in other words, statistically significant difference (p < 0.05) in sensory characteristics was determined only among monovarietal spirits produced with stones in year 2012 (w2), and produced without stones in year 2011 (wo1). in these cases it was noted that: i) in the processing of plums with stones – plum spirits obtained from požegača were assessed by a significantly higher monovarietal plum spirits – volatile profiles 1387 score than the spirits of čačanska rodna and stanley cultivars, among which there was no significant difference; ii) in the processing of plums without stones – no significant difference was found between the spirits of čačanska rodna and požegača, both with significantly higher score than the spirit of stanley cultivar. cluster analysis as results of the cluster analysis, appropriate dendrograms were obtained. based on the content of volatile compounds, all experimentally produced plum spirits (12 samples) can be grouped into three main clusters (fig. 2). the first cluster includes all plum spirits produced from the fruits of the čačanska rodna cultivar. results indicate that, according to the contents of analysed volatile compounds, čačanska rodna spirits were different from those produced from parent cultivars. in the third cluster, which contains plum spirits with the highest contents of methanol and ethyl lactate, two subclasters were observed: in the first were st w2 and st wo2, and the second subclaster included plum spirit from požegača (po w1). fig. 2. dendrogram obtained by the cluster analysis for all 12 plum spirits based on the contents of 24 volatile components čr – čačanska rodna, st – stanley, po – požegača; w – processing with stones, wo – processing without stones; 1 – year 2011, 2 – year 2012. dendrogram based on the cluster analysis of sensory grades of the plum spirits produced (fig. 3) differs from the dendrogram based on the content of volatile compounds. the spirit samples are more grouped in clusters based on the processing method (with or without stones) than on the cultivar used for their production. 1388 popović et al. fig. 3. dendrogram obtained by the cluster analysis based on the sensory characteristics of 12 plum spirits; čr – čačanska rodna, st – stanley, po – požegača; w – processing with stones, wo – processing without stones; 1 – year 2011, 2 – year 2012. cluster 1 contained all samples produced without stones, but also the two spirits produced with stones (po w2 and čr w1) in which the presence of stones in mashes did not show a negative impact on the sensory characteristics of the spirit. all the plum spirits of cluster 1 were distinguished by the pleasant fruity odour; sensory grades in this cluster ranged from 17.33 to 17.88. the spirits in other clusters were characterized with fruity odour, although less pronounced, and partially masked by the stone-like ton: cluster 2 (st w1) and cluster 3 (st w2, čr w2 and po w1). such sensory characteristics are common for plum spirits obtained by spontaneous alcoholic fermentation of mashes with stones. because of heavy odour, sensory grades of the plum spirits in this group ranged from 16.93 to 17.48. conclusion based on the content of volatile compounds from the spirit obtained, the fruits of the plum cultivar čačanska rodna can be regarded as a suitable raw material for the production of quality plum spirit. plum spirits produced from the čačanska rodna cultivar are distinguished by the content of some important volatile compounds from spirits made in the same manner from the parent cultivars (stanley and požegača). the significant differences in sensory characteristics of the monovarietal spirits is not influenced by the cultivar only, but also by the processing method and the production year. in the years with significant differences in the sensory assessments of monovarietal plum spirits, it was est monovarietal plum spirits – volatile profiles 1389 ablished that the čačanska rodna plum spirits could be of similar sensory characteristics as the spirit of the stanley cultivar (in the processing with stones), i.e., with similar characteristics as spirits from požegača (in the processing without stones). supplementary material additional data are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. this study was funded by the ministry of education, science and technological development of the republic of serbia (project tr 31093). и з в о д хемијска и сензорна карактеризација шљивовица добијених од сорте чачанска родна и њених родитељских сората брaнкo t. пoпoвић1, oлгa в. mиtрoвић1, aлeксaндaр п. лeпoсaвић1, свetлaнa a. пaунoвић1, дaркo р. jeврemoвић1, нинoслaв j. никићeвић2 и вeлe в. teшeвић3 1институт за воћарство чачак, крaљa пeтрa i 9, 32000 чaчaк, 2пољопривредни факултет, универзитет у београду, немањина 6, 11080 земун и 3универзитет у београду – хемијски факултет, студeнтски трг 12–16, 11000 бeoгрaд током двогодишњих испитивања, анализирани су садржаји 24 важније испарљиве компоненте и сензорне карактеристике шљивовица произведених од спонтано ферментисаних кљукова шљиве са коштицама или без њих сорте чачанска родна и њених родитељских сората – стенлеја и пожегаче. сорта шљиве значајно утиче на хемијски састав шљивовица. шљивовице добијене од сорте чачанска родна садржале су мање метанола, 1-хексанола, етилацетата и ацеталдехида, а више 3-метил-1-бутанола и 2-фенилетанола него шљивовице произведене од родитељских сората. садржаји 1-пропанола, 1-бутанола и хексанске киселине у ракијама од сорте чачанска родна били су нижи од садржаја у шљивовицама од сорте пожегача, а виши него у шљивовицама добијеним од сорте стенлеј. поред сорте, на сензорни квалитет шљивовица је значајно утицало и присуство коштица у кљуку током прераде шљива у ракију. при преради шљива са коштицама, најбоље је оцењена шљивовица сорте пожегача (17,88), док је при преради без коштица највишу оцену добила шљивовица сорте чачанска родна (17,78). шљивовице сорте стенлеј добиле су најниже сензорне оцене, без обзира на начин прераде. на основу добијених резултата може се закључити да је сорта чaчaнскa рoднa пoгoдна сирoвина зa прoизвoдњу квaлитeтнe рaкиje шљивoвицe. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice functional properties of pumpkin (cucurbita pepo) seed protein isolate and hydrolysate j. serb. chem. soc. 81 (1) 35–46 (2016) udc 582.98+66.094.941:577.15:541.18.051.3: jscs–4825 577.112.004.12 original scientific paper 35 functional properties of pumpkin (cucurbita pepo) seed protein isolate and hydrolysate sandra đ. bučko*, jaroslav m. katona, ljiljana m. popović, žužana g. vaštag and lidija b. petrović university of novi sad, faculty of technology novi sad, bul. cara lazara 1, 21000 novi sad, serbia (received 15 june, revised 1 september, accepted 23 september 2015) abstract: pumpkin seed protein isolate (pspi) was enzymatically hydrolysed by pepsin to obtain pumpkin seed protein hydrolysate, psph. investigation of the solubility, interfacial and emulsifying properties of both pspi and psph was conducted under different conditions of ph (3–8) and ionic strength (0–1 mol dm-3 nacl). pspi had the lowest solubility, i.e., isoelectric point (pi) at ph 5. psph had higher solubility than pspi over the whole range of tested ph and ionic strength values. the decreases in the surface and interfacial tension evidenced that both pspi and psph adsorb at the air/protein solution and oil/ /protein interfaces of the solution. emulsions (20 % oil in water) stabilized by 10 g dm-3 pspi or psph solution were prepared at ph 3, 5 and 8 and ionic strength of 0 and 0.5 mol dm-3 nacl. psph stabilized emulsions from coalescence at all tested ph and ionic strength values. pspi was able to stabilize emulsions at ph 3 and 0 mol dm-3 nacl, and at ph 8 regardless of ionic strength, while emulsions at ph 5 and both 0 and 0.5 mol dm-3 nacl and at ph 3 when the ionic strength was increased separated into an oil and a serum layer immediately after preparation. all emulsions were susceptible to creaming instability. keywords: plant proteins; natural emulsifiers; enzymatic hydrolysis; oilseed proteins. introduction proteins from vegetable origin are an alternative to animal proteins for food and cosmetics applications due to the renewability of the raw material and widespread and variety of sources (especially legumes, cereals and oilseeds).1 oilseeds, despite the fact that they are primarily grown as a source of oil, are rich in proteins. moreover, after oil extraction, the protein content in an oil cake, which is a by-product of the oil extraction process, can increase up to 60 %, making it a valuable material for protein extraction.2 * corresponding author. e-mail: sandranj@uns.ac.rs doi: 10.2298/jsc150615081b 36 bučko et al. as one of the oilseeds, pumpkin (cucurbita sp.) seed has 36 % of proteins,3 while the protein content in the oil cake can increase up to 65 %.4 the major fraction of these proteins is represented by cucurbitins. cucurbitins are 12s globulins composed of six similar subunits with molecular weights of 54 kda, and thus, the total molecular weight of cucurbitin is about 325 kda.5,6 cucurbitins are accompanied by 2s albumins, proteins with a molecular weight of 12.5 kda. albumins are composed of two chains, a small and a large one, with molecular weights of 4.8 and 7.9 kda, respectively.7,8 the 12s and 2s protein fractions together make 59 % of total crude protein content in pumpkin seed.9 pumpkin seed proteins are desirable ingredients in food products because they are reported to be health improving on several levels, i.e., they were found to have anti-microbial and anti-carcinogenic effects, to inhibit mechanisms of blood coagulation and to alleviate the detrimental effects associated with protein malnutrition.10,11 thus, plant proteins are increasingly used as unconventional sources of proteins to perform functional roles in food formulations.12 however, the low solubility of plant proteins in acidic media complicates their utilization in foodstuffs of moderate acidity (citric beverages, dressings, etc.),13 especially when the required functional properties, e.g., foaming and emulsifying properties, depend on solubility.14 the solubility of pumpkin seed proteins was reported to be very low (< 20 %) at ph < 5.15 one of the most efficient means of increasing protein solubility as well as improving the functional properties of oilseed proteins is to subject them to enzymatic hydrolysis.2 functional properties of particular interest include improved solubility, particularly at ph near the isoelectric point (pi), enhanced emulsifying properties, as well as enriched biological activities.16 namely, modification of proteins by partial enzymatic hydrolysis is accompanied by: 1) a decrease in molecular weight, 2) an increase in number of ionisable groups and 3) exposure of previously concealed hydrophobic groups at the interface. these effects can well modify the conformation and structure of proteins, thus changing the solubility, surface characteristics and emulsifying properties.12,17,18 hitherto, only a few investigations on pumpkin seed protein hydrolysates have been conducted. vaštag et al.4 reported pumpkin seed protein hydrolysates to have antioxidant and ace-inhibitory activities and investigation of peričin et al.19 showed that enzymatic hydrolysis is an effective tool for obtaining highly soluble pumpkin seed proteins. the aim of this study was to investigate the effect of different environmental conditions (ph and ionic strength) on functional properties such as solubility, interfacial and emulsifying properties of pumpkin seed protein isolate (pspi) in comparison to pumpkin seed protein hydrolysate (psph). pumpkin seed protein’s functional properties 37 experimental materials pumpkin (cucurbita pepo) seed oil cake was obtained from “agrojapra”, bosnia and herzegovina. it was stored at a temperature of 4 °c and ground in a coffee grinder before use. sunflower oil was obtained from “vital”, serbia. demineralised water was used as a solvent. pepsin (0.7 fip-u mg-1) was obtained from sigma (st. louis, mo, usa). all other used chemicals were obtained from “centrohem d.o.o.”. serbia, and were of at least extra pure quality. buffer solutions were prepared by mixing 0.2 mol dm-3 disodium hydrogenphosphate and 0.1 mol dm-3 citric acid in proportions defined for each ph. pspi preparation pumpkin seed protein isolate (pspi) was prepared by alkali extraction with isoelectric precipitation. firstly, the ground pumpkin seed oil cake was defatted with hexane (mass ratio 1:5) in two stages and air-dried at room temperature. the defatted pumpkin cake was suspended in alkali solution at ph 10.00, which was set by 1 mol dm-3 naoh, at room temperature to allow protein dissolution. after 30 min of gentle stirring, the slurry was filtered. the dissolved proteins in the filtrate were precipitated by adjusting ph to 5.00 with 1 mol dm-3 hcl. the precipitate was separated from the liquid phase by centrifugation (sorvalltm rc 5b centrifuge) at 4 °c and 10000 rpm for 20 min and dried at 30 °c for 48 h. finally, the dried protein precipitate was ground in a coffee grinder to obtain pspi powder. enzymatic hydrolysis pspi suspension (10 g dm-3) was prepared by suspending the required amount of pspi in a solution of ph 3, which was set and controlled by the addition of 1 mol dm-3 hcl. enzymatic hydrolysis was performed in a batch reactor at 37 °c and an enzyme to substrate ratio of 0.02 g per 1 g. the reaction conditions were set so that degree of hydrolysis was 19±1 % at the end of reaction. enzymatic hydrolysis was completed after 90 min. the hydrolysed suspension was then vacuum filtered and dried using a “büchi 190” spray drier at an inlet temperature of 120 °c and outlet temperature of 70 °c. determination of the degree of hydrolysis (dh) the same volumes of hydrolysed suspension and trichloroacetic acid (0.44 mol dm-3) were mixed and incubated at 4 °c for 30 min. thereafter, the mixture was centrifuged (eppendorf mini spin plus, 14500 rpm, 10 min).4 the obtained 0.22 mol dm-3 trichloroacetic acid soluble protein fraction and the hydrolysate mixture without addition of trichloroacetic acid were each analyzed to determine the protein content by the method of lowry et al.20, using bovine serum albumin as the standard protein. the dh was calculated as the ratio of the 0.22 mol dm-3 trichloroacetic acid soluble proteins to total proteins in the hydrolysate, expressed as a percentage. determination of pspi and psph solubility pspi and psph suspensions of different concentrations (csusp, 10–100 g dm -3) were prepared by suspending the required amount of pspi in buffer solutions of different values of ph (3–8) and ionic strength, ic (0–1 mol dm -3). the suspensions were placed in a water bath at 50 °c for 1 h under constant stirring, in order to enable dissolution of the proteins. soluble proteins were separated from undissolved particles by sartorius membrane filtration (filter pore size 0.45 μm) to obtain pspi or psph solutions. the concentration of dissolved proteins in the protein solution, csol, was determined by the lowry et al. 20 method and was expressed as g dm-3. the pspi and psph solubility, s, was calculated as: 38 bučko et al. sol susp 100 c s c  (1) tensiometry the surface tension (air–water interface) and interfacial tension (oil–water interface) of pspi solutions were determined at 25 °c according to the du noüy ring method using a sigma 703d tensiometer (ksv instruments ltd., finland). the ring was immersed in pspi solution (20 ml) and the surface was left to equilibrate for 10 min. for the interfacial tension measurements, after ring immersion in the solution, 20 ml of sunflower oil was carefully added on top of the solution surface and the interface was left to equilibrate for 10 min. upon surface–interface equilibration, the surface/interfacial tension was measured. the reported values of surface and interfacial tension are the mean values of at least three measurements. surface tension of buffer solutions of ph 3–8 was 71.7±0.4 mn m-1. the interfacial tension of the buffer solutions of ph 3–7 was 28.4±0.9 mn m-1, whereas, it was 25.7±1.0 mn m-1 at ph 8. preparation and characterization of emulsions emulsions of 20 mass% sunflower oil in water were prepared by dispersing 6 g of sunflower oil in 24 g of a continuous phase by means of an ultraturrax t–25 (janke & kunkel, germany) homogenizer at 10000 rpm for 10 min at 25 °c. the compositions of the continuous phase were varied by changing the ionic strength (0 and 0.5 mol dm-3) and ph (3, 5 and 8), while the concentration of the pspi and psph solution was kept constant at 10 g dm-3. the volume weighted mean diameter (d4,3) of oil the droplets in the emulsions was determined using a mastersizer micro particle analyzer (malvern instruments ltd., uk). buffer solutions which corresponded to the measured emulsions were used to collect the background data. emulsions were dosed so that the obscuration stayed between 10 and 20 %. during the measurements, the pump rotation speed was kept at 1500 rpm. the reported d4,3 values are the average value of at least three measurements. stability of emulsions was evaluated by the creaming test. emulsions were transferred into 10 ml sealed graduated glass cylinders immediately after preparation and were left for 14 days at room temperature to observe their creaming behaviour. during time, the emulsions separate into an emulsion cream (top) and an emulsion serum (bottom) layer. changes in a creaming index, ci, were visually monitored, where the creaming index is: s e 100 v ci v  (2) where vs and ve are the volumes of the serum layer and the emulsion, respectively. results and discussion solubility the solubility characteristics of protein are among the most important functional properties since many functional performances of proteins depend upon their capacity to go into solution initially.2 therefore, the solubility of pspi and psph as a function of ph were investigated first (fig. 1). figure 1 shows that pspi had the lowest solubility at ph 5, which is reported to be the isoelectric point for the majority of food proteins.13 pspi solubility increased as the ph of the solution is increased towards more alkaline or decreased towards more acidic conditions. pumpkin seed protein’s functional properties 39 fig. 1. influence of ph on the solubility of pspi and psph; csusp = 10 g dm-3, ic = 0 mol dm -3. the enzymatic hydrolysis improved the solubility of pspi at all the tested ph values. it is noteworthy that the lower was the solubility of pspi, the more it was increased by enzymatic hydrolysis, which was especially true for ph values close to the pi of pspi (fig. 1). similar results were observed for proteins from oilseed flour mixture, chickpea, peanut, brassica carinata and barley and for sodium caseinate.16,21–24 the increase in protein solubility as a result of enzymatic hydrolysis could be attributed to the reduction of the molecular size and the increase in hydrophilic character of proteins, exposing more polar groups to the aqueous environment.2,12,21 the influence of ionic strength on pspi and psph solubility at three different ph values (3, 5 and 8) is shown in fig. 2. depending on the ph of pspi and psph solutions, two distinct effects, salting-in and salting-out, could be observed. the salting-out effect, i.e., a decrease in solubility with increasing ionic strength was observed at ph 3 for pspi and at ph 3 and 5 for psph. on the contrary, the addition of nacl up to 0.5 mol dm–3 for pspi and 1 mol dm–3 for psph at ph 8 brought about increase in solubility of about 10 %, indicating a slight salting-in effect. influence of ionic strength on the solubility of proteins is the result of the complex interplay of various effects, such as electrostatic interactions, ion specific effects, hydrophobic effects, etc.24,25 interfacial properties interfacial properties of pspi and psph solutions of different ph values, ionic strengths and protein concentrations were investigated. influence of ph on surface and interfacial tension of pspi and psph solutions is shown in fig. 3. pspi had the lowest interfacial tension at ph 5, i.e., at the isoelectric point. namely, low protein solubility at pi, which is a result of increased hydrophobic interactions, force protein molecules to minimize unfavourable interactions by aligning themselves at the interface.26 on the contrary, on increasing the ph above or 40 bučko et al. decreasing it below pi, the protein molecules become more hydrated and solubility increases. consequently, protein drive towards the interface is mitigated as evidenced by the increase in the surface tension of pspi solutions (fig. 3). fig. 2. influence of ionic strength on the solubility, s, of pspi (solid lines) and psph (dash lines); csusp = 10 g dm -3. fig. 3. the influence of ph on the surface and interfacial tension of pspi (solid lines) and psph (dash lines); ic = 0 mol dm -3, nacl, csol = 1 g dm -3. filled symbols represent surface and the open symbols interfacial tension. influence of psph on surface and interfacial tension was comparable to that of pspi except at ph > 6 when psph was more effective in decreasing surface and interfacial tension, indicating that the hydrophobic amino acids might pumpkin seed protein’s functional properties 41 become exposed during hydrolysis, resulting in unfolded structures and increased hydrophobicity of the hydrolysates.16,17 influence of the pspi and psph solution concentration on the surface and interfacial tension at three different ph values (3, 5 and 8) is shown in fig. 4a and 4b, respectively. as expected, the higher is the concentration of the pspi or psph solution, the lower are the surface and interfacial tension, regardless of ph. nevertheless, psph showed more pronounced decrease in surface and interfacial tension, especially at lower solution concentrations (csol < 0.1 g dm–3), when compared to pspi. fig. 4. influence of solution concentration, csol, on the surface and interfacial tension of: a) pspi; and b) psph solution; ic = 0 mol dm -3 nacl. filled symbols represent the surface and open symbols the interfacial tension. 42 bučko et al. the influence of the ionic strength on the surface and interfacial tension of 1 g dm–3 pspi and psph solutions at ph 3, 5 and 8 are presented in fig. 5. increasing the ionic strength had a minimal effect on the surface and interfacial tension of pspi and psph solutions (±1.5 mn m–1) at all tested ph values, except for the pspi solution at ph 3. since the addition of nacl at ph 3 significantly decreased pspi solubility (fig. 2), i.e., increased hydrophobic interactions and decreased protein hydration, it was expected that the pspi solution would be more effective in decreasing the surface and interfacial tension upon increasing the ionic strength at ph 3. in addition, the lowest surface and interfacial tension of psph solution at ph 8 could be because of its lowest solubility at ph 8 (see fig. 1). fig. 5. influence of ionic strength on surface and interfacial tension of pspi (solid lines) and psph (dash lines); csol = 1 g dm -3. filled symbols represent the surface and open symbols the interfacial tension. emulsifying properties of pspi and psph emulsions of 20 % sunflower oil in protein solution (10 g dm–3) were prepared at ph 3, 5 and 8 without or with 0.5 mol dm–3 nacl. the volume weighted mean droplet diameter, d4,3, of the pspi and psph stabilized emulsions without and with 0.5 mol dm–3 nacl in the continuous phase is illustrated in fig. 6a and b, respectively. stable emulsions when psph was used as emulsifier were obtained at all tested ph and ionic strength values while the pspi stabilized emulsions were only stable at ph 3 and without nacl and at ph 8 regardless of the ionic strength. pspi failed to stabilize emulsions at ph 5 regardless of the ionic strength and at ph 3 when the ionic strength was increased and hence, the pumpkin seed protein’s functional properties 43 oil separated immediately after emulsion preparation. it is noteworthy that oil separation occurred even though interfacial study showed that both pspi and psph adsorb at the air–protein and oil–protein solution interfaces under all tested conditions (ph, csol and ionic strength), as evidenced by the decrease in the surface and interfacial tension (figs. 3–5). the inability of pspi to stabilize emulsions at ph 5 is attributed to insufficient electrostatic repulsion between the emulsion droplets, which led to droplet aggregation/flocculation and coalescence. namely, proteins contain many ionisable groups that alter the electrical characteristics of the interface of oil droplets once adsorbed.27 these ionisable groups could be positively or negatively charged depending on the ph of the solution. at pi, the net protein charge is zero and thus electrostatic repulsion between droplets of the emulsion is minimal, resulting in emulsion instability. on the other hand, as the ph increases above or decreases below the pi value, negative or positive fig. 6. mean volume weighted droplet diameter, d4,3, of emulsions stabilized by 10 g dm-3 pspi and psph: a) without nacl, and b) with 0.5 mol dm-3 nacl in the continuous phase. 44 bučko et al. charge takes preponderance leading to enhancement of the emulsion stability due to increased electrostatic repulsion between the droplets. the d4,3 values of the emulsions stabilized by either pspi or psph depended on ph of the continuous phase (fig. 6a and b). however, the d4,3 values for the pspi stabilized emulsions (ph 3 without nacl and ph 8 without or with 0.5 mol dm–3 nacl) were smaller than the d4,3 for emulsions stabilized by psph under the same environmental conditions (ph and ionic strength). all emulsions were susceptible to creaming instability. creaming index, ci, of the emulsions stabilized by pspi and psph are shown in fig. 7a and b, res fig. 7. creaming index, ci, of emulsions stabilized by 10 g dm-3 of: a) pspi; and b) psph during 14 days of storage. filled symbols indicate ic = 0 mol dm-3 nacl, and open symbols ic = 0.5 mol dm-3 nacl. pumpkin seed protein’s functional properties 45 pectively. all emulsions separated to a cream and a serum layer within 20 min after preparation as a consequence of relatively large droplet size and low viscosity of the continuous phase. the ci of the psph stabilized emulsions at ph 3 when 0.5 mol dm–3 of nacl was added to the continuous phase and at ph 5 regardless of ionic strength were lower than the ci of pspi stabilized emulsions under the same conditions of ph and ionic strength, indicating improved stability of the emulsions with psph in the continuous phase. conclusions the effect of enzymatic hydrolysis on the functional properties of pspi at different values of ph (3–8) and ionic strength (0–1 mol dm–3) was investigated. the solubility of psph was significantly increased when compared to solubility of pspi over whole range of tested ph and ionic strength values. solubility was especially improved at ph values close to the pi of pspi. both pspi and psph decreased surface and interfacial tension. stable psph emulsions were obtained irrespective of the ph and ionic strength of the continuous phase, in contrast to pspi, which gave stable emulsions only at ph 3 and 8 in 0 mol dm–3 nacl and at ph 8 in 0.5 mol dm–3 nacl. the d4,3 values of emulsions stabilized by psph were larger when compared to those of pspi stabilized emulsions. the obtained results suggest that due to its high solubility, psph may be better suited than pspi for many food formulations, especially in conditions of acidic ph and increased ionic strength. acknowledgements. this work was financed by ministry of education, science and technological development of republic of serbia, grant no iii 46010. it was realized within the cost cm 1101 and mp1106 frameworks. и з в о д функционалне особине протеинских изолата и хидролизата добијених из семена тикве (cucurbita pepo) сандра ђ. бучко, јарослав м. катона, љиљана m. поповић, жужана г. ваштаг и лидија б. петровић универзитет у новом саду, технолошки факултет нови сад, бул. цара лазара 1, 21000 нови сад протеински хидролизат семена уљане тикве (psph) добијен је путем ензиматске хидролизе протеинског изолата семена уљане тикве (pspi) пепсином. испитиван је утицај услова средине као што су ph (3–8) и јонска јачина (0–1 mol dm-3) на растворљивост и површинска и емулгујућа својства pspi и psph. pspi је најмање растворљив на ph изоелектричне тачке (pi), тј. на ph 5. у односу на pspi, растворљивост psph је повећана при свим испитиваним условима ph и јонске јачине. присуство pspi или psph у раствору изазвало је смањивање површинског и међуповршинског напона што указује на то да се и изолат и хидролизат адсорбују на међуповршинама ваздух/протеински раствор и уље/протеински раствор. припремљене емулзије са 20 % уља у води на ph 3, 5 и 8, и при јонској јачини од 0 и 0,5 mol dm-3 стабилизоване су раствором pspi или psph (10 g dm-3). psph је спречио коалесценцију емулзија при свим испитиваним условима ph и јонске јачине. pspi је стабилизовао емулзије на ph 3 при 0 mol dm-3 nacl и на ph 8 без обзира 46 bučko et al. на јонску јачину док су се емулзије припремљене на ph 5 и 0 и 0,5 mol dm-3 nacl и на ph 3 при повећаној јонској јачини раздвојиле на уљану и серум фазу одмах након припреме. све емулзије су биле подложне гравитационој нестабилности. (примљено 15. јуна, ревидирано 1. септембра, прихваћено 23. септембра 2015) references 1. a. moure, j. sineiro, h. domínguez, j. c. parajó, food res. int. 39 (2006) 945 2. c. radha, p. r. kumar, v. prakash, food chem. 106 (2007) 1166 3. l. quanhong, l. caili, food chem. 92 (2005) 701 4. ž. vaštag, lj. popović, s. popović, v. krimer, d. peričin, food chem. 124 (2011) 1316 5. r. blagrove, g. lilley, eur. j. biochem. 103 (1980) 577 6. p. colman, e. suzuki, a. van donkelaar, eur. j. biochem. 103 (1980) 585 7. e. f. fang, j. h. wong, p. lin, t. b. ng, febs lett. 584 (2010) 4089 8. a. g. vassiliou, g. m. neumann, r. condron, g. m. polya, plant sci. 134 (1998) 141 9. g. fruhwirth, a. hermetter, eur. j. lipid sci. technol. 109 (2007) 1128 10. m. yadav, s. jain, r. tomar, g. b. k. s. prasad, h. yadav, nutr. res. rev. 23 (2010) 184 11. p. p. s. tomar, k. nikhil, a. singh, p. selvakumar, p. roy, a. k. sharma, biochem. biophys. res. commun. 448 (2014) 349 12. g. chabanon, i. chevalot, x. framboisier, s. chenu, i. marc, process biochem. 42 (2007) 1419 13. m. yuliana, c. t. truong, l. h. huynh, q. p. ho, y.-h. ju, lwt food sci. technol. 55 (2014) 621 14. s. e. m. ortiz, j. r. wagner, food res. int. 35 (2002) 511 15. l. rezig, f. chibani, m. chouaibi, m. dalgalarrondo, k. hessini, j. guéguen, s. hamdi, j. agric. food. chem. 61 (2013) 7715 16. y. luo, k. pan, q. zhong, food chem. 155 (2014) 146 17. r. adjonu, g. doran, p. torley, s. agboola, j. food eng. 122 (2014) 15 18. a. m. ghribi, i. m. gafsi, a. sila, c. blecker, s. danthine, h. attia, a. bougatef, s. besbes, food chem. 187 (2015) 322 19. d. peričin, lj. radulović-popović, ž. vaštag, s. mađarev-popović, s. trivić, food chem. 115 (2009) 753 20. o. h. lowry, n. j. rosenbrough, a. l. fair, r. j. randall, j. biol. chem. 193 (1951) 265 21. m. del m. yust, j. pedroche, m. del c. millán-linares, j. m. alcaide-hidalgo, f. millán, food chem. 122 (2010) 1212 22. s. n. jamdar, v. rajalakshmi, m. d. pednekar, f. juan, v. yardi, a. sharma, food chem. 121 (2010) 178 23. j. pedroche, m. m. yust, h. lqari, j. girón-calle, m. alaiz, j. vioque, f. millán, food chem. 88 (2004) 337 24. e. yalçın, s. çelik, food chem. 104 (2007) 1641 25. r. w. maurer, s. i. sandler, a. m. lenhoff, biophys. chem. 156 (2011) 72 26. e. m. papalamprou, g. i. doxastakis, v. kiosseoglou, j. sci. food agric. 90 (2010) 304 27. r. s. h. lam, m. t. nickerson, food chem. 141 (2013) 975. {synthesis of novel phthalimido oxime esters and evaluation of their cytotoxicity} j. serb. chem. soc. 85 (1) 1–8 (2020) udc 547.584+547.574.2–326+542.913:576+ jscs–5278 615.9:57–188 original scientific paper 1 synthesis of novel phthalimido oxime pseudoesters and evaluation of their cytotoxicity asma mehrez1, ibtissem chakroun2, dalila mtat1, hédi ben mansour3 and ridha touati1* 1laboratory of asymmetric organic synthesis and homogeneous catalysis (ur11es56), faculty of sciences of monastir, avenue of the environment, 5019 monastir, tunisia, 2laboratory for analysis, treatment and recovery of environmental pollutants and products, faculty of pharmacy, avicenne street, 5000 monastir, tunisia and 3research unit analyzes and processes applied to the environment (ur17es32), higher institute of applied sciences and technology of mahdia, 5111 mahdia, tunisia (received 2 march 2018, revised 16 july 2019, accepted 1 august 2019) abstract: a series of novel optically pure oxime pseudoesters derivatives were synthesized by the reaction of substitute keto oximes with various n-substituted α-amino acids chlorides in the presence of triethylamine and dichloromethane at 0 °c, and their structures were characterized by ir and 1d-nmr methods. the synthesized compounds were tested for their ability to inhibit the proliferation of human colon cancer cells and human epithelial cells. some of them were revealed to have a significant cytotoxic effect. keywords: oxime esters; α-amino acids; stereoselective; cytotoxic; biological activity; enantiomer. introduction cancer remains a serious human health problem despite considerable progress in the understanding of its biology and pharmacology. the main problem is that cancer is not one disease, but a group of diseases affecting different organs and systems of the body. cancer develops due to abnormal and uncontrolled cell division, frequently at a rate greater than that of most normal body cells.1 for some types of disseminated cancers, chemotherapy is the only effective therapy because it distributes anticancer drugs through the circulatory system. oxime pseudoesters are a small, but important, class of biologically useful compounds for the synthesis of fragrances,2 crop protection, and therapeutic studies.3 they are useful building blocks in peptide synthesis.4 oxime pseudoesters are * corresponding author. e-mail: ridhato@yahoo.fr https://doi.org/10.2298/jsc180302082m ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 2 mehrez et al. selective covalent inhibitors of serine hydrolase retinoblastoma-binding protein 9 (rbbp9) and cleave dna under photolytic conditions.5,6 they also possess fungicidal,7 herbicidal,8 insecticidal and antitumor activity.9 oxime pseudoesters of dihydrocoumaric acid have been synthesized and they were reported to have antibacterial activity.10 aromatic benzophenone oxime esters and dibenzosuberone oxime esters are pharmacologically important.11 vanillin derived piperidin-4-one oxime esters have been tested for their antioxidant and antimicrobial potential.12 the oxime pseudoesters derived from nafimidone have been tested as potential anticonvulsant compounds.13 several methods have been developed for the preparation of oxime pseudoesters derivatives.3 the most common method is the condensation of acid chlorides with oximes under basic conditions or the use of acid anhydrides in presence of strong acids.2,6,10,12,13 oxime pseudoesters can be prepared using α,β-unsaturated aldehydes and oximes employing a n-heterocyclic carbene as a redox esterification catalyst,14 or by treatment of alkylor aryl-substituted oximes with aliphatic or aromatic acids in the presence of the n-[3-(dimethylamino)propyl]-n′-ethylcarbodiimide hydrochloride (edci) reagent.15 a large number of studies on their synthesis and biological activities have been reported during the last thirty years.16 however, no attention was paid to the stereoselective synthesis of chiral oxime pseudoesters derivatives. hence, in continuation of our research aimed at the preparation of natural and non-natural compounds of biomedical importance,17 and in connection with ongoing investigations on the reactivity of natural amino acids,18 herein, an efficient and easy methodology is reported for the synthesis of a series of new optically pure oxime pseudoesters 5a–k starting from the commercially available acetophenone derivatives 1a–f and natural amino acids 3a–d, which are of considerable interest as chiral pool agents since they are easily accessible and inexpensive enantiomerically pure compounds. experimental reagent grade chemicals and solvents were purchased from commercial supplier and used without purification. tlc was performed on silica gel f254 plates (merck). silica gel (100–200 mesh) was used for column chromatographic purification. melting points are uncorrected and were measured in open capillary tubes, using a rolex melting point apparatus. ir spectra were recorded as kbr pellets on perkin elmer rx spectrometer. 1h-nmr and 13c -nmr spectral data were recorded on advance bruker 300 spectrometer (300 mhz) with cdcl3 as solvent and tms as internal standard. j values are in hz. mass spectra were obtained by agilent 5973network mass selective detector (ei). general procedure for the preparation of oximes 2a–f acidic hydroxylamine (nh2oh·hcl, 0.1 mol) was added dropwise to a stirred solution of substituted acetophenone (0.02 mol) in 95 % etoh (150 ml) and pyridine (8 ml, 0.1 mol) ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. synthesis of oxime pseudoesters 3 at room temperature. the resulting mixture was refluxed for 0.5–2 h (until the starting material was completely consumed as indicated by tlc), and cooled in cold water for1 h. the precipitate was collected by suction, washed with warm water (3×50 ml) and dried in a vacuum oven. the crude compound was recrystallized in ethanol to give a white solid. synthesis of n-phthaloyl-l-amino acids 4a–d a solution of the required amino acid (1 equiv.) in toluene was added to a solution of phthalic anhydride (1 equiv.) and triethylamine (1.2 equiv.). the reaction mixture was then refluxed on a water bath for about 4–5 h. after completion of the reaction, the resulting solution was separated. the organic phase was washed with water until neutral, dried over mgso4 and filtered. the filtrate was evaporated and the residue purified by column chromatography on silica gel to give the required compounds (4a–d). synthesis of oxime pseudoesters 5a–k a mixture of an n-phthaloyl-l-amino acid 4a–d (1 equiv.) and thionyl chloride (2 ml, slow addition) were mixed together and the contents were heated at 55 °c for 4 h. the reaction mixture was cooled to room temperature and kept in an ice bath. then, a solution of oxime 2 (1 equiv.), triethylamine (1.2 equiv.) in dichloromethane (ch2cl2, 30 ml) was added subsequently to the reaction mixture and the contents were stirred at room temperature for 2 h. on completion of the reaction, checked by thin-layer chromatography (tlc) analysis, the solvent was removed under reduced pressure and extracted with dichloromethane (3×30 ml). the organic layer was dried over anhydrous mgso4 and concentrated under reduced pressure. the thus obtained residue was purified by silica gel chromatography (hexane/ethyl acetate) to afford the desired oxime pseudoesters derivatives (5a–k). cell lines and culture medium the human colon carcinoma cells (caco-2; ecacc 86010202) and the epidermoid carcinoma epithelial cells (hep-2; atcc ccl-23) were cultured in dulbecco’s modified eagle medium (dmem) supplemented with 10 % of fetal bovine serum, 1 % non-essential amino acids and 1 % penicillin/streptomycin (invitrogen). at 85–90 % confluence, the cells were harvested using a 0.25 % trypsin/edta solution and sub-cultured onto 96-well plates according to the experimental requirements. cytotoxicity screening assay the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) colorimetric assay described earlier19 was used to screen the cytotoxic activity of the isolated compound. briefly, the caco-2 and hep-2 cell lines (1×105 cells/well) were grown overnight on 96-well flat bottom cell culture plates, incubated 24 h. when a partial monolayer had formed, the supernatant was flicked off, the monolayer washed once with medium and 100 μl of different concentrations (10, 5, 2.5 and 1.25 mg ml-1) of pure compound were added to the cells.in the microtitre plates. after 24 h, the cells were washed and treated with 0.01 ml mtt reagent (invtrogen) prepared in 5.0 mg ml-1 phosphate buffered saline (pbs) per well. the plates were incubated at 37 °c in a 5 % co2 atmosphere for 4 h, and 0.1 ml dimethyl sulfoxide (dmso) was added. after overnight incubation at 37 °c, the absorbance was measured at 550 nm using an elisa reader (thermo scientific multiskan fc) and was compared with the control cultures without compound. the results were generated from 3 independent experiments and each experiment was performed in triplicate. the percentage growth inhibition was calculated using following formula: cell inhibition, % = 100–100{(at–ab)/(ac–ab)} ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 4 mehrez et al. where, at = absorbance value of the test compound, ab = absorbance value of the blank and ac = absorbance value of the control. stock solutions (5 mg/ml) of pure compounds were prepared in dimethyl sulfoxide (dmso) and the final concentration of this solvent was kept constant at 0.25 %. serial dilutions with culture media were prepared just prior to the test. statistical analysis the results are expressed as mean ± sem. the data were statistically analyzed by oneway analysis of variance (anova) to determine differences among the groups and the tukey test as a post-hoc. all the statistical analysis was conducted using statistical package for social science (spss for windows; v. 19.0, usa) and differences were considered statistically significant at p < 0.05. analytical and spectral data of the synthesized compounds are given in the supplementary material to this paper. results and discussion the substituted oximes 2a–f were synthesized according to the literature procedure6b,20 shown in scheme 1. the condensation of acetophenone derivatives 1a–f with hydroxylamine hydrochloride and pyridine gave white shiny coloured oximes 2a–f in 90 % yield. o o o no o r1 o oh h 2 a-f toluene, et3n, reflux 1) socl2, 55°c, 4h ch3 o r ch3 n oh r r= h, ch3, och3, f, cl, br 1 a-f nh2oh.hcl, 100°c 4 a-d 5 a-k n oo o o nr1 h ch3 r 2) oxime 2, et3n, 0°c, ch2cl2 95% etoh, pyridine r1 o oh nh2 h 3 a-d 5a: r= ch3, r1= ch3 5e: r= br, r1= ch3 5i: r= h, r1= ch2c6h3(oh)2 5b: r= och3, r1= ch3 5f: r= ch3 , r1= ch2ch(ch3)2 5j: r= och3, r1= ch2c6h3(oh)2 5c: r= f, r1= ch3 5g: r= cl, r1= ch2ch(ch3)2 5k: r= cl, r1= ch2c6h3(oh)2 5d: r= cl, r1= ch3 5h: r= ch3, r1= ch(ch3)ch2ch3 scheme 1. synthesis of oxime pseudoesters 5a–k. the α-n-phthalimido amino acids (compounds 4a–d) were synthesized according to a method described in the literature18a by allowing phthalic anhydride to react with a number of commercially available amino acids in refluxing apolar solvents, such as toluene, in the presence of triethylamine and separation of the formed water (scheme 1). ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. synthesis of oxime pseudoesters 5 treatment of 4a–d with thionyl chloride followed by treatment with keto oxime in anhydrous dichloromethane in the presence of et3n at 0 °c to room temperature provided the corresponding n-substituted phthaloyl derivatives 5a–k with an average yield of 76 % in two steps and after purification. their structures were established by ir, 1h-nmr, 13c-nmr and mass spectrometry. the antiproliferative potential of the synthesized compounds 5a–d, 5f and 5h–j was determined in vitro against two cancer cell lines, i.e., hep-2 and caco-2. the cytotoxicity values were obtained as inhibition of different concentrations and the results are summarized in tables i and ii. table i. cytotoxic activity of some derivatives against caco-2 cells; the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) colorimetric assay described earlier19 was used to screen the cytotoxic activity of the isolated compound compound concentration, mg ml-1 10 5 2.5 1.25 5a < 20 % < 20 % < 20 % < 20 % 5b 59.67±1.91 40.98±1.22 22.56±2.03 25.17±1.15 5c 52.94±1.22 43.01±2.16 41.37±0.9 35.01±0.85 5d 56.19±0.78 44.41±1.08 37.96±1.2 29.06±0.63 5f < 20 % < 20 % < 20 % < 20 % 5h < 20 % < 20 % < 20 % < 20 % 5i 41.03±3.54 40.15±1.56 28.92±1.94 14.02±0.76 5j < 20 % < 20 % < 20 % < 20 % table ii. cytotoxic activity of some derivatives against hep-2 cells compound concentration, mg ml-1 10 5 2.5 1.25 5a < 20 % < 20 % < 20 % < 20 % 5b 48.22±1.12 20.15±0.86 11.02±1.61 7.89±0.74 5c 57.85±2.13 43.21±1.92 21.11±0.83 27.02±2.35 5d 61.02±1.69 56.12±2.01 31.12±0.77 10.25±0.92 5f < 20 % < 20 % < 20 % < 20 % 5h < 20 % < 20 % < 20 % < 20 % 5i 53.91±1.6 50.77±2.51 17.01±1.38 9.49±1.02 5j < 20 % < 20 % < 20 % < 20 % the results demonstrated that only four compounds, 5b–d and 5i, have moderate potency of around 40 % inhibition at 10 mg ml–1 against hep-2 and caco-2, while the other compounds, 5a, 5f, 5h and 5j were inactive against the two cancer cell lines (inhibition effect < 20 %). it was observed that when the methoxy group was attached at the para position of the phenyl ring (compound 5b), the activity reduced to 48.22 % against the hep-2 cell line. replacing the substituent at the para position by an electron withdrawing group caused a decrease in the anticancer activity as compared to compound 5b against the caco-2 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 6 mehrez et al. cell line. this could be justified by the fact that compounds bearing electron withdrawing groups, such as fluoro (5c) and chloro (5d) substituents at the para position of the phenyl ring exhibited activity at inhibitory ratios values 52.94 and 56.19 %, respectively, against the caco-2 cell line and 57.85 and 61.02 %, respectively, against the hep-2 cell line. compound 5i also showed activity against hep-2 at 53.91 and 41.03 % against caco-2 cell lines. it could be concluded that the cytotoxicities of the resulting oxime pseudoesters derivatives are significantly correlated with the nature of the substituent group. conclusions in summary, a new series of optically pure phthalimido oxime pseudoesters derivatives were prepared and their cytotoxic activities against two human cancer cells lines, caco-2 and hep-2, evaluated. some derivatives exhibited strong cytotoxic activity and therefore, further structural modifications and in vivo anti -tumor activity studies are to be undertaken. the present findings could provide new evidence showing the relationship between chemical structure and biological activity and may be useful for the design of novel chemotherapeutic drugs. supplementary material analytical and spectral data of the synthesized compounds are available electronically at the pages of journal website: http://www.shd-pub.org.rs/index.php/jscs, or from the corresponding author on request. и з в о д синтеза нових псеудоестара фталимидо оксима и процена њихове цитотоксичности asma mehrez1, ibtissem chakroun2, dalila mtat1, hédi ben mansour3 и ridha touati1 1 laboratory of asymmetric organic synthesis and homogeneous catalysis (ur11es56), faculty of sciences of monastir, avenue of the environment, 5019 monastir, tunisia, 2 laboratory for analysis, treatment and recovery of environmental pollutants and products, faculty of pharmacy, avicenne street, 5000 monastir, tunisia и 3 research unit analyses and processes applied to the environment (ur17es32), higher institute of applied sciences and technology of mahdia, 5111 mahdia, tunisia синтетисана је серија нових, оптички чистих деривата псеудоестара оксима, реакцијом супституисаних кето-оксима са различитим хлоридима n-супституисаних α-амино киселина, у присуству триетил-амина, у дихлорметану на 0 °c. њихове структуре су утврђене ir и 1d-nmr спектроскопским методама. испитана је активност синтетисаних једињења према инхибицији пролиферације хуманог канцера дебелог црева и хуманих епителних ћелија. нека од испитиваних једињења показују значајан цитотоксичан ефекат. 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(cc) 2019 scs. 8 mehrez et al. 18. a) a. essersi, r. touati, b. ben hassine, lett. org. chem. 7 (2010) 69 (http://dx.doi.org/10.2174/157017810790533869); b) a. ould aliyenne, j. kraiıem, y. kacem, b. ben hassine, c. r. chim. 10 (2007) 251 (http://dx.doi.org/10.13140/rg.2.1.1067.0247); c) y. kacem, a. bouraoui, v. ratovelomanana-vidal, j. p. genêt, b. ben hassine, c. r. chim. 5 (2002) 611 (http://dx.doi.org/s1631-0748(02)01418-2) 19. m. krifa, a. della, i. bouhlel, j. robert, a. cherif, d. barillier, r. mosrati, l. chekirghedira, h. ben mansour, environ sci. pollut. res. 20 (2013) 3842 (http://dx.doi.org/10.1007/s11356-012-1322-1) 20. w. w. tan, b. wu, y. wei, n. yoshikai, org. synth. 95 (2018) 1 (https://doi.org/10.15227/orgsyn.95.0001). ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. {synthesis and structural analysis of polynuclear silver(i) complexes with 4,7-phenanthroline} j. serb. chem. soc. 84 (7) 689–699 (2019) udc 547.677.5+546.571:543.42:548.0–77 jscs–5219 original scientific paper 689 synthesis and structural analysis of polynuclear silver(i) complexes with 4,7-phenanthroline ivana m. stanojević#, nada d. savić1*#, aurélien crochet2, katharina m. fromm2, miloš i. djuran3# and biljana đ. glišić1**# university of niš, faculty of agriculture, kosančićeva 4, 37000 kruševac, serbia, 1university of kragujevac, faculty of science, department of chemistry, radoja domanovića 12, 34000 kragujevac, serbia, 2department of chemistry, university of fribourg, chemin du musée 9, ch-1700 fribourg, switzerland and 3serbian academy of sciences and arts, knez mihailova 35, 11000 belgrade, serbia (received 26 february, revised 8 march, accepted 18 march 2019) abstract: new polynuclear silver(i) complexes, [ag(cf3so3)(4,7 -phen)(ch3cn)]n (1) and [ag(po2f2)(4,7-phen)]n (2), were synthesized by the reaction of 4,7-phenanthroline (4,7-phen) and the corresponding agx salt (x = cf3so3 and pf6 -) in 1:2 mole ratio, respectively, in methanol/acetone (1:1 volume ratio) at room temperature. the characterization of the complexes was established on the basis of elemental microanalysis, ir and nmr (1h and 13c) spectroscopic techniques, while their crystal structures were determined by single-crystal x-ray diffraction analysis. the results of spectroscopic and crystallographic analyses revealed that in these complexes, 4,7-phen behaves as a bridging ligand between two metal ions, while the remaining coordination sites of the ag(i) ions are occupied by the oxygen atom of cf3so3 and an acetonitrile nitrogen atom in 1 or by two oxygen atoms from two po2f2 -, formed after hydrolysis of pf6 -, in 2. in the solid state, both complexes are coordination polymers in which the geometry around the ag(i) ions is distorted tetrahedral. keywords: silver(i) complexes; 4,7-phenanthroline; polynuclear complexes; spectroscopy; x-ray crystallography. introduction silver(i) complexes with aromatic nitrogen-containing heterocycles (n-heterocycles) are of great importance in the field of medicinal chemistry and the coordination polymer design.1,2 generally, silver(i) complexes with these ligands are known to possess significant antibacterial activity against a wide range of gram-positive and gram-negative bacteria and have shown remarkable activity against different candida species.3–14 moreover, they have manifested cytotoxic *,** corresponding authors. e-mail: (*)nada.savic@kg.ac.rs; (**)biljana.glisic@pmf.kg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc190226024s 690 stanojević et al. activity against different human tumor cell line, being more active and less toxic to humans than the clinically used platinum(ii) complexes.15 their effectiveness is thought to be the consequence of the presence of a weak ag−n bond, which can be easily cleaved in the interaction with thiol-containing proteins and dna, the process that is a prerequisite for their action.3 besides the significant antimicrobial and antiproliferative activities of silver(i) complexes with aromatic n-heterocycles, the ag(i) ions coordinated by this type of ligands are favorable building blocks for coordination polymers which may find interesting applications for the development of innovative materials, such as liquid crystals.1,16,17 coordination polymers based on ag(i) ions are attracting great attention due to the flexibility of the coordination sphere of this metal ion.1,16,17 thus, it can adopt coordination numbers between two and six and form complexes of various geometries such as linear, bent, trigonal, t-shaped, tetrahedral, trigonal pyramidal and octahedral. besides that, weak contacts such as ag⋅⋅⋅ag, ag⋅⋅⋅π and ag⋅⋅⋅solvent/counterion interactions significantly affect the geometry and topology of the silver(i)-based coordination polymers in the solid state.1,18 in the design of silver(i) coordination polymers having different possible applications, various bridging and chelating n-heterocyclic ligands have been used. among them, phenanthrolines have been investigated due to their coordination diversity which allowed the tuning of the nuclearity and biological activities in a series of silver(i) complexes.5,13,14 very recently, five silver(i) complexes with 4,7-phenanthroline ligand (4,7-phen), [ag(no3)(4,7-phen)]n, [ag(clo4)(4,7-phen)]n, [ag(cf3coo)(4,7-phen)]n, [ag2(h2o)0.58(4,7-phen)3] (sbf6)2 and {[ag2(h2o)(4,7-phen)2](bf4)2}n, have been synthesized and biologically evaluated, showing higher selectivity towards candida spp. in comparison to bacteria, while being only moderately cytotoxic against healthy human fibroblasts.14 moreover, [ag(no3)(4,7-phen)]n and [ag(cf3coo)(4,7-phen)]n complexes were effective in vivo rescuing zebrafish embryos from lethal c. albicans infection and reducing the fungal burden by preventing fungal filamentation.14 in the present study, 4,7-phen reacted with two agx salts (x = cf3so3– and pf6–), yielding new polynuclear silver(i) complexes, [ag(cf3so3)(4,7 -phen)(ch3cn)]n (1) and [ag(po2f2)(4,7-phen)]n (2). these complexes were fully characterized by spectroscopy (ir, 1hand 13c-nmr) and single-crystal x-ray crystallography. experimental reagents silver(i) salts (agcf3so3 and agpf6), 4,7-phenanthroline (4,7-phen), methanol, acetone, acetonitrile, dimethyl sulfoxide (dmso) and deuterated dimethyl sulfoxide (dmso-d6) silver(i) complexes with 4,7-phenanthroline 691 were purchased from the sigma–aldrich chemical co. all the employed chemicals were of analytical reagent grade and used without further purification. measurements elemental microanalysis of the silver(i) complexes for carbon, hydrogen and nitrogen was performed at the adolphe merkle institute of the university of fribourg. the nmr spectra were recorded at 25 °c on a varian gemini 2000 spectrometer (1h at 200 mhz, 13c at 50 mhz). 5.0 mg of 4,7-phen and the corresponding silver(i) complex was dissolved in 0.6 ml of dmso-d6 and transferred into a 5 mm nmr tube. chemical shifts are expressed in ppm (δ / ppm) and scalar couplings are reported in hertz (j / hz). chemical shifts were calibrated relative to those of the solvent. the ir spectra were recorded as kbr pellets on a perkin–elmer spectrum one ft-ir spectrometer over the wavenumber range of 4000–450 cm-1. analytical and spectral data of the compounds are given in supplementary material to this paper. synthesis of complexes 1 and 2 10.0 ml of methanolic solution of the corresponding silver(i) salt (0.50 mmol, 128.4 mg of agcf3so3 for 1 and 126.4 mg of agpf6 for 2) was added dropwise to a solution of 4,7 -phen (0.25 mmol, 45.0 mg) in 10.0 ml of warm acetone with stirring at room temperature. the stirring was continued for 3 h in the dark at room temperature, the white precipitate was filtered off and dissolved in acetonitrile. the obtained solution was left to evaporate slowly at room temperature. after 3–5 days, colorless crystals of 1 and 2 suitable for single crystal x-ray analysis were formed. these crystals were filtered off and dried in the dark at room temperature. yield (calculated on the basis of the n-heterocyclic ligand): 73 % (87.3 mg) for 1 and 66 % (64.2 mg) for 2. crystallographic data collection and refinement of the structure crystal data and details of the structure determinations are listed in table i. a suitable crystal was mounted on a mylar loop in oil on a stoe ipds 2 diffractometer. the crystals were kept at 250(2) k for 1 and 293(2) k for 2 during data collection. using olex2,19 the structures were solved with the shelxt20 structure solution program using intrinsic phasing and refined with the shelxl21 refinement package using least squares minimization. drawings were prepared with mercury computer graphics program.22 table i. details of the crystal structure determination of [ag(cf3so3)(4,7-phen)(ch3cn)]n (1) and [ag(po2f2)(4,7-phen)]n (2) complexes property 1 2 empirical formula c15h11agf3n3o3s c12h8agf2n2o2p formula weight 478.20 389.04 crystal system, space group monoclinic, p21/c monoclinic, p21/c a / å 13.6834(11) 10.3939(4) b / å 8.0480(5) 14.7587(5) c / å 15.5345(13) 8.0940(5) α / ° – – β / ° 103.199(6) 97.683(4) γ / ° – – v / å3 1665.5(2) 1230.48(10) f000 944 760 ζ 4 4 692 stanojević et al. table i. continued property 1 2 x-radiation, λ / å mo-kα 0.71073 mo-kα 0.71073 data collect. temperature, k 250(2) 293(2) calculated density, mg m-3 1.907 2.100 absorption coefficient, mm-1 1.389 1.794 crystal size, mm3 0.270 × 0.190 × 0.110 0.260 × 0.153 × 0.040 2θ range, ° 5.3 to 50.3 3.9 to 50.3 index ranges h, k, l -16 ... 16, -9 ... 9, -18 ... 18 -12 ... 12, -17 ... 17, -9 ... 9 no. of collected & indep. reflections 20622, 2962 15604, 2192 rint 0.0842 0.0315 data / restraints / parameters 2962 / 34 / 237 2192 / 0 / 181 goodness-on-fit on f2 1.066 1.047 final r indices [i ≥ 2σ(i)] 0.0597, 0.1595 0.0269, 0.0687 final r indices (all data) 0.0679, 0.1660 0.0325, 0.0711 difference density: max, min / e å-3 0.94, –1.86 0.67, –0.49 ccdc number 1899714 1899713 results and discussion synthesis and structural features of complexes 1 and 2 the silver(i) complexes with 4,7-phenanthroline (4,7-phen) were synthesized according to the route presented in scheme 1. the reactions between 4,7 -phen and agx (x = cf3so3– and pf6–) in 1:2 mole ratio were performed in methanol/acetone (1:1 volume ratio) at room temperature yielding the polynuclear [ag(cf3so3)(4,7-phen)(ch3cn)]n (1) and [ag(po2f2)(4,7-phen)]n (2) complexes. the composition and structural formula of both silver(i) complexes were consistent with the elemental analysis, ir and solution nmr (1h and 13c) spectroscopic results and were also supported by single-crystal x-ray diffraction analyses. in these complexes, 4,7-phen acts as a bridging ligand between two metal ions, being in accordance with its coordination mode in the previously synthesized silver(i) complexes with this n-heterocycle.14 in complex 1, the cf3so3– is monodentately coordinated to the ag(i) ion, while the fouth coordination site is occupied by the acetonitile nitrogen atom. instead of the expected pf6– in 2, this complex contains two bridging po2f2– that are monodentately coordinated to ag(i) ion via oxygen atoms. it can be assumed that po2f2– was formed by the hydrolysis of pf6–, a process previously described in the literature:23,24 pf6– + h2o → pof4– + 2hf (1) pof4– + h2o → po2f2– + 2hf (2) description of the single crystal structure the molecular structures of silver(i) complexes 1 and 2 with the anisotropic displacement ellipsoids and the atom numbering scheme are shown in fig. 1, silver(i) complexes with 4,7-phenanthroline 693 while the selected bond distances, å and angles, °, with the estimated standard deviations are given in table ii. scheme 1. schematic presentation of the synthesis of [ag(cf3so3)(4,7-phen)(ch3cn)]n (1) and [ag(po2f2)(4,7-phen)]n (2) complexes.the solid products of these reactions were recrystallized in acetonitrile. the numbering scheme of carbon atoms in 4,7-phen is in agreement with iupac recommendations for fused ring systems and does not match the one applied in the x-ray analysis of silver(i) complexes. a b fig. 1. molecular structures of: a) [ag(cf3so3)(4,7-phen)(ch3cn)]n (1) and b) [ag(po2f2)(4,7-phen)]n (2) complexes. displacement ellipsoids are drawn at 50 % probability level and h atoms are represented by spheres of arbitrary size. symmetry codes for complex 1: #1: 1–x, 1/2+y, 3/2–z and for 2: #1: 1–x, -1/2+y, 1/2–z, #2: x, 3/2–y, 1/2+z. 694 stanojević et al. table ii. selected bond distances and valence angles (°) in silver(i) complexes 1 and 2; symmetry code: (#2) x, –y+3/2, z–1/2 1 2 bond bond distance, å bond bond distance, å ag1—n1 2.305(5) ag1—n1 2.240(3) ag1—n2 2.251(5) ag1—n2 2.252(3) ag1—n3 2.284(7) ag1—o1 2.500(3) ag1—o1 2.505(6) ag1—o2 2.499(3) valence angles, ° valence angles, ° n1—ag1—n2 130.94(17) n1—ag1—n2 143.77(10) n2—ag1—n3 128.3(2) n1—ag1—o1 108.67(10) n1—ag1—n3 96.5(2) n1—ag1—o2 115.62(9) n1—ag1—o1 98.1(2) n2—ag1—o1 90.84(10) n2—ag1—o1 94.7(2) n2—ag1—o2 95.47(10) n3—ag1—o1 97.8(3) o1—ag1—o2 85.80(11) c1—n1—ag1 120.9(4) c1—n1—ag1 121.2(2) c5—n1—ag1 121.6(4) c5—n1—ag1 121.2(2) c12—n2—ag1 121.2(4) c12—n2—ag1 117.0(2) c8—n2—ag1 121.8(4) c8—n2—ag1 123.7(2) c14—n3—ag1 162.2(9) p1—o1—ag1 122.28(16) s1—o1—ag1 145.9(4) p1#2—o2—ag1 151.58(19) in the solid state, both complexes 1 and 2 are coordination polymers. in 1, each ag(i) ion is surrounded by two 4,7-phen, one acetonitrile and one monodentately coordinated trifluoromethanesulfonate (triflate) anion (fig. 1a). an extended view of polynuclear silver(i) complex 1 is shown in fig. 2a. this complex has a distorted tetrahedral geometry, what can be concluded from the value of τ4 parameter25 of 0.71, τ4 = [360° – (β + α)]/141°, where β and α are the largest angles around the ag(i) ion (β = n1—ag1—n2 = 130.9(2)° and α = n2—ag1—n3 = 128.3(2)°). the ag1–n1/n2(4,7-phen) bond distances in 1 (table i) adopt values of 2.305(5) and 2.251(5) å, respectively, and are comparable with those observed in the other pseudo tetrahedral silver(i) complexes with aromatic n-heterocycles.8–14 the ag1–n3 (acetonitrile) bond distance of 2.284(7) å falls in the normal range of 2.18–2.33 å.26 on the other hand, the ag1–o1 bond distance of 2.505(6) å is much longer than usual covalent silver(i)–oxygen bonds of approximately 2.3 å.8 in complex 1, two ag(i) ions are connected by one 4,7-phen, which behaves as a bridging ligand. the intramolecular ag⋅⋅⋅ag interaction is not observed in this complex, considering the fact that ag∙∙∙ag distance of 7.689 å is much longer than the commonly ag∙∙∙ag bond range of 2.853–3.290 å.27 similar to 1, complex 2 has a distorted tetrahedral geometry (τ4 = 0,71; fig. 1b and table ii). in this coordination polymer, each ag(i) ion is coordinated by two 4,7-phen and two po2f2– (fig. 1). two ag(i) ions are bridged by one 4,7 -phen and one po2f2–, with d(ag–n) = 2.240(3) and 2.252(3) å and d(ag–o) = silver(i) complexes with 4,7-phenanthroline 695 2.500(3) and 2.499(3) å (an extended view of 2 is presented in fig. 2b). the ag– –n(4,7-phen) bond lengths in 2 are slightly shorter than in 1, while the ag–o bond distances are comparable in these two silver(i) complexes (table ii). a b fig. 2. an extended view of polynuclear silver(i) complexes: a) 1 and b) 2. the coordination mode of 4,7-phenanthroline in silver(i) complexes 1 and 2 is the same as in the previously characterized silver(i) complexes with this aromatic n-heterocycle.14 almost all of the synthesized silver(i) complexes with 4,7-phen are polynuclear species, with the exception of [ag2(h2o)0.58(4,7 -phen)3](sbf6)2 complex, which is obtained in the reaction of agsbf6 with an equimolar amount of 4,7-phen in the ethanolic solution.14 contrary to 4,7-phen, its structural isomer 1,7-phenanthroline affords mononuclear silver(i) complexes, namely [ag(no3)(1,7-phen)2] and [ag(1,7-phen)2]x (x = clo4–, cf3so3–, bf4– and sbf6–), in which it is monodentately coordinated via the less sterically hindered n7 nitrogen atom.12 696 stanojević et al. spectroscopic characterization the ir and nmr (1h and 13c) spectroscopic data for silver(i) complexes 1 and 2 are given in supplementary material. the ir spectra of the complexes show the bands which can be attributed to the coordinated 4,7-phen, cf3so3– and po2f2– ligands. thus, in the ir spectrum of 1 with coordinated triflate, a number of strong absorptions in the 1300–1000 cm–1 region can be observed. the bands at 1263, 1255 and 1031 cm–1 can be assigned to the asymmetric and symmetric stretching modes of the –so3 group of the triflate anion.28,29 the splitting of the band corresponding to asymmetric stretching vibration of –so3 group is an indication of the monodentate coordination of the triflate in 1.29 the two bands at 1247 and 1168 cm–1 are due to the symmetric and asymmetric stretching modes of –cf3 group, respectively. additionally, the medium intensity bands at 635 and 517 cm–1, and at 754 and 594 cm–1 can be ascribed to the symmetric and asymmetric deformations of –so3 and –cf3 groups in cf3so3–, respectively.29 the hydrolysis of pf6– and the coordination of po2f2– to ag(i) in 2 can be also confirmed from the ir spectrum of this complex. beside the very strong band at 838 cm–1 which is attributed to the ν(pf) mode, two bands at 1305 and 1149 cm–1 due to the ν(po) indicate that the hydrolysis of pf6– to po2f2– occurred in the investigated reaction.23 solution state 1hand 13c-nmr spectra were recorded in deuterated dmso with the aim to confirm the coordination of 4,7-phen to the ag(i) ion. the spectra of the complexes 1 and 2 were compared with those for the free ligand. in the aromatic region, 1h-nmr spectra of the complexes contain the same number of signals as that of the 4,7-phen, indicating that symmetric species are present in solution and that the ligand is coordinated to the ag(i) ion via both donor nitrogen atoms (n4 and n7). however, the resonances for these protons are only slightly shifted downfield compared to those of the free 4,7-phen (up to +0.05 for h5/h6 in 2). only small shifts of the resonances of the silver(i) complexes with respect to those for the corresponding ligand seem to be characteristic spectroscopic feature of the silver(i) complexes in solution and was assumed to be the consequence of the fast ligand exchange on the nmr timescale.6 in addition, a singlet at 2.07 ppm, attributed to the acetonitrile protons, is observed in the spectrum of 1. the 13c-nmr spectra were found to be almost identical for the both silver(i) complexes, excluding resonances assigned to the carbon atoms of the coordinated acetonitrile in 1. similar to the proton resonances, upon 4,7-phen coordination to ag(i) ion, the signals of its carbon atoms remain almost unaffected. conclusion we have shown that the reactions between 4,7-phen and agx salts (x = cf3so3– and pf6–) in 1:2 mole ratio, respectively, in methanol/acetone lead to the formation of polynuclear silver(i) complexes, [ag(cf3so3) (4,7-phen)(ch3cn)]n silver(i) complexes with 4,7-phenanthroline 697 and [ag(po2f2)(4,7-phen)]n. the crystallographic results revealed that these complexes have a distorted tetrahedral geometry with 4,7-phen being bridging ligand between two ag(i) ions. the present results are in accordance with those previously reported for the reactions of the same n-heterocyclic ligand with different agx salts (x = no3–, cf3coo–, bf4–, clo4– and sbf6–), most of them leading to the formation of polynuclear silver(i) species with different geometry of ag(i) metal ion.14 the only exception was a dinuclear [ag2(h2o)0.58(4,7 -phen)3](sbf6)2 complex, obtained in the reaction of agsbf6 with 4,7-phen in ethanol.14 this study confirms that the nuclearity and geometry of the silver(i) complexes with aromatic n-heterocyclic ligands strongly depend on the reaction conditions, such as starting silver(i) compound, the nature of n-heterocyclic ligand and reaction solvent. all these together should be carefully considered during preparation of new silver(i) complexes for different applications in medicinal and supramolecular chemistry. supplementary material analytical and spectral data are available electronically from http://www.shd.org.rs/ /jscs/, or from the corresponding author upon request. acknowledgements. this research has been financially supported by the ministry of education, science and technological development of the republic of serbia, under grant no. 172036, the supramedchem@balkans.net scopes institutional partnership (project no. iz74z0_160515) and the serbian academy of sciences and arts (project no. f128). и з в о д синтеза и структурна анализа полинуклеарних комплекса сребра(i) са 4,7-фенантролином ивана м. станојевић, нада д. савић1, aurélien crochet2, katharina m. fromm2, милош и. ђуран3 и биљана ђ. глишић1 универзитет у нишу, пољопривредни факултет, косанчићева 4, 37000 крушевац, 1универзитет у крагујевцу, природно–математички факултет, институт за хемију, радоја домановића 12, 34000 крагујевац, 2department of chemistry, university of fribourg, chemin du musée 9, ch-1700 fribourg, switzerland и 3српска академија наука и уметности, кнез михаилова 35, 11000 београд полинуклеарни комплекси сребра(i), [ag(cf3so3)(4,7-phen)(ch3cn)]n (1) и [ag(po2f2)(4,7-phen)]n (2), синтетисани су у реакцијама између 4,7-фенантролина (4,7 -phen) и одговарајућих agx соли (x = cf3so3 и pf6 ) у молском односу 1:2 у смеши метанол/ацетон (запремински однос 1:1) на собној температури. ови комплекси су окарактерисани применом елементалне микроанализе, ir и nmr ( 1 h и 13 c) спектроскопије, док је њихова кристална структура одређена применом методе дифракције x-зрака са монокристала. резултати спектроскопских и кристалографских испитивања показују да је у овим комплексима, 4,7-phen мостни лиганд између два јона метала, док се за преостала два координациона места координује атом кисеоника из cf3so3 и атом азота из ацетонитрила у комплексу 1, односно атоми кисеоника из два po2f2 , добијена хидролизом pf6 , у комплексу 2. у чврстом стању, комплекси сребра(i) су координациони полимери, у којима је геометрија сребро(i) јона дисторгована тетраедарска. 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(2004) 2086 (https://doi.org/10.1002/ejic.200300835) 27. a. melaiye, z. sun, k. hindi, a. milsted, d. ely, d. h. reneker, c. a. tessier, w. j. youngs, j. am. chem. soc. 127 (2005) 2285 (https://doi.org/10.1021/ja040226s) 28. d. johnston, d. f. shriver, inorg. chem. 32 (1993) 1045 (https://doi.org/10.1021/ic00058a050) 29. g. a. van albada, w. j. j. smeets, a. l. spek, j. reedijk, inorg. chim. acta 260 (1997) 151 (https://doi.org/10.1016/s0020-1693(96)05554-5). {furofuran lignans of artemisia genus: isolation, biosynthesis and biological activity} j. serb. chem. soc. 85 (5) 575–600 (2020) jscs–5323 review 575 review furofuran lignans of artemisia genus: isolation, biosynthesis and biological activity jovana d. ickovski, jovana lj. pavlović, milan n. mitić, ivan r. palić, danijela a. kostić, goran m. petrović and gordana s. stojanović*# department of chemistry, faculty of science and mathematics, višegradska 33, 18000 niš, serbia (received 10 december 2019, revised 20 january, accepted 31 january 2020) abstract: since ancient times, medicinal plants and pharmacologically active products obtained from different natural sources play an important role in human health. plants belonging to the genus artemisia possess a great biological potential and it is a well-studied genus in the fields such as systematics (including molecular phylogenetics) and genome organization. many species of the genus (e.g., a. absinthium, a. annua, a. vulgaris, a. abrotanum, a. arborescens) are widely exploited, because of their high economic value as medicines, food and ornamentals. withal, in such a large genus, some hiatus must inevitably exist, concerning attainments and potentials that individual species possess. most of the studies are focused on bioactivity and pharmacology of sesquiterpene lactones. lignans are unjustly neglected, even though they as well exhibit a wide range of bioactivities. motivated by that fact, we tried to consolidate findings on bioactive lignans accumulated through the years, with the logical perspectives on further work on isolation and identification of new bioactive lignans and the exploitation of lignans as substances of potential pharmacological interest. keywords: artemisia; lignan; sesamin; sesartemin; diayangambin; epiyangambin. contents 1. introduction 1.1. genus artemisia l.: an overview 1.2. lignans 1.3. lignans of artemisia genus 2. lignans biosynthesis 3. isolation of lignans * corresponding author. e-mail: gocast@pmf.ni.ac.rs https://doi.org/10.2298/jsc191210009i ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 576 ickovski et al. 4. biological activity of furofuran lignans 5. conclusion 1. introduction 1.1. genus artemisia l.: an overview the genus artemisia l. is the largest of subtribe artemisiinae less. and tribe anthemideae cass., and one of the largest and most widely distributed genera of the family asteraceae, which has been divided into four subgenera (artemisia, absinthium, dracunculus and seriphidium),1–4 whose number is increased with the proposal of another subgenus endemic to north america (tridentatae).5 it is a heterogenous genus that comprises around 600 species at specific and subspecific levels, present in all continents but antarctica, mostly distributed in the northern hemisphere (temperate zones of europe, asia and north america), with no more than 25 species in the southern hemisphere.1 in addition to the widespread distribution and a large representation of species within the genus artemisia, the occurrence of the endemic species in certain areas is quite high. some examples are the whole subgenus tridentatae (rydb.) mcarthur in the western united states of america, where some of its species dominate landscapes, a. afra jacq. in south africa, a. argentea l’hér. in madeira, a. canariensis less. (a. thuscula cav.) in the canary islands, a. gorgonum webb. in hook in cape verde, a. granatensis boiss. in the spanish sierra nevada, a. magellanica sch. bip. in argentina, a. mauiensis skottsb. in hawaii (usa), a. melanolepis in the iranian mount damavand, a. molinieri quézel, barbero & r. loisel in only two locations in south-east france and a. negrei ouyahya in morocco.1 withal the genus has been the object of numerous systematic, including molecular phylogenetics4–11 and taxonomic studies.12,13 the genus has also been thoroughly studied from the phytochemical,14–18 pharmacological19–22 and biotechnological point of view.23–29 many of artemisia species have been frequently utilized for many various purposes such as medicines (a. cina, a. santonica l., a. maritime, a. herba-alba, a. pallens wallich ex besser, a. afra, a. ludoviciana nutt. for their antihelminic activity and a. annua, a. apiacea. hance, a. lancea vaniot, a. afra, a. abrotanum for their antimalarial activities),30–36 food (edible plants, condiments and ingredients of beverages: a. absinthium, a. dracunculus, a. genipi and related species and a. vulgaris)1,37,38 and ornaments (a. arborescens and a. vulgaris).39–41 an intensive investigation of the phytochemicals of the genus artemisia reveals that the artemisia species comprise mainly terpenoids, flavonoids, coumarins, caffeoylquinic acids, sterols and acetylenes.42–50 the literature review revealed that most of the attention was paid to bioactive constituents of the essential oils, with a major focus on sesquiterpene lactones with potential pharmacological and medicinal activity.51–55 despite the vast number of phytochemical compo________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ furofuran lignans of artemisia genus 577 sition related studies of species belonging to genus artemisia, majority of them are still based on the investigation of sesquiterpene lactones, as a consequence of their pharmacological activity.14,56–60 artemisinin, a cadinane-type sesquiterpene lactone with a 4,6-endoperoxide function, is an antimalarial drug derived from a. annua l.61 santonin, a sesquiterpene lactone, isolated from various asian species of artemisia genus, especially a. china and a. maritima, is responsible for antihelmintic activity.62 arglabin, a guaianolide type of sesquiterpene lactone, isolated from a. glabella kar. et kir. and a. myriantha, shows promising antitumor activity against different tumor cell lines.63 on the other side, lignans are still insufficiently explored. these compounds are found in diverse species of the plant kingdom, including members of pteridophytes, gymnosperms and angiosperms.64 although lignans exhibit a wide variety of bioactivities on plants, insects and mammals,65–69 they are of especial interest due to the unique antitumor-associated activities70–74 and reduction of lifestyle-related diseases (anti-inflammatory, immunosuppression, cardiovascular and antioxidant).75–79 the plant lignans most commonly distributed in foods are lariciresinol, matairesinol, pinoresinol and secoisolariciresinol. several other lignans are present in some foods, including medioresinol (in sesame seeds, rye and lemons), syringaresinol (in grains), sesamin and the lignan precursor sesamolin (in sesame seeds).80–82 the amount of lignans in food is generally low, with the exceptions of flaxseed and sesame seeds, which have a lignan content a hundred times higher than other dietary sources.82,83 the specific distribution and the low amount of production in plants, some of which are endangered species, restrain the efficient and stable production of beneficial lignans.84 therewithal, plant sources of lignans are frequently limited because of the high cost of plant collection, poor cultivation systems and long growth phase.85–89 an exhaustive literature survey on phytochemical reports of the genus artemisia reveals that the artemisia species comprises mainly furofuran lignans (2,6-diarylfurofurans).90–98 1.2. lignans lignans represent a large group of naturally occurring phenolic compounds, widely distributed within the plant kingdom. the term lignan originated from haworth,99 to describe a group of secondary plant metabolites, which molecular backbone consists of two phenylpropanoid (c6-c3) units. lignans are phenylpropane dimers linked via β-β′ (8-8′) carbon atoms, with a different degree of oxidation in the side-chain and a different substitution pattern in the aromatic moieties.100 the lignans are bioactive, non-nutrient, non-caloric phenolic plant compounds, and they should not be confused with lignins.82 lignans are stereospecific dimers of monolignols, coniferyl or cinnamyl alcohol, while lignins are racemic polymers built from the hydroxycinnamic alcohols, coniferyl alcohol and ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 578 ickovski et al. sinapyl alcohol, with minor amounts of p-coumaryl alcohol.82,101–103 based on the type of carbon skeleton, cyclization pattern and the way in which oxygen is incorporated in the molecule, lignans are classified into six subgroups: dibenzylbutanes, dibenzylbutyrolactones, arylnaphthalenes, dibenzocyclooctadienes, substituted tetrahydrofurans and 2,6-diarylfurofurans.100 in addition, these lignans can be further classified into three categories depending on the oxidation state of the c9(c9′) positions, which are located at the terminal of the propyl side chain: lignans with 9(9′)-oxygen, lignans without 9(9′)-oxygen and dicarboxylic acid lignans (fig. 1). fig. 1. lignans classification based on the type of carbon skeleton, cyclization pattern and oxidation state of the c9(c9′) positions. apart from the fact that lignans are structurally diverse, they show substantial diversity in the terms of enantiomeric composition.104 naturally occurring lignans have been found to exist exclusively as one enantiomer, or as enantiomeric mixtures with various enantiomeric compositions. the enantiomeric composition of the plant lignans in trees and medicinal herbs and shrubs is commonly known, and usually only one of the enantiomers occurs in a certain species.105–107 1.3. lignans of artemisia genus lignans are a large and diverse class of natural products composing of phenylpropanoid dimers in which c6-c3 units are linked by the central carbon of their propyl side chains. the furofuran lignans represent one of the major subclasses of the lignan family. due to their structural diversity and broad bioactivities, natural furofuran lignans have attracted increasing research attention. as previously mentioned, a literature survey on the type of lignans in members of the genus artemisia revealed that furofuran lignans are characteristic for artemisia species (lignan’s profile mainly consists of furofuran lignans). furofuran lignans have been ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ furofuran lignans of artemisia genus 579 found throughout the plants from roots, stems, leaves, bulbs, barks to seeds. research progress on the naturally occurring furofuran lignans within the plant species of artemisia genus reported in the literature (chemical structures, names, corresponding sources and references) is summarized in fig. 2. and table i. fig. 2. structures of lignans isolated form artemisia species. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 580 ickovski et al. table i. the lignans isolated from artemisia species plant plant part lignans ref. a. absinthium l. aerial parts 9, 1, 28, 27, 18 94 a. absinthium l. fresh roots 1, 2, 3, 4, 26, 27, 28, 9, 11, 12, 22, 14 92 a. absinthium l. aerial parts 28, 1, 27, 23, 18, 20, 26 108 a. absinthium l. fresh roots 6, 9, 1 91 a. arborescens l. aerial parts 6, 26, 27, 8, 11, 1, 15, 31, 25 92,95 a. canariensis less. fresh roots 6, 8, 1, 26, 27, 11 91 a. caruifolia buch.-ham. ex roxb. aerial parts 16, 17, 18, 19 97 a. gorgonum webb. aerial parts 21, 13, 14, 8, 5, 6 93 a. gorgonum webb. fresh roots 6, 8, 1, 26, 14, 11 92 a. jacutica drob. fresh roots 6, 8, 1, 26 92 a. macrocephala jacq. ex bess. fresh roots 1, 26 92 a. minor jacq. ex bess. aerial parts 23, 24 98 a. sieversiana willd. fresh roots 1, 26, 27, 28, 9, 8, 12, 11, 22, 14, 6 92 a. sieversiana willd. aerial parts 27, 28, 7, 29, 30, 10 96,90 a. austro-yunnanensis whole plant 25, 23 109 2. lignans biosynthesis phenylpropanoid metabolism is a convoluted network of biosynthetic pathways, which lead to the synthesis of a vast number of secondary metabolites. the plant shikimate pathway is the entry to the biosynthesis of phenylpropanoids, where just a few intermediates represent the core unit for the further biosynthesis of secondary metabolites, including flavonoids, isoflavonoids, lignins and lignans. the shikimate pathway results in the biosynthesis of chorismate, which is the branch point for the synthesis of aromatic amino acids tryptophan on the one hand and phenylalanine and tyrosine on the other hand.110,111 chorismic acid is transformed into prephenic acid via a claisen rearrangement, which transfers the phosphoenolpyruvate derived side-chain so that it becomes directly bonded to the carbocycle and thus builds up the basic carbon skeleton of phenylalanine. decarboxylative aromatization of prephenic acid yields phenylpyruvic acid and pyridoxal phosphate-dependent transamination leads to l-phenylalanine.112,113 the conversation of phenylalanine to the hydroxycinnamic acids (p-coumaric, ferulic and sinapic acids) and the monolignols (p-coumaryl, coniferyl and sinapyl alcohols) is the start point of phenylpropanoid pathway. phenylalanine ammonialyase and tyrosine ammonia-lyase catalyze the non-oxidative deamination of phenylalanine to trans-cinnamate and direct the carbon flow from the shikimate pathway to the various branches of the general phenylpropanoid metabolism.112 subsequent steps, i.e., hydroxylation of cinnamic acid by cinnamate 4-hydroxylase which leads to the biosynthesis of p-coumaric acid and activation of coumaric acid by 4-coumaroyl coa-ligase which leads to the biosynthesis of p-coumaroyl-coa, are mandatory and provide the basis for all subsequent branches and resulting metabolites.113,114 p-coumaroyl-coa is a precursor for the biosyn________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ furofuran lignans of artemisia genus 581 thesis of p-coumaryl alcohol and coniferyl alcohol. as part of monolignol biosynthesis (building blocks of lignans and lignins), p-coumaroyl-coa can undergo two types of modifications: reduction of the carboxyl group on the propane side chain to alcohol and substitution of the phenyl ring. the two predominant monolignols are coniferyl alcohol and sinapyl alcohol.114 although lignans and neolignans are abundant class of phytochemicals, little is known about the specific biosynthetic steps leading to the biosynthesis of complex lignans. during the years, the majority of the studies were devoted to understanding the biosynthesis of podophyllotoxin, thanks to which the biosynthesis of lignans with 9(9′)-oxygen is very well studied. these type of lignans are formed by enantioselective dimerization of two coniferyl alcohol units with the aid of a dirigent protein to give rise to pinoresinol (25, furofuran). pinoresinol (25) is then reduced to secoisolariciresinol (dibenzylbutane) by pinoresinol/lariciresinol reductase, via lariciresinol (furan), which is in turn oxidized to afford matairesinol (dibenzylbutyrolactone) by secoisolariciresinol dehydrogenase. the conversion from coniferyl alcohol to secoisolariciresinol has been demonstrated in various plant species (forsythia, linum and podophyllum), which strongly suggests that this is the general biosynthetic pathway of lignans.84,105,115 lignans and neolignans are normally found in optically active forms. they are composed of only one enantiomer or both, but with one of them being in excess. this implies that lignan biosynthesis is under strict enantioselective control.86 for example, (+)-pinoresinol (25) is found in f. suspense116,117 and (–)-secoisolariciresinol and (–)-matairesinol occur in f. intermedia.118 lignans are generally believed to be formed by a phenolic oxidative coupling process119 more precisely, by a large number of distinct radical coupling modes of phenoxyl radical. those coupling modes can be either stereoselective and/or regiospecific in coupling origin.120 the first demonstration of phenoxyl radical coupling control was reported during the investigation of (+)-pinoresinol (25) formation from coniferyl alcohol in forsythia species.121,122 it was suggested that the dirigent protein bind and orient coniferyl alcohol-derived radicals in such a way as to enable 8,8′ coupling at the si-si face with subsequent intramolecular cyclization to afford (+)-pinoresinol (25).122 since the initial discovery of this protein from f. intermedia, homology searches in sequence databases have revealed the existence of additional genes encoding putative dirigent proteins, from a variety of species. one of the proposed biosynthetic routes123 starts with coniferyl alcohol and subsequent formation of (+)-pinoresinol (25). the enzyme pinoresinol/lariciresinol reductase converts this compound to (+)-lariciresinol and then to (–)-secoisolariciresinol. the enzyme secoisolariciresinol dehydrogenase converts into (–) -matairesinol. the conversion from (–)-matairesinol to podophyllotoxin is likely to be similar to the route shown in scheme 1. matairesinol is metabolized to arc ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 582 ickovski et al. scheme 1. (adapted from literature86,101,114) part of the shikimate and phenylpropanoid biosynthetic pathways and possible biosynthetic pathways for various types of lignans. the ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ furofuran lignans of artemisia genus 583 enzymes involved in the shikimate pathway are: dahp synthase (ec 2.5.1.54), 3-dehydroquinate synthase (ec 4.2.3.4), 3-dehydroquinate dehydratase (ec 4.2.1.10), shikimate dehydrogenase (ec 1.1.1.25), shikimate kinase (ec 2.7.1.71), 5-enolpyruvylshikimate 3-phosphate synthase (ec 2.5.1.19), chorismate synthase (ec 4.2.3.5), chorismate mutase (ec 5.4.99.5), prephenate aminotransferase (ec 2.6.1.78) and arogenate dehydratase (ec 4.2.1.91). the enzymes involved in phenylpropanoid pathway are: phenylalanine ammonia lyase (ec 4.3.1.24), cinnamic acid 4-hydroxylase (ec 1.14.13.11), 4-coumaric acid:coa ligase (ec 6.2.1.12), cinnamoyl-coa:nadp oxidoreductase (ec. 1.2.1.44), hydroxycinnamoyl-coa shikimate/quinatehydroxy-cinnamoyl transferase (htc), p-coumaroyl-coa 3′-hydroxylase (ec 1.14.14.1), caffeoyl-coa o-methyltransferase (ec 2.1.1.104), cinnamyl alcohol dehydrogenase (ec 1.1.1.195), aldehyde/coniferyl alcohol 5-hydroxylase (ec 1.14.13), 5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcohol o-methyltransferase (ec 2.1.1.68). tigenin by matairesinol o-methyltransferase via methylation of a phenolic hydroxyl group in various plants including f. koreana, carthamus tinctorius and anthriscus sylvestris.124,125 in linum, anthriscus and podophyllum plants, matairesinol is also converted into hinokinin, yatein, or ptox via multiple biosynthetic pathways, although all of the relevant enzymes have not yet been identified.86,107 in sesamum plants pinoresinol (25) is metabolized into piperitol, followed by further conversion into (+)-sesamin (6) by a cytochrome p450 family enzymes.86,107,126 3. isolation of lignans lignans are natural products with highly diverse structures, which affects their separation. lignan aglycones are the most prevalent natural form of this compounds, so the high hydrophobicity of this compounds, as well as the separation itself can be influenced by skeletal substitution, the position of the substituent, partition coefficient, isomerism and size of the molecule. for example, in aryltetraline lignans podophyllotoxine and α-peltatine the position of oh group is decisive factor: if it is in 7α position the substance is less hydrophobic, than in the case of substitution in position 6 on the aromatic ring. in the case of hydroxyl group glycosylation, hydrophilicity rises significantly.127 the introduction of an additional hydroxyl group on position 7′ (matairesinol transformation to hydroxymatairesinol) leads to a significant increase of polarity. a similar effect occurs during glucosylation (mono and diglucoside of secoisolariciresinol). size of the molecule has a significant influence on the separation of the lignan molecules. the higher molecular mass of an aglycone the lower mobility, though in this case the chromatographic behavior can hardly be predicted. solvent extraction is a traditional method for extracting lignans from plant sources. however, other less polar components present in most plant tissues may interfere with the subsequent separation of lignans if a polar solvent is used. therefore, the sequential solvent extraction is recommended for efficient separation of lignan compounds. extraction in soxhlet extractor is a widely used method. it ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 584 ickovski et al. can be used for sequential extraction, with solvents with increasing polarity, which is usually started with non-polar organic solvents such as petroleum ether, hexane or dichloromethane.127,128 during this ”pre-extraction”, the extraction of a part of lignans can occur as well. preparation of lignan extracts with a low content of ballast substances by a common extraction is practically impossible; lipophilic solvents extract not only undesirable substances but also lignans which are without oh groups or possibly with maximum one hydroxyl group.129–131 after removing lipidic substances, polar solvents (ethanol, methanol and acetone) are used for the preparation of the total extract. in some cases, the addition of polar solvents such as water to the sample may increase the recovery of more polar compounds such as lignan glycosides. lignans of low or medium polarity can be efficiently extracted with a less polar solvent. direct extraction with a hot polar solvent, appropriate for lignans of low polarity, has also been used for extraction of some plant lignans.119,130,132,133 a recently introduced method for extraction of plant lignans. the accelerated solvent extraction, is carried out at higher temperature and pressure and under inert nitrogen atmosphere. this method may enable fast and convenient extraction using relatively small amounts of solvents128,134,135. it has been successfully used for extraction of lignans from the wood of certain trees (picea abies, pineceae).128,136–138 lignans in some plant materials require special pretreatments before extraction. polar lignans, present in the plant as ester-linked oligomers or polymers,139,140 seem to be readily soluble in aqueous methanol or ethanol. nonetheless, the subsequent hydrolysis is required to release free aglycone. furthermore, additional hydrolysis steps, enzymatic or non-enzymatic, can be used for the release of free aglycone.137,138 percolation at room temperature is also used for lignan extraction (e.g., lignans from the twigs of magnolia thailandica were defatted with hexane and continuously extracted with mixture of dichloromethane and methanol).141 purification of total extracts with lignan content is quite time-consuming and laborious. methanol extracts are usually concentrated, diluted with water, this suspension fractioned with n-hexane and consequently with chloroform,142 dichloromethane143,144 or ethyl acetate,145 to obtain a lignan fraction. for example, syringaresinol (23) from the crude extract of m. thailandica was obtained as follows: crude extract was subjected to silica gel column chromatography with ethyl acetate–hexane and methanol–ethyl acetate mixtures to give seven fractions. fraction which contained syringaresinol (23) was fractionated on a silica gel column with ethyl acetate-hexane mixture to yield six subfractions. syringaresinol (23) was gathered from one of the subfractions via crystallization by ethanol.141 the details of the previously employed extraction and chromatographical methods for isolation of artemisia genus plant lignans are summarized in table ii. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ furofuran lignans of artemisia genus 585 table ii. details of previously employed analytical techniques for isolation of lignans from different plant sources plant part isolated lignans extraction isolation technique ref. a. absinthium aerial parts 28, 1, 27, 23, 18, 20, 26 ethanol, 95 % successive extraction with ether, chcl3, ethyl acetate. chromatography: silica gel column (petroleum ether:ethyl acetate) gives 6 fractions. fractionation (fraction 4): silica gel column chromatography (petroleum ether:ethyl acetate, 10:1 → 1:2) gives 14 sub-fractions. separation (sub-fractions 8, 10, 13): open column chromatography (ch3oh:h2o, 40:60 → 90:10), semi-preparative hplc (acetonitrile:h2o, 50:50), sephadex lh-20 column chromatography (methanol). 108 a. arborescens aerial parts 6, 26, 27, 8, 1, 15, 25 maceration (methanol) chromatography; silica gel column chromatography (hexane:ether, 2:1, ether, ether:ch3oh, 6:1) gives 3 fractions. medium pressure column chromatography: hexane:ether, 1:2. 95 a. absinthium fresh roots 1, 2, 3, 4 petrol (60–80 °c):ether, 2:1 chromatography: resin was dissolved in ether, tlc on silica gel with ether:petrol, 4:1. fractionation: silica gel column (petrol:ether, ether 100:0 → 0:100 and ch3oh:ether, 3:97 → 10:90 %). the lignan containing fractions (petrol:ether–ch3oh:ether, 50–10 %) were also subjected to preparative tlc. 92 a. caruifolia aerial parts 6, 1, 16, 17, 18, 19 refluxing (methanol). methanol extract was partitioned with chcl3 and h2o chromatography (chcl3 extract): silica gel column (hexane:ethyl acetate, 7:3→3:1 and ethylacetate:ethanol:h2o, 6:2:1), gives 4 fractions. open column chromatography (fraction 1) with 60–100 % methanol gives 3 sub-fractions. preparative tlc (sub-fractions): sio2, benzene:acetone, 9:1 gives sesamin (6) and sesartemin (1). open column chromatography (fraction 2 and 3) with 40–60 % methanol. hplc preparative chromatography of sub-fractions gives caruilignans. 97 ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 586 ickovski et al. table ii. continued plant part isolated lignans extraction isolation technique ref. a. absinthium aerial parts 27, 1 maceration (ch2cl2, 2 days) chromatography: silica gel, ch2cl2:ch3oh, 98:2 → 40:60, gives 4 fractions. semipreparative hplc: isocratic, h2o:acetonitrile, 40:60. 46 9, 1, 28, 27, 18 ethanol, 70 % fractionation: petroleum ether, ch2cl2, ethyl acetate and butanol, successively. chromatography (ch2cl2 fraction): silica gel column chromatography with petroleum ether:ethyl acetate, 10:0 → 5:1 and ch2cl2:ch3oh, 100:0 → 5:1. sephadex lh-20 column chromatography with chcl3:ch3oh, 1:1. 94 4. biological activity of furofuran lignans yamauchi et al.146 synthesized nine oxygenated furofuran lignans and found that the tertiary hydroxy group on the furofuran ring affected the degree of antioxidant activity. pinoresinol (25), sesamin (6) and their glycosides are metabolized by intestinal microflora to yield enterodiol and enterolactone which are supposed to protect against estrogen-dependent cancers,147,148 and which are known as enterolignans or mammalian lignans.149–151 these metabolized lignans elicited their estrogen-like activity in mammals. for example, enterolignans bind to the mammalian estrogen receptors, which are key regulatory factors in the sexual maturation of genital organs.152,153 enterolignans, combined with other intestinal flora-generating metabolites of isoflavones and coumestans, are also called phytoestrogens.84 in human intestinal caco 2 cells, pinoresinol (25) decreased the production of inflammatory factors, such as interleukin-6 and prostaglandin e2, following the down-regulation of cox-2, an inducible prostaglandin synthase that is responsible for the synthesis of prostaglandin h.154 sesamin (6) reduced signaling downstream of mitogen-activated protein kinase, and potently reduce breast tumor growth.155 lee et al.156 demonstrate the role of magnolin (13) as a metastatic inhibitor in lung cancer cells. epieudesmin (22) has been shown to have antineoplastic activity against the murine p388 lymphocytic leukemia cell line and several human cancer cell lines (bxpc-3, mcf-7, sf268, nci-h460, km20l2 and du-145).157 yangambin (26) prevents the cardiovascular collapse observed during anaphylactic and endotoxic/septic shocks, as well as the vascular and cardiac hypo________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ furofuran lignans of artemisia genus 587 responsiveness to catecholamines in endotoxic shock.77 diayangambin (28)158 and fargesin (11)159 have been reported to exert anti-inflammatory activity. the findings of serra et al.160 indicate that yangambin (26) shows an antagonistic action on ltb4 receptors and suggest that it may be useful in the treatment of some allergic inflammatory responses. phillyrin (29) has an anti-obesity effect in nutritive obesity mice.161 cyclic adenosine monophosphate (amp) is found to be the second messenger inside cells, so compounds that act to alter cyclic amp metabolism have been the subject of many studies. nikaido et al. presented in their paper that pinoresinol (25) and pinoresinol-β-d-glucoside showed cyclic amp phosphordiesterase inhibitory activity.162 kobusin (5), fargesin (11) and epieudesmin (22) were assayed for inhibitory activity against nitric oxide production in lps stimulated raw 264.7 cell, but all lignans were inactive.163 rimando et al.164 studied furofuran lignans epiyangambin (27), diayangambin (28), diasesartemin (4) and epiaschantin (9) for their phytotoxicity. diayangambin (28) was the most phytotoxic to lactuca sativa, showing strong inhibitory activity. diayangambin (28) was more active than epiyangambin (27) and diasesartemin (4) in inhibiting the growth of agrostis stolonifera. all of these compounds inhibited all phases of onion root cell division. fargesin (11) and sesamin (6), which have very similar structures to epiyangambin (27), diayangambin (28), diasesartemin (4) and epiaschantin (9), were shown to inhibit germination of peanut and cucumber.165 sesamin (6) is used as an antioxidant.166 the antioxidative propensity of sesamin (6) is likely to be involved in protecting the liver from oxidation by alcohols, lipids and oxygen radicals.84,167–169 sesamin (6) and its metabolites exhibited antihypertensive activities.76,170–172 sesamin (6) is also an insecticide.173 sesamin (6), pinoresinol (25) and kobusin (5) have various biological activities, which include synergistic effects with pyrethrum insecticides174–177 and inhibitors of δ5 desaturases in mammals.178 sesamin (6) has an anti-inflammatory effect by specifically inhibiting δ5 desaturase in polyunsaturated fatty acid biosynthesis.179 sesamin (6), pinoresinol (25) and kobusin (5) also have significant plant protective properties as antioxidants, as well as having important roles in health protection.180 this lignans, when provided in the diet, can reduce serum cholesterol level,181 as well as increase vitamin e activities182,183 and the availability of γ-tocopherol in vivo.184 the lignans epiyangambin (27) and sesartemin (1) reduce spontaneous locomotor activity and isolation-induced aggression in mice.185 epiyangambin (27) and epimagnolin (14) possessed strong selective inhibition of paf-induced platelet aggregation.186 epiyangambin (27) and yangambin (26) competitively inhibited platelet activating factor (paf)-induced rabbit platelet aggregation in a dose-dependent manner, but they had no effect on the platelet aggregation induced by collagen, thrombin or adp.187 these results indicated ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 588 ickovski et al. that both lignans were potent and selective antagonists of paf.188,189 sesamin (6) feeding is associated with reduced serum levels of triacylglycerol,190–192 cholesterol193,194 and phospholipid190 in rodents. dietary sesamin (6) also reduces hepatic concentrations of triacylglycerol191,193 and cholesterol190,194 but increases phospholipid levels accompanying liver hypertrophy,190,191,195 although temporarily.194 kiso196 found that sesamin (6) was absorbed by the route of the portal vein and metabolized to monoor dicatechol metabolite by drug metabolizing enzymes in the liver cells. it is suggested that sesamin (6) ingestion regulated the transcription levels of hepatic metabolizing enzymes for lipids and alcohol. ashakumary et al.197 demonstrated that dietary sesamin (6) greatly increased the hepatic activity of fatty acid oxidation enzymes, including carnitine palmitoyltransferase, acyl-coa dehydrogenase, acyl-coa oxidase, 3-hydroxyacyl-coa dehydrogenase, enoyl-coa hydratase and 3-ketoacyl-coa thiolase. sesamin (6) also increased the activity of 2,4-dienoyl-coa reductase and δ3,δ2 -enoyl-coa isomerase, enzymes involved in the auxiliary pathway for β-oxidation of unsaturated fatty acids. furofuran lignans: sesamin (6), aschantin (8), sesartemin (1) and yangambin (26) showed weak activity against staphylococcus aureus. sesartemin (1) and yangambin (26) also showed weak activity against escherichia coli, while sesamin (6) and aschantin (8) were inactive.198 epiaschantin (9) exhibited moderate antimicrobial activity against strains of gram -positive bacteria staphylococcus aureus, bacillus subtilis, escherichia coli and the yeast candida albicans.199 kawamura et al.200 investigated the antifungal activity of epieudesmin (22) against trametes versicolor and fomitopsis palustris. lignan showed antifungal activity. macrae et al.201 tested a number of lignans and found that antiviral activity is specific to a certain classes of lignans. episesartemin b (3), sesartemin (1), epiyangambin (27) and yangambin (26) were all without antiviral effect, although these lignans are known to have a number of biological activities.202 three diepoxy-pinoresinol glycosides, one diepoxy-syringaresinol glycoside, pinoresinol (25) and syringaresinol (23) were tested for inhibitory activity against tobacco mosaic virus. pinoresinol-4′o-[4′′,6′′o-(e)-diferuloyl]-β-d-glucopyranoside, pinoresinol-4′o-[3′′,6′′o-(e)-diferuloyl]-β-d-glucopyranoside and syringaresinol-4′o-[4′′,6′′o-(e)-diferuloyl]-β-d-glucopyranoside exhibited moderate activities in inhibiting the multiplication of the tobacco mosaic virus203. ortet et al.57 evaluated in vitro cytotoxicity of eudesmin (21), magnolin (13), epimagnolin (14), aschantin (8), kobusin (5) and sesamin (6) against various human and murine tumor and normal cells and antimalarial activity against chloroquine-resistant plasmodium falciparum. tested compounds showed no cytotoxic activity against human tumor cells. with the exception of the sesamin (6), all other lignans showed weak cytotoxic activity against murine normal cells. furthermore, ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ furofuran lignans of artemisia genus 589 the cytotoxicity of sesamin (6) on mammalian normal cells was unnoticeable. epimagnolin (14), aschantin (8), kobusin (5) and sesamin (6) showed mild antiplasmodial activities. the cytotoxic activity of caruilignan a (16), caruilignan b (17), caruilignan c (18), caruilignan d (19), sesamin (6), and sesartemin (1) were tested using meth-a (sarcoma) and llc (lowis lung carcinoma) cell lines. caruilignan a (16), caruilignan b (17), caruilignan c (18), sesamin (6) and sesartemin (1) were found to be cytotoxic only against the meth-a cell line.97 fargesin (11), epieudesmin (22) and sesamin (6) were effective against trypomastigotes, but these compounds were highly toxic to mammalian cells and no parasite selectivity could be identified.204 5. conclusion this review represents furofuran lignans, their isolation from the plants of the genus artemisia, along with their biological activity. extensive literature survey, revealed that lignans have been obtained just from ten species of the artemisia genus: a. absinthium, a. arborescens, a. canariensis, a. caruifolia, a. gorgonum, a. jacutica, a. macrocephala, a. minor, a. sieversiana, a. austroyunnanensis, although the genus includes a large number of species. despite the isolation and characterization of numerous lignans from different plant species, there is a lot of an unfinished work left on this class of secondary metabolites, especially within the plant species of artemisia genus, primarily because lignans represent a huge source of potentially bioactive compounds. further work on the isolation of lignans and determination of differences in the lignan patterns among artemisia species belonging to a different section of the genus could have chemotaxonomic significance. lignans are characterized by the stereoselective oxidative coupling of two phenylpropane units; the presence of chiral centres is an interesting challenge that needs to be overcome for the synthesis of these compounds. although during the last decade, the total synthesis of several biologically active lignans has been achieved, there is still much left to learn about the lignan biosynthetic pathways. abbreviations adp – adenosine diphosphate amp – adenosine monophosphate bxpc-3 – human pancreatic cancer cell line coa – coenzyme a cox-2 – cyclooxygenase-2 du-145 – human prostate cancer cell line ec 1.1.1.195 – cinnamyl alcohol dehydrogenase ec 1.1.1.25 – shikimate dehydrogenase ec 1.14.13 – aldehyde/coniferyl alcohol 5-hydroxylase ec 1.14.13.11 –cinnamic acid 4-hydroxylase ec 1.14.14.1 – p-coumaroyl-coa 3′-hydroxylase ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 590 ickovski et al. ec. 1.2.1.44 – cinnamoyl-coa:nadp oxidoreductase ec 2.1.1.104 – caffeoyl-coa o-methyltransferase ec 2.1.1.68 – 5-hydroxyconiferaldehyde/5-hydroxyconiferyl alcohol o-methyltransferase ec 2.5.1.19 – 5-enolpyruvylshikimate 3-phosphate synthase ec 2.5.1.54 – dahp synthase ec 2.6.1.78 – prephenate aminotransferase ec 2.7.1.71 – shikimate kinase ec 4.2.1.10 – 3-dehydroquinate dehydratase ec 4.2.1.91 – arogenate dehydratase ec 4.2.3.4 – 3-dehydroquinate synthase ec 4.2.3.5 – chorismate synthase ec 4.3.1.24 – phenylalanine ammonia lyase ec 5.4.99.5 – chorismate mutase ec 6.2.1.12 – 4-coumaric acid:coa ligase htc – hydroxycinnamoyl-coa shikimate/quinatehydroxy-cinnamoyl transferase km20l2 – human colon tumor cell line llc – lowis lung carcinoma cell line lps – lipopolysaccharide ltb4 – leukotriene b4 receptors mcf-7 – human breast cancer cell line meth-a – sarcoma cell line nci-h460 – human lung cancer cell line p388 – murine lymphocytic leukemia cell line paf – platelet activating factor ptox – podophyllotoxin raw 264.7 – murine macrophage cell line sf268 – human brain tumor cell line acknowledgment. support of the ministry of education, science and technological development of serbia (project no. 172047) is gratefully acknowledged. и з в о д фуранофурански лигнани рода artemisia: изоловање, биосинтеза и биолошка активност јована д. ицковски, јована љ. павловић, милан н. митић, иван р. палић, данијела а. костић, горан м. петровић и гордана с. стојановић департман за хемију, природно–математички факултет, универзитет у нишу, вишеградска 33, 18000 ниш од давнина лековите биљке и фармаколошки активни производи добијени из различитих природних извора играју важну улогу у здрављу људи. биљке које припадају роду artemisia поседују велики биолошки потенцијал и прилично су добро проучаван род у областима као што су систематика (укључујући молекуларну филогенетику) и организација генома. многе врсте овог рода (a. absinthium, a. annua, a. vulgaris, a. abrotanum, a. arborescens) су широко искоришћаване, због своје велике економске вредности, као лекови, храна и украси. ипак, у тако великом роду неизбежно постоје многобројне разлике, које се тичу употребљивости и потенцијала појединих врста. већина студија усмерена је на биоактивност и фармакологију сесквитерпенских лактона. лигнани су неправедно занемарени, иако и они имају веома значајна својства. мотивисани том чињени________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ furofuran lignans of artemisia genus 591 цом покушали смо да објединимо резултате о биоактивним лигнанима, који су објављивани током година, а са логичним циљем даљег рада на изоловању и идентификовању нових биолошки активних лигнана и њиховој, потенцијалној, фармаколошкој експлоатацији. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {anti-inflammatory activity of synthetic and natural glucoraphanin} j. serb. chem. soc. 84 (5) 445–453 (2019) udc 635.4:547.458.34:616.006.03/04:615.276 jscs–5197 original scientific paper 445 anti-inflammatory activity of synthetic and natural glucoraphanin quan v. vo1,2*, pham c. nam3**, thuc n. dinh4, adam mechler5 and thi t. v. tran6 1department for management of science and technology development, ton duc thang university, ho chi minh city, vietnam, 2faculty of applied sciences, ton duc thang university, ho chi minh city, vietnam, 3department of chemical engineering, university of da nang – university of science and technology, vietnam, 4faculty of natural sciences, hong duc university, thanh hoa, vietnam, 5la trobe institute for molecular science, la trobe university, melbourne, australia and 6department of chemistry, hue university of science, 77 nguyen hue, hue, vietnam (received 18 may, revised 13 november, accepted 3 december 2018) abstract: glucoraphanin is one of the best known glucosinolates because of its health benefits. the compound is known to eliminate carcinogens in tissue and hence is frequently studied for its cancer preventative properties. in this work, the total synthesis of αand β-glucoraphanin epimers was attempted. β-glucoraphanin potassium salt was successfully synthesized in high overall yield, whereas the α-epimer was found to be unstable as it decomposed in the final step of the total synthesis. the anti-inflammatory activity of the synthetic glucoraphanin was determined by inhibition of the release of tumor necrosis factor alpha (tnf-α) secretion in lipopolysaccharide-stimulated thp-1 cells. it was shown that in the presence of either the synthetic or natural glucoraphanin, tnf-α secretion was significantly reduced (≈52 % inhibition) at a concentration of 15 μm. keywords: broccoli; tnf-α; sulforaphane; glucosinolates; synthesis; thp-1. introduction glucoraphanin (4-methylsulfinylbutyl glucosinolate, grp) is the most abundant glucosinolate in common culinary brassica species, such as broccoli (brassica oleracea)1 or hoary cress (cardaria draba).2 when ingested, it is hydrolyzed by the enzyme myrosinase to sulforaphane that imparts numerous health benefits: it was shown to eliminate carcinogens in living tissue3,4 and it is an inducer of phase 2 enzymes (glutathione s-transferase and quinone reductase)5 that are linked to cancer protection.6,7 consistently it is possible, and desirable, *,** corresponding authors. e-mail: (*)vovanquan@tdtu.edu.vn; (**)pcnam@dut.udn.vn https://doi.org/10.2298/jsc180518108v ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 446 vo et al. to create a grp-based nutraceutical that retains the health advantages of sulforaphane and natural grp. the evaluation of the biosafety and anti-tumor activity of purified and semi -purified grp was reported previously.8,9 the studies were performed on male f344 rats by testing the effect of grp on ethoxyresorufin o-deethylase and/or nqo1 activity. the response to grp was similar for the purified and semi-purified grp preparations, suggesting that semi-purified preparations could be used in supplements and to fortify foods. however, there are no reports of control studies of grp bioactivity using synthetic grp that is a better defined source with much reduced and easily identifiable impurities. furthermore, there are indications that the activity of grp is more diverse and/or it exerts a protective function via an anti-inflammatory effect, similar to the pathway reported for aromatic and indole glucosinolates (gls).10,11 there is a link between chronic inflammation and carcinogenesis,12–14 and thus a study of anti-inflammatory activity could be a simple and inexpensive step to initially evaluate the anticancer activity of glucoraphanin. therefore, in this study, a comprehensive study of the total synthesis of αand β-grp potassium salts and the measurements of their anti-inflammatory activity in comparison with other typical glucosinolates are reported. experimental general methods melting points (m.p.) were recorded on a hot stage apparatus and are uncorrected. optical rotations were measured at the stated temperatures in the stated solvent on a polarimeter at the sodium d-line (589 nm); [α]d values are given in 10-1° cm2 g-1. infrared spectra (νmax) were recorded on a ft-ir spectrometer. the samples were analyzed as kbr discs (for solids) or as thin films on nacl plates (for liquids/oils). unless otherwise specified, the proton (1h) and carbon (13c) nmr spectra were recorded on a 300 mhz spectrometer operating at 300 mhz for protons and 75 mhz for carbon nuclei. chemical shifts were recorded as δ values in ppm. the spectra were acquired in deuterated chloroform (cdcl3), methanol-d4 (cd3od) or deuterium oxide (d2o) at 300 k unless otherwise stated. for the 1h-nmr spectra recorded in cdcl3, cd3od and d2o, the peaks due to residual chcl3, cd3od and d2o (δh 7.24, 3.28 and 4.65 ppm, respectively) were used as the internal reference, while the central peaks (δc 77.0 and 49.0 ppm) of cdcl3 and cd3od were used as the reference for the proton -decoupled 13c-nmr spectra. low-resolution mass spectra were measured on a mass spectrometer at 300 °c at a scan rate of 5500 m/z per s using water/methanol/acetic acid in a volume ratio of either 0/99/1 or 50/50/1 as the mobile phase. accurate mass measurement was by mass spectrometry with a heated electrospray ionization (hesi) source. the mass spectrometer was operated with full scan (50–1000 amu) in the positive or negative ft mode (at a resolution of 100,000). the analyte was dissolved in water/methanol/acetic acid in a volume ratio of 0/99/1 or 50/50/1 and infused via syringe pump at a rate of 5 µl/min. the heated capillary was maintained at 320 °c with a source heater temperature of 350 °c and the sheath, auxiliary and sweep gases were at 40, 15 and 8 units, respectively. the source voltage was set to 4.2 kv. the solvents were dried over standard drying agents and freshly distilled before use. ethyl acetate and hexane used for chromatography were distilled prior to use. all solvents ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. anti-inflammatory activity of glucoraphanin 447 were purified by distillation. reactions were monitored by thin layer chromatography (tlc) on silica gel 60 f254 plates with detection by ultra-violet (uv) fluorescence or charring with a basic potassium permanganate stain. flash column chromatography was performed on silica gel 60 particle size 0.040–0.063 mm (230–400 mesh). general procedures for the synthetic compounds 1-thio-αor β-d-glucopyranoze 2,3,4,5-tetraacetate 1-[(1z)-n-hydroxy-5-(methylsulfinyl)pentanimidate] (2a or 2b). to a suspension of 1 (150 mg, 0.9 mmol) in dcm (10 ml) was added pyridine (0.09 ml, 0.95 mmol) and then n-chlorosuccinimide (120 mg, 0.63 mmol). the mixture was stirred for 2.5 h at r.t. under a nitrogen atmosphere, then 1-thio-α-d -glucopyranose 2,3,4,6-tetraacetate or 1-thio-β-d-glucopyranose 2,3,4,6-tetraacetate (0.33 g, 0.9 mmol) in dcm (5 ml) was added. the resulting mixture was treated with triethylamine (0.75 ml, 5.4 mmol). the reaction mixture was stirred for 2 h at r.t. under a nitrogen atmosphere then acidified with aqueous 1 m h2so4 (7 ml/mmol of sugar). the mixture was left to stand for about 10 min and then separated. the aqueous phase was extracted with dcm (3×30 ml). the combined organic layers were dried over mgso4, filtered and the filtrate was concentrated under reduced pressure. compound 2a (189 mg, 40 %) was obtained as a foam by flash column chromatography on silica gel eluting with 10 % meoh/dcm. the characterization data for 2a are given in the supplementary material to this paper. compound 2b (150 mg, 47 %) was obtained as a foam by flash column chromatography eluting with 90 % dcm/meoh. the characterization data for compound 2b were identical to literature values.10 potassium salt of 1-thio-αor β-d-glucopyranoze 2,3,4,5-tetraacetate 1-[(1z)-5-(methylsulfinyl)-n-(sulfooxy)pentanimidate] (3a or 3b). to a stirred solution of the thiohydroximate (2a or 2b, 120 mg, 0.2 mmol) in dry pyridine (5 ml) was added pyridine–sulfur trioxide complex (95.0 mg, 0.6 mmol). after stirring at r.t. under n2 for 24 h, an additional portion of the pyridine–sulfur trioxide complex (19.0 mg, 0.1 mmol) was added and stirring was continued for 2 h. subsequently, a solution of khco3 (850 mg, 8.4 mmol) in water (10 ml) was added, the mixture stirred for 30 min and then concentrated under reduced pressure. the residue was dissolved in water and extracted with chloroform (3×40 ml) and then 20 % meoh/ /chcl3 (2×30 ml). the organic layers were dried (mgso4), filtered and concentrated under reduced pressure. to remove excess pyridine, the mixture was co-distilled several times with toluene. compound 3a was obtained as a white solid (74 mg, 50 %). compound 3b was obtained by flash chromatography eluting with 80 % dcm/meoh as a colorless solid (103 mg, 73 %). the characterization data for compound 3b were identical to literature values.10 β-glucoraphanin potassium salt (4b). the β-grp potassium salt 4b was prepared from 3b following literature methods and the data were identical to literature values.10 spectral and analytical data of the compounds are given in supplementary material to this paper. anti-inflammatory assays the anti-inflammatory assays were conducted following the literature.11,15 human monocytic leukaemia thp-1 cells were obtained from the american type culture collection. the cells were grown in 10 % heat-deactivated fetal bovine serum and invitrogen rpmi-1640 containing 2 mm l-glutamine. the cytokine (tnf-α) elisa kit including the reagents was obtained from bd bioscience (r&d systems). all compounds were dissolved in sterile distilled water then further diluted in invitrogen dmem (dulbecco’s modified eagle medium). the cells were grown in a 75 ml flask and ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 448 vo et al. maintained at 37 °c in a humidified 5 % co2 atmosphere. the experiments were performed once the cells had reached 105 cells ml-1. phorbol 12-myristate 13-acetate (pma) was dissolved in dmso to a concentration of 1 mg ml-1 and then further diluted before use. the cells were plated out to a cell density of 10×104 cell ml-1, at 100 µl well-1 in a 96-well plate then treated with pma to a final concentration of 50 nm for 24 h at 37° under a humidified 5 % co2 atmosphere. lipopolysaccharides (lps) were dissolved in sterile water to a concentration of 5 mg ml-1 and then further diluted to the working stock of 10 µg ml-1. the thp-1 cells were challenged with various compounds at a concentration ranging from 0.1–15 µm. they were stimulated with lps at a final concentration of 50 ng ml-1. the supernatants were collected after 4 h incubation and stored at –20 °c until enzyme-linked immunosorbent assay (elisa) analysis. a sandwich elisa was used to screen the supernatants for the release of cytokine tnf-α. the elisa plates were coated with a capture antibody (1:250) which was diluted in coating buffer and left at 4 °c overnight. the elisa plates were aspirated and washed 3 times with 1×pbst (phosphate buffered saline with tween-20 (0.05 % tween-20, ph 7.4) before adding 200 μl well-1 assay diluent and incubated at room temperature for 1 h. standards were prepared by 2-fold serial dilutions to the range from 500–7.8 pg ml-1 in assay buffer diluent. standards and sample were added in quadruplicate into appropriate wells and incubated at room temperature for 2 h. after the 2 h incubation, the plates were aspirated and washed for a total of 5 washes. the detection antibody and hrp reagent were added (100 μl well-1) and incubated at room temperature for 1 h. the plates were aspirated and washed again, this time for a total of 7 washes and were soaked for 30 s between each wash. the substrate solutions were added (100 μl well-1) and incubated at room temperature for 30 min in the dark. the reaction was stopped by adding 50 μl well-1 of kit stop solution then read at 450 nm with a plate reader within 30 min with a λ correction at 570 nm. isolation and purification of grp from broccoli seeds natural grp was isolated from broccoli seeds and purified following the literature.16 to approximately 9 g of broccoli seeds 90 ml of boiling water was added, and the mixture boiled for 5 min. the bulk of the water was decanted and the seeds transferred to a mortar with 15 ml of water. the seeds were ground to a paste. the resultant slurry was transferred to a 200 ml volumetric flask with deionized water, made to the mark and sonicated for 5 min. the extract was filtered under vacuum through whatman no. 4 filter paper. mega bond elut c18 cartridges (3 g) were activated with methanol and washed with water. mega bond elut nh2 cartridges (3 g) were activated with methanol and equilibrated with 1 % acetic acid in water. the c18 and nh3+ cartridges were connected in series and 30 ml of the extract loaded onto the c18 cartridge. the cartridges were washed with 18 ml of deionized water, the c18 cartridge discarded and the nh3+ cartridge washed with 18 ml of methanol. the glucosinolate fraction was removed from the nh3+ cartridge with 30 ml of freshly prepared 2 % solution of concentrated nh4oh solution in methanol. the solution was evaporated to dryness under a stream of nitrogen at room temperature. grp was obtained from the crude residue by hplc (mobile phase 1 vol. % ch3cn, 99 % aqueous 0.1 % formic acid)16 as a colorless liquid (35.7 mg). the nmr and ms data of the isolated grp matched with the literature values.10,17 the isolated grp was then used for anti-inflammatory assays. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. anti-inflammatory activity of glucoraphanin 449 results and discussion synthesis of αor β-glucoraphanin potassium salts it was shown that the oxime 1 could be synthesized from the 5-chloropentanol (scheme 1).10 based on previous work, the thiohydroxymates 2a or 2b were formed by the coupling of oxime 1 and 1-thio-α-d-glucopyranose 2,3,4,6 -tetraacetate18 (or 1-thio-β-d-glucopyranose 2,3,4,6-tetraacetate11) following the vo method.10 thus, oxime 1 was treated with n-chlorosuccinimide (ncs) in the presence of pyridine in dichloromethane (dcm) to form the hydroximoyl chloride, which was directly coupled with the α-thiol (or β-thiol) in triethylamine to yield the thiohydroxymates 2a (or 2b) in 47 % (or 40 %) yield in a one-pot reaction (scheme 1). scheme 1. synthesis of αor β-grp potassium salts. sulfation of 2a (or 2b) was accomplished with pyridine-sulfur trioxide complex in pyridine (pyr) (scheme 1).19 the resulting potassium salts 3a or 3b (73 % and 40 % yields) were isolated by flash column chromatography on silica gel. de-o-acetylations were performed by dissolving 3a (or 3b) in meoh in the presence of meok as catalyst. the final β-grp potassium salt 4b (17 % overall yield over seven steps) was successfully purified by flash column chromatography on silica gel.10 unfortunately, the final α-epimer could not be obtained. the mass spectrogram of the reaction solution showed no peak for the [m–k]– of the α-grp potassium salt, which was usually observed as the base peak ion in the spectra of α-gls.18 attempts to achieve the α-isomer by using different de-o-acetylation conditions20 were unsuccessful. thus, only the potassium salt of 1-thio-αor β-d-glucopyranoze 2,3,4,5-tetraacetate 1-[(1z)-5-(methylsulfinyl)-n-(sulfooxy)pentanimidate] 3a was obtained for the first time in 11.4 % yield over 6 steps. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 450 vo et al. anti-inflammatory activity of the synthetic β-grp to compare the anti-inflammatory activity of the synthetic β-grp (4b) with natural grp and typical indole and aromatic glucosinolates,11,21 the anti-inflammatory properties of the synthetic β-grp were tested via an in vitro assay developed based on the thp-1 cell line following the literature.11,15,21 the results are summarized in fig. 1 and table i. fig. 1. comparison of tnf-α released by lps alone and in addition to gls at different concentrations. lps stimulates the release of tnf-α. the % inhibition is calculated from the difference between tnf-α released with lps alone and in combination with the gls (from the average of the three replicates). moderate activity was observed for the majority of the gls at low micromolar levels,17 a11, b21. it was shown that in the presence of synthetic β-grp, tnf-α secretion was significantly inhibited (> 50 % inhibition) at a concentration of 15 μm, while synthetic β-grp exhibited higher inhibition than neoglucobrassicin at all testing concentrations, reaching nearly the same point as glucobrassicin but higher than 4-methoxyglucobrassicin at a concentration of 15 μm (> 50 %, fig. 1).11 comparison with the positive control (catechin) showed that at concentrations lower than 10 μm, most of the synthetic gls showed lower inhibition than catechin, but at a concentration of 15 μm, β-grp exhibited higher activity than catechin (52 % inhibition for β-grp compared with 48 % inhibition for catechin). the trend was almost opposite in comparison with potassium 1-thio-β-d-glucopyranose 1-[[c(z)]-3,4-dimethoxy-n-(sulfooxy)benzenecarboximidate] (the glucosin________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. anti-inflammatory activity of glucoraphanin 451 olate with the highest anti-inflammatory activity in the aromatic glucosinolate family18). the results showed that synthetic β-grp reached nearly the same inhibition of natural grp at a concentration of 15 μm (52 % inhibition by β-grp, 54 % inhibition by natural grp); the trends were similar at higher investigated concentrations. thus, it was clearly demonstrated that the synthetic β-grp has significant anti-inflammatory activity at low concentrations (≈50 % inhibition at a concentration of 15 μm). the results of the biological activity of synthetic grp, consistent with previous studies,8,9 provide ample evidence for the potential of synhetic grp for medicinal uses. table i. effects of synthetic gls on tnf-α secretion in lps-stimulated thp-1 cells treatment content of tnf-α secretion (sd)a, pg mg-1 lps (50 μg l-1) 488.68 (15.81)b lps + 15.00 μm catechin 255.94 (33.77) lps + 10.00 μm catechin 273.02 (21.53) lps + 1.00 μm catechin 301.41 (20.14) lps + 0.10 μm catechin 373.30 (25.14) lps (50 μg l-1) 236.16 (65.15)b lps + 15.00 μm β-grp 113.07 (39.42) lps + 10.00 μm β-grp 137.63 (52.90) lps + 1.00 μm β-grp 178.98 (47.48) lps + 0.10 μm β-grp 204.98 (52.34) lps (50 μg l-1) 253.15 (45.53)b lps + 15.00 μm grp 115.38 (14.45) lps + 10.00 μm grp 137.69 (32.82) lps + 1.00 μm grp 170.26 (41.76) lps + 0.10 μm grp 199.33 (29.51) athe results are for 3 different experiments run in duplicate; bp ≤ 0.07, compared with control conclusions the total synthesis of αor β-grp (potassium salts) was attempted. while the β-epimer was successfully synthesized in high overall yield (17 % over seven steps), α-grp was found to be unstable as it decomposed in the final step. by observing the inhibition of the release of tnf-α in lps-stimulated thp-1 cells, it was shown that the synthetic β-grp and natural grp have similar, significant anti-inflammatory activity at low concentrations. the obtained results raise the possibility of developing a grp-based nutraceutical for therapeutic and/or preventive medicine purposes. supplementary material analytical and spectral data of the compounds are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. this research is funded by the vietnam national foundation for science and technology development (nafosted) under grant no. 104.06–2016.03. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 452 vo et al. и з в о д анти-инфламаторна активност синтетичког и природног глукорафанина quan v. vo1,2, pham c. nam3, thuc n. dinh4, adam mechler5 и thi t. v. tran6 1 department for management of science and technology development, ton duc thang university, ho chi minh city, vietnam, 2 faculty of applied sciences, ton duc thang university, ho chi minh city, vietnam, 3 department of chemical engineering, university of da nang – university of science and technology, vietnam, 4 faculty of natural sciences, hong duc university, thanh hoa, vietnam, 5 la trobe institute for molecular science, la trobe university, melbourne, australia и 6 department of chemistry, hue university of science, 77 nguyen hue, hue, vietnam глукорафанин је због својих корисних лековитих карактеристика један од најпознатијих глукозинолата. познато је да деловањем једињења долази до уклањања канцерогена у ткивима, и услед тога су често изучаване превентивна антиканцерска својства једињења. успешно је синтетисана калијумова со β-глукорафанина, у високом укупном приносу, док је утврђено да је α-епимер нестабилан и да се разграђује у финалном кораку синтезе. антиинфламаторна активност глукорафанина је испитана одређивањем инхибиције ослобађања фактора некрозе тумора-алфа (tnf-α) из thp-1 ћелија стимулисаних липополисахаридима. показано је да је у присуству синтетичког или природног глукорафанина, при концентрацијама 15 μm значајно инхибирана (≈ 52 % инхибиције) секреција tnf-α. 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice formation of conjugated double bonds in soybean oil with iodine as a catalyst j. serb. chem. soc. 81 (2) 141–151 (2016) udc 665.33+547.226:544.478:543.422.25 jscs–4834 original scientific paper 141 formation of conjugated double bonds in soybean oil with iodine as a catalyst jie yan1, jinlan yang1, rifu yang2*, haifen he1, qihai liu1 and hailin lin1 1college of chemistry and chemical engineering, zhongkai university of agriculture and engineering, guangzhou, 510225, china and 2school of physics and optoelectronics, south china university of technology, guangzhou, 510640, china (received 4 may, revised 30 june, accepted 23 september 2015) abstract: a method for the iodine-catalyzed formation of conjugated double bonds in soybean oil was developed, and the conjugated product was analyzed by uv, ir and 1h-nmr. the results indicated that the optimal conditions for the isomerisation included a temperature of 180 °c, a catalyst loading of 0.5 mass % and a reaction time of 3 h. under these conditions, the concentration of conjugated linoleum acid was 1.51 mol l-1 and the clna concentration reached 0.225 mol l-1 with a conversion rate of 99.9 %. the reaction predominantly produced trans–trans, trans–cis and cis–trans isomers. it was also revealed that the isomerisation of linolenic acid was much faster than that of linoleic acid. the method possessed the advantages of a short procedure, a high conversion rate, and no conversion into methyl esters of the raw material and it is an environmentally friendly technology that does not require the use solvents. keywords: soybean oil; conjugation; iodine; linoleic acid; linolenic acid. introduction for a long time, petroleum has been used as a raw material of chemicals. however, due to the exhaustion of petroleum resources, alternative feedstocks must be found. agricultural-based chemicals, as one of the most attractive alternatives, have gained much attention during the last few years. vegetable oils are easily utilized because of their unsaturated double bonds and are sustainable environmentally friendly chemical feedstocks. conjugated vegetable oils have found their way into many industrial applications because of their improved drying property.1–5 in recent years, the physiological functions of the conjugated linoleic acid (cla) were identified, and studies on the formation of conjugated double bonds in oils or fatty acids have become a major focus of the international chemistry community. over the past * corresponding author. e-mail: yangrifu@scut.edu.cn doi: 10.2298/jsc150504082y _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 142 yan et al. few decades, there have been numerous reports on the preparation of conjugated fatty acids and oils.6–16 dejarlais et al. found that the isomerisation of methyl esters affording conjugated double bonds were much faster than those of glycosides using the sodium salt of dimethyl sulfoxide as the catalyst.15 larock et al. prepared conjugated oils and fatty acids with homogeneous transition metals as catalysts.9 basu and kasar described an isomerization method of safflower oil and methyl linoleate using cationic rhodium complexes as catalysts.12 radlove et al. performed a nickel–carbon catalyst method that could reach yields of 30–45 % in 3–6 h at 170 °c.16 they also mentioned that some investigators found that sulfur dioxide could be used as an isomerisation catalyst for linseed and soybean oils under certain conditions. simakova et al. primarily focused on the development of heterogeneous catalytic approaches.17 many of these methods have drawbacks, including being difficult to scale-up and having poor product yields, unwanted side reactions, high catalyst costs, the requirement of pressure, and long reaction times. iodine is inexpensive and non-toxic. in recent years, iodine has been used as a catalyst for various organic reactions, including isomerization.18–22 there have also been reports concerning oil isomerization affording conjugated double bonds with iodine as the catalyst. gangidi22 prepared cla isomers from soybean oil by photo-isomerization of linoleic acid with the help of iodine within the temperature range 35–40 °c under irradiation from a mercury lamp for 120 h. jain and proctor23,24 obtained cla-rich soybean oil also by a photochemical reaction with iodine as the catalyst, maintaining the oil temperature between 22 and 25 °c. however, photochemical methods have common shortcomings of low reaction rates, long reaction times and low yields. they are also not easy to scale-up to an industrial level. for these reasons, the main purpose of this work was to develop an efficient procedure for oil isomerization affording conjugated double bonds using iodine as the catalyst. the new method has a high conversion rate, short reaction time, and is easy to operate under industrial conditions. additionally, it is an environmentally sensitive technology that does not require the use of organic solvents. materials and methods materials and equipment transgenic soybean oil was purchased from the cofco corporation. the mass fractions of linoleic acid (la) and linolenic acid (lna) determined by gc were 50.88 and 6.86 %, respectively. analytical grade iodine was purchased from tianjin zhongxing chemical reagent factory. analytical grade hexane was purchased from sinopharm chemical reagent co., ltd. the standard products of cla were purchased from nu-chek-prep inc. nitrogen (99.99 % purity) was obtained from guangzhou chun jiang industrial gas co., ltd. all other chemical reagents were analytical grade. the following instruments were used in this study: a uv spectrophotometer (2550, shimadzu, japan), a fourier transform infrared spectrometer (spectrum 100, perkinelmer, inc., _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ isomerisation reaction of soybean oil 143 usa), a magnetic agitator with heat collection and temperature control (df-101s, gongyi yingyu instrument factory, china), and a superconducting nmr spectrometer (avance av400, bruker, germany). isomerisation reaction for the isomerisation reaction, 100 g of soybean oil and a specified amount of iodine (in solid form) were added to a 250 ml three-necked round-bottom flask fitted with a thermometer, a reflux condenser, and a high purity nitrogen gas delivery tube. the mixture was purged with nitrogen for ≈10 min to remove the oxygen, and the flask was lowered into a silicone oil bath, heated and stirred magnetically under the protection of nitrogen. the reactor vessel was covered with protective foil. the reaction products were then cooled to room temperature and washed 3 times with 10–15 ml of 0.2 m sodium thiosulfate and of distilled water. the sample was obtained after drying with anhydrous sodium sulfate. determination of the cla and the conjugated linolenic acid (clna) concentrations standard cla was dissolved in n-hexane to prepare a series of standard solutions that ranged from 4 to 14 mg l-1 in concentration. using n-hexane as a reference solvent, the maximum absorbance at 234 nm was measured to obtain a standard curve. according to the curve, an equation was determined: a = 0.1047c – 0.046, r2 = 0.9992, where a is the absorbance and c is the concentration of cla in mg l-1. the product was diluted with n-hexane, and the absorbance was measured at 234 nm. if the absorbance was greater than 1.0, the sample was diluted further. the raw material was also diluted and determined to be the product. the difference of the two absorbencies was used to calculate the cla concentrations according to the regression equation. the clna concentrations were determined according to the iupac method.25 the percentage conversion was also estimated from the 1h-nmr spectra using the glycerol backbone peaks at chemical shifts of 4.15 and 4.30 ppm, according to the literature.26 removal of i2 sodium thiosulfate,27 granulated charcoal and starch26 are reported to remove iodine in the presence of hexane. sodium thiosulfate was adopted in this study, owing to its capability to remove iodine quickly.27 titrimetry was adopted to determine the optimal sodium thiosulfate concentration in the eluate. thus, the residual iodine in the oil could be estimated. the results showed that the residual iodine in oil was not more than 0.2 % after washing three times with ≈10 ml 0.1 m sodium thiosulfate. another method to remove iodine, which utilized the volatility of iodine, was performed at 180 °c under reduced pressure for 1 h. condensate iodine remained in the flask mouth. however, the dark color of the oil manifested the poor effect. the third method was to wash with a potassium iodide–water solution, due to its high iodine solubility. determination of the eluate showed that no iodine was removed from the oil after washing three times with ≈10 ml 0.1 mol l-1 potassium iodide–water solution. to remove iodine as much as possible, 0.2 m sodium thiosulfate was used. a slightly yellow oil was obtained by washing three times with 10–15 ml. spectroscopy analysis the structures of the products were characterized by uv–vis, ftir and nmr analyses. for the uv analysis, approximately 0.1 g of the raw material and the product were diluted with n-hexane by a factor of approximately 5000. the samples were scanned from 210–300 nm, using n-hexane as the reference. for the ftir analysis, the raw material and product were spread onto potassium bromide tablets. the samples were scanned from 4000–400 cm-1. for _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 144 yan et al. the nuclear magnetic resonance spectroscopy analysis, a superconducting pulse fourier transform nmr spectrometer was utilized. cdcl3 was used as the solvent, the magnet was 400 mhz, the temperature was 45 °c, and the chemical shift of chloroform, δ = 7.27 ppm, was used as the reference. results and discussion effect of the single factors on the isomerisation reaction the effect of the catalyst loading on the concentration of the conjugated oil is shown in fig. 1. thus, the concentration of the conjugated oil first increased with increasing catalyst loading concentrations up to 0.5 mass %. at a catalyst usage of 0.5 mass %, the concentrations of cla and clna were 1.05 and 0.167 mol l–1, respectively. these values did not increase appreciably at higher catalyst loadings. the product yield increased due to the higher collision probability between the catalyst and the reactant as the catalyst dosage was increased. however, larger amounts of catalyst would lead to higher costs, and the color of the product was deeper at increased iodine usage. fig. 1. effect of catalyst loading on conjugation reaction (at 16 °c for 3 h). the effect of temperature on concentration of the conjugated oils is shown in fig. 2. as can be seen, the concentration of the conjugated oils was very low when the temperature was 80 °c, but the product yield increased at higher temperatures. the concentration of cla increased to its maximum of 1.42 mol l–1 at a temperature of 180 °c. the concentration of clna reached 0.225 mol l–1 when the temperature was 130 °c, corresponding to a conversion of 99.9 %, and then decreased at higher temperatures. theoretically, at higher temperatures, the number of activated molecules per unit volume increased, the viscosity of the system decreased, and the collision opportunities between the catalyst and reactant increased, all of which served to accelerate the reaction. the effect of the reaction time on the conjugation reaction is shown in fig. 3, from which it can be seen that the concentration of cla and clna first _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ isomerisation reaction of soybean oil 145 increased and then decreased with increasing reaction time. the concentration of cla first increased remarkably with increasing time, but the product yield began to decrease at reaction times greater than 3 h; therefore, it is important to control the reaction time strictly according to the conjugated products required. however, the concentration of clna reached a maximum 0.204 mol l–1 at 1.5 h. it should be noted that the mass fraction of lna in the raw material was only 6.88 %, which was much lower than that of la at 50.88 %. therefore, to obtain the same conversion ratio, the former reaction time was relatively shorter than the latter one. fig. 2. effect of temperature on conjugation reaction (with a catalyst usage 0.5 % for 3 h). fig. 3. effect of reaction time on conjugation reaction (with a catalyst usage 0.5 % at 180 °c). orthogonal experiment based on the single factor experiments described above, an orthogonal test was adopted to determine the optimal conditions, using the concentration of cla as the index. as shown in table i, under the tested conditions of the experiments, _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 146 yan et al. the most important factor was the reaction temperature. the optimal conditions were a temperature of 180 °c, a catalyst loading of 0.5 mass % and a reaction time of 3 h. reproducible tests under the optimal conditions showed that the product concentration was 1.51 mol l–1, with a conversion rate of approximately 92 %. table i. results of orthogonal experiments serial no. catalyst amount, % temperature, °c time, h product concentration, mol l-1 1 0.55 170 2.5 1.225 2 0.55 180 3 1.497 3 0.55 190 3.5 0.945 4 0.50 170 3 1.318 5 0.50 180 3.5 1.399 6 0.50 190 2.5 1.127 7 0.45 170 3.5 1.321 8 0.45 180 2.5 1.290 9 0.45 190 3 1.176 k1 1.222 1.288 1.214 k2 1.281 1.395 1.330 k3 1.262 1.082 1.222 r 0.059 0.313 0.116 a photochemical method for obtaining cla-rich soybean oil in the presence of iodine as a sensitizer, as reported in literature,22–24 has the advantages of using a temperature of no more than 40 °c and an iodine loading of less than 0.25 %. however, the reaction rate is very slow. irradiating soybean oil for 240 h with 0.25 % iodine gave a 54 % conversion of la to cla.23 other reports indicated that using a similar approach, soybean oil conjugation could be performed rapidly with a 500 w projector lamp,26 and a pilot-scale apparatus capable of yielding 20 % cla in 12 h.28 however, this method is difficult to scale up to an industrial level. moreover, some methods require large quantities of an organic solvent26 or a complicated pre-treatment, such as conversion into methyl esters.29 compared with a photochemical method, in view of industrialization, the method performed in the present study seems to be more favorable, resulting in a conversion of more than 90 % in 3 hours. comparison of isomerisation of la and lna the conjugation of la and lna was assumed to be completed in one-step and described by a first-order reaction. the data in fig. 3 prior to 90 min was used to study the kinetics of the reaction. thus, the rate curve and equation ln (c0/c) = kt were obtained from these data, where c is the concentration of la or lna at any time t, c0 is the initial concentration and k is the rate constant. as shown in fig. 4, the rate constant of lna conjugation was much larger than that of la; the conjugation of lna was clearly faster than that of la. in addition, _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ isomerisation reaction of soybean oil 147 the concentration of lna was low in the raw material and therefore, it was quickly consumed in the reaction. according to figs. 1–3, in its conjugation, lna was completely consumed within 3 h at 130 °c using 0.5 mass % iodine and a reaction time of 3 h. under these conditions, the cla yield was not high. figure 4 also shows that the original hypothesis of the la conjugation was correct as illustrated by its perfect fitting curve. however, the fitting curve for lna isomerisation was not appropriate. it was not clear that the three double bonds of lna were to form a dior triene conjugation in the first step. furthermore, when the reaction continued after exhaustion of lna, as shown in figs. 1–3, the concentration of clna in the system was gradually reduced. further study on this phenomenon and its mechanism of isomerisation is required. fig. 4 curve of ln (c0/c) as function of time. structural characterization of the product ultraviolet absorbance analysis. the uv absorption spectra of the raw soybean oil (a) and the products formed at different temperatures (b, c and d) are shown in fig. 5. the absorption peaks at 234 and 268 nm correspond to the maximum of the absorption peaks for cla and clna, respectively. as seen from the plots, the spectrum of soybean oil did not contain any absorption peaks between 220–300 nm. therefore, soybean oil does not contain a conjugated structure. the spectra b, c and d indicated that conjugation has been formed. in addition, as the temperature was increased from 100 to 180°c, the absorbance of the product at 234 nm increased, corresponding to the increase in the cla yield. however, the absorbance at 268 nm first increased and then decreased, indicating that the concentration of clna also first increased but then decreased with increasing temperature. this result was consistent with the phenomenon described above. infrared spectrum analysis. the infrared absorption spectra of the raw material (a) and the products (b) are shown in fig. 6. as shown in the spectra, the _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 148 yan et al. raw oil did not produce any characteristic absorption peak from 900–1000 cm–1, while the reaction product produced very significant peaks at 986, 967 and 948 cm–1. the peak at 986 cm–1 revealed the presence of trans–trans dienes,30 the 986 and 948 cm–1 peaks revealed cis–trans or trans–cis dienes,11 and the 967 cm–1 peak corresponded to trans monoenes.30 fig. 5. uv absorption spectrum of raw soybean oil (a) and reaction products (b, c, d). fig. 6. ir absorption spectra of the raw soybean oil (a) and the reaction products (b). proton nuclear magnetic resonance analysis. the 1h-nmr spectra of raw soybean oil and the reaction products after formation of conjugation are shown in fig. 7a and b, respectively. the degree of unsaturation and information regarding the cis–trans isomers of the conjugated oil were determined according to the chemical shifts and coupling constants of the protons from the spectra.31 peaks at approximately 5.30 ppm reveal non-conjugated vinylic protons, and they shift to _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ isomerisation reaction of soybean oil 149 5.30–6.29 ppm after conjugation.9 the chemical shifts of interest in spectrum a included δ = 5.31 ppm, –ch=ch–; δ = 2.72 ppm, =ch–ch2–ch= and δ = 2.08 ppm, =ch–ch2–, and spectrum b included δ = 6.24 ppm, z-=ch–ch=ch; δ = = 5.92 ppm, e-=ch–ch=ch; δ = 5.63 ppm, e-=ch–ch=ch; δ = 5.52 ppm, e-=ch–ch=ch and δ = 5.33 ppm, z-=ch–ch=ch. from the integration of these peaks, the percent conjugation could be estimated. it was evident that iodine-catalyzed isomerization affording conjugated double bonds had occurred. the main composition of the conjugated oil included trans–trans, trans–cis and cis–trans isomers. fig. 7. 1h-nmr spectra of raw soybean oil (a) and reaction products (b). conclusions the iodine-catalyzed soybean oil isomerization affording conjugated double bonds possesses the advantages of a short procedure, a high conversion rate, no conversion into methyl esters, and easy transition to industrialization. additionally, it is an environmentally friendly technology not requiring solvents. the optimal conditions included a temperature of 180 °c, a catalyst loading of 0.5 mass %, and a reaction time of 3 h. under these conditions, the concentration of cla and percent conversion were 1.51 mol l–1 and 92 %, respectively. clna _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 150 yan et al. reached 0.225 mol l–1 with a conversion of 99.9 % when the temperature was 130 °c, at a catalyst loading of 0.5 wt. %. the main configurations of cla and clna in the final products included trans–trans, trans–cis and cis–trans isomers, based on uv, ir and 1h-nmr analysis. however, the reaction mechanism, especially the decrease in the concentration of clna, requires further study. acknowledgment. the research was supported by the national natural science foundation of china (project no. 31071560). и з в о д грађење конјугованих двоструких веза у сојином уљу у присуству јода као катализатора jie yan1, jinlan yang1, rifu yang2, haifen he1, qihai liu1 и hailin lin1 1 college of chemistry and chemical engineering, zhongkai university of agriculture and engineering, guangzhou и 2school of physics и optoelectronics, south china university of technology, guangzhou, china развијен је поступак изомеризације сојиног уља у присуству јода као катализатора, а добијени конјуговани производ је анализиран методама uv, ir и 1h-nmr. оптимални услови за изомеризацију су били следећи: температура 180 °c, почетна концентрација катализатора 0,5 мас. % и реакционо време 3 h. под тим условима је концентрација конјуговане линолне киселине била 1,51 mol l-1, уз 92 % конверзије. clna је достигла концентрацију од 0,225 mol l-1 при температури од 130 °c, 0,5 мас. % катализатора и 3 h реакције, а степен конверзије је био 99,9 %. током реакције су претежно настали trans– –trans, trans–cis и cis–trans изомери, али је при истој температури растао удео trans– –trans изомера са временом реакције. такође, брзина изомеризације линолеинске киселине је била знатно већа него линолне. предност описаног поступка је у кратком времену трајања, високом степену конверзије, одсуству превођења у метил естре полазног материјала и у еколошкој чистоћи, пошто се не користе растварачи. 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31. m. s. l. k. jie, m. k. pasha, m. s. alam, lipids 32 (1997) 1041. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {degradation of benzodiazepines using water falling film dielectric barrier discharge reactor} j. serb. chem. soc. 82 (7–8) 933–942 (2017) udc 547.53’235.4+542.92+542.943: jscs–5013 615.005.936.5:628.3:543.544.5.068.7 original scientific paper 933 degradation of benzodiazepines using water falling film dielectric barrier discharge reactor vesna m. radulović1*, goran m. roglić2 and dragan d. manojlović2 1galenika a.d. belgrade, batajnički drum b.b. 11080 belgrade, serbia 2faculty of chemistry, university of belgrade, studentski trg 12–16, 11000 belgrade, serbia (received 18 january, revised 22 april, accepted 25 april 2017) abstract: classical methods of wastewater treatment are often not suitable for the treatment of pharmaceutical waste. the previous studies have shown that the use of the advanced oxidation procedures (aop) can lead to a more efficient degradation of various biologically active compounds, which are active pharmaceutical ingredients of applied drugs. the aim of this paper is the application of the plasma technology on the degradation of a two active pharmaceutical ingredients (apis, diazepam and alprazolam) and the finished products (bensedin® and ksalol®) using the dielectric barrier discharge (dbd) reactor for aop. we studied the degradation rate of these pharmaceuticals, depending on the number of passes through the reactor. this degradation method was efficient 61 % for diazepam and 95 % alprazolam. we also examined the influence of the ph adjustment between the passes of apis through the dbd reactor. the degradation rate of apis and the finished products was monitored by the high performance liquid chromatography (hplc) technique, using a photodiode array detector. the concentration of the dissolved ozone was determined using the iodometric procedure. keywords: alprazolam; diazepam; oxidation; pollutants. introduction modern society faces the increasing use of pharmaceutical products and, consequently, the increasing amounts of these products end up in the natural environment.1–10 what is difficult to control is the generation of pharmaceutical waste made by the users of pharmaceutical products. unlike other pollutants whose amounts in the environment have been reduced by the introducing of stricter laws, it is very difficult to apply equivalent standards when medicinal products are considered, taking into account all the benefits they brought to human and animal populations. in the light of the facts * corresponding authors. e-mail: veca.rad@gmail.com https://doi.org/10.2298/jsc170118050r (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 934 radulović, roglić and manojlović above, the amounts of pharmaceutical waste may also be expected to increase in the future. pharmaceutical waste is generated either during the process of an active pharmaceutical ingredient (api)/finished product manufacturing, or from the apis/finished products that are past their expiry dates. the finished products containing diazepam and alprazolam or any other benzodiazepine as their active ingredients, belong to a group of the most commonly used pharmaceutical products. in nearly all developing countries these are among the top twenty most commonly used pharmaceutical products.11 by their nature, the pharmaceutical products are the biologically active compounds. the parameters used to monitor the effect of the biologically active compounds on the living organisms are biodegradation and bioaccumulation. pharmaceutical waste belongs to the group of primary pollutants, being toxic for both humans and animals; it is poorly degradable12 and is not readily removed. a number of investigations shown the standard wastewater treatment methods as unreliable in the treatment of the pharmaceutical waste. such methods are generally non-destructive to diazepam and to the structurally similar substances.8,13–15 the application of advanced oxidation processes (aop) may result in a more effective degradation of such substances.16,17 these processes involve the creation of radicals (e.g., hydroxyl radicals) and their reaction with the sample. one of the most recent aop technologies is the plasma-based technology, which finds the increasing application in the water treatment for the purpose of removing a variety of different pollutants.18–23 one of the methods to generate plasma is the dielectric barrier discharge (dbd). dbd reactor, as a system for ozone generating, was recently developed and described.24 this reactor has been proved to be applicable and very effective in a variety of fields, such as the removal of phenol and chlorophenols from water,25 the degradation of phenol solutions,26 decolorization of reactive textile dyes,27 simultaneous removal of nox and so2 from flue gas in a coal-combustion power plant,28 degradation of herbicides,29,30 removal of ibuprofen from aqueous solutions,31 degradation of textile azo dye,32 as well as the degradation of anionic surfactants. 33 herein, we report for the first time, the degradation of diazepam and alprazolam, as well as of their respective finished products bensedin® and ksalol®, depending on the number of passes through the dbd reactor. experimental for the purpose of this paper, the apis (diazepam and alprazolam) and the finished products (bensedin® and ksalol®) of galenika a.d., belgrade, serbia, were used. all solutions were prepared immediately before their use. (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ degradation of benzodiazepines 935 diazepam solution a solution of 50 mg l-1 concentration was prepared by dissolving 100 mg of diazepam in 2 l of water (double-distilled), and adding 0.5 ml of concentrated hydrochloric acid (merck p.a.), due to its poor solubility. bensedin® solution bensedin® product containing 5 mg of diazepam, as active ingredient, was used. twenty tablets were dissolved in 2 l of water, adding 0.5 ml of concentrated hydrochloric acid in order to obtain the same concentration of diazepam (50 mg l-1). alprazolam solution a solution of 50 mg l-1 was prepared by dissolving 100 mg of alprazolam in 2 l of water, and adding 1.7 ml of concentrated hydrochloric acid due to its poor solubility. ksalol® solution ksalol® product containing 1 mg of alprazolam, as active ingredient, was used. one hundred tablets were dissolved in 2 l of water, adding 1.7 ml of concentrated hydrochloric acid, in order to obtain the same concentration of alprazolam (50 mg l-1). the reagents used for high performance liquid chromatography (hplc) analysis were: acetonitrile (j. t. baker, hplc grade), methanol (j. t. baker, hplc grade), potassium dihydrogen phosphate (merck), sodium hydroxide (fluka), ammonium acetate (fluka) and glacial acetic acid (merck). iodometric procedure prior to the experiment, the concentration of dissolved ozone was determined using the standard iodometric procedure. the concentration of dissolved ozone was measured by sampling from the upper reservoir and calculated for one pass through the ozone generator.24,25 in the ozonated water sample 5 ml of 20 vol. % solution of sulfuric acid (merck) and potassium iodide (sigma-aldrich) were added in excess. the sample was left for 10 min in a dark place and titrated with 0.1 mol l-1 solution of sodium thiosulfate. the titrant was added until the yellow color of liberated iodine faded. after the addition of 4 ml of starch indicator solution titration was continued until the blue colour disappeared. degradation procedure degradation of apis (diazepam and alprazolam) and their respective finished products (bensedin® and ksalol®) was carried out by means of an aop, using the non-thermal plasma reactor based on coaxial dbd.24,25 this coaxial dbd reactor is of fairly simple structure. the both coaxial electrodes are coated with glass as a dielectric and located at the discharge side. the system comprises off three reactors connected in parallel. the length of one dbd reactor, i.e., the length of an electrode is 40 cm and the distance between the electrodes is 3 mm. the electrode diameter is 20 mm. solutions of apis and of their respective finished products were pumped to the top of the reactor through a hollow central electrode using a peristaltic pump. from the top of the reactor, the solutions flowed as a thin film over the glass that was coating the inner electrode. the treated solutions were collected in a receptacle at the bottom of the reactor. the total flow rate through the system of three parallel dbd reactors was 210 ml min-1. one series of passes of diazepam and alprazolam solutions was carried out without the ph adjustment (ph 3.00). the treated samples were recirculated 12 times for diazepam and (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 936 radulović, roglić and manojlović alprazolam, with a 100 ml aliquot withdrawn after each pass of the solution through the dbd reactor. after a single series of passes of bensedin® and ksalol®, where the initial ph was 3.00, and was not modified, the treated sample was recirculated 12 times. after each pass of the solution through the dbd reactor a 100 ml aliquot was withdrawn. for a series of experiments with diazepam and alprazolam with the ph adjustment, one series of passes was carried out with an initial ph of 3.00. the treated sample was recirculated 7 times for diazepam and alprazolam. after each pass of the solution through the dbd reactor a 100 ml aliquot was withdrawn, while the ph of the remaining solution was adjusted to 3.00 (using 0.1 mol l-1 hydrochloric acid), and the solution returned for recirculation. analytical procedure the determination of degradation rate was performed by hplc technique using a photodiode array detector (hewlett packard 1100, belgrade, serbia). a zorbax sb-c8 column (250 mm×4.6 mm, 5μm) was used at 30 °c for chromatographic separation in diazepam and bensedin®. the mobile phase was obtained by mixing acetonitrile, methanol and 0.34 g/l aqueous solution of potassium dihydrogen phosphate (its ph being previously adjusted to 5 by the addition of sodium hydroxide solution) at the volume ratio of 22:34:44. the flow rate of the column was 1 ml/min in the isocratic mode, the injection volume being 100 µl. the detection wavelength was set to 254 nm. a zorbax sb-phenyl (250 mm×4.6 mm, 5μm) column was used at 40 °c for chromatographic separation in alprazolam and ksalol®. the mobile phase consisted of a buffer solution (a 0.77 g/l aqueous solution of ammonium acetate in water, its ph being adjusted to 4.2 using glacial acetic acid) and methanol at the volume ratio of 44:56, as the component a and a buffer solution (0.77 g/l aqueous solution of ammonium acetate in water, its ph being adjusted to 4.2 using glacial acetic acid) and methanol at the volume ratio of 5:95 as the component b. the flow rate of the column was 2 ml min-1 in the gradient mode: 2 % b, 15 min, 2–99 %, 20 min, 99 % b, 5 min. the injection volume was 100 µl. the detection wavelength was set to 254 nm. results and discussion as the emission of ozone takes place in a closed system there are no losses of ozone during the generation and dissolution in water.25 d. manojlović et al.25 applied this reactor for the removal of phenol from water samples. depending on the experimental conditions, the concentration of dissolved ozone was about 7-40 mg l-1. these values were higher than 0.2 mg l–1 obtained by using the reactor based on the corona needle-to-plate discharge.25 in our experiment, after approximately 1 h of work, the determined concentration of dissolved ozone in water was 12.9 mg l–1. in the first part of the paper we presented the degradation rate of diazepam and alprazolam apis, as well as of their respective finished products bensedin® and ksalol®, depending on the number of passes through the dbd reactor. the rate of degradation of starting compounds was determined by means of hplc technique, using the standard compendial method. the results are presented in tables i and ii. (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ degradation of benzodiazepines 937 table i. increase of the degradation rate (%) of active ingredient (diazepam and alprazolam) by passing through the dbd reactor number of passes active ingredient diazepam alprazolam 0 0 0 1 11.00 13.06 2 21.40 20.95 3 28.87 32.39 4 31.18 42.89 5 38.00 51.21 6 39.96 64.05 7 43.76 81.15 8 46.94 84.95 9 52.07 89.31 10 51.97 91.47 11 55.87 93.89 12 60.76 95.17 table ii. increase of the degradation rate (%) of active ingredient (diazepam and alprazolam) in bensedin® and ksalol® by passing through the dbd reactor number of passes active ingredient diazepam in bensedin® alprazolam in ksalol® 0 0 0 1 2.59 1.92 2 4.62 3.45 3 7.44 4.90 4 8.35 5.65 5 9.01 6.24 6 10.69 7.34 7 11.76 7.68 8 12.91 9.84 9 14.28 10.34 10 14.63 9.91 11 15.41 11.17 12 32.23 11.83 dbd reactor we used 24,25 can produce oxidative species such as ozone, the ultraviolet (uv) radiation, free electrons, short-lived radicals and ions26,27. the trend of decrease in diazepam and alprazolam concentration, in our experiment, may be explained by the fact that the fall of concentration decreases the possibility of the effective collision of the test substance molecules with the oxidizing agent. on the other hand, each subsequent pass of solution through the dbd reactor increases the amount of degradation products, which may also further react with the oxidizing agent, additionally diminishing the possibility of the effective collision of the test substance molecules with the oxidizing agent. (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 938 radulović, roglić and manojlović what is evident in alprazolam is a far higher rate of degradation, achieved with fewer passes through the dbd reactor. considering that the experimental conditions were identical, the explanation of this phenomenon might be related to the alprazolam structure. it has been shown that during benzodiazepine oxidation a cleavage in nitrogen-carbon double bond occurs most commonly.34 besides that, the alprazolam carbon c4 (fig. 1)35 is more susceptible to oxidation than the diazepam carbon c3 (fig. 1),36 and the probability of the effective collision, that would result in oxidation, is by far higher in alprazolam. fig. 1. diazepam and alprazolam structure. the hydrolysis of the benzodiazepines ring is the most frequently observed degradation routes for benzodiazepines.35 the hydrolysis of diazepam in both acidic and basic media was observed, and it may occur in two ways. however, either of these ways results in the same end products of hydrolysis.34 in the case of alprazolam, the hydrolysis, even under several different conditions, was not a major degradation source.35 the aromatic diazo ring of alprazolam probably protects it from hydrolysis, ensuring its better stability in both acidic and alkaline environment.34 some authors describe the degradation of this kind of compounds using a photodegradation method, such as uv irradiation,34 light-stress exposure testing with a medium-pressure metal halide lamp and mirrors35 or photolysis under conditions simulating the natural sunlight.36 this kind of degradation could produce some oxidative species.37 it is shown in the table ii that the rate of diazepam and alprazolam degradation in bensedin® and ksalol® finished products was not identical when compared to the degradation of the apis themselves. with the same number of passes, a lower percentage of the api degradation in the finished product was observed, which may be ascribed to the oxidation agent being spent on the excipients contained in the finished products. the oxidative species produced during (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ degradation of benzodiazepines 939 the dbd reactor operation are extremely reactive, nonselective, producing an almost instantaneous reaction. the results presented in the table ii show an abrupt rise in the rate of diazepam degradation in bensedin® at one point, indicating that the oxidation of excipients already occurred, after which the considerable degradation of diazepam began. this phenomenon was also described in some other papers. after phenol was treated by dbd reactor, the lower removal from samples prepared with danube river water could be explained by the protective effect of fulvic and humic acids which also reacted with ozone. it could be concluded that ozone reacted with different organic components present in the water.24,25 liu et al. reported removal of carbamazepine in an aqueous solution using ex situ and in situ discharge dbd reactors.18 the ozone concentration was fixed at the same value of 40 mg l–1 for the two systems. the absorbance decrease, measured on uv detector, shows that the degradation of carbamazepine in ex situ discharge was rapid (decrease of absorbance for about 80 %) according to the degradation in the in situ discharge, where degradation was very slow (decrease of absorbance for about 40 %).18 magureanu et al. investigated the decomposition of pentoxifylline in aqueous solution, using a dielectric barrier discharge (dbd), where oxygen was introduced through a separate entrance in the upper lid of the reactor.19 after plasma treatment (for concentration of 50 mg l–1) about 90 % removal of pentoxifylline was achieved.19 after ozonization in dbd reactor, the percentage of removed phenol, 2chlorophenol, 4-chlorophenol and 2,4-dichlorophenol, 2,6-dichlorophenol from bidistilled water was between 84.9 and 99.9 %.25,26 while working with diazepam and alprazolam samples, used in our experiment, an increase of the solution ph value was observed after the sample passing through the dbd reactor. such observations were contrary to the previous observations on this reactor, when the ph of distilled water decreased as well, which might be explained by the cleavage of water molecules, i.e., by the participation of hydroxyl groups in the ozone generation.24 in the case of diazepam and alprazolam, it might be assumed that during the molecule disintegration, a protonation of certain negatively charged atoms, produced by bonds cleavage, occurs along with oh ions formation. that was the reason for performing the experiment with the ph adjustment to the initial value (3.00) after each pass through the dbd reactor and the testing of the solution ph influence on the rate of degradation. the initial ph value of the solution was 3.00. after each pass a 100 ml aliquot of solution was withdrawn, while the remaining solution was acidified to ph 3.00. the ph was measured after each pass through the dbd reactor and was, with minor fluctuations, approximately about 4.5 of ph units. diazepam and alprazolam samples were passed through the dbd reactor for 7 times. (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ 940 radulović, roglić and manojlović table iii shows the rate of diazepam degradation depending on the number of passes through the dbd reactor and ph adjustment between the passes. table iii. increase of the degradation rate (%) of diazepam and alprazolam (with corrected ph) by passing through the dbd reactor number of passes active ingredient diazepam alprazolam 0 0 0 1 12.28 15.04 2 23.09 25.11 3 31.24 42.62 4 37.87 49.45 5 42.67 54.07 6 49.73 63.36 7 53.15 74.91 the results which are presented in the tables tables i and iii show that the acidification of a solution, after each pass, has no important impact on the rates of diazepam and alprazolam degradation. conclusions the rate of diazepam and alprazolam degradation, depending on the number of passes through the dbd reactor, was tested, as well as the influence of excipients contained in bensedin® and ksalol® finished products, on the rate of degradation. based on the results obtained, it may be concluded that dbd for aop has been proved as a highly effective method for the removal of tested apis from the water, since high degradation rate was obtained for the otherwise poorly degradable compounds (diazepam: 61 %, alprazolam: 95 %). this method has not been proved effective enough in the degradation of finished products, containing the above mentioned active substances, due to a great consumption of oxidative species (e.g., ozone) on degradation of the excipients contained in the finished products. a large amount of energy was spent on these readily biodegradable compounds, which reduced the cost-effectiveness of this procedure. it has been demonstrated that the ph adjustment, between the passes of diazepam and alprazolam sample through the dbd reactor, has no important influence on the rate of degradation process. acknowledgments. this research was supported by the ministry of education, science and technological development of the republic of serbia, grant number 172030. (cc) 2017 scs. all rights reserved. _________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ degradation of benzodiazepines 941 и з в о д деградација бензодиазепина у реактору са диелектричним баријерним пражњењем кроз танки водени филм весна м. радуловић1, горан м. роглић2 и драган д. манојловић2 1галеника a.д. београд, батајнички друм б.б. 11080 београд и 2хемијски факултет, универзитет у београду, студентски трг 12-16, 11000 београд класичне методе пречишћавања отпадних вода често нису погодне за третирање фармацеутског отпада. досадашња истраживања су показала да употреба унапређених оксидационих поступака (aop) може довести до ефикасније деградације различитих билошки активних једињења, која представљају активне фармацеутске супстанце примењиваних лекова. циљ овог рада је примена плазма технологије за деградацију две активне фармацеутске супстанце (диазепам и алпразолам) и готових производа (bensedin® and ksalol®) у реактору са диелектричним баријерним пражњењем (dbd), као унапређеног оксидационог процеса (aop). испитиван је степен деградације ових фармацеутика, у зависности од броја пролаза кроз реактор. овај метод деградације се показао ефикасним: за диазепам 61 %, а за алпразолам 95 %. испитиван је и утицај ph подешавања између пролаза apis кроз dbd реактор. степен деградације api и готових производа праћен је hplc техником помоћу фотодиодног детектора. концентрација раствореног озона одређена је јодометријским поступком. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {degradation of polycyclic aromatic hydrocarbons in contaminated soil by immobilized laccase} j. serb. chem. soc. 83 (5) 549–559 (2018) udc 542.92.000.57+547.591:504.53.054+ jscs–5095 547.681+547.814 original scientific paper 549 degradation of polycyclic aromatic hydrocarbons in contaminated soil by immobilized laccase xin wang, shi yu sun, zi jun ni, zhao xing li and jia bao* school of science, shenyang university of technology, shenyang 110870, china (received 4 october 2017, revised 15 january, accepted 22 january 2018) abstract: the biodegradation of polycyclic aromatic hydrocarbons (pahs) in soils by laccase is reported. however, the low laccase activities of free laccase have limited its applications in environmental bioremediation. in this study, polluted soil was made with 3:1 volume ratio of soil to pah solution. subsequently, the adsorption–cross-link composite immobilization method was applied to immobilize laccase derived from fungi onto nylon net and chitosan, respectively. these two kinds of immobilized laccase were used in the degradation of pyrene (pyr) and benzo[a]pyrene (bap), and their degradation efficiencies under different temperature and ph conditions were investigated. consequently, the optimal laboratory experimental parameters were determined as follows: first, compared with free laccase, the degradation rates of pyr and bap by immobilized laccase increased by around 10–30 %; second, the degradation efficiency of chitosan as a carrier of immobilized laccase was much better than that of nylon net as a carrier of the immobilized laccase; finally, when the temperature was set at 40 °c and the ph was set at 4, the degradation efficiency achieved by immobilized laccase was the best. keywords: immobilized laccase; polycyclic aromatic hydrocarbons (pahs); soil remediation; nylon net; chitosan. introduction polycyclic aromatic hydrocarbons (pahs) are hydrocarbons that contain more than two benzene rings,1 including over 150 kinds of compounds, such as naphthalene, anthracene, phenanthrene and pyrene. they are considered toxic chemicals that are mainly formed during the incomplete combustion of coal, crude oil, gas, or other organic substances. numerous studies2,3 have shown that most of the low molecular weight pahs have one, two, or three aromatic rings, and they are acutely toxic. most of the high molecular weight pahs have more than three aromatic rings, which are highly mutagenic, teratogenic, and carcinogenic to * corresponding author. e-mail: baojia@sut.edu.cn https://doi.org/10.2298/jsc171004022w 550 wang et al. humans and animals. some studies have reported a potential relationship between the death from lung cancer of humans and pahs.4 for instance, in a study conducted in the henan province, feng et al.5 revealed that the accumulation of pahs is the highest in roots and the lowest in grains, and that the consumption of wheat grain caused potential risk of cancer. some researchers found that the incidence of lung cancer was also closely related to air pollution by benzo[a]pyrene.6 in addition, jung et al.7 maintained that exposure to pahs in the air around new york city, especially in the warmer seasons, could increase the risk of cancer and mutations. the common sources of pahs found in the environment are both natural and anthropogenic. natural sources include forest and pasture fires, oil leakages, volcanic eruptions and plant secretions. anthropogenic sources of pahs include the burning of fossil fuel, coal tar, wood and garbage, municipal solid waste incineration, petroleum spills and used lubrication oil.8,9 hitherto, pahs were considered to be everywhere and pollute air, soil, or water, and their migration pathways into the environment include volatilization, photooxidation, chemical oxidation, adsorption on soil particles, leaching and microbial degradation. pahs are resistant pollutants. generally, the degradation rate of pahs are reduced with increasing molecular weight.10 therefore, under natural conditions, compared with high molecular weight pahs, low molecular weight pahs could be degraded rapidly. furthermore, with increasing molecular weight, the toxicity and durability of pahs will also increase. due to their high toxicity and environmental persistence, pahs are classified as carcinogens in many organizations, including the united states environmental protection agency (usepa), the international agency for research on cancer (iarc), and the national occupation safety and health administration (osha), etc. resulting from these classifications, safety issues related to the degradation of these compounds have been attracting more and more research interests.11–13 laccase (ec 1.10.3.2), also named phenolase, is a kind of cu-containing polyphenol oxidase. it is a protein in rhus vernicifera that can catalyze the immobilization of coatings and was first identified in 1883 by yoshida, a famous japanese scholar.14 more than a decade later, keshavarzifard et al.15 also found the enzyme in fungi and named it ‘laccase’. laccase is generally divided into plant laccase, fungal laccase, and bacterial laccase. at present, most of current research is based upon fungal laccase, with white-rot fungus being the most important producer of fungal laccase. the relative molecular masses of fungal laccase are between 50 and 130 kda, and its isoelectric points are from three to six. laccase has strong stability and broad substrate specificity. polyphenols (a structural class of mainly natural, but also synthetic or semisynthetic, organic chemicals characterized by the presence of large multiples of phenol structural units), aromatic amines, benzyl mercaptan, and methoxybenzene can be used as the object of laccase oxidation. compared with the direct degradation of pol degradation of polycyclic aromatic hydrocarbons by immobilized laccase 551 lutants by microorganisms, enzyme-catalyzed reactions have high decomposition efficiencies, low toxicity, a wide application range, and simple operation. simultaneously with the development of modern biological technology, it has also become possible to obtain large amounts of cheap laccase by effective separation, purification, gene expression, and recombinant technology. in recent years, due to its abilities to degrade phenolic compounds, laccase has been widely applied to different biotechnological processes. laccase from fungus has also been recognized as a potential candidate material for the bioremediation of pahs.16–18 over the past decade, immobilized enzyme technology has rapidly developed, with more and more new carriers applied for the immobilization of various enzymes.19–21 new carrier materials based on immobilized laccase could increase the stability and repetition for the utilization of laccase, and reduce the cost of its degradation.15 immobilized laccase has been mainly used in the production of food, pharmaceuticals, and other biological degradation techniques.22–24 in this study, two methods using immobilized laccase were applied to the remediation of pahs in soils, under different temperature and ph conditions. the objectives of this study were: i) to screen optimal carriers for the immobilization of laccase, which should be cheap and readily available, and widely applied, and ii) to effectively improve the activity of laccase by choosing suitable immobilization methods, and then provide a new way for the degradation of pahs in organically polluted soils using immobilized laccase. experimental materials and methods fungal laccase was purchased from the beijing institute of microbiology of the chinese academy of sciences. the soil was collected from non-polluted surface soil (0–20 cm depth) at the ecological station of the chinese academy of sciences. nylon mesh (100 mesh), chitosan (deacetylation degree 90.3 %), glutaraldehyde and other reagents were purchased from the tianjin bo di chemical co., ltd. (china). a uv721 uv–vis spectrophotometer from the shanghai precision science instrument co., ltd was used. an hp 1090-іі liquid chromatograph and a diode array detector (dad) were used for the determination of the degradation rate of the pahs. methods preparation of crude laccase. a certain quantity of straws (each about 3 cm long) was weighed into a conical flask and then water was added and the straws soaked for one day. the wet straws were sterilized at a high temperature and high pressure for 30 min. after cooling, the straws were used as the raw material to produce the enzyme culture medium. white-rot fungi (lenzites betulinus) were grafted into this culture medium and fostered in an incubator at 26 °c, then 100 ml aseptic liquid was added and soaked at 26 °c and 120 rpm agitation for 24 h. to remove the impurities, the mixture was centrifuged for 15 min at 4000 rpm and the supernatant was collected as crude laccase. immobilization procedure. two kinds of carriers were used to immobilize laccase and the adsorption–cross-link method was adopted to immobilize laccase. the cross-linking agent was glutaraldehyde. 552 wang et al. nylon net, a hydrophilic carrier, was the adopted carrier material, and linked by amide bonds. nylon net has the advantage of low cost, stable chemical properties and good operability. the nylon net was cut into small pieces (1 cm×1 cm), and then soaked in formaldehyde solution with cacl2 (19 %) and h2o (19 %) for 15 min. the nylon net was placed into hcl (5 mol l-1) for 40 min, after which the sample was washed and dehydrated, and then the ph of the nylon net was adjusted to neutral before repeated dehydration. then, the nylon net was immersed in a glutaraldehyde solution (5 %) for 6 h, placed in 30 units of laccase for 8 h and then flushed with phosphate buffer to reduce the ph to 7. the laccase recovery rate was 49 % under these conditions. the other carrier was chitosan. chitosan was obtained by deacetylation of chitin, which is rich in free amino groups. chitosan is an admirable carrier for laccase immobilization with many advantages, such as abundant resources, good biocompatibility, good hydrophilicity and resistance to microbial decomposition.25 specific operational steps in detail were as follows: chitosan was dissolved in glacial acetic acid until chitosan precipitation. naoh (2 mol l-1) was taken into the glacial acetic acid, and the ph of the chitosan solution was adjusted to neutral before dehydration. carrier (0.1 g) was accurately weighed and added to a glutaraldehyde solution (15 ml, 5 %) for eight hours, and subsequently 30 units of laccase was added to the glutaraldehyde solution. after 12 h, the immobilized carrier was cleaned with phosphate buffer to reduce the ph to 7. under these conditions, the enzyme recovery rate was 50 %. determination of free laccase activity. 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), abts, was used to determine the activity of free and immobilized laccase. the reaction solution (1 ml) was made of hoac–naoac (500 μl, 50 mm, ph 4), h2o (390 μl), abts (100 μl, 500 μm), and laccase liquid (10 μl). the reaction solution was placed in the uv–vis spectrophotometer and the temperature was set as 28 ℃. then the increase in absorbance during 3 min at 420 nm was used to calculate the free laccase activity (ε420 = 36000 l/(mol cm)). the free laccase activity was calculated by the following formula: 420 0 1000δ δ ε =l av u t v l , 1s s = l u v u m the culture condition of the first one in liquid, the second one in solid, when the condition was solid culture, the unit of activity of laccase enzyme was u/g, in which the v1 was the sterile extraction solution added during extraction, ms was the quality of the added straw culture medium. δa was increment of absorbance during three minutes, v was reaction solution (1 ml), δt was 3 min, v0 was laccase liquid (0.01 ml). l was the inner diameter of cuvette, l = 1 cm, and ms was the quality of the medium. one unit (u) of laccase activity was defined as the amount of laccase used for catalytic oxidation of abts (1 μm) after 1 min. determination of immobilized laccase activity. immobilized laccase (0.01 g) was accurately weighted and added into beaker, reaction solution (1.98 ml) was made of hoac– –naoac (1000 μl, 50 mm, ph 4), h2o (780 μl), abts (200 μl, 500 μm), and reaction solution was mixed for 10 min at room temperature. next, the supernatant was put into the uv– vis spectrophotometer and the reaction started. a free laccase activity was determined as an increase in the absorbance at 420 nm during 3 min. measurement data was used to calculate the immobilized laccase activity. the activity was calculated by the following formula: i 420 0 1000δ δ ε = av u t m degradation of polycyclic aromatic hydrocarbons by immobilized laccase 553 ui was the activity of immobilized laccase v was 2 ml of reaction solution and m0 was 0.01 g immobilized laccase. recovery of enzyme activity of immobilized enzyme. when laccase was immobilized, the stability of laccase would be improved obviously, but the enzyme activity would have some loss. the inactivation of laccase could be expressed by relative enzyme activity, that is, the recovery rate of immobilized enzyme activity. the specific calculation formula was as follows: i f 100 = u a u in the formula, a was the recovery rate of enzyme activity, ui was the total activity of immobilized enzyme and uf was the total activity of the added free enzyme. preparation of contaminated soil. an amount of 0.1 g of pollutants, pyrene (pyr) and benzo[a]pyrene (bap), was separately weighed and added into 500 ml volumetric flask containing methanol. then, it was fully dissolved in the ultrasonic bath and constant volume. when a 15 ml sample was added into 250 ml erlenmeyer flask containing 30 g of soil, the pollutant concentration was 100 µg/g. after the soil was dried naturally, 90 ml seed medium was added to the triangle bottle to make mud, then the ratio of soil to water was 3:1. methods for analysis and determination of soil samples. the soil samples were centrifuged, freeze-dried and sieved, then packed in bags and placed in freezers. 1 g soil sample and 20 ml of dichloromethane extract liquid was taken into 100 ml glass centrifuge tube, and then oscillated continuously for 2 h (changing water to keep water temperature not more than 35 ℃). after centrifugation for 5 min with 3500 rpm, the upper liquid was taken out and constant volume with dichloromethane to 20 ml. 1 ml liquid was filtered by silica gel column, then eluted with n-hexane and dichloromethane (1:1), dried with nitrogen and constant volume with methanol to 1 ml to be tested. determination of the residue. the collected water samples and 10 ml dichloromethane were transferred into 100-ml separating funnels, shaken for 5 min and left to stand for 3 min. the lower organic phase was transferred into a kd concentrator and the process was repeated a further two times. then, the organic phase was concentrated to 1 ml at a temperature of 55 °c, dried with nitrogen, made up to 1 ml with methanol and finally transferred to a liquid chromatographic sample bottle for measurement. experimental design. the laccase immobilized by the two methods was used to repair soil contaminated by pahs. mud and soil were in volume ratio of 3:1, the solution was hoac–naoac buffer (ph 4), and the initial concentration of pollutants was 100 g ml-1. the immobilized laccase added into the soil was 1 g per 50 ml, and free laccase was used as the control. some of the environmental factors encountered in the experiment need to be studied further. the first one was different temperatures, i.e., 20 °c (room temperature) and 40 °c, with the ph set to 4. secondly, the ph value was set at 4 or 6, with the temperature at 40 °c. the running time of an experiment was 72 h. hplc conditions the mobile phase was prepared with methanol and distilled water. an alkyl silica gel column was used. the detection uv wavelength was set to 240 nm for pyr and 290 nm for bap. the column temperature was 40 °c and the flow rate was 0.800 ml min-1. 554 wang et al. data processing microsoft excel software was applied to process all the experimental data. spss 17.0 was applied for the statistical analysis, and p < 0.05 was considered as a significant difference. results and discussion the recovery rates of immobilized laccase and free laccase as shown in fig. 1 in the case of different environmental conditions or different carriers, the recovery rates of laccase had obvious differences. from the point of recovery rates, immobilized laccase had greater activity than free lac (a) (b) fig. 1. the recovery of the immobilized laccase and free laccase under different conditions: ph (a) and temperature (b). carrier 1 was nylon net; carrier 2 was chitosan. degradation of polycyclic aromatic hydrocarbons by immobilized laccase 555 case, in addition, compared with nylon net, the laccase was immobilized on chitosan had higher degradation rates of pyr and bap. however, the laccase lost about 50 % of activity during the immobilization process. with the constant development of immobilized technology and the deepening of the study of laccase, the application of immobilized laccase has demonstrated a positive contribution to the literature. at present, immobilized laccase has been employed in areas of the decolorization of dyes,26,27 degradation of various pollutants,19,28 and biosensing.25,29 hitherto, most of the studies that have been performed on immobilized laccase were aimed at the degradation of organic contaminants in water.26,27,30 in this study, fungal laccase was immobilized on nylon net and chitosan, and then the immobilized laccase was applied to the degradation of pyrene (pyr) and benzopyrene (bap) in contaminated soil. the results indicated that the methods adopted in this study were effective. in addition, laccase has been successfully immobilized on many different materials, such as silica gel,31 non-porous acrylate beads27 and magnetic chelator particles.32 in the present study, nylon net and chitosan were used as immobilization carriers and have advantages of being cheap, practical and readily available. this could reduce the cost of the application and simplify the operational processes of immobilized laccase. previous studies33 have shown that through immobilization, the loss of laccase activity decreased greatly, and the activity of laccase was generally considered stable. under different ph values, it could be seen that the laccase can maintain a certain activity in near neutral or acid conditions. the influence of ph on the immobilized laccase degradation of pyr and bap after immobilization, the two kinds of immobilized laccase were used to repair pahs-contaminated soil. in this part, the ph value was set at 4 or 6, with the temperature at 40 °c. the running time of the experiment was 72 h. as shown in figs. 2 and 3, when the temperature was set at 40 °c, both immobilized laccase and free laccase could effectively degrade pyr and bap under different ph during 72 h. the degradation rate of pyr exceeded 80 % (fig. 2), and the degradation rate of bap exceeded 40 % (fig. 3). compared with ph 6, two kinds of immobilized laccase had higher degradation rates of the pollutants at ph 4. according to the statistical analysis, the two kinds of immobilized laccase had significantly different degradation effects on pyr and bap at different ph values. the experimental results were similar to those obtained in other studies.34 the influence of temperature on immobilized laccase degradation of pyr and bap when the ph was set at 4, the two kinds of immobilized laccase had different degradation effects on pyr and bap under different temperatures (fig. 4). at 20 or 40 °c, both the degradation rates of pyr and bap exceeded 80 and 50 %, and it could also be seen that the immobilized laccase had a better degradation 556 wang et al. effect than free laccase. zafra et al.35 found that the optimum temperature range was 15–20 °c for immobilized laccase, and that laccase was easy to inactivate at high temperatures. fig. 2. the influence of ph on the ability of the immobilized and free laccase to degrade pyr; different letters indicate significant difference in the degradation rate at different ph values (p < 0.05). fig. 3. the influence of ph on the ability of the immobilized and free laccase to degrade bap; different letters indicate significant difference in the degradation rate at different ph values (p<0.05). degradation of polycyclic aromatic hydrocarbons by immobilized laccase 557 (a) (b) fig. 4. the influence of temperature on the ability of the immobilized and free laccase to degrade pyr (a) and bap (b); the different letters indicate significant difference in the degradation rate at different temperatures (p < 0.05). conclusions in summary, according to the present study, it could be seen that immobilized laccase and free laccase had a great degradation capacity for pyr and bap. when the temperature was set at 20 or 40 °c and ph was set at 4 or 6, the degradation of immobilized laccase was improved. moreover, several factors should be considered in the application process. first, considering the time of remediation, if a short-term remediation can be achieved with free laccase, it might consider558 wang et al. ably reduce the cost. if the remediation is long-term, immobilized laccase should be used due to their better stability. secondly, it is important to select the most suitable method of immobilization of laccase to reduce the loss of laccase as much as possible. the proper immobilization method with a good performance of the enzyme carrier could improve the efficiency of the enzyme. finally, the existing enzyme carrier was processed and modified to ensure that it has a superior immobilization performance, which would be a trend in the development of enzyme carriers. as the application of immobilized laccase continues to expand, more new laccase carriers that meet the technological requirements and economic changes will be developed in the years to come. и з в о д разлагање полицикличних ароматичних угљоводоника у контаминираном земљишту имобилизованом лаказом xin wang, shi yu sun, zi jun ni, zhao xing li и jia bao school of science, shenyang university of technology, shenyang, china у раду је описан поступак биоразлагања полицикличних ароматичних угљоводоника (pah) у земљишту применом лаказе. мала активност слободне лаказе ограничава њену примену у биоремедијацији животне средине. у овом раду је коришћена лаказа из гљива, имобилизована методом адсорпције/умрежавања на најлонској мрежи или на хитозану, за разградњу pah једињења у загађеном земљишту. имобилизована лаказа је коришћена за разлагање пирена (pyr) и бензо[a]пирена (bap), а ефикасност разлагања је праћена на различитим температурама и ph. резултати су показали да имобилизована лаказа разлаже pyr и bap 10–30 % ефикасније од слободне лаказе. такође, ефикасност разлагања је већа када је лаказа имобилизована на хитозану него на најлонском носачу. имобилизована лаказа је најефикаснија на 40 °c и на ph 4. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis of sulfonamides bearing 1,3,5-triarylpyrazoline and 4-thiazolidinone moieties as novel antimicrobial agents} j. serb. chem. soc. 85 (2) 155–162 (2020) udc 547.541.521+547.772.2’78:542.913: jscs–5290 615.272’285 original scientific paper 155 synthesis of sulfonamides bearing 1,3,5-triarylpyrazoline and 4-thiazolidinone moieties as novel antimicrobial agents thi-dan thach1,2, thi tuong-vi le1, huu thien-an nguyen2-4, chi-hien dang2,4**, van-su dang5 and thanh-danh nguyen3,4** 1tra vinh university, tra vinh city, tra vinh province, vietnam, 2graduate university of science and technology, vietnam, academy of science and technology, 18 hoang quoc viet, cau giay, hanoi, vietnam, 3institute of research and development, duy tan university, da nang city, vietnam, 4institute of chemical technology, vietnam academy of science and technology, 1 mac dinh chi street, district 1, ho chi minh city, vietnam and 5department of chemical technology, ho chi minh city university of food industry, vietnam (received 21 june 2018, revised 8 april 2019, accepted 13 june 2019) abstract: two series of sulfonamides were synthesized from 4-hydrazinylbenzenesulfonamide as the key starting material. 1,3,5-triarylpyrazoline sulfonamides (2a–i) were obtained by cyclocondensation of various chalcones in 53– –64 % yields, while 4-thiazolidinone derivatives (4a–e) were synthesized by cyclocondensation between mercaptoacetic acid and different phenylhydrazones in 43–62 % yields. the synthesized compounds were characterized based on ftir, 1h-nmr, 13c-nmr and hrms data. the sulfonamides were evaluated for their in vitro antimicrobial activities against four bacterial strains (e. coli, p. aeruginosa, b. subtillis and s aureus), two filamentous fungal strains (a. niger and f. oxysporum) and two yeast strains (c. albicans and s. cerevisiae). seven pyrazolines, 2a–c and 2e–h, exhibited significant inhibition of different microbial strains. among them, compound 2b displayed good antifungal activity against a. niger (mic value at 12.5 μg ml-1) over the reference drug. keywords: synthesis; sulfonamide; pyrazoline; thiazolidinone; antimicrobial agents. introduction sulfonamides have been particularly considered as essential scaffolds for developing new medicines. the sulfonamides possess a wide range of biological activities such as anti-inflammatory,1 antidepressant,2 antimicrobial3,4 and anticancer.5 in bacterial cells, antibacterial sulfonamides act as competitive inhibitors of the enzyme, dihydropteroate synthase that is involved in folate synthesis.6 *,** corresponding authors. e-mail: (*)dangchihien@gmail.com; (**)danh5463bd@yahoo.com https://doi.org/10.2298/jsc180621057t ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 156 thach et al. several sulfonamide drugs developed from aromatic compounds have been used in antimicrobial therapeutic applications. for instance, sulfanilamide and sulfamethoxazole (fig. 1) are the simplest antimicrobial therapy drugs which were commonly used in treatments against gram-positive and gram-negative bacteria, as well as in the treatments of fungi and protozoa infections. these sulfonamides are selectively toxic to bacteria via blocking of the synthesis of folic acid.7,8 however, resistance to these sulfonamides has increased during past years because the susceptible pathogens might develop an ability to take up folic acid from their environment.9 fig. 1. structures of antimicrobial therapy sulfa drugs. pyrazolines are one of the important heterocycles containing two nitrogen atoms in the five-membered ring. these derivatives have been found to possess a broad spectrum of pharmacological actions, such as antimicrobial,10,11 anticonvulsant,12 anti-inflammatory13 and antiviral.14 numerous studies have shown that pyrazoline derivatives possess interesting antimicrobial activity against many fungal and bacterial strains. for example, zampieri et al.15 reported synthesized pyrazoline derivatives bearing imidazole moieties with high activity against two strains, candida albicans and mycobacterium tuberculosis h37rv. in particular, 1,3,5-substituted pyrazolines were documented as potential antibacterial agents.16–18 additionally, 4-thiazolidinones, derivatives of thiazolidine with a carbonyl group at the 4-position, are an important scaffold known to be associated with many biological applications including anticancer, antimicrobial, antiviral and anti-inflammatory.19–22 in connection with our ongoing research program involving the synthesis and biological evaluation on heterocycles,23–25 the biological importance of sulfonamides prompted us to synthesize derivatives bearing pyrazoline and 4-thiazolidinone moieties, as well as to evaluate their antimicrobial activity. experimental chemistry all the materials were purchased from acros (belgium) or aldrich. the solvents were purchased from fluka and used without further purification. melting points were measured with an electrothermal model 9200 (uk). the ir spectra were recorded on a bruker equinox 55 ir spectrophotometer. the esi-ms were taken on an aligent 1100 lc-msd instrument ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. synthesis of antimicrobial sulfonamides 157 with an ms detector (mass range 100–2000 m/z, energy 3.5 kv, dried gas 8.0 l min-1). 1h (500 mhz) and 13c-nmr (125 mhz) spectra were recorded on a bruker avance 500 nmr spectrometer using acetone-d6, cdcl3 and dmso-d6 as solvents and tetramethylsilane (tms) as internal standard. chemical shifts are reported in δ relative to tms. analytical and spectral data are given in supplementary material to this paper. general procedure for the synthesis of dihydro-1h-pyrazol-1-ylbenzenesulfonamides (2) a stirred solution of chalcones 1 (3 mmol) and 4-hydrazinylbenzenesulfonamide (699 mg; 3 mmol) in anhydrous methanol (30 ml) was refluxed for 12 h, monitoring by tlc. after completion of the reaction, the mixture was cooled overnight at 0 °c. the separated solid was filtered and washed with hexane (30 ml). the residue was purified by recrystallization from ethanol and dried under the vacuum to afford the pure compounds 2. general procedure for the synthesis of 4-thiazolidinone sulfonamides (4) a stirred mixture of phenylhydrazones (1 mmol) and mercaptoacetic acid (1 ml) were refluxed for 2 h. after completion of the reaction, monitored by tlc, ethyl acetate (10 ml) was added into the mixture. the organic residue was washed with nahco3 solution (3×20 ml), water and dried by anhydrous na2so4. then the solvent was evaporated and the crude product purified by column chromatography on silica gel 60 (230–400 mesh, e merck, darmstadt, germany) with n-hexane/ethyl acetate (30:70 volume ratio) as the eluent. antimicrobial assay the antimicrobial activity and the minimal inhibitory concentration (mic) of the synthesized compounds were determined by the broth dilution method in 96-well microtiter microplates, as reported previously.28 briefly, stock solutions of the synthesized compounds at concentration of 2 mg ml-1 were prepared by dissolving 2 mg of the test compound in 1 ml dimethyl sulfoxide (dmso). the stock solutions were serially diluted in 1 ml of corresponding sterile media (eugon broth (difco, usa) for bacteria and mycophil (difco, usa) for yeast). to obtain concentrations of 12.5–50 μg ml-1 and inoculated with a suspension (100 μl) of the respective microorganism. four bacterial strains including two gram negative bacteria (escherichia coli atcc 25922 and pseudomonas aeruginosa atcc 10145) and two gram positive bacteria (bacillus subtillis atcc 11774 and staphylococcus aureus subsp. aureus atcc 11632) were used for the evaluation of antibacterial activity. two filamentous fungal strains (aspergillus niger (atcc 6275) and fusarium oxysporum (atcc 7601)) and two yeast strains (candida albicans atcc 7754 and saccharomyces cerevisiae (vtcc–y–62)) were used for evaluating the antifungal properties of the synthesized compounds. tetracycline and streptomycin (sigma) were used as positive controls of the tests with gram negative and gram positive, respectively, and nystatin (sigma) was used as positive control for the test of filamentous fungi and yeast. pure dmso was used as the negative control. the mic value for a sample is expressed as the lowest concentration that inhibits the tested microbial growth. results and discussion chemistry in the present strategy, the series of triarylpyrazoline sulfonamides contains a benzensulfonamide ring attached to a five-membered pyrazoline ring at position 1 and two aryl rings bearing varied functional groups attached to the pyrazoline ring at positions 3 and 5. these derivatives (2a–i) were synthesized in meth________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 158 thach et al. anol by cyclocondensation of 4-hydrazinylbenzenesulfonamide and the corresponding chalcones (1a–i), which were prepared from the appropriate aromatic aldehyde and the corresponding ketone in base catalyst/ethanol at room temperature for 5 h (scheme 1). the present results showed that reaction time of the cyclocondensation (12 h) was much shorter than that of the previous report (36 h)13 while the yields were similar (53–64 %). scheme 1. synthesis of 1,3,5-triarylpyrazoline sulfonamides. reagents: i) etoh, koh aq. (2 %), rt, 4 h; ii) 4-hydrazinylbenzenesulfonamide, meoh, reflux, 12 h. the structures of triarylpyrazolines were unambiguously characterized by ir, nmr and mass spectral data. the 1h-nmr spectra showed characteristic singlet around 4.63–6.98 ppm for two protons of the –so2nh2 moiety in the structures. in addition, signals belonging to the aliphatic protons of the pyrazoline ring, ha, hb and hx appeared at around 3.15–3.27, 3.87–4.09 and 5.33–5.83 ppm, respectively. the other signals are in complete agreement with the assigned structures. the synthetic route of 4-thiazolidinone sulfonamides is illustrated in scheme 2. the hydrazone derivatives (3a–e) were prepared in good yields (63.0–74.0 %) via acid catalyzed condensation of the appropriate aromatic aldehyde and 4-hydrazinylbenzenesulfonamide in methanol for 4 h. cyclocondensation of the appropriate phenylhydrazones (3a–e) with a large excess of mercaptoacetic acid afforded the respective 4-thiazolidinone sulfonamides (4a–e). the ir data of 4a–e showed bands at around 3270 and 1700 cm–1 assigned to nh and co stretching, respectively. in 1h-nmr spectra, protons of amines including nh and nh2 moieties are characterized by singlets at around 8.59–8.70 ppm and 7.04–7.06 ppm, respectively while the appearance of signals around 3.74–3.82, 3.84–3.95 and scheme 2. synthesis of 4-thiazolidinone sulfonamides. reagents: i) ch3cooh, methanol, reflux, 4 h; ii) hsch2cooh, reflux, 2 h. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. synthesis of antimicrobial sulfonamides 159 5.83–5.90 ppm, confirmed aliphatic protons of 4-thiazolidinone ring (ha, hb and hx, respectively). antimicrobial activity the pyrazoline and 4-thiazolidinone derivatives were screened for their activity against four bacterial strains, including two gram negative (e. coli and p. aeruginosa) and two gram positive (b. subtillis and s aureus) bacteria, two filamentous fungal strains (a. niger and f. oxysporum) and two yeast strains (c. albicans and s. cerevisiae). the samples as the standard references including tetracycline and streptomycin were used for the tests of gram negative and gram positive bacteria, respectively while nystatin was used for test of fungi and yeast. the data in average mic values are given in table i. it indicated that most of the pyrazoline sulfonamides exhibited high activity against three bacterial strains, e. coli, b. subtillis and s. aureus, both tested fungal strains and a yeast, c. albicans, whereas the 4-thiazolidinone derivatives did not show antimicrobial activity at the tested concentration (50 μg ml–1). table i. antimicrobial activity (mic / μg ml-1) of sulfonamide derivatives; t – tetracycline; s – streptomycin; n – nystatin compd. microorganism bacterial gram (–) bacterial gram (+) fungi yeast e. coli p. aeruginosa b. subtillis s. aureus a. niger f. oxysporum s. cerevisiae c. albicans 2a >50 >50 >50 25 25 25 >50 >50 2b >50 >50 12.5 12.5 12.5 50 >50 >50 2c 50 >50 25 25 >50 >50 >50 25 2d >50 >50 >50 >50 >50 >50 >50 >50 2e 25 >50 25 25 >50 >50 >50 50 2f >50 >50 50 25 >50 >50 >50 >50 2g >50 >50 >50 >50 25 >50 >50 >50 2h >50 >50 >50 >50 25 >50 >50 >50 2i >50 >50 >50 >50 >50 >50 >50 >50 4a >50 >50 >50 >50 >50 >50 >50 >50 4b >50 >50 >50 >50 >50 >50 >50 >50 4c >50 >50 >50 >50 >50 >50 >50 >50 4d >50 >50 >50 >50 >50 >50 >50 >50 4e >50 >50 >50 >50 >50 >50 >50 >50 t 5.5 11.0 – – – – – – s – – 7.2 11.4 – – – – n – – – – 23.1 11.6 5.8 11.6 amongst the synthesized pyrazolines, compound 2b having 2-hydroxy substitution on benzene ring exhibited similar antibacterial activity to both gram positive bacteria (mic values at 12.5 μg ml–1) in comparison with streptomycin and superior antifungal activity to a. niger (mic value at 12.5 μg ml–1) in res________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 160 thach et al. pect to nystatin (mic value at 23.1 μg ml–1). it is noteworthy that only derivatives 2c and 2e containing 4-tolyl substitut attached to the pyrazoline ring at position 5 showed activity against e. coli (mic values at 50 and 25 μg ml–1, respectively) and yeast, c. albicans (mic values at 25 and 50 μg ml–1, respectively) while three pyrazoline compounds 2g, 2h and 2i bearing 4-methoxyphenyl substituent at position 3 did not show antibacterial activity at the tested concentration. the results reveal that substituents in the structure of triarylpyrazoline sulfonamides have important effects on the antimicrobial activity. in general, a 4-methylphenyl substituent attached to pyrazoline ring exhibited activity over the 4-methoxyphenyl substituent and the pyrazolines containing hydroxy group can be important antibacterial agents. it is essential for the next research to prepare new antimicrobial triarylpyrazoline sulfonamides with excellent activity. conclusions in summary, 1,3,5-triarylpyrazoline and 4-thiazolidinone sulfonamides were prepared from 4-hydrazinylbenzenesulfonamide in good yields. all the synthesized sulfonamides including novel compounds 2f, 2h, 4c and 4d, were well characterized by spectral analyses and investigated for their antimicrobial activity. seven pyrazoline sulfonamides 2a–c and 2e–h exhibited high activity against the different strains, whereas the 4-thiazolidinone derivatives did not display antimicrobial activity at the tested concentration. among them, compound 2b was found to be a potential antibacterial and antifungal agent compared with the references, streptomycin and nystatin, respectively. supplementary material experimental data for synthesis of compounds 1a–i and 3a–e, nmr and hrms spectra of compounds 2 and 4 are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. the authors are thankful to the tra vinh university, tra vinh city for financial assistance (no. 243/hđ.đhtv-khcn). и з в о д синтеза нових деривата сулфонамида који садрже 1,3,5-триарилпиразолинске и 4-тиазолидинонске структурне делове, као антимикробних агенаса thi-dan thach1,2, thi tuong-vi le1, huu thien-an nguyen2-4, chi-hien dang2,4, van-su dang5 и thanh-danh nguyen3,4 1 tra vinh university, tra vinh city, tra vinh province, vietnam, 2 graduate university of science and technology, vietnam, academy of science and technology, 18 hoang quoc viet, cau giay, hanoi, vietnam, 3 institute of research and development, duy tan university, da nang city, vietnam, 4 institute of chemical technology, vietnam academy of science and technology, 1 mac dinh chi street, district 1, ho chi minh city, vietnam и 5 department of chemical technology, ho chi minh city university of food industry, vietnam две серије сулфонамида су синтетисане полазећи од 4-хидразинилбензенсулфонамида као полазног једињења. 1,3,5-триарилпиразолин-сулфонамиди (2a–i) добијени су реакцијом ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. synthesis of antimicrobial sulfonamides 161 циклокондензације различитих халкона у приносу 53–64 %, док су 4-тиазолидинонски деривати (4a–e) добијени реакцијом циклокондензације 2-меркаптосирћетне киселине са различитим фенилхидразонима у приносу 43–62 %. синтетисана једињења су окарактерисана ftir, 1hи 13c-nmr и масеним спектрима. испитана је in vitro антимикробна активност према четири бактерије (e. coli, p. aeruginosa, b. subtillis и s. aureus), два филаментна соја гљивица (a. niger и f. oxysporum) и два соја квасца (c. albicans и s. cerevisiae). седам пиразолина 2a–c и 2e–h показују значајну инхибицију различитих микробних сојева. од њих, једињење 2b показује добру антифунгалну активност према гљивици a. niger (mic 12,5 μg ml-1), у поређењу са референтним леком. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis and dyeing performance of some amphiphilic naphthalimide azo disperse dyes on polyester fabrics} j. serb. chem. soc. 85 (10) 1253–1264 (2020) udc 667.27+677.494.674:547.651: jscs–5372 677.027.625.122 original scientific paper 1253 synthesis and dyeing performance of some amphiphilic naphthalimide azo disperse dyes on polyester fabrics umar salami ameuru1, mohammed kabir yakubu1*, kasali ademola bello1, peter obinna nkeonye1 and azim ziyaei halimehjani2** 1department of polymer and textile engineering, ahmadu bello university, zaria, nigeria and 2faculty of chemistry, kharazmi university, 49 mofateh st., tehran, iran (received 23 january, revised 24 march 2019, accepted 20 august 2020) abstract: a series of monoazo disperse dyes were synthesized by coupling diazotized 4-amino-n-dodecyl-1,8-naphthalimide with n,n-dialkyl anilines and naphthol derivatives. the synthesized intermediates and the dyes were characterized using ftir, 1h-nmr, 13c-nmr, mass spectroscopy and elemental analysis (chn). visible absorption spectra of the dyes were examined in solvents of different polarities. the electronic absorption spectra cover a wavelength (λmax) range of 515–535 nm in dmf at uniformly absorption intensity between 1.59–3.00×104 l mol-1 cm-1. the dyes gave deep and bright intense hues of light violet, maroon, pink and neon red on polyester fabrics. the dyes generally showed good washing and perspiration rating but poor to moderate light fastness properties on woven polyester fabric and could be recommended for commercial outlets. keywords: 1,8-naphthalimide derivatives; azo dyes; solvatochromic effects; naphthols; fastness properties. introduction azo compounds are the most widely used class of industrial synthesized organic dyes due to their versatile applications in various fields, such as the dyeing of textiles, biological–pharmacological activities and advanced application in organic synthesis.1–3 most of the heterocyclic dyes are derived from diazo components containing five-membered rings with one or more nitrogen heteroatoms, with the ring being fused into another aromatic ring.4 azodyes containing heterocyclic rings result in brighter and often deeper shade than their benzene analogues. they are very important in applications such as disperse dyes for polyester fabrics, reprography, functional dyes and nonlinear optical systems, photodynamic therapy and lasers.5–10 azo dyes based on naphthalimides have a *,** corresponding authors. e-mail: (*)mkyakubu@abu.edu.ng; (**)ziyaei@khu.ac.ir https://doi.org/10.2298/jsc190123049a ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 1254 ameuru et al. violet to blue hue. this phenomenon results from the high maximum absorption of their dyes. the strength of the electron withdrawing inductive effect of naphthalimide systems is greater than that of benzene ring systems containing nitro groups.11 pioneering work on heterocyclic derivatives of naphthalimides, phenylazophthalimides and 1,8-naphthalic anhydrides has been performed to assess their role as intermediates for dye preparation.12–15 4-aminonaphthalimide compounds have an amino group in their 4-position, which tends to act as a donor group, and two carbonyl groups, which act as acceptor groups. these substituents bring about a considerable bathochromic effect that led to their usefulness as the diazo component for the synthesis of azo dyes.16 naphthalimide derivatives have obtained great industrial importance due to their excellent fastness of some of their dyes.17 aminonaphthalimides are known to produce fluorescent yellow dyes for synthetic polymer fibres. the dyes with an alkyl group in n-substituted naphthalimides can be converted into disperse dyes that are used in the coloration of synthetic fibres.18 generally, monoazo disperse dyes derived from naphthalimides show exceptionally good thermal stability and good dyeing fastness properties.19 however, the 4-amino-n-alkyl(ester)-1,8-naphthalimide derivatives of monoazo disperse dyes have relatively low heat fastness properties. conventional disperse dyes are hardly soluble in water due to the lack of polar groups in their structures. this leads to milling of the dye into fine particle size and addition of dispersion in water using dispersing agents during application, hence gave rise to prolong application time. in the present study, a series of monoazo disperse dyes with in situ incorporation of amphiphiles to enhance their solubility was synthesized. this would eliminate the milling and additions of dispersing agents associated with the application of known disperse dyes. experimental materials and methods all the chemicals used in the synthesis were of commercial grade and used as received. the structures of all the newly synthesized compounds were confirmed by thin-layer chromatography (tlc), melting point, ft-ir, 1hand 13c-nmr spectroscopy, mass spectra (ms) and elemental analysis (chn). the purity of the synthesized compounds was checked by tlc using ethyl acetate–n-hexane (90:10 volume ratio). the tlc was performed on silica gel 60 f254, 0.2 mm coated aluminium sheets. the melting points were recorded on a barnstead electrothermal 9200 apparatus and are uncorrected. the infra-red spectra were recorded on a perkin-elmer spectrum rx1 ft-ir spectrometer, 1hand 13c-nmr spectra were obtained on a 300 mhz bruker instrument using deuterated chloroform (cdcl3) and dimethylsulphoxide (dmso-d6) as solvent. chemical shifts are reported in parts per million (ppm) downfield from internal tetramethysilane (tms). elemental analyses (chn) were recorded on perkin–elmer ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ amphiphilic naphthalimide azo disperse dyes 1255 pe 2400 series ii and the mass spectra were obtained using an agilent technologies 5975c vl msd mass spectrometer. analytical and spectral data are given in supplementary material to this paper. synthesis of 5-nitroacenaphthene (2) acenaphthene 1 (15.4 g, 0.1 mol) was dissolved in hot glacial acetic acid (57.2 ml) and then cooled under vigorous stirring to separate as fine as possible crystalline acenaphthene. concentrated nitric acid (65 %, d = 1.40 g ml-1, 9.84 g) was added drop wise to this mixture over 1 h at 22–27 °c and then further stirred for 1 h at 25 °c. the solution was warmed gradually over 30 min to 68 °c then cooled to form crystals that were filtered, washed with 1:1 acetic acid–water and water to give yellow needles (yield 85 %).20 synthesis of 4-nitronaphthalene-1,8-dicarboxylic anhydride (3) 5-nitroacenaphthene (24.87 g, 0.125 mol) was dissolved in hot glacial acetic acid (248.75 ml), sodium dichromate (158.5 g) was added over 3 h at 65–70 °c. the solution was warmed gradually to 98–100 °c over 30 min and further refluxed for 5 h. the reaction mixture was treated with water (0.6 l) and cooled to room temperature. the precipitate was filtered and washed with dilute hcl (10 %). in addition, the solid was boiled with 200 ml of 5 % na2co3 solution for 30 min and filtered. the filtrate was acidified and the separated crystals dried at 120 °c for 4 h to obtain 4-nitronaphthalene-1,8-dicarboxylic anhydride, which was recrystallized from concentrated hno3 (65 %, d = 1.40 g ml-1) to afford colourless needle crystals (yield 74 %).21-22 synthesis of 4-nitro-n-dodecyl-1,8-naphthalimide (4) a suspension of 4-nitronaphthalene-1,8-dicarboxylic anhydride (0.02 mol, 4.86 g) and dodecylamine (0.03 mol, 5.55 g) in ethanol (99.5 %, 102 ml) was stirred under reflux for 7 h. the reaction mixture was cooled to precipitate. the precipitate was filtered and recrystallized from ethanol to give off-white crystals (yield 74 %).23 synthesis of 4-amino n-dodecyl-1,8-naphthalimide (5) a mixture of 4-nitro-n-dodecyl-1,8-naphthalimide (18 mmol, 6.88 g) and stannous chloride (90 mmol, 20.25 g) in ethanol (81 ml) was refluxed for 1 h with hydrochloric acid (36 %, 10.1 ml) until the reaction was completed. the mixture was transferred into 100 cm3 of water and the observed precipitate was filtered. the crude product was purified by recrystalization from ethanol (99.5 %) to give orange crystals (yield 84 %).23 procedure for the synthesis of dyes (7a–d) (z)-2-dodecyl-6-((2-hydroxynaphthalen-1-yl)diazenyl)-benzo[de]isoquinoline-1,3(2h) -dione (7a). sodium nitrite (1.5 mmol, 0.104 g) was slowly added under constant stirring to cold concentrated sulphuric acid (98 %, 1.1 ml) at below 10 °c. the temperature of the reaction mixture was gradually increased to 65 °c using a water bath until all the sodium nitrite had dissolved. the solution was then cooled externally to 5 °c and diluted with a mixture of propionic acid and glacial acetic acid (10ml, 1.5:8.5 volume ratio). finely ground powder of 4-amino-n-dodecyl-1,8-naphthalimide (1.5 mmol) was added portion wise below 10 °c, and then the mixture was stirred for 3 h. the obtained clear diazonium salt solution was used immediately in coupling reactions with 2-naphthol (1.5 mmol) dissolved in ethanol by its addition over 30–40 min under vigorous stirring. the mixture was stirred further for 2 h at 5 °c. the ph of the solution was adjusted to 4–5 using 10 % sodium acetate and stirred for 1 h. the resulting product was then collected by filtration, washed with warm water and then cold water and dried. the crude product was purified by recrystallization from dmso several ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 1256 ameuru et al. times to obtained dye 7a (yield 80.9 %). the remaining dmso was removed from the product by washing the precipitate several times with water and aqueous ethanol (50 %). (z)-6-((2,3-dihydroxnaphthalen-1-yl)diazenyl)-2-dodecyl-1h-benzo[de]isoquinoline -1,3(2h)-dione (7b). the same procedures as for 7a were used, except that 2,3-dihydroxynaphthalene was used for the coupling. (z)-6-((4-(dimethylamino)phenyl)diazenyl)-2-dodecyl-1h-benzo[de]isoquinoline-1,3(2h) -dione (7c). the same procedures as in 7a were used, except that n,n-dimethylaniline was used for the coupling. (z)-6-((4-(diethylamino)phenyl)diazenyl)-2-dodecyl-1h-benzo[de]isoquinoline-1,3(2h) -dione (7d). the same procedures as for 7a were used, except that n,n-diethylaniline was used for the coupling. dyeing procedure a kms-ire laboratory dyeing machine – elite, was used with a material to solution ratio of 1:50, 2 % shade on weight of the fabric (owf) was used for dyeing. the dye was dissolved in dmf (5 ml) and water (45 ml) was added under constant stirring and the ph adjusted to 5 using glacial acetic acid. the dye suspension (50 ml) was transferred into a beaker provided with a lid and a screw cap. before closing the lid and tightening the metal cap over the beaker, a wetted pattern of polyester fabric was rolled into the beaker. the beaker was then placed vertically on a rotatory carrier inside the tank and the clamp plate was firmly tightened. the rotatory carrier was then allowed to rotate in the glycerine-bath and the temperature was raised to 130 °c at a rate of 2 °c min-1. the dyeing was continued for 1 h under pressure. after cooling to 70 °c, the beaker was removed from the bath. the dyed pattern was removed from the beaker|, thoroughly washed with water and dried at room temperature. reduction clearing the dyed fabric was treated by stirring in an aqueous solution containing sodium dithionite (2 g l-1) and sodium hydroxide (2 g l-1) for 45 min at 70 °c. the fabric was then washed with water and dried. colour measurement a gretag macbeth colour-eye 7000a spectrophotometer was used for colour measurement of the dyed polyester fabrics using a d65 source, 10° collection angle and small area view aperture (3 mm×8 mm). fastness properties of the dyed polyester fabrics wash fastness. the dyed samples were tested for fastness to washing treatment according to the international standard organization (iso 3). the composite samples were treated with a solution containing 5 g l-1 detergent, 2 g l-1 sodium carbonate and liquor ratio 50:1 for 30 min at 60 °c. the change in colour of the tested samples and the degree of staining of the adjacent undyed fabrics were rated against the grey scales. the grey scales are in the range 1–5, where 1 is poor and 5 is excellent. light fastness. the photostability was evaluated by exposing the dyed fabric samples to a xenon arc lamp according to (iso 105 b02:2014). the conditions for the test were: black panel temperature 60 °c, dry bulb temperature 43 °c, relative humidity 30 %, duration of test 48 h. after testing, the tested samples were rated against standard blue wool scale (grade 1–8) and the indications in this case are 1 is poor and 8 excellent. perspiration fastness. colour fastness to perspiration was determined according to iso 105 – eo4, 2013 by treating the dyed fabrics (10 cm× 4 cm) in an alkaline solution of freshly ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ amphiphilic naphthalimide azo disperse dyes 1257 prepared 0.5 g l-1 1-histidine monohydrochloride monohydrate, 5 g l-1 sodium chloride, 2.5 g l-1 disodium hydrogen orthophosphate and 0.1 m sodium hydroxide volume ratio of 50:1. the acid perspiration test solution contained 0.5 g l-1 1-histidine monohydrochloride monohydrate, 5 g l-1 sodium chloride, 2.2 g l-1 disodium hydrogen orthophosphate and 0.1 m acetic acid solution. the tested fabric was rinsed, dried and then rated against the grey scales for colour changes. results and discussion synthesis of intermediates and the dyes compounds 2–5 were prepared starting from acenaphthene 1 according to the literature, as depicted in scheme 1.23 the structure of the intermediates and dyes were confirmed by various techniques including ft-ir, nmr, elemental analysis (chn) and mass spectroscopy. the dyes 7a and 7b showed absorption band at 3387–3375 cm–1 due to the presence of an oh group. scheme 1. synthesis of the intermediates and the dyes. all the dyes showed absorption bands at 3106–2947 and 2922–2919 cm–1 due to the presence of c–h stretching vibration of aromatic c–h, methyl and methylene groups. the absorption band at 1663–1646 cm–1 corresponded to c=o groups. the absorption band at 1647–1604 cm–1 confirmed the presence of c=c groups. the signal at 1599–1579 cm–1 is attributed to –n=n– stretch, while ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 1258 ameuru et al. the signal at 1388–1349 cm–1 is due to c–n. the 1h-nmr spectra of the dyes displayed signals at 0.82–0.86 and 1.22–1.74 ppm for the methyl and methylene protons of the aliphatic region. the signal at 4.03–4.17 ppm shows is downfield due to n–ch2 protons of the aliphatic group. the diazo group was substituted in the alpha position of 2-naphthol and 2,3-naphthalenediol in dyes 7a and 7b. due to this, the dyes 7a and 7b showed signals at 16.45–16.91 ppm for oh protons in 1h-nmr spectra. this amount of deshielding can be attributed to intramolecular hydrogen bonding of the diazo group and the –oh group. the aromatic protons showed signals at 6.55–8.91 ppm and the number of protons is in agreement with the aromatic protons in the structure of dyes. the presence of two signals at 6.7 and 7.9 ppm as a doublet in the 1h-nmr spectra of 7c and 7d exactly confirms that the diazo group is substituted at the para position on the n,n-(dialkyl)aniline ring. in addition, mass analysis exactly confirmed the molar mass of the dyes. spectral characteristics of the synthesized dyes the absorption spectra of the synthesized dyes were measured in various solvents. absorption maxima, molar extinction coefficients of each dye in various solvents are listed in table i. wavelengths of maximum absorption (λmax) and molar extinction coefficient ranged from 515–535 nm and 1.59×104–3.00×104 l mol–1 cm for the synthesized dyes in dmf, respectively. the wavelength (λmax) shifts in different solvents are due to solvatochromic effects resulting from changes in the dielectric constant of the solvent. in many dyes, however, the ground state is less polar than the excited state and thus, a polar solvent will tend to stabilize the excited state more than the ground state, giving rise to bathochromic shift.24 it was observed that in chloroform, ethanol and ethanol plus a drop of hcl, the absorption spectral of the dyes did not change significantly. table i. uv–vis spectroscopic data for the dyes dye εmax(dmf) 104 mol-1 cm-1 λmax / nm dmf chloroform ethanol a ethanol +hcl b change (b–a) 7a 1.94 520 515 518 518 0 7b 2.26 515 515 512 512 0 7c 3.00 522 510 510 520 10 7d 1.59 535 525 525 530 5 generally, the colour of naphthalimide azo dyes depends on the delocalization of electrons through the conjugated chromogen depicted by the energy difference between structures i and ii, as shown in scheme ii. this agrees with bojinov et al.25 the bathochromic effect of these compounds increases when the energy difference between resonance structures is low. the contribution of the high energy ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ amphiphilic naphthalimide azo disperse dyes 1259 stabilized structure ii could be initially enhanced by the presence of a conjugated electron acceptor (i.e., a naphthalimide residue) and secondly by an electron donor on the coupling component (i.e., dialkylamino group), which results in a considerable bathochromic shift. n o o r .. n ch3 ch3 n n n o -o r n+ ch3 ch3 n n i ii scheme ii. delocalization of electrons through the conjugated chromogen. dye 7a, obtained by diazotisation of 4-amino-n-dodecyl-1,8-naphthalimide and coupling to 2-naphthol, absorbed at 520 nm in dmf. when the hydroxyl group was introduced into the ortho-position of the coupling component (2,3-dihydroxnaphthalene), the resulting dye 7b absorbed at 515 nm in the same solvent and thus dye 7b was hypsochromic by 5 nm when compared with dye 7a. dye 7c, obtained by diazotisation of 4-amino-n-dodecyl-1,8-naphthalimide and coupled with n,n-dimethylaniline, absorbed at 522 nm in dmf. when the coupling component was replaced by n,n-diethylaniline (dye 7d), it absorbed at 535 nm. dye 7d showed a bathochromic shift of 13 nm when compared with dye 7c, which may be attributed to the higher electron donor capacity of the ethyl groups compared to the methyl groups. dyeing and fastness properties of the synthesized dyes the synthesized dyes were applied to polyester fabrics using a high temperature dyeing method. visual evaluation of the dyeing on the polyester fabrics showed that the nature of the alkyl residue in the amine group of the coupling component affects both the intensity of dyeing and the shade obtained. the highest intensity was shown by the dyes that contain methyl and ethyl groups. these substituents increase the electron density on the tertiary nitrogen atom and stabilize the polar forms, especially when there are no substituents on the phenyl ring causing steric effects.26 an electron donor substituent on the ring of the coupling components also considerably affects the colour of the dyeing, especially those containing a substituent in the ortho position to the azo band, such as dye 7a. this phenomenon is described in terms of structures iii and iv, scheme iii. the results of the wash fastness ratings are summarized in table ii. the dyes gave good levelness and fibber penetration on polyester. the wash fastness rating is good with rating of 3 for dye 7b and rating of 4 for dyes 7a and 7c. dye 7d gave outstanding wash rating of 5. the good wash fastness obtained on polyester ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 1260 ameuru et al. may be due to the crystalline structure of the polyester that disallowed the migration of dye out of the fabric after entering the fabric: a) to the absence of solubilising groups that renders solubility and washability of dye-out of the fabrics. b) to the good intra-fibber diffusion of the dye molecules inside the fabric. scheme iii. structures iii and iv. table ii. fastness properties of the synthesized dyes on polyester fabrics dye light fastness washing perspiration acid alkaline 7a 2 4 5 4 7b 5 3 5 4 7c 1–2 4 4 4 7d 4 5 4 5 the dyed fabrics have a poor to moderate light fastness rating, as shown in table ii. the dye 7a and 7c exhibited poor light fastness ratings while dye 7d were marginally superior to the others. the dye 7b gave a very good light fastness rating. disperse dyes have a tendency to deposit on the fibber surface during dyeing as well as during cooling and therefore, in order to obtain a satisfactory combination of light and sublimation fastness, a judicious choice of substituent is required.27 several reports suggest that fading of azo dyes is mainly a consequence of decomposition of the –n=n– moiety, either by oxidation, reduction or photolysis.28 the rates of these processes should be sensitive to the chemical structure of the dye, the type of substrate and treatment conditions. the dyed substrate employed in this study was polyester fabric and thus, the fading process likely occurs by oxidation.29 the ease of oxidation of azo linkages should be a function of electron density. therefore, electron donating substituents on this moiety should increase the fading rate while electron withdrawing groups should decrease the rate. this agrees with the observed results (table ii) that demon________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ amphiphilic naphthalimide azo disperse dyes 1261 strate that the presence of a hydroxyl group in some of the synthesized dyes caused a decrease in light fastness to 2. the introduction of polar groups can adversely affect the dyeing properties of a dye, but the hydroxyl and alkyl group appear to be particularly useful in increasing polarity without excessive undesirable side effects. a free hydroxyl group and also a short alkyl chain favour the formation of compact aggregates of dyes, which confer low surface activity, which cause a dye to spread as multilayers on the surface of the fabric; a multilayer is less susceptible to fading. this may account for why the dyes from n,n-dimethylaniline and n,n-diethylaniline have acceptable results in light fastness. colour fading can be caused by the reaction between dyes on garments and the constituents of human perspiration, such as skin waste. it varies for different individuals and conditions. there are cases where more colour fading and alteration is caused, depending on the conditions under which garments are worn. coupled with the eventual accumulation and concentration of perspiration, reaction with sunlight is regarded as the cause for such occurrences, casting an influence on certain elements in the dye structure. to forecast such occurrences, methods for examining the components of perspiration under different conditions (acid and alkaline ph values) were investigated by the standards as shown in table ii. the fastness to perspiration was generally very good both in acidic and alkaline medium with rating of 4 and 5. this may be attributed to the crystalline structure of the polyester which disallowed the migration of dye out of the fabric when it had entered the fabric.23 colour measurement properties the colour parameter of the dyed polyester fabrics are given in terms of the cielab value (table iii) and the following cielab coordinates were measured, lightness (l*), chroma (c*), hue angle from 0 to 360° (h), a* value representing the degree of redness (positive) and greenness (negative) and b* representing the degree of yellowness (positive) and blueness (negative). the k/s values are used to represent the depth of dyeing as they are proportional to the amount of colorant present in the dyed fabrics.29 the colour hue of the dyes 7a–d on the polyester fabrics shifted in the reddish–yellowish direction according to the positive values of a* and b*, respectively. the k/s values listed in the table table iii. colorimetric and spectrophotometric data of the synthesized dyes on polyester fabrics dye no. l* a* b* c* h / ° k/s 7a 44.88 49.88 10.9 51.06 12.32 8.07 7b 43.89 41.94 355.47 357.94 83.27 6.55 7c 37.03 40.89 10.76 42.28 14.74 10.71 7d 37.07 35.77 355.83 357.63 84.25 13.61 ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 1262 ameuru et al. iii show that amphiphilic naphthalimide disperse dyes showed the best build-up for polyester fabric and have high affinity for the polyester fabrics. the k/s values of the dyes under investigation showed that the dyes had good affinity to polyester fabrics. dyes 7c and 7d were characterized by higher values of k/s, which indicates outstanding results when compared with their analogues. conclusions in this research, an efficient and simple protocol for the syntheses of monoazo disperse dyes based on amphiphilic naphthalimide in good yields was developed. data obtained by various analytical techniques proved the synthesis of the claimed novel dyestuffs. it was found that on changing the coupling components, the bathochromic and hypsochromic properties of the dyes varied, which could be related to the structure of the replaced groups. introduction of electron donor and electron acceptor groups in the coupler had influencing effects on the bathochromic and hypsochromic properties of the dyes. the dyes were applied on polyester fabrics by the hthp method and gave violet, maroon, pink and neon red with poor to moderate light fastness and good to very good washing and perspiration fastness properties. the kubelka–munk (k/s) value, which is representative of the colour depth of the dyed fabric, was also evaluated. the value of the coordinates l*, a* and b* represent lightness/darkness, red/green and yellow/blue tones of colours, respectively. from the values of a* and b*, it is clear that the synthesized dyes have positive values, which indicates yellowish red colour. most of the dyes have fairly high values of l*, confirming their colour brilliancies. the chroma or chromatic purity (c*) of the dyeing were equally high. the net effect of high chroma and high l* is the good colour brilliance of the dyes. supplementary material additional data are available electronically at the pages of journal website: https:// //www.shd-pub.org.rs/index.php/jscs/index, or from the corresponding author on request. acknowledgements. we are grateful to the faculty of chemistry of kharazmi university for supporting this work. the authors also thank the staff of the faculty for their kind collaboration. и з в о д синтеза неких амфифилних нафталимидних азо дисперзних боја и њихова способност бојења полиестарских влакана umar salami ameuru,1 mohammed kabir yakubu1, kasali ademola bello1, peter obinna nkeonye1 и azim ziyaei halimehjani2 1 department of polymer and textile engineering, ahmadu bello university, zaria, nigeria и 2 faculty of chemistry, kharazmi university, 49 mofateh st., tehran, iran синтетисана је серија дисперзних моноазо боја, купловањем диазонијум соли добијене из 4-амино-n-додецил-1, 8-нафталимида са n,n-диалкиланилинима или дериватима нафтола. синтетисани интермедијери и боје су окарактерисани ft-ir, 1h и ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ amphiphilic naphthalimide azo disperse dyes 1263 13c-nмr спектроскопијом, масеном спектрометријом и елементалном анализом (chn). електронски апсорпциони спектри, у опсега таласне дужине (λmax) 515–535 nm имају јединствену абсорбанцу у интервалу 1,59–3,00×104 l mol-1 cm-1. боје дају дубоке и светле интензивне нијансе љубичасте, бордо, розе и неон-црвене боје на полиестарским влакнима. боје дају генерално добре особине приликом прања и знојења, али имају лошу до просечну постојаност на светлу на полиестарској тканини и могу бити препоручене за комерцијалну употребу. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis of substituted allyl acetates from heterocyclic dienes by pd-promoted arylation-acetoxylation cascade} j. serb. chem. soc. 82 (12) 1335–1341 (2017) udc 547.384+542.913:547..7+ jscs–5044 547.944/.945:66.095.11 original scientific paper 1335 synthesis of substituted allyl acetates from heterocyclic dienes by a pd-promoted arylation–acetoxylation cascade milena r. simić**#, miloš r. petković, predrag m. jovanović, gordana d. tasić# and vladimir m. savić*# university of belgrade, faculty of pharmacy, department of organic chemistry, vojvode stepe 450, 11221 belgrade, serbia (received 17 march, revised 3 april, accepted 10 april 2017) abstract: pd-catalysed arylation–acetoxylation cascade, a previously reported methodology, was applied in the functionalisation of unsymmetrical dienes. both explored classes of compounds, isoquinoline and β-carboline-derived dienes, afforded single regioisomers. although further improvements of the process are necessary, primarily due to lower yields, the described functionalisation of the studied compounds might be useful in the synthesis of emetine and related naturally occurring compounds. keywords: heterocycles; synthesis; emetine-like compounds; pd-catalysis; regioselectivity. introduction allylic acetates are an important class of organic compounds employed in various chemical transformations.1–5 their reactions promoted by different transition metals are of particular interest.6–13 amongst them, pd-catalysed processes stand as the most intensively studied methodologies frequently used in the synthesis of various organic molecules, including naturally occurring substances.14–22 the most common way to access allylic acetates utilizes allyl alcohols as starting materials, which are widely accessible via addition of vinyl-nucleophiles on aldehydes or ketones or simple nucleophilic additions on α,β-unsaturated equivalents.23–26 some alternative processes usually based on the acetoxylation of alkenes or related compounds have also been developed.27–31 to this end, the arylation of dienes/allenes with concomitant acetoxylation promoted by pd-complexes as a way to produce substituted allyl acetates, scheme 1, was recently reported.32,33 *,** corresponding authors. e-mail: (*)vladimir.savic@pharmacy.bg.ac.rs; (**)milena@pharmacy.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc170317046s ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1336 simić et al. although the product itself is a pd-substrate, the reaction conditions favoured the formation of the acetate in moderate to good yields. scheme 1. pd-promoted arylation/acetoxylation of dienes or allenes. results and discussion in order to explore further this methodology, the present interest lay in functionalisation of heterocyclic, unsymmetrical diene 4, which would be potentially useful to access emetine 1 and its derivatives, such as dehydroemetine 2 or biologically active compound 3.34–36 in addition, related to these are also protoemetine or protoemetinol.36 a potential synthetic strategy towards these natural compounds is outlined in scheme 2. functionalisation of the diene moiety followed by the acetate to methyl transformation would provide a core skeleton, while further structural manipulation would lead towards the target compounds.37–40 obviously, the first issue with this approach is the regioselectivity of the arylation/acetoxylation process and initial results related to this concern are outlined in this report. starting from isoquinoline, diene 4 was prepared in several high yielding steps as reported previously.41,42 scheme 2. emetine and some related derivatives. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. functionalisation of heterocyclic dienes 1337 synthesis of corresponding allylic acetate was performed under typical conditions employing pd(oac)2/pph3 as the catalytic system and an excess of naoac (5 equivalents) in dmso as a solvent (scheme 3). a single product was isolated in 32 % yield after column chromatography. analysis of the 1h/13c -nmr spectra confirmed the structure of the isolated derivative with the ir and mass spectra corroborating its formation. full signal assignment in the 1h-nmr (fig. 1) and 13c-nmr (supplementary material to this paper) spectra was based on 2d experiments (h−h/c correlations), while the reaction regioselectivity was unambiguously established by examining the noesy and hmbc spectra (supplementary material). scheme 3. synthesis of heterocyclic allyl acetate. fig. 1. 1h-nmr spectrum of allyl acetate 5. analysis of noesy spectrum showed weak interactions between h-1/h-2′ and h-4/h-3′ hydrogens, supporting the proposed structure (fig. 2). additionally, examination of hmbc spectrum (supplementary material) led to same conclusion since it showed correlations c-4/h-3′ and c-1/ h-2′ (fig. 2). the isolation of only one regioisomeric product came as a surprise but detailed analysis of the 1h-nmr spectrum (200 mhz) of the crude reaction mixture, including careful tlc analysis did not reveal the presence of any other distinctive compound. furthermore, several additional reactions, as outlined in scheme 4, were performed. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1338 simić et al. fig. 2. key correlations in the noesy/hmbc spectra of allyl acetate 5. scheme 4. synthesis of isoquinoline derived allyl acetates. all transformations were performed as already described producing a single isolatable isomer in all reactions in yields of up to 47 %. the final example was performed using β-carboline derived diene 8 that, under the described conditions, produced acetate 9, a compound structurally related to naturally occurring pseudotubulosine (scheme 5).36 the indole n–h bond does not seem to influence the reaction pathway and the product was isolated in a yield similar to those outlined in scheme 4. scheme 5. synthesis of the β-carboline derived allyl acetate. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. functionalisation of heterocyclic dienes 1339 although the formation of the other regioisomer in all the studied reactions cannot be ruled out completely, it was not observed during the reaction or work -up and purification procedure. at present, it is difficult to rationalise the exclusive formation of the observed products. it is not clear whether the other isomer was not formed at all or it was formed but decomposed during the reaction course. the fact that the yields were relatively low might suggest the second options but it remains to be explored further. experimental general the nmr spectra were recorded on a bruker avance iii (500 mhz) spectrometer. chemical shifts are given in parts per million (δ) downfield from tetramethylsilane as the internal standard. deuterochloroform was used as the solvent. mass spectral data were recorded using an agilent msd tof spectrometer coupled with agilent 1200 hplc or agilent technologies 5975c ms coupled with agilent technologies 6890n gc. the ir spectra were recorded on an ir thermo scientific nicolet is10 (4950) spectrometer. silica gel 60 (230– –400 mesh) was employed for the flash chromatography while thin layer chromatography was performed using alumina plates with 0.25 mm silica layer (kieselgel 60 f254, merck). the compounds were visualised by staining with alkaline potassium permanganate solution. the solvents were purified by distillation before use. general procedure for the synthesis of allyl acetate from dienes and aryl iodide a mixture of diene (0.1 mmol) and aryl iodide (0.15 mmol, 1.5 eq), pd(oac)2 (10 mol %), ph3p (20 mol %) and naoac (0.5 mmol, 5 eq) in dmso (2 ml) was heated at 85–90 °c (bath temperature) under a nitrogen atmosphere for 12 h. the reaction mixture was then cooled to room temperature, et2o (20 ml) was added and the mixture washed with h2o (3×5 ml). the organic layer was dried over anhydrous na2so4, filtered and concentrated under reduced pressure. the crude residue was purified by flash chromatography (petroleum ether, diethyl ether) to afford the required product. [2-(4-methoxybenzyl)-1,6,7,11b-tetrahydro-4h-pyrido[2,1-a]isoquinolin-3-yl]methyl acetate (5). the compound was synthesised from diene 4 and 1-iodo-4-methoxybenzene following the general procedure. flash chromatography (sio2, 3:2 diethyl ether/petroleum ether volume ratio) afforded 5 (32 %) as a yellow oil. [2-(3,4-dimethylbenzyl)-1,6,7,11b-tetrahydro-4h-pyrido[2,1-a]isoquinolin-3-yl]methyl acetate (7a). the compound was synthesised from diene 4 and 1-iodo-3,4-dimethylbenzene following the general procedure. flash chromatography (sio2, 1:1 petroleum ether/diethyl ether volume ratio) afforded the 7a (30 %) as a pale yellow oil. [2-(4-methylbenzyl)-1,6,7,11b-tetrahydro-4h-pyrido[2,1-a]isoquinolin-3-yl]methyl acetate (7b). the compound was synthesised from diene 4 and 4-iodotoluene following the general procedure. flash chromatography (sio2, 2:3 petroleum ether/diethyl ether volume ratio) afforded the product 7b (47 %) as a pale yellow oil. [2-(naphthalen-1-ylmethyl)-1,6,7,11b-tetrahydro-4h-pyrido[2,1-a]isoquinolin-3-yl]methyl acetate (7c). the compound was synthesised from diene 4 and 1-iodonaphthalene following the general procedure. flash chromatography (sio2, 1:1 petroleum ether/diethyl ether volume ratio) afforded 7c (32 %) as a pale yellow oil. [2-(4-methylbenzyl)-1,4,6,7,12,12b-hexahydroindolo[2,3-a]quinolizin-3-yl]methyl acetate (9). compound was synthesised from diene 8 and 4-iodotoluene following the general ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2017 scs. 1340 simić et al. procedure. flash chromatography (sio2, 2:3 diethyl ether/petroleum ether volume ratio) afforded 9 (34 %) as a yellow oil. conclusion in conclusion one can say the reactions of unsymmetrical heterocyclic dienes in the arylation–acetoxylation cascade afforded allyl acetate highly regioselective products. although further improvement of the process is necessary, the described methodology may be potentially useful in the synthesis of a series of natural products or their derivatives. supplementary material analytical and spectral data of the synthesized compounds are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. financial support from the ministry of education, science and technological development of the republic of serbia (grant no. 172009) is greatly appreciated. и з в о д синтеза алилних ацетата из хетероцикличних диена каскадом ариловања и ацетоксиловања промовисаном паладијумом милена р.симић, милош р. петковић, предраг м. јовановић, гордана д. тасић и владимир м. савић фармацеутски факултет, катедра за органску хемију, војводе степе 450, 11221 београд у овом раду проучаване су функционализације несиметричних диена у pd-катализованим процесима заснованим на каскади ариловање/ацетоксиловање. диенски изохинолински деривати, као и деривати који садрже β-карболински структурни фрагмент у поменутим трансформацијама дају само један региоизомер. мада процес захтева даљу оптимизацију, пре свега због нижих приноса, методологија може наћи примену у синтези еметина и сродних природних производа. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice j. serb. chem. soc. 80 (3) 333–345 (2016) udc 543.544.3+543.51:547.562.1+ jscs–4850 544.032.72:504.4.054(282.243.74) original scientific paper 333 seasonal variations of bisphenol a in the danube river by the municipality of novi sad, serbia maja milanović1*, jan sudji1, nevena grujić letić1, jelena radonić2#, maja turk sekulić2#, mirjana vojinović miloradov2 and nataša milić1 1faculty of medicine, university of novi sad, hajduk veljkova 3, 21000 novi sad, serbia and 2faculty of technical sciences, university of novi sad, trg dositeja obradovića 6, 21000 novi sad, serbia (received 21 july, revised 5 november, accepted 16 november 2015) abstract: seasonal variations of bisphenol a (bpa, iupac: 4,4′-(propane-2,2 -diyl)diphenol) were investigated in the danube river along the novi sad bank, serbia using solid-phase extraction followed by the gas chromatographic mass spectrometric method. the obtained results confirmed the presence of bpa above the limit of quantification (6 ng l-1) in 22 out of 32 the water samples at all eight sampling sites. the bpa concentration varied from <6 to 693 ng l-1. the mean bpa concentration for summer (220 ng l-1) significantly differed from those obtained for autumn (39 ng l-1), winter (6 ng l-1) and spring (41 ng l-1). the risk quotients, expressed as the ratio of the 95th percentile of the measured environmental concentrations for each season and the predicted no-effect concentration were higher than one (>1) for autumn, spring and summer. the high potential risk that is attributed to the elevated summer concentrations is probably the result of increased human activities and weather conditions. keywords: bisphenol a; danube river; endocrine disrupter; gc–ms; surface water. introduction bisphenol a (bpa, 4,4′-(propane-2,2-diyl)diphenol) is one of the most investigated and discussed endocrine disrupting compounds (edcs), and has received much attention as an emerging chemical of concern in order to protect wildlife and human health.1 endocrine disrupters (eds) can interfere with hormonal action and with human cell functions mainly through interaction with specific receptors and can induce numerous adverse health effects. therefore, the occurrence of bpa in the environment could have potential harmful effects on * corresponding author. e-mail: majam021@yahoo.com # serbian chemical society member. doi: 10.2298/jsc150721095m 334 milanović et al. the reproductive system, growth, digestion and metabolic processes of aquatic organisms, such as fish or mussels.2 moreover, its effects on the human endocrine system through direct exposure via drinking water (bpa was used in polycarbonate plastic bottles) and its function have been discussed.3 bpa is also believed to pose a potential risk in the development of various types of cancers.4,5 bpa is an organic compound extensively used as an intermediate in the production of polycarbonate plastics and epoxy resins, unsaturated polyester–styrene resins and flame-retardants. it is used in a broad range of products for the modern consumer society, including reusable baby bottles, toys, food and drink storage containers, protective coatings for metal cans, adhesives and paints, electronic components, personal computers, cds and dvds, sports equipment, medical devices, eyeglass lenses, dental sealants and dental bonding agents, etc.6,7 more than 3.6 million tons of bpa are synthesized worldwide annually and its production is constantly increasing owing to a growing demand for plastic products.8 the first country that recognized the importance and necessity to legislate and regulate the environmental issue of bpa was canada.1 although, the european commission and the us government have banned bpa use in baby bottles and packaging items for children aged 0–3, bpa is still under discussion to be prioritized for regulation by the european union and the united states because of its possible adverse impact on human health and the environment. various declarations and statements have been issued (the berlaymont declaration, the ramazzini statement, european environmental bureau, world health organization, etc.) on this challenging topic, and the debate is still on -going on how to regulate and reduce the bpa concentrations effectively in consumer products and aquatic systems in order to protect humans and wildlife.1 recent studies confirmed the presence of bpa in different human biological matrices, such as urine,9 breast milk10 and hair.11 the presence of bpa in food, at levels up to 1858 µg kg–1,12 is associated with the manufacture of various polycarbonate plastic products (packages or food contact materials) or it is leached from the final products as a result of the conducted hydrolysis of polymers at high temperature, or under acid/basic conditions.3,13,14 the main source of the bpa occurrence in the aquatic environment is municipal and industrial wastewater (mainly from chemical plants15). bpa concentration in surface water is reported to be between <0.001 and 92 µg l–1.15 despite its rapid degradation, favored by aerobic conditions, with a half-life of about 0.5–5 days in the aquatic environment, the constant release into the surface water makes bpa a pseudo-persistent pollutant in the environment.16–18 although, european legislation set environmental quality standards for priority and certain other pollutants, bpa permitted levels, together with some pharmaceuticals, personal care products, algal toxins and various endocrine disrupters bisphenol a in the danube 335 have not been regulated by any legislative bodies yet because of the insufficient reliable data on their safety or risks.16,19 in the western balkan region, few extensive multinational monitoring programs have been realized in the last decade. first comprehensive information on the occurrence of different emerging contaminants including edcs along the danube river was conducted during the joint danube survey 1 (in 2001) and the joint danube survey 2 (in 2007), organized by the international commission for the protection of the danube river (icpdr). during the joint danube survey 2, 34 organic compounds were screened along the entire course and the highest concentrations of emerging contaminants were recorded around the budapest area. bpa was detected in only few samples (15 out of 52) in concentration up to 68 ng l–1.20 moreover, within the emco project, some classes of emerging contaminants were determined in municipal and industrial wastewater derived from bosnia and hercegovina, croatia and serbia.21 this study confirmed the occurrence of bpa in municipal wastewater in the western balkan region and the mean concentration of bpa was 510 ng l–1.21 besides these findings, monitoring data on water status in serbia are still scarce and limited in terms of sampling sites and number of compounds. recently, screening analyses of the wastewater discharged into the danube along the novi sad municipality were performed and more than 130 chemical contaminants were detected.22 furthermore, non-target screening analysis of danube water samples in the area of belgrade confirmed the presence of a wide range of organic contaminants.23 the primary goal of this study, conducted for the first time, was to examine the occurrence of bpa along the danube river in the area of novi sad (serbia). the obtained results were also used to evaluate the seasonal variations of this endocrine disrupter and to assess the potential risk of the determined bpa concentrations. experimental chemicals the standard bisphenol a, iupac: 2,2-bis-(4-hydroxyphenyl)propane, was obtained from sigma aldrich (deisenhofen, germany). methanol, dichloromethane and ethyl acetate were of hplc grade (lgc promochem, wesel, germany). the silylation agent n,o-bis(trimethylsilyl)trifluoroacetamide (bstfa, iupac: trimethylsilyl(1e)-2,2,2-trifluoro-n-trimethylsilylethanimidate) was obtained from fluka (sigma aldrich, deisenhofen, germany) and ultra pure water was used. the regenerated cellulose filters (0.45 μm) and cartridges were obtained from agilent technologies. all glassware was rinsed with methanol and dried at 100 °c for 1 h before use. 336 milanović et al. collection of water samples a map of the showing the sampling sites with code for the samples is given in fig. s-1 of the supplementary material to this paper. details about the collection of the samples and the main characteristics of the studied area are also given in the supplementary material. sample preparation the samples were prepared using the method previously developed by ballesteros et al.24 a solid-phase extraction procedure (spe) with agilent bond elut plexa (200 mg per 6 ml) type of cartridges was used for sample pre-concentration and removal of the existing interferents. spe was performed on an agilent vacuum manifold for 12 columns connected to a water vacuum pump. the ph values of filtered water samples were adjusted to ph 3 with 1 m hydrochloric acid to increase the extraction efficiency.24,25 the cartridges were activated with 5 ml mixture of dichloromethane and methanol (50:50, v/v) followed by 5 ml of pure methanol and 15 ml of ultra pure water (ph 3). the final extraction volume of each sample was 500 ml. the analytes were eluted with 5 ml of methanol. after isolation, the eluate was gently evaporated to dryness at 40 °c under a nitrogen stream (5.0 purity) and then the residue of each sample was reconstituted in 50 µl of ethyl acetate. a silylation step was performed using 50 µl of bstfa prior to the gas chromatography-mass spectrometry (gc–ms) analysis following the method obtained by schönfelder et al. (fig. 1).26 a fully automated derivatization procedure using amber glass gc vials was performed in order to avoid degradation of the reagent and derivatives by light and humidity. just before the gc-ms analysis, the bstfa reagent was injected into the vial with an analyte and the mixture was heated for 20 min at 50 °c. after cooling of the mixture, the derivatized form of the analyte was immediately injected into the gc–ms. fig. 1. derivatization of bisphenol a. gc–ms analysis the analyses were realized using an agilent gc 7890a coupled with 5975c vl mass detector employing the method developed by zafra et al.27 the system was used in the scan (complete spectrum) and in selected ion monitoring (sim) modes. the separation was realized on a fused silica capillary column db-5 ms (60 m, 0.25 mm i.d., 0.25 µm film thickness; j & w scientific, folsom, ca, usa). the column stationary phase was the low polarity phenyl arylene polymer, virtually equivalent to (5 %-phenyl)-methylpolysiloxane. the ms quad temperature was set at 150 °c and the electron impact (ei) ion source temperature of the ms was at 230 °c. a 2-µl aliquot of the silylated samples were automatically injected using the split bisphenol a in the danube 337 less injection mode at 250 °c. a programmed gc oven temperature was applied. the initial oven temperature was set at 50 °c and held for 1 min. then, the temperature was increased to 150 °c (held 1 min) and 270 °c via ramps of 25 and 10 °c min-1, respectively, and maintained at 270 °c for 2 min. high purity helium (99.99 %) was used as the carrier gas at a flow rate of 1.0916 ml min-1. the characteristic ions listed in table i for the sim mode operation were used for quantification of bpa in the water samples. table i. sim mode characterization analyte monitored ions (m/z) retention time, min quantifier qualifier bpa 357 373 and 207 15.114 the bpa in the water samples was quantified with the external standard bis(trimethylsilyl)bisphenol a. a stock solution for calibration standards and quality control was prepared by dissolving an appropriate amount of bpa in methanol. the spiked blank water samples with appropriate bpa concentrations were extracted and silylated following the previously described sample treatment procedure. the linearity was examined over the range 10–100 ng ml-1 of bpa. each concentration level was injected in triplicate, and the calibration curves were constructed by considering the relative abundance as a function of the bpa concentration. a new set of bpa calibration standards was prepared for each season (in november 2012, march 2013, may 2013 and september 2013). the appropriate calibration curve was applied to all samples analyzed within the same season. the correlation coefficient (r2) of all calibration curves was more than 0.998 and each curve was checked (and corrected if required) at a concentration level near the determined bpa concentrations in samples (6–50 ng ml-1). quality control samples that were run in each season were prepared in the same manner. the relative standard deviation of the response factors of the calibration curves for the same standard concentration was less than 10 %, which is in accordance with the requirements of commission decision 2002/657/ec (european commission, 2002)28 and the slope of the calibration curves did not differ significantly from zero. the limit of quantification (loq) was set at s/n ratios ≥10. all instrumental and procedural blanks were far below the loq of 6 ng l-1. the identification of bpa was confirmed using fiehn and nist08 mass spectral libraries. a characteristic chromatogram of the examined water samples containing bpa, after extraction and the derivatization procedure, is presented in fig. s-2 of the supplementary material. statistical analyses statistical analyses were performed using originpro 8 sro (originlab corporation, northampton, ma, usa). the comparison of the mean values of the measured concentrations during different seasons was performed by the one-way analysis of variance (anova) using the tukey test and the results were considered significant at p < 0.05. results and discussion occurrence and seasonal variations of bpa in the surface water during the one-year trial, contamination of the novi sad municipality by bpa was confirmed at each sampling site. bpa was found with a high frequency of the detection (68.75 %) in 22 out of 32 water samples above the loq (6 ng l–1). 338 milanović et al. the bpa concentration varied from 11 to 693 ng l–1 and the mean bpa concentration was 77 ng l–1. the obtained bpa levels in the surface water varied depending on the sampling sites, possible sources and routes of pollution during the four seasons (table ii). the seasonal occurrence of bpa in the surface water at the chosen sites along the novi sad section of the danube is shown in fig. 2. table ii. bpa concentrations in the danube samples in the municipality of novi sad; n.d.: < loq sample range, ng l-1 mean, ng l-1 frequency of detection, % (n = 4) ri n.d.–77 29 75 gc n.d.–304 90 50 gc2 n.d.–177 73 75 ro n.d.–693 186 50 rp n.d.–140 63 75 dm n.d.–41 21 75 dl n.d.–130 42 75 dr n.d.–383 108 75 fig. 2. seasonal variations of the bpa concentration in the danube surface water. bisphenol a in the danube 339 in autumn, bpa was detected above the loq (6 ng l–1) in 7 out of 8 collected samples at concentration levels up to 98 ng l–1. the maximum detected concentration (98 ng l–1) was in the rp sample, while the lowest concentration was recorded at the ri sampling site (13 ng l–1). the high detection rate of 87.5 % during the first campaign confirmed the presence of bpa in the danube surface water in the novi sad area. in the winter period, bpa was only detected in two samples (out of eight), rp and dm, at concentrations of 13 and 33 ng l–1, respectively. the low detection rate and extensive reduction of the bpa concentration in winter were assumed to be due to the dilution effect because of the weather conditions and high river flow rate. however, the presence of bpa at the dm sampling site away from direct anthropogenic impact was unexpected during the winter. probably, it was the result of the specific water flow and is attributed to the pollution effect of the dtd canal. on contrary, bpa was detected in almost all spring samples (7 out of 8). again, as in the autumn, the maximum detected bpa concentration was at the rp site (140 ng l–1), while the lowest concentration was in the dm sample (11 ng l–1). the major contributor to the high concentration of bpa in autumn and in spring at the locality rp could be the more intense surface runoff over waste disposal sites due to the increased volume of rainfall, as well as frequent hospital procedures during these periods. during the summer, the detected bpa levels were above the loq in six samples (out of 8) at high concentrations, ranging from 77 ng l–1 in the ri sample to 693 ng l–1 in the ro sample. although, bpa has a short half-life in surface water due to its rapid photo and microbial degradation,16 which was favored in the summer, due to the higher temperatures, stronger sunlight and increased activity of microorganisms, the overload of bpa contamination tended to resist degradation in the surface water and was transported much further along the danube. unexpectedly high bpa concentration at ri site (77 ng/l) was probably influenced by increased anthropogenic activity in upstream weekend settlements. similar to the autumn and spring, septic tanks and unauthorized discharges from upstream settlements contributed to the presence of bpa at the ri site in the summer. the highest concentration in the summer was observed at the ro site (693 ng l–1). at the same site in autumn and winter, the bpa levels were below the loq, demonstrating that the determined levels in spring and summer were probably attributed to occasional discharges. one of the possible sources of bpa contamination at the ro site could be overhaul of the thermal power plant, such as cleaning of the equipment, replacement of working fluids, painting, etc., which is common during the summer. the elevated bpa levels detected along the danube banks (at the dl and dr sites) in comparison to the middle of the river (at the dm site) are in accordance with the water flow regime. 340 milanović et al. in the middle of the river, due to intensive horizontal and vertical turbulent flow, the degree of bpa dilution is significant. on the other hand, in the laminar flow along the coast, diffusion of pollutants is limited and the degree of dilution is considerably smaller. the mean bpa concentration for the summer (220 ng l–1) significantly differed from the mean bpa levels for autumn (39 ng l–1), winter (6 ng l–1) and spring (41 ng l–1). there was no significant difference between the mean bpa concentrations detected for autumn, winter and spring. these seasonal differences could be the result of variations in the environmental conditions, such as rainfall and temperature, and/or different usage patterns of bpa-related products. during the summer period, traffic, sports and entertainment activities are intensified and among these, the consumption of beverages in plastic bottles is increased as well as the use of bpa-containing paints for boats. additionally, the summer is the typical low water level season. all of this probably contributed to direct contamination of the danube by solid waste and excretion, which significantly affected the water quality in the surface layers. in a study performed in brazil, similar results regarding the seasonal variation of bpa concentrations were obtained.32 considering the 26 samples collected at the eight sampling sites along the atibaia river (são paulo state, brazil), bpa was found in 56 % of samples in the four campaigns and the highest concentrations were recorded in the dry period. on the contrary, in a study performed in china, the highest concentration of bpa in the surface water was found in the winter.33 a possible explanation for the different seasonal occurrence of bpa might be the low water level of the huangpu river from november to april and the flooding period that occurs during the summer in china.33 however, the obtained mean bpa concentration for the five campaigns in china (95 ng l–1)33 was similar to the mean bpa value (77 ng l–1) found in the present study. the bpa levels obtained in the present study were also compared with available data related to european rivers. the detected concentrations of bpa in this investigation were in the range of <6 to 693 ng l–1 (the mean bpa concentration was 77 ng l–1). although, the levels found in freshwater streams of germany were higher (up to 1927 ng l–1),34 bpa only appeared in samples from october, while no bpa was detected in december, april and september. however, bpa was quantified in dutch surface waters at levels up to 330 ng l–1, with the highest concentration being found in the summer, similar to the present study.35 in the seine river and in the rhine surface water, bpa was found in a lower range (11–15436 and 42 to 229 ng l–1,25 respectively). the maximum bpa concentration in the present study (693 ng l–1) was higher than the previously recorded maximum bpa concentration in the danube surface water (68 ng l–1) measured during the joint danube survey 2.20 however, in the same bisphenol a in the danube 341 study, bpa was detected in the danube tributaries at concentrations up to 490 ng l–1.20 the relatively high bpa concentrations found in the investigated danube river stretch in serbia could be explained by the fact that novi sad still has no urban wastewater treatment plant and that most of the industries around novi sad municipality are without wastewater treatment facilities. possible environmental implication the octanol–water partition coefficient, log kow, for bpa is 3.64, which suggests that bpa has modest capacity for bioaccumulation.37 the presence of the bpa endocrine disrupter might have adverse effects on aquatic organisms38 and the consumption of bpa contaminated freshwater fish35 is probably one of the main routes of contamination of humans.16 following the european union guidelines, the environmental risk assessment is evaluated by comparing the predicted environmental concentration (pec) or by measuring the environmental concentration (mec) with the sensitivity of the aquatic ecosystem for bpa (predicted no-effect concentration, pnec). the ratio pec/pnec (or mec/pnec), known as the risk quotient (rq), should not exceed 1, otherwise, a risk to the aquatic environment is assumed.39 the pnec values are based on eco-toxicity data from single species laboratory tests combined with safety factors or even on derived provisional pnec values.40 based on literature data, the aquatic pnec value for bpa varies from 0.06 µg l–1 estimated by wright-walters et al.18 using a non-parametric methodology to 0.175 µg l–1 in canada,41 1.5 µg l–1 in the european union according to the european union risk assessment report,42 1.6 µg l–1 in japan43 and even to 64 µg l–1 derived by staples et al.44 the possible negative implications of bpa on the danube surface water were evaluated using the 95th percentile of the measured bpa concentrations along the danube section by the novi sad municipality together with a pnec value to calculate the rq for each season. all sampling points were considered as one locality due to the relatively small distance between the sites. the strictest standard, i.e., the lowest pnec value of water based on the wright-walters and co-workers methodology18 was applied in order to reduce the uncertainties of standardized acute toxicity tests in risk assessment approach. the calculated ratio ranged from 0.44 in winter to 9.73 in summer. the mec/pnec ratio was below 1 only in winter (fig. 3). the high rq in summer is attributed to the elevated bpa level at the ro site (693 ng l–1). considering this value as an exception, probably due to an occasional discharge at this site, the rq was recalculated using the mean summer bpa concentration (220 ng l–1). however, the obtained mec/pnec ratio was still above 1 (3.67). moreover, the rq calculated by dividing the mean bpa concentration derived in this study (77 ng 342 milanović et al. l–1) with the pnec value was above one, indicating that potential risk of bpa in this danube section exists. fig. 3. mec/pnec value of bpa during different seasons. this risk assessment approach was based on an evaluation of the toxic potential of a single plasticizer to the aquatic environment without taking into account the possibility and effects of bpa acting additively with other eds. conclusions this research investigated the occurrence and seasonal variations of bpa as an endocrine disrupter in the danube surface water of the novi sad section, serbia using spe followed by the gc–ms method. bpa was found at all 8 sampling sites during the one-year trial. in 22 out of 32 water samples, bpa was detected above the loq (6 ng l–1), in concentrations varying from 11 to 693 ng l–1. the highest concentrations of this compound were observed in the summer and significantly differed from the determined mean bpa levels for autumn, winter and spring. these seasonal variations might have resulted from environmental factors, such as the low water level, and increased anthropogenic activities. the calculated risk quotient was below one only in the winter. the obtained results indicate that the aquatic environment of the danube river by novi sad municipality is not sufficiently protected from possible adverse effects to bpa exposure and that attention should be paid to bpa pollution control in order to protect drinking water supply sources, which are very close to the riverbanks. supplementary material details about samples collection and the main characteristics of the studied area, as well as a representative gc–ms chromatogram are available electronically from http:// //www.shd.org.rs/jscs/, or from the corresponding author on request. bisphenol a in the danube 343 acknowledgements. this research was financially supported by the ministry of education, science and technological development of the republic of serbia (iii46009) and nato science for peace programme (esp.eap.sfpp 984087). и з в о д сезонске варијације бисфенола а у површинској води дунава, на подручју новог сада маја милановић1, јан суђи1, невена грујић летић1, јелена радонић2, маја турк секулић2, мирјана војиновић милорадов2 и наташа милић1 1медицински факултет, универзитет у новом саду, хајдук вељкова 3, 21000 нови сад и 2факултет техничких наука, универзитет у новом саду, трг доситеја обрадовића 6, 21000 нови сад сезонске промене концентрација бисфенола а у површинској води дунава, на територији новог сада, анализиране су применом гасне хроматографије са масеном спектрометријом. бисфенол а детектован је изнад лимита квантификације (6 ng l-1) у 22 узорка воде (од укупно 32 узорка) у опсегу концентрација 11–693 ng l-1. средња вредност концентрације бисфенола а у летњем периоду (220 ng l-1) статистички је значајно виша од средњих концентрација измерених у јесен (39 ng l-1), зиму (6 ng l-1) и пролеће (41 ng l-1) и указује на већу контаминацију дунава током летњег периода. процењен ризик по животну средину, изражен као однос 95. перцентила измерених концентрација бисфенола а у површинској води током четири годишња доба и концентрације за коју је предвиђено да нема биолошке ефекте, био је мањи од јединице само током зиме. високе концентрације бисфенола а у летњем периоду које су последица повећане људске активности и временских услова представљају потенцијални ризик по животну средину на подручју новог сада. 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(2016) 333–345 collection of water samples the surface water samples (1 l) were collected in autumn (november 2012), winter (march 2013), spring (may 2013) and summer (september 2013), in amber glass bottles at eight representative locations (ri, gc, gc2, rp, ro, dm, dl, dr) along the danube river of the novi sad municipality, serbia and stored at 4 °c until the analysis were performed. samples were collected on the same day in every season and the meteorological conditions were the same for all localities within a particular season. all 32 water samples were taken at a depth of 50 cm. the position of sampling points in relation to the flow of the river could be seen in fig. s-1. the sampling sites were selected according to the recommendations of the icpdr’s experts (the international commission for the protection of the danube-icpdr, www.icpdr.org), in order to estimate the level of violation of the ecological status of the danube by the municipality of novi sad. location ri was chosen in order to determine the status of the danube prior to any local urban and canal tributary impact. sampling points gc, gc2, ro and rp were positioned 100 m downstream, i.e., transversely from the four sewage discharges of the city. the locations gc and gc2 were selected to determine the influence of two biggest untreated municipal wastewater discharges (with capacity of 400 l s–1 during the dry period). the location ro was chosen to determine the influence of untreated wastewater from the oil refinery and the rain and urban runoff water from the industrial (thermal-heating plant, oil refinery) and suburban underdeveloped area. sampling point rp was selected in order to explore the * corresponding author. e-mail: majam021@yahoo.com supplementary material s115 impact of untreated municipal wastewater from numerous illegal settlements with waste disposal sites and unauthorized traffic, as well as the influence of wastewater coming from two clinical centers. the sampling locations dm (central part of the river) and dl and dr (along the bank) were chosen as control sites in order to estimate the possible existence and impact intensity of the municipality of novi sad on the river water quality after some period (estimation of the impact of dilution, sorption and sedimentation processes). fig. s-1. map of the danube river sampling sites. all the samples were filtered through regenerated cellulose 0.45 μm membrane filters (agilent technologies, germany) and extracted within 24 h in the laboratory. main characteristics of the studied area the danube flows through ten countries from the west to the east, passing through four capital cities before emptying into the black sea, and its drainage basin extends into the territories of nine more nations. constantly, it is influenced by nutrients and other polluting substances. therefore, continuous water quality monitoring of the danube followed by pollution reduction steps is obligatory for all countries of the danube basin. novi sad, the second largest town in serbia, is located on the banks of the danube and the dtd canal. during the nato bombing in 1999, novi sad was faced with the destruction of all bridges and the oil refinery and suffered an economic downturn.29 however, during the last decade, the industry has been recovering. s116 milosavljević et al. the river pollution in the area of novi sad is caused by direct and indirect discharge of urban and industrial wastes and run-off. the acute problem is the emission of more than 95 % of the total industrial wastewater without any treatment. the dominating industries in this area are chemical and food industries and building materials production. the biggest polluters such as the oil refinery, meat processing plants and the production of chemicals and cosmetics are situated on the danube and dtd canal banks.30 additionally, novi sad has joint collector for both industrial and municipal wastewaters and no urban wastewater treatment plant. the gc and gc2 sampling point are influenced by discharges of the two main sewers of the town, cepelin and beogradski kej. furthermore, the municipality water supply system uses mainly drinking water abstraction points petrovaradinska ada (petrovaradin fluvial island) and ratno ostrvo (war island), that are positioned only a few kilometers downstream from these main municipal wastewater discharges.22 the ro sampling site is located near the main water supply source ratno ostrvo and is 100 m downstream from the discharge site of the sewage collector for collecting industrial wastewater from the big local oil refinery complex as well as rain and urban run-off water from the industrial zone and the underdeveloped suburban area with septic tanks. the most dangerous potential pollution sources near ro are the oil refinery and a thermal power plant that are situated only a few hundred meters upstream.31 additionally, the influence of the extensive agricultural production in the backland cannot be ignored. the rp location is situated on the right riverbank near the water source supply petrovaradinska ada. in the vicinity, there are numerous illegal settlements with septic tanks and waste disposal sites, unauthorized traffic and an old industrial complex.22 sampling points dl, dm and dr are outside direct anthropogenic influence. the intensity impact of the municipality and the possible harmful effect of the dtd canal on the river water quality were examined at these sites. previously, it was concluded that the dtd canal has the worse water quality in novi sad, where increased concentrations of organic compounds, mineral forms of nitrogen phenols as well as mercury, manganese, copper and iron were recorded.30 the ri sampling site is located upstream of the city before municipal discharge points. however, suburban and weekend settlements without a sewage system, together with septic tanks and an old industrial complex nearby could influence the surface water quality in this area. according to all, the sampling sites (fig. s-1) were chosen for seasonal analysis of bpa at kilometer 1255 of the danube river in order to identify the primary source of the bpa emission (gc, gc2, ro, rp), the possible influence of incoming pollution (ri), the global impact of the city on the river status (dm, dr, dl) as well as the seasonal fluctuation of the bpa concentration. the pos supplementary material s117 sible pollution by the dtd canal was examined at the dl, dm and dr sampling points. representative gc–ms chromatogram of a sample fig. s-2. representative gc–ms chromatogram of a danube water sample extract after clean up and the derivatization procedure (sim mode). << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {3,4-dihydro-2h-1,3-benzoxazines and their oxo-derivatives-chemistry and bioactivities} j. serb. chem. soc. 86 (3) 213–246 (2021) survey jscs–5417 213 survey 3,4-dihydro-2h-1,3-benzoxazines and their oxo-derivatives – chemistry and bioactivities nabaweya abd el-salam sharaf el-din* department of pharmaceutical chemistry, faculty of pharmacy, tanta university, el giesh street, 31527, tanta, egypt (received 30 may, revised 3 december 2018, accepted 11 january 2019) abstract: 3,4-dihydro-2h-1,3-benzoxazines derivatives are a significant class of heterocycles with particular awareness due to their remarkable biological activities in humans, plants and animals, and also their natural occurrence. alteration in the benzoxazine skeleton and their comparative chemical simplicity and accessibility, make these compounds suitable sources of other bioactive compounds, resulting in the discovery of a wide set of these compounds that have broad biological activity, such as antifungal, antibacterial, anti-hiv, anticancer, anticonvulsant, anti-inflammatory, etc. subsequently, this review gives herein a brief overview of the chemistry and bioactivities of derivatives of 3,4 -dihydro-2h-1,3-benzoxazine monomers and their oxo-derivatives. keywords: 1,3-benzoxazines; synthesis; reactions; biological activities. contents 1. introduction 2. synthesis of 1,3-benzoxazine derivatives 2.1. synthesis of 3,4-dihydro-2h-1,3-benzoxazines 2.1.1. one-step mannich condensation 2.1.2. two-step mannich condensation 2.1.3. three-step mannich reaction 2.2. synthesis of a sulfone-scaffold benzoxazine monomer 2.3. synthesis of bio-based benzoxazine monomer 2.4. synthesis of 4h-1,3-benzoxazin-2-ones 2.5. synthesis of 2h-1,3-benzoxazin-4-one 2.6. synthesis of 1,3-benzoxazine-2,4(3h)-diones 3. chemistry of 1,3-benzoxazine derivatives 3.1. unusual behavior of ortho-functional 3.2. ring opening of benzoxazines * e-mail: nsharafd@gmail.com https://doi.org/10.2298/jsc180530001s ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 214 sharaf el-din 4. reactions of 1,3-benzoxazine derivatives 4.1. hydrolysis with hcl 4.2. salt formation 4.2.1. reactions of 4h-1,3-benzoxazin-4-onium salts 4.3. reaction with alkyl halides 4.4. nucleophilic substitution reaction 4.4.1. reaction with pyridine n-oxide 4.4.2. reaction with organometalic compounds 5. biological activities 5.1. antiviral therapy 5.2. anti-tuberculosis activity 5.3. fungicidal and pesticide activities 5.4. anticonvulsive activities 5.5. antibacterial activities 5.6. anticancer activities 5.7. antihypertensive activities 5.8. antimalarial activities 5.9. antidiabetic and hypolipidaemic activity 5.10. receptor antagonist activity 5.11. antidepressant activity 5.12. anti-platelet aggregation activity 5.13. miscellaneous activities 6. conclusions 1. introduction 1,3-benzoxazine is a bicyclic skeleton in which an oxazine ring is annulated with a benzene ring. a number of isomeric structures are possible depending on the positions and the degree of oxidation of the ring system. the two isomeric structures 1 and 2 (2hand 4h-1,3-benzoxazine) in addition to 2,3-dihydro-2h -1,3-benzoxazine (3) are illustrated in fig. 1.1 this survey is focused on 3,4 -dihydro-2h-1,3-benzoxazines and their oxo-derivatives. 1 2 3 2h-1,3-benzoxazine 4h-1,3-benzoxazine 3,4-dihydro-2h-1,3-benzoxazine fig. 1. chemical structures of 1,3-benzoxazines. 3,4-dihydro-2h-1,3-benzoxazines exist in two basic conformations, the semi-chair (a) and semi-boat (b) structures, as shown in fig. 2. according to the orientation of the substituent at the nitrogen atom, each conformation exists in another two forms.2 on the other hand, the dihydro-1,3-benzoxazine monomers are synthesized not only by traditional mannich condensation methods3,4 of phenol, amine, and formaldehyde, but also by cycloaddition5,6 and other methods. interestingly, ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 2h-1,3-benzoxazine derivatives synthesis and bioactivities 215 several works have been performed to investigate the reactant ratios,7,8 reactant structures,9,10 solvent effect,11 temperatures of reaction,12 and reaction duration.13 o n r on r hh hh h h h h o n r o n r h h h h h h h h half chair (a) half boat (b) side view side view fig. 2. conformations of 3,4-dihydro-2h-1,3-benzoxazines.2 all previous studies14,15 demonstrated that these factors play an important role in the synthesis and the properties of benzoxazine (such as, low yield and poor purity), resulting in limitation of the development of benzoxazine chemistry. consequently, these problems need further efforts and studies.10,16 furthermore, the benzoxazine nucleus is not only present in many pharmacologically active molecules, medicinally significant derivatives and natural products, but also they have been used as intermediates for the synthesis of other heterocyclic-scaffold bioactive compounds.17 furthermore, several 1,3-benzoxazines (fig. 3) show interesting biological and pharmaceutical properties.18,19 moreover, these derivatives are very valuable in the chemistry of natural products due to the formation of acetal glycosides in plant20 which act as a plant’s own resistance factor towards insects, pests, fungi and other microbial diseases.21 in this frame, the collected data in this survey is focused on the 3,4-dihydro-2h -1,3-benzoxazine monomers and their one-derivatives chemistry and bioactivities. fig. 3. 1,3-benzoxazines with biological and pharmaceutical properties. 2. synthesis of 1,3-benzoxazine derivatives 2.1. synthesis of 3,4-dihydro-2h-1,3-benzoxazines 3,4-dihydro-2h-1,3-benzoxazines have been synthesized through the one-pot mannich reaction of a substituted phenol with formaldehyde and aliphatic or aromatic monoamines/diamines (scheme 1).22 the importance of the role of the basicity of the amine on the rate of the reaction was found.13,23 thus a weakly basic amine will react faster than a strongly basic amine.24 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 216 sharaf el-din scheme 1. synthesis of 1,3-benzoxazines by a mannich reaction. this synthetic method could be performed either in solvent,25 such as dioxane/water,26 absolute ethanol,27,28 methanol,29 etc., or solventless.30,31 the use of an organic solvent increases the cost of the products and causes some environmental problems. furthermore, the solvent residue in the products leads to problems during the handling of the benzoxazine synthesis. to overcome these drawbacks, the solventless synthesis was developed under melt condition.32 the reaction mechanism and kinetics of this method were suggested by liu and ishida for the preparation large quantities of benzoxazine monomers.24 moreover, the influence of substituent attached to phenol or aniline on the stability of the oxazine ring and the equilibrium constant has been investigated and studied in the literature.24,26 in the solventless method30,33 all the reactants are mixed together, heated, and maintained at above their melting point using paraformaldehyde to maintain the reaction stoichiometry.30 additionally, in the case of reactants with high melting points, it is necessary to use toluene or 1,4-dioxane as solvents.34 better yield and purity were obtained using two-step methods by reaction of an aliphatic amine and formaldehyde at low temperature first before adding the phenol derivative.32 on the other hand, the kinetics and details of 3,4-dihydro-3-phenyl-2h-1,3 -benzoxazine synthesis by the mannich reaction were investigated.16 it was observed that n-hydroxymethyl aniline (hma) is considered the key intermediate. hma then reacts with phenol to give a second intermediate (4) that reacts with formaldehyde to form benzoxazines. however, hma reacts with other intermediates and reactants to form byproducts, as shown in scheme 2. thus, this research observed that the formation and the mechanism of benzoxazines synthesis besides the formation of byproducts will need further investigations.16 in addition, due to the presence of water, polar solvents and the high temperatures used, the formation of oligomers are considered the main drawbacks in benzoxazine synthesis by the mannich reaction.10,32 to minimize the previous drawbacks in the synthetic methods to 3,4 -dihydro-3-phenyl-2h-1,3-benzoxazine monomers via mannich condensation, numerous efforts have focused on two approaches: the use of a suitable synthetic method or the use a catalyst. herein, the different synthetic approaches for these derivatives have been studied as described in scheme 3a–c.35 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 2h-1,3-benzoxazine derivatives synthesis and bioactivities 217 nh2 hn oh oh n h o n hn oh n n n (3) phenol hcho hc hophenolhcho 4 scheme 2. possible pathways for the synthesis of benzoxazines by the mannich reaction.16 from the previous, there are three general synthetic methods for the preparation of benzoxazine monomers, one-pot, two-step and three-step mannich reactions. 2.1.1. one-step mannich condensation traditionally, benzoxazine synthesis was realized using the one-pot multi -component reactions of burke.3 this method has been generalized and studied because of its simplicity and diversity of substituents on both the phenol and the amine. for example, nitro,35 halogenic,36 cyano,37 aldehyde,38 carboxy,39 alkenyl,40 maleimide41 groups, etc. could be adopted onto benzoxazine by using functional phenols/amines, leading to the production of a large variety of functional benzoxazines. furthermore, by the use of bisphenol and/or diamine compounds, bifunctional benzoxazines could be obtained.42 another advantage of the solvent-free, one-pot method is that it avoids solvent residues, which may cause serious defects during processing, saves on solvent and its recovery costs, and there is no worry about the solubility of raw materials in an organic solvent.32 as example, compound 5 was prepared in a one-step mannich reaction (fig. 4).43 moreover, one-pot reactions are simple, easy to handle and avoid isolation and purification of intermediates, maximize the yield, minimize solvent, and enhance the greenness of the transformations.44 consequently, they have become a popular tool in the synthesis of complex heterocyclic molecules.45 a modification of the one-pot mannich reaction was developed via the oxidative hydroxylation of arylboronic acids and subsequent coupling with paraformaldehyde and amines in good to excellent yields with a variety of functional groups, scheme 4.46 the synthesis of dihydro-1,3-benzoxazines were obtained via one-pot condensation of αor β-naphthol, aniline and formaldehyde using thiamine hydrochloride as catalyst.47 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 218 sharaf el-din (a) (b) scheme 3. illustration of the one-pot (a), two-pot (b) and three-pot (c) synthetic method for the preparation of benzoxazine. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 2h-1,3-benzoxazine derivatives synthesis and bioactivities 219 o n n o r (1)5 fig. 4. structure of compound 5. scheme 4. synthesis of 1,3-benzoxazines using arylboronic acids. the preparation of a novel tetrafunctional oxazine monomer (6) containing 1,3-benzoxazine and fluorene-oxazine was performed through a one-step mannich condensation reaction (fig. 5).48 o n o n o n o n ch3 ch3 h3c h3c ( 2 )5 fig. 5. structure of compound 6. additionally, benzoxazine monomers were synthesized in high purity and good yield through one-pot reactions from the reaction of bisphenol a with paraformaldehyde and isomeric butylamines, as indicated in scheme 5.49 the brønsted acidic ionic liquid [hmim]bf4 was used as a nonvolatile ecofriendly solvent and catalytic reagent for the one-pot green synthesis of isoxazolyl-3,4-dihydro-2h-1,3-benzoxazines (7, fig. 6). this method afforded excellent yields in short reaction times, and avoids multistep synthesis.50 moreover, 3,4-dihydro-2h-1,3-benzoxazines were synthesized in one pot by the directed ortho-lithiation of phenols.51 2.1.2. two-step mannich condensation on the other hand, the two-step synthesis, first described by holly and cope,52 is performed in solvent. the reaction by this method proceeds by first ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 220 sharaf el-din scheme 5. synthesis of benzoxazines from bisphenol a. adding amine to formaldehyde at lower temperatures to form an n,n-dihydroxymethylamine, which then reacts with the labile hydrogen of the hydroxyl group on the ortho-position of the phenol at an elevated temperature to form the oxazine ring.53 the slow reaction rate and the large amount of solvent required for the synthesis due to the poor solubility of the reacting compounds are considered the disadvantages of this procedure, in addition to increasing the costs of the products and creating environmental problems. to overcome these drawbacks, the solvent-free synthesis was developed.32 o n n o c h3 ( 3 )7 fig. 6. structure of compound 7. thereafter, the two-step reaction involved the formation of perhydrotriazine (intermediate) in the reaction of formaldehyde with benzylamine. this intermediate reacts with phenol and formaldehyde in acidic condition to give benzoxazines (scheme 6).13 this method has been generalized with the proposed mechanism in the literature.10 additionally, another way was reported through the formation of bis(alkoxymethyl)alkylamine as intermediate, which was obtained in the reaction of alkyl amine with alcohol (scheme 7).54 notably, these methods cannot be used in the presence of a primary amine similar as one-pot methods. however, these methods were enabled in cases of reactive phenolic compounds, such as hydroxybenzaldehyde and hydroxybenzoic ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 2h-1,3-benzoxazine derivatives synthesis and bioactivities 221 acid, allowing a primary amine to be used. due to the diversity of substituents on both the phenol and the amine, a large variety of functional benzoxazines could be produced. scheme 6. synthesis of benzoxazines with 1,3,5-hexahydrotriazine. scheme 7. synthesis of benzoxazines with bis(alkoxymethyl)alkylamine. in the synthesis of benzoxazine in a two-step reaction, the first step involved the formation of 1,3,5-tris(pentafluorophenyl)perhydro-1,3,5-triazine, then the reaction between the acid-promoted cleavage of the perhydrotriazine with substituted phenol and formaldehyde occurred. the latter step is considered the rate determining step reaction.55 furthermore, 1,3,5-triphenylhexahydro-1,3,5-triazine (8, fig. 7) was formed as intermediate during the solventless synthesis of benzoxazines. this triazine could be used as an amine source instead of the direct use of a primary amine.10,56 n n n ( 4 )8 fig. 7. structure of compound 8. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 222 sharaf el-din 2.1.3. three-step mannich reaction brurke suggested a reaction pathway for a mannich condensation, i.e., initially, n,n-dihydroxymethylamine (9) is formed that is then converted into a n-hydroxymethyl mannich base (10), which finally reacts with phenol to generate 1,3-benzoxazines (scheme 8).42,57,58 h2n r1 h2c o nr1 oh oh oh n r oh o n r + 2 step 1 step 2 step 3 r r substituted phenols ( 1 ) ( 2 ) 1 1 9 10 scheme 8. three-step mannich reaction for the synthesis of 1,3-benzoxazines. moreover, a three-step method was developed by imine formation between salicylaldehyde and the selected primary amine as first step. the second step is the reduction of this imine into secondary amine and finally, ring closure using formaldehyde.59 the advantage of this method is the ability to control each step and the usage of amines that are incompatible in classical methods. furthermore, the use of this method avoids the formation of undesirable oligomeric or polymeric species, thus leading to a simple workup and improving the yield and purity of the final product. as an example, salicylaldehyde59 or 4-aminophenol can be used as the phenol or amine source, respectively. in addition, free phenol-containing benzoxazines can also be synthesized easily by this method. moreover, asymmetrical benzoxazine derivatives can easily be obtained by choosing a suitable salicylaldehyde.60 furthermore, 1,3-benzoxazine derivatives were formed via dehydration of methylene glycol to formaldehyde, which reacts with a mannich base as indicated in scheme 9.26 scheme 9. three-step mannich reaction for the synthesis of 1,3-benzoxazines. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 2h-1,3-benzoxazine derivatives synthesis and bioactivities 223 salicylaldehyde was condensed with primary aromatic amines to give imine compounds which on reduction with nabh4, yielded intermediate 11 at room temperature (fig. 8). compound 11 subsequently undergoes ring-closure reaction with paraformaldehyde in toluene at 60 °c to give benzoxazine monomer by a three-pot method. 27,58 n h oh r ( 5 )11 fig. 8. structure of compound 11. furthermore, the kinetics of the reaction between 2-[(phenylamino)methyl]phenol (phenol-aniline based mannich base) and formaldehyde to benzoxazine has been studied. the results showed that the reaction occurs rapidly and the reverse reaction occurs via hydrolysis of the benzoxazine to the mannich base.26 moreover, a di-functional benzoxazine was prepared in the reaction of 1,2-bis(ortho-hydroxybenzylamino)ethane (12, fig. 9) with formaldehyde.61 the advantage of this synthesis is the flexible substitution of functional groups on the oxazine ring. in addition, another substitution on the oxazine ring could be achieved by ring closure of salicylaldehyde with various aldehydes (aliphatic or aromatic) instead of formaldehyde.62 moreover, the oxazine ring could be closed by the reaction of salicylaldehyde not only with aldehydes but also with methylene bromide.63 furthermore, this method enhances the formation of benzoxazine monomer only because its intramolecular cyclization permits the reaction conditions to moderate, leading to the minimization of side reactions caused by high temperatures. however, in the case of a one-pot method, sometimes relatively high temperature are required to close the oxazine ring leading to the formation of undesirable oligomeric or polymeric species. 12 ( ) o h o h n h n h fig. 9. structure of compound 12. on the other hand, for further limitation on the drawbacks of mannich methods, many catalysts have been used for the growing number of benzoxazines syntheses. as example, 2,3-diaryl-3,4-dihydro-2h-1,3-benzoxazines have been prepared in high yields from o-(arylaminomethyl)phenols and aromatic aldehydes in the presence of sncl4.27 in addition, the condensation of hexakis(methoxymethyl)melamine (hmmm) with monoor di-substituted phenols in p-xylene ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 224 sharaf el-din catalyzed by di-nonylnaphthalenedisulfonic acid64 gave 1,3-benzoxazines. finally, i2/h2o2-promoted intramolecular c–o bond formation reaction of a variety of 1-(aminoalkyl)-2-naphthols or 2-(aminoalkyl)phenols yielded the corresponding 1,3-oxazines. the reaction is simple, economic, and proceeds at room temperature in ethanol as solvent.65 r r 1 2( 7 ) no n o 13 fig. 10. structure of compound 13. 2.2. synthesis of a sulfone-scaffold benzoxazine monomer on the other hand, the formation of byproducts (oligomers or polymers) in mannich reactions has been considered beneficial in many industrial usages in spite of being considered a drawback in the preparation of benzoxazine monomers. thus, 3,4-dihydro-2h-1,3-benzoxazines can yield polymeric structures through ring-opening of the cyclic monomers. these polymeric structures are commercially important and widely applied in the areas of coatings, adhesives, microelectronics, aerospace, etc.66,67 one example of commercially importance are polysulfones (psu), a class of polymers with excellent features, e.g., thermal stability, durability in harsh conditions, oxidation, ph and temperature resistance, ease of process ability and good film properties.68,69 sulfone-scaffold 3,4-dihydro-2h-1,3-benzoxazines (14) were prepared in high purity from 4,4′-diaminodiphenyl sulfone (15),70,71 4,4′-sulfonylbisphenol (16)25 or polysufone72 and paraformaldehyde and phenol using a high boiling point, nonpolar solvent, see fig. 11. s o o o n o n phph h2n o o s nh2 ohs o o ho ( 7 ) ( 8 ) ( 6 )14 16 15 fig. 11. sulfone-based 1,3-benzoxazines, 4,4′-diaminodiphenyl sulfone and 4,4′-sulfonylbisphenol. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 2h-1,3-benzoxazine derivatives synthesis and bioactivities 225 2.3. synthesis of bio-based benzoxazine monomer interestingly, the raw materials for the synthesis of benzoxazine derivatives are almost always derived from petroleum oil. with the fast consumption of petroleum oil and the increasingly serious environmental pollution, the utilization of bio-based feedstock for the green preparation of these derivatives has gained more attention in all domains.73,74 in the synthesis of benzoxazines, renewable starting materials are used due to their availability, low toxicity, and relatively low cost. thus, naturally occurring phenols, such as chavicol,75 guaiacol,76 cardanol77,78and lignocelluloses,79,80 are used in the synthesis of 1,3-benzoxazines (fig. 12). fig. 12. the structures of some naturally occurring phenols. 2.4. synthesis of 4h-1,3-benzoxazin-2-ones the benzoxazinones were prepared in the one-pot reaction of 2-naphthol, an aldehyde and urea in the presence of various catalysts such as iodine (scheme 10), p2o5 and yb(otf)3,81 cellulose sulfuric acid,82 cyanuric chloride,83 phosphomolybdic acid,84 pyridinium-based ionic liquid,85 thiamine hydrochloride,86 zinc triflate,87 montmorillonite k10,88 zinc oxide,89 tmscl/nai,90 guanidine hydrochloride,91 etc. scheme 10. one-pot mannich reaction using iodine as catalyst. on the other hand, by condensation of (aminoalkyl)naphthols with phosgene92 or 1,1′-carbonyl diimidazole93 in the presence of triethylamine, 2h-1,3 -oxazin-2-one derivatives were produced in moderate yields. in addition, 1,3 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 226 sharaf el-din -benzoxazin-2-ones (17, fig. 13) were prepared by reaction of 2-hydroxyphenylsubstituted enones and isocyanates using bisguanidinium salt as catalyst.94 o n r h r o o 1 ( 8 ) r 17 fig. 13. structure of compounds 17. furthermore, 2h-1,3-benzoxazine-2-ones were synthesized in the reaction of substituted salicylaldehydes with a primary amine and an aldehyde. as example, spiropyrans based on benzoxazinone (18) were synthesized in the reaction of compound (a) with compound (b) using protonated acetic acid [mec(oh)2]+clo4– as catalyst, as indicated in scheme 11.95 oh c l o h 2n r oh cl n r n o oh2c r oh no2r o c l n o oh 2c r cl o n r o r o 2n cl + ( a ) ( b ) ( a ) + [mec(oh) ] clo + _ ( 4 ) 1 1 2 4 18 scheme 11. synthesis of spiropyrans based on 2h-1,3-benzoxazin-2-one. in addition, 3,4-dihydro-2h-1,3-oxazin-2-ones were synthesized by intramolecular cyclization of aryl carbamates, which were produced from the reaction of aryl isocyanate and the corresponding 2-(nitroethenyl)phenol under basic conditions.96 via the reaction of salicylaldehyde/2-hydroxyacetophenone or its hydrazones and substituted urea or substituted semicarbazide under solventless microwave irradiation,97 2h-1,3-benzoxazin-2-ones were synthesized in a one-pot method (scheme 12). ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 2h-1,3-benzoxazine derivatives synthesis and bioactivities 227 scheme 12. synthesis of 2h-1,3-benzoxazin-2-ones from salicylaldehyde/2-hydroxyacetophenone. 2.5. synthesis of 2h-1,3-benzoxazin-4-one in the condensation of an acid halide with salicylamides in the presence of pyridine using boiling xylene as solvent, substituted 2h-1,3-benzoxazin-4-ones were formed in one-step. they were also formed in a two-step method by refluxing the salicylamide with aroyl chloride in pyridine followed by cyclization of the isolated intermediate by hydrogen chloride.98 furthermore, by carbonylation-cyclization of ortho-halophenols and cyanamide99 or by treatment of the corresponding 2-hydroxycarboxamides with a formaldehyde/formic acid mixture,100 the corresponding 4h-1,3-benzoxazin-4 -ones were synthesized. 2-(trichloromethyl) and 2-(dichloromethylene)-2h-1,3-benzoxazine derivatives were obtained via intramolecular cyclization of n-(α-aryloxytrichloroethyl)imidoyl chlorides through dehydrochlorination.101 additionally, 2aryl-2-(trifluoromethyl)-2,3-dihydro-4h-benzoxazin-4-ones were synthesized via intramolecular thermal cyclization of 3-alkoxyphenyl n-(1-aryl-2,2,2-trifluoroethylidene)carbamates, which were produced in the reaction of 1-aryl-2,2,2-trifluoroethyl isocyanates with 3-alkoxyphenols (scheme 13).102 r f 3c cl n c o oh r et3n o o n cf3 r r o r nco cf3 r o nh r o cf3 r toluen + 1 1 1 1 e scheme 13. synthesis of 2-aryl-2-(trifluoromethyl)-2,3-dihydro-4h-benzoxazin-4-ones. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 228 sharaf el-din moreover, 2,3-dihydro-4h-1,3-benzoxazin-4-ones have been synthesized by intermolecular cyclization reactions of o-halobenzamides, lioh and dichloromethane using copper-catalyzed tandem reaction.103 2.6. synthesis of 2h-1,3-benzoxazine-2,4 (3h)-diones the 2h-1,3-benzoxazine-2,4(3h)-diones were synthesized from the reaction of acardic acids with triphosgene,104 from the reaction of phthaloyl chlorides with acetone oxime105 or from the reaction of salicylate esters with isocyanates.106 reaction of 2-hydroxybenzonitrile with isocyanates107 using triethylamine as catalyst has been performed to obtain the target compounds as in scheme 14. scheme 14. synthesis of 2h-1,3-benzoxazine-2,4(3h)-diones from 2-hydroxybenzonitrile and isocyanates. 3. chemistry of 1,3-benzoxazine derivatives 3.1. unusual behavior of ortho-functional benzoxazines the formation of intramolecular five-membered ring h-bond between the nh of the amide group and the oxygen of the oxazine ring (fig. 14) is considered as unusual behavior of ortho-functional benzoxazines.108 fig. 14. the intramolecular five-membered ring h-bond in benzoxazines. furthermore, it was observed that, o-methyl-substituted benzoxazine dimers, as shown in fig. 15,109,110 trimers or tetramers exhibit intramolecular hydrogen bonding.111 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 2h-1,3-benzoxazine derivatives synthesis and bioactivities 229 interestingly, the o-substituted benzoxazine dimers are used as novel ligands for rare earth metal ions, e.g., the ce(iii) ion. it was found that, the substituted groups on the para-positions of benzoxazine dimers do not affect the formation of complexes.112 h hh h fig. 15. the molecular structure of a pair of methyl benzoxazine dimers. 3.2. ring opening of benzoxazines the dihydro-derivatives are more stable than the 1,3-benzoxazines towards acidic agents. the ring opening ability depends on the basicity of the oxygen and nitrogen atoms.113 in compounds with an active hydrogen, such as indoles, carbazole, imides, and aliphatic nitro compounds, even phenol (scheme 15),114,115 thiols (scheme 16)116 or carboxylic acids,117 auto-ring opening occurs as shown in schemes 15–17. the benzoxazines ring opening begins with protonation of oxygen and nitrogen atoms,118 as indicated in scheme 17. r 1 r2 r3 r4 r6 1 r r r r r r r r r r r r r r 5 2 5 3 1 5 6 3 6 6 1 2 3 2 scheme 15. the mechanism of benzoxazines ring opening. scheme 16. the auto-ring opening reaction of 1,3-benzoxazines. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 230 sharaf el-din scheme 17. the benzoxazines ring opening by protonation of oxygen and nitrogen atoms. furthermore, ring opening is promoted by irradiation with uv radiation (scheme 18),119,120 resulting in the formation of two chromophoric systems (the 3h-indolium cation and the 4-nitrophenolate anion moiety).119 r1 no2 r r r no2 1 2 2 hv/ heat _ scheme 18. ring opening of 1,3-oxazine ring upon irradiation. in addition, the ring opening reaction of substituted benzoxazine would readily occur by heating due to the resonance stabilization of the iminium ion, as indicated in scheme 19.121 scheme 19. the resonance stabilization of the iminium ion. 3.3. ring-chain tautomerism the 1-(substituted phenyl)-3-aryl-2,3-dihydro-1h-naphth[1,2-e][1,3]oxazines undergo ring–chain tautomerism, resulting in predominately the trans(19) over the cis-configuration (20) through compound (21), as shown in scheme 20.122 (19) (21) (20) scheme 20. naphthoxazines epimerization. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 2h-1,3-benzoxazine derivatives synthesis and bioactivities 231 4. reactions of 1,3-benzoxazine derivatives 4.1. hydrolysis with hcl the benzoxazine derivatives (22) are hydrolyzed by hcl to give 2-aminopyridine (23) or n-2-pyridylsalicylamide (24) depending on the concentration of the acid, as indicated in scheme 21.123 dil.hcl conc. hcl (33) (31) (32) cooh o h nh 2 o n o n o h o n h n + (24) (22) (23) scheme 21. effect of acids on benzoxazine derivatives. 4.2. salt formation the formation salts of 1,3-benzoxazines has been realized by acidic cyclization of disalicylamide124 or by acylation of (o-aminophenyl)diphenylmethanol with carboxylic acids in the presence of perchloric acid,125 producing l,3-benzoxazinium perchlorate 25, fig. 16. (25) fig. 16. structure of compound 25. 4.2.1. reactions of 4-oxo-4h-1,3-benzoxazinium salts interestingly, 4-oxo-4h-1,3-benzoxazinium perchlorate 26 reacts with the dialdehyde potassium 3,5-diformyl-2,4-dihydroxybenzoate 27 in glacial acetic acid yielding the spiropyran of the 1,3-benzoxazine series through the formation of the intermediate styryl salt 28. this intermediate has been isolated and then cyclized under the action of triethylamine in anhydrous diethyl ether to yield compound 29, as shown in scheme 22.126,127 4.3. reaction with alkyl halides 1,3-benzoxazine-2,4-dione was reacted with alkyl halide in the presence of k2co3128,129 yielding n-substituted derivatives (scheme 23). 4.4. nucleophilic substitution reaction 4.4.1. reaction with pyridine n-oxide 2,3-dihydro-2,2-dimethyl-3-(2-pyridyl)-4h-1,3-benzoxazin-4-one (30) was produced by refluxing the 4-chloro-derivative of benzoxazine (31) with two ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 232 sharaf el-din moles of pyridine n-oxide in dichloromethane through a nucleophilic substitution reaction followed by rearrangement123 (scheme 24). o n+ ch3 o rr co2k oh oh oo co2k oh oh o n o o r r et3n o n o r o co2k oh o r cl o _ + ( 1 6 ) ( 15 ) (18 ) acoh (17) 2 2 2 4 (26)(27) (28) (29) scheme 22. reaction of 4-oxo-4h-1,3-benzoxazinium salts with 3,5-diformyl-2,4-dihydroxybenzoate. scheme 23. reaction of 1,3-benzoxazine-2,4-dione with alkyl halides. (30) (31) scheme 24. reaction of substituted-1,3-benzoxazines with pyridine n-oxide. 4.4.2. reaction with organometalic compounds 2,3-dihydro-2,2-dimethyl-1,3-benzoxazin-4-one derivatives react with organometallic compounds by nucleophilic substitution as shown in scheme 25.130 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 2h-1,3-benzoxazine derivatives synthesis and bioactivities 233 scheme 25. reaction of 1,3-benzoxazin-4-ones with organometalic compounds. 5. biological activities benzoxazinone and its derivatives are a significant class of heterocyclic compounds, because many of these derivatives display diverse biological activities. 5. 1. antiviral therapy elbasvir (32, fig. 17)131,132 is potent inhibitor of the hcv ns5a protein and is used in combination with grazoprevir for the treatment of the hepatitis c virus (hcv) ns3/4a.133 in addition, grazoprevir/elbasvir plus ribavirin were examined as a new treatment option for patients after failure of triple therapy containing an earlier-generation protease inhibitor.134 fig. 17. structure of elbasvir (32). 5.2. anti-tuberculosis activity the antimycobacterial activity of various substituted 3-phenyl-2h-1,3-benzoxazine-2,4(3h)-dithiones and 3-phenyl-2h-benzoxazine-2,4(3h)-diones have been studied using a quantum molecular similarity approach. the replacement of the oxo-group by the thioxo-group in position 4 on the benzoxazin-2,4-dione ring increases the activity, as well as the similar replacement in position 2.135,136 in vitro antimycobacterial activity against mycobacterium tuberculosis, m. avium and two strains of m. kansasii were studied. furthermore, the antimycobacterial activity increased with replacement of the carbonyl group by the thiocarbonyl group in the starting 3-(4-alkylphenyl)-2h-1,3-benzoxazine-2,4(3h)-diones.137,138 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 234 sharaf el-din 5.3. fungicidal and pesticide activities a series of 2,3-disubstituted-3,4-dihydro-2h-1,3-benzoxazines was prepared by reaction of aza-acetalizations of aromatic aldehydes with 2-(n-substituted aminomethyl)phenols in the presence of trimethylsilyl chloride (tmscl). the fungicidal activities were evaluated, and some of these compounds exhibited activity against rhizoctonia solani.62 additionally, a series of 2,3-diaryl-3,4 -dihydro-2h-1,3-benzoxazines was prepared in high yields from o-arylaminomethylphenols and aromatic aldehydes in the presence of sncl4. their fungicidal activities were investigated. some of the products showed good fungicidal activities against r. solani.27 furthermore, novel naphtho[1,2-e][1,3]oxazines bearing an arylsulfonamide moiety were synthesized and evaluated for their anticancer and antifungal activities.139 moreover, substituted 8-hydroxy-3-phenyl-2h-1,3-benzoxazine-2,4-(3h) -diones were synthesized by cyclization of the corresponding dihydroxy-n-phenylbenzamides with methyl chloroformate. thionation of the compounds was performed using lawesson's reagent. all compounds were tested in vitro for their antifungal activity against eight test strains. the compounds showed moderate activity.140 in addition, the compounds 3,4-dihydro-4-methyl-3-nonyl-2h-1,3-benzoxazines and 3-decyl-3,4-dihydro-4-methyl-2h-1,3-benzoxazines were studied and investigated as pesticides.141 5.4. anticovulsive activities 2,4-dioxo-2h-1,3-benzoxazine-3(4h)-butanoic acid (bxdba) shows good anticonvulsive activity and its ability to block bicuculline-induced convulsions suggests that it could be a gabaa mimetic drug.142,143 5.5. antibacterial activities substituted n-[(benzylamino)thioxomethyl]-2-hydroxybenzamides were synthesized using sodium bicarbonate and benzyl amine with 2-thioxo-substituted-1,3-benzoxazines. these derivatives were investigated as antibacterial and antifungal agents.144 moreover, a series of 3,3′-(1,2-ethanediyl)-bis[3,4-dihydro-2h-1,3-benzoxazine derivatives (33, fig. 18) was synthesized via an eco-friendly mannich-type condensation-cyclization reaction of phenols or naphthols with formaldehyde and primary amines in water at ambient temperature. in vitro antimicrobial activity of the synthesized compounds was assessed against six pathogenic fungi, two gram-negative and two gram-positive bacteria. some of the screened compounds showed significant in vitro antimicrobial effects.145 benzofuranyl-1,3-benzoxazines and benzofuranyl-1,3-benzoxazin-2-ones were synthesized via coupling benzofuran with 1,3-benzoxazines and 1,3-benzo________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 2h-1,3-benzoxazine derivatives synthesis and bioactivities 235 xazin-2-ones through –conh– and –coch2– bridges, respectively. the antimicrobial activity of these compounds was reported.146 r2 r1 r2 r1 ( 11 ) n o o n (33) fig. 18. structure of compound 33. 5.6. anticancer activities furthermore, naphtho[1,2-e][1,3]oxazines bearing a arylsulfonamide moiety, synthesized via a one-pot method, showed remarkable activities against mcf-7 (breast) and hct116 (colon) cancers.139 in addition, 1,3-benzoxazines having a flavone moiety at the 3-position also showed activities against mcf-7.147 2h-1,3-oxazine-2,6(3h)-dione (3-oxauracil) exhibited cytotoxic activity against the tested cancer cell lines (pancreatic, colon, neuroendocrine and nonsmall cell lung). these derivatives were studied as an inhibitor of selected neoplastic cell growth in vivo.148 in addition, a series of modified hexacyclic camptothecin derivatives containing a 1,3-oxazine ring was synthesized. all compounds were assayed in vitro against nine human cancer cell lines. some of these compounds showed about 13-fold greater potency than camptothecin, and about six-fold greater potency than topotecan toward hepg-2. furthermore, the n-alkyl-substituted derivatives were more potent than the n-aryland n-benzyl-substituted compounds.149 the synthesis of 6-aryl, 8-aryl, and 8-aryl-6-chloro-2-morpholino-1,3-benzoxazines with potent activity against pi3k and dna-pk was studied. a compound with the 8-(naphthalen-1-yl) scaffold showed strong anti-proliferative activity against a498 renal cancer cells, which warrants further investigation.150 5.7. antihypertinsive activities the antihypertensive and cardiovascular properties of a new potassium channel opener, tcv-295 (34), were studied in rats and dogs. in conscious, spontaneously hypertensive rats (shr), tcv-295 reduced blood pressure (bp) with a low dose dependence and with slow onset of action being observed.151 an efficient process for potassium channel opener tcv-295, based on 4-(2 -pyridyl)-2h-1,3-benzoxazine ring formation from 2-(o-hydroxybenzoyl)pyridine derivative by the nh4i/piperidine/2,2-dimethoxypropane system and subsequent selective pyridine-n-oxidation using dimethyldioxirane, was examined.152 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 236 sharaf el-din in addition, to explore k+ channel openers, a series of 1,3-benzoxazine derivatives with a 2-pyridine-1-oxide group at c4 (34, fig. 19) was synthesized by one-pot 1,3-benzoxazine skeleton formation and using a palladium(0)-catalyzed carbon–carbon bond formation reaction of imino-triflates with organozinc reagents. the compounds were tested for vaso-relaxant activity using bacl2-induced and high kcl-induced contraction of rat aorta to identify potential k+ channel openers, and also for oral hypotensive effects in spontaneously hypertensive rats.130 ( 12 ) n + o n o (34) fig. 19. structure of compound 34. 5.8. antimalarial activities a series of 6-(2-chloroquinolin-3-yl)-4-(substituted phenyl)-6h-1,3-oxazin2-amines was synthesized and evaluated in vitro for antimalarial efficacy against chloroquine sensitive (mrc-02) and chloroquine resistant (rkl9) strains of plasmodium falciparum.153 the antimalarial activities of the resulting benzoxazines, their isosteric tetrahydroquinazoline derivatives, and febrifugine-based quinazolin-4-ones were examined in vitro (against p. falciparum) and in vivo (against p. berghei).138 5.9. antidiabetic and hypolipidaemic activity a series of 5-{{4-[2-(4-oxo-2h-1,3-benzoxazin-3(4h)-yl)ethoxy]phenyl} methyl}thiazolidine-2,4-diones was synthesized and investigated for their plasma glucose and plasma triglyceride lowering activity. in addition the synthesized 2,4-thiazolidinedione derivatives of 1,3-benzoxazinone were evaluated for their antidiabetic and hypolipidaemic potential. for example, drf-2519 (35, fig. 20) showed potent dual ppar activation.154 r1 ( 13 )o o o n s o n o na + (35) fig. 20. structure of compound 35. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 2h-1,3-benzoxazine derivatives synthesis and bioactivities 237 5.10. receptor antagonist activity the synthesis and pharmacology of benzoxazines (36, fig. 21) were investigated as highly selective antagonists at m4 muscarinic receptors.155 n o h 3c o n h r o o ch3 ( 14 )(36) fig. 21. structure of compound 36. 5.11. antidepressant activity it was found that 1,3-benzoxazine-2,4-diones (37, fig. 22) have binding affinities for the 5-ht1a and 5-ht7 receptors.128 furthermore, the benzoxazine derivative caroxazone (38, fig. 23), was investigated in vitro and in vivo as antidepressant (ro 11-1163) and as a specific and short-acting mao-a inhibitor.156 o n o o n n ( 15 ) r (37) fig. 22. structure of compound 37. fig. 23. structure of compound 38. 5.12. anti-platelet aggregation activity a series of 2,8-disubstituted benzoxazinones (39, fig. 24) was synthesized and studied as anti-platelet aggregation agents via inhibition of superoxide anion generation and inhibition of neutrophile elastase release assays. it was found that, the synthesized compounds were more potent than aspirin on arachidonic acidinduced platelet aggregation.157,158 5.13. miscellaneous activities in addition, other benzoxazine compounds have anti-inflammatory activities, e.g., compounds 40 and 41,30 and analgesic and antipyretic properties, such as ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 238 sharaf el-din chlorthenoxazin (42), fig. 25.19,30 furthermore, these derivatives are used as specific inhibitors of the tissue factor (tf)/factor via (via)-induced pathway of coagulation, as reported in the literature.159 r1 r2 ( 17 ) o n o o n (39) fig. 24. structure of compound 39. r 1 r 1 r2 ( 18 ) ( 19 ) o o n o o n (40) (41) o nh o cl r ( 20 )(42) fig. 25. structures of compounds 40–42. conclusions in conclusion, the synthetic potential and transformations of 3,4-dihydro-2h -1,3-benzoxazines remain largely of interest. the 3,4-dihydro-2h-1,3-benzoxazines are flexible and tough, which lead the molecules to have diverse workable site for substitution. in addition, they exhibit a wide range of biological activities, such as herbicides and agricultural microbiocides, and they show diverse pharmacological activities, such as antitumor agents, antiretroviral therapy, anti-tubercular activity, antibacterial activity, anti-inflammatory activity, anti-convulsant activity, etc. on the other hand, the growth of drug resistance is considered a major problem in medicine and to overcome this status, the synthesis of new classes of compounds is a requisite. consequently, the data collected in this review could be used to provide novel benzoxazine derivatives that could be utilized for the development of new compounds to overcome resistance of drugs for various diseases. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 2h-1,3-benzoxazine derivatives synthesis and bioactivities 239 и з в о д хемија и биолошка активност 3,4-дихидро-2h-1,3-бензоксазина и њихових оксо-дериватa nabaweya abd el-salam sharaf el-din department of pharmaceutical chemistry, faculty of pharmacy, tanta university, el giesh street, 31527, tanta, egypt деривати 3,4-дихидро-2h-1,3-бензоксазина су природни производи и значајна класа хетероцикличних једињења посебно због њихове изузетне активности у хуманој медицини, фитофармацији и ветерини. услед могућности за надградњу бензоксазинске структуре, компаративне хемијске једноставности и доступности, ова једињења су подесан извор за нова биоактивна једињења. резултати тога су истраживање и открића велике групе ових једињења која показују широк опсег биолошких активности, као што су антифунгална, антибактеријска, анти-хив, антиканцерска, релаксациона, антиинфламанторна и др. овај прегледни чланак даје кратак приказ деривата 3,4-дихидро-2h-1,3-бензоксазина и њихових оксо-деривата, хемијску реактивност и биоактивност. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {molecular modeling studies of hiv-1 non-nucleoside reverse transcriptase inhibitors using 3d-qsar, virtual screening, and docking simulations} j. serb. chem. soc. 84 (3) 303–316 (2019) udc 616.98:578.828+66.011:539.16:615.2 jscs–5185 original scientific paper 303 molecular modeling studies of hiv-1 non-nucleoside reverse transcriptase inhibitors using 3d-qsar, virtual screening and docking simulations jian-bo tong*, shang-shang qin, shan lei and yang wang shaanxi key laboratory of chemical additives for industry, shaanxi university of science and technology, xi’an 710021, p. r. china (received 4 september, revised 15 october, accepted 15 november 2018) abstract: acquired immunodeficiency syndrome (aids) is a significant human health threat around the world and therefore, the study of anti-hiv drug design has become an important task for today’s society. in this paper, a three-dimensional quantitative structure–activity relationships study (3d-qsar) was conducted on 72 hiv-1 non-nucleoside reverse transcriptase inhibitors (nnrtis) using topomer comparative molecular field analysis (topomer comfa). the multiple correlation coefficients of fitting, cross-validation, and external validation were 0.899, 0.788 and 0.942, respectively. the results indicated that the obtained model had both a favorable estimation stability and a good prediction capability. topomer search was used to search appropriate r groups from the zinc database, thereby, 14 new compounds were designed, and 12 of the new compounds were predicted to be more active than the template molecule. these results strongly suggest that the topomer search was effective in screening and could be a useful guide in the design of new hiv-1 drugs. the ligands of the template molecule and the new designed compounds were used for molecular docking to study the interaction of these compounds with the protein receptor. the results showed that the ligands would generally form hydrogen-bonding interactions with the residues ala28, asp29, gly49 and ile50 of the protein receptor, thereby providing additional insights for the designing of even more effective drugs. keywords: 3d-qsar; nnrtis; topomer comfa; topomer search; new drug design; molecular docking. introduction aids caused by human immunodeficiency virus type-1 (hiv-1) has been threatening human health and no drug could hitherto cure it completely.1–3 there are two types of hiv that infect humans: hiv-1 and hiv-2. hiv-1 is the more virulent form because it is more easily transmitted.3 there are millions of people *corresponding author e-mail: jianbotong@aliyun.com https://doi.org/10.2298/jsc180904098t 304 tong et al. living with hiv, and its rapid spread and high fatality rate resulted in large economic losses and social impacts.4 while over 20 antiretroviral drugs have been approved for use in hiv-infected patients,5 many of these drugs are becoming less effective because of resistance resulting from changes in the mutation-prone hiv. therefore, the successful management of hiv infection requires new patient-friendly and effective therapies. the host proteins involved in the viral replication cycle have been used as drug targets when designing inhibitors to prevent the spread of infection. these targets include reverse transcriptase, protease, integrase, polymerase, glycoprotein, as well as the host cell receptor (cd4) and coreceptors (ccr5 and cxcr46). nnrtis are among the most potent and promising of the anti-aids agents that specifically target the hiv-1 reverse transcriptase (rt). the hiv-1 rt is an asymmetric heterodimer, comprised of a p66 subunit (560 amino acids) and a p51 subunit (440 amino acids)7 that has been used as an effective target for antiretroviral drugs. however, the effectiveness of nnrtis8,9 drugs is hampered by the rapid emergence of drug-resistant viruses and severe side effects associated with long-term use. therefore, there is a need to develop additional, highly potent nnrtis with a broad spectrum of antiviral activity and improved pharmacokinetic properties. consequently, more efficient strategies that facilitate and shorten the drug discovery process would be extremely beneficial.10 the availability of computational techniques based on qsars provide a possible means of accelerating the drug design process, since a qsar is a technique that attempts to summarize chemical and biological information in a form that allows the rapid generation of relationships between chemical structures and biological activity.11 the success of a qsar study depends on a proper selection of the variables and on a useful representation of the information. the best variables are those for which small changes in the value result in significant changes in activity. a 3d-qsar model better reflects the interactions between a substrate and the receptor than does a 2d-qsar model. comparative molecular field analysis (comfa)12 is a widely-used method of 3d-qsar investigation. in this paper, topomer comfa,13,14 a second generation of comfa, was employed to construct a 3d-qsar model utilizing data for 72 hiv-1 nnrtis to analyze the chemical–biological interactions governing their activities. the topomer comfa model was combined with the topomer search15 technology to conduct a ligandbased virtual screening of possible compounds in order to lay the foundation of improved drug design. materials and methods preparation of data set a total of 72 dihydro(alkylthio)benzyloxopyrimidines (s-dabos), a class of highly potent nnrtis, was selected from the published literature.16 the full data set was randomly divided into two parts: a training set of 57 compounds (compounds 1 to 57) to build the 3d-qsar 3d-qsar of hiv-1 nnrtis 305 model and a test set of 15 compounds (compounds 58 to 72) for use in the evaluation of the model. the biological activity was characterized by the half-maximal (50 %) inhibitory concentration (ic50). the ic50 values in μm range were converted to the m range and then converted to a logarithmic scale (pic50 = –log ic50).17 the chemical structures and their pic50 values are listed in table i. table i. the structures and activity values of the s-dabo derivatives; compounds 58–72 were chosen as the test set n n o h r3 r1 r2 no. r1 r2 r3 pic50 no. r1 r2 r3 pic50 1 me 2-naphthyl s-sec-bu 4.23 37 me 2,6-di-cl-ph s-tert-bu 5.96 2 h 1-naphthyl s-cyclopentyl 4.31 38 h 2,6-di-f-ph s-me 6.10 3 me 1-naphthyl s-cyclopentyl 4.35 39 me 2-cl-ph s-sec-bu 6.10 4 me 4-f-ph s-sec-bu 4.59 40 me 2-f-ph s-sec-bu 6.10 5 me 4-cl-ph s-sec-bu 4.77 41 me 3-no2-ph s-sec-bu 6.10 6 h 1-naphthyl s-sec-bu 4.79 42 h 2-f-ph s-sec-bu 6.22 7 h 2-naphthyl s-sec-bu 4.83 43 h 3-no2-ph s-sec-bu 6.22 8 h 4-f-ph s-sec-bu 4.83 44 h 2,6-di-cl-ph s-tert-bu 6.22 9 h 4-cl-ph s-sec-bu 5.02 45 h 2,6-di-cl-ph s-n-bu 6.30 10 h ph s-tert-bu 5.07 46 h 2,6-di-cl-ph s-cyclopentyl 6.40 11 h 3-me-ph s-tert-bu 5.09 47 h 2,6-di-f-ph s-n-bu 6.70 12 me 3-me-ph s-sec-bu 5.27 48 h 2,6-di-f-ph s-tert-bu 6.70 13 me 2,6-di-cl-ph s-cyclohexyl 5.31 49 h 2,6-di-cl-ph s-sec-bu 6.70 14 me ph s-me 5.31 50 me 2,6-di-cl-ph s-sec-bu 6.92 15 me ph s-sec-bu 5.32 51 h 2,6-di-f-ph s-sec-bu 7.00 16 me 3-me-ph s-tert-bu 5.34 52 me 2,6-di-f-ph s-sec-bu 7.00 17 me ph s-cyclohexyl 5.37 53 h 2,6-di-f-ph s-cyclohexyl 7.05 18 h 3-cl-ph s-sec-bu 5.42 54 me 2,6-di-f-ph s-tert-bu 7.05 19 me 4-no2-ph s-sec-bu 5.44 55 h 2,6-di-f-ph s-cyclopentyl 7.10 20 me 3-me-ph s-cyclopentyl 5.47 56 me 2,6-di-f-ph s-cyclopentyl 7.10 21 h 2-cl-ph s-sec-bu 5.49 57 h 2,6-di-f-ph s-iso-pr 7.30 22 me 3-f-ph s-sec-bu 5.52 58 me 1-naphthyl s-sec-bu 4.35 23 h 2,6-di-cl-ph s-me 5.52 59 h 2-naphthyl s-cyclohexyl 4.48 24 h ph s-cyclohexyl 5.52 60 h ph s-sec-bu 5.27 25 h 3-me-ph s-iso-pr 5.54 61 me ph s-cyclopentyl 5.47 26 h ph s-cyclopentyl 5.55 62 h 3-me-ph s-cyclopentyl 5.59 27 h 3-me-ph s-cyclohexyl 5.59 63 me ph s-iso-pr 5.60 28 me 3-me-ph s-me 5.60 64 h 3-me-ph s-sec-bu 5.62 29 me 3-me-ph s-iso-pr 5.60 65 me 3-cl-ph s-sec-bu 5.74 30 h 4-no2-ph s-sec-bu 5.62 66 h 3-f-ph s-sec-bu 5.92 31 me 3-me-ph s-cyclohexyl 5.66 67 h 2-no2-ph s-sec-bu 6.22 32 me ph s-tert-bu 5.72 68 h 2,6-di-cl-ph s-cyclohexyl 6.40 33 me 2,6-di-cl-ph s-cyclopentyl 5.80 69 me 2,6-di-f-ph s-me 6.70 34 h 2,6-di-cl-ph s-iso-pr 5.89 70 me 2,6-di-f-ph s-n-bu 7.05 306 tong et al. table i. continued no. r1 r2 r3 pic50 no. r1 r2 r3 pic50 35 me 2,6-di-cl-ph s-iso-pr 5.94 71 me 2,6-di-f-ph s-cyclohexyl 7.15 36 me 2,6-di-cl-ph s-n-bu 5.94 72 me 2,6-di-f-ph s-iso-pr 7.30 molecular structure construction the 3d structures of these compounds were generated using sketch molecule of the sybyl-x 2.0 package. all molecular structures used in both the training set and the test set were optimized using tripos force field18 and gasteiger–hückel charges.19 the structural energy minimization was terminated using the powell gradient algorithm with a convergence criterion of 0.005 kcal* mol-1, reached after a maximum of 1000 iterations.20 generally, the lowest energy conformer of the most active compound was selected as a molecular template, which in this study was compound 57. topomer comfa analysis the topomer comfa model was developed using sybyl-x 2.0. the topomer comfa is the second generation of comfa, and is a 3d-qsar technique that automates the creation of models for predicting the biological activity or property of compounds.21,22 in topomer comfa, the molecules are cut into two or more fragments, and all the fragments are aligned automatically,23 the choice of splitting modes or the identification of the best r-group is the key step for the topomer comfa. steric and electrostatic interaction energies were calculated using the carbon sp3 probe. the topomer comfa model was built by partial least square (pls)24 based on the optimal number of principal components (n) and evaluated by leave-one-out (loo) cross-validation.25 the bioactivity of the test set molecules were calculated by the model in order to evaluate the predictive ability of the model. in the process of topomer comfa, the mode of cutting would affect the quality of the model. in this study, each of the training set structures was broken into three fragments shown as ra (yellow), rb (red) and rc (blue) groups (fig. 1). fig. 1. cutting style of molecule 57. the partial least-squares (pls) statistical method, which is an extension of multiple regression analysis, was used to build the topomer comfa model. the leave-one-out (loo) cross-validation approach was used to acquire the optimal number of components (nc), the correlation coefficient (q2), the non-cross-validation correlation coefficient (r2), the standard error of estimate (see), and the fischer ratio value (f). the 3d contour maps are represented by field contribution maps using the stdev*coeff field type. 2extq indicates external validation statistics of the model. q2, r2 and 2extq are calculated according to the formulas:26 2 obs pred2 2 obs mean ( ) 1 ( ) y y q y y − = − −   * 1 kcal = 4184 j 3d-qsar of hiv-1 nnrtis 307 2 obs cvpred2 2 obs mean ( ) 1 ( ) y y r y y − = − −   test 2 test=12 ext test 2 trave=1 ( ) 1 ) ii ii y y q ( y y − = − −   where ymean is the average activity value of the entire data set, while yobs, ypred, ycvpred, ytest and ytrave represent observed values, predicted values, cross-validated activity values, test predicted activity values and training predicted activity values, respectively. often, high q2, 2extq and r2 values are considered as strong evidence of high predictive ability of the model. molecular virtual screening topomer search is a fast 3d ligand-based virtual screening tool that can search large libraries of compounds for fragments that are similar to the chemical structures of known lead compounds. in addition to pharmacophoric properties, topomer search uses topomeric fields to compare molecules, and it allows screening for whole molecules, r groups, or scaffolds, using topomer-based similarity.27 the topomer distance was used to estimate the similarity between the query fragment and molecular fragments being screened after 3-d reassembling, with lower values demonstrating greater similarity. this principle is explained as follows: the molecules in the database are incised into fragments, which are compared with the topomer similarity of the r groups of the training molecules. then, the topomer comfa model is used to predict their contributions to activity. finally, a series of r groups is obtained. in this paper, compound 57 with the highest activity was chosen as the basic scaffold, and r1, r2 and r3 groups acted as the query to search in the zinc (2012) database (130,000 compounds) for similar fragments. topomer distance (topdist) was set as 18528 to evaluate the binding degree, and other parameters were defaulted by sybyl-x 2.0. molecular docking research into the mechanism of the interaction between an enzyme and ligands is properly meaningful for the design of new drugs. molecular docking technology can effectively explore the interaction between an enzyme and ligands.29 docking and scoring technology is applied to drug discovery for predicting the biological activity. generally, van der waals forces, hydrogen bonds, hydrophobic interactions, and electrostatic interactions are regarded as the primary factors in a docking study. the molecular docking studies in this work were performed using surflex-dock of sybyl-x 2.0. docking and scoring technology was applied to drug discovery for predicting the biological activity. the crystal structure (pdb code: 1qbt)30 was taken from the rcsb protein data bank.31 the 1qbt was prepared by adding hydrogen and charges, treating the terminal residues and extracting the ligand. next, the 3d molecular model of the biomacromolecule was generated. all the ligands were prepared in accordance with the method used for the training molecules. the number of the maximum output poses was set as 20, and other parameters (additional starting conformations per molecule, angstroms to expand search grid and maximum number of rotatable bonds per molecule) were set, and the sybyl-x 2.0 default values were considered for calculation. the output poses were evaluated by scoring functions, including total score, g-score, d-score, chem -score, pmf-score and c-score32 (consensus score) which reflect the scoring consistency of the five other scores. generally, the higher the total score value is, the greater is the selectivity of the output pose. 308 tong et al. results and discussion topomer comfa statistical results the statistical results obtained from the topomer comfa models are summarized in table ii. the multiple correlation coefficient of fitting (r2), cross-validation (q2) and external validation ( 2extq ) are 0.899, 0.788 and 0.942, respectively. meanwhile, the standard estimated error (see) is 0.271 and the f value is 66.584. when q2 is larger than 0.5 and r2 is greater than 0.6, it is generally believed that the model has statistical significance.33 the biological activity predicted by the topomer comfa model for all the molecules are shown in table iii. the linear regression between the experimental pic50 and the predicted pic50, for both the training set and the test set is shown in fig. 2, where the values are distributed near the 45° diagonal. this is further evidence of the strength of the results stemming from the topomer comfa model. table ii. the statistical results of topomer comfa; n – optimal number of components; q2 – the multiple correlation coefficient of cross validation; r2 – the multiple correlation coefficient of fitting; see – standard estimated error; f – f test value; 2estq – the multiple correlation coefficient of external validation field parameter n q2 r2 see f 2estq se 6 0.788 0.899 0.271 66.584 0.942 table iii. experimental and predicted pic50 values and corresponding residual values for all compounds no. pic50 exp, m pic50 pred, m residual, m no. pic50 exp, m pic50 pred, m residual, m 1 4.23 4.49 0.26 37 5.96 5.97 0.01 2 4.31 4.43 0.12 38 6.10 6.59 0.49 3 4.35 4.41 0.06 39 6.10 5.85 –0.25 4 4.59 4.53 –0.06 40 6.10 5.96 –0.14 5 4.77 5.03 0.26 41 6.10 6.13 0.03 6 4.79 4.61 –0.15 42 6.22 6.20 –0.02 7 4.83 4.51 –0.32 43 6.22 6.16 –0.06 8 4.83 4.77 –0.06 44 6.22 5.99 –0.23 9 5.02 5.05 0.03 45 6.30 6.01 –0.29 10 5.07 5.36 0.29 46 6.40 6.15 –0.25 11 5.09 5.47 0.38 47 6.70 6.85 0.15 12 5.27 5.78 0.51 48 6.70 6.83 0.13 13 5.31 5.98 0.67 49 6.70 6.33 –0.37 14 5.31 5.10 –0.21 50 6.92 6.31 –0.61 15 5.32 5.68 0.36 51 7.00 7.17 0.17 16 5.34 5.44 0.10 52 7.00 7.14 0.14 17 5.37 5.35 –0.02 53 7.05 6.84 –0.19 18 5.42 5.59 0.17 54 7.05 6.80 –0.25 19 5.44 5.34 –0.10 55 7.10 6.99 –0.11 3d-qsar of hiv-1 nnrtis 309 table iii. continued no. pic50 exp, m pic50 pred, m residual, m no. pic50 exp, m pic50 pred, m residual, m 20 5.47 5.60 0.13 56 7.10 6.96 –0.14 21 5.49 5.88 0.31 57 7.30 6.94 –0.36 22 5.52 5.48 –0.04 58 4.35 4.59 0.24 23 5.52 5.75 0.23 59 4.48 4.19 –0.29 24 5.52 5.38 –0.14 60 5.27 5.70 0.43 25 5.54 5.58 0.04 61 5.47 5.50 0.03 26 5.55 5.52 –0.03 62 5.59 5.62 0.03 27 5.59 5.48 –0.11 63 5.60 5.45 –0.15 28 5.60 5.20 –0.4 64 5.62 5.81 0.19 29 5.60 5.55 –0.05 65 5.74 5.56 –0.18 30 5.62 5.36 –0.26 66 5.92 5.51 –0.41 31 5.66 5.45 –0.21 67 6.22 5.74 –0.48 32 5.72 5.34 –0.38 68 6.40 6.01 –0.39 33 5.80 6.13 0.33 69 6.70 6.56 –0.14 34 5.89 6.10 0.21 70 7.05 6.82 –0.23 35 5.94 6.08 0.14 71 7.15 6.82 –0.33 36 5.94 5.99 0.05 72 7.30 6.91 –0.39 4.0 4.5 5.0 5.5 6.0 6.5 7.0 4.0 4.5 5.0 5.5 6.0 6.5 7.0 the training set n=57 the test set n=15 pr ed . p ic 50 exp. pic 50 fig. 2. linear regression between the experimental and predicted pic50 values for the 72 inhibitors. contour maps of the topomer comfa model the 3d-qsar contour maps of the topomer comfa model are shown in fig. 3a–f with compound 57 as the template structure. the topomer comfa results were graphically interpreted by field contribution maps using the “compound filed*coeff” type of field. through different colors, the contour maps display information about various factors that increase or decrease the molecular bioactivitiy generated by steric and electrostatic fields effects. the steric interactions of the r1, r2 and r3 groups are represented by green and yellow contours 310 tong et al. in fig. 3a–c, while the electrostatic interactions of the r1, r2 and r3 groups are denoted by red and blue contours in fig. 3d–f. the green contours represent regions where a large or bulky substituent results in higher activity. the contrary is true for the yellow contours. the red isopleths indicate regions where a negatively charged substituent results in higher activity and the blue isopleths indicate regions where a positively charged substituent results in higher activity. fig. 3. 3d contours generated by the topomer comfa model: a) steric field map of r1; b) steric field map of r2; c) steric field map of r3; d) electrostatic field map of r1; e) electrostatic field map of r2; f) electrostatic field map of r3 (green and yellow contours represent steric favorable and unfavorable regions, respectively. blue and red contours represent regions that favor electropositive and electronegative groups, respectively). as shown in fig. 3a and d, the big green contour at the r1 position indicates that a bulky substituent at this position would increase the activity. in contrast, the electrostatic fields at the r1 position has no effect on activity, which could be seen from the activities of compounds 16 (r1=me) > 11 (r1=h), 39 (r1=me) > > 21 (r1=h) and 65 (r1=me) > 18 (r1=h). in fig. 3b, there is a large yellow contour around the benzene ring of r2 group, indicating that an r2 substituent of greater volume decreases the activity. for example, the inhibitor activity of 15 (r2 = ph) > 1 (r2 = 2-naphthyl), 20 (r2 = 3-me-ph) > 3 (r2 = 1-naphthyl), and 68 (r2 = 2,6-di-cl-ph) > 59 (r2 = 2-naphthyl). in fig. 3e, the large red contour and a small blue contour at the r2 position indicate that introduction of electropositive substitutions at this position decreases the activity. for example, the biological activity of 22 (r2 = 3-f-ph) > 15 (r2 = ph), 49 (r2 = 2,6-di-cl-ph) > > 21 (r2 = 2-cl-ph) and 72 (r2 = 2,6-di-f-ph) > 35 (r2 = 2,6-di-cl-ph). in fig. 3c and f, a large green contour, a small yellow contour, a big blue contour and a small red contour at the r3 position, indicate that a large electropositive substituent at this position could increase the activity. for example, the biological 3d-qsar of hiv-1 nnrtis 311 activity of 31 (r3 = s-cyclohexyl) > 20 (r3 = s-cyclopentyl), 50 (r3 = s-sec-bu) > 36 (r3 = s-n-bu) and 16 (r3 = s-tert-bu) > 12 (r3 = s-sec-bu). molecular screening and molecular design the results of molecular screening using the topomer search technology were further evaluated through the use of topomer distance (topdist) and the contribution values of r-groups (topcomfa_r). in general, an r group with higher priority contribution value was used to replace the r group of the template molecule in the same limit of the topdist. in this study, the most active compound, 57, was chosen as the basic scaffold. five thousand r1, r2 and r3 groups were screened from the drug-like molecules in zinc database using similar fragments. finally, two r2 and seven r3 groups were found that had higher topcomfa_r contributions than the r-groups of the template molecule. in this work, the two r2 groups and seven r3 groups with higher contribution values were employed to substitute alternately for the r2 and r3 of the molecular template. then 14 new molecules were designed. all of the compounds were optimized using the method that had been applied to the training molecules. the topomer comfa model was then used to predict the activities of these new structures. the 14 new compounds and their predicted activities are given in table iv. it can be seen from the data that there are 12 new compounds with activities that are higher than that of the template molecule. close examination of the data in table iv shows that the 12 new compounds have higher activities primarily because of the small volume and electronegativity of substituent at the r2 position of the molecules. moreover, a bulky substituent in r3 contributes to the activity of the 14 new compounds. this observation is consistent with the analysis of the 3d contour of the topomer comfa model. the result that the activities of compounds 2 and 4 are lower than that of the template molecule 57 may be due to one or both of the following causes: a) the mismatch between the r2 and r3 groups and b) unfavorable interactions in the molecule. table iv. structures and predicted pic50 values of the newly designed molecules no. structure predicted pic50 no. structure predicted pic50 1-1a n n nh sn h o oh so hn o 7.36 2-1a hn no oh n hn o s nh o s 7.48 1-2 n n nh s o o oh hn o o 7.26 2-2a hn no oh n hn o so o 7.39 312 tong et al. table iv. continued no. structure predicted pic50 no. structure predicted pic50 1-3a n n nh so o oh hn o o 7.35 2-3a hn no oh n hn o s o o 7.48 1-4 n n nh so o oh hn o 7.21 2-4a hn no oh n hn o s o 7.33 1-5a n n nh so o oh hn o o 7.34 2-5a hn no oh n hn o s o o 7.47 1-6a n n nh so o oh hn o o o 7.32 2-6a hn no oh n hn o s o o o 7.45 1-7a n n nh so o oh hn o o 7.35 2-7a hn no oh n hn o s o o 7.47 acompounds with higher activity than that of the template molecule docking study to validate the docking reliability, the crystal structure of protein (pdb code: 1qbt) with the cognate ligand was redocked. as the reference ligand, the cognate ligand was taken out of its protein–ligand complex (1qbt) and redocked in its binding site. as can be seen from fig. 4a, the redocked ligand and the reference ligand can be almost completely superimposed. their rotational positions are very similar. the result shows that the docking method is rational and reliable. the prototype molecule generated in this docking study is displayed in fig. 4b. in fig. 5, the ligand is represented by sticks, the amino acid residues by lines, and the hydrogen bonds by red dotted lines, and it reveals that the key residues asp25, asp30, gly48, and ile50 in chain a and asp25, asp30, gly48 and ile50 in chain b interact with the inhibitor through hydrogen bonds. considering the location of the cognate ligand as the binding site, the training set molecules and new designed molecules were docked into the receptor. the higher the total score, the better the selectivity of the output pose in the same c-score. therefore, the best output pose of each molecule with full marks in the c-score was selected. the pose of template molecule 57 was chosen to explain 3d-qsar of hiv-1 nnrtis 313 the binding mode between the protein receptor and inhibitors, and the docking results are shown in fig. 6a. as can be seen from fig. 6a, the template molecule forms hydrogen-bonding interactions with ala28, asp29, gly49 and ile50 in chain a of the protein receptor and with gly49 and ile50 in the chain b of the protein receptor. the total score is 7.0064, the crash score is –0.6330 and the polar score is 1.8215. fig. 4. a) superimposition of the redocked reference ligand (the blue stick model) and the reference ligand (the purple stick model); b) the protomol (the green region represents the prototype molecule). fig. 5. the hydrogen-bond interactions (the ligand is represented by sticks, the amino acid residues by lines and the hydrogen bonds dotted lines). in order to illustrate further the binding relationship between the ligands and the protein receptor, the new designed molecules were also subjected to the docking study. the total scores are given in table v. the docking results for molecules 1-4, 1-6 and 2-6, as examples for the newly designed inhibitors, are analyzed in fig. 6b–d. in fig. 6b, compound 1-4 has hydrogen-bonding interactions with asp30, thr31, gly49 and ile50 in chain a, and arg8, gly49 and ile50 in chain b. the total score, crash and polar are 8.1039, –0.8966, 4.0317, respectively. seven active sites (asp25 in chain a, and asp25, ala28, asp29, ile47, gly48 314 tong et al. and gly49 in chain b) of the protein receptor have hydrogen-bonding interactions with the ligand (compound 1-6) in fig. 6c. the total score, crash and polar are 8.5314, –1.1183, 6.5322, respectively. as shown in fig. 6d, hydrogen-bonding interactions between the ligand (compound 2-6) and the protein receptor are generated between asp25 in chain a, and asp25, ala28, asp29 and asp30 in chain b. the total score, crash and polar are 8.8812, –1.3272, 4.9789, respectively. table v. total-score and predicted pic50 of newly designed molecules no. pred. total score no. pred. total score 1-1 7.36 6.8876 2-1 7.48 6.1404 1-2 7.26 7.1161 2-2 7.39 7.5404 1-3 7.35 7.6069 2-3 7.48 6.7080 1-4 7.21 8.1039 2-4 7.33 6.7009 1-5 7.34 7.8423 2-5 7.47 8.0498 1-6 7.32 8.5314 2-6 7.45 8.8812 1-7 7.35 7.9667 2-7 7.47 8.1133 fig. 6. a) the hydrogen-bond interaction between 1qbt and compond: a) 57, b) 1-4, c) 1-6 and d) 2-6 (the red dotted lines represent hydrogen bonding). the results indicate that generally ligands would form hydrogen-bonding interactions with ala28, asp29, gly49 and ile50 of the protein receptor. conclusions a series of s-dabo derivatives as non-nucleoside reverse transcriptase inhibitors were studied by computer-aided drug design processes. the topomer comfa 3d-qsar method generated a model with good internal and external prediction capability from a training set of 57 s-dabo derivatives. a test set of 3d-qsar of hiv-1 nnrtis 315 15 molecules was employed to validate the external predictive ability of the model. the models can be extrapolated to predict novel and more potent inhibitors, and the contour maps obtained from topomer comfa analyses can provide a useful insight for structure-based design for designing new chemical entities with high hiv-1 inhibitory activity. then, 14 new hiv-1 inhibitors were designed adopting the topomer search technology, of which 12 had higher activities than that of the template molecule. for a better understanding of the binding modes of inhibitors at the active site of hiv-1 protein, molecular docking analyses of representative compounds were performed. the docking results indicate that the ligands would generally form hydrogen-bonding interactions with the residues ala28, asp29, gly49 and ile50 of the protein receptor. this study could serve as a basis for the development of hiv-1 nnrtis, and provide a theoretical reference for the synthesis of new drugs. acknowledgements. this work was supported by the national natural science funds of china [21475081], the natural science foundation of shaanxi province of china [2015jm2057] and the graduate innovation fund of shaanxi university of science and technology. we are thankful to dr donald g. barnes for carefully reviewing this manuscript. и з в о д студија молекулским моделовањем инхибитора ненуклеозидне реверсне транскриптазе за hiv-1 користећи 3d-qsar, виртуелни скрининг и симулације докингом jian-bo tong, shang-shang qin, shan lei и yang wang shaanxi key laboratory of chemical additives for industry, shaanxi university of science and technology, xi’an 710021, p. r. china синдром стечене имунодефицијенције (сида) је значајна претња здрављу људи у свету, зато је студија дизајна анти-hiv лекова постала значајан задатак савременог друштва. у овом раду је проведена студија тродимензионалног односа структуре и активности (3d-qsar) на 72 hiv-1 инхибитора ненуклеозидне реверсне транскриптазе (nnrtis) користећи topomer упоредну анализу молекулског поља (topomer comfa). коефицијенти фитовања вишеструке корелације, унакрсне и спољашње евалуације су били 0,899, 0,788, односно 0,942. резултати указују да добијени модел има и повољну стабилност у процењивању и добру способност предвиђања. topomer search је употребљен за тражење одговарајућих r група из zinc базе података, одакле је дизајнирано 14, а за 12 oд нових једињења је предсказано да би била активнија од темплатног молекула. ови резултати снажно сугеришу да је topomer search ефикасан у скринингу и може бити користан водич дизајну нових hiv-1 лекова. лиганди темплатног молекула и новосинтетисана једињења су искоришћени за молекулски докинг у циљу проучавања интеракција ових једињења са протеинским рецептором. резултати показују да ће генерално лиганди формирати водонично-везивне интеракције са остацима ala28, asp29, gly49 и ile50 протеина, чиме се добија додатни увид за дизајн још ефикаснијих лекова. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {hptlc-direct bioautography-guided isolation of isogeranic acid as the main antibacterial constituent of artemisia santonicum essential oil} j. serb. chem. soc. 84 (12) 1355–1365 (2019) udc 582.998.1:665.52/.54+547–32: jscs–5269 615.28–188:615.33 original scientific paper 1355 hptlc-direct bioautography-guided isolation of isogeranic acid as the main antibacterial constituent of artemisia santonicum essential oil jovana stanković1#, miroslav novaković1#, vele tešević2#, ana ćirić3, marina soković3, gordana zdunić4, zora dajić-stevanović5 and dejan gođevac1*# 1institute of chemistry, technology and metallurgy, university of belgrade, njegoševa 12, 11000 belgrade, serbia, 2university of belgrade – faculty of chemistry, studentski trg 12–16, 11000 belgrade, serbia, 3institute for biological research “siniša stanković”, university of belgrade, department of plant physiology, bulevar despota stefana 142, 11000 belgrade, serbia, 4institute for medicinal plant research “dr. josif pančić”, tadeuša košćuška 1, 11000 belgrade, serbia and 5faculty of agriculture, university of belgrade, nemanjina 6, 11080 zemun, serbia (received 13 may, revised 3 august, accepted 7 october 2019) abstract: this study was performed to determine the main antibacterial compounds of the essential oil (ео) of saltmarsh plant artemisia santonicum (asteraceae). the combination of hptlc and direct bioautography was used for the activity guided isolation of isogeranic acid as the main antibacterial constituent with remarkable antimicrobial activity, although it was the minor component of the eo, present only in 0.2 %, as calculated from gc/fid. its structure was determined by 1dand 2d-nmr and gc–ms techniques. antibacterial activity of isogeranic acid against all tested bacteria was significantly higher than eo and even than both controls streptomycin and ampicillin. in further investigation of antibiofilm and antiquorum sensing activity eo exhibited the best inhibition of the biofilm formation at 1/8 minimal inhibitory concentration (mic) and isogeranic acid at 1/2 mic. both eo and isogeranic acid possessed pyocyanin inhibitory activity showing the reduction of pigment at 60.6 and 62.8 %, respectively, at 1/2 mic concentrations. keywords: antimicrobial activity, direct bioautography, antibiofilm, antiquorum potential. introduction genus artemisia belongs to family asteraceae. plants of this genus have long ethnopharmacological tradition and they were used in folk medicines of * corresponding author. e-mail: dgodjev@chem.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc190513106s 1356 stanković et al. various countries. a. absinthium was used as antiparasitic plant and against gastric problems, especially prepared in alcoholic beverages (absinthe products); a. abyssinica as an anthelmintic, antirheumatic and antibacterial agent; a. afra against coughs, bronchitis, asthma, diabetes; a. annua as famous anti-malaric; a. biennis as antiseptic; a. argui as herbal medicine for liver, spleen and kidney; a. campestris as anti-inflammatory, anti-rheumatic and antibacterial agent.1 the most abundant secondary metabolites isolated from artemisia species are coumarins, flavones, flavonols, phenolic acids and sesquiterpenes.2 famous sesquiterpene lactone, artemisinin is by far the best known compound from artemisia species. chinese scientist tu youyou isolated artemisinin from a. annua, found its great antimalarial activity and got nobel prize in 2015. nowadays it is considered promising as anticancer agent.3 there are several reports about antioxidant, antibacterial, antifungal, antimalarial, and antidiabetic activities of different artemisia species. in the previous investigations of a. santonicum essential oil (eo) exhibited antibacterial effect against 24 of 25 bacterial strains, but constituents responsible for this activity have not been discovered yet.4 high performance thin layer chromatography (hptlc) in combination with bioautography is well known technique for the determination of active components, especially antimicrobial components of complex mixtures (extracts and eos).5 quorum sensing (qs) involves biofilm formation, bioluminescence, conjugation and virulence, antibiotic production, competence, conjugation, swarming, motility and sporulation. the biofilm, as well as inter-cellular microbial communication challenge many problems and is a growing threat to global public health throughout the world. the biofilm causes recalcitrant results in blood stream, urinary tract infection sand contamination leading to food spoilage.6,7 one of the possible approaches to treat bacteria-caused diseases is investigation of intra-cellular microbial communication and mechanisms involved in microbial pathogenesis.8,9 many microorganisms are using qs mechanisms in regulation of virulence. the interruption of this bacterial communication gives a possibility of controlling bacterial infections by natural products. the most commonly studied objects are: pseudomonas aeruginosa (pao1), chromobacterium violaceum (cv026), aliivibrio fischeri, escherichia coli, salmonella enterica, acinetobacter sp., aeromonas sp., yersinia enterocollitica, etc.10 pseudomonas aeruginosa is one of the resistant bacteria to antibiotic treatment. it may become multi -drug resistant using quorum sensing to coordinate the formation of biofilms, swarming motility, exopolysaccaride production, virulence, and cell aggregation.11 the aim of the present investigation was to determine antimicrobial, antibiofilm, and antiquorum sensing activity of the a. santonicum eo, find its active components, and isolate them. isolation of isogeranic acid from a. santonicum essential oil 1357 experimental general methods anisaldehyde–sulphuric acid, used as spray reagent, was freshly made according to the literature procedure.12 aqueous 3 % solution of p-iodonitrotetrazolium violet (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride; sigma) was used as spray reagent for bioautography. solvents used for the dry flash chromatography (toluene, ethyl acetate and n-hexane) were freshly distilled. hptlc silica gel 60 f254 aluminum foil plates were obtained from merck. hptlc was conducted with a camag hptlc system (muttenz, switzerland) composed of linomat 5, an adc 2 automatic developing chamber, and a tlc visualiser. gc/ /fid and gc–ms analyses were carried out with an agilent 7890a apparatus equipped with a 5975c msd, fid, and a hp-5msi fused-silica capillary column (30 m×0.25 mm, film thickness 0.25 μm). the oven temperature was programmed from 60 to 315 °c at 3 °c/min then held isothermal for 5 min. injector: 250 °c; fid detector: 300 °c; carrier gas, he (1.0 ml/min at 210 °c); injection volume, 1 μl; split ratio, 3:1. ei-ms (70 ev), m/z range 40–550. compound identification. identification of all compounds in analysis was performed by comparison of their linear retention indices (relative to c8–c36 n-alkanes on the hp-5msi column) and ms spectra with those of authentic standards from nist11 database. all nmr spectra (1h, 13c-dept, cosy, tocsy, noesy, hsqc, hmbc) were recorded on a bruker avance iii 500 spectrometer at 500.26 mhz for 1h and 125.80 mhz for 13c, with chloroform-d as solvent and tms as reference. temperature was 298 k. analytical and spectral data are given in supplementary material to this paper. plant material and isolation of eo the plant material was collected in august 2017 from a saltmarsh in northern serbia, near the city of novi bečej. the plant material was authenticated by prof. zora dajić stevanović and the voucher specimen beou17468 was deposited at the herbarium of the institute of botany and botanical garden “jevremovac”, belgrade, serbia. the eo was isolated by hydrodistillation of the aerial parts of dried a. santonicum using a clevenger-type apparatus for 3h. hydrodistillation of a. santonicum was performed six times (620 g of plant material in total) and eos from all distillations were joined together. the quantity of 1331 mg of the essential oil was obtained in total, representing 0.20 mass %, based on dry plant material. hptlc-direct bioautography hptlc was performed on 10 cm×10 cm aluminum foil backed silica gel 60 f254 plates. eo was dissolved in methanol to give a 100 µl/ml solution. the position of the starting line was 1.50 cm from the left side and 0.80 cm from the bottom. distance between tracks was 1.54 cm. on the plates 10 cm×10 cm, the oil sample aliquots of 2, 4 and 8 µl were applied. after the sample application, the plates were developed in a saturated chamber (adc2 camag) using toluene/ethyl acetate (92:8 volume ratio) solvent system. saturation time was 10 min. time of development was 20 min and separation distance 8 cm. after separation, the mobile phase was removed from the plates by drying under a cold air stream for 4 min. four plates were used for bioautography, and one plate was sprayed with anisaldehyde reagent to mark the bands. the developed plate was dipped into ethanolic anisaldehyde reagent by camag chromatogram immersion device iii and then heated at 100 ºc for 5 min, until the bands became visible. bioautography was performed according to horvath et al.13 the dried plates were sprayed with freshly prepared bacterial suspension (prepared at the day of experiment, 1.0×106 cfu/ml) in nutrient broth (tryptic soy broth; biolife italiana s.r.l., milano, italia). the plates were incubated in a water-vapor chamber for 24 h at 37 °c and then sprayed 1358 stanković et al. with aqueous sol. 3 % of p-iodonitrotetrazolium violet, stored for another 3 h and sprayed with 70 % etoh to stop bacterial growth. microbial growth inhibition appeared as clear zones against a pink background. the rf values of the spots showing inhibition were determined.13 the following gram-negative bacteria were used: escherichia coli (atcc 25922), pseudomonas aeruginosa (atcc 27853), and the following gram-positive bacteria: micrococcus lutens (atcc 10240) and listeria monocytogenes (nctc 7973). the organisms were obtained from the mycological laboratory, department of plant physiology, institute for biological research “siniša stanković”, belgrade, serbia. isolation of isogeranic acid dry-column flash chromatography fractionation of the eo was performed in a sintered glass column (300 mm×32 mm) packed with silica gel (100 g merck, <0.08 mm) using water pump vacuum. the column was equilibrated with 750 ml of the mobile phase toluene/ethyl acetate (70:30 volume ratio) solvent system. the eo (m = 812 mg) was diluted with the mobile phase (1:1) before applying to the column. the elution was isocratic and 25 fractions of around 10 ml were collected. after the hptlc-direct bioautography analysis of eo, the position of the active component (isogeranic acid), i.e., its rf value was determined. ordinary tlc chromatography of all of the fractions from dry flash chromatography revealed the fractions 7 and 8 which contained a spot of the same rf value which exhibited antibacterial activity. these fractions, were joined, evaporated on the rotary vacuum evaporator (ika-werke, rv10, staufen, germany) without heating, and further separated on the second dry-column flash chromatography packed with the silica gel (sio2, <0.08 mm, merck), with the same dimensions as the first one. the elution was gradient, starting with the toluene–ethyl acetate (98:2 volume ratio) and ending with the toluene–ethyl acetate (95:5 volume ratio). forty fractions were collected and the volume of each fraction was 10 ml. the last two fractions, which contained an “active spot”, were grouped and evaporated on the rotary vacuum evaporator without heating, and the final separation was performed on the third dry-column flash chromatography in a sintered glass column (85 mm× 20 mm) packed with silica gel (sio2, <0.08 mm, merck). the elution was isocratic with the mixture of solvents hexane/ethyl acetate (85:15 volume ratio). volume of each fraction was 3 ml, and nineteen fractions where collected. the isogeranic acid (1.15 mg) was obtained from fractions 13–19 after evaporation on the rotary vacuum evaporator without heating. antibacterial activity for the evaluation of antibacterial activity of the essential oil gram-negative bacteria: escherichia coli (atcc 25922), enterobacter cloacae (human isolate), pseudomonas aeruginosa (atcc 27853) and salmonella enterica subsp. enterica serovar typhimurium (atcc 13311), and gram-positive bacteria: listeria monocytogenes (nctc 7973), bacillus cereus (clinical isolate), micrococcus lutens (atcc 10240) and staphylococcus aureus (atcc 11632) were used. the following bacteria: e.coli (atcc 25922), p. aeruginosa (atcc 27853), l. monocytogenes (nctc 7973) and m. lutens (atcc 10240) were tested for the antibacterial activity investigation of isogeranic acid. the antibacterial assay was carried out by a microdilution method14,15 using procedure described by rasheda et al.16 ranges of concentrations of the essential oil and isogeranic acid were 1.99–24.88 mg/ml and 0.05–0.075 mg/ml, respectively. streptomycin (sigma–aldrich s6501) and ampicillin (sigma–aldrich a9393) were used as positive controls (1 mg/ml in sterile physiological saline). isolation of isogeranic acid from a. santonicum essential oil 1359 biofilm formation p. aeruginosa pa01 used in this study is from the collection of the mycoteca, institute for biological research “siniša stanković”, belgrade, serbia. bacteria were routinely grown in luria-bertani (lb) medium (1 % nacl, 1 % tryptone, 0.5 % yeast extract) with shaking (220 rpm) and cultured at 37 °c. the effect of different subinhibitory concentrations of the essential oil (ranging from 1/8 to 1/2 of mic; mic was 4.0 mg/ml) and isogeranic acid (ranging from 1/8 to 1/2 of mic; mic was 0.075 mg/ml) on biofilm forming ability was tested on polystyrene flat-bottomed microtitre 96 well plates as described by mileski et al.17 briefly, 100 µl of overnight culture of p. aeruginosa (inoculum size was 108 cfu/ml) was added to each well of the plates in the presence of 100 µl sub-inhibitory concentrations (sub-mic) of extracts (1/2, 1/4 and 1/8 mic) or 100 μl medium (control). after incubation for 24 h at 37 °c, each well was washed twice with sterile pbs (ph 7.4), dried, stained for 10 min with 0.1 % crystal violet in order to determine the biofilm mass. after drying, 200 µl of 95 % ethanol was added to solubilize the dye that had stained the biofilm cells. the excess stain was washed off with distilled h2o. after 10 min, the content of the wells was homogenized and the absorbance at λ = 625 nm read on a sunrise™-tecan elisa reader (mannedorf, switzerland). the experiment was done in triplicate and repeated two times and values were presented as a mean value ± se. inhibition of twitching and flagella motility of p. aeruginosa inhibition of twitching and flagella motility of p. aeruginosa was determined by the procedure of mileski et al.17 the sub-mic concentration of essential oil and isogeranic acid (2.00 and 0.0375 mg/ml) was mixed into 10 ml of molten mh (mueller–hinton) agar medium and poured immediately over the surface of a solidified lb agar plate as an overlay. the plate was point inoculated with an overnight culture of pao1 once the overlaid agar had solidified and was incubated at 37 °c for 3 days. the extent of swimming was determined by measuring the area of the colony.18 the experiment was done in triplicate and repeated two times. the colony diameters were measured three times in different direction and values were presented as a mean values ± se. inhibition of synthesis of p. aeruginosa pa01 pyocyanin overnight culture of p. aeruginosa pa01 was diluted to od 600 nm 0.2. inhibition of synthesis of p. aeruginosa pa01 pyocyanin was determined by the procedure of mileski et al.17 the oil and isogeranic acid were added in sub-mic concentrations (2.00 and 0.0375 mg/ml) to p. aeruginosa and incubated at 37 °c for 24 h. absorbance of the extracted organic layer was measured at 520 nm using a shimadzu uv1601 spectrophotometer (kyoto, japan).18 the experiment was done in triplicate and repeated two times. the values for optical density were presented as a mean value ± se. results and discussion chemical composition of the essential oil the chemical composition of the isolated eo was established by gc–ms/ /fid. the relative amounts of the components are shown in table s-i (supplementary material). the number of 75 compounds were characterized, corresponding to 90.1 % of the total. oxygenated monoterpenes were identified as the major class of compounds (67.5 %). among the oxygenated monoterpenes the most abundant were 1,8-cineole (18.8 %), chrysanthenone (13.3 %), cis-thujone 1360 stanković et al. (8.4 %), trans-sabinyl acetate (3.3 %), and camphor (3.3 %). monoterpene hydrocarbons were present in amount of 8.2 %, sesquiterpene hydrocarbons 5.6 %, oxygenated sesquiterpenes 3.3 %, normonoterpenes 2.6 %, homomonoterpenes 1.0 %, and aromatics 1.9 %. camphor and cis-thujone were previously reported as major constituents of a. santonicum eo.4,19 hptlc-direct bioautography hptlc-direct bioautography was performed in order to find the antibacterial constituents of the eo. the mobile phase toluene–ethyl acetate (92:8 volume ratio) was chosen, and hptlc separation on silica gel plates was performed. hptlc-direct bioautography on gram-negative e. coli and p. aeruginosa, and gram-positive l. monocytogenes and m. lutens exhibited a white zone on violet background with the rf value of 0.08 using aqueous sol. 3 % of p-iodonitrotetrazolium violet for visualization (fig. 1). another hptlc plate was visualized using anisaldehyde reagent, where the active component appeared as a pink colored band. the rf value and the color of the band was taken into account for further bioactivity-guided isolation. fig. 1. hptlc of the essential oil treated with: a – anisaldehyde and hptlc-direct bioautography: b – e. coli, c – p. aeruginosa, d – l. monocytogenes and e – m. lutens. isolation and identification of isogeranic acid the a. santonicum eo was fractionated by three consecutive dry-column silica gel flash chromatographies, using toluene/ethyl acetate and hexane/ethyl acetate solvent systems as mobile phases. isogeranic acid as an active component (rf 0.08), was isolated and its structure was elucidated using 1h, 13c-dept and 2d-nmr (figs, s-2–s-7 and table s-ii, supplementary material) and gc–ms. noesy correlations revealed z configuration (fig. 2). gc/fid chromatograms of the eo and pure isogeranic acid are given in fig. 3. the content of isogeranic acid in the eo was found to be 0.2 %, according to gc/fid. isogeranic acid is a rarely investigated compound, only found in few eos.20,21 isolation of isogeranic acid from a. santonicum essential oil 1361 fig. 2. the structure of isogeranic acid with the noesy correlations. fig. 3. a – gc/ms data; b – gc/fid; c – ei/ms spectrum of isogeranic acid. antibacterial activity of the essential oil and isogeranic acid antibacterial activity of the eo, isogeranic acid and known antibiotics used as controls are presented in table s-iii of the supplementary material. eo showed moderate antibacterial activity in the range of 2–25 mg/ml for mic, with the most pronounced effect on b. cereus and p. aeruginosa, while pure isogeranic acid exhibited strong antibacterial activity against all tested bacteria, in the range 0.05–0.075 mg/ml, higher than streptomycin and ampicillin (table s-iii). anti-qs activity of the essential oil and isogeranic acid on swimming plates, the motile strain pao1 was used as the 100 % standard (control) for motility, while the petri dishes with the same strain plus oil and isogeranic acid were compared with the control.22 inhibition of the biofilm formation. the mic values of the eo and isogeranic acid against p. aeruginosa were determined by the microdilution method (4.00 and 0.075 mg/ml, respectively). the effect on biofilm formation of p. aeruginosa was tested at lower values than the mic, i.e., 1/2 mic, 1/4 mic and 1/8 mic. a concentration of 1/8 mic of the eo allowed 42.54 % of inhibition of biofilm formation, while 1/4 mic allowed activity of 1.52 % inhibition of biofilm 1362 stanković et al. formation. concentration of 1/2 mic did not influence biofilm formation. a concentration of isogeranic acid of 1/2 mic exhibited the best activity with 46.77 % of inhibition of biofilm formation. according to the obtained data a concluding remark could be highlighted: eo and isogeranic acid exhibited non-dose dependent activity on biofilm formation at subinhibitory concentrations, but certain activity undoubtedly exists. both controls ampicillin and streptomyin exhibited the best activity for 1/8 mic (table s-iv, supplementary material). finally, isogeranic acid exhibited higher potential for inhibition of the biofilm formation than eo and lower than both positive controls. twitching and flagella motility. tested oil and isogeranic acid completely reduced the twitching motility of p. aeruginosa. the normal colonies of p. aeruginosa, were flat with a rough appearance displaying irregular colony edges (fig. 4e) and a hazy zone surrounding the colony. p. aeruginosa solution with oil and isogeranic acid was incapable of producing such a twitching zone and had almost round, smooth, regular colony edges, the flagella were completely reduced both in size and in numbers (fig. 4a and b). ampicillin and streptomycin reduced the flagellae completely (fig. 4c and d). the eo, isogeranic acid and antibiotics also influence the white color of colony, in contrast with green control. fig. 4. light microscopy of colony edges of p. aeruginosa in twitching motility plates, grown in the presence or absence of a. santonicum essential oil and isogeranic acid; magnification: 100×. the eo and isogeranic acid provoked lower colony diameter (11.00 and 10.67 mm, respectively) than control of p. aeruginosa untreated (29.34 mm). ampicillin and streptomycin reduced colony diameter in higher content (8.33 and 6.00 mm) (table s-v, supplementary material). the bacterial colonies grown in the presence of 1/2 mic of oil and isogeranic acid; p. aeruginosa colony in the presence of ampicillin (1/2 mic) had a reduced protrusion; p. aeruginosa colony in the presence of streptomycin with a reduced protrusion; p. aeruginosa produced a flat, widely spread, irregularly shaped colony in the absence of eo and isogeranic acid. pyocyanin production. tested eo and isogeranic acid showed pyocyanin inhibitory activity. the oil showed a reduction of pigment at 60.6 %, isogeranic acid 62.8 % while ampicillin and streptomycin decreased pyocyanin in higher isolation of isogeranic acid from a. santonicum essential oil 1363 amount (32.5 and 23.8 %) in comparasion to untreated control (79.3 %, fig. 5, table s-vi, supplementary material). these values for the reduction of pigment refer to 1/2 mic concentrations (mg/ml). fig. 5. reduction of the pyocyanin production of p. aeruginosa pao1 by a. santonicum essential oil, isogeranic acid, streptomycin and ampicillin tested at 1/2 mic (mg/ml). conclusion activity guided isolation from the eo of saltmarsh plant artemisia santonicum (asteraceae) revealed isogeranic acid as a minor, but highly active ingredient with remarkable antimicrobial, antibiofilm, and antiquorum activities. its antibacterial activity against all tested bacteria was significantly higher than eo and even than both controls streptomycin and ampicillin. both eo and isogeranic acid possess pyocyanin inhibitory activity. supplementary material additional data are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. the authors acknowledge their gratitude to the ministry of education, science and technological development of serbia for financial support, project nos. 172053, 173032 and tr 31089. 1364 stanković et al. и з в о д hptlc директно-биоаутографски вођено изоловање изогеранилне киселине као главне антибактеријске компоненте етарског уља artemisia santonicum joвaнa сtaнkoвић1, mирoслaв нoвakoвић1, вeлe teшeвић2, aнa ћирић3, maринa сokoвић3, гордана здунић4, зора дајић-стевановић5 и дејан гођевац1 1институт за хемију, технологију и металургију, универзитет у београду, његошева 12, 11000 београд, 2хемијски факултет, универзитет у београду, студентски трг 12–16, 11000 београд, 3институт за биолошка истраживања “синиша станковић”, универзитет у београду, одељење за физиологију биљака, булевар деспота стефана 142, 11000 београд, 4институт за медицинска истраживања “др јосиф панчић”, тадеуша кошћушка 1, 11000 београд и 5пољопривредни факултет, универзитет у београду, немањина 6, 11080 земун ово истраживање је спроведено у циљу одређивања главних антибактеријских компоненти етарског уља слатинске биљне врсте artemisia santonicum (asteraceae). комбинација hptlc методе и методе директне биоаутографије је коришћена за активношћу вођено изоловање изогеранилне киселине као главне компоненте са значајном антимикробном, антибиофилм и антикворум активношћу иако је била присутна у етарском уљу са само 0,2 %, израчунато из gc/fid. њена структура је одређена 1d и 2d nmr и gc–ms техникама. антибактеријска активност изогеранилне киселине на све тестиране бактерије је била значајно боља од етарског уља, и од обе контроле, стрептомицина и ампицилина. етарско уље је показало најбољу инхибицију формирања биофилма у концентрацији 1/8 минималне инфибиторске концнетрације (mic вредности), а изогеранилна киселина у концентрацији 1/2 mic вредности. и етарско уље и изогеранилна киселина су имале пиоцијанин инхибиторну активност показавши смањење пигмента на 60,6 и 62,8 %, у концентрацији 1/2 mic вредности, редом. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {arsenic removal from water using a one-pot synthesized low cost mesoporous fe-mn modified biosorbent} j. serb. chem. soc. 84 (3) 327–342 (2019) udc 504.4.054:628.161.2:546.19+546.72’711: jscs–5187 66.081.000.57 original scientific paper 327 arsenic removal from water using a one-pot synthesized low-cost mesoporous fe–mn-modified biosorbent jasmina nikić1, malcolm watson1#, aleksandra tubić1#, marijana kragulj isakovski1#, snežana maletić1#, emilijan mohora2 and jasmina agbaba1*# 1university of novi sad, faculty of sciences, department of chemistry, biochemistry and environmental protection, trg dositeja obradovića 3, 21000 novi sad, serbia and 2singidunum university, faculty of applied ecology futura, požeška 83a, 11030 beograd, serbia (received 9 august, revised 8 november, accepted 13 november 2018) abstract: this paper investigates the removal of arsenic from water using an environmentally friendly modified biosorbent, chitosan coated with fe–mn binary oxide (chit-femn), simply prepared with an one-pot low-cost procedure by simultaneous oxidation and coprecipitation. the sorbent was characterized by sem, eds, xrd, ftir, bet specific surface area, and point of zero charge (phpzc) measurements. the kinetic data fitted a pseudo-second order model for both as(iii) and as(v), suggesting chemical adsorption on the sorbent surface and that intra-particle diffusion is not the only rate-limiting step during adsorption. the adsorption isotherms were best fit to the freundlich model, and the non-monolayer adsorption model for arsenic on chit-femn is therefore proposed. below ph 9, the effect of ph on as(iii) and as(v) removal by chit-femn was insignificant, with as removals remaining above 85 %. cland no3 had negligible influences on as(iii) and as(v) removal, whereas po4 3-, sio3 2-, co3 2and so4 2were observed to compete with arsenic species for adsorption sites. the adsorbent was successfully applied to remove arsenic from real arsenic contaminated groundwater samples to below 10 µg l-1 suggesting that chit-femn is a promising candidate for the low cost removal of both as(v) and as(iii) during drinking water treatment. keywords: adsorption; chitosan; mechanism; competitive ions; groundwater. introduction arsenic is considered as one of the most hazardous elements for living organisms and its presence in natural waters used as drinking water resources can cause serious public health problems. arsenic contamination of groundwater has been reported in many countries around the world including serbia.1 it is well *corresponding author. e-mail: jasmina.agbaba@dh.uns.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc180809099n 328 nikić et al. -known that long term consumption of arsenic polluted drinking water may cause cancer and other related diseases. therefore, the world health organization (who) recommends a maximum allowable concentration (mac) for arsenic in drinking water of 10 µg l–1. this value has been endorsed by the european union and other health organizations,2 and is also applicable in the republic of serbia. however, many poorer countries still follow the earlier limit of 50 µg l–1.3 arsenic can be removed from water through oxidation, coagulation, adsorption, membrane technology and ion exchange technologies.4 however, adsorption technologies are regarded as the most suitable techniques because the process is inexpensive, simple to operate, safe to handle and can be used at different scales ranging from household modules to community plants.5 different adsorbents are available for arsenic removal, however iron based adsorbents are still widely in use, because of their low cost and high affinity for arsenic. moreover, in recent years iron oxides have been modified in order to get simultaneous oxidation of as(iii) and adsorption of as(v).6–8 in particular, fe-mn binary oxide materials have attracted a lot of attention as they combine the oxidation potential of mno2 to convert as(iii) to as(v) and the adsorption capacity of iron oxy-hydroxides, therefore providing simultaneous removal of both arsenic species.9–11 however, most of these composite materials are in the form of powders and cannot be separated easily from aqueous solution which make the process difficult to apply. in addition, these fine powders cannot be used in columns due to their low hydraulic properties. thus, in order to overcome such problems some authors have immobilized powdered fe–mn binary oxide onto different carriers. chang et al.12 incorporated fe–mn binary oxide onto diatomite (fmbo–diatomite) and the resultant sorbent showed high oxidation and adsorption ability for as(iii). in other work, anion exchanger-supported fe–mn binary oxide was also shown to have great potential in as(iii) removal from contaminated waters.13 rye et al.14 reported the synthesis of granular activated carbon (gac)–femn and used batch studies to investigate its potential for as(iii) and as(v) removal. given the continued need to solve the problems with arsenic in drinking water sources as efficiently and economically as possible, the search for suitable adsorbents continues. in this work, we use the inexpensive, biodegradable and nontoxic biopolymer chitosan as a carrier for fe–mn binary oxide and apply a simple one-pot method to prepare one such sorbent, chit-femn, for arsenic removal from water. batch experiments and ftir analyses were applied to study the as(iii) and as(v) adsorption mechanisms and capacities, and the influence of ph and the effect of competitive ions was also investigated. finally, the local arsenic contaminated drinking water source was treated with chit-femn to demonstrate its real life applicability. arsenic removal from water using fe–mn biosorbent 329 experimental materials medium molecular weight chitosan was obtained from sigma aldrich. all chemicals and reagents used in this study were of analytical grade. ferric sulphate (feso4·7h2o, poch poland s.a) and kmno4 (centrohem, serbia) were used for the adsorbent preparation. stock solutions of as(iii) and as(v) of 1 g l-1 were prepared by dissolving as2o3 and as2o5 (obtained from alfa aesar, thermo fisher scientific) in deionized water. deionized water was astm type i, with a conductivity of less than 0.056 µs cm-1, a ph value of 7.3 and a toc < 0.5 mg l-1. adsorbent preparation the chitosan coated with fe–mn binary oxide was prepared in a single one-pot process, after modification of the method proposed by zhang et al.15 the key modification was in the application of chitosan as a carrier for the fe–mn binary oxide. 10 g of raw chitosan was added to 200 ml kmno4 solution (0.015 m), prior to spraying 200 ml feso4 solution (0.045 m) into the kmno4 solution while stirring vigorously. 5 m naoh solution was added simultaneously to maintain the solution ph between ph 7.0 and 8.0. the suspension was continuously stirred for 1 h and stabilised at room temperature for 24 h. the suspension was then filtered, washed several times with fresh ultra-pure water to neutral ph and dried at 110°c for 4 h. the dried sorbent was crushed and stored in a desiccator before use. adsorbent characterization scanning electron microscopy (sem) images were obtained by hitachi tm3030 (japan) and the elemental contents were analyzed by energy dispersive x-ray spectroscopy (eds). x-ray diffraction (xrd) patterns were obtained by philips pw automated x-ray powder diffractometer (usa). the specific surface area was obtained by nitrogen adsorption using the brunauer–emmett–teller (bet) method and the mesopore and micropore volumes were determined using the bjh and t-test methods, using a quantachrome autosorb tmiq surface area analyzer. the point of zero charge (phpzc) was determined according to the method described by zhang et al.16 fourier transform infrared (ftir) spectra of sorbents before and after arsenic adsorption were recorded by infrared spectrophotometer (ftir nexus 670, thermonicolet, usa). samples for ftir determination were ground with spectral grade kbr in an agate mortar. adsorption experiments arsenic adsorption experiments were performed in 40 ml glass bottles containing 20 ml aqueous arsenic solution with adsorbent doses of 0.5 g l-1. the solution ph was adjusted to 7.0±0.2 by adding 0.1 m hno3 and/or naoh; ionic strength was adjusted to 0.01 m with nano3. the suspensions were shaken on an orbital shaker at 180 rpm at 22±1 °c. adsorption kinetics were studied with fixed as(iii) or as(v) concentrations (0.5 mg l-1) at specific time intervals: 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 7, 9, 12, 18 and 24 h. adsorption isotherms were obtained by varying the initial arsenic concentration (0.1–1 mg l-1). the effect of solution ph on the arsenic adsorption was investigated with a fixed as(iii) or as(v) concentration (0.2 mg l-1) by varying the initial ph from 3 to 11, and the release of fe and mn to the final solutions was also determined. to investigate the influence of coexisting anions such as phosphates, silicates, carbonates, sulphates, chlorides and nitrates, their corresponding sodium salts were introduced into the as(iii)/as(v) solutions. similarly, humic acid at concentrations of 4–12 mg c l-1 was used to study the influence of natural organic matter (nom). in all batch tests, after adsorption, the samples were filtered through a 0.45 µm cellulose membrane filter and 330 nikić et al. the concentration of arsenic in the filtrate was determined by atomic absorption spectrophotometry (aas). chemical analysis ph measurements were carried out using an inolab ph/ion 735 instrument. arsenic concentrations were determined by graphite furnace atomic absorption spectrophotometry (perkin elmer aanalyst 700) according to epa method 7010.17 the practical quantitation limit (pql) for the arsenic measurements was 0.5 µg l-1. concentrations of iron and manganese were determined by flame atomic absorption spectrophotometry (perkin elmer aanalyst 700) according to epa method 7000b.18 the pql for fe and mn were 0.068 and 0.034 mg l-1, respectively. dissolved organic carbon (doc) contents were analysed after filtration through a 0.45 µm membrane filter on an elementar liquitocii, using pt catalysed combustion at 850 °c to oxidize the carbon, in accordance with the standard method sprs iso 8245:2007.19 the pql of the method was 0.5 mg c l-1. results and discussion adsorbent characterization the sem images of chit and chit-femn are given in fig. s-1 of the supplementary material to this paper. the surface of chit-femn is covered with a large number of tiny fe–mn binary oxide aggregates, while the surface of chit features relatively smooth larger granules. this indicates that fe–mn binary oxide was well dispersed on the chitosan surface, with the high degree of dispersion increasing the number of active sites on the chitosan which are available for adsorption. the elements present on the surface of chit and chit-femn were investigated by eds analysis (fig. s-2 of the supplementary material). the chitosan surface consists of carbon, oxygen and nitrogen at 48.68, 29.34 and 21.98 mass %, respectively (fig. s-2a). after modification, fe and mn are distributed on the surface and the oxygen content increased up to 41.88 %, establishing the presence of fe–mn binary oxide particles on the chit surface. the analysis of the surface of chit-femn revealed that the fe and mn is present at an fe/mn mole ratio of about 2.81, very close to the desired 3:1 ratio (fig. s-2b). the bet surface area of the synthesized chit-femn was 1.99 m2 g–1, almost 4 times higher than the non-impregnated chit (0.58 m2 g–1). this increase is in agreement with the sem imagery where the finer structures on the surface of chit-femn are readily apparent, and are similar to results obtained in other studies, whereby gupta et al.20 found that the bet surface area of chitosan coated with iron flakes (icf) was 1.44 m2 g–1, while the specific surface area of iron doped chitosan granules was found to be 1.48 m2 g–1.21 furthermore, the mesopore volume of the chit-femn sorbent was approximately 3 times higher (0.014 cm3 g–1) than that of chitosan (0.005 cm3 g–1), with the t-test method showing there are no micropores on chit or chit-femn. the results of the t-test method and the pore diameters of chit and chit-femn (15.5 and 14.2 nm) mean that according to the iupac classification22 they may be considered as mesoporous arsenic removal from water using fe–mn biosorbent 331 adsorbents (pore diameter in the range 2 to 50 nm). the points of zero charge of chit and chit-femn were ph 7.3 and 5.8, respectively. x-ray diffraction patterns of chit and chit-femn, in the 2θ range of 10–80°, are given in fig. s-3 of the supplementary material. the most significant difference observed between the two xrd patterns is the decreasing crystallinity of the compound after modification with fe–mn binary oxide. the xrd pattern of chitosan shows typical crystalline diffraction peaks at 2θ = 19.9°23 which weaken and become wider after modification indicating a non-crystalline nature and suggesting the fe–mn oxide particles are in a more stable amorphous form.15,16 this is in agreement with neto el al.24 who reported that diffractograms of chitosan beads crosslinked with glutaraldehyde (ch -cl) present a much lower crystallinity index when chitosan beads are used for immobilization of fe(iii) (ch-fecl). adsorption kinetics the adsorption kinetics of as(iii)/as(v) on chit and chit-femn are shown in fig. 1a and b. it can be seen that the adsorption process is time dependent and can be divided into two steps. in the first step of the process, adsorption of as(iii)/as(v) onto chit and chit-femn is fast due the large number of available sites on the sorbent surfaces. the adsorption rate is slower in the subsequent step because the concentration gradients reduce due to the accumulation of as ions on the surface. equilibrium of as(iii) and as(v) on chit was reached after 12 h (720 min, fig. 1a). in the case of adsorption of as(iii) and as(v) on chit-femn, equilibrium was reached after 18 h (1080 min, fig. 1b). fig. 1. adsorption kinetics fitted with pseudo-first and pseudo-second order for as(iii) and as(v) adsorption on: a) chit and b) chit-femn. initial as(iii)/as(v) concentration: 0.5 mg l-1, sorbent dose: 0.5 g l-1, ph 7.0±0.2. 332 nikić et al. in order to elucidate the adsorption mechanism, pseudo-first, pseudo-second order and elovich models were initially used to fit the kinetic data.25 fit quality was determined using the coefficient of determination (r2) and the sum of the squared errors (sse) to calculate the error deviation between the experimental and predicted data, for linear analysis (table i). table i. kinetic parameters for the adsorption of as(iii) and as(v) onto chit and chit-femn adsorbents model parameter chit chit-femn as(iii) as(v) as(iii) as(v) pseudo-first order parameters k1 / min -1 0.00169 0.00641 0.00389 0.00392 qe / µg g -1 239 124 807 457 sse 0.307 2.29 4.61 5.51 r2 0.9537 0.7456 0.8936 0.8766 pseudo-second order parameters k2 / min -1 0.0000205 0.0000927 0.00000671 0.0000288 qe / µg g -1 308 725 840 833 sse 0.274 0.183 0.0137 0.00161 r2 0.9990 0.9642 0.9963 0.9996 elovich α / µg g-1 min-1 0.00160 0.113 0.000487 0.0122 β / µg g-1 56 40.9 158 112 sse 2351 10853 2082 10243 r2 0.9710 0.9516 0.9827 0.9678 for sse, a value closer to 0.0 indicates the model has a smaller random error component and that the fit is thus useful for prediction. in our work, lower sse values and good coefficients of determination were observed for the second order kinetic model, for both as(iii) and as(v) adsorption, indicating that the as(iii) and as(v) sorption on chit-femn may be due to chemisorptions.14 since the pseudo-second-order model well describes the reaction kinetics, it is limited in accuracy by considering the adsorption as a single, one-step binding process. however, the intraparticle diffusion model can provide a more comprehensive view of adsorption as a series of distinct steps. this model was therefore also employed to fit the kinetic data and the corresponding parameters of this model are present in tables s-i of the supplementary material. according to the model, if intraparticle diffusion is the rate-controlling step, the plot of qt (sorption capacity at time t) against t0.5 should be linear and pass through the origin. from fig. 2, it can be clearly observed that none of the plots in their entirety pass through the origin, indicating that the mechanism of intraparticle diffusion is not the only limiting step of the adsorption process and that other interaction mechanisms must be acting simultaneously. moreover, at each graph qt vs. t0.5, we observe two or three linear section with different gradients, which can be attributed to certain processes involved in the arsenic adsorption. the first segment (the initial steep part of the curves) corresponds to the rapid diffusion of arsenic through arsenic removal from water using fe–mn biosorbent 333 the water matrix to the outer surface of the adsorbent (external diffusion). the second segment describes a gradual adsorption in which intraparticle diffusion limits the rate of the adsorption process. the third section, with the smallest slope, corresponds to the state of adsorption equilibrium, when the speed of the adsorption process is significantly slowed down due to the lower residual concentration of the arsenic. thus, the rapid adsorption of arsenic from an aqueous solution corresponding to external diffusion and slower arsenic adsorption, caused by intraparticle diffusion, limits the entire adsorption process on the synthesized adsorbents. similar observation was reported in study neto et al.24 fig. 2. intraparticle mass transfer plot for: a) as(iii) and b) as(v) adsorption on chit and chit-femn. initial as concentration: 0.5 mg l-1, sorbent dose: 0.5 g l-1, ph 7.0±0.2. adsorption isotherms in order to evaluate the adsorption capacities of chit and chit-femn for as(iii) and as(v), the equilibrium data were fitted with the freundlich (fig. 3) and langmuir models.26 fig. 3. freundlich sorption isotherms for: a) as(iii) and b) as(v) adsorption on chit and chit-femn. initial as concentration: 0.1–1 mg l-1, sorbent dose: 0.5 g l-1, ph 7.0±0.2. 334 nikić et al. the values of the model parameters determined by linear regression analysis are presented in table ii. table ii. freundlich and langmuir isotherm parameters for the adsorption of arsenic on chit and chit-femn model parameter chit chit-femn as(iii) as(v) as(iii) as(v) freundlich kf / µg g -1 (µg l-1)n 0.000366 1.034 61.6 63.8 1/n 2.499 1.21 0.655 0.798 sse 0.0926 0.0455 0.00689 0.00638 r2 0.9574 0.9499 0.9031 0.9912 langmuir qmax / µg g -1 880 1225 3914 3893 kl / l µg -1 0.00000542 0.0000571 0.0164 0.0187 rl 0.99-1.00 0.95-0.99 0.06-0.38 0.05-0.35 sse 1.221 1.013 0.0518 0.0211 r2 0.7234 0.8862 0.7094 0.9883 the freundlich isotherm exhibited higher coefficient of determination (r2) and lower sse values than the langmuir model for both as(iii) and as(v). the better fitting of the adsorption data to freundlich isotherm (fig. 3) indicated that the adsorption of both arsenic species preferably followed multilayer and heterogeneous adsorption process. the sem images of chit and chit-femn showed the chit and chit-femn having rough structure development, therefore adsorbent was non -homogeneous. the kf values from the freundlich model show the much higher affinity of chit-femn for both as(iii) and as(v) in comparison with chit. this confirms the fact that the impregnation of fe–mn binary oxide onto chitosan significantly increases the arsenic adsorption capacity of chitosan. the 1/n values are less than 1, suggesting that the chemisorption of both arsenic species was the predominant adsorption process onto chit-femn. in contrast, for the raw chitosan, the values of the freundlich exponent 1/n for as(iii) and as(v) were 1.12 and 3.21, indicating that the physical adsorption is favoured.27 according to the langmuir isotherm model, the maximum adsorption capacities, qmax, for chit were 0.88 and 1.22 mg g–1 for as(iii) and as(v), respectively, with much higher qmax values observed (3.91 and 3.89 mg g–1 for as(iii) and as(v), respectively) for chit-femn. the adsorption capacities reported in this current work are an improvement on the work of gupta et al.20 who found that the adsorption capacity of iron-doped chitosan granules (icb) was 2.32 and 2.24 mg g–1 for as(iii) and as(iii), respectively. other authors have coated chitosan with a variety of different metals, including molybdate, titanium dioxide and iron(iii)28–32 these authors also reported comparable adsorption capacities, ranging from 2.0 to 6.5 mg g–1 for as(iii) and from 0.43 to 2.1 mg g–1 for as(v). the values of rl, an essential parameter of langmuir isotherm, calculated at different initial concentrations of as(iii) and as(v), were all between 0 and 1 arsenic removal from water using fe–mn biosorbent 335 (table ii), indicating that the adsorption onto chit-femn is favourable for both as(iii) and as(v). effect of solution ph the ph of the aqueous solution, the most important parameter in adsorption studies, strongly affects the adsorption properties of iron-based sorbents. hence, the influence of solution ph (from 3 to 11) on the removal of as(iii) and as(v) by chit-femn was investigated and the results are presented in fig. 4. fig. 4. effect of initial solution ph on as(iii) and as(v) removal by chit-femn, initial as concentration: 0.5 mg l-1, sorbent dose: 0.5 g l-1. the adsorption of arsenate was effective over the ph range 3–8, with removals not falling below 85 %. however, removal efficacy dropped upon further ph increases. a similar trend was observed for as(iii) adsorption. the results can be explained by the point of zero charge (phpzc) of the sorbent and the degree of protonation of arsenate in aqueous solution, which is a function of ph. the dissociation constants of aqueous arsenate are pka1 = 2.3, pka2 = 6.8 and pka3 = = 11.6, resulting in arsenate species varying from h2aso3-, haso32-and aso33 when the ph increases from acidic to alkaline (i.e., ph 3–11 in this work). the phpzc of the synthesized chit-femn was determined to be ph 5.8. it is well known that solid surfaces are positively charged at ph below their phpzc and negatively charged at ph above their phpzc.33 hence at ph < phpzc as(v) adsorption was promoted due to electrostatic attraction between the positively charged adsorbent surface and anionic as(v) species. however, the adsorption of as(v) decreased at ph > phpzc due to the electrostatic repulsion between the negatively charged adsorbent surface and negatively charged arsenic species. the removal of as(iii) was very similar to as(v), remaining almost constant at better than 85 % removal in the ph range 3–8, then decreasing with further increases above ph 8. the pk1 value of h3aso3 is 9.2, indicating that as(iii) exists as a neutral species at ph < 9.2 and as anions at ph > 9.2. the lower removal above ph 8 is therefore to be expected, given the electrostatic repulsion from the neg336 nikić et al. ative charge of the chit-femn surface. below ph 8, in the absence of favourable electrostatic interactions, the good removal of as(iii) may be due to the oxidation reaction of as(iii) to as(v) and sorption by iron oxide.33,34 the oxidation of as(iii) to as(v) by mn oxides has been shown to be favourable at lower ph.13 furthermore, as discussed below, the oxidation of as(iii) in the presence of manganese dioxide is accompanied by the reductive dissolution of mno2, which leads to the release of mn2+. these cations are then adsorbed on the sorbent surface, giving it a positive charge which facilitates the adsorption of arsenate oxyanions in a neutral or alkaline environment.16,33,34 effect of competitive ions in general, ions such as phosphates, bicarbonates, silicates, sulphates, chlorides and nitrates can coexist with arsenic in groundwaters, and may interfere with arsenic removal via competitive binding or complexation. the effect of these anions on the adsorption of as(iii) and as(v) by chit-femn sorbents was examined at three different concentration levels (0.1, 0.5 and 1 mm) and the results are given in fig. 5. fig. 5. effect of coexisting anions on: a) as(iii) and b) as(v) removal by chit-femn; initial as concentration: 0.5 mg l-1, sorbent dose: 0.5 g l-1, ph 7.0±0.2. the presence of chlorides and nitrates had only a slight influence on as(iii) and as(v) removals. po43– caused the greatest percentage decrease in arsenic removal at each concentration level, followed by sio32–, co32– and so42–. increasing the po43– concentration from 0.1 to 1 mm reduced as(iii) removals from 92 to 51 % (fig. 5a). similarly, the as(v) uptake by chit-femn was reduced from 95 to 55 % (fig. 5b). for sio32–, an obvious decrease in arsenic removal was observed at 1 mm. as(iii) and as(v) removals by chit-femn decreased from 92 to 59 % and from 95 to 60 %, respectively. the arsenic, silicate and arsenic removal from water using fe–mn biosorbent 337 phosphate oxyanions can all form inner-sphere complexes with the hydroxyl groups on the surface of adsorbents.35 thus the lower arsenic removals in the presence of phosphates and silicates can be attributed to the competition between po43– or sio32– and arsenic for adsorption sites. the decrease in as(iii) and as(v) adsorption in the presence of co32– may result from the formation of arseno–carbonate complexes, including as(co3)2–, as(co3)(oh)2– and asco3+, which would prevent arsenic from forming inner-sphere complexes on the adsorbent surface.36 the negative effect of the higher so42– concentration (1 mm) on as(iii) and as(v) removal by chit-femn may be attributed to the competition for active sites on the sorbent surface. in contrast, chloride and nitrate can only form outer-sphere complexes with ferric hydroxides, explaining the insignificant effect of these anions on arsenic removal. hence, the order of interference for arsenite and arsenate adsorption on chit-femn for the anions studied is phosphates > silicates > carbonates >sulphates >nitrates > chlorides. effect of humic acid natural organic matter, which is not only present everywhere in natural aquatic and soil environments but also highly reactive toward metals and surfaces, may interfere with the arsenic adsorption on the surfaces of minerals and therefore increase the mobility of arsenic. the effect of the presence of humic acid (4 to 12 mg doc l–1) on arsenic sorption by the chit-femn binary oxides was therefore investigated and the results are given in fig. s-4 of the supplementary material. humic acid at the concentrations of up to 12 mg l–1 had no obvious influence on as(v) removal. however, increasing the concentration of humic acid from 0 to 12 mg l–1 decreased the percentage removal of as(iii) by chit-femn from 93 to 70 %. ftir analysis a ftir spectroscopic analysis was carried out to investigate the adsorption mechanism of as(iii) and as(v) onto chit-femn. the ftir spectra of chit-femn before and after sorption with as(iii)/as(v) are shown in fig. 6. all spectra exhibited bands at 3376 and 3434 cm–1, which is characteristic of the –oh and –nh stretching vibrations. peak at 2920 cm–1 could be attributed to –ch stretching vibration in –ch and –ch2. the peaks at 1634, 1540 and 1320 cm–1 are attributed to stretching vibration (amide-i), n-h stretching vibration (amide-ii) and the c–n stretching vibration, respectively. the peak at 1384 cm–1 is due to symmetrical deformation of methyl (ch3) groups.37 the peak at 1070 cm–1 corresponds to the co stretching vibration in –coh. after reaction with as(v), this peak disappears and a new peak appeared at 825 cm–1, corresponding to stretching vibration of as–o–fe groups. this indicates that the as(v) is bound as a surface complex and not as a precipitated solid phase.38 similarly, after the adsorption of as(iii), a small band at 825 cm–1 appeared. this peak can be ascribed as an as–o stretching vib338 nikić et al. ration, which is characteristic of as(v) adsorption. thus, this observation suggests that the part of the initial as(iii) present was first oxidized to as(v) and then adsorbed onto the surface of the fe–mn oxides.39 fig. 6. ftir spectra of chit-femn before and after as(iii) and as(v) adsorption. in oxidative adsorption process for as(iii) 1m of mno2(s) can oxidize 1 m of as(iii), and subsequently releases 1 m of mn2+ into the aqueous phase.39 therefore, to further confirm the oxidation of as(iii) on chit-femn, the release pattern of soluble mn2+ from chit-femn was determined after reaction with as(iii) and as(v) at different solution ph. the concentration of dissolved iron was below the maximum allowed concentration (mac) in all cases, indicating that the chit-femn is stable under the conditions investigated (ph 3 to 11). in the case of as(v) adsorption, the release of mn was also low (a maximum concentration of 0.098 mg l–1 at ph 3). however, the significant amounts of mn were released after as(iii) adsorption, up to 1.82 mg l–1 at ph 3. the greater release of manganese after as(iii) adsorption can be attributed to the reductive dissolution of mno2 during as(iii) oxidation.33,34 above ph 7, the release of mn and fe was not observed. xu et al.40 suggest that at this ph, the redox reaction which oxidizes as(iii) does not progress far enough to generate excess mn2+, with the as(iii) adsorbing directly onto the adsorbent without oxidation. the data presented above suggests that the mechanism of arsenic adsorption proceeds by the ligands exchange to form inner sphere complexes. the results for the ph dependence and the impact of ionic strength (nitrates investigated as competitive anions) show very little variation, which is suggestive of inner sphere complexes, according to goldberg and johnston41 and maliyekkal et al.42 while goldberg and johnston41 also reported that although arsenate forms inner sphere complexes with fe oxides, arsenite can form both inner and outer sphere complexes, and the leaching of mn from our sorbent during adsorption of as(iii) arsenic removal from water using fe–mn biosorbent 339 confirms the oxidation of as(iii) prior to adsorption,33,34 such that even during as(iii) removal, the mechansim involves the adsorption of as(v). finally, kong et al.43 investigated the adsorption of arsenic on a similar sorbent, nanoscale fe–mn binary oxides loaded on zeolite, and also proposed a ligand exhange mechanism forming inner-sphere comlexes for both as(iii) and as(v). applicability of the adsorbent to naturally arsenic contaminated groundwater groundwater used for water supply from kikinda, serbia (table s-ii of the supplementary material), was treated with chit-femn in order to investigate its potential to remove arsenic from naturally contaminated water sources. the initial as concentration in this water is 35 µg l–1, but it contains relatively high concentrations of natural organic matter, phosphate and sulphate, and has a high ph of 8.29. despite the presence of these competitive anions and the unfavourable ph conditions, a chit-femn sorbent dose of 1.0 g l–1 was sufficient to satisfy the who drinking water standards, indicating the strong suitability of this sorbent for full scale water treatment applications. conclusion chitosan coated with fe–mn binary oxide, an environmentally friendly biosorbent, was prepared in a single step process by the simultaneous redox and co-precipitation method. the kinetic study indicates that the adsorption of as(iii) and as(v) on chit-femn can be well described by the pseudo-second-order kinetic model. maximal adsorption capacities of chit-femn for as(iii) and as(v) was 3.91 and 3.89 mg g–1, respectively. the surface complexation is the major mechanism for as(iii) and as(v) removal by chit-femn. the removal efficiencies for both as(iii) and as(v) were not significantly affected over a broad ph range (removals ranged from 99 to 88 % between ph 3 and 8), indicating that the sorbent is suitable for use in all typical groundwaters. chit-femn also performed well with above 80 % removals for both as(iii) and as(v) in the presence of less than 8 mg doc l–1. the presence of chlorides, nitrates, and sulphates had only a slight influence on as(iii) and as(v) removal, whereas the presence of phosphates, silicates and carbonates significantly reduced the removal of both as(iii) and as(v), especially at high concentrations. results show that this modified biosorbent has the potential for application in the treatment of arsenic contaminated water, with investigations under dynamic conditions necessary in the future. supplementary material additional data are available electronically at the pages of journal website: http:// //www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. the authors gratefully acknowledge the support of the ministry of education, science and technological development of the republic of serbia (project no. iii43005 and project no. tr37004). 340 nikić et al. и з в о д уклањање арсена из воде применом биосорбента модификованог fe–mn бинарним оксидом јасмина никић1, malcolm watson1, александра тубић1, маријана крагуљ исаковски1, снежана малетић1, емилијан мохора2 и јасмина агбаба1 1универзитет у новом саду, природно–математички факултет, департман за хемију, биохемију и заштиту животне средине, трг доситеја обрадовића 3, 21000 нови сад и 2универзитет сингидунум, факултет за примењену екологију футура, пожешка 83а, 11030 београд у раду је приказано уклањање арсена из воде применом биосорбента (chitosan), који је модификован са fe–mn бинарним оксидом. модификација fe–mn бинарним оксидом (chit-femn) изведена је у једном степену, симултаним процесима оксидације и копреципитације. новосинтетисани сорбент је карактерисан различитим техникама, укључујући sem/eds, xrd, ftir и bet анализу и одређивање тачке нултог наелектрисања. сорпционо понашање as(iii) и as(v) на синтетисаном сорбенту испитивано је кинетичким и равнотежним експериментима, у шаржном систему. установљено је да модел псеудо -другог реда најбоље описује кинетику адсорпционог процеса оба облика арсена на chit -femn, а да само унутарчестична дифузија није ограничавајући корак адсорпционог процеса. максимални адсорпциони капацитети chit-femn за as(iii) и as(v) одређени ленгмировим моделом износили су 3,91 и 3,89 mg g -1 , редом. промена ph вредности (од 3 до 8), није у већој мери утицала на степен уклањања as(iii) и as(v) (>85 %), указујући да је сорбент прикладан за примену у подземним водама, без додатне корекције ph вредности. међу испитиваним компетитирајућим јонима, фосфати, а затим силикати и карбонати, су испољили највећи инхибиторни утицај на сорпцију as(iii) и as(v) на chitfemn, док утицај хлорида, нитрата и сулфата није био од већег значаја. испитивања спроведена на реалним узорцима подземне воде указују на то да се применом модификованог биосорбента концентрација арсена може смањити испод максимално дозвољене концентрације од 10 μg l -1 . 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {porous amphiphilic biogel from facile chemo-biosynthetic route} j. serb. chem. soc. 85 (3) 353–367 (2020) udc 544.7+577.11:542.913.000.57:539.24 jscs–5306 original scientific paper 353 porous amphiphilic biogel from a facile chemo-biosynthetic route syed mohammad daniel syed mohamed1, m. suffian m. annuar1*, thorsten heidelberg2, nor faezah ansari3 and nor hidayah ismail4 1institute of biological sciences, faculty of science, university of malaya, 50603, kuala lumpur, malaysia, 2department of chemistry, faculty of science, university of malaya, 50603, kuala lumpur, malaysia, 3department of biotechnology, kulliyyah of science, international islamic university malaysia, bandar indera mahkota, kuantan, 25200, pahang, malaysia and 4research cluster office, institute of research management and services, university of malaya, 50603, kuala lumpur, malaysia (received 25 june, revised 5 september, accepted 16 september 2019) abstract: grafting of medium-chain-length poly-3-hydroxyalkanoates (mcl -pha) with glycerol 1,3-diglycerolate diacrylate (gdd) in acetone was performed using benzoyl peroxide as the initiator. a detailed mechanism scheme provides significant improvement to previous literature. radical-mediated grafting generated α–β carbon inter-linking of mcl-pha and gdd, resulting in a macromolecular structure with gel properties. the thermal properties of the copolymer for different graft yields were investigated as a function of initiator concentration, gdd monomer concentration, incubation period and temperature. the water absorption and porosity of the gel were significantly improved relative to neat mcl-pha. keywords: biogel; chemo-biosynthetic; biopolymer; radical grafting. introduction polyhydroxyalkanoates (pha) are well-known biopolymers with attractive biocompatibility. they are accumulated within certain bacterial species in the form of granules when the microorganisms experience imbalanced growth conditions, viz., simultaneous excess carbon source and limitation of nutrients such as nitrogen and phosphorus.1–4 two categories of pha could be differentiated, i.e., short-chain-length polyhydroxyalkanoates or scl-pha, comprising of monomers with fourto five-carbon atom length, and medium-chain-length polyhydroxyalkanoates or mcl-pha, made up of 6to 14-carbon atom length monomers.2 modification and functionalization of pha, intended for tuning the features, are * corresponding author. e-mail: suffian_annuar@um.edu.my https://doi.org/10.2298/jsc190625101s ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 354 syed mohamed et al. important for certain applications. functionalization of pha on the side chain, for example, can alter the polymer interaction behaviour by introducing elements of hydrophilicity.5 one of the functionalization techniques is grafting. graft copolymerization of pha forms a modified segmented copolymer with interesting properties, particularly in terms of wettability and thermo-mechanical strength. the grafting processes can be realised in several ways, including chemical, radiation, and plasma discharge methods.6–8 the current grafting methods for many polymers are equally applicable in the case of pha functionalization. for example, benzoyl peroxide-initiated graft polymerization of 2-hydroxyethylmethacrylate (hema) onto poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (phbhv) enhances the crystallinity and wettability of the biopolymer.9 grafting applies radical intermediates during the reaction that mediate the polymerization of a vinyl monomer as grafted branches on the main polymer chain through “grafting onto”, “grafting through” and “grafting from” techniques.7,10 free radical grafting via radical initiators is widely used for the modification of polymers. benzoyl peroxide is one of the initiators extensively used to functionalize bacterial polyesters.7,9,11–15 it is a simple and robust method. benzoyl peroxide has been reported to be more efficient compared to other common thermal initiators, such as azo-bis-isobutyronitrile (aibn)16 and other peroxyl initiators.17 when the concentration of the propagating radical balances the rate of radical termination, polymers with high number average molecular weight and low dispersity can be obtained. the catalyst determines the equilibrium constant between the active and dormant species, which in turn determine the polymerization rate.18,19 in this study, medium-chain-length poly-3-hydroxyalkanoates (mcl-pha), obtained from bacterial fermentation, was graft copolymerized with glycerol 1,3 -diglycerolate diacrylate (gdd) by free radical polymerization. the grafted product, pha-g-gdd, was prepared through a thermal incubation process with all reaction components mixed together in a selected organic solvent. the effects of different initiator concentrations, in this case benzoyl peroxide (bpo), were investigated alongside incubation temperature and time, and initial gdd concentration. the pha-g-gdd copolymer was characterized and a mechanism of the grafting reaction was proposed. experimental materials lauric acid (c12h24o2, mw 200.23, cas 143-07-7) for synthesis was purchased from merck as a sole carbon source in fermentation medium to produce medium-chain-length poly -3-hydroxyalkanoates (mcl-pha) by pseudomonas putida bet001.20,21 the mcl-pha was obtained by solvent extraction and purified through repeated methanol precipitation and washing steps.21 ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ radical grafting of mcl-pha 355 benzoyl peroxide (bpo) (c4h10o4, mw 242.23, cas 94-36-0), (with 25 % h2o, used as received) for synthesis was purchased from merck millipore (darmstadt, germany) and applied as the sole radical initiator of the grafting reaction. glycerol 1,3-diglycerolate diacrylate (gdd, c15h24o9, mw 348.35, cas 60453-84-1) was purchased from sigma–aldrich (saint louis, ca, usa) and used as received. monomer composition of mcl-pha the monomer content of the mcl-pha was determined by gas chromatography. a sufficient amount of the sample was subjected to methanolysis. two milliliters of dichloromethane (dcm) and 2 ml mixture of methanol and sulphuric acid (1:1 volume ratio) were added to the sample. the mixture was incubated at 100 °c for 2 h and 20 min in a heating block. distilled water was added, and the mixture vortexed for about one minute before being left standing overnight or at least four hours for phase separation. the organic bottom layer containing the methylated products was transferred into a vial and mixed with sodium sulphate (na2so4) to remove excess water. the organic layer was auto-injected into a fused silica capillary column (30 m length×0.32 mm internal diameter×0.25 μm film, supelco spbtm-1, bellefonte, pa, usa) fitted within a gas chromatography machine (trace gc ultra, thermo scientific, rodano, milan, italy) with flame ionization detector. during the process, helium was used as a carrier gas at the rate of 48.3 ml min-1 and 0.41 bar pressure.21 four types of monomers, i.e., 3-hydroxyhexanoate (3hhx), 3-hydroxyoctanoate (3ho), 3-hydroxydecanoate (3hd) and 3-hydroxydodecanoate (3hdd), at 4, 37, 38 and 21 mol %, respectively, hence, alternatively known as p(3ho-co-3hhx-co-3hd-co-3hdd) were identified and quantified in the mcl-pha samples. preparation of pha-g-gdd copolymers copolymers were prepared by incubation of 50 g dm-3 mcl-pha and 0.14 mol dm-3 gdd in acetone at varying concentrations of bpo. oxygen was purged from the solution with nitrogen gas for ten minutes and the vial was subsequently capped to introduce airtight conditions. the mixture was incubated within a heating block at 70 and 90 °c. the mixture was left to cool to ambient temperature (25±1 °c) post-incubation before adding methanol to allow precipitation of the gel product, and separate the non-grafted gdd monomer and gdd homo -oligomers in the soluble fraction at the same time. successful grafting was indicated by the increase in the mass of precipitated product over the initial mass of mcl-pha used (eq. (1)),9,11,13,14 calculated as follows: graft yield f i i 100 w w w − = (1) where wf is the final mass of the grafted pha after reaction, and wi is the initial mass of pha before reaction. characterisation of the pha-g-gdd copolymers fourier transform infrared-attenuated total reflectance (ftir-atr) spectroscopy. ftir -atr was used to record the spectra on perkin–elmer spectrum 400 ft-ir and ft-nir spectrometer (perkin–elmer inc., wellesley, ma, usa), equipped with pike gladiatr hovering monolithic diamond atr accessory (pike technologies inc., usa) at room temperature. the samples were placed on the monolithic diamond atr probe and fastened against the diamond crystal plate using a force adaptor. the spectra were recorded between 4000 and 450 cm-1 using cuts of 0.5 cm×0.5 cm films. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 356 syed mohamed et al. simultaneous thermal analysis (sta) and differential scanning calorimetry (dsc). the applied thermal analysis was destructive simultaneous thermal analysis (sta) of astm -e2550-11 thermal stability method. the machine used was a perkin–elmer sta 6000 (perkin–elmer inc., wellesley, ma, usa) running on tandem differential scanning calorimetry (dsc) and thermogravimetric analysis (tga). the sample was prepared in the form of a film. the analysis was programmed to initialize from 30 °c until 800 °c at a rate of 10 °c min-1 under a nitrogen gas stream of 10 ml min-1. proton nuclear magnetic resonance (1h-nmr). a sufficient quantity of grafted sample was washed with deuterated chloroform (cdcl3). the solvent was filtered to separate the undissolved component using a borosilicate glass syringe equipped with 0.22 μm polytetrafluoroethylene (ptfe) disposable filter (11807–25; sartorius stedim, gӧttingen, germany). the filtrate fraction with the dissolved component was subjected to nmr analysis. the spectrum was acquired using a jeol jnm-gsx 270 ft-nmr spectrometer (joel, tokyo, japan) at 400 mhz against tetramethylsilane (tms) as internal reference. for mcl-pha, the sample was simply dissolved in cdcl3 and filtered. carbon solid-state nuclear magnetic resonance (13c-nmr). a sufficient quantity of undissolved grafted sample was washed with chloroform to remove the residue, dried and pulverized for solid-state analysis. the spectrum was acquired using a jeol jnm-ecx500 nmr spectrometer (joel, tokyo, japan) at 500 mhz. water absorption and porosity studies. the samples were cut into small cubes with measured height, length and breadth, and then immersed in deionised water overnight. for the porosity study, the cubes were immersed in 95 % ethanol solution for an hour before being left overnight immersed in deionised water.11,20,22 gravimetric measurement was used to record the weight change for each of the samples in order to determine the degree of swelling from water absorption (eq. (2)). to calculate the porosity of the samples (eq. (3)), the solvent replacement method was used. the calculations involved are as follows: degree of swelling w d d 100 w w w − = (2) porosity w d100 w w vρ − = (3) where wd is the weight of the sample before immersing (dry), ww is the weight of the sample after immersing (wet), v is the volume of the sample and ρ is the density of the solvent used, in this case, denatured 95 % ethanol, which is 790 kg m-3. results and discussion characterisation of pha-g-gdd copolymers ftir-atr spectroscopy. ester vibrations were detected for both the individual mcl-pha and gdd samples. the carbonyl absorptions were observed at 1726 and 1718 cm–1, respectively, while the corresponding c=o bond signals appeared at 1162 and 1189 cm–1. in addition, the vibration signalling wavelength for symmetric –ch2– of the samples were observed at 2858–2855 cm–1, and 2926–2925 cm–1 for asymmetric –ch3. no asymmetric –ch3 signalling was detected in the pure gdd samples (fig. 1). ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ radical grafting of mcl-pha 357 fig. 1. ftir spectra of grafted materials obtained from reaction mixture at 70 (left) and 90 °c (right). a & g – neat mcl-pha; b & h – pha-g-gdd with 3.0×10–6 mol dm–3 bpo; c & i – pha-g-gdd with 5.0×10–6 mol dm–3 bpo; d & j – pha-g-gdd with 10–5 mol dm-3 bpo; e & k – pha-g-gdd with 1.5×10–5 mol dm–3 bpo; f & l – gdd monomer. in the grafted copolymer, a signal from the presence of a hydroxyl group introduced by the gdd monomers was evident as the copolymers exhibited new broad signals of –oh group shifted to 3432–3431 cm–1. in addition, a strong shift at about 1151 cm–1 signifying ester bond signal, available in both mcl-pha and gdd monomer, was present in abundance for the grafted product samples compared to the neat mcl-pha samples. ether bond presents exclusively in pure gdd samples was also evidenced with a strong signal at about 1091 cm–1. it can be concluded that grafting of mcl-pha with gdd was successful. 1h-nmr. a representative 1h-nmr spectrum for the grafted products is shown in fig. 2. the spectrum shows all typical signals for neat pha. the signal indicating a successful grafting process was found between 1.8 and 1.9 ppm. the signal was assigned to overlapping signals of methine and methylene hydrogen atoms (ch) and (ch2) on the backbone of polyacrylate, labelled as h and i. the peak also signifies the hydrogen atoms at the grafting position of pha, reflecting α and β positions to the carbonyl of the terminal fatty acid of pha, labelled as f and g in fig. 2, respectively. additionally, another new signal of interest was evi________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 358 syed mohamed et al. dent between 3.7 and 3.8 ppm. this is associated with hydrogens from methylene and methine groups of the triglycerol core of gdd, labelled as j and k. fig. 2. 1h-nmr spectrum for pha-g-gdd copolymer. a broad signal, overlapping with the chloroform peak at 7.3 ppm (labelled as z), most likely indicates the presence of a benzene ring, originating from the phenyl radical, which is generated by the initiator bpo and attaches to the very first monomer during the polymerization process. extensive cross-linking in the copolymer, due to the divalent structure of gdd, leads to a gel-like material, which is insoluble in the solvent applied for recovery and purification in this study i.e. methanol. however, some of the grafted mcl-pha copolymer could be dissolved in hydrophobic solvent, such as chloroform, and subsequently investigated for nmr studies. this separated component represents a fraction of the grafted copolymer with a relatively lower molecular weight from less extensive cross-linking, thereby providing an organic solvent–soluble material for structural authentication. 13c-nmr. solid-state 13c-nmr authentication was also performed since the major fraction of the grafting product exhibited gel-like morphology, which is virtually insoluble in any organic solvent. however, the signal resolution was rather poor with several combined peaks especially related to the aliphatic components of both mcl-pha and gdd. nevertheless, an additional signal of minute ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ radical grafting of mcl-pha 359 amount of carbonyl ketone groups was observed at around 210 ppm on the spectrum (see supplementary material to this paper, fig. s-5), indicating oxidation of secondary alcohol hydroxyl group within gdd, occurring as a side reaction due to the cascading radical reaction environment. elaboration on the spectrum signals is made available in the supplementary material. mechanism of mcl-pha grafting with gdd from structural studies, a detailed grafting reaction mechanism was proposed, which presented a significant improvement to previous literature.11,13 while the proposed reaction scheme still follows a typical three-step radical polymerization, which includes initiation, propagation and termination phases, it introduces a thorough revision of participating reactive components in the reaction. firstly, the grafting process involving mcl-pha requires an unsaturated terminal monomer unit, referring to a double bond between αand β-carbon atoms, most likely originating from thermal degradation of mcl-pha. there are several possibilities to introduce the double bond via elimination processes, as corroborated by other similar studies23,24 and shown in fig. 3. fig. 3. possible routes of mcl-pha degradation that contribute to alkenyl end groups able to participate in grafting reaction via proton abstraction. grafting of mcl-pha with gdd to obtain pha-g-gdd copolymer starts with the dissociation of bpo into a benzene radical. it is proposed that the radicals start the initiation step by attacking the β-carbon of the double bond to introduce a radical on the α-carbon. the reaction is possible at either end of gdd molecules. in the process, the benzene ring is covalently bonded to the monomer to become part of the terminal monomer unit of the copolymer. although initiation could also occur at the unsaturated terminal fatty acid of mcl-pha, the probability is small owing to low concentration density of mcl-pha molecules compared to the acrylate monomer. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 360 syed mohamed et al. nevertheless, grafting reaction with mcl-pha does not hinder the propagation of the vinyl polymerization (fig. 4). fig. 4. initiation and propagation mechanism scheme involving species with alkenyl reactive group r groups that are available within the biogel. the chain process of the polymerization is continuously repeated to produce a growing macromolecule consisting of a mixture of gdd monomers, acrylate monomers and mcl-pha with random connection pattern. the cascade of growing copolymer consistently assembles radicalized α-carbon to be bound to β-carbon of monomers, thereby transferring the radical to the α-carbon of the newly attached monomer.25 concurrently, the same process is presumably occurring at the other end of gdd monomers as well. the bivalent nature of gdd, thereby, is giving rise to a cross-linked copolymer. this cross-linking affects the properties of the resulting gel, accounting for almost zero solubility in aqueous solution and ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ radical grafting of mcl-pha 361 a rigid shape. further complexity of the macromolecule arose from hydroxyl groups in gdd monomer being exposed to dehydration to form carbonyl groups, albeit in minute amount due to the radical transfer propagation process. termination step occurs when two different growing macroradical copolymers react with each other, thereby losing their radical character that is associated with unpaired electrons. termination by disproportionation is more likely than combination of radicals, as shown in fig. 5. fig. 5. termination step. during the grafting process, gdd monomers may also react with each other to form a densely cross-linked homopolymer gel following the same mechanism that applies for the grafting process. owing to the divalent character of gdd and the nature of the vinyl polymerization, a high molecular weight is achieved. the crosslinking converts most of the monomers into a highly crosslinked gel, which consists of only a few interwoven polymer networks. the separation of these is practically impossible. however, gdd-polymers with low crosslinking, owing to incorporation of substantial contents of mono-valent acrylates, may be separated from grafted mcl-pha based on their solubility in methanol, while highly crosslinked gels and polymers containing higher portions of mcl-pha are expected to form a precipitate. in general, the grafted material may comprise of mcl-pha with varying degrees of grafting component based on different reaction parameters, depending ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 362 syed mohamed et al. on the initiator concentration, temperature and the concentration of gdd monomer itself. thermal properties of pha-g-gdd copolymer thermal properties of pha-g-gdd samples were determined using tga and dsc analyses. the grafted samples showed changes in terms of their thermal degradation behaviour, fig. 6. a b c d fig. 6. tga analysis for pha, pha-g-gdd and gdd samples for different initiator concentrations, a & b weight percentage curves, and c & d derivative weight percentage curves. a and c represent samples incubated at 70 °c, while b and d represent samples incubated at 90 °c. neat pha samples degraded earlier at around 260 °c compared to the other samples (fig. 6a–d). the gdd sample was the most stable among all, with higher degradation temperature at around 300 °c. the thermal curves of pha-g -gdd samples from 90 °c incubation temperature were more similar to the gdd curve (fig. 6), indicating that these samples were more stable compared to those incubated at 70 °c (fig. 6). the shapes of the curves were closely related due to molecular composition similarity. from dsc analysis, the tm of the grafted ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ radical grafting of mcl-pha 363 samples was in the range of 53.0 – 55.5 °c, which was slightly higher compared to neat mcl-pha at 52.7 °c, due to the presence of gdd monomer (table i). table i. thermal properties of pha and pha-g-gdd for different initiator concentrations and incubation temperature, initial rate of reaction, graft yield percentages after two hours incubation, water uptake swelling and porosity percentages tincub / °c sample initial rate of reaction, % min-1a thermal analysis graft yield, % water uptake (swelling), % porosity, % tm / °c td / °c mcl-pha – 52.7 294.4 – ~ 0 ~ 0 70 bpo, mol dm-3 pha-g-gdd 3.0×10-6 1.0 55.5 297.9 72 ± 5 2.4 ± 0.2 7 ± 1 5.0×10-6 1.8 53.0 305.3 73 ± 11 4.6 ± 0.2 9 ± 1 10-5 2.6 53.7 298.1 77 ± 2 8.2 ± 1.1 10 ± 1 1.5×10-5 3.0 54.5 292.8 90 ± 9 6.6 ± 0.1 11 ± 1 90 bpo, mol dm-3 pha-g-gdd 3.0×10-6 4.7 53.5 295.6 73 ± 4 6.5 ± 0.9 12 ± 1 5.0×10-6 4.8 54.7 295.0 86 ± 6 7.2 ± 1.0 10 ± 2 10-5 4.6 53.8 291.6 88 ± 3 7.3 ± 0.4 10 ± 1 1.5×10-5 4.4 53.9 298.7 85 ± 3 9.7 ± 0.5 8 ± 1 amaximum standard deviation ±10 % generally, the thermograms were only slightly different from each other, since the grafting parameters, viz., mcl-pha concentration and gdd concentration were the same throughout the sample preparation reaction. on the other hand, the increase in initiator concentration contributed to the increase in graft yield (table i), hence resulting in more thermostable functionalized product with higher gdd concentration that were successfully grafted to the mcl-pha backbone. the findings also agreed with the results from tga analysis (fig. 6). in terms of water absorption ability, the grafted samples exhibited increased in swelling percentage with higher initiator concentration used, although grafted samples from lower incubation temperature showed slightly lower water absorption. neat pha material showed negligible water absorption due to its strong hydrophobicity. for samples obtained from grafting reaction at 70 °c and different initiator concentrations, approximately similar porosity percentages were determined except at the lowest concentration of bpo used (table i) attributed to lower degree of grafting. for grafted samples from 90 °c incubation, the increase in starting radical initiator concentration resulted in lower porosity percentages (table i). neat pha material showed no evidence of porosity. it is suggested that the increase in initial concentration of bpo and incubation temperature may have contributed to more extensive grafting of pha that resulted in lower porosity percentages of the resulting materials. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 364 syed mohamed et al. effects of starting initiator concentration, incubation temperature and time the grafting reaction of mcl-pha with gdd made use of bpo as the sole initiator. as seen in fig. 7a, at 70 °c incubation, the graft yield (%) increased with increasing starting bpo concentration. a similar trend was observed in fig. 7b for an incubation temperature of 90 °c. at an incubation temperature of 70 °c, the rate of increase in graft yield was gradual for lower starting concentrations of initiator, i.e., 3.0×10–6 and 5.0×10–6 mol dm–3. when the initial concentration was increased to 1.0×10–5 and 1.5×10–5 mol dm–3, a steep increase in the graft yield with time was observed (fig. 7a). on the other hand, when incubation temperature was at 90 °c, similar fast rates of graft yield were observed for all used starting initiator concentrations (fig. 7b). nevertheless, for both temperatures, the grafting reaction eventually reached a plateau indicating termination of reaction following exhaustion of grafting sites and/or depletion of the radical initiator. a fig. 7. graft yield as a function of incubation time for: a) 70 and b) 90 °c incubation temperature. initial bpo concentrations at 3.0×10-6, 5×10-6, 1.0×10-5 and 1.5×10-5 mol dm-3 for both temperatures. b effects of initial gdd concentration the plots in fig. 8 show the graft yields for different gdd concentrations as a function of time. the initiator reaction is considered a fast one and hence, is expected to enter the termination phase as the concentrations of the radicals ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ radical grafting of mcl-pha 365 and/or reactive sites start to deplete. with higher initial gdd concentration, higher graft yields were also observed. a similar trend was evident for different starting initiator concentrations, as shown in fig. 7. fig. 8. graft yield as a function of incubation time for different gdd concentrations at 70 °c incubation. initial mcl-pha and bpo concentrations were 50 g l-1 and 10-5 mol dm-3, respectively. initial rate of reaction the initial rates of grafting at two different temperatures, i.e., 70 and 90 °c, for different starting initiator concentrations are given in table i. at 70 °c, the initial rate was increasing gradually from 1.0 to 3.0 % min–1 as bpo concentration was increased, suggesting that the bpo dissociation has yet to reach its maximum level. however, at 90 °c, the initial rate of reaction was almost constant within a narrow range of 4.4 to 4.8 % min–1 for all initial bpo concentrations studied (table i). it is suggested that at this temperature, generation of radical initiator from its parent molecule was relatively faster than at 70 °c and hence the higher initial rate of grafting. this is supported by the fact that half-life of bpo is one hour at 91 °c. conclusions the graft copolymerization of mcl-pha with gdd was successfully performed using benzoyl peroxide as the sole initiator. elucidation of its mechanism indicates that both species could be incorporated into the same backbone of mcl -pha polymer consisting of α–β carbon linkages due to the random nature of radical polymerization involved. the grafted product yields an amphiphilic copolymer with improved wettability, thus potentially refining its facility for cellular interaction. in addition, grafting of mcl-pha to yield p(3ho-co-3hhx-co -3hd-co-3hdd)-g-gdd adds to the available repertoire of functional materials. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 366 syed mohamed et al. acknowledgment. this work was supported by the universiti malaya under grant no. br008-2015. и з в о д порозни амфифилни биогелови добијени једноставним био-синтетским поступком syed mohammad daniel syed mohamed1, m. suffian m. annuar1, thorsten heidelberg2, nor faezah ansari3 и nor hidayah ismail4 1institute of biological sciences, faculty of science, university of malaya, 50603, kuala lumpur, malaysia, 2department of chemistry, faculty of science, university of malaya, 50603, kuala lumpur, malaysia, 3department of biotechnology, kulliyyah of science, international islamic university malaysia, bandar indera mahkota, kuantan, 25200, pahang, malaysia и 4research cluster office, institute of research management and services, university of malaya, 50603, kuala lumpur, malaysia реакција калемљења глицерол-1,3-диглицерoлат-диакрилата (триглицерол-диакрилата) (gdd) на поли(3-хидрокси-алканоат) средње дужине бочних алкил ланаца (mcl-pha) је извођена у раствору ацетона у присуству бензиол-пероксида, као иницијатора. механизам реакције калемљења на полимерне ланце је детаљно приказан и знатно побољшан у односу на претходно описане у литератури. слободним радикалима иницирано калемљење омогућава међусобно повезивање mcl-pha и gdd молекула преко �α–β� угљеничне везе, при чему настаје умрежена структура полимера са својствима гела. термичка својства кополимера са различитим садржајем калемљених грана су анализирана у зависности од концентрације иницијатора и gdd мономера, као и времена извођења реакције полимеризације и температуре. показано је да су апсорпција воде и порозност синтетисаних гелова знатно повећани у поређењу са полазним полимерном, mcl-pha. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {novel silver(i) compounds with 1-adamantanamine} j. serb. chem. soc. 83 (6) 699–705 (2018) udc 549.753.312+546.571:543.422.25:57–188 jscs–5105 original scientific paper 699 novel silver(i) compounds with 1-adamantanamine dejan jeremić1#, milena đorđević2, srđan miletić3#, ljubica andjelković4, dušan sladić2# and ilija brčeski2*# 1innovation center of the faculty of chemistry, university of belgrade, studentski trg 12–16, 11000 belgrade, serbia, 2faculty of chemistry, university of belgrade, studentski trg 12–16, 11000 belgrade, serbia, 3center for remediation, institute of chemistry, technology and metallurgy, university of belgrade, njegoševa 12, 11000 belgrade, serbia and 4department of chemistry, institute of chemistry, technology and metallurgy, university of belgrade, njegoševa 12, 11000 belgrade, serbia (received 14 november 2017, revised 11 april, accepted 16 april 2018) abstract: in this work, three novel silver(i) complexes with an almost completely rigid and bulky monodentate ligand, 1-adamantanamine, were synthesized. the aliphatic amine, 1-adamantanamine, is the sole electron donor ligand in these complexes. in addition to spectroscopic characterization, the basic biological activities of the new compounds were investigated and their minimum inhibitory concentrations were determined. the antifungal and antibacterial activities indicate that these compounds could potentially be applied as new therapeutics. keywords: amantadine; silver(i) complexes; rigid and bulky ligand; nmr spectroscopy; biological activity. introduction adamantane is a hydrocarbon that consists of three connected cyclohexane rings, forming a cage-like structure. although there are numbers of isomers with the formula c10h16, adamantane is the most stable one.1 moreover, it is peculiar since it is rigid considering its “armchair” configuration, but practically without strain. the lipophilic adamantyl group is interesting from the aspect of medicinal chemistry, since it is present in several compounds that are currently used for the treatment of neurodegenerative disorders, viral infections and type 2 diabetes.2 adamantane polymers have been patented as antiviral agents against hiv.3 it is important to emphasize that in many cases, the adamantyl group has improved the pharmacological properties of a parent compound, without increasing the toxicity. * corresponding author. e-mail: ibrceski@chem.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc171114041j ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 700 jeremić et al. different derivatives of adamantane, such as memantine, dopamantine and rimantadine have been utilized as therapeutics. amantadine (1-adamantanamine), which can be obtained in a well-established way from adamantane,4–6 was used as an antiviral drug against different strains of influenza7 and in the treatment of parkinson’s disease.8,9 it is well known that ligands having electron-donating atom(s) often show increased cytotoxicity due to their enhanced coordination capacity.10,11 various research groups are devoted to development of novel coordination compounds that contain 1-adamantanamine as a ligand in order to improve the performances and potentially overcome the shortcomings of known therapeutics. so far, platinum, titanium and zinc complexes with adamantylamine ligand were synthesized, characterized and studied from the perspectives of therapy and biological interest.12–15 for example, platinum(iv) complexes with adamantylamine were thoroughly tested for their biological activity and have the potential to provide a significantly improved therapy for many types of tumors.13 to the best of our knowledge, a complex of 1-adamantanamine with silver(i) has not been described, although some similar compounds could be found in the literature.16 in addition, in these complexes, 1-adamantanamine is the sole electron donor ligand. since silver(i) ion is very well recognized for its antibacterial activity,17 three novel silver(i) complexes with bulky, rigid 1-adamantanamine ligand were synthesized and characterized by spectroscopic measurements. the antifungal and antibacterial activities of these compounds were investigated and their minimum inhibitory concentrations were determined. experimental an appropriate silver(i) salt was added dropwise during 30 min under stirring to a methanolic solution of 1-adamantanamine. the mixture was stirred for 2 h in total darkness. the mole ratio of the reactants was 2.1:1 in favor of the amine. the obtained clear solution was left to crystallize for 72 h in the dark. the formed photosensitive crystals were suction-filtered in the dark and dried in a light-protected desiccator. the synthetic procedure can be summarized by scheme 1. scheme 1. overall synthesis of the silver(i) complexes. total silver content for the three novel compounds was determined by means of the atomic absorption method epa 272.1, direct aspiration. the infrared (ir) spectra were recorded in the wavenumber range 4000–480 cm-1 on a nicolet 6700 ft-ir instrument (thermo scientific) using the kbr technique. the 1h-nmr spectra were recorded on a varian gemini 2000 instrument at 200 mhz and the 13c-nmr spectra were recorded on a bruker avance iii ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ new silver(i) complexes with adamantylamine 701 instrument at 50 mhz. the agar-dilution method18 was used for the determination of the minimal inhibitory concentration (mic) values of the synthesized complexes. the microorganisms were prepared in nutrient-rich agar plates without the tested substances to obtain single cell colonies, which were then transferred into nutrient-rich agar plates containing the tested substances (10 μl, range: 0.02 to 50 μmol l-1). the inoculated agar-plates were incubated for 24 h and then the colonies were counted. results and discussion three novel silver(i) complexes, bis(1-adamantanamine)silver(i) nitrate hemimethanolate, bis(1-adamantanamine)silver(i) perchlorate hemimethanolate and bis(1-adamantanamine)silver(i) d-camphor-10-sulphonate hemipentamethanolate (fig. 1), were synthesized and characterized by atomic absorption, ir and 1hand 13c-nmr measurements. fig. 1. the presumed structure of bis(1-adamantanamine)silver(i) d-camphor-10-sulphonate. elemental microanalysis measurements are not reliable for this type of compounds, since the studied complexes showed incomplete combustion. moreover, reported sublimation and vaporization enthalpies showed significant discrepancies.19–24 the highly symmetric, globular ligand, 1-adamantanamine, shows a tendency to sublime. in order to determine accurately the combustion enthalpy of adamantane, its derivatives and complexes, the samples should be combusted in a calorimetric bomb under high pressure.25 thus, atomic absorption measurements were performed as a substitute. the calculated value for the total silver in bis(1adamantanamine)silver(i) nitrate hemimethanolate is 22.09 %, while the determined value was 21.9±0.1 %. in the case of bis(1-adamantanamine)silver(i) perchlorate hemimethanolate, the calculated value for the total silver is 20.51 % and the determined value was 20.56±0.1 %. for the bis(1-adamantanamine)silver(i) d-camphor-10-sulphonate complex, the calculated and determined values were 14.94 % ([ag(1-adnh2)2]csa·2.5ch3oh) and 14.77± 0.1 %, respectively. the infrared spectrum of 1-adamantanamine showed characteristic bands for primary amines: asymmetric and symmetric stretching bands (nh2 group) at 3329.8 and 3252.7 cm–1, respectively, n–h bend vibrations at 1587.7 cm–1, c–n stretch (aliphatic amines) in the region of 1250–1020 cm–1, located at 1098.8 cm–1, ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 702 jeremić et al. and n–h wag vibration was found at 812.2 cm–1. moreover, in this spectrum two strong bands were observed at 2907.5 and 2848.7 cm–1, presenting asymmetric and symmetric stretching bands, respectively (ch and ch2 groups). in the ir spectrum of the synthesized complex of 1-adamantanamine with silver nitrate, the following bands were observed: 3435.2 cm–1, wide stretching band; 1591.4 cm–1, weak bending band that arises from the nh2 group; 1348.1 cm–1, strong stretching band of the no3– ion; 2910.6 and 2847.1 cm–1, strong stretching bands originating from the ch2 groups and 1450.7 cm–1, weak bending band, which is derived from the ch groups. in the ir spectrum of the synthesized complex of 1-adamantanamine with silver camphorsulfonate, the following bands were registered: 3457.1 cm–1, wide stretching band; 1597.3 cm–1, weak bending band originating from the nh2 group; 2903.5 and 2846.5 cm–1, strong stretching bands that originate from the ch2 groups, 1451.6 and 1367.7 cm–1, weak bending bands that originate from the ch groups; 1740.6 cm–1, strong stretching band that comes from the c=o group; 1197.6 and 1047.0 cm–1 strong bands of the so3– group. in the ir spectrum of the synthesized complex of 1-adamantanamine with silver perchlorate, the following bands were detected: 3426.0 cm–1, wide stretching band; 1584.2 cm–1, weak bending band that originates from the nh2 group; 1097.0 and 621.5 cm–1 strong stretching bands that arise from clo4–, 2906.7 and 2846.5 cm–1, strong stretching bands that come from the ch2 group and 1450.7 and 1363.6 cm–1, weak bending bands originating from the ch group. due to relatively low concentration of methanol, its ir bands were overlapped with the bands that belong to the nh2 and ch2 groups and counter ions. in summary, two peaks assigned to stretching modes of nh2 group (3329.8 and 3252.7 cm–1) in the non-coordinated ligand had become one broad peak located around 3440 cm–1 in the spectra of all the investigated complexes, indicating that the coordination to the metal ion was successful. evidence for complexation of silver(i) to l-adamantanamine could also be obtained from the nmr spectra. in the 1h-nmr spectrum of l-adamantanamine, two broad signals of carbon-bound hydrogen atoms could be seen – one at 2.05 ppm, corresponding to the methine protons, and the other at 1.59 ppm, corresponding to the methylene protons. upon complexation, the methine proton signals and the methylene proton signals from hydrogens at carbons 4, 6 and 10 remain at the same chemical shift (as in the ligand), while the methylene proton signals from the hydrogens at carbons 2, 8 and 9 are shifted downfield, to 1.66–1.70 ppm (adequate numeration is shown in fig. 2). in the 13c-nmr spectra, there is an important downfield shift of the quaternary carbon 1, from 47.2 ppm in the ligand to 48.8–50.1 ppm in the complex salts. this deshielding is the consequence of the electron-withdrawing effect of the silver(i) ion. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ new silver(i) complexes with adamantylamine 703 fig. 2. numeration of 1-adamantanamine. determination of mic values was performed and all obtained complexes showed activity to the tested microorganisms. the antifungal activities are comparable with those of the test compound nystatin. unfortunately, 1-adamantanamine is not soluble in water. therefore, mic values for the ligand were not determined. the diminished activity of the studied complexes (table i) compared to simple silver(i) salts was expected, considering the lower concentration of free silver(i) ions due to complexation. on the contrary, only in one case, i.e. aspergillus niger, mic values determined for [ag(1-adnh2)2]csa·2.5ch3oh showed significant increases in activity. although the percentage of silver(i) in bis(1-adamantanamine)silver(i) d-camphor-10-sulphonate complex is lower than in nitrate and perchlorate analogues, [ag(1-adnh2)2]csa·2.5ch3oh showed comparable activity in the case of candida albicans. the increased activity of the camphorsulphonate complex was probably caused by the greater lipophilicity of the complex. table i. mic values (μmol l-1) of the complexes and reference compounds compound staphylococcus aureus (atcc 25923) escherichia coli (atcc 25922) micrococcus luteus (atcc 10240) candida albicans (atcc 24433) aspergillus niger (atcc 10249) streptomycin <0.001 0.019 0.019 >0.617 >0.617 nystatin – – – 5.399 2.483 agno3 0.188 0.188 0.188 0.765 0.005 agclo4·h2o 0.124 0.244 0.124 0.124 0.124 agcsa 0.144 0.590 0.590 0.289 1.150 [ag(1-adnh2)2]no3·0.5ch3oh 0.471 31.962 31.962 31.962 31.962 [ag(1-adnh2)2]clo4·0.5ch3oh 29.420 29.420 29.420 29.420 4.393 [ag(1-adnh2)2]csa·2.5ch3oh 0.819 1.639 1.639 21.956 0.410 the antiviral effect of adamantane amines is well known, as well as the antibacterial effect of silver(i) ions.26 the demonstrated antifungal and antibacterial activities of the newly synthesized complex compounds may have significance for their further application. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 704 jeremić et al. conclusions in this work, although being photosensitive, three novel compounds with a monodentate ligand, the almost completely rigid and bulky aliphatic amine (1-adamantanamine), were successfully prepared for the first time in a simple and low-cost way. all three compounds were characterized by atomic absorption, ir, and 1hand 13c-nmr measurements. all three characterization methods evidenced that the complexation had been successfully achieved. two characteristic bands that appear in the ir spectrum of the non-coordinated ligand became one broader peak positioned at higher energies in the spectra of the investigated complexes. in the nmr spectra, important downfield shifts were observed upon complexation. since this amine is a bulky and rigid monodentate ligand, and silver(i) mostly forms linear compounds, the obtained complexes had coordination number 2. it should be noted that in these compounds, the amine ligand is the sole electron donor. the measured antifungal and antibacterial activities of the synthesized compounds, may have an impact on their further applications as therapeutics. special attention should be paid to the nitrate and perchlorate complexes, although the lipophilic camphorsulphonate analogue gave satisfactory results despite having a lower percentage of silver(i). the obtained compounds are relatively unstable. however, these complexes could be observed as model systems in order to produce new derivatives with desired biological properties belonging to this class of compounds. acknowledgement. this work was financially supported by the ministry of education, science and technological development of the republic of serbia (grants no. 172017, 172035 and iii 43004). и з в о д нова једињења сребра(i) са 1-адамантанамином дејан јеремић1, милена ђорђевић2, срђан милетић3, љубица анђелковић4, душан сладић2 и илија брчески2 1иновациони центар хемијског факултета, универзитет у београду, студентски трг 12–16, 11000 београд, 2хемијски факултет, универзитет у београду, студентски трг 12–16, 11000 београд, 3центар за ремедијацију, институт за хемију, технологију и металургију, његошева 12, 11000 београд и 4центар за хемију, институт за хемију, технологију и металургију, његошева 12, 11000 београд у овом раду су синтетисана три нова комплекса сребра(i) са јако ригидним и релативно великим монодентатним лигандом, 1-адамантанамином. алифатични амин, 1-адамантанамин, једини је електрондонорски лиганд у овим комплексима. поред спектроскопске карактеризације, испитиване су и основне биолошке активности и одређене су минималне инхибиторне концентрације. антифунгалне и антибактеријске активности ових једињења указују да она потенцијално могу бити примењења као нови терапеутици. (примљено 14. новембра 2017, ревидирано 11. априла, прихваћено 16. априла 2018) ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ new silver(i) complexes with adamantylamine 705 references 1. а. senning, elsevier's dictionary of chemoetymology, elsevier, amsterdam, 2006 2. j. liu, d. obando, v. liao, t. lifa, r. codd, eur. j. med. chem. 46 (2011) 1949 3. а. boukrinskaia, а. serbin, о. bogdan, l. stotskaya, i. alymova, y. klimochkin, u.s. 5 880 154 (2009) 4. i. moiseev, r. doroshenko, v. ivanova, pharm. chem. j. 10 (1976) 450 5. h. stetter, j. mayer, m. schwarz, k. wulf, chem. ber. 93 (1960) 226 6. j. watts, p. marvin, u.s. 3 310 469 (1967) 7. t. maugh, science 206 (1979) 1058 8. l. sonnberg, the complete pill guide: everything you need to know about generic and brand-name prescription drugs, barnes & noble publishing, new york, 2003 9. t. blanpied, r. clarke, 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a. v. blokhin, m. b. charapennikau, a. g. kabo, v. m. sevruk, thermochim. acta 345 (2000) 125 20. r. h. boyd, s. n. sanwal, s. shary-tehrany, d. mcnally, j. phys. chem. 75 (1971) 1264 21. a. van roon, j. r. parsons, h. a. j. govers, j. chromatogr. a 955 (2002) 105 22. j. reiser, e. mcgregor, j. jones, r. enick, g. holder, fluid phase equilib. 117 (1996) 160 23. i. mokbel, k. ruzicka, v. majer, v. ruzicka, m. ribeiro, j. jose, m. zabransky, fluid phase equilib. 169 (2000) 191 24. w. z. florian, phys. chem. 61 (1968) 319 25. a. b. bazyleva, a. v. blokhin, g. j. kabo, m. b. charapennikau, v. n. emel’yanenko, s. p. verevkin, v. diky, j. phys. chem. b 115 (2011) 10064 26. x. jing, c. ma, y. ohigashi, f. oliveira, t. jardetzky, l. pinto, r. lamb, proc. natl. acad. sci. u.s.a. 105 (2008) 10967. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice effect of an industrial chemical waste on the uptake volume 83 (2018) 1 2018 list of referees editorial board of the journal is grateful to the following referees for reviewing the manuscripts during 2018: messaoudi abdelatif, université de batna 1, algeria borivoj adnađević, faculty of physical chemistry university of belgrade, serbia vladimir ajdačić, faculty of chemistry, university of belgrade, serbia rossano amadelli, chemistry department, università degli studi di ferrara, italy filip andrić, faculty of chemistry, university of belgrade, serbia ivan anđelković, innovation center of the faculty of chemistry, university of belgrade, serbia boban anđelković, faculty of chemistry, departmet of organic chemistry, university of belgrade, serbia mališa antić, faculty of agriculture, university of belgrade, serbia davor antanasijević, faculty of technology and metallurgy, university of belgrade, serbia ozgur ozgur arar, department of chemistry, ege university, i̇zmir, turkey stevan armaković, department of physics, faculty of sciences, university of novi sad, serbia mehmet aslanoglu, department of chemistry, harran university, turkey ayten ates, cumhuriyet university, sivas, turkey marija baranac-stojanović, faculty of chemistry, university of belgrade, serbia jiří barek, charles university, prague, czech republic ivona baričević-jones, faculty institute of cancer sciences, manchester academic health science centre, university of manchester, uk nemanja barać, innovation center of the faculty of technology and metallurgy, university of belgrade, serbia zvezdana baščarević, institute for multidisciplinary research, university of belgrade, serbia esra bayram, department of pharmaceutical chemistry, faculty of pharmacy, yeditepe university, istanbul, turkey ivana n. beara, department of chemistry, biochemistry and environmental protection, faculty of sciences university of novi sad, serbia snežana belanović simić, faculty of forestry, university of belgrade, serbia dejan bezbradica, faculty of technology and metallurgy, university of belgrade, serbia martin bilek, university of hradec kralove, czech republic srđan bjedov, faculty of science, university of novi sad, serbia jelena bogdanović-pristov, institute for multidisciplinary research, university of belgrade, serbia nevenka bošković-vragolović, faculty of technology and metallurgy, university of belgrade, serbia nataša božić, institute of chemistry, technology and metallurgy, university of belgrade, serbia ioannis bratsos, demokritos national centre for scientific research, department of physical chemistry, athens, greece ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 2 journal of the serbian chemical society mihael bučko, defence university, military academy, belgrade, serbia joaquin campos rosa, departamento de química farmacéutica y orgánica, facultad de farmacia, granada, spain meryem çamur, department of chemistry, kirklareli university, turkey susana casal, ispup, university of porto, portugal josef caslavsky, faculty of chemistry, brno university of technology, czech republic aurelien crochet, fribourg center for nanomaterials, frimat machine pool responsible, university of fribourg, germany jános csanádi, faculty of science, university of novi sad, serbia olga cvetković, institute of chemistry, technology and metallurgy, university of belgrade, serbia ilija cvijetić, inovation center of faculty of chemistry, university of belgrade, serbia božidar čobeljić, faculty of chemistry, university of belgrade, serbia ivan damljanović, department of chemistry, faculty of science, university of kragujevac, serbia ljiljana damjanović vasilić, faculty of physical chemistry, university of belgrade, serbia tamara dapčević-hadnađev, institute of food technology, university of novi sad, novi sad, serbia özlem darcansoy i̇şeri, baskent universitesi, transplantasyon ve gen bilimleri enstitüsü, ankara, turkey aniruddha das, university college of science, technology & agriculture, university of calcutta, india aleksandar dekanski, institute of chemistry, technology and metallurgy, university of belgrade, serbia mircea darabantu, babes-bolyai university, department of chemistry, cluj-napoca, romania srimoyee dasgupta, colburn laboratory, university of delaware, newark, usa vesna despotović, faculty of sciences, university of novi sad, serbia david dixon, department of chemistry, the university of alabama, tuscaloosa, al, usa biljana dojnov, institute of chemistry, technology and metallurgy, university of belgrade, serbia ljubica dokić, department of carbohydrate food engineering, university of novi sad, serbia tomislav došlić, faculty of civil engineering, university of zagreb, croatia gordana dražić, faculty for applied ecology futura, singidunum university, belgrade, serbia branko dunjić, faculty of technology and metallurgy, university of belgrade, serbia iris đorđević, faculty of veterinary medicine, university of belgrade, serbia neda đorđević, laboratory of molecular biology and endocrinology, institute of nuclear sciences vinča, university of belgrade, serbiae miloš i. đuran, department of chemistry, faculty of science, university of kragujevac, serbia predrag đurđević, department of chemistry, faculty of science, university of kragujevac, serbia ivana đuričić, faculty of phrmacy, university of belgrade, serbia saša đurović, institute of general and physical chemistry, university of belgrade, serbia hamdi elagab, faculty of science and artsalmandaq, albaha university, saudi arabia nazan yalcin erik, cumhuriyet university, department of geological engineering, sivas, turkey mihajlo etinski, faculty of physical chemistry, university of belgrade, serbia raúl figueroa martínez, universidade de vigo, vigo, spain marta fik, faculty of chemistry at adam mickiewicz university in poznań, poland boris furtula, department of chemistry, faculty of science, university of kragujevac, serbia gordana gajić, institute for biological research „sinisa stankovic”, university of belgrade, serbia ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 2018 list of referees 3 carlos garcía-delgado, universidad autonoma de madrid, department of agricultural chemistry and food sciences, madrid, spain uroš gašić, faculty of chemistry, university of belgrade, serbia vladimir gein, perm pharmaceutical academy, perm, russia anton georgiev, department of organic and physical chemistry, sofia, university of chemical technology and metallurgy, bulgaria nikola gligorijević, institute for the application of nuclear energy, university of belgrade, serbia biljana dj. glišić, department of chemistry, faculty of science, university of kragujevac, serbia snežana gojković, faculty of technology and metallurgy, university of belgrade, serbia dragan govedarica, faculty of technology, university of novi sad, serbia branimir grgur, faculty of technology and metallurgy, university of belgrade, serbia sanja grgurić šipka, faculty of chemistry, university of belgrade. serbia sonja grubišić, faculty of chemistry, university of belgrade. serbia maja gruden, faculty of chemistry, university of belgrade. serbia alisa gruden-movsesijan, institute for the application of nuclear energy-inep, belgrade, serbia svetlana grujić, faculty of technology and metallurgy, university of belgrade, serbia michel gruselle, institut parisien de chimie moleculaire, paris, france lourdes gude, departamento de química orgánica y química inorgánica, universidad de alcalá, alcalá de henares (madrid), spain tay meng guan, faculty of resource science and technology, universiti malaysia sarawak, kota samarahan, sarawak, malaysia milica hadnađev kostić, faculty of technology, university of novi sad, serbia amir hossein haghighaty, department of petroleum, science and research branch, islamic azad university, tehran, iran radmila hercigonja, faculty of physical chemistry, university of belgrade. serbia ivanka holclajtner-antunović, faculty of physical chemistry, university of belgrade, serbia tingjun hou, institute of functional nano & soft materials; soochow university, china mila ilić, institute of chemistry, technology and metallurgy, university of belgrade, serbia tatjana ilić-tomić, institute of molecular genetics and genetic engineering; university of belgrade, serbia jasna ivanović, faculty of technology and metallurgy, university of belgrade, serbia gorica ivaniš, faculty of technology and metallurgy, university of belgrade, serbia goran janjić, institute of chemistry, technology and metallurgy, university of belgrade, serbi milovan janković, faculty of technology, university of novi sad, serbia nenad janković, department of chemistry, faculty of science, university of kragujevac, serbia lidija jevrić, faculty of technology, university of novi sad, serbia branimir jovančićević, faculty of chemistry, university of belgrade, serbia vesna jovanović, faculty of chemistry, university of belgrade, serbia branislav jović, faculty of sciences, department of chemistry, biochemistry and environmental protection, university of novi sad, serbia jae-kyung jung, college of pharmacy and medicinal research center (mrc), chungbuk national university, cheongju, republic of korea ivan juranić, faculty of chemistry, university of belgrade, serbia zorica juranić, institute of oncology and radiology of serbia, belgrade, serbia tatjana kaluđerović radoičić, faculty of technology and metallurgy, university of belgrade, serbia željko kamberović, faculty of technology and metallurgy, university of belgrade, serbia ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 4 journal of the serbian chemical society ivanka karadžić, department of chemistry, school of medicine, university of belgrade, serbia hassan karimi-maleh, university of advanced technology, kerman, iran mirjana kijevčanin, faculty of technology and metallurgy, university of belgrade, serbia gergana kirova, faculty of pharmacy, medical university of plovdiv, bulgaria nevenka kopjar, institute for medical research and occupational health, zagreb, croatia nikola knežević, faculty of technology and metallurgy, university of belgrade, serbia miroslav komljenović, institute for multidisciplinary research, university of belgrade, serbia aleksandar kostić, faculty of mining and geology, university of belgrade, serbia mirjana kostić, faculty of technology and metallurgy, university of belgrade, serbia nenad kostić, texas a&m university, commerce, usa ivana kovačević, faculty of sciences, university of novi sad, serbia danina krajišnik, faculty of pharmacy, university of belgrade, serbia maciej kuligowski, poznan university of life sciences, poznan, poland ilya kuselman, independent consultant on metrology, modiin, israel kenneth laali, department of chemistry, university of north florida, jacksonville, fl usa uroš lačnjevac, institute for multidisciplinary research, university of belgrade, serbia steva lević, faculty of agriculture, university of belgrade, serbia katarzyna lewandowska, faculty of chemistry, nicolaus copernicus university, torun, poland ping li, qufu normal university, qufu, china anđelija malenović, department of drug analysis, faculty of pharmacy, university of belgrade serbia nedeljko manojlović, department of pharmacy, faculty of science university of kragujevac, serbia sheik mansoor, c. abdul hakeem college, melvisharam, india silvija markic, university og bremen, germany zoran marković, state university of novi pazar, serbia violeta marković, department of chemistry, faculty of science, university of kragujevac, serbia sanja martinović, institute of chemistry, technology and metallurgy, university of belgrade, serbia veselin maslak, faculty of chemistry, university of belgrade, serbia thomas mavromoustakos, department of chemistry, national and kapodistrian university of athens, panepistimiopolis, greece mina medić, vinča institute of nuclear sciences, university of belgrade, serbia luis mendoza-huizar, universidad autónoma del estado de hidalgo, ciudad del conocimiento, mexico nevena mihailović, institute for the application of nuclear energy, university of belgrade, serbia dušan mijin, faculty of technology and metallurgy, university of belgrade, serbia jelena miladinović, faculty of technology and metallurgy, university of belgrade, serbia nikola milašinović, department of forensics, academy of criminalistic and police studies, belgrade, serbia srđan miletić, faculty of chemistry, university of belgrade, serbia uroš miljić, faculty of technology, university of novi sad, serbia dušica milojković-opsenica, faculty of chemistry, university of belgrade, serbia slobodan milonjić, vinča institute of nuclear sciences, university of belgrade, serbia nenad milosavić, department of medicine, columbia university, new york, usa aleksandra milutinović-nikolić, institute of chemistry, tehnology and metallurgy, university of belgrade, serbia tarek mohamed, department of chemistry, faculty of science al-azhar university, cairo, egypt takeshi nakano, research center for environmental preservation, osaka university, japan ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 2018 list of referees 5 pham cam nam, danang university of science and technology, da nang, vietnam aleksandra nastasović, institute of chemistry, tehnology and metallurgy, university of belgrade, serbia bojana nedić vasiljević, faculty of physical chemistry, university of belgrade, serbia olgica nedić, institute for the application of nuclear energy, university of belgrade, serbia jovan nedeljković, institute of nuclear sciences vinča, belgrade, serbia katarina nikolić, faculty of chemistry, university of belgrade, serbia milan nikolić, faculty of chemistry, university of belgrade, serbia jasmina novaković, apotex inc, toronto, ontario, canada sylwia onacik-gür, szkola glowna gospodarstwa wiejskiego, department of food technology, warsaw, poland antonije onjia, vinča institute of nuclear sciences, university of belgrade, serbia dejan opsenica, faculty of chemistry, university of belgrade, serbia astrid ortner, karl‐franzens‐university, graz, austria katarzyna owczarek, department of biochemistry, medical university of lodz, poland sanja panić, faculty of technology, university of novi sad, serbia jelena pavličević, faculty of technology, university of novi sad, serbia nataša pejić, faculty of pharmacy, university of belgrade, serbia franc perdih, faculty of chemistry and chemical technology, ljubljana, slovenia marija pergal, institute of chemistry, technology and metallurgy, university of belgrade, serbia aleksandra perić-gujić, faculty of technology and metallurgy, university of belgrade, serbia milena petković, faculty of physical chemistry university of belgrade, serbia biljana petrović, department of chemistry, faculty of science, university of kragujevac, serbia dušan petrović, forschungszentrum jülich gmbh rada petrović, faculty of technology and metallurgy, university of belgrade, serbia lato pezo, institute of general and physical chemistry, university of beograd, serbia rada pjanović, faculty of technology and metallurgy, university of belgrade, serbia marijana ponjavić, faculty of technology and metallurgy, university of belgrade, serbia alexandru popa, institute of chemistry timişoara, timisoara, romania aleksandra popović, faculty of agriculture, university of novi sad, serbia marija popović-nikolić, faculty of pharmacy university of belgrade, serbia bojan radak, vinča institute of nuclear sciences, university of belgrade, serbia maja radetic, faculty of technology and metallurgy, university of belgrade, serbia marija radoičić, vinča institute of nuclear sciences, university of belgrade, serbia serbia zagorka radojević, ims institute, belgrade, serbia karlo raić, faculty of technology and metallurgy, university of belgrade, serbia ljubinka rajaković, faculty of technology and metallurgy, university of belgrade, serbia snežana rajković, faculty of science, department of chemistry, university of kragujevac, serbia slavica ražić, faculty of pharmacy university of belgrade, serbia damoder reddy motati, union university, jackson, tennessee, usa ana rilak, department of chemistry, faculty of science, university of kragujevac, serbia ivan ristić, faculty of technology, university of novi sad, serbia dušica rodić, faculty of sciences, university of novi sad, serbia jelena rogan, faculty of technology and metallurgy, university of belgrade, serbia goran roglić, faculty of chemistry, university of belgrade, serbia ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 6 journal of the serbian chemical society norio sakai, faculty of science and technology, tokyo university of science (rikadai), chiba, japan tatjana savić, vinča institute of nuclear sciences, university of belgrade, serbia vladimir savić, faculty of pharmacy university of belgrade, serbia jelena savović, faculty of pharmacy university of belgrade, serbia ulrich schubert, tu vienna, institute of materials chemistry, vienna, austria milan senćanski, vinča institute of nuclear sciences, university of belgrade, serbia l. senthilkumar, bharathiar university, tamilnadu, india mario saparrat, facultad de ciencias agrarias y forestales, universidad nacional de la plata, argentina siroos shakeri, department of agriculture, payame noor university (pnu), tehran, iran ignacio sirés sadornil, facultat de química, universitat de barcelona, spain ivan spasojević, institute for multidisciplinary research, university of belgrade, serbia pavle spasojević, innovation centre of faculty of technology and metallurgy, serbia bojana srećo zelenović, faculty of sciences, university of novi sad, serbia dragomir stanisavljev, faculty of physical chemistry, university of belgrade, serbia dalibor stanković, vinča institute of nuclear sciences, university of belgrade, serbia slavka stanković, faculty of technology and metallurgy, university of belgrade, serbia sanja stevanović, institute of chemistry, technology and metallurgy, department of electrochemistry, university of belgrade, serbia dušica b. stojanović, faculty of technology and metallurgy, university of belgrade, serbia milovan stojanović, institute of chemistry, technology and metallurgy, department of chemistry, university of belgrade, serbia ksenija stojanović, faculty of chemistry, university of belgrade, serbia jasmina stojkovska, faculty of technology and metallurgy, university of belgrade, serbia marina stojanovska, institute of chemistry, faculty of natural science and mathematics, ss cyril and methodius university, skopje, macedonia jadranka stojanovski, department of information science, university of zadar, croatia yumiko suzuki, faculty of science & technology, sophia university, japan luca szalay, eötvös loránd university, faculty of science, institute of chemistry, budapest, hungary arkadiusz szterk, national medicines institute, warsaw, poland zoran šaponjić, vinča institute of nuclear sciences, belgrade, serbia biljana šmit, faculty of science, university of kragujevac, serbia bogdan šolaja, faculty of chemistry, university of belgrade, serbia dragana šoronja-simović, department of carbohydrate food engineering, university of novi sad, serbia maja šumar ristović, faculty of chemistry, university of belgrade, serbia ljubiša šutulović, retired, belgrade serbia lubomir švorc, faculty of chemical and food technology, slovak university of technology, bratislava, slovak republic anja terzić, institute for testing of materials ims institute, belgrade, serbia vele tešević, faculty of chemistry, university of belgrade, serbia biljana tomasević, faculty of chemistry, university of belgrade, serbia miloš tomić, faculty of technology and metallurgy, university of belgrade, serbia zoran tomić, vinča institute of nuclear sciences, belgrade, serbia miloš trajković, faculty of science and engineering, university of groningen, netherlands ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 2018 list of referees 7 polonca trebše, faculty of health sciences, university of ljubljana, slovenia katarina trivunac, faculty of technology and metallurgy, university of belgrade, serbia nazar trotsko, faculty of pharmacy with medical analytics division, medical university, lublin, poland alexander n. turanov, institute of solid state physics, russian academy of sciences, chernogolovka, moscow oblast, russia ayşe uzgören-baran, hacettepe university department of chemistry, hacettepe university, ankara, turkey milica vasić, ims institute, belgrade, serbia vesna vasić, vinča institute of nuclear sciences, belgrade, serbia vlada veljković, faculty of technology, university of niš, leskovac, serbia tatjana ž. verbić, faculty of chemistry, university of belgrade, serbia vesna vitnik, department of chemistry, institute of chemistry, technology and metallurgy, university of belgrade, serbia vesna vodnik, vinča institute of nuclear sciences, belgrade, serbia tatjana volkov-husović, faculty of technology and metallurgy, university of belgrade, serbia ljubodrag vujisić, faculty of chemistry, university of belgrade, serbia milica vujković, faculty of physical chemistry, university of belgrade, serbia jelena vuksanović, faculty of technology and metallurgy, university of belgrade, serbia arzu yıldırım, faculty of arts and science, giresun university, turkey yuanyuan yue, school of chemistry and chemical engineering, henan normal university, xinxiang, pr china george a. zachariadis, aristotle university of thessaloniki, greece jing jing zhang, department of chemistry, university of illinois at urbana, illinois, usa zoran zeković, faculty of technology, university of novi sad, serbia peng zhou, school of life science and technology, university of electronic science and technology of china, chengdou, sichuan, pr china matija zlatar, institute of chemistry, technology and metallurgy, university of belgrade, serbia fedor zubkov, people's friendship university of russia, moscow, russia gregor žerjav, department of environmental sciences and engineering, national institute of chemistry, ljubljana, slovenia marija živković, faculty of science, university of kragujevac, serbia irena žižović, faculty of technology and metallurgy, university of belgrade, serbia ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ {permeability of gas mixtures in the glassy polymers with and without plasticization} j. serb. chem. soc. 86 (4) 341–353 (2021) survey jscs–5425 341 survey permeability of gas mixtures in glassy polymers with and without plasticization masoud saberi* department of chemical engineering, bushehr branch, islamic azad university, bushehr, iran (received 15 july, revised 31 july, accepted 4 august 2020) abstract: in this research, the solubility, permeability and diffusivity of gas mixtures through glassy polymers were comprehensively studied. the diffusivity of the components in the mixture was assumed to be a function of the concentration of all components in the mixture. then, the permeability of pure species was expanded to the gas mixtures and to check the validity, the model was fitted to the experimental data for permeation of co2/ch4 through different glassy membranes and the parameters of the model were calculated. afterwards, the obtained parameters were used for predicting the permeability of co2 and ch4 in the mixture. the results showed that the solubility, diffusivity, and the permeability of co2 in the glassy polymers are suppressed in the presence of ch4 as well as plasticization. moreover, the diffusivity (d) for pure co2 is significantly pressure dependent in the presence of plasticization whereas with the increase in the ch4 fraction, this dependency decreases due to the reduction in the plasticization. keywords: gas separation; membrane; plasticization; solubility; diffusivity. introduction polymeric membranes are widely used in the natural gas separation process. for removal of carbon dioxide (co2), glassy polymeric membranes are often preferred over rubbery polymeric membranes because of their higher co2/ch4 or co2/n2 selectivity.1–6 although some types of glassy membranes have a good performance in co2 separation, the performance of these membranes can be hindered by the plasticization phenomenon.7–12 therefore, co2 permeability increases with the feed pressure.7–13 on the other hand, permeability of pure inert gases, such as ch4 or n2, has a decreasing trend with the pressure.14–16 thus, the ideal selectivity of co2/n2 or co2/ch4 increases with feed pressure.13–16 however, the behavior of mixed gas feeds is significantly different from pure * e-mail: msd.saberi@gmail.com https://doi.org/10.2298/jsc200715046s ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 342 saberi species. in the presence of plasticization, the permeabilities of both co2 and n2 or ch4 increase. but n2 or ch4 generally have larger increases than co2, resulting in decreasing selectivity.1,10,14,17,18 thus, the actual selectivity is lower than ideal selectivity at a special pressure.1,14,19 raymond et al.19 reported that for mixed gas feed of co2 and ch4 with equal composition, the actual selectivity at 5 atm* is well predicted by pure gases, whereas at 20 atm, the actual selectivity was much lower than ideal selectivity. it was due to plasticization of membranes at 20 atm pressure. in addition, ideal selectivity of co2 and ch4 for polyimide (6fda-mpd) was reported to be about 60 at a feed pressure of 17.5 atm, whereas the actual selectivity for feed with equal composition of these gases was observed about 4.20 therefore, for a proper prediction of transport behavior for gas mixtures, especially in the presence of plasticization, it is essential to represent accurately the experimental results. then, an accurate and simple model is required to be used for all the different behaviors of gases in glassy polymers. different approaches were developed to describe the solubility and transport of gases and vapors in glassy polymers. among these models, the dual mode sorption (dms) and non-equilibrium lattice fluid (nelf) models are well-known models. it should be mentioned that, although nelf model has been extended for all permeability behavior of gaseous in glassy polymers, it is used less than the dms model because of its complexity and long calculation times. dms, a model with empirical parameters, is widely used mainly due to its remarkable simplicity. although, different models with different assumptions have been developed based on this theory to investigate the permeability of pure and mixed gases in glassy polymers, less attention has been paid for predicting permeation of mixed gases through glassy polymers in the presence of plasticization. in our previous works, we extended a model for permeation of gas mixtures in glassy polymers based on dms model with no predictive capability.21,22 in the present study, a comprehensive model based on the dms model was developed to predict the permeation behavior of mixed gases through glassy polymers with and without plasticization using pure data for solubility and permeability. to achieve this aim, the diffusivity of all species in the mixture is assumed to be a function of the concentration of all components in the mixture. then, for determining the parameters and evaluation of the accuracy of the model, the predictions of the model were compared against experimental data for the permeation of different groups of gas mixture in different glassy polymers. * 1 atm = 101325 pa ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ gas permeability in glassy polymers 343 theory and background solubility based up on the dms model, two mechanisms of sorption occur in glassy polymers: i) ordinary dissolution based on the henry’s law and ii) “hole-filling” according to the langmuir theory. the equilibrium isotherm for a pure gas a based on the dms model is expressed as:21-23 a da ha a 'ha a a a a/ (1 )= + = + +dac c c k p c b p b p (1) where c is the gas concentrations in the polymer (cm3 stp/cm3 polymer), cd is the henry’s solubility, represents ordinary dissolution, ch is langmuir solubility, represents sorption in microvoids or holes, kd is henry’s law solubility coefficient (cm3 stp/cm3 polymer.atm), c'h is the hole saturation constant (cm3 stp/cm3 polymer), b is the hole affinity constant (atm-1) and p is pressure (atm). the solubility coefficient of gas a in polymeric membranes is defined as:21,22 sa = ca/pa (2) koros et al. extended the dms model for gas mixture systems and the sorption of components a and b of a binary gas mixture is expressed as:23 a da a 'ha a a a a b b= / (1 )+ + +c k p c b p b p b p (3) b db b 'hb b b a a b b= / (1 )+ + +c k p c b p b p b p (4) permeability based on the partial immobilization model (pim), a fraction f of the sorbed gases in the langmuir sites are mobile and the remainder (1−f) are immobile whereas the whole gas dissolved in the henry’s region is mobile. the total concentration of the mobile part of the adsorbed gas is cm with a diffusion coefficient d. f is the immobilization factor and depends on the nature of penetrant–polymer system as well as the system temperature.24,25 the flux (n) of component i is expressed as follows:21 i i mi( ) ∂ = − ∂ c n d x (5) where:21 mi di i hi di i 'i hi i i i i/ (1 )= + = + +c c f c k p f c b p b p (6) for the diffusivity of species i, a simple exponential relationship with the penetrant mobile concentration was found effective and is given by:26,27 di = di0exp(βicmi) (7) where di0 is the diffusion coefficient of pure gas at zero penetrant concentration, and βi is the plasticization factor. it should be noted that eq. (7) could be used for all gases, including plasticizer or not. in the absence of plasticization (i.e., βi = 0), diffusivity will be constant and would not change with pressure. then, eqs. (5)–(7) yield the following expression for the flux of the penetrant gas in glassy polymers: ( )m 2 m 1 0 m mexp dβ= −  i i ci i i i ic d n c c l (8) ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 344 saberi where subscripts 2 and 1 represent the upstream and downstream conditions and when the downstream pressure is considered zero, cma1 = 0. toni et al. considered the two mobility coefficients related to the concentration of both penetrants,28 whereas the diffusivities for components a and b in the binary gas mixture were assumed to be related to the concentration of both penetrants and obtained by: da = da0exp (βacma+ βbcmb) (9) db = db0exp(βacma+ βbcmb) (10) where da0, db0, βa and βb were obtained from pure state, and: ma da a ha da a a 'ha a a a a b b/ (1 )= + = + + +c c f c k p f c b p b p b p (11) mb db b hb db b 'b hb b b a a b b/ (1 )= + = + + +c c f c k p f c b p b p b p (12) again, combining eq. (5) and eqs. (9)–(12) and integrating, yields the following expression for the flux of components a and b in glassy polymers: ( )ma2 ma1 a0 a a ma b mb maexp dβ β= − + c c d n c c c l (13) ( )mb2 mb1 b0 b a ma b mb mbexp dβ β= − + c c d n c c c l (14) it is worth mentioning that for integrating eq. (13), cma and cmb should be written in term pa and for eq. (14) should be written in term pb. furthermore, under steady state conditions, the permeability and selectivity are given by:22 2 1 = − i i i i n l p p p (15) where l is the membrane thickness. results and discussion the mathematical procedure to predict the permeation of mixed gas through glassy polymeric membranes is as follows: 1. calculation of parameters of the dms model (eq. (1)) for pure species by fitting this equation to the experimental data of the isotherms. 2. using the obtained parameters from step 1, fitting eq. (8) to the experimental data for permeability of pure species and the calculation of parameters β, f and d0 for the pure species. 3. using the obtained parameters of steps 1 and 2 in conjunction with eqs. (13) and (14), for the prediction of the permeability of the species in the gas mixture. it is worth noting that the parameters of the dms model and the non-linear proposed models are obtained by the least squares regression technique using matlab software. to validate the model, comparing with the experimental data for the permeation of co2/ch4 mixtures through different glassy membranes including poly________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ gas permeability in glassy polymers 345 sulfone (psf), polyetherimide (pei), polyhydroxyether (ph), polyarylate (par) and cellulose acetate (ca) are investigated.15,29–31 solubility in order to study the permeability behavior of co2/ch4 gaseous mixture, the parameters of the sorption isotherm of pure co2 and ch4 in the glassy polymers is required. the dms parameters for co2 and ch4 in the different glassy polymers, which were obtained by fitting the dms model to the experimental data, are reported in table i. then, by consideration the parameters of table i, and using eqs. (3) and (4), the solubility of the species in the gas mixture were predicted. table i. dms parameters for pure co2 and ch4 in the different glassy polymers at 35 °c polymer gas kd / (cm3 stp/(cm3 atm)) c'h / (cm3 stp/cm3) b / atm-1 reference psf co2 0.664 17.91 0.326 29 ch4 0.161 9.86 0.070 ph co2 0.289 10.01 0.184 29 ch4 0.051 2.70 0.067 pei co2 0.758 25.02 0.366 29 ch4 0.207 7.31 0.136 par co2 0.631 22.69 0.215 29 ch4 0.181 6.45 0.100 ca co2 1.362 22.58 0.248 15 ch4 0.190 2.504 0.132 as mentioned in a previous work,21,22 the solubility–pressure isotherm for co2 and ch4 and their mixtures in glassy polymers, at lower pressures severely increases and with increasing pressure, a decrease in the sorption slope occurred. for higher pressures, this slope is almost constant and the sorption isotherm changes linearly, like the sorption of gases in rubbery polymers. this trend of sorption is because at low pressures, gas molecules adsorbed in the henry and langmuir sites and for higher pressures langmuir sites will be occupied. for gas mixtures, the presence of the second component (i.e., ch4) inhibited the sorption of first component (co2) by occupation of some sites of the langmuir portion. then, the sorption of co2 is suppressed by the presence of ch4 in the mixture (fig. 1). the solubility selectivity of co2/ch4 vs. pressure is shown in fig. 2. solubility selectivity is found to be significantly higher in mixtures compared to the pure condition. this could be attributed to competitive sorption whereby the solubility of co2 decreases in the presence of ch4 as well as ch4. it should be mentioned that the decrease in ch4 solubility is more than that of co2 solubility due to higher hole affinity constant of co2 (bco2>bch4) resulting in an increase in the solubility selectivity. as could be observed, the ideal solubility selectivity ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 346 saberi increases with increasing ch4 fraction at constant pressure as reported by vopicka et al.20 fig. 1. solubility of: a) co2 and b) ch4 in ca glassy polymer. fig. 2. solubility selectivity of co2/ch4 in ca glassy polymer. permeation without plasticization the permeability of pure co2 and ch4 in different glassy polymers was fitted using experimental data from the literature30 and the parameters of the model, including β, f and d0 for co2 and ch4, are reported in table ii (also determined in the literature30). the permeability–pressure plots have a decreasing and/or constant trend in all cases. in these cases, there is no plasticization (β = 0), then, the diffusivity is constant. in this case when there was no plasticization, the decreasing and/or constant trends for permeability is related to the solubility coefficient and is controlled by the immobilization factor (f), which shows the mobile parts of the sorbed gas in the langmuir region. the predictions of the model for co2 and ch4 gases of 50/50 volume ratio mixture in different glassy membranes using eqs. (13) and (14) compared to the experimental data from the literature31 are shown in fig. 4a and b. at a glance, almost a small suppression in permeability in gas mixture is observed compared to the pure species. as mentioned above, solubility of species in the presence of ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ gas permeability in glassy polymers 347 second component is reduced compared to pure species due to occupation of langmuir sites, which resulted in a reduction in the diffusivity as well as permeability. an acceptable prediction for all cases could be observed. table ii. parameters of eq. (8) for permeation without plasticization (β = 0)30 d0×108 / cm2 s-1 f gas polymer 4.53 0.118 co2 psf 0.690 0.174 ch4 0.877 0.094 co2 ph 0.246 0.072 ch4 1.14 0.063 co2 pei 0.113 0.073 ch4 6.90 0.126 co2 par 1.30 0.160 ch4 fig. 3. permeability of pure: a) co2 and b) ch4 in different glassy polymers without plasticization (experimental data from the literature30). fig. 4. permeability of: a) co2 and b) ch4 gases of 50/50 volume ratio mixture in different glassy polymers without plasticization (experimental data from the literature31). permeation with plasticization permeability. the permeability behavior of pure co2 and ch4 through the ca membrane is shown in fig. 5. these figures present experimental data from ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 348 saberi the literature15 with predictions of the model, calculated by eq. (8), using the parameters β, f and d0 for co2 and ch4 listed in table iii. for pure co2, the permeability increases with increasing pressure due to the higher degree of plasticization of the ca membrane. due to the high sorption of co2, which is a condensable gas, the polymer matrix swells and the interaction between adjacent segments of the polymer chain reduces. therefore, due to the increase in segmental mobility and the free volume of polymer matrix, the diffusivity increases with increasing pressure. on the other hand, the solubility coefficient decreases with increasing pressure. since, the increase in the diffusivity overcomes the decrease in solubility coefficient, co2 permeability increases with increasing pressure. for ch4, which has low solubility in the membrane, the permeability decreases with increasing pressure. in this case, plasticization does not occur, and diffusivity is constant. on the other hand, the solubility coefficient decreases with pressure. then, the permeability decreases with increasing pressure. fig. 5. permeability of pure co2 and ch4 penetrants in the ca membrane (experimental data from the literature15). table iii. infinite dilution diffusivity and plasticization factor for the various penetrants in the ca membrane d0×107 / cm2 s-1 f β gas polymer 1.45 0.06 0.031 co2 ca 0.29 0.38 0 ch4 in addition, comparing the experimental data for the permeability of co2 in the gas mixture feed with different compositions from the literature15 and the predictions of the model using parameters from table ii is shown in fig. 6. for a feed with 46.1 % co2 and the rest ch4, the permeability decreases with increasing pressure up to about 30 atm and then increases and 30 atm is called the “plasticization pressure”. the aforementioned solubility coefficient decreases with increasing pressure and in the presence of co2 as a plasticizer component, the diffusivity increases with increasing pressure. for a feed with 46.1 % co2, at pressures lower than 30 atm, the decrease in the solubility coefficient overcomes the increase in the diffusivity whereas at higher pressures, the ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ gas permeability in glassy polymers 349 increase in the diffusivity dominates. indeed, by adding ch4 as the second component to the feed, some sites for sorption of co2 are occupied by ch4 molecules so that the solubility of co2 in the mixture declines compared to pure co2. by suppression in the co2 solubility, diffusivity of co2 lowers at a specific pressure, consequently co2-induced plasticization decreases. it means that ch4 in the feed acts as anti-plasticizer. for higher fractions of ch4 in the feed, the effect of anti-plasticization increases and the permeability with the increase in the pressure decreases. therefore, by introducing ch4 to the feed, co2-induced plasticization is suppressed. as can be seen, the prediction of the model for permeability behavior is almost acceptable. fig. 6. permeability of co2 in mixtures with different compositions vs. pressure, comparison between the experimental data from the literature15 and the model prediction. moreover, the experimental data and the predictions of the model for ch4 in gas mixture feeds with different compositions using parameters in table ii are compared in fig. 7. as observed, for a feed with 53.9 % ch4, the permeability of ch4 passes through a minimum similar to the permeability of co2 in fig. 3. this behavior is due to the presence of co2, which causes the membrane to plasticize. in addition, for feeds with higher fractions of ch4, plasticization decreases due to the reduction of co2 sorption and diffusion, and hence, for feeds with the fractions higher than 53.9 % of ch4, the ch4 permeability decreases and/or is constant with increasing in pressure. furthermore, with increasing ch4 fraction in the feed, the ch4 permeability at specific pressures is reduced following suppression fig. 7. permeability of ch4 in mixtures of different compositions vs. pressure, comparison between the experimental data from the literature15 and the model calculation. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 350 saberi of plasticization. diffusivity the estimated diffusivity vs. pressure for co2 and ch4 in the pure state and in the gas mixture derived from eqs. (9) and (10) using parameters from tables i and ii is illustrated in fig. 8a and b. for pure co2, stronger dependency of d on pressure was observed, so that d increases significantly with increasing pressure due to higher degree of plasticization. for feeds with different fractions of ch4, because of the reduction in the plasticization, the effect of pressure on d for co2 became very weak and the dependency of d on pressure decreases with increasing ch4 fraction. for pure ch4, d is constant and did not change with increasing pressure. by adding 9.7 % co2 to the feed, a very weak dependency of d on pressure was observed and this dependency increased with increasing co2 fraction due to the increase in the plasticization, so that for feeds with 46.1 % co2, d for ch4 increased significantly. additionally, at a specific pressure, d for ch4 decreases with increasing ch4 fraction. it should be mentioned that although with increasing ch4 fraction in the feed, the ch4 sorption increases, the swelling and the plasticization affect decreases due to reduction in co2 sorption. the latter reason overcomes the results in the reduction in the diffusivity of ch4 with increasing ch4 fraction at a specific pressure. fig. 8. diffusivity of: a) co2 and b) ch4 in the pure state and as mixtures in the ca membrane. conclusions the permeation behavior of mixed gases through glassy membranes was significantly different from pure species, especially in the presence of the plasticizeation phenomenon. the presence of the second component, such as ch4 or n2, along with co2 in the feed led to a decrease in the co2 solubility resulting in a decrease in diffusivity, permeability and the plasticization effect. this research was focused on gas mixtures and a model was developed for the prediction the ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ gas permeability in glassy polymers 351 permeability of the species in mixed gases through glassy polymers with and without plasticization. then, by comparing the proposed model for the experimental data of permeation of pure co2 and ch4 through the different glassy polymer membranes, the parameters of the model were calculated. then, these parameters were used for predicting the permeability of gases in the mixtures. the results showed that the presence of ch4 in the feed reduces the permeability of co2 as well as the plasticization. moreover, the results show that d for pure co2 significantly changes with pressure and with the addition of ch4 to the feed, this dependency decreased. for a feed with 53.9 % ch4 (46.1 % co2) the d value for ch4 increased with increasing pressure but for higher fractions of ch4 in the feed, this dependency almost disappeared. acknowledgements. this research was supported by islamic azad university, bushehr branch. и з в о д пропустљивост смеше гасова кроз полимере у стакластом стању са пластификацијом и без ње masoud saberi department of chemical engineering, bushehr branch, islamic azad university, bushehr, iran у овом истраживању проучавана је растворљивост, пропустљивост и дифузивност смеша гасова кроз полимере у стакластом стању. претпоставља се да је дифузивност компонената у мешавини функција концентрације свих компоненти у смеши. затим се пропустљивост чистих компонената проширује на смеше гасова и за проверу ваљаности се проверава модел фитовањем експерименталних података за пермеабилност co2/ch4 кроз различите мембране у стакластом стању и израчунавају се параметри модела. након тога, тако добијени параметри се користе за предвиђање пропустљивости co2 и ch4 у смеши. резултати показују да су растворљивост, дифузивност, а такође и пропустљивост co2 кроз полимере у стакластом стању смањени у присуству ch4 и пластификатора. штавише, дифузивност за чисти co2 значајно зависи од притиска у присуству пластификатора док се с повећањем удела ch4 та зависност смањује због смањења пластификације. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {nitrogen-modified nanoporous activated carbon from eucalyptus leaves for ultrasound-assisted removal of basic dyes using derivative spectrophotometric method} j. serb. chem. soc. 82 (5) 651–668 (2017) udc 667.2+546.131+661.183.2:544.723.2+ jscs–5102 582.883.4:543.4/.5 original scientific paper 651 nitrogen-modified nanoporous activated carbon from eucalyptus leaves for ultrasound-assisted removal of basic dyes using derivative spectrophotometric method aisan khaligh1*, hassan zavvar mousavi1, alimorad rashidi2 and hamid shirkhanloo3 1department of chemistry, semnan university, semnan 35131-1911, iran, 2nanotechnology research center, research institute of petroleum industry (ripi), west entrance blvd., olympic village, tehran 14857-33111, iran and 3research institute of petroleum industry (ripi), west entrance blvd., olympic village, tehran 14857-33111, iran (received 27 august, revised 17 october, accepted 25 october 2017) abstract: the nanoporous activated carbon (ac) was prepared from the eucalyptus leaves via chemical activation with koh, then treated with nitric acid/ /urea (noac) and finally used as a new adsorbent for simultaneous ultrasound-assisted removal of basic red 46 (br46) and basic yellow 13 (by13) dyes from binary aqueous solutions. the noac nano-adsorbent was characterized with sem, tem, raman, bet, ftir, chn, phpzc and boehm titration analysis. both of the ac and noac samples had superior bet surface area of 2222 and 1572 m2 g-1 with average micropore volume of 0.81 and 0.50 cm3 g-1, respectively. first order derivative spectrophotometric method was used for analysis of by13 in binary mixtures. small amount of the adsorbent (30 mg) was capable to remove high percentage of dyes (>99 %) in a very short time (8 min). the adsorption of dyes follows the langmuir isotherm and the pseudo -second-order kinetics. the adsorption capacities of noac for single solutions of br46 and by13 were 1111 and 1250 mg g-1 as well as for binary solutions were 769 and 909 mg g-1, respectively. the adsorption thermodynamics were also explored. exhausted noac was regenerated using hcl (2 m) and reused for five adsorption-desorption cycles with high performance. keywords: low-cost adsorbent; cationic dyes; fast adsorption; isotherm; kinetics; thermodynamics. introduction industry dyes are becoming worldwide sources of environmental pollution, and their existence has severely affected the aquatic organisms and life cycle. one of the most problematic classes of dyes are the basic dyes called cationic * corresponding author. e-mail: akhalighv@gmail.com https://doi.org/10.2298/jsc170827112k 652 khaligh et al. dyes.1 toxic cationic dyes of basic red 46 (br46, single azo class) and basic yellow 13 (by13, azomethine class) are widely used in acrylic, nylon, silk, leather and wool dyeing process. these dyes have carcinogenic, mutagenic, and teratogenic properties which may be related to the presence of nitrogen and positive charge on their structures.2 dyes are particularly difficult to remove by conventional waste treatment methods since they are recalcitrant organic molecules, resistant to aerobic digestion and are stable to light, heat and oxidizing agents. herein, the removal of dyes from water in an economical way remains an important challenge to scientists. processes such as coagulation/flocculation, membrane filtration, ion exchange and adsorption are also used for treating dye containing wastewater.3 the later method gain major superiority due to its proven efficiency, the ease of use and the insensitivity to toxic material without resulting in the formation of harmful substances as well as the availability of a wide range of adsorbents.4 adsorption by the activated carbon has been widely used for wastewater treatment due to its exceptionally high surface area and micropore volume, well -developed internal microporosity, favourable pore size distribution, and high adsorption efficiency together with the availability of low-cost resources such as plant residues. recently, an enormous range of waste agricultural materials have been used as activated carbon precursors including rice husk, fruit stone, palm shell, almond shell, coconut shell, pistachio-nut shell, sugar cane bagasse, spent tea leaves, and reedy grass, etc.5 in general, raw materials are initially treated to form charcoal. then, physical or chemical activation of the charcoal results in activated carbons.6 recently, much attention has been paid to the surface modification of the activated carbons,7 the conventional protocols used for the modification are oxidation, nitrogenation, sulfuration and coordinated ligand anchora.7 the introduction of nitrogen functional groups onto the surface of ac and replacing them with the existing oxygen groups has become a subject of great interest nowadays.7,8 in this trend, oxidation pretreatment of the ac can enhance the incurporation of nitrogen into the carbon.9 nitrogen modification of ac is an effective method which increases its basicity, polarity of its surface and hence the specific interaction with polar adsorbates. in order to incorporate of nitrogen into the carbon structure, carbons are treated with ammonia, urea, dicyanodiamine, n,n -dimethylformamide, melamine or their derivatives, in the temperature range from 350 to 900 °c, in different periods of time under nitrogen.9,10 it has been reported that the basic nitrogen functional groups such as amine groups (–nh2, –nh) can be introduced by the urea treatment of ac at low temperatures range, from 350 to 500 °c.10 the presence of the heterocyclic nitrogen compounds incorporated in the carbon matrix with free pairs of electrons increase the electron donor capacity of the solid. therefore, the nitrogen-containing acs are the dyes removal using modified actvated carbon 653 effective adsorbents of positively charged pollutants such as heavy metal ions and cationic dyes through the formation of donor–acceptor complexes.9 nitrogen-modified acs have successfully been applied in the water decontamination treatments for the removal of heavy metal ions (cu(ii), pb(ii), hg(ii) and cd(ii)),7,11,12 anions (cn−, clo4−, aso43−),7,11 organic substances (benzoic acid, phenol, atrazine),7,13 and natural organic matter. herein, their adsorption was generally enhanced on nitrogenated acs versus virgin acs. the uv–vis spectrophotometric method is usually used as a simple, economic, rapid and accurate technique for dye assessments.14 however, the simultaneous analysis of dyes in binary solution by the spectrophotometric methods can be very complex due to the overlapping absorption bands of the dyes and spectral interferences. derivative spectrophotometry is an analytical technique of great utility for overcomming this limitation. the derivatization of the zero order spectrum can lead to the separation of overlapped signals and to the elimination of background caused by the presence of the other compounds in a sample.2,15 in this study, a low-cost nanoporous activated carbon with a large surface area was prepared from eucalyptus leaves through koh activation, then treated with nitric acid and urea (noac) and finally used as a new nanoadsorbent for the simultaneous ultrasound-assisted removal of basic red 46 (br46) and basic yellow 13 (by13) dyes from binary solutions. eucalyptus leaves, as a raw material for the production of activated carbon, can be considered as one of the best candidates among the agricultural wastes because it is cheap and quite abundant, especially in arid and semiarid areas like semnan, iran. the first derivative spectrophotometric method was used for analysis of by13 in binary mixtures. the influence of several parameters such as ph, temperature, initial concentration of dyes, adsorbent dosage and ultrasonication time for the maximum removal of both dyes was investigated, using the batch adsorption method. furthermore, the characterization of dye adsorption was described by the kinetics models, adsorption isotherms and thermodynamic parameters. experimental apparatus and characterization methods a double beam uv–vis spectrophotometer (shimadzu, model pc 1650-uv, japan) with two matched 1-cm quartz cells was used for the analysis of the studied dyes. the ph measurements were carried out using a digital ph meter (metrohm, model 744, herisau, switzerland). the ultrasonic bath with heating system (tecno-gaz spa ultra sonic system) and a centrifuge (hettich, model eba 20, germany) were used in the batch adsorption experiments. the surface morphology of the adsorbent was characterized using a scanning electron microscopy (sem, phillips, pw3710, netherland) and a transmission electron microscopy (tem, cm30, philips, the netherlands). the textural parameters of the prepared samples including surface area, pore volume, and pore size distribution were determined by the nitrogen adsorption–desorption isotherms at 70 k using a micrometrics asap 2010 system (micrometric instruments co, cleveland, oh, usa). the specific surface area was calculated by 654 khaligh et al. the bet (brunauer–emmett–teller) method. the total pore volume was obtained from the volume of nitrogen adsorbed at the relative pressure of 0.99. the micropore analysis was performed by the t-plot method (a technique which allows determining the microporous volumes and the specific surface area of a sample by comparison with a reference adsorption isotherm of a nonporous material having the same surface chemistry) and mesopore volume was determined from desorption branch of the isotherm by the bjh (barrett, joyner and halenda) method. the pore size distribution was determined as well. the average pore diameter was also given by 4vtotal/sbet. prior to the analysis the samples were degassed under vacuum at 200 °c for 2 h. raman spectroscopy was carried out with an almega thermo nicolet and 532 nm ar-ion laser excitation source in order to investigate the structural properties and bonding in the virgin and modified acs. the elemental composition of the samples was measured using the chn elemental analyzer (euro vector s.p.a, model ea 3000, italy). the oxygen content was calculated as a difference between 100 % and the sum of c+h+n. the fourier transform infrared (ft-ir) spectra were recorded in the range from 4000 to 400 cm−1 using a bruker ifs 88 spectrometer (bruker optik gmbh, ettlingen, germany) with kbr pelleting method. the surface acidic and basic groups of the prepared samples were estimated using boehm titration method. 0.5 g of the activated carbon samples were added in test tubes containing 25 ml of naoh, na2co3, nahco3, or hcl (0.05 m). the test tubes were shaken for 24 h at 25 °c and filtrated. then, 5 ml of the filtrate was pipetted and the excess of base or acid was titrated with hcl or naoh (0.1 m) solution. the metrohm ph-meter was employed to monitor the titration. the numbers of acidic sites and basic sites were calculated using the titration result.16 the phpzc (point of zero charge) of the sorbents was determined using the known method as was detailed before.17 chemical reagents and solutions all chemicals, with analytical grade purity available, were purchased from merck (darmstadt, germany). basic red 46 (br46) and basic yellow 13 (by13), as model dyes, were used without further purification. the chemical structure and properties of the dyes are shown in table s-i of supplementary material to this paper. deionised water (di-water) from a millipore continental water system (bedford, ma, usa) was used for the preparation of the aqueous solutions. the stock solution of each dye (1000 mg l-1) was prepared by dissolving 250 mg of the dye in 250 ml of di-water. the experimental and working standard solutions were prepared daily by the dilution of the stock solutions. preparation of nanoporous activated carbon the eucalyptus leaves (el) as a raw material for the preparation of ac were obtained from semnan city, north central iran. the collected el was washed with distilled water to remove all the dirt particles, then dried at room temperature for 24 h, and finally ground to powder in a laboratory blender to attain particle size of 0.4–0.8 mm. the el-based activated carbon was prepared via two steps; carbonization of the dried precursor under an inert atmosphere followed by the chemical activation of the char impregnated with potassium hydroxide. koh was used as the chemical activating agent as it is one of the most effective compounds for the production of activated carbons. details are as follows: the resulting el powder was first carbonized at 600 °c for 1 h under n2 flow (200 ml min -1) in a stainless steel tube furnace (1500 mm×50 mm) at a heating rate of 5 °c min-1 up to 600 °c. the prepared el-char was then cooled to room temperature under n2 flow and removed from the reactor. nanoporous activated carbon was prepared by mixing the el-char with koh solution (koh:char weight ratio of 2.5:1) at room temperature for 1 h. the slurry was dried overnight dyes removal using modified actvated carbon 655 at 120 °c. then, the impregnated sample was pyrolyzed in the stainless steel tube furnace under n2 flow (200 ml min -1) at the activation temperature of 850 °c, the holding time of 1 h and the heating rate of 5 °c min-1. after the activation process, the obtained ac was cooled down to room temperature under n2 flow. then, in order to obtain the pure ac and remove any residual organic and mineral matter, the product was mixed with hcl solution (50 vol.%) for 2 h and then washed with hot and cold deionised water until ph of 6–7 was attained. finally, el-based nanoporous ac was oven dried at 100 °c. nitrogen modification of nanoporous activated carbon the ac surface was first treated with hno3 solution. 3 g of nanoporous ac was immersed in a flask containing 100 ml of aqueous hno3 solution (50 vol.%) and stirred under reflux at 60 °c for 3 h. the oxidized ac (oac) was then cooled to room temperature, washed repeatedly with di-water until the filtrate was neutral and finally dried overnight at 70 °c. in order to prepare the nitrogen-modified nonporous ac (noac), urea was used as a nitrogen-rich precursor. 1 g of oac was impregnated with 10 ml of urea solution (1 m) at room temperature for 2 h. the mixture was oven dried at 80 °c. then, the dried sample was annealed by using stainless steel tube furnace at 450 °c for 1 h in the n2 atmosphere (200 ml min-1) at a heating rate of 5 °c min-1 up to 450 °c. the obtained product was cooled down to room temperature under n2 flow, washed with boiling di-water to remove the unreacted urea and finally oven dried at 60 °c for 6 h. ultrasound-assisted adsorption procedure ultrasound-assisted adsorption experiments were conducted using the batch method to elucidate the effect of various parameters such as the solution ph, the ultrasonication time, the adsorbent dose, the temperature and the initial dye concentration on the removal of dyes from binary solutions. the necessary amount of noac was added to a 250 ml erlenmeyer flask containing 100 ml of binary dye solution with the initial concentration of 150 mg l-1 (from each of br46 and by13) at desired ph value. the solution ph was adjusted by adding the negligible volumes of naoh or hcl diluted solutions. the flask was immersed in an ultrasonic bath (40 khz, 130 w) at 25 °c. an aliquot of the sample solution was withdrawn at the pre-determined time intervals and centrifuged at 3000g for 1 min. finally the solution was analyzed for the final concentration of dyes by using the uv–vis spectrophotometer. the removal percentage (r / %) and the solid phase dye concentration, qe, mg g -1, was calculated. adsorption isotherm for single and binary dye solutions, the isotherm studies were carried out at different dye concentrations (table s-ii of the supplementary material), ph 9 and 25 °c with 30 mg noac and 8 min ultrasonication time, according to the above adsorption procedure. adsorption kinetics kinetics of the dyes adsorption from binary solutions by noac were also investigated according to the adsorption procedure using different ultrasonication times (2–8 min) at the optimum values of ph, the adsorbent dosage and the temperature, obtained from the batch optimization procedure, i.e., 9, 30 mg and 25 °c, respectively. adsorption thermodynamics thermodynamic studies at different temperatures (15–55 °c) were carried out by adding 20 mg of noac into 100 ml of binary dye solutions (150 mg l-1). the solution ph and ultrasonication time were adjusted at 9 and 8 min, respectively. 656 khaligh et al. desorption studies initially, the batch adsorption tests were carried out on the fresh noac with 100 ml of 150 mg l-1 binary dyes solution at ph 9, containing 30 mg of noac. after mixing for 8 min with ultrasonic bath (40 khz, 130 w, 25 °c), the mixtures were centrifuged (1 min, 3000g) and the residual concentrations of dyes in the supernatant solutions were similarly analyzed using uv–vis spectrophotometer. the spent noac separated from the solution was washed with di-water to remove the unabsorbed dyes and then oven dried at 70 °c for 3 h. to choose the effective solvent for desorption of the retained dyes on noac, 30 mg of the loaded adsorbent was separately added into 100 ml of various acid-base solutions (hcl, naoh, h2so4, ch3cooh) with different concentrations (1–3 mol l-1). the resulting mixtures were agitated for the same time duration as the adsorption tests (i.e., 8 min) in the ultrasonic bath (40 khz, 130 w, 25 °c). after desorption, the concentrations of by13 and br46 desorbed, cde (mg l -1), were similarly determined using uv–vis spectrophotometer. the desorption percentage was also calculated. results and discussion characterization of adsorbent tem image (fig. 1a) of noac shows the porous structure of this sample with pore sizes less than 50 nm. moreover, as can be seen in fig. 1, the carbon layers shift to the graphene sheet as indicated by the arrow. this structure causes an increase in the surface area and the pore volume of the nanoadsorbent. the sem image (fig. 1b) also confirmed the porous morphology of the sorbent. the activation stage produced the extensive external surfaces with quite irregular cavities and pores. the nitrogen adsorption isotherms for the pristine and the nitrogen-modified nanoporous ac samples are displayed in fig. 1c. the corresponding isotherm curves of both samples exhibit isotherms of type i, which is the characteristic of the microporous materials. the bjh pore size distribution curves (fig. 1d) for both samples confirm the presence of the microporosity and also of small mesopores in the range of 2–5 nm. the textural properties of ac and noac are presented in table i. the results show that the el-based nanoporous ac have the extremely high bet surface area of 2222 m2 g–1 and the significant micropore volume of 0.83 cm3 g–1. the obtained bet surface area is attributed to the decline of o2, h2 and n2 contents of the precursor, during successive activation steps, creating a nanoporous carbon adsorbent with high porosity and surface area. after the modification, the surface area decreased to 1572 m2 g–1 with the decrease of the micropore volume. this is due to the oxidation-nitrogenation reactions, which produced functional groups on the surface of ac. figs. 1e and f show the raman spectra of ac and noac. as it can be seen in fig. 2e, the virgin ac shows two broad peaks at about 1345 and 1594 cm–1, assigned to d-band and g-band, respectively. for noac (fig. 1f), d and g bands were slightly shifted up to 1350 and 1596 cm–1, respectively, which may be related to the changes in the hydrogen content of ac sample. the id/ig ratio dyes removal using modified actvated carbon 657 (the intensity ratio of the d and g bands) for ac and noac was found to be 1.46 and 1.43, respectively, which confirms the presence of the defective and the amorphous carbon structures in the both samples. moreover, a shoulder which appears at around 1110–1140 cm–1 is attributed to the presence of some sp2-hybridized structures in both samples. fig. 1. a) tem image of noac, b) sem image of noac, c) nitrogen adsorption isotherms of ac and noac, d) bjh pore size distribution of ac and noac, e) raman spectrum of ac, and f) raman spectrum of noac. 658 khaligh et al. table i. textural properties of pristine and nitrogen-modified ac sample bet suface area m2 g-1 total pore volume cm3 g-1 micropore volume cm3 g-1 mesopore volume cm3 g-1 average pore diameter, 4vtotal/sbet, nm average particle size, nm ac 863 2.67 52.2 0.42 0.84 5.59 noac 626 2.62 42.2 0.27 0.41 5.59 c, h, n, o contents of the pristine and modified ac samples were presented in table ii. from the results, compared with the pristine ac, oac had higher o content, originating from the oxidation reaction. also, it is obvious that the urea treatment of the oac led to an incorporation of a significant amount of nitrogen, instead of the surface oxygen groups and therefore noac has the low o content, compared to oac. table ii. elemental analysis of pristine and modified ac samples (content, wt. %); o content = 100 – chn content element sample o n h c 11.57 0.62 2.74 85.07 ac 23.15 0.85 1.78 74.22 oac 15.15 5.98 1.85 77.02 noac oxidation and nitrogenation of ac were further confirmed using the boehm titration method. as can be seen in table iii, the oxidation of ac increased total acidity, especially the value of carboxylic acid groups. modification of oac with urea resulted in the replacement of the surface oxygen groups with the nitrogen functional groups such as amine groups, so the basicity increased and the total acidity decreased. the ft-ir spectra of the modified ac samples were also recorded. details about the ft-ir spectra are presented in supplementary material (fig. s-1a and b). table iii. content of functional groups of pristine and modified ac samples (mmol g-1) determined by boehm titration method sample group phenolic lactonic carboxylic total acidic basic ac 0.210 0.096 0.184 0.480 0.425 oac 0.510 0.643 1.024 2.177 0.231 noac 0.129 0.105 0.124 0.357 1.826 for the determination of phpzc of the synthesized samples, the final ph values were plotted versus their corresponding initial ph values (fig. s-2 of the supplementary material). the phpzc was taken as the point at which the curve crossed the line; phfinal equals to phinitial. from the results, phpzc of ac, oac dyes removal using modified actvated carbon 659 and noac were 6.3, 5.5 and 7.9, respectively. it is obvious that the phpzc of noac is higher than 7 (in the basic range), due to having the nitrogen functional groups that are naturally basic; while ac and oac were acidic, due to the acidic oxygen functionalities. the analysis of the single solutions and simultaneous analysis of the binary solutions after preparation of the single solutions of by13 and br46 with the concentration of 10 mg l–1, the zero order absorption spectra of these solutions were recorded between 200 and 800 nm (fig. 2a). the maximum absorbance of br46 and by13 dyes in their single solutions were obtained at 530 and 411 nm, respectively. as shown in fig. 2a, the absorption spectrum of by13 dye in binary solution overlapped. hence, it was not possible to estimate the amount of by13 dye by the direct absorbance measurement in binary solutions. this problem was solved using the derivative spectrophotometric method. the first-order derivative spectrophotometry (fig. 2b) showed that the wavelength of 385 nm should be used for the analysis of by13 in the binary dye solution, where the absorbance of br46 is zero. therefore, the first and the zero order derivative spectrophotometric methods were utilized for the analysis of by13 and br46 in binary solutions at 385 (1d385) and 530 nm (0d530), respectively, and then calibration curves were made in the concentration range of 2–20 mg l–1 of the binary dyes solution (r2 of 0.9995 and 0.9990 was found, respectively). fig. 2. spectra of by13 and br46 in single and binary solutions: a) zero order spectra; b) first order derivative spectra (initial dye concentration of 10 mg l-1). in order to evaluate the accuracy of the zero and the first order derivative methods for determining of the br46 and by13 concentrations in binary solutions, the recovery studies were done. for this, the binary dye solutions with 660 khaligh et al. different concentrations of both components were prepared; then the absorbance spectra of these solutions were taken and differentiated (fig. s-3 of the supplementary material). the calibration curve of each dye was made and the concentration of the br46 and by13 dyes was easily determined (see table s-iii of the supplementary material). the recoveries, and the errors between the measured (cm) and theoretical (ct) concentrations were calculated. from table s-iii, the high and reasonable recoveries (95–103 %) and the low error values (<5 %) show the efficiency of this method for the accurate analysis of both dye concentrations in binary solutions. optimization of noac-based adsorption process for simultaneous removal of br46 and by13 dyes in order to obtain the maximum removal percentages of both dyes, the effects of different parameters such as ph, adsorbent dose, sonication time, initial dye concentration and temperature on the simultaneous dye removal were studied and optimized using the batch adsorption method (fig. 3a–d). fig. 3. effect of different parameters on the adsorption of by13 and br46 dyes onto noac: a) solution ph for both the ac and noac adsorbents, b) ultrasonication time, c) adsorbent dosage and d) temperature; conditions: 100 ml of 150 mg l-1 dyes solution, a) t = 6 min, m = 30 mg, t = 25 ºc; b) ph 9, m = 30 mg, t = 25 ºc; c) ph 9, t = 8 min, t = 25 ºc; d) ph 9, m = 30 mg, t = 8 min. dyes removal using modified actvated carbon 661 effect of the solution ph the influence of the solution ph on the simultaneous removal of by13 and br46 dyes from binary solution by the pristine and the nitrogen modified ac samples was separately studied over the ph range of 2–10 in conditions of 150 mg l–1 of the binary dyes solution, 30 mg adsorbent, 6 min ultrasonication time and 25 °c, according to the general adsorption procedure. as depicted in fig. 3a, using noac adsorbent (phpzc 7.9) the removal percentages of both dyes were improved with the increase of solution ph from 2 to 9 and then constant to 10. consequently, ph 9 was chosen as optimum for all further studies. the electrostatic interactions control the adsorption of ionic compounds significantly. for the low ph values (ph < phpzc), since the surface charge of the adsorbent is positive (because of the protonation of nitrogen and oxygen-containing groups), the electrostatic repulsion between the cationic dye molecules and the positively charged adsorbent surface decreases the adsorption of both dyes. for high ph values (ph > phpzc), the surface of the adsorbent is charged negatively (due to the deprotonation reaction) and the adsorption of dyes increases due to the electrostatic attraction between the negatively charged adsorbent surface and the cationic dye molecules. as shown in fig. 3a, using the pristine ac (phpzc 6.3) the similar observations were also obtained, whereas, the removal percentages of the cationic dyes were low and the quantitative removal values (< 60 %) were observed in the ph range of 7–10 (ph > phpzc). this is due to the lower density of oxygen-containing groups on ac, which deprotonated at this ph range. so, it can be concluded that physisorption is the main mechanism for dye adsorption by pristine ac. effect of ultrasonic-assisted adsorption time the ultrasound irradiation is well known to accelerate a chemical process, due to the phenomenon of the acoustic cavitation. recently an ultrasonic assisted adsorption process has been developed to favour the kinetic of the mass-transfer process of the adsorbate to the adsorbent and to reduce the time required for adsorption.18 in this study, the different ultrasonication times from 2 to 12 min were investigated with 100 ml of 150 mg l–1 binary dyes solution (ph 9) containing 30 mg of noac at 25 °c. fig. 3b indicates that with the increase in time, the adsorption rate of the both br46 and by13 dyes over the adsorbent increased and the adsorption process reaches the equilibrium in only 8 min and then remained constant up to 12 min. from the results, 8 min ultrasonication time was required to bring the complete saturation of the active sites of adsorbent, which is excellent for the synthesized noac; this was therefore selected as the optimum ultrasonication time for further experiments. 662 khaligh et al. effect of adsorbent amount to optimize the adsorbent dosage for the removal of by13 and br46 from aqueous solutions, the adsorption experiments were performed using different adsorbent amounts (5–35 mg) at the optimum ph and contact time, according to the general procedure. as shown in fig. 3c, the removal percentage of cationic dyes increased from 45.3 to 98.5 % for br46 and from 56.1 to 99.6 % for by13, with the increase of the adsorbent dose from 5 to 30 mg. this result is due to the increase in the adsorbent surface area and the availability of more adsorption sites stemming from the increased dose. however, no significant changes in the removal efficiency were observed for higher adsorbent doses (>30 mg) due to the fact that the dyes concentration on the surface of the adsorbent and in the solution came to equilibrium with each other. accordingly, 30 mg of noac was used in all subsequent experiments. effect of temperature the batch adsorption of by13 and br46 were carried out at five different temperatures in the range of 288–328 k with 100 ml of 150 mg l–1 binary dyes solution and 20 mg of noac at ph 9, and the ultrasonication time of 8 min. as can be seen in fig. 3d, the removal efficiencies of both dyes by noac increase with the rise in solution temperature from 15 to 55 °c; suggesting that the adsorption process is endothermic. the kinetic energy of dyes increases with increasing temperature and this situation leads to an increased tendency of dyes to towards the adsorbent surface and consequently more adsorption onto noac. adsorption isotherms the equilibrium isotherm plays an important role in the predictive modelling for the analysis and the design of adsorption systems. in this study, the dye removal process was analyzed using the well-known langmuir, freundlich and tempkin models, according to the procedure given in “experimental” with the various initial dye concentrations and constant values of the other parameters, for both the single and binary solutions. the isotherm constants calculated from the linear forms of the langmuir, freundlich and tempkin isotherms, along with the regression coefficient values (r2) for three experimental conditions were listed in table iv. the comparison of the correlation coefficients (r2) of the linear isotherm plots indicated that for the adsorption of dyes onto noac in the single and binary solutions, the langmuir isotherm yielded a much better fit than the other models at all dye concentrations (fig. s-4 of the supplementary material). it was observed that the equilibrium uptake amounts of by13 and br46 dyes in binary mixture onto noac decreased considerably with the increase of the concentration of the other dye, resulting in their antagonistic effect. as it can be seen from table s-iv, the dyes removal using modified actvated carbon 663 values of rl were in the range of 0–1, confirming the favourable uptake of both dyes onto noac. therefore, the uptake of cationic dyes by noac in single and binary systems preferably followed the monolayer adsorption process. table iv. isotherm parameter values for removal of by13 and br46 by noac from single and binary solutions (sample volume 100 ml, ph 9, m = 30 mg, t = 8 min, t = 25 °c) system dye c0 mg l-1 langmuir freundlich temkin qmax mg g-1 kl l mg-1 r 2 kf n r 2 kt l mg-1 bt kj mol-1 r 2 single by 200–400 1250 0.186 0.993 528.48 5.72 0.985 21.33 149.97 0.962 br 200–400 1111 0.188 0.995 519.47 6.90 0.985 26.05 138.05 0.966 binary (1)a by 150–350 909 0.324 0.997 523.22 8.83 0.961 177.24 751.24 0.929 br 150–350 769 0.342 0.999 454.86 8.74 0.990 466.85 71.10 0.989 binary (2)b by 150–350 833.3 0.265 0.993 509.28 8.63 0.978 757.48 75.98 0.967 br 150–350 555.6 0.414 0.999 511.68 22.03 0.431 47.1×107 25.31 0.403 abinary system with constant concentration of the other dye :150 mg l-1; bbinary system with variable concentrations of the other dye: 150–350 mg l-1 comparison of the developed noac-based adsorption method with others a comparison of the maximum adsorption capacity and the main adsorption factors of the developed noac-based batch adsorption method with the other published adsorption methods for the removal of br46 and by13 dyes from single and binary systems2,14,22–27 were reported in table v. obviously, the adsorption capacity of noac used in this study is significantly high for single and binary systems. this may be attributed to the acid-base character of the carbon surface, its high surface area, the micropore volume and the pore size distribution. furthermore, the type and the concentration of the active sites of adsorbents are responsible for the variation in maximum adsorption capacities between the adsorbents. as can be further seen from table v, the considerably short contact time (8 min) along with the economic consumption of the small amount of adsorbent (30 mg) for the developed noac-based adsorption process is a big improvement for the dye removal from single and binary solutions. therefore, noac can be considered to be an efficient and a potential adsorbent for the removal of br46 and by13 from single and binary solutions. adsorption kinetics the characteristic constants of the adsorption kinetics were determined using the pseudo-first-order, the pseudo-second-order and the intraparticle diffusion models at various ultrasonication times (2–6 min) and the optimized values of the other parameters, according to the procedure described in “experimental”. the calculated parameters and their corresponding r2 values are presented in table vi. it can be seen that the pseudo-second-order kinetic model provides a good correlation coefficients (r2 close to 1) which suggests that the simultaneous 664 khaligh et al. adsorption of by13 and br46 on noac can be better represented by the pseudo-second order model (fig. s-5). this model is based on the assumption that the rate limiting step may be a chemical sorption involving the valence forces through sharing or exchange of electrons between the adsorbent and the adsorbate. table v. comparison of the optimized conditions and maximum adsorption capacity of the noac-based adsorption method with other reported methods for removal of br46 and by13 from single and binary solutions dye adsorbent system optimized conditions qmax / mg g -1 ref. dye concentration, mg l-1 adsorbent dose, g l-1 contact time min by13 br46 br46, by28 bentonite sa bb 100–400 1.00 120 – s: 333 b: 208 2 bb41, br18 and br46 graphene oxide s b 50 0.16 60 – s: 476 b: 625 14 br46 cerbera odollam ac s 60 0.20 120 – 456 22 br46 ni oxide nps-diatomite s 25–55 0.05 60 – 124.35 23 br 46, by28 boron industry waste s 50–300 2.00 60 – 74.73 24 by13 wheat bran s 50–1000 4.00 30 69.06 – 25 by13 apricot stone ac s 50–1000 6.00 35 134.59 – 26 by13 sepiolite s 25–500 0.50 180 62.5 – 27 br 46, by13 noac s 200–400 0.30 8 1111 1250 this study b 150–350 0.30 8 769 909 asingle; bbinary table vi. kinetic parameter values for removal of by13 and br46 by noac from binary solutions (100 ml of 150 mg l-1 dyes solution, ph 9, m = 30 mg, t = 2–6 min, t = 25 °c) kinetic model constant metal ion by13 br46 pseudo-first order q1 / mg g -1 335.49 348.94 k1 / min −1 0.263 0.219 r2 0.981 0.978 pseudo-second order q2 / mg g -1 555.56 526.32 k2 / g mg -1 min-1 10.41×10-4 9.49×10-4 r2 0.9935 0.9909 intra-particle diffusion ci 305.40 270.32 ki / mg min -1/2 g-1 3.755 3.874 r2 0.9561 0.9480 dyes removal using modified actvated carbon 665 adsorption thermodynamics the thermodynamic behaviour of the simultaneous adsorption of by13 and br46 on noac were further investigated in the temperature ranges of 288–328 k, according to the procedure given in the “experimental”. the thermodynamic parameters such as gibbs energy (δg), enthalpy (δh) and entropy change (δs0) were calculated.21 the thermodynamic parameters are presented in table vii. for the adsorption of both dyes onto noac, the obtained δh values were positive which indicates the endothermic nature of the adsorption process, which is in good agreement with the results that show that the adsorption of dyes increased with the temperature. the negative δg values showed the spontaneous nature of the adsorption process. also, the positive values of δs show the increased randomness at the solid/solution interface. table vii. thermodynamic parameters for removal of by13 and br46 by noac from binary solutions (100 ml of 150 mg l-1 dyes solution, ph=9, m=30 mg, t= 8 min) dye parameter t / k 288 298 308 318 328 by13 ∆h / kj mol-1 36.51 36.51 36.51 36.51 36.51 ∆g / kj mol-1 –5.513 –6.825 –8.125 –9.640 –11.418 ∆s / kj mol-1 k-1 0.146 0.146 0.146 0.146 0.146 br46 ∆h / kj mol-1 34.57 34.57 34.57 34.57 34.57 ∆g / kj mol-1 –2.499 –5.133 –8.329 –12.365 –17.334 ∆s / kj mol-1 k-1 0.136 0.136 0.136 0.136 0.136 desorption and regeneration studies an ideal adsorbent should exhibit not only the high sorption capacity, but also the easy regeneration and stability during several adsorption–desorption cycling, which can significantly reduce the overall cost of the adsorbent. desorption studies also help to elucidate the nature of adsorption and the recycling of the spent adsorbent and the dye.20 first, the best desorption solvent was selected according to the procedure explained in the “experimental” and then the number of the adsorption-desorption cycles using this solvent were investigated. fig. s-6a and b show the effects of various solvents for desorption of by13 and br46 dyes from noac, respectively. among these, hcl (2.0–3.0 mol l–1) and h2so4 (3.0 mol l–1) solutions provided higher desorption efficiency (r ≈ 100 %) for both by13 and br46. thus, in order to use low concentration of an organic solvent, hcl (2.0 mol l–1) was specified as the best solvent for the quantitative desorption of dyes. this indicates that the adsorption of dyes onto noac is performed through ion exchange. in an acidic medium due to the protonation, the electrostatic interactions between the adsorbent surface and the cationic dyes become weaker, and then the adsorbed dyes leave the adsorption site. 666 khaligh et al. in order to investigate the long-term stability of noac, it was subjected to several adsorptions and desorption cycles under the optimized conditions, as described above. the adsorption process was started with fresh noac and after the desorption step, using 100 ml of hcl (2.0 mol l–1), the regenerated adsorbent was reused for the subsequent adsorption-desorption cycle. as shown in fig. 4, the noac nanosorbent can be used for 5 adsorption–desorption cycles without the decrease in the removal percentages of by13 and br46. at the sixth cycle, the removal percentages of by13 and br46 were decreased to 96 and 94 %, respectively. however, after 16 adsorption-desorption cycles, the removal percentages of by13 and br46 were decreased to 16% and 45%, respectively. fig. 4. reusability of the noac under the optimized conditions. conclusions in the present paper, the eucalyptus leaves-based nanoporous activated carbon was prepared via the chemical activation with koh, oxidized with nitric acid, then modified with urea, and finally introduced as a new and efficient adsorbent for simultaneous ultrasound assisted removal of br46 and by13 dyes from binary solutions. the ac and noac both generated the high specific surface area and the high micro pore volume. the first-order derivative spectrophotometric method was successfully applied for the determination of by13 in binary solutions. the optimum conditions of batch adsorption were found to be ph 9, initial br and by concentrations of 150 mg l–1, noac dosage of 30 mg, sonication time of 8 min, and temperature 25 °c. the data indicated that the adsorption kinetics of dyes on noac followed the pseudo-second order. the equilibrium data were correlated considerably well by the langmuir adsorption isotherm in both the single and binary mixtures. based on the thermodynamic studies, the adsorption of dyes was both endothermic and spontaneous. the rem dyes removal using modified actvated carbon 667 arkable advantages of the developed noac-based adsorption process include its high removal performance (>98.5 %) for a very short time and the economic consumption of only 30 mg of noac adsorbent which has superior adsorption capacities and can also be regenerated and reused for 5 adsorption-desorption cycles. in conclusion, noac can be effectively used as an adsorbent for the removal of cationic dyes from single and binary systems. supplementary material aditional data and information are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. the authors wish to thank semnan university research council, semnan, iran, and iranian research institute of petroleum industry (ripi) for financial support of this work. и з в о д нитрогенизовани активни угаљ од еукалиптусовог лишћа са нанопорама за ултразвучно уклањање основних боја уз коришћење деривативне спектроскопије aisan khaligh1, hassan zavvar mousavi1, alimorad rashidi2 и hamid shirkhanloo3 1 department of chemistry, semnan university, semnan 35131-1911, iran, 2 nanotechnology research center, research institute of petroleum industry (ripi), west entrance blvd., olympic village, tehran 14857-33111, iran и 3 research institute of petroleum industry (ripi), west entrance blvd., olympic village, tehran 14857-33111, iran активни угаљ од еукалиптусовог лишћа са нанопорама (ac) је припремљен од еукалиптусовог лишћа хемијском активацијом помоћу koh, а затим третиран азотном киселином/уреом (noac) и коначно употребљен као нови адсорбенс за истовремено ултразвучно уклањање основне црвене 46 (br46) и основне жуте 13 (by13) боје из бинарних водених раствора. наноадсорбенс noac је oкарактерисан sem, tem, раман, bet, ftir, cxn, phpzc и boehm титрационим анализама. и ac и noac узорци су имали супериорну bet површину од 2222 и 1572 m 2 g -1 са просечном запремином микропора од 0,81 и 0,50 cm 3 g -1 , редом. за анлизу by13 бинарних смеша коришћена је деривативна спектроскопија првог реда. мала количина адсорбента (30 mg) је била способна да уклони висок проценат боја (>99 %) за врло кратко време (8 min). адсорпција боја је пратила лангмирову изотерму и кинетику псеудопрвог реда. адсорпциони капацитети noac за просте растворе br46 и by13 били су 1111 и 1250 mg g -1 , а за бинарне растворе 769 и 909 mg g -1 , редом. испитана је и адсорпциона термодинамика. истрошени noac је регенерисан коришћењем hcl (2 m) и поново употребљен за пет циклуса адсорпција– –десорпција уз веома успешне перформансе. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {optimization of phenol biodegradation by immobilized bacillus subtilis isolated from hydrocarbons contaminated water using the factorial design methodology} j. serb. chem. soc. 84 (7) 679–688 (2019) udc 547.562+504.4.054:582.261: jscs–5218 542.92.000.57 original scientific paper 679 optimization of phenol biodegradation by immobilized bacillus subtilis isolated from hydrocarbons-contaminated water using the factorial design methodology hamida hamdi* and amina hellal ecole nationale polytechnique, laboratoire des sciences et techniques de l’environnement, 10 avenue hacen badi, bp182 el harrach, 16200 algiers, algeria (received 4 december 2018, revised 8 march, accepted 11 march 2019) abstract: the ability of newly isolated bacteria, identified as bacillus subtilis immobilized on alginate hydrogel beads, to degrade phenol was investigated under different parameters, such as phenol concentration, bead diameter and inoculums size, and was optimized using full factorial design methodology. a mathematical model that governs the degradation of phenol by the immobilized system was obtained and it fitted the experimental data very well. the model indicated that within the range of variables employed, all the parameters and their interactions influenced the biodegradation process, whereby the phenol concentration was the most significant factor. b. subtilis revealed a very high degradation activity and could be grown using phenol as the sole source of carbon. phenol was degraded by the new bacteria in 8 h under the optimum conditions obtained by the desirability function: 100 mg l-1 phenol concentration, 3 mm beads diameter and 244.5 mg of cell dry per liter biomass size, with a desirability value of 91.25 %. keywords: alginate beads; biodegradation; immobilized system; phenol. introduction environmental pollution is one of the major problems and the most important in the world. the development in agriculture, energy sources, and chemical industries is necessary in order to fulfill the needs and demands of the ever-growing human population. almost all processes employed by man for the production of goods and services lead to the production of environmental pollutants.1,2 several physico–chemical methods for removing organic pollutants have been proposed, but these methods are not cost effective for large-scale applications. nowadays, biological methods are widely used as low-cost alternative treatment methods, which offer the possibility of complete mineralization of organic compounds.3 * corresponding author. e-mail: hamida.hamdi@g.enp.edu.dz https://doi.org/10.2298/jsc181204022h 680 hamdi and hellal some of these pollutants are biologically recalcitrant and inhibitory organics, which greatly reduces the ability of microorganism to biodegrade the compounds during treatment or in nature.4,5 for this reason, the use of degradation of the pollutants by free system is not preferable. immobilized systems in which cells are entrapped in polymeric matrices6 have received increasing attention in the field of contaminant treatment and may be an effective and economical technique. immobilization provides higher cell concentrations, ease of use in continuous reactors, shorter start-up period for the bioreactor, and greater stability by protecting cells from direct exposure to toxic compounds compared with freely suspended cells and hence they can be stored for long periods without losing their degradative abilities.6–8 the rate and efficiency of the biodegradation of pollutants by immobilized systems depend on several factors. among these parameters are initial concentration of pollutant, bead diameter and biomass size.9,10 the factorial experimental design has been used for studying, developing and optimizing a wide range of engineering systems.11,12 factorial designs are widely used to investigate the effects and influence of experimental factors and the interactions between them.12 the advantages of factorial experiments include the relatively low cost, a reduced number of experiments, and increased possibilities to evaluate interactions among the variables.12,13 in this study, the biodegradation of phenol by immobilized system using a newly isolated bacteria strain (isolated and identified in the authors’ laboratory), was optimized by application of a factorial design methodology. this mathematical method was used to determine the main and the interactive effects of different variables on the immobilized system and to predict a model that evaluates the process. experimental culture media the growth medium (gm) of the strain consisted of peptone, 10.0 g l-1; beef extract, 3.0 g l-1 and nacl, 5.0 g l-1, at a ph of 7.0, autoclaved at 121 °c for 15 min. the mineral culture medium (mm) that contained the necessary nutrients for the growth of microorganisms was composed of: kh2po4, 1.5 g l-1; k2hpo4, 0.5 g l-1; nacl, 0.5 g l-1; mgso4·7h2o, 0.5 g l-1; nh4no3, 3.0 g l-1; feso4·7h2o, 0.02 g l-1 and cacl2·2h2o, 0.02 g l-1.8 the mm was used for the isolation and the biodegradation tests with phenol as the sole carbon and energy source. solidified with agar, this medium (mma) was used for preservation of the strains. the nutrient agar (na) contained 28 g of powdered nutrient agar dissolved in 1 l of distilled water and autoclaved at 121 °c for 15 min. isolation, selection and purification of new bacterial strains samples of water contaminated by hydrocarbons were collected from the petroleum refinery situated at hassi massoud, south of algeria. the contaminated water was inoculated on agar mineral culture medium (mma) supplemented with 100 mg l-1 of phenol and incubated for 7 days. the development of colonies was optimization of the phenol degradation by immobilized system 681 monitored every day. well-isolated colonies were subcultured on gm. this procedure was repeated several times. phenol tolerant bacterial strains were preserved on mma at 4 °c. the purity of the cultures was regularly checked during preservation. identification and characterization of the isolated strain the genera and/or species of the most active selected strain were identified by performing gram staining, morphological characteristics and biochemical tests (using api system tests). dry-cell-weight determination inoculums size was measured using a spectrophotometer (shimadzu uv–vis 1240) at 600 nm and the concentration, expressed in cell dry weight, were determined from previously established curves. phenol dosage phenol was quantified by a colorimetric method based on the condensation of 4-aminoantipyrine with phenol in the presence of an oxidizing agent, potassium ferricyanide, in an alkaline medium to give a red complex.14,15 the absorbance was measured at 510 nm using a shimadzu uv–vis 1240) spectrophotometer. immobilization of bacteria on calcium alginate beads first, the pure strain was cultivated into 100 ml of gm at 37 °c. bacterial cells were then harvested at the stationary phase by centrifugation (6000 rpm at 4 °c for 10 min), washed three times with the phosphate buffer solution and suspended in sterile mm. the bio-beads of calcium alginate were obtained by inclusion of microorganisms in sodium alginate and then application of the extrusion technique. this method involves the preparation of an aqueous solution of sodium alginate (3 mass%) in which microorganisms were incorporated. the suspension containing the cells with the sodium alginate solution was extruded as drops into 100 ml of a 0.1 m calcium chloride solution. the formed beads were transferred into a solution of 0.2 m cacl2 and incubated at 37 °c for 2 h to allow for complete replacement of the sodium ions by calcium to ensure a better stability. after washing with sterile distilled water, the beads were stored at 4 °c for preservation.3 biodegradation tests biodegradation tests by the immobilized system were performed in a batch bioreactor at 37 °c under aerobic conditions. a control test without bacteria allowed for verification of whether the phenomenon of adsorption or other interactions between the alginate beads and the phenol occurred. the reduced phenol was less than 2 %, which is the result of the chemical method used for the determination of phenol. factorial design methodology factorial design methodology was used to determine the main and interactive effects of different variables on the immobilized system and to predict a model to evaluate the process. the principle steps of factorial designed experiments are determination of the response that reflects the aim of the study, factors and their levels and choice of the experimental design and statistical analysis of the data. a full 23 factorial design was performed to evaluate the importance of three factors: the initial phenol concentration (x1), the diameter beads (x2), and the inoculums size expressed in dry cell weight (x3) on the response (y), which is the time, expressed in h, required for total phenol removal. the number of experiments conducted is considered as 23. 682 hamdi and hellal the high and low levels defined for the 23 factorial design are listed in table i. the low and high levels for the factors were selected according to some preliminary experiments. table i. factors and levels used in the factorial design independent variable coded variables level low (–1) center (0) high (+1) x1 concentration of phenol, mg l -1 100 500 900 x2 diameter beads, mm 3 4 5 x3 dry cell weight, mg l -1 244.5 317.7 390.9 the results were analyzed with jmp® 8 software, and the main effects and interactions between factors were determined. results and discussion isolation and selection of phenol degrading strains ninety bacterial strains were isolated and purified from contaminated waters. among these strains, 44 were able to degrade phenol. of these 44 strains, only 7 started growth from the second day. the rest of the strains started development from the third day. the period of appearance of the colonies constitutes a lag phase necessary for the adaptation of these strains to the new carbon source. the faster the growth of the strain, the better its adaptation to the pollutant. for this reason, one strain taken of the seven most active strains was chosen for the study. identification and characterization of phenol degrading strain the identification and characterization of the isolated bacterial was performed using preliminary analysis: morphological and cultural characteristics (table s-i of the supplementary material to this paper) and biochemical tests (table s-ii of the supplementary material). the identification of the strain by the preliminary analysis confirmed that the strain was b. subtilis species. biodegradation of phenol by the immobilized bacteria factorial design methodology. the design matrix of the coded values for factors and the response y, measured in each factorial experiment, is shown in table ii. the response represents the time for total degradation. the data listed in table ii indicate a wide variation in the response y, from 8 to 103, in the 10 trials. application of factorial design methodology as an optimization technique requires the selections of a model at the beginning. the model employed for 23 factorial designs with interaction is represented by eq. (1):16–18 y = a0 + a1x1 + a2x2 + a3x3 + a12x1x2 + a13x1x3 + a23x2x3 + + a123x1x2x3 (1) optimization of the phenol degradation by immobilized system 683 table ii. experimental values for the total time of degradation experiment number x1 x2 x3 y (response) 1 –1 –1 –1 08 2 +1 –1 –1 78 3 –1 +1 –1 09 4 +1 +1 –1 75 5 –1 –1 +1 10.5 6 +1 –1 +1 77 7 –1 +1 +1 13 8 +1 +1 +1 103 9 0 0 0 46 10 0 0 0 46 in eq. (1), y is the estimated response that represents the time of total degradation, a0 is the independent coefficient (a constant term), ai (i = 1, 2, 3) are the linear coefficients for the variables: phenol concentration, bead diameter and inoculums size, respectively, aij represents the coefficients of the interaction parameters xi and xj with i < j. the coefficients ai, aij and aijk were determined using jmp® 8 software.17,19 substitution of coefficient ai, aij and aijk in eq. (1) by their values, the mathematical model selected is the following: y = 46.55 + 36.5625x1 + 3.3125x2 +4.187 x3 + 2.4375x1x2 + + 2.5625x1x3 + 3.8125x2x3 +3.4375x1x2x3 (2) eq. (2) showed how the experimental variables and their interactions influence the time of total degradation.13,20 the phenol concentration (x1) had the greatest effect on y, followed by the inoculum size, bead diameter–inoculum size interaction (x2x3), phenol concentration–bead diameter–inoculum size interaction (x1x2x3), bead diameter (x2) and phenol concentration–bead diameter interaction (x1x2). according to the obtained model, all parameters had a positive effect on the response. the positive sign shows that there is a direct relation between the parameter and the dependent variable. thus, an increase in the phenol concentration, bead diameter or inoculums size increases the time of total degradation. the predicted values vs. the experimental values of the time of total degradation of phenol are shown in fig. 1. the model presented a high square correlation coefficient (r2) of 99.993 % and an adjusted square correlation coefficient (r2 adj) of 99.9697 %, which confirmed that the chosen of 23 factorial design with the interaction model was found to be adequate for the prediction within the range of variables employed.18,21–23 the student’s t-test the significance of the regression coefficients was determined by applying a student’s t-test (table iii).13,16 684 hamdi and hellal fig. 1. comparison of the experimental and the estimated responses at the levels of the variable. table iii. estimated regression coefficients for the time of total degradation parameter estimate coefficients standard error t-value p-valuea constant 46.55 0.194454 239.39 ˂.0001* x1 36.5625 0.217407 168.18 ˂.0001* x2 3.3125 0.217407 15.24 0.0043* x3 4.1875 0.217407 19.26 0.0027* x1x2 2.4375 0.217407 11.21 0.0079* x1x3 2.5625 0.217407 11.79 0.0071* x2x3 3.8125 0.217407 17.54 0.0032* x1x2x3 3.4375 0.217407 15.81 0.0040* ap-value < 0.05 the student’s t-test and p-value were used to determine the significance of the regression coefficients of the parameters. with the 95 % confidence level and two degrees of freedom, the value of t-critic is equal to 2.92. the coefficient of the regression is statistically significant if the corresponding t-value is higher than 2.92. it was observed from table iii that the linear effects of phenol concentration, bead diameter, and inoculum size and the interaction effect between these factors were significant. the values for each effect are shown in the pareto chart by the horizontal columns (fig. 3). the importance of the data can also be judged by their p-value, which is the probability value that is used to determine the statistically significant effects in the model, with values closer to zero denoting greater significance. for the 95 % confidence level, the p-value should be less than or equal to 0.05 for the effect to be considered statistically significant.24,25 hence, the final model is given in eq. (3): y=46.55+36.5625 x1 + 3.3125 x2 +4.1875 x3 + 2.4375 x1x2 +2.5625x1x3 + 3.8125 x2x3 +3.4375x1x2x3 (3) optimization of the phenol degradation by immobilized system 685 interaction plots the plots of the interaction effects are shown in fig. 2. an interaction is effective when the change in the response from low to high levels of a factor is dependent on the level of a second factor, i.e., when the lines do not run parallel.13 thus, significant interactions between the parameters could be concluded. fig. 2. interaction plot for the time of total degradation (x1: phenol concentration, mg l-1; x2: diameter of beads, mm; x3: dry-cell weight, mg l-1). the standardized pareto chart (fig. 3) depicts the main effect of the independent variables and interactions on the time of total degradation. the length of each bar in the graph indicates the effect of these factors and the level of their effects on the response.26,27 fig.3. pareto chart of the standardized effects. this graphic (fig. 3) shows both the magnitude and the importance of the effects (variables and interactions).26 according to fig. 3, it could be inferred that the main factors (x1, x2 and x3) and their interactions (x1x2, x1x3, x2x3 and x1x2x3) were significant at the 0.05 level. the concentration of phenol represents the most significant effect on the time of total degradation. 686 hamdi and hellal analysis of variance (anova) the adequacy of the model was checked using analysis of variance (anova), as shown in table iv.18,23 table iv. analysis of variance (anova) source sum of squares df mean square f-value t-f model 11233.469 7 1604.78 4244.5 19.4 residual (error) 0.756 2 0.38 prob ˃ f correlation total 11234.225 9 0.0002 the f-value of the model obtained (4244.5) was higher than the tabular value t-f (f0.05,5,4 = 19.4), suggesting that the model is highly significant. estimation of optimal design conditions by the method of desirability function. the optimization by the desirability function improved the performance of the analytical process and discovering the conditions at which the best response is obtained.28 the optimum condition for the degradation of phenol by the immobilized process was obtained from the desirability plot (fig. 4). x1 x2 x3 desirability x d es ira bi lit y y / h fig. 4. desirability functions for the optimization of the response (x1: phenol concentration, mg l-1); x2: bead diameter, mm; x3: dry-cell weight, mg l-1). the best combination of factor settings for achieving the optimum response (minimum time of total degradation) was found to be phenol concentration 100 mg l–1, bead diameter 3 mm and inoculums size 244.5 mg l–1 for the predicted response of 7.86 h and a desirability value of 91.25 % (fig. 4). conclusions in conclusion, a new bacterial strain capable of degrading phenol was isolated from hydrocarbons contaminated water, collected from the petroleum refin optimization of the phenol degradation by immobilized system 687 ery, situated in the south of algeria. it was identified as b. subtilis species based on morphological and cultural characteristics and biochemical tests the b. subtilis species presented a very high activity for phenol biodegradation. the best time of complete degradation by immobilized cells determined using factorial design methodology was 7.86 h, which corresponds to the optimum conditions: phenol concentration 100 mg l–1, bead diameter 3 mm and biomass size 244.5 mg l–1. the concentration of phenol represented the most significant effect on the total time of degradation. full factorial design methodology can be an efficient method for testing the effect of operating conditions and optimize the degradation of phenol by the studied immobilized process. supplementary material additional data are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. и з в о д оптимизација биодеградације фенола имобилизованом бактеријом bacillus subtilis изолованом из воде контаминиране угљоводоницима применом методе факторијалног дизајна hamida hamdi и amina hellal ecole nationale polytechnique, laboratoire des sciences et techniques de l’environnement, 16200 algiers, algeria испитана је примена новоизоловане бактерије bacillus subtilis, имобилизоване на честицама алгинатног хидрогела, за деградацију фенола, варирајући концентрацију фенола, пречник честица и величину инокулума, а за оптимизацију је коришћена метода потпуног факторијалног дизајна. израђен је математички модел разлагања фенола имобилизованим системом и он се добро уклапао са експерименталним резултатима. према моделу, све променљиве које су тестиране, као и њихов примењени опсег, су утицале на процес биодеградације, а концентрација фенола је била најутицајнији фактор. показано је да b. subtilis испољава јаку деградациону активност и да може расти користећи фенол као извор угљеника. ова бактерија разлаже фенол у року од 8 h са 91,25 % ефикасности, под следећим оптималним условима: концентрација фенола 100 mg l-1, пречник честица 3 mm и 244,5 mg сувих ћелија по литру биомасе. 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paper 369 preparation of composite zein/natural resin nanoparticles ljiljana spasojević1, jaroslav katona1*, sandra bučko1, lidija petrović1, jelena milinković budinčić1, jadranka fraj1, altynay sharipova2 and saule aidarova2,3 1university of novi sad, faculty of technology novi sad, bul. cara lazara 1, 21000 novi sad, serbia, 2kazakh national research technical university, almaty, 050013, kazakhstan and 3kazakh–british technical university, almaty, 050000, kazakhstan (received 1 april, revised 1 july, accepted 5 july 2019) abstract: the aim of this work was to investigate a possibility of preparing composite zein/natural resin (shellac and rosin) nanoparticles by antisolvent co–precipitation from their aqueous ethanol solutions. influence of zein/resin mass ratio (1/0, 0.8/0.2, 0.5/0.5, 0.4/0.6 and 0/1) and ph (2–12) on particle size, d, and zeta potential, ζ, of the prepared particles was studied. the functional properties of zein/rosin composite nanoparticles were evaluated by studying carvacrol encapsulation. it was shown that the antisolvent precipitation can be successfully used to prepare the shellac and rosin nanoparticles, as well as the composite zein/shellac and the zein/rosin nanoparticles. colloidal properties, d and ζ, of the obtained nanoparticles are influenced by the zein/ /resin mass ratio and the ph of nanoparticles’ dispersions. the isoelectric point of composite nanoparticles can be modulated by varying the zein/resin mass ratio. it was found that the zein/rosin nanoparticles are suitable for carvacrol encapsulation, where carvacrol release is enhanced by increasing the rosin share in the composite zein/rosin nanoparticles. keywords: zein; shellac; rosin; biopolymer; encapsulation. introduction there has been a great interest in developing novel biopolymer nanoparticles, with tuned properties, which are aimed at different niche applications such as encapsulation, drug delivery, and agro-formulations.1–3 to this purpose, various procedures are developed, and a multitude of biomaterials are used for preparing biopolymer nanoparticles. in this regard, zein is especially interesting due to its commercial availability, gras status, plant origin, and unique physicochemical properties. * corresponding author. e-mail: jkatona@uns.ac.rs https://doi.org/10.2298/jsc190401071s ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 370 spasojević et al. zein is a corn protein, comprising 50 % of all corn proteins. raw zein consists of α, β, γ and δ protein fractions, differing in their structure and molecular weight, with α-zein being the most abundant. commercial zein, however, is almost exclusively α-zein, consisting of two polypeptide chains of 19 kg mol–1 and 22 kg mol–1.4,5 zein is a prolamin. it is therefore insoluble in water, and soluble in concentrated aqueous ethanol (60–90 % ethanol). it is also soluble in a range of other primary (e.g., acetic acid, glycerol, and propylene glycol), secondary (e.g., mixtures of water and acetone, isopropanol, or methanol) and ternary (e.g., mixtures of water, lower aliphatic alcohol and acetaldehyde or benzene) solvents.4,6 zein is a film forming material, and it is commercially used for preparing edible films to extend shelf life of fresh fruits.7 on the other hand, zein nanoparticles are readily prepared by antisolvent precipitation, where zein solution, typically in aqueous ethanol, is sheared into a bulk of water to precipitate zein.8 zein nanoparticles were evaluated for encapsulation of different bioactive materials,9,10 as stabilizers for pickering emulsions,11 or, e.g., as white pigments for liquid products.12 composite zein nanoparticles, where zein is combined with another biomaterial (e.g., gum arabic, caseinate, propylene glycol alginate, carboxymethyl chitosan) were also prepared, in order to adjust various properties of nanoparticles (e.g., colloidal stability, wetting properties, encapsulation efficiency etc.).13–16 composite nanoparticles are usually prepared by shearing aqueous ethanol zein solution into a water solution of a biopolymer. in such composite particles, zein core is typically surrounded by a biopolymer or a polymeric surfactant layer, where, during anti-solvent precipitation, zein firstly precipitates to form nanoparticles, and subsequently a biopolymer attaches to the surface of the particles via electrostatic or hydrophobic forces. on the other hand, when fast diffusing, small surfactant molecules are used (e.g., tween 20), zein–surfactant interaction and complex formation take place prior to zein precipitation, and zein–surfactant complexes aggregate to form composite nanoparticles.17 this strategy for preparing zein-based composite nanoparticles, works only for water soluble biopolymers or surfactants. there is, however, also an interest in preparing composite nanoparticles of zein with water insoluble compounds, such as natural resins shellac and rosin (i.e., colophony). shellac is the purified product of the natural material lac, which is secreted by parasitic insect kerria lacca. it is a complex mixture of esters and polyesters of polyhydroxy acids. it has gras status, and is used in food industry as a coating for citrus fruits or confectionaries.18 rosin is derived from the oleoresin of pine trees. it consists mainly of resin acids (90 %) that are diterpene monocarboxylic acids. it is widely used in industry as a component of varnishes, adhesives or drug coatings.19 both shellac and rosin are insoluble in water, and well soluble in concentrated aqueous ethanol. to the best of our knowledge, pre________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ preparation of composite zein/resin nanoparticles 371 paration of composite zein/shellac and zein/rosin nanoparticles was thus far not investigated. the aim of this work was therefore to investigate the possibility of preparing composite zein/shellac and zein/rosin nanoparticles by antisolvent co-precipitation of zein/shellac and zein/rosin, from their aqueous ethanol solutions. the influence of zein/resin mass ratio on colloidal properties (particle size and zeta potential) of composite nanoparticles, as well as on encapsulation of carvacrol, was investigated. experimental zein, shellac, and carvacrol (5-isopropyl-2-methylphenol) were obtained from sigma– –aldrich, usa. colophony (rosin) was obtained from fluka chemie ag, switzerland. nonionic surfactants tween 20 (polyoxyethylenesorbitan monolaureate) and tween 80 (polyoxyethylenesorbitan monooleate) were obtained from serva electrophoresis gmbh, germany. ethanol (96 %) was purchased from reahem, serbia, sodium hydroxide was obtained from centrohem, serbia and 36 % hydrochloride acid was obtained from zorkapharm, serbia, and all were of p.a. quality. demineralized water was used in all experiments. preparation of stock solutions 25 g of 2 mass % zein, shellac and rosin solutions in 90 vol. % aqueous ethanol were prepared, in 100 ml flask with the ground joint, by constant stirring on a magnetic stirrer during 1 h. the solutions were left over night in the sealed container at room temperature to ensure full dissolution of zein and resins. the solutions were subsequently filtered through quantitative filter paper to remove any undissolved particles. 2 mass % zein/shellac (0.8/0.2 and 0.5/0.5 mass ratio) and zein/rosin (0.8/0.2 and 0.4/0.6 mass ratio) stock solutions were prepared in the similar manner, where zein and resin (in an appropriate mass ratio) were firstly jointly dissolved in 90 vol. % aqueous ethanol, while the rest of the procedure was the same as for the pure zein, shellac and rosin nanoparticles. preparation of nanoparticles dispersions of zein, shellac, rosin, as well as composite zein/shellac and zein/rosin nanoparticles in water were prepared by antisolvent precipitation,8 where 20 ml of a stock solution was dropwise added to 80 ml of water, in a 200 ml flask with the ground joint, with constant stirring on a magnetic stirrer. the stirring was continued 30 min after all of the stock solution was added to water, in order to ensure complete precipitation of zein. ethanol was removed from the dispersions by rotary evaporation (rotavapor r300, büchi, switzerland), and thereby aqueous dispersions of zein nanoparticles were obtained. thus obtained dispersions were diluted to 0.1 % concentration of nanoparticles, and ph of the dispersions was then adjusted (ph 2–11) by 0.1 m hcl or 0.1 m naoh. the dispersions were left over night at room temperature, when ph was checked and, if necessary, adjusted, prior to further use. particles size and zeta potential measurements zeta potential (ζ) and average intensity diameter (d) of zein nanoparticles were measured by dynamic light scattering (dls), using zetasizer nano zs (malvern instruments ltd, uk), at 25 °c. folded capillary cell (dts 1060) was used for zeta potential measurements, and disposable polystyrene cuvette (dts 0012) for particle size measurements. zeta potential and ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 372 spasojević et al. particle size were measured at least three times for each sample, and average values were reported. solubilization of carvacrol maximum additive concentration (mac) of carvacrol in 1 % tween 20 and 1 % tween 80 solutions was determined spectrophotometrically (halo db-20s uv–vis double beam, dynamica, switzerland) in order to evaluate suitability of using tween solutions as a sink medium during testing the release of encapsulated carvacrol. carvacrol was added in 15 μl aliquots to 100 ml of 1 % tween 20 and 1 % tween 80 solutions in an extraction funnel, to obtain mixtures with varying carvacrol concentrations (from 0.015 to 0.18 vol. % v/v). the mixtures were vigorously shaken, and absorbance at 275 nm was measured. the mixtures became turbid and the absorbance sharply increased when mac was reached, indicating the saturation of tween micelles with solubilized carvacrol and emulsification of unsolubilized carvacrol. the emulsion droplets scatter light and cause an apparent increase in absorbance. encapsulation of carvarcol carvacrol was added (5 mass %, based on zein/rosin mass) to 2 mass % zein/rosin solution in 90 vol. % aqueous ethanol, with zein/rosin mass ratio 1/0, 0.8/0.2 and 0.6/0.4. thus prepared mixture was dropwise added to a bulk of water to prepare the zein/rosin nanoparticles encapsulating carvacrol, in the same fashion as preparing the pure zein/resin nanoparticles. thus prepared suspensions of nanocapsules were ultrafiltered (l/s economy drive, masterflex, usa, peristaltic pump; vivaflow 200 crossflow cassette with 10000 hy membrane, sartorius, germany; at trans membrane pressure of 3 bar) in order to remove ethanol and non-encapsulated carvacrol. 100 ml of nanocapsule suspensions were ultrafiltered, until 60 ml of permeate was collected. the concentrate was then diluted with water to obtain 100 ml of purified nanocapsule suspension. the ultrafiltration step was then repeated for three more times to further purify the suspension. 3 ml of the purified nanocapsule suspension were diluted with 3 ml of 2 % tween 20 solution. the mixture was then left on a magnetic stirrer during 24 h to allow the release of carvacrol into the sink medium. the nanocapsules were then precipitated by adding 0.01 m naoh or 0.01 m hcl. the precipitate was separated from supernatant by 0.45 μm filtration (cellulose nitrate membrane filter, sartorius, germany), and carvacrol content in the supernatant was determined spectrophotometrically (halo db-20s uv–vis double beam, dynamica, switzerland) by measuring absorbance at 275 nm. results and discussion zein, shellac and rosin share a common property of being soluble in concentrated aqueous ethanol, but insoluble in water. therefore, antisolvent precipitation of these biomaterials takes place when their aqueous ethanol solutions are added to a bulk of water, and consequently nanoparticles of zein, shellac and rosin may be prepared.8 colloidal properties of zein, shellac and rosin nanoparticles dispersions of zein (z), shellac (s) and rosin (r) nanoparticles were prepared by the antisolvent precipitation. the influence of ph on zeta potential of the nanoparticles is shown in fig. 1. it can be seen that zein nanoparticles are positively charged at ph lower than 6, while at higher ph they acquire negative ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ preparation of composite zein/resin nanoparticles 373 charge. the point of zero charge is around six, in agreement with the literature data, where commonly reported isoelectric point for zein nanoparticles is 6.2.20 zein powder dissolves at highly alkaline ph > 11 and forms transparent solutions, as it was visually observed. stable dispersions of shellac and rosin nanoparticles were prepared at ph 4–9 and ph 3–8, respectively. throughout these phs, both shellac and rosin nanoparticles have negative charge, fig. 1. the negative charge comes from ionized carboxylic groups of esters and polyesters of polyhydroxy acids in shellac (mainly aleuritic, butolic, shellolic and jalaric acid), and from resin acids in rosin (mainly isomeric abietic acids).19,21 shellac and rosin nanoparticles precipitate at ph 3 and 2, respectively, as zeta potential approaches zero. isoelectric point of zein, shellac and rosin nanoparticles, as obtained by zeta potential measurements and visual observations are given in fig. 2. shellac and rosin nanoparticles dissolve at phs higher than 9 and 8, respectively. fig. 1. influence of ph on zeta potential of zein (z), shellac (s) and rosin (r) nanoparticles. fig. 2. isoelectric point of zein (z), shellac (s) and rosin (r) nanoparticles. the influence of ph on particle size of zein (z), shellac (s) and rosin (r) particles prepared by the antisolvent precipitation is shown in fig. 3. it can be seen that ph has no influence on size of shellac nanoparticles, where the particles with d = 100 nm were obtained at all phs tested. on the other hand, ph influences the size of both zein and rosin particles. zein nanoparticles with the small________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 374 spasojević et al. est particle size of d ≈ 100 nm were obtained at the lowest and the highest phs tested, ph 3 and 4 and ph 9 and 10, respectively. particle size increases as ph approaches pi of zein, due to a decrease in particle charge, which favours particle agglomeration. in the like manner, the largest rosin nanoparticles with d = 370 nm were prepared at ph 3, which is close to pi of rosin. at ph 4, 5 and 6, rosin nanoparticles with d = 330 nm are obtained, and in this ph range the particle size does not change. at still higher phs, ph 7 and 8, the particle size decreases to d = = 300 nm and d = 220 nm, respectively. the decrease in particle size at ph 7 and 8 is attributed to the increased rosin dissolution at the increased ph values.22 there are three competing mechanisms during antisolvent precipitation which influence size of the obtained particles.8 these are: 1) breakup of aqueous ethanol solution droplets by shear forces on impacting bulk of water; 2) coalescence of the droplets in analogy to emulsions; 3) solidification of dissolved material during solvent attrition. dynamics of these three processes is influenced by different parameters such as ethanol concentration in aqueous ethanol solution, concentration of a material dissolved in aqueous ethanol, applied shear forces, etc. fig. 3. influence of ph on particle size of zein (z), shellac (s) and rosin (r) nanoparticles. colloidal properties of composite zein/shellac and zein/rosin nanoparticles the suspensions of composite zein/resin particles, prepared by the antisolvent precipitation of dissolved biopolymer mixtures in water, were diluted and suspensions’ ph was adjusted. the images of zein/shellac dispersions at various phs (3–11) and at different zein/shellac mass ratio (1/0, 0.8/0.2, 0.5/0.5 and 0/1) are shown in fig. 4. the dispersed particles scatter light and make dispersions turbid. 1/0 z/s samples at ph 11 and all other z/s samples at ph 10 and 11 appear transparent, indicating the dissolution of z/s particles, where both, zein and shellac are known to dissolve at aqueous alkaline.18,23 the precipitation of z/s particles was observed at ph 6 for 1/0 z/s, at ph 5 and 6 for 0.8/0.2, at ph 5 for 0.5/0.5 z/s, and at ph 3 for 0/1 z/s particles. the precipitation took place due to the low particle charge and thereby to the decreased electrostatic repulsion between the particles in the ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ preparation of composite zein/resin nanoparticles 375 vicinity of isoelectric point of the particles, which makes the particles colloidally unstable. the influence of ph on zeta potential of z/s particles of different zein/ /shellac mass ratio is shown in fig 5a. it can be seen that the point of zero charge shifts to lower ph values, as mass ratio of shellac in composite z/s particles increases, in agreement with the visual observations. we were unable to prepare the z/s particles at z/s ratio >0.5/0.5 due to the excessive coagulation during the antisolvent precipitation. the influence of ph on particle size of z/s particles of different z/s mass ratio is shown in fig. 5b. it can be seen that the prepared z/s dispersions contained nano–sized particles, where particles size increases as ph approaches pi of the corresponding z/s particles. the influence of ph on particle size becomes more prominent as the zein portion in z/s particles increases. fig. 4. dispersions of composite zein/shellac (z/s) nanoparticles of different z/s mass ratio (1/0, 0.8/0.2, 0.5/0.5 and 0/1), and of different ph (3–11). fig. 5. influence of ph on zeta potential (a) and particles size (b) of composite zein/shellac (z/s) nanoparticles of different z/s mass ratio (1/0, 0.8/0.2, 0.5/0.5 and 0/1). ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 376 spasojević et al. the dispersions of composite zein/rosin particles at different z/r mass ratio and at different ph are shown in fig. 6. similarly to z/s dispersions, z/r dispersions appear turbid, unless particles are precipitated or dissolved. it can be seen that by increasing the rosin share, z/r particles dissolve at lower ph, where 1/0, 0.8/0.2 and 0.4/0.6 z/r particles dissolve, by visual judgment, at ph 11, 10 and 8, respectively. in the like manner, the point of zero charge of z/r particles decreases on an increase in rosin share, as evidenced by the zeta potential measurements, supported by the visual observations, figs. 6 and 7a. the prepared z/r dispersions contained nano–sized particles, where composite z/r nanoparticles proved to be much smaller than pure rosin nanoparticles, fig. 7b. fig. 6. dispersions of composite zein/ /rosin (z/r) nanoparticles of different z/r mass ratio (1/0, 0.8/0.2, 0.4/0.6 and 0/1), and of different ph (2–12). fig. 7. influence of ph on zeta potential (a) and particles size (b) of composite zein/rosin (z/r) nanoparticles of different z/r mass ratio (1/0, 0.8/0.2, 0.4/0.6 and 0/1). as seen in figs. 4, 5b, 6 and 7b, the composite z/r nanoparticles dissolve at lower ph compared to z/s nanoparticles, closer to neutral ph, which is desirable for spontaneous release of active compounds in natural mediums. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ preparation of composite zein/resin nanoparticles 377 within stable dispersions, at ph 3–4 and 8–9, both z/s (fig. 5b) and z/r (fig. 7b) nanoparticles are about the same size, or smaller than other previously reported nanoparticles based on zein.7,24,25 carvacrol was also encapsulated in pure zein nanoparticles, with diameter of 293.6 nm at ph 4,9 which is about twice larger than nanoparticles prepared in this paper. smaller particles are prepared from less concentrated stock solutions. same authors also report the average diameter decrease at ph 10.9 encapsulation of carvacrol in composite zein/rosin nanoparticles encapsulation of carvacrol in composite zein/rosin nanoparticles, prepared at different z/r mass ratios, is further investigated. carvacrol is a hydrophobic compound, insoluble in water. in order to study the release of encapsulated carvacrol in water, an appropriate method had to be identified. hydrophobic compounds are known to be soluble in the presence of surfactants above cmc, which are often used to improve the solubility of hydrophobic compounds in water.26 therefore, the solubilization of carvacrol in 1 % tween 20 and 1 % tween 80 water solutions are studied spectrophotometrically, in order to investigate if the tween solutions are suitable to be used as the test medium for the carvacrol release from composite zein/rosin nanoparticles. influence of carvacrol concentration on absorbance of 1 % micellar solutions of tween 20 and tween 80 is shown in fig. 8. it can be seen that at low carvacrol concentrations, the solutions remain optically transparent, where the absorbance of both tween 20 and tween 80 solutions remains close to zero, indicating that all of the added carvacrol is solubilized in tween micelles. on further carvacrol addition, the absorbance of tween solutions sharply increases when a maximum additive concentration (mac) is reached. it was found that mac for 1 % tween 20 and 1 % tween 80 is 0.86 and 1.16 g l–1, respectively. the sharp increase in the absorbance is due to the emulsification of unsolubilized carvacrol. namely, at carvacrol concentrations higher than mac, all tween micelles are saturated with carvacrol, and any further addition of carvacrol leads to carvacrol emulsification and droplet formation. the emulsified droplets scatter light and the mixtures become optically turbid, which consequently leads to an increase in measured absorbance.26 the obtained mac values shows that both 1 % tween 20 and 1 % tween 80 solutions successfully solubilized carvacrol, and that both solutions can be used as the test medium for the release of encapsulated carvacrol. 1 % tween 20 was chosen for carvacrol release experiments, because of better correlation coefficient (r2) between absorbance value and concentration of carvacrol solubilized, compared to r2 when 1 % tween 80 is used (data not shown). carvacrol was encapsulated in composite z/r nanoparticles with different z/r mass ratio (1/0, 0.8/0.2, 0.6/0.4 and 0/1). the influence of rosin share in composite zein/rosin nanoparticles on carvacrol released after 24 h in 1 % tween ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 378 spasojević et al. 20 solution is shown in fig. 9. it can be seen that the composition of z/r nanoparticles influence the release, where on the increasing rosin share more carvacrol is released. fig. 8. influence of carvacrol concentration on absorbance of 1 % tween 20 and 1 % tween 80 solutions in water. the arrows indicate the maximum additive concentration (mac). fig. 9. influence of rosin share in composite zein/rosin nanoparticles on the released carvacrol. conclusion the antisolvent precipitation was successfully used to prepare shellac and rosin nanoparticles, as well as the composite zein/shellac (z/s) and the zein/rosin (z/r) nanoparticles with different zein/resin mass ratio (1/0, 0.8/0.2, 0.5/0.5 and 0/1 for z/s, and 1/0, 0.8/0.2, 0.4/0.6 and 0/1 for z/r nanoparticles). particle size, zeta potential and isoelectric point of the obtained nanoparticles was influenced by the zein/resin mass ratio, as well as by the dispersion’s ph. it was found that the zein/rosin nanoparticles are suitable for carvacrol encapsulation, where an increase in the rosin share in composite zein/rosin nanoparticles contributes to the carvacrol release. acknowledgments. this work was financed by ministry of education, science and technological development of republic of serbia (grant no. iii 46010/3), provincial secretariat for higher education and scientific research of the autonomous province of vojvodina (grant no 142-451-3680/2017-01/01) and ministry of education and science of the republic of kazakhstan (grant no. 2018/ap05131968). it was done within cost action ca15216 framework. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ preparation of composite zein/resin nanoparticles 379 и з в о д припрема композитних наночестица зеин/природна смола јарослав катона1, љиљана спасојевић1, сандра бучко1, лидија петровић1, јелена милинковић будинчић1, јадранка фрај1, altynay sharipova2 и saule aidarova2,3 1 универзиет у новом саду, технолошки факултет нови сад, бул. цара лазара 1, 21000 нови сад, 2 kazakh national research technical university, almaty, 050013, kazakhstan и 3 kazakh–british technical university, almaty, 050000, kazakhstan циљ рада био је испитивање могућности припреме композитних наночестица зеина и природних смола (шелака и калафонијума), растворених у етанолу, применом копреципитације у нерастварачу. проучаван је утицај масеног удела зеин/смола (1/0, 0,8/0,2, 0,5/0,5, 0,4/0,6 и 0/1) и рн вредности (2–12) на величину и зета потенцијал припремљених наночестица. могућност инкапсулације карвакрола у наночестицама зеин/калафонијум испитивана је у циљу карактерисања њихових функционалних особина. показано је да је применом преципитације у нерастварачу могуће припремити наночестице шелака и калафонијума, као и композитне наночестице зеин/шелак и зеин/калафонијум. такође, резултати су показали да масени однос зеин/смола и рн вредност суспензије наночестица, утичу на колоидне особине, односно на пречник и зета потенцијал наночестица. на изоелектричну тачку наночестица може се утицати променом односа зеин/смола. показано је да су наночестице зеин/ /калафонијум погодне за инкапсулацију карвакрола, те да се отпуштање карвакрола из честица повећава са повећањем удела калафонијума у саставу наночестица. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {accelerated solvent extraction of carrot response surface methodology optimization} j. serb. chem. soc. 83 (11) 1223–1228 (2018) udc 633.43+66.061.3:547.565: jscs–5145 544.032.732:615.279 short communication 1223 short communication accelerated solvent extraction of bioactive compounds from carrot – optimization of response surface methodology vanja šeregelj1#, vesna tumbas šaponjac1*#, anamarija mandić2, gordana ćetković1#, jasna čanadanović-brunet1#, jelena vulić1# and slađana stajčić1# 1faculty of technology, university of novi sad, bulevar cara lazara 1, 21000 novi sad, serbia and 2institute of food technology, university of novi sad, bulevar cara lazara 1, 21000 novi sad, serbia (received 19 march, revised 9 august, accepted 10 august 2018) abstract: carrot is considered to be rich in bioactive antioxidants, both lipophilic (carotenoids) and hydrophilic (phenolic compounds). in the present study, the conditions for accelerated solvent extraction (ase) of bioactive compounds from carrots (daucus carota l.) were optimized using response surface methodology (rsm). box–behnken design was employed for the experimental design to obtain the optimized combination of extraction temperature, time, and number of extraction cycles. total carotenoid content (tcar), total polyphenol content (tph), free radical scavenging activity (sa) and reducing power (rp) of the obtained extracts were used as responses for the optimization. considering the four quality indicators, the ideal extraction conditions were found to be: 120 °c, 60 min and three extraction cycles. under these conditions, predicted values of 28.84 mg β-carotene/100 g for tcar; 530.81 mg gae/100 g for tph; 2572.29 μmol te/100 g for sa and 1336.26 μmol te/100 g for rp were obtained with high desirability (0.975) and no significant difference (p < 0.05) with the experimental values. keywords: carotenoids; polyphenols; scavenging activity; reducing power; extraction. introduction bioactive compounds in plants have gained popularity for their beneficial effects on human health.1 storage root of carrot (daucus carota l.) provides an important source of carotenoids and polyphenols in human diets. the extraction process is the first step towards recovery of natural antioxidants from plants. * corresponding author. e-mail: vesnat@uns.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc180531068s ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 1224 šeregelj et al. optimum extraction methods depend on the characteristics of the bioactive compounds and the diversity of the tissue plant structures.2 therefore, optimization of the extraction conditions is a starting point in obtaining the highest yields of target compounds. accelerated solvent extraction (ase) is an automated extraction technique that uses elevated pressure and temperature to achieve efficient extraction in a very short time, using lower solvent volume and resulting in higher extraction yields.3 mustafa et al. reported that both time and temperature and the interaction between these two factors significantly affect the extraction yield of carotenes.4 the aim of this study was to optimize the extraction conditions, i.e., time, temperature and number of extraction cycles, for isolation of bioactive compounds from carrots using the ase technique, taking into account the total carotenoid and polyphenol contents, free radical scavenging activity and reducing power. experimental plant material fresh carrots (daucus carota l.) were purchased from a local supermarket. after washing, fresh carrots were chopped in a kitchen blender (b 800 e, gorenje, slovenia), freeze -dried at –40 °c (martin crist alpha 2-4, osterode, germany), ground and stored at –20 °c until use. experimental design the optimization of the extraction conditions was established by response surface methodology (rsm). the experimental plan was based on three variables at three levels, referred to as box–behnken design. the design consisted of 15 experimental runs, including three replicates at the central point.5 the independent variables were extraction time (x1: 20–60 min), temperature (x2: 40–120 °c) and number of extraction cycles (x3: 1–3). the coded values of the independent variables were –1, 0 and 1. the actual values were chosen from the preliminary studies, and the corresponding coded values of three independent variables are given in table i. accelerated solvent extraction procedure a dionex ase 350 (thermo scientific, waltham, ma, usa) system was used for the extraction of the carrots using 100 % ethanol. for this purpose, a stainless-steel dionex cell was filled with a diatomaceous earth (to reduce the volume of the extraction solvent) and the carrot sample (0.5 g) in the ratio 4:1. to prevent the collection of suspended particles in the extract, a cellulose filter was placed at the bottom of the cell. finally, the cell was placed in the cell tray and used for extraction under the conditions obtained from the rsm guided experimental design. glass vials were used to collect the extracts, which were stored at −20 °c until further use. total carotenoid content (tcar) the contents of carotenoids in the carrot extracts were analyzed spectrophotometrically by the method of nagata and yamashita6 using the extraction solvent as blank. the content of total carotenoids was calculated using the equation: tcar (mg β-carotene per100 ml) = 0.216a663–1.22a645–0.304a505+0.452a453 (1) ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. accelerated solvent extraction of bioactive compounds from carrot 1225 where a663, a645, a505 and a452 represent the absorbance measured at 663, 645, 505 and 453 nm, respectively. the total carotenoid content is expressed as mg of β-carotene equivalents per 100 g sample. total polyphenol content (tph) the total polyphenol content in carrot extracts was determined spectrophotometrically by folin–ciocalteau method adapted to microscale.7 the results are expressed as gallic acid equivalents (gae) per 100 g sample. radical scavenging activity by dpph assay (sa) the levels of free radical scavenging activity (sa) of the carrot extracts on the 2,2 -diphenyl-1-picrylhydrazyl (dpph•) radical were measured spectrophotometrically in a 96 -well microplate, according to girones-vilaplana et al.8 the sa values were calculated using the following equation: sa = 100(ac − as)/ac (2) where ac is the absorbance of the control and as is the absorbance in the presence of extracts. the results are expressed as µmol trolox equivalents (te) per 100 g of sample. reducing power (rp) the reducing power of the extracts was determined by the method of oyaizu9 adapted for a 96-well microplate. a calibration curve was made with trolox and the results are expressed as µmol te per 100 g of sample. results and discussion in order to understand the effect of the extraction parameters on the efficiency of carotenoids and polyphenols extraction, and antioxidant activity of carrot extracts, experimental design was prepared and evaluated by rsm (table i and figs. s-1 and s-2 and table s-i of the supplementary material to this paper). the medium time (40 min), lowest temperature (40 °c) and one extraction cycle (experiment 9) were the least suitable for the isolation of carotenoids, while the longest extraction (60 min), medium temperature (80 °c) and 3 cycles (experiment 8) yielded the highest amount of tcar. mustafa et al. obtained the highest yield of αand β-carotene in extracts of carrot by-products using the following ase extraction conditions: 60 °c and 10 min extraction time (5 cycles of 2 min each).4 however, the carrot samples were fresh and the pressure was constant (50 bar), while in the present study, the carrot samples were freeze-dried and the pressure varied in dependence on the temperature. the amount of tcar in the carrot extracts ranged from 8.42–29.01 mg 100 g–1, which corresponds with the reports of mustafa et al.4 for the content of αand β-carotene in carrot by-products (10.3–27 mg 100 g–1). the highest tph amount was obtained using the highest temperature (120 °c), with 3 cycles and a moderate duration of the process (40 min, experiment 12), whereas the lowest tph was obtained when the extraction was performed longer (60 min), in 2 cycles and at a relatively low temperature (40 °c, experiment 2). liu et al. reported that elevated temperature improves diffusion rates and solubility in extraction solvents.10 according to ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 1226 šeregelj et al. herrero et al. ase with high temperature is an effective way to increase the recovery of bioactive compounds.11 longer extraction times, higher extraction temperature, and more extraction cycles also resulted in higher sa and rp of the extracts. both sa and rp reached the highest values (2615.99 and 1324.80 μmol te 100 g–1, respectively) when the extraction was performed using the parameters: 120 °c, 60 min and 2 cycles (experiment 4). table i. experimental design, total carotenoid (tcar) and polyphenol (tph) contents, radical scavenging activity (sa) and reducing power (rp) of carrot extracts; the results are presented as mean values of three replicates ± sd run t / min (x1) t / °c (x2) na (x3) tcarb tphc sad rpe 1 20 (–1) 40 (–1) 2 (0) 8.98±0.01 94.21±0.98 404.30±1.59 278.05±0.48 2 60 (+1) 40 (–1) 2 (0) 11.07±0.02 78.51±0.13 473.96±10.38 274.82±0.30 3 20 (–1) 120 (+1) 2 (0) 24.55± 0.01 322.71±0.69 1135.87±3.01 997.85±6.53 4 60 (+1) 120 (+1) 2 (0) 24.20±0.01 470.01±3.03 2615.99±23.41 1324.80±5.27 5 20 (–1) 80 (0) 1 (–1) 22.01±0.01 127.79±0.37 619.30±6.90 569.97±1.91 6 60 (+1) 80 (0) 1 (–1) 23.72±0.01 157.11±0.43 532.43±1.29 730.67±8.40 7 20 (–1) 80 (0) 3 (+1) 24.24±0.01 154.88±0.07 658.47±1.12 483.99±2.28 8 60 (+1) 80 (0) 3 (+1) 30.57±0.01 232.15±0.37 949.60±0.02 649.73±0.85 9 40 (0) 40 (–1) 1 (–1) 8.42±0.01 87.26±0.50 733.54±1.07 192.66±1.40 10 40 (0) 120 (+1) 1 (–1) 24.14±0.03 454.65±0.60 2356.49±1.41 921.57±0.81 11 40 (0) 40 (–1) 3 (+1) 12.57±0.002 107.79±0.82 657.94±3.26 129.56±1.00 12 40 (0) 120 (+1) 3 (+1) 29.01±0.02 472.74±0.84 2071.03±10.79 1040.04±6.07 13 40 (0) 80 (0) 2 (0) 26.02±0.02 144.14±0.19 824.66±2.28 469.38±1.86 14 40 (0) 80 (0) 2 (0) 26.95±0.01 143.26±0.01 997.41±2.11 429.32±0.73 15 40 (0) 80 (0) 2 (0) 27.50±0.01 208.80±0.21 837.78±1.72 514.45±2.13 anumber of cycles; bmg β-carotene/100 g; cmg gae/100 g; dμmol te/100 g; eμmol te/100 g the experimental values of all quality indicators obtained in the optimization experiments (table i) were analyzed by single and multi-response optimization and the results are reported in table ii and figs. s-1 and s-2. table ii. single and multi response optimization of the extraction parameters optimization variable code variable value optimal response x1 x2 x3 x1 x2 x3 tcara tphb sac rpd single response (tcar) 0 0.21 0.95 40 88.4 3 30.80 – – – single response (tph) 1 1 –1 60 120 1 – 472.78 – – single response (sa) 1 1 1 60 120 3 – – 2572.28 – single response (rp) 1 1 0.59 60 120 3 – – – 1325.67 multi response 1 1 1 60 120 3 28.84 530.81 2572.29 1336.26 amg β-carotene/100 g; bmg gae/100 g; cμmol te/100 g; dμmol te/100 g it was found that single-cycle extraction applying a high temperature (120 °c) for a longer time (60 min) ensures the maximum extraction of carotenoids. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. accelerated solvent extraction of bioactive compounds from carrot 1227 the optimal conditions maximizing tph were lower temperature and duration, in several cycles. considering sample 4 that showed the highest sa and rp, it could be concluded that the higher temperature (120 °c) and longer extraction time (60 min) are the main contributors to these responses. the simultaneous optimization of multiple responses is the main concern for industrial applications, especially in view of energy cost reduction. the optimal extraction conditions for all observed responses were 120 °c for 60 min with three extraction cycles. conclusions response surface methodology (rsm) and box–behnken design were developed to determine the optimum process parameters of carrot ase extraction. the optimal conditions to obtain the highest extraction yield of carotenoids and polyphenols in carrot extracts, as well as maximum antioxidant activity were: 120 °c, 60 min in 3 extraction cycles. under the optimal conditions, the experimental values were in agreement with the predicted values. supplementary material analysis of variance (anova) of the modelled responses, as well as singleand multi -response optimization of the influence of extraction parameters on the total carotenoid contents (tcar), total polyphenol contents (tph), scavenging activity (sa) and reducing power (rp) of carrot extracts are available electronically at the pages of journal website: http:// //www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. this work was supported by the ministry of education, science and technological development of the republic of serbia under grant no. tr 31044 and cost action 15136. и з в о д оптимизација екстракције биоактивних једињења из шаргарепе растварачима под притиском методом одзивних површина вања шерегељ1, весна тумбас шапоњац1, анамарија мандић2, гордана ћетковић1, јасна чанадановић-брунет1, јелена вулић1 и слађана стајчић1 1технолошки факултет нови сад, универзитет у новом саду, булевар цара лазара 1, 21000 нови сад и 2научни институт за прехрамбене технологије, универзитет у новом саду, булевар цара лазара 1, 21000 нови сад шаргарепа се сматра богатим извором биоактивних антиоксидативних једињења, и липофилних (каротеноиди) и хидрофилних (полифеноли). у овом раду извршена је оптимизација екстракције биоактивних компонената из шаргарепе (daucus carota l.) растварачима под притиском, употребом методе одзивних површина (rsm). експерименти су планирани употребом box–behnken дизајна у циљу одређивања оптималне комбинације екстракционе температуре, времена и броја циклуса екстракције. садржај укупних каротеноида (tcar), укупних полифенола (tph), способност хватања радикала (sa) и редукциона способност (rp) добијених екстраката коришћени су као одзиви за оптимизацију. узимајући у обзир ове одзиве добијени су следећи оптимални услови екстракције: 120 °c, 60 min и три екстракциона циклуса. под овим експерименталним условима предвиђене вредности tcar (28,84 mg β-каротена на 100 g), tph (530,81 mg ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 1228 šeregelj et al. gae на 100 g), sa (2572,29 μmol te на 100 g) и rp (1336,26 μmol te на 100 g) су добијене са високим фактором пожељности (0,975) и без статистички значајне разлике (p < 0,05) у поређењу са експериментално добијеним вредностима. (примљено 19. марта, ревидирано 9. августа, прихваћено 10. августа 2018) references 1. c. o. perera, g. m. yen, int. j. food prop. 10 (2007) 201 2. v. t. nguyen, recovering bioactive compounds from agricultural wastes, wiley, new york, 2017 3. j.-h. kang, s. kim, b. k. moon, food chem. 205 (2016) 140 4. a. mustafa, l. m. trevino, c. turner, molecules 17 (2012) 1809 5. v. tumbas šaponjac, j. čanadanović-brunet, g. ćetković, m. jakišić, s. djilas, j. vulić, s. stajčić, molecules 21 (2016) 584 6. m. nagata, i. yamashita, j. jpn. soc. food sci. technol. 39 (1992) 925 7. e. gonzález-molina, d. a. moreno, c. garcía-viguera, j. agric. food chem. 56 (2008) 8. а. girones-vilaplana, p. mena, d. a. moreno, c. garcia-viguera, j. sci. food agric. 94 (2014) 1090 9. m. oyaizu, jpn. j. nutr. 44 (1986) 307 10. h. liu, y. zhang, q. li, y. zou, j. shao, s. lan, j. liq. chromatogr. relat. technol. 34 (2011) 2653 11. m. herrero, m. castro-puyana, j. a. mendiola, e. ibañez, trac, trends anal. chem. 43 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {molecular design of orthogonal stacking system at the complex interface of htra pdz domain with its peptide ligands} j. serb. chem. soc. 84 (12) 1367–1379 (2019) udc 577.112+66–977:547.466.1:576:577.15 jscs–5270 original scientific paper 1367 molecular design of orthogonal stacking system at the complex interface of htra pdz domain with its peptide ligands dingwa zhang1, deyong he1, xiaoliang pan2, yaping xu1 and lijun liu1* 1school of chemistry and chemical engineering, jinggangshan university, ji’an 343009, china and 2school of mechanical and electrical engineering, jinggangshan university, ji’an 343009, china (received 21 december 2018, revised 2 march, accepted 11 april 2019) abstract: the high temperature requirement a (htra) protease plays a crucial role in protein quality control and cell fate. the enzyme contains a catalytic protease domain and a regulatory pdz domain; the latter determines the substrate specificity of the former by specifically binding to the c-terminal hydrophobic stretch of its partner proteins. previously, a pentapeptide ligand h3c1 was identified as the potential binder of htra pdz domain using phage display technique. here, an orthogonal π–cation–π stacking system at the crystal domain–peptide complex interface was analysed by integrating theoretical calculations and experimental assays. it was demonstrated that there is a strong (positive) synergistic effect between the two wings of the stacking system; breaking of cation–π interaction in one wing can largely impair the interaction strength of another wing. the π-electron contributes primarily to the synergistic effect, although geometric property is also (marginally) responsible for it. next, the systematic combinations between the four aromatic amino acids (phe, tyr, trp and his) plus one non-aromatic amino acid (ala) at the two wings of π–cation–π stacking were investigated. it was found that two aromatic substitutions (phe-4tyr and phe-4trp) at a wing can considerably and moderately improve peptide affinity by 3.2and 1.5-fold, respectively, whereas the non-aromatic mutations at each wing and at both of them can significantly reduce the affinity with kd increase from 1.8 (wild type) to 34 μm and 160 μm (single-point mutations), as well as 210 μm (double-point mutation), suggesting that just breaking of one wing can substantially undermine the synergism of orthogonal π–cation–π stacking. keywords: htra protease; htra pdz domain; peptide ligand; orthogonal π–cation–π stacking; synergistic effect. * corresponding author. e-mail: lij_liu@163.com https://doi.org/10.2298/jsc181221029z 1368 zhang et al. introduction the high temperature requirement a (htra) protease is a key enzyme involved in protein quality control, stress response, and cell fate,1 which is also functionally related to a variety of biological events such as cell proliferation, migration and apoptosis.2 the enzyme was initially identified in e. coli by two phenotypes of corresponding null mutants and named accordingly. mutants either did not grow at elevated temperatures (htra for high temperature requirement) or failed to digest misfolded protein in the periplasm (degp).3 subsequently, prokaryotic htra has been linked to the tolerance against various folding stresses as well as to pathogenicity. the protein is characterized by the presence of a catalytic serine protease domain (pd) followed by a regulatory post-synaptic density 95, drosophila discs large, zona occludens-1 (pdz) domain, which has been shown to act as substrate specificity determinant by binding to the c-terminal hydrophobic stretch of its protein partners, leading to structural change in pd domain and enzyme activation.4 htra pdz–partner interaction has been recognized as a new and attractive druggable target; molecular disruption of the interaction can inactivate the serine protease, which can be a potential therapeutic strategy for a variety of relevant diseases such as cancer, arthritis, neurodegenerative disorder and macular degeneration.5,6 previously, runyon et al. have identified a pentapeptide ligand h3c1 (phe-4-gly-3-arg-2-trp-1-val0-cooh) of htra pdz domain using phage display technique.7 the h3c1 peptide is bound in the active pocket of htra pdz domain to competitively block the domain interaction with its cognate partners. however, the peptide can only bind weakly to the domain with a moderate affinity. later, liu et al. described a rational halogenation strategy to improve biological activity for the peptide ligand, which introduced a geometrically satisfactory halogen bond at the domain–peptide complex interface by systematically optimizing the combination of halogen types and their substitution positions at the indole moiety of peptide trp-1 residue.8,9 however, chemical synthesis of non-natural halogenated peptides is technically sophisticated. in addition, the designed halogen bond is vulnerable to its protein environment as this type of noncovalent force is structurally exquisite and highly specific.10 runyon et al. fused h3c1 peptide into the c-terminus of htra pdz domain and then solved the crystal structure of the fusion protein system (pdb: 2p3w),7 which characterizes a homodimer of two h3c1 peptide-fused htra pdz domains in biological assembly form (fig. 1a), where the c-terminal h3c1 peptide of one domain is tightly bound to the active pocket of another (fig. 1b). it was revealed that the h3c1 peptide contains two aromatic residues trp-1 and phe-4; they can form an orthogonal π–cation–π stacking with the arg360 residue of htra pdz domain (fig. 1c). previously, we have successfully designed several protein–peptide interactions using computational modeling and affinity assay, design of π–cation–π stacking system 1369 including amphiphysin sh3–ppii peptide11 and egfr–mig-6 peptide.12 here, an attempt was made to investigate the synergistic effect between the two wings of the stacking by systematically mutating the two aromatic residues to other three natural aromatic amino acids (phe, tyr, trp and/or his) and one nonaromatic amino acid (ala), as well as their impacts on the domain–peptide binding. instead of halogenation strategy,13 the mutation does not introduce nonnatural amino acids and thus the designed peptide mutants can be readily obtained via standard phase peptide synthesis. in addition, the stacking interaction is insensitive to its protein environment and has only low geometric requirement. these features largely extend the applicability of the mutation strategy in rational peptide design. it was demonstrated that combinatorial optimization of the synergistic effect can effectively enhance the interaction potency of htra pdz domain with its peptide ligands, and fluorescence spectroscopy assays to substantiate the computational findings were performed. fig. 1. a) homodimer of two h3c1 peptide-fused htra pdz domains in the crystal structure of biological assembly (pdb: 2p3w). b) complex structure of htra pdz domain with h3c1 peptide extracted from the biological assembly. c) an orthogonal π–cation–π stacking is formed across the complex interface by domain positively charged residue arg360 and peptide aromatic residues trp-1 and phe-4. the stacking consists of two approximately perpendicular wings 1 and 2; each represents a cation–π interaction. 1370 zhang et al. experimental geometric description of orthogonal π–cation–π stacking system the geometric configuration of orthogonal π–cation–π stacking across the complex interface of htra pdz domain with h3c1 peptide is schematically illustrated in fig. 2. the wings 1 and 2 represent two perpendicular cation–π interactions of domain arg360 residue with peptide trp-1 and phe-4 residues, respectively. the angle between wings 1 and 2 is δ. the guanidyl group of domain arg360 residue, the indolyl group of peptide trp-1 residue and the phenyl group of peptide phe-4 residue are assigned within planes 1, 2 and 3, respectively. the dihedral angle between planes 1 and 2 is θ1, and between planes 1 and 3 is θ2. the distance between arg360 cζ atom and trp-1 aromatic ring center is d1, and between arg360 cζ atom and phe-4 aromatic ring center is d2. fig. 2. schematic representation of the geometric parameters of orthogonal π–cation–π stacking across the complex interface of htra pdz domain with h3c1 peptide. quantum mechanics/molecular mechanics analysis the residues trp-1 and phe-4 of h3c1 peptide in crystal complex structure with htra pdz domain (pdb: 2p3w) can be computationally mutated to other aromatic amino acids by using the rotamer-based scwrl4 program.14 the mutated complex structures were then subjected to structural optimization with a two-layered hybrid quantum mechanics/molecular mechanics (qm/mm).15 this method enables different levels of theory to be applied to different parts of a large biomolecular system. the qm/mm calculations were carried out using oniom algorithm.16 a scheme described in our previous work17 was employed to partition the pdz–peptide system, that is, the domain cationic residue arg360 as well as the peptide aromatic residues trp-1 and phe-4 involved in the π–cation–π stacking were included in high -level qm layer, while rest of the system was in low-level mm layer. hydrogen atoms were used as link atoms to saturate the dangling bonds. the qm layer was modeled by the semiempirical am1 theory,18 while the mm layer was described with biomolecular amber force field.19 other methods such as pm620 and b3lyp21 were not considered here since we have previously demonstrated that the am1/amber hybrid combination is comparable with or even better than that of first principle-based scheme, but has a low computational cost.22 the qm/mm calculations were carried out with gaussian 09 suite of programs;23 the inputs and outputs were prepared/examined using gaussview gui.24 design of π–cation–π stacking system 1371 the intermolecular interaction energy (∆u) of pdz–peptide complex was estimated with a strategy described by zhou et al.25-27 this was accomplished by performing single-point energy calculation twice; one on the complex system (ucomplex) and another on the same system but its members (protein and peptide) were separated from each other by a sufficiently large distance (udomain and upeptide). in this way, the interaction energy can be expressed as ∆u = ucomplex – (udomain + upeptide).28-30 the poisson-boltzmann/surface area (pb/sa) method was used to account for solvent effect associated with the pdz–peptide binding.31 in pb/sa procedure, the change of total desolvation gibbs energy (∆gslv) was estimated from the polar (∆gplr) and nonpolar (∆gnplr) desolvation energies. the polar aspect was calculated by finite difference solutions to the nonlinear poisson–boltzmann equation in delphi program,32 while nonpolar contribution was determined by summing up the weighted surface area of solute molecule, i.e., ∆gnplr = γ∆a, where γ = 0.0072 kcal*/(mol·å2) and ∆a is the change in solute’s surface area upon the pdz–peptide binding.33–35 the change of total binding gibbs energy of a peptide ligand to its domain receptor can be expressed as follows:36 δg = ∆uint + ∆gplr + ∆gnplr (1) ab initio electron correlation calculation the π–cation–π stacking region (i.e., pdz residue arg360 and peptide residues trp-1 and phe-4) was split from the whole qm/mm-optimized domain–peptide complex system, which was then used to derive the change of interaction energy (δe) of each wing of the stacking at a high electron correlation theory level of møller–plesset second order perturbation (mp2) in conjunction with a dunning’s correlation consistent basis set aug-cc-pvdz. this electron-correlation theory has been shown to accurately capture the dispersive energy involved in noncovalent complex system.37 in addition, the basis set superposition error (bsse) was eliminated by the standard counterpoise method of boys and bernardi.37 the electron correlation calculations were carried out in gaussian 09 suite of programs.23 fluorescence polarization assay the h3c1 peptide and its five mutants (see table i) were synthesized by gbiochem using fmoc solid-phase chemistry. the binding affinity between the pdz and peptide was determined using a fluorescence polarization (fp) protocol modified from our previous work.12 synthetic peptides were labeled with conjugated fluorescein (fitc). titrations were performed by monitoring fp as a function of increasing protein amounts of htra pdz domain (residues 354–453) added to 10 μm fitc-peptides in a buffer containing 50 mm tris-hcl, 100 mm nacl, 5 mm edta and 1 mm dithiothreitol (dtt). fp analysis was measured using a perkin–elmer spectrofluorimeter. the dissociation constants (kd) were determined by fitting titration curves to the equation: ( ) ( ) 0 pdz d pdz d / 1 / ∞+= + f f c k f c k (2) where the cpdz is the pdz protein concentration at each measurement point, f is the observed fp value at a given protein concentration, f0 is the fp value of free peptide, and f∞ is the maximal fp value saturated with protein. each assay was performed in duplicate. * 1 kcal = 4186 j 1372 zhang et al. results and discussion synergistic effect of geometric and energetic properties between two wings the orthogonal π–cation–π stacking at the complex interface of htra pdz domain with its h3c1 peptide ligand contains two individual cation-π interactions that are approximately perpendicular to each other (δ = 76°). the positively charged guanidyl group of domain residue arg360 serves as the central cation that can separately form a t-shaped cation–π interaction with the aromatic indolyl group of peptide trp-1 residue (wing 1) and a parallel cation–π interaction with the aromatic phenyl group of peptide phe-4 residue (wing 2, fig. 1). considering that the two interactions are tightly bound together and share a common cation, they are expected to exert direct influence on each other. here, the peptide π-electron residues trp-1 and phe-4 are separately mutated to non-aromatic amino acid ala (i.e., trp-1ala or phe-4ala mutation) to examine the synergistic effect of geometric and energetic properties between the two orthogonal wings. firstly, the three complex structures of htra pdz domain with wild-type h3c1 peptide (crystal structure) as well as its trp-1ala and phe-4ala mutants (scwrl4 -modeled structures) were separately subjected to qm/mm optimization to reach at conformation-refined state. next, the π–cation–π stacking region was stripped from the optimized complex structures, and the interaction energies δe of domain residue arg360 with peptide residues -1 (trp-1 or ala-1) and -4 (phe-4 or ala-4) were separately calculated using ab initio electron correlation mp2/aug -cc-pvdz theory with bsse correction. the impacts of peptide mutation phe-4ala (in wing 2) or trp-1ala (in wing 1) on the change of interaction energy, δe, distance d and dihedral angle θ of wings 1 and 2 are investigated, respectively. as can be seen, mutation of aromatic residue to ala in one wing would considerably impair the interaction strength of another wing, although the mutation is not involved in the calculated wing itself. for example, the peptide residue trp-1 only participates in the cation–π interaction of wing 1, but the interaction energy of wing 2 is reduced largely upon the trp-1ala mutation, with δe2 change from –6.7 to –4.2 kcal/mol for wing 2. a similar phenomenon can be observed in wing 2 phe-4ala mutation on wing 1 interaction energy (with δe1 change from –4.8 to –2.9 kcal/mol). in addition, the two mutations phe-4ala (in wing 2) and trp-1ala (in wing 1) have also a moderate effect on the geometric configuration of non-self-wing; which causes the wing distance to increase from 5.64 to 6.37 å (d1) and from 4.12 to 4.97 å (d2), respectively, and induces the wing dihedral angle shifting from 72 to 67° (θ1) and from 12 to 16° (θ2), respectively. both the energetic and geometric analyses suggest that there is a strong synergistic effect between the two wings of orthogonal π–cation–π stacking, that is to say, presence of cation-π interaction in one wing can substantially enhance the interaction strength in another wing. this design of π–cation–π stacking system 1373 is expected because the π-electron conjugated effect and delocalization energy can be maximized only by forming the complete stacking system. the synergistic effect can also be found in the binding of peptide ligand to htra pdz domain. the change of total binding gibbs energies, δg, of wild-type c3h1 peptide and its two single-point mutations trp-1ala and phe-4ala as well as one double-point mutation trp-1ala/phe-4ala were calculated with qm/mm and pb/sa. as shown in fig. 3, the wild-type peptide binds to domain with δg of –12.5 kcal/mol, and the single-point mutation can considerably impair the binding potency to –8.4 (trp-1ala) or –9.1 (phe-4ala) kcal/mol. the trp-1 residue should not only participate in cation–π interaction, but also tightly pack against its surroundings to form other nonbonded chemical forces such as van der waals and hydrophobic contacts, although the wing 1 seems to be tighter than wing 2 as the former is formed by a short, parallel cation–π stacking, while the latter is defined by a long, t-shaped stacking. simultaneous double-point mutation of the two residues (trp-1ala/phe-4ala) can further reduce the binding potency to –8.0 kcal/mol, which is quite modest relative to single-point mutations, suggesting that just a single mutation on one wing can substantially undermine the synergism of π–cation–π stacking. fig. 3. the impacts of peptide single-point mutation trp-1ala or phe-4ala and double-point mutation trp-1ala/phe-4ala on the change of total binding gibbs energy, δg, of peptide ligands to htra pdz domain. combinatorial optimization of the synergistic effect between two wings in order to explore the synergistic effect on domain–peptide binding, the systematic combinations between the four aromatic amino acids (trp, phe, tyr and 1374 zhang et al. his) plus one non-aromatic amino acid (ala) at the residues -1 and -4 of c3h1 peptide were investigated, totally resulting in 25 (5×5) peptide ligands, in which one is wild-type c3h1 peptide, fifteen are peptide mutants with aromatic amino acid substitutions at one or both of the residues –1 and –4 (and thus can form the complete π–cation–π stacking), eight are peptide single-point mutations with an aromatic substitution at one residue and a non-aromatic ala substitution at another (and thus can only form one wing cation–π interaction), and one is a peptide double-point mutation with two ala substitutions at both the residues -1 and -4 (and thus cannot form any wing cation-π interaction). these mutant domain– –peptide complex structures were computationally modeled using scwrl4 method14 based on the crystal structure of htra pdz domain in complex with wild-type c3h1 peptide (pdb: 2p3w), refined with qm/mm optimization, and their binding energies (δg) were then derived by qm/mm and pb/sa analyses. the systematic peptide binding energy profile is visualized as a heatmap in fig. 4a. at first glance, a distinct difference between the 16 aromatic-substituted peptides (surrounded by a frame in the heatmap) and others 9 ala-substituted peptides; the former generally has higher binding potency as compared to the latter, imparting that formation of the complete π–cation–π stacking is critical for domain–peptide binding, and breaking of just one wing cation–π interaction would largely impair the peptide binding potency due to the synergistic effect between two wings. at residue-1, the trp is most favorable for the binding. a previous competitive assay also identified that the wild-type trp-1 residue plays a crucial role in the domain–peptide recognition,7 indicating that the residue should fig. 4. a) heatmap of peptide binding energy profile regarding the systematic combinations between the four aromatic amino acids (trp, phe, tyr and his) plus one non-aromatic amino acid (ala) at the residues -1 and -4 of c3h1 peptide. the block surrounded by a frame represents those aromatic amino acid combinations that can form the orthogonal π–cation–π stacking. b) superposition of the residues trp-1 and tyr-4 of affinity-improved peptide phe-4tyr mutant onto the residues trp-1 and phe-4 of wild-type c3h1 peptide. the structures of both the wild-type and mutant domain–peptide complexes are refined by qm/mm optimization. design of π–cation–π stacking system 1375 not only participate in π–cation–π stacking, but also interact with its protein environment to provide additional contribution to the binding. for residue –4, the tyr and trp substitutions can improve peptide binding capability from wild-type phe. tyrosine has a polar phenolic hydroxyl group, while tryptophan is π-electron rich system; both of them can effectively interact with the guanidinium cation of domain arg360 residue. the his substitution at either residue -1 or -4 would reduce peptide affinity, albeit the decreased affinity is moderate, suggesting that histidine is not a good choice for the π–cation–π stacking system since its imidazolyl moiety may be partially protonized (and thus cause electrostatic repulsion with arg 360 cation). next, the binding affinity of wild-type c3h1 peptide and its two predicted affinity-improved peptide mutants phe-4tyr and phe-4trp as well as its three ala-substituted mutants trp-1ala, phe-4ala and trp-1ala/phe-4ala (served as controls) to the recombinant protein of htra pdz domain was determined using fluorescence polarization assays (fig. 5). the obtained affinity kd values as well as calculated geometric and energetic parameters for the π-cation-π stacking of these domain–peptide complexes are listed in table i. fig. 5. the normalized binding curves of wild-type c3h1 peptide and its five designed mutants to htra pdz domain. as might be expected, the wild-type peptide can bind to domain with a moderate affinity (kd = 1.8 μm), which is basically in line with a previously reported value (kd = 1.1 μm) for the peptide measured using isothermal titration calorimetry (itc).7 the phe-4tyr and phe-4trp mutations can considerably and moderately improve the affinity by 3.2-fold and 1.5-fold (kd change from 1.8 to 0.57 and 1.2 μm), respectively; this is basically consistent with theoretical prediction 1376 zhang et al. (δg change from –12.5 to –14.2 and –13.7 kcal/mol, respectively). however, separate breaking of wings 1 and 2 can largely reduce peptide affinity with kd change from 1.8 (wild type) to 160 μm and 34 μm (single-point mutations trp -1ala and phe-4ala), respectively, and simultaneous breaking of the two wings can further (moderately) reduce the affinity to 210 μm (double-point mutation trp-1ala/phe-4ala), confirming that the completeness of π–cation–π stacking system is fundamentally important to guarantee the high affinity of peptide ligands. table i. geometric and energetic parameters of orthogonal π–cation–π stacking system formed across the complex interface of htra pdz domain with wild-type c3h1 peptide and its five designed mutants wing 1 wing 2 δ ° δgb kcal mol-1 kdc μm residue -1 d1 å θ1 ° δe1a kcal mol-1 residue -4 d2 å θ2 ° δe2a kcal mol-1 trp-1d 5.64 72 –4.7 phe-4d 4.12 12 –6.7 76 –12.5 1.8±0.4 trp-1 5.37 75 –5.2 tyr-4 3.98 14 –7.6 83 –14.2 0.57±0.18 trp-1 5.49 70 –5.0 trp-4 4.03 19 –7.2 79 –13.7 1.2±0.3 trp-1e 6.37 67 –2.9 ala-4e – – –2.1 – –9.1 34±7 ala-1e – – –1.3 phe-4e 4.97 16 –4.2 – –8.4 160±30 ala-1f – – –1.1 ala-1f – – –1.7 – –8.0 210±50 aδe, the interaction energy of htra pdz domain residue arg360 with peptide residue -1 or -4, calculated using ab initio electron correlation mp2/aug-cc-pvdz with bsse correction; bδu, the total binding energy of peptide ligand to htra pdz domain, calculated using qm/mm and pb/sa; ckd, the binding affinity (dissociation constant), determined by fluorescence spectroscopy assay; dwild-type c3h1 peptide; econtrol (only one wing cation–π interaction formed); fcontrol (no wing cation–π interaction formed) the geometric profile is changed moderately as compared to energetic and affinity properties upon the mutation, where the distances d1 and d2 seem to vary more significantly than dihedral angles θ1 and θ2. it is worth noting that the angles δ (76, 83 and 79°) of three π–cation–π stackings in wild-type peptide as well as its phe-4tyr and phe-4trp mutants are all roughly close to 90°, indicating that the orthogonality of these stacking systems can be maintained during the aromatic substitution. superposition of the residues trp-1 and tyr-4 of high-affinity peptide phe-4tyr mutant onto the residues trp-1 and phe-4 of wild-type peptide is shown in fig. 4b. as can be seen, the location and configuration of the two residues are well overlapped between the two peptides, although the mutation considerably improves peptide affinity kd from 1.8 to 0.57 μm, indicating that the stacking geometry and orthogonality are not vulnerable to the affinity-improved mutation. conclusion an orthogonal π–cation–π stacking system was identified at the co-crystallized complex interface of htra pdz domain with its pentapeptide ligand c3h1. the stacking system was characterized rigorously via crystal structure dissection, design of π–cation–π stacking system 1377 qm/mm calculation and energetics analysis, revealing a strong synergistic effect involved in the system; breaking of one cation–π interaction would largely impair the interaction strength of another. the π-electron contributes primarily to the synergistic effect, although geometric property is also responsible for it. a number of c3h1 mutants were designed based on the computational investigations, from which two mutants were identified to have increased affinity relative to the wild-type peptide, which may be used as lead molecular entities to develop htra pdz-targeted agents for disease therapy. acknowledgements. this work was supported by the national science foundation of china (nos. 21565016, 51762022 and 51564024), the jiangxi provincial science and technology support key project (no. 20152acg70021), the jiangxi provincial natural science foundation (no. 2010gzh0073), the jiangxi provincial department of education’s item of science and technology (no. gjj150761), the jiangxi provincial science and technology support program (no. 20123bbg70218), the jiangxi provincial educational reform research program (no. jxjg-14-9-31), the jinggangshan university natural science item (no. jz0813), and the jinggangshan university startup fund for doctor research (no. jzb11035). и з в о д молекулски дизајн ортогоналног система паковања на додирној површини између htra pdz домена и његових пептидних лиганада dingwa zhang1, deyong he1, xiaoliang pan2, yaping xu1 и lijun liu1 1school of chemistry and chemical engineering, jinggangshan university, ji’an 343009, china и 2school of mechanical and electrical engineering, jinggangshan university, ji’an 343009, china протеаза a зависна од високе температуре (htra) има кључну улогу у контроли квалитета протеина и судбине ћелије. ензим се састоји од каталитичког протеазног домена и регулаторног pdz домена, који одређује супстратну специфичност протеазе након везивања с-терминалног хидрофобног дела протеина-партнера. пентапептидни лиганд h3c1 је раније идентификован као потенцијални везујући партнер за htra pdz домен користећи технику експримирања у фагу. у овом раду је анализиран ортогонални π-катјон–π систем паковања на међуповршини кристалног комплекса домен–пептид користећи теоријска израчунавања и експерименталне податке. показано је да постоји јак (позитиван) синергистички ефекат између два крака система паковања; прекид у катјон–π интеракцији у једном краку у великој мери спречава интеракцију у другом. π-eлектрон је најодговорнији за синергистички ефекат, мада и геометрија има делимичан допринос. даље је систематично испитана комбинација четири ароматичне аминокиселине (phe, tyr, trp и his) и једне неароматичне (ala) на два крака π–катјон–π паковања. нађено је да присуство две ароматичне аминокиселине (phe-4tyr и phe-4trp) на краку може повећати афинитет према пептиду 3,2, односно 1,5 пута, док мутације са неароматичним аминокиселинама на једном или оба краја доводе до значајног смањења афинитета, повећавајући kd од 1,8 (немутирана форма) до 34 μm и 160 μm (једна тачкаста мутација), односно 210 μm (две мутације), указујући да промена у једном краку може значајно умањити синергизам ортогоналног π–катјон–π паковања. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {electrogenerated based-promoted synthesis of nanoparticles 5-benzoyl-4-(aryl)-2-hydroxy-6-trifluoromethyl-1,4-dihydro-pyridine-3-carbonitriles by three-component condensation of aryl aldehydes, malononitrile and 4,4,4-trifluoro-1-phenylbuta-1,3-dione} j. serb. chem. soc. 85 (1) 79–87 (2020) udc 547.822.1:539.24+544.653.1:547 jscs–5284 original scientific paper 79 electrogenerated base-promoted synthesis of 4-aryl-5-benzoyl-2 -hydroxy-6-(trifluoromethyl)-1,4-dihydropyridine-3 -carbonitriles nanoparticles by three-component condensation of aromatic aldehydes, malononitrile and 4,4,4-trifluoro-1 -phenylbutane-1,3-dione esmaeil goodarzi and behrooz mirza* department of chemistry, karaj branch, islamic azad university, karaj, iran (received 16 march, revised 28 may, accepted 26 june 2019) abstract: an electrochemical strategy to the synthesis of novel 4-aryl-5-benzoyl-2-hydroxy-6-(trifluoromethyl)-1,4-dihydropyridine-3-carbonitriles nanoparticles via three-component reaction of aromatic aldehydes, malononitrile and 4,4,4-trifluoro-1-phenylbutane-1,3-dione in water/ethanol in an undivided cell in the presence of sodium bromide as an electrolyte is described. this method has several advantages, such as high to excellent product yields (65– –85 %), atom economy, environment friendly, and no need for chromatographic separations. keywords: multi-component; electrosynthesis; 1,4-dihydropyridine; nanosized; aromatic aldehydes. introduction modern synthetic design demands high efficiency in terms of minimization of synthetic steps together with maximization of complexity.1 one of the ways to fulfill these goals is the development and use of multicomponent reactions that consist of several simultaneous bond-forming reactions and allow the highly efficient synthesis of complex molecules starting from simple substrates in a onepot manner.2–4 the electrocatalytic multicomponent reaction is known as an important approach to address this issue, in which three or more starting materials are combined together in an electrochemical cell in the presence of an appropriate electrolyte and working electrodes to generate the target products.5,6 the noteworthy growth in studies in organic electrochemistry during recent years has made electrosynthesis one of the most competitive protocols of modern organic chemistry and provides organic chemists with a novel and versatile synthetic * corresponding author .e-mail: b_mirza@azad.ac.ir https://doi.org/10.2298/jsc190316063g ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 80 goodarzi and mirza device of great promise.7 electrochemical procedures aimed at the synthesis of organic compounds are valuable for large-scale processes due to their catalytic nature and the use of an inexpensive and environmentally responsible chemical reagent, namely electricity.8,9 the electrosynthesis of heterocyclic compounds can be performed at ambient temperature and pressure, which it is considered a further advantage of this approach. derivatives of 1,4-dihydropyridine (dhp) represent an important class of bioactive molecules, well known for their role as calcium channel modulators and used extensively for the treatment of hypertension.10–12 polyfunctionalized 1,4-dihydropyridines have also shown a variety of biological and pharmacological activities, such as anti-allergic, antitumor, antibacterial. anticonvulsant, antianalgesic, anti-inflammatory, antihypertensive, cardiovascular disease and stress protective activities.13,14 due to their unique physical, chemical, and biological properties, fluorinated organic compounds,15–17 have attracted much attention. among various fluorine substituents, the trifluoromethyl group is one of the most important structural fragments, because of its important role in modulating the chemical, physical and biochemical properties of organic molecules.18 the trifluoromethyl fragment is a part of many biologically active molecules, such as celecoxib (nonsteroidal anti-inflammatory drug),19 efavirenz (hiv rt inhibitor),20 mefloquine (antimalarial agent),21 and sorafenib (oral multikinase inhibitor).22 drug structures with a high surface–volume ratio display substantial improvement of solubility, which results in stronger therapeutic effects. thus, nano or micro-sized drugs, due to their high surface–volume ratio, result in increases of the drug adsorption and improvement of the curative characteristics. accordingly, the development of several new methods to synthesize nano-sized drugs is a significant challenge for both the chemist and pharmacist. several methods, including micronization, modification of polymorphic configuration, expansion of oil-based solutions, smart application of co-solvents, application of stabilizing agents, micro-emulsions, and creation of solid dispersions, have been offered for the synthesis of nano-sized drug compounds.23 recently, an electrocatalytic reaction of aromatic aldehydes, malononitrile and 4,4,4-trifluoro-1-phenylbutane-1,3-dione in alcoholic solvent to produce nanoparticles of 2-amino-4-aryl-4h-pyran derivatives was reported24 (scheme 1). electrolysis etoh, r.t our previous work h o + cf3 o o nc cnar + nabr o c nh2 cn f3c ph o ar scheme 1. electrocatalytic reaction of aromatic aldehydes, malononitrile and 4,4,4-trifluoro-1-phenylbutane-1,3-dione. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. electrosythesis of 1,4 -dihydropyridines nanoparticles 81 considering the above reports, and in continuation of our studies on the electro synthesis of heterocyclic compounds,23–26 herein a convenient and facile synthesis of 4-aryl-5-benzoyl-2-hydroxy-6-(trifluoromethyl)-1,4-dihydropyridine-3 -carbonitriles 4 is designed based on the electrochemically induced three-component reaction of aromatic aldehydes 1, malononitrile 2, and 4,4,4-trifluoro-1 -phenylbutane-1,3-dione 3, in water/ethanol (1:9) solvent in an undivided cell without a base or any additive catalyst (scheme 2). h o + cf3 o o etoh,h2o, 50 0c 1 2 3 nc cn n h c oh cn 4 f3c ph o ar + ar electrolysis o c nh2 cn 5 f3c ph o ar nabr not observed scheme 2. electrocatalytic synthesis of nanoparticles of 1,4-dihydropyridine derivatives. experimental all chemicals and solvents were purchased from merck or sigma–aldrich. melting points of the target products were measured using an ia 9100 melting point apparatus. elemental analyses were performed using a costech ecs 4010 chns-o analyzer. controlled -current coulometry and preparative electrolysis were realized via a sama potentiostat/galvanoastat (isfahan, iran). the electrodes used in this work were an iron cathode (5 cm2) and a graphite and magnesium anode (5 cm2). 1h-nmr spectra were achieved in dmso-d6 with a bruker-avance aqs 500 mhz spectrometer. the 13c-nmr spectra were recorded in dmsod6 on a bruker-avance spectrometer 125 mhz. mass spectra were determined on an agilent technology (hp) mass spectrometer operating at an ionization potential of 70 ev. analytical and spectral data of the compounds are given as supplementary material to this paper. general procedure for the synthesis of nanoparticle of 4-aryl-5-benzoyl-2-hydroxy-6-(trifluoromethyl)-1,4-dihydropyridine-3-carbonitriles a mixture of the required aromatic aldehyde (1, 1 mmol), malononitrile (2, 1 mmol), 4,4,4-trifluoro-1-phenylbutane-1,3-dione (3, 1 mmol) and sodium bromide (0.5 mmol, 0.035 g) (as the supporting electrolyte) in water/ethanol (1:9, 25 ml) was electrolyzed in an undivided cell supplied with a magnetic stirrer, a mg anode, and a fe cathode, at 50 °c and constant current density of 30 ma/cm2 (i = 150 ma, electrode surface 5 cm2). the progress of the reaction was monitored by thin layer chromatography (tlc, n-hexane/ethyl acetate = 3/2). after completion of the reaction, the mixture was cooled to room temperature and then concentrated to one fifth of its initial volume under reduced pressure. the solid product was ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 82 goodarzi and mirza collected by filtration and washed with water (2×5 ml), ethanol (2×5 ml), cold diethyl ether (5 ml) to afford the pure product. results and discussion: the reaction of 4-nitro benzaldehyde with malononitrile and 4,4,4-trifluoro1-phenylbutane-1,3-dione in an undivided cell at a current density of 30 ma cm– 2 (i = 150 ma, electrode surface 5 cm2) at room temperature was selected as a model and the effects of the solvent were investigated, in order to optimize the reaction conditions. the results are summarized in table i. as can be seen from this table, on using ethanol or methanol as the solvent, only a trace amount of the product 4 was formed and the major product was 524 (entries 1 and 2). also on using water/ethanol (1:9), the product 4 is formed in good yield, without observation of product 5 (entry 5). table i. effect of the solvent on the electrocatalytic reaction of 4-nitrobenzaldehyde, malononitrile and 4,4,4-trifluoro-1-phenylbutane-1,3-dione. time 90 min; for all reactions, aromatic aldehyde (1 mmol), malononitrile (1 mmol), 4,4,4-trifluoro-1-phenylbutane-1,3-dione (1 mmol), nabr (0.5 mmol), 20 ml of solvent, iron cathode (5 cm2), magnesium anode (5 cm2) were used entry solvent yield a, % 4 5 1 etoh trace 80 2 meoh trace 70 3 h2o 30 25 4 ch3cn 40 – 5 etoh/h2ob 85 – 6 meoh/h2ob 65 – 7 ch3cn/h2ob 45 – aisolated yield; bvolume ratio of 1:9 furthermore, the model reaction was examined under other factors, such as current, anode type, and temperature, and results are given in table ii. it can be seen from table ii that the best conditions for minimizing the synthesis time and maximizing the yield of the nanosized particles of 1,4-dihydropyridine production is ethanol/water at a current density of 30 ma cm–2 (i = 150 ma, electrode surface 5 cm2) at 50 °c. afterwards, the synthesized products were evaluated by scanning electron microscopy (sem), fig. 1. fortunately, it was found that the 1,4-dihydropyridine derivatives were of nanoscale size. the presence of mg2+in the solution may prevent the aggregation of the products and promote the formation of nanoparticles.9,27 to study the scope and generality of the reaction, a series of aromatic aldehydes were employed. the results are given in table iii. in all cases, the aromatic ring of the aromatic aldehydes substituted with either electron-donating or electron-withdrawing groups underwent the reaction smoothly and gave the res________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. electrosythesis of 1,4 -dihydropyridines nanoparticles 83 pective products in good yields. it could also be concluded that the aromatic ring of the aromatic aldehydes bearing electron-withdrawing groups required shorter times and gave higher yields (table iii). table ii. the effect of the current used in the reaction of 4-nitrobenzaldehyde, malononitrile and 4,4,4-trifluoro-1-phenylbutane-1,3-dione in water/ethanol on the formation of 1,4-dihydropyridine (4a) nanoparticles; for all reactions, aromatic aldehyde (1 mmol), malononitrile (1 mmol), 4,4,4-trifluoro-1-phenylbutane-1,3-dione (1 mmol), nabr (0.5 mmol), 20 ml water/ /ethanol (1:9), iron cathode (5 cm2) entry temperature, °c current, ma time, min electricity passed, f mol-1 yieldc, % 1a r.t 30 140 2.6 40 2a 35 30 120 2.2 50 3a 50 30 100 1.8 55 4a r.t 50 120 3.7 50 5a 35 50 120 3.7 55 6a 50 50 100 3.1 60 7a r.t 100 120 7.5 55 8a 35 100 120 7.5 60 9a 50 100 100 6.2 70 10a 35 150 100 9.3 70 11a 50 150 90 8.3 85 12a 60 150 100 9.3 85 13a 50 200 100 12.4 80 14a 60 200 100 12.4 80 15b 50 150 90 8.3 78 16b 60 200 100 12.4 75 amagnesium anode; bgraphite (5 cm2) anode. cisolated yield fig. 1. sem image of nanoparticles 1,4-dihydropyridine derivatives. compounds 4a–h are new and their structures were deduced by elemental and spectral analysis. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 84 goodarzi and mirza table iii. results obtained from synthesis of nanoparticles of 4-aryl-5-benzoyl-2-hydroxy -6-(trifluoromethyl)-1,4-dihydropyridine-3-carbonitriles (4a–h); 0.5 mmol of nabr, iron cathode (5 cm2), water/ethanol (1:9) used as solvent, magnesium (5 cm2) used as anode, and 100 ma current at r.t. entry ar time, min yielda, % m.p. / °c 4a 4-no2-c6h4 90 85 173–175 4b 3-no2-c6h4 100 70 174–176 4c 4-oh-c6h5 95 65 175–177 4d 4cl-c6h4 110 70 182–184 4e 2,4-dimethoxy-c6h3 120 72 172–174 4f 3,5-dimethoxy-c6h3 115 65 165–167 4g 4-br-c6h4 90 70 174–176 4h 2-ch3-c6h4 100 65 171–173 aisolated yield for all reactions for example, the 1h-nmr spectrum of compound 4a exhibited a singlet signal at 4.49 ppm for ch protons. the aromatic protons and nh proton were observed at δ 7.32–7.92 ppm. in addition, the proton of the hydroxy group resonated at 9.65 ppm as a broad singlet. when the 1h-nmr spectrum was recorded after addition of some d2o to the dmso-d6 solution of 4a, the signals related to nh and oh disappeared due to rapid exchange with d2o. the 13c-nmr spectrum of compound 4a showed 16 signals, which is consistent with the proposed structure. a possible mechanism for the formation of the products 4a–h is proposed in scheme 3. + + 3 cnnc o arh c arh nc c 1 2 5 ph o oh cf3 c ar h cn cn 5 n o c o ph f3c ar cn nh h 6 cathode : etoh + e in solution: ch2(cn)2 +eto ch(cn)2 +etoh eto +1/2 h2 -h2o n h c oh cn f3c ph o ar n h c o cn f3c ph o ar o c o ph f3c ar cn h2n h 7 oh n h c o cn f3c ph o ar ho -h2o 8 9 4 h2o scheme 3. the mechanism proposed for the formation of 4-aryl-5-benzoyl-2-hydroxy-6-(trifluoromethyl)-1,4-dihydropyridine-3-carbonitriles. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. electrosythesis of 1,4 -dihydropyridines nanoparticles 85 in the first step of the catalytic condensation cycle, deprotonation of an alcohol at the cathode leads to the formation of the corresponding alkoxide anion. its subsequent reaction in solution with malononitrile gives rise to the malononitrile anion. then knoevenagel condensation of aromatic aldehydes 1 with the malononitrile anion 2 occurs in the solution with the elimination of water and the formation of the corresponding arylidenemalononitrile 5. then the nucleophilic addition of the enolizable 4,4,4-trifluoro-1-phenylbutane-1,3-dione 3 to arylidene malononitrile 5 leads to intermediate 6 and then intermediate 6 could be hydrolyzed by water to form 7 and then through intramolecular condensation yield the cyclic product 8. this intermediate loses a molecule of water and tautomerization to product 4 under the reaction condition. conclusions in conclusion, an efficient, convenient electrochemical way to the synthesis of novel nanosized 1,4-dihydropyridine derivatives has been presented. from the green chemistry point of view, the application of electro-synthetic method has some significant advantages, i.e., clean synthesis, one-step reaction, using electricity as an alternative source of energy instead of an oxidative reagent, technical feasibility, and high atom economy are prominent advantageous of this green approach. supplementary material analytical and spectral data of the compounds are available electronically from http:// //www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. this study was supported by the islamic azad university, branch of karaj. и з в о д електрохемијска синтеза наночестица 4-арил-5-бензоил-2-хидрокси -6-(трифлуорометил)-1,4-дихидропиридин-3-карбонитрила потпомогнута базом и изведена трокомпонентном кондензацијом ароматичног алдехида, малононитрила и 4,4,4-трифлуоро-1-фенилбутан-1,3-диона esmaeil goodarzi и behrooz mirza department of chemistry, karaj branch, islamic azad university, karaj, iran описан је електрохемијски поступак синтезе нових наночестица 4-арил-5-бензоил -2-хидрокси-6-(трифлуорометил)-1,4-дихидропиридин-3-карбонитрила трокомпонентном реакцијом ароматичног алдехида, малононитрила и 4,4,4-трифлуоро-1-фенилбутан-1,3-диона у смеши етанола и воде у једноделној електрохемијској ћелији у натријум-бромиду као електролиту. ова метода има неколико предности као што су висок принос (65–85 %), висок степен конверзије атома, односно мала количина споредних производа, еколошка прихватљивост и то што нема потребе за хроматографском сепарацијом. (примљено 16. марта, ревидирано 28. маја, прихваћено 26. јуна 2019) ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 86 goodarzi and mirza references 1. b. m. trost, science 254 (1991)1471 (https://dx.doi.org/ 10.1126/science.1962206) 2. h. bienayme, c. hulme, g. oddon, p. schmidt, chem. eur. j. 6 (2000) 3321 (https://dx.doi.org/10.1002/1521-3765(20000915)6:18<3321::aidchem3321>3.0.co;2-a) 3. a. j. von wangelin, h. neumann, d. gördes, s. klaus, d. strübing, m. beller, chem. eur. j. 9 (2003) 4286 (https://dx.doi.org/ 10.1002/chem.200305048) 4. r. v. a. orru, m. de greef, synthesis (2003) 1471 (https://dx.doi.org/ 10.1055/s-200340507) 5. m. n. elinson, a. s. dorofeev, f. m. miloserdov g. i. nikishin, mol. diversity 13 (2009) 47 (https://dx.doi.org/ 10.1007/s11030-008-9100-1) 6. m. n. elinson, a. i. ilovsaiky, a. s. dorofeev, v. m. merkulova, n. o. stepanov, f. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {novel (−)-goniofufurone mimics: synthesis, antiproliferative activity and sar analysis} j. serb. chem. soc. 84 (12) 1345–1353 (2019) udc 547.724+547.455.623:547.466.1: jscs–5268 576+615.9 original scientific paper 1345 novel (–)-goniofufurone mimics: synthesis, antiproliferative activity and sar analysis bojana srećo zelenović1#, slađana kekezović1#, mirjana popsavin1#, vesna kojić2, goran benedeković1# and velimir popsavin1,3*# 1department of chemistry, biochemistry and environmental protection, faculty of sciences, university of novi sad, trg dositeja obradovića 3, 21000 novi sad, serbia, 2oncology institute of vojvodina, put dr goldmana 4, 21204 sremska kamenica, serbia and 3serbian academy of sciences and arts, knez mihajlova 35, 11000 belgrade, serbia (received 12 september, accepted 23 september 2019) abstract: divergent syntheses of novel (–)-goniofufurone mimics with an alkoxymethyl group as the side chain have been accomplished from d-glucose in nine synthetic steps and in overall yields 6.7–8.7 %. their in vitro antiproliferative activity was evaluated against eight human tumour cell lines as well as a single normal cell line. all analogues demonstrated powerful to good antiproliferative effects toward all malignant cell lines under evaluation. against the hl-60 cell line, all mimics showed increased activities being 27to 1604-fold more potent than the lead compound, (–)-goniofufurone. remarkably, the majority of synthesized analogues displayed higher or similar activity to the commercial antitumour agent doxorubicin (dox) against a549 cell line. the most potent compound exhibited 196-fold stronger cytotoxicity than dox in the culture of this cell line. keywords: d-glucose; antitumour agents; goniofufurone mimics; cytotoxic lactones, structure–activity relationships. introduction (–)-goniofufurone (1) is a synthetic styryl lactone, which represents the opposite enantiomer of naturally occurring (+)-goniofufurone (ent-1, fig. 1). after isolation of (+)-goniofufurone from the stem bark of gonithalamus giganteus (annonaceae)1 in 1990, and the confirmation of the absolute stereochemistry of both 1 and ent-1,2,3 many syntheses of these compounds with unique and intriguing structures have been published.4–18 both, 1 and ent-1, demonstrated a remarkable antiproliferative activity toward several human tumour cell lines.19,20 synthetic molecule 1 and some of * corresponding author. e-mail: velimir.popsavin@dh.uns.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc190912104s 1346 srećo zelenović et al. its analogues showed more powerful cytotoxicity than the natural product ent-1 and the corresponding analogues from the (+)-series.21 previous studies in our laboratory showed that dephenylated (–)-goniofufurone derivatives bearing an ether group with c9and c10-hydrocarbon chains (compounds 6 and 7) exhibited in vitro antitumour activity against several human cancer cell lines.22 fig. 1. design of (+)-goniofufurone analogues 2–10: i) enantiomerization of ent-1; ii) dephenylation of 1; iii) o-alkylation of ho-7 in 2. as an extension of this work, herein the synthesis and in vitro antitumour activity of six novel (–)-goniofufurone mimics (3–5 and 8–10) and full experimental details for the preparation of 6 and 7 are reported. the preparation and cytotoxicity of known23,24 dephenylated (–)-goniofufurone analogue 2 is also disclosed. experimental general procedures melting points were determined on a büchi 510, or on a hot stage microscope nagema phmk 05 apparatus and are not corrected. optical rotations were measured on an autopol iv (rudolph research) automatic polarimeter. the ir spectra were recorded with a ftir nexus 670 (thermo-nicolet) spectrophotometer. the nmr spectra were recorded on a bruker ac 250 e or a bruker avance iii 400 mhz instrument and the chemical shifts are expressed in ppm downfield from tetramethylsilane. low resolution mass spectra were recorded on a finnigan-mat 8230 (ci) mass spectrometer. high-resolution mass spectra were taken on a micromass lct ka111 spectrometer or on ltq orbitrapxl (thermo fisher scientific inc., usa) mass spectrometer. tlc was performed on dc alufolien kieselgel 60 f254 (e. merck). flash column chromatography was performed using kieselgel 60 (0.040–0.063 mm, e. merck). all organic extracts were dried with anhydrous na2so4. organic solutions were concentrated on a rotary evaporator under diminished pressure at a bath temperature below 35 °c. synthetic procedures general procedure for the synthesis of the intermediates 12–19. to a solution of compound 11 (1 eq) in dry et2o (2 ml) were added ag2o (2.6 eq), agotf (0.3 eq) and the corresponding alkyl bromide (3 eq). the mixture was stirred under reflux for 8–32 h (table i). after completion of the reaction, which was detected by tlc, the mixture was purified by flash column chromatography (eluents in table i). the characterization data for 12–19 are given in the supplementary material to this paper. novel (−)-goniofufurone mimics 1347 general procedure for the synthesis of the analogues 2–10. a solution of starting compound 11–19 (0.1 mmol) in dry etoh (2 ml) was hydrogenated over 10 % pd/c (≈10 % of pd) at room temperature for 18–24 h (table ii). after completion of the reaction (tlc), the mixture was purified by flash column chromatography (eluents in table ii). the characterization data for 2–10 are presented in the supplementary material. cytotoxic activity cell cultures. human myelogenous leukaemia (k562), promyelocytic leukaemia (hl -60), t cell leukaemia (jurkat), burkitt’s lymphoma (raji) and er+ breast adenocarcinoma (mcf-7) malignant cells were grown in a suspension in rpmi 1640 nutrient medium, while er− breast adenocarcinoma (mda-mb 231), cervix carcinoma (hela), lung adenocarcinoma epithelial cells (a549), and normal foetal lung fibroblasts (mrc-5) were cultured as a monolayer in dmem medium. both media were supplemented with 10 % of foetal calf serum (fts, nivns) and antibiotics (100 iu ml-1 of penicillin and 100 mg mg-1 of streptomycin). the cell lines were cultured in flasks (costar, 25 ml) at 37 °c in an atmosphere of 100 % humidity and 5 % of co2 (heraeus). exponentially growing viable cells were used throughout the assays. mtt test.25 cells were harvested, counted using trypan blue and plated into 96-well microtiter plates (costar) at the optimal seeding density of 5×103 cells per well to assure a logarithmic growth rate throughout the assay period. viable cells were placed in a volume of 90 μl per well, and preincubated in complete medium at 37 °c for 24 h to allow cell stabilization prior to the addition of the substances. the tested substances, at 10-fold the required final concentration, were added (10 μl well-1) to all wells, except for the control ones, and the microplates were incubated for 72 h. the wells containing cells without tested substances were used as controls. mtt solution (10 μl) was added to all wells 3 h before the end of incubation period. mtt was dissolved in medium at 5 mg ml-1 and filtered to sterilize and remove the small amount of insoluble residue present in some batches of mtt. acidified 2-propanol (100 μl of 0.04 m hcl in 2-propanol) was added to all wells and mixed thoroughly to dissolve the dark blue crystals of formazan. after a few minutes at room temperature, to ensure that all crystals were dissolved, the plates were read on a spectrophotometer plate reader (multiscan mcc340, labsystems) at 540 and 690 nm. the wells without cells containing complete medium and mtt acted as blanks. results and discussion chemistry the syntheses of intermediates 12–19 is presented in table i. starting compound 11 was prepared from d-glucose in seven synthetic steps as previously reported.22 o-alkylation of alcohol 11 with an excess of hexyl bromide in ether, in the presence of silver(i) oxide and silver(i) triflate as catalysts, gave the expected 7-o-hexyl derivative 12 in 69 % yield (table i, entry 1). compound 11 under the similar experimental conditions reacted with different alkyl bromides (c7–c13) to afford the corresponding ether derivatives 13–19 in good yields (table i, entries 2–8). hydrogenolytic removal of the benzyl ether protective group in 12–19, under standard reaction conditions furnished the target (–)-goniofufurone mimics 3–10 1348 srećo zelenović et al. in good to excellent yields (table ii, entries 2–9). finally, under similar reaction conditions, alcohol 11 gave the known23,24 diol 2 in 97 % yield (entry 1). table i. preparation of intermediates 12–19 entry r reaction time, h eluent for fcca product (yield, %) 1 c6h13 18 3:2 light petroleum/et2o 12 (69) 2 c7h15 15 1:1 light petroleum/et2o 13 (71) 3 c8h17 8 1:1 light petroleum/et2o 14 (77) 4 c9h19 28 3:2 light petroleum/et2o 15 (33) 5 c10h21 32 3:2 light petroleum/et2o 16 (54) 6 c11h23 22.5 1:1 light petroleum/et2o 17 (78) 7 c12h25 12 3:2 light petroleum/et2o 18 (73) 8 c13h27 17.5 7:3 light petroleum/et2o 19 (69) afcc – flash column chromatography table ii. preparation of final products 2–10 entry starting compound r reaction time, h eluent for fcca product (yield, %) 1 11 h 20 7:3 ch2cl2/etoac 2 (97) 2 12 c6h13 24 7:3 light petroleum/et2o 3 (96) 3 13 c7h15 20 7:3 light petroleum/et2o 4 (91) 4 14 c8h17 18 7:3 light petroleum/et2o 5 (84) 5 15 c9h19 18 et2o 6 (82) 6 16 c10h21 21 9:1 ch2cl2/etoac 7 (80) 7 17 c11h23 20 7:3 light petroleum/et2o 8 (86) 8 18 c12h25 20 3:2 light petroleum/et2o 9 (72) 9 19 c13h27 21 7:3 light petroleum/et2o 10 (88) afcc – flash column chromatography in vitro antiproliferative activity the biological activities of synthesized compounds 2–10 were evaluated by an in vitro cytotoxicity test against a panel of eight human malignant cell lines, including human myelogenous leukaemia (k562), human promyelocytic leukaemia (hl-60), t cell leukaemia (jurkat), burkitt's lymphoma (raji), er+ breast adenocarcinoma (mcf-7), er− breast adenocarcinoma (mda-mb 231), cervix carcinoma (hela) and lung adenocarcinoma epithelial cells (a549) and novel (−)-goniofufurone mimics 1349 against single normal cell line, foetal lung fibroblasts (mrc-5). cell growth inhibition was evaluated using the standard mtt colorimetric assay after exposure of cells to the test compounds for 72 h.25 (–)-goniofufurone (1), analogue 2 and the commercial antitumour agent doxorubicin (dox) were used as positive controls. according to the resulting ic50 values (table iii), of all cell lines tested, four were sensitive to all of the synthesized analogues 2–10 (k562, hl-60, hela and a549). of the remaining four cell lines, jurkat and raji cells were sensitive to eight, while mcf-7 and mda-mb 231 cell lines were sensitive to seven of nine synthesized analogues. the most active compound in a549 cell culture is analogue 10 (ic50 = 0.025 µm) that exhibited 94and 196-fold higher potency when compared to lead 1 and dox, respectively. simultaneously, analogue 10 represents the most active compound described in this paper. table iii. in vitro cytotoxicity of (–)-goniofufurone (1), dox and the analogues 2–10 after 72 h compound ic50 / µma k562 hl-60 jurkat raji mcf-7 mda-mb 231 hela a549 mrc-5 1 2.96 >100 2.49 23.42 51.27 >100 >100 2.36 >100 2 2.69 9.97 9.51 7.40 9.64 0.24 5.22 31.45 59.88 3 0.70 4.91 8.87 1.11 12.34 15.62 3.54 2.43 >100 4 1.02 1.10 11.53 5.98 2.38 9.76 0.56 4.43 >100 5 0.74 0.68 19.78 4.25 0.34 28.70 3.41 4.19 >100 6 8.61b 1.53b 6.64b 7.25 >100 >100 9.59b 0.92 >100b 7 1.25b 0.14b >100b 76.36 89.36 >100 0.30b 29.05 >100b 8 0.18 1.83 16.26 2.79 2.28 26.57 4.11 7.72 >100 9 3.46 8.25 8.02 3.52 5.31 7.63 2.25 3.96 >100 10 4.87 3.96 4.29 4.88 15.36 36.47 10.32 0.025 >100 dox 0.25 0.92 0.03 2.98 0.20 0.09 0.07 4.91 0.10 aic50 is the concentration of compound required to inhibit the cell growth by 50 % compared to an untreated control. the values are means of three independent experiments. the coefficients of variation were less than 10 %; btaken from the literature22 all the synthesized analogues exhibited strong antiproliferative effects on k562 cells with ic50 values in the range of 0.18–8.61 µm. the highest potency against this cell line was recorded after treatment with analogue 8 (ic50 = 0.18 µm), which is approximately as active as dox (ic50 = 0.25 µm), but 16-fold more active than lead 1. all analogues (2–10) demonstrated powerful to good antiproliferative effects towards hl-60 cells (ic50 values in the range 0.14–9.97 µm), in contrast to lead 1, which was completely inactive against this cell line. moreover, all analogues with an alkoxymethyl grou(3–10) showed better antiproliferative effects against the hl-60 cell line than diol 2 (ic50 = 9.97 µm). 1350 srećo zelenović et al. the most active molecule against hl-60 cell line was analogue 5 (ic50 = = 0.68 µm), which exhibited similar activity as dox, but was 330-fold more potent than lead 1. analogue 3 was the most active compound in the culture of raji cells that exhibited over 2.5-, 6.5and 21-fold higher potency than control compounds, dox, 2 and 1, respectively. all novel ether analogues (3–5 and 8–10) demonstrated 4(4, ic50 = 5.98 µm) to 21-fold (3, ic50 = 1.11 µm) higher activity than lead 1, against raji cells. the parent compound 1 showed poor activity (ic50 = 51.27 µm) against mcf-7 cells, as did the previously synthesized compounds 6 and 7, which were practically inactive to these cells (ic50 >100 µm and ic50 = 89.36 µm, respectively). however, all new analogues (3–5 and 8–10) and compound 2 exhibited good cytotoxic effects toward this cell line with ic50 values in the range 0.34– 15.36 µm, being essentially 3–150-fold more active than 1. the most active compound against the mcf-7 cell line was the ether analogue 5. this molecule exhibited submicromolar cytotoxicity (ic50 = 0.34 µm) although its potency was slightly lower than the activity of dox (ic50 = 0.20 µm). lead compound 1 was inactive against hela and mda-mb 231 cells, but all novel analogues and dephenylated analogue 2 showed good (0.56–10.32 µm) to moderate inhibitory activity (7.63–36.47 µm), respectively, against these cell lines. all the synthesized ether analogues (3–10) as well as lead compound 1, were completely inactive toward normal mrc-5 cells. only molecule 2 showed low cytotoxicity (ic50 = 59.88 µm) against this cell line. on the contrary, the commercial antitumour agent dox exhibited a potent cytotoxicity against this cell line. sar analysis as shown in table iii, replacement of the α-hydroxylbenzyl group in 1 with an alkoxymethyl chain significantly increased the activities of the resulting analogues 3–10. (for a graphical presentation see, fig. s-35a of the supplementary material). as is further evident from table iii, the introduction of a hydrophobic group, by o-alkylation of oh-7 in 2, resulted in analogues 3–10 of improved cytotoxicity toward the majority of cell lines under evaluation (fig. s-35b of the supplementary material). finally, it was found that the length of the side chain of the analogues is not crucial for antiproliferative activity of the analogues 3–10 (table iii and fig. s -35c of the supplementary material). this is in contrast with previous findings,22 which suggested that oneor two-carbon homologation of the side chain increases the activity of the resulting homologues against most of the cells tested. however, the results of sar analysis presented in this work are more reliable novel (−)-goniofufurone mimics 1351 because they were conducted on seven pairs of analogues, unlike the previous sar study that was realized with a lower number of compounds. conclusions to summarize, nine novel (–)-goniofufurone analogues were designed and synthesized from d-glucose as the starting compound. the newly synthesized molecules were evaluated for their antiproliferative activity against eight human malignant cell lines. eight of the nine synthesized analogues showed submicromolar anticancer activity against at least one of the tested cell lines and five of them were more potent than dox (10, ic50 = 0.025 µm, and 6, ic50 = 0.92 µm against a549; 7, ic50 = 0.14 µm, and 5, ic50 = 0.68 µm against hl-60 and 8, ic50 = 0.18 µm against k562). sar analysis showed that the replacement of α-hydroxylbenzyl group in 1 with an alkoxymethyl chain, as well as o-alkylation of oh-7 in 2, may improve the cytotoxicity of the analogues towards the majority of the cell lines under evaluation. all analogues were devoid of any toxicity against a normal human cell line (mrc-5). it is believed that this approach could be used in the search for novel, more potent and selective antitumour agents derived from lead 1. supplementary material analytical and spectral data, as well as additional experimental data, are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. this work was supported by research grants from the ministry of education, science and technological development of the republic of serbia (grant no. oi 172006). this work also received funding from the serbian academy of sciences and arts under the strategic projects programme (grant agreement no 01-2019-f65). и з в о д нови миметици (−)-гониофуфурона: синтеза, антипролиферативна активност и sar анализа бојана м. срећо зеленовић1, слађана м. кекезовић1, мирјана попсавин1, весна в. којић2, горан и. бенедековић1 и велимир попсавин1,3 1департман за хемију, биохемију и заштиту животне средине, природно-математички факултет, трг доситеја обрадовића 3, 21000 нови сад, 2онколошки институт војводине, пут др голдмана 4, 21204 сремска каменица и 3српска академија наука и уметности, кнез михајлова 35, 11000 београд дивергентна синтеза нових миметика (–)-гониофуфурона, са алкоксиметил групом у бочном низу, остварена је полазећи из d-глукозе у девет синтетских фаза са укупним приносима од 6,7 до 8,7 %. њихова in vitro антипролиферативна активност је испитана према осам хуманих туморских и једној нормалној ћелијској линији. сви аналози су испољили снажне или добре антипролиферативне ефекте према већини испитиваних ћелијских линија. према ћелијској линији hl-60, сви миметици су испољили повећану активност и били су 27–1604 пута потентнији од водећег једињења (–)-гониофуфурона. такође, већина синтетизованих аналога је показала бољу или сличну активност као комерцијани антитуморски агенс доксорубицин (dox) према a549 ћелијама. најак1352 srećo zelenović et al. тивније једињење је испољило чак 196 пута снажнију цитотоксичност од dox, према овој ћелијској линији. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {application of principal component and hierarchical cluster analyses in classification of the serbian bottled waters and the comparison with waters from some european countries} j. serb. chem. soc. 82 (6) 711–721 (2017) udc 628.1.033+683.51:543.3:54.061/.063(497.11) jscs–4998 original scientific paper 711 application of principal component and hierarchical cluster analyses in the classification of serbian bottled waters and a comparison with waters from some other european countries jelena đ. cvejanov* and biljana d. škrbić university of novi sad, faculty of technology, bulevar cara lazara 1, 21000 novi sad, serbia (received 19 february, revised 2 march, accepted 3 march 2017) abstract: the contents of major ions in bottled waters were analyzed by principal component (pca) and hierarchical cluster (hca) analysis in order to investigate if these techniques could provide the information necessary for classifications of the water brands marketed in serbia. data on the contents of ca2+, mg2+, na+, k+, cl-, so42-, hco3and total dissolved solids (tds) of 33 bottled waters was used as the input data set. the waters were separated into three main clusters according to their levels of tds, na+ and hco3-; sub-clustering revealed a group of soft waters with the lowest total hardness. based on the determined chemical parameters, the serbian waters were further compared with available literature data on bottled waters from some other european countries. to the best of our knowledge, this is the first report applying chemometric classification of bottled waters from different european countries, thereby representing a unique attempt in contrast to previous studies reporting the results primarily on a country-to-country scale. the diverse character of serbian bottled waters was demonstrated as well as the usefulness of pca and hca in the fast classification of the water brands based on their main chemical parameters. keywords: chemometrics; anions in bottled water; cations in bottled water; total dissolved solids. introduction water is essential for any living organism; without water, there would not be plant or animal life in the form that is known. the water consumed by human beings comes in various forms. bottled water is widely consumed because it is readily available, tastes better, and contains fewer impurities.1 bottled waters contain many essential macroand micro-elements that are responsible for the maintenance of underlying biochemical and physiological processes in the * corresponding author. e-mail: cvejanov@tf.uns.ac.rs doi: 10.2298/jsc170219034c 712 cvejanov and škrbić human body, as the main ions in mineral waters are the main constituents of body fluids and cells. the chemical composition of the bottled water is dependent on the environment from which it originates. the technological processing line from the producing well to the final bottling might alter the original water composition, but there are only few parameters affected (iron and sulphur compounds, nitrite, nitrate and ammonium).2 the concentrations of the major dissolved components (such as, ca2+, mg2+, k+, na+ and cl–) in bottled water are unaffected by technological processing and could be considered to partially represent the original groundwater in the exploited aquifers.2 several studies have been performed to analyze in more detail the composition and variation of the major and trace elements in bottled waters from different countries, presenting specific interpretations of the results, primarily on a more detailed, country-to-country scale.3–12 bertoldi et al. provided descriptive statistics on the chemical composition of 571 european bottled mineral waters marketed in 23 european countries;13 however, similarities of the bottled water brands available on different markets have not hitherto been considered, even though it would provide useful information for consumers worldwide. the objective of this study was to use principal component analysis (pca) and hierarchical cluster analysis (hca) to investigate the natural variation of the main chemical composition parameters of bottled waters. thirty-three bottled waters produced in serbia were classified based on the major ions in order to assess the water diversity, knowing that serbia is one of the richest european countries regarding the availability of water resources. currently, there are about 30 factories producing about 60 million l of bottled waters per year. bottled water consumption in serbia is about 75 l a year per inhabitant, which might be considered low with respect to the yearly italian consumption rate of 200 l per inhabitant and to the european average rate of 150 litres per capita.2,5 in this study, the serbian bottled water brands were first characterized according to the existing eu directive 2009/54/ec on natural mineral waters14 taking into account the major ion contents and then they were evaluated by pca and hca in order to obtain their statistically based classification. furthermore, the serbian waters were compared with the relevant available literature data on bottled waters from croatia, estonia, italy, portugal and slovenia, thereby providing the first simultaneous comparison of the bottled waters brands marketed in different countries and evaluating the similarities/differences among them. experimental data set used for chemometric classification of the serbian bottled waters consisted of 7 variables for 33 waters available on the serbian market. this set (“set 1”) gathered 31 water brands in serbia; two brands in the data set (voda vrnjci and bivoda) were represented with waters of two mineralization degrees (i.e., one water of low mineral content and one of high bottled waters assessment by chemometrics 713 mineral content, later coded “2”). eight most frequently used chemical composition parameters were taken for the chemometric evaluation of the serbian bottled waters: total dissolved solids – tds, and the contents of ca2+, mg2+, na+, k+, hco3-, cland so42-; their levels are presented in table i. table i. main chemical composition parameters (concentration in mg l-1) of 33 bottled waters from the serbian market no brand tds ca2+ mg2+ na+ k+ hco3clso42 1 jazak 389 76.5 43.3 7.7 3.4 427 4.9 29.3 2 aqua viva 377 83.0 19.0 9.9 1.6 317 11.5 21.2 3 knjaz miloš 1175 107 45.0 282 17.5 1256 13.0 39.0 4 mivela 1621 26.6 335 121 9.1 2079 12.7 2.0 5 rosa 58 10.0 0.9 2.7 0.5 42 0.5 5.3 6 vlasina 48 5.7 2.7 2.3 0.3 32 0.05 3.7 7 vrnjačko vrelo 261 7.2 44.0 26.1 1.0 287 4.8 5.6 8 zlatibor 272 62.7 30.5 4.51 0.9 13 1.4 17.6 9 iva 245 58.4 20.2 2.9 0.8 268 2.4 6.8 10 aqua heba 3200 65.0 14.0 1059 56.0 3110 57.0 198 11 baš baš 248 48.3 11.1 15.9 0.8 198 11.1 16.5 12 voda voda 376 76.2 14.5 40.4 3.1 391 7.9 12.2 13 voda vrnjcia 295 36.8 22.3 40.2 3.4 292 5.0 18.5 14 aqua stilo 830 156 65.0 71.0 3.0 723 98 104 15 bistrica 3282 87.7 20.7 1160 54.0 3233 46.6 173 16 bivodaa 232 37.2 8.7 23.0 2.0 165 14.9 21.0 17 duboka 847 238 18.9 56.6 5.2 966 15.0 11.0 18 eko voda 293 48.0 15.1 26.6 2.9 189 21.8 42.1 19 golijska ledena 133 37.9 3.9 0.5 0.2 116 3.5 5.5 20 karađorđe 1510 129 80.0 312 40.0 1560 56.0 50.0 21 moja voda 163 33.7 7.9 9.9 0.8 119 8.3 12.1 22 vujić voda 358 95.0 20.0 2.7 0.8 397 2.2 5.7 23 la fantana 248 39.6 32.5 1.8 0.6 263 4.4 18.0 24 aqua gala 300 59.0 30.3 18.2 2.0 377 1.0 13.0 25 prolom voda 150 2.0 0.05 48 0.3 80 2.0 2.5 26 tron voda 364 83.0 38.7 1.8 0.6 401 1.4 22.2 27 voda kopaonik 1115 28.3 12.8 409 7.4 1183 18.9 0.3 28 aqua balkanika 394 78.5 28.3 33.9 2.1 440 5.2 18.0 29 eva 202 47.8 15.2 3.1 1.0 200 1.9 15.8 30 dar voda 637 90.6 22.6 92.4 17.2 521 28.4 80.5 31 minaqua 1181 22.2 19.9 412 3.6 768 287 0.4 32 voda vrnjci2a 1174 76.3 55.4 241 35.1 1177 15.5 29.1 33 bivoda2a 3401 85.4 20.6 1216 52.0 3290 54.1 173 mean 769 64.8 33.9 174 10.0 754 24.8 35.5 median 365 59.0 20.2 26.6 2.1 391 8.30 17.6 minimum 48 2.00 0.05 0.5 0.2 13.3 0.05 0.3 maximum 3401 238 335 1216 56.0 3290 287 198 skewness 2.02 1.68 4.88 2.44 1.97 1.88 4.35 2.26 kurtosis 3.45 4.73 26.0 5.11 2.55 2.73 21.3 4.23 ain the formed data set, the two brands, voda vrnjci and bivoda, were represented with two different bottled waters regarding the mineralization degree, i.e., with one water of low and one of high mineral content, later coded “2” 714 cvejanov and škrbić for the majority of the waters (coded 1–26, table i), the data on the major ions were taken from the manufacturers’ specifications, while the analytical results obtained elsewhere15 were taken for the additional 7 brands (coded 27–11, table i). these analytical results were obtained by different techniques:15,16 contents of ca2+, mg2+, na+ and k+ were determined by inductively coupled plasma atomic emission spectroscopy (with respective limits of detection (lod) values in mg l-1: 0.005, 0.005, 0.02 and 0.05), cland so42by ion chromatography (both with lod values of 0.01 mg l-1) and hco3by the titration (alkalinity) method (with a limit of detection of 1 mg l-1); the repeatability of the measurements was acceptable (below 5 %). good agreement between the specified values and the analytical results was found by random comparison of data for the same brands. in order to compare the selected major ions in the serbian bottled waters with the ones in the waters marketed in different countries, comparable literature-based data were considered: 39 bottled waters from portugal (samples nos. 34–72),3 14 from croatia (samples nos. 73– –86),4 5 from estonia (samples nos. 87–91),5 37 from italy (samples nos. 100–136)6 and 22 from slovenia (samples nos. 137–158).7 hence, the second data set (i.e., “set 2”) used for comparison of the bottled waters from different countries consisted of the 7 major ion contents in 150 bottled waters. for chemometric analysis, both data sets were arranged as follows: the rows refer to the bottled water samples, the while columns contain the ion concentrations. chemometric analysis basic univariate statistics have the capability of extracting precise characteristics from an examined population of data but information obtained in this way is necessarily one-sided and therefore limited.17 for a more comprehensive insight into the data sets formed in this study, multivariate statistical analyses were required. application of multivariate (chemometric) analysis to complex data sets has attracted high scientific interest in recent years and they are now used in a wide range of application.18-24 several chemometric-based studies have been performed to analyze bottled waters utilizing different physico-chemical parameters.3,9,12 in this study, pca and hca were used to elaborate the multidimensional data sets formed from the contents of major ions in the considered bottled waters (previously log -transformed and standardized). pca can be summarized as a tool for transforming the original measured variables into new uncorrelated variables, i.e., principal components, pcs. each pc is a linear combination of the original variables. the results of pca are presented in terms of the variable loadings and sample scores, which could be superimposed graphically in the form of a biplot graph. the number of extracted pcs from the data set was determined in accordance to the kaiser rule.25 in order to interpret the significance of the retained pcs in terms of original variables, only the loadings with absolute values greater than 60 % of the maximum loading for a particular pc were considered. hca was used to group bottled waters into statistically determined groups (i.e., clusters). it differs from other classification tools (for example discriminant analysis) since the number and characteristics of the groups, which are derived from the data, are not usually known in advance. in this study, the ward method as an amalgamation rule and the euclidean distance as a measure of distance were used for hca. throughout the study, the chemometric analyses were accomplished within the statistica 6.0 computing environment (statsoft inc.). bottled waters assessment by chemometrics 715 results and discussion classification of the serbian bottled waters in this study, chemometric characterization (classification) of 33 serbian bottled waters (31 brands) was performed by means of eight important chemical composition parameters (see table i). the skewness of all parameters was positive (table i), indicating a right-skewed distribution with most values concentrated on the left of the means and the extreme values to their right. a kurtosis near 3 (obtained for hco3– and k+ (table i) and also for tds) showed a distribution similar to normal, while those greater than 3 (particularly high values of kurtosis were obtained for mg2+ and cl–, table i) suggested a distribution sharper than normal with thicker tails and a high probability for extreme values. hence, due to presence of the extreme values (i.e., outliers), the median values were used for the further discussion instead of simple means. concerning the median values, the major cations in the serbian bottled waters could be ordered in decreasing order as follows: ca2+ > na+ > mg2+ > k+. the prevailing anion was hco3– followed by so42– and cl–. this is in accordance with the most prevailing types of mineral waters in the large geotectonic units present in serbia: hco3–na type in the pannonian basin; hco3–na or hco3–ca–mg type in the dinarides; hco3–na–cl or hco3–na–so4–cl in the serbian–macedonian massif; hco3–ca or hco3–na–so4–cl type in the carpatho-balkanides.26 to gain a better insight into the selected serbian bottled waters, they were classified following the criterion of the eu directive 2009/54/ec.14 the results of such classifications in accordance to the main cations and anions, as well as according to the tds values, are presented in table ii. pca was used to reduce the dimensionality of the data set, identify the underlying structure of the data and the distributional structure of bottled waters in relation to the main chemical composition parameters. the application of the kaiser rule led to the retention of two pcs with eigenvalues larger than one, which totally explained 79.6 % of total data variance. the first pc, pc1, accounted for 63.4 % of the data variance, and it was related to the concentrations of na+, tds, k+, hco3– and cl– (with loadings of 0.966, 0.909, 0.870, 0.859 and 0.828, respectively); this observation suggested that higher contents of na+, k+, hco3– and cl– could be found in serbian waters with higher mineral contents (i.e., higher tds). this is in agreement with the study of peh et al.,4 who reported dominant na–k signature characterizing croatian bottled waters of the mineral type, as well as their enrichment in cl–. the second pc, pc2, was responsible for an additional 16.2 % of the data variance and it was related to the concentration of ca2+, mg2+ and so42– (with loadings of 0.895, 0.732 and 0.708, respectively). according to the parameters grouping, pc1 could be described as the component related to water mineralization (or dissolved salt content, i.e., salinity), while pc2 716 cvejanov and škrbić table ii. classification of the serbian bottled waters according to eu directive 2009/54/eec14 mineral water type criterion brand name %b cation and anion classification contains bicarbonate > 600 knjaz miloš, mivela, aqua heba, aqua stilo, bistrica, duboka, karađorđe, voda kopaonik, minaqua, voda vrnjci 2, bivoda 2 33 contains sulphate > 200 aqua heba (≈ 200) 3 contains chloride > 200 minaqua 3 contains calcium > 150 aqua stilo, duboka 6 contains magnesium > 50 mivela, voda vrnjci 2 6 contains iron > 1 − contains sodium > 200 knjaz miloš, aqua heba, bistrica, karađorđe, voda kopaonik, minaqua, voda vrnjci 2, bivoda 2 24 suitable for low sodium diets < 20 jazak, aqua viva, rosa, vlasina, zlatibor, iva, baš baš, golijska ledena, moja voda, vujić voda, la fantana, aqua gala, tron voda, eva 45 tds classification very low mineral content < 50 vlasina 3 low mineral content 50–500 jazak, aqua viva, rosa, vrnjačko vrelo, zlatibor, iva, baš baš, voda voda, voda vrnjci, bi voda, eko voda, golijska ledena, moja voda, vujić voda, la fantana, aqua gala, prolom, tron, aqua balkanika, eva 61 intermediate mineral content 500–1500 knjaz miloš, aqua stilo, duboka, voda kopaonik, dar voda, minaqua, voda vrnjci 2 21 rich in mineral salts > 1500 mivela, aqua heba, bistrica, karađorđe, bivoda2 15 aall in mg l-1; bpercentage of the particular type of water relative to the total number of bottled waters considered in this work (n = 33) might be related to the cations responsible for water hardness. the results of pca in a two-dimensional biplot plane are depicted in fig. 1. the first group of parameters correlating significantly with pc1 had an impact on the classification of bottled waters along pc1: waters located on the right side from the vertical dotted line (i.e., samples distributed along the positive side of pc1, fig. 1) had tds values above 600 mg l–1 and these waters could be classified as intermediate and rich in mineral salts, whereas those on the left side of the vertical line (distributed along the negative side of pc1) had tds values lower than 500 mg l–1 and could be classified as low mineral content waters (table ii). the second group of parameters correlating with pc2 influenced further the classification of the low mineralized waters along pc2, highlighting three brands of waters (prolom voda no. 25, vlasina no. 6, rosa no. 5) separated from the others in fig. 1. these waters had the lowest total hardness values (5.2–28.8 mg l–1) calculated based on the concentrations of ca2+ ([ca2+]) and mg2+ ([mg2+]) bottled waters assessment by chemometrics 717 as 2.5[ca2+]+4.1⋅[mg2+].12 such waters with total hardness less than 50 mg l–1 could be classified as soft.11 fig. 1. the pca biplot of pc1 vs. pc2 obtained for 33 serbian bottled water (“set 1”). additionally, hca was used to find the natural grouping of the serbian bottled water brands in accordance with the selected main chemical composition parameters. the results of hca are presented in the form of a dendrogram (fig. 2). based on eight parameters, the hca classified 33 serbian bottled waters into the following main clusters. the first cluster (cluster “i-outliers”, fig. 2) comprised three brands with the highest values of tds (>3000 mg l–1, median 3282 mg l–1), hco3– (>3000 mg l–1, median 3233 mg l–1) and na+(>1000 mg l–1, median 1160 mg l–1), having also among the highest content of cl– (46.6, 54.1, 27.0 mg l–1). the second cluster (“ii”) grouped nine brands that could also be found on the right side of pc1 (fig. 1) with intermediate values of tds (637– –1621 mg l–1, median 1175 mg l–1), hco3– (521–2079 mg l–1, median 1177 mg l–1) and na+ (92.4–412 mg l–1, median 241 mg l–1), having also pronounced cl– content (12.7–287 mg l–1, median 18.9 mg l–1). the third main cluster (“iii”, fig. 2) contained the majority of the analyzed brands (21, i.e., 64 %), which all could be classified as low in mineral salts, having tds values less than 500 mg l–1 (table ii), i.e., in the range 48–395 mg l–1, with median 261 mg l–1; additional common features for this cluster of waters were hco3– levels (from 48 to 395 mg l–1 with median of 264 mg l–1) less than 600 mg l–1 (the 718 cvejanov and škrbić limit for the classification of water as “contains bicarbonate”, table ii); na+ contents less than 50 mg l–1 (0.5–40.5 mg l–1 with median of 10 mg l–1) and cl– contents less than 12 mg l–1 (except bi voda – no. 16 and eko voda – no. 18, see table 1). within cluster “iii”, further sub-clustering was observed; one of the sub-clusters, “iiia-soft” (fig. 2), gathered three waters with the lowest mg2+ levels (0.05–2.7 mg l–1), the lowest total hardness (5.2–28.8 mg l–1; the total hardness of the remaining waters ranged from 110 to 1442 mg l–1) and the lowest k+ levels (0.3–0.5 mg l–1). fig. 2. the hca dendrogram for the 33 serbian bottled waters (“set 1”). hence, the two applied multivariate chemometric tools gave results in good agreement (fig. 1). in addition to the hca dendrogram, pca biplots visualized the relationship among the chemical compositional parameters and mineral water brands. the result of this study indicated that tds and the contents of hco3– and na+ are the most important variables for classification and separation of the investigated water brands. in a similar manner, güler9 and oyebog et al.11 mentioned tds as the most discriminating variable in the grouping of water brands. comparison of the serbian bottled waters with those marketed in other countries hca was applied to the data set (“set 2”) consisting of available data for 7 major ions in 150 bottled waters produced in different countries, including 33 bottled waters assessment by chemometrics 719 previously characterized serbian waters. the groups (clusters) obtained by hca contained waters with similar macrocomponent patterns listed in table iii. a description of the clusters (in terms of the origin of the waters within the particular cluster) is given in table iv, from which it could be seen that the majority of the bottled water brands from croatia, italy, serbia and slovenia were grouped in cluster “1a”. the serbian waters in this cluster were those from cluster “iii” mentioned above (fig. 2). the exceptions were 3 waters that clustered together with the majority of the portuguese brands in cluster “1b” (table iii). these three waters belong to the sub-cluster “iiia-soft” (fig. 2) with the lowest total hardness among the 33 considered serbian brands. in cluster “2” (tables iii and iv), there were 12 serbian waters of high and intermediate levels of tds, hco3– and na+ that were previously classified in clusters “i – outliers” and “ii” (fig. 2). thus, the allocation of the serbian waters in all three main clusters (tables iii and iv) suggested diversity of the marketed bottled waters, contrary to the slovenian and croatian waters not grouped in cluster “1b” with the lowest median values of the ca2+ and mg2+ contents. table iii. the content of the clusters (marked 1a, 1b and 2) obtained by hierarchical cluster analysis (hca) of the data “set 2” containing the concentration of 7 major ions in 150 bottled water brand marketed in different european countries. the clusters’ content is given in the form of the bottled water sample numbers explained previously in the experimental. the samples numbered 1–33 are from serbia, 34–72 from portugal,3 73–86 from croatia,4 87–91 from estonia,5 100–136 from italy6 and 137–158 from slovenia7 1 2 (n = 50) 1a (n = 66) 1b (n = 34) 1, 2, 7–9, 11–13, 16, 18, 19, 21–24, 26, 28, 29, 37, 58, 59, 64, 75–80, 82, 86, 85, 86, 89–100, 102, 107, 108, 109, 112, 115, 117, 118, 120, 123, 127–132, 134, 136, 140, 145, 146, 149–158 5, 6, 25, 34, 38–41, 43, 46, 48–51, 54, 55, 57, 60–63, 65–72, 91, 103, 106, 110, 113 3, 4, 10, 14, 15, 17,20, 27, 30–33, 35, 36, 42, 44, 45, 47, 52, 53, 56, 73, 74, 81, 84, 87, 88, 101, 104, 105, 111, 114, 116, 119, 121, 122, 124–126, 133, 135, 137–139, 141–144, 147, 148 table iv. origin of the bottled water samples grouped in the main hca clusters from table iii: number of samples and percentage (in parentheses) of the bottled water from one country grouped in a particular cluster clusters croatia estonia italy portugal serbia slovenia total number of samples 2 4 (29) 2 (40) 14 (38) 9 (23) 12 (36) 9 (41) 50 1b 0 (0) 1 (20) 4 (11) 26 (67) 3 (9) 0 (0) 34 1a 10 (71) 2 (40) 19 (51) 4 (10) 18 (54) 13 (59) 66 total number of samples 14 5 37 39 33 22 150 720 cvejanov and škrbić conclusion the main chemical composition parameters of 33 bottled waters available on the serbian market were presented in the light of relevant eu legislation and they were evaluated by means of multivariate chemometric tools. the applied chemometric methods, i.e., pca and hca, were shown to be effective tools in the identification of the principal interrelationships of the major ions of the studied bottled waters, thereby indicating their similarities and dissimilarities. high degree of correlations among examined chemical composition parameters was revealed: the group of parameters reflecting the water total mineralization (tds, hco3–, na+) was separated from the group of parameters reflecting the total hardness of the waters (ca2+, mg2+). additionally, it is noteworthy that the application of pca and hca allowed a simple and meaningful classification of serbian water brands into three main clusters primarily based on the parameters reflecting the water mineralization (i.e., tds, hco3–, and na+). moreover, the brands with the lowest total hardness were identified, providing valuable information for consumers. simultaneous comparison of the waters from the serbian markets with those marketed in croatia, estonia, italy, portugal and slovenia by hca revealed diversity of the domestic brands, which may represent competitive choices on the global market. acknowledgement. the results presented herein were obtained within project no. 172050 supported by the ministry of education, science and technological development of the republic of serbia. и з в о д примена анализе главних компонената и кластера у класификацији флашираних вода из србије и њиховом поређењу са водама из изабраних европских земаља јелена ђ. цвејанов и биљана д. шкрбић универзитет у новом саду, технолошки факултет нови сад, булевар цара лазара 1, 21000 нови сад садржаји основних јона у флашираној води су обрађени применом анализе главних компонената и хијерархијеске кластер анализе са циљем испитивања могућности примене ових хемометријских техника при класификацији вода са српског тржишта. подаци о хемијском саставу и то садржаји ca2+, mg2+, na+, k+, cl-, so4 2-, hco3 и суви остатак у 33 флаширане воде, чинили су улазну базу података. уочене су три главне групе вода у складу на садржајима сувог остатка , na+ and hco3 -; даље груписање у оквиру једне од главних група указало је на групу „меких“ вода са најмањим степеном тврдоће. уочене групе дају информације корисне за потрошаче при избору вода на тржишту. додатно су ове српске воде на основу одређених хемијских параметара упоређене са релевантним подацима доступним у литератури о флашираним водама из иностранства; нова улазна база података у овом случају састојала се од садржаја 7 главних јона у 150 узорака флаширане воде из србије и иностранства. према нашим сазнањима, ово је први рад у ком се примењује истовремена (хемометријска) класификација флашираних вода из различитих земаља применом хијерархијске кластер анализе, представљајући јединствен покушај у односу на досадашње студије које су приказивале bottled waters assessment by chemometrics 721 првенствено резултате који се односе на једну земљу. резултати су указали на разноликост флашираних вода из србије, као и на корисност анализе главних компонената и хијерархијске кластер анализе за једноставну и брзу класификацију флашираних вода са тржишта на основу главних хемијских параметара. 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice j. serb. chem. soc. 84 (12) 1469–1478 (2019) 1469 contents of volume 84 number 1 organic chemistry e. s. ramadan, h. a. rasheed and e. s. h. el ashry: synthesis and antimicrobial screening of novel 1,3-dioxolanes linked to n-5 of 5h-1,2,4-triazino[5,6-b]indole -3-thiol ........................................................................................................................ 1 biochemistry and biotechnology e. hodžić, m. balaban, n. šuškalo, s. galijašević, d. hasanagić and b. kukavica: antioxidative response of melissa officinalis l. and valeriana officinalis l. leaves exposed to exogenous melatonin and excessive zinc and cadmium levels ................. 11 theoretical chemistry z. mei, f. zhao, s. xu and x. ju: a simple relationship of bond dissociation energy and average charge separation to impact sensitivity for nitro explosives .......................... 27 electrochemistry f. kesri, a. m. affoune and i. djaghout: effects of thiourea on the kinetics and electrochemical nucleation of tin electrodeposition from stannous chloride bath in acidic medium ....................................................................................................................... 41 analytical chemistry b. g. savić, i. j. mihajlović, s. m. milutinović, m. m. seović, ž. m. nikolić, m. s. tošić and t. p. brdarić: validation and uncertainty estimation of an analytical method for the determination of phenolic compounds in concrete ................................................ 55 materials n. yang, p. ning, k. li and j. wang: a new method of processing co2 and magnesite slag simultaneously ..................................................................................................... 69 environmental t. t. c. truong, k. takaomi and h. m. bui: chitosan/zeolite composite membranes for the elimination of trace metal ions in the evacuation permeability process ................ 83 r. karkalić, m. mandić, j. nikodinovic-runic, d. jovanović, z. luković and s. vojnović: antimicrobial and anti-biofilm activity and biological decontamination efficiency of ed-1 emulsion ........................................................................................................ 99 letters to the editor m. v. rodić: comments on dft calculation, biological activity, anion sensing studies and crystal structure of (e)-4-chloro-2-((pyridin-2-ylimino)-methyl)phenol by nuray yıldırım, neslihan demir, gökhan alpaslan, bahadir boyacıoğlu, mustafa yıldız and huseyin ünver, published in the journal of the serbian chemical society, volume 83, issue 6, 2018, pp. 707–721 ........................................................ 111 1470 journal of the serbian chemical society m. yildiz: discussion on the comments on dft calculation, biological activity, anion sensing studies and crystal structure of (e)-4-chloro-2-((pyridin-2-ylimino) -methyl)phenol by nuray yıldırım, neslihan demir, gökhan alpaslan, bahadir boyacıoğlu, mustafa yıldız and huseyin ünver, published in the journal of the serbian chemical society, volume 83, issue 6, 2018, pp. 707–721 ........................... 117 number 2 organic chemistry m. yusuf and s. thakur: multicomponent synthesis of new bis(pyranopyrazoles) and their antimicrobial–antioxidant evaluations ................................................................ 119 biochemistry and biotechnology a. v. buntić, o. s. stajković-srbinović, d. i. delić, s. i. dimitrijević-branković and m. d. milić: the production of cellulase from the waste tobacco residues remaining after polyphenols and nicotine extraction and bacterial pre-treatment ........................ 129 m. ljekočević, m. jadranin, j. stanković, b. popović, n. nikićević, a. petrović and v. tešević: phenolic composition and dpph radical scavenging activity of plum wine produced from three plum cultivars ............................................................................ 141 theoretical chemistry r. c. silva, j. g. c. poiani, r. s. ramos, j. s. costa, c. h. t. p. silva, d. s. b. brasil and c. b. r. santos: ligandand structure-based virtual screening of 16-((diisobutylamino)methyl)-6α-hydroxyvouacapane-7β,17β-lactone, a compound with potential anti-prostate cancer activity .............................................................................. 153 electrochemistry m. safaei, h. beitollahi, m. reza shishehbore, s. tajik and r. hosseinzadeh: electrocatalytic determination of captopril using a carbon paste electrode modified with n(ferrocenyl-methylidene)fluorene-2-amine and graphene/zno nanocomposite ......... 175 o. shmychkova, t. luk’yanenko, l. dmirtikova and a. velichenko: modified lead dioxide for organic wastewater treatment: physicochemical properties and electrocatalytic activity ................................................................................................................. 187 analytical chemistry e. makrlík and p. vaňura: calcium ionophore i as very effective extraction agent for trivalent europium and americium .............................................................................. 199 materials v. gupta and j. anandkumar: synthesis of crosslinked pva-ceramic composite membrane for phenol removal from aqueous solution ........................................................ 211 environmental s. rajan, a. pattanaik, v. kumaresan, p. bhatt, s. gunasekaran, j. arockiaraj, m. pasupuleti, v. p. beškoski and p. chakraborty: characterization of some naphthalene using bacteria isolated from contaminated cooum riverine sediment of the bay of bengal (india) ............................................................................................................. 225 volume 84: contents 1471 number 3 organic chemistry d. ashok, r. kavitha, s. gundu and m. sarasija: microwave-assisted synthesis and antimicrobial evaluation of 6-[3-aryl-1-phenyl-4′,5′-dihydro[4,5′-bi-1h-pyrazol]-3′ -yl]-2h-chromen-5-ols ................................................................................................ 237 g. tang, x. liu, x. gong, x. lin, x. lai, d. wang and s. ji: studies on the chemical compositions of hyptis suaveolens (l.) poit. .............................................................. 245 biochemistry and biotechnology d. r. đokić-stojanović, z. b. todorović, d. z. troter, o. s. stamenković, lj. m. veselinović, m. v. zdujić, d. d. manojlović and v. b. veljković: influence of various cosolvents on the calcium oxide-catalyzed ethanolysis of sunflower oil .................... 253 inorganic chemistry c. kanagavalli, m. sankarganesh, j. dhaveethu raja and m. kalanithi: spectral, nlo and antimicrobial studies of co(ii), ni(ii) and cu(ii) complexes of schiff base ligands of 2-amino-6-nitrobenzothiazole .................................................................... 267 m. sankarganesh, n. revathi, j. dhaveethu raja, k. sakthikumar, g. g. vinoth kumar, j. rajesh, m. rajalakshmi and l. mitu: computational, antimicrobial, dna binding and anticancer activities of pyrimidine incorporated ligand and its copper(ii) and zinc(ii) complexes ...................................................................................................... 277 theoretical chemistry x. tan and x. lu: ab initio study of the mechanism of formation of a spiro-sn-heterocyclic ring compound by the cycloaddition reaction of h2c=sn: and ethylene .......... 293 j.-b. tong, s.-s. qin, s. lei and y. wang: molecular modeling studies of hiv-1 non -nucleoside reverse transcriptase inhibitors using 3d-qsar, virtual screening and docking simulations .................................................................................................... 303 polymers s. naz and r. saeed: ionic-interaction of aqueous and alcoholic poly(vinyl alcohol) in the presence of protons ............................................................................................... 317 environmental j. nikić, m. watson, a. tubić, m. kragulj isakovski, s. maletić, e. mohora and j. agbaba: arsenic removal from water using a one-pot synthesized low-cost mesoporous fe–mn-modified biosorbent ............................................................................ 327 number 4 organic chemistry l. rekovic, l. kosychova, i. bratkovskaja and r. vidziunaite: synthesis and spectral characterization of novel 1,5-benzodiazepine oxime derivatives ................................ 343 r. venkataramana, v. r. r. chittireddy, d. ashok and k. karunakar rao: microwave -assisted synthesis of 2,8-di(alkyl/aryl)-4,6-dichloro-2h,8h-pyrano[3,2-g]chromene-3,7-dicarbaldehydes and their antimicrobial activity ........................................ 355 biochemistry and biotechnology g. n. zavišić, s. m. petričević, s. m. ristić, m. g. rikalović, n. m. jovanović-lješković, j. m. begović and i. d. strahinić: probiotic potential of lactobacillus fermentum g-4 originating from the meconium of newborns .............................................................. 365 1472 journal of the serbian chemical society b. stojanović, lj. radović, d. natić, m. dodevska, g. vraštanović-pavičević, m. balaban, s. lević, t. petrović and v. antić: influence of a storage conditions on migration of bisphenol a from epoxy–phenolic coating to canned meat products ........... 377 theoretical chemistry a. dadras, a. benvidi, m. namazian, s. abbasi, m. d. tezerjani, m. roozegari and r. tabaraki: determination of the pka for caffeic acid in mixed solvent using the net analyte signal method and the ab initio theory ............................................................ 391 y. driouche and d. messadi: quantitative structure–retention relationship model for predicting retention indices of constituents of essential oils of thymus vulgaris (lamiaceae) (short communication) ........................................................................... 405 electrochemistry i. a. pašti: contemporary approaches in development of new materials for electrochemical energy conversion (extended abstract)................................................................. 417 metallic materials and metallurgy n. gajić, ž. kamberović, z. anđić, m. korać, j. trpčevská and m. stamatović: improving the synthesis process of tribological materials based on tin sulphides by adding graphite as additive ..................................................................................................... 423 environmental o. a. urucu, e. k. yetimoglu, s. donmez and s. deniz: undecanol–ethanol–water ternary system-based microextraction for the detection of cadmium .............................. 435 number 5 organic chemistry q. v. vo, p. c. nam, t. n. dinh, a. mechler and t. t. v. tran: anti-inflammatory activity of synthetic and natural glucoraphanin .......................................................... 445 biochemistry and biotechnology b. davidović-plavšić, n. lukić, a. nikolić-kokić and b. kukavica: effects of hemazin sc 500 (terbuthylazine) on antioxidative enzymes in human erythrocytes in vitro .... 455 inorganic chemistry m. v. rodić, m. m. radanović, lj. s. vojinović-ješić, s. k. belošević, ž. k. jaćimović and v. m. leovac: synthesis and crystal structure of copper(ii) complexes with pyridoxal s-methylisothiosemicarbazone bearing a new coordination mode ............. 467 physical chemistry a. k. ryskaliyeva, m. e. baltabayev and k. t. abaeva: empirical method for predicting the enthalpy changes of combustion of amides (short communication) ..................... 477 electrochemistry m. m. petrović, s. m. najdanović, m. m. kostić, m. d. radović vučić, n. d. velinov, d. v. bojić and a. lj. bojić: effect of electrochemical parameters and working electrode material on the characteristics of bismuth (iii) oxide obtained by electrodeposition and thermal oxidation (extended abstract) ........................................................ 483 analytical chemistry m. payehghadr, h. h. qezelje, f. nourifard, a. attaran and m. kalhor: preconcentration of ultra-traces of cu(ii) in water samples using sba-15 sorbent modified volume 84: contents 1473 with a thiocarbohydrazide ligand prior to determination by flame atomic absorption spectrometry ................................................................................................................ 489 metallic materials and metallurgy j. šćepanović, v. asanović, d. radonjić, d. vuksanović, s. herenda, f. korać and f. bikić: mechanical properties and corrosion behaviour of al–si alloys for ic engine .......... 503 environmental n. ali, c. b. yeoh, se. lau and m. g. tay: an enhanced treatment efficiency for diluted palm oil mill effluent using a photo-electro-fenton hybrid system ............................. 517 i. ćipranić, r. marković, s. đorđievski, z. stevanović and m. stevanović: the impact of coal ash and slag dump on the quality of surface and ground waters – a case study (note) .......................................................................................................................... 527 number 6 organic chemistry j. tadić, m. mihajlović, m. jovanović and d. mijin: continuous flow synthesis of some 6and 1,6-substituted 3-cyano-4-methyl-2-pyridones ................................................ 531 biochemistry and biotechnology v. lazić, i. vukoje, b. milićević, b. spremo-potparević, l. živković, d. topalović, v. bajić, d. sredojević and j. m. nedeljković: efficiency of the interfacial charge transfer complex between tio2 nanoparticles and caffeic acid against dna damage in vitro: a combinatorial analysis ............................................................................... 539 v. b. tatipamula and g. s. vedula: antimicrobial and anti-tubercular activities of isolates and semi-synthetic derivatives of lichen ramalina leiodea (nyl.) nyl. ......... 555 inorganic chemistry u. yildiz and b. coban: chemical and photo-induced nuclease activity of a novel minor groove dna binder cu(ii) complex ........................................................................... 563 theoretical chemistry s. kherouf, n. bouarra, a. bouakkadia and d. messadi: modeling of linear and nonlinear quantitative structure property relationships of the aqueous solubility of phenol derivatives ....................................................................................................... 575 r. a. da costa, s. g. silva, s. de o. silva, j. n. cruz, w. a. da costa, l. s. n. dos santos brasil, r. c. silva, c. b. r. santos, c. n. alves and d. de s. barros brasil: theoretical study via dft for the prediction of 13cand 1h-nmr data for two diterpenoids derived from the root of salvia grandifolia (short communication) ...... 591 thermodynamics j. safarov, u. ashurova, b. ahmadov and e. hassel: vapour pressure of 1-butanol and diesel b0 binary fuel blends ....................................................................................... 599 materials l. xu, s. wang, p. u. okoye, j. wang, s. li, l. zhang, a. zhang and t. tang: water glass derived catalyst for the synthesis of glycerol carbonate via the transesterification reaction between glycerol and dimethyl carbonate ............................................ 609 environmental i. ivančev-tumbas, m. bogunović, m. česen, a. tubić and e. heath: influence of water matrix on benzophenone degradation by uv-irradiation ............................................ 623 euchems news ................................................................................................................... 633 1474 journal of the serbian chemical society number 7 organic chemistry i. i. jevtić, j. z. penjišević, m. d. ivanović and s. v. kostić-rajačić: synthetic route towards potential bivalent ligands possessing opioid and d2/d3 pharmacophores ..... 639 m. a. abbasi, m. s. ramzan, a.-ur-rehman, s. z. siddiqui, m. hassan, h. raza, s. a. ali shah, b. mirza and s.-y. seo: structure–activity relationship and in silico study of unique bi-heterocycles: 5-[(2-amino-1,3-thiazol-4-yl)methyl]-1,3,4-oxadiazole-2 -thiol derivatives ......................................................................................................... 649 biochemistry and biotechnology k. hanousek čiča, m. pezer, j. mrvčić, d. stanzer, j. čačić, v. jurak, m. krajnović and j. gajdoš kljusurić: identification of phenolic and alcoholic compounds in wine spirits and their classification by use of multivariate analysis .................................... 663 h. hamdi and a. hellal: optimization of phenol biodegradation by immobilized bacillus subtilis isolated from hydrocarbons-contaminated water using the factorial design methodology. .............................................................................................................. 679 inorganic chemistry i. m. stanojević, n. d. savić, a. crochet, k. m. fromm, m. i. djuran and b. đ. glišić: synthesis and structural analysis of polynuclear silver(i) complexes with 4,7-phenanthroline .................................................................................................................... 689 physical chemistry s. d. abkenar, m. hassannezhad, m. hosseini and m. r. ganjali: efficient removal of malachite green from aqueous solution by adsorption on carbon nanotubes modified with znfe2o4 nanoparticles ......................................................................... 701 electrochemistry j. wan, c. jin, b. liu, z. she, m. gao and z. wang: electrochemical oxidation of sulfamethoxazole using ti/sno2–sb/co–pbo2 electrode through ann-pso ................... 713 materials i. o. mladenović, j. s. lamovec, v. b. jović, m. obradov, d. g. vasiljević radović, n. d. nikolić and v. j. radojević: mechanical characterization of copper coatings electrodeposited onto different substrates with and without ultrasound assistance ..... 729 environmental d. marković, j. vasiljević, b. golja, b. tomšič, b. simončič and m. radetić: biodegradation of cotton fabric impregnated with tio2 nanoparticles ....................................... 743 e. yabalak, i̇. topaloğlu and a. m. gizir: application of the eco-friendly subcritical water oxidation method in the degradation of epichlorohydrin................................... 757 number 8 dedicated to academician miljenko perić on the occasion of his 70th birthday editorial ............................................................................................................................. 769 m. zlatar and m. gruden: calculation of the jahn–teller parameters with dft (authors’ review) ........................................................................................................................ 779 s. v. jerosimić, m. lj. mitić and m. z. milovanović: sccs– radical: renner–teller effect and spin–orbit coupling in the x 2πu electronic state .................................................. 801 f. dinkelbach and c. m. marian: vibronic and spin–orbit coupling effects in the absorption spectra of pyrazine: a quantum chemical approach ....................................... 819 volume 84: contents 1475 f. bohle and s. grimme: efficient structural and energetic screening of fullerene encapsulation in a large supramolecular double decker macrocycle .................................... 837 m. mladenović: six-dimensional potential energy surface and rotation–vibration energy levels of hnco in the ground electronic state ............................................................ 845 b. d. ostojić and d. s. đorđević: the role of molecular properties of mononitrofluoranthenes to their mutagenic activity: insight from ab initio and dft calculations ........ 861 d. dimić, d. milenković, z. marković and j. dimitrić marković: the reactivity of dopamine precursors and metabolites towards abts•–: an experimental and theoretical study ............................................................................................................................ 877 b. ž. milovanović, m. r. etinski and m. m. petković: hydrogen transfer reaction: bond formation and bond cleavage through the eyes of interacting quantum atoms ............ 891 i. a. pašti, a. s. dobrota, n. m. gavrilov, g. ćirić-marjanović and s. v. mentus: effects of alkali metal cations on oxygen reduction on n-containing carbons viewed as the interplay between capacitive and electrocatalytic properties: experiment and theory ... 901 s. stojadinović and r. vasilić: spectroscopic study of micro-discharges during plasma electrolytic oxidation of al–zn–si alloy ..................................................................... 915 number 9 organic chemistry j. z. penjišević, d. b. andrić, v. b. šukalović, g. m. roglić, v. šoškić and s. v. kostić -rajačić: synthesis of novel 2-(piperazino-1-yl-alkyl)-1h-benzimidazole derivates and assessment of their interactions with the d2 dopamine receptor .......................... 925 f. bihelovic, z. ferjancic and z. joncev: synthesis of two novel c-19 analogues of (±)-alstoscholarisine a (short communication) .......................................................... 935 biochemistry and biotechnology n. gligorijević, m. šunderić, a. vilotić, m. baralić and o. nedić: quantitation of active alpha-2-macroglobulin by trypsin protease zymography ............................................ 943 j. gvozdenović-jeremić, e. vert-wong and lj. mojović: antiosteogenic effect of arsenic trioxide, cholecalciferol, lovastatin or their combination in vitro. .............................. 951 inorganic chemistry m. antonijević nikolić, b. dražić, j. antić stanković and s. tanasković: new mixed -ligand ni(ii) and zn(ii) macrocyclic complexes with bridged (endo,endo)-bicyclo[2.2.1]hept-5-ene-2,3-dicarboxylate: synthesis, characterization, antimicrobial and cytotoxic activity ......................................................................................................... 961 theoretical chemistry z. kazeminejad, a. shiroudi, k. pourshamsian, f. hatamjafari and a. r. oliaey: understanding the isomerization kinetics in the gas phase of a triazole-3-thione derivative: a theoretical approach (short communication) ............................................... 975 physical chemistry m. vraneš, a. tot, j. panić, s. papović, s. gadžurić and d. četojević-simin: towards edible ionic liquids – cholinium taurate ..................................................................... 991 electrochemistry s. z. mohammadi, s. tajik and h. beitollahi: screen printed carbon electrode modified with magnetic core shell manganese ferrite nanoparticles for electrochemical detection of amlodipine ............................................................................................... 1005 1476 journal of the serbian chemical society analytical chemistry k. pastor, v. vujasinović, a. marjanović jeromela, dj. vujić, dj. jovanović and m. ačanski: gas chromatography–mass spectrometry system applied to determine botanical origin of various types of edible vegetable oils ........................................... 1017 materials l. almásy, a.-m. putz, q. tian, g. p. kopitsa, t. v. khamova, r. barabás, m. rigó, a. bóta, a. wacha, m. mirica, b. ţăranu and c. savii: hybrid mesoporous silica with controlled drug release ................................................................................................ 1027 environmental j. mitrović, m. radović vučić, m. kostić, n. velinov, s. najdanović, d. bojić and a. bojić: sulfate radical-based degradation of antraquinone textile dye in a plug flow photoreactor ................................................................................................................ 1041 number 10 e. e. kiss and s. n. panić: accelerated physical and chemical transformations in ceramics processing (survey) .......................................................................................... 1055 organic chemistry m. gucma, w. marek gołębiewski, k. żelechowski and m. krawczyk: studies on the [2+3] cycloaddition reaction of nitrile oxides to abietic acid esters ............................ 1073 f. mohamadpour: glutamic acid as green and bio-based α-amino acid catalyst promoted one-pot access to polyfunctionalized dihydro-2-oxypyrroles ..................................... 1083 biochemistry and biotechnology f. matpan bekler, s. yalaz, r. gül güven and k. güven: optimization of the thermostable alkaline and ca-dependent α-amylase production from bacillus paralicheniformis by statistical modeling .................................................................................... 1093 i. t. drvenica, a. z. stančić, a. m. kalušević, s. b. marković, j. j. dragišić maksimović, v. a. nedović, b. m. bugarski and v. lj. ilić: maltose-mediated, long-term stabilization of freezeand spray-dried forms of bovine and porcine hemoglobin .............. 1105 inorganic chemistry n. đ. pantelić, m. lerbs, k. wolf, l. a. wessjohann and g. n. kaluđerović: in vitro anticancer evaluation of novel triphenyltin(iv) compounds with some n-acetyl-snaphthoquinonylcysteine derivatives .......................................................................... 1119 theoretical chemistry a. s. vorob’ev, a. v. isakov, a. y. galashev and y. p. zaikov: dft-based calculations of silicon complex structures in kf–kcl–k2sif6 and kf–kcl–k2sif6–sio2 melts ...... 1129 physical chemistry a. a. fodeke and o. o. olayera: thermodynamics of adsorption of malachite green hydrochloride on treated and untreated corncob charcoal ........................................... 1143 electrochemistry m. h. mehmood, m. tariq, a. hassan, a. raziq, a. rahim and j. khan: facile solvothermal synthesis of pt–cu nanocatalyst with improved electrocatalytic activity toward methanol oxidation .......................................................................................... 1155 volume 84: contents 1477 thermodynamics n. setoodeh, p. darvishi and a. ameri: a thermodynamic approach for correlating the solubility of drug compounds in supercritical co2 based on peng–robinson and soave–redlich–kwong equations of state coupled with van der waals mixing rules ... 1169 environmental d. shi, g. sun and y. cui: study on the removal of no from flue gas by wet scrubbing using naclo3 .............................................................................................................. 1183 number 11 in memoriam to professor konstantin i. popov editorial: prof. dr konstantin i. popov (1941–2019) ......................................................... 1193 s. s. djokić, ž. antić, n. s. djokić and t. thundat: electroless deposition of fe–ni alloys from acidic and alkaline solutions using hypophosphite as a reducing agent ............. 1199 n. d. nikolić, p. m. živković, m. g. pavlović and z. baščarević: overpotential controls the morphology of electrolytically produced copper dendritic forms ......................... 1209 m. bučko, m. v. tomić, m. maksimović and j. b. bajat: the importance of using hydrogen evolution inhibitor during the zn and zn–mn electrodeposition from ethaline ... 1221 n. s. marinković and r. r. adžić: infrared spectroelectrochemical configurations for in situ measurements ....................................................................................................... 1235 s. hadži jordanov: far-reaching governance of electrode potential: the case of priority in metal exploitation (letter to the editor) .................................................................. 1249 b. n. grgur, m. m. gvozdenović, b. z. jugović and t. lj. trišović: characteristics of the citrate-based zinc–polyaniline secondary cell with supercapattery behaviour ............ 1261 v. d. jović, b. m. jović, n. r. elezović and lj. gajić-krstajić: corrected accelerated service life test of electrodeposited nisn alloys and ni as cathodes for industrial alkaline water electrolysis ........................................................................................... 1271 m. stevanović, m. djošić, a. janković, k. y. rhee and v. mišković-stanković: electrophoretically deposited hydroxyapatite-based composite coatings loaded with silver and gentamicin as antibacterial agents (review) ........................................................ 1287 m. r. pantović pavlović, m. m. pavlović, s. eraković, t. barudžija, j. s. stevanović, n. ignjatović and v. v. panić: relationship between the properties of an interlayer formed by in situ ti anodization and anaphoretically deposited hydroxyapatite ......... 1305 j. lović, j. lađarević, d. mijin, m. jadranin, s. d. petrović and m. avramov ivić: electrochemical stability of metformin in nahco3 and na2so4 water solution at au, gc and irox electrodes ........................................................................................ 1319 v. s. cvetković, n. j. jovićević, n. m. vukićević and j. n. jovićević: aluminum/zirconium alloys obtained by al underpotential deposition onto zr from low temperature alcl3+nacl molten salts ............................................................................... 1329 number 12 organic chemistry b. srećo zelenović, s. kekezović, m. popsavin, v. kojić, g. benedeković and v. popsavin: novel (–)-goniofufurone mimics: synthesis, antiproliferative activity and sar analysis ............................................................................................................... 1345 1478 journal of the serbian chemical society j. stanković, m. novaković, v. tešević, a. ćirić, m. soković, g. zdunić, z. dajić-stevanović and d. gođevac: hptlc-direct bioautography-guided isolation of isogeranic acid as the main antibacterial constituent of artemisia santonicum essential oil ........ 1355 biochemistry and biotechnology d. zhang, d. he, x. pan, y. xu and l. liu: molecular design of orthogonal stacking system at the complex interface of htra pdz domain with its peptide ligands.......... 1367 b. t. popović, o. v. mitrović, a. p. leposavić, s. a. paunović, d. r. jevremović, n. j. nikićević and v. v. tešević: chemical and sensory characterization of plum spirits obtained from cultivar čačanska rodna and its parent cultivars ................................ 1381 inorganic chemistry lj. t. suručić, a. b. nastasović, a. e. onjia, g. v. janjić and a. а. rakić: design of an amino-functionalized chelating macroporous copolymer poly(gma-co-egdma) for the sorption of cu(ii) ions ..................................................................................... 1391 theoretical chemistry m. zine, a. bouakkadia, l. lourici and d. messadi: quantitative structure–property relationship studies for the prediction of the vapor pressure of volatile organic compounds ........................................................................................................................ 1405 electrochemistry d. e. garcia-rodriguez, c. h. rios reyes and l. h. mendoza huizar: tuning the copper cluster’s size on hopg by electrodeposition from perchlorate aqueous solutions. an afm study ............................................................................................................ 1415 polymers j. l. fraj, l. b. petrović, j. r. milinković budinčić, j. m. katona, s. đ. bučko and lj. m. spasojević: properties of double w/o/w emulsions containing vitamin c and e stabilized with a gelatin/sodium caseinate complex .................................................... 1427 environmental m. brborić, b. vrana, j. radonić, m. vojinović miloradov and m. turk sekulić: spatial distribution of pahs in riverbed sediments of the danube river in serbia: anthropogenic and natural sources .................................................................................... 1439 m. j. lazarević, v. n. despotović, d. v. šojić merkulov, n. d. banić, n. l. finčur, d. d. četojević-simin, m. i. čomor and b. f. abramović: photodegradation of selected pesticides: photocatalytic activity of bare and pani-modified tio2 under simulated solar irradiation ........................................................................................................... 1455 contents of volume 84 ....................................................................................................... 1469 author index 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice j. serb. chem. soc. 83 (10) 1177–1192 (2018) udc 54–028.42(079)+517.9+542.9:37.04:377 jscs–5142 original scientific paper 1177 the macroscopic, submicroscopic and symbolic level in explanations of a chemical reaction provided by thirteen-year olds dragica d. trivic*# and vesna d. milanovic# university of belgrade – faculty of chemistry, studentski trg 12–16, belgrade, serbia (received 20 december, revised 4 july, accepted 5 july 2018) abstract: the aim of this research was to investigate whether pupils aged 13, at the end of their first year of studying chemistry, are capable of perceiving the macroscopic and the submicroscopic level of the previously learned contents on chemical reactions, and whether they relate them to the chemical equations (the symbolic representation). another aim was to establish how much demonstration experiments contribute to a better linking of the mentioned levels. the research featured 69 pupils of the seventh grade from three primary schools. the pre-test was conducted, following which experiments were demonstrated, and the post -testing was carried out in the end. after the intervention, a total of 12 pupils were interviewed about which aspects of chemical reactions they thought of based on the chemical equations. a statistically significant difference in two out of five requirements of the post-test, compared to the pre-test, indicate that the demonstration experiments may contribute to a better linking of three levels of representing chemical reactions. however, when one compares the pupils’ answers in the test and in the interview, it can be observed that the correct answers in the test are not always based on understanding the concepts in connection with the chemical reaction. keywords: chemical reaction; chemical equation; demonstration experiment; primary school. introduction identifying pupils’ problems when it comes to understanding the basic concepts in chemistry is important for planning the teaching process that will serve to avoid those problems, or to overcome them if they have already appeared. they may be hidden because pupils’ success at solving problem tasks is not always the result of deep conceptual understanding but may be due to a reproduction of a familiar algorithm used to solve a particular problem.1 for this * corresponding author. e-mail: dtrivic@chem.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc171220055t ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1178 trivic and milanovic reason, it is necessary to examine what kind of reasoning pupils use while solving various tasks from the key areas of chemistry.2 researches point to the fact that pupils think about a chemical reaction as a process of adding or mixing molecules, without any clear idea of the mechanism of the given chemical reaction.3,4 also, pupils’ answers indicate that, when they recognise that it is a matter of a chemical reaction, at the same time they do not view it at the level of atoms and molecules and do not recognise that the law of conservation of mass applies, while some recognise only a physical change instead of a chemical reaction.3,4 for pupils of different age, the request to simultaneously review chemical reactions at three levels of representation is rather challenging: the macroscopic level, where on the basis of observation one may describe substances and changes in their properties, for example, reactions that occur between substances in solution in which one of the products is insoluble, the submicroscopic/particle level, that is, the level of atoms, molecules and ions, and the symbolic level, where the structure of the given substance and interactions between atoms, molecules and ions are represented by means of chemical symbols, formulas and chemical equations.5 the connections between these different levels of presenting chemical reactions are that which is not obvious to pupils and what distinguishes their reasoning from that of chemists.6,7 striving to interpret chemical reactions at the micro level, pupils resort to memorising chemical equations – the symbolic level of presenting chemical reactions.8 even though they, within the framework of many laboratory exercises and observing experiments, write equations of chemical reactions and mathematically determine coefficients (which should provide information to them about quantitative relations in chemical reactions at the submicroscopic level), they do not necessarily perceive the interconnectedness of these curricular contents and activities.9 demonstrations of chemical reactions are a part of usual curricular activities, the aim being for observations at the macroscopic level to facilitate pupils’ understanding of chemical reactions.10 researches have shown that the most difficult thing for pupils is to think about the structure of substances (the submicroscopic level) and to interpret at that level the properties and changes of substances that they perceive at the macroscopic level.9,11–15 a chemical equation, as one of the key models in chemistry, serves to explain, quantitatively and qualitatively describe a chemical reaction, linking the macroscopic and the submicroscopic level of a chemical reaction.16 researches suggest that the understanding of secondary school pupils and college students is predominantly in the realm of the symbolic,1,17–19 which may be due to the abstract nature of the particulate structure of a substance20, and consequently the inadequate mental models of students.21 the ability to present a chemical reaction at the symbolic level does not guarantee the ability to understand a reaction at the level of particles. in one research, of the 65 % of secondary school pupils ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ the explanations of a chemical reaction provided by thirteen-year olds 1179 who correctly determined the coefficients in a chemical reaction, more than half of them could not present that chemical reaction at the particle level.22 also, pupils were not sure how the symbolic level was connected with the perceived properties and changes of substances at the macroscopic level or chemical bonds in substances presented by means of chemical formulas.9,23 when determining the coefficients in chemical equations, they often follow certain algorithmic procedures without any deeper consideration of the meaning of coefficients. on the other hand, a research conducted on a sample of secondary school pupils aged 15–16 showed that pupils’ ability to use multiple levels of representation can be improved.24 according to the curriculum for primary school in serbia, the pupils start studying chemistry as a separate teaching subject in the seventh grade of primary school (aged 13). the chemistry curriculum for the seventh grade encompasses five topics: chemistry and its importance, basic chemical concepts, structure of substance, homogeneous mixtures – solutions, and chemical reactions and calculations. the themes structure of substance and chemical reactions and calculations comprise three levels of representing chemical concepts. the question that arises is what mental representations for chemical reactions are formed by thirteen-year-old pupils and what connections between various levels of representation (the macroscopic, submicroscopic and symbolic level) they can establish and understand. demonstration experiments are considered to be an important support for the purpose of forming concepts in chemistry teaching. for successful chemistry teaching, it is important to review their role and contribution to the linking of the macroscopic, submicroscopic and symbolic levels at which chemical reactions are reviewed. experimental the aim of the research the aim of this research was to investigate whether pupils aged 13, at the end of their first year of studying chemistry, are capable of perceiving the macroscopic and the submicroscopic level of the previously learned contents on chemical reactions, and whether they relate them to the chemical equations (the symbolic representation). in addition to this, we wished to establish to what extent the observation of demonstration experiments stimulates pupils to review chemical reactions at the level of the structure of reactants and products (atoms, ions and molecules), and how they link observations at the macroscopic level with various ways of representing substances and chemical changes by means of models and chemical symbols, formulas and chemical equations. from the aims thus defined, there arose the following research questions: 1. which connections between various levels of representing chemical reactions (the macroscopic, submicroscopic and symbolic level) do thirteen-year-old pupils establish and understand? ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1180 trivic and milanovic 2. what is the contribution of demonstration experiments to understanding chemical reactions by thirteen-year-olds? 3. what information on a chemical reaction on the basis of a chemical equation do thirteen-year-olds accept? the sample the research involved the participation of pupils from three seventh grade classes from three different schools on the territory of belgrade (one class from each school). the sample encompassed a total of 69 pupils (42 boys and 27 girls). all the pupils did a test. of the overall number of pupils in the sample, 12 were selected by random choice (four pupils from each class) and were interviewed. before commencing the research, the management of every school and chemistry teachers were acquainted with the aim of the research and the manner of its realisation. the school managements and chemistry teachers gave their agreement for conducting the research, and a contract of cooperation was signed by the dean of the faculty of chemistry and the principals of the schools involved. the pupils’ participation in the research was voluntary, and their score in the test did not influence their chemistry marks. design and procedure one classroom period (lasting 45 min) was conducted with each class comprising the sample. the classroom period, which is designed as review of the previously learned contents on chemical reactions, consisted of three parts (see table i). in the first and the third segment of the work, the pupils were given the same test to solve. during the second segment of the work, the teacher demonstrated three experiments related to three tasks comprising the test. after observing the demonstration, the pupils talked with the teachers about the properties and changes of the substances used in the experiments observed. also, the pupils wrote the chemical equations of the reactions. the following experiments were demonstrated: 1. obtaining hydrogen and hydrogen combustion; 2. magnesium combustion; 3. obtaining carbon dioxide and its reaction with a barium hydroxide solution. these demonstrations involve the macroscopic visible indicators that chemical reactions occur, which are explained in all currently used chemistry textbooks for the seventh grade. after the classroom period, four pupils from each class were interviewed. table i. research design classroom period segment duration, min activity i 12 pre-testing ii 21 experiment demonstration iii 12 post-testing after the intervention 4 × 7 to 8 interview instruments for the purpose of conducting the research, we used a test (supplementary material to this paper, addendum 1) and the interview protocol (supplementary material, addendum 2). the same test was used for the pre-testing and for the post-testing. the test comprises three tasks, containing a total of five requirements. the tasks were associated with three levels of representing chemical contents, previously elaborated under the curriculum themes: structure of substance and chemical reactions and calculations. this means that different levels of ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ the explanations of a chemical reaction provided by thirteen-year olds 1181 representations were known to the students before this research. the three tasks in the test relate to the three experiments that were demonstrated to the pupils after the first test and subsequently discussed with them. the purpose of the pre-test was to establish the level of the pupils’ knowledge about chemical reactions at the end of the seventh grade, after they had dealt with the entire chemistry curriculum envisaged for that year of primary school. the post-test, using the very same tasks, was conducted in order to examine whether the pupils have made any progress when it came to connect the macroscopic, submicroscopic and symbolic levels of presenting chemical reactions, after observing the demonstration experiments and discussing them, compared to the pre-testing. these demonstrations are directly related to the requirements in the test. in this way, it was possible to monitor the students’ reasoning associated with the demonstrations and the influence of them on linking macroscopic, submicroscopic and symbolic levels of representing chemical reactions. everything was done during one classroom period, in order to establish the effects that can be expected from this period of chemistry instruction. using the interviews, data were gathered on what aspects of chemical reactions the pupils thought about and what information they accepted from the equations of chemical reactions. the validity of the test in terms of the aim of the research was checked by the chemistry teachers working in the schools encircled by the sample. the teachers’ estimate was that the test corresponded to the chemistry curriculum for the seventh grade of primary school. results and discussion the parameters of the results distribution in the pre-test and the post-test are presented in table ii. table ii. the parameters of the results distribution in the pre-test and the post-test (the maximum number of points was 5) test n min max mean sd p skewness kurtosis pre-test 69 0 4 1.93 1.18 38.6 –0.022 –1.086 post-test 69 0 5 2.87 1.27 57.4 –0.235 –0.482 the value of the t-test indicates that the difference between the arithmetical means in the two tests is statistically significant (table iii). table iii. the paired samples t-test (pair 1, pre-post) paired samples test; paired differences 95 % confidence interval of the difference t df sig. (2-tailed) mean sd se mean lower upper –0.94 1.862 0.224 –1.39 –0.50 –4.202 68 0.000 table iv presents the percentage of the pupils’ correct answers to each task in the pre-test and the post-test, as well as the values of the t-test, which was used to evaluate the statistical significance of the difference in the percentage of correct answers in the two tests. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1182 trivic and milanovic table iv. the percentage of correct answers to the tasks in the pre-test and the post-test task number contribution, % number contribution, % t-test of correct answers in the pre-test of correct answers in the post-test 1a 44 63.8 55 79.7 2.00a 1b 26 37.7 25 36.2 –0.18 2a 17 24.6 20 29.0 0.56 2b 37 53.6 41 59.4 0.66 3 9 13.0 39 56.5 5.20b aconfidence interval 95 %; bconfidence interval 99 % a statistically significant difference in the pupils’ results in the two tests related to item 1а (table iv) points to the fact that, after observing a demonstration of the reaction of hydrogen combustion and a discussion about the observed change in terms of the particles involved, the pupils are more capable of relating the chemical change described in the task to the corresponding chemical equation. the pupils who did not provide the correct answer in the pre-test but did so in the post-test did not know that the reactants were in the form of two -atom molecules, or did not pay attention to the coefficients in the equation of that chemical reaction (in other words, they neglected the law of conservation of mass). however, the number of answers to item 1b in the pre-test and the post -test points to the fact that, even after being presented a demonstration and discussing the experiment, a little less than two-thirds of the pupils still failed to link the change with the corresponding reactant molecules and the product in this reaction. they still do not show that they are capable of reviewing a chemical reaction at the submicroscopic level, and the number of correct answers to this task was even somewhat lower in the post-test. when one compares the pupils’ achievements in the post-test pertaining to items 1a and 1b, it is evident that a little more than half of those pupils who provided a correct answer to item 1a failed to do so in the case of item 1b. the lower number of correct answers is partly the consequence of the pupils’ failure to understand the meaning of the index in chemical formulas and the coefficients in chemical equations, and also of their failure to join the description of an experiment and the symbolic notation with the submicroscopic level. the respondents’ better score in the tasks that are related to the symbolic level of presenting chemical reactions, than in those related to the interpretation of chemical reactions at the submicroscopic level has also been established in other researches featuring pupils from an older age group.22,25 in item 2a, a chemical change was described – the combustion of magnesium. to provide help, the atomic numbers of magnesium and oxygen were given. the pupils were expected to write a chemical equation on the basis of the information provided, and in doing so, to apply the knowledge from the lessons on the ionic bond, valence and the synthesis reaction they have dealt with during ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ the explanations of a chemical reaction provided by thirteen-year olds 1183 the school year. the requirement to write a chemical equation was fulfilled by a smaller number of pupils related to item 1a, which required of them to recognise the correct chemical equation among those given. an error frequently made by the pupils involved the wrong index in the formula of magnesium oxide (those are the answers mgo2, mg2o). the replies of 10 pupils in the pre-test showed that they thought the atomic numbers of magnesium and oxygen were actually their indexes (they did not pay attention to the way in which the numbers were written next to the symbol of the corresponding element), and some of them thought that the product of the coefficient and the index of the reactant had to be equal to the atomic number. those pupils did not repeat this mistake in the post-test. however, while dealing with this reaction at the submicroscopic level, in item 2b, on the basis of the model of the structure of atoms and ions, the pupils were more successful than in item 1b. the number of correct answers related to this task in the post-test increased in relation to the pre-test, but the difference in the percentage of the correct answers in the two tests is not statistically significant. the percentage of the correct answers related to the item 3 was statistically significantly higher in the post-test in relation to the pre-test. solving the task was based on a picture showing the apparatus for the unfolding of two chemical reactions (the macroscopic level of representation). observing the demonstration of the experiment and the resulting discussion contributed to a statistically significant increase in the number of the correct answers to the item three in the post-test in relation to the pre-test. this result has important implications for the practice sessions, where there are not enough demonstration experiments and the students are observing the given experiment in textbooks. the test results indicate that the observation of experiments has a considerably greater influence on the pupils’ knowledge than illustrations of experiments in textbooks. interview analysis the actual level of knowledge supporting the increase in test scores was to be shown by the interviews conducted after the second test with 12 randomly selected pupils (four pupils from each class). table v details their answers in both tests and their chemistry marks, expressed on a scale of 1 (the lowest and failing grade) to 5 (the best grade). the pupils were marked by letters from a to l. table vi presents the pupils’ answers as to what a chemical reaction equation represents and what information it provides. the pupils’ answers show that the majority of them notice the qualitative meanings of chemical reaction equations, and that fewer of them also notice the quantitative meanings. from the answers of some of the pupils, one can see their efforts to link the symbolic and the submicroscopic levels, which refers to the structure of reactants and the products of chemical reactions. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1184 trivic and milanovic table v. the answers of the pupils interviewed in the pre-test and the post-test, and their chemistry marks; the correct answers are marked by capital letters in bold print. the incorrect answers were marked by small letters, not in bold print. in task 2a, the letter n marks an incorrect answer, the letter t a correct answer, whereas the sign / indicates that the pupil in question skipped this item pupil answers in the pre-test no. of correct answers answers in the post-test no. of correct answers chemistry mark 1a 1b 2a 2b 3 1a 1b 2a 2b 3 a b c n b b 1 b d т a a 3 4 b b d n c c 2 b d т c a 4 4 c b d n b b 1 b d т b а 3 4 d a a n b b 0 b a т b а 3 3 e b d t b d 2 b d n c а 3 4 f a d t c b 2 b d т c b 3 3 g d b / b c 1 b b n b b 2 2 h а b / c c 2 b b т c d 4 5 i b c t c b 3 b c т c а 4 5 j b a t c b 3 b b т c а 5 5 k b c t c a 4 b c т c а 4 5 l b c t c a 4 b c т c а 4 5 table vi. the pupils’ answers to the questions: what does a chemical reaction equation represent? what information does a chemical reaction equation provide?; the numbers in brackets indicate the number of points scored in the pre-test and the post-test, and the pupil’s mark in chemistry pupil answer a (1; 3; 4) provides information on what reacts and what is obtained. b (2; 4; 4) i can see molecules. c (1; 3; 4) how many atoms, that is, reactants, were used to achieve equality. d (0; 3; 3) how a substance is created. for example, how water is created from hydrogen and an oxide. e (2; 3; 4) i can see the reactants and the products. substances that react and create some new substance. f (2; 3; 3) i see valences, how many moles there are, the relations in which reactants react. g (1; 2; 2) i can see the elements. h (2; 4; 5) i can see the change that occurs when two elements react with each other and produce a new compound. i (3; 4; 5) one can see the reactants and the products. j (3; 5; 5) i can see how two elements form a bond. i can see how many atoms a compound contains, and of what atoms that compound consists. k (4; 4; 5) i can see how two elements react and a compound is obtained, how many atoms an element contains, i can see the formulas. l (4; 4; 5) how to obtain some compound. from the pupils’ answers to the question what the coefficients in a chemical reaction equation represent (table vii), it is evident that they do not understand the coefficients as the stoichiometric ratios of reactants and products (with the ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ the explanations of a chemical reaction provided by thirteen-year olds 1185 exception of pupils d and f). to most of the pupils interviewed, the coefficients are numbers used to balance the chemical equations. also, pupils tend to confuse the meanings of the terms coefficient and index, as evidenced by the answers of pupils g, h, i and k. table vii. the pupils’ answers to the question: what do the coefficients in a chemical reaction equation represent?; the numbers in brackets indicate the number of points scored in the pre-test and the post-test, and the pupil’s mark in chemistry pupil answer a (1; 3; 4) how much there is of that substance... how to determine what the equation should be like at the end, how everything is to be correctly determined. b (2; 4; 4) those are the numbers written in front so that everything should be equalised. c (1; 3; 4) we use them to determine the equation. d (0; 3; 3) how much we need of which substance in order to produce water, for example, how much hydrogen we need and how much oxygen. e (2; 3; 4) the coefficient represents multiplied molecules. f (2; 3; 3) they represent the number of moles. g (1; 2; 2) that is the number showing how many elements there are. h (2; 4; 5) that is the number in front of a compound or element. through those numbers i can see how many atoms an element has. i (3; 4; 5) they represent the number of atoms in a molecule. j (3; 5; 5) they represent how many elements there are in a compound and how many compounds are produced when two reactants react. k (4; 4; 5) they indicate how many atoms there are. if it says н2, then there are two hydrogen atoms. l (4; 4; 5) we see how many molecules there are. for example, if it says 3н2о, it means there are three molecules of water. to the third question, why it is important to determine the coefficients in a chemical equation, most pupils answered it is in order to balance the equation (a, b, c, f, h). the answers of pupils d, i, j and k implicitly indicate that there is a connection between coefficients and stoichiometric ratios, but they were not well formulated terminologically. pupil g has no idea why coefficients are important, whereas pupil l confuses the meanings of the terms coefficient and index. the only complete answer was provided by pupil e: “for example, regarding the reaction of the production of water, from the equation where the coefficients have been determined we see that it takes twice as much hydrogen to produce water, which you can’t see from an equation where the coefficients have not been determined. that is why we determine them, so that we know the quantity of a substance that is needed.” from the pupils’ answers to the question what is the difference between a coefficient and an index (table viii), it is evident that pupils a, d and f confuse the meanings of these terms. pupil h states that the difference lies in the place of writing the coefficient and the index in relation to the formula of the substance, ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1186 trivic and milanovic whereas pupil k mentions the place of writing them and provides an explanation from which one cannot see the difference in the meaning of coefficient and index. table viii. the pupils’ answers to the question: what is the difference between a coefficient and an index? the numbers in brackets indicate the number of points scored in the pre-test and the post-test, and the pupil’s mark in chemistry pupil answer a (1; 3; 4) the index... that is, the coefficient, shows how many atoms there are in that substance, and as for the index... i don’t know what index is. b (2; 4; 4) when we have an index, that can be because of the valence, and then... we can know the index because of the valence, and then you add the coefficient to equalise that. c (1; 3; 4) the coefficient applies for that whole part (points at the formula), and the index only applies to one atom. d (0; 3; 3) the coefficient is natural, and we add the index for the formula to be right. e (2; 3; 4) the index is how many atoms we have in a molecule. the coefficient marks how much of that substance we have. specifically, in the example of the equation for a chemical reaction producing water, the coefficient 2 in front of hydrogen means that we have two molecules. f (2; 3; 3) (points to the equation for a chemical reaction producing water) the coefficient is this little number 2 that stands to the right of oxygen and hydrogen, and the indexes are the big 2’s in front of hydrogen and water. g (1; 2; 2) the coefficient is the number of how much hydrogen there is and stands on the left (points to the equation for a chemical reaction producing water). and i’ve forgotten what the index is. h (2; 4; 5) the coefficient is placed in front, and the index is the small number afterwards. i (3; 4; 5) the index determines the number of atoms in a molecule, for example о2, and the coefficient also determines the number of atoms, only it is placed in front and applies to all the atoms in that molecule, and the index is just for one atom. j (3; 5; 5) the coefficient is always written in front, and it can mark how many particles, that is molecules, atoms, there are, and the index is determined by those particles, how many there are. k (4; 4; 5) the coefficient stands in front and the index after a symbol. the coefficient tells me how many atoms there are, and the index tells me the same, i suppose. l (4; 4; 5) the index stands after the atom and determines... i mean, it also determines, but some molecules have two atoms, and that is why the index is written, and the coefficient is written in front and applies to the whole compound, how many atoms it has, and the index is just for that atom that is in front of it. the majority of the pupils did not mention the quantitative relation between reactants and products when they answered about the meaning of chemical equation (table vi). however, they considered the ratios of substances that mutually react when they were explicitly asked about that (table ix). the answers presented in table ix point to the fact that determining the coefficients is an activity that pupils conduct by following the algorithm. three out of twelve pupils (i, g and d) answered that from a chemical reaction equation they cannot see the regrouping of the atoms of the reactants (table x). ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ the explanations of a chemical reaction provided by thirteen-year olds 1187 table ix. the pupils’ answers to the question: can you see, from the equation of a chemical reaction, the ratios of substances that mutually react?; the numbers in brackets indicate the number of points scored in the pre-test and the post-test, and the pupil’s mark in chemistry pupil answer a (1; 3; 4) i can see how much there is of what. b (2; 4; 4) i don’t know. c (1; 3; 4) it can’t be determined precisely. d (0; 3; 3) i can see, for example, how much hydrogen and oxygen there is in an equation so that water can be produced. e (2; 3; 4) until the coefficients are determined, we can see how much of what there is in a compound, and we see what the reactants are and how many of them there are, which create that one compound. f (2; 3; 3) presumably i can, on the basis of a compound. for example, how many atoms an element in the compound has. g (1; 2; 2) you can see how much there is of what, for example, how many atoms there are. h (2; 4; 5) in an equation we can see how many atoms of one reactant there are, and how many of the other. i (3; 4; 5) you can see the ratio of the substances reacting. i see that four atoms of hydrogen react with two atoms of oxygen. j (3; 5; 5) i can, by means of coefficients and indexes. k (4; 4; 5) the quantitative aspect can be seen by means of the coefficients, but the ratio cannot be seen from the equation of a chemical reaction. l (4; 4; 5) we can, on the basis of indexes and coefficients. table x. the pupils’ answers to the question: can you see the regrouping of the atoms from a chemical reaction equation?; the numbers in brackets indicate the number of points scored in the pre-test and the post-test, and the pupil’s mark in chemistry pupil answer a (1; 3; 4) i can see it from the reactants and the product. b (2; 4; 4) i see that hydrogen was separated from oxygen, and then the coefficient was added in order to equalise it. c (1; 3; 4) if we see that there is н2 and also о2, then we have to regroup so that it is equalised. d (0; 3; 3) i really don’t know. e (2; 3; 4) i see molecules before and after the reaction. f (2; 3; 3) (points at the equation of the chemical reaction producing baco3 from ba(oh)2 and co2) i see that barium was in a compound with oxygen and hydrogen, and after the reaction with carbon and oxygen. g (1; 2; 2) i don’t know how to see that regrouping. h (2; 4; 5) i can see how they change places. i (3; 4; 5) i can’t see that. j (3; 5; 5) i can see with what an element formed a compound before the reaction, and with what after the reaction. k (4; 4; 5) i see what is produced. l (4; 4; 5) i see from the formula. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1188 trivic and milanovic on the basis of a chemical reaction equation, the pupils’ answers to the question if they imagined particles colliding shows that they only perceive the regrouping of atoms through the initial and the final state of the reaction in question, without reviewing the movement of particles and their collisions (table xi). pupils c and i were reminded of the process of dissolving, probably under the influence of the animation of this process presented during one of the previous lessons. according to their answers, they did not distinguish the physical and chemical changes. moreover, their explanations of the process of dissolving were incorrect. table xi. the pupils’ answers to the question: on the basis of a chemical reaction equation, do you imagine particles colliding?; the numbers in brackets indicate the number of points scored in the pre-test and the post-test, and the pupil’s mark in chemistry pupil answer а (1; 3; 4) it did interest me, but no one explained that to us and i have no idea about it. b (2; 4; 4) i have no idea about it. c (1; 3; 4) i tried to imagine. for example, if we place bluestone in water and it dissolves and colours the water blue, that is, it switches from the solid aggregate state to liquid. d (0; 3; 3) i have never imagined that. e (2; 3; 4) not really. i only know that it is necessary for them to collide for a reaction to happen, that’s what it says in the book. f (2; 3; 3) i don’t know. g (1; 2; 2) i have never imagined it. h (2; 4; 5) i’ve no idea. i (3; 4; 5) well, i can describe it, our teacher showed us various photos, what that looks like. when a substance dissolves in water, then the molecules of water disintegrate, and as for the substance dissolved in water, all its particles dissolve in water, for the molecules of water took those particles, those bits, and that was how it dissolved. j (3; 5; 5) i haven’t imagined it. k (4; 4; 5) i have never imagined it. l (4; 4; 5) well, for example, we have one oxygen and two hydrogens colliding in order to get water. of the 12 pupils interviewed, item 1a was solved correctly by 8 of them in the pre-test, while all the pupils gave the correct answer in the post-test. progress was made by pupils d, f, g and h. however, the answers given by pupils d and f during the interview indicate that they still confuse the meaning of coefficients and indexes (table viii). pupils g and h think that in chemical equations “coefficients mark the number of atoms”, which is probably the result of an application of algorithm in the process of determining coefficients. in dealing with item 1b, only pupil j made progress. the answers that this pupil gave during the interview indicate that he understands the terms coefficient ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ the explanations of a chemical reaction provided by thirteen-year olds 1189 and index, but he has a problem when it comes to expressing this in words. pupil g, who has a mark 2 in chemistry and whose answers in the interview indicate that his knowledge of chemistry is poor, solved task 1b correctly on both occasions. task 2a, wherein the pupils were required to write the equation of the chemical reaction of magnesium combustion, was correctly solved by six of the pupils interviewed in the pre–test, and in the post-test 10 of them provided the correct solution. the incorrect answers in the pre-test were as follows: pupils a and b: 6mg2 + o6 → 6mg2o; pupil d: mg + o2 → mgo2; pupil c: 2mg + 2o2 → 2mgo2. in the answers given during the interview, these pupils stated that “the equation must be balanced”, but they made mistakes when it came to applying the knowledge of valence during the process of compiling a formula of the compound formed by those elements. pupil c does not understand that coefficients in a chemical equation represent the stoichiometric ratios. in the post -test, these four pupils wrote the correct chemical equations of magnesium combustion. two of the pupils interviewed did not provide the answer to item 2a in the pre-test. one of those two pupils wrote the correct chemical equation in the post-test, and the other correctly presented the reactants and the product of the chemical reaction in terms of symbols and formulas, but did not determine the coefficients. dealing with item 2b, of the 12 pupils interviewed, seven provided the correct answer in the pre-test, and eight in the post-est. progress was made by pupil e. of the 12 pupils interviewed, two of them (k and l) gave the correct answer to the third task in the pre-test, and nine of them did so in the post-test. progress was made by pupils a, b, c, d, e, i and j. conclusion researches that have been conducted so far have mostly related to the problems faced by secondary-school pupils and older students when it comes to linking the macroscopic, submicroscopic and symbolic levels of presenting chemical reactions. according to the chemistry curriculum for primary schools in serbia, pupils are first taught the concept of a chemical reaction at the age of thirteen and, which is important for the purpose of the further planning of the teaching process, to identify their first notions of a chemical reaction, especially in the view of the fact that they are expected to interpret that concept at various levels. on the other hand, it was important to ascertain to what extent the observing experiments helped pupils at that age to understand the concept of a chemical reaction, and also whether, through the application of this method, they improved their ability to interpret a chemical reaction at various levels. the results obtained ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1190 trivic and milanovic by administering the same test before and after watching demonstration experiments showed a statistically significant difference in the pupils’ achievements, when they were asked to identify the correctly written chemical equation and to identify a chemical reaction/chemical reactions at the macroscopic level. however, watching demonstration experiments neither did influence the pupils’ ability to write chemical reaction equations, nor did it help them interpret chemical reactions at the submicroscopic level. this is in line with the comments of some researchers.9 the results of this research correspond to the results achieved by older pupils, when it came to linking the various levels of presenting chemical reactions. comparing the pupils’ answers given in the test with those given in the course of the interview indicates that they can be successful at writing indexes in chemical formulas and at determining coefficients in chemical equations, while remaining unsure of the actual meaning of the terms coefficient and index. this indicates that pupils can solve tasks successfully by using algorithms, and that their success is not always connected with in-depth conceptual understanding. the ideas of thirteen-year-old pupils of how particles behave during a chemical reaction are unclear. most often, they review a chemical reaction only through the equation, from the point of view of the initial and the final state, without any idea of how the reaction actually proceeds. the answer given by the thirteen-year olds during the interview reveal that after the demonstrations and a discussion, and after they had successfully solved the tasks in the post-test, the problems related to the meaning of indexes in chemical formulas and coefficients in chemical equations still remained. in other words, the fact that they had better results in the post-test does not mean that they have a better understanding of chemical reactions and that they are capable of linking the macroscopic, submicroscopic and symbolic levels of presenting chemical reactions. that should be taken into consideration in the teaching practice in the future, especially the fact that the pupils’ success at recognising the chemical equations and writing them does not necessarily mean that they perceive a chemical reaction at the level of the structure of the given substance, at the level of the particles that make it up and at the level of the chemical bonds. in other words, focusing on explaining chemical reactions on the basis of chemical equations does not necessarily mean that the pupils will have an idea of what goes on during the reaction at the submicroscopic level. most researches have dealt with the problems faced by students, especially freshmen, but if we manage to eliminate the problems that beginners encounter in the study of chemistry and lead them to form a functional system of chemical concepts, we could expect them to be more successful in their education later on. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ the explanations of a chemical reaction provided by thirteen-year olds 1191 the limitations of the research one limitation of this research is the insufficient sample, which prevents generalisations. also, when pupils were randomly selected to be interviewed, it turned out that in that segment of the sample three-quarters of the respondents had a mark of 5 (the best grade) or 4 in chemistry. that is why the problems pertaining to the reasoning of those pupils whose marks in chemistry are lower were not sufficiently manifested. also, in the further research the students whose score decreases in the post-test should be chosen for the interview and the reasons for that should be examined. acknowledgements. this research is the result of the work on the project “the theory and practice of science in society: multidisciplinary, educational and intergenerational perspectives”, no. 179048, the realization of which is financed by the ministry of education, science and technological development of the republic of serbia. и з в о д макроскопски, субмикроскопски и симболички ниво у објашњењима хемијске реакције тринаестогодишњака драгица д. тривић и весна д. милановић универзитет у београду – хемијски факултет, студентски трг 12–16, београд циљ овог истраживања је да се испита да ли ученици узраста 13 година на крају седмог разреда сагледавају макроскопски и субмикроскопски ниво представљања претходно наученог садржаја о хемијским реакцијама, и да ли та два нивоа повезују с хемијским једначинама (симболичким представљањем хемијских реакција). други циљ је био испитивање доприноса демонстрационих огледа у бољем повезивању наведених нивоа. у истраживању је учествовало 69 ученика седмог разреда из три основне школе. изведено је почетно тестирање, затим су демонстрирани огледи и на крају је изведено завршно тестирање. након сваког часа, интервјуисано је 12 ученика о томе о којим аспектима хемијских реакција размишљају на основу једначина хемијских реакција. статистички значајна разлика на два од пет захтева завршног тестирања, у односу на почетно тестирање, указује да демонстрациони огледи могу допринети бољем повезивању макроскопског, субмикроскопског и симболичког нивоа представљања хемијских реакција. међутим, када се упореде ученички одговори на тесту и интервјуу уочава се да иза тачних одговора на тесту не стоји увек разумевање појмова у вези с хемијском реакцијом. (примљено 20. децембра 2017, ревидирано 4. јула 2018, прихваћено 5. јула 2018) references 1. s. c. nurrenbern, m. pickering, j. chem. educ. 64 (1987) 508 2. m. weinrich, v. talanquer, chem. educ. res. pract. 16 (2015) 561 3. b. anderson, stud. sci. educ. 18 (1990) 53 4. j. j. hesse, c. w. anderson, j. res. sci. teach. 29 (1992) 277 5. a. h. johnstone, chem. educ. res. pract. 1 (2000) 9 6. r. kozma, in innovations in science and mathematics education: advanced designs for technologies of learning, m. jacobson, r. kozma, eds., erlbaum, mahwah, nj, 2000, pp. 11–46 7. d. f. treagust, g. chittleborough, t. l. mamiala, int. j. sci. educ. 25 (2003) 1353 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1192 trivic and milanovic 8. d. l. gabel, j. chem. educ. 76 (1999) 548 9. m. e. hinton, m. b. nakhleh, chem. educ. 4 (1999) 158 10. h. k. wu, sci. educ. 87 (2003) 868 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treagust, m. mocerino, res. sci. educ. 38 (2008) 237 25. v. talanquer, j. chem. educ. 90 (2013) 832. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice microwave assisted synthesis of 2-(2-(tetrazolo[1,5-a]quinolin-4-yl)-2,3-dihydro-1h-1,5-benzodiazepin-4-yl) substituted phenols and evaluation of their antimicrobial activity j. serb. chem. soc. 81 (8) 851–858 (2016) udc 547.53’235+547.79’831–361:537.5– jscs–4891 962+542.913:615.28–288 original scientific paper 851 microwave assisted synthesis of 2-(2-(tetrazolo[1,5-a]quino lin-4-yl)-2,3-dihydro-1h-1,5-benzodiazepin-4-yl) substituted phenols and evaluation of their antimicrobial activity dongamanti ashok*, velagapuri hanumantha rao and rangu kavitha department of chemistry, osmania university, hyderabad-500 007, india (received 4 may 2015, revised 15 february, accepted 21 may 2016) abstract: a series of new benzodiazepines 4a–h were synthesized by michael addition of chalcones 3a–h with o-phenylenediamine (opda) in presence of sodium acetate under conventional heating and microwave irradiation. the structures of the newly synthesized benzodiazepines were established based on ir, 1hand 13c-nmr and mass spectral data and tested for their antimicrobial activity. keywords: antimicrobial activity; benzodiazepines; chalcones; michael addition; microwave irradiation; tetrazoloquinoline aldehyde. introduction the recent literature is enriched with progressive findings concerning the synthesis and pharmacological properties of benzodiazepines. benzodiazepines; were found to possess a wide range of biological activities and therapeutic uses.1 in addition to their well-known anxiolytic, anticonvulsant, sedative, and musclerelaxant activities found in therapeutics,2 benzodiazepines also exhibit activities as antibiotics,3 anti-hiv agents4 and farnesyltransferase inhibitors.5 on the other hand, quinoline derivatives are also known to exhibit antiallergic,6 anticonvulsant,7 antimicrobial8 and antimalarial9 activities. many chalcone derivatives have been reported to show antimalarial10 and anticancer11 activities. synthesis of quinolinyl chalcones is scarcely reported in the literature whereas chalcones derived from tetrazolo[1,5-a]quinoline-4-carboxaldehyde are not reported so far. moreover, fusion of tetrazole, which is considered as a planar acidic heterocyclic analogue of a carboxylic function, has the ability to increase potency and bioavailability of quinolinyl chalcones. the tetrazole group, which is considered as an analogue to the carboxylic group, as a pharmacophore possesses a wide range of biological activities. several substituted tetrazoles were shown to * corresponding author. e-mail: ashokdou@gmail.com doi: 10.2298/jsc150504041a _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 852 ashok, rao and kavitha possess anticonvulsant,12 anti-inflammatory,13 cns depressant,14 anti-hiv15 and antifertility16 properties. some drugs containing quinoline, chalcone or benzodiazepine moiety, which are readily available on the market, are shown in fig. s-1 of the supplementary material to this paper. encouraged by the biological activities of benzodiazepines and tetrazolo[1,5 -a]quinoline moieties and in continuation of on-going endeavors towards the synthesis of biologically potent heterocyclic compounds, the synthesis of new benzodiazepines 4a–h was undertaken. experimental materials all used chemicals were obtained commercially, mostly from sigma–aldrich, and used without further purification. equipment melting points were determined by the open capillary method using an electrical melting point apparatus and are uncorrected. microwave reactions were performed in a multisynth series microwave system (milestone). the ir spectra were recorded as kbr pellets on a shimadzu ft-ir-8400s spectrophotometer. 1hand 13c-nmr spectra were recorded on a bruker dpx 400 mhz spectrophotometer using tms as the internal standard and dmso -d6/cdcl3 as solvent. the mass spectra were recorded on a gcms-qp 1000 ex mass spectrometer and thin layer chromatography (tlc) was performed to check the purity of the compounds, the spot being located under uv light and iodine vapor. general procedure for the synthesis of (e)-1-(2-hydroxyphenyl)-3-(tetrazolo[1,5-a]quinolin-4-yl)prop-2-en-1-ones (3a–h) conventional method a. to a mixture of a substituted 2-hydroxyacetophenone 1 (0.01 mol) and tetrazolo[1,5-a]quinoline-4-carboxaldehyde 2 (0.01 mol) in ethanol (30 ml) was added koh (0.005 mol) and stirred for overnight at room temperature. the progress of the reaction was monitored by tlc. after completion, the reaction mixture was poured into crushed ice and neutralized with dilute hcl. the thus obtained yellow solid was filtered, washed with water and dried. recrystallization with methanol afforded the pure chalcones 3a–h. yields: 56–68 %. microwave method b. to a mixture of substituted 2-hydroxyacetophenone 1 (0.01 mol) and tetrazolo[1,5-a]quinoline-4-carboxaldehyde 2 (0.01 mol) in ethanol (10 ml) in a 30 ml glass vial equipped with z-cap, was added koh (0.005 mol) and the mixture was then irradiated for 15–17 min. at 125 °c, using an irradiation power of 180 w. the °progress of the reaction was monitored by tlc. after completion of the reaction, the vial was cooled, diluted with crushed ice and neutralized with dilute hcl. the thus obtained yellow solid was filtered, washed with water and dried. recrystallization with methanol afforded the pure chalcones 3a–h. yields: 80–85%. general procedure for the synthesis of 2-(2-(tetrazolo[1,5-a]quinolin-4-yl)-2,3-dihydro1h-1,5-benzodiazepin-4-yl) substituted phenols (4a–h) conventional method a. to a solution of chalcone 3a–h (0.01 mol) in etoh (5 ml), containing sodium acetate (0.01 mol), o-phenylenediamine (opda, 0.01 mol) was added and the reaction mixture was heated at 80–90 °c for 2–3 h. the progress of the reaction was monitored by tlc. after the completion of the reaction, ice water was added. a solid product _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis of tetrazolo quinoline-benzodiazepines hybrids 853 separated hat was filtered, washed with water and dried. the crude products were recrystallized from meoh: chcl3 (1:1). yields: 60–70 %. microwave method b. to a solution of chalcones 3a–h (0.01 mol) in etoh (5 ml), containing sodium acetate (0.01 mol) in a 10 ml glass vial equipped with a cap, opda (0.01 mol) was added and the mixture was then irradiated for 2–4 min. at 130 °c, using an irradiation power of 180 w. the progress of the reaction was monitored by tlc for each 30 s of time interval. after the completion of the reaction, ice water was added. a solid product separated was filtered, washed with water and dried. recrystallization from meoh:chcl3 (1:1). yields: 80–86 %. spectral data are given in the supplementary material to this paper. biological assay antibacterial activity. all the synthesized compounds 3a–h and 4a–h were screened for their antibacterial activity against gram-positive (staphylococcus aureus and bacillus facealis) and gram-negative bacteria (escherichia coli and klebsiella pneumoniae). the bacterial cultures were grown in nutrient agar media and subcultured for better growth (log phase cultures) in a liquid nutrient broth medium and further subcultured onto petri plates for the experiments. both cultures were diluted with sterilized saline to bring the final size of the inoculum to approximately 105–106 cfu ml-1. the compounds were diluted in acetone, dmso and diethyl ether for the biological assays. of the three solvents, diethyl ether was taken as being the best. the bacterial culture inoculum was placed on the media and incubated at 37 °c for 24 h to 48 h along with the chemical discs dipped and placed over the media. the zones of bacterial growth inhibition were measured using the diameter of the zone as a unit to measure the antibacterial activity. all the experiments were performed in triplicate and the results are expressed as zone of inhibition in mm. the results were compared with the activity of the standard antibiotic ciproflaxin (20 and 40 µg ml-1). for disc diffusion method, a test compound was introduced into the disc and then allowed to dry. once the disc was completely saturated with the test compound, it was introduced into the upper layer of the medium containing the bacterial inoculum. the petri dishes were incubated overnight at 37 °c for 24 h. bioactivity was determined by measuring the diameter of the inhibition zones (diz) in mm. the antibacterial activity was evaluated and the results are presented in table i. antifungal activity. the antifungal activities of the synthesized compounds 3a–h and 4a–h were tested against three pathogenic fungi (a. niger, a. flavus and fusarium oxysporum) by the poison plate technique. the test compounds were dissolved in diethyl ether (10 ml) before mixing with potato dextrose agar medium (pda, 90 ml). the final concentration of compounds in the medium was 50 µg ml. the above-mentioned types of fungi were incubated in pda at 25±1 °c for 3–4 days to obtain good mycelium growth for the antifungal assay, then a mycelia disk of approximately 0.45 cm diameter cut from the culture medium was picked out with a sterilized inoculation needle and inoculated in the center of pda plate. the inoculated plates were incubated at 25±1 °c for 5 days. diethyl ether in sterilized distilled water was used as control, while hymexazole was used as positive control for all the treatment, three replicates were performed. the radial growth of the fungal colonies was measured on the fourth day and the data were statistically analyzed. the in vitro inhibition effects of the test compounds on the fungi were calculated by the given formula cv =a–b/a, where a represents the diameter of fungi growth on untreated pda, b represents the diameter of fungi on _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 854 ashok, rao and kavitha treated pda, and cv represents the rate of inhibition. the antifungal activity was evaluated and the results are presented in table ii and compared with that of the standard drug amphotericin-b. results in recent years, reports on microwave assisted synthesis revealed that it is a safe, rapid, economic and environmental friendly procedure.17 owing to increased regulatory pressure in research and industry, tremendous efforts have been made to reduce the amount of pollutants produced, including organic solvents in chemical synthesis. to enforce such practices, the discovery and invention of new synthetic methods are required. the synthesis of new derivatives of benzodiazepines was performed as outlined in scheme 1. the chalcones were prepared by reacting substituted 2-hydroxyacetophenones 1a–h with tetrazolo[1,5-a]quinoline-4-carboxaldehyde (2) in the presence of koh by conventional and microwave irradiation using the claisen–schmidt condensation.17 the reaction of (e)-1-(2-hydroxy substituted phenyl)-3-(tetrazolo[1,5-a]quinolin-4-yl)prop-2-en-1-ones 3a–h with o-phenylenediamine in etoh was performed by conventional as well as microwave irradiation in the presence of sodium acetate to give 2-(2-(tetrazolo[1,5-a]quinolin-4 -yl)-2,3-dihydro-1h-1,5-benzodiazepin-4-yl) substituted phenols 4a–h. the formation of benzodiazepines 4a–h was confirmed by ir, nmr and mass analyses. scheme 1. synthesis of 2-(2-(tetrazolo[1,5-a]quinolin-4-yl)-2,3-dihydro-1h-1,5-benzodiazepin-4-yl) substituted phenols 4a–h. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis of tetrazolo quinoline-benzodiazepines hybrids 855 it was found that the synthesis of benzodiazepines 4a–h by the conventional method took a longer time (2–3 h) and gave lower yields, when compared to microwave irradiation technique in which the reaction proceeded smoothly with excellent yields, within a few minutes (2–3 min). the 1h-nmr spectra of benzodiazepine 4a displayed three characteristic signals in the aliphatic region corresponding to the diastereotopic protons (ha, hb and hx). the ha proton, which is cis to hx, resonated upfield in the δ range 3.38–3.46 ppm as doublet of doublets (dd), while the hb proton, which is trans to hx, resonated downfield in the δ range of 3.80–3.88 ppm (dd). the hx proton, which is vicinal to two methylene protons (ha and hb), also resonated as a doublet of doublets in the δ region 6.16–6.24 ppm. the cyclization of chalcones into benzodiazepines was further supported by the 13c-nmr spectrum of 4a, in which the ch2 and ch carbons resonated at δ 32.12 and 65.40 ppm, respectively.18 these values are in close agreement with the reported values for benzodiazepine carbons ch2 and ch. the combination of 1h-nmr and 13c-nmr data provided strong evidence in support of the structures assigned to the benzodiazepine derivatives. the mass spectrum of 4a showed a molecular ion peak at m/z 407 [m+h]+. the nmr and ms data were found to be satisfactory for the structures assigned to compounds 4a–h. antibacterial activity all the synthesized compounds 3a–h and 4a–h were screened for their antibacterial activity against gram-positive (s. aureus and b. faecalis) and gram -negative bacterial strains (e. coli and k. pneumoniae) at 20 and 40 µg ml–1 concentrations. the zone of inhibition (in mm) was compared with those of the standard drug ciprofloxacin. all the compounds showed relatively better activity against gram-positive bacterial strains than gram-negative bacterial strains (table i). the compounds 3a, 3b, 3f, 4a, 4b and 4f showed promising activity against gram-positive bacterial strains. compounds 4a (r1, r2, r3 = h), 4b (r1 = ch3, r2, r3 = h) and 4f (r1, r2 = h, r3 = och3) showed maximum zones of inhibition. compound 4f exhibited the most potent antibacterial activity against gram-negative bacterial strains. the remaining compounds showed moderate activity against gram-negative bacterial strains. it could be concluded that the compounds with an electron donating methyl and methoxy groups displayed a more potent activity. antifungal activity the antifungal activities of the synthesized compounds 3a–h and 4a–h were tested against three pathogenic fungi (a. niger, a. flavus and f. oxysporum) at 50 µg ml–1 concentration. the zones of inhibition (in mm) were compared with those _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 856 ashok, rao and kavitha table i. antibacterial activity of 3a–h and 4a–h compound gram-positive bacterial strains gram-negative bacterial strains b. faecalis s. aureus k. pneumoniae e. coli c / µg ml-1 20 40 20 40 20 40 20 40 3a 15 28 14 27 12 23 11 20 3b 16 30 15 26 8 16 10 21 3c 13 22 11 20 9 16 9 17 3d 10 19 10 15 9 19 6 13 3e 9 17 9 15 8 13 6 12 3f 16 28 16 28 7 15 8 17 3g 9 15 9 15 10 19 9 14 3h 11 19 13 25 7 15 5 10 4a 17 30 16 29 9 17 9 16 4b 17 31 15 28 11 22 10 19 4c 11 20 7 15 9 12 5 12 4d 10 19 9 20 14 22 8 17 4e 11 20 10 17 7 16 8 17 4f 16 30 15 27 22 35 16 34 4g 9 17 8 13 13 20 5 10 4h 11 23 12 18 24 34 17 33 ciprofloxacin 16 30 15 28 23 35 18 35 of the standard drug amphotericin-b (table ii). all the compounds showed moderate to good activity against the tested fungal strains. compounds 3a, 3f, 4c and 4h showed maximum activity against a. niger, compounds 3c and 4c were active against a. flavus and compound 4f showed promising activity against f. oxysporum. the antifungal activity results revealed the electron donating methyl and methoxy groups played a significant role for the inhibitory potency of the compounds against the fungal strains. table ii. antifungal activity of 3a–h and 4a–h; c = 50 µg ml-1 compound fungi a. niger a. flavus f. oxysporum 3a 15 6 7 3b 8 7 8 3c 6 14 5 3d 8 9 6 3e 3 4 8 3f 15 5 3 3g 10 2 9 3h 6 5 2 4a 11 10 8 4b 10 9 7 4c 16 15 10 4d 6 10 8 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ synthesis of tetrazolo quinoline-benzodiazepines hybrids 857 table ii. continued compound fungi a. niger a. flavus f. oxysporum 4e 9 8 6 4f 10 10 15 4g 7 8 4 4h 15 10 11 amphotericin-b 16 15 16 conclusions an easy, high yielding, convenient method for the synthesis of 2-(2-(tetrazolo[1,5-a]quinolin-4-yl)-2,3-dihydro-1h-1,5-benzodiazepin-4-yl)substituted phenols 4a–h from (e)-1-(2-hydroxy substituted phenyl)-3-(tetrazolo[1,5-a]quinolin -4-yl)prop-2-en-1-ones 3a–h in conventional and microwave irradiation routes is reported. the microwave irradiation process proved to be a simple environmentally friendly technique with high yields and high rate of acceleration. all the new compounds were screened for their antimicrobial activity. it was observed that compounds 3a, 3b, 3f, 4a, 4b, and 4f exhibited broad spectrum of antibacterial activity, and compounds 3a, 3f, 4c, 4f and 4h shown broad spectrum of antifungal activity against all the tested strains compared to the standard drugs with their respective concentrations. supplementary material spectral data of the synthesized compounds are available electronically from http:// //www.sbd.org.rs/jscs/ or from the corresponding author on request. acknowledgements. the authors are grateful to the head of department of chemistry, osmania university, hyderabad, for providing the laboratory facilities. one of the authors, v. h. rao, is grateful to csir, new delhi, for an award of csir–srf. и з в о д синтеза 2-(2-(тетразоло[1,5-а]кинолин-4-yл)-2,3-дихидро-1h-1,5-бензодиазепин-4-ил) супституисаних фенола под микроталасима и испитивање њихове антимикробне активности dongamanti ashok, velagapuri hanumantha rao и rangu kavitha department of chemistry, osmania university, hyderabad-500 007, india синтетисана је серија бензодиазепина 4a–h реакцијом мајклове адиције, полазећи од халкона 3a–h и o-фенилендиамина (opda), у присуству натријум-ацетата, под условима класичног термичког загревања и озрачивањем микроталасима. структура синтетисаних бензодиазепина 4a–h утврђена је на основу података ic и nmr (1hи 13c-) спектроскопије и масене спектрометрије и испитана је антимикробна активност синтетисаних једињења. (примљено 4. маја 2015, ревидирано 15. фебруара, прихваћено 21. маја 2016) _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 858 ashok, rao and kavitha references 1. m. j. barker, k. m. greenwood, m. jackson, s. f. crowe, arch. clin. neuropsych. 19 (2004) 437 2. l. h. strenbach, j. med. chem. 22 (1979) 1 3. s. fotso, t. m. zabriskie, p. j. proteau, p. m. flatt, d. a. santosa, sulastri, t. mahmud, j. nat. prod. 72 (2009) 690 4. k. vandyck, m. d. cummings, o. nyanguile, c. w. boutton, s. vendeville, d. mcgowan, b. devogelaere, k. amssoms, s. last, k. rombauts, a. tahri, p. lory, l. hu, d. a. beauchamp, k. simmen, p. raboisson, j. med. chem. 52 (2009) 4099 5. s. fletcher, e. p. keaney, c. g. cummings, m. a. blaskovich, m. a. hast, m. p. glenn, s. chang, c. j. bucher, r. j. floyd, w. p. katt, m. h. gelb, w. c. van voorhis, l. s. beese, s. m. 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i. rivera vera, s. chuprakov, v. gevorgyan, org. lett. 12 (2010) 2166 16. p. shanmugapandiyan, a. ramesh, j. pharm. chem. 2 (2008) 1 17. d. ashok, v. hanumantha rao, p. sreenivas, heterocycl. commun. 19 (2013)363 18. a. a. nabila, s. a. b. amal, m. s. k. makarem, j. saudi chem. soc. 16 (2012) 19. m. cutolo, c. pizzorni, a. sulli, autoimmun. rev. 11 (2011) 84. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {spectral, nlo and antimicrobial studies of co(ii), ni(ii) and cu(ii) complexes of schiff base ligands of 2-amino-6-nitrobenzothiazole} j. serb. chem. soc. 84 (3) 267–275 (2019) udc 546.732’742’562:542.9+547.571’551+ jscs–5182 543.422.25:57–188 original scientific paper 267 spectral, nlo and antimicrobial studies of co(ii), ni(ii) and cu(ii) complexes of schiff base ligands of 2-amino-6-nitrobenzothiazole chinnaraj kanagavalli1, murugesan sankarganesh2, jeyaraj dhaveethu raja2 and manivannan kalanithi3* 1department of chemistry, theni college of arts and science, veerapandi, theni-625534, tamil nadu, india, 2chemistry research centre, mohamed sathak engineering college, kilakarai, ramanathapuram-623806, tamil nadu, india and 3pg and research centre of chemistry, jayaraj annapackium college for women, periyakulam, theni-62560, tamil nadu, india (received 18 may, revised 2 november, accepted 16 november 2018) abstract: novel series of co(ii), ni(ii) and cu(ii) transition metal complexes were synthesized from the schiff base ligands hl1–hl4 derived from 2-amino-6nitrobenzothiazole with various aromatic aldehydes, i.e., 4-methylbenzaldehyde, 4ethylbenzaldehyde, 2-hydroxybenzaldehyde and 4-hydroxybenzaldehyde, respectively. the ligands hl1–hl4 and their metal (ii) complexes were characterized by atomic absorption spectroscopy (aas), magnetic susceptibility and molar conductance measurements, and uv–vis, ft-ir and nmr spectroscopic techniques. the spectroscopic studies revealed that the complexes of hl1–hl3 coordinated in an octahedral environment and l4 in square planar/tetrahedral geometry. the ligands hl1–hl4 and their metal(ii) complexes were screened for their antimicrobial activities against staphylococcus aureus, escherichia coli, pseudomonas aeruginosa and candida albicans. the results suggest that the complexes of hl4 possess greater inhibition activity towards c. albicans. the nonlinear optics (nlo) activities of ligands hl1–hl4 were determined. the obtained results showed that ligands hl1 and hl2 have greater second harmonic generation (shg) efficiency than hl3and hl4. keywords: aldehydes; metal(ii) complexes; spectroscopic; aas; biological activity. introduction in the last five decades, schiff bases have emerged as the most preferred compounds in the field of coordination chemistry and medicinal chemistry. the azomethine group of schiff bases makes it a potential donor for the formation of complexes. besides the chelating ability of schiff bases of heterocyclic com *corresponding author e-mail: kalacharle@gmail.com https://doi.org/10.2298/jsc180521101k 268 kanagavalli et al. pounds containing 5and 6-membered rings, they also find potential applications in the field of therapeutics.1 benzothiazole is a privileged bicyclic ring system with significant biological properties, such as antimicrobial2–5 and anticancer.6 transition metal complexes of schiff bases of 2-amino-6-nitrobenzothiazole enabled a study of the different ligation behaviors of schiff bases. in this respect, a structure activity relationship may also be studied. in recent years, organic materials have been studied for their nonlinear optical (nlo) properties, which are of use in optical communications. a large number of organic compounds have been tested for their nlo property in order to design effective communication materials.7 in this context, the synthesis of the schiff bases of 2-amino 6-nitrobenzothiazole with various aromatic aldehydes and also of the corresponding co(ii), ni(ii) and cu(ii) metal complexes are reported. the antimicrobial activity and nlo property of the synthesized compounds are compared. experimental all the chemicals used in the present work, viz. 2-amino-6-nitrobenzothiazole, 4-methylbenzaldehyde, 4-ethylbenzaldehyde, 4-hydroxybenzaldehyde, 2-hydroxybenzaldehyde, and cobalt(ii), nickel(ii) and copper(ii) chlorides were of analytical reagent grade. commercial solvents were distilled and then used for the preparation of the ligands and metal(ii) complexes. elemental analyses and esi-ms were performed at saif, csir-cdri-lucknow, india. molar conductivities in dmso (10-3 mol dm-3) were measured at room temperature on an elico cm-180 digital conductivity meter. magnetic susceptibility measurements of the complexes were realized on a gouy balance using copper sulfate pentahydrate as the calibrant. the ir spectra were recorded on a shimadzu ft-ir ir affinity-1 spectrophotometer in the 4000–400 cm-1 range using kbr pellets. the nmr spectra were recorded on a bruker drx – 300 mhz nmr spectrometer in dmso-d6 and cdcl3 as solvents with tetramethylsilane (tms) as the internal reference. the absorption spectra were recorded using a shimadzu uv -1800 spectrophotometer between 200–1100 nm by using a suitable solvent. the shg (second harmonic generation) conversion efficiency of the ligands was determined by a modified version of powder technique in iisc, bangalore, india. synthesis of ligands hl1–hl4 an ethanolic solution (30 ml) of 2-amino-6-nitrobenzothiazole (0.050 mol) was mixed with ethanolic solution (20 ml) of aldehyde (0.050 mol; 4-methylbenzaldehyde, 4-ethylbenzaldehyde, 4-hydroxybenzaldehyde and 2-hydroxybenzaldehyde) and the mixture was stirred. completion of the reaction was monitored by tlc. the precipitated yellow colored schiff bases were filtered, washed and dried. the scheme of the synthesis is shown in fig. 1. synthesis of metal(ii) complexes all the complexes were prepared from hydrated metal salts. to a solution of 20 mmol of schiff bases (hl1–hl4) in 25 ml ethanol was added to 10 mmol of mcl2·nh2o (m = co(ii), ni(ii) or cu(ii); n = 6, 6 and 2, respectively). the solution was stirred for 2 h and the resulting solid product was filtered, washed with ethanol and dried under vacuum. estimation of percentage of metal the percentage of metal in the complexes was estimated by atomic absorption spectroscopy at the bsr laboratory, jayaraj annapackiam college for women, india. a complex (5 metal(ii) complexes of schiff-base ligands of 2-amino-6-nitrobenzothiazole 269 mg) was dissolved in 2 ml of dmso and the solution is made up to 50 ml with double distilled (dd) water. standard solutions (1000, 100, 10 and 1 ppm, respectively) of metal salts of cobalt, nickel and copper were prepared in dd water. different metal filters were used for running the analysis for estimation. comparing with the amount of metal ion present in the standard solutions, the amount of metal ion in the metal complexes was estimated graphically. fig. 1. scheme of the synthesis of ligands hl1–hl4. antimicrobial activity screening antimicrobial activity of the compounds was tested in vitro by the well diffusion method8 against the bacteria staphylococcus aureus, escherichia coli and pseudomonas aeruginosa using agar nutrient as the medium. antifungal activity was evaluated against candida albicans cultured on potato dextrose agar as medium. all the mentioned bacterial strains were incubated in nutrient broth (nb) at 37 °c for 24 h and fungal isolate was incubated in pda broth at 28 °c for 2 to 3 days. wells each of 5 mm in diameter were made in muller–hinton agar using a cork borer. the test solution was prepared at 10-3 mol l-1 concentration in dmso and then 100 µl of the solution was transferred into each well. the plates were incubated for 24 h at 37 °c and examined for a clear inhibition zone around the well. standard drugs ampicillin and ketoconazole were used for antibacterial and antifungal testing, respectively. nlo properties of ligands hl1–hl4 in a nonlinear process, the oscillation of electrons becomes inharmonic and the re-emitted light may differ in frequency and amplitude from the incident light. inorganic materials have dominated the field of nlo, for example, lithium niobate (linbo3) has been used for electro-optic modulation.9 in organic materials, electrons are more accessible and nlo -effects arise from the interaction between light and electrons within individual molecular units. especially, organic structures with large delocalized π-systems have proven to be useful. the reason for this is that π-electrons are more easily affected by an external optical field as they are relatively loosely bound to the nucleus, and that the delocalized orbitals may extend over the entire molecule enabling large and fast polarization.10,11 in the present work, the second harmonic generation (shg) efficiency of the ligands was determined by a modified version of the powder technique developed by kurtz and perry.12 the efficiency of the sample was compared with microcrystalline powder of potassium dihydrogen phosphate (kdp) and urea. the input energy used in this particular setup was 2.2 mj per pulse. 270 kanagavalli et al. results and discussion the schiff bases hl1–hl4 were obtained as low melting solids and stored in vacuum desiccators. the ligands form stable complexes in ethanol medium. the percentage of metal in the complexes was estimated by atomic absorption spectroscopy technique. the analytical data of the hl1 to hl4 and their metal(ii) complexes together with their physical properties are given in the table i. all the ligands coordinate in a bidentate fashion. the metal(ii) complexes are partially soluble in ethanol, chloroform and acetonitrile but soluble in polar solvents, such as dmf and dmso. the low values of conductance in dmso indicate that the complexes are non-electrolytes.13 the estimated percentage data of metal ion (ii) in the complexes were in good agreement with the calculated data for a 1:2 (m:l) ratio. table i physical characterization and molar conductance of hl1–hl4 and their metal(ii) complexes compound fw / g mol-1 calculated (observed) metal content, % color λm / s cm 2 mol-1 hl1 297 – yellow – [co(hl1)2cl2] 723.8 8.14 (7.67) light green 07 [ni(hl1)2cl2] 723.6 8.11 (7.62) dark green 06 [cu(hl1)2cl2] 728.4 8.72 (8.06) brown 07 hl2 311 – yellow – [co(hl2)2cl2] 751.8 7.83 (7.51) green 04 [ni(hl2)2cl2] 751.6 7.80 (7.38) green 05 [cu(hl2)2cl2] 756.4 8.40 (7.92) brown 05 hl3 299 – yellow – [co(hl3)2cl2] 727.8 8.09 (7.81) green 09 [ni(hl3)2cl2] 727.6 8.06 (7.58) dark green 06 [cu(hl3)2cl2] 732.4 8.67 (8.12) brown 10 hl4 299 – yellow – [co(hl4)2] 655.5 8.97 (8.44) light green 07 [ni(hl4)2] 656.6 8.93 (8.42) green 05 [cu(hl4)2] 661.5 9.60 (9.11) brown 07 ir spectra in the ir spectrum of the free ligands (hl1–hl4) the frequencies at 1653– –1649 cm–1 are assigned to the azomethine group (ν(ch=n)). the bands at 1523–517 cm–1 are attributed to the stretching vibration of the c=n group of the thiazole ring (ν(c=n)). in the spectra of the metal(ii) complexes of hl1–hl3, the frequencies at 1653–1649 cm–1 and 1523–1517 cm–1 are shifted to lower values (1651–1504 cm–1 and 1517–1493 cm–1) confirming the coordination of the azomethine nitrogen and ring nitrogen14 of the benzothiazole group, respectively, to the metal ion. in the spectra of metal(ii) complexes of hl1–hl4, the frequencies at 653–516 cm–1 are assigned to the m–n stretching mode.15–17 the metal(ii) complexes of schiff-base ligands of 2-amino-6-nitrobenzothiazole 271 spectrum of the ligand hl4 shows a band at 3454 cm–1 due to phenolic oh group. the absence of this band in the spectrum of metal(ii) complexes evidences the coordination of deprotonated phenolic group to the metal(ii) ion. in the spectra of the metal(ii) complexes of hl4, the stretching frequencies at 712–742 cm–1 are assigned to m–o vibrations.18 nmr spectra the 1h-nmr and 13c-nmr data for the schiff base ligands hl1–hl4 are given in the supplementary material to this paper. electronic absorption spectra the uv–vis spectra of the complexes were recorded in dmso solution. the copper(ii) complexes of hl1–hl3 exhibit a d–d band in the region 10000–9267 cm–1. this band may be assigned to a 2b1g→2a1g transition, characteristic for a distorted octahedral structure.19 in addition, the complexes of co(ii) and ni(ii) exhibited two bands at 14684 and 16977 cm–1 and at 9354 and 10405 cm–1, assigned to 4t1g(f)→4a2g(f), 4t1g(f)→4t2g(f) and 3a2g(f)→3t2g(f), 3a2g(f)→3t1g(f) transitions, respectively, suggesting octahedral coordination. the copper(ii) complex of hl4 shows a band at 12650 cm–1, assignable to a 2b1g→2a1g transition, characteristic for a distorted square–planar geometry.20 the electronic spectrum of cobalt(ii) complex of hl4 shows band at 15,880 cm–1, which may be tentatively assigned to a 4a2(f)→4t1(p) transition for tetrahedral geometry.21 the nickel(ii) complex exhibits an absorption band at 13897 cm−1 due to a 3t1(f)→3t1(p) transition, corresponding to tetrahedral geometry. magnetic moments of metal(ii) complexes magnetic susceptibility measurements gave sufficient data to characterize the structure of the metal complexes. magnetic moment measurements of compounds were performed at 25 °c. the magnetic moment values of the copper(ii) complex of hl4 was 1.75 µb, suggesting a square planar environment.22 the tetrahedral structure of the nickel(ii) complex of hl4 was evidenced by the magnetic moment value 2.81 µb. the magnetic moment value of cobalt(ii) complex of hl4 was 4.35 µb, suggesting a tetrahedral structure. the high spin nature and the octahedral structure of cobalt(ii) complexes of hl1–hl3 was further evidenced by the magnetic moment values of 4.79–5.40 µb. the magnetic moment values of the copper(ii) complexes of hl1–hl3 were in the range 1.81–2.08 µb, indicating octahedral coordination. that the magnetic moment values were higher than the spin only value (1.73 µb) might be due to an orbital contribution. the octahedral structure of the nickel(ii) complexes of hl1–hl3 was further supported by the magnetic moment values 3.05–3.10 µb.22 based on the above discussion, the proposed structures of the complexes are shown in fig. 2. 272 kanagavalli et al. x = ch3, c2h5, oh m = co(ii), ni(ii) and cu(ii) hl1 to hl3 m = co(ii), ni(ii) and cu(ii) hl4 m cl n n cl nn s s x x o2n no2 m on no s n no 2 ns o2n fig. 2. proposed geometry of the complexes. antimicrobial screening the ligands and the complexes exhibited antifungal activity. from the zone of inhibition, it was shown that some of the ligands and complexes have very low inhibition activity towards escherichia coli, staphylococcus aureus, pseudomonas aeruginosa and candida albicans. some of the metal complexes were found to be more potent than the ligands against bacterial and fungal strains. the different substituents in the schiff bases had very low effects on the antimicrobial activity. the metal complexes of hl2 exhibited no antifungal activity. the data showing the zone of inhibition are given in the table ii. it is observed that, in a table ii. antimicrobial activity data of hl1–hl4 and their metal(ii) complexes (zone of inhibition in mm); r = resistant compound e. coli p. aeruginosa s. aureus c. albicans hl1 4 5 3 10 [co(hl1)2cl2] r r r 11 [ni(hl1)2cl2] r r 12 r [cu(hl1)2cl2] r 10 12 12 hl2 r r r r [co(hl2)2cl2] r 10 r r [ni(hl2)2cl2] r r r r [cu(hl2)2cl2] r r r 10 hl3 r r 12 12 [co(hl3)2cl2] 10 12 r 11 [ni(hl3)2cl2] 8 r r 14 [cu(hl3)2cl2] r 12 10 14 hl4 r r 12 12 [co(hl4)2] 10 12 r 14 [ni(hl4)2] 8 r r 14 [cu(hl4)2] r 12 14 14 metal(ii) complexes of schiff-base ligands of 2-amino-6-nitrobenzothiazole 273 complex, the positive charge of the metal is partially shared with the donor atoms present in the ligand and there may be π-electron delocalization over all the chelating system.23,24 this increases the lipophilic character of the metal chelate and favors its permeation through the lipoid layer of the bacterial membranes. so the metal complex could become more active than the free ligand. therefore, the metal complexes showed greater antifungal activities than the uncoordinated ligand and free metal ion. the bacterial strains are resistant towards the alkyl substituted ligands hl1 and hl2 and their metal complexes. the activity may be related to the length of carbon chain25 present and they do not show an appreciable antifungal activity. the hydroxyl-substituted ligands hl3 and hl4 showed a moderate antibacterial and antifungal activity. from sar studies, it is known that electron withdrawing groups increase the antibacterial activity and this supports the resistant nature of all the ligands and complexes towards most of the antibacterial strains.26 although the ligand contains the nitro group in the aromatic ring that is electron withdrawing group, the antimicrobial activity was suppressed due to electron releasing groups, such as alkyl and hydroxyl groups. the solubility of the compound, type of the strain and the coordinating nature of the schiff base determine the inhibition activity. the hydrogen bonding ability27 of the azomethine group with the microbial strain may improve the antimicrobial activity of the synthesized compounds. in this regard, the complexes of hl3 and hl4 with a hydroxyl group showed better inhibition compared to the metal complexes of hl1 and hl2. some of the complexes were inactive against e. coli, which may be due to the bulkiness of the complexes.2 nlo activity the synthesized ligands possess nlo properties. the methyl-substituted ligands (hl1 and hl2) show greater shg efficiency than the hydroxyl-substituted ligands. the more pronounced electron donating nature of alkyl groups makes ligands more polarized and show enhanced nlo activity.28 the ligand hl3 is more nlo active than hl4, due to the para position of the hydroxyl group. the shg efficiency was found to be lower when compared with urea and kdp.29–31 the activity data is given in table iii. table iii. nlo properties of the ligands ligand shg efficiency, %, with respect to kdp urea hl1 15 8 hl2 20 10 hl3 14 6 hl4 13 5 274 kanagavalli et al. conclusions in this work, transition metal(ii) complexes of schiff bases of 2-amino-6-nitrobenzothiazole were synthesized and characterized. the ligands and their metal complexes were studied by various physical and chemical techniques, i.e., metal estimation by aas, uv–vis, 1h-nmr and ir. all the synthesized ligands and complexes were screened for their antimicrobial activity. all the metal (co(ii), ni(ii) and cu(ii)) complexes of the synthesized ligands hl1–hl3 show octahedral geometry. distorted square planar geometry is proposed for the copper complex of hl4. the other complexes of hl4 coordinate in a tetrahedral fashion. ligands and complexes possess lower antimicrobial activity than the standard drugs. the synthesized compounds possess very small antibacterial activity and appreciable antifungal activity. all the ligands were found to possess nlo activity. the alkyl-substituted schiff bases (hl1 and hl2) have greater shg efficiency than the hydroxyl substituted schiff bases hl3 and hl4. supplementary material characterization data of the ligands and complexes are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/ or from the corresponding author on request. acknowledgements. the authors express their sincere thanks to the college management, principal and bsr laboratory, department of chemistry, jayaraj annapackiam college for women, periyakulam, india, for providing the necessary research facilities. и з в о д спектроскопска, nlo и антимикробна испитивања co(ii), ni(ii) и cu(ii) комплекса са шифовим базама 2-амино-6-нитробензотиазола као лигандима chinnaraj kanagavalli1, murugesan sankarganesh2, jeyaraj dhaveethu raja2 и manivannan kalanithi3 1 department of chemistry, theni college of arts and science, veerapandi, theni-625534, tamil nadu, india, 2 chemistry research centre, mohamed sathak engineering college, kilakarai, ramanathapuram-623806, tamil nadu, india и 3 pg and research centre of chemistry, jayaraj annapackium college for women, periyakulam, theni-62560, tamil nadu, india синтетисана је нова серија комплекса прелазних метала, co(ii), ni(ii) и cu(ii), са шифовим базама као лигандима (hl1–hl4) који су добијени из 2-амино-6-нитробензотиазола и различитих ароматичних алдехида, као што су 4-метилбензалдехид, 4-етилбензалдехид, 2-хидроксибензалдехид и 4-хидроксибензалдехид. лиганди (hl1–hl4) и њихови комплекси су окарактерисани помоћу aas, мерења магнетне и моларне проводљивости, као и uv–vis, ft-ir и nmr спектроскопских техника. на основу спектроскопских испитивања нађено је да лиганди hl1–hl4 граде комплексе октаедарске геометрије, док у случају hl4 лиганда настају комплекси који имају и квадратно-планарну, односно тетраедарску геомтерију. лиганди hl1–hl4 и одговарајући метал(ii)-комплекси су испитивани на антимикробну активност према микроорганизмима staphylococcus aureus, escherichia coli, pseudomonas aeruginosa и candida albicans. добијени резултати ових испитивања су показали да комплекси са metal(ii) complexes of schiff-base ligands of 2-amino-6-nitrobenzothiazole 275 лигандом hl4 показују већу инхибиторску активност према c. albicans. одређена је nlo активност лиганада hl1–hl4. добијени резултати су 11 показали да је у случају лиганада hl1 и hl2 shg ефикасност већа у односу на лиганде hl3 и hl4. 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(2017) s1878 29. m. n uddin, d. a chowdhury, k. hossain, j. chin. chem. soc. 59 (2012) 1520 30. m. n. uddin, d. a chowdhury, m. t. islam, f. hoque, orbital: electron j. chem. 4 (2012) 273 31. n. chattopadhyay, a. mallick, s. sengupta, j. photochem. photobiol. a 177 (2006) 55. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice j. serb. chem. soc. 83 (10) 1071–1097 (2018) udc 547.7+547.78:544.275–128: jscs–5134 615.9+542.9:57–188 review 1071 review [bmim]pf6: an efficient tool for the synthesis of diverse bioactive heterocycles gurpreet kaur, aditi sharma and bubun banerjee* department of chemistry, indus international university, village and post office bathu, district una. himachal pradesh-174301, india (received 3 january, revised 3 july, accepted 16 july 2018) abstract: heterocycles are the privileged structural subunit of many marketed drug molecules. on the other hand, the last decade has seen tremendous applications of the ionic liquid [bmim]pf6 (1-butyl-3-methyl-1h-imidazolium hexafluorophosphate) as an efficient, cheap, commercially available, low toxic reaction medium for various organic transformations. the present review summarizes recent reported applications of [bmim]pf6 as an efficient reaction medium for the synthesis of diverse biologically relevant heterocycles. keywords: [bmim]pf6; heterocycles; hexafluorophosphate; imidazolium; p-ionic liquid. contents 1. introduction 2. synthesis of n-heterocycles 2.1. synthesis of aziridines 2.2. synthesis of n-substituted phthalimides 2.3. synthesis of spiro[azetidine-2,3′-(3h)indole] derivatives 2.4. synthesis of 3-methylene-2,3-dihydro-1h-quinolin-4-ones 2.5. synthesis of 1,3,5-triarylpyrazoles 2.6. synthesis of spiro[indoline-pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidine]triones 2.7. synthesis of dispiropyrrolidine-bisoxindole derivatives 2.8. synthesis of 2,3-dihydroquinazolin-4(1h)-ones 2.9. synthesis of 2,4-diamino-6-aryl-1,3,5-triazines 2.10. synthesis of 4-(1h-1,2,3-triazol-1-yl)-1,2,5-oxadiazol-3-amine derivatives 2.11. synthesis of 3,4-dihydropyrimidin-2(1h)-ones 2.12. synthesis of quinolines 2.13. synthesis of anthraquinone fused n-heterocycles 2.14. synthesis of novel n-heterocycles via ring-closing metathesis reaction * corresponding author. e-mail: banerjeebubun@gmail.com dedicated to prof. kamal usaf sadek, mania university, mania, egypt. https://doi.org/10.2298/jsc180103052k ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1072 kaur, sharma and banerjee 3. synthesis of o-heterocycles 3.1. synthesis of 2,3-disubstituted benzo[b]furans 3.2. synthesis of coumarin derivatives 3.3. synthesis of pyrano[4,3-b]pyran-5-one derivative 3.4. synthesis of epoxyisobenzofuran-1,3-dione 3.5. synthesis of xanthenes 3.6. synthesis of 2-amino-3-cyano-benzochromenes 3.7. synthesis of 1,3-dioxane derivatives 3.8. synthesis of chromanone derivatives 3.9. synthesis of lactone and lactam derivatives 4. synthesis of n,o-heterocycles 4.1. synthesis of spiro[chromeno[2,3-d]pyrimidine-5,3′-indoline]tetraones 4.2. synthesis of pyrimidine containing isoxazolines 4.3. synthesis of isoxazolidines 4.4. synthesis of benzoxazine and quinazoline 5. synthesis of s-heterocycles 5.1. synthesis of 2-aminothiophenes 6. synthesis of n,s-heterocycles 6.1. synthesis of spiro[3h-indole-3,2′-thiazolidine]-2,4′(1h)-diones 6.2. synthesis of 2-phenylthiazoles 6.3. synthesis of benzothiazole derivatives 6.4. synthesis of 1,5-benzothiazepine-4-ones 7. conclusions 1. introduction heterocycles are the skeleton of the majority of hitherto known organic compounds.1,2 many synthetic compounds containing heterocycles possess immense biological activities that include anti-microbial,3 anti-malarial,4 anti-cancer,5 cytotoxic,6 anti-inflammatory,7 anti-oxidant,8 anti-hyperglycemic and anti-dyslipidemic,9 along with anti-neurodegenerative disorders such as alzheimer’s and parkinson disease and many more.10–12 among the other significant parameters, screening of suitable reaction medium plays the key role during organic transformations.13,14 worldwide, scientists are trying to modify the reaction media to increase the efficiency of the reaction and reduce their toxicity level as well. in recent times, a wide range of ionic liquids have been employed as reaction media due to their inherent features that include high thermal stability, ability to dissolve a large number of organic and inorganic compounds, non-volatility, low inflammability, easy reusability etc.15–29 the organic and ionic environment in ionic liquids renders almost all kinds of interactions with reactants. this may cause a reduction in activation energies, either by stabilizing the transition states or by destabilizing the reactants.30,31 moreover, both ionic and van der waals interactions with solutes generate internal pressure in an ionic liquid, which eventually accelerates the chemical reaction by promoting an accumulation of reactants in the cavities of the solvent.32–34 recyclability of ionic liquid makes a protocol cost effective as well. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ diverse bioactive heterocycles 1073 due to these above-mentioned advantages, ionic liquids are being used in many organic transformations as both reaction medium as well as promoter even in the absence of any other catalyst.35–40 recently, among the others, ionic liquids based on imidazolium salts, have gained significant attention as efficient reaction media and have found immense applications in various organic synthesis that include hydrogenations,41 friedel– –crafts reactions,42 heck reactions,43 bishler–napieralski reactions,44 henry reaction45 and many more.46 some of the ionic liquids based on the 1-butyl-3 -methylimidazolium cation are presented in fig. 1. among these, during few years, the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim]pf6) has shown tremendous applications in various organic transformations.47–50 n n bf4 [bmim]bf4 n n pf6 [bmim]pf6 n n x [bmim]x n n clo4 [bmim]clo4 n n ooccf3 [bmim]cf3coo n n alcl4 [bmim]alcl4 n n n(so2cf3)2 [bmim]ntf2 n n n3 [bmim]n3 x = cl or br n n oh [bmim]oh n n oocch3 [bmim]ch3coo + + + + + + + + + + fig. 1. some of the 1-butyl-3-methylimidazolium based ionic liquids. the present review deals with the applications of the ionic liquid [bmim]pf6 for the synthesis of diverse bioactive heterocycles, and when possible, gives a comparison of its efficiency with the rest of the hitherto reported congeners. 2. synthesis of n-heterocylces 2.1. synthesis of aziridines aziridines are used as an important precursor for the synthesis of a wide range of nitrogen-containing heterocycles.51,52 a simple, efficient and stereo-selective method was developed for the synthesis of aziridines (3) in the reaction between various imines (1) and ethyl diazoacetate (2) under catalyst-free conditions in the ionic liquid [bmim]pf6 at room temperature (scheme 1).53 after completion of the reaction, the ionic liquid was recovered easily and reused five times without any loss in its activity. mild reaction conditions, good yields and high product selectivity are some of the advantages of this protocol. in the same year, another method was reported for the synthesis of cis-diastereoselective aryl aziridines (3) via three-component reactions of aldehydes (4), various amines (5) and ethyl diazoacetate (2) using bi(otf)3 or sc(otf)3 as catalyst in the same ionic liquid at 27 °c (scheme 2).54 the use of a catalyst red________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1074 kaur, sharma and banerjee uces the reaction time as compared to the catalyst-free conditions (scheme 1). ambient reaction condition, wide range of substrate tolerance, reusability of the media, high atom economy, good to excellent yields are some of the major benefits of this developed protocol. scheme 1. [bmim]pf6-mediated synthesis of aziridines at room temperature. scheme 2. [bmim]pf6-mediated triflate salts catalyzed three-component synthesis of aziridines. 2.2. synthesis of n-substituted phthalimides the ionic liquid [bmim]pf6 was found to be an efficient alternative to classical solvents for the synthesis of n-substituted phthalimides.55–57 a series of n-substituted phthalimides (7) was synthesized in good yields via the reaction of succinic anhydride (6) and various aryl amines (5) under catalyst-free conditions in [bmim]pf6 at 80 °c (scheme 3).55 the ionic liquid was recovered and reused many times. in the next year, along with succinic anhydride, le et al.56 also employed maleic and phthalic anhydride for the synthesis of n-aryl phthalimides in the ionic liquid [bmim]pf6 at 140 °c. under these optimized conditions, reactions with aliphatic amines also proceeded smoothly to produce the desired products in high yields. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ diverse bioactive heterocycles 1075 scheme 3. [bmim]pf6-mediated synthesis of n-substituted phthalimides. 2.3. synthesis of spiro[azetidine-2,3′-(3h)indole] derivatives azetidinones are very common in many biologically active compounds having significant biological efficacies that include anti-bacterial, anti-fungal,58 and anti -inflammatory59 activities. a series of novel spiro[azetidine-2,3′-(3h)indole] derivatives (11) was synthesized by the reactions of isatins (8), 4-amino-4h-1,2,4-triazole (9) and acetyl chloride (10) or chloroacetyl chloride (10a) in ionic liquid [bmim]pf6 using triethylamine as catalyst at 60–70 °c (scheme 4).60 the ionic liquid was recovered easily and reused twice without any loss in its activity. the insecticidal activities of the synthesized compounds were tested against periplaneta americana and few were found to possess prominent efficacy. scheme 4. [bmim]pf6-mediated synthesis of spiro[azetidine-2,3′-(3h)indole derivatives. 2.4. synthesis of 3-methylene-2,3-dihydro-1h-quinolin-4-ones the ionic liquid [bmim]pf6 was found to be an efficient medium for the palladium-catalyzed cyclocarbonylation reaction of o-iodoanilines (12), various allenes (13) and carbon monoxide (14) to afford the corresponding 3-methylene -2,3-dihydro-1h-quinolin-4-ones (15) in the presence of a catalytic amount of ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1076 kaur, sharma and banerjee 1,4-bis(diphenylphosphino)butane (dppb) and diisopropylethylamine [(ipr)2net] as promoter at 90 °c (scheme 5).61 the entire medium containing the catalyst as well as the promoter was successfully recovered and reused more than four times without significant loss in activities. scheme 5. [bmim]pf6-mediated synthesis of 3-methylene-2,3-dihydro-1h-quinolin-4-ones. 2.5. synthesis of 1,3,5-triarylpyrazoles heterocycles containing the pyrazole moiety have exhibited diverse biological activities that include anti-depressant, anti-convulsant,62 anti-inflammatory and anti-arthritic63 activities. a simple, efficient, and environmentally benign protocol was developed for the synthesis of 1,3,5-triarylpyrazoles (18) via the one-pot oxidative addition of chalcones (16) and arylhydrazines (17) using cu(otf)2 as catalyst in [bmim]pf6 at 130 °c (scheme 6).64 during optimization, scheme 6. [bmim]pf6-mediated synthesis of 1,3,5-triarylpyrazoles. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ diverse bioactive heterocycles 1077 it was found that other metal triflates such as sc(otf)3, ce(otf)3, agotf, zn(otf)2 and yb(otf)3 as catalyst in [bmim]pf6 produced lower yields. on the other hand, cu(otf)2 yielded lower product in [bmim]bf4 and [bmim]br compared to in [bmim]pf6. the catalyst along with the reaction media was successfully recovered and reused four times without loss in catalytic activity. the synthesized compounds were found to possess antiproliferative efficacies. 2.6. synthesis of spiro[indoline-pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidine]triones a simple, efficient and practical protocol was developed for the synthesis of a series of spiro[indoline-pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidine]trione derivatives (21) via one-pot four-component reactions between isatins (8), barbituric acids (19), phenylhydrazines (17) and 3-oxo-3-phenylpropanenitrile (20) using alum kal(so4)2·12h2o as a reusable catalyst in [bmim]pf6 at 100 °c (scheme 7).65 scheme 7. [bmim]pf6-mediated synthesis of spiro[indoline-pyrazolo[4′,3′:5,6]pyrido [2,3-d]pyrimidine]triones. during optimization, other 1-butyl-3-methylimidazolium based ionic liquids such as [bmim]bf4, [bmim]cf3co2, [bmim]br, [bmim]cl were also screened using alum as catalyst and among them, [bmim]pf6 came out to be superior in terms of both reaction time as well as product yields. mild reaction conditions, short reaction time, excellent yields, varieties of substrates, high atom economy and reusability of medium are some of the major advantages in this protocol. in the same year, under the same reaction conditions shirvan et al.66 also synthesized a variety of spiro[indoline-pyrazolo[4′,3′:5,6]pyrido[2,3-d]pyrimidine]tri________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1078 kaur, sharma and banerjee ones staring from substituted isatins, barbituric acids, and 1,3-diphenyl-1h-pyrazol-5-amines. 2.7. synthesis of dispiropyrrolidine-bisoxindole derivatives spiro-oxindoles are very common in pharmacologically important compounds.67 a simple, facile and efficient catalyst-free one-pot three-component protocol was reported for the synthesis of a series of novel dispiropyrrolidine-bisoxindole derivatives (24) via the cycloaddition between isatins (8), sarcosine (22) and 3-(aroylmethylene)-1,3-dihydro-2h-indol-2-one (23) in [bmim]pf6 at 80 °c (scheme 8).68 ionic liquid was recovered successfully and reused three times without significant loss in its activity. scheme 8. [bmim]pf6-mediated catalyst-free synthesis of dispiropyrrolidine-bisoxindoles. 2.8. synthesis of 2,3-dihydroquinazolin-4(1h)-ones now-a-days, performing reactions in absence of any added catalysts is one of the thrusting areas.69–71 a series of 2,3-dihydroquinazolin-4(1h)-one derivatives (26) was synthesized in moderate to high yields via the condensation between anthranilamides (25) and various aldehydes (4) in [bmim]pf6 under catalyst-free conditions at 75 °c (scheme 9).72 mild reaction conditions, short scheme 9. [bmim]pf6-mediated catalyst-free synthesis of 2,3-dihydroquinazolin-4(1h)-ones. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ diverse bioactive heterocycles 1079 reaction times, good to excellent yields make this protocol attractive. a wide range of aldehydes that include electron donating as well as electron withdrawing substituent are well tolerated under the optimized reaction conditions affording the required products in good yields. during optimization, a number of other ionic liquids were also tested, whereby [bmim]pf6 was found to be superior for this transformation. after completion of the reaction, the ionic liquid [bmim]pf6 was successfully recovered and reused for the several runs without loss of its activity. 2.9. synthesis of 2,4-diamino-6-aryl-1,3,5-triazines a simple, rapid and efficient microwave-assisted environmentally benign protocol was developed for the synthesis of 2,4-diamino-6-aryl-1,3,5-triazines (29) via the reaction of aromatic nitriles (27) and dicyanodiamide (28) using koh as catalyst in [bmim]pf6 at 130 °c (scheme 10).73 in the absence of microwave, i.e., under conventional heating at the same temperature, the reaction took more than 8 h to complete and yields of the corresponding products were also lower. [bmim]pf6 was found to be superior to [bmim]bf4 for this conversion. after completion of the reaction, the ionic liquid was successfully recovered and recycled five times without significant loss in its activity. scheme 10. [bmim]pf6-mediated synthesis of 2,4-diamino-6-aryl-1,3,5-triazines under microwave conditions. 2.10. synthesis of 4-(1h-1,2,3-triazol-1-yl)-1,2,5-oxadiazol-3-amine derivatives seregin et al.74 synthesized 4-(1h-1,2,3-triazol-1-yl)-1,2,5-oxadiazol-3-amines (32) via the 1,3-dipolar cycloaddition between 4-amino-3-azidofurazan (30) and butynediol (31) or propargyl alcohol (31a) in [bmim]pf6 under catalyst-free conditions at 80 °c (scheme 11). the use of the ionic liquid [bmim]bf4 produced lower yields. 2.11. synthesis of 3,4-dihydropyrimidin-2(1h)-ones dihydropyrimidinones possess significant biological efficacies that include anti-viral, anti-bacterial, anti-hypertensive and anti-tumour activity.75 a series of 3,4-dihydropyrimidin-2(1h)-ones (35) were synthesized in good yields via the one-pot, three-component biginelli reaction76 of various aromatic aldehydes (4), ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1080 kaur, sharma and banerjee urea (33) and ethyl acetoacetate (34) using a catalytic amount of ionic liquid [bmim]pf6 under solvent-free conditions at 100 °c (scheme 12).77 for this transformation, the ionic liquid [bmim]bf4 was found to be as efficient as [bmim]pf6, whereas [bmim]cl afforded lower yields. scheme 11. [bmim]pf6-mediated synthesis of 4-(1h-1,2,3-triazol-1-yl)-1,2,5-oxadiazol-3 -amines. scheme 12. [bmim]pf6-catalyzed synthesis of 3,4-dihydropyrimidin-2(1h)-ones. 2.12. synthesis of quinolines a mild, simple and efficient protocol was developed for the synthesis of a variety of quinoline derivatives (38) by following the friedländer annulation reaction78 of o-aminobenzophenone (36) and various ketones (34, 37a-e) using gadolinium triflate [gd(otf)3] as an inexpensive, moisture-stable lewis acid catalyst in [bmim]pf6 at 60 °c (scheme 13).79 after completion of the reaction, the catalyst containing ionic liquid was successfully recovered and reused several times without appreciable loss in product formation. mild reaction conditions, operational simplicity, very short reaction time; high yields of products are some of the major advantages of this developed protocol. 2.13. synthesis of anthraquinone fused n-heterocycles a number of anthraquinone fused n-heterocycles (41) were synthesized via the ring-closure metathesis reactions of 1,4-dihydroxyanthraquinone (39) and various diamines (40) in the presence of a catalytic amount of cucl2 in the ionic liquid [bmim]pf6 at room temperature (scheme 14).80 the reaction was also performed in conventional solvents, such as dimethylformamide and dichloromethane, but lower yields were obtained. the ionic liquid containing catalyst was successfully recovered and reused five times without significant loss in its activity. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ diverse bioactive heterocycles 1081 scheme 13. [bmim]pf6-mediated synthesis of a variety of quinoline derivatives. scheme 14. [bmim]pf6-mediated synthesis of anthraquinone fused n-heterocycles. 2.14. synthesis of novel n-heterocycles via ring-closing metathesis reactions the ionic liquid [bmim]pf6 was found to be an effective medium for ring -closing metathesis using grubbs catalysts.81,82 a simple and straightforward protocol was demonstrated for the ring-closing metathesis of 1,5-diallyl-3-benzyl-5-isobutylimidazolidine-2,4-dione (42) to afford the corresponding novel 2-benzyl-8a-isobutyl-8,8a-dihydroimidazo[1,5-a]pyridine-1,3(2h,5h)-dione (43) using ruthenium based grubbs catalyst in [bmim]pf6 at 50 ºc (scheme 15).83 scheme 15. [bmim]pf6-mediated ring-closing metathesis reaction using a ruthenium catalyst. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1082 kaur, sharma and banerjee after completion of reaction, the ionic liquid containing the ruthenium catalyst was successfully recovered and thrice reused for the same reaction. 3. synthesis of o-heterocycles 3.1. synthesis of 2,3-disubstituted benzo[b]furans benzo[b]furan-containing heterocycles possess immense pharmaceutical efficacies that include anti-fungal84 and anti-tumor85 activity. a combination of cui and 1-butyl-3-methylimidazolium acetate ([bmim]oac) in [bmim]pf6 was shown to be an efficient catalytic system for the synthesis of a series of 2,3 -disubstituted benzo[b]furan derivatives (47) via one-pot three-component tandem reactions of salicylaldehyde (44), alkynes (45) and various aliphatic secondary amines (46), such as morpholine, dibenzylamine, piperidine etc., at 80 °c (scheme 16).86 scheme 16. [bmim]pf6-mediated synthesis of 2,3-disubstituted benzo[b]furans. reactions with aromatic secondary amines, such as n-benzylaniline or n-methylaniline, and aromatic primary amines, such as aniline, instead of aliphatic secondary amines did not afford the corresponding benzo[b]furan derivatives. in this reaction, it was also observed that aryl alkynes are more effective and produced higher yields than aliphatic alkynes. after completion of the reaction, the ionic liquid containing cui and [bmim]oac was recovered and recycled five times without any significant loss in its catalytic activity. 3.2. synthesis of coumarin derivatives coumarins are very common in naturally occurring heterocycles possessing a wide range of pharmaceutical activities that include anti-bacterial, anti-hiv, anti-viral, anti-coagulant, anti-oxidant and anti-cancer activities.87–90 a variety of 3-substituted coumarins (49) were synthesized via the knoevenagel condensation of salicylaldehyde (44) and dialkyl malonate (48 or 48a) using sodium methoxide as catalyst in the ionic liquid [bmim]pf6 at 95 °c (scheme 17).91 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ diverse bioactive heterocycles 1083 several other butylmethylimidazolium-based ionic liquids, such as [bmim]cl, [bmim]bf4, [bmim]br and [bmim]alcl4, were also screened for the same transformation. among them [bmim]pf6 was found to be superior in terms of both reaction time and product yield. scheme 17. [bmim]pf6-mediated synthesis of 3-substituted coumarin derivatives. 3.3. synthesis of pyrano[4,3-b]pyran-5-one derivative 2-isobutyl-3-isopropyl-7-phenyl-2h,5h-pyrano[4,3-b]pyran-5-one (52) was synthesized via the domino reaction between 3-isopropyl-6-methylhept-3-en-2one (50) and 4-hydroxy-6-phenyl-2h-pyran-2-one (51) in the presence of a catalytic amount of β-alanine as catalyst in [bmim]pf6 under microwave irradiation at 110 °c (scheme 18).92 scheme 18. [bmim]pf6-mediated synthesis of a pyrano[4,3-b]pyran-5-one derivative. 3.4. synthesis of epoxyisobenzofuran-1,3-dione a simple and efficient method was developed for the synthesis of epoxyisobenzofuran-1,3-dione 55 via the diels–alder reaction of furan (53) and furan -2,5-dione (54) using zni2 as promoter in [bmim]pf6 at room temperature (scheme 19).93 scheme 19. [bmim]pf6-mediated synthesis of an epoxyisobenzofuran-1,3-dione. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1084 kaur, sharma and banerjee 3.5. synthesis of xanthenes xanthenes, in particular, 1,8-dioxo-octahydroxanthene moieties, have gained significant attention due to their potent pharmacological efficacies, such as anti -microbial, anti-cancer and enzyme inhibitory activity.94–96 a simple, rapid and efficient protocol was reported for the synthesis of 1,8-dioxo-octahydroxanthenes (56) via one-pot pseudo three-component reactions of aromatic aldehydes (4) and dimedone (37b) or 1,3-cyclohexanedione (37b′) in the presence of a catalytic amount of [bmim]pf6 under microwave irradiation and solvent-free conditions (scheme 20).97 using the same optimized reaction conditions, a series of benzoxanthenes (59, scheme 21) and chromene derivatives (60, scheme 22) were also synthesized starting from various aromatic aldehydes (4), 1,3-cyclohexanediones (37b or 37b′) and β-naphthol (57) or 4-hydroxycoumarin (58), respectively. very short reaction times, solvent-free conditions, high atom economy, reusability of the media and good to excellent yields are some of the major advantages of this protocol. a range of aldehydes that include electron donating and withdrawing scheme 20. [bmim]pf6-catalyzed synthesis of 1,8-dioxo-octahydroxanthenes under solvent-free conditions. scheme 21. [bmim]pf6-catalyzed synthesis of benzoxanthenes under solvent-free conditions. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ diverse bioactive heterocycles 1085 substituent are well tolerated under the optimized reaction conditions and excellent yields were attained. all the synthesized compounds were screened for their anti-oxidant properties and some of them were found to possess significant anti-oxidant efficacies. scheme 22. [bmim]pf6-catalyzed synthesis of chromenes under solvent-free conditions. another efficient protocol was developed for the synthesis of 2,3-dihydro-1hxanthen-1-one derivatives (61) via the cycloaddition of 1,3-cyclohexanediones (37b or 37b′) and salicylaldehyde (44) using glycine as promoter in [bmim]pf6 at 25 °c (scheme 23).98 conventional solvents, such as acetonitrile, dmf, dmso, etc., were also screened for this reaction but lower yields were attained. under the optimized conditions, other amino acids, such as l-histidine, l-lysine, l-alanine, as catalyst produced lower yields. the [bmim]pf6 containing glycine was recovered and recycled four times without any loss in its catalytic activity. scheme 23. [bmim]pf6-mediated glycine-catalysed synthesis of 2,3-dihydro-1h-xanthen-1-ones. 3.6. synthesis of 2-amino-3-cyano-benzochromenes 2-amino-3-cyano-pyrans and related derivatives possess immense biological activities.99–101 a simple and facile protocol was developed for the efficient synthesis of 2-amino-3-cyano-benzochromenes (63) via one-pot three-component condensation between aromatic aldehydes (4), various substituted β-naphthols (57) and malononitrile (62) in [bmim]pf6 at 80 °c (scheme 24).102 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1086 kaur, sharma and banerjee scheme 24. [bmim]pf6-mediated synthesis of 2-amino-3-cyano-benzochromenes. other ionic liquids, such as [bmim]br, [bmim]bf4, n-butylpyridinium bromide, cetylpyridinium chloride and n-butylpyridinium dodecyl sulphate, were also screened for this reaction but lower yields were attained. after completion of reaction, [bmim]pf6 was successfully recovered and recycled several times. some of the synthesized compounds possess prominent anti-proliferative activity. a range of aromatic aldehydes that include electron donating and withdrawing substituent are well tolerated under the optimized reaction conditions and produced excellent yields. operational simplicity, high atom economy, excellent yields, catalyst-free reaction conditions are some of the major benefits of this developed protocol. 3.7. synthesis of 1,3-dioxane derivatives a series of 1,3-dioxane derivatives (66) was synthesized in excellent yields via the condensation of olefins (64) and paraformaldehyde (65) in the presence of a catalytic amount of inbr3 in [bmim]pf6 at 25 °c (scheme 25).103 the ionic liquid containing the catalyst was recovered and successfully recycled up to the fourth run with no appreciable loss in product formation. scheme 25. [bmim]pf6-mediated synthesis of 1,3-dioxane derivatives. 3.8. synthesis of chromanone derivatives a simple and facile microwave-assisted intramolecular stetter reaction104 was demonstrated with (e)-methyl 4-(2-formylphenoxy)but-2-enoate (67) to pre________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ diverse bioactive heterocycles 1087 pare the corresponding chromanone derivatives (68). the reaction was realized under the catalytic combination of thiazolium salt and [bmim]pf6 in basic medium at 80 °c (scheme 26).105 the ionic liquid containing catalyst was recovered and recycled several times. scheme 26. [bmim]pf6-catalyzed microwave-assisted synthesis of chromanone derivatives. 3.9. synthesis of lactone and lactam derivatives the ionic liquid [bmim]pf6 was found to be an efficient reaction medium for the palladium-catalyzed cyclocarbonylation of 2-allylphenols (69, scheme 27), 2-vinylphenols (71, scheme 28), and 2-aminostyrenes (73, scheme 29) using scheme 27. [bmim]pf6-mediated synthesis of seven-membered lactones. scheme 28. [bmim]pf6-mediated synthesis of six membered-lactones. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1088 kaur, sharma and banerjee carbon monoxide (14) to afford the corresponding lactones (70 and 72) or lactams (74), respectively, in good yields in the presence of 1,4-bis(diphenylphosphino)butane (dppb) as promoter at 90 °c. the ionic liquid containing the palladium catalyst and ligand was recovered and successfully recycled for several runs.106 scheme 29. [bmim]pf6-mediated synthesis of six membered-lactams. 4. synthesis of n,o-heterocycles 4.1. synthesis of spiro[chromeno[2,3-d]pyrimidine-5,3′-indoline]tetraones a simple and facile protocol was developed for the efficient synthesis of a series of spiro[chromeno[2,3-d]pyrimidine-5,3′-indoline]tetraones (75) via one-pot three-component reactions of substituted isatins (8), dimedone (37b) and barbituric acid derivatives (19) in the presence of a catalytic amount of montmorillonite k-10 in [bmim]pf6 at 100 °c (scheme 30).107 during optimization, [bmim]pf6 was found to be superior to other butylmethylimidazolium-based ionic liquids, such as [bmim]bf4, [bmim]br, [bmim]cf3coo and [bmim]cl. very short reaction times, a wide range of substrate tolerance, high atom economy, reusability of the media, excellent yields are some of the major advantages of this developed protocol. 4.2. synthesis of pyrimidine containing isoxazolines a series of pyrimidine-containing isoxazoline derivatives (78) was synthesized via the reactions of 3,4-dihydropyrimidin-2(1h)-ones (76) or 3,4-dihydro -2(1h)-pyrimidinethiones (76a) with hydroxylamine (77) using potassium hydroxide as a promoter in water–[bmim]pf6 as a biphasic medium at ambient temperature. the reaction medium was successfully recovered and recycled ten times with no appreciable loss in yields (scheme 31).108 short reaction times, a wide range of substrate tolerance, ambient conditions and good to excellent yields are some of the major benefits of this developed protocol. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ diverse bioactive heterocycles 1089 scheme 30. [bmim]pf6-mediated synthesis of spiro[chromeno[2,3-d]pyrimidine-5,3′-indoline] derivatives. scheme 31. [bmim]pf6-mediated synthesis of pyrimidine-containing isoxazolines. 4.3. synthesis of isoxazolidines the ionic liquid [bmim]pf6 was found to be an efficient medium for the 1,3 -dipolar intermolecular cycloaddition of aldehydes (4), phenylhydroxylamine (79) and various electron deficient olefins (80) to afford the corresponding stereospecific isoxazolidines (81) under catalyst-free conditions at room temperature (scheme 32).109 under the same reaction conditions, [bmim]bf4 also afforded the same products with comparable yields. a catalyst-free reaction, a wide range of substrate tolerance, high atom economy, operational simplicity and good to excellent yields are some of the advantages of this protocol. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1090 kaur, sharma and banerjee scheme 32. [bmim]pf6-mediated synthesis of isoxazolidines at room temperature. 4.4. synthesis of benzoxazine and quinazoline the ionic liquid [bmim]pf6 was found to be a safe and recyclable reaction medium for the efficient synthesis of 1,2,3,4-tetrahydro-2-phenylquinazoline (84) and 1,4-dihydro-2-phenyl-2h-3,1-benzoxazines (85) from the reaction of benzaldehyde (4) and 2-aminobenzyl amine (82) or 2-aminobenzyl alcohols (83), respectively, at room temperature (scheme 33).110 scheme 33. [bmim]pf6-mediated synthesis of benzoxazine and quinazoline at room temperature. 5. synthesis of s-heterocycles 5.1. synthesis of 2-aminothiophenes hu et al.111 demonstrated the ionic liquid [bmim]pf6-mediated gewald synthesis112 to afford the corresponding 2-aminothiophenes (87 and 88) starting from various carbonyl compounds (37d and 86), malononitrile (62) and sulphur in the presence of a catalytic amount of ethylenediammonium diacetate (edda) at 50 °c. after completion of the reaction, the ionic liquid containing catalyst was recovered and reused several times without any loss in its activity (scheme 34). ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ diverse bioactive heterocycles 1091 [bmim]pf6, 50 oc o cn cn + s h3c ch3 o s s h3c h3c cn nh2 o nh2 cn 20 mol% edda 3 h, 87% [bmim]pf6, 50 oc 20 mol% edda 6 h, 69% 62 37d 86 8788 scheme 34. [bmim]pf6-mediated synthesis of 2-aminothiophenes. scheme 35. [bmim]pf6-mediated synthesis of spiro[3h-indole-3,2′-thiazolidine]-2,4′(1h) -diones. 6. synthesis of n,s-heterocycles 6.1. synthesis of spiro[3h-indole-3,2′-thiazolidine]-2,4′(1h)-diones a simple, facile and environmentally sustainable protocol was developed for the synthesis of novel spiro[3h-indole-3,2′-thiazolidine]-2,4′(1h)-dione derivatives (90) via the one-pot three-component condensation of isatins (8), 4-amino-4h-1,2,4-triazole (9) and 2-sulfanylpropanoic acid (89) under catalyst -free conditions in [bmim]pf6 at 80 °c (scheme 35).113 catalyst-free reaction conditions, high atom economy, good to excellent yields are some of the major benefits of this method. 6.2. synthesis of 2-phenylthiazoles a series of 2-phenylthiazoles (93) was synthesized via the cycloaddition of α-tosyloxyketones (91) and thiobenzamide (92) in [bmim]pf6 as an efficient and reusable ionic liquid under catalyst-free conditions at room temperature (scheme 36).114 catalyst-free ambient reaction conditions, operational simplicity, good yields are some of the advantages of this method. after completion of the reaction, the ionic liquid was successfully recovered and reused without any loss in its activity. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 1092 kaur, sharma and banerjee [bmim]pf6, rt, 2 h 10 entries, 71-83% o r ots r1 + s nh2 n s r r1 r1 = h; r = c6h5, 4-ch3c6h4, 4-och3c6h4, 4-clc6h4, 4-brc6h4, 3-furyl, 3-thienyl r1 = cooc2h5; r = c6h5, ch3, oc2h5 91 92 93 catalyst-free scheme 36. [bmim]pf6-mediated synthesis of 2-phenylthiazoles at room temperature. 6.3. synthesis of benzothiazole derivatives [bmim]pf6 efficiently catalyzed the reaction of various aldehydes (4) and 2-aminothiophenol (94) to afford the corresponding benzothiazoles (95) in aqueous media under reflux conditions (scheme 37).115 scheme 37. [bmim]pf6-catalyzed synthesis of benzothiazole derivatives. 6.4. synthesis of 1,5-benzothiazepine-4-ones a facile and convenient protocol was developed for the regioselective synthesis of a series of 1,5-benzothiazepin-4-one derivatives (97) starting from substituted 2-aminobenzenethiol (94) and methyl (±)-trans-3-(4-methoxy/(benzyloxy)phenyl) -glycidate (96) in [bmim]pf6 under catalyst-free conditions at 60 °c (scheme 38).116 scheme 38. [bmim]pf6-mediated synthesis of 1,5-benzothiazepine-4-ones. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ diverse bioactive heterocycles 1093 catalyst-free reaction conditions, operational simplicity, reusability of media and good to excellent yields are some of the major advantages of this developed protocol. 7. conclusions the ionic liquid [bmim]pf6 (1-butyl-3-methyl-1h-imidazolium hexafluorophosphate) has been successfully employed as an efficient, commercially available, cheap, low toxicity and recyclable reaction medium for various organic transformations. during optimization, on many occasions, it was found that the efficiency of [bmim]pf6 was better than those of other imidazolium-based ionic liquids, such as [bmim]bf4, [bmim]oh, [bmim]br, [bmim]cl, [bmim]clo4, [bmim]ch3coo, [bmim]ntf2, [bmim]n3, [bmim]alcl4, etc. in many situations, the addition of another catalyst was not required for the [bmim]pf6 -mediated transformations. the weak electrostatic interactions of hexafluorophosphate with the imidazolium cation provide good thermal and electrochemical stability of [bmim]pf6. other favourable physical and chemical properties, such as mild properties, low volatility, lack of inflammability, commercial availability and excellent solubility with many organic compounds make this ionic liquid superior. after completion of the reaction, in the majority of cases, the ionic liquid was successfully recovered and recycled several times without significant loss in its efficacy. the present review summarizes the applications of [bmim]pf6 as an efficient, cheap, commercially available and reusable ionic liquid for the hitherto reported synthesis of structurally diverse bioactive heterocycles. acknowledgements. the authors are grateful to dr. sudhir kartha, chancellor, indus international university, una, himachal pradesh, india, for his active support throughout and the kartha education society, mumbai, india, for the financial help. the authors are also grateful to prof. dr. györgy keglevich, department of organic chemistry and technology, budapest university of technology and economics, budapest, hungary, for his wholehearted guidance throughout. и з в о д [bmim]pf6: ефикасан медијум за синтезу различитих биоактивних хетероцикличних једињења gurpreet kaur, aditi sharma и bubun banerjee department of chemistry, indus international university, village and post office bathu, district una. himachal pradesh-174301, india хетероцикли су од изузетне важности због тога што чине важне структурне елементе многих лекова присутних на тржишту. истовремено, у последњој деценији забележен је изузетан пораст у примени јонске течности [bmim]pf6 (1-бутил-3-метил-1h-имидазолијум хексафлуорофосфат) као ефикасног, јефтиног, комерцијално доступног и мање токсичног реакционог медијума за различите трансформације у органској хемији. овај прегледни чланак даје приказ најновије примене [bmim]pf6 као ефикасног реакционог медијума за синтезу различитих биолошки важних хетероцикличних једињења. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {quantitative structure-activity relationship modelling of influenza м2 ion channels inhibitors} j. serb. chem. soc. 86 (7–8) 625–637 (2021) original scientific paper jscs–5449 625 quantitative structure–activity relationship modelling of influenza м2 ion channels inhibitors ivanka g. stankova1*, radoslav l. chayrov1, michaela schmidtke2, dancho l. danalev3**, liudmila n. ognichenko4, anatoly g. artemenko4, valery a. shapkin5 and victor e. kuz'min4 1department of chemistry, south-west university “neofitrilski”, blagoevgrad, 2700, bulgaria, 2friedrich schiller university, department of virology and antiviral therapy, jena, 207745, germany, 3university of chemical technology and metallurgy, biotechnology department, 1756 sofia, 8 blvd. kliment ohridski, bulgaria, 4a.v. bogatsky physico -chemical institute of ukrainian national academy of sciences, department of molecular structure and chemoinformatics, 86, lustdorfskaya doroga, odessa, 65080, ukraine and 5department of department of theoretical foundations of chemistry, odessa national polytechnic university, 1, shevchenko ave., odessa 65044, ukraine (received 9 may 2020, revised 15 march, accepted 5 may 2021) abstract: a series of adamantane derivatives (rimantadine and amantadine) incorporating amino-acid residues are investigated by simplex representation of molecular structure (sirms) approach in order to found correlation between chemical structures of investigated compounds and obtained data for antiviral activity and cytotoxicity. the obtained data from qsar analysis show that adamantane derivatives containing amino acids with short aliphatic non-polar residues in the lateral chain will have good antiviral activity against the tested virus a/h3n2, strain hong kong/68 with low cytotoxicity. qsar experiments and in vitro data also show good correlation and reveal that modified adamantine derivatives including guanidated in the lateral chain amino acid and β -amino acids as substituents show low to none activity. keywords: qsar study; molecular simplex; adamantine derivatives; amantadine; rimantadine; amino acids. introduction adamantane derivatives have been used successfully for the prevention and treatment of influenza a virus infection for more than 30 years.1,2 the aminoadamantanes (amantadine and rimantadine) block m2 proton channel and thus stop virus replication. however, they are no longer effective because of widespread drug resistance. s31n is the predominant and amantadine-resistant m2 mutant, present in almost all of the circulating influenza a strains as well as in the pan *,** corresponding authors. e-mail: (*)ivastankova@abv.bg; (**)ddanalev@uctm.edu https://doi.org/10.2298/jsc200509036s ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 626 stankova et al. demic 2009 h1n1 and the highly pathogenic h5n1 flu strains.3 structural and biochemical studies of the s31n mutant showed that replacing 31ser, which is located in the helix–helix interface, with the bulkier asn results insubstantially weaker helix–helix packing. since the pocket is composed of residues from two adjacent tm helices, the stability and physical properties of the pocket depend on the dynamics and conformation of helical packing.4 the m2 proton channel from influenza a virus, a prototype for a class of viral ion channels known as viroporins, conducts protons along a chain of water molecules and ionizable sidechains, including 37his. drugs inhibit proton conduction by binding to an aqueous cavity adjacent to m2's proton-selective filter, thereby blocking access of proton to the filter, and altering the energetic landscape of the channel and the energetics of proton-binding to 37his. according to gaiday et al. studied cage compounds inhibit the m2 ion channel by binding to the 37his residue. the adamantane cage fits into a pocket formed by 41trp residue, while the hydrogen bond is formed between hydrogen atom of ammonium nitrogen and the nitrogen of histidine residue.6 one of the possible approaches to restore the antiviral properties of this class of compounds is to incorporate more than one functional group in their molecule. amino acids and peptides are promising alternative for such kind of modification because of their multi functionality. herein, we report the qsar analysis of some adamantanes modified with different substituents and the role of specific modifications on the biological activity. experimental chemical synthesis compounds aimed to this study were previously synthesized according to scheme 1. the synthetic protocol of target compounds was as follow: 3 mmol of 2-(1h-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (tbtu) was dissolved in 15 ml ch2cl2. further, the corresponding boc-αn-protected amino acid (3 mmol) and dipea (3.1 mmol) were added to a solution of tbtu. the obtained mixture was stirred at room temperature for 30 min and 3 mmol of rimantadine or amantadine and 3 mmol n,n-dimethylaminopyridine (dmap) were also added. this mixture was stirred at room temperature for another 3 h, and then evaporated to dryness. after evaporation the obtained oil was purified by flash chromatography in system ethyl acetate:n-hexan (50:50 volume ratio). 1 eq. of boc-αn-aaa-amantadine or boc-αn-aaa-rimantadine was dissolved in 10-fold excess of trifuoroacetic acid (tfa) at 0 °c. the reaction mixture was stirred until fully deprotection of boc-group under chromatographic control in systems chloroform-methanol (95:5 volume ratio). the excess of tfa evaporated and the obtained oil was dissolved in 10 ml methanol. further 25 % ammonium hydroxide was added until the ph reached around 8. the solvent was evaporated under vacuum. tha obtained crystals were dissolved in ethyl acetate and washed with 3×25 ml water. the organic layers were combined, dried on anhydrous na2so4 and solvent removed under vacuum. the yield of all compounds as well as 1hand 13c-nmr analysis confirming their structures are given in chayrov et al.17 for guanidated analogues synthesis the following procedure was used. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. m2 ion channels inhibitors – qsar study 627 scheme 1. synthesis of amino acid derivatives of adamantine; r = –nh2 is amantadine (am), r = ch3chnh2 is rimantadine (rim), aaa = amino acid; for amantadine aaa = ala; phe; phe(4-f); val; for rimantadine aaa = ala; gly; ile; leu; phe; d-phe(4-f); l-phe(4-f); val; beta-ala; tyr. 0.67 mmol of boc-deprotected aaa-rimantadine or aaa-amantadine was dissolved in 2 ml of acetonitrile. further to the obtained solution 1.00 mmol of 1h-pyrazole-1-carboxamidine hydrochloride and 2.0 mmol triethylamine were added. the reaction was run for 48 h at room temperature. at the end of the reaction time the solvent was evaporated under vacuum. the obtained crude oil was dissolved in 25 ml chloroform and washed several times with 5 % nahso4 (ph 3). the organic layers were combined, dried with anhydrous na2so4 and solvent was evaporated under vacuum. the residue was crystalized in methanol/diethyl ether. biological studies the results obtained by the realized biological tests17 are presented in table i. table i. data of biological tests of studied compounds; cc is cytotoxic concentration; hntc is high-nontoxic concentration no. structural formula of compound (abbreviation) cc50 / μm hntc / μm ic50 / μm log (ic50 / μm) mdck cells mdck cells against a/hong kong/68 average class numb. average class numb. average – 1 rim o nh2 f (d-phe(4-f)-rim) 19.91 1 6.23 1 100.00 2.00 2 rim o nh2 oh (l-tyr-rim) > 100 0 9.97 1 0.52 –0.28 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 628 stankova et al. table i. continued no. structural formula of compound (abbreviation) cc50 / μm hntc / μm ic50 / μm log (ic50 / μm) mdck cells mdck cells against a/hong kong/68 average class numb. average class numb. average – 3 rim o nh2 (l-phe-rim) 21.93 1 4.75 1 100.00 2.00 4 rim o nh2 (l-val-rim) 70.33 1 10.20 1 100.00 2.00 5 rim o nh2 (l-leu-rim) > 100 0 11.66 1 0.75 –0.12 6 rim o nh2 (l-ile-rim) 58.35 1 5.33 1 100.00 2.00 7 rim o nh2 (l-gly-rim) > 100 0 13.89 1 0.11 –0.96 8 rim o nh2 (l-ala-rim) > 100 0 14.65 1 1.53 0.18 9 rim o nh2 (β-ala-rim) > 100 0 20.78 1 15.72 1.20 10 rim o nh oh nh2 nh (gua-l-tyr-rim) > 100 0 36.69 1 8.19 0.91 11 rim o nh n nh h h (gua-l-ala-rim) > 100 0 > 100 0 41.90 1.62 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. m2 ion channels inhibitors – qsar study 629 table i. continued no. structural formula of compound (abbreviation) cc50 / μm hntc / μm ic50 / μm log (ic50 / μm) mdck cells mdck cells against a/hong kong/68 average class numb. average class numb. average – 12 rim o nh nh nh2 (gua-l-β-ala-rim) > 100 0 > 100 0 100.00 2.00 13 am o nh2 f (l-phe(4-f)-am) 83.61 1 15.35 1 0.81 –0.09 14 am o nh2 oh (l-tyr-am) > 100 0 > 100 0 3.93 0.59 15 am o nh2 (l-phe-am) 89.40 1 15.10 1 0.75 –0.12 16 am o nh2 (l-val-am) > 100 0 > 100 0 1.32 0.12 17 am o nh2 (l-ala-am) > 100 0 > 100 0 1.41 0.15 18 am o nh oh nh2 nh (gua-l-tyr-am) > 100 0 > 100 0 59.71 1.78 19 am o nh n nh h h (gua-l-val-am) > 100 0 > 100 0 17.13 1.23 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 630 stankova et al. table i. continued no. structural formula of compound (abbreviation) cc50 / μm hntc / μm ic50 / μm log (ic50 / μm) mdck cells mdck cells against a/hong kong/68 average class numb. average class numb. average – 20 am o nh n nh h h (gua-l-ala-am) > 100 0 > 100 0 18.37 1.26 21 am-h > 100 0 n/a 0 0.39 –0.41 22 rim-h – – – – 0.06 –1.25 qsar calculations in the present study the simplex representation of molecular structure (sirms) approach7,8 was used for calculation of structural descriptors for all investigated compounds. the simplex approach is based on isolating and counting the number of molecular fragments (pairs, triples, quadruples of atoms). any molecule in the framework of sirms can be represented as a system of different specific fragments (simplexes) of fixed composition and topology. various atomic characteristics can be used for the vertex differentiation in the simplex, such as the uniqueness of the atom (atom nature or a more detailed type), electronegativity, partial charge, lipophilicity, electronic polarizability (refraction), h-bond donor/acceptor potential, van der waals interactions, etc. for atomic characteristics having real values (electronegativity, lipophilicity etc.) at the preliminary stage the range of values is divided into a certain number of groups g (g is a tuning parameter of models and can vary, as a rule from 3 to 7; see the example showing how using vertexes differentiation by atomic charges in supplementary materials). in addition, electronegativity, refraction, molecular weight and octan-1-ol–water partition coefficient (log p) are calculated as integral characteristics that describe the whole molecule. the calculation of descriptors was carried out at the 2d level of molecular structure representation. the 2d-qsar models are the most popular in structure-property studies.7,8 in this case only molecular topology is taken into account, i.e. all information is extracted from the structural formula. the relationships between the calculated molecular descriptors and the studied properties of investigated molecules were established by methods of partial least squares (pls)9 and random forest (rf).10 the removal of highly correlated and constant descriptors, the trend vector method,11 genetic algorithm (ga)12 and the automatic variable selection (avs) strategy7 have all been used to select the descriptors in pls. the removal of highly correlated descriptors is not necessary for pls analysis since descriptors are reduced to a series of uncorrelated latent variables. this procedure frequently helps to obtain more adequate models. during this procedure one descriptor from each pair having a pair correlation coefficient r satisfying |r|>0.90 is eliminated. it was previously discovered13 that descriptors involved in the best trend vector models (several decades of models with approximately identical quality) form a good subset for their subsequent usage in pls. the noise elimination can be one of the more probable ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. m2 ion channels inhibitors – qsar study 631 explanations of the success of the trend vector procedure. ga is used as a tool for the selection of adequate pls models. we used the small set of following algorithm parameters as mutation rate = 0.3, crossingover rate = 0.7, type of crossingover is double. descriptors, from the best model obtained by a preliminary avs procedure, are normally used as the starting “population”. in this work we run ga only once. the ga is definitely not a tool for the elucidation of the global maximum or minimum, and very often subsequent avs procedures and different enumerative techniques allow one to increase the quality of the pls models obtained. according to the qsar/qspr oecd principles,14 applicability domain (ad) of developed models was estimated. a ellipsoid model of structural space,7 williams plot15 and tree ad approach16 were used for ad estimation. within the framework of sirms approach it is possible to define the relative influence of the different physical and chemical factors on the character of the molecules interaction with the biological target.7,8 for this purpose it is necessary to sum and compare absolute values of pls regression coefficients of descriptors for all groups used for atom differentiation. the clear interpretation is one of the advantages of sirms approach.8 on the basis of developed qsar models the influence of each atom over a particular property can be calculated. the contribution of each atom in the molecule can be defined as the ratio of the sum of pls regression coefficients for all simplexes containing this atom to the number of atoms in the simplex. the atomic contribution depends on the number of simplexes that include this atom. the number of simplexes is not constant. it varies in different molecules depending on other constituents. thus, this contribution is non-additive. the analysis of such information allows selecting different fragments which have negative or positive influence on a considered property. the descriptors calculation and data analysis were performed using hitqsar software,7 which was developed in the department of molecular structure and chemoinformatics of the a. v. bogatsky physico-chemical institute of national academy of sciences of ukraine. results and discussion during the first step of synthetic scheme boc-αn-protected amino acid are bonded to rimantadine or amantadine. next step is reaction of boc-group deprotection with treatment with 10 fold excess of tfa. finally target compounds are obtained as a free bases on their amino functions by treatment with 25 % ammonium hydroxide till ph around 8. thus using structures and results from biological investigations of target rimantadine and amantadine analogues qsar analysis was carried out and the effects of the substituent on the in vitro cytotoxicity (cc50), hntc and antiviral activity (against human influenza virus a/h3n2 strain/hong kong/68) were also investigated.17 in this study we used a dataset consisting of 22 derivatives (13 are rimantadine derivatives and 9 – amantadine derivatives, table i). in addition, the derivatives tyr-rimantadine, ala-rimantadine, β-ala-rimantadine, and ala-amantadine, val-amantadine, which exhibit the lowest cytotoxicity were guanidated using 1h-pyrazole-1-carboxamidine reagent (scheme 1).18 all compounds were tested against human influenza virus a/h3n2 strain hong kong/68 and their activity is described by chayrov et al.17 the antiviral ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 632 stankova et al. activity against influenza virus strain a/h3n2 strain hong kong/68 in vitro of new analogs of amantadine and rimantadine conjugated with amino acids reveal that the highest antiviral activity combined with low cytotoxicity was demonstrated by the rimantadine derivative conjugated with the simplest in structure glycine. moreover, glycyl-rimantadine presented a high stability profile after incubation in human plasma for 24 h. interestingly, the analogues of amantadine with the amino acids l-phenylalanine and l-(4-f)-phenylalanine exhibited high activity although lower than those of the amantadine in the same concentration. in addition, amantadine and rimantadine analogues conjugated with guanidine showed low toxicity but they also exhibited low activity. the various aliphatic and aromatic amino acids as substitutes in the rimantadine molecule have no significant effect on antiviral activity. the use of β-alanine reduces antiviral activity. guanidation of the rimantadine and amantadine analogues do not increase antiviral activity unlike guanidated oseltamivir analogues. using sirms approach for calculation of structural descriptors a total of about 2000 different structural characteristics were calculated for the investigated compounds. the ranges of dividing the atom properties into intervals are, as already mentioned above, tuning characteristics and the following schemes were used in the calculation of simplex descriptors: electronegativity: a < 2.19 ≤ b < 2.5 ≤ c < 3 ≤ ≤ d, refraction: a < 1.5 ≤ b < 3 ≤ c < 8 ≤ d, atomic charge: a < –0.16 ≤ b < < –0.10 ≤ c < –0.04 ≤ d < 0.01 ≤ e < 0.07 ≤ f < 0.13 ≤ g, lipophilicity: a < –1.51 ≤ b < –0.96 ≤ c < –0.42 ≤ d < 0.13 ≤ e < 0.68 ≤ f < 1.23 ≤ g, vdw attraction: a < 50 ≤ b < 100 ≤ c < 250 ≤ d < 400 ≤ e < 650 ≤ f < 2000 ≤ g, vdw repulsion: a < 20.000 ≤ b < 32.000 ≤ c < 50.000 ≤ d < 100.000. all atoms corresponding to the simplex vertices were also divided into three groups: a – acceptors of potential h-bond, d – donors of potential h-bond and i – indifferent ones. on the first stage of this study, 2d qsar model reflecting the structural influence of investigated compounds on their antiviral activity against human influenza (virus a/h3n2 strain hong kong/68) was developed. this model (model a1) was used for interpretation, i.e., for estimation of influence of different structural factors on investigated activity (on value of log ic50), but this model was not intended for prediction of activity. model a1 was built using the pls method with two latent variables (based on 9 descriptors in the final) and with the following statistical characteristics: determination coefficient for the training set r2 = = 0.88, coefficient of determination for cross-validation (leave-one-out) q2 = 0.77, standard error s(ws) = 0.35, s(cv) = 0.52 for training set and cross-validation, respectively. a “randomization” procedure (y-scrambling) was used to confirm the “non-randomness” of the developed qsar model.7 the statistical characteristics obtained using the y-scrambling procedure were lower in indices than in ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. m2 ion channels inhibitors – qsar study 633 the final model: r2 (y-scr) = 0.26 ± 0.03, q2 (y-scr) = 0.10 ± 0.03. thus, the non -randomness of the established relationship between the structure of the investigated compounds and their antiviral activity can be stated (see supplementary material to this paper). the analysis of obtained model a1 testifies that, electrostatic (atomic charges) factors and lipophilicity have the largest influence on changing of antiviral activity (41 and 37 %, respectively). the relative influence of atom nature is 22 %. as already mentioned above, on the basis of developed qsar models the contribution of each atom in the molecule can be calculated. the analysis of such information allows selecting different fragments which have negative or positive influence on a considered property. the sequence of mean relative influence of various substituents (r) for amantadine and rimantadine derivatives on antiviral activity (log ic50) is shown on fig. 1. n rh n r h o nh nh nh2 o nh n nh h h o nh2 o nh oh nh2 nh (r1) < (r2) < (r3) < (r4) < o nh2 o nh2 o nh2 f o nh2 < (r5)< (r6)< (r7)< (r8) < o nh2 oh o nh2 o nh2 o nh2 < (r9) < (r10) < (r11) < (r12) fig. 1. averaged relative influence of substituents (r) for amantadine and rimantadine derivatives on antiinfluenza activity (log ic50) by the model a1. in addition, the influence of identical substituents for amantadine and rimantadine derivatives was estimated separately: a) for amantadine: r2 < r4 < r5 < r6 <r7 < r9 < r10; ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 634 stankova et al. b) for rimantadine:r2 < r5 < r6 <r7 < r4 < r10 < r9. in general, the sequence of changes in the relative influence of the substituents is the same for derivatives of amantadine and rimantadine, a noticable difference can only be noted for hydroxyl-containing substituents r4 and r9. averaged relative influence of rimand am-centers on antiinfluenza activity by the model a1 equals 0.75 and –0.19, respectively. rf model was developed for estimation of the predictive ability of the qsar model. rf models were constructed according to the described original rf algorithm.10 rf is an ensemble of single decision trees.16 each tree has been grown as: a) a bootstrap sample, which will be a training set for the current tree, is produced from the whole training set of n compounds. compounds which are not in the current tree training set are placed in an out-of-bag (oob) set (oob set size is ∼n/3); b) the best split by classification and regression tree (cart) algorithm10 among the m randomly selected descriptors from whole set of m ones in each node is chosen. the value of m is just one tuning parameter for which rf models are sensitive; c) each tree is grown to the largest possible extent (there is no pruning). rf model (model a2) was built using the descriptor’s set from model a1. obtained model (trees count = 50, randomly selected descriptors count = 3) has following statistical characteristics: determination coefficient for the training set r2(ws) = 0.95, coefficient of determination for out-of-bag (oob) set r2(oob) = = 0.65, rmse(ws) = 0.26, rmse(oob) = 0.62. the characteristics of the oob just reflect the predictive ability of the qsar model, which can be considered satisfactory. the greater accuracy of prediction is observed for compounds with medium activity (see supplementary material). further in this study, qsar models reflecting the structural influence of the investigated compounds on their cytotoxicity (сс50) and hntc were developed. the cytotoxicity data (сс50) show (table i) that among the 21 compounds, only 6 exhibit cytotoxicity (сс50 values are less than 100). for hntc data were found that among the 21 compounds, only 12 exhibit activity (property values are less than 100). thus, the original activity values were coded as follows: 1-active (property < < 100) and 0-inactive (property > 100). in these cases rf method was used for decision of classification tasks. for the first case since the dataset is unbalanced, i.e., the count of active and inactive molecules is significantly different, a special procedure for balance was used. the count of inactive molecules was constant (15 molecules) and the count of active ones was duplicated. in the first series, 6 active molecules (all of active and inactive – 21 molecules) were used for developed model b1 (trees count = 100, randomly selected descriptors count = 5); in the second series the count of active molecules was increased twofold, i.e., 12 active molecules (a total of 27 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. m2 ion channels inhibitors – qsar study 635 molecules) were used for developed model b2 (trees count = 150, randomly selected descriptors count = 13). the resulting qsar models for the training set showed an unmistakable classification. the predictive ability of the qsar models was evaluated using the “out-of-bag” (oob) procedure. the quality of the classification models was assessed according to the following statistical characteristics: matthew’s correlation coefficient (mcc), specify (spc), accuracy (acc) and sensitivity (sen): ( ) ( )( )( )( ) tp tn fp fn mcc tp fp tp fn tn fp tn fn ⋅ + ⋅ = + + + + ( ) tn spc tn fp = + ( ) ( ) tp tn acc tp tn fp fn + = + + + ; ( ) tp sen tp fn = + where tp – true positive, tn – true negative, fp – false positive, fn – false negative. the obtained data are presented in table ii. table ii. statistical parameters for classification models for cc50 (models b1 and b2) and hntc (model b3) for “out-of-bag” set model set mcc acc spc sen b1 6 + 15 = 21 0.77 0.90 0.88 1 b2 12 + 15 = 27 0.86 0.93 0.93 0.92 b3 12 + 9 = 21 0.81 0.90 0.89 0.91 as it can be seen from table ii, the balancing of models leads to a significant quality improvement. model b2 could be considered as the most appropriate. for hntc data the duplication procedure was not used, since the dataset is more balanced (active molecules 12 and inactive 9). model b3 (trees count = = 150, randomly selected descriptors count = 20) was also built for hntc dataset, statistical parameters are shown in table ii. as can be seen from this table, the statistical characteristics of models b1– –b3 are quite acceptable. it should be noted that all molecules are in domain applicability (da) of models a1, a2 and b1–b3. conclusion the realized qsar studies have found good compliance between theoretical predictions and in vitro revealed activity and cytotoxicity. compounds 2, 5 and 7 have both good antiviral activity against tested virus a/h3n2, strain hong kong/68 combined with low cytotoxicity which is in a perfect correlation with qsar pre________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 636 stankova et al. diction studies. all guanidated derivatives have lower activity according to both qsar investigation and in vitro assay. based on the obtained results we can conclude that more bulky side chain residues are not tolerated by the mechanism of interaction with m2 ion channels of influenza virus. unfortunately, the same conclusion is revealed for structures creating less conformational freedom including β-amino acids as moiety in the lateral chain of amantadine and rimantadine. supplementary material additional data are available electronically at the pages of journal website: https:// //www.shd-pub.org.rs/index.php/jscs/index, or from the corresponding author on request. и з в о д моделовање квантитативног односа структура–активност инхибитора m2 јонског канала вируса грипа ivanka g. stankova1, radoslav l. chayrov1, michaela schmidtke2, dancho l. danalev3, liudmila n. ognichenko4, anatoly g. artemenko4, valery a. shapkin5 и victor e. kuz'min4 1 department of chemistry, south-west university “neofitrilski”, blagoevgrad, 2700, bulgaria, 2 friedrich schiller university, department of virology and antiviral therapy, jena, 207745, germany, 3 university of chemical technology and metallurgy, bio-technology department, 1756 sofia, 8 blvd. kliment ohridski, bulgaria, 4 a.v. bogatsky physico-chemical institute of ukrainian national academy of sciences, department of molecular structure and chemoinformatics, 86, lustdorfskaya doroga, odessa, 65080, ukraine и 4 department of department of theoretical foundations of chemistry, odessa national polytechnic university, 1, shevchenko ave., odessa 65044, ukraine серија нових деривата адамантана који садрже фрагменте аминокиселина испитана је користећи метод симплекс представљања молекулских структура (енг. simplex representation of molecular structure, sirms) са циљем да се утврди однос између хемијске структуре испитиваних једињења и резултата антивиралне и цитотоксичне активности. добијени резултати qsar анализе показују да ће деривати адамантана који садрже аминокиселине кратких неполарних алифатичних група у бочном низу имати добре антивиралне активности према a/h3n2 соју hong kong/68 вируса као и малу цитотоксичност. qsar експерименти и резултати in vitro активности показују добру корелацију и указују да деривати који садрже гуанидо фрагмент у бочном низу и β-аминокиселину као супституент, имају малу или не показују активност. 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rev. med. chem. 12 (2012) 1273 (https://doi.org/10.2174/138955712802761997). ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice the impact of instructional strategy based on the triplet model of content representation on elimination of students’ misconceptions regarding inorganic reactions j. serb. chem. soc. 81 (6) 717–728 (2016) udc 378.4+377.1+54:011.8:371.212 jscs–4880 original scientific paper 717 the impact of instructional strategy based on the triplet model of content representation on elimination of students’ misconceptions regarding inorganic reactions dušica d. milenković1*#, mirjana d. segedinac#, tamara n. hrin# and saša horvat faculty of sciences, university of novi sad, trg d. obradovića 3, 21000 novi sad, serbia (received 12 august, revised 9 december 2015, accepted 17 february 2016) abstract: recently we reported on the efficient teaching instruction based on integration of macroscopic, submicroscopic and symbolic levels of knowledge representation, from the standpoints of performance and mental effort. based on obtained results a goal of this study was set – to identify misconceptions in the group of students trained in traditional manner and to determine whether the same misconceptions occur among students in the treatment group in the field of inorganic reactions. a two-tier test was used in the research, administered by 189 second grade high school students. analysis was performed by calculating the frequencies of choosing distractors in the tasks. results revealed a total of nine misconceptions in the group of students trained in traditional manner, while only one misconception remained in the treatment group, after implementing the intervention program. it can be concluded that the applied intervention strategy proved to be very efficient in elimination of majority of misconceptions in the examined group of students. keywords: macroscopic; submicroscopic; symbolic; two-tier test; intervention strategy. introduction the existence of students’ home-made and school-made misconceptions has been widely described in the literature. namely, in the last 30 years researchers have extensively worked on identification of common misconceptions among students at various levels of education.1–8 as a result, the extensive material on students' misconceptions for almost every area of chemistry has been collected. beside their identification, it has been worked on the collection of information about their features such as persistence. for instance, similar misunderstandings about thermal phenomena occur among adolescents, adults and natural scient * corresponding author. e-mail: dusica.milenkovic@dh.uns.ac.rs # serbian chemical society member. doi: 10.2298/jsc150812021m _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 718 milenković et al. ists,9 while well-known misconception that boiling water bubbles consist of air is present among students of different ages. thus, papageorgiou and johnson10 reported previously mentioned misconception for the age 10–11, johnson11,12 for the age 11–14, hatzinikita and koulaidis13 for the age 10–18, goodwin14 for postgraduate science students, chang15 for trainee teachers. allen16 states that misconceptions are likely to form a conceptual network, which contributes significantly to their persistence. accordingly, removal of miconceptions is very slow and lengthy process and development of effective teaching models that could lead to the elimination of misconceptions, thus presents a significant challenge for educational researchers. it is worth noting that attempts to replace students’ misconception with scientifically accepted ideas met with partial success.16 on the other hand, development of such models is essential, since only by correcting misconceptions, existing knowledge structures can be further upgraded in scientifically acceptable form. according to posner et al.,17 in order to transform misconceptions into scientifically acceptable concepts, there must be dissatisfaction with the existing concepts, or the inability to use them to explain new problems or situations. furthermore, to be adopted, new concepts have to be more sensible, plausible and fruitful than the existing concepts. the teacher and teaching methods, including chemical models and representations play an important role in finding explanations for abstract concepts. nowadays, it is well-known and widely accepted that teaching which is based on the inter-correlation of levels of content representation represents a very important component of a meaningful understanding of chemical concepts.18 the first was johnstone19 who suggested a model of three levels of chemistry representation of matter, later referred to as: chemistry triplet, triplet relationship or johnstone’s triangle. these three levels are commonly named: macroscopic (perceptually available observations), submicroscopic (particles inaccessible to direct sensory observations) and symbolic level (representation of macroscopic or submicroscopic level). an ability to move between different levels presents an essential skill necessary for understanding natural phenomena.20 the chemistry triplet represents one of the most extensively investigated areas in chemistry education, however, it should be mentioned that most research studies in this field were concerned mainly with identifying problems and misunderstandings, primarily in the submicroscopic level of content representation,21–23 while a much smaller number of studies was devoted to finding effective strategies for their elimination. in relation to that, georgiadou and tsaparlis24 proposed the so-called three-cycle method, which is based on a separate study of three levels of content representation. one-half of the total available teaching time is devoted to the macro-cycle, and then the second and the third cycle (symbolic and submicroscopic levels) are gradually introduced in the teaching process. in this research, it _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ elimination of students’ misconceptions 719 was showed that the greatest positive effect in the teaching process had a group of students trained by using this particular model. bunce and gabel25 examined how the performance of students would be affected by the instruction based on three levels of representation of matter compared to instruction whereas only two representations were used (macroscopic and symbolic). it was obtained that students’ achievements on the selected topics were greatly affected by the use of particulate nature of matter, that is, submicroscopic level. one of the teaching models that is based on the idea of johnstone’s triangle suggests that students first in the most basic form should study chemical concepts parallel in all three levels, and then following a rule of the spiral curriculum, teaching contents should be built up in all three levels, from grade to grade. according to this model, more students learn in the three levels of representation, more they will be able to connect information into a meaningful whole.26 jaber and boujaoude27 and milenković, segedinac and hrin28 suggest that carefully planned instruction based on macro–submicro–symbolic teaching significantly increases students’ performance. therefore, from all the above described studies it can be clearly noted that processing the contents in three levels of representation with special emphasis on the submicroscopic level is necessary for a meaningful understanding of chemical concepts and formation of a flexible system of chemical knowledge. methodology aim of research the main goal of this research was to identify misconceptions in the field of inorganic reactions among students trained in traditional manner and to examine whether the same misconceptions occur among students trained with the use of multiple levels of knowledge representation, i.e., to determine whether the described intervention strategy provides the elimination of misconceptions among treatment group of students. sample the primary study sample included 195 high school students (10th school year), from 8 intact classes from two public urban general type grammar schools in novi sad, serbia. the age range of the participants was 16–17 years. upon applying the initial test for group equalization by students’ performances achieved on the test, classes were divided into four experimental and four control classes thus forming two statistically equivalent groups i.e. e (experimental) and c (control) group (t1 = 4.70 > t(α = 0.1) = 1.29; t2 = 2.40 > t(α= 0.1) = = 1.29). a total of 189 students (46.6 % male; 53.4 % female) were subjected to final testing while 6 students (1 from e and 5 from c group) were not present on the day of the final testing. students from 8 classes were instructed by two licensed chemistry teachers. each of the teachers taught in two experimental and two control classes. these two teachers participated in the study on a voluntary basis and were selected by authors of this research for possessing the following required competences: i) similar teaching experience, ii) achievement of optimal educational results and iii) familiarity with the concept of multiple levels of knowledge representation. before conducting the research, the authors have had several meetings with selected teachers to make them additionally informed about the levels of knowledge _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 720 milenković et al. representation and its application in the teaching process, but also to ensure that selected teachers are competent and motivated to participate in demanding and complex research. the selected teachers are very committed to improvement of teaching process. they are active participants of numerous seminars for teachers of chemistry, while as mentors-practitioners participate in the practical training of students, future teachers of chemistry. in order to provide additional validity of the study, all classes, both experimental and control, were performed under supervision of one of the authors of the paper. data collection in the republic of serbia grammar school education takes four years following a compulsory eight-year primary education. in general type grammar school chemistry is obligatory subject for all students. they are studying chemistry for four years (school year 9–12), with 2 classes (90 min) per week. during the first year, students are studying general chemistry, during the second year inorganic chemistry, during the third year organic chemistry, and during the fourth, final year, biologically important organic compounds. practical training (laboratory practice) is not separately presented within curriculum, but it is an integral part in each teaching unit in a form of demonstration experiments performed by teacher. this study was largely based on demonstration experiments which are predicted by the national curriculum. the research was conducted during the 2012−2013 school year. during the first semester both e and c groups were trained in the traditional manner with identical obligatory demonstration experiments. in this phase of the study, authors had no impact on the work of selected chemistry teachers. at the end of the first semester, the previously mentioned initial testing was conducted, with the goal of group equalization, and formation of experimental and control classes. in the next phase of the research, students in c group were trained in unchanged manner, while e groups students had been demonstrated identical experiments as their peers in c group, however, their analysis and discussion flowed taking into account multiple levels of knowledge representation. students in group c were trained in traditional manner, which is characterized by weak inter-correlations among levels of representations. in group e, upon demonstration, teacher encourages students to present their observations (formation of gas, colored compounds, sedimentation, sound effect etc.), and then encourages them to explain the observed changes at the level of particles. at that point, teacher has a task to guide students toward correct response helping them to find the path to the correct answer. to achieve that, teacher uses molecular models, three-dimensional representations, charts and drawings and asks questions to held discussion at the level of particles. the additional role of teacher is to activate as many students as well as to involve them in active discussion. when students manage to realize the connection between visible and particulate, teacher directs them towards symbolic level, requesting them to represent the observed changes in form of chemical equations. in doing so, teacher helps students to understand the meaning of the symbols and to realize the symbolic relation with the macroscopic and submicroscopic level. at the end of the second semester the final testing was conducted, and the results of that test are presented in this paper. diagnostic instrument multiple-choice questions (alone or in combination with other methods) are very popular technique for identifying students’ misconceptions.29-31 however, the effect of guessing the correct answer in these tasks is non-negligible, for instance, in the tasks with four options, the possibility of guessing the correct answer is 25 %. therefore, in recent decades, researches have increasingly begun to apply the so-called two-tier diagnostic tests with tasks that com_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ elimination of students’ misconceptions 721 prise two parts.32-37 first part of the task contains a question related to a problem statement (macroscopic or symbolic level), while other part provides possible explanations (submicroscopic level). in this research, final test contained 15 two-tiered tasks, each part of which comprised four offered answers, only one of which is correct. thereby, the possibility of guessing the correct answer was reduced to only 6.25 %. the items on the final test were related to experiments suggested by national curriculum that belong to the following topics: i) group 14 elements, ii) group 15 elements, iii) group 16 elements, iv) group 17 elements and v) transition metals. preparation of the final test items required a prior detailed analysis and evaluation of students' responses obtained by initial testing upon which the authors classified the distractors with high choosing frequency to define areas of conceptual difficulties. identified problems were related to poor understanding of the properties and behavior of submicroscopic particles resulting in the projection of visible macroscopic properties to submicroscopic particles, then poor understanding of the process of oxidation, but also with literal and incorrect interpretation of chemical symbols and formulas. on that basis, tasks on the final test were prepared to contain mentioned misunderstood concepts. items were evaluated for both correct and incorrect response combinations selected. on the basis of incorrectly chosen answers, possible misconceptions have been considered. criteria for evaluation of conceptual understanding within the analysis of misconceptions, frequency of selecting the individual answers have been considered. according to the literature, a response represented as a distractor can be considered a misconception if it is chosen by more than 20 % of the respondents.33,38,39 on the other hand, correct answers given by approximately 75 % of the students (for items with four options) can serve as an indicator of the satisfactory conceptual understanding (scu). frequency of choosing the correct answer in a range 50–74 % represent a roughly adequate performance (rap). furthermore, 25–49 % frequency indicates inadequate performance (ip), while obtained frequency less than 25 % represents quite inadequate performance (qip).40 test psychometrics according to the data available in literature, test assurance parameters were evaluated on the combined sample (n = 189) and included calculations of: cronbach α, item-total correlations, item difficulty and item discrimination indices. since two-tier items were considered as paired items, mentioned parameters were calculated for a total of 15 items, and data were converted to binary data (1 point for both tiers correct and 0 points for incorrect or partially correct tiers). the collected data were processed in microsoft office excel and ibm spss statistics 20. the reliability of the applied test was examined by two methods: i) internal consistency using cronbach α parameter and ii) item-total correlation, using the pearson's correlation coefficient. the obtained cronbach α value (α = 0.89) exceeded benchmark of 0.70,41 indicating very good reliability. results of item-total correlation (r is in a range 0.31–0.70) indicate that all items contribute to the reliability of the test. namely, based on values obtained for pearson correlation coefficient, two tasks (14 and 15) could be described as appropriate (0.30 < r < 0.39), while the remaining thirteen as quite appropriate (r > 0.40) for further analysis. in addition, the mean difficulty index for 15 items is 0.39, while item difficulty indices range from 0.18–0.56. difficulty index values for three items are less than 0.30 which characterize them as difficult, while remaining 12 items have difficulty index ranging from 0.30– _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 722 milenković et al. –0.56 and therefore can be considered as moderately difficult. discrimination index was calculated using 27 % cut-off. for the index of test discrimination, a value of 0.70 has been obtained, indicating very good discrimination. for individual tasks, it varies in the range of 0.33 to 0.96. according to towns,42 discrimination index greater than 0.40 is an indicator of excellent item with high discrimination. in this test, 13 tasks meet this criterion. discrimination indices in the range 0.20–0.40 indicate that the item is good and there are two such tasks on the test. item analysis showed that this test does not contain items with 0.00 < id < 0.20 (the tasks that are unacceptable and need to be discarded or revised), nor the tasks with the negative value of index discrimination (the tasks that are flawed or not keyed correctly). item validity was established through expert validation. the expert group consisted of seven experts: 2 university professors and 3 researchers in the field of chemical education, 1 university professor of pedagogy and 1 chemistry teacher in secondary education. results and discussion the percentage of correct answers on the final test (for e and c group) only for the first tier and combined for both tiers are shown in table i. as can be observed from the table, achievements of e group students were significantly higher in comparison to the achievements of c group students regardless of whether only the content results were considered (t = 10.15; p = 0.00) or results for both tiers (t = 7.41; p = 0.00). on the other hand, it is important to note that students’ achievements in the content part are higher than the achievements for both tiers in both examined groups. table i. the percentage of choosing the correct answers for both tiers task e c first tier both tiers first tier both tiers 1. 74.47 72.34 41.05 31.58 2. 81.91 61.70 50.53 30.53 3. 86.17 77.66 49.47 34.74 4. 63.83 60.64 43.16 16.84 5. 72.34 58.51 45.26 23.16 6. 68.09 45.74 40.00 14.74 7. 85.11 68.09 31.58 15.79 8. 75.53 71.28 37.89 31.58 9. 72.34 56.38 41.05 25.26 10. 88.30 74.47 48.42 22.11 11. 68.09 51.06 36.84 11.58 12. 80.85 79.79 34.74 18.95 13. 45.74 35.11 25.26 9.47 14. 87.23 26.60 31.58 9.47 15. 92.55 27.66 33.68 10.53 mean 76.17 57.80 39.37 20.42 _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ elimination of students’ misconceptions 723 identification of misconceptions table ii summarizes all identified misconceptions, with specified distractor choosing frequencies, which indicate the existence of misconception. table ii. comparative review of misconception by groups misconception task distractor indicating a misconception frequency of choosing distractors, % frequency of choosing correct answer, % evaluation of understanding e c e c e c attributing macroscopic properties to submicroscopic particles 1 chloride ions are green 8.5 24.2 80.9 48.4 scu ip 4 collisions of molecules in the solid phase are explosive 10.6 21.1 63.8 38.9 rap ip 6 micropores of activated carbon contain gas molecules of which have an unpleasant odor 12.8 26.3 66.0 34.7 rap ip 12 potassium ions are blue 10.6 21.1 81.9 38.9 scu ip oxidation-reduction reactions 3 in the burning process, red phosphorus molecules decompose to atoms 5.3 26.3 80.9 47.4 scu ip 13 permanganate ion readily loses an electron in an alkaline environment 26.6 30.5 47.9 25.3 ip ip interpretation of symbolic representations 9 molecules of hydrogen are formed in reaction 12.8 24.2 60.6 37.9 rap ip 5 13.8 24.2 70.2 34.7 rap ip particles 8 molecule of potassium permanganate is stable at increased temperatures 2.1 23.2 77.7 45.3 scu ip the first type of misconceptions involved attributing macroscopic properties to submicroscopic particles. this type of misconception arises in the tasks: 1, 4, 6 and 12. the first task was related to the chemical reaction of calcium carbonate and dilute hydrochloric acid. in the first tier of the task, students were expected to recognize a visible change that occurs during the reaction, while in the second tier they were expected to choose a logical explanation for the visible change selected in the first tier. based on the obtained results it can be noted that large percentage of c group students believe that chloride ions are green. on the basis of this response it can be assumed that students have transferred macroscopic property of chlorine _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 724 milenković et al. gas to its submicroscopic particles (chloride ions). this is in a line with previous research,26,31,43,44 which reported that students tend to attribute visible macroscopic properties of a substance to its submicroscopic particles in a number of examples. in contrast to the group c, the results show that the e group students do not possess the aforementioned misconception, but scu of the examined content. the task no. 4 was related to the obtaining ammonia by reaction of ammonium chloride and calcium hydroxide. in the c group there was a large percentage of students who believed that the collisions of molecules in the solid phase are explosive. it has been reported that students believe that molecules of solids are harder than molecules of liquids and gases, and larger in size (cited by horton45) therefore, c group students in this study could believe that collisions of such particles are accompanied by the loud sound. e group students elected this distractor in significantly lower percentage. the task no. 6 was related to the experiment “testing the properties of activated carbon”. in this task e group students achieved significantly higher achievement than their peers in the group c as well, showing rap. on the contrary, a large percentage of c group students believed that the activated carbon in its micro-pores contains gas, molecules of which have an unpleasant odor. four questions on the test were related to the experiment “chemical chameleon” (the reaction of potassium permanganate and sucrose in an alkaline medium). in the first tier of the tasks students were expected to possess descriptive knowledge about colors observed at different stages of the experiment, while in the second tier they were expected to explain the observations by the existence of certain particles in solution. in the task number 12, students were expected to relate purple color of potassium permanganate to the presence of permanganate ions. however, a large percentage of c group students did not have a satisfactory level of descriptive knowledge about the color of an aqueous solution of potassium permanganate, while in the second tier 21.1 % of the students stated that potassium ions are blue, which once again confirmed misconception observed in the task 1. the following type of misconception referred to oxidation-reduction reactions. the third task was related to reaction of combustion of red phosphorus. a large number of students from the e group identified the correct answer that air oxygen oxidizes phosphorus, to form molecules of phosphorus(v) oxide, while a large number of c group students considered that in this reaction phosphorus molecules decompose into atoms. the explanation for this could be found in the fact that in the above reaction only one reactant (red phosphorus) is visible, while oxygen is not directly perceptually available. this is in line with the conclusions reached by schmidt,31 who examined misconceptions in the field of combustion reactions. namely, the author has found that students believe that oxygen sup_________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ elimination of students’ misconceptions 725 ports combustion process, but it does not participate. besides, it is found that students consider combustion rather as process of releasing heat, which destroys the substance, than type of reaction (cited by horton45). in the thirteenth task, which refers to the experiment “chemical chameleon”, a large percentage of both group students believed that oxidation of potassium permanganate takes place in the reaction. based on obtained results it can be concluded that students of both groups did not sufficiently mastered the concept of oxidation-reduction reactions. a large number of research studies, which dealt with misconceptions in the field of oxidation–reduction reactions were primarily focused on the difficulties that students have in identifying the oxidation-reduction reaction.46,47 according to rosental and sanger48 the cause of such misunderstandings could be found in the existence of many definitions of oxidation-reduction reactions in chemistry textbooks and their application by teachers. the above-mentioned authors, state several definitions of oxidation and reduction, which can be considered the cause of misconceptions among students: i) method of electrons (oxidation – losing electrons, reduction – receiving electrons), ii) method of oxidation number (oxidation–oxidation number increases, reduction–oxidation number decreases), iii) method of oxygen (oxidation – number of oxygen atoms increases, reduction – number of oxygen atoms decreases) and iv) method of hydrogen (oxidation – number of hydrogen atoms decreases, reduction-number of hydrogen atoms increases), etc. since the same problem is present within education system in the republic of serbia, it could be considered as one of the possible causes of poor performance in this task. the results showed that e group students have achieved ip in this area as well. difficulties related to the interpretation of symbolic representations were found in the tasks number 5 and 9. these tasks are related to the knowledge of chemical equations and require developed capacity of their interpretation. in the fifth task, it was necessary to identify the equation which describes the reaction of ammonium chloride and calcium hydroxide, and in the ninth task equation which describes thermal decomposition of potassium permanganate. in both tasks in the level of explanation a large percentage of c group students stated that molecules of hydrogen are product of the reaction, although in the task number 5 only a small percentage of students selected the equation in which h2 was shown as product of reaction, while in the ninth task, h2 was not even listed as a reaction product within the offered equations. considering task number 8, one misconception was observed. this task shows that students have ambiguities regarding structure of potassium-permanganate. namely, similar to other studies36,49 which found that students thought that sodium-chloride exists as molecules, this study showed that students believed that fundamental particles of potassium-permanganate are also molecules. according to othman et al.36 this is one of the prevalent conception that _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ 726 milenković et al. bonded substances are commonly in the form of molecules. this misconception was observed only in the group c, while e group students did not exhibit this misconception, on the contrary, they showed scu. at the end, we should briefly comment on tasks number 14 and 15 which, as mentioned earlier, refer to the experiment “chemical chameleon”. namely, almost all offered distractors in these tasks, for both groups were selected with a percentage higher than the value which indicate the existence of a misconception. thus, it can be concluded that examined students poorly mastered these concepts, because of which they were choosing answers randomly. since guessing was most likely the method of selecting answers in these two tasks, they will not be commented in this paper. however, based on overall results presented, it can be concluded that e group students have acquired better understanding of the tested contents. conclusions and limitations results of the study have indicated that the instruction based on the intercorrelation of levels of content representation were efficient in the elimination of a large number of misunderstandings commonly encountered among students trained in the traditional manner, in the field of inorganic reactions. these results suggested that students who were trained through the application of triplet model of knowledge representation were able to effectively cope with some abstract concepts that could be labeled as common misconceptions. however, it is important to note that certain misconceptions still retained among treatment group of students, and these are misconceptions related to the oxidation–reduction reactions. therefore, further work with special emphasis on redox reactions is necessary. regarding the limitations of the study, it is important to mention the type of questions applied in the test. namely, in this study exclusively multiple-choice tasks in the form of a two-tier tasks have been used. although these tasks have a number of advantages over traditional multiple choice tasks, such as reduced possibility of guessing the correct answer and the introduction of an additional tier in the task with meaningful distractors, which assesses understanding of the content, one must take into account the fact that in such tasks answers are offered in advance, which can enable students to recall the correct response, instead of using complex cognitive processes, critical thinking skills and linguistic expression. finally, considering teaching practice, it is important that teachers become aware of the misconceptions that students possess and to devote more effort to apply appropriate methods and instructions suggested by various researchers to prevent misconceptions, which is of a great importance since proper formation of basic chemical concepts affects students’ future learning. _________________________________________________________________________________________________________________________ (cc) 2016 scs. all rights reserved. available on line at www.shd.org.rs/jscs/ elimination of students’ misconceptions 727 acknowledgements. this material is based upon research supported by the ministry of education, science and technological development of the republic of serbia, under grant no. 179010. и з в о д утицај инструкционе стратегије засноване на триплетном моделу репрезентације садржаја на елиминацију ученичких мисконцепција везаних за неорганске реакције душица д. миленковић, мирјана д. сегединац, тамара н. хрин и саша хорват природно–математички факултет, универзитет у новом саду, трг д. обрадовића 3, 21000 нови сад недавно је извешћено о ефикасној наставној инструкцији заснованој на интеграцији макроскопског, субмикроскопског и симболичког нивоа репрезентације знања, са становишта перформанси и менталног напора. на основу добијених резултата, постављен је циљ овог истраживања, а то је да се идентификују мисконцепције код групе ученика обучаваних на традиционалан начин и утврди да ли се исте мисконцепције јављају и међу ученицима експерименталне групе у области неорганских реакција. у истраживању је коришћен тест који је решавало 189 ученика другог разреда гимназије. анализа је спроведена рачунањем фреквенција бирања дистрактора у задацима. резултати су указали на постојање укупно 9 мисконцепција у групи ученика обучаваних на традиционалан начин, док је само једна мисконцепција уочена у експерименталној групи, након спровођења интервентног програма. може се извести закључак да се примењена инструкциона стратегија показала као веома ефикасна у елиминацији највећег броја мисконцепција код испитиване групе ученика. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {modelling of hydrochemical and hydromechanical parameters' synergism in the process of solid deposit creation in geothermal and other hard waters} j. serb. chem. soc. 83 (5) 625–640 (2018) udc 553.7:628.032+66.094.2:510.644: jscs–5100 556:54(497.11) original scientific paper 625 modelling of hydrochemical and hydromechanical parameters’ synergism in the process of solid deposit creation in geothermal and other hard waters dragan b. milićević1*, ljiljana n. anđelković2, marjan p. mitić2, jelena m. purenović3 and milovan m. purenović4 1university of niš, faculty of civil engineering and architecture, niš, serbia, 2university of priština, faculty of technical sciences, kosovska mitrovica, serbia, 3university of kragujevac, faculty of technical sciences, čačak, serbia and 4university of niš, faculty of science and mathematics, department of chemistry, niš, serbia (received 31 july, revised 11 september, accepted 12 september 2017) abstract: this paper presents the experimental research results, regarding the effect of hydromechanical parameters and based on the relative reduction of the starting hardness of geothermal water of sijarinska banja and niška banja as well as the water from the medijana spring in niš. the measurements were conducted on a laboratory pilot plant/ facility with glass pipes of diameter 2, 4, 6, 8 and 10 mm and with water flow controlled by a digital peristaltic pump with a flow interval from 2 to 5000 ml min-1. the effect of the hydrodynamic parameters on the change of input hardness of geothermal and other hard waters and the process of solid deposit creation were modelled by an empirical model based on simple linear regression analysis, multiple linear regression model and the neural network. the high accuracy of all applied models unequivocally proves that a synergism of hydrochemical and hydrodynamic parameters exists in the process of creation of solid deposit – limescale, thanks to which the starting hypothesis is confirmed. keywords: water hardness; deposit and scale formation; hydrochemical and hydromechanical parameters. introduction geothermal, underground and other waters of high mineralization and great hardness, present a complex composite heterogeneous system, which plays a very important role, especially in the distribution of such waters to the user. whether it is the use of the waters in question in spa resorts and rehabilitation objects, or in a boiler or in other heating or cooling systems, it is surely not possible to prevent the formation of solid deposit – limescale.1 deposit in these systems reduces * corresponding author. e-mail: dragan.milicevic@gaf.ni.ac.rs https://doi.org/10.2298/jsc170731015m 626 milićević et al. the passage of heat, which increases the energy consumption, creates the conditions for corrosion under deposit, reduces the water flow velocity and disrupts the hydrodynamic regime, which leads to the decreasing of capacity of the entire system.2 there have been a plenty of reasons for solid deposit formation, because of the distinctively high mineralization of those waters with calcium and magnesium salts. from a chemical point of view, the synergy and presence of certain impurities in dissolved and colloid state have been clarified. this research will not consider the hydrochemical parameters of balance disruption of calcium and magnesium bicarbonates,3 namely the changes which lead to the formation of the carbonate anion co2 firstly and later the carbonates of calcium and magnesium (caco3 and mgco3) and the appearance of the solid deposit formation phenomenon – limescale, but it will analyse the effect of parameters on solid deposit formation – limescale. in practice, it has been noted that limescale forms in heat supply or water supply systems where the water flow velocity is the smallest, in pipelines of small pipe diameter, or in places where water flows quite slow. this fact indicates that in the analysis of the causes of compact solid deposits, hydromechanical parameters cannot be bypassed, among which, except for the linear velocity of water flow, a probably important part belongs to flow criteria and pipe diameters, through which the flow process takes place. the research is based on a hypothesis that in the process of solid deposit – limescale formation a synergism of hydrochemical and hydromechanical parameters is present, and as a general hydrochemical parameter, the change of the input water hardness is observed. the basic idea of the research is that the pilot facility, supplied with a peristaltic pump with a digitally regulated flow through a set of pipes of various diameters, enables a clip flow of geothermal and other hard waters and after that, through a change of real hardness, the effect of hydrodynamical parameters on the process of formation of solid deposit – limescale is observed. the research on the effects of hydromechanical parameters on the relative decrease of starting hardness have been conducted on samples of geothermal water of sijarinska banja and niška banja and water from the spring medijana, niš.4 the analysis of the received experimental results in the past research was completed individually for each of the geothermal and other hard waters used in the research.1,4 the received results have unequivocally confirmed the existence of a linear connection between the relative mass output flow of hardness, namely the relative change in hardness per unit of time on the exit of the pipe and the linear velocity, for different pipe diameters.1 for each of the geothermal and other hard water used in research, a family of lines was received. that family of lines represents the dependence of mass output flow of hardness on the linear velocity for different pipe diameters.1 hydrochemical and hydromechanical parameters' synergism in deposit formation 627 in this paper, we will analyse the received experimental results in research of hydromechanical parameter effects on the change of hardness of geothermal and other hard waters with different flow velocities on different glass pipe cross sections, in the direction of generalizing results, independently of the origins of the geothermal and other hard waters. the results of this research should answer some disputable questions of theoretical character with the useful suggestions for practice and design of the systems for distribution of geothermal, underground and other hard waters. experimental materials and methods in order to measure the effect of hydromechanical parameters on the change of water hardness of geothermal and other hard waters and the process of solid deposit formation, water samples from sijarinska banja, off the aragon spring, from niška banja, and off the drinking water spring medijana were taken. the waters of sijarinska and niška banja contain metasilicic and metaboric acid in considerate amounts, and the water from the medijana spring contains the mentioned substances in very small quantities4. the water of sijarinska banja belongs to the category of sodium-hydrocarbonate, sulfide hypertherms, while the water of niška banja belongs to the category of slightly radioactive, calcium magnesium hydrocarbonate, oligomineral homeotherms. laboratory pilot installation for realization of the predicted program and the research methodology, a pilot facility was planned (fig. 1). the pilot facility consists of a set of glass pipes, 1000 mm of length whose diameters are 2, 4, 6, 8 and 10 mm. all of the pipes are made by extraction of pyrex glass, so that the same conditions of roughness are created on the inner walls on all pipes. the flows and linear speeds are defined by a digital peristaltic pump dose – it p910, ibs integra biosciences agch 7000 chur switzerland with water flow ranging from 0.6 to 5000 ml min-1. the water temperature in the receiving reservoir with heater is controlled with the help of a thermostat with a constant temperature of 40 °c. in front of the pipe set a glass splitting dish is placed, whose volume is designed. it even allows flow through the system at high flow fig. 1. pilot installation for hydraulic investigation of linear flow velocity of geothermal and other hard waters and other hydromechanical parameters influence on solid deposits formation;4 1 – peristaltic pump, 2 – set of different diameters pipes, 3 – distribution splitting dish, 4 – degassing valve, 5 – silicon hose of the peristaltic pump, 6 – vacuum part of the peristaltic pump silicon hose, 7 – intake prochrome 80 l reservoir with heater, 8 – dashboard with thermostat and switcher, 9 – electrical energy supply, 10 – metal supporting frame. 628 milićević et al. rates. on the splitting dish, a venting valve is placed, so that the creation of a gas phase in the solid liquid system is avoided at a piston flow through the glass pipe set. experimental procedure immediately before the experiment the examined water sample is filtered through blue ribbon paper and the starting water hardness is determined by the standard methodology. the water is poured into the open water heater, 80 dm3 of volume, and the water is heated to 40 °c. for a pipe of the chosen diameter the given flow is adjusted. the taps on the other glass pipes are shut so that water flows only through the pipe of the chosen diameter with a steady flow. after the passing of constant time of 2 h, the pump is stopped, the water sample is taken, filtered through the blue ribbon and the residual hardness is measured by the standard method. at the end of the examination, for the pipe of the chosen diameter with single flow and finished measuring of residual water hardness, it is preceded to measuring on the same pipe but with different flows. after that, the same process as described is repeated for the remaining glass pipes out of the set. that way, the starting and final water hardness is determined for all pipe diameters and assigned flows. analysis of experimental results analysis of results with use of the multiple linear regression model. the dependence of a single occurrence on two or more independent occurrences can be examined with multiple regression, whose task is to find as many factors (independent variables) which have an effect on the examined occurrence (dependent variable) as possible. it is very important to carefully choose the variables which will be included in the model. the model of multiple linear regression with n variables can be written as: y = a0 + a1x1 + a2x2 + a3x3 + ... + anxn + ε (1) where: y – dependent variable; x1, x2,..., xn – independent variables; a0 – intercept; a1, a2, ..., an – regression coefficient; ε – latent variable (accidental error). this model gives the best possible prediction of values of the dependent variable for any combination of values of independent variables, if all assumptions are completed. the assumptions on which the multiple regression model is based on are the following: between the variables exists a linear dependence, which is especially important for the relation of independent variables with the dependent variable, multicollinearity between variables is small or nonexistent, all variables are continuous and have a variation interval, dispersion or a variance that makes sense, meaning that the most of the observations are not a single variation or interval and that in the base there is at least three to five times more observation data than there are variables, because otherwise the regression coefficients would be unreliable. based on sizes of the regression coefficients, it could be assumed what the relative effect or importance of each independent variable in the explanation of variations of the dependent variable is, or which volume of change of the dependent variable could be expected for each unit of change of each independent variable. multiple regression also show, through the correlation index r, how strong the correspondence of the dependent variable with all independent variables is, and through the determination index, r2, how well the independent variables combined explain the variations, or how a reason could be assigned to them for the variation of the dependent variable and what is the percentage of variability of the dependent variable explained by the variability of independent variables. before the explanation of multiple regression results, testing of their statistical importance is needed. if the free form and all regression coefficients, ai (i = 0,1,2,...,n) are statis hydrochemical and hydromechanical parameters' synergism in deposit formation 629 tically important, then the correlation index, r, will surely be important. vice versa that does not have to happen because it is possible that because of a large number of variables a statistically important r is received, and that coefficients ai (i = 0,1,2,...,n) are not important. if the free form and the regression coefficients, ai (i = 0,1,2,...,n) and the correlation index, r, are not statistically important, it is concluded that none of the independent variables have a real connection with the dependent variable, or that the received model has no practical value. in practice, for describing physic-chemical processes a model of multiple linear regression with n variables without the intercept, a0 is used, if leaving out the intercept has no effect on the model relevancy, because the existence of a intercept is most often opposite of the physical reality, and the linear regression model with an intercept does not correctly describe the modelled processes. data analysis with use of the neuron network. the interest in the neuron network concept has increased over the last few years,5 because of the ability to predict models based on neuron networks along with their adjustability.5 the basic advantage of modelling with the use of neuron networks is the fact that it is enough to have the data on the input and output parameters with adequate training of the network to successfully generalize a model of satisfactory accuracy which enables precise prediction of output values for a new set of input data.5 this advantage led to a wide application of neuron networks in various engineering disciplines.5 for application of this concept on modelling of chemical and biochemical processes usually the feed forward neuron network with a back propagation algorithm is used, composed of three elementary neuron layers; input, hidden and output. the number of neurons in the input layer is defined by the number of independently variable parameters, and the number of neurons in the output layer is defined by the number of dependently variable parameters, the prediction of which is wanted. there are no set rules to choose the optimal number of neurons in the hidden layer, or namely the optimal configuration of the neuron network. the number of neurons in the hidden layer is determined by the experience or the trial and error process.5 in the procedure of examining the influence of the change in the number of neurons in the hidden layer, the following recommendations can be applied on the quality model to select the range of variation of their number:5 = + +nm n m a (2) 1≥ n tw (3) where: n – number of input variables, m –number of output variables, a – parameter which changes from 1 to 10, n – the total number of points used in modelling and tw – total number of synaptic weights in the neuron network after choosing the number of hidden neurons in the network and optimal configuration of the network, a choice of learning algorithm and its parameters is made. the choice of a learning algorithm, which sets the strengths or the synapse weights between certain neurons depends on the possibility of its application, relying on the difference in the nonlinearity of the observed problem. during the training of the neuron network of the chosen configuration, the synaptic weights of certain neurons are changed with the aim of minimizing the mean squared error of data meant for the training, simultaneously the mean squared error of the data meant for check-up is calculated and when it starts to increase the training of the network is stopped.5 630 milićević et al. the result of the neuron network model can be interpreted through the interpretation of the output result, the interpretation of weights in the network, and sensitivity analysis (interpretation of importance of the input variables). in most cases, the aim of training of the neuron network is to receive the smallest possible mistake for the value of the dependent variable calculated by the model in relation to experimental values of the dependent variable. as a measure of adequacy of the experimental result reproduction the mean squared error, mse is used most often: ( )2 1 1 = = − n ij j j mse p p n (4) where: pij – value of dependent variable received by neuron network calculation, pj – experimental value of the dependent variable and n – number of experiments. for the best match the mse tends towards 0, when the calculated values completely match the experimental values of the dependent variables. it should be noted that in practice a rule on the size of the error which could be generally applied does not exit.5 since the weights in the network are in certain an indicator of the importance of variables in the neuron network, analysis of their values after the network training phase proves useful. greater values of the weights show that values of a certain variable in the network have a bigger influence on the calculation of the output value. results and discussion table s-i of the supplementary material to this paper shows the results of examination of hydromechanical parameter effects on the change of hardness of geothermal and other hard waters and the solid deposit formation process for geothermal water of sijarinska banja and niška banja and water from medijana spring, completed according to the previously described experimental procedure. the beginning and final water sample hardness for each pipe section and flow are determined experimentally. for each pipe diameter d / mm and the corresponding pipe section, a / mm2 and flow q / l min–1, a linear water velocity, υ / m s–1 and reynolds number, re, are calculated, along with the flow rate multiplied by a ratio of output and input hardness, g0 / l min–1 expressed through the relative change of the input hardness, ci / mg l–1 as caco3 per unit time on the exit, according to the formula: 00 i = c g q c (5) experimental results modelling with the goal of confirming the starting hypothesis, the effect of hydrodynamic parameters on the change of input hardness of geothermal and other hard waters and the process of solid deposit formation are modelled by an empirical model based on simple linear regression analysis, the multiple linear regression model and the neuron network model. hydrochemical and hydromechanical parameters' synergism in deposit formation 631 the aim of the relatively simple empirical model based on simple linear regression analysis is to define which hydromechanical parameter has an effect on the change of input hardness of geothermal and other hard waters, and to establish the functional dependence between them, while the goal of the more complex model of multiple linear regression and the neuron network model is to verify the established functional dependence of hydrochemical and hydromechanical parameters in the solid deposit formation process, which will verify the starting hypothesis. for calculation of linear regression models the datafit v8.1.69 software by oakdale engineering was used and for the neuron network model development the matlab r12 software by mathworks was used. it should be mentioned that all models which are presented in this paper are valid only for the range of experimental data used for modelling and for water temperature of 40 °c, on which experimental measuring was performed. empirical model based on linear regression analysis the appropriate calculated relative mass flows of output hardness, g0, and linear velocity, υ (table s-i), are grouped for each pipe diameter, d, and are individually fitted with a single linear regression model without a free form, which can be shown with the formula: g0 = aυ (6) where: a – linear regression coefficient. the linear regression model dependences of the relative mass flow of hardness from the linear velocity for different pipe diameters is shown in table i. table i. parameters of the linear regression model for different pipe diameters d / mm a linear regression model r2 standard error of the estimate 2 0.19155 g0 = 0.19155υ 0.9973 0.033 4 0.77285 g0 = 0.77285υ 0.9979 0.030 6 1.70469 g0 = 1.70469υ 0.9822 0.079 8 3.00524 g0 = 3.00524υ 0.9887 0.054 10 4.72197 g0 = 4.72197υ 0.9677 0.088 the values of the correlation coefficient, r2, is close to 1 and small values of the standard error of the estimate for the defined regression models for all pipe diameters (table i), unequivocally affirm that a strong linear connection exists between the relative mass flow, that is the relative change of hardness per unit time on the exit, and the linear velocity, for different pipe diameters, for the examined geothermal and other hard waters, are independent from their origin. the received linear regression models for different pipe diameters for the examined geothermal and other hard waters are shown on fig. 2. 632 milićević et al. as can be seen in fig. 2, families of straight lines which represent the dependences of the relative mass flow of hardness on the output from the linear velocity for different pipe diameters are received. with further analysis it was established that a connection between the regression coefficient, a in formula (6) and the square of the pipe diameter, d2, are shown in table i, and that the connection can be the best defined by a single linear regression model without a intercept: a = bd2 (7) where: b – linear regression coefficient. fig. 2. dependences of relative mass flow of hardness on the exit on the linear velocity for different pipe diameters. a single linear regression model (7) of dependence with coefficient, a, in formula (6) and square of pipe diameter, d2, is received in the form: a = 0.0472d2 (8) for which the correlation coefficient is r2 = 0.99996 and standard error of the estimate = 0.01214, which shows that the model is highly accurate and confirms a very strong connection between the regression coefficient value, a, in formula (6) and square of the pipe diameter, d2. based on the formulas (6) and (8), the relative mass flow of output hardness can be defined by the formula: g0 = 0.0472υ d2 (9) based on the formulas (5) and (9) the output hardness can be defined by the empirical model: 20 i0.0472 υ =c c d q (10) hydrochemical and hydromechanical parameters' synergism in deposit formation 633 the received empirical model, based on single linear regression analysis of experimental results, is highly accurate (r2 = 0.99658, standard error of the estimate = 4.34177, mse = 18.851). regarding that υ and q are co-dependent variables, based on the empirical model (10), it can be concluded that the variability of the output hardness, c0 for different variations of input influential parameters (ciqd2), which confirms the functional dependence of the output hardness, c0 is not just dependant on the input hardness, ci, but also from the hydrodynamical parameters, specifically the linear pipe flow, q, and square of pipe diameter, d2, meaning that: c0 = f(ciqd2) (11) the defined functional co-dependence (11) confirms the starting hypothesis that in the process of hard deposit formation – limescale, exists a synergism of the change of input water hardness, as an adopted primary hydrochemical parameter and hydromechanical parameters, specifically pipe flow and pipe diameter. model of multiple linear regression with three independent variables for the analysis of the influence the hydrodynamical parameters have on the change of hardness of geothermal and other hard waters and the process of solid deposit formation, a model of multiple linear regression with three independent variables, can be written, based on formula (1) and the defined functional dependence (10), in the form: c0 = a0 + a1ci + a2q + a3d2 (12) results of the regression analysis for the adopted model of multiple linear regression with three independent variables are shown in table ii. the results presented in table ii show that the multiple linear regression model with three independent variables describes the experimental data very well and the connection between the variables is quite strong. the coefficient of multiple determination, r2 equals 0.9970, meaning that the model explains 99.70 % of variability of the output hardness for different values of input parameters. the standard error of the estimate, which shows the standard deviation of residuals, equals 3.76911. the residual sum of squares, which shows the sum of squares of the difference between the experimental data and the data, generated by the regression model equals 1065.46592, and finally the mean square error, mse, which shows the adequacy of the experimental result reproduction equals 14.206. the first regression coefficient, a1, is interpreted as an increase of the output hardness, c0, by 0.99969 mg l–1 as caco3 if the input hardness, ci increases by 1 mg l–1 as caco3, and the remaining independent variables stay unchanged, the second regression coefficient, a2, is interpreted as an average decrease of the output hardness, c0, by –2.66662 mg/l caco3 if the flow, q increases by 1 l min–1, and the remaining independent variables stay unchanged, and the fourth regression coefficient is interpreted as an average increase of the output hardness, 634 milićević et al. c0 by 0.02490 mg l–1 as caco3 if the square of the pipe diameter, d2, increases by 1 mm, and the remaining independent variables stay unchanged. it can be concluded that the output hardness, c0, is in direct proportion with the input water hardness, ci, and the square of the pipe diameter, d2, but inversely proportional to the pipe flow, q. table ii. results of regression analysis for the multiple linear regression model with three independent variables sum of residuals = 2.47581510848249e-10 average residual = 3.1339431752943e-12 residual sum of squares (absolute) = 1065.46592144379 residual sum of squares (relative) = 1065.46592144379 standard error of the estimate = 3.76911293090526 coefficient of multiple determination (r^2) = 0.9969843461 proportion of variance explained = 99.69843461% adjusted coefficient of multiple determination (ra^2) = 0.99686372 durbin–watson statistic = 2.02209675423985 regression variable results variable value standard error t ratio prob(t) a0 –4.88548 1.36644 –3.57534 0.00062 a1 0.99969 0.00667 149.89886 0.00000 a2 –2.66662 0.77606 –3.43611 0.00097 a3 0.02490 0.01632 1.52588 0.13124 variance analysis source df sum of squares mean square f ratio prob(f) regression 3 352246.274 117415.425 8265.076 0 error 75 1065.466 14.206 total 78 353311.740 the estimated intercept a0 and the regression coefficients a1 and a2 have high values of the t ratio (t ratioa0 = –3.57534 > t0.05(75) = 1.667, t ratio a1 = 149.89886 > t0.05(75) = 1.667 and t ratio a2 = –3.43611 > t0.05(75) = 1.667), and small values of prob(t) (0.00062, 0 and 0.00097), which indicates that there is no possibility that their values can be zero, meaning that these regression coefficients are statistically important and cannot be removed from the regression model without decreasing the validity of the model. the estimated regression coefficient a3 has a relatively low value of the t ratio (t ratioa3 = 1.52588 > t0.05(75) = 1.667 and a relatively high value of prob(t) (0.13124), which indicates that a small possibility exists (13.12 %) that the coefficient value can be zero. the high quality of the applied regression model is shown in the regression model diagram presented in fig. 3 where it can be seen that the data are ran hydrochemical and hydromechanical parameters' synergism in deposit formation 635 domly distributed above and below the regression plane and that the applied model adjusts well to the experimental data. (a) (b) (c) fig. 3. scatter plots of multiple linear regression model results; scatter plot of input ci and output hardness c0 (a); scatter plot of flow q and output hardness c0 (b); scatter plot of square of the pipe diameter d2 and output hardness c0 (c). it can be concluded that the received results make statistical and scientific sense, and that the values of certain variables are not opposite to the physical reality, meaning that the multiple linear regression model with three independent variables correctly describes the modelled processes. the received model is highly accurate and it can describe the variability of the output hardness, co for different variations of the influential input parameters (ciqd2) well, which confirms the starting hypothesis and the functional dependence (11) of the output hardness, c0 from the input water hardness, ci and hydrodynamical parameters: pipe flow, q, and the square of pipe diameter, d2. results of the neuron network model with three independent variables since the neuron network model based on the defined functional dependence (11) should mould the influence of the three independent variables, namely the 636 milićević et al. input values (ciqd2) on a single dependent variable, namely the output value, c0, the number of neurons in the input layer, will equal 3 and in the output layer will equal 1. although the aim of this paper was not the choice of the optimal neuron network configuration for its use in the modelling of the solid deposit formation process, with the use of recommendations defined by formula 2 the number of neurons in the hidden layer is varied from 3 to 12, with the satisfied condition defined by formula (3) that the n/tw ratio is larger than one. in the used software package there are more defined learning algorithms, and in this paper for all configurations of the neuron network one of the most common algorithms in literature was used.5 it was the levenberg–marquardt method (trainlm – matlab network training function), with an adopted degree of the learning rate from 0.25 and the neuron network momentum coefficient for the chosen learning algorithm of 0.5. for its application in modelling the change of hardness of geothermal and other hard waters and the solid deposit formation process, the experimental data (table s-i) are separated into three groups in the used software: the data for learning, the data for check-up and the data for testing of the neuron network. in the analysis of the optimal neuron network configuration, it was proved that the increase of the number of neurons in the hidden layer leads to an improvement in the predictable properties of the neuron network. with an increase of the number of neurons, the mean square error decreases and has the lowest value when the number of neurons equals 9. further increase of the number of neurons does not contribute to the improvement of the network prediction, so the adopted networks configuration is the configuration with one hidden layer of 9 neurons, or 3-9-1 neurons. the results of moulding the experimental data with the neuron network model with an adopted configuration of 3-9-1 neurons and an adopted learning algorithm are shown in fig. 4. as it was assumed, during the training of the neuron network on the experimental data, the neuron network model could adequately show the change of hardness of geothermal and other hard waters as well as the process of solid deposit formation depending on the hydrodynamical parameters. the received values of the correlation coefficient, r, of 0.99916, or the determination coefficient, r2, of 0.9983 point to the great agreement of the experimental data and the data received with the neuron network model, based on the set experimental data for learning. the models received by the application of the neuron network methodology typically have a great prediction value on the set of data for learning, and a significantly smaller value on the test set of data or on the set of new, unknown data.5 however for the neuron network model with the adopted configuration of 3-9-1 neurons and the adopted learning algorithm for the data set for check-up, the data set for testing of the neuron network, as well as the complete set of hydrochemical and hydromechanical parameters' synergism in deposit formation 637 experimental data the received values of the correlation coefficient, r, of 0.99907, 0.99544 and 0.9988 or the determination coefficient, r2, of 0.99814, 0.99090 and 0.99760, respectively, indicate the good correlation of the experimental data and the data received by the neuron network model. fig. 4. neural network model regression. the received neuron network model has a very high accuracy and it can describe the variability of the output hardness, c0 for different variations of the influential input parameters (ciqd2), which confirms the starting hypothesis and the functional dependence (10) of the output hardness, c0 from the input water hardness, ci and hydrodynamical parameters: pipe flow, q, and square of the pipe diameter, d2. for the exact application of the neuron network model in the moulding of the solid deposit formation processes in the observed range of data (table s-i) it is sugessted a choice of the optimal neuron network configuration, the learning algorithm and its parameters (the learning rate and momentum coefficient) which will provide the highest accuracy of the system. 638 milićević et al. summary of experimental results modelling all applied models show that the connection between variables is very strong and describe the experimental data well, which is confirmed by the multiple determination coefficient, r2, close to one in all models and the low value of the mean square error, mse, which equals 18.851 in the empirical model, 14.206 in the multiple linear regression model, and 10.7903 in the neuron network model. the accuracy of the applied models could best be shown by application of the model on the experimental data (table s-i). in fig. 5 the results of predictions of the experimental data for all applied models are shown. fig. 5. scatter plot of predicted and measured output hardness. the diagram shown in fig. 5 confirms the accuracy of the applied models because for all applied models the small dispersal of prediction values appears for the output hardness around the line of cp.o = cm.o, which represents the absolutely correct prediction, or that the prediction values of the output hardness, cp.o are very close to the experimentally determined values, cm.o for all applied models. discussion in this research the changes of water hardness, as one of the hydrochemical parameters, are put in function of hydromechanical parameters, so that the insight could be received into the synergism between the hydrochemical and hydromechanical parameter in the solid deposit formation process. the solid deposit formation process is highly complex and depends on the hydrochemical and hydrodynamical parameter. as a starting general hydrochemical parameter in this research, the input water hardness was adopted. the completed program and research methodology confirmed the set foundations and hydrochemical and hydromechanical parameters' synergism in deposit formation 639 pointed towards the synergism model between the hydrochemical parameters of solid deposit formation and the hydrodynamical parameters. with an empirical model based on single linear regression, the multiple linear regression model and the neuron network model with three independent variables, it was confirmed that except the linear velocity as an independent variable, there are also other independent variables which influence the change of the input hardness, as the dependent variables in the solid deposit formation process. in all applied models, the output hardness, c0 was coupled as a dependent variables with four independent variables: input hardness, ci; pipe flow, q and square of pipe diameter, d2. the applied models describe the experimental data well, and the connection between the variables is very strong, which is confirmed by the multiple determination coefficient, r2 close to one and a small value of the mean square error, mse in all models. the quality of the applied models unequivocally confirms that a multiply defined synergism of the hydrochemical and the hydrodynamical parameters exists, which in turn confirms the starting hypothesis and creates a starting point for a more variable analysis of the complex phenomena of the solid deposit formation and the explanation of the processes happening in the heterogeneous solidliquid system. conclusion the basic indicator of the alternation of the hydrochemical parameters is the change of hardness which is followed by the different flows and the different flow velocities through the glass pipes of various intersections. by modelling the experimental data, with the use of different model types expected changes of hydrochemical parameters, were observed on the influence of hydromechanical parameters. the empirical model based on the single linear regression analysis, the multiple regression model and the neuron networks model with three independent variables includes hydromechanical parameters: pipe flow and pipe diameter. results of all applied models have shown a very high value of the multiple determination coefficient, r2 close to one, with a small standard error and they have confirmed a good adaptability of the mathematical model to the experimental data. with the high accuracy of all applied models it was unequivocally confirmed that a multiply defined synergism exists between the hydrochemical and the hydrodynamical parameters in the solid deposit – limescale formation process, which in turn confirms the starting hypothesis. the applied modelling describes the influence of the hydrodynamical parameters well, and the connection with the change of water hardness enables an all encompassing view of the synergism of the hydrochemical and the hydromechanical parameters. 640 milićević et al. supplementary material results of completed examination for geothermal water are available electronically at the pages of the journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. this work was funded by the ministry of education, science and technological development of the republic of serbia (project no. 37018 and no. 33046). и з в о д моделовање синергизма хидрохемијских и хидромеханичких параметара у процесу стварања чврстих депозита у геотермалним и другим тврдим водама драган б. милићевић1, љиљана н. анђелковић2, марјан п. митић2, јелена м. пуреновић3 и милован м. пуреновић4 1универзитет у нишу, грађевинско-архитектонски факултет, ниш, 2универзитет у приштини, факултет техничких наука, косовска митровица, 3универзитет у крагујевцу, факултет техничких наука, чачак и 4универзитет у нишу, природно-математички факултет, департман за хемију, ниш у раду су приказани резултати експерименталних истраживања утицаја хидромеханичких параметара на релативно смањење полазне тврдоће геотермалне воде сијаринске бање и нишке бање и воде из изворишта медијана, ниш. експериментална истраживања су изведена на лабораторијском пилот постројењу са стакленим цевима пречника 2, 4, 6, 8 и 10 mm, на коме је проток контролисан дигиталном перисталтичком пумпом са интервалом протока од 2 до 5000 ml min-1. утицај хидродинамичких параметара на промену улазне тврдоће геотермалних и других тврдих вода и процес стварања чврстих депозита су моделовани емпиријским моделом базираним на простој линеарној регресионој анализи, моделом вишеструке линеране регресије и неуронском мрежом. високом тачношћу свих примењених модела недвосмислено је потврђено да постоји вишеструко изражени синергизам хидрохемијских и хидродинамичких параметара у процесу стварања чврстих депозита – каменца, чиме је полазна хипотеза истраживања потврђена. (примљено 31. јула, ревидирано 11. септембра, прихваћено 12. септембра 2017) references 1. lj. anđelković, m. purenović, m. ranđelović, d. milićević, a. zarubica, m. mitić, s. tomović, chem. ind. chem. eng. q. 20 (2014) 197 2. b. đukić, s. smiljanić, a. došić, zaštita materijala 53 (2012) 151 (in serbian) 3. n. stanković, m. purenović, m. ranđelović, j. purenović, hem. ind. 65 (2011) 43 4. lj. anđelković, phd thesis, faculty of technical sciences in kosovska mitrovica, 2013 (in serbian) 5. a. jokić, j. grahovac, j. dodić, s. dodić, s. popov, z. zavargo, d. vučurović, hem. ind. 66 (2012) 211 (in serbian). << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {photodegradation of selected pesticides: photocatalytic activity of bare and pani-modified tio2 under simulated solar irradiation} j. serb. chem. soc. 84 (12) 1455–1468 (2019) udc 632.95+546.824–31:544.526.2:576+615.9 jscs–5277 original scientific paper 1455 photodegradation of selected pesticides: photocatalytic activity of bare and pani-modified tio2 under simulated solar irradiation marina j. lazarević1#, vesna n. despotović1#, daniela v. šojić merkulov1*#, nemanja d. banić1#, nina l. finčur1#, dragana d. četojević-simin2, mirjana i. čomor3 and biljana f. abramović1# 1university of novi sad, faculty of sciences, department of chemistry, biochemistry and environmental protection, trg d. obradovića 3, 21000 novi sad, serbia, 2university of novi sad, faculty of medicine, oncology institute of vojvodina, dr goldmana 4, 21204 sremska kamenica, serbia and 3university of belgrade, vinča institute of nuclear sciences, p. o. box 522, 11001 belgrade, serbia (received 1 march, revised 2 august, accepted 3 august 2019) abstract: in this paper the efficiency of photocatalytic degradation of different pesticides was investigated using bare tio2 and tio2 nanoparticles modified with polyaniline under simulated solar irradiation. sulcotrione showed the highest percentage degradation and further experiments were related to this herbicide. mineralization and cytotoxicity of the starting compound and intermediate species formed during the decomposition in double distilled water (ddw), as well as the efficiency of removal from various environmental waters were studied. the contents of the most abundant ions present in the river danube were simulated in ddw and their influence was evaluated. it was found that cytotoxicity was in all cases below 11 % and the efficiency of photocatalytic degradation in environmental waters was decreased compared with ddw. furthermore, addition of different scavengers revealed that the main path of degradation is through holes, while the presence of h2o2 decreased and kbro3 increased the efficiency of photocatalytic degradation compared with the system without the mentioned electron acceptors. keywords: pesticides; bare tio2 nanoparticle; tio2/polyaniline nanocomposite; cytotoxicity; environmental waters; radical, hole and electron scavengers. introduction in order to protect crops from specific types of pests, today in agriculture, the usual practice is to use different pesticides. bearing in mind the importance and extensive use of pesticides in agriculture, such chemicals could reach the * corresponding author. e-mail: daniela.sojic@dh.uns.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc190301083l 1456 lazarević et al. environmental waters as the consequence of a combination of run-off, accidental spillages, spray drift, drainage, or atmospheric deposition.1 since the appearance of pyrethroids, neonicotinoid insecticides belong to the fastest growing insecticides on the market. in the past, the stability of neonicotinoids in the environment was extensively studied. for thiacloprid, it was found that it is resistant to water hydrolysis at neutral and acidic ph values. furthermore, thiacloprid showed significant photostability under the influence of simulated solar radiation.2–5 the herbicide clomazone is applied for the suppression of various types of weeds in different cultivation fields.6 it has the potential to pollute water because of its high water solubility.7 residues of clomazone were found in samples taken from river waters in rice cultivation areas.6 quinmerac belongs to the quinoline group, which is a relatively new class of highly selective herbicides, with recent commercial use.8 this herbicide is relatively well soluble in water and hardly degradable.9 according to a report from the swedish board of agriculture and the swedish environmental protection agency,10 quinmerac has been detected in ground, surface and drinking water. in addition, quinmerac can potentially cause pollution of the entire ecology through the food chain thereby having negative consequences also to human health.11 sulcotrione belongs to the class of triketone herbicides and is used to control dicotyledonous weeds and barnyard grass in maize production fields. beside good properties in the control of weeds, this herbicide is a potential pollutant, wherein sulcotrione and its degradation product (2-chloro-4-(methylosulfonyl) benzoic acid were detected in high concentrations in water from the klodnica river in poland.12 results of some studies have indicated that sulcotrione and/or its degradation products are toxic toward microorganism vibrio fischeri,13 genotoxic towards allium cepa root meristem14 and vicia faba seedlings.15 considering that pesticides have a negative influence on living organisms, the need for their monitoring and control has become more obvious in recent years. pollution of the aquatic environment due to the presence of pesticides has encouraged researchers to find ways to prevent such pollution.16 clear and perceivable advantages were found using advanced oxidation processes (aops) in the treatment of pesticides.17 among different aops, heterogeneous photocatalysis represents a low-cost, versatile, environmentally friendly, and one of the most promising green chemistry method for the removal of different contaminants.18 in order to prevent the harmful effect which arises as a consequence of the presence of hazardous pollutants, photocatalytic degradation of water contaminants is used to detoxify or treat wastewater.19 the main disadvantage of photocatalytic degradation using tio2 as a photocatalyst is the occurrence of electron−hole pair recombination, which as a consequence decreases the quantum efficiency. in addition, the wide band gap, of photodegradation behaviour of selected pesticides 1457 tio2, which is 3.0 ev for rutile, and 3.2 ev for anatase, narrows the absorption of photons to the ultraviolet region (wavelength < 390 nm). this reduces the practical use of the mentioned processes in the presence of tio2 for solar light harvesting.20 in the past decades, several studies were based on the combination of tio2 nanoparticles with polyaniline (pani) due to the increased visible light absorption property as a result of the synergistic action between the conducting polymers and tio2.21–24 the potential applicability of tio2/pani (tp) nanocomposites synthesized with different molar tio2:pani ratios (tp-50, tp-100, and tp -150)22 as photocatalysts was checked following the photodegradation reaction of organic compounds: pharmaceuticals (propranolol and amitriptyline) and pesticides (sulcotrione and clomazone) using uv irradiation. it was found that the photocatalytic degradation of sulcotrione was the best of all the investigated compounds and tp-150 was the most efficient catalyst in the mineralization of pesticides.25 furthermore, the results of the uv/vis reflectance spectra22 indicate that mentioned tp nanocomposites could be activated by uv and visible light simultaneously, which opens the possibility of their application as photocatalysts. as a consequence, the aim of this work was to investigate the photodegradation behaviour of selected pesticides (thiacloprid, clomazone, quinmerac, and sulcotrione; fig. s-1 of the supplementary material to this paper) in aquatic systems using simulated solar irradiation in the presence of bare tio2, as well as tp nanocomposites. in addition, due to the highest percentage of photodegradation and mineralization of sulcotrione using bare tio2, further investigations were related to sulcotrione. namely, the cytotoxicity of the starting compound and intermediate species formed during the decomposition in double distilled water (ddw), as well as efficiency of sulcotrione removal from various environmental waters (danube, tisa and begej rivers, as well as lake moharač and sot lake) were studied. moreover, the most abundant ions that were identified in the water from the river danube were added to ddw in order to evaluate their influence. finally, in order to investigate the influence of active radicals, holes and electrons on the efficiency of sulcotrione photodegradation using bare tio2 under simulated solar irradiation, different scavengers were added to the reaction mixture. the kinetics of the photodegradation was monitored by ultrafast liquid chromatography with a diode array detector (uflc–dad). the process of mineralization was accompanied by determination of total organic carbon (toc). in order to evaluate the hazardousness to the environment of the photocatalytic treatment of waters containing sulcotrione and the formed intermediates, mammalian cell lines (rat hepatoma (h-4-ii-e), mouse neuroblastoma (neuro-2a), human colon adenocarcinoma (ht-29) and human foetal lung (mrc-5) were used for toxicity assessment. 1458 lazarević et al. experimental chemicals, water samples and catalysts all the used chemicals are listed in the supplementary material. the collection of the environmental water samples and their physicochemical characteristics are given in table s-i of the supplementary material. the tio2 catalysts, bare and modified with polyaniline in different tio2:pani mole ratios (tp-50, tp-100 and tp-150) were prepared as previously described.22 details are given in šojić merkulov et al.25 the formation of nanocomposites was proved using raman spectroscopy and tem measurements.22 photodegradation procedure the photocatalytic degradation was realised in a previously described cell.26 for the halogen lamp, the visible light intensity was 63.85 mw cm-2 and uv radiation intensity was 0.22 mw cm-2. the radiation energy fluxes were measured using a delta ohm hd 2102.2 (padova, italy) radiometer, which was fitted with a lp 471 uv (spectral range 315–400 nm) and a lp 471 rad (spectral range 400–1050 nm) sensor. additionally, the lamp output was calculated to be ca. 1.7×10-9 einstein cm-3 min-1 using potassium ferrioxalate actinometry.27 experiments were performed using 20 cm3 of 50 µmol dm-3 pesticide (thiacloprid, clomazone, quinmerac and sulcotrione) suspension containing 0.5 mg cm-3 of catalyst (bare tio2 or tp). the aqueous suspension of the catalyst was sonicated (50 hz) in the dark for 15 min before irradiation, in order to uniformly disperse the particles of photocatalyst and to attain adsorption equilibrium. before irradiation, the thus obtained suspension was set at 25.0 °c in a stream of o2 (3.0 cm3 min-1). during irradiation, the mixture was stirred at a constant rate under continuous gas flow. all experiments were performed at natural ph (for thiacloprid ≈5.7, clomazone ≈3.5, quinmerac ≈4.6 and sulcotrione ≈3), except when studying the effect of the quality of environmental waters on the photocatalytic degradation of sulcotrione. analytical procedures experimental conditions regarding uflc–dad, total organic carbon (toc) and ph measurements can be found in the supplementary material. toxicity tests examination of the cytotoxic effect on the growth of cell lines is described in the supplementary material. the cell growth was evaluated by the colorimetric sulforhodamine b (srb) assay of skehan et al.,28 modified by četojević-simin et al.29 results and discussion photocatalytic oxidation of selected pesticides using tio2/polyaniline nanocomposites recently, novel modified tio2 nanostructures have gained great attention because of their extended adsorption range to the visible light region. several methods of tio2 modification have been proposed in order to achieve the utilization of visible light. conductive polymers, such as polyaniline, have been used as the sensitizer in polymer-sensitized tio2 nanocomposites due to their unique electrical, optical, and photoelectric properties and the advantages of easy synthesis and low photodegradation behaviour of selected pesticides 1459 cost. owing to its good thermal conductivity and thermostability, nanoscale polyaniline can be used as a substitute product of the solar materials in photovoltaic devices.23 under uv irradiation, conjugated polymers promote the separation efficiency of photoinduced electron–hole pairs due to the high mobility of photoinduced holes in the conjugated polymers. under visible light irradiation, conjugated polymers in hybrid materials are excited to produce photoinduced electron– –hole pairs and then the photoinduced electrons are injected into the conduction band (cb) of tio2. namely, the electrons from tp nanocomposites coupled with the polymers can be excited from the highest occupied molecular orbital (homo) to the lowest unoccupied molecular orbital of the polymers under visible light irradiation. then the excited electrons (e–) can be injected into the conduction band of tio2 and react with electron acceptors such as oxygen adsorbed on the tio2 surface to generate oxidative species, such as hydroxyl radicals and superoxide radical anions. the surface of tio2 is generally a good adsorber of oxygen, which acts as an electron scavenger, due to the position of the cb of tio2 and the potential of o2/o2•– formation, while, in the case of tp nanocomposites, the polymer is a hole scavenger. if bare tio2 nanoparticles were used as a photocatalyst, the hole scavengers would be organic pollutants (thiacloprid, clomazone, quinmerac and sulcotrione) and the excited electrons are used for the formation of superoxide radical ions, hydrogen peroxide, and hydroxyl radicals.30 the photoinduced holes (h+) are left in the homo of the polymers. therefore, the photocatalytic activity of tio2 under visible light could be enhanced by using conductive polymers as stable photosensitizers to modify tio2 nanoparticles.23 radoičić et al.22 reported that presence of the pani-emeraldine salt in the case of tp-50; pani-emeraldine salt and pani-emeraldine base-like segments in the case of tp-100 and branched oligomers in the case of tp-150 as confirmed by raman spectroscopy. moreover, the surface of tio2 is most likely hindered in the case of tp-50 sample because of high content of pani. additionally, in the case of tp-150, tio2 surface is probably only partially covered due to the lowest content of pani.22 the photocatalytic activities of tp nanocomposites in the process of thiacloprid degradation are enhanced as the content of pani in the nanocomposite decreases, with a maximum value for tp-150 (13 %). in the case of tp-50, the tio2 surface is hindered with positively charged with pani -emeraldine salt, which probably leads to repulsion of the thiacloprid molecule (the pka value for thiacloprid is not applicable). as the content of pani decreases beside emeraldine salt, emeraldine base-like segments are also present, and the interaction with thiacloprid is better. the highest efficiency of photocatalytic degradation of thiacloprid can be noticed in the presence of tp-150, where only branched oligomers are present. moreover, examination of the photodegradation of selected pesticides under simulated solar irradiation suggests that tp 1460 lazarević et al. nanocomposites showed higher photocatalytic activity for elimination of clomazone and quinmerac compared with bare tio2. namely, in the presence of bare tio2, tp-50, tp-100 and tp-150 nanocomposites 10, 28, 25 and 26 % of clomazone was removed and 0.5, 18, 27 and 8 % of quinmerac from the suspension after 240 min of irradiation, respectively (fig. 1). tp nanocomposites showed very similar activity in the case of photocatalytic degradation of clomazone. slight better activity showed tp-50 probably due to electrostatic or hydrogen bonding between positively charged pani-emeraldine salt (−nh•+−) and free electron pair on the nitrogen atom. the highest efficiency of photocatalytic degradation of quinmerac was noticed in the presence of tp-100. this may be also explained by electrostatic interaction of emeraldine salt with the nitrogen atom and by protonation of emeraldine base-like segments. furthermore, the surface of tio2 is less hindered with pani groups, which enable interaction of carboxyl group with the oh groups on the surface of tio2. while pka value for clomazone is also not applicable, for quinmerac, the pka value is 4.31. this means that at ph ∼5, the deprotonated form is mostly present in the suspension whereby at this ph the nanocomposites are protonated (phpzc is 5.2) and therefore there is attraction between the anion of quinmerac and the nanocomposites. in contrast to mentioned pesticides, tio2 modified with polyaniline showed a decrease in the efficiency of sulcotrione photocatalytic degradation compared with bare tio2. namely, about 26 % of the compound was removed using tp-50 and tp-100, while 35 % was removed using tp-150. fig. 1. efficiency of selected pesticides elimination using bare tio2 and tp nanocomposites after 240 min of simulated solar irradiation. experimental conditions: c0 = 50 µmol dm-3; catalyst loading 0.5 mg cm-3. the results can be viewed in different ways, concerning the optical properties of the nanocomposites, bearing in mind the mechanism of light absorption, the molecular structure of the selected pesticides and the pani layer in the nanocomposites.22 it could be assumed that the sulcotrione molecules have a better interaction with oh groups on the surface of tio2 than with positively charged pani-emeraldine salt. tp-150 showed a slightly better activity than tp photodegradation behaviour of selected pesticides 1461 -50 and tp-100, due to the smaller content of pani, more available oh groups on the surface of tio2, and the presence of only branched emeraldine oligomers. besides, 52 % of sulcotrione was eliminated from the aquatic suspension using bare tio2. namely, the initial ph value of the suspension in ddw was ∼3 whereby the higher degradation rate may probably be attributed to interaction of sulcotrione anion (pka = 3.13) with the protonated sites of the catalyst (the phpzc for bare tio2 is 5.2). complete mineralization of the organic target compound to co2 and inorganic anions is the desirable result for every treatment process. the process of mineralization of selected pesticides was studied by toc measurements (table s-ii, supplementary material). it was found that in the cases of thiacloprid, clomazone, and quinmerac photodegradation, mineralization practically did not occur, while 40 % of sulcotrione was mineralized using bare tio2 after 240 min of irradiation. furthermore, mineralization was slower than removal of sulcotrione, which could be explained by the formation of organic intermediates during the photocatalytic process. since bare tio2 was the most efficient in the photocatalytic degradation of sulcotrione, cytotoxicity experiments and efficiency of photocatalytic degradation of sulcotrione in environmental waters were performed using the mentioned catalyst under simulated solar irradiation. toxicity assessment degradation of pesticides in the environment could potentially generate many by-products that are sometimes persistent and requires better knowledge on their reactivity and toxicity. in order to evaluate the environmental hazard of water containing sulcotrione and the formed reaction intermediates using bare tio2 under simulated solar irradiation, in vitro growth of h-4-ii-e, neuro-2a, ht-29 and mrc-5 cell lines was evaluated (fig. s-2, supplementary material). blank tests of an aqueous solution of 5 μmol dm−3 sulcotrione and of a filtered aqueous suspension of 0.05 mg cm–3 bare tio2 were also performed (fig. s-3, supplementary material). as can be seen, the effect of photodegraded mixtures on the growth of selected cell lines was dependent on the time of irradiation and type of cell line. based on the results, it appears that both growth inhibition and growth stimulation were observed within 240 min of irradiation using sulcotrione photodegraded mixtures in the case of all cell lines. the obtained results showed maximum inhibition of cell growth of 11 % after 30 min of irradiation and the maximum stimulation of 5 % obtained after 240 min of irradiation, for the ht-29 cell line. the inhibition of cell growth was influenced by the parent compound (blank test of sulcotrione with ~5 % growth inhibition, fig. s-3, supplementary material) and the mixture of sulcotrione and its photocatalytic degradation intermediates (fig. s-2, supplementary material) after different irradiation times. in the case of the cell line mrc-5, growth inhibition or stimulation was 1462 lazarević et al. negligible for the blank tests of sulcotrione and bare tio2 and also for the photodegradation mixtures of the obtained intermediates was below 2 %. the highest inhibition of cell growth for the blank test of sulcotrione (≈6 %) was noticed in the case of the neuro-2a cell line. however, during irradiation, less toxic intermediates were formed with stimulation or inhibition of growth of up to 2 %. bare tio2 showed the highest toxicity, i.e., cell growth inhibition of ≈9 % for the cell line h-4-ii-e (blank test, fig. s-3, supplementary material). for the same cell line, the blank test of sulcotrione showed a growth inhibition of ≈4 %. furthermore, the photodegraded mixture in the presence of bare tio2 showed only growth inhibition with a maximum value of ≈7 % after 120 min of irradiation. at other irradiation times, inhibition growth was less than 6 % with minimum inhibition of ≈1 % after 60 min. in addition, the toxicity was reduced, compared with blank tests, for the cell lines neuro-2a and h-4-ii-e, in the process of photocatalytic degradation of sulcotrione. however, cytotoxicity in all cases was substantially lower compared to cytotoxic drugs and the known toxicant, hgcl2.31 photocatalytic activity of bare tio2 in environmental waters and evaluation of different ions influence it is well known that the composition of environmental waters can influence the efficiency of photocatalytic degradation of pesticides. in this context, it must also be considered that real water might contain substances that could compete with the target pollutant for the oxidizing agent (•oh), such as other organic and inorganic compounds and natural organic matter. this usually causes a decrease in the photodegradation efficiency.32 due to this, photocatalytic degradation of sulcotrione in environmental waters was investigated (the danube, tisa and begej rivers, as well as lake moharač and sot lake) using bare tio2. examination of sulcotrione photodegradation under simulate solar irradiation suggested that a herbicide removal of 15 % was only achieved in water from sot lake after 240 min of irradiation (fig. 2). on the other hand, it was found that degradation of the herbicide was the most efficient in ddw. fig. 2. influence of water type on the efficiency of sulcotrione photocatalytic degradation. experimental conditions: c0(sulcotrione) = 50 µmol dm-3; bare tio2 loading 0.5 mg cm-3. photodegradation behaviour of selected pesticides 1463 this behaviour could be explained by the presence of inorganic and organic matter in the examined environmental waters that affect the efficiency of the photodegradation process. another reason might be the different ph values for ddw (≈3) in comparison to environmental waters (≈8). namely, the higher degradation rate at the lower ph may probably be attributed to the more effective binding of the sulcotrione molecule to the protonated sites of the catalyst. bearing in mind that the photodegradation efficiency in the water from the river danube was among the least efficient, ions in the concentrations for this water were added to ddw in order to simulate and evaluate their influence. first simulation of ions was investigated without adjusting the ph value of the suspension and the obtained results are presented in fig. 3a. in the case of the addition of hco3–, so42– and ca2+, no photodegradation of sulcotrione was observed. it is assumed that so42–, ca2+, sr2+ and ba2+ decrease the efficiency of the photocatalytic degradation of sulcotrione by decreasing the adsorption of the substrate on the surface of the catalyst.33,34 moreover, hco3– can react with •oh (reaction (1)): hco3– + •oh → co3• + h2o (1) thereby decreasing the efficiency of removal. another reason for the negligible photocatalytic degradation, in the case of addition of hco3– and also ca2+ is the ph value ∼8, at which there is repulsion between deprotonated bare tio2 and sulcotrione. fig. 3. influence of the addition of different ions on photocatalytic degradation efficiency of sulcotrione at: a) natural ph and b) ph 8. experimental conditions: c0(sulcotrione) = 50 µmol dm-3; bare tio2 loading: 0.5 mg cm-3. however, addition of humic acid to the system leads to improvement of the efficiency of sulcotrione photocatalytic degradation compared to ddw at natural ph ∼3, when 63 and 52 % of sulcotrione was removed, respectively. according to wu et al.,35 humic acid can promote the generation of •oh, which increases the efficiency of sulcotrione removal. 1464 lazarević et al. in the same way, all ions and humic acid were added separately to suspension and ph value was adjusted at 8, which corresponds to ph value of environmental waters (fig. 3b). the obtained results show that in almost all cases photocatalytic degradation of sulcotrione did not occur. slight degradation of 10 % after 240 min of irradiation was observed in the case of hco3– addition. from the obtained results, it may be concluded that the ph value has a strong influence on the efficiency of sulcotrione photocatalytic degradation. effect of •oh scavengers and holes information about the influence of active radicals and holes on the efficiency of photocatalytic degradation is very important in view of practical application. the results of a previous work clearly indicated that processes on the surface of catalyst play the main role in the photodegradation of sulcotrione. namely, the disappearance of sulcotrione under uv light using tio2 degussa p25 occurs via •oh and holes,36 but in the case of tp-150, the main path of degradation is through holes and o2•–, whereby surface •oh is only partly involved in the removal of sulcotrione.25 in the present work (fig. 4), the effects of addition of naf, tert-butanol and edta as scavengers of surface •oh, bulk •oh and holes, respectively, were investigated. it was found that edta has a remarkable influence on the photodegradation using bare tio2 under simulated solar irradiation. furthermore, surface •oh partly participate in the photocatalytic degradation of sulcotrione, whereby the main path of degradation is through holes. from fig. 4, it could be seen that bulk •oh have no influence on the efficiency of sulcotrione photocatalytic degradation. fig. 4. effect of active radicals and hole scavengers (10 mmol dm-3) on the efficiency of sulcotrione (c0 = 50 µmol dm-3) photocatalytic degradation using bare tio2 (0.5 mg cm-3) under simulated solar irradiation. effect of electron acceptors therefore, several approaches exist in order to improve the photocatalytic efficiency of tio2. one of the strategies for inhibiting electron−hole recom photodegradation behaviour of selected pesticides 1465 bination is to add other (irreversible) electron acceptors (h2o2 and kbro3), besides o2, to the reaction.20 the effect of h2o2 addition was investigated in the concentration range 1.0−5.0 mmol dm−3 (fig. 5a), whereby it could be noticed that the addition of h2o2 to the system did not improve efficiency of sulcotrione photocatalytic degradation. with increasing of h2o2 concentration, the efficiency of sulcotrione photocatalytic degradation decreased. one of the reasons may be that h2o2 could also act as an •oh scavenger, thereby generating less reactive hydroperoxyl radicals ho2•.37 the second reason may be that h2o2 can react with tio2 and form a peroxo compound that negatively influences the efficiency of photocatalytic degradation.38 furthermore, excess h2o2 might absorb uv light leaving less available for photocatalysis.39 t / min t / min fig. 5. effect of: a) h2o2 and b) kbro3 on the efficiency of sulcotrione (c0 = 50 µmol dm-3) photocatalytic degradation using bare tio2 (0.5 mg cm-3) under simulated solar irradiation. in the same way, the influence of kbro3 on the efficiency of photocatalytic degradation of sulcotrione was investigated (fig. 5b). it can be seen that at all kbro3 concentrations the efficiency of sulcotrione photodegradation was improved during 60 min of irradiation. this phenomenon may probably be due to reaction between bro3– and conduction band e−, which results in reduced recombination between e− and h+.37 besides, electrons can reduce bromates, leading to formation of reactive oxidizing radicals such as bro2• and hobr (reactions (2) and (3)), thereby increasing the efficiency of sulcotrione degradation:40 bro3– + 2h+ + e− → bro2• + h2o (2) bro3– + 6h+ + 6e− → [bro2–, hobr] → br− + 3h2o (3) conclusions the obtained results indicate that tp nanocomposites showed the highest photocatalytic activity for elimination of clomazone compared with bare tio2. however, sulcotrione using bare tio2 showed the highest efficiency of photo1466 lazarević et al. catalytic degradation and mineralization of all investigated organic compounds and therefore, sulcotrione was chosen for further investigations. moreover, cytotoxic experiments revealed that both growth inhibition and stimulation of different cell lines were observed, which were below 11 % in all cases. a comparison of sulcotrione removal in different types of water showed that the efficiency of sulcotrione photocatalytic degradation in environmental waters was lower compared with that in ddw. moreover, the content of ions present in water from the river danube showed that the ph value mainly influences the efficiency of sulcotrione removal. in addition, results with scavengers indicated that •oh partly participate in the photocatalytic degradation of sulcotrione, although the main path of degradation was through holes. furthermore, the addition of h2o2 as an electron acceptor decreased, while addition of kbro3 increased the efficiency of sulcotrione removal. acknowledgements. the authors acknowledge the financial support of the ministry of education, science and technological development of the republic of serbia (project no. 172042 and 172056). besides, the authors thank msc suzana ćebić, and msc dajana demonjić for their technical assistance. supplementary material additional data are available electronically at the pages of journal website: http:// //www.shd-pub.org.rs/index.php/jscs, or from the corresponding author on request. и з в о д фоторазградња одабраних пестицида: фотокаталитичка активност чистог и полианилином модификованог tio2 у присуству симулираног сунчевог зрачења марина ј. лазаревић1, весна н. деспотовић1, даниела в. шојић меркулов1, немања д. банић1, нина л. финчур1, драгана д. четојевић-симин2, мирјана и. чомор3 и биљана ф. абрамовић1 1унвиверзитет у новом саду, природно–математички факултет, департман за хемију, биохемију и заштиту животне средине, трг д. обрадовића 3, 21000 нови сад, 2универзитет у новом саду, медицински факултет, институт за онкологију војводине, др голдмана 4, 21204 сремска каменица и 3институт за нуклеарне науке винча, , п. пр. 522, 11001 београд у овом раду је испитана ефикасност фотокаталитичке разградње различитих пестицида коришћењем tio2 и модификованих tio2 наночестица полианилином коришћењем симулираног сунчевог зрачења. сулкотрион је показао највећи проценат разградње и даљи експерименти су се односили на овај хербицид. проучаване су минерализација и цитотоксичност полазног једињења и интермедијера формираних током разградње у бидестилованој води (bdv) као и ефикасност уклањања сулкотриона из различитих природних вода. садржај најзаступљенијих јона присутних у води из реке дунав су додати у bdv и процењен је њихов утицај. утврђено је да је цитотоксичност у свим случајевима испод 11 % и да је ефикасност фотокаталитичке разградње у природним водама мања у односу на bdv. надаље, додатак различитих хватача је показало да се разградња у највећој мери одиграва преко шупљина, док је присуство h2o2 смањило а kbro3 повећало ефикасност фотокаталитичке разградње у односу на систем без поменутих електрон-акцептора. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthetic route towards potential bivalent ligands possessing opioid and d2/d3 pharmacophores} j. serb. chem. soc. 84 (7) 639–647 (2019) udc 547.822’861.3:66.095.253:615.1/3 jscs–5215 original scientific paper 639 synthetic route towards potential bivalent ligands possessing opioid and d2/d3 pharmacophores ivana i. jevtić1, jelena z. penjišević1, milovan d. ivanović2 and slađana v. kostić-rajačić1* 1ictm – department of chemistry, university of belgrade, njegoševa 12, 11000 belgrade, serbia and 2faculty of chemistry, university of belgrade, studentski trg 12–16, 11000 belgrade, serbia (received 2 october, revised 5 november, accepted 29 november 2018) abstract: a scalable, cost-efficient and simple synthetic pathway towards potential bivalent opioid/dopamine receptor ligands was developed and optimized. three novel compounds that contain both opioid and dopamine pharmacophores linked by the four methylene group chain were synthesized in 33, 35 and 39 % overall yield after a four-step synthetic route starting from three commercially available n-aryl piperazines. the anilino piperidine precursor was easily prepared in three steps, as previously published, starting from 4piperidone. the synthesis presented in this paper could be of interest for heterocyclic and general organic chemistry. the newly designed compounds possessing two pharmacophores, opioid and d2/d3, are potentially useful pharmacological probes. of particular interest would be the simultaneous binding to both opioid and d2/d3 receptors, and the resulting pharmacological responses may be useful for the further understanding of tolerance and dependence phenomena in opioid clinical use and/or abuse. keywords: piperidine; piperazine; heterocycles; n-alkylation; analgesics; dopaminergic. introduction 4-anilido-piperidines and n-aryl piperazines are pharmacologically important moieties.1–6 both of these pharmacophores are seen separately in numerous compounds possessing high pharmacological activities, many of which have found their way into clinical use. 4-anilido-piperidines are best known for their analgesic activity since many of them act as μ-opioid receptor agonists in the central nervous system (cns).1–4 opioid analgesics are especially effective for the treatment of strong chronic and acute pain, when other standard analgesics, such as non-steroidal anti-inflammatory drugs (nsaids) are ineffective. there * corresponding author. e-mail: srkostic@chem.bg.ac.rs https://doi.org/10.2298/jsc181002105j 640 jevtić et al. are several highly potent opioid analgesics with a 4-anilido-piperidine core in clinical use, including fentanyl (actiq® and abstral®), sufentanil (sufenta®), alfentanil (alfenta®) and remifentanil (ultiva®). the serious drawback when opioid usage is considered are side effects, among which the most important are tolerance and dependence due to prolonged treatment of chronic pain, with the most dangerous outcome being respiratory arrest due to cns depression as a result of overdosing with opioids. the n-aryl piperazine scaffold is present in many compounds that are active towards the dopaminergic signalling system in the cns, especially d2 and d3 receptor subtypes.5 several antipsychotic drugs that are used in clinical practice for treatment of schizophrenia and bipolar disorders are compounds that contain the piperazine moiety that act as d2 antagonists, such as geodon®, clozaril®, or partial agonists, such as abilify®. d2 and d3 agonists however are clinically used for the treatment of parkinson’s disease, for example pronoran®. recently it was discovered that activation of μ-opioid receptors influences the dopaminergic signal pathway, which in turn is believed to have a reinforcing effect on some psychoactive drugs. it was found that the drugs affecting the dopaminergic system can also influence the effect of opioid agonists on cognitive functions in animal models.7–14 these results are expected to be useful for further understanding of the tolerance and dependence phenomena in opioids addiction. in the past several decades, a large number of anilido piperidines have been prepared, particularly analogues with diverse substituents on the piperidine nitrogen. interestingly, it seems that none of the substituents included a piperazine moiety.15,16 in view of the fact that many piperazines are significant d2/d3 receptor pharmacophores, the aim of this study was to develop a synthetic route towards novel compounds as potential bivalent, opioid–dopaminergic ligands, having the general structure 1 (fig. 1). apparently, none of the bivalent, opioid– dopaminergic ligands have hitherto been reported. fig. 1. the general structure of bivalent ligand containing 4-anilido-piperidine and n-aryl piperazine moieties. synthesis of bivalent opioid-dopamine receptor ligands 641 designing multi-target drugs has become a growing area of medicinal chemistry in recent years. the aim of such drug design is to provide the structures that will be superior in efficacy and safety, relative to single target drugs. introducing two pharmacophores, such 4-anilido-piperidine and n-aryl piperazine in one molecule could lead to interesting results in the pharmacological testing of such compounds, regarding their opioid and dopamine receptor activity. therefore, the development of a synthetic pathway towards bivalent opioid/dopamine receptor ligands could be of interest to synthetic, heterocyclic and medicinal chemistry in general. experimental general information unless otherwise stated all solvents were freshly distilled under argon prior to use. all reagents purchased from a commercial vendor were used as supplied. 1hand 13c-nmr spectra were recorded on a bruker avance iii spectrometer, at 500 mhz for protons (1h) and at 126 mhz for carbons (13c). chemical shifts are given in parts per million from tetramethylsilane (tms) as the internal standard in cdcl3. the 2d nmr spectra (hsqc) were recorded at 500 mhz. coupling constants (j) are reported in hz. unless otherwise stated, all spectra were recorded at 25 °c. high resolution mass spectra (hrms) were obtained with a heated esi (hesi)-orbitrap spectrometer. spectral and analytical data are given in supplementary material to this paper. all reactions were monitored by thin layer chromatography (tlc). flash and dry -column flash chromatography17,18 were performed using silica gel (10−18 or 18–32 μm, icn-woelm). melting points were obtained at a heating rate of 4 °c min-1 and are uncorrected. ir spectra were recorded using a thermo scientific nicolet 6700 fourier-transform spectrometer operated in the atr mode. structures of all new compounds were determined by methods of 1d, 2d nmr and ir spectroscopy. structures of the three final compounds were additionally confirmed by high resolution mass spectrometry (hrms). syntheses general procedure for the synthesis of anilido-piperidines 8a and b. to a magnetically stirred solution of norfentanyl 4 (300 mg, 1.3 mmol) and k2co3 (360 mg, 2.6 mmol) in mecn (12 ml) was added bromochloro alkane (13 mmol). the mixture was allowed to stir at 25 °c. the reaction was monitored by tlc with a mixture of ch2cl2/meoh = 95:5 as the eluent. after 24 h, et2o was added, and the mixture was filtered, then concentrated by rotary evaporator. 10 % hbr (0.4 ml) was added, and mixture was extracted with 2×20 ml of n-hexane. aqueous layer was then neutralised with 1.5 m solution of k2co3 (ph ≈11) and extracted with 2×20 ml of ch2cl2. the organic layers were combined and concentrated on a rotary evaporator. the crude product was purified by dry-column flash chromatography (sio2; ch2cl2/meoh = 8:2 to 0:10). general procedure for the synthesis of aryl piperazino carboxamides 7a–c. to a magnetically stirred solution of aryl piperazine hydrochloride 12a–c (3.5 mmol) in ch2cl2 (10 ml), were added et3n (425 mg, 4.2 mmol) and 4-chloro-butyrilchloride (740 mg, 5.25 mmol) at 0 °c. the mixture was then allowed to stir. the reaction was monitored by tlc, with mixture of ch2cl2/meoh = 95:5 as the eluent. after 40 min stirring at 0 °c and an additional 20 min 642 jevtić et al. at 25 °c, the reaction was complete. meoh (10 ml) was then added, and the mixture was concentrated on a rotary evaporator. a solution of k2co3 (1.5 m) was added (ph ≈11) and the mixture was extracted with 2×25 ml of ch2cl2. the organic layers were collected and concentrated on a rotary evaporator. the crude product was used in the next step without further purification. general procedure for the synthesis of the aryl piperazino carboxamide-anilino-piperidine adducts (13a–c). to a magnetically stirred solution of anilino-piperidine 6 (300 mg, 1.7 mmol) and k2co3 (2 equiv.) in mecn (12 ml), was added aryl piperazino carboxamide 7a–c (2.5 mmol). the mixture was stirred at 70 °c. the reaction was monitored by tlc, with mixture of ch2cl2/meoh = 95:5 as the eluent. the reaction was completed after 8 h of reflux and additional stirring for 10 h at 25 °c. the mixture was then concentrated on a rotary evaporator and then 50 ml of ch2cl2 was added and the mixture extracted with 2×25 ml of brine. the organic phase was concentrated on a rotary evaporator. the crude product was purified by dry-column flash chromatography (sio2; ch2cl2/meoh = 1:0 to 95:5). general procedure for the synthesis of the aryl piperazino-anilino-piperidine adducts (5a–c). to a magnetically stirred suspension of aryl piperazino-anilino-piperidine adduct 13a–c (1.0 mmol) in dry thf (12 ml) was added 1m solution of bh3 in dry thf (34 mg, 2.5 mmol) at 0 °c. mixture was allowed to stir at 25 °c. after the spontaneous boiling stopped, the mixture was heated at 70 °c. the reaction was monitored by tlc with mixture of ch2cl2/meoh = 95:5 as the eluent. after 4 h of reflux, water (1 ml) was added dropwise, followed by the addition of 5.5 m hcl (2 ml) at 25 °c. reflux was continued for an additional 4 h. mixture was then concentrated on a rotary evaporator, 50 ml of ch2cl2 was added, the layers were separated, and the organic phase was washed with 2×25 ml of brine. the organic phase was concentrated on a rotary evaporator. the crude product was purified by dry-column flash chromatography (sio2; ch2cl2/meoh = 1:0 to 95:5). general procedure for the synthesis of aryl piperazino-anilido-piperidine adducts (1a–c). to a magnetically stirred solution of aryl piperazino-anilino-piperidine adduct 5a–c (0.6 mmol) in ch2cl2 (10 ml) was added et3n (91 mg, 0.9 mmol) followed by the addition of propionyl chloride (etcocl, 166 mg, 1.8 mmol). the mixture was then allowed to stir at 25 °c. the reaction was monitored by tlc with a mixture of ch2cl2/meoh = 95:5 as the eluent. after 3.5 h, meoh (10 ml) was added, and the mixture was concentrated on a rotary evaporator. a solution of k2co3 (1.5 m) was added (ph ≈11) and the mixture was extracted with 2×25 ml of ch2cl2. the organic layers were collected, washed with 2×25 ml of brine, and concentrated on a rotary evaporator. the crude product was purified by dry-column flash chromatography (sio2; ch2cl2/meoh = 1:0 to 95:5). results and discussion the synthesis of bivalent ligands of the general structure 1 was initially attempted according to the retrosynthetic route a presented in scheme 1. this approach was anticipated to include two simple alkylation steps of readily available piperazine and piperidine derivatives. while n-aryl piperazines are commercially available, the norfentanyl 4 was obtained according to a known literature procedure.19–22 for the starting material in the first alkylation step, norfentanyl 4 was selected as it was more readily available than the n-aryl piperazine 2. however, it was expected that both secondary amines would react similarly in this reaction. the alkylating agents were synthesis of bivalent opioid-dopamine receptor ligands 643 linear α,ω-bromochloroalkanes having 3–6 methylene groups. unexpectedly, 1,4-bromochlorobutane and 1,5-bromochloropentane mainly gave the respective spiro-bicyclic quaternary ammonium salts 8a and b. apparently, the entropic factor favoured the formation of five and six member rings, rather than the linear alkylation products. since the spiro-bicyclic products were obtained under all attempted conditions, an alternative approach was sought. for further investigation of the synthetic route target compound of general formula 1, it was selected to have four methylene groups in the alkyl chain, since it could not be prepared by the previous method (scheme 2) because of quaternary ammonium ion 8a formation. scheme 1. initial retrosynthetic route (route a) and optimized retrosynthetic route (route b) for the synthesis of 4-anilido-piperidine and n-aryl piperazine containing bivalent ligands. scheme 2. initial synthetic route towards bivalent ligands 1: alkylation of 4 with alkylating agents of different chain lengths. according to the retrosynthetic route b, replacing the two successive alkylation steps of the previous synthetic route with acylation and alkylation steps seemed to be a promising alternative for connecting the two pharmacophores as 644 jevtić et al. no quaternisation is possible with an amide nitrogen. indeed, this approach gave the desired ligands of general structure 1 (scheme 3). n o n h nh n nh n n r r= h, 12a; r= 3-ome,12b; r= 2,3-cl, 12c. cl o cl1.5 equiv 1.2 equiv et3n, ch2cl2, 0 °c-r.t., 1 h hh cl n n r o cl n o 1.2 equiv boc2o, 1.5 equiv et3n ch2cl2, r.t., 24 h o o 2.0 equiv phnh2, 0.7 equiv nabh3cn, 3.0 equiv acoh meoh, r.t., 24 h oo 13 equiv tfa ch2cl2, 0 °c-r.t., 3 h n n r o n h n n h nh a b c 1.5 equiv 7a-c, 2.0 equiv k2co3 mecn, reflux, 8 h, r.t.,10 h 9 10 11 6 7a-c 6 13a= 49 % 13b= 45 % 13c= 55% n n r n h n n n n n o 1. 5.0 equiv bh3, thf, reflux, 4 h 2. 20% hcl, reflux, 4 h 3.0 equiv etcocl, 1.5 equiv et3n ch2cl2, r.t., 3.5 h 14a-c 1a = 83% meo cl cl r r = 1b = 86 % 1c = 84 % 98% 70% 86 % 96-98% n n r o n n n o n r + 15-20% h h cl 5a = 85 % 5b = 84 % 5c = 83 % scheme 3. synthesis of 4-anilido-piperidine/n-aryl piperazine adducts 1a–c. as a synthon for a four methylene linker, 4-chlorobutyryl chloride was selected.23 acylation of n-aryl piperazine hydrochloride 12a–c with 4-chlorobutyryl chloride under standard acylation conditions24 successfully provided carboxamides 7a–c in almost quantitative yields (scheme 3b). the required anilinopiperidine 6, was prepared according to scheme 3a using a known protocol.25,26 subsequent alkylation of 6 with carboxamides 7a–c provided intermediates 13a–c in moderate yields (45–55 %). the reaction was slow, requiring synthesis of bivalent opioid-dopamine receptor ligands 645 refluxing acetonitrile for several hours. higher temperatures and longer reaction times were necessary for completion of the alkylation reaction, due to the relatively low alkylation reactivity of alkyl chlorides compared to other alkylating reagents. a significant amount of side product 14a–c, which originated from alkylation of the aniline nitrogen, was detected in all three examples, while no quarterisation was observed. the aniline nitrogen is less nucleophilic than that of alkyl amines, however not sufficiently to afford chemoselective alkylation of just the secondary amino group in 6. since there is no efficient way to selectively protect the aniline nitrogen in the presence of other amino groups, the moderate yields of the desired alkylation products 13a–c made this step the least effective in this reaction route. the structure of the dialkylated side product was confirmed in the case of 14b as a representative, and the spectra are presented in the supplementary material. clean and complete reduction of the amide bond with borane afforded 5a–c in high yields. the final products 1a–c were obtained by propionylation of the aniline nitrogen and isolated in high yields, after purification and in overall yields of nearly 35 % starting from 12 (scheme 3b and c). compounds 1a–c may have significant potential in pharmacological testing and docking studies as novel bivalent ligands for opioid and dopamine d2/d3 receptors. synthesis of analogues with different chain lengths as well as their pharmacological testing and theoretical modelling, is part of ongoing research. conclusions three novel compounds that contain both opioid and d2/d3 receptors pharmacophores connected by four methylene group linkers were synthesized via a scalable, cost-efficient and simple synthetic pathway. starting from three different n-aryl piperazines 12a–c and anilino-piperidine 6, the final products 1a–c were obtained in nearly 35 % overall yield after four steps employing two acylation as well as alkylation and reduction steps. the actual pharmacological activity of the synthesized bivalent ligands cannot be predicted. however, there is a significant possibility that opioid and/or dopamine receptor activity will be revealed in various pharmacological tests. potentially, these results may help advance the understanding of the interaction between dopaminergic and opioid signal pathways as well as related phenomena. therefore, the development of the synthetic pathway towards bivalent opioid/dopamine receptor ligands could be of interest to synthetic, heterocyclic and medicinal chemistry in general. supplementary material spectral and analytical data of the synthesized compounds are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. the authors acknowledge the ministry of education, science and technological development of the republic of serbia for financial support, grant no. 172032. 646 jevtić et al. и з в о д синтетички пут за синтезу потенцијалних, бивалентних лиганада који поседују опиоидну и допамин д2/д3 фармакофору ивана и. јевтић1, јелена з. пењишевић1, милован д. ивановић2 и слађана в. костић-рајачић1 1ихтм – центар за хемију, универзитет у београду, његошева 12, 11000 београд и 2хемијски факултет, универзитет у београду, студентски трг 12–16, 11000 београд три нова једињења, која садрже опиоидну и допаминску фармакофору, спојене преко четири метиленске групе, синтетисанa су у четири фазе у укупном приносу од приближно 35 %, полазећи од три комерцијално доступна n-арилпиперазина. анилинопиперидински прекурсор је добијен у три једноставне реакционе фазе, полазећи од 4-пиперидона, према познатој литературној методи. синтеза приказана у овом раду може бити од значаја за хетероцикличну и органску хемију у целини. новосинтетисана једињења која поседују две фармакофоре, опиоидну и д2/д3, су потенцијално корисни супстрати за фармаколошка испитивања. од посебног интереса може бити истовремено везивање за опиоидне и д2/д3 рецепторе, а резултујући фармаколошки одговор био би користан у даљем разумевању толеранције и зависности, као феномена везаних за клиничку употребу и/или злоупотребу опијата. 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manjarrez alvarez, herminia i. pérez méndez*, aida solís oba, lucía ortega cabello, maría t. lara carvajal, omar e. valencia ledezma and rubria m. martínez-casares departamento de sistemas biológicos, universidad autónoma metropolitana unidad xochimilco, calzada del hueso 1100, colonia villa quietud, c. p. 04960, alcaldía coyoacán, cdmx, méxico (received 6 august 2018, revised 15 july 2019, accepted 19 august 2019) abstract: the aim of this research was to evaluate the redox system of nocardia corallina b-276 in the biotransformation of 1-phenyl-1-propanone (1a), 2-hydroxy-1-phenylethanone (2a) and methyl (2-chlorophenyl)(oxo)acetate (3a) into 1-phenylpropan-1-ol (1b), 1-phenyl-1,2-ethanediol (2b) and methyl (2-chlorophenyl)(hydroxy)acetate (3b). the biomass of n. corallina was obtained in a liquid medium with an initial ph of 8.50, but the ph changed during the 96 h of the culture media, the final ph was between 4.74 and 7.62. the n. corallina biomass biocatalyzed the enantioselective reduction of 1a–3a to the corresponding alcohols. whereas, during the process of oxidation of the rac-alcohols 1b–3b, 1b was oxidized in enantioselective way, the oxidation of 2b was not selective, but 3b was biotransformed mainly to (r)-3b. these results are indicative that n. corallina produced reductases and oxidases, whereby the biocatalytic activity was influenced by the final ph of the culture media, the reaction time and structure of the substrate. keywords: actinomycetes; oxidation–reduction; enantioselectivity; ph influence. introduction chirality is a key factor in the effectiveness of biologically active compounds, because each enantiomer can perform a different biological function, and thus, the production of enantiomerically pure compounds would be relevant that avoid or diminish the probability of side effects, toxicity or high doses of chiral drugs.1–3 a vast majority of the drugs developed nowadays contain chiral centers, and according to global industry analysts nearly 95 % of drugs will be chiral by 2020, and hence, the fda demanded manufacturers of chiral drugs to investigate the properties (physicochemical, pharmacokinetic, etc.) of all enantio * corresponding author. e-mail: hperez@correo.xoc.uam.mx https://doi.org/10.2298/jsc180806089m ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 280 manjarrez alvarez et al. mers/diastereomers to determine their individual safety and efficacy. due to this, the development of efficient chiral synthesis technologies is a challenge for many academic and industrial researchers.4,5 the application of enzymes in the synthesis of compounds for the manufacture of chemicals, agrochemicals, pharmaceuticals, cosmetics, flavors and fragrances is an attractive alternative to conventional synthetic methods, because of their high levels of chemo-, regioand stereo-selectivity, reduced formation of by-products and waste, lower energy consumption and decrease in the environmental impact.1–3 enantiomerically pure alcohols are amongst the most important building blocks for the production of chiral pharmaceuticals, flavors, agrochemicals and functional materials. they can be obtained by either asymmetric reduction of the respective prochiral ketones or kinetic resolution of the racemic alcohols. the bioreduction of ketones is more valuable than chemical methods for the synthesis of chiral alcohols, due to its remarkable chemo-, regioand stereo-selectivity together with mild reaction conditions; and environmental compatibility.6,7 pure enantiomers of 1-phenylpropan-1-ol (1b, fig. 1) have been used in the preparation of interleukin-2 inducible t cell kinase (itk) inhibitors8 and antiandrogen.9 enantiomerically pure 1,2-diols are important intermediates for various synthetic applications; for example, (r)-1-phenyl-1,2-ethanediol can be used as a building block for the preparation of (r)-norfluoxetine, (r)-fluoxetine and β-lactam antibiotics.10 methyl (r)-(2-chlorophenyl)(hydroxy)acetate is a key chiral intermediate for the synthesis of vicagrel and (s)-clopidogrel,11 which are drugs with antiplatelet activity. clopidogrel has been widely administered to atherosclerotic patients with the risk of a heart attack or stroke caused by blood clots and is the second-best-selling drug in the world, with global sales of $10 billion per year.12 r1 r2 r1 r2 1a ch2ch3 h 1b ch2ch3 h 2a ch2oh h 2b ch2oh h 3a cooch3 cl 3b cooch3 cl fig. 1. biotransformation of ketones 1a–3a and alcohols 1b–3b with n. corallina. nocardia corallina b-276 (atcc 31338) is a versatile microorganism that catalyzes redox processes, such as the oxidation of aldehydes, allylic and benzylic alcohols to obtain carboxylic acids or ketones, and enantioselective reduction of ketones to secondary alcohols in an ecofriendly manner, with good yields.13–17 the aim of this research was to evaluate the redox system of nocardia corallina b-276 in the biotransformation of 1-phenyl-1-propanone (1a), 2-hydroxy-1-phenylethanone (2a), methyl (2-chlorophenyl)(oxo)acetate (3a), 1-phen________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ biotransformation of ketones and alcohols 281 ylpropan-1-ol (1b), 1-phenyl-1,2-ethanediol (2b) and methyl (2-chlorophenyl)(hydroxy)acetate (3b) (fig. 1). experimental compounds 1(a, b) and 2(a, b), (r)-(2-chlorophenyl)(hydroxy)acetic acid and rac-(2 -chlorophenyl)(hydroxy)acetic acid were purchased from aldrich (st. louis, missouri, usa), 3a, rac-3b and (r)-3b were prepared according to previously reported methods18 and characterized by their infrared spectra (ft-ir) recorded on a perkin–elmer paragon 1600 ft as liquid films. hydrogen and carbon nuclear magnetic resonance (1h-nmr and 13c-nmr) spectra were recorded on an agilent dd2 600 mhz instrument, in cdcl3 using tetramethylsilane as the internal reference. conversion was determined by gc analysis using a hewlett– –packard hp 6890 gas chromatograph (waldbronn, germany) equipped with a flame ionization detector. the enantiomeric excess (ee) was determined by hplc analysis, performed on an agilent 1100 liquid chromatograph (hannover, germany) equipped with a diode array detector; and by tlc on silica gel 60 gf254 (merck, darmstadt, germany). organism and growth the cells of nocardia corallina b-276 (atcc 31338, were grown at 28–30 °c for three days on petri dishes containing 15 g l-1 of agar, 3 g l-1 of beef extract and 5 g l-1 of peptone (bioxon, oaxaca, méxico). then, the cells were inoculated in liquid media, which consisted on three solutions: solution a (feso4·7h2o 0.05 g l-1, k2hpo4 1.74 g l-1, (nh4)2so4 2.00 g l-1, yeast extract 1.00 g l-1); solution b (mgso4 1.50 g l-1) and solution c (glucose 2.00 g l-1, j. t. baker, usa); the ph was adjusted to 8.50±0.5. the culture was incubated in an orbital shaker at 28–30 °c, 150 rpm for 96 h.19 the cells were collected by centrifugation at 4500 rpm for 15 min, and the ph of the supernatant was measured. the cells were washed twice with potassium phosphate buffer (0.1 mol l-1, ph 7.00) and centrifuged at 4500 rpm for 15 min. biotransformation of 1-phenyl-1-propanone (1a) and 1-phenylpropan-1-ol (1b) the n. corallina cells were incubated in a 50 ml phosphate buffer (0.1 mol l-1, ph 7.00) for 30 min at 28–30 °c in an orbital shaker (150 rpm), then 1a or 1b was added separately to the whole cells with a substrate:cells mass ratio of 1:500 using, 0.6 vol. % of n,n -dimethylformamide. the mixture was shaken under the same conditions, and experiments were performed in triplicate at different final ph of the culture media and times. the sample was centrifuged at 4500 rpm for 15 min and then extracted with ethyl acetate (3×15 ml), and the organic layer was concentrated to dryness.15 the products 1a or 1b were identified by ft -ir and nmr and the spectra were in full accordance with literature.20,21 the product was dissolved in 0.5 ml of hplc 2-propanol and analyzed by gc using a supelcowax™-10 column (30m×0.25mm, 0.25 µm) at 180 °c with n2 as carrier gas at 0.8 ml min-1, the retention times were tr(1a) = 4.39 min and tr(1b) = 5.63 min. it was then analyzed by hplc equipped with two columns: chiralcel ob-h (25.0 cm×0.46 cm, 0.5 µm) column; the mobile phase was hexane:2-propanol (90:10), with a flow rate of 0.5 ml min-1, λ = 220 nm at 25 °c, the retention times were tr(r)-(1b) = 9.6 min, tr(s)-(1b) = 11.4 min, and tr(1a) = 20.4 min: chiralcel od (25.0 cm×0.46 cm, 10 µm) column; the mobile phase was hexane:2-propanol (97:3) at a flow rate of 1.0 ml min-1, λ = 220 nm at 25 °c. the retention times were tr(r)-(1b) = 12.7 min, tr(s)-(1b) = 14.9 min, and tr(1a) = 7.7 min. the absolute configuration of 1b was assigned according to the literature.22 ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 282 manjarrez alvarez et al. biotransformation of 2-hydroxy-1-phenylethanone (2a) and 1-phenyl-1,2-ethanediol (2b) the n. corallina cells were incubated in 50 ml phosphate buffer (0.1 mol l-1, ph 7.00), 30 min, at 28–30 °c in an orbital shaker (150 rpm), and 2a or 2b was added to the whole cells at a substrate:cells mass ratio of 1:100 for 2a and 1:400 for 2b, using 0.6 vol. % of n,n -dimethylformamide and shaken under the same conditions. the experiments were performed in triplicate at different final ph values of the culture media and times. the sample was centrifuged at 4500 rpm for 15 min and then extracted with ethyl acetate (4×15 ml), and the organic layer was concentrated to dryness. the product was dissolved in 0.5 ml of hplc grade 2-propanol. the gc analysis was performed with a hp-5 column (30 m×0.33 mm, 0.25 µm) (hewlett–packard, germany) at 80–200 °c, with n2 as the carrier gas at 1.0 ml min-1. the oven temperature was ramped from 80–200 °c at 10 °c min-1, held for 3 min, decreased to 80 °c at 25 °c min-1 and held for 2 min. the retention times were tr(2a) = 4.90 min and tr(2b) = = 5.79 min. then, the analyse was realized by hplc using a chiracel ob-h (25.0 cm×0.46 cm, 0.5 µm) column (daicel chemical industries, tokyo, japan). the mobile phase was hexane:2-propanol (90:10), 0.5 ml min-1, λ = 220 nm, 24 °c. for the reduction of 2a, the retention times were tr(r)-2b = 15.10 min, tr(s)-2b= 18.90 min and tr(2a) = 28.90 min. for the oxidation of 2b, the mobile phase was hexane:2-propanol (90:10), 0.8 ml min-1, λ = 260 nm, 24 °c. the retention times were tr(r)-2b = 9.04 min, tr(s)-2b = 11.38 min and tr(2a) = = 17.12 min, the absolute configuration of 2b was assigned according to the literature.23 biotransformation of methyl (2-chlorophenyl)(oxo)acetate (3a) and methyl (2-chlorophenyl)(hydroxy)acetate (3b) the n. corallina cells were incubated in 50 ml phosphate buffer (0.1 mol l-1, ph 7.00), 30 min at 28–30 °c in an orbital shaker (150 rpm), and then 3a or 3b was added to the whole cells, with a substrate:cells mass ratio of 1:500, using 0.6 vol. % of n,n-dimethylformamide. the mixture was shaken under the same conditions, and experiments were performed in triplicate at different final ph values of the culture media and times. the samples were centrifuged at 4500 rpm for 15 min and then extracted with ethyl acetate (3×15 ml), and the organic layer was concentrated to dryness. the product was dissolved in 0.5 ml of hplc grade 2-propanol and analyzed by gc using a supelcowaxtm-10 column (30 m×0.25 mm, 0.25 µm) at 230 °c, with n2 as carrier gas at a flow rate of 1.0 ml min-1. the retention times were tr(3a) = 4.6 min and tr(3b) = 6.1 min. then the product was analyzed by hplc using a chiralcel od (25.0 cm×0.46 cm, 10 µm) column; the mobile phase was hexane:2-propanol (90:10) at a flow rate of 1.0 ml min-1, λ = 235 nm at 25 °c. to assign the retention times of the enantiomers of rac-3b, methyl (r)-(2-chlorophenyl)(hydroxy)acetate was previously prepared in order to obtain the retention time of (r)-3b (10.2 min), then rac-3b was mixed with (r)-3b to enrich the enantiomer r and confirm the retention time of (r)-3b within the mixture. the retention times were tr(r) 3b = 10.2 min, tr(s)-3b= 8.9 min, and tr(3a)= 7.0 min. results and discussion nocardia corallina b-276 biomass the n. corallina biomass was produced in a liquid medium with an initial ph of 8.50. after 96 h, it was observed that in several experiments the final ph of the culture media was between 4.74 and 7.62. several authors have reported variations in the initial and final ph of the culture media during microorganism growth, and they found that the production of metabolites was dependent on ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ biotransformation of ketones and alcohols 283 these ph variations.24–26 for example, pediococcus damnosus ncfb 1832 was grown at initial ph 6.7 uncontrolled and the highest production of pediocin pd-1 was allowed at a final ph 4.14.24 the highest hydrogen production with clostridium beijerinckii l9 and clostridium tyrobutyricum fya102 was achieved under uncontrolled ph conditions (initial ph of 6.4–6.6 and final ph of 4–4.2).25 glycerol fermentation for the production of 2,3-butanediol (2,3-bd) by klebsiella pneumoniae was sensitive to ph, a decrease in the ph was the most important condition for high 2,3-bd productivity, while experiments with constant ph and avoiding ph fluctuations resulted in low amounts of 2,3-bd produced from glycerol.26 it has likewise been observed that the production and catalytic activity of enzymes depends on the ph value, depending on the dissociation constant of the ionizing groups present at the active site of the enzyme, such as aspartic acid that ionizes at ph 3–6 or histidine with a ionization ph of 5–8.27–29 the outcome of a given reaction with whole-cell not only depends on the strain being used and the substrate structure but also on the biological status of the cells, such as age and growth conditions. therefore, the predictability of whole-cell alcohol oxidations and the potential production pattern with respect to regio-, chemoand enantio-selectivity is generally low.30 therefore, controlling ph of the medium may have a negative effect over the biocatalytic activity of the microorganism, because there are interactions between the constituent amino acids on the active site of the enzyme and the substrate, where most amino acids perform acid–base catalysis, and such catalysis can also affect the activity and selectivity of oxidation–reduction reactions.24–26,31,32 based on these results, in this work it was decided not to control the ph during the growth of n. corallina and to evaluate its redox biocatalytic behaviour at the final ph of the culture media. to characterize the compounds 1(a, b) and 2(a, b), their spectra were compared with spectra data obtained for the compounds purchased from aldrich. the ir and nmr spectra, as well as gc and hplc chromatograms of 1-phenyl-1 -propanone (1a), 2-hydroxy-1-phenylethanone (2a), methyl (2-chlorophenyl)(oxo)acetate (3a) into 1-phenylpropan-1-ol (1b), 1-phenyl-1,2-ethanediol (2b) and methyl (2-chlorophenyl)(hydroxy)acetate (3b), as well as the biotransformation experiments are presented in the supplementary material to this paper. reduction of ketones 1a, 2a and 3a with n. corallina biomass the reduction of the ketones 1a, 2a and 3a to the corresponding alcohols 1b, 2b and 3b was performed using as a source of biocatalyst the n. corallina cells obtained with uncontrolled ph of the culture medium. the biotransformations were performed at different times and final ph values, and the results are presented in fig. 2 and in the supplementary material, figs. s-1–s-3. the aromatic ketone 1a without substituent was reduced in an enantioselective way (fig. s-1), the specificity of the n. corallina redox system was depen________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 284 manjarrez alvarez et al. dent on the final ph of the culture media. at final ph of the culture medium of 5.36 and 5.67, the reduction of 1a to the corresponding alcohol 1b was 98 and 80 % respectively, however a decrease of ee was observed from 80 to 10 %. however, at a final ph of 7.62, total conversion and enantioselectivity was observed regardless of the reaction time. in every experiment, only the (r)-1b enantiomer was obtained. then, it was decided to study the biotransformation of 1a with n. corallina biomass obtained at final ph of 5.36 because in fig. s-1 an influence of the reaction time was noted. in the first 24 h, it could be observed that 1a was completely transformed to (r)-1b (fig. 2); but after 48 h, a slight decrease in the quantity of (r)-1b from 90 to 75 % and an increase in the amount of (s)-1b from 10 to 25 % were observed, probably due to a competition between the oxidation and reduction reactions. between 72 and 96 h, oxidation of the alcohol 1b to the ketone 1a was observed, but this reaction was not enantioselective because the proportion of (r)-1b/(s)-1b was constant (72/28, fig. 2). fig. 2. biotransformation of 1a with n. corallina biomass, obtained with uncontrolled ph of the culture medium (ph 5.36). in the fig. s-2, it could be observed that the conversion, as well as the ee, of compound 2a to compound 2b at different times using substrate to cell ratio of 1:100 was dependent on the media ph, which was mostly acidic. thus, a low conversion was observed (39 and 60 %), however, at 20 and 144 h, enantioselectivity was evident with production of the (r) enantiomer regardless the ph (98 %). the production of the (r) enantiomer in the reduction of compound 2a was previously reported with different microorganisms.27,28,33,34 the low conversion could have been because the regeneration of nad+ by lactate dehydrogenase was slow, thus becoming the limiting factor in the reduction of compound 2a because of the side reaction between compound 2a and nad+; thus being a factor to be examined in later studies when the amount of substrate is considered.35,36 ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ biotransformation of ketones and alcohols 285 in the case of methyl(2-chlorophenyl)(oxo)acetate (3a, fig. s-3), it was reduced quantitatively at the two final ph values (5.80 and 7.30), which produced mainly the alcohol (r)-3b, although at the lower ph, the enantioselectivity was highest (62 % for ph 5.8 and 22 % for ph 7.30) at 24 h, but at 48 h and ph 5.80, the ee remained constant and at ph 7.30, the enantiomeric excess increased from 22 to 50 %. the reductase activity and enantioselectivity of the n. corallina cells were mainly influenced by the final ph of the culture media, and the structure of the substrate, fig. 2 and figs. s-1–s-3. on the one hand, the ph can affect the ionization of the active site of the enzyme as well as the substrate, e.g., as observed in figs. s-1 and s-2, the ph optimum to achieve maximum reductase activity was approximately at ph 7.62. it should be noticed that in a whole cell catalysis, such as the one studied in this research, there may be other enzymes that could have affinity for the substrate.35,37 considering the structure of 1a, the alkyl group induced the biotransformation with high selectivity and an excellent conversion. in contrast, it was easier for 2a to generate hydrogen ions, which probably competed with nad+ during the regeneration of the oxide reductase making the bioconversion less effective compared to 1a. regarding 3a, the presence of an ester group in the side chain makes the enzyme more active,38 which is the reason for the complete transformion of 3a to 3b. the results of the biotransformation of 1a were different from those obtained with 2a and 3a at 24 h because in the case of 1a (fig. s-1), the conversion and enantioselectivity were less influenced by the final ph. at ph values of 5.36 and 7.62, the conversions were 98 %, and only the enantiomer (r)-1b was present, the biotransformation 2a (fig. s-2) at final ph of 4.74 conversions were 39 %, and again only the enantiomer (r)-2b was observed. in contrast, the reduction of 3a (fig. s-3), the final ph were 5.80 and 7.30 and the transformation was almost quantitative and the enantioselectivity was medium. resolution of rac-alcohols 1b–3b by selective oxidation with n. corallina the oxidations of the alcohols 1b–3b to the corresponding ketones 1a–3a were performed using the n. corallina cells obtained with uncontrolled ph of the culture medium as the biocatalyst. the biotransformations were performed at different times and final ph values. the results are given in figs. 3 and 4, and s-4 and s-5 of the supplementary material. in fig. s-4, it could be observed that only (s)-1b was oxidized to ketone 1a, while (r)-1b remained in the enantiopure form if the final ph was 7.62 (24 h), with a slightly variation in the percent ketone was observed on extending the reaction time to 36 h. a competitive reduction may be involved to enrich the (r)-alcohol. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 286 manjarrez alvarez et al. fig. 3. biotransformation of 2b with n. corallina biomass, final ph 5.99. fig. 4. biotransformation of 3b with n. corallina biomass obtained with uncontrolled ph of the culture medium (5.90 and 7.30), substrate:cells ratio (1:500). in fig. s-5, the conversion, as well as the ee from compound 2b to compound 2a, at different final ph values and times using a substrate:cell ratio 1:400 can be seen. depending on the media, the final ph after the incubation of the bacteria and different conversions can be observed at 48 h, i.e., from 12 % at ph 6.18 to 42 % at ph 6.8 with a low ee for both ph values. these results may be due to byproduct formation, since at ph 6.8 after 96 h, a quantitative conversion was achieved with a higher ee, while after 120 h, the conversion diminished but the highest ee was observed. after 96 h at ph values of 5.99, 6.28 and 6.80, different conversions and ee values can be observed. the highest conversion and ee were obtained at ph 6.28, this could be because at the same time that n. corallina oxi________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ biotransformation of ketones and alcohols 287 dizes alcohol 2b, another enzyme, such as glycerol dehydrogenase, acts on the same substrate.35 as could be seen in fig. 3, at ph 5.99 after 24 h, a stereoinversion process occurs and the ee remains constant after 48 h. a first attempt to obtain diols and ketones using n. corallina whole cells was achieved when it was determined that the conversion is low in acidic ph although deracemization was observed. observing both reactions, it could be concluded that the ph value is an essential factor because either the substrate can be ionized or the active site can be modified, as was the case of glycerol dehydrogenase and its cofactors, thus affecting the binding of the substrate to the enzyme. during the expected oxidation of 3b (fig. 4) the quantity of ketone was insignificant, but an increase in the quantity of (r)-3b was observed. several authors found that the activity of reductases from different microorganisms was higher with α-ketoesters than with ketones, but the enantioselectivity was higher with ketones,39,40 which is similar to the results obtained with n. corallina. this indicates that the oxidation of 3b is slower than the reduction of 3a. when the final ph of the culture medium is slightly acidic (ph 5.90), n. corallina biotransforms 3b to the ketone (3 %), with 32 % ee of (r) in the remnant alcohol at 24 h. on extending the reaction time to 48 h, the ketone was not observed and only a slight increment to 37 % ee of (r) was obtained. at a ph 7.30, 1 % of 3a was obtained at 24 h but the ketone was not detected at 48 h. in addition, at 24 and 48 h, 17 and 40 % ee of (r) were observed. this could be explained by the stereoinversion of the alcohol (s) to (r) via the ketone. it was also observed that n. corallina can perform the bio-oxidation of rac-3b to 3a followed by reduction of 3a to 3b, leading to an enantio-enrichment of (r)-3b, with a substrate:cells ratio of 1:500, during 48 h. these results fully demonstrated the redox ability of n. corallina to obtain enantiopure alcohols. increasing the substrate:cells ratio and the time to 1:1000 and at 120 h, at ph 7.57, the conversion to ketone (3a) was 92 % and residual 3b was obtained with an ee of 10 %. thus under these conditions, n. corallina oxidizes rac-3b, inhibiting the inverse reaction. the substituents of the carbonyl (1a–3a) exerted an important influence over the biocatalytic activity of the n. corallina enzymes, due to steric hindrance and electronic effects.41 in the case of the aromatic ketone 2a substituted in the side chain by –oh, the behaviour of the redox system was different, because the reduction was enantioselective independently of the final ph of the culture medium, but the oxidation of rac-2b was not a favourable reaction. of the three compounds of pharmaceutical importance, 3a was the most interesting compound due to the ester group (α-ketoester) in the side chain. with 1a and 2a, the reduction was enantioselective, but with 3a the reduction was almost quantitative but with ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 288 manjarrez alvarez et al. low ee; besides, during the expected oxidation of 3b the quantity of ketone was insignificant, but an increase in the quantity of (r)-3b was observed. conclusions the biocatalytic activity of the redox system of n. corallina, reductases and oxidases, was dependent of the course of the growth of the microorganism, such as the final ph value, time of reaction and structure of the substrate. n. corallina was able to reduce ketones 1a–3a, oxidize the alcohols (s)-1b, (s)-2b and 3b to the corresponding ketones. this allows for the enrichment of (r)-alcohols by biocatalytic synthesis with n. corallina biomass, obtained with uncontrolled ph of the culture media during the growth. supplementary material analytical and spectral data of the synthesized compounds are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. the authors would like to thank the department of biological systems of the metropolitan autonomous university, xochimilco unit, for the support. и з в о д примена редокс-система nocardia corallina b-276 у енантиоселективној биотрансформацији кетона и алкохола norberto manjarrez alvarez, herminia i. pérez méndez, aida solís oba, lucía ortega cabello, maría t. lara carvajal, omar e. valencia ledezma и rubria m. martínez-casares departamento de sistemas biológicos, universidad autónoma metropolitana unidad xochimilco, calzada del hueso 1100, colonia villa quietud, c. p. 04960, alcaldía coyoacán, cdmx, méxico циљ овог рада је био испитивање редокс-система nocardia corallina b-276 у реакцији биотрансформације 1-фенил-1-пропанона (1a), 2-хидрокси-1-фенилетанона (2a) и метил -(2-хлорфенил)(оксо)ацетата (3a) у 1-пропан-1-ол (1b), 1-фенил-1,2-етандиол (2b) и метил (2-хлорфенил)(хидрокси)ацетат (3b). биомаса n. corallina добијена у воденом раствору при почетном ph 8,50 се мењала током 96 h и коначан ph раствора је био између 4,74 и 7,62. биомаса n. corallina врши биокатализу енантиоселективне редукције 1a–3a до одговарајућих алкохола. такође, током реакције рацемских алкохола 1b–3b, код 1b енантиоселективно је оксидован један од енантиомера, оксидација 2b није селективна, док се 3b углавном преводи у (r)-3b. добијени резултати указују да n. corallina производи редуктазе и оксидазе, док је биокаталитичка активност под утицајем крајњег ph медија, реакционог времена и структуре супстрата. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis, characterization and anthelmintic activity evaluation of pyrimidine derivatives bearing carboxamide and sulphonamide moieties} j. serb. chem. soc. 83 (4) 401–409 (2018) udc 547.853+542.913:547.447–327+ jscs–5084 547.541.521:615.284 original scientific paper 401 synthesis, characterization and anthelmintic activity evaluation of pyrimidine derivatives bearing carboxamide and sulphonamide moieties david i. ugwu1,2*, uchechukwu c. okoro1 and narendra k. mishra2 1department of pure and industrial chemistry, university of nigeria, nsukka, 410002, nigeria and 2department of chemistry, indian institute of technology, kanpur, 208016, india (received 27 january, revised 5 october, accepted 9 october 2017) abstract: pyrimidines, sulphonamides and carboxamides have shown a large number of pharmacological properties against different types of diseases including helminthiasis. seventeen new pyrimidine derivatives bearing sulphonamide and carboxamide were synthesized and investigated for their in vitro anthelmintic properties. substituted benzenesulphonyl chlorides 15a–c were treated with various amino acids (16a–h) to obtain benzenesulphonamide derivatives 17a–l. compounds 17a–f were subsequently treated with benzoyl chloride to obtain the n-benzoylated derivatives 19a–f. further reactions of compounds 19a–f and 17g–l with 4or 2-aminopyrimidine (20) using boric acid as a catalyst gave the required sulphonamide carboxamide derivatives 21a–q in excellent yields. the compounds were isolated in their analytical grade and characterized using ftir, 1h-nmr, 13c-nmr and hrms. the in vitro anthelmintic studies showed that all the synthesized compounds possessed anthelmintic property. compounds 21a–c, e, g, m and p showed mean paralyzing times of 15, 19, 14, 18, 19, 19 and 18 min, respectively, at 100 mg ml-1 compared to 10 min for albendazole. compounds 21a–c, g and m had mean death times of 18, 24, 16, 20 and 25 min, respectively, at 100 mg ml-1 compared to 13 min for albendazole. keywords: catalysis; anthelmintics; synthesis; carboxamides; sulphonamides. introduction helminthiasis is causing untold misery to infected individuals. anthelmintics are a group of anti-parasitic drugs that expel parasitic worms and other internal parasite from the body by either stunning or killing them without causing significant damage to the host.1 resistance to benzimidazoles (1) and closantel (2), scheme 1, used to treat helminthiasis have been reported. there are genetic features in parasitic helminthes that favour the development of anthelmintic resis * corresponding author. e-mail: zuchukwu.ugwu@unn.edu.ng https://doi.org/10.2298/jsc170127109u ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 402 ugwu, okoro and mishra ch 3 s hn hn o o h 3 c n h 3 c o o hn n hn o h 3 c o o ohn ns n o o h 3 c ch 3 nh 3 c o n o o o h 3 c ch 3 n ch 3 o h 3 c o o h 3 c ch 3 n ch 3 o no o o ch 3 h 3 c n h 3 c o ch 3 o o scheme 1. the structures of some anthelmintics. tance.2 frequent usage of the same group of anthelmintic drugs, use of anthelmintics in sub-optimal doses, prophylactic mass treatment of domestic animals and continuous use of a single drug have contributed to the widespread development of anthelmintic resistance.3 the spread of resistance to triclabendazole, the main drug used in the treatment of fluke infections, is of global concern.4 ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ synthesis of new anthelmintic agents 403 pyrimidines have attracted attention as an important class of heterocyclic compound in chemotherapy. alloxan (3), a diabetogenic agent,5 sulfadiazine (4), sulfamerazine (5) and sulfadimidine (6), a potent agent against urinary tract infections and cerebrospinal meningitis6 all contain the pyrimidine moiety. the biological importance of the pyrimidine ring was further highlighted by the reported use of sulphonamide–trimethoprim (7) combinations for the treatment of opportunistic infections in patients with aids,7 5-iododeoxyuridine (8), for the treatment of viral infections,8 and zidovudine (9) as a potent inhibitor of in vivo replication and cytopathic effects of hiv.9 the wide range of biological activities of pyrimidines are well reported in the literature.10–19 hunziker20 in 1967 reported pyrantel pamoate (10) as a depolarizing neuromuscular blocking agent that causes spastic paralysis in helminthes when employed in the treatment of infestations with pinworms and roundworms. this report suggests the possible use of pyrimidine derivatives as anthelmintic agents. in addition to their traditional uses as antibacterial agents,21 sulphonamides have also being widely used as diuretics,22 anticonvulsant,23 anticancer,24 antiretroviral,25 antihypertensive,26 and antimalarial27 agents amongst others. mrozik and matawan as early as 1976 patented the synthesis of substituted benzenesulphonamides that possessed anthelmintic properties.28 mohan et al.29 reported the synthesis of some new methylpyrimidine sulphonamides that possessed anthelmintic activity comparable to that of piperazine citrate. vijaya et al.30 reported the synthesis of benzothiazole derived benzenesulphonamides that possessed anthelmintic activity comparable to that of albendazole. kumar et al. reported some benzenesulphonamide derivatives that possessed mean paralyzing times comparable to that of mebendazole.31 babu and selvakumar also reported some isoindole derivatives of benzenesulphonamide that could cause paralysis in 42 min at 10 mg per group.32 these findings point to the fact that sulphonamide derivatives could play a leading role in the fight against helminthiasis. carboxamides are ubiquitous functional groups in most drug molecules. albendazole (11), mebendazole (12), nitazoxanide (13) and emodepside (14) are all carboxamide-containing molecules used as anthelmintic (scheme 1). kumar and joshi33 also reported carboxamide derivatives of 3h-1,5-benzodiazepine as promising anthelmintic with some derivatives possessing an activity better than that of piperazine citrate. in view of the need for new anthelmintic agents,2,4 in this work, the boric acid-catalysed syntheses of pyrimidine-derived carboxamides bearing a sulphonamide functionality are reported. the synergy arising from the successful incorporation of pyrimidine ring, carboxamide and sulphonamide pharmacophore was exploited in this research. it was expected that since the pharmacophores have individual anthelmintic activity, their successful incorporation in one molecule would improve the anthelmintic activity of the compound. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 404 ugwu, okoro and mishra experimental all reactions requiring inert atmosphere were performed under a nitrogen atmosphere. drying of solvents was achieved using molecular sieves for 48 h. all reagents were purchased from commercial suppliers, aldrich, merck, fluka, avra, sd fine and alfa aesar. thin layer chromatography was performed using silica plates purchased from avra. the plates were visualized under uv light (popular india, india). the ft-ir spectra of the compounds were run in a perkin–elmer spectrum version 10.03.06 and the bands are presented in wavenumbers. proton and carbon-13 nmr spectroscopy were run in dmso-d6 and cd3od, unless otherwise stated on either jeol 500 or 400 mhz instruments. the 1h-nmr and 13c-nmr spectra were recorded at a frequency of 400 and 100 mhz, respectively, using the 400 mhz instrument and at 500 and 126 mhz, respectively, using the 500 mhz instrument. the chemical shifts are reported in ppm with reference to tetramethylsilane. mass spectrometry was performed using micro-tof electrospray time of flight (esi-tof) mass spectrometer, sodium formate was used as the calibrant. some of the mass spectra were recorded in the negative mode and others in the positive mode. all experiments were realised at prof. sandeep verma’s laboratory, department of chemistry, indian institute of technology, kanpur, india. melting points were determined using a digital melting point apparatus and are uncorrected. general procedure for the synthesis of substituted benzenesulphonamides 17a–l sodium carbonate (na2co3, 1.590 g, 15 mmol) was added to a solution of an amino acid (16a–h, 12.5 mmol) in water (15 ml) with continuous stirring until all the solutes had dissolved. the solution was cooled to –5 °c and the appropriate benzenesulphonyl chloride (15a–c, 15 mmol) was added in four portions over a period of 1 h. the slurry was further stirred at room temperature for about 4 h. the progress of the reaction was monitored using tlc (meoh/dcm, 1:9). upon completion of the reaction, the mixture was acidified using 20 % aqueous hydrochloric acid to ph 2. the crystals were filtered via suction and washed with ph 2.2 buffer. the pure products 17a–l were dried over self-indicating fused silica gel in a desiccator. general procedure for the synthesis of n-benzoyl derivatives of benzenesulphonamides 19a–f the appropriate benzenesulphonamide (17a–f, 1.0 mmol) was dissolved in naoh (10 %, 10 ml) in a 50 ml round bottom flask. benzoyl chloride (18, 1.1 mmol, 0.2 ml) was transferred into the solution of the appropriate benzenesulphonamide and stirred at room temperature. the reaction progress was monitored by tlc (3 % meoh in dcm) to the disappearance of the benzenesulphonamide spot. upon completion of the reaction, the solution was transferred into a beaker containing crushed ice and then acidified to a ph of 3 with concentrated hydrochloric acid. the solid was collected via suction filtration and transferred into a beaker containing ccl4 (10 ml), covered with a watch glass and boiled for 10 min. the mixture was allowed to cool slightly and then filtered. the obtained product 19a–f was washed with 10– –20 ml of ccl4 and dried over fused self-indicating silica gel in a desiccator. general procedure for the synthesis of the novel pyrimidine derivatives 21a–q to a suspension of n-benzoyl substituted benzenesulphonamide (19a–f) or substituted benzenesulphonamide (17g–l, 1.0 mmol) in dry toluene (40 ml), in a flask equipped with a dean–stark apparatus for azeotropic removal of water, was added 4or 2-aminopyrimidine (20, 1.0 mmol) and boric acid (0.1 mmol) at room temperature and then refluxed for 8 h. on completion of the reaction, as monitored by tlc, the reaction mixture was precipitated to amides by the addition of about 40 ml n-hexane.34 the pyrimidine derivative was obtained ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ synthesis of new anthelmintic agents 405 via suction filtration, washed with n-hexane and dried over fused silica gel or concentrated using a rotary evaporator and dried over vacuum in the case of the oily products. the 1h-nmr and 13c-nmr spectra were recorded at a frequency of 400 and 100 mhz, respectively, using the 400 mhz instrument and at 500 and 126 mhz, respectively, using the 500 mhz instrument. anthelmintics studies the anthelmintic activity studies were performed against giardia duodenalis at 2 mg ml-1 concentration using the garg and atal method.35 suspensions of the samples were prepared by triturating the synthesized compounds (100 mg) with tween 80 (0.5 %) and distilled water and the resulting mixtures were stirred using a mechanical stirrer for 30 min. the suspensions were diluted to contain 0.2 % of the test samples. a suspension of the reference drug, albendazole, was prepared at the same concentration in a similar way. three sets of g. duodenalis of almost similar sizes (2 inch in length) were placed in petri plates of 4″ diameter containing 50 ml of a suspension of a test sample or the reference drug at room temperature. another set of g. duodenalis was kept as control in 50 ml suspension of distilled water and tween 80 (0.5 %). the paralyzing and death times were noted and their mean was calculated for triplicate sets. the death time was ascertained by placing the earthworms in warm water (50 °c), which stimulated the movement if the worm was alive. results and discussion the reaction of substituted benzenesulphonyl chloride (15a–c) with various amino acids (16a–h) in the presence of sodium carbonate gave various benzenesulphonamides (17a–k). further reactions of compounds 17a–f with benzoyl chloride (18) in the presence of sodium hydroxide gave n-benzoyl derivatives (19a–f). the boric acid catalysed reaction of compounds 19a–f and 17g–l with 4 and 2-aminopyrimidine (20) gave new pyrimidine derivatives (21a–q), as shown in scheme 2. the analytical and spectral data of the synthesized compounds are given in the supplementary material to this paper. the spectral characterizations are in agreement with the structures. in the ftir spectra, the diagnostic bands at 3412–3217 cm–1 were assigned to nh stretching vibrations. the bands between 1700–1661 cm–1 (two bands per molecule), 1623–1601 cm–1 (two bands per molecule) and 1586–1456 cm–1 (two bands per molecule) were assigned to c=o, c=n and no2 stretching, respectively. compound 21c showed two bands at the nh region which were assigned to the nh of pyrimidine and of the indole ring. among the hydroxyl derivatives, the bands between 3312–3203 cm–1 were assigned to the oh group. in the proline derivatives 21j–q, the oh band disappeared. in both the 4-hydroxyproline and proline derivatives 21g–q, there was only one c=o band between 1749–1659 cm–1. in the 1h-nmr spectra, the peaks at 8.71–8.55 ppm appearing as a doublet were assigned to the 2h of the pyrimidine ring. the lone hydrogen of the pyrimidine appeared at 8.45–8.30 ppm as a singlet. in compound 21c, the nh proton of indole appeared at 10.66 ppm. in the 4-hydroxyproline derivatives, the singlet at 8.35–8.28 ppm was assigned to the 1h of the pyrimidine ring; while the doublet at ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 406 ugwu, okoro and mishra r s cl o o + nh2 r 1 o oh i. na2co3, 0 o c, 1 h rt, 4 h ii. hcl r s nh o o r 1 oh o cl o i. naoh (10%) ii. hcl o2n s n o o r 1 oh o o b(oh) 3, dry toluene ii. n-hexane 15a-c 16a-h 17a-f 18 19a-f 20 21a-f a= no 2, b= ch3, c=h 2 h or r s o o n o oh r 1 17g-l 21g, i, l, m, p b(oh) 3, dry toluene ii. n-hexane 20 nn nh2 or nn nh2 r s n o o r 1 nh o o n n r s o o n o nh r 1 n n r s o o n o nh r 1 n n 21h, j, k, n, o, q 16a: r1=h; 16b: r1=bn; 16c: r1=2-indolyl; 16d: r1=i-bu; 16e: r1=s-bu; 16f: r1=i-pr; 16g=l-4-hydroxyproline and 16h= l-proline; 17a–f: r=no2; 17a: r 1=h; 17b: r1=c8h9; 17c: r1=2-indolyl; 17d: r1=i-bu; 17e: r1=s-bu; 17f: r1=i-pr; 17g: r=no2, r 1=oh; 17h: r= no2, r 1=h; 17i: r=ch3, r 1=oh; 17j: r=ch3, r 1=h; 17k: r=h, r1=oh; 17l: r=h, r1=h; 19a–f: r=no2; 19a: r 1=h; 19b: r1=bn; 19c: r1=2-indolyl; 19d: r1=i-bu; 19e: r1=s-bu; 19f: r1=i-pr; 21a–f: r=no2; 21a: r 1=h; 21b: r1=bn; 21c: r1=2-indolyl; 21d: r1=i-bu; 21e: r1=s-bu; 21f: r1=i-pr; 21g: r=no2, r 1=oh; 21i: r=ch3, r 1=oh; 21l: r=h, r1=oh; 21m: r=no2, r 1=h; 21p: r=h, r1=h; 21h: r=no2, r 1=oh; 21j: r=ch3, r 1=oh; 21k: r=h, r1=oh; 21n: r=no2, r 1=h; 21o: r=ch3, r 1=h; 21q: r=h, r1=h scheme 2. synthetic route to the pyrimidine derivatives. 8.25–8.08 ppm were assigned to the 2h of the pyrimidine. in the 2-aminopyrimidine derivatives, the pyrimidine rings appeared as a triplet and two doublets between 8.76–8.08 ppm. the oh peak appeared as a singlet at 4.31–4.17 ppm. 13c-nmr spectra of the derivatives, excepting the proline and 4-hydroxyproline derivatives 21g–q, showed two peaks between 174.99–163.88 ppm assigned to the c=o carbons. the peaks at 158.24–152.02 ppm were assigned to c=n carbons. the c–oh peak of the 4-hydroxypyrimidine derivatives appeared at 69.31–68.91 ppm. the results of the anthelmintic activity are presented in table i. the anthelmintic activities increased as the concentration increased. all the synthesized compounds possessed anthelmintic activity with a mean paralyzing time ranging from 25–75, 18–60 and 14–41 min at 25, 50 and 100 mg ml–1, respectively, compared to 28, 20 and 10 min for albendazole. the mean death time ranged from 30–109, 20–97 and 16–63 min, compared to 35, 25 and 13 min for albendazole at 25, 50 and 100 mg ml–1, respectively. compounds 21a and c had comparable anthelmintic activity to that of albendazole. among the 4-hydroxyproline derivatives 21g–l, the trend of activities showed that 4-hydroxy-1-[(4 -nitrophenyl)sulfonyl]-n-(pyrimidin-4-yl)pyrrolidine-2-carboxamide (21g) was the most active. the results showed that with the exception of the 4-aminopyrimidine derivative 21g, which was more active than the 2-aminopyrimidine ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ synthesis of new anthelmintic agents 407 derivative 21h, the 2-aminopyrimidines were more active than the 4-aminopyrimidine derivatives in the 4-hydroxyproline series. among the proline derivatives, the 4-aminopyrimidines were more active than the 2-aminopyrimidines. considering the substitution in the benzene ring, the p-nitro derivatives were the most active. the trend among the 4-aminopyrimidines was no2>ch3>h, while that of the 2-aminopyrimidines was h>no2>ch3. the trend of the activities imply that substitution at the para position of the benzene ring improved activity among the 4-aminopyrimidine derivatives, whereas the unsubstituted derivatives had better activity among the 2-aminopyrimidine derivatives. compounds 21a, 21c, 21g and 21m are worthy of further development as anthelmintic agents given their similar anthelmintic activities to that of albendazole. table i. in vitro anthelmintic activities of the new derivatives sample mean paralyzing time, min mean death time, min c / mg ml-1 25 50 100 25 50 100 21a 25 22 15 36 25 18 21b 39 29 19 64 44 24 21c 22 18 14 30 20 16 21d 47 41 28 79 67 42 21e 47 34 18 79 68 42 21f 46 38 20 73 49 27 21g 37 26 19 59 34 20 21h 48 38 21 83 65 30 21i 55 40 23 89 77 45 21j 49 44 22 74 56 29 21k 48 36 20 76 61 28 21l 56 42 24 92 75 49 21m 39 31 19 63 39 25 21n 48 40 26 90 78 42 21o 75 62 41 109 95 59 21p 39 31 18 63 52 29 21q 75 60 41 103 97 63 albendazole 28 20 10 35 25 13 conclusions in conclusion, novel pyrimidine derivatives bearing sulphonamide and carboxamide moieties were successfully synthesized through boric acid catalysed direct amidation of various benzenesulphonamides and 4and 2-aminopyrimidine in good to excellent yield. this synthetic approach provided further highlights into the utility of boric acid in direct amidation reactions of unactivated carboxylic acids. the procedure reported herein is simple, economical, efficient and environmentally friendly. additionally, the derivatives were precipitated in their analytical grade without the necessity for chromatographic purification. four of ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ 408 ugwu, okoro and mishra the seventeen new derivatives had comparable anthelmintic activity to that of albendazole and as such could be further developed as alternative anthelmintic agents to combat the resistance that will certainly follow the use of monotherapy in helminthiasis. supplementary material analytical and spectral data of the synthesized compounds are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. и з в о д синтеза, карактеризација и испитивање антихелминтичке активности деривата пиримидина који садрже карбоксамидне и сулфонамидне функционалне групе david i. ugwu1,2, uchechukwu c. okoro1 и narendra k. mishra2 1 department of pure and industrial chemistry, university of nigeria, nsukka, 410002, nigeria and 2 department of chemistry, indian institute of technology, kanpur, 208016, india пиримидини, сулфонамиди и карбоксамиди показују велики број фармаколошких особина према различитим узрочницима болести, међу којима су и глисте. синтетисано је седамнаест нових деривата пиримидина, који садрже сулфонамидне и карбоксамидне функционалне групе, и испитана је њихова in vitro активност према црвима. супституисани бензенсулфонил хлориди 15a–c у реакцији са различитим амино-киселинама (16a–h) дају бензенсулфонамидне деривате 17a–l. једињења 17a–f су у реакцији са бензоил-хлоридом дали n-бензоиловане деривате 19a–f. у наставку, добијени деривати 19a–f и 17g–l са 4или 2-аминопиримидином (20), у присуству борне киселине као катализатора, као производе дају сулфонамидне и карбоксамидне деривате 21a–q, у одличном приносу. једињења су изолована и окарактерисана помоћу ftir, 1h-nmr, 13c-nmr и hrms. испитивањем in vitro активности према црвима показано је да су сва једињења активна. једињења 21a–c, e, g, m и p показују средње време изазивања парализе 15, 19, 14, 18, 19, 19 и 18 min при 100 mg ml-1 редом, што је блиско активности коју показује албендазол (10 min). једињења 21a–c, g и m показују средње време парализе 18, 24, 16, 20, односно 25 min при 100 mg ml-1. 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synth. 81 (2005) 262 35. l. c. garg, c. k. atal, indian j. pharm. sci. 59 (1963) 245. ________________________________________________________________________________________________________________________ (cc) 2018 scs. available on line at www.shd.org.rs/jscs/ << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {ab initio study of mechanism of forming a spiro-sn-heterocyclic ring compound by cycloaddition reaction of h2c=sn: and ethylene} j. serb. chem. soc. 84 (3) 293–301 (2019) udc 546.812:66.095.252.091.7:537.872 jscs–5184 original scientific paper 293 ab initio study of the mechanism of formation of a spiro-sn-heterocyclic ring compound by the cycloaddition reaction of h2c=sn: and ethylene xiaojun tan1* and xiuhui lu2** 1school of biological science and technology, university of jinan, jinan 250022, p. r. china and 2school of chemistry and chemical engineering, university of jinan, jinan 250022, p. r. china (received 3 june, revised 2 september, accepted 10 september 2018) abstract: x2c=sn: (x = h, me, f, cl, br, ph, ar…) are new species of chemistry. the cycloaddition reactions of x2c=sn: is a new study field of stannylene chemistry. the mechanism of cycloaddition reaction of singlet h2c=sn: with ethylene is studied for the first time using the mp2/genecp (c, h in 6-311++g; sn in lanl2dz) method in this paper. from the potential energy profile, it could be predicted that the reaction has one dominant reaction channel. the reaction rule presented is that the 5p unoccupied orbital of tin in h2c=sn: sidewise overlaps with the bonding π orbital of ethylene resulting in the formation of an intermediate. the instability of the intermediate makes it isomerise to a four-membered ring stannylene. as the 5p unoccupied orbital of the sn atom in the four-membered ring stannylene and the π orbital of ethylene form a π→p donor–acceptor bond, the four-membered ring stannylene further combines with ethylene to form another intermediate, and this intermediate further isomerises to a spiro-sn-heterocyclic ring compound. the sn in the spiro-sn-heterocyclic ring compound is combined with adjacent atoms by sp3 hybridization. the results of this study reveal the mechanism of cycloaddition reaction of x2c=sn: with symmetric π-bond compounds. keywords: h2c=sn:; four-membered ring stannylene; spiro-sn-heterocyclic ring compound; potential energy profile. introduction the unsaturated olefins of the group iv elements (c, si, ge) are all active intermediates,1–6 their cycloaddition reaction have been studied7–13 and with progress of these studies, the study on cycloaddition reactions of unsaturated stannylene should also be put on the agenda. however, there have hitherto been no published reports concerning cycloaddition reactions of unsaturated stan *,** corresponding authors. e-mail: (*)jndxswxy@163.com; (**)lxh@ujn.edu.cn https://doi.org/10.2298/jsc180603072t 294 tan and lu nylene. unsaturated stannylenes (i.e., x2c=sn:) are a new chemical species and new study field of stannylene chemistry. in particular, the mechanism of their cycloaddition reaction and the rules of cycloaddition reactions between x2c=sn: and symmetric π-bonded compounds should be studied. in this paper h2c=sn: and ethylene were selected as model molecules, and the cycloaddition reaction mechanism (considering h transfer simultaneously) was investigated and analyzed theoretically. the research result indicates the laws of cycloaddition reaction between x2c=sn: and symmetric π-bonded compounds, which are significant for the synthesis of a small-ring containing sn and spiro-sn-heterocyclic compounds. the study extended the research area and enriched the research content of stannylene chemistry. calculation method the method of the second-order perturbation theory (mp2)14 and gaussian 09 package were used to optimize the structure of h2c=sn: and its cycloaddition reaction with ethylene, its transition state form at the mp2/genecp (c, h in 6-311++g**; sn in lanl2dz level) of theory. in order to further confirm the correctness of the relevant species and obtain the thermodynamic function for the species, vibration analysis was included. finally, the intrinsic reaction coordinate (irc)15,16 was also calculated for all the transition states to determine the reaction paths and directions. results and discussion as theoretical research shows that the ground state of h2c=sn: (r1) is a singlet state, its cycloaddition reaction with ethylene (r2) has the following three possible routes: + + + (1) (2) (3) r1 r2 p1 r1 r2 p2 p2.1 p2 r2 p3 the geometrical parameters of the intermediates (int1 and int3), transition states (ts1, ts2, ts2.1 and ts3) and products (p1, p2, p2.1 and p3) that appear in the above three reactions are given in fig. 1, the energies are listed in table i and the entropy, enthalpy and gibbs energy values are listed in table ii. the potential energy profile of the above three reactions are shown in fig. 2. forming a spiro-sn-heterocyclic ring compound 295 r1 r2 int1 ts1 p1 sn c(1) c(2) c(3) c(1) c(1) c(1) c(2)c(2)c(2) c(3)c(3) c(3) sn snsn 2.866 2.866 1.353 81.5 2.151 2.199 1.504 118.8 2.1112.111 1.568 146.4 c(1)snc(2)c(3)=88.0∠ 2.011 1.985 1.339 ts2 p2 ts2.1 p2.1 c(1) c(1) sn sn c(2)c(2) c(3) 2.076 2.388 1.418 79.5 c(1)snc(2)c(3)=50.0 c(3) ∠ c(1)snc(2)c(3)=24.6∠ 2.217 2.217 1.546 65.6 2.157 2.335 1.541 1.986 2.170 1.559 58.5 72.3 sn sn c(1) c(1) c(2)c(2) c(3) c(3) int3 ts3 p3 c(1) c(1) c(1) c(2)c(2) c(2) c(3) c(3) c(3) sn sn sn c(4) c(5) c(4) c(4) c(5) c(5)1.349 3.030 2.922 85.8 1.4852.241 2.168 113.2 1.554 2.126 2.126 138.2 fig. 1. optimized mp2/genecp (c, h in 6-311++g**; sn in lanl2dz) geometrical parameters and the atomic numbering for the species in cycloaddition reaction between h2c=sn: and ethylene. bond lengths and bond angles are in angstrom and degree, respectively. table i. the electronic structure energy (eese) and relative energies (er) for the species from the mp2/genecp (c, h in 6-311++g**; sn in lanl2dz) method at 298 k and 101325 pa reaction species mp2/genecp eese / ha er / kj mol-1 (1)a r1+r2 –120.76486 0.0 int1 –120.78058 –41.3 ts1 (int1-p1) –120.72718 98.9 p1 –120.73150 87.6 (2)a r1+r2 –120.76486 0.0 int1 –120.78058 –41.3 ts2(int1-p2) –120.76248 6.2 p2 –120.82512 –158.2 ts2.1(p2-p2.1) –120.71765 123.9 p2.1 –120.75656 21.8 (3)b p2+r2 –199.17164 0.0 int3 –199.18360 –31.4 ts3(int3-p3) –199.16216 24.9 p3 –199.16806 9.4 aer = eese–eese(r1+r2), ber = eese– eese(p2+r2) 296 tan and lu table ii. entropy, enthalpy and gibbs free energy for the species from mp2/genecp (c, h in 6-311++g**; sn in lanl2dz) methods at the 298 k and 101325 pa reaction species h / ha s×104 / ha g / ha (1) r1+r2 –120.68436 1.83010 –120.73895 int1 –120.69667 1.31074 –120.73577 ts1 (int1-p1) –120.64511 1.19512 –120.68076 p1 –120.64842 1.23904 –120.68538 (2) ts2(int1-p2) –120.67918 1.18527 –120.71454 p2 –120.73864 1.17524 –120.77369 ts2.1(p2-p2.1) –120.63781 1.17213 –120.67277 p2.1 –120.67517 1.20096 –120.71099 (3) p2+r2 –199.03032 2.01150 –199.09031 int3 –199.03946 1.52138 –199.08484 ts3(int3-p3) –199.01940 1.35050 –199.05968 p3 –199.02440 1.40081 –199.06618 r1+r2 int1 p2+r2 0.0 -41.3 0.0 reaction(1) e r (k j/ m ol ) 50 0 -50 -100 -150 100 150 ts1 98.9 p1 87.6 ts2 6.2 p2 -158.2 int3 -31.4 ts3 24.9 p3 9.4 reaction(2) reaction(3) ts2.1 123.9 p2.1 21.8 fig. 2. the potential energy surface for the cycloaddition reactions between h2c=sn: and ethylene as calculated with mp2/genecp (c, h in 6-311++g** ; sn in lanl2dz). the unique imaginary frequency of the transition states ts1, ts2, ts2.1, and ts3 obtained through vibrational analysis are 220.7i, 419.7i, 784.5i and 120.8i, respectively, and therefore, these transition states could be affirmed as the forming a spiro-sn-heterocyclic ring compound 297 genuine ones. irc (with a step-size of 0.1 amu–1/2 bohr) analysis confirmed that ts1 connects int1 and p1, ts2 connects int1 and p2, ts2.1 connects p2 and p2.1, and ts3 connects int3 and p3. according to fig. 2, it could be seen that reaction (1) consists of two steps: the first one is that the two reactants (r1 and r2) form an intermediate (int1). according to fig. 2 and table ii, the reaction is a barrier-free exothermic reaction, and the changes of molar constant volume heat of reaction (δrum) and molar heat of reaction (δrhm) at normal temperature and pressure are –41.3 and –32.3 kj mol–1, respectively, and the change of molar gibbs energy of the reaction (δrgm) is –8.3 kj mol–1. the second is that int1 isomerises to a three-membered sn-heterocyclic ring product p1 via transition state ts1 with an energy barrier of 140.2 kj mol–1. according to fig. 2 and table ii, the reaction is endothermic, and the δrum and δrhm values at normal temperature and pressure are 128.9 and 126.7 kj mol–1, respectively, and the δrgm value is 140.6 kj mol–1. hence, int1→p1 is thermodynamically forbidden at normal temperature and pressure, and reaction (1) will end in int1. according to fig. 2, it could be seen that reaction (2) consists of three steps: the first one is that the two reactants (r1 and r2) form an intermediate (int1) (the situation is the same as reaction (1)). the second is that the int1 isomerizes to a four-membered ring stannylene (p2) via transition state ts2 with an energy barrier of 47.5 kj mol–1. according to fig. 2 and table ii, the reaction is exothermic, and δrum and δrhm the values at normal temperature and pressure are –116.9 and –110.2 kj mol–1, respectively, and the δrgm value is –91.2 kj mol–1. the third is that the p2 undergoes h transfer via transition state ts2.1 with an energy barrier of 282.1 kj mol–1, resulting in the formation of product p2.1. according to fig. 2 and table ii, the reaction is an endothermic reaction, and the δrum and δrhm values at normal temperature and pressure are 180.0 and 166.6 kj mol–1, respectively, and the δrgm value is 164.6 kj mol–1. thus, p2→p2.1 is thermodynamically forbidden at normal temperature and pressure, and reaction (2) will end in p2. comparing reaction (2) with reaction (1), int1→p1 is thermodynamically forbidden, and thus, reaction (2) will be the dominant reaction channel. in reaction (3), the four-membered ring stannylene (p2) further reacts with ethylene (r2) to form a spiro-sn-heterocyclic ring, compound (p3). according to fig. 2, it could be seen that the process of reaction (3) is based on the p2 formed in reaction (2) reacting further with ethylene (r2) to form an intermediate (int3). according to fig. 2 and table ii, the reaction is a barrierless exothermic reaction, and the δrum and δrhm values at normal temperature and pressure are –31.4 and –24.0 kj mol–1, respectively, and the δrgm value is –14.4 kj mol–1. then intermediate (int3) isomerizes to a spiro-sn-heterocyclic ring compound (p3) via a transition state (ts3) with an energy barrier of 56.3 kj mol–1. accord298 tan and lu ing to fig. 2 and table ii, the reaction is an endothermic reaction, and the δrum and δrhm values at normal temperature and pressure are 40.8 and 39.5 kj mol–1, respectively, and the δrgm value is 63.4 kj mol–1. according to fig. 2, reaction (3) and p2→p2.1 in reaction (2) are competitive reactions. as p2→p2.1 is thermodynamically forbidden, reaction (3) will be the dominant reaction channel. in reaction (3), since δrgm value of p2+r2→p3 is 49.0 kj mol–1, it is a thermodynamically forbidden reaction at normal temperature and normal pressure. in order to realize the reaction, according to the following thermodynamic formula: 2 1 2 1( ) ( ) dδ − δ = δ p p g p g p v p at a temperature of 298 k, p2+r2→p3 is allowed to proceed, and the pressure of the reaction system must be greater than 150325 pa (1.5 atm). according to all the analyses, reaction (3) should be the dominant reaction channel of the cycloaddition reaction between singlet h2c=sn: and ethylene, namely: ts2 r2 ts3r1 r2 int1 p2 int3 p3++ → ⎯⎯⎯→ ⎯⎯⎯→ ⎯⎯⎯→ in this reaction, the frontier molecular orbitals of r1, r2 and p2 are shown in fig. 3. according to fig. 3, the mechanism of reaction (3) could be explained with the frontier molecular orbital diagrams (see figs. 4 and 5). according to figs. 1 and 4, when h2c=sn: (r1) initially interacts with ethylene, the 5p unoccupied orbital of tin sidewise overlaps with the bonding π-orbital of ethylene, leading to the formation of an intermediate (int1). as the reaction proceeds, the sn–c(2) bond (int1: 2.886 å, ts2: 2.388 å, p2: 2.217 å), ∠c(1)snc(2)c(3) (int1: 88.0°,ts2: 50.0°, p2: 24.6°) and ∠c(1)snc(2) (int1: 81.5°, ts2: 79.5°, p2: 65.6°) gradually decrease, and the c(1)–sn and c(2)–c(3) bond (int1: 2.011 and 1.353 å; ts2: 2.076 and 1.418 å; p2: 2.217 and 1.546 å) gradually lengthen. before the transition state ts2, sn and c(2) form a covalent bond. after the transition state ts2, c(1) and c(3) form a covalent bond. thus, int1 isomerizes to a four-membered ring stannylene (p2) via transition state ts2. as 5pπ π 5p sp homo of r1 lumo of r1 homo of r2 homo of p2 lumo of p2 fig. 3. the frontier molecular orbitals of r1, r2, p2. forming a spiro-sn-heterocyclic ring compound 299 p2 is still an active molecular species, p2 may further react with ethylene to form a spiro-sn-heterocyclic ring compound (p3). the mechanism of this reaction could be explained with figs. 1 and 5. according to the rule of molecular orbital symmetry adaptation, when p2 interacts with ethylene (r2), the 5p unoccupied orbital of the sn atom in p2 insert the p orbital of ethylene forming a π→p donor–acceptor bond, leading to the formation of an intermediate (int3). as the reaction proceeds, the sn–c(4) and sn–c(5) bond lengths gradually decrease (int3: 3.030 and 2.922 å; ts3: 2.241 and 2.168 å; p3: 2.126 and 2.126 å), the ∠c(2)snc(4) angle (int3: 85.8°, ts3: 113.2°, p3: 138.2°) gradually increases, the c(4)–c(5) bond gradually lengthens (int3: 1.349 å, ts3: 1.485 å, p3: 1.554 å). before the transition state ts3, a covalent bond is formed between sn and c(4) and between sn and c(5). after the transition state ts3, int3 further isomerizes to a spiro-sn-heterocyclic ring compound (p3) via transition state ts3. sn in the spiro-sn-heterocyclic ring compound is combined with adjacent atoms by sp3 hybridization. 5p + r1 sn π r2 + fig. 4. a schematic interaction diagram for the frontier orbitals of h2c＝sn: (r1) and c2h4 (r2). sp + 5p p2 sn + π r2 fig. 5. a schematic diagram for the frontier orbitals of p2 and c2h4 (r2). 300 tan and lu conclusions according to the potential energy profile, the cycloaddition reaction between singlet h2c=sn: and ethylene obtained with the mp2/genecp (c, h in 6-311++g**; sn in lanl2dz) method can be predicted. this reaction has one dominant channel. it consists of four steps: 1) the two reactants first form an intermediate (int1) through a barrier-free exothermic reaction of 41.3 kj mol–1; 2) the intermediate (int1) isomerizes to a four-membered ring stannylene (p2) via transition state ts2 with an energy barrier of 47.5 kj mol–1; 3) the four -membered ring stannylene (p2) further reacts with ethylene (r2) to form another intermediate int3 through a barrier-free exothermic reaction of 31.4 kj mol–1; 4) intermediate (int3) isomerizes to a spiro-sn-heterocyclic ring compound (p3) via transition state ts3 with an energy barrier of 56.3 kj mol–1. at a temperature of 298 k, the reaction is carried out, and the pressure of the reaction system needs to be greater than 150325 pa (1.5 atm). the 5p unoccupied orbital of sn in x2c=sn: is involved in cycloaddition reaction of x2c=sn: and the symmetric π-bonded compounds. the 5p unoccupied orbital of tin in h2c=sn: sidewise overlaps with bonding π-orbital of ethylene resulting in the formation of an intermediate. the instability of the intermediate makes it isomerise to a four-membered ring stannylene. as the 5p unoccupied orbital of the sn atom in the four-membered ring stannylene and the π-orbital of symmetric π-bonded compounds forms a π→p donor–acceptor bond, the four-membered ring stannylene further combines with symmetric π-bonded compounds to form another intermediate, and this intermediate further isomerises to a spiro-sn-heterocyclic ring compound. sn in the spiro-sn-heterocyclic ring compound is combined with adjacent sp3 hybridized atoms. и з в о д ab initio проучавање механизма настајања спиро-sn-хетероцикличних једињења циклоадиционом реакцијом h2c=sn: и етилена xiaojun tan1 и xiuhui lu2 1 school of biological science and technology, university of jinan, jinan 250022, p. r. china и 2 school of chemistry and chemical engineering, university of jinan, jinan 250022, p. r. china x2c=sn: (x = h, me, f, cl, br, ph, ar…) су нове хемијске врсте. циклоадиционе рекције врста x2c=sn: су ново поље у хемији станилена. механизам циклоадиционе реакције синглетног h2c=sn: са етиленом је у овом чланку по први пут проучаван коришћењем mp2/genecp (c, h са 6-311++g; sn са lanl2dz) метода. из профила потенцијалне енергије може се предсказати да реакција има један доминантан реакциони пут. представљено је реакционо правило да се 5p незаузета орбитала калаја у h2c=sn: бочно преклапа са везивном π-орбиталом етилена формирајући одговарајући интермедијер. нестабилност интермедијера чини да се он изомеризује у четворочлани прстен станилена. пошто 5p незаузета орбитала sn атома у четворочланом прстену станилена и π-орбитала етилена формирају π→p донорско–акцепторску везу, четворочлани прстен станилена се даље комбинује са етиленом дајући нови интермедијер, а тај се даље изомеризује у спиро-sn-хетероциклично једињење. sn у спиро-sn-хетеро forming a spiro-sn-heterocyclic ring compound 301 цикличном једињењу се комбинује са суседним sp3 хибридизованим атомима. резултат ове студије открива механизам циклоадиционе реакције x2c=sn: са симетричним π везивним једињењима. (примљено 3. јуна, ревидирано 2. септембра, прихваћено 10. септембра 2018) references 1. p. j. stang, chem. rev. 78 (1978) 383 2. p. j. stang, acc. chem. res. 15 (1982) 348 3. h. leclercq, i. dubois, j. mol. spectrosc. 76 (1979) 39 4. r. srinivas, d. sulzle, h. schwarz, j. am. chem. soc. 113 (1991) 52 5. w. h. harper, e. a. ferrall, r. k. hilliard, s. m. stogner, r. s. grev, d. j. clouthier, j. am. chem. soc. 119 (1997) 8361 6. d. a. hostutler, t. c. smith, h. y. li, d. j. clouthier, j. chem. phys. 111 (1999) 950 7. x. h. lu, y. h. xu, h. b. yu, w. r. wu, j. phys. chem., a 109 (2005) 6970 8. x. h. lu, z. x. lian, y. q. li, j. serb. chem. soc. 76 (2011) 743 9. x. h. lu, j. h. han, z. x. lian, y. q. li, j. serb. chem. soc. 76 (2011) 1395 10. x. h. lu, l. y. shi, y. q. li, z. n. wang, j. serb. chem. soc. 77 (2012) 75 11. x. h. lu, j. j. ming, j. serb. chem. soc. 81 (2016) 633 12. x. h. lu, y. h. xu, l. y. shi, j. f. han, z. x. lian, j. organomet. chem. 694 (2009) 4062 13. x. h. lu, l. y. shi, j. j. ming, arabian j. chem. 9 (2016) 163 14. l. a. curtis, k. raghavachari, j. a. pople, j. chem. phys. 98 (1993) 1293 15. k. fukui, j. phys. chem. 74 (1970) 4161 16. k. ishida, k. morokuma, a. komornicki, j. chem. phys. 66 (1977) 2153. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {design of amino-functionalized chelated macroporous copolymer [poly(gma-edgma)] for the sorption of cu (ii) ions} j. serb. chem. soc. 84 (12) 1391–1404 (2019) udc 541.572.54+546.562–71:539.217+ jscs–5272 66.081:678–13 original scientific paper 1391 design of an amino-functionalized chelating macroporous copolymer poly(gma-co-egdma) for the sorption of cu(ii) ions ljiljana t. suručić1, aleksandra b. nastasović2, antonije e. onjia3, goran v. janjić2 and aleksandra а. rakić4* 1university of banja luka, faculty of medicine, save mrkalja 14; banja luka, republic of srpska, bosnia and herzegovina, 2university of belgrade, institute of chemistry, technology and metallurgy, njegoševa 12, belgrade, serbia, 3university of belgrade, faculty of technology and metallurgy, karnegijeva 4, belgrade, serbia and 4university of belgrade, faculty of physical chemistry, studentski trg 12–16, belgrade, serbia (received 21 january, revised 12 april, accepted 15 april 2019) abstract: polymer-based, highly porous nanocomposites with functionalized ligands attached to the core structure are extremely efficient in the detection, removal and recovery of metals through the process of sorption. quantumchemical models could be helpful for sorption process analyses. the sorption of cu(ii) ions by amino-functionalized chelating macroporous copolymers poly(gma-co-egdma)-amine and sorption selectivity of the subject copolymers, ethylenediamine (en), diethylenetriamine (dien) and triethylenetetramine (trien), were successfully modelled by quantum chemical calculations. considering the crystal structures from csd and experimental conditions during the formation of metal complexes, the most frequent mononuclear complexes are those with the tetradentate teta ligand, while binuclear complexes are formed when the metal ion is in large excess. although the en-copolymer was the most effective functionalized one, higher maximum sorption capacities (qmax) were observed for the dienand trien-copolymers, due to their abilities to form binuclear complexes. the enthalpy term has the greatest contribution to the total gibbs energy change of reaction for the formation of mononuclear cu(ii) complexes (δgaq), while the solvation energy of the reaction has the greatest contribution in the formation of binuclear complexes. the results of the study indicate that small amines with the ability to form binuclear complex are the best choice for functionalization of the considered copolymer. keywords: sorption of metal ions; amino-functionalized copolymer; dft modelling; coordination isomers; csd. * corresponding author. e-mail: saska@ffh.bg.ac.rs https://doi.org/10.2298/jsc190125031s 1392 suručić et al. introduction the earth’s crust naturally contains heavy metal compounds, the concentration of which is increased in places such as bedrocks, or can be found as ores in rocks. unfortunately, heavy metals are constantly being released into the environment from anthropogenic sources: metalliferous mining and smelting (as, cd, pb, hg), industry (as, cd, cr, co, cu, hg, ni, zn), atmospheric deposition (as, cd, cr, cu, pb, hg, u), agriculture (as, cd, cu, pb, si, u, zn) and waste disposal (as, cd, cr, cu, pb, hg, zn).1–3 consequently, the amounts of heavy metals deposited on the earth’s surface are considerably higher compared to natural background concentrations. heavy metals accumulate over time and since they are non-decomposable or non-biodegradable, they are the long-term health and environmental hazards. very rarely, acute effects appear when considerable amounts of heavy metals are accidentally released into the environment.4 various methods of heavy metal removal have been developed and are still being developed: physicochemical (membrane filtration, chemical precipitation, ionexchange, adsorption), electrochemical (electrocoagulation, electroflotation, electrodeposition) biological5 (biosorption, by bacteria and microorganism, activated sludge process, biofilter, anaerobic digestion, stabilization ponds) and current (membrane filtration, photo-catalysis, nanotechnology) methods. process of sorption is efficient in the detection and removal of heavy metals.6 recently, nanocomposite carbon-7 (activated carbon, carbon nanotubes, fullerenes, graphene), polymer-8,9 silica-10–20 and natural-based21–24 nanocomposites, highly porous materials designed for heavy metal adsorption, are being intensively developed.6 the features of the above-mentioned composites are environmental stability, high specific surface area and strength of the core (carbon, polymer and silica), and in synergy with the reactivity of attached ligands to specific metal ions make them efficient, specific and sensitive metal ions sorbents. this allows fast and cheap detection, complete removal or recovery of metals from contaminated waters. although essential, cu oligoelement is important, above all, for the activity of enzymes in living organisms,25,26 it becomes very toxic27 when present in excessive levels. this metal is classified as highly hazardous to human health and the environment with various long-term detrimental effects. there is a great interest in cu(ii) remediation in terms of phase transfer or its transformation into harmless chemical forms. in order to protect the quality of soil and water pollution, many countries instituted rigorous environmental regulations.28 mesoporous silica with attached 3-{[3-(methoxycarbonyl)benzylidene]hydrazinyl}benzoic acid,29 3-{[(4-ethoxy-2-mercaptophenyl)imino]methyl}salicylic acid,30 3-{[2-(2-hydroxy-1-naphthoyl)hydrazono]methyl}benzoic acid,31 4-tert-butyl-2 -hydroxybenzaldehyde thiosemicarbazone,32 3-[[[4-[[(3-carboxyphenyl)methylene]amino]-2,5-dihydro-5-oxo-1h-pyrayol-3-yl]imino]methyl]benzoic acid,33 2,2’-[(4,5-dimethyl-1,2-phenylene)bis(nitrilomethylidene)bis[phenol],34 modelling of metal ion sorption 1393 2,2’-[(1,2-ethanediylbis(thio-2,1-phenylenenitrilomethylidene)]bis[phenol],35 2,3 -dihydro-5,6-bis[[(2-hydroxyphenyl)methylene]amino]-2-thioxo-4(1h)-pyrimidinone,36 e,e,e,e-3,3′,3″,3″′-[2,3,6,7-naphthalenetetrayltetrakis(nitrilomethylidyne)tetrakis[2-hydroxybenzoic acid],37 2,2’-(1,8-octanediylidenedinitrilo)bis[4,6-dimethylphenol]38 are used as optical nanosensors for selective adsorption, detection and removal of cu(ii) ions.36 the mentioned materials are very sensitive, changing their colour when cu(ii) ions are present even in traces. any additional procedure or equipment besides the silica complexes is not necessary for the detection of cu(ii) ions. the physicochemical method of remediation through precipitation of cu(ii) ions using chelating agents has proved to be very efficient in fulfilling environmental regulation guidelines.39 chelating homopolymers and copolymers are highly selective sorbents of heavy metal ions from aqueous solutions.40 efficient and selective polymers consist of cross-linked polymer structures (core) and functional groups (ligands) that have the ability to form adequate interactions with heavy metals. these functional groups are the key for the selective chelation of heavy metals. within functional groups, coordination bonding with metal ions is achieved through n, o, p and s atoms.41,42 macroporous copolymers based on glycidyl methacrylate (gma) are especially interesting, since they contain an epoxide ring in their structures. optionally, the ease of opening the epoxide ring offers the possibility for the introduction of a ligand with an iminodiacetate, thiol, pyrazole or amino group.43 the gma molecule is a convenient basis for the relatively simple formation of different metal ions and organic compound sorbents. therefore, the structure of a gma-based copolymer suggests promising possibilities of numerous applications referring to environmental issues.41,42,44–49 an incredible number of possible structures that could be synthesized, demands a theoretical evaluation of the most appropriate ligand for the sorption of a particular compound. recently, through the process of polymerization of gma monomer together with ethylene glycol dimethacrylate (egdma), a macroporous copolymer poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly(gma-co-egdma)) has been synthesised. the final products were obtained through functionalization of the copolymer poly(gma-co-egdma) with three different amines (scheme 1): ethylenediamine in poly(gma-co -egdma)-en, diethylenetriamine in poly(gma-co-egdma)-dien and triethylenetetramine in poly(gma-co-egdma)-trien. these three amino-functionalized copolymer samples showed differences in the efficacy of cu(ii) sorption from aqueous solutions, which is expressed as the polymer capacity of sorption in mmol of adsorbed ions per gram of the polymer, q (mmol g–1). the process of sorption itself is a rapid one, while the capacity of sorption depends on the ligand. the sorption capacity rises from -en to -dien (around 1.75 times larger), 1394 suručić et al. but there is no difference for -dien and -trien copolymer, i.e., the values are practically identical.40 scheme 1. the structure of chelated copolymer poly(gma-co -egdma), functionalized through the reaction of amines (en, dien and trien) with the epoxide ring of the original copolymer. the aim of this study was to explain experimentally the obtained trend for cu(ii) by applying theoretical techniques to predict the selectivity of the ligands. the complex molecular system of each of the three copolymers is comprised of a recurring structural unit. therefore, it is possible to discuss cu(ii) ion and copolymer reactions within the macromolecule at the unit segment level. the unit segment would be the cu(ii) ion with coordinated amino ligands. a large number of such segments (up to several thousands) that can be found in the solution where the sorption occurs include a sorbent/metal ion system. the unit segment, crucial for metal–ligand bonding is, in itself, a complex of metal ions and amino ligands, the geometry and stability of which can be predicted by statistical analysis of data taken from the crystal structures and quantum chemical calculations. methodology possible coordination modes of triethylenetetramine (trien) ligand in crystal structures, extracted from cambridge structural database (csd, version 5.38, november 2016),51 were investigated by applying statistical analysis of the obtained data. the quantum chemical calculations were performed in the gaussian09 program52 applying the b3lyp functional. the 6-311++g** basis set was used for non-metal elements and the lanl2dz basis set for cu2+. the initial complex geometries of possible isomers were optimized at the same level of theory, and the parameters obtained by optimization were used to evaluate gibbs free energies for the reactions of complex formation. in these reactions (eq. (1)), the hydroxyamine obtained in the ring opening reaction (marked as l), reacts with the hexaaqua complex, forming the corresponding complex with the coordinated amine: a[cu(h2o)6]2+ + l → [cua(h2o)6a-n(l)]2+ + nh2o (1) in eq. (1), l represents enoh, dienoh or trienoh ligand (scheme 1), n represents the hapticity of ligands, while a equals 1 for mononuclear and 2 for binuclear complexes. the modelling of metal ion sorption 1395 smd method and the aforementioned level of theory were applied for the solvation energy (δes) calculations (eq. (2)): δes(r) = ∑es(product) – ∑es(reactants) (2) the gibbs energy change of reaction (1) in aqueous solution (δgaq) was estimated according to the following equation: δgaq = δgg + δes(r) (3) δgg, the gibbs energy change of the reaction (1) in vacuum (eq. (4)), includes the enthalpy change (δh) and entropy change (δs), connected through the following equation: δgg = δh – tδs (4) the enthalpy change of the reaction is determined by eq. (5): δh = δee + δet + pδv (5) in which δee stands for the electronic energy change, δet is the thermal energy change, and pδv is the work performed in a reaction. results and discussion crystallographic analysis of the structures from csd crystal structure analysis is a very useful tool used in conformational analysis and in supramolecular chemistry. in this particular case, the primary interest was to gather information from the crystal structure analysis about coordination geometries of complexes with cu(ii) ion and the trien ligand, as well as the abundance of each geometry observed. considering the complexes of cu(ii) and the substituted trien ligand from csd, the most frequent ones are mononuclear complexes of the cu(ii) ion with tetradentate trien ligand geometries, while the second most abundant are binuclear complexes in which two n atoms of the trien ligand are coordinated to each metal ion. taking into consideration the experimental conditions during trien complex formation, it was observed that binuclear binding occurs when the metal ion is in excess compared to the trien ligand (more than two times). the abundance of mononuclear complexes of cu(ii) and the tetradentate trien ligand are distributed as follows: by far the most frequent are those with square pyramidal (71 %), followed by octahedral (26 %) and square planar (3 %) complex geometry. when only the cu(ii) complexes with unsubstituted trien ligand (17 crystal structures) are taken into account, a similar trend was observed. mononuclear complexes with the tetradentate trien ligand are the most frequent (10 structures), while binuclear (2 structures) and polynuclear complexes (5 structures) with the tetradentate trien ligand are less abundant (fig. 1). structures with a bridging trien ligand were not found. complexes with a square-pyramidal geometry (15 structures) are much more numerous than complexes with a square-planar geometry (2 structures). the values of the torsion angle t (n1–n2–n3–n4 angle) are in the range from 0 to 20°, indicating a planar or trans conformation of the trien ligand. 1396 suručić et al. fig. 1. the fragments of crystal structures with the square-pyramidal cu(ii) complex (refcode aratao), the square-planar cu(ii) complex (refcode pajsey), and the mixed binuclear complex (with cu(ii) and zn(ii) metal ion) with a tetradentate trien ligand (refcode zackap). experimental conditions during the sorption of cu(ii) ions for functionalized copolymers as previously concluded, the quantity ratio of metal ions and ligands influences the mode in which the ligand is coordinated to the metal. for that purpose, both the quantity of cu(ii) ions and the corresponding copolymer amino-functionalized groups were determined (table i), in sorption studies of the cu(ii) ion.42 poly(gma-co-egdma)-en contains 1.12 mmol, poly(gma-co-egdma) -dien 0.81 mmol and poly(gma-co-egdma)-trien) 0.65 mmol amino-functionalized groups.42 considering the number of amino-functionalized groups (fg) in the copolymer, the quantity of the metal ion is significantly higher in all three cases (table i). the cu(ii) ion is in an excess, so the complete quantity of the cu(ii) ion would not be sorbed. if the cu(ii) ions formed the mononuclear enoh, dienoh or trienoh complexes, the maximum sorption (qmax = f(t)) would be different. according to the number of amino-functionalized groups, en-functionalized copolymer should have the highest and the trien-functionalized one should have the lowest qmax value. however, dienand trien-functionalized copolymers have greater qmax values. one of the reasons could be the ability of these ligands to form binuclear complexes. the en-functionalized copolymer is the most effectively functionalized one (53 %), followed by the dien(38 %) and trien-functionalized copolymer (31 %), which is inverse to their molecular lengths. similar qmax values of dienand trien-ligands are quite unlikely given the number of functionalized groups. however, the shortest amine (en) more easily penetrates inside the initial poly(gma-co-egdma) copolymer, thus functionalizing a larger number of epoxide rings. in aqueous solutions cu(ii) ion exists as a hexaaqua complex. the sorption of this metal strongly depends on the ability of its hexaaqua complex to penetrate near the amino-functionalized groups of the corresponding copolymer. this complex is more voluminous and less flexible than the considered amines (en, modelling of metal ion sorption 1397 dien and trien), resulting in its lower penetrability than the amines. indicators are the theoretical qmax values (table i), which are in cases of en-functionalized and dien-functionalized copolymers significantly higher than the experimentally determined qmax values. table i. the quantities of cu(ii) ions and amino-functionalized groups (in mmol) in the corresponding copolymer, obtained in the process of the experimentally conducted sorption of cu(ii) ions. theoretically qmax(theor) and experimentally qmax(exp) determined values of the maximum sorption of cu(ii) ion, expressed in mmol g-1 copolymer n(cu2+) n(fg)a qmax(theor)b mononuclear qmax(theor)c binuclear qmax (exp) poly(gma-co-egdma)-en 2.5 2.24 2.24 4.48 1.40 poly(gma-co-egdma)-dien 2.5 1.62 1.62 3.24 2.45 poly(gma-co-egdma)-trien 2.5 1.30 1.30 2.60 2.40 athe quantities of functionalized groups (fg) are calculated per g of the copolymer sample used in the experiments; btheoretical qmax values calculated per g of the copolymer, considering that mononuclear cu(ii) complexes are formed; cthe theoretical qmax values calculated per g of the copolymer, considering that binuclear cu(ii) complexes are formed quantum chemical calculations for trienoh complexes to confirm the above-proposed assumption, quantum chemical calculations were performed to evaluate the gibbs energy change (δgaq) that accompanies the formation of mononuclear and binuclear cu(ii) complexes with the trienoh ligand in aqueous solution. for this purpose, the complex geometries obtained through optimization of four possible isomers of mononuclear complexes and one binuclear cu(ii) complex were used. depending on the mutual positions of coordinated oxygen atoms from aqua and trienoh ligands, initial isomers of octahedral complexes with pentadentate trienoh ligand (n1,n2,n3,n4,otrienoh) could be a cis or trans isomer. upon the optimization, dissociation of a water molecule (wd) from an octahedral mononuclear complex occurs. the newly formed complexes take square pyramidal geometry (fig. 2). fig. 2. optimization of possible isomers of mononuclear cu(ii) complexes with the trienoh ligand. 1398 suručić et al. the apical position is occupied by an oh group ([cu(n1,n2,n3,n4,o -trienoh)]2+ complex), or by the nh group of the trienoh ligand ([cu(n1,n3,n4,o,n2-trienoh)]2+ complex): a) cis-[cu(h2o)2(n1,n2,n3,n4-trienoh)]2+ → [cu(h2o)(n1,n2,n3,n4-trienoh)]2+ + h2o b) trans-[cu(h2o)2(n1,n2,n3,n4-trienoh)]2+ → [cu(h2o)(n1,n2,n3,n4-trienoh)]2+ + h2o c) cis-[cu(h2o)(n1,n2,n3,n4,o-trienoh)]2+ → [cu(n1,n3,n4,o,n2-trienoh)]2+ + h2o d) trans-[cu(h2o)(n1,n2,n3,n4,o-trienoh)]2+ → [cu(n1, n2,n3,n4,o-trienoh)]2+ + h2o the tetradentate n1,n2,n3,n4-trienoh ligand in octahedral mononuclear complexes leaves a space for two aqua ligands in the cis or trans positions. optimized complexes take square pyramidal geometry, likewise in complexes with pentadentate trienoh ligand (fig. 2). one of the aqua ligands is released, while the other one remains in the apical position of the square pyramidal ([cu(h2o)(n1,n2,n3,n4-trienoh)]2+) complex. as far as the coordinated n atoms are concerned, they are in equatorial position (trans geometry of a tetradentate ligand). the structures obtained by the optimization were used to calculate the gibbs free energy change in the formation reaction of these complexes out of the cu(ii) hexaaqua complex (eq. (1)). interestingly, the results of the optimization are in agreement with the results of the crystallographic analysis, which is strong confirmation that the square pyramidal geometry of the cu(ii) complexes is the most frequent one in crystal structures. the enthalpy of the formation reaction of cu(ii) complexes with the trienoh ligand, corresponding to the sum of electronic energy change (δee), thermal energy change (δet), and work (pδv), has the greatest contribution to the total gibbs energy change in aqueous solution (δgaq). an exothermal process is indicated by the negative sign of enthalpy. on the other hand, contributions from the entropy (tδs term) and solvation energy (δgaq−δgg) are significantly lower than those from the enthalpy term. the formation of a complex without coordinated oh-group is energetically more favourable, by only about 5 % (table ii). the structure the geometry of which is based on crystallographic data was used as a model system for quantum chemical calculations for binuclear cu(ii) complexes with the trienoh ligand. the first two n atoms (n1 and n2) are coordinated to one cu(ii) ion in the square pyramidal complex, while the other modelling of metal ion sorption 1399 two n atoms (n3 and n4) along with the oh group are coordinated to the second cu(ii) ion (fig. 3). upon the optimization, one aqua ligand was dissociated from the cu(ii) ion to which the n1 and n2 atoms of the trienoh ligand are coordinated, leading to a change towards the square planar geometry of that cu(ii) ion. the other cu(ii) ion remains in the square pyramidal geometry. the enthalpy change of this reaction has positive sign (endothermal reaction), which is compensated by the entropic term (tδs). however, the greatest contribution to the spontaneous nature of the reaction originates from the solvation energy of the reaction. table ii. the calculated results of the thermodynamic functions (kcal* mol-1) for the thermo-chemical analysis in the process of the formation of cu(ii) complexes with the trienoh ligand optimized metal complex δee δet pδv tδs δgg δgaq mononuclear trienoh complexes trans-[cu(h2o)(n1,n2,n3,n4-trienoh)]2+ –411.5 21.3 –1.2 23.3 –414.7 –410.9 trans-[cu(h2o)(n1,n2,n3,n4-trienoh)]2+ –411.5 21.3 –1.2 23.3 –414.7 –410.9 [cu(n1,n3,n4,o,n2-trienoh)]2+ –389.9 4.1 –0.6 13.4 –399.8 –395.9 [cu(n1,n2,n3,n4,o-trienoh)]2+ –396.4 4.1 –0.6 13.2 –406.2 –402.2 binuclear trienoh complex [cu(h2o)2(n1,n2:n3,n4,o-trienoh)(h2o)2cu]4+ 135.4 –8.8 3.0 130.5 –1.0 –344.8 fig. 3. the optimization of the binuclear cu(ii) complex with the trienoh ligand. [cu(h2o)3(n1,n2:n3,n4,o-trienoh)(h2o)2cu]4+ → [cu(h2o)2(n1,n2:n3,n4,o-trienoh)(h2o)2cu]4+ + h2o quantum chemical calculations for enoh and dienoh complexes as in the trienoh complexes, the initial structures of all possible isomers of mononuclear enoh and dienoh complexes of cu(ii) ion were optimized, with the geometries of all possible coordination isomers (table iii). the optimized structures of the complexes with the highest values of the gibbs energy changes for reactions of the complex formation in an aqueous solution (δgaq) are shown in fig. 4. unlike the most stable dienoh complex, the most stable structure of an enoh complex does not contain a coordinated oh group. both enoh and dienoh complexes have higher δgaq values than those of trienoh complexes, while dienoh complex displayed the highest δgaq value. this trend could be * 1 kcal = 4184 j 1400 suručić et al. explained by a larger number of chelate rings in the dienoh complex, compared to the enoh complex. although the most stable trienoh complex has the greatest number of chelate rings, it is the least stable complex, since chelate rings are condensate and, as such, under a higher ring strain than the rings of the most stable dienoh complex. fig. 4. optimized cu(ii) complexes with the enoh ([cu(h2o)3(n1,n2 -enoh)]2+) and dienoh (trans -[cu(h2o)(n1,n2,n3,o-dienoh)]2+) ligands having the highest values of the gibbs energy change (δgaq) for the reaction of metal complex formation in aqueous solutions. table iii. the calculated results of the thermodynamic functions (kcal mol-1) for the thermochemical analysis in the process of formation the mononuclear cu(ii) complexes with enoh or dienoh ligand optimization of the metal complexes δee δet pδv tδs δgg δgaq mononuclear enoh complexes mer-[cu(h2o)3(n1,n2,o-enoh)]2+→ mer-[cu(h2o)2(n1,n2,o-enoh)]2++h2o –372.8 36.8 –0.6 –29.0 –365.6 –361.7 fac-[cu(h2o)3(n1,n2,ii-enoh)]2+→ mer-[cu(h2o)2(n1,n2,o-enoh)]2++h2o –372.8 36.8 –0.6 –29.0 –365.6 –361.7 [cu(h2o)4(n1,n2-enoh)]2+→ [cu(h2o)4(n1,n2-enoh)]2+ –404.3 71.2 0 –48.1 –381.2 –375.4 [cu(h2o)3(n1,n2-enoh)]2+→ [cu(h2o)3(n1,n2-enoh)]2+ –388.6 9.3 0 –39.4 –418.6 –414.8 mononuclear dienoh complexes trans-[cu(h2o)2(n1,n2,n3,o-dienoh)]2+ → trans-[cu(h2o)(n1,n2,n3,o-dienoh)]2++h2o –388.3 20.6 –0.6 63.4 –431.6 –429.0 cis-α-[cu(h2o)2(n1,n2,n3,o-dienoh)]2+ → cis-β-[cu(h2o)(n1,n2,n3,o-dienoh)]2++h2o –385.1 20.6 –0.6 35.8 –400.9 –398.1 cis-β-[cu(h2o)2(n1,n2,n3,o-dienoh)]2+ → cis-β-[cu(h2o)(n1,n2,n3,o-dienoh)]2++h2o –385.1 20.6 –0.6 35.8 –400.9 –398.1 mer-[cu(h2o)3(n1,n2,n3-dienoh)]2+ → [cu(h2o)(n1,n2,n3-dienoh)]2++2h2o –364.2 20.2 –1.2 20.4 –365.5 –362.9 fac-[cu(h2o)3(n1,n2,n3-dienoh)]2+ → [cu(h2o)(n1,n2,n3-dienoh)]2++2h2o –364.2 20.2 –1.2 20.4 –365.5 –362.9 conclusions by comparing the gibbs energy change (δgaq) for mononuclear and binuclear trienoh complex, it could be concluded that the formation of mononuclear complexes is a slightly more favourable (spontaneous) process. these results are in agreement with the combined potentiometric–spectrophotometric study of a modelling of metal ion sorption 1401 cu(trien) complex,52 which showed that the mononuclear complex with a tetradentate trien ligand is dominant. this type of complex already occurs at ph 2.5, reaching its maximum at ph 5. in that study, a binuclear complex was not experimentally detected, which could be explained by the fact that the synthesis is conducted in a solution in which the quantity of ligands is four times that of cu(ii) ions. however, it is possible to calculate the equilibrium constant of the reaction in the process of complex formation from the gibbs energy change (δg = −rtln k). the functional dependence of this equilibrium constant versus the [cu(h2o)6]2+ complex concentration is linear in the reaction where a mononuclear complex is formed, or square for binuclear complex formation. in accordance with the le chatelier principle, an increase in metal ion concentration shifts the equilibrium towards trienoh complex formation. as a surplus in concentration of cu(ii)aqua complex versus trienoh ligand is more pronounced, the proportion of binuclear complex in the product increases. therefore, it is reasonable to assume that, in case of a trien-functionalized complex, the adsorption of cu(ii) ion occurs as a result of the formation of binuclear complexes, since the concentration of cu(ii) ions is about four times higher than the quantity of ligands (functionalized groups of copolymers). for the same reasons, the formation of binuclear complexes with a dien-functionalized copolymer could be expected, but in this case, the concentration of cu(ii) ions is three times that of ligands. finally, the results of this study clearly indicate that amines with three nitrogen ligator atoms are preferable (due to the possibility of binuclear complex formation). however, the more ligator atoms it contains, the more difficult it is for the amine to diffuse inside the polymer. on the other hand, a higher number of ligator atoms increases the strain of chelate rings and reduces the stability of amino-functionalized complex with the sorbed ion. acknowledgements. this work was supported by the ministry of education, science and technological development of the republic of serbia (project no. oi 172043, 172023 and iii 43009). numerical simulations were run on the paradox supercomputing facility at the scientific computing laboratory of the institute of physics belgrade, supported in part by the ministry of education, science and technological development of the republic of serbia. 1402 suručić et al. и з в о д дизајн аминофункционализованих хелатних макропорозних кополимера поли(гма-ко-егдма) за сорпцију cu(ii) јона љиљана т. суручић1, александра б. настасовић2, антоније е. оњиа3, горан в. јањић3 и александра а. ракић4 1универзитет у бањој луци, медицински факултет, саве мркаља 14, бања лука, република српска, босна и херцеговина, 1универзитет у београду, институт за хемију, технологију и металургијy, његошева 12, београд, 3универзитет у београду, технолошко–металуршки факултет, карнегијева 4, београд и 4универзитет у београду, факултет за физичку хемију, студентски трг 12–16, београд квантнохемијско моделовање се показало као корисна алатка за разјашњавање процеса сорпције, као и за предвиђање структуре насталих производа те сорпције. уз помоћ квантнохемијских прорачуна успешно су моделаовани процеси сорпције cu(ii) јона и селективност тих сорпција на амино-функционализованим хелатним кополимерима поли(глицидил-метакрилат-ко-етиленгликол-диметакрилат) (поли(gma-ко -еgdma)) са различитим бројем амино група у свом саставу (етилен (-en), диетилентриамин (-dien) и триетилентетраамин (-trien) кополимери). узимајући у обзир кристалне структуре из csd и експерименталне услове приликом формирања комплекса са cu(ii) јоном, дошли смо до закључака да су најчешће структуре мононуклеарних комплекса са тетрадентатно везаним лигандима, али да се бинуклеарни комплекси јављају када је јон металa у вишку у односу на кополимер. без обзира на то што је кополимер са en-лигандима најефикасније функционализован, најбоља сорпција cu(ii) јона (qmax) се, ипак одиграва на dienи trien-кополимерима. овакав резултат је последица боље способности dien и trien лиганада за грађење бинуклеарних комплекса са cu(ii) јонима. највећи допринос укупној промени гибсове енергије реакције грађења мононуклеарног cu(ii) комплекса (δgaq) потиче од енталпијског члана. међутим, пресудну улогу на формирање бинуклеарних комплекса има солватациона енергија. резултати наше студије показују да су краћи амини са могућношћу формирања бинуклеарних комплекса најбољи избор приликом функционализације разматраних кополимера за потребе сорпције cu(ii) јона. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {electrochemical oxidation of sulfamethoxazole using ti/sno2-sb/co-pbo2 electrode through ann-pso} j. serb. chem. soc. 84 (7) 713–727 (2019) udc 66.087.3+547.787:544.6.076.32–034: jscs–5221 4–31+546.73:510.644 original scientific paper 713 electrochemical oxidation of sulfamethoxazole using ti/sno2–sb/co–pbo2 electrode through ann-pso jiteng wan, chunji jin*, banghai liu, zonglian she, mengchun gao and zhengyang wang college of environmental science and engineering, ocean university of china, no.238 songling road, qingdao, shandong province 266100, china (received 10 august 2018, revised 7 march, accepted 15 march 2019) abstract: even in a trace amounts, the presence of antibiotics in aqueous solution is getting more and more attention. accordingly, appropriate technologies are needed to efficiently remove these compounds from aqueous environments. in this study, we have examined the electrochemical oxidation (eo) of sulfamethoxazole (smx) on a co modified pbo2 electrode. the process of eo of smx in aqueous solution followed the pseudo-first-order kinetics, and the removal efficiency of smx reached the maximum value of 95.1 % within 60 min. the effects of major factors on smx oxidation kinetics were studied in detail by single-factor experiments, namely current density (1–20 ma cm-2), solution ph value (2–10), initial concentration of smx (10–500 mg l-1) and concentration of electrolytes (0.05–0.4 mol l-1). an artificial neural network (ann) model was used to simulate this eo process. based on the obtained model, particle swarm optimization (pso) was used to optimize the operating parameters. the maximum removal efficiency of smx was obtained at the optimized conditions (e.g., current density of 12.37 ma cm-2, initial ph value of 4.78, initial smx concentration of 74.45 mg l-1, electrolyte concentration of 0.24 mol l-1 and electrolysis time of 51.49 min). the validation results indicated that this method can ideally be used to optimize the related parameters and predict the anticipated results with acceptable accuracy. keywords: sulfamethoxazole; electrochemical oxidation; artificial neural networks; particle swarm optimization. introduction synthetic antimicrobial agents in aqueous environments are attracting more and more attention from the scientific community due to their potentially hazardous impacts on the aquatic environment and human health. widespread detection of these compounds in water bodies, even at minute concentrations, has already raised public health concerns. the adverse health effects, such as enhancement of * corresponding author. e-mail: 17806230627@163.com https://doi.org/10.2298/jsc180810025w 714 wan et al. antibiotic resistance, have been increasingly reported, but the consequences caused by long-lasting exposure to trace amounts of such synthetic antimicrobial agents remain uncertain.1–5 in this study, sulfamethoxazole (smx), a typical broad-spectrum sulfonamide antibiotic used to combat gram-positive and gram -negative aerobic bacteria, was selected as a model pollutant.6,7 in recent years, smx has been detected in surface water, hospital effluents, and drinking water, thus posing a potential environmental risk.8–10 besides, its antibacterial property impedes effective abatement using conventional biological treatment processes.5,11 therefore, effective methods are sorely needed to prevent dangerous accumulation of smx in aqueous environments. the electrochemical oxidation (eo) process has been proved to be an effective method for the removal of a broad spectrum of organic matters due to its ease of operation, low cost and environmental friendliness.12–14 in eo, organic pollutants are either removed by direct oxidization (e.g., electrons are transferred to the anode directly) or by indirect oxidization (i.e., some hydroxyl radicals are generated and act as mediators in the degradation process).15–19 eo is a thoroughly studied advanced oxidation process (aop) for all kinds of wastewater applications, with most studies focused on the anode material, operating factors and design of the eo reactor.18–21 the performance of eo is highly dependent on the activity and stability of the anode. various anodes such as, pbo2, dimensionally stable anodes, and boron-doped diamond (bdd) have shown outstanding removal efficiency for smx.7,19,22 however, the possibility of releasing lead ions during the eo process is a major drawback of the pbo2 anode. the bdd anode seems to be the best choice, as it exhibits the highest oxygen evolution reaction overpotential and better oxidation performance than alternative approaches. in addition, it has been reported that bdd anodes almost completely mineralize smx and its intermediates, achieving high smx removal efficiency (≈100 %), chemical oxygen demand (cod > 90 %) and total organic carbon (toc > 90 %).23 however, the price of bdd anodes is relatively high ($15,000–22,000/m2), which limits its industrial applications.24 as an engineering method, modeling can help to comprehensively understand the eo process.25 nevertheless, few studies have focused specifically on optimizing relevant parameters and predicting results to reach optimal conditions. the characteristics of current density, ph value, the initial concentration of smx, electrolyte concentration and degradation time also play key roles in the eo of the antibiotic in aqueous solution. the mechanism of eo is very complicated and the relationships among these parameters are nonlinear. it is difficult to explain such problems by using conventional mathematical models. an artificial neural network (ann) is a universal and highly flexible mathematical tool for the modeling and analysis of some complicated problems in which the data relationships are unknown. compared to the conventional modeling methods, anns electrochemical oxidation of sulfamethoxazole 715 perform better in extracting a pattern in nonlinear, complex and noisy data sets to detect the trends with higher accuracy. anns have proved useful in a variety of biological, medical and economic aspects.26–28 particle swarm optimization (pso), which was originally inspired by the regularity of the behavior of bird flocks, obtains the optimal solution by sharing information among the groups.29,30 with advantages such as less parameters, ease of adjustment, local and global integration and fast convergence, pso has been successsfully applied to many cases including ann training, function optimization and pattern classification.31 the present study describes an efficient method for the degradation of smx in aqueous solution. the effects of the major factors on the kinetics were investigated in detail, particularly current density, electrolyte concentration, solution ph value, and initial concentration of smx. an ann was developed to model the smx removal through the eo process. moreover, the ann model coupled with pso was applied to predict the optimized parameters required for maximizing the smx removal efficiency. the outcomes of this study may contribute to the further application of eo for the removal of synthetic antimicrobial agents in aqueous medium. experimental material and reagents all reagents and materials were analytical grade and all solutions were prepared with deionized water (resistivity = 18 mω cm at 25 °c) using a milli-qtm system (millipore corp., bedford, ma, usa). titanium foil with a reaction area of 37.5 cm2 (50 mm×75 mm) was selected as the metal matrix. smx was purchased from sigma-aldrich (saint louis, mo, usa). preparation of electrodes in this study, the anode contained three coatings. first, before use, the titanium plates were chemically degreased in a naoh solution (5 %) at 90 °c for 1 h and then etched in a boiling oxalic acid solution (10 %) for 2 h. second, the sno2–sb2o3 bottom coating was prepared by the sol–gel method. the titanium plate was symmetrically brushed with the sol– –gel, consisting of sbcl3, sncl4·4h2o and isopropyl alcohol, dried under vacuum for 10 min at 130 °c and annealed for 15 min at 515 °c. this process was repeated 15 times, followed by annealing for 60 min. ultimately, the co-doped modified β-pbo2 active coating was electrodeposited in an acidic electrolyte containing 0.5 m pb(no3)2, 0.1 m hno3, 0.01 m naf and 0.006 m co(no3)2 ·6h2o. electrochemical oxidation experiments the eo processes of smx were performed in an electrochemical system with an effective volume of 100 ml. a 1.5-mm-thick co-doped modified pbo2 electrode with dimensions of 7.5 cm (length)×5 cm (width) was selected as the anode, and same-sized stainless steel plates (ss304) were used as cathode. the electrode spacing was maintained at 1.5 cm. a 1000-ml reservoir contained the test solution, which was kept in a constant state of flow by pumping it through the electrochemical system before being returned to the reservoir (scheme 1). in each experiment, an smx aqueous solution (10–500 mg l-1) containing 0.05–0.4 m nacl electrolyte solution was used and the current density was adjusted in the range from 1 to 20 ma 716 wan et al. cm-2 with alternating current power. in each experiment, samples were taken from the reservoir at predetermined time intervals. scheme 1. configuration of the electrochemical oxidation experiments. analytical methods the concentration of smx was determined by high-performance liquid chromatography (hplc) on an agilent hplc system (agilent technologies, santa clara, ca, usa) equipped with an agilent eclipse xrd-c18 column (4.5 mm×250 mm with 5 μm pore size) using 36 mm koh as eluent at a flow rate of 1.0 ml l-1. the ph value was determined with a 766 laboratory ph meter (knick international, berlin, germany). ann-pso modelling and optimization a standard ann consists mainly of network topology, activation functions between neurons, and learning algorithms. in this study, a three-layer network with a back propagation algorithm was designed to build the predictive mathematical model. the input layer consists of five neurons, including current density, ph value of the solution, the initial smx concentration, electrolyte concentration and electrolysis time, with the tangent sigmoid (eq. (1)) as the activation function. the output layer comprises one neuron, described as the smx removal efficiency, with a linear function (eq. (2)) as the transfer function: 1 ( ) = 1+e x f x (1) f(x) = x (2) although there are formulas for reference, the stability of the network structure is particularly sensitive to the number of neurons. therefore, this study compares the mean square error (mse) when training the networks under different hidden layer neurons to determine the optimal number of neurons. (eq. (3)): ( ) 2 ,cal ,exp 1 n i i i x x mse n = − = (3) since the unit data and the order of magnitude of the sample data are different, the input data for developing the ann model were normalized in the range of −1 to 1, using eq. (4). electrochemical oxidation of sulfamethoxazole 717 ii max min 2 = 1 x x x m − − (4) based on the obtained model, pso was used to optimize the operating parameters. the hybrid program comprising the ann and pso is shown in scheme 2. in this study, matlab version r2012a (mathworks, inc., natick, ma, usa) was used to perform all calculations and analyze the experimental data. scheme 2. flow chart of the ann in combination with pso. results and discussion kinetics of the electrochemical oxidation of smx the effects of the applied current density (1–20 ma cm–2), ph of the solution (2–10), the initial concentration of smx (10–500 mg l–1) and electrolyte concentration (0.05–0.4 mol l–1) on the removal of smx were systematically evaluated. based on the results, the kinetics of the eo of smx were calculated according to the pseudo-first-order kinetics model, as described in eq. (5): 0ln t c = kt c (5) where c0 / mg·l–1 is the initial sulfamethoxazole concentration; ct / mg·l–1 is the sulfamethoxazole concentration at time t / min. the kinetic parameters, rate constant, k, and half-life, t1/2, and coefficient r2 are summarized in table i. operating parameters influencing the electrochemical oxidation of smx effects of current density. in the process of eo, the current density plays a very important role as it determines the removal rate of pollutants by controlling electron transport and the generation of reactive oxidants.7,15,19,20 the current density was variously set to 1, 5, 10, 15 and 20 ma cm–2, and the removal efficiency of smx during the degradation at each current density is shown in fig. 1. these 718 wan et al. results reveal that the increase of the current density has a positive effect on the smx removal efficiency. when the current density was 10 ma cm–2, the smx removal efficiency was 92.3 % after 60 min, and reached 95.1 % when the current density was increased to 15 ma cm–2. in addition, when the current density was increased to a certain level, its influence on the smx removal efficiency would gradually become insignificant. for instance, as the current density was increased from 15 to 20 ma cm–2, the smx removal rate was only increased by 0.9 %. these findings were consistent with the previous reports that increasing current density enhances the pollutant removal efficiency.23,32 table i. efficiency and kinetics of smx electrochemical degradation j / ma cm-2 degradation efficiency, % k / 10 -2 min-1 t1/2 / min r 2 1 72.1 1.97 35.2 0.936 5 77.9 2.41 28.8 0.961 10 92.3 4.14 16.7 0.994 15 95.1 5.08 13.6 0.995 20 96.0 5.34 13.0 0.995 initial ph 2 90.8 3.73 18.6 0.989 4 89.3 3.38 20.5 0.981 6 87.2 3.16 21.9 0.981 8 78.8 2.38 29.1 0.964 10 72.2 1.99 34.8 0.96 csmx (initial) / mg l -1 10 94.1 4.66 14.9 0.995 50 89.4 3.59 19.3 0.961 100 82.7 2.98 23.3 0.970 200 78.8 2.49 27.8 0.943 500 79.9 2.1 33.0 0.950 celectrolyte / mol l -1 0.05 88.1 3.59 19.3 0.992 0.1 91.2 4.10 16.9 0.995 0.2 93.9 4.67 14.8 0.995 0.3 94.5 3.43 13.9 0.995 0.4 87.2 3.38 20.5 0.970 as shown in table i, the coefficient of determination (r2) was always >0.94, suggesting that the removal of smx by eo followed the pseudo-first-order kinetics pattern. at the same time, at the current densities of 1, 5, 10, 15 and 20 ma cm–2, the degradation kinetic constants were 1.97×10–2, 2.41×10–2, 4.14×10–2, 5.08×10–2 and 5.34×10–2 min–1, respectively. however, when the current density exceeded 15 ma cm–2, the removal efficiency of smx and the kinetic constant were not significantly increased. the results can be interpreted from two aspects: ⅰ) the anodic oxidation takes place on the surface of the electrode, and the mass electrochemical oxidation of sulfamethoxazole 719 transfer efficiency is limited. once the current density is reaching the limiting current density, the rate of degradation is only related to mass transfer; ⅱ) the undesirable parasitic reactions such as oh– to o2 or to •oh2 were enhanced with the increasing current density.15,16 fig. 1. effects of the current density on the dynamics of the electrochemical oxidation of smx. effects of the ph value of the solution. it is well known that the ph value has a significant impact on the eo of organic pollutants.14,18,33 the removal efficiency of smx during degradation was determined at various solution ph values (2, 4, 6, 8 and 10), and the results are shown in fig. 2. under acidic conditions (ph of 2, 4 and 6), the smx removal efficiency was 90.8, 89.3 and 87.2 %, res fig. 2. effects of the initial ph value on the dynamics of the electrochemical oxidation of smx. 720 wan et al. pectively, while, under alkaline conditions, the maximum removal efficiency of smx was 78.8 %. as shown in table i, the kinetic constant (k) values changed significantly with the various ph values evaluated. for example, at an initial ph value of 2, the k value (3.73×10–2 min–1) was about 1.9 times higher than that (1.99×10–2 min–1) at an initial ph value of 10. the acid dissociation constant (pka) of smx is reported to be 1.8, suggesting that acidic conditions have a positive impact on the diffusivity of smx.22 on the other hand, the oxygen evolutions on the anode surface were decreased under acidic condition (eq. (6) and (7)) and the polymerization reactions were also accelerated under this condition.34 furthermore, the active chlorine was present in the form of cl2 at the ph range from 0 to 3.3, as hclo at the ph range from 3.3 to 7.2, and mainly as clo– at ph higher than 7.2. among these chlorine forms, the oxidizing organic matter of hclo is significantly stronger than that of the other two forms: 2oh– → o2+ 2h+ + 2e– (6) 2h2o → o2+ 4h+ + 4e– (7) effects of initial smx concentration. the removal efficiency of smx during degradation was determined at various initial smx concentrations (10, 50, 100, 200 and 500 mg l–1) and the results are shown in fig. 3. fig. 3. effects of the initial smx concentration on the dynamics of the electrochemical oxidation of smx. they reveal that as the initial concentration of smx increased from 10 to 500 mg l–1, the removal efficiency of smx decreased from 94.0 to 69.9 %, meanwhile, the k values were consistent with the tendency. these findings can be explained by the following aspects: ⅰ) direct oxidation was controlled by mass transfer. besides, smx and some intermediate products can be adsorbed on the electrochemical oxidation of sulfamethoxazole 721 surface of the electrode, which contaminate the electrode and also hinders the diffusion of other pollutants to the electrode surface;33,35 ⅱ) the number of hydroxyl radicals and active sites on the electrode surface at a given current density have been identified. as the concentration of smx increases, more and more intermediates are formed, and these compounds compete with smx for hydroxyl radicals and active sites on the electrode surface.18 effects of electrolyte concentration. in order to promote the transfer of electrons and ionic conduction, nacl was added to smx solution. the removal efficiency of smx during degradation was determined at various electrolyte concentrations (0.05, 0.1, 0.2, 0.3 and 0.4 mol l–1) and the results are shown in fig. 4. the results indicated that an increase in the electrolyte concentration from 0.05 to 0.2 mol l–1 improved the smx removal efficiency and k values, and reached the maximum removal efficiency at the nacl concentration of 0.2 mol l–1 after 60 min. thus, it is clear that increasing the concentration of electrolyte in a certain concentration range was beneficial to the degradation of smx. this is probably due to the addition of electrolyte, which can effectively reduce the solution resistance and increase the conductivity, thereby increasing the amount of •oh generated on the electrode surface and accelerating the degradation of smx. besides, in an electrochemical system, increasing chlorides under high applied potential will produce more active chlorine compounds, such as cl2 (eq. (8)), hclo (eq. (9)) and ocl– (eq. (10)) which are active oxide species, leading to the rapid elimination of organic matter:36 2cl– → cl2 + 2e– (8) cl2 +h2o → hocl + h+ + cl– (9) hocl → h+ + ocl– (10) fig. 4. effects of the electrolyte concentration on the dynamics of the electrochemical oxidation of smx. 722 wan et al. however, the smx removal efficiency decreased from 94.5 to 87.2 % when the nacl was further increased from 0.3 to 0.4 mol l–1. these results suggest that if the optimum concentration of electrolyte is exceeded, the removal rate will not increase but rather decrease, likely due to the formation of a layer salt on the surface of the anode, which exerted a negative influence on the direct and indirect oxidation.37 ann modelling and optimization of parameters establishment of ann model. the 125 data sets used in this experiment were divided into two groups. one group consists of 110 data sets, which are used for the training of the network and are called training sample sets. the other group is called the test network and was evaluated with the remaining 15 data sets. different hidden layer nodes (5–20) were selected to establish a network and determine the optimal number of neurons by training the mse for training the network using different neuron number conditions. the relationship between the mse and the number of neurons is illustrated in fig. 5. the results show that the network with fewer neurons or more than 11 in the hidden layer cannot effectively converge, thus the topology of 5–11–1 seem to be the optimal topology to develop the model. fig. 5. relationship between the mse and number of neurons in the hidden layer. evaluation of the ann model. the relationship between the number of epochs and the mse was evaluated. the results shown in fig. 6 reveal that the training of the ann achieved convergence after 645 epochs with the minimum mse, suggesting that the proposed ann model successfully predicted the results of smx removal through the eo process. electrochemical oxidation of sulfamethoxazole 723 fig. 6. the relationship between the number of epochs and mse. to further validate the current ann model, the regression analysis and the standardized residual analysis were also conducted. as shown in fig. 7, the coefficient of determination for the training, test and all data sets were 0.9845, 0.9644 and 0.9826, respectively. the representation of the predicted smx removal efficiency versus experimental data for both training and test data shows the good predictive ability of the proposed ann model. the high value of the coefficient of determination suggests that the current ann model is suitable for the prediction of data which has not been used during the training of the model. in addition, the histograms (fig. 8) of the standardized residual of the model is subject to normal distribution, which confirms that the ann model has high accuracy to predict results.38 fig. 7. the ann model predicted versus actual data. optimization of parameters by ann-pso. based on the obtained ann model, the optimization of experimental parameters was performed. with the objective of maximizing the removal efficiency of smx, the pso technique was 724 wan et al. used to optimize the input layer of the ann model. the values of the pso parameters used in the optimization simulation are as follows: number of particles in swarm = 20, cognitive component = 2, social component = 2, maximum velocity = 1, minimum inertia weight = -1, and maximum inertia weight = 0.9. as shown in fig. 9, the maximum removal efficiency was obtained after 100 generations. fig. 8 standardized residual distribution of predicted and experimental values. fig. 9. fitting curve of pso. the operating conditions of the optimized solution were obtained at a current density of 12.37 ma cm–2, an smx concentration of 74.45 mg l–1, a reaction time of 51.49 min, a ph of 4.78, and a nacl dose of 0.24 mol l–1. the ann -pso prediction of smx removal efficiency under the optimized process conditions was 93.70 %. in order to effectively verify the prediction results, the above-mentioned optimal conditions were used for the test, and the validation tests were performed in three parallel tests. the results of the verification tests are listed in table ii, and the actual smx removal efficiency of 92.03±2.30 % was achieved under the same optimized conditions. the absolute error between electrochemical oxidation of sulfamethoxazole 725 the verification test results and the predicted value was 1.67 %. thus, the validation results indicated that this method can be ideally used to optimize the related parameters and predict the anticipated results with acceptable accuracy table ii. the results of the verification test no. of verification test j ma cm-2 ph csmx mg l-1 ce mol l-1 t min removal efficiency, % relative standard deviation, % 1 12.37 4.78 74.45 0.24 51.49 91.9% 1.27 2 12.37 4.78 74.45 0.24 51.49 89.8% 2.75 3 12.37 4.78 74.45 0.24 51.49 94.4% 0.49 conclusion in this study, the co modified pbo2 electrode was used to degrade the smx in aqueous solution. the eo of smx in aqueous solution followed the pseudo -first-order kinetics pattern, and the oxidation process was more favorable at high current density, acidic ph and low initial smx concentration. once the electrolyte exceeds a certain concentration, the removal rate does not increase but instead decreases. an ann with 5–11–1 topology was used to develop a predictive model for simulating this eo process. the training of the ann achieved convergence after 645 epochs, with the minimum mse. the correlation coefficient for the training, test and all data sets are 0.9845, 0.9644 and 0.9826, respectively, indicating an excellent fit between the predicted and experimental values. the optimized operation conditions (i.e., a current density of 12.37 ma cm–2, an initial ph of 4.78, an initial smx concentration of 74.45 mg l–1, an electrolyte concentration of 0.24 mol l–1, and an electrolysis time of 51.49 min) were obtained through pso. the results of the verification tests suggest that the proposed ann -pso model can successfully model and optimize the smx removal process. acknowledgement. supported by the fundamental research funds for the central universities. и з в о д испитивање електрохемијске оксидације сулфаметоксазола на pbo2 електроди модификованој кобалтом применом модела вештачких неуронских мрежа и оптимизације засноване на моделу ројева честица jiteng wan, chunji jin, banghai liu, zonglian she, mengchun gao и zhengyang wang college of environmental science and engineering, ocean university of china, оo.238 songling road, qingdao, shandong province 266100, china присуство антибиотика у водотоковима, чак и у траговима, изазива све већу пажњу. у складу с тим, неопходне су одговарајуће технологије за ефикасно уклањање ових једињења из водених раствора. у овом истраживању је испитивана електрохемијска оксидација сулфаметоксазола (smx) на електроди од pbo2 која је модификована кобалтом. нађено је да оксидација smx следи кинетику псеудо-првог реда и да ефикасност уклањања smx достиже максималну вредност од 95,1 % у току 60 min. ефекат 726 wan et al. главних фактора на кинетику оксидације smx је испитиван у једнофакторним експериментима у којима је густина струје износила 1–20 ma cm –2 , вредност ph раствора 2–10, почетна концентрација smx 10-500 mg l –1 и концентрација електролита 0,05–0,4 mol l –1 . за симулацију електрохемијске оксидације smx коришћен је модел вештачке неуронске мреже, а затим је урађена оптимизација радних параметара заснована на моделу ројева честица. показано је да се максимална ефикасност уклањања smx добија под оптимизованим радним условима, тј. при густини струје од 12,37 ma cm -2 , почетној вредности ph од 4,78, почетној концентрацији smx од 74,45 mg l –1 , концентрацији електролита oд 0,24 mol l –1 и трајању електролизе oд 51,49 min. резултати провере ваљаности добијених параметара показали су да се описана метода може успешно користити за оптимизацију сродних параметара и предвиђање очекиваних резултата са прихватљивом тачношћу. 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scientific outputs of countries formed from former yugoslav republics and some other countries for the period 2008–2012 verica roglić-korica and slobodan k. milonjić#* vinča institute of nuclear sciences, university of belgrade, belgrade, serbia (received 17 april, accepted 8 may 2017) abstract: scientific outputs of six countries (republics of the former yugoslavia – the socialist federal republic of yugoslavia): serbia, croatia, slovenia, bosnia and herzegovina, former yugoslav republic of macedonia and montenegro were discussed in this paper. for the purpose of comparison, the data for seven neighbouring countries of the former yugoslavia (albania, greece, bulgaria, romania, hungary, austria and italy) were given as well. also, the data for the group of eight (g8) countries (the united states, canada, the united kingdom, france, italy, germany, japan and the russian federation) and some european countries similar in population to ex-yugoslav republics were included. to gain a more complete picture on the outputs in scientific production (e.g., publications and patents), data on several developed european countries, as well as the countries from other continents were given. the analysis, which included 33 countries, was made based on different bibliometric indicators for the period of five years (2008–2012). the data were collected from international databases. keywords: bibliometric analysis; scientific productivity; ex-yugoslav republics. introduction bibliometric analysis is a useful method for the characterization of scientific research. it provides the best available measures of scientific “output” by the world’s countries. there are plenty of articles devoted to the scientific production of individual countries in the literature. only a few of them are mentioned here.1-3 however, a small amount of data on scientific outputs of the countries arising from the former yugoslav republics can be found. * corresponding authors. e-mail: smiloni@vinca.rs # serbian chemical society member. https://doi.org/10.2298/jsc170417061r available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs 1076 roglić-korica and milonjić kutlača et al.4 presented a comprehensive comparative analysis of 13 south east european countries (including all republics of the former yugoslavia) according to their scientific outputs in the period of 2005–2010. an analysis of the scientific production in the republic of serbia, for the last several years, was made by ivanovic and coworkers.5,6 kastrin et al.7 made a comprehensive analysis of the performance of the slovenian research community between the years 1970 and 2015 with the focus on productivity, collaboration, internationality and interdisciplinarity. they demonstrated a fast increase in scientific productivity after the year 1992, soon after slovenia won the independence from yugoslavia, its climax reached in 2011 and then a steady decline. the average number of publications per author increased a lot in the period 1990–2010, stabilizing in the years 2011–2015. they concluded that political and financial instability has always had an impact (mostly negative) on the scientific productivity. igić8 also investigated the influence of the civil war during disintegration of the former yugoslavia on scientific outputs, as measured by changes in numbers of articles published in peer-reviewed journals. the annual numbers of articles from each republic were determined from 1988 to 2000. lewison and igic9 investigated scientific outputs from serbia, croatia and slovenia, and the patterns of co -authorship between them and five western countries and with each other. they studied the influence of the war on scientific outputs, as measured by the changes in the number of papers indexed in the science citation index from various parts of the former yugoslavia. the number of papers from each of these states was determined from 1985 to 1996. recently, ivanović et al.10 have shown that the yugoslav wars affected the republics’ productivities and scientific cooperation in different ways. the most affected republics by wars and social crisis were serbia and bosnia & herzegovina, while the least affected ones were slovenia and fyr macedonia. also, there are a few studies11–15 on the scientific productivity of researchers from the republic of croatia, a constituent republic of the former yugoslavia up to 1991. this letter offers a comparative analysis of the scientific research outputs of serbia, croatia, slovenia, bosnia and herzegovina, macedonia and montenegro for the period 2008-2012, i.e., after these states declared independence. the last of them, montenegro, reached its statehood after the referendum held in 2006. integration of slovenia and croatia into the european union resulted in significant increase in their research and development, due to granted eu projects/programs. methods the study is based on data obtained from online available databases of: scimago, sjr-scimago journal & country rank, arwu – the academic ranking of world universities, the world bank, and oecd – the organization for economic co-operation and development, for the period from 2008 to 2012. available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs a comparative analysis of scientific outputs (2008–2012) 1077 results and discussions the data on population, gdp (gross domestic product) per capita, as well as the research and development expenditure for 33 selected countries for the period 2008–2012 are given in table i. of all given countries, montenegro has the smallest population (0.620 million) and china the largest (1,337,689 million). gdp per capita, as a measure of the level of economic development of a country, ranges from us $ 1,326 (india) to 91,701 (norway). table i. population, gross domestic product per capita (current us dollars) and r&d expenditure from 2008 to 2012, according to: http://data.worldbank.org/indicator/ /sp.pop.totl and http://data.worldbank.org/indicator/ny.gdp.pcap.cd no. country population 2008–2012 (in million) gdp per capita 2008–2012 (in us $) r&d expenditure (% of gdp) 1 serbia 7.289 5,645 0.35 2 croatia 4.366 14,147 0.82 3 bosnia & herzegovina 3.847 4,559 0.33 4 slovenia 2.044 24,097 1.87 5 macedonia, fyr 2.102 4,614 0.44 6 montenegro 0.620 6,944 0.41 7 albania 3.155 3,995 0.15 8 bulgaria 7.479 6,762 0.53 9 romania 21.429 8,264 0.51 10 hungary 9.990 13,646 1.17 11 greece 11.282 26,483 0.68 12 italy 60.430 35,309 1.24 13 austria 8.396 47,452 2.72 14 united kingdom 62.291 38,437 1.80 15 sweden 9.372 51,609 3.57 16 denmark 5.545 58,281 2.99 17 finland 5.363 46,982 3.84 18 netherlands 16.610 48,777 1.81 19. norway 4.892 91,701 1.68 20. spain 45.985 31,565 1.38 21 switzerland 7.825 73,459 2.99 22 france 65.035 41,192 2.21 23 germany 81.895 42,021 2.78 24 portugal 10.595 21,961 1.58 25 poland 38.308 12,715 0.67 26 russian federation 142.548 11,669 1.15 27 usa 309.139 47,352 2.87 28 canada 34.107 46,949 1.86 29 brazil 195.211 10,378 1.15 30 korea rep. 49.465 20,301 3.56 31 japan 127.618 42,683 3.42 32 india 1,203.710 1,326 0.84 33 china 1,337.689 4,648 1.58 available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs 1078 roglić-korica and milonjić it is evident that the former yugoslav republics, except croatia and slovenia, compared with other countries have low gross domestic product per capita. such a low gdp per capita can also be seen for the three neighbouring countries of ex -yugoslavia (albania, bulgaria and romania). therefore, a conclusion, based on the data given, is that the balkan countries are the least developed ones. in the same table (the fifth column), data on the gross domestic expenditure on research and development, as a percentage of gdp, are given (r&d expenditure). according to the presented figures for all countries, the lowest research and development expenditure was recorded in albania (0.15 %) and the highest in finland (3.84 %). as for the countries formed from the former yugoslavia, the highest r&d expenditure is in slovenia and croatia, while in other countries (former yugoslav republics), it is less than 0.5 %. these data correlate well with the scientific outputs in these countries. the inadequate budget for r&d with the already lowest gross national income per capita inevitably reflects in the modest productivity of researchers in these countries. also, the inadequate budget (less than 1 % of gdp) can be seen in most neighbouring countries of the former yugoslavia (albania, bulgaria, romania and greece). poland and india form a group of countries with gdp less than 1 %. slovenia, hungary, italy, the united kingdom, the netherlands, norway, spain, portugal, the russian federation, canada, brazil, and china belong to the group of countries with gdp between 1 and 2 %. the countries spending between 2 and 3 % of gdp on r&d are austria, finland, switzerland, france, germany and the usa, while sweden, finland, the republic of korea and japan spend more than 3 % of gdp. table ii compares the total number of publications and their citations for all studied countries over the study period. citation, however, is not a direct measure of the quality and the academic significance of an article but reflects its visibility and impact on the scientific community16,17. the highest citation in the case of serbia5 is connected with the articles that were products of international cooperation with corresponding authors with no address in serbia. the articles co -authored by at least one researcher from serbia in 2006–2013, cited more than 300 times (in total, eight articles), are characterized by the number of co-authors usually more than 10, but the first and the corresponding author are not from serbia.5 for a better comparison, the ratio of citations to the number of publications (calculated by dividing data from column 4 by those from column 3) is given in column 5. of all 6 countries emerged from the former yugoslavia, the highest citations/publication ratio has slovenia. also, the ratios of these countries are much lower than those of the others. such low ratios are noticed for albania, romania, the russian federation, india and china. in the same table (next to the last column), the data on self-citation are gathered as well, given in percent. self -citations range from 10.4 % (albania) to 54.38 % (china). available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs a comparative analysis of scientific outputs (2008–2012) 1079 table ii. number of publications, citations, citations per publications, self-citations and hindex, from 2008 to 2012, according to: scimago (2007), sjr-scimago journal & country rank. retrieved november 12–18, 2014, from http://www.scimagojr.com no. country publications citations cit./publ. self-citations a % h-indexa (order) 1 serbia 27,194b 108,118 3.49 27.08 86 (29) 2 croatia 28,497 112,329 5.79 22.24 161 (26) 3 bosnia & herzegovina 3,337 7,849 3.48 14.71 49 (31) 4 slovenia 24,569 140,265 8.73 19.69 172 (25) 5 macedonia, fyr 2,961 9,163 5.59 12.18 67 (30) 6 montenegro 981 3,320 2.62 22.16 23 (33) 7 albania 1,066 2,284 4.21 10.4 40 (32) 8 bulgaria 18,307 83,877 7.65 16.73 154 (27) 9 romania 57,802 170,836 4.59 25.25 153 (28) 10 hungary 44,963 284,434 11.4 16.34 277 (23) 11 greece 85,635 579,729 11.1 17.02 295 (22) 12 italy 411,261 3,286,702 14.1 23.31 654 (7) 13 austria 94,481 798,529 15.2 13.79 416 (14) 14 united kingdom 769,857 6,794,401 17.5 23.58 934 (2) 15 sweden 145,830 1,455,129 19.4 15.7 567 (10) 16 denmark 93,463 966,366 19.8 14.4 476 (12) 17 finland 76,909 682,709 17.3 15.87 407 (15) 18 netherlands 234,413 2,501,187 19.7 16.55 636 (8) 19 norway 75,329 625,790 15.3 16.16 362 (17) 20 spain 354,267 2,573,254 12.4 24.84 531 (11) 21 switzerland 170,147 1,925,206 20.8 13.65 629 (9) 22 france 512,152 3,154,317 14.9 22.72 742 (4) 23 germany 706,143 5,886,941 15.4 26.00 815 (3) 24 portugal 77,754 513,491 10.3 19.9 269 (24) 25 poland 151,069 680,400 7.58 26.13 336 (21) 26 russian federation 198,281 632,834 5.73 29.72 355 (18) 27 usa 2,751,023 24,399,854 19.5 47.71 1,518 (1) 28 canada 423,586 3,663,476 17.0 19.02 725 (5) 29 brazil 247,654 1,115,495 7.86 33.98 342 (19) 30 korea rep. 299,069 1,688,568 8.76 22.2 375 (16) 31 japan 615,605 3,630,154 12.2 28.91 694 (6) 32 india 400,406 1,584,765 6.52 34.56 341 (20) 33 china 1,674,843 6,127,888 4.71 54.38 436 (13) aaccording to: self-citations and h-index for the period 1996–2013; b27,879 according to daily newspaper “politika” from 7 may, 2017 all high-quality papers have been published in international journals and in english. a lot of countries have their own journals in their native languages. therefore, the articles of most researchers from these countries have been published in their own language; examples are china and the russian federation. according to the russian science citation index (rsci), a bibliographic database of scientific publications in russian established in 2005, the total number of available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs 1080 roglić-korica and milonjić publications in russia, between 2008 and 2012, is 6,829,000. rsci accumulates more than 20 million publications of russian authors, as well as the information about citing these publications from more than 5,000 russian journals (only 154 of them included in web of science in 2014)18. this explains high self-citations in these countries. hence, preferential us citing of us papers is not a surprising thing. the last column in table ii contains hirsch index (h-index) data for each studied country. h-indices go from 23 (montenegro) to 1,518 (the us). among the countries arose from the former yugoslavia, the highest h-index have slovenia and croatia. the ranking of the studied countries according to the h-index is given in parentheses. as evident, at the very top is the us (h = 1,518) and on the bottom montenegro (h = 23). all g8 countries have high h-index and are ranked from 1 to 7. the exception is russia with h-index of 18. it is useful to compare the scientific output (number of publications) with the number of inhabitants and the number of researches in particular country. table iii (column 3) provides the number of researchers per million inhabitants for all studied countries, for the period 2008–2012. as evident, the lowest number of researchers has india (137) and the highest has finland (7,685). in the case of the former yugoslav republics, the lowest number of researchers (217) has bosnia and herzegovina and the highest has slovenia (3,653). all g8 countries, as well as the eu ones have high number of researchers/millions of inhabitants. albania, with 147 researchers/millions of inhabitants, is according to this criterion, closely behind india. in the same table, column 4, data on the number of publications per 1000 inhabitants are given, while the column 5 in the same table contains the data on the number of publications per researcher. the data given in these two columns enable comparison of all countries according to the uniform criteria. of all countries studied, the number of publications per 1000 inhabitants is the lowest for india (0.33) and albania (0.34). the highest number of publications per 1000 inhabitants can be observed for switzerland (21.7). the g8 countries, apart from russia, have high number of publications per 1000 inhabitants. this indicator ranges from 6.80 (italy) to 21.7 (switzerland). the number of publications per 1000 inhabitants for the countries formed from the former yugoslavia goes from 0.87 (bosnia and herzegovina) to 12.0 (slovenia). only for slovenia and croatia this number is higher than 6, while in the case of other countries it is lower than 5. albania, bulgaria, romania and hungary, the neighbouring countries of the former yugoslavia, have low number of publications per 1000 inhabitants (< 5). this ratio for china is also low (1.25) in contrast to all developed countries that have high number of publications per 1000 inhabitants. as for the number of publications per researcher (table iii, column 5), the lowest number according to this criterion is evident for russia (0.45), with switzavailable on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs a comparative analysis of scientific outputs (2008–2012) 1081 erland (6.55) in the lead. the number of publications per researcher for the countries emerged from the former yugoslavia ranges from 2.08 (montenegro) to 4.16 (croatia). contrary to the previous criteria, the data based on this one, for all studied countries, do not vary greatly. one should have in mind that we took the account of the total number of publications making no distinction in quality between them, i.e., impact factors of journals in which these papers were published. table iii. number of researchers per million inhabitants, publications/1000 inhabitants, publications/researcher, r&d expenditure/inhabitant, and r&d expenditure /researcher, for the period 2008 to 2012 according to: http://data.woldbank.org/indicator/sp.pop.scie.rd.p6 no. country no. of researchers per million inhabitants publications per 1000 inhabitants publications per researcher r&d expend./inhab. us $ r&d expend./researcher us $×10-4 1 serbia 1,037 3.73 3.60 20 1.90 2 croatia 1,567 6.53 4.16 116 7.40 3 bosnia & herzegovina 217 0.87 4.00 15 6.93 4 slovenia 3,653 12.0 3.29 451 12.34 5 macedonia, fyr 613 1.41 2.30 20 3.31 6 montenegro 760 1.58 2.08 28 3.75 7 albania 147 0.34 2.30 6 4.08 8 bulgaria 1,515 2.45 1.62 36 2.36 9 romania 904 2.70 2.98 42 4.66 10 hungary 2,394 4.50 1.88 160 6.67 11 greece 2,232 7.59 3.40 180 8.07 12 italy 1,719 6.80 3.96 438 25.5 13 austria 4,187 11.2 2.69 1,291 30.8 14 united kingdom 4,020 12.4 3.07 692 17.2 15 sweden 5,247 15.6 2.96 1,842 35.1 16 denmark 6,462 16.8 2.61 1,743 27.0 17 finland 7,685 14.3 1.86 1,804 23.5 18 netherlands 3,015 14.1 4.68 883 29.3 19 norway 5,047 15.4 2.85 1,540 30.5 20 spain 2,918 7.70 2.64 436 14.9 21 switzerland 3,320 21.7 6.55 2,196 66.2 22 france 3,708 7.88 2.12 910 24.6 23 germany 3,832 8.62 2.25 1,168 30.5 24 portugal 4,079 7.34 1.80 347 8.51 25 poland 1,633 3.94 2.41 85 5.22 26 russian federation 3,112 1.39 0.45 134 4.31 27 usa 4,019 8.90 2.21 1,359 33.8 28 canada 4,470 12.4 2.78 873 19.5 29 brazil 667 1.27 1.90 119 17.9 30 korea rep. 5,172 6.05 1.17 723 14.0 31 japan 5,184 4.82 0.93 1,460 28.2 32 india 137 0.33 2.43 11 8.13 33 china 1,031 1.25 1.21 73 7.12 available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs 1082 roglić-korica and milonjić table iii, columns 6 and 7, compares the data on the gross domestic expenditure on research and development per capita (column 6) and per researcher (column 7), for each studied country. the lowest r&d expenditure per capita is evident for albania (us $ 6) and the highest for switzerland (us $ 2,196). for the countries formed from ex-yugoslav republics, the highest expenditure can be seen for slovenia (us $ 451) and croatia (us $ 116), and the lowest for bosnia and herzegovina (us $ 15). the other countries (serbia, fyr macedonia and montenegro) have very low r&d expenditure per capita. concerning other countries, r&d expenditure per capita in india is also very low (us $ 11). the g8 countries as well as other developed countries spend significant amounts (from several hundred to several thousand us $) on r&d per capita. this certainly results in top research productivity and quality in these countries. the last column of table iii presents the data on r&d expenditure (in us $) per researcher. a comparison shows that, of all countries formed from the former yugoslavia, the lowest r&d expenditure can be noted for serbia and the highest for slovenia. according to the data for all studied countries, the r&d expenditure per researcher is the lowest in serbia (us $ 1.90×104) and the highest in switzerland (us $ 66.2×104). in high-developed countries, g8, the expenditure is high as expected starting from us $ 17.2×106–30.5×106. the exception is russia with expenditure of us $ 4.31×104. table iv (column 3) shows the data on the number of patents for all selected countries in the period between 2008 and 2012. montenegro is the only country in which no patent was filed in this period. the number of patents filed ranges from 1 (albania) to 1,146,059 (the us). for a better comparison of these data, the numbers of patents per million inhabitants are given in column 4 of the same table. of all countries, which came into being from the former yugoslavia, the highest number of patents per million inhabitants can be seen for slovenia (73.4) and croatia (19.5). for other countries of the same group, the figures are more than modest and range from 0 (montenegro) to 4.2 (serbia). neighbouring countries of the former yugoslavia have also a modest number of patents per million inhabitants – albania (0.32) and romania (5.32). low numbers of patents per million inhabitants are also evident for india (4.59) and brazil (5.17). all g8 countries, apart from the russian federation (with 9.25), have high number of patents per million inhabitants, going from 180 (italy) to 3,707 (the us). all high-developed countries have high number of patents per million inhabitants. this indicator confirms the technological level of a country. it should be mentioned that, besides the us, the following countries have more than 1000 patents per million inhabitants: switzerland (1,105), the republic of korea (1,177) and japan (1,745). finally, the data on the number of universities among the world top 500 ones (in 2012 and 2015) for all selected countries are given in columns 5 and 6, table iv. available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs a comparative analysis of scientific outputs (2008–2012) 1083 table iv. number of patents, patents per million inhabitants from 2008 to 2012, and academic ranking of world universities, according to: arwu – the academic ranking of world universities; top 500 universities shanghai ranking-2012.mht; top 500 universities shanghai ranking-2015.mht; www.uspto.gov/go/taf/cst_all.htm no. country no. of patents 2008–2012 no. of patents (2008–2012) per mill. inhab. arwu 2012 arwu 2015 1 serbia 30 4.12 1 1 2 croatia 85 19.5 1 3 bosnia & herzegovina 5 1.30 4 slovenia 150 73.4 1 1 5 macedonia, fyr 3 1.43 6 montenegro 0 0 7 albania 1 0.32 8 bulgaria 183 24.5 9 romania 114 5.32 10 hungary 442 44.2 2 2 11 greece 273 24.2 2 2 12 italy 10,886 180 20 20 13 austria 4,161 496 7 6 14 united kingdom 23,647 380 42 39 15 sweden 8,212 876 11 11 16 denmark 3,687 665 4 5 17 finland 5,298 988 5 6 18 netherlands 9,441 568 13 12 19 norway 1,923 393 4 3 20 spain 2,606 56.7 11 13 21 switzerland 8,650 1,105 7 7 22 france 23,598 363 20 22 23 germany 62,078 758 40 42 24 portugal 166 15.7 3 3 25 poland 344 8.98 2 2 26 russian federation 1,318 9.25 2 2 27 usa 1,146,069 3,707 149 146 28 canada 26,246 770 22 20 29 brazil 1,010 5.17 6 6 30 korea rep. 58,211 1,177 10 12 31 japan 222,751 1,745 21 18 32 india 5,522 4.59 1 1 33 china 16,574 12.4 42 39 the list of ranked universities has been regularly updated and released by the shanghai institute of education. ranking is based on the number of staff winning nobel prizes, number of highly cited researchers, articles published in journals of science and nature, as well as on the number of articles published. these criteria are compared with the number of full-time staff members in each institution the university is composed of (collages and institutes). among the countries formed from the former yugoslavia, only serbia, croatia and slovenia had 1 university available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs 1084 roglić-korica and milonjić on this list in 2012. in 2015, serbia and slovenia kept their place. of all studied countries (33), only bosnia and herzegovina, fyr macedonia, montenegro, albania, bulgaria and romania have no university on the list. these data also indicate poor scientific outputs of universities and research staff in these countries. all the member countries of g8 except the russian federation have from 20 (italy) to 149 (the us) universities among the 500 best ranked ones in 2012. two russian universities are on the list. it is interesting that india has only 1 university on the list. conclusions in this paper, a comparative analysis of the scientific production of authors from six countries, former yugoslav republics, was given. the analysis also included seven countries, neighbours of the former yugoslavia, several developed european countries, as well as countries from other continents. the total number of 33 countries was analyzed over the five-year period (2008–2012). the analysis revealed that all countries, which emerged from the former yugoslavia, except slovenia and croatia significantly lagged behind other selected countries. most developed countries (g8), apart from the russian federation, as well as developing countries are the leaders in quality and production of scientific articles. also, small (according to population) northern europe countries play an important role in world science. research and development expenditure (% of gdp) in the countries arose from the former yugoslavia is the lowest (except in slovenia and croatia) and amounts to < 0.5 %, compared with other analyzed countries. in order to improve the situation in these countries, the investment in science must be increased (at least up to 1 % of gdp) and the conditions for research and development including infrastructure should be improved. и з в о д упоредна анализа научне продукције држава насталих из република бивше југославије и неких других земаља, за период 2008–2012. верица роглић-корица и слободан к. милоњић институт за нуклеарне науке “винча“, универзитет у београду, београд дискутовани су научни доприноси шест држава (некадашњих република бивше југославије): србије, хрватске, словеније, босне и херцеговине, македоније и црне горе. ради упоређења са другим државама, дати су и подаци за седам држава (албанија, грчка, бугарска, румунија, мађарска, аустрија и италија) суседа бивше југославије. tакође су наведени и подаци групе г8 држава (уједињено краљевство, канада, сад, француска, италија, немачка, јапан и русија) као и неких европских земаља чији је број становника сличан броју становника некадашњих република бивше југославије. ради потпунијег сагледавања научног доприноса појединих држава наведени су подаци о научној продукцији неколико развијених европских држава као и држава са осталих континената. aнализа обухвата укупно 33 државе и урађена је на основу различитих available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs a comparative analysis of scientific outputs (2008–2012) 1085 индикатора, а односи се на петогодишњи период (2008–2012.). подаци су сакупљени из међународних база података. 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40 (2006) 345 15. b. klaic, croat. med. j. 38 (1997) 88 16. j. c. furlan, m. g. fehlings, j. neurotrauma 23 (2006) 156 17. a. baltusen, c. h. kindler, anesthesia analgesia 98 (2004) 443 a. guskov, d. kosyakov, i. selivanova, scientometrics 107 (2016) 28718. available on line at www.shd.org.rs/jscs/_________________________________________________________________________________________________________________________ (cc) 2017 scs << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {alkylamino and aralkylamino derivatives of avarone and its mimetic as selective agents against non-small cell lung cancer cells, their antibacterial and antifungal potential} j. serb. chem. soc. 83 (11) 1193–1207 (2018) udc 542.913:547.21.024–304.2+547.53.024– jscs–5143 304.2:547.567:615.277+615.281 original scientific paper 1193 alkylamino and aralkylamino derivatives of avarone and its mimetic as selective agents against non-small cell lung cancer cells, their antibacterial and antifungal potential marko jeremić1#, jelena dinić2, milica pešić2, marija stepanović2, irena novaković3#, dejan šegan4 and dušan sladić4#* 1innovation center of the faculty of chemistry, university of belgrade, studentski trg 12–16, 11000 belgrade, serbia, 2institute for biological research “siniša stanković”, university of belgrade, despota stefana 142, 11060 belgrade, serbia, 3institute of chemistry, technology and metallurgy, university of belgrade, center for chemistry, njegoševa 12, 11000 belgrade, serbia and 4faculty of chemistry, university of belgrade, studentski trg 12–16, 11000 belgrade, serbia (received 27 june, accepted 23 july 2018) abstract: in this paper, the synthesis of fourteen alkylamino and arylamino derivatives of sesquiterpene quinone avarone and its model compound tert-butylquinone is described. branched, cyclic, allylic and benzylic alkylamino/arylamino groups were introduced into the quinone moiety. for all the obtained derivatives, their biological activity and redox properties were studied. the cytotoxic activity of the synthesized derivatives towards multidrug resistant (mdr) human non-small cell lung carcinoma nci-h460/r cells, their sensitive counterpart nci-h460 and human normal keratinocytes (hacat) was investigated. the antimicrobial activity towards gram-positive and gram-negative bacteria, and fungal cultures was determined. some of the synthesized derivatives showed selectivity for cancer cells, including mdr cells. regarding their cell death induction potential, the most promising compounds were allylamino derivatives, preferentially triggering apoptosis, with high selectivity for cancer cells, including mdr cells. several compounds showed promising antimicrobial activity, comparable to those of commercial antibiotic and antimycotic agents. keywords: quinones; anticancer activity; multidrug resistant; apoptosis; antimicrobial activity; cyclic voltammetry. introduction cancer is one of the leading causes of death worldwide, with over 8 million deaths in 2015.1 therefore, a great amount of resources and effort have been * corresponding author. e-mail: dsladic@chem.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc180627062j ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 1194 jeremić et al. invested in developing novel strategies and drugs for combating this disease. tackling this serious issue includes various forms of therapies, such as radiotherapy, chemotherapy, immunotherapy, surgery as well as prevention of cancer by reducing the risk of carcinogenesis.2 chemotherapy involves the application of drugs or cocktails of drugs in order to eliminate developed cancer cells. the limitations of chemotherapy are low efficacy, lack of selectivity, severe toxicity, metastasis and development of drug resistance.3 the phenomenon of multi-drug resistance (mdr) is a particular problem, caused by the fast proliferation and metabolism of cancer cells together with a proneness to mutations, which enables cancer cells to overcome the toxicity of the applied drugs, resulting in resistant forms of cancer.4 quinones are a class of organic compounds that have a versatile array of activities, some of which are herbicidal,5 antimalarial,6 antiviral,7 antipsoriatic,8 antileishmanial9 and cytotoxic activity.10–12 biological activity of quinones originates from dual mode of action. it is a combination of 1,4-michael addition of cellular nucleophiles to conjugate enone system and the generation of reactive oxygen species (ros) in redox cycling reactions of quinone and its hydroquinone pair via a semiquinone anion radical.13 quinone/hydroquinone core-containing compounds are very abundant in nature, being involved in crucially important processes, such as photosynthesis,14 the mitochondrial electron transport chain15 and protection from oxidizing species.16 natural products of marine origin are of particular interest for their diversity of structures and a myriad of activities, together with the vast possibilities for their modifications.17–19 as a continuation of previous work on alkylamino, aralkylamino and amino acid derivatives of avarone and its mimetic tert-butylbenzoquinone (tbq),20,21 the synthesis and investigation of the cytotoxic activity of a series of alkylamino and aralkylamino derivatives of avarone and tert-butylbenzoquinone on three cell lines, i.e., non-small cell lung cancer cells, both the sensitive nci-h460 and multi-drug resistant nci-h460/r, and healthy human keratinocytes hacat are reported herein. since in a previous work, benzylamino derivatives of tert-butylquinone showed good selectivity for tumour cells, including mdr cells, benzylamino derivatives of avarone were synthesized in the present study. other amines selected for derivatization of both quinones were allylamines due to similar electronic effects of the allyl and benzyl group, and amines leading to sterically more compressed derivatives, i.e., sec-butylamine and pyrrolidine. additionally, their antibacterial and antifungal activities were determined. cyclic voltammetry was used to study the redox behaviour of newly synthesized compounds in the system quinone–semiquinone–hydroquinone, in order to gain information on the structure−activity relationship. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. alkylamino and aralkylamino derivatives of avarone 1195 results and discussion chemistry as a continuation of previous work, fourteen aminoquinones 3–10a and 3–10b were synthesized (preparation of compounds 6a and 6b was presented in a previous paper20). treatment of the parent quinones tert-butylquinone (1) and avarone (2) with various amines produced the corresponding two regioisomers of aminoquinones via 1,4-michael addition reactions (scheme 1). scheme 1. synthesis of the quinone derivatives. as expected, with tert-butylquinone, the 2,6-disubstituted quinone products were dominant over the 2,5-disubstituted products. on the other hand, with avarone, only in the reaction with pyrrolidine was the 2,6-disubstituted product dominant while in the reaction with allylamine, the 2,5-disubstituted product was the main product and with the other two nucleophiles, similar amounts of two products were obtained. racemic sec-butylamine was used in the reactions. the nmr spectra of the products obtained in its reaction with avarone indicate the presence of only one diastereoisomer. unfortunately, the configuration of the asymmetric carbon could not be determined, since all attempts to crystallize the products were unsuccessful. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 1196 jeremić et al. half-wave potentials were recorded at a glassy carbon disk (3 mm diameter) in dmso towards a silver wire immersed in the electrolyte solution containing 0.01 m silver ions as the reference electrode, and ferrocene as the reference compound. the results are given in table i and shown in fig. s-1 of the supplementary material to this paper. table i. voltammetric half-peak potentials and standard redox potentials of the synthesized compounds (v vs. silver/silver chloride electrode) compound ec1 ea1 ec2 ea2 e01/fc 3a –1.210 –1.126 –1.890 –1.741 –1.170 3b –1.218 –1.154 –1.868 –1.744 –1.186 4a –1.180 –1.121 –1.812 –1.712 –1.149 4b –1.210 –1.152 –1.848 –1.733 –1.181 5a –1.276 –1.214 –1.876 –1.694 –1.244 5b –1.276 –1.214 –1.865 –1.749 –1.243 6a20 –1.128 –1.053 –1.800 –1.619 –1.159 6b20 –1.141 –1.071 –1.803 –1.593 –1.174 7a –1.193 –1.127 –1.881 –1.774 –1.158 7b –1.201 –1.133 –1.859 –1.713 –1.168 8a –1.170 –1.113 –1.830 –1.741 –1.142 8b –1.177 –1.117 –1.799 –1.689 –1.150 9a –1.267 –1.163 –1.883 –1.710 –1.217 9b –1.259 –1.177 –1.861 –1.704 –1.218 10a –1.115 –1.043 –1.865 –1.733 –1.147 10b –1.123 –1.054 –1.840 –1.685 –1.157 typical quinone electrochemical behaviour was observed for all the synthesized compounds, i.e., two waves that could be attributed to the reduction of quinone to the semiquinone radical and, subsequently, to the hydroquinone dianion (fig. s-1). first reduction wave was fully reversible, indicating a diffusion controlled process. however, second reduction process was not reversible, having higher peak separation potentials. the derivatization did not perturb the nature of these two redox processes, but rather shifted the peak potentials. as expected, all the derivatives had more negative reduction potential than the parent quinone. it could be assumed that amino substituents with electron-donating ability destabilize the intermediate semiquinone relative to the corresponding quinone. this effect leads to a negative shift in the peak potential. since the inductive effect of the alkyl part of the alkylamino substituent influences the electron-donating ability of the substituent, the pyrrolidino derivatives have the most negative reduction potentials, while benzylamino and allylamino derivatives have the least negative one. as previously described,20 the position of the substituent plays a particular role in shifting the peak potential. it was observed that the 4′-derivatives generally had a peak potential more negative by about 10 to 20 mv than the corresponding 3′-derivatives. the probable reason is that the arrangement is more ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. alkylamino and aralkylamino derivatives of avarone 1197 favourable when the electron-donating amino and alkyl substituents in semiquinone anion radical are both in the meta position to the negative oxygen. anticancer activity derivatives of tbq were compared with corresponding avarone derivatives regarding their growth inhibition activity against human cancer and normal cell lines. the cell growth inhibition activity was studied in non-small cell lung carcinoma cells – nci-h460/r, their multidrug resistant (mdr) counterpart – nci -h460/r and human keratinocytes – hacat cells. the differences in response between cancer and normal cells, evaluated by the mtt assay after 72 h treatment, are presented in table ii and fig. s-2 of the supplementary material. table ii. growth inhibition activity (ic50 in µm) of tbq, avarone and their derivatives in human non-small cell lung carcinoma cell lines (nci-h460 – sensitive and nci-h460/r – mdr variant) and human normal keratinocytes (hacat); the ic50 values were calculated from a minimum of three independent experiments (average ± standard deviation) compound cell line nci-h460 nci-h460/r hacat cddpa 5.2±0.4 1.7±0.1 0.7±0.1 120a >100 72±8 95±4 220a,b 84±9 24±4 37±8 3ab,c 10.5±0.4 16.3±1.3 24.4±1.5 3bb,c 36.6±1.8 96.3±3.9 72.5±1.5 4a 9.6±0.1 12.6±0.4 8.5±0.4 4b 18.3±0.6 21.8±0.2 23.9±0.7 5ac 40.9±0.4 65.9±2.6 36.8±4 5bc 44.4±4.0 85.7±2.6 26.8±2.4 6a20b 14±2 10±2 21±1 6b20b 19±4 15±2 28±2 7a 3.5±0.04 3.8±0.1 4±0.1 7b 37.5±1.1 44.4±0.9 40.6±2 8a 2.8±0.03 3.0±0.1 3.5±0.1 8b 18±1.3 24.2±0.7 19.2±0.4 9a 28.2±3.7 20.4±1.2 19.3±0.8 9bb 8.9±1.5 7.1±0.2 15.1±0.3 10ac 54.4±2.2 26.3±2.6 12±1 10b >100 >100 42.2±3.0 aselectivity towards mdr cells (higher efficacy in nci-h460/r compared to nci-h460 i.e., ic50 of sensitive cancer cells ≥ 1.5 fold than ic50 of corresponding mdr cells); bselectivity towards cancer cells (ic50 of either sensitive or mdr cancer cells ≤ 1.5 fold than ic50 of normal cells. the dose response curves are shown in fig. s-2); cresistance (lower efficacy in nci-h460/r compared to nci-h460 i.e., ic50 of sensitive cancer cells ≤ 1.5 fold than ic50 of the corresponding mdr cells) the growth inhibition abilities of parent compounds tbq and avarone, as well as cddp, an fda-approved drug for non-small cell lung carcinoma treatment,22 were investigated in our previous study.20 cddp showed stronger effect ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 1198 jeremić et al. in mdr cancer cells in comparison with their sensitive counterparts, but it was not selective against cancer cells due to the pronounced activity obtained in normal human keratinocytes. tbq was largely ineffective in all cell lines while avarone showed high selectivity towards mdr cells. the derivatives had generally a higher activity than the parent compounds. avarone 2,6-disubstituted sec-butylamino and allylamino derivatives (7a and 8a, respectively) exhibited the highest activity in all tested cell lines with ic50 values below 5 µm and, importantly, their activity was not affected by the presence of mdr phenotype. corresponding 2,5-disubstituted derivatives 7b and 8b showed the same pattern of activity (without selectivity against cancer cells) but their efficacy was significantly diminished as their ic50 values were around one order of magnitude higher than those obtained by 7a and 8a. similarly, 2,6-disubstituted allylamino derivative 4a with ic50 values near 10 µm was more efficient than 4b but without selectivity to cancer cells. benzylamino avarone derivative 10b was the least active compound in all cell lines, while the ic50 values for corresponding 10a and two pyrrolidino tert-butylquinone derivatives 5a and 5b were above 25 µm in cancer cells. these four derivatives were more active in normal cells. compounds 3a and 9b exhibited both high cytotoxicity and selectivity towards cancer cell lines and most notably mdr phenotype did not reduce their activity. although direct correlation between redox properties and cytotoxicity of derivatives with different substituents could not be established, it should be noticed that out of two regioisomers, the isomer with a less negative cathodic potential of the first wave, i.e., the isomer that is more prone to the formation of semiquinone radicals, is more active. in the tert-butylquinone series, the 6-derivatives were always more active to tumour cells than the 5-derivatives. the introduction of the bulky sec-butylamino group into the 6-position, i.e., closer to the tert-butyl group, did not have much influence on the activity to tumour cells, but it decreased the toxicity to normal cells, and hence, contrary to the 6-(butylamino) derivative, the corresponding sec-butylamino substituted quinone showed a pronounced selectivity to tumour cell lines. this did not apply for a more remote 5 substituent. the introduction of allylamino group led to products with higher cytotoxicity than similar derivatives with linear saturated alkylamino substituents, but the selectivity for tumour cells displayed by 6-(ethylamino) derivative was lost.20 5-(allylamino) and 5-(benzylamino) products were the only 5-substituted derivatives that showed a relatively high cytotoxicity, but only the benzylamino derivative showed selectivity for tumour cell lines. the pyrrolidino derivatives were only moderately active, without selectivity for tumour cells. if the whole set of compounds is considered, for a strong cytotoxic activity against tumour cells, an unsaturated or bulky substituent should be present in position 6. for achieving selectivity, the set of sub________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. alkylamino and aralkylamino derivatives of avarone 1199 stituents was narrowed to bulky groups, and even more to aralkyl groups (benzylamino, phenethylamino) if selectivity to mdr cells is considered. as for avarone derivatives, the 3′-derivatives (2,6-disubstitution) were always much more active than the 4′-derivatives (2,5-disubstitution), with the exception of the pyrrolidino derivatives when the inverse was true. within this series, the introduction of branching or unsaturation into alkylamino substituent increased the cytotoxicity regardless of the position of the substituent. 3′-(sec -butylamino) and 3′-(allylamino) derivatives showed ic50 values in the range 3–4 μm. unfortunately, the selectivity for mdr cells, shown by 3′-(butylamino)avarone,20 was lost. the benzylamino derivatives were less active to tumour cells than the phenethylamino ones. the 4′-pyrrolidino derivative (the more active isomer) showed strong activity to the tumour cell lines, with an ic50 value of 7.1 μm against the multi-drug resistant cells, and selectivity for tumour cells. the 3′-derivative showed moderate activity, without selectivity. summarizing these results, the necessary prerequisite for an avarone derivative to have a strong activity against tumour cells is a substituent with a relatively low number of carbon atoms at the position 3′ or pyrrolidine at position 4′. of these substituents, only unbranched saturated butylamino derivative and heterocyclic pyrrolidino derivative showed the desired selectivity. apoptosis and necrosis are the two main forms of cell death. apoptosis is regarded as a programmed process, with minimal level of atp required for the assembly of a apoptosome complex and activation of caspases, while necrosis is often referred to as the complete decay of cell metabolism.23 they can be distinguished from one another by various morphological and biochemical characteristics, although there is no clear-cut distinction between these two (hence, other forms of cell death, i.e., necroptosis, aponecrosis, etc.).24 the most obvious characteristics of apoptosis are cell shrinkage, dna fragmentation, condensation of chromatin, formation of apoptotic bodies, disruption of mitochondrial redox processes and generation of atp. cells undergoing apoptosis do not release their constituents into the surrounding tissue because the integrity of the cell membrane is preserved. in addition, macrophages quickly phagocytose apoptotic bodies and the surrounding cells do not produce anti-inflammatory cytokines.25 therefore, no inflammatory reaction occurs during the apoptotic process. necrosis, on the other hand, is usually followed by inflammation, because one of the main characteristics of necrosis is the loss of membrane integrity and the release of the cytoplasmic content into the surrounding area, leading to inflammation.26 inflammation, for its part, can damage cells and even cause cancer.27 bearing this in mind, apoptosis is clearly the preferred type of cell death, considering the removal of either healthy, aging or tumour cells. besides inflammation, the other characteristics of necrosis are cell swelling and complete abolition of atp pro________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 1200 jeremić et al. duction. however, many types of cancer employ anti-apoptotic mechanisms for their survival, so necrosis could be useful in promoting the death of tumour cells. the cell death inducing capacity of 25 µm tbq and the avarone derivatives was analyzed after 72 h treatment (table iii, fig. s-3 of the supplementary material). both pyrrolidino tert-butylquinone derivatives (5a and 5b) showed significant activity towards sensitive cancer cells, triggering both apoptosis and necrosis, which was not observed in normal cells. the allylamino derivatives 4a and 4b induced apoptotic type of cell death more prominently in both cancer cell lines than in hacat cells. the sec-butylamino derivatives 3a and 3b also induced apoptosis, most effectively in nci-h460 cells. cells treated with the avarone pyrrolidino derivatives 9a and 9b underwent necrosis as the predominant cell death type. consistent with the cell growth analysis, the inverted efficacy pattern of 9a and 9b as well as the selectivity of 9b towards cancer cells was confirmed by cell death induction. compounds 8a and 8b (allylamino derivatives of avarone) also predominantly induced necrosis in cancer cells with 8a being the significantly more potent derivative. sec-butylamino derivative 7a was considerably more active against mdr cancer cells in triggering necrotic cell death compared to 7b. a similar but reduced effect was also observed in nci-h460 and hacat cells. importantly, according to the cell death analysis, 7a and 8a were shown to be selective against cancer cells. table iii. cell death induction by 25 μm tbq, avarone and their derivatives in human non -small cell lung carcinoma cell lines (nci-h460 – sensitive and nci-h460/r – mdr variant) and human normal keratinocytes (hacat)). cddp is included as a positive control. the values represent percentages of viable, apoptotic and necrotic cells compound viable cells early apoptosis late apoptosis necrosis av–pi– av+pi– av+pi+ av–pi+ nci-h460 control 95.5 2 1.1 1.3 cddp 66 1.6 5.2 27.2 120 95.2 0.5 0.5 3.8 220 74 3.4 16.3 6.3 3a 43.8 21.9 28.2 6.1 3b 85.6 3.5 4.9 6 4a 5.1 24.9 61.4 8.6 4b 4.9 23.1 63.9 8.1 5a 67.4 4.7 11.4 16.5 5b 51 5.3 18.1 25.6 7a 53.9 6.6 5.7 33.8 7b 81.4 0.7 3.9 13.9 8a 28.6 12.5 18 40.9 8b 72.1 3.5 7 17.4 9a 83.6 2 3.5 10.9 9b 31 3.4 10 55.6 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. alkylamino and aralkylamino derivatives of avarone 1201 table iii. continued compound viable cells early apoptosis late apoptosis necrosis av˗pi˗ av+pi˗ av+pi+ av˗pi+ nci-h460/r control 95.6 1.6 1.9 0.9 cddp 55.3 3.8 13.3 27.6 120 95.2 0.5 1.2 3.1 220 65.8 2.6 22.7 8.9 3a 68.4 9.2 17.5 4.9 3b 93 2.3 3.2 1.5 4a 7.3 4.1 64.4 24.2 4b 11.8 6.3 63.4 18.5 5a 91.8 2 4.1 2.1 5b 84.8 2 5.5 7.7 7a 38.4 3.8 9.8 48 7b 96.3 1.5 1.4 0.8 8a 21.7 6.6 19.2 52.5 8b 55.1 4.4 7.1 33.4 9a 58.7 5.2 7.7 28.4 9b 26.9 3.9 10.9 58.3 hacat control 93.6 2.9 1 2.4 cddp 53.5 1.4 9 36.1 120 86.8 1.9 3.8 7.5 220 78.3 2.5 10.1 9.1 3a 64 14.2 9.7 12.1 3b 82 6.8 5.5 5.7 4a 61.5 15.9 8.1 14.5 4b 33.5 18.8 18.9 28.8 5a 85.9 4.8 4.9 4.4 5b 85.3 4.9 4 5.8 7a 72.3 1.1 1 25.6 7b 95.7 0.5 0.6 3.2 8a 73.8 2.4 4.1 19.7 8b 89.3 0.6 0.8 9.3 9a 97.2 0.3 0.3 2.2 9b 83.7 0.9 1.1 14.3 the cell death-inducing activity of compounds 4a and 4b significantly stand out from the others in the series. the majority of the cells treated with 4a or 4b underwent apoptotic cell death, targeting late apoptosis preferentially. most importantly, this pair were very selective toward cancer cell lines, both sensitive and resistant ones, particularly the 4a derivative with over 60 % of viable cells in healthy hacat cell lines. this distinction could be attributed to the allyl moiety. the probable reason is the additional generation of ros via allyl radicals, formed by the decomposition of the semiquinone radical generated by the one________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 1202 jeremić et al. electron reduction of allylamino quinone (the other product would be amino quinone) and/or the formation of cytotoxic acrolein by the oxidative metabolism of allylamino quinones.28 considering that allylamino tbq derivatives preferentially induce (late) apoptosis, and that cancer cells are usually already in a state of oxidative stress,29 this seems a plausible explanation for the selective action. antimicrobial activity compounds 3–10a and 3–10b were tested for their antimicrobial activity against gram-positive and gram-negative bacteria, as well as fungal strains, and compared to the commercial antibiotics amikacin and antimycotic nystatin. the results are given in tables iv and v. most of the derivatives showed weak activity in comparison to amikacin, having at least an order of a magnitude higher mic. the most active tbq derivatives were allylamino derivatives 4a and 4b, with activity comparable to amikacin for e. coli. this pair had almost identical mic for all strains except m. luteus (atcc 10240 and 4698) and s. enterica. the pair 3a and 3b showed generally weaker activity than 4a and 4b, with 3a being only slightly more active against b. subtilis, m. luteus (atcc 10240 and 4698) and e. coli, and 3b for k. rhizophila. the pair 5a and 5b displayed the weakest activity of all the tested tbq compounds, with some activity only towards k. rhizophila, e. coli and m. luteus, atcc 4698, (only 5a). the avarone counterparts showed no activity, as expected from previous results.20 the probable reason for inactivity is insufficient hydrophilicity. this conclusion is corroborated by the fact that several amino acid derivatives of avarone showed strong antibacterial activity.21 in general, in order to show a relatively broad activity comparable to amikacin, tert-butylquinone derivatives should have a non-branched medium length alkylamino group or an aralkyl group in position 3′. it is interesting that among the avarone amino acid derivatives, the most active were those with aromatic amino acids, implying that there is an aromatic binding site in a putative target. all the tested compounds, except 10a and 10b, showed stronger activity toward c. albicans than nystatin, with 3a, 4a, 4b and 5a even having a two orders of a magnitude lower mic (less than 50 μm). towards a. brasiliensis, the activities were generally comparable to that of nystatin, with 4a, 4b and 5a displaying stronger activity. considering s. cerevisiae, 4a, 5a, 7b, 9a and 9b possess stronger activity than amikacin. of all the tested compounds, 4a showed the strongest antifungal activity, while 10a and 10b showed no activity at all. based on these results and those from a previous paper,20 it could be concluded that in order to achieve a strong anti-candida activity, a compound should be a 6-substituted 2-tert-butyl-1,4-benzoquinone (exception are allylamino derivatives, where both isomers have a similar activity), ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. alkylamino and aralkylamino derivatives of avarone 1203 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 1204 jeremić et al. with at least three carbon atoms in the side chain. the avarone derivatives were much less active, but in contrast to antibacterial effects, most of them had some activity, although much lower than the amino acid derivatives.21 the amino acid derivatives of tbq had weak antimicrobial activity, probably due to their excessive hydrophilicity. table v. antifungal in vitro activity (mic/mm) compound fungi candida albicans (atcc 10231) aspergillus brasiliensis (atcc 16404) saccharomyces cerevisiae (atcc 9763) 3a 0.042 1.33 1.33 3b 0.166 2.656 2.656 4a 0.047 0.36 0.36 4b 0.047 0.716 1.427 5a 0.042 0.339 0.673 5b 0.673 2.679 2.679 *6a 2.333 2.333 2.333 6b20 – – – 7a 0.41 1.631 0.817 7b 0.41 1.631 1.631 8a 0.852 1.701 3.401 8b 1.701 1.701 3.401 9a 1.638 1.638 0.82 9b 1.638 1.638 0.411 10a – – – 10b – – – nystatin 2.7 1.35 1.35 experimental general synthetic procedure the parent quinones were obtained from hydroquinones according to a previously described procedure.20 quinones (300mg; 1.83 mmol 1; 0.96 mmol 2) were dissolved in ethanol (50 ml). amine hydrochloride salts (in large excess, 22×) were prepared as aqueous solutions. the ph of the solution was adjusted to 7–8 by the addition of solid sodium bicarbonate, and the solution was added to quinone. water and ethanol were added to the reaction mixture to a final ratio water:ethanol of 1:1, and total volume of 300 ml. the reaction mixture was stirred at 60–70 °c for 3 h. ethanol was removed by vacuum evaporation, and the reaction mixture was extracted two times by dichloromethane, with half the volume of the aqueous phase each time. the organic phase was separated, dried with anhydrous calcium chloride, and the solvent was removed by evaporation under vacuum. the crude products were separated by column or low-bar chromatography and purified by preparative thin-layer chromatography, with the indicated solvents. numbering scheme for assignment of signals in nmr spectra of all compounds is given in scheme s-1. cyclic voltammetry electrochemical behaviour of synthesized aminoquinones was studied as previously described.20 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. alkylamino and aralkylamino derivatives of avarone 1205 biological activity cytotoxic, antibacterial and antifungal activity of synthesized compounds were analyzed according to a previous study.20 conclusions among the 14 newly synthesized compounds, a potential to be antitumor agents was shown for sec-butylamino derivatives of tert-butylquinone (3a and b) because of their selectivity for tumour cells, allylamino derivatives of tert-butylquinone (4a and b) because of their selective induction of apoptosis in tumour cells, including mdr cells, 3′-(sec-butylamino)avarone (7a) and 3′-(allylamino)avarone (8a) because of their higher cytotoxic activity than cisplatin, as well as 4′-pyrrolidinoavarone (9b) because of selectivity to tumour cells, including mdr cells. some derivatives showed promising antimicrobial properties: sec-butylamino (3a) and allylamino (4a and b) derivatives of tert-butylquinone, because of an activity against e. coli similar to that of amikacin, and strong antifungal activity to c. albicans, which also applies to the pyrrolidino derivative of tert -butylquinone (5a). supplementary material additional experimental results, as well as spectroscopic and analytical data, are available electronically on the pages of the journal’s website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. the authors are grateful to the ministry of education, science and technological development of the republic of serbia for financial support (grant nos. oi172055 and iii41031). извод алкиламино и аралкиламино деривати аварона и његовог миметика као селективни агенси према ћелијама неситноћелијског карцинома плућа, њихов антибактеријски и антифунгални потенцијал mарко јеремић1, jелена динић2, mилица пешић2, mарија степановић2, ирена новаковић3, дејан шеган4 и душан сладић4 1иновациони центар хемијског факултета, универзитет у београду, студентски трг 12–16, 11000 београд, 2институт за биолошка истраживања „синиша станковић“, универзитет у београду, деспота стефана 142, 11060 београд, 3институт за хемију, технологију и металургију, универзитет у београду, центар за хемију, његошева 12, 11000 београд и 4хемијски факултет, универзитет у београду, студентски трг 12–16, 11000 београд у овом раду, описана је синтеза четрнаест алкиламино и аралкиламино деривата сесквитерпенског хинона аварона и његовог модел-једињења, терц-бутилхинона. алкиламино/аралкиламино групе које су уведене у хинонски остатак биле су разграната, циклична, алилна и бензилна. за све добијене деривате одређена је биолошка активност и редокс особине. испитана је цитотоксична активност синтетисаних деривата према резистентним ћелијама неситноћелијског карцинома плућа (nci-h460/r), њиховом осетљивом пандану (nci-h460) и нормалним хуманим кератиноцитима (hacat). одређена је и антимикробна активност према грам-позитивним и грам-негативним бактеријама и културама гљивица. неки од синтетисаних деривата су показали селективност ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2018 scs. 1206 jeremić et al. према ћелијама канцера, укључујући и резистентне ћелије. што се тиче потенцијала за индукцију ћелијске смрти, деривати који највише обећавају су алиламино деривати, који преференцијално активирају апоптозу, са великом селективношћу према ћелијама канцера, укључујући и резистентне ћелије. неколико једињења је показало обећавајућу антимикробну активност, упоредиву са комерцијалним антибиотицима и антимикотицима. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {properties of double w/o/w emulsions containing vitamin c and e stabilized with gelatin/sodium caseinate complex} j. serb. chem. soc. 84 (12) 1427–1438 (2019) udc 577.112+544.032.1:547.253.3+ jscs–5275 547.963.2+54–188 original scientific paper 1427 properties of double w/o/w emulsions containing vitamin c and e stabilized with a gelatin/sodium caseinate complex jadranka l. fraj*, lidija b. petrović, jelena r. milinković budinčić, jaroslav m. katona, sandra đ. bučko and ljiljana m. spasojević faculty of technology novi sad, university of novi sad, bulevar cara lazara 1, 21000 novi sad, serbia (received 4 june, revised 10 july, accepted 11 july 2019) abstract: double emulsions are complex liquid dispersion systems in which the droplets of one dispersed liquid are further dispersed in another liquid, producing w/o/w or o/w/o emulsions. w/o/w emulsions are the most studied systems because they have great potential application. however, despite all the advantages, that these systems offer, it is very difficult to obtain stable formulations, and this is the reason for their limited practical application. the use of biopolymers to stabilize double emulsions could give rise to pharmaceutical and food applications. based on previous studies, appropriate concentrations of gelatin and sodium caseinate (nacas) were selected to investigate the possibility of stabilization of double w/o/w emulsions by this system, if they are present in the outer aqueous phase. the investigations showed that interactions between gelatin and nacas in the outer water phase, as well as the composition of the mixtures of lipohilic emulsifiers used for the primary w/o emulsions preparation, influences the droplets size and sedimentation stability of double emulsions. the most stable emulsions were obtained at a nacas concentration when an insoluble coacervate forms (0.5 mass %) and at concentrations higher then this, when soluble negatively charged complexes adsorb at the oil/water interface. keywords: double emulsions; biopolymers; proteins; interactions; coacervation. introduction double (or multiple) emulsions are complex liquid dispersion systems in which the droplets of one dispersed liquid (water in oil or oil in water) are further dispersed in another liquid (water or oil, respectively), producing w/o/w or o/w/o emulsions. w/o/w emulsions are the most studied systems because they have great potential application, first of all in the food, pharmaceutical and cosmetic industries. in the food industry, w/o/w multiple emulsions can improve * corresponding author. e-mail: jadrankam@gmail.com https://doi.org/10.2298/jsc190515075f 1428 fraj et al. the solubility of a certain active matter and the production of low calorie and reduced fat products, serve as protective liquid reservoirs for molecules sensitive to external environmental reactivity, such as oxidation, light or enzymes, and serve as entrapment reservoirs for masking undesired flavors and odorants.1–3 application in the cosmetic industry include aqueous preparations that provide a “good feel”, slow release of active materials, and deposition of water soluble agents onto the skin from wash-off systems.4,5 most applications are related to the pharmaceutical industry, such as enhancement of the chemotherapeutic effect of anticancer drugs, drug immobilization and protection of insulin from enzymatic degradation.6–8 however, despite all the advantages, that these systems offer, it is very difficult to obtain stable formulations, and for this reason, their practical application is limited. compared with simple emulsions of only two phases and one interface, many more destabilization processes need to be taken into consideration for multiple emulsions. four possible mechanisms lead to the instability of w/o/w emulsions:9 − coalescence of the internal aqueous droplets, − coalescence of the oil droplets, − rupture of the oil film resulting in the loss of the internal aqueous droplets, and − passage of the water and water soluble substances through the oil layer between water phases. this can occur in two various ways: via reverse micellar transport by the lipophilic emulsifier and by simple diffusion across the oil phase connected with the osmotic difference between two water phases. two different emulsifiers are necessary for the stabilization of w/o/w emulsions: the first with a low hydrophilic–lipophilic balance (hlb) for the w/o interface and the second one with a high hlb for the o/w interface. most of the current research in this field is about improving the stability of these systems, and most of the proposed techniques can be classified into three groups: − techniques that include improving stability of the primary w/o emulsion, − techniques that include changes of the oil phase properties, such as increasing viscosity and − techniques that include improvement of the stability of the interfaces by using polymeric emulsifiers or natural biopolymers. during years of investigations to improve the stability and to control sustained and prolonged release of active materials, monomeric surfactants have been progressively replaced by polymeric emulsifiers.10 polymeric amphiphilic molecules, synthetically tailor made and naturally occurring ones, are known to possess surface activity and to improve the interfacial coverage during emulsification. the use of biopolymers to stabilize double emulsions could give rise to pharmaceutical and food applications. the use of proteins has long been adopted by scientists exploring the stability of double emulsions. gelatin,11 milk pro double emulsions stabilized by proteins complex 1429 teins,12 bovine serum albumin,13 caseins14,15 and other proteins have been mentioned and evaluated. however, a single protein is far from an ideal emulsion stabilizer as the prepared emulsions are sensitive to temperature, salt and ph.16,17 the method proposed to compensate the deficiency of single proteins is to form a multilayer on the surface of oil droplets through electrostatic interactions between biopolymers. there are many published scientific papers in which complexes, formed as results of interactions between proteins and polysaccharides were applied for emulsion stabilization.18–20 however, to our best knowledge, there are only a few studies investigating the possibility of emulsion stabilization by mixtures of two proteins, which are simultaneously systems of two polyelectrolytes.21,22 due to its unique hydrophilic character, gelatin is really the only protein that can be properly categorized as a hydrocolloid.23 it does have some emulsifying ability, but its more characteristic roles in formulations are as a stabilizer, film former and gelling agent. according to this, gelatin has a wide range of applications, especially in the food and pharmaceutical industries. sodium caseinate (nacas) has been widely used as an ingredient in food systems due to its functional and nutritional properties. it is prepared from coagulated casein micelles and possesses considerable negative charge at around neutral ph.24 nacas adsorbs readily at the oil/water interface, thus stabilizing emulsion droplets through a combination of both electrostatic and steric mechanisms25 however, nacas stabilized emulsions are unstable at ph values close to its isoelectric point (iep, ≈4.6) due to a reduction in electrostatic repulsion between the oil droplets.26 in some previous study, the mechanism of interactions between gelatin and sodium caseinate was investigated in detail and explained whereby it was concluded that at certain proteins mass ratio, complex coacervation occurs.27 the main objective of the present paper was to investigate the possibility of the application of mixtures of gelatin and sodium caseinate for the stabilization of double w/o/w emulsions. based on previous results, the appropriate concentrations of both proteins in the outer aqueous phase of emulsions were selected. experimental materials the experiments were realized using two proteins, acid-processed gelatin, type a (300 bloom), from bovine skin, with an iep at ph 7.43, and casein sodium salt (nacas), from bovine milk, with an iep at ph 4.6,26 both product of sigma, usa. for primary stabilization of 20 % w/o emulsions, the oil soluble surfactants polyglycerol polyricinoleate (pgpr) donated by jaffa crvenka, serbia, and decaglycerol decaoleate (caprol 10g10o) from abitec, usa, were used. as the oil phase, medium chain triglycerides (mct) of caprylic/capric fatty acids (saboderm tcc) produced by sabo spa, italy, were used. vitamin c, ascorbic acid, and vitamin e, tocopherol acetate, both obtained from alfa aesar, germany, were used as model substances for encapsulation. distilled water was used as the aqueous phase. 1430 fraj et al. preparation of solutions the stock solutions of lipophilic emulsifiers were prepared by dissolution of an appropriate amount of emulsifier in the oil phase. binary mixtures of emulsifiers pgpr and caprol 10g10o, at different mass ratios (1:0, 1:1 and 1:2), were prepared by mixing stock solutions and addition of desired amount of oil phase to obtain a concentration of the emulsifiers of 3 vol. %. stock solution of 1.7142 mass % gelatin was prepared by adding 1/5 of the total amount of water to a given mass of protein and then left for 15 min at the room temperature to swell. then, the rest amount of water was added under mild stirring at 40 °c. stock solution of 10 % nacas was prepared by dispersing a given mass of protein in water, left for 1 h at room temperature to allow hydration, then gently stirred and heated (up to 40 °c) until complete dissolution. solutions of lower concentrations were obtained by dilution of the stock solution. preparation of double w/o/w emulsions double emulsions were prepared by a two-step procedure using a homogenizer, ultra turrax t25, ika, germany, and a magnetic stirrer, c-mag hs7, ika, germany. in the first step, primary w/o emulsions were prepared at a water–oil mass ratio of 20:80. the aqueous phase was a 25 vol. % solution of vitamin c in deionized water, while the continuous phases were a 1.6 vol. % solution of vitamin e in 3 vol. % solutions of binary mixtures of lipophilic emulsifiers pgpr and caprol 10g10o, at the announced mass ratios, in mct. the emulsions were prepared by dispersing the water phase in the continuous phase at 40 °c by means of homogenizer, ultra turrax t25, at 20000 rpm during 10 min. the second step involved the dispersion of the primary emulsion (30 g) into 1.7142 mass % gelatin solution (70 g), using the same homogenizer, at 5000 rpm during 10 min, at 30 °c. in this way, 30 % w/o/w emulsions were obtained. the final 20 % w/o/w emulsions were obtained by the slow addition of nacas solutions of appropriate concentrations, to the 30 % w/o/w emulsions, during 20 min under mild stirring on a magnetic stirrer at 40 °c. thus, the final double emulsions represent 20 % emulsions of primary w/o emulsions in mixtures of 1 mass % gelatin and various nacas concentrations (0.01, 0.1, 0.5, 1 and 3 %). droplet size analysis the droplet size of the double emulsions were determined by microphotography analysis using qwin software. microphotographs were taken on an optical microscope bel 3000 bioptica, italy. droplet mean diameter, expressed as volume–surface mean value dvs/µm, was calculated from the experimental data, eq. (1): 3 i i vs 2 i i /μm =   n d d n d (1) where di is the droplet diameter and ni is the number of droplets. emulsions stability test for stability test, the double emulsions were transferred into 10 ml graduated cylinders and stored at room temperature for 14 days. the emulsions were observed for changes in homogeneity and phase separation during storage. continuous phase separation was visually monitored at certain time intervals. the total height of the emulsion, he, and the height of the separated continuous phase, hc, were measured. the extent of the phase separation was characterized by the creaming index, h (eq. (2)). a higher value of the creaming index indicates a worse emulsion stability: double emulsions stabilized by proteins complex 1431 c e / % 100= h h h (2) results and discussion the investigation of the interaction in mixtures of two oppositely charged proteins, gelatin and sodium caseinate,27 showed the presence of complex coacervation resulting in soluble (charged) and insoluble (uncharged) complex formation. the formation of an insoluble, neutral complex coacervate always appeared at a gelatin:nacas mass ratio of 2:1. since these interactions had an influence on the system properties, in the present work, the possibility of the stabilization of double emulsions by these mixtures was investigated. the gelatin concentration was 1 mass % while the nacas concentrations were chosen to cover the region before insoluble coacervate formation (0.01 and 0.1 %), concentration when insoluble coacervate forms (0.5 %), and region after insoluble coacervate formation (1 and 3 %). in addition, based on some previous studies, mixtures of lipophilic emulsifiers pgpr and caprol 10g10o (at the announced mass ratios) were selected for primary emulsions stabilization.28 microscopic observation of the emulsion samples showed the presence of inner water droplets inside oil droplets, i.e., the existence of double w/o/w emulsions. the microphotograph of the emulsion stabilized with mixtures of 1 mass % gelatin and 0.5 % nacas showed the presence of a coacervate layer around the oil droplets (fig. 1). for better contrast, a solution of methylene blue was added before microscopy. fig. 1. microphotograph of a 20 % w/o/w emulsion stabilized with a mixture of gelatin:nacas at a mass ratio 2:1, at a magnitude 40×. influence of the gelatin/nacas interaction on the mean diameter of the droplets in 20 % w/o/w emulsions since the formed complexes showed different behavior in the bulk, as well as at the interface,27 it was expected that changes in emulsions properties would 1432 fraj et al. occur during storage. changes in the mean diameters of the droplets of 20 % w/o/w emulsions stabilized with 1 mass % of gelatin and various nacas concentrations during storage at room temperature are shown in fig. 2. a b c fig. 2. mean diameters of the droplets of 20 % w/o/w emulsions stabilized with 1 mass % gelatin and various nacas concentrations as a function of time. the primary emulsions were stabilized with mixtures of pgpr:caprol 10g10o at mass ratios of 1:2 (a), 1:1 (b) and 1:0 (c) and total concentrations of 3 %. it could be noticed that in freshly prepared emulsions, mean diameters of the droplets were in interval from 11.12 to 13.45 µm. the mass ratio of caprol 10g10o in the mixture with pgpr, which has an influence on the droplet diameter of primary emulsions,28 also affects the droplet diameter of the double emulsions. namely, on dispersing the primary emulsion in the gelatin solution, its molecules adsorb at the interface forming a film around the oil droplets. at the same time, excess pgpr molecules present in the oil phase, also adsorb at the outer oil/water interface, additionally stabilizing the formed gelatin film. the increase in caprol 10g10o mass ratio in the mixture reduces the concentration of pgpr molecules, thus inducing a slight increase in the mean diameter of the droplets. during storage, the influence of gelatin/nacas interactions, as well as the concentration of pgpr molecules in the oil phase, on the properties of the emulsions was clearly noticeable. namely, in single emulsions, the change in droplet double emulsions stabilized by proteins complex 1433 size is mostly associated with a coalescence process. on the other hand, in double emulsions, a number of different factors can affect such changes. changes in the mean diameters of the droplet of double emulsions, in which primary emulsions were stabilized with pgpr:caprol 10g10o mixtures at mass ratio 1:2, are presented in fig. 2a. the decrease in droplets mean diameter was noticeable in all emulsion samples after two days of storage. one of the instability factors in such systems might be water diffusion between the two sides of oil layer due to the osmotic gradient, since the inner aqueous phase was hypertonic vitamin c solution. this induces flow of water in the direction of decreasing osmotic gradient, i.e., from the outer to inner water phase, and swelling of the internal water drops. when the critical limit is reached, the oil layer breaks down and the inner water phase migrates into the outer water phase resulting in double emulsion breakdown and the size of the oil droplets decreases.29,30 these changes were confirmed by microscopic observation (not shown) where the existence of double emulsions had not been observed. low stability of this emulsions series is a consequence of bad properties of the adsorption layer on the oil drops, where protein complexes as well as lipophilic emulsifiers adsorb.18 in the emulsion stabilized with the gelatin:nacas complex at a mass ratio of 2:1, a slight increase in the diameter of the droplets after seven days was noticed. this could be a consequence of the coalescence of the droplets since they were stuck by the coacervate layer. changes in the mean diameter of the droplets in a series of double emulsions with a primary emulsion containing pgpr: caprol 10g10o at a 1:1 mass ratio are shown in fig. 2b. these results show an increase in the mean diamater of the droplets in emulsions with nacas concentrations of 0.01 and 0.5 mass %, while in the others, the mean diameter of the droplets decreases. the addition of the lower amount of nacas (0.01 mass%) does not significantly destroy the gelatin layer around oil drops, thereby slowing the diffusion of water from the outer phase and swelling of the inner water drops, i.e., the breakdown of the double emulsion is slower. microscopy of this emulsion still showed the existence of a double emulsion after two days of storage. compared to the previous series of emulsions, here the improved emulsifying properties of the mixture with a higher mass ratio of pgpr are expressed. on the other hand, the added amount of nacas is not sufficient to cover the entire surface of the oil drops and to form compact coacervate layer and hence, one protein molecule is adsorbed on two or more oil drops. for this reason, the emulsion droplets are interconnected through so-called macromolecular bridges, i.e., bridging flocculation occur. as a consequence of flocculation, there is coalescence leading to an increase in the mean diameter of the droplets.31 the bridging flocculation appears in systems in which the concentration of the added macromolecule is so low that the process of droplets collision is faster than adsorption of the macromolecules on to the droplets.32 in the emulsion with a nacas concentration of 0.5 %, the presence of inner 1434 fraj et al. water droplets after two days of storage is also noticeable, which could be attributed to a compact coacervate film on the oil droplets. this sticky coacervate increases the coalescence process inducing an increase in the mean diameter of the droplets. in all other emulsions, there was a decrease in the mean diameter oh the droplets and a breakdown of the double emulsions. the results of the droplets mean diameter measurement in the double emulsions in which the primary w/o emulsions were stabilized with pure pgpr (fig. 2c) show that only in the sample with an nacn concentration of 0.01 % was an increase in the mean diameter of droplets evidenced as a consequence of bridging flocculation. in the other emulsions, there were no significant changes in this parameter, which indicates stability of the adsorbed layer and the synergistic effect of pgpr molecules and the gelatin/nacn complexes formed at the interface. namely, at an nacn concentration of 0.1 %, its molecules bind to the gelatin molecules adsorbed on the oil droplets thereby forming a compact layer and a retardation of the migration of the water between the two water phases. at a nacn concentration of 0.5 %, emulsion stability is a result of a compact coacervate layer around the oil droplets. further increase in nacn concentration induces dissolution of the gelatin/nacn complexes, but these complexes are surface active28 i.e., have a tendency to adsorb at the oil/water interface, thereby stabilizing the dispersed system. in addition, in this area of nacn concentrations, a significant increase in the system viscosity was noticed, which retards droplets movement, and therefore, their collision and coalescence.33,34 in all emulsion samples, the existence of double emulsions after two days of storage at the room temperature was confirmed. after this period, in the emulsion with 0.01 % of nacn, a decrease in the mean diameter of the droplets occurs, as a consequence of worse properties of the adsorption layer. in the other emulsion samples, there were no significant changes in the mean diameter of the droplets, which indicates that the synergistic action of the lipophilic emulsifier pgpr and gelatin/nacn complex can produce double w/o/w emulsions stable for 7 days. influence of the gelatin/nacn interaction on 20 % w/o/w emulsions sedimentation stability along with the dispersed analysis of the 20 % w/o/w emulsions, their sedimentation stability was observed. the emulsions were transferred into graduated cylinders immediately after preparation and their phase separation was visually monitored at the room temperature during 14 days. changes of the creaming index (h) with time and cylinders filled with emulsions after 14 days of storage are shown in fig. 3. it can be noticed that in all emulsions phase separation occurs in the first 24 h after preparation and subsequently, there were no significant changes in the creaming index. the separation of the serum layer appears due to the difference in density between the continouos and dispersed phases. at nacas concentrations double emulsions stabilized by proteins complex 1435 (1) (2) fig. 3. creaming index of 20 % w/o/w emulsions stabilized with mixtures of 1 mass % gelatin and various nacas concentrations as a function of time (1); cylinders filled with the emulsions after 14 days of storage at room temperature (2). the primary emulsions were stabilized with mixtures of pgpr:caprol 10g10o at mass ratios of 1:2 (a), 1:1 (b) and 1:0 (c) at a total concentrations of 3 %. of 0.01 and 0.1 %, a sharp boundary between the layers was clearly visible, which is mainly due to bridging flocculation, i.e., flow of the flocculated droplets of the emulsion to the top of cylinder, while smaller and unflocculated ones remain in the serum layer.31,35 in emulsions stabilized with insoluble gelatin/ /nacas coacervate (at nacas concentration of 0.5 %), there is also sharp 1436 fraj et al. boundary between phases. since the formed coacervate is electro neutral,27 electrostatic repulsion between droplets is reduced, which enables their closer packing resulting in the separation of the floccules. on further increase in nacas concentration, the creaming index is lower and the boundary between phases less sharp, which is a consequence of an increase in the viscosity of the continuous phase.27 namely, it is known that an increase in the viscosity of the continuous phase improves the sedimentation stability of emulsions due to retarded movement of the droplets.33 in addition, the increase in the total negative charge on the surface of the droplets with increasing nacas concentration, also has a positive influence on emulsions stability by increasing the electrostatic repulsion between the droplets. it is also important to stress that in no emulsion was separation of the oil phase noticed. conclusions investigations of the properties of double 20 % w/o/w emulsions containing vitamin c and e showed that interactions between gelatin and nacas in the outer water phase, as well as the composition of the mixtures of lipohilic emulsifiers used for the preparation of the primary w/o emulsions, influences the droplet size and sedimentation stability of the emulsions. increasing amount of pgpr in the mixtures with caprol 10g10o had a positive effect on the stability of the emulsions with time. an investigation of the influence of gelatin and nacas interactions on properties of the emulsions showed that stable emulsions were obtained at the nacas concentration when an insoluble coacervate forms (0.5 %) and at concentrations higher then this, when soluble negatively charged complexes adsorb at the oil/water interface. further research will be focused on examining the possibilities of cross-linking the adsorbed proteins layer in order to obtain microcapsules in powder form suitable for the simultaneous encapsulation of hydrosoluble and liposoluble active substances. acknowledgement. this work was supported by the ministry of education, science and technological development of the republic of serbia, project no. iii46010. и з в о д особине двоструких w/o/w емулзија са витаминима це и е стабилизованих желатин/натријум-казеинат комплексима јадранка л. фрај, лидија б. петровић, јелена р. милинковић будинчић, јарослав м. катона, сандра ђ. бучко и љиљана м. спасојевић технолошки факултет нови сад, универзитет у новом саду, булевар цара лазара 1, 21000 нови сад двоструке емулзије су комплексни течни дисперзни системи, код којих је један дисперзни систем даље диспергован у другој течној фази, формирајући w/o/w или о/w/o емулзије. w/o/w емулзије су најчешће испитивани системи због њихове потенцијално веома широке примене. ипак, и поред низа предности које ови системи имају, њихова примена је још увек доста ограничена, с обзиром на то да је тешко добити стабилан систем. примена double emulsions stabilized by proteins complex 1437 биополимера за стабилизацију двоструких емулзија омогућава њихову ширу примену у прехрамбеној и фармацеутској индустрији. на основу наших претходних истраживања, изабрали смо одговарајуће концентрације желатина и натријум-казеината да испитамо могућност стабилизације двоструких w/o/w емулзија овим системом биополимера, када су они присутни у спољашњој воденој фази. резултати ових истраживања су показали да интеракција између желатина и натријум-казеината у спољашњој воденој фази, као и састав смеше липофилних емулгатора коришћених за припрему примарних w/o емулзија, утичу на величину честица и седиментациону стабилност двоструких емулзија. најстабилније емулзије су добијене при концентрацији натријум-казеината када долази до формирања нерастворног коацервата (0,5 %) и вишим, када долази до адсорпције негативно наелектрисаних комплекса на граници фаза. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice j. serb. chem. soc. 84 (12) 1479–1485 (2019) 1479 author index abaeva, k. t., 477 abbas dadras, a., 391 abbasi, m. a., 649 abbasi, s., 391 abkenar, s. d., 701 abramović, b. f., 1455 ačanski, m., 1017 adžić, r., 1235 affoune, a. m., 41 agbaba, j., 327 ahmadov, b., 599 ali benvidi, a., 391 ali, n., 517 almasy, l., 1027 alves, c. n., 591 ameri, a., 1169 anandkumar, j., 211 anđić, z., 423 andrić, d. b., 925 antić, v., 377 antić, ž., 1199 antonijević nikolić, m., 961 arockiaraj, j., 225 asanović, v., 503 ashok, d., 237, 355 ashurova, u., 599 attaran, a., 489 avramov ivić, m., 1319 bajat, j. b., 1221 bajić, v., 539 balaban, m., 11, 377 baltabayev, m. e., 477 banić, n. d., 1455 barabas, r., 1027 baralić, m., 943 barudžija, t., 1305 baščarević, z., 1209 begović, j. m., 365 beitollahi, h., 175, 1005 bekler, f. m., 1093 belošević, s. k., 467 benedeković, g., 1345 beškoski, v. p., 225 bhatt, p., 225 bihelovic, f., 935 bikić, f., 503 bogunović, m., 623 bohle, f., 837 bojić, a. lj., 483, 1041 bojić, d. v., 483, 1041 bota, a., 1027 bouakkadia, a., 575 bouakkadia, a., 1405 bouarra, n., 575 brasil, d. do s. b., 153, 591 brasil, l. do s. n. dos s., 591 bratkovskaja, i., 343 brborić, m., 1439 brdarić, t., 55 bučko, m., 1221 bučko, s. đ., 1427 bugarski, b., 1105 bui, h. m., 83 buntić, a. v., 129 chakraborty, p., 225 chittireddy, v. r. r., 355 coban, b., 563 costa, j. s., 153 crochet, a., 689 cruz, j. n., 591 cui, y., 1183 cvetković, v. s., 1329 1480 journal of the serbian chemical society ćipranić, i., 527 ćirić, a., 1355 ćirić-marjanović, g., 901 čačić, j., 663 česen, m., 623 četojević-simin, d. d., 991, 1455 čomor, m. i., 1455 da costa, r. a., 591 da costa, w. a., 591 dajić-stevanović, z., 1355 darvishi, p., 1169 davidović-plavšić, b., 455 delić, d. i., 129 deniz, s., 435 despotović, v. n., 1455 dimić, d., 877 dimitrijević-branković, s. i., 129 dinh, t. n., 445 dinkelbach, f., 819 djaghout, i., 41 djokić, n., 1199 djokić, s., 1199 djošić, m., 1287 djuran, m. i., 689 dmitrikova, l., 187 dobrota, a. s., 901 dodevska, m., 377 dönmez, s., 435 dragišić maksimović, j., 1105 dražić, b., 961 driouche, y., 405 drvenica, i., 1105 đokić-stojanović, d. r., 253 đorđević, d., 861 đorđievski, s., 527 el ashry, e. s. h., 1 elezović, n. r., 1271 eraković, s., 1305 etinski, m., 891 ferjancic, z., 935 finčur, n. l., 1455 fodeke, a. a., 1143 fraj, l. j., 1427 fromm, k. m., 689 gadžurić, s., 991 gajdoš kljusurić, j., 663 gajić, n., 423 gajić-krstajić, lj., 1271 galashev, a., 1129 galijašević, s., 11 ganjali, m. r., 701 gao, m., 713 garcia-rodriguez, d. e., 1415 gavrilov, n. m., 901 gengqiu tang, gizir, a. m., 757 gligorijević, n., 943 glišić, b. dj., 689 gođevac, d., 1355 golebiewski, w. m., 1073 golja, b., 743 gong, x., 245 grgur, b. n., 1261 grimme, s., 837 gruden, m., 779 gucma, m., 1073 gunasekaran, s., 225 gundu, s., 237 gupta, v., 211 güven, k., 1093 güven, r. g., 1093 gvozdenović, m. m., 1261 gvozdenović-jeremić, j., 951 hadži jordanov, s., 1249 hamdi, h., 679 hanousek čiča, k., 663 hasanagić, d., 11 hassan, a., 1155 hassan, m., 649 hassannezhad, m., 701 hassel, e., 599 hatamjafari, f., 975 he, d., 1367 heath, e., 623 hellal, a., 679 herenda, s., 503 hodžić, e., 11 hosseini, m., 701 volume 84: author index 1481 hosseinzadeh, r., 175 ignjatović, n., 1305 ilić, v., 1105 isakov, a., 1129 isakovski, m. k., 327 ivančev-tumbas, i., 623 ivanović, m. d., 639 jaćimović, ž. k., 467 jadranin, m., 141, 1319 janjić, g. v., 1391 janković, a., 1287 jasmina nikić, j., 327 jerosimić, s. v., 801 jevremović, d. r., 1381 jevtić, i. i., 639 ji, s., 245 jin, c., 713 joncev, z., 935 jovanović, d., 99 jovanović, dj., 1017 jovanović, m. b., 531 jovanović-lješković, n. m., 365 jović, b. m., 1271 jović, v. b., 729 jovićević, j. n., 1329 jovićević, n., 1329 ju, x., 27 jugović, b. z., 1261 jurak, v., 663 kalanithi, m., 267 kalhor, m., 489 kaluđerović, g. n., 1119 kalušević, a., 1105 kamberović, ž., 423 kanagavalli, c., 267 karkalić, r., 99 katona, j. m., 1427 kavitha, r., 237 kazeminejad, z., 975 kekezović, s., 1345 kesri, f., 41 khamova, t. v., 1027 khan, j., 1155 kherouf, s., 575 kiss, e., 1055 kojić, v., 1345 kopitsa, g. p., 1027 korać, f., 503 korać, m., 423 kostić, m. m., 483, 1041 kostić-rajačić, s. v., 639, 925 kosychova, l., 343 krajnović, m., 663 krawczyk, m., 1073 kukavica, b., 11, 455 kumar, g. g. v., 277 kumaresan, v., 225 lađarević, j., 1319 lai, x., 245 lamovec, j. s., 729 lau, s., 517 lazarević, m. j., 1455 lazić, v., 539 lei, s., 303 leovac, v. m., 467 leposavić, a. p., 1381 lerbs, m., 1119 lević, s., 377 li, k., 69 li, s., 609 lin, x., 245 liu, b., 713 liu, l., 1367 liu, x., 245 lourici, l., 1405 lović, j., 1319 lu, x., 293 lukić, n., 455 luković, z., 99 luk'yanenko, t., 187 ljekočević, m., 141 makrlík, e., 199 maksimović, m., 1221 maletić, s., 327 mandić, m., 99 manojlović, d. d., 253 marian, c. m., 819 marinković, n., 1235 marjanović jeromela, a., 1017 marković, d., 743 marković, j. d., 877 1482 journal of the serbian chemical society marković, r., 527 marković, s., 1105 markovic, z., 877 mechler, a., 445 mehmood, m. h., 1155 mei, z., 27 mendoza-huizar, l. h., 1415 mentus, s., 901 messadi, d., 405, 575, 1405 mihajlović, i., 55 mihajlović, m. a., 531 mijin, d. z., 531, 1319 milenković, d., 877 milić, m. d.. 129 milićević, b., 539 milinković budinčić, j. r., 1427 milovanović, b., 891 milovanović, m., 801 milutinović, s., 55 mirica, m. c., 1027 mirza, b., 649 mišković-stanković, v., 1287 mitić, m., 801 mitrović, j., 1041 mitrović, o. v., 1381 mitu, l., 277 mladenović, i. o., 729 mladenović, m., 845 mohamadpour, f., 1083 mohammadi, s. z., 1005 mohora, e., 327 mojović, lj., 951 mrvčić, j., 663 najdanović, s. m., 483, 1041 nam, p. c., 445 namazian, m., 391 natić, d., 377 naz, s., 317 nedeljković, j., 539 nedić, o., 943 nedović, v., 1105 nestorović, a. b., 1391 nikićević, n. j., 141, 1381 nikodinovic-runic, j., 99 nikolić, n. d., 729, 1209 nikolić, ž., 55 nikolić-kokić, a., 455 ning, p., 69 nourifard, f., 489 novaković, m., 1355 obradov, m., 729 okoye, p. u., 609 olayera, o. o., 1143 oliaey, a. r., 975 onjia, a. e., 1391 ostojić, b., 861 pan, x., 1367 panić, j., 991 panić, s., 1055 panić, v. v., 1305 pantelić, n., 1119 pantović pavlović, m. r., 1305 papović, s., 991 pašti, i. a., 417, 901 pastor, k., 1017 pasupuleti, m., 225 pattanaik, a., 225 paunović, s. a., 1381 pavlović, m. g., 1209 pavlović, m. m., 1305 payehghadr, m., 489 penjišević, j. p., 639 penjišević, j. z., 925 petković, m., 891 petričević, s. m., 365 petrović, a., 141 petrović, l. b., 1427 petrović, m. m., 483 petrović, s., 1319 petrović, t., 377 pezer, m., 663 poiani, j. g. c., 153 popović, b. t., 141, 1381 popsavin, m., 1345 popsavin, v., 1345 pourshamsian, k., 975 putz, a.-m., 1027 qezelje, h. h., 489 qiang tian, q., 1027 qin, s.-s., 303 radanović, m. m., 467 volume 84: author index 1483 radetić, r., 743 radojević, v. j., 729 radonić, j., 1439 radonjić, d., 503 radović vučić, m. d., 483, 1041 radović, lj., 377 rahim, a., 1155 raja, j. d., 267, 277 rajalakshmi, m., 277 rajan, s., 225 rajesh, j., 277 rakić, a. a., 1391 ramadan, e. s., 1 ramos, r. s., 153 ramzan, m. s., 649 rao, k. k., 355 rasheed, h. a., 1 raza, h., 649 raziq, a., 1155 rehman, a. u., 649 rekovic, l., 343 revathi, n., 277 rhee, k. y., 1287 rigo, m., 1027 rikalović, m. g., 365 rios-reyes, c. h., 1415 ristić, s. m., 365 rodić, m. v., 111, 467 roglić, g. m., 925 roozgari, m., 391 ryskaliyeva, a. k., 477 saeed, r., 317 safaei, m., 175 safarov, j., 599 sakthikumar, k., 277 sankarganesh, m., 267, 277 santos, c. b. r., 153, 591 sarasija, m., 237 savić, b. g., 55 savić, n. d., 689 savii, c., 1027 šćepanović, j., 503 seo, s.-y., 649 seović, m., 55 setoodeh, n., 1169 shah, s. a. a., 649 she, z., 713 shi, d., 1183 shiroudi, a., 975 shishehbore, m. r., 175 shmychkova, o., 187 siddiqui, s. z., 649 silva, c. h. t. p., 153 silva, r. c., 153, 591 silva, s. de o., 591 silva, s. g., 591 simončić, b., 743 šojić merkulov, d. v., 1455 soković, m., 1355 šoškić, v., 925 spasojević, lj. m., 1427 spremo-potparević, b., 539 sredojević, d., 539 stajković-srbinović, o. s., 129 stamatović, m., 423 stamenković, o. s., 253 stančić, a., 1105 stanković, j. a., 961 stanković, j., 141 stanković, j., 1355 stanojević, i., 689 stanzer, d., 663 stevanović, j. s., 1305 stevanović, m., 527, 1287 stevanović, z., 527 stojadinović, s., 915 stojanović, b., 377 strahinić, i. d., 365 šukalović, v. b., 925 sun, g., 1183 šunderić, m., 943 suručić, lj., 1391 šuškalo, n., 11 tabaraki, r., 391 tadić, j. d., 531 tajik, s., 175, 1005 takaomi, k, 83 tan, x., 293 tanasković, s., 961 tang, t., 609 taranu, b. o., 1027 tariq, m., 1155 tatipamula, v. b., 555 tay, m. g., 517 1484 journal of the serbian chemical society tešević, v. v., 141, 1355, 1381 tezerjani, m. d., 391 thakur, s., 119 thundat, t., 1199 todorović, z. b., 253 tomić, m. v., 1221 tomšič, b., 743 tong, j.-b., 303 topaloğlu, i̇., 757 topalović, d., 539 tošić, m., 55 tot, a., 991 tran, t. t. v., 445 trišović, t. lj., 1261 troter, d. z., 253 trpčevska, j., 423 truong, t. t. c., 83 tubić, a., 327, 623 turk sekulić, m., 1439 urucu, o. a., 435 vaňura, p., 199 vasilić, r., 915 vasiljević, j., 743 vasiljević-radović, d. g., 729 vedula, g. s., 555 velichenko, a., 187 velinov, n. d., 483, 1041 veljković, v. b., 253 venkataramana, r., 355 vert-wong, k., 951 veselinović, lj. m., 253 vidziunaite, r., 343 vilotić, a., 943 vo, q. v., 445 vojinović miloradov, m., 1439 vojinović-ješić, lj. s., 467 vojnovic, s., 99 vorob’ev, a. s., 1129 vrana, b., 1439 vraneš, m., 991 vraštanović-pavičević, g., 377 vujasinović, v., 1017 vujić, d., 1017 vukićević, n. m., 1329 vukoje, i., 539 vukšanović, d., 503 wacha, a., 1027 wan, j., 713 wang, d., 245 wang, j. y., 69 wang, j., 609 wang, s., 609 wang, y., 303 wang, z., 713 watson, m., 327 wessjohann, l., 1119 wolf, k., 1119 xu, l., 609 xu, s., 27 xu, y., 1367 yabalak, e., 757 yalaz, s., 1093 yang, n., 69 yeoh, c. b., 517 yetimoğlu, e. k., 435 yildiz, u., 563 yildriz, m., 117 yusuf, m., 119 zaikov, y. p., 1129 zavišić, g. n., 365 zdujić, m. z., 253 zdunić, g., 1355 zelechowski, k., 1073 zelenović srećo, b., 1345 zhang, a., 609 zhang, d., 1367 zhang, l., 609 zhao, f., 27 zine, m., 1405 zlatar, m., 779 živković, l., 539 živković, p. m., 1209 volume 84: author index 1485 subject index of vol. 84 and list of referees in 2019 are given in the electronic form at the internet address of the journal of the serbian chemical society: http://www.shd.org.rs/jscs end of volume 84. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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<feff04120438043a043e0440043804410442043e043204430439044204350020044604560020043f043004400430043c043504420440043800200434043b044f0020044104420432043e04400435043d043d044f00200434043e043a0443043c0435043d044204560432002000410064006f006200650020005000440046002c0020044f043a04560020043d04300439043a04400430044904350020043f045604340445043e0434044f0442044c00200434043b044f0020043204380441043e043a043e044f043a04560441043d043e0433043e0020043f0435044004350434043404400443043a043e0432043e0433043e0020043404400443043a0443002e00200020042104420432043e04400435043d045600200434043e043a0443043c0435043d0442043800200050004400460020043c043e0436043d04300020043204560434043a0440043804420438002004430020004100630072006f006200610074002004420430002000410064006f00620065002000520065006100640065007200200035002e0030002004300431043e0020043f04560437043d04560448043e04570020043204350440044104560457002e> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {efficient removal of malachite green from aqueous solution by adsorption on carbon nanotubes modified with znfe2o4 nanoparticles} j. serb. chem. soc. 84 (7) 701–712 (2019) udc 547.632.5:544.723+546.26:54–145.2+ jscs–5220 539.12 original scientific paper 701 efficient removal of malachite green from aqueous solution by adsorption on carbon nanotubes modified with znfe2o4 nanoparticles shiva dehghan abkenar1*, morassa hassannezhad2, morteza hosseini3 and mohammad reza ganjali2,4** 1department of chemistry, savadkooh branch, islamic azad university, savadkooh, iran, 2center of excellence in electrochemistry, faculty of chemistry, university of tehran, tehran, iran, 3department of life science engineering, faculty of new sciences & technologies, university of tehran, tehran, iran and 4biosensorresearch center, endocrinology and metabolism molecular-cellular sciences institute, tehran university of medical sciences, tehran, iran (received 28 december 2018, revised 17 april, accepted 25 april 2019) abstract. in this study, multiwall carbon nanotubes modified with spinel zinc ferrite nanoparticle (znfe2o4/mwcnts) were used as a solid phase adsorbent for the removal of malachite green (mg) from aqueous media. the synthesized nanocomposite was characterized by different methods, such as fourier transform infrared spectroscopy (ft-ir), scanning electron microscopy (sem) and x-ray diffraction (xrd). batch adsorption experiments to determine the optimal adsorption conditions and different factors that influence the adsorption efficiency (i.e., ph, amount of adsorbent, contact time, and the initial concentration of mg) were also evaluated and optimized. the data were satisfactorily fitted to the langmuir model and a maximum adsorption capacity of 116.2 mg g-1 was obtained at a ph of 7.5. in addition, adsorption kinetics studies were performed. the adsorption of the model dye (mg) was found to reach equilibrium after 60 min, following a pseudo-second-order kinetic model. furthermore, an external magnetic field could easily separate the nanoparticles from water with a high separation efficiency. keywords: dye removal; malachite green; modified multiwall carbon nanotubes; spinel zinc ferrite nanoparticle. introduction water is one of the necessities required for the sustenance and continuation of life. it is therefore imperative that a water supply of good quality be available for various activities. however, this is becoming increasingly difficult considering the large-scale pollution caused by industrial, agricultural and domestic acti *,** corresponding authors. e-mail: (*)dehghan54@yahoo.com; (**)ganjali@gmail.com https://doi.org/10.2298/jsc181228038d 702 abkenar et al. vities. these activities generate wastewater that contains both inorganic and organic pollutants. some of the common pollutants are phenols, dyes, detergents, insecticides, pesticides and heavy metals.1 removal of dyes from waste waters has recently received considerable attention and a range of methods, such as adsorption, flocculation, oxidation and electrolysis, has been developed.2–4 among these approaches, adsorption techniques offer simple, yet effective and economical tools for treating wastewaters.5,6 the unique physicochemical qualities of nanomaterials, including their considerable surface area and the large number of defective sites present in their structure, are expected to result in enhanced uptake and adsorption properties, in comparison to their micro-sized counterparts.7–10 malachite green (mg) is a triarylmethane dye that has been controversially applied in the aquaculture industry to treat and prevent protozoa and fungal infections.11 mg is potentially carcinogenic, mutagenic, teratogenic, and toxic and thus it is classified as a class ii health hazardous material by the occupational safety and health administration (osha, usa).12 the acute oral ld50 (lethal dose, 50 %) values of mg in rats have been reported to be 275–520 mg kg–1 body weight. although mg has been banned in most countries, it is still used in many parts of the world because of its high efficacy, low cost, and availability, as well as the existence of less restrictive laws for non-aquaculture purposes.11 in recent years, multiwall carbon nanotubes (mwcnts) have attracted considerable attention due to their unique structure and properties, including large surface areas, high aspect ratios, nanosized stability and rich surface chemical functionalities. however, mwcnts are very hydrophobic and easily aggregate in aqueous solution because of strong van der waals interaction forces between mwcnts, which may hinder effective adsorption behaviors and reduce the adsorption capacity.13,14 therefore, decoration of mwcnts by the introduction of inorganic nanoparticles is an effective way to enhance their dispersibility and performance.15 spinel ferrites nanoparticles, with the general formula mfe2o4 (m = fe, co, cu, mn, zn, etc.), are of the most popular magnetic materials in analytical chemistry and can improve electrochemical, optical and magnetic properties of carbon based nanomaterials, such as carbon nanotubes (cnts),16 graphene oxide17 and graphene quantum dots.18 zinc ferrite nanoparticle (znfe2o4 np), as one of the spinel ferrite compounds; have attracted increasing interest because of their good biocompatibility, low toxicity, electrical properties, easy synthesis and high adsorption ability.19 in order to effectively utilize the advantages of both ferrite nanoparticles and carbon nanotubes, various kinds of modified cnts have been synthesized by decorating cnts with nanoparticles, such as cofe2o4,20 nife2o421 and mnfe2o4.22 efficient removal of malachite green from aqueous solutions 703 in the present study, multiwall carbon nanotubes modified with spinel zinc ferrite nanoparticle (znfe2o4/mwcnts) were synthesized and then assessed as the adsorbent for mg removal from water. the znfe2o4/mwcnts could be easily separated by an external magnetic field. influencing factors, including ph, time and the initial concentration, on the mg adsorption efficiency were investigated. the adsorption kinetics were studied. in addition, the adsorption efficiency and mechanism were assessed based on isothermal studies. experimental instruments spectrophotometric measurements were realized with a perkin elmer (lambda 25) double-beam spectrophotometer for monitoring the mg concentration. all the ph values were measured with a lab 850 benchtop ph meter. the ir spectra were taken using a fourier transform infrared spectrometer (ft-ir; perkin elmer, usa). scanning electron microscopy (sem) images were taken with a kyky-em3200ldigital scanning electron microscope operated at 26 kv. the morphological analysis by x-ray diffraction was performed on an xpert mpd advanced diffractometer using cu(kα) radiation at room temperature in the 2θ range from 4 to 120° at a scanning rate of 0.02° s-1. chemicals and reagents reagent-grade compounds were procured from fluka and merck and double distilled water (ddw) was used in all steps of the study. the glassware used for trace analysis were maintained in diluted nitric acid for at least one day and then washed repeatedly with ddw, prior to application. multi-walled carbon nanotubes (purity >95 %) were purchased from shenzhen nanotech port co. and zinc nitrate (zn(no3)2·6h2o) and ferric nitrate (fe(no3)3·9h2o) were obtained from sigma–aldrich (st. louis, mo, usa). the mg stock solution was obtained through dissolving the dye powder in ddw. the other solutions were obtained through diluting stock solution to the desired concentrations. synthesis of znfe2o4/mwcnts nanocomposite the znfe2o4 nanoparticles on carboxyl functionalized mwcnts were fabricated by a hydrothermal process. in order to synthesize carboxyl functionalized mwcnts, the pure mwcnts were refluxed in a mixture of concentrated sulfuric acid and nitric acid (3:1 volume ratio) at 70 °c for 6 h. after cooling to room temperature, the product was washed with double distilled water several times until the ph of the filtrate was ≈7 and finally dried at 60 °c for 24 h. the prepared carboxyl functionalized mwcnts were used as templates for the formation of the znfe2o4/mwcnts nanocomposite. then, certain quantities of the carboxyl functionalized mwcnts was weighed and dispersed in mixture of 15 ml ethanol and 5 ml acetonitrile, through sonication in an ultrasound bath for 50 min. thereafter, 0.35 g of fe(no3)3⋅9h2o and 0.12 g zn(no3)2·6h2o were added to the solution while stirring for 60 min at room temperature. during this process, positively charged fe3+ and zn2+ can be absorbed on the hydroxyl and carboxyl groups on the surface of the negatively charged carboxyl functionalized mwcnts by electrostatic attraction. then 1 ml of naoh (0.1 m) solution was added into the above solution under stirring. upon the addition of naoh solution, the hydrolysis of fe3+ and zn2+ leads to the formation of znfe2o4 nanoparticles deposited on the surface of the mwcnts. after 1 h of stirring, the formed dark brown suspension was cen704 abkenar et al. trifuged and the solid product was separated, washed with a water and ethanol mixture. the formed dark brown precipitate was dispersed in 25 ml water while sonicating, transferred to a 50 ml autoclave, and treated at 180 °c for 12 h. the resulting powder was denoted as znfe2o4/mwcnts nanocomposite. mg adsorption experiments the znfe2o4/mwcnts were used to adsorb malachite green from water samples and all experiments were performed in triplicate. equilibrium experiments involved adding 0.010 g of znfe2o4/mwcnts to 10 ml of a 50 mg l-1 of mg solution (ph 7.5). the mixtures were gently shaken for 60 min under ambient conditions and subsequently, the magnetic nanoparticles with adsorbed dyes were separated from the mixture via a permanent hand-held magnet. before, during and after the adsorption experiments, the mg concentration was determined through spectrophotometry at 616.5 nm. from the obtained data, the dye removal efficiency was calculated using the following equations: 0 0 / % 100 t c c r c − = (1) where c0 and ct are the initial mg concentration and the concentration at the end of the experiment at time t. the experiments also involved evaluating the effects of ph, the amount of znfe2o4/ /mwcnts, contact time, and initial mg concentration on the adsorption efficiency. furthermore, the kinetics of the adsorption phenomenon was evaluated through monitoring the adsorption capacity at various intervals. to obtain the adsorption isotherms, the dye solutions within the initial concentrations in the range of 10–200 mg l-1 were used in adsorption experiments until equilibrium conditions. the amounts of the adsorbed mg (qe) of dye were calculated using the following equation: 0 ee c c q v m − = (2) where c0 and ce represent the initial and equilibrium mg concentrations, m is the mass of znfe2o4/mwcnts, and v is the solution volume. results and discussion characterization of nanocomposite ft–ir was used to ascertain the presence of carboxyl functionality on mwcnts, and to check the presence of znfe2o4 nanoparticles on the surface of mwcnts (fig. 1). in the spectrum of carboxyl functionalized mwcnts (curve a) the peaks at 1640 and 1382 cm–1 correspond to c=o and c–o stretching, respectively. the two weak peaks at 2935 and 2840 cm–1 correspond to the –ch stretching mode and a broad band peak at 3440 cm–1 is attributed to the carboxylate groups on the external surface of mwcnts.23 compared with the spectrum of znfe2o4/mwcnts (curve b), the peaks at around 620 and 1153 cm–1, which are assigned to the deformation of different types of c–h bonds and formation of cooh groups,24,25 were shifted to higher wavenumbers. this observation indicates the bonding of znfe2o4 to c–o–h groups on the surface of efficient removal of malachite green from aqueous solutions 705 mwcnts. in curve b, the new bands at 1385 and 820 cm–1 are due to the stretching of zn–o–fe bonds of the tetrahedral building units forming the spinel ferrite structure. in addition, the peak at 590 cm–1 could be attributed to the stretching vibration of the fe–o bond.23 fig. 1. ft-ir spectra of carboxyl functionalized mwcnts (a) and znfe2o4/ /mwcnts (b). the phase and crystalline structure of the pure mwcnts as well as the synthesized znfe2o4/mwcnts nanocomposite was investigated by their powder x-ray diffraction pattern, shown in fig. 2. the xrd pattern of pure mwcnts shows one obvious peak at 2θ of 24°, which corresponds to the (002) plane of fig. 2. xrd pattern of pure mwcnts and znfe2o4/mwcnts. 706 abkenar et al. mwcnts. for the xrd pattern of the znfe2o4/mwcnts nanocomposite, the presence of six diffraction peaks at 2θ values of 30, 35.65, 39.25, 43, 56 and 57°, corresponding to the reflection planes (220), (311), (222), (400), (422) and (511), respectively, confirm the formation of the spinel structure of znfe2o4.26 the xrd result indicates that the synthesized nanocomposite contained mwcnts and znfe2o4 nanoparticles. in order to obtain information about the surface morphologies of the mwcnts before and after the modification process, sem images were taken. as shown in fig. 3a, pure mwcnts have almost homogeneous structures with a smooth surface. however, the znfe2o4 nanoparticles are well distributed on the surface of mwcnts, producing no change in the morphology of the multiwalled carbon nanotubes, only modifying it (fig. 3b). a) b) fig. 3. sem images of: a) pure mwcnts and b) znfe2o4/mwcnts. effect of ph the ph of the dye solution plays a crucial role in the whole adsorption process because the ph may affect both the aqueous chemistry and surface binding sites of the adsorbent. in order to determine the best ph, studies were performed in the ph range of 3.0 to 10.0 with fixed initial concentration and contact time onto an exactly weighed amount of adsorbent (10 mg). the results are given in fig. s-1 of the supplementary material to this paper. mg is known to exist in the cationic form in aqueous media and is hence attracted to negatively charged surfaces. according to the results, the adsorption of mg was increased at higher ph values until 8.0 and then decreased on increasing the ph. hence, ph 7.5 was chosen for subsequent experiments. this phenomenon could be explained based on added negative charges on the surface of the nanoparticles, which could enhance the electrostatic interaction of nanoparticles and the cationic dye mg. at acidic ph, the decrease in adsorption could be explained by the fact that h + may compete with dye ions for the adsorption sites of adsorbent, thus inhibiting the efficient removal of malachite green from aqueous solutions 707 adsorption of dye27 and in alkaline ph, oh– could be absorbed by mg, and the resulting electrostatic repulsion could negatively influence the adsorption. effect of the amount of adsorbent on the removal efficiency the effect of the amount of znfe2o4/mwcnts on the removal of mg reaching the optimal adsorption efficiency was determined by varying the adsorbent amount in the range of 0.002 to 0.015 g, in 10 ml sample containing 50 mg l–1 mg at ph 7.5. based on the results, the removal efficiency increased on increasing the amount of znfe2o4/mwcnts, which corresponds to the presence of more adsorption sites and their availability. the solution containing 0.006 g adsorbent had a removal efficiency of 90 % and the adsorption reached maximum with 0.010 g of adsorbent with maximum percentage removal of about 93 %. effect of contact time the influence of contact time on the adsorption efficiency was studied at an initial mg concentration of 50 mg l–1 and the amount of the adsorbent of 0.010 g. the concentration of dye in the solution at different times (after 5, 10, 20, 40 and 60 min) was spectrophotometrically determined. the results (fig. s-2 of the supplementary material) clearly indicate that the adsorption capacity initially rapidly increases, and then continues to increase at a relatively slow speed and after about 60 min, the total dye content of the sample was adsorbed. to determine the adsorption mechanism, a pseudo first-order and a pseudo-second-order kinetic model were used to fit the experimental data. the pseudo-first-order model is described by the lagergren equation:28 1e elog( ) log 2.303t k t q q q− = − (3) where k1 is the pseudo-first-order rate constant, and qe and qt are the amount of absorbed mg at equilibrium and at time t, respectively. the pseudo-second-order model, on the other hand, is expressed as:29 22 e e 1 t t t q k q q = + (4) where k2 / g mg–1 min–1 is the rate constant of the pseudo-second-order adsorption, and qe and qt are as given above. the kinetic constants were obtained through linear regression for the two models (fig. s-3 of the supplementary material) and the results are presented in table i. the correlation coefficient (r2) of the pseudo-first-order kinetic model was rather low and the qe values (qe,cal) calculated from this model did not show good agreement with the experimental data (qe,exp), reflecting the fact that the model is not appropriate. in the case of the pseudo-second-order model, on the other hand, r2 was 0.9976 and the qe,cal values were in good agreement with the 708 abkenar et al. qe,exp values, indicating the applicability of the kinetic model to the adsorption of mg onto znfe2o4/mwcnts. the present findings are in good agreement with earlier reports.30–35 table i. adsorption kinetic parameters of mg adsorption on znfe2o4/mwcnts pseudo-first order pseudo-second order experimental data k1 / min-1 qe,cal / mg g-1 r2 k2 / g mg-1 min-1 qe,cal / mg g-1 r2 qe,exp / mg g-1 0.0110 4.63 0.9598 0.01635 48.78 0.9976 48.52 effect of the initial mg concentration the effect of the initial concentration of mg in the solution on the adsorption capacity was studied at different mg concentrations in the range of 10–200 mg l–1. the experiment was performed with a fixed adsorbent dosage, together with a firmly maintained temperature, under the optimum ph condition. the adsorption capacity for mg dye increased with increasing initial mg dye concentration in solution. a plot of adsorption capacity vs. mg concentration is illustrated in fig. 4. obviously, increasing concentration enhances the interaction between the dye and adsorbent, which could be attributed to the driving force created by the strong concentration gradients. adsorption increases while the surface of the adsorbent is not saturated. naturally, after all the adsorption sites are occupied, further increasing in the mg concentration does not increase the adsorption efficiency. in this study, langmuir and freundlich models were applied to fit the adsorption properties of the nanoparticles. the former model is based on assuming that the homogeneous sites of the adsorbent are covered by a monolayer of the dye molecule and that no species can be adsorbed after saturation is reached.36 the freundlich isotherm model, on the other hand, uses an empirical equation to describe heterogeneous systems.37 fig 4. the effect of mg concentration on the dye adsorption capacity of znfe2o4/mwcnts. the linearized expression of the langmuir model is given by: efficient removal of malachite green from aqueous solutions 709 e e e m m 1c c q bq q = + (5) in which qm is the highest adsorption capacity (reached when a monolayer completely covers the surface) and b expresses the equilibrium constant (l mg–1). the linearized form of the freundlich is expressed as: e f e f 1 log log logq k c n = + (6) kf and 1/nf being a rough indicator of the adsorption capacity, and the adsorption intensity, respectively. the slope 1/nf ranges between 0–1 and reflects the adsorption intensity or surface heterogeneity. closer to zero, 1/nf values reflect higher heterogeneity of the surface.38 these constants, determined using linear regression analysis, are given in table ii, illustrating the langmuir isotherm as fitting since it exhibits a higher r2 value compared to the freundlich model. the maximum adsorption capacity was found to be 116.2 mg g–1. it could be seen form table iii that the adsorption capacity of znfe2o4/mwcnts is higher than that of many other previously reported adsorbents. table ii. isotherm parameters for the adsorption of mg on znfe2o4/mwcnts dye langmuir freundlich qm / mg g-1 b / l mg-1 r2 kf / mg g-1 1/n r2 mg 116.2 0. 0143 0.9933 3.57 0.6433 0.9851 table iii. comparison of the maximum capacity factor of mg on various adsorbents adsorbent capacity factor, mg g-1 ref. znfe2o4/mwcnts 116.2 this work cofe2o4–sio2 75.5 2 chitosan ionic liquid beads 8.07 1 cellulose modified with phthalic anhydride 111.0 30 activated carbon/cofe2o4 composite 89.29 31 zno nanorod-loaded activated carbon 66.68 32 graphene oxide /cellulose bead 30.09 33 copper sulfide nanorods loaded on activated carbon 145.98 34 almond gum 196.07 35 conclusions in this study, the multi-walled carbon nanotubes modified with spinel ferrite znfe2o4 nanoparticles was prepared and employed as an adsorbent with great efficiency for mg removal from water. the equilibrium data were analyzed using the langmuir and freundlich isotherm models. the results showed the adsorption of mg on znfe2o4/mwcnts follows the langmuir adsorption 710 abkenar et al. model with a maximum adsorption capacity of 116.2 mg g–1. adsorption kinetics of the process was found to follow a pseudo-second-order kinetic model. in addition, the cost of nanoparticle preparation is low, the functionalization is available easily, and the process of purifying water pollution is clean and safe using magnetic nanomaterials. all of these results indicate that this methodology could be used as a highly effective method for the removal of malachite green from aqueous solutions. supplementary material additional data are available electronically from journal web site http:// //www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. the financial support of this work by the research council of the islamic azad university of the savadkooh branch and of the university of tehran is gratefully acknowledged. и з в о д ефикасно уклањање малахитно зеленог из водених раствора адсорпцијом на угљеничним нанотубама модификованим znfe2o4 наночестицама shiva dehghan abkenar1, morassa hassannezhad2, morteza hosseini3 и mohammad reza ganjali2,4 1 department of chemistry, savadkooh branch, islamic azad university, savadkooh, iran, 2 center of excellence in electrochemistry, faculty of chemistry, university of tehran, tehran, iran, 3 department of life science engineering, faculty of new sciences & technologies, university of tehran, tehran, iran и 4 biosensorresearch center, endocrinology and metabolism molecular-cellular sciences institute, tehran university of medical sciences, tehran, iran у овом раду су вишеслојне угљеничне нанотубе модификоване спинелним цинк -феритним наночестицама (znfe2o4/mwcnts) коришћене као чврсти адсорбент за уклањање малахитно зеленог (mg) из водене средине. синтетисани нанокомпозит је окарактерисан различитим методама, као што су инфрацрвена спектроскопија са фуријеовом трансформацијом (ft-ir), сканирајућа електронска микроскопија (sem) и дифракција x-зрачења. оптимални услови адсорпције одређени су експериментима равнотежне адсорпције. испитиван је и оптимизован утицај различитих фактора на адсорпциону ефикасност (тј. ph, количина адсорбента, време контакта, и полазна концентрација mg), подаци су успешно фитовани ленгмировим моделом и одређен је максимални адсорпциони капацитет од 116,2 mg g-1 на ph 7,5. такође, извршено је испитивање адсорпционе кинетике. одређено је да се адсорпциона равнотежа у случају модел молекула боје (mg) успоставља након 60 min, пратећи кинетички модел псеудо-другог реда. додатно, спољашње магнетно поље успешно одваја наночестице од воде са високом ефикасношћу сепарације. 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2221(https://doi.org/10.1021/ja02268a002) 37. h. m. f. freundlich, j. phys. chem. 57 (1906) 385. 38. f. haghseresht, g. lu, energy fuels 12 (1998) 1100 (https://doi.org/10.1021/ef9801165). {synthesis of proton exchange membranes for dual chambered microbial fuel cells} j. serb. chem. soc. 83 (5) 611–623 (2018) udc 544.726+546.212:532.14:66.081+ jscs–5099 678.744+546.185–32 original scientific paper 611 synthesis of proton exchange membranes for dual-chambered microbial fuel cells sandeep dharmadhikari, prabir ghosh and manivannan ramachandran* department of chemical engineering, national institute of technology, raipur – 492010, chhattisgarh, india (received 2 september, revised 5 december 2017, accepted 9 january 2018) abstract: proton exchange membranes (pems) were synthesized using three different compositions of poly(oxyethylene) (poe), poly(vinyl alcohol) (pva), chitosan (cs) and phosphoric acid (pa) in weight ratios of 1:1:1:1, 1:2:1:1 and 1:3:1:1 by physical blending and the casting method. water uptake of the membrane increases with increasing concentration of pva. a higher percentage of water uptake signifies a higher ion exchange capacity (iec) of the synthesized membrane. the synthesized membranes were evaluated in microbial fuel cells (mfcs) and the performance observed. the synthesized membranes were characterized for identification of precursor material and inter polymer interactions using x-ray diffraction (xrd) and fourier transform infrared (ftir) spectroscopy analysis, respectively. the removal of chemical oxygen demand (cod) depends on the microbial activity in the anodic chamber. in the present study, the composition of the membrane was optimized and compared with other membranes that had been synthesized by different compositions of all materials. cod removal in the mfc-3 setup connected with an m-3 membrane was found to be 88 %. keywords: ion exchange capacity; chemical oxygen demand; water uptake; power density; current density; open circuit voltage. introduction microbial fuel cells (mfcs) are used for power generation from wastewater through microbial oxidation of organic contaminants.1 proton exchange membranes (pem) physically separate the anode from cathode.2 commercial membranes, such as nafion®, are highly conductive due to their structural properties. the material nafion® consists of three parts viz., a poly(tetrafluoroethylene) backbone, side chains connected with the molecular backbone and ion clusters consisting of sulfonic ions. nafion has high ion exchange capacity and proton con * corresponding author. e-mail: rmani.che@nitrr.ac.in https://doi.org/10.2298/jsc170902016d 612 dharmadhikari, ghosh and ramachandran ductivity due to the above-mentioned functional groups.3 there are many advantages of nafion 117 membrane in fuel cell applications. however, several problems associated with nafion still exist, including oxygen leakage from the cathode to the anode, substrate losses and biofouling,4 conductivity at low water content and poor mechanical strength at high temperatures.3–5 the proton exchange membranes are associated with some other major problems, such as surface layer losses from the membranes and the migration of protons. an efficient proton exchange membrane possesses a high ion exchange capacity in aqueous condition.6,7 due to these drawbacks, alternate approaches have been developed, such as opting by mo2 (where m = zr, si, ti),8 by non-fluorinated polymer electrolytes,9 and by inorganic and organic composite membranes.10 jiang and tang11 discussed the effects of layer-by-layer (lbl) self-assembly of polyelectrolyte bilayers on methanol permeability and proton conductivity of nafion membranes and studied the crossover properties of modified nafion membranes for the application in direct methanol fuel cells (dmfcs). magnetic ion-conducting nanoparticles were prepared by hasanabadi et al.12 for pem applications. the surface of the γ-fe2o3 nanoparticles was modified via silanation followed by the oxidation of the terminal mercapto groups to form functional sulfonate groups and studied the effect of sulfonation on proton conductivity, activation energy, methanol permeability and selectivity. sadrabadi et al.13 described the preparation and characterization of novel ion exchange polymer nanocomposites for fuel cells applications. montmorillonite (mmt) nanoclay was modified using amino benzotriazole (bta) via ion exchange. they found that nafion/bta–mmt-3.0 wt. % membranes were able to provide over three times more power output than nafion-117. erkatal et al.14 demonstrated a novel proton conducting membrane consisting of poly(vinyl alcohol) (pva), poly(2 -acrylamido-2-methylpropane sulfonic acid) (pamps) and 1,2,4-triazole, which was fabricated by physical blending, casting and solvent evaporation techniques. the in situ chemical cross-linking was performed using glutaraldehyde (ga). momlouk and scott15 synthesized a composite membrane with bpo4 (boron phosphate) with an excess quantity of po4 (bpox) and investigated the enhancement of ion exchange capacity in high temperature ranges. in this study, sulfonic and sulfuric acid were replaced by phosphoric acid due to its good chemical and electrochemical properties. poly(vinyl alcohol) (pva) and phosphoric acid (pa) mixtures were used in previous literature, whereby good mechanical strength but a low open circuit voltage (ocv) of 436 mv and a very low current density of 1 ma cm–2 were evidenced.16,17 daries et al.18 reported the synthesis of a proton exchange membrane consisting of pva, sulfamic acid (sa) and poly(oxyethylene) (poe). the mfc connected with this membrane had a maximum ocv value of 516 mv. the maximum power and current density of pva–sa–poe separator was found to proton exchange membranes for microbial fuel cells 613 be 7.16 w m–3 and 1.72 a m–3, respectively, but it was low when compared with nafion 117 under identical conditions.18 ghasemi et al. reported the synthesis of a pem using sulfonated poly(ether ether ketone) (speek) and evaluated the performance of the mfc. the power production of the mfc with speek was 77.3 mw m–2, which is still lower than the nafion 117 value of 106.7 mw m–2.19 hence, there is a need to focus on the synthesis of a membrane with a suitable composition for mfc applications. the present study was focused on the synthesis of proton exchange membranes using water-soluble polymers with different weight ratios. polymers such as poly(oxyethylene) (poe), used as plasticizer,18 and poly(vinyl alcohol) (pva) were used to increase the water transport due to their hydrophilic properties.20 phosphoric acid (pa), an alternative to sulfamic acid18 and sulfonic acid,15 has specific properties, such as excellent thermal, chemical and electrochemical stability in fuel cell applications. chitosan (cs) is a low-cost biopolymer that can provide chemical stability and electronic properties to membranes.20 the synthesized pems were characterized using x-ray diffraction (xrd) and fourier transform infrared (ftir) spectroscopy for the identification of precursor material and inter polymer interactions, respectively. the water uptake and ion exchange capacity of the synthesized membranes were quantified to optimize the composition of the membrane. the synthesized membranes were used in a mfc and the performance of the same was monitored with respect to open circuit voltage (ocv), power and current density, and compared with the conventional nafion 117 membrane. chemical oxygen demand (cod) removal of wastewater was measured using the synthesized membranes in the mfcs. experimental materials poly(oxyethylene) (poe-600) was purchased from sigma–aldrich, india, poly(vinyl alcohol) (pva), potassium hydrogen phosphate and sodium hydroxide were purchased from merck, india; sulfuric acid, propan-2-one and phosphoric acid (pa) were purchased from fisher scientific, india; chitosan (cs) was purchased from srl pvt. ltd., india, glutaraldehyde (ga) was purchased from loba chemie, india; d-glucose anhydrous purified, potassium manganite (vii), calcium chloride, magnesium sulfate heptahydrate, ammonium chloride, n-methyl-2-pyrrolidone (nmp) and n,n-dimethylmethanamide (dmf) were purchased from rankem, india. all the reagents and chemicals were used as received. unless otherwise mentioned, distilled water was used for all the experiments. synthesis of membranes the quantity of poe, pva, cs and pa used for the synthesis of membranes in the weight ratio of 1:1:1:1 (m-1), 1:2:1:1 (m-2) and 1:3:1:1 (m-3). n-methyl-2-pyrrolidone (nmp) and n,n-dimethylmethanamide (dmf), used as organic solvents, were mixed in the volume ratio of 1:1. the mixture was used for dissolving poe, pva, cs and pa according to the various compositions. the mixture was maintained at a temperature of 80 °c using a hot plate with continuous stirring for 4 h, in order to dissolve everything completely in the sol614 dharmadhikari, ghosh and ramachandran ution. finally, the prepared solution was cast into three different glass petri dishes up to the mark in order to maintain the thickness of all synthesized membranes and dried in an oven at 50 °c for 24 h. the thickness of the synthesized pems were measured using a digital screw gauge (mituotoyo, japan) and were found to be 0.2±0.001 mm. the dried membranes were immersed into a cross-linking solution consisting of 1.5 wt. % glutaraldehyde (ga), 0.5 wt. % of sulfuric acid, 48 wt. % of acetone and the remaining distilled water for 3 h. subsequently, the cross-linked membranes were washed several times with distilled water to remove traces of glutaraldehyde.21 construction of mfcs laboratory scale dual chambered batch microbial fuel cells were fabricated for this study. the two chambers, with a capacity of 1000 ml each, were made up of polyacrylic material with dimensions of each compartment 12.5 cm (height)×9 cm (length)×9 (width). both the chambers were connected using a flange, in which the proton exchange membrane was placed. rubber gaskets were used to prevent leakages and silicone gel was used for binding the gaskets. carbon electrodes in the form of rod were used as anode and cathode. the dimensions of the electrode were 1.5 cm diameter and 12.5 cm effective height. power and current density were calculated based on an anode area of 60.6 cm2. one chamber was closed with an airtight lid, which served as the anode, while the other chamber was left open to the atmosphere, acted as the cathode. the effective diameter of the membrane connecting the two chambers was 2.5 cm. the setups of microbial fuel cell mfc-1, mfc-2, mfc-3 and mfc-4 were with m-1, m-2, m-3 and nafion-117 pem, respectively. the microbial fuel cell mfc-4 setup with nafion 117 was taken as a reference. the anodic compartment was filled with 300 ml of sludge. the initial chemical oxygen demand (cod) value of sludge was 11400 mg l-1. synthetic wastewater was prepared as per the composition reported in the literature.22 the amount of wastewater filled in anodic chamber was 700 ml. initial cod and ph of synthetic wastewater was 560 mg l-1 and 7.3, respectively. cod of the mixture was found to be 5760 mg l-1, which was taken as reference for the calculation purpose. potassium permanganate (kmno4) of 0.2 g l-1 concentration was filled in the cathodic chamber, as reported in the literature.23 potassium permanganate was used as a catholyte for enhancing the reduction of h+ in the cathode compartment.23 experiments were performed at a room temperature of 28 °c. the closed chamber acted as an anode for anaerobic treatment. the initial ph of potassium permanganate solution was measured as 7.2. during the study, ph of the solution was maintained at 7, by adding few drops of 1 m naoh or 0.5 m h2so4, as required.24 characterization scanning electron microscopy (sem). morphological analysis of the synthesized membrane surface was realized using sem (jeol 6380a). the synthesized membranes were cut into small portion and kept in sample bags prior to sem. x-ray diffraction (xrd). in order to identify the materials used in the synthesis of the pem membranes, x-ray diffraction (xrd, panalytical 3kw x’pert powder) analysis was performed using cukα with a scanning rate of 5° min-1 and 2θ ranging from 5 to 80°. fourier transform infrared spectroscopy (ftir). the synthesized membranes were examined using ftir (bruker, alpha model) for identification of polymer interactions. a small piece of synthesized membrane was used for the analysis in the atr mode. the spectra were obtained between 4000 and 500 cm-1. proton exchange membranes for microbial fuel cells 615 water uptake. the membranes were dried using a closed hot air oven at 50 °c for 15 min to remove the moisture content. the membranes were weighed using an analytical balance (sartorius, bsa 2245-cw) with a readability of 0.1 mg. the dried membranes were then soaked in deionized water for 24 h. membrane surfaces were wiped with tissue paper and then the wet membrane weighed. water uptake of the synthesized membranes was calculated using eq. (1): w d d water uptake 100 − = w w w (1) where wd is the weight of the dried membrane and ww is the weight of the wet membrane.25-27 swelling ratio was found to be lesser than 5 %, which is reasonable as reported in the literature.35 ion exchange capacity (iec). the iec of the synthesized membrane was measured using the standard procedure reported in the literature.28,29 the synthesized membranes were immersed in 1 m hcl for 24 h and washed with distilled water to remove excess acid. subsequently, the membrane was soaked in 1 m nacl solution for 24 h. the nacl solution was titrated with 0.01 m naoh to determine the concentration of the exchanged protons with phenolphthalein as the indicator. the iec of the synthesized membrane was calculated using eq. (2): = abiec m (2) where iec is the ion exchange capacity in meq g-1, a is the volume of naoh consumed in ml, b is the concentration of naoh in molarity and m is the weight of the dried membrane in g.30 calculations of the power and current density the potential difference across the mfcs was measured using a digital multimeter without any load. the current was measured by connecting various resistances (10, 20, 47, 100, 427 and 1000 ω) to the mfcs, in accordance to the ohm law. the current density was calculated by dividing the current by the anode area. the power density was calculated using the following eq. (3): 2 = ri p a (3) where r is the applied external resistance, i is the current and a is the surface area of anode.12 polarization curves were plotted for the membranes as per the standard procedure mentioned in the literature.31-33 measurement of cod. the cod in wastewater samples were measured by the closed reflux method using the standard procedure mentioned elsewhere.34 the samples were heated at 150 °c for 2 h in a thermo-reactor (drb200). after cooling, the sample was titrated against ferrous ammonium sulfate (fas) with ferroin as the indicator. cod in the sample was calculated as follows: 1 sample 8000( ) (mg l )− − = a b m cod v (4) where a is the volume of fas required for the titration of the blank (ml), b is the volume of fas required for the titration of the sample (ml), m is the concentration of fas in mol dm-3, and vsample is the volume of sample (ml).34 616 dharmadhikari, ghosh and ramachandran results and discussion scanning electron microscopy (sem) the sem surface images of three membranes m-1, m-2 and m-3, synthesized using various recipes, are shown in fig. 1a–c. the surface morphology shows that all constituents of the membranes were homogeneously distributed throughout the membranes without any major migration or phase segregation.35 the sem images confirmed that pores were not significant in the synthesized membranes. a b c fig. 1. sem images of pems synthesized using various recipes: a) m-1, b) m-2 and c) m-3. x-ray diffraction (xrd) analysis the xrd pattern of membranes synthesized using various recipes are shown in fig. 2. the strong crystalline peak at 19.2° and the weak peak at 40.2° confirm the presence of poly(vinyl alcohol).36 the peak at 10° corresponds to the presence of chitosan in the synthesized membranes.37 the peak at 23.34° corresponds to poe-600.38 the h3po4 present in all the membranes was of non crystalline form and hence the peak was not observed in the xrd patterns.39 fig. 2. xrd patterns of membranes synthesized using various recipes. fourier transform infrared spectroscopy (ftir) analysis the ftir transmittance spectra of the synthesized membranes using different compositions are presented in fig. 3. the broad peak seen between 3500 and 3000 cm–1 represents –oh stretching and c–h stretching, which confirmed proton exchange membranes for microbial fuel cells 617 the presence of pva.14 peak at 1670 cm–1 corresponds to carboxylic group (c=o), while the peak at 1590 cm–1 (–nh2) represents carbonyl groups; both the peaks confirmed the presence of chitosan at the edges.40 the c–h stretching observed at 2887 cm−1 was due to the presence of poe–600.38 the phosphate group peaks were observed between 650–550 cm–1 and 1300–910 cm–1.41 fig. 3. ftir spectra of all synthesized membranes using various recipes. water uptake the water uptake by all the synthesized membranes are shown in fig. 4 and the values for m-1, m-2 and m-3 were 30.20, 33.15 and 36 %, respectively. the water uptake value increased with increasing pva concentration. water uptake of the nafion-117 membrane was 30 %, which matches well with the reported literature.42 compared with the water uptake by pristine nafion-117, the values of water uptake for synthesized membranes were slightly higher. this could be due to the hydrophilic nature of peg and pva.24 pva is more hydrophilic in nature due to the –oh groups.43 the content of pva in the m-3 membrane was higher and hence, the water uptake of m-3 membrane was higher than for the m-1 and m-2 membranes. the higher value of water uptake signifies a higher ion exchange capacity (iec).42 fig 4. water uptake and iec vs. pva concentration of the synthesized membranes. 618 dharmadhikari, ghosh and ramachandran ion exchange capacity (iec) the iec is a measure of relative concentration of acid groups within pems.44 the ion content is characterized by the mass of dry membrane per molar equivalents of ion conductor. fig. 4 shows the ion exchange capacities of the three synthesized membranes, i.e., 1, 1.2 and 1.52 meq g–1, respectively, according to increasing the concentration of pva. a membrane that had a higher concentration of pva yielded a higher iec value. peg provided the backbone for a pem; cs has a good electronic property and hence enhanced the iec.20 the iec for the nafion-117 membrane was 0.91 meq g–1, which is comparable with the literature value.42 the iec values of the synthesized membrane were higher than the iec of the nafion 117 membrane. performance of the mfcs power density and polarization curves. the power density curves of the microbial fuel cells with various synthesized membranes, while the other factors were constant are presented in fig 5, from which it could be seen that the power density trend for mfc-1, mfc-2 and mfc-3 passes through a maxima and then drops because of microbial oxidation of organic and inorganic contaminant present in wastewater. this trend matches well with the reported literature.19 fig. 5. power density curves of the synthesized membranes and nafion-117 membrane in mfcs setups. the performance of mfc-3 with an m-3 membrane showed a better performance when compared with the other mfcs with an m-1 or m-2 membrane. fouling prevents the exchange of protons in dual chambered mfcs.45–47 the maximum values of power density are given in table i. the maximum values correspond to the membrane without fouling. however, the power densities of m-1, m-2, m-3 and nafion 117 were reduced to 42.5, 46.3, 56.9 and 25.9 mw m–2, respectively. the reduction in the power density might be due to fouling of the membrane. the polarization curves, voltage vs. current density,48 of various synthesized membranes in mfcs are shown fig. 6. the reduction in the over potentials, proton exchange membranes for microbial fuel cells 619 particularly the ohmic overpotential, was caused by the higher ion exchange capacity of the m-3 membrane. the over potential of m-2 and m-1 membranes were less than the over potentials of m-3, which resulted in a better performance of the membrane and a lower potential loss. table i. summary of the performance factors of various synthesized membranes and a nafion-117 membrane in mfcs. mfc membranea pmaxb / mw m-2 imaxc / ma m-2 open circuit voltaged, v cod removal e, % mfc-1 m-1 48.1 99.0 0.89 82 mfc-2 m-2 55.1 104.0 0.94 86 mfc-3 m-3 63.3 107.3 1.07 88 mfc-4 nafion 117 29.7 74.4 0.615 78 adata are expressed as type of membranes; bmaximum power density; cmaximum current density; dmaximum open circuit density; emaximum cod removal fig. 6. polarization curves of the synthesized membranes and the nafion-117 membrane in mfcs setups. the open circuit voltage (ocv) of the mfcs with different membranes was measured at steady state (ss) condition and the results are shown in fig. 7. in a typical plot of ocv vs. time, there are three stages, viz., microbial growth, energy production and microbial death.49 there was a rapid increase in ocv in the first stage, which was due to microbial growth and colonization, a stable period of fig. 7. ocv of various synthesized membrane in mfcs vs. time. 620 dharmadhikari, ghosh and ramachandran energy production in the mfc and the death of the microbial cells that led to a decrease in the ocv.49 in the case of mfc-3 with m-3, the energy production stage was significant when compared to those of mfc-1, mfc-2 and mfc-4 with m-1, m-2 and nafion-117, respectively. mfc-3 generated a more stable ocv among all the mfcs. the ocvs of mfc-1, mfc-2, mfc-3 and mfc-4 were 0.89, 0.94, 1.07 and 0.615 v, respectively. the reason for this order might be the ion exchange capacity of the membranes. cod removal. the cod removal (%) over time is shown in fig. 8. the mfcs connected with m-1, m-2 and m-3 and nafion-117 membrane yielded 82, 86, 88 and 78 % cod removal, respectively. oxygen diffuses relatively rapidly through the m-1 membrane (synthesized with low amount of pva) from the cathode to the anode when compared with m-3 (high pva). this transfer of oxygen might be due to the crossover problem of the membrane. as the oxygen transfer was greater in the membrane synthesized with low amount of pva, the availability of an anaerobic bacterial community in the anodic chamber decreases. hence, the cod reduction was lower with the m-1 membrane. however, in the case of membrane synthesized with a high pva content (m-3), the diffusion of oxygen was lower and hence, the cod reduction was high. the cod removal results were in agreement with the microbial activity in anodic chamber and proved the efficiency of the membrane efficiency, which was in agreement with the iec results. the cod removal (%) of the mfc-3 setup was higher than were those of the other setups. mfc-3 was efficient when compared to mfc-1, mfc-2 and mfc-4. the values of maximum power density, current density, open circuit voltage (ocv) and cod removal (%) of mfc-1, mfc-2 and mfc-3. connected with the m-1, m-2 and m-3 membranes, are summarized in table i. among the three mfc setups, mfc-3 performed better by yielding higher power density, current density, open circuit voltage (ocv) and cod removal. m-3 membrane has higher water uptake and ion exchange capacity when compared with m-2 and m-1 membranes. fig. 8. cod removal of wastewater in anodic compartment of mfcs vs. time. proton exchange membranes for microbial fuel cells 621 conclusions the membranes synthesized in this study significantly improved the power density of the mfcs. the improvement in the mfc power density is a direct consequence of the improved ion exchange capacity of the membranes. these membranes acted as an effective barrier to bacterial migration from the anode to the cathode, which would otherwise have reduced the performance of the mfcs. the mfc-3 setup using the m-3 membrane exhibited the highest value of power density, current density and ocv. the cod removal of mfc-3 with the m-3 membrane yielded a higher value when compared with the other mfcs. the m-3 membrane was more effective and efficient when compared with the other three membranes (m-1, m-2 and nafion-117). the improved performance of m-3 membrane was due to its higher quantity of pva. и з в о д синтеза протон-измењивачких мембрана за двокоморне микробне горивне ћелије sandeep dharmadhikari1, prabir ghosh1 и manivannan ramachandran1 1 department of chemical engineering, national institute of technology, raipur – 492010, chhattisgarh, india протон-измењивачке мембране (pem) су синтетисане полазећи од три различита састава поли(етилен-оксида) (peo), поли(винил-алкохола) (pva), хитозана (cs) и фосфорне киселине (pa) у масеном односу 1: 1:1:1; 1:2:1:1 и 1:3:1:1, користећи поступак физичког мешања и изливања из раствора. апсорпција воде полимерних мембрана је расла са повећањем садржаја pva. већи проценат апсопције воде значио је уједно и већи капацитет измене јона (cec) синтетисаних мембрана. синтетисане мембране су окарактерисане у погледу састава и интеракција између полимених компоненти помоћу дифракције x-зрака и инфрацрвене спектроскопије са фоуријеовим трансформацијама, редом. затим су перформансе полимерних мембрана проверене у микробним горивним ћелијама (mfc). показано је да проценат смањења хемијске потрошње кисеоника (cod / %) зависи од микробне активности у анодној комори. основни циљ ових истраживања је био да се одреди оптимални састав мембране на основу поређења својстава са осталим синтетисаним мембранама различитог састава и једном комерцијалном мембраном. установљено је да је смањење cod у mfc-3 микробној горивној ћелији са м-3 полимерном мембраном износило 88 %. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {editorial prof. dr konstantin i. popov (1941–2019)} udc 929 konstantin i. popov editorial prof. dr konstantin i. popov (1941–2019) this issue is dedicated to late prof. dr konstantin i. popov, former full professor of electrometallurgy at the department of physical chemistry and electrochemistry, at the faculty of technology and metallurgy, university of belgrade, in honor of his huge contribution to university education, and investigation of various aspects of the processes of electrochemical deposition and dissolution of metals. konstantin i. popov was born in sokobanja, serbia, on the 28th of august 1941. his school years were difficult, as he changed many schools moving from town to town. however, he managed to finish secondary school few years before his generation and he became one of the youngest students to enroll faculty of technology and metallurgy. he was only twenty years old, when he obtained his bsc degree in 1961, which is the age when regular students are still freshmen. following year he joined the faculty of technology and metallurgy as one of the youngest teaching assistants ever. he was younger than most of his students, for the next few years at faculty. professor konstantin i. popov received his msc (1966), and phd (1971) degrees in the field of electrochemical science, from the faculty of technology and metallurgy, university of belgrade. he was appointed to assistant professor in 1975, associate professor in 1982 and full professor in 1988 (all appointments at the faculty of technology and metallurgy, department of physical chemistry and electrochemistry). at the same time, professor popov closely collaborated with the institute of chemistry, technology and metallurgy (ictm), department of electrochemistry, university of belgrade, and after his retirement in 2006, he spent two years working as a scientific adviser at ictm, university of belgrade. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 1194 during 1966 he spent some time at institute for electrochemistry of academy of science of soviet union in moscow, on improving, which is curiosity, since professor popov was half russian by his father igor, who died during wwii, and unfortunately his son could not remember him. during his rich academic carrier, professor popov has published over 230 research papers, twelve book chapters (see selected references), and coauthored the two books from the area of electrochemical deposition and dissolution of metals (fundamental aspects of electrometallurgy; kluwer academic/plenum publishers, 2002, and k. i. popov, s. s. djokić, n. d. nikolić, v. d. jović, morphology of electrochemically and chemically deposited metals, springer international publishing, 2016). in addition, professor popov has published several textbooks devoted to the electrochemistry/electrometallurgy fields in the serbian language which are used by the university students and professionals (see selected references). he published many papers in the journal of serbian chemical society, to mutual benefit, spreading his ideas to the scientific community and contributing to the journal to improve its status. he had numerous presentations, including invited lectures, at national and international conferences. professor popov successfully supervised more than 80 students in their bsc, msc and phd research studies. the main scientific interests of professor popov included various fundamental and applied aspects of electrochemical deposition and dissolution of metals, constant and periodically changing regimes of electrolysis, production and characterization of metal powders by electrolysis, the current density distribution effects, bright coatings, and open porous electrodes (the honeycomb-like structures). in his scientific and educational career professor popov sought to understand the essence and find simple, but accurate and reliable solution. professor popov taught on following courses: fundamentals of electrometallurgy, metal and non-metal coatings (graduated studies) and electroplating and electrochemical deposition and dissolution of metals in diffusion-controlled processes (msc studies). the students loved him, among other things, because he could separate the essential from the irrelevant and emphasize (and repeat several times if necessary) what they needed to know even when they have passed his class. his books, textbooks and papers were very specifically written and focused on the topic they are dealing with, without superfluous details and descriptions, with a solid mathematical background. he was brilliant in all fields of interest and his contribution to investigation of phenomena of metals deposition and dissolution was world widely recognized, which could be illustrated with following quotes: “...take another gigantic leap along the timeline of electrochemical discovery and application. consider michael faraday, that london superstar who in 1834 discovered the relation between the amount of electricity consumed and ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 1195 the amount of metal produced in solid form from some invisible particles in solution. in 1995, more than a century later, despic and popov wrote an article that described electroforming of (almost) anything from its ion solution: powders or dendrites, whiskers or pyramids, in laminar shapes of any chosen composition (including that of semiconductors), or indeed in nanometer sizes. this is what has become of faraday’s electrodeposition at cutting edge, as well as in practical applications such as electrodissolution to shape metal parts in the making of roll royce cars.” – the fifth paragraph from the first chapter in the book “modern electrochemistry” by john o'mara bockris and amulya k. n. ready; in the same chapter also mentioned are galvani (in the first paragraph), volta (in the second paragraph), debye, hückle and mamantov. “...until the work of popov et al. electrometallurgy has been regarded as largely empirical, an activity in which there was much art and little science. this will all change with the publication of this book.” – foreword for the book “fundamental aspects of electrometallurgy” (or, as the professor used to call it: “the green book”) by popov, djokić and grgur; foreword was written by john o'mara bockris, one of the greatest scientist of xx century in the field of electrochemistry. professor popov was a member of the serbian chemical society and serbian society of corrosion and materials protection. he was a recipient of several national awards, including medal of serbian chemical society for enduring and outstanding contributions to science. professor popov was also an emeritus scientist of the republic of serbia. he was also awarded by chamber of commerce of belgrade for invention in 2002, and he became a merited member of the serbian chemical society in 2004. belgrade, november 2019 guest editors miomir g. pavlović nebojša d. nikolić acknowledgement. guest editors would like to acknowledge the considerable help of predrag m. živković, faculty of technology and metallurgy, university of belgrade, during the preparation of this editorial. selected publications 1. k. i. popov, s. s. djokić, b. n. grgur, fundamental aspects of electrometallurgy, kluwer academic/plenum publishers, new york, 2002. 2. k.i. popov, s. s. djokić, n. d. nikolić, v. d. jović, morphology of electrochemically and chemically deposited metals, springer international publishing, 2016. 3. k. popov, m. maksimović, o. tatić-janjić, praktikum iz elektrohemije, tehnološko– metalurški fakultet, beograd, 1989 (in serbian). ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 1196 4. konstantin i. popov, branimir n. grgur, osnovi elektrometalurgije, tehnološko– metalurški fakultet, beograd, 2002 (in serbian). 5. спасоје ђорђевић, mиодраг mаксимовић, mиомир павловић, kонстантин попов, галванотехника, техничка књига, београд, 1998 (in serbian). 6. r. despić, k. i. popov, “transport controlled deposition and dissolution of metals”, in modern aspects of electrochemistry, vol. 7., b. e. conway and j. o’m. bockris, eds., plenum press, new york, 1972, ch. 4, pp. 199–313. 7. k.i. popov, m.d. maksimović, “theory of the effect of electrodeposition at periodically changing rate on the morphology of metal deposition”, in modern aspects of electrochemistry, vol. 19, j. o’m. bockris and b. e. conway, eds., plenum press, new york, 1989, ch. 3, pp. 193–250. 8. k. i. popov, m. g. pavlović, “electrodeposition of metal powders with controlled grain size and morphology”, in modern aspects of electrochemistry, vol. 24, r. w. white, j. o’m. bockris and b. e. conway, eds., plenum press, new york, 1993, ch. 6, pp. 299– 391. 9. k. i. popov, n. v. krstajić, m. i. čekerevac, “the mechanism of formation of coarse and disperse electrodeposits”, in modern aspects of electrochemistry, r. e. white, b. e. conway and j. o’m. bockris, eds., plenum press, new york, 1996, ch. 3, pp. 261–311. 10. n. d. nikolić, z. rakočević, k. i. popov, “nanostructural analysis of bright metal surfaces in relation to their reflectivities”, in modern aspects of electrochemistry, vol. 38, b. e. conway, c. g. vayenas, r. e. white and m. e. gamboa-adelco, eds., kluwer academic/plenum publishers, new york, 2005, ch. 5, pp. 425–474. 11. n. d. nikolić, k. i. popov, “hydrogen co-deposition effects on the structure of electrodeposited copper”, in electrodeposition: theory and practice, series: modern aspects of electrochemistry, vol. 48, s. s. djokić, ed., springer, 2010, ch. 1, pp. 1–70. 12. k. i. popov, p. m. živković, n. d. nikolić, “the effect of morphology of activated electrodes on their electrochemical activity”, in electrodeposition: theory and practice, series: modern aspects of electrochemistry, vol. 48, s. s. djokić, ed., springer, 2010, ch. 4, pp. 163–213. 13. k. i. popov, n. d. nikolić, “general theory of disperse metal electrodeposits formation”, in electrochemical production of metal powders, modern aspects of electrochemistry, vol. 54, s. s. djokić, ed., springer, 2012, ch. 1, pp. 1–62. 14. v. d. jović, n. d. nikolić, u. č. lačnjevac, b. m. jović, k. i. popov, “morphology of different electrodeposited pure metal powders”, in electrochemical production of metal powders, modern aspects of electrochemistry, vol. 54, s. s. djokić, ed., springer, 2012, ch. 2, pp. 63–123. 15. n. d. nikolić, k. i. popov, “electrodeposition of copper powders and their properties”, in electrochemical production of metal powders, modern aspects of electrochemistry, vol. 54, s. s. djokić, ed., springer, 2012, pp. 125–185. 16. n. d. nikolić, k. i. popov, “a new approach to the understanding of the mechanism of lead electrodeposition”, in electrodeposition and surface finishing, modern aspects of electrochemistry, vol. 57, s. s. djokić, ed., springer, 2014, pp. 85–132. 17. m. g. pavlović, k. i. popov, “metal powder production by electrolysis”, electrochemistry encyclopedia, z. nagy, ed., e. b. yeager, center for electrochemical sciences (yces) and the chemical engineering department, case western reserve university, cleveland, oh, http://knowledge.electrochem.org/encycl/art-p04-metalpowder.htm (december, 2005) ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 1197 18. k. i. popov, m. g. pavlović and p. m. živković, “current density distribution in electrochemical cells”, electrochemistry encyclopedia, zoltan nagy, ed., ernest b. yeager, center for electrochemical sciences (yces) and the chemical engineering department, case western reserve university, cleveland, oh, usa, http://knowledge.electrochem.org/encycl/art-d03-curr-distr.htm (february, 2011). ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {identification of phenolic and alcoholic compounds in wine spirits and their classification by use of multivariate analysis} j. serb. chem. soc. 84 (7) 663–677 (2019) udc 663.251+663.551.5:543.39+547.828+ jscs–5217 547.42 original scientific paper 663 identification of phenolic and alcoholic compounds in wine spirits and their classification by use of multivariate analysis karla hanousek čiča1, martina pezer1,2, jasna mrvčić1*, damir stanzer1, jasna čačić3, vesna jurak2, mirela krajnović3 and jasenka gajdoš kljusurić1 1university of zagreb, faculty of food technology and biotechnology, pierottijeva 6, 10000 zagreb, croatia, 2badel 1862 d.d., wines and alcoholic beverages producer, ulica grada gospića 7, 10000 zagreb, croatia and 3croatian association of drink producers, kučerina 64, 10000 zagreb, croatia (received 15 january, revised 3 march, accepted 4 march 2019) abstract: during the ageing period wine spirits are changing their color, chemical composition and sensory characteristics. these changes should be simply monitored. the aim of this study was to develop partial least squares regression (pls) models for higher alcohols and phenols in wine spirits as well as to show the feasibility of the nir spectroscopy combined with chemometric tools to distinguish wine spirits and brandies with different ageing degree. to get the reference values, the usual methods for the analysis of spirits drinks were used. ethanol, esters, acids, methanol and higher alcohols were studied. wine spirits and brandies phenol composition was determined by liquid chromatography. principal component analysis (pca) was used to classify the wine spirits and brandies according to their phenolic and higher alcohols composition. moreover, the partial least squares regression (pls regression) was used to calibrate and predict expected contents of higher alcohols and phenols in the wine spirits. success of the classification of samples by ageing based on individual alcohols was 93.8 %, while success of the classification based on individual phenols raised to 100 %. this efficiency of the prediction was evaluated by use of linear discriminator analysis (lda). keywords: distillate; ageing; nir spectroscopy; principal component analysis. introduction wine spirits (a spirit drink produced exclusively by the distillation of a wine at less than 86 vol.%) are commonly used for the brandy production. brandy is a spirit drink produced from wine spirit, whether or not wine distillate has been added, distilled at less than 94.8 vol.% and matured for at least one year in oak receptacles, or for at least six months in oak casks with a capacity of less than * corresponding author. e-mail: jmrvcic@pbf.hr https://doi.org/10.2298/jsc190115020h 664 hanousek čiča et al. 1000 l, to obtain characteristic taste, flavor and attractive color. no addition of ethyl alcohol of agricultural origin or distillates of agricultural origin is permitted. brandy may only contain added caramel as a means to adapt color and should not be flavored. the first stage in the brandy production is the selection of wine spirits for further processing, mostly based on classical sensory analysis. after selection, wine spirits used in brandy production are subjected to ageing process in wooden barrels for certain time, depending on traditional practices and legislative.1 during the ageing period, chemical composition of wine spirits is altered, due to extraction of wood compounds (mostly phenols and sugars) and oxidation process, contributing to spirit aroma, taste and colour.2,3 since these reactions are slow, ageing of high-quality brandies takes several years, resulting in the low production efficiency and high cost. for economic reasons, producers sometimes use caramel and vanillin to imitate color and aroma equal to the aged brandies or apply methods for accelerating distillate ageing (ageing in a wooden barrel exposed to warmth and light or ageing in a wooden barrel with wooden chips). although legislative allows this way of production, these products are fraud to consumers and are example of unfair market competition. another complex group of compounds that are primarily produced during fermentation and distillation, but also during ageing of spirits, are volatiles (higher alcohols, esters, aldehydes). the volatile compounds are responsible for the quality and safety of spirits, depending on their composition, concentration and sensorial properties, essential for customer's acceptance.4 phenolic compounds and volatiles that give specific aroma to spirit drinks can be used to classify these drinks by type, origin, country and even provenience region.4 the widely used method for the phenolic and volatile compounds analysis is chromatography.5 prior to chromatographic analysis which are time-consuming, relatively expensive and require skilled personnel, various extraction methods have been widely used, such as liquid-liquid extraction. from aforementioned, there is a need to establish a rapid method to validate the authenticity and quality of these alcoholic beverages. such an analytical method should accomplish a fast data acquisition, carry out data treatment accurately with relatively low costs and measure sample as intact, without any additional preparation. spectroscopic techniques combined with chemometric data analysis are non-destructive methods that provide relative rapid and low cost alternative to traditional chemical composition and sensory analysis.5,6 welke et al. used hs-gc×gc/tofms associated with multivariate analysis (fisher ratio, pca and lda) to investigate the volatile composition of wines.7 this proved to be an interesting approach to differentiate wines according to their original grape cultivars and also to find potential markers of these grape cultivars. nir technique has been used for determination of food and beverage quality.8 nir and chemometrics were proposed to classify 69 samples of distilled spirits rapid distinguishing of brandy with different ageing degree 665 with respect to type (whiskey, brandy, rum and vodka) and presence/absence of adulterants.9 uv–vis and near infrared (nir) spectroscopy demonstrated the possibility of grouping single-malt whiskies according to their geographic area of production.10 a distinguishing of commercial samples of slovak, belgian, german, czech and british juniper-flavored spirit drinks based on spectroscopic methods combined with the principal component analysis (pca), followed by the pca-linear discriminant analysis (pca-lda) were presented by sádecká et al.11 the same authors geographically classified czech, hungarian and slovak plum spirit drinks by synchronous fluorescence spectroscopy.12 jakubikova et al. classified 67 fruit spirits (apple, apricot, pear and plum spirits) by type using nir combined with multivariate analysis.13 although there are numerous reports on the use of nir spectroscopy in food and beverage analysis,9,10,12–15 the potential of nir spectroscopy in the wine spirit/brandy ageing degree estimation have not been studied. thus, the aim of this study was to develop partial least squares regression (pls) models for higher alcohols and phenols in wine spirits as well as to show the feasibility of the nir spectroscopy combined with chemometric tools to distinguish wine spirits and brandies with different ageing degree. the multivariate analysis of obtained spectroscopic and chromatographic data was performed. principal component analysis (pca) was used to classify the wine spirits and brandies as spirit drinks produced from the wine spirits based on their phenolic and higher alcohols composition. moreover, the partial least squares regression (pls regression) was used to calibrate and predict expected contents of higher alcohols in the wine spirits and their ageing. this strategy can be used as a screening analysis for rapid classification of spirit drinks and could become an effective authentication tool when coupled to chemometrics. experimental samples in these study, 13 brands of wine spirits with different ageing period (ws1-ws13) acquired from the small scale manufacturer and 3 commercial brandies (brandy 1–3, colored) produced in croatia were analyzed. manufacturer defined samples as: ws 1, 3, 11, 12, 13 not -aged, colorless wine spirit; ws 4 and 7 colored samples briefly aged (2–3 years) and ws 2, 5, 6, 8, 9, 10 aged, colored samples. wine spirits aged in lightly charred oak barrels whose volumes were 225, 300 and 330 l. all samples were characterized by ethanol content, total acidity, total esters and higher alcohols composition. according to color, only the colored samples were submitted to phenolic composition determination by hplc, while nir spectrum was obtained for all samples. samples were stored in dark at room temperature until analysis. determination of wine spirits physicochemical parameters determination of alcoholic content of samples is based on density determination by pycnometer method.16 total acidity and total esters were determined in accordance with regulation.17 666 hanousek čiča et al. determination of wine spirits volatile and polyphenol compounds major volatile compounds were analyzed by gas chromatography (gc)18 while qualitative and quantitative analyses of polyphenol composition of wine spirits and brandies were performed by hplc.19 a detailed methods description is given as supplementary material to this paper. near infrared spectroscopy (nir spectroscopy) the nir spectrophotometer, control development, inc., nir-128-1.7-usb/6.25/50 µm, with installed control development software spec32 with halogen light source (hl-2000) was used. the ranges of this spectrophotometer are 11062–5885 cm-1 or 904–1699 nm with a spectral resolution of 6.25 cm-1. ten different spectral measurements were conducted for each wine spirit and brandy. as spectral data pre-processing method the savitzky–golay smoothing was used.20 multivariate analysis all statistical analyses were carried out using software (statistica, v. 8.1, statsoft inc., usa). analysis of variance (anova) with use of tukey’s test (p < 0.05) was applied to determine the significance of differences among the wine spirits and brandies. all conducted results, as nir spectroscopy (796 data per scan) and contents of observed alcohols (10 parameters) as well as the polyphenolic content (9 parameters) of wine spirits and brandies, were submitted to principal component analysis (pca) in order to interpret measured content of the observed different wine spirits and brandies and changes in the observed alcohols as well as the polyphenolic content vs. associated nir spectra. multivariate tools as pca are aimed to derive a small number of independent linear combinations (principal components, pcs) for the observed set of variables, retaining as much as possible information. scatter plot of the 1st principal component versus the 2nd principal component was used to present the results of this study. the pc loadings explain which parameters were responsible for such separation of the observed samples.21 partial least regression models (pls) were developed based on the nir spectroscopy of wine spirits and brandies. the data matrix used in the modeling consisted of 160 rows (10 spectra for each ws and brandy) and 796 columns (nir spectra wavelengths, pace 1 nm). for each sample, a set of 6 spectra was chosen to serve as a training dataset (96 spectra in total) and the rest of 4 spectra were used for the testing dataset (64 spectra in total). all pls models were evaluated on the values of coefficient of determination (r2), root mean square errors of cross calibration and validation (rmsec and rmsev, respectively), ratio of performance to deviation (rpd) and ratio of error range (rer). in the pls models were used nir spectra to calibrate and validate the expected contents of higher alcohols and phenolic compounds in wine spirits. desirable parameters used for estimation of pls model efficiency are lower root mean square errors with higher r2, rpd and rer.22 a good model will range from 0.83 to 0.9 for the r2; 5–6.4 for the rpd value and the preferable rer is over 10.23 applied linear discriminant analysis (lda) was used to evaluate the efficiency of sample separation based on type (wine spirit or brandy). lda is a supervised pattern recognition technique with the task of inferring a function from labelled training data. the training data were randomized wine spirits and brandies, and their phenolic components and alcohols. rapid distinguishing of brandy with different ageing degree 667 results and discussion wine spirits chemical analysis wine spirits chemical composition depends on the compounds present in wine being sufficiently volatile to distill. the most abundant volatile components in distillate are derived from yeast metabolism (esters, higher alcohols, aldehydes and acids) and grapes volatile which give distillates fruity and floral notes. there are two main categories of flavor‐active esters in fermented beverages: acetate esters as well as medium‐chain fatty acid ethyl esters. fusel alcohols produced during fermentation and contribute to essential aroma and flavors include propan1-ol, 3-methylbutan-1-ol (isoamyl alcohol), 2-methylpropan-1-ol (isobutanol), 2methylbutan-1-ol (active amyl alcohol), 2-phenylethan-1-ol.24 terpenes like geraniol, nerol, linalool, citronellol, nerolidol, β-damascenone and vitispiranes are key odorant compounds in distilled wine spirit derived from grapes.25 during the ageing process, the interaction between distillate components, oxygen and the substances derived from wood occurs, causing many changes of the chemical and sensory characteristic of the distillate. physicochemical parameters and volatile compounds identified by gas chromatography in the tested fresh and aged distillate and brandies are listed in table i. table i. anova results and mean values of content of ethanol, esters and acids observed in all wine spirits and brandies; different letters in the same column indicate significant differences according to the tukey’s test (p < 0.05); a.a sample content of ethanol vol% content, g/(100 l absolute alcohol) total esters total acids methanol higher alcohols 3-methylbutan-1-ol 2-methylpropan-1-ol propan-1-ol butan -2-ol butan -1-ol ws1 76.6a 40.4a 11.8a 104.6a 112.5a 81.6a 20.3a 10.2a 0.2a 0.2a ws3 81.8a 7.0b 7.7a 26.5b 176.8c 122.6b 29.3a 22.8c 1.1c 1.0c ws11 77.1a 15.3b 37.2b 53.9b 212b 166.4b 21.9a 21.6c 1.7c 0.4a ws12 75.2a 17b 83.0c 49.5b 284.2b 211.4c 54.8b 14.9a 1.9c 1.2c ws13 77.0a 26.7a 19.4a 56b 212.1b 156b 32.4a 21.7c 1.3c 0.7c ws4 67.0b 10.7b 9.0a 0.2c 245.1b 187.2b 34.1a 22.5c 0.0a 1.3c ws7 64.7b 63.1c 52.7b 112.2a 243b 169.7b 47.1b 25.3b 0.0a 0.9c ws2 71.9a 28.5a 36.7b 140.8a 268.7b 180.5b 50.7b 28.5b 4.3b 4.7b ws5 73.6a 144.2c 134.5c 42.6b 375.9d 242.3c 59.9b 36.1b 32.5d 5.1b ws6 67.2b 77.6d 26.2b 46.7b 378.8d 286.3c 67.4b 19.4c 3.9b 1.8c ws8 70.0a 117.6c 81.4c 79.4c 370.2d 243.5c 60.4b 37.0b 26.4d 2.9b ws9 68.5a 4.5b 54.0b 13.8b 385.5d 279.8c 66.9b 24.1b 10.8b 3.9b ws10 39.9c 69.8d 38.0b 106.4a 259b 181.9b 50.3b 25.9b 0.0a 0.9c brandy 1 41.3c 33.2a 5.8a 187e 165.9c 108.4b 28.0a 29.1b 0.0a 0.4a brandy 2 39.2c 38.6a 163.3c 79.7c 259.7b 171.8b 46.6b 32.4b 7.6b 1.3c brandy 3 41.1c 87.7d 217.7d 70.4c 316.6d 224.7c 52.9b 22.8c 14.8b 1.4c the ethanol concentration of the tested samples varied from about 65 to 80 vol% in accordance with the industry practice to be placed on ageing distillate with a high ethanol degree. higher ethanol degree provides better lignin degrad668 hanousek čiča et al. ation and better extraction of compounds from the wood. the ethanol concentration differences between the samples originate from different ethanol concentration achieved by distillation, but oscillations in the ethanol content of the distillate may occur also due to evaporation of alcohol and water, depending on the cellar temperature and humidity, as well as ethanol oxidation to ethanal and acetic acid.26 in the samples analyzed, total esters (ethyl acetate, ethyl lactate, ethyl hexanoate, ethyl octanoate and ethyl decanoate) varied from 4.5–144 g/(100 l absolute alcohol (a.a.)). using univariate statistics, such as anova approaches, differences (p > 0.05) in the total esters were not observed between not-aged and aged samples. however, some differences can be noticed. in aged samples, esters show a mean content of ≈ 74.5 g/(100 l a.a.), higher than the mean values obtained for the not-aged samples (≈ 21 g/(100 l a.a.)). during the ageing process the content of esters changes; some of esters are formed and others change their amount.27 total ester content increases mostly due to ethanol oxidation and acetic acid formation. ethyl acetate is the most abundant acetate in the distillates derived from the yeast metabolism, as well as esterification process during the ageing. additionally, distillates usually contain other odorous esters, especially fatty acid ethyl esters (ethyl butanoate, ethyl hexanoate; ethyl octanoate) as well as acetates of higher alcohols, mostly 3-methylbutyl acetate (isoamyl acetate). except for the ageing process, the presence and concentration of esters depend on other variables like grape variety, soil type, climate conditions, yeast strain and must fermentation conditions, as well as type of distillation and presence of yeast cells in the wine at the time of distillation.27 literature data indicate that the amount of acid during ageing increases26 due to ethyl alcohol oxidation stimulating the wood hydrolysis of components and liberation of phenolic acids. regard to tested samples there was no difference (p > 0.05) in total acid content with respect to the ageing process (table i). difference can be explained by different initial amount of acids in wine and in distillate before ageing which is characteristic of the quality of raw materials as well as grapes varietal characteristics. similar results were obtained for the methanol. methanol concentration depends on quality of raw materials, type of distillation and proper fractions separation. in distillate, methanol is found in concentrations ranging from 30–70 g/(100 l a.a.) while the total acid concentration ranges from 20–100 g/(100 l a.a.).28 higher molecular weight alcohols present in distillates in optimal concentration contribute to the sensory quality of the distillate. they are products of yeast amino acids metabolism and because of that yeast species, fermentation conditions and distillation process are affecting their content in the not-aged distillate. according to the results given in the table i, there was statistically significant difference in higher alcohol concentration in the not-aged (a,b,c) and rapid distinguishing of brandy with different ageing degree 669 aged samples (d). concentrations of all measured alcohols, especially isoamyl alcohol, were higher in aged samples. concentration of higher alcohol in aged distillates was approximately 375 g/(100 l a.a.). according to tsakiris et al.28 concentrations of higher alcohols in brandy is usually in the range of 250–500 g/(100 l a.a.). the relationship between phenolic compounds and the ageing period is well -known.29,30 during the ageing process distillate is enriching with extractable phenolic compounds that are released from the wooden cask. duration of ageing period, as well as type of wood, are among the most important factors that determine phenolic composition and sensory characteristics of the resulting aged distillate.31 the phenolic compounds identified and quantified by hplc in the analyzed wine spirits and brandies are listed in table ii. since phenolics originate exclusively from wood, and are not present in fresh distillate, they could be used as wood ageing markers. based on phenolic compounds, there is a significant difference (p > 0.05) between wine spirits aged in wooden cask for a longer period (ws 2, 5, 6, 8, 9) and wine spirits aged for a shorter period (ws 4, 7) or not-aged spirits/adulterated (ws 10). these differences result from the physical and chemical changes (extraction and oxidation processes) during wine distillate ageing, but there is also a strong influence of the chemical composition of the wood itself and its heat treatment/toasting level.32 the phenolic aldehydes are produced by thermodegradation of the terminal monomer units of lignin: the cinammic aldehydes convert to benzoic aldehydes, and then they are oxidized to corresponding phenolic acids.33 table ii. anova results and mean values (mg/(100 ml) of phenolic compounds hplc analysis) observed in different wine spirits and brandies; different letters in the same column indicate significant differences according to the tukey’s test (p < 0.05); nd – not detected; ga – gallic acid; dga – gallic acid derivate; va – vanillic acid; syr – syringic acid; v – vanillin; syal – syringaldehyde; coal – coniferaldehyde; ea – ellagic acid ; dea – ellagic acid derivate sample content of phenolic compounds, mg/(100 ml) ga dga va syr v syal coal ea dea ws4 nd nd nd nd 0.02a nd nd 0.20b nd ws7 0.02b nd 0.01a 0.05a 0.03a 0.15a 0.51b 1.08d nd ws2 0.13a nd 0.04a 0.05a 0.02a 0.08a nd 0.95a nd ws5 0.79c nd 0.36b 0.45b 0.44b 0.78c 0.54b 6.61c 0.97b ws6 0.58c nd 0.28b 0.30b 0.27c 0.63c 0.46b 5.07c 0.86b ws8 0.62c 0.06a 0.48b 0.58c 0.53b 1.33d 0.19c 5.03c nd ws9 0.30a 0.18b nd 0.08a nd 0.21a 0.26c 2.05d nd ws10 nd 0.15b nd nd nd nd nd nd nd brandy 1 nd nd 0.17b 0.06a 0.35b 0.02b 0.06a 0.13b nd brandy 2 nd nd nd nd 0.09a nd nd nd nd brandy 3 0.38c nd 0.48b 0.20b 1.10d 0.43c 0.62b 1.99d nd 670 hanousek čiča et al. wine spirits aged in wooden cask for a longer period are richer in phenolic acids than wine spirits aged for a shorter period (table ii). in the former, all the phenolic acids are present in the higher concentrations: ellagic acid (ea) is the most abundant compound, followed by gallic acid (ga), syringic acid (syr) and finally, vanillic acid (va). although ellagic acid was found in the almost all analyzed samples, higher concentrations were observed in wine spirits 5, 6 and 8 followed by wine spirits 9 and 7 and brandy 3. the content of gallic acid in analyzed wine spirits was significantly higher in wine spirits 5, 6 and 8 and brandy 3. in contrast, in wine spirits 4 and 10 and brandies 1 and 2 gallic acid was not found. vanillic and syringic acid were present in significant concentrations also in wine spirits 5, 6, 8 and brandy 3. vanillin is the phenolic aldehyde which greatly influences the aroma of spirits because of its low threshold value (320 µg l-1) and adds positive vanilla notes.34 consequently, vanillin could be added afterwards by the producers in order to adapt brandies aroma. this was the case with brandy 2 where only vanillin was detected. because of the absence of other phenolics it could be concluded that this sample is not-aged. moreover, ratio of syringaldehyde to vanillin above 1 indicates spirits produced by ageing in oak barrels over a long period as in sample ws5, ws6 and ws8 (ratio ≈ 1.7–2.5, fig. 1). brandy 1–3 do not meet this criterion, which indicates the manufacturer’s habit to add extra vanillin quantities to their products. also, gallic acid/vanillin ratio indicates quality of spirits and it is the largest for same samples ws5, ws6 and ws8. ellagitannin degradation during the heat treatment of the wood and ellagitannin hydrolysis during the ageing process are the major sources of ellagic acid in brandy.32 the ellagic acid content is particularly important for the evaluation of brandy authenticity, as wood ageing marker, the differentiation of brandies according to the botanical species and the geographical origin of wood29 as well as for the taste, flavor and color of the final products. gallic acid concentration in aged spirits depends on hydrolysis of wood digallic acid and the toast level of casks, since gallic acid is degraded at high temperatures. consequently, this compound is more abundant in spirits aged in casks with light or medium toast level.29,32 furthermore, concentration of vanillic acid also decreased with higher temperatures during the toasting process. vanillic acid can be directly extracted from oak wood or be formed by oxidation of vanillin during the ageing period, whereas syringic acid is formed during toasting by the oxidetion of the corresponding aldehyde.30 syringaldehyde is predominant, probably due to higher accumulation in the wood as a consequence of higher thermal stability than the vanillin and coniferaldehyde. according to panossian et al.35 adulterated, not -aged spirits/brandies are easy to recognize by the absence of sinapaldehyde, syringaldehyde and coniferaldehyde. rapid distinguishing of brandy with different ageing degree 671 fig. 1. principal component analysis of clustering of brandies from the wine spirits (ws) based on the phenolic composition (a) and on higher molecular weight alcohols (b). nir spectra although all tested samples had similar nir spectra differences in the intensity of some peaks were observed (supplementary material, fig. s-1). significant differences were observed in two different nir spectra regions, first: 904 to 935 nm and the second identified specific region from 1400 to 1699 nm. the vibrations in in the first specific range of the nir spectra are related to the third ch and roh overtone, second overtone, and a combination of stretch and determination of the oh stretch of h2o,15 which are the bands and groups expected in phenolic and aromatic compounds. spectral differences based on vibration differences of observed samples; in the range from 1400 to 1699 nm present the vib672 hanousek čiča et al. ration of the c–h and o–h bonds corresponding to the water and phenolic and aromatic absorbance.5 those spectra performance is used in the pls modelling of alcohols and phenols in the wine spirits as well as to detect and predict the ageing of spirits and brandies. chemometric data analysis in the current work, the performance of three different multivariate statistical techniques was assessed: pca, pls and lda. principal component analysis (pca) allowed identification of significant parameters in the total data matrix which consisted of 42 rows (samples were measured in triplicate, and the average presented the fourth value) and 815 columns, number of columns was reduced identifying 336 significant nir data (absorbance units at wavelengths from 904–935 and 1400–1699 nm), 5 physicochemical parameters and 9 phenolic compounds per each row). the clustering of the samples was investigated by visualization of discriminating the analyzed wine spirits and brandies based on the phenolic composition (fig. 1a) and higher alcohols (fig. 1b). each component of a pca model is characterized by two complementary sets of attributes: loadings and scores. loadings describe the data structure in terms of variable correlations. the scores describe the properties of the samples (differences or similarities). when the phenolic compounds are observed, explained are 78.3 % of all variances in the observed data matrix and the brandies separated from the wine spirits in the second quadrant. when the content of total and partial alcohols is observed, the plot of scores on pc1 against pc2 with the cumulative contribution over 78 % was the first step to visualize the main trends in the sample set. the aged spirits are dominant on the right side of the chart while the briefly and non-mellowed spirits are positioned in the second and third quadrant. the grouping is mostly caused by the alcohol content (ethanol, butan-2-ol and butan-1-ol) with the pc2 = 24.2 %, and 2-methylpropan-1-ol and propan-1-ol with the dominant contribution in the pc1 (54.6 %). in order to study applicability of nir spectroscopy in calibration and validation of alcohol and phenol content as well as aging process, partial least square models method was used (supplementary material, table s-i). the pls modelling was performed on the entire nir range (904–1699 nm) to observe the absorption bands related to alcohol(s) and phenolic compounds (c–h stretch; c–h3 stretch or compounds containing c–h aromatic groups; the o–h overtone of water and related r–oh). row nir spectra vs. content of individual phenols and alcohols in wine spirits, as well as the information of ageing of wine spirits and brandies were included in the modeling. the spectral data undergo spectral pre-processing, the smoothing by savitzky–golay. the final rapid distinguishing of brandy with different ageing degree 673 input data matrix for pls models consists of 64 rows and 811 columns. based on developed psl models 2/3 of data were used in calibration and 1/3 for validation. to evaluate the efficacy of the models, the root mean square errors of calibration (rmsec) and cross validation (rmsecv) as well as the relative errors of prediction (rep) were calculated.36 the r2 values in the calibration models ranged from 0.82–0.98. fearn38 explained that mentioned parameters (rmsecv, rpd and rer) are useful indices of the model efficiency and they are most commonly used parameters in assessing the applicability of the model in process control and prediction of parameters.15 fragoso et al.39 reported ft-mir spectroscopy and chemometrics as a rapid method to quantify phenolic compounds all during the red winemaking process. the calibration models yield good calibration statistics for the different parameters evaluated (r2 > 0.95 and rpd > 4.0 for tpc; r2 > 0.90 and rpd > 3.0 for ta; r2 < 0.8 and rpd < 3.0 for ct). it was concluded by the authors that ft -mir spectroscopy together with multivariate calibration could be a rapid and valuable tool for wineries to carry out the monitoring of phenolic compound extraction during winemaking. r2, for individual alcohols calibration and validation (prediction) are higher than for individual phenols as well as rpd and rer, respectively. the highest values are achieved for aging (r2 = 0.99; rpd ≈ 6.8 and rer ≈ 15) with preferable values showing the potential to use nir spectra in quantitative prediction of this parameter (ageing). with successful prediction of ageing process it should be possible to detect potential fraud. the price of spirits proportionally increases with ageing. de villiers and co-workers40 were successful in classification of wines according to the grape variety using discriminant analysis (da). evaluation of the classification performance regarding the examined samples (wine spirits and brandies) was provided by use of lda (fig. 2), containing 16 variables (ethanol; esters; acids; 2-methylpropan-1-ol; propan-1-ol; butan-2-ol; butan-1-ol; gallic acid; gallic acid derivate; ellagic acid; ellagic acid derivate; vanillic acid; syringic acid; vanillin; syringaldehyde; coniferaldehyde). first two roots (f1+f2) for both classifications (based on individual alcohols and phenols) were found to explain 100 % of the properties of different wine spirits and brandies (fig. 2). the classification based on individual phenols in spirits and brandies are totally successful, while the classification based on individual alcohols was successful for 93.8 % with the less effectiveness for samples that are briefly aged (66 %). phenols are reliable parameters that should be used in identification of aged samples. thus, applied lda showed to be a suitable and simple supervised statistical approach to assess the ageing process of spirits and brandies based just on nir spectra. multivariate tools or chemometrics proved to be a useful tool in classifications, even of complex samples as wine spirits and spirits that differ in their phenolic composition and alcohol composition. 674 hanousek čiča et al. fig. 2. linear discriminator analysis classification of wine spirits (ws) and brandies based on aging using individual alcohols (a) and phenols (b). conclusions nir spectroscopy was used in present study to analyse raw wine spirit and wine spirit and brandy with different ageing times. the principal component analysis based on the phenolic composition and higher molecular weight alcohols has been proven as a good method for classification of brandies of different ages. chemometrics showed to be a powerful mathematical technique when it is related to spectroscopic techniques for the analysis of phenolic compounds and alcohols in wine spirits and brandy. such modelling allows valuable information from large data sets to be obtained, underpinning the application of methods based on nir spectroscopy that is a fast and easy-to-operate techniques. the rer values (ranged from 4.3 to 20.8) suggest that nir spectroscopy could be an operative method for the measurement of important parameters as alcohols in wine spirits/brandies and for quantitative prediction of those parameters. supplementary material additional data are available electronically from http:// www.shd.org.rs/jscs/, or from the corresponding author upon request. acknowledgment. this study was funded by the ministry of science, education and sports of the republic of croatia. rapid distinguishing of brandy with different ageing degree 675 и з в о д идентификација фенолних и алкохолних једињења у винским пићима и њихова класификација применом мултиваријантне анализе karla hanousek čiča1, martina pezer1,2, jasna mrvčić1, damir stanzer1, jasna čačić3, vesna jurak2, mirela krajnović2 и jasenka gajdoš kljusurić1 1 university of zagreb, faculty of food technology and biotechnology, zagreb, croatia, 2 badel 1862 d.d., wines and alcoholic beverages producer, 10000 zagreb, croatia и 3 croatian association of drink producers, zagreb, croatia винска пића мењају своју боју, хемијски састав и укус током сазревања, а промене би требало лако пратити. циљ ове студије је био да развије парцијални регресиони модел најмањих квадрата (pls) за више алкохоле и феноле у винским пићима и ракијама, као и да докаже применљивост nir спектроскопије комбиноване са хемометријским приступом за препознавање пића различите старости. референтне вредности су добијене коришћењем уобичајених метода за анализу алкохолних пића. одређивани су етанол, естри, киселине, метанол и виши алкохоли. садржај фенолних једињења је одређен течном хроматографијом. класификација пића је урађена на основу садржаја фенолних једињења и виших алкохола користећи методу анализе главних компонената (pca). pls регресиони модел је послужио за калибрацију и предвиђање очекиваног садржаја ових једињења у винским пићима. успешност класификације узорака по основу старења је била 93,8 %, мерена према садржају појединачних алкохола, а 100 % мерена према садржају фенола. ефикасност предвиђања је процењена користећи линеарну дискриминантну анализу (lda). 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(https://doi.org/10.1007/s00217-005-1169-5). {a simple computational approach for pka calculation of organosulfur compounds} j. serb. chem. soc. 86 (2) 165–170 (2021) short communication jscs–5412 165 short communication a simple computational approach for pka calculation of organosulfur compounds syed tahir ali*, aneesa choudhary1, syed majid khalil2 and arif zubair3 1department of chemistry, federal urdu university of arts science and technology, karachi, pakistan, 2bosch pharmaceutical private limited, plot no. 209, sector 23, korangi industrial area, karachi, pakistan and 3department of environmental science, federal urdu university of arts science and technology, karachi, pakistan (received 18 may, accepted 7 july 2020) abstract: the present work is related to predicting the pka values of organosulfur compounds through the density functional theory (dft). in this study, 22 organosulfur compounds were considered to calculate the theoretical pka values. the main emphasis was given on the substitution of different groups on the sulfur atom. the computations were performed in the presence of dimethyl sulfoxide (dmso) as solvent. experimentally, the order of increase of acidity is; sulfides < sulfoxides < sulfones. the herein computed pka values also follow the same order. the theoretical pka values were computed using the dft method b3lyp, with the basis sets 6-31g(d), 6-31+g(d,p) and the iefpcm bulk solvation model. the majority of the pka values computed through the diffuse function basis set were in excellent agreement with the experimental ones. hence this computational approach, b3lyp/6-31+g(d,p)/iefpcm, could be utilized to predict the pka values of these types of organosulfur compounds. keywords: dft method; diffuse function basis set; dmso solvent. introduction the acid dissociation constant (pka) is an important property of many organic compounds and it is strongly related to their applications. fast and accurate methods for determining aqueous pka values of organic compounds would have a wide range of applications. aside from experimental measurements, theoretical determination of the acidity of a compound has been an important and challenging objective of computational chemistry.1,2 the computations (theoretical calculations) are a reconfirmation of experimental results. a * corresponding author. e-mail: stahir.ali@fuuast.edu.pk https://doi.org/10.2298/jsc200518042a ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 166 ali et al. computation provides an idea of the structural information of a molecule in vacuum (gas phase), which is difficult to obtain through experiments. theoretical calculations are also helpful in providing information for the determination of the preferred protonation site when more than one site is available.3 organosulfur compounds have many important applications, which have already been reported in literature.4–7 in this communication, we are presenting a very easy computational approach for theoretical calculation of pka values. this theoretical model is employed for three types of organosulfur compounds, i.e., sulfides, sulfoxides and sulfones. different kinds of substituents were selected that were attached on both sides of the sulfur atom. the structures of compounds considered for pka calculation are shown in fig. 1. the significance of this computational model is that it could be uniformly applied for sulfides, sulfoxides and sulfones. fig. 1. compounds considered for the theoretical pka calculations. this computational protocol was developed in previous studies related to pka calculations.8,9 in these studies, it was shown that the theoretically calculated pka values could be utilized to resolve discrepancies in experimental pka values. these computational studies were performed with different solvation models but only water was used as the solvent. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ theoretical calculation of dissociation constant 167 experimental the experimental pka values of different derivatives of sulfide, sulfoxide and sulfone were obtained from literature.10-13 these experimental pka values are determined in dmso as solvent. the series starts with the simple forms, i.e., dimethyl sulfide, dimethyl sulfoxide and dimethyl sulfone. other derivatives were set by variation of different groups (see fig. 1). first, the geometries of all the considered compounds were drawn with the help of gaussview 6.14 then the molecular modeling software gaussian 1615 was employed for all quantum calculations. for the computation of the pka values, the gibbs energy changes in the gas phase (∆ggas) were calculated through the dft method, b3lyp, with the basis sets 6-31g(d) and 6-31+g(d,p). solvation free energy changes (∆gsolv) in dmso have been obtained by single point computations on gas phase geometries, using the bulk solvation model – integral equation formalism polarizable continuum model (iefpcm). the calculations of pka values is performed by using a well known thermodynamic cycle (scheme 1; eqs. (1) and (2)).16 the gibbs energy of the gas phase proton17 was taken from the sackur–tetrode equation as ggas(h+) = –6.28 kcal* mol-1; for the gibbs energy change of hydration of the proton, the experimental value,18 δgsolv(h+) = –270.0 kcal mol-1, was used. the usual correction term of 1.9 kcal mol-1 was applied for standard state conversion between 1 atm in the gas phase and 1 mol l-1 in solution.19 scheme 1. the thermodynamic cycle utilized for eqs. (1) and (2). pka = δg/(2.303rt) (1) δg = δggas + δgsolv(a-) + δgsolv(h+) – δgsolv(ha) (2) results and discussion the data set of experimental pka values shows that changing the methyl group with a phenyl or a benzyl group increases the acidity of the considered organosulfur compounds. electron withdrawing substituents also have the same effect (see table i). addition of oxygen atoms on sulfur also increases the acidity from sulfide to sulfoxides and sulfones (see fig. 2). initially all structures were fully optimized with frequency calculations, by the b3lyp method using the basis set, 6-31g(d). solvation energies were obtained by single point computations with the same basis set. the calculated pka values obtained through this computational approach and the experimental pka values are summarized in table i. * 1 kcal = 4184 j ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 168 ali et al. table i. comparison of experimental and calculated pka values sulfide sulfoxide sulfone comp. exp. calcd.a calcd.b comp. exp. calcd.a calcd.b comp. exp. calcd.a calcd.b 1a 45.0 55.7 45.7 2a 35.1 46.9 35.1 3a 31.1 38.2 31.3 1b 42.4 51.3 43.2 2b 29.0 35.3 28.6 3b 25.4 30.2 24.4 1c 30.8 37.6 32.0 2c 33.0 53.5 34.1 3c 29.0 36.5 28.6 1d 26.7 32.0 27.7 2d 27.2 33.8 27.6 3d 23.4 29.8 24.1 1e 20.8 24.5 18.6 2e 24.5 30.3 25.3 3e 22.3 32.1 23.4 1f 18.7 24.9 21.7 2f 15.1 21.9 12.8 3f 12.0 15.2 9.4 1g 16.9 25.2 15.4 3g 11.4 24.5 17.0 1h 11.8 15.7 8.7 3h 7.1 10.1 3.0 mad 6.8 1.7 9.6 0.83 6.8 1.9 r2 0.99 0.98 0.83 0.99 0.89 0.91 ab3lyp/6-31g(d)/iecpcm; bb3lyp/6-31+g(d,p)/iecpcm fig. 2. addition of oxygen atoms increases the acidity. a comparison of the experimental and computed pka values shows that the pka values computed through b3lyp/6-31g(d)/iefpcm procedure are large than experimental pka values. the mean absolute deviations (mad) are also large (<7pka – units) in sulfide, (<9pka – units) in sulfoxide and (<7pka – units) in sulfone. however, the correlation coefficients (r2) are somehow better in each series (>0.8). addition of a diffuse function in the basis set subsequently improved the results. the mad values decreased significantly in each series (<2pka – unit) and the r2 values also improved (>0.9). the data obtained through the b3lyp/6 -31+g(d,p)/iefpcm computational approach shows an excellent agreement between the experimental and computed pka values (see table i). hence, this computational model is excellent in predicting the pka values of these kinds of organosulfur compounds. all investigated data obtained through b3lyp/6 -31+g(d,p)/iefpcm computational model are presented in fig. 3. conclusions the dft method was employed to calculate theoretical pka values of organosulfur compounds using two different basis sets. the diffuse function basis set provided the best calculated pka values and these are in excellent agreement with ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ theoretical calculation of dissociation constant 169 fig. 3. plot of experimental and computed pka through b3lyp/6-31+g(d,p)/iefpcm. the majority of the experimental pka values. oxidation of sulfur and substitution of electron withdrawing and aromatic groups increase the acidity of considered organosulfur compounds. the predicted pka values showed the same phenomenon regarding the acidity of organosulfur compounds. finally it was concluded that the proposed computational approach b3lyp/6-31+g(d,p)/iefpcm is predictive and could be utilized to calculate theoretical pka values of these kinds of small organosulfur compounds. for large and flexible organosulfur compounds, conformational analysis will be required. acknowledgement. the authors are thankful to higher education commission (hec), pakistan, for financial support. и з в о д једноставан рачунарски приступ израчунавању pka органосумпорних једињења syed tahir ali, aneesa choudhary1, syed majid khali2 и arif zubair3 1 department of chemistry, federal urdu university of arts science and technology, karachi, pakistan, 2 bosch pharmaceutical private limited, plot no. 209, sector 23, korangi industrial area, karachi, pakistan и 3 department of environmental science, federal urdu university of arts science and technology, karachi, pakistan овај рад се односи на предвиђање pka вредности органосумпорних једињења помоћу теорије функционала густине (dft). у овој студији су разматрана 22 органосумпорна једињења за израчунавање теоријских pka вредности. нагласак је стављен на супституцију различитих група на атому сумпора. израчунавања су урађена за присуство диметил-сулфоксида (dmso) као растварача. експеримeнтални редослед пораста киселости је: сулфиди < сулфоксиди < сулфони. наше израчунате pka вредности такође следе овај поредак. теоријске pka вредности израчунате су користећи dft метод b3lyp, са базисима 6-31g(d), 6-31+g(d,p) и iefpcm помоћни солватациони модел. већина pka вредности, израчунатих помоћу дифузног базиса, су у изврсној сагласности са експерименталним. отуда се овај рачунарски приступ, b3lyp/6-31+g(d,p)/iefpcm, може користити за предвиђање pka вредности овог типа органосумпорних једињења. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {grape seed flour of different grape pomaces: fatty acid profile, soluble sugar profile and nutritional value} j. serb. chem. soc. 85 (3) 305–319 (2020) udc 581.84 +634.84+664.76:547.466.3: jscs–5302 664.1:641.1 original scientific paper 305 grape seed flour of different grape pomaces: fatty acid profile, soluble sugar profile and nutritional value danijel d. milinčić1, aleksandar ž. kostić1, bojana d. špirović trifunović1, živoslav lj. tešić2#, tomislav b. tosti2#, aleksandra m. dramićanin2#, miroljub b. barać1 and mirjana b. pešić1* 1faculty of agriculture, department of food technology and biochemistry, university of belgrade, nemanjina 6, 11081 zemun-belgrade, serbia and 2faculty of chemistry, university of belgrade, p.o. box 51, 11158, belgrade, serbia (received 13 july, revised 29 october, accepted 30 october 2019) abstract: the aim of this study was to determine fatty acid and soluble sugar profiles of the grape seed flour originated from non-fermented dried pomace of international and autochthonous grape varieties in order to estimate their potential nutritional value. the grape seed flours were obtained from the grapes harvested in technological maturity. it has been shown that grape seed flours contained significant quantities of unsaturated fatty acids (ufas), especially linoleic fatty acids, whose content ranged from 61.15 83.47 %. oleic acid mostly contributed to the content of monounsaturated fatty acids, while the stearic acid was the most abundant saturated fatty acid (sfa). among polyunsaturated fatty acids, mainly ω-6 fas, were the most represented. the tested grape seed flours had the high ufa/sfa ratio (3.6311.09), low atherogenicity (0.04-0.13) and thrombogenicity (0.16-0.47) indices. fifteen different sugars were found in analysed samples with the total concentration ranging from 40588 to 91319 mg/kg seed with fructose and glucose as the most abundant. principal component analysis based on the content of fas and soluble sugars revealed unique composition of the seed flour of prokupac variety. these findings indicate that the tested grape seed flours is a good source of nutritionally valuable fas and sugars that can play an important role in the formulation of a new functional food products. keywords: vitis vinifera, marc, soluble carbohydrates, long-chain organic acids, index of atherogenicity, index of thrombogenicity. introduction grape is the most extensively cultivated fruit crop in the world, commonly used for wine production. however, the use of grapes in the winemaking industry * corresponding author. e-mail: mpesic@agrif.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc190713117m ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 306 milinčić et al. leads to accumulation of large quantities of seed and skin by-product (known as pomace) which is approximately 10–30 % of the total grape mass that has been processed.1 depending on the winemaking process, the composition of the pomace is different. in the production of red wines, the entire disintegrated grape mass is included in the alcoholic fermentation while for the production of rosé and white wines only the juice is fermented.1 it is known that grape berry contains nutritional and health promoting compounds such as carbohydrates, fatty acids, vitamins, minerals and polyphenols.2 during wine production most of these prominent compounds are extracted into grape juice or wine, but significant amount remains trapped in the grape pomace.3 recently, numerous studies have shown that the revalorisation of these by-products is possible and that interesting as well as useful products, for the food industry, can be obtained.4–6 the value -added products containing specific compounds, antioxidants (such as polyphenols), minerals, dietary fibres or minimally processed products such as grape pomace flours have been proposed for enrichment of food.1,6 grape seeds, constituents of grape pomace, have been used for decades in production of grape seed oil and are widely commercialized in some countries. according to previous research, unsaturated fatty acids such as linoleic and oleic are dominant in the grape seed oil, while among the saturated fatty acids mainly palmitic and stearic fatty acid are present.7–9 high content of essential fatty acids in the seed oil makes it suitable as functional food ingredients, since they contribute to reduced risk of various diseases.10 also, grape seed oil is very deficient in ω-3-fatty acids.8,9 besides the oil, yedro et al.11 observes that grape seed as a lignocellulosic residue consisting mostly of three fractions such as hemicellulose, cellulose and lignin, whereas beres et al.1 suggest that seed contains about 40 % of the fibre, as well as significant amounts of polyphenols, mainly catechin and proanthocyanidins. so far, for the isolation of the target compounds from the grape seed the complex extraction procedures that require a lot of equipment, time and are less suitable for an industrial application, have been used.6 more and more studies are emphasizing the importance of the use of whole grape seed or its flour and powder, where different groups of chemical constituents are combined together and enable intense fortification, which may result in better functional properties of value-added products.6,12 therefore, the whole grape seed is an interesting by-product that can be used for fortification and incorporation of nutrients into food. nowadays, there are several categories of foods such as cereal,4,12 dairy13 or meat products14 that are successfully enriched with grape seed flour and powder. studies on enrichment of products such as bread,4 biscuits12,15 or pancakes,5 with grape seed flour pay special attention to polyphenols due to their antioxidative properties and role in the sensorial acceptability of the product as well as on dietary fibres. nevertheless, other compounds present in the seeds like lipids or ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ fatty acids and sugars of grape seeds 307 components concentrated on the surface of the seed such as sugars also affect the nutritional value of the final product and can have an important function and impact on human health.1,6 namely, on the surface of seed separated from the pomace, immediately after grape pressing, some amount of soluble sugars remain, because the seeds were in permanent contact with the pulp. it is known that most of the total soluble grape sugars are concentrated in pulp or grape juice.16 in grapes, depending on the variety and climatic conditions, the soluble sugar content may vary from 12 to 28 %,17 with glucose and fructose as predominant. besides, in most of the berry cultivars sucrose is present in traces.2,18 thus, it is very important to define fatty acids (fas) and soluble sugar profiles of grape seed flours which are intended to be an integral part of food product. knowing that the composition of grape seeds depends on several factors, such as variety, location, harvest time, etc., the aim of this work was to determine fatty acid and soluble sugar profiles of the grape seed flour originated from non -fermented dried pomace of international and autochthonous grape varieties aiming to estimate their potential nutritional value. in that sense, sfas, ufas, monounsaturated fatty acids (mufas), polyunsaturated fatty acids (pufas), ufa/sfa ratio, index of atherogenicity (ia) and index of thrombogenicity (it) were calculated. in order to obtain a more detailed insight into the structure of the data and identify similarities and specificities of grouping of objects principal component analysis (pca) and hierarchical cluster analysis (hca) were also performed based on the contents of fatty acids, as well as the soluble sugar content detected in various samples of grape seed flour. the obtained results will be valuable for profiling a new functional food product enriched with whole grape seed products. experimental chemicals and materials supelco 37 component fame mix standard was purchased from supelco (bellefonte, usa). sugar standards were obtained from tokyo chemical industry, tci (europe, belgium). other chemicals and solvents were of analytical grade. technological parameters of analysed grapes technological parameters were determined on sample of 50 berries of each analysed grapes. total soluble solids (tss) was analysed by refractometer (atc 0-32 brix, huixia supply co.,ltd, china), titrable acidity (ta) expressed as g/l of tartaric acid was estimated by aoac method 942.1519 and ph was determined on ph meter (consort, belgium). preparation of grape seed flour in total, the seven samples of seeds from different grape varieties, four red varieties: ,,hamburg”, ,,prokupac”, ,,merlot”, ,,cabernet sauvignon” and three white varieties: ,,smederevka”, ,,riesling italien” and ,,tamjanika” were examined. samples were obtained from vinery located in aleksandrovac, center of župa district, serbia. various grape varieties were harvested in the technological stage of maturity suitable for the production of wine. fresh ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 308 milinčić et al. grape pomace samples of all grape variety were collected after pressing. then, pomaces were immediately dried in an drying oven (thermo scientific haraeus, ma, usa) at 60 °c for 72 h (final water content about 15 %). thereafter, the seeds were manually separated from the skin and were ground in a small laboratory coffee grinder (bosch mkm 6003 uc, bsh hausgeräte gmbh, munich, germany). the grape seed flours were maintained at –20 °c in vacuum-packed plastic containers until further analysis. preparation of grape seed flour samples for gc and hpaec analysis extraction of lipids from seeds flour (approximately 0.5 g), was carried out using 10 ml hexane in ultrasonic bath for 30 min at 40 °c. thereafter, the extraction was continued with stirring on a magnetic stirrer at 40 °c for 1 h. then, lipid extracts were filtered through whatman no.1 filter paper and supernatant was collected. extractions were carried out in duplicate and both supernatants were combined and evaporated to dryness by rotary evaporator (heidolph, laborota 4000, schwabach, germany) under reduced pressure at 40 °c. after evaporation, the residues were dissolved in 6 ml hexane and used for further gc analysis. for soluble sugar analysis, defatted seed flour (approximately 0.1 g) was extracted with 10 ml 80 % methanol containing 0.1 % hcl. samples were stirred for 1h on a mechanical shaker (thys 2, mlw labortechnik gmbh, seelbach, germany) at room temperature and additionally, for another 1h, treated on a water bath at 38 °c. then, samples were centrifuged at 4000 rpm for 10 min and supernatant was collected. thereafter, supernatants were evaporated to dryness and residues dissolved in 10 ml milli-q water. gc analysis of fas fas composition of different grape seed lipid extracts were determined using capillary gas chromatography (gc instrument agilent technologies 6890 (usa) with flame ionization detector (gc-fid) previously described by kostić et al.20 the fame’s were determined using capillary gas column sp-2560 (length 100 m, i.d. 0.25 mm, film thickness 0.20 μm, supelco, bellefonte, pa, usa). the following conditions were applied: injector temperature, 250 °c; detector temperature, 260 ºc; carrier gas, helium at flow rate of 5 ml/min; injection volume 1 μl; injector split ratio set at 20:1; the column temperature was: 50 °c, 5 min to 240 °c, 20 min with temperature rate of 4 °c/min. the analysis run was 72.5 min. the identification and quantification of the fa was done by the fame mix standard. fatty acid content was expressed in relative quantities as mass % of total detected fatty acids. from these data, the nutritional quality parameters such as sfas, ufas, mufas, pufas and ufa/sfa ratio were determined. additionally, index of atherogenicity (ia) and index of thrombogenicity (it) were calculated according to eqs. (1) and (2):21 4 ia mufa 6 3 a b c e ω ω + + + = + +   (1) it 0.5 mufa 0.5 6 3 3 3 6/ b c d e ω ω ω ω + + + = + + +     (2) a – the content of c12:0; b – the content of c14:0; c – the content of c16:0; d – the content of c18:0; mufa – the content of mufa, ω6 – the content of ω6 fatty acid; ω3 – the content of ω3 fatty acids; e – the content of trans-fatty acids. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ fatty acids and sugars of grape seeds 309 high-performance anion-exchange chromatography with pulsed amperometric detection (hpaec/pad) for sugars analysis composition of the soluble sugar of defatted seed flour of different grape varieties were determined using hpaec/pad, according methodology previously described by gašić et al.22 briefly, dionex ics 3000 dp liquid chromatography system (dionex, sunnyvale, ca, usa) equipped with a quaternary gradient pump (dionex), ics as-dv 50 autosampler (dionex) and carbo pac®pa100 pellicular anion-exchange column (4×250 mm, particle size – 8.5 μm, pore size – microporous,<10 å (dionex), was used for sugar analysis at 30 °c. the electrochemical detector consisted of gold as the working and ag/agcl as the reference electrodes. the mobile phase consisted of the following reagents: 600 mm sodium hydroxide (a), 500 mm sodium acetate (b) and ultrapure water (c). the linear gradient (flow rate, 0.7 ml/min) was: 0–5 min, 15 % a, 85 % c; 5.0–5.1 min, 15 % a, 2 % b, 83 % c; 5.1–12.0 min, 15 % a, 2 % b, 83 % c; 12.0–12.1 min, 15 % a, 4 % b, 81 % c; 12.1–20.0 min 15 % a, 4 % b, 81 % c; 20.0–20.1 min 20 % a; 20 % b; 60 % c; 20.1–30.0 min 20 % a; 20 % b; 60 % c. before the analyses, the system was preconditioned with 15 % a, 85 % c, for 15 min. the sample injection volume was 25 μl. the quantification of carbohydrate concentration was obtained from the calibration curves of pure compounds as already reported by gašić et al.22 statistical analysis results were reported as means of three measurements ± standard deviation. differences between mean values were estimated using tukey’s-test, at level of significance p < 0.05 in statistica software v. 6.0 (statsoft co., tulsa, ok, usa). principal component (pca) and hierarchical cluster (hca) analyses were performed in the software package pls toolbox, v. 6.2.1, matlab 7.12.0 (r2011a). all data were auto-scaled before the multivariate analysis. results and discussion technological parameters of analysed grapes sugars, expressed as soluble solid contents, acids and ph of grape pulp usually used to estimate technological maturity of grapes. these are very important parameters for quality of wine as well as quality of seed extracts.9,23 technological parameters depend on the cultivar, production area and viticultural practices.24–26 as can be seen in table i, the total soluble solids, titrable acidity and ph of analysed grape berries varied from 19.3 to 25.5 °bx, 6.56 to 10.78 g/l of tartaric acid and 3.36 to 3.85, respectively. these data were comparable to other results obtain for grapes harvested in technological maturity.23–26 fatty acid profile of grape seed oils results obtained for the relative content of fas in different grape seed oils are presented in table ii. on the basis of the obtained results, in total, twelve fatty acids were identified in grape seed oil samples. fas such as linoleic, oleic, stearic and palmitic were found in all examined samples. the linoleic acid, as unsaturated fatty acid, was dominant and ranged from 61.15±0.36 to 83.47±1.15 % depending on the variety. the highest concentration of linoleic acid was detected in seed oil of international grape varieties such as hamburg and riesling ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 310 milinčić et al. italian. among the indigenous varieties tamjanika seed was the best source of this ufa. table i. technological parameters of analysed grape berries (tss; ta; ph); the results in the table were presented as mean±standard deviations (mean±sd; n = 3); the same letters in the same row are not significantly different according to tukey’s test, p ˂ 0.05. abbreviations: tss – total soluble solids); ta – titrable acidity sample tss, g of sucrose / 100 g ta, g of tartaric acid/l ph °bx smederevka 19.5±0.3a 10.78±0.94a 3.36±0.02a italien riesling 23.6±0.2b 6.56±0.1b 3.81±0.01b tamjanika 22.3±0.5c 7.03±0.94b 3.83±0.01b hamburg 20.2±0.2ae 7.50±0.1bc 3.76±0.02c prokupac 19.3±0.1a 8.43±0.1cd 3.85±0.01b merlot 25.5±0.1d 9.37±0.2de 3.56±0.01d cabernet sauvignon 21.1±0.9e 10.31±1.88ae 3.40±0.005a table ii. fatty acid composition of analysed grape seed flours; n.d. – stands for not detected; the same letters in the same row are not significantly different according to tukey’s test, p ˂ 0.05. abbreviations: (c16:0) – palmitic; (c16:1) – palmitoleic; (c17:0) – heptadecanoic; (c18:0) – stearic; (c18:1ω9c) – oleic; (c18:2ω6c) – linoleic; (c18:2ω6t) – linoleaidic; (c20:2) – cis-11,14-eicosadienoic; (c20:3ω6) – cis-8,11,14-eicosatrienoic; (c20:3ω3) – cis11,14,17-eicosatrienoic; (c22:0) – behenic; (c22:1ω9) – erucic fatty acid smederevk a italien riesling tamjanika hamburg prokupac merlot cabernet sauvignon fatty acid content, % c16:0 4.38±0.1ad 3.57±0.09b 3.36±0.07b 3.41±0.08b 7.94±0.18c 4.69±0.27a 4.14±0.07d c16:1 n.d. n.d. 0.40±0.001 n.d. n.d. n.d. n.d. c17:0 n.d. n.d. 0.86±0.04 n.d. n.d. n.d. n.d. c18:0 6.53±0.11a 4.80±0.09b 4.58±0.25bc 4.10±0.07c 8.27±0.13d 5.83±0.21e 6.59±0.32a c18:1ω9 c 8.57±0.17 a 8.24±0.18ab 7.74±0.4be 6.14±0.17cf 9.72±0.24d 7.10±0.1e 6.40±0.26f c18:2ω6 c 73.61±1.05 a 83.39±1.26b 83.07±1.09 b 83.47±1.15 b 61.15±0.36 c 76.98±1.17 a 75.61±2.62 a c18:2ω6t n.d. n.d. n.d. n.d. 2.15±0.06a 1.24±0.05b 1.40±0.07c c20:2 0.90±0.03 n.d. n.d. n.d. n.d. n.d. n.d. c20:3ω6 1.00±0.01a n.d. n.d. 0.35±0.01b 2.09±0.1c 0.80±0.02d 1.19±0.02e c20:3ω3 1.47±0.04a n.d. n.d. 0.89±0.04b n.d. n.d. 1.44±0.03a c22:0 2.06±0.05a n.d. n.d. 0.76±0.02b 5.4±0.25c 1.36±0.03d 1.97±0.01a c22:1ω9 1.43±0.03a n.d. n.d. 0.87±0.05b 3.25±0.12c 2.01±0.09d 1.25±0.01a similar results were obtained by other authors who agreed that linoleic acid is the most abundant fa in oil of grape seeds of grape varieties grown in serbia27,28 or other countries.7–9,29–31 thus, malićanin et al.27 found that the content of linoleic acid in seeds of the cabernet sauvignon variety grown in serbia was in range from 73.10–75.30 %, whereas in prokupac variety ranged from 69– ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ fatty acids and sugars of grape seeds 311 –81 %.28 lachman et al.9 obtained results for content of linoleic acid in range from 68.10 to 78.18 % depending on the year of harvest, whereas beveridge et al.30 recorded the highest content of linoleic acid in the seed oil of merlot variety. oleic acid mostly contributed to the content of mufas and its content varied from 6.14±0.17 to 9.72±0.24 %, while the stearic acid was the most abundant sfa in all analysed samples with content from 4.1±0.07 to 8.27±0.13 %. both fas were registered in the highest amount in indigenous variety prokupac. however, significant differences among the results for oleic fatty acid in grape seed oil could be found in the literature. for example, these results are in agreement with those obtained by lachman et al.,9 while pardo et al.29, registered significantly higher amount of oleic acid in the seed oil of red grape varieties grown in spain (16.07–24.88 %) and malićinin et al.27 for seeds of cabernet sauvignon grown in serbia (12.60 to 13.80 %). results for content of palmitic and stearic acids fall within similar ranges reported by beveridge et al.30 (6.35 to 8.62 % for palmitic and 3.60 to 5.26 % for stearic acids, depending on extraction solvents and conditions), fernandes et al.8 (6.17 to 8.50 % and 4.09 to 5.91 %, respectively) or zdunić et al.,28 obtained for clones of the prokupac variety (3 to 8 % for stearic acid and 2 to 4 % for palmitic acid). however, according to other reports, palmitic acid is the dominant sfa in most of grape seed oils, which is not correlated with this study.7–9,29,31 furthermore, seed oil of variety prokupac contained significant amount of linoleaidic, erucic, behnic and cis-8,11,14-eicosatrienoic fatty acids compared to that of other analyzed varieties. other identified fas, known as „rear fatty acids“ were present in traces and their content can often serve as a marker for the characterization of different grape varieties. for example, palmitoleic and heptadecanoic fas were identified only in the seed oil of variety tamjanika. additionally, the presence of cis-11,14-eicosadienoic fa was recorded in smederevka seed oil. soluble sugar profile of defatted grape seed flours the composition of soluble sugars extracted from defatted grape seed flours are presented in table iii. according to results of hpaec analysis, the concentration of fifteen different sugars was determined in all analyzed samples. their total concentration varied from 40588 to 91319 mg/kg seed. monosaccharides were dominant and their concentration was in ranges from 39090 to 89659 mg/kg seed, while disaccharides and trisaccharides are present in traces. in addition to the predominantly presence of monosaccharides, such as glucose and fructose, it is important to note that the sucrose concentration was higher compared to other detected diand tri-saccharides. the residual carbohydrates, that remain in the pomace after the disintegration and pressing of grapes, are mainly water soluble (monosaccharides and oligosaccharides) and water insoluble structural polysaccharides from the cell wall.32 in most studies the monosaccharide composition of ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 312 milinčić et al. table iii. the soluble sugar profile of defatted grape seed flours (mg/kg seed); the same letters in the same row are not significantly different according to tukey’s test, p ˂ 0.05. abbreviation: monosaccharides (ms); disaccharides (ds); trisaccharides (ts); glucose (glc); fructose (fru); saccharose (sac); trehalose (tre); maltose (mal); arabinose (ara); turanose (tur); gentiobiose (gent); isomaltose (ism); panose (pan); isomaltotriose (ismt); maltotriose (malt); melibiose (mel); rafinose (raf); melesitose (mele) sugar smederevka italien riesling tamjanika hamburg prokupac merlot cabernet sauvignon sugar concentration, mg/kg (mean ± standard deviation) ms ara 23±1a 18±1.3b 11±0.6c 22±1.4a 35±2d 3±0.3e 17±0.7b glc 46777± 896a 37827± 829b 41415± 636c 38351± 682b 31456± 342d 19350± 348e 33316± 427f fru 42859± 1526a 37731± 618b 42911± 915a 41259± 263a 35562± 339b 19737± 516c 32818± 694d σ 89659 75576 84337 79632 67053 39090 66151 ds tre 11±0.7ae 9±0.7a 155±8b 32±2cf 387±10d 45±3c 24±1.1ef ism 24±1.2a 10±0.9b 36±0.8c 36±2.2c 26±2.3a 42±0.9d 39±1.9cd mel 13±0.8a 4±0.3be 9±0.3c 3±0.2b 4±0.3be 7±0.3d 5±0.4e sac 955±11a 3737±140b 2445±34c 4146±59d 4413±26e 1009±18a 1763±64f gent 1±0.1a 2±0.2b 1±0.2a n.d. 2±0.3b 3±0.3c n.d. tur 116±5ad 122±9a 241±12b 178±11c 106±8ad 94±3d 200±13c mal 59±4a 74±8b 91±7c 68±6ab 65±2.9ab 41±3d 70±3ab σ 1179 3958 2978 4463 5003 1241 2101 ts raf 339±8a 249±20b 340±9a 249±11b 335±14a 181±6c 277±9b mele 15±0.6a 16±0.8a 19±0.8b 14±0.9a 19±1.2b 6±0.3c 22±1.9d ismt 20±1.1a 93±6b 10±0.7ce 5±1c 83±7d 11±0.4ac 19±1.2ae pan 84±5ae 55±4ac 309±13b 28±2c 448±24d 47±2d 100±7e malt 23±1.1a 26±2ac 16±0.9b 30±2.2ce 7±0.4d 12±0.4b 32±2.3e σ 481 439 694 326 892 257 450 total 91319 79973 88009 84421 72948 40588 68702 the grape pomace, after intensive hydrolysis of complex lignocellulosic polysaccharides, was analyzed. however, according to our knowledge, the concentration of soluble sugars in the defatted seed flour obtained from non-fermented dried pomace have been rarely investigated until now, but the different results for total soluble sugar concentration in grape pomace, obtained after grape pressing, could be found in the literature. according to beres et al.,33 the carbohydrate concentration of grape pomace flour of the red pinot variety was 196800 mg/kg, while sousa et al.,34 reported that the pomace flour of benitaka variety contained 292000 mg/kg of carbohydrate with a respectable amount of glucose and fructose, 79500 and 89100 mg/kg, respectively. also, according to corbin et al.,32 the content of water soluble carbohydrates in pomace of varieties cabernet sauvignon and sauvignon blanc were 4.6 and 37.6 mass %, with the dominant pre________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ fatty acids and sugars of grape seeds 313 sence of glucose and fructose. in addition, wang et al.35 found the total sugar concentration of 368000 mg/kg in grape pomace. gonzález-centeno et al.,36 showed that the fresh pomace of ten different grape varieties after pressing and maceration have soluble sugar concentration in range of 20000 to 62000 mg/kg, with the sugar concentration of those of varieties cabernet sauvignon and merlot, 23000 and 24000 mg/kg, respectively. these data suggest that significant amount of soluble sugars remaining in grape pomaces after pressing of grapes which resulted to their significant amount on the surface of pomace seeds and consequently in the grape seed flour. thus, the apart from fatty acid profiles, it is also desirable to determine the content of soluble sugars in grape seed flour intended for enrichment of the food product. dominant detected monosaccharides such as glucose or fructose are typical energy components, and their quantity is not negligible. nutritional quality parameter of grape seed flours nutritional quality parameter such as sfas, mufas, pufas and ratio of ufas/sfas, were determined and given in table iv. the content of sfas was in range from 8.27 (hamburg) to 21.61 % (prokupac). the content of mufas in seed of all investigated samples were 7.01 (hamburg)–12.97 % (prokupac), while the content of pufas were in range from 65.39 (prokupac) to 84.71 % (hamburg). according to the obtained results, grape seeds of different varieties can be an important source of various fas, primarily pufas with high prevalence of ω-6 fas. this research also confirmed that grape seeds are deficient in the content of ω-3 fas. table iv. nutritional quality parameters of grape seed flours; sfas – saturated fatty acids; ufas – unsaturated fatty acids; mufas – monounsaturated fatty acids; pufas – polyunsaturated fatty acids; ufa/sfa – the ratio of the content of ufa to the content of sfa; ia – index of atherogenicity; it – index of thrombogenicity nutritional parameter smederevka italien riesling tamjanika hamburg prokupac merlot cabernet sauvignon fatty acid content, % sfas 12.97 8.37 8.80 8.27 21.61 11.88 12.7 ufas 86.98 91.63 91.21 91.72 78.36 88.13 87.29 mufas 10 8.27 8.14 7.01 12.97 9.11 7.65 pufas 76.98 83.39 83.07 84.71 65.39 79.02 79.64 ufa/sfa, ratio; ia index; it index ufa/sfa 6.71 10.95 10.36 11.09 3.63 7.42 6.87 ia 0.05 0.04 0.04 0.04 0.13 0.07 0.06 it 0.23 0.18 0.17 0.16 0.47 0.27 0.26 it is known that sfas increase low-density lipoprotein (ldl) cholesterol and thus increases the possibility of the risk of cardiovascular disease30, while ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 314 milinčić et al. the inclusion of products that have high content of mufas and primarily pufas in the diet, have hypocholesterolemic potential, i.e., protective effect against coronary heart and artery diseases.10,37 the availability of essential ω-3 and ω-6 fas in nutrition is associated with the normal physiological functions of the membrane and the regulatory cell signals.10 therefore, according to baydar et al.,31 grape seeds that are rich in linoleic acid can be a highly valuable source of dietary fat. by comparing the content of sfas and ufas, it was found that the ufa/sfa ratio, has significantly higher values than 1.6 for all tested samples (from 3.63 to 11.09) indicating that grape seeds can be characterized as potentially good dietary supplement (who/fao, 2003).38 additionally, the values for it and ia are defined, which enable a better classification of different foods.37 ia showing the inhibition of the aggregation of plaque and the reduction of esterified fas, cholesterol, and phospholipids, which prevents the appearance of coronary diseases. it is showing the tendency to form clots in the blood vessels.39 analysed grape seeds had low atherogenic (0.04–0.13) and thrombogenic indices (0.16–0.47). the highest indices were obtained for seed oil of prokupac variety and the lowest for that of hamburg and tamjanika varieties. the obtained ia values were similar to that of sunflower oil (0.07) but lower than those of olive (0.19) and oat oils (0.17–0.19), whereas it values were in the range of those obtained for the same oils (olive, 0.4, sunflower, 0.20 and oat, 0.30–0.34).30 these findings indicated that the tested grape seed flours could be a good source of nutritionally valuable fas that plays an important role in the prevention of cardiovascular diseases. principal component analysis (pca) pca based on contents of sfas and ufas in various samples of grape seed results in three-component model explaining 90.70 % of the total variance among data. the results obtained by analysing the first two principal components (table ii) are shown in score and loading plots (fig. 1a and b). fig. 1. score (a) and loading (b) plot. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ fatty acids and sugars of grape seeds 315 the score plot (fig. 1a) shows the separation of three groups of objects. seeds of samples tamjanika and prokupac are separated from other samples and form groups i and ii. the third group consists of other analysed samples (fig. 1a, table ii). palmitoleic and heptadecanoic acids have the strongest positive influence along the pc2 axis on the separation of seed sample tamjanika, which is in accordance with the fact that content of these acids is the highest in this sample (fig. 1, table i), while erucic, cis-11,14-eicosadienoic, cis-8,11,14-eicosatrienoic and cis-11,14,17-eicosatrienoic fas, which were not identified in this sample, have a negative influence in the separation of this sample along the pc2 axis. the highest content of oleic, palmitic, linolelaidic, behenic, stearic, erucic and cis-8,11,14-eicosatrienoic fas in the prokupac variety have the strongest positive effect along the pc1 axis on the separation of this variety. linoleic acid has a negative influence along the pc1 axis, as its concentration is the lowest in the prokupac sample, compared to all others, while acids cis-11,14-eicosadienoic and cis-11,14,17-eicosatrienoic were not identified in this sample. as for the third group of objects (fig. 1a), by comparing the results of pca and hca analyses (fig. 2a) and based on the contents of linoleic and palmitic acids it can be concluded that there is a similarity between samples merlot and cabernet sauvignon (table i), as well as between samples hamburg and italian riesling (table ii) which separated them in two different sub-clusters (fig. 2a). further, hca on distance 5 results in the separation of samples of three clusters. the first cluster consists of all analysed samples except pokupac and tamjanika. fig. 2. a) hca dendrogram of fatty acids; b) hca dendrogram of soluble sugars; abbreviations: m – merlot, p – prokupac, c.s. – cabernet sauvignon, h – hamburg, i.r. –italian riesling, t – tamjanika, s – smederevka. pca based on sugar concentrations in various samples of grape seed result in three-component model that explains 77.63 % of total variance among data. results obtained by analysing the first two principal components (table iii) are ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 316 milinčić et al. shown in score and loading plots (fig. 3 a and b). three groups of objects can be seen in the score plot (fig. 3a). samples merlot and prokupac are separated from other samples (group iii) and comprise group i and group ii (fig. 3a, table ii). fig. 3. score (a) and loading (b) plot. rafinose, melesitose, maltose, fructose, glucose, and turanose have the most positive influence along the pc1 axis on the separation of sample merlot in which the concentration of these sugars is the lowest (fig. 3, table iii), while melibiose and isomaltose, the concentration of which is the highest in this sample, have a negative influence along the pc1 axis. gentiobiose, the concentration of which is the highest in merlot, has the most positive effect on the separation of this sample along pc2 axis. the separation of sample prokupac along the pc2 axis is most positively influenced by trehalose, isomaltotriose, panose, saccharose and arabinose, which concentrations are the highest in this sample. maltotriose, the concentration of which is the lowest in sample prokupac, has the strongest negative effect along the pc2 axis. the separation of the third group of objects (fig. 3a), which consists of other analysed seed samples, is influenced by rafinose, melesitose, maltose, fructose, glucose, and turanose. the results obtained by the hierarchical cluster analysis are shown in dendrogram (fig. 2b). hca at distance 6 results in the separation of samples in three clusters. the first cluster consists of sample merlot, the second one of prokupac, and the third of other analysed samples, which is in accordance with the results of pca. dendrogram also shows that within the thirds cluster, at the distance 5, two sub -clusters can be separated. the first sub-cluster consists of samples smederevka and tamjanika, while the other consists of samples cabernet sauvignon, hamburg, and italian riesling. conclusion the grape seed flours were obtained from the grapes harvested in technological maturity. all of tested grape seed flours showed favorable fa compo________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ fatty acids and sugars of grape seeds 317 sitions, i.e., low levels of sfas, and high level of ufas, especially linoleic fatty acids (61.15 – 83.47 %). the highest content of linoleic acid was detected in grape varieties hamburg, riesling italian and tamjanika. the oleic acid was the most abundant mufa, whereas the stearic acid was the most represented sfa. both fatty acids were registered in the highest amounts in variety prokupac. furthermore, seed oil of variety prokupac contained significant amount of linoleaidic, erucic, behenic and cis-8,11,14-eicosatrienoic fas. the significant amount of soluble sugars was registered in all analysed grape seed flours ranged from 40588 to 91319 mg/kg seed. fifteen different sugars were found in samples among which glucose and fructose were the major ones. the pca of the content of fatty acids revealed the separation of two autochthonous grape varieties, prokupac and tamjanika from each other and from the other analyzed samples, whereas pca of the soluble sugar concentration differentiated prokupac and merlot as separate groups from the other grape varieties. unique fatty acids and soluble sugar profiles of seed flour of autochthonous grape variety prokupac together with good nutritional quality parameters indicate to its possible use as a new functional food ingredient. acknowledgement. this investigation was supported by the ministry of education, science and technological development of republic of serbia through grants no. tr31069 and oi172017. извод kоштице гpожђа различитих комина: маснo-киселински профил, профил растворљивих шећера и нутритивна вредност данијел д. милинчић1, александар ж. костић1, бојана д. шпировић трифуновић1, живослав љ. тешић2, томислав б. тости2, александра драмићанин2, мирољуб, б. бараћ1 и мирјана б. пешић1 1пољоприведни факутет, инситут за прехамбну технологију и биохемију, унивезитет у београду немањина 6, 11081 земун-београд и 2хемијски факултет, унивезитет у београду, п. пр. 51, 11158 београд циљ овог истраживања је да се одреди масно-киселински профил и профил растворљивих шећера брашна семенки грожђа добијеног од нефементисане осушене комине интернационалних и аутохтоних сорти грожђа у циљу одређивања њихове потенцијалне нутритивне вредности. брашно је добијено од сменки грожђа убраног у технолошкој зрелости. показано је да брашно коштица грожђа садржи значајну количину незасићених масних киселина (ufa), посебно линолне, чији се садржај кретао од 61,15–3,47 %. олеинска киселина је највише допринела садржају мононезасићених масних киселина, док је стеаринска киселина била најзаступљенија засићена масна киселина (sfa). од полинезасићених масних киселина претежно су ω-6 fa детектоване. анализирана брашна коштице грожђа имала су висок однос ufa/sfa (3,63–11,09), низак атерогени (0,04–0,13) и тромбогени (0,16–0,47) индекс. у анализираним узорцима је пронађено петнаест различитих шећера, чији се укупни садржај кретао од 40588 дo 91319 mg/kg коштице, са моносахаридима, глукозом и фруктозом као најзаступљенијим. анализа главних компоненти на основу садржаја fa и растворљивих шећера открила је јединствен састав брашна семенки сорте прокупац. ова истраживања ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 318 milinčić et al. указују да анализирана брашна грожђа могу бити добар извор нутритивно вредних масних киселина и шећера који могу имати важну улогу у формулацији нових функционалних прехрамбених производа. 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {in silico studies on smoothened human receptor and its antagonists in search of anticancer effects} j. serb. chem. soc. 85 (3) 335–346 (2020) udc 612.398:577.354:542.97+519.8:615.277 jscs–5304 original scientific paper 335 in silico studies on smoothened human receptor and its antagonists in search of anticancer effects ana borota*, sorin avram, ramona curpan, alina bora, daniela varga, liliana halip and luminita crisan** “coriolan dragulescu” institute of chemistry, romanian academy, 24 mihai viteazul avenue, ro-300223, timisoara, romania (received 3 april, revised 5 july, accepted 6 august 2019) abstract: lately, the cancers related with abnormal hedgehog (hh) signalling pathway are targeted by smoothened (smo) receptor inhibitors that are rapidly developing. still, the problems of known inhibitors such as severe side effects, weak potency against solid tumors or even the acquired resistance need to be overcome by developing new suitable inhibitors. to explore the structural requirements of antagonists needed for smo receptor inhibition, pharmacophore mapping, 3d-qsar models, database screening and docking studies were performed. the best selected pharmacophore hypothesis based on which statistically significant atom-based 3d-qsar model was developed (r2 = = 0.856, q2 = 0.611 and pearson-r = 0.817), was further subjected to dataset screening in order to evaluate its ability to prioritize active compounds over decoys. the efficiency of one four-points pharmacophore hypothesis (aahr.524) was observed based on good evaluation metrics such as the area under the curve (0.795), and weighted average precision (0.835), suggesting that the model is trustworthy in predicting novel inhibitors against smo receptor. keywords: pharmacophore; 3d-qsar; docking; smo inhibitors. introduction the smoothened (smo) receptor is a g-protein-coupled receptor (gpcr) -like protein and it is one of the relevant components of the hedgehog (hh) signalling pathway1. gpcr are seven transmembrane receptors which constitute the largest family of human proteins2 that regulate a various multitude of intracellular signalling cascades.3 late breakthroughs in gpcrs biochemistry4,5 have enhanced our knowledge of drug action on these significant targets, which will lead to the design of better therapeutics in the future. *,** corresponding authors. e-mail: (*)ana_borota@acad-icht.tm.edu.ro; (**)lumi_crisan@acad-icht.tm.edu.ro https://doi.org/10.2298/jsc190403085b ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 336 borota et al. it is known that ~25 % of cancers present aberrant hh pathway activation.6 regarding the implication of hh pathway in cancer, three models of action have been proposed.7 the first type is ligand independent and refers to mutations in hh pathway components, that have been associated with medulloblastoma and basal cell carcinomas.8,9 the ii (autocrine) and iii (paracrine) types of mechanisms are ligand dependent and include: lung,10 pancreatic,11 colorectal,12 breast,13 gastrointestinal tract,14 prostate,15 glioma tumours9 and hematological malignancies.16 the growth of these types of tumours can be suppressed by different pathway inhibitors, such as smo receptor antagonists.17 indeed, a number of smo antagonists (such as: cur61414, ipi-926, bms-833923, pf-04449913, leq-506 and tak-441) have demonstrated anticancer activity and have entered the clinical trials. hitherto, fda approved two smo antagonists (lde225/ /sonidegib and gdc-0449/vismodegib) for the treatment of basal cell carcinoma.18 unfortunately, these two inhibitors have shown severe side effects, along with weak potency against solid tumours, and, moreover, an issue related to acquired resistance has been identified.1,19 to overcome these downsides, many studies have focused on designing new and diverse smo inhibitors, with desirable selectivity against the target of interest.20 in this regard, a recently synthetized and biologically evaluated series of 26 compounds21 as smo antagonists (table i) was used for computational investigation. ligandand structure-based approaches were involved in order to understand which are the essential features responsible for the inhibition of smo receptor. phase software (schrödinger)22 was used for the generation of ligandbased pharmacophore models and 3d-qsar module was employed to validate the models. an external validation of the best pharmacophore hypothesis was achieved based on virtual screening (vs) and statistical evaluation of the results. the importance of stereochemical aspects involved in ligand-receptor interactions were highlighted based on the rigid and flexible docking studies performed with glide23 and induced fit24 programs. computational methods pharmacophore generation protocol in the current study, a dataset of 26 compounds newly synthesized and biologically tested against smo receptor, by lu and collaborators21 (table s-i of the supplementary material to this paper) was the subject of computational analysis for pharmacophore generation and docking studies. the preparation of the ligands for in silico studies along with the steps followed for the generation of pharmacophore hypotheses are presented in detailed manner in the supplementary material. phase22 with the option: “develop common pharmacophore hypotheses” was used for the generation and validation of the pharmacophore hypotheses by the involvement of the atom-based qsar module. the following features: hydrogen bond donor/acceptor, hydrophobic, negative, positive and aromatic rings were taken in account for the generation of the pharmacophore hypotheses. in order to select the best hypothesis, an atom-based 3d-qsar ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ computational studies on smo inhibitors 337 analysis was carried out using three partial least-squares (pls) factors. the test set compounds that represent roughly 23 % of the dataset were selected to cover the same range of activity as the compounds from the training set.25 the multidimensional descriptor space between the training and test set was checked26 using 2d fingerprints. the well-known 2d tanimoto and euclidean distance coefficients, and the median values for important 2d properties of molecules were calculated (supplementary material). the best selected pharmacophore hypothesis was employed in vs experiments using the “advanced pharmacophore screening” option of phase software (the inter-site distance matching tolerance was of 2å for minimum four site points) to test its ability to distinguish active compounds or positives from inactive ones or negatives (assumed to be the decoys). for this purpose, an external validation dataset was assembled, and it was realized following the protocol described in the supplementary material. the vs results were evaluated using an in-house developed program, “evaluation” tool in cheminformatics (etici 1.6).27 the following statistical metrics (eqs. (1)–(5)) were used for the evaluation: receiver operating characteristics (roc) curve and its corresponding area under the curve (auc)28 boltzmann enhanced discrimination of roc (bedroc)29, precision (ppv)30, accuracy (acc), sensitivity (se), specificity (sp)31 and the true positive (tp) at 1, 2, 5, 10 % of false positives (fps):32 + = 1 1 = 1 + tp fn i i auc fpr tp fn −  (1) tp ppv tp fp = + (2) tp se tp fn = + (3) tn sp tn fn = + (4) tp tn acc tp tn fp fn + = + + + (5) where: tp indicates the correctly predicted actives; fn designates the incorrectly identified inactives; fpr is false positive rate; tn represents the correctly predicted inactives or negatives and fp denotes the incorrectly predicted actives or positives. auc parameter is used to evaluate the models performance in classification, to discriminate actives from inactives and it takes values in the range 0 to 1. the closer auc value is to 1 the better a model discerns true positive from false positive results.28 bedroc can be considered as a “virtual screening usefulness scale” while roc assesses “improvement over random scale”. the difference between the two aforementioned evaluation parameters is that roc relates to a uniform distribution and bedroc refers to an exponential distribution.29 for imbalance class when the number of actives is lower than a number of inactives, the precision parameter was replaced with weighted average precision.33 docking simulations protocol the best resolution x-ray structure of smo receptor was extracted from pdb (pdb id: 4jkv) and prepared for docking studies with the “protein preparation wizard” module from schrödinger suite. briefly, the following steps were employed: the bond orders were assigned; the chemical components dictionary (ccd) database was used; hydrogens were added; ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 338 borota et al. create zero-order bonds to metals parameter was selected; disulfide bonds were created; waters beyond 5 å were deleted and missing side-chains were added to the protein. the proper ionization state of the co-crystallized ligand (ly2940680), at physiological ph of 7.0–7.4 was generated and the h-bond assignment was performed using propka module, setting ph to 7.0. finally, the protein was energetically minimized with opls_2005 force field until an rmsd of 0.3 å for heavy atoms was reached. a molecular docking study was carried out with “glide”23 and “induced fit”24 protocol included in schrödinger suite. “glide” module23 was used in standard precision (sp) mode with default settings, i.e., scaling factor of 0.8, partial charge cut-off of 0.15 and only the flexibility of ligands was considered. “induced fit” docking protocol24 accounts also for protein flexibility and it was carried out with the following settings: sp option, receptor and ligand van der waals scaling of 0.50. results and discussion analysing the results generated by phase,22 22 categories of four features common pharmacophore hypotheses (cph) were identified. cph include the same features placed together in similar spatial arrangements. all hypotheses received a good survival score (values > 3) and were subjected to qsar model development. a qsar model is reliable if a number of statistical parameters meet certain condition as follows: r2 (squared correlation coefficients for the training set) > 0.6; q2 (squared correlation coefficients for the test set) > 0.5;26 pearson-r (correlation between the predicted and observed activity for the test set) > 0.5; p (the significance level of variance ratio) < 0.05; sd (standard deviation) and rmse (root mean square error) should have low values, while f (variance ratio) should have high value.22,34 the best hypothesis named aahr.524 with a survival score of 5.263 has statistical significant results (table i). table i. the statistical parameters for the aahr.524 hypothesis obtained pls factor sd r2 p f rmse q2 pearson-r 1 0.255 0.601 6.00e-05 27.100 0.308 0.470 0.760 2 0.195 0.779 2.72e-06 29.900 0.303 0.486 0.769 3 0.161 0.859 4.90e-07 32.500 0.263 0.611 0.817 for aahr.524 hypothesis the distances between pharmacophore sites are shown in fig. 1 and scatter plots of experimental versus predicted bioactivity values for training and test set are presented in fig. 2. in order to highlight favorable and unfavorable regions for ligand–protein interactions, the atom-based 3d-qsar model regarding hydrogen bond donor, hydrophobic and electron withdrawing properties was mapped against the pharmacophore model (fig. 3). additionally, the aahr.524 hypothesis was validated in virtual screening (vs) experiments using phase advanced pharmacophore screening (fig. 4). in fig. 3 the combined effects for all the features retrieved in the workspace ligands along with the overlapping of the best fitted and the less active ligands are depicted. the favorable regions for biological activity ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ computational studies on smo inhibitors 339 are represented by blue cubes, while the red cubes indicate detrimental regions. for the aahr.524 hypothesis, the presence of blue cubes in the region of the –nh– group of compound 17 (fig. 3b) indicates that hydrogen bond donor property of this group is favorable for activity. this finding is also supported by glide docking results which highlight the contribution of this amino group in the formation of a hydrogen bond with residue tyr 394 of the smo binding site. fig. 1. aahr.524 pharmacophore hypothesis depicted with the best fitted compound 17. the distances between the following features are presented: hydrogen bond acceptor (a1), hydrogen bond acceptor (a3), aromatic ring (r12) and one hydrophobic region (h8). fig. 2. correlation plots of the experimental versus phase predicted activity of training set (circles) and test set (triangles) for aahr.524 hypothesis. the pyridine ring of compound 17 is engaged in hydrophobic interactions with the binding site, while in the less active ligand (compound 22) this feature is missing (fig. 3c). this finding was also confirmed by docking studies. regarding the electron withdrawing property (fig. 3a), the glide docking study revealed that the oxygen atom of the carbonyl group of compound 17 can act as hydrogen bond acceptor for arg400 residue. in order to assess the efficiency of vs experiments, it is necessary to test their ability to distinguish active compounds or positives from inactive ones or negatives (assumed to be the decoys). in this regard, different evaluation operators were employed. namely, the receiver operation characteristics (roc) curve28 (fig. 4) was used for the visualization of the true positive rate (tpr) versus the false positive rate (fpr) and the area under the curve (auc) was calculated. in our experiment, a good scores for: auc of 0.795 (±0.027), se (the ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 340 borota et al. fraction of actives correctly predicted) of 0.587, sp (the fraction of inactives correctly predicted) of 0.647, acc (the fraction of correct predictions) of 0.640, wppv (weighted average precision) of 0.835 and bedroc (α = 20) of 0.766, were obtained, suggesting that the aahr.524 pharmacophore hypothesis provides a good class separation. additionally, good enrichment factors, tp at x of fps were obtained: 19.047 % at x = 1 %; 30.476 % at x = 2 %; 42.857 % at x = 5 %; 56.190% at x = 10 %. fig. 3. superposition of compound 17 (best fitted over aahr.524 hypothesis) represented by grey carbon atoms with compound 22 (least active ligand) depicted by green carbon atoms: a) electron withdrawing property; b) hydrogen bond donor property; c) hydrophobic property. fig. 4. the receiver operation characteristics (roc) curve, involving the aahr.524 hypothesis against the validation data set. in fig. 4 is depicted the roc curve, the diagonal represents the random sampling of compounds (actives and decoys) and it separates two classification areas: the correctly predicted compounds are shown above, while the incorrectly ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ computational studies on smo inhibitors 341 classified compounds occupy the underneath area. tpr represents the fraction of correctly classified positives (actives) from the total of positives, while fpr expresses the fraction of the incorrectly classified negatives (decoys) from the total negatives.28,35 following ligand-based analysis, the structure-based study, represented by molecular docking, bring additional light on ligand-protein interaction profile. in order to validate the reasonability of choosing the two docking software for our study (glide (fig. 5a) and induced fit (fig. 5b)), we have undertaken a control experiment. the co-crystallized inhibitor ly2940680 was docked to its native x-ray structure, 4jkv, and the best docked pose was superimposed with the native binding pose from the experimental structure 4jkv. a b fig. 5. superposition of the pose of the crystal structure 4jkv (with the inhibitor ly2940680), and the pose of the same ligand obtained by docking with glide sp (a) superposition of the pose of the crystal structure 4jkv (with the inhibitor ly2940680), and the pose of the same ligand obtained by docking with induced fit sp (b). the rmsd values were also calculated. according to gohlke et al.,35 an rmsd value less than 2.0 å is broadly accepted as “effectively” docked structure. the docking protocol with glide and induced fit have provided very good rmsd values of 0.192 (fig. 5a), and 0.700, respectively (fig. 5b). a comparative study made by wang et al.36 on the binding profile and interactions of the ligands: antagonists and agonists at the human smo receptor, reveals some specific features. the binding sites of antagonists, e.g., ly2940680, cyclopamine and antaxv and the agonist sag1.5 consist mostly of residues from the extracellular linker domain and loops. in particular, asp219 (of the extracellular linker domain) it involved in the formation of hydrogen bonds with ligands: ly2940680, antaxv and sag1.5.36 also, an important residue from the helix v is arg400 (arg5.39) which forms hydrogen bonds with antagonists: ly2940680 and antaxv.36,37 according to the latest studies, the smo receptor ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 342 borota et al. contains two distinct binding sites. one ligand-binding site is located in heptahelical transmembrane domain, and the second, occupied by a cholesterol molecule, is placed in the extracellular cysteine-rich domain.38 thus, the smo inhibitors were classified in two categories: 1) antagonists that bind mainly to the extracellular loops and 2) antagonists that enter deeper into the cavity formed by the transmembrane helices domain.20 the large binding site of smo receptor allows flexibility to the ligands leading to several distinct binding modes.20,36–38 although, similar disposition of the ligand in situ were retrieved by the two docking methods employed (fig. 6c), the orientation of the ligand is reversed in the binding site, hence resulting in different interactions with the amino acids of the protein (fig. 6a and b). two interaction patterns for compound 17 were observed depending on the docking software used, glide (fig. 6a) or induced fit (fig. 6b). fig. 6. representation of the interactions map of the best docked pose of compound 17 with the amino acids of smo human binding site: glide docking (a); induced fit docking (b); their superposition in situs (c). following the most important features characterizing the interaction of compound 17 with the binding site of smo receptor resulted from docking with glide are presented: i) the oxygen atom of the carbonyl group acts like hydrogen bond acceptor for arg400 and gln 477; ii) the aliphatic –nh– group is hydrogen bond donor to tyr394; iii) the dimethyl-pyridine ring is involved in π–π stacking ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ computational studies on smo inhibitors 343 interactions with trp281 and phe391; iv) the chlorine atom is involved in alkyl interactions with ile234, leu522 and trp281 (fig. 6a). the following main interactions were found when the compound was docked with induced fit protocol: i) the n= atom of dimethylpyridine is acting as hydrogen bond acceptor for asn219; ii) the –nh= group of chloropyridine is functioning as hydrogen bond donor to glu518; and iii) the pyridine ring is involved in π–π stacking interactions with trp281 and his470 residues (fig. 6b). also, the ligand rings are involved in multiple hydrophobic interactions with residues of the binding site, e.g., pro513, trp281, val386, leu325, met525, his470 and ser387 (fig. 6a and b), leading to the stabilization of the ligand into the binding pocket. the amino acids arg400 and tyr394 which interact with compound 17, are also known to be important for the interaction with other smo antagonists. the relevance of these two amino acids in the antagonistic binding is supported by crystallographic studies.36–38 it can be seen from the fig. 6b that three pharmacophore features of the four of aahr.524 hypothesis are recapitulated in the best docking pose resulted from induced fit, only the interaction with the acceptor (a3) is missing. conclusions in order to discover the common features of inhibitors of smo receptor with potential anticancer benefits, ligand-based pharmacophore models were first generated. the best pharmacophore hypothesis turned out to be aahr.524, with the following characteristics: two hydrogen bond acceptors (a), one hydrophobic (h) site and one aromatic ring (r). the pharmacophore hypothesis was found to be statistically significant with the correlation coefficient, r2 of 0.859 for the training set and the correlation coefficient, q2 of 0.611 for the test set. virtual screening experiments and molecular docking were involved in the evaluation of the models and to gain insights into the ligand-receptor interaction profile. considering the large size of the binding site, as well as the different binding patterns of inhibitors to the site, our pharmacophore model being built on a congeneric series of inhibitors it is assumed to lead to increased specificity and reduced diversity in virtual screening experiments. however, we can say that the pharmacophore model was validated through a virtual screening experiment with reliable values of the evaluation metrics, e.g., auc of 0.795, and enrichment factors. in addition, it can be seen that our docking results reproduce the main interactions with key residues (e.g., asn219, arg400, tyr394 and asp384) found in the x-ray crystal structure (pdb id: 4jkv). between the two docking approaches used: glide and induced fit, the last one which accounts also for protein flexibility has led to better results. the validated pharmacophore model obtained suggests that it could ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 344 borota et al. be reliably used in predicting novel, promising inhibitors against smo receptor with potential anticancer activity. supplementary material additional data are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. this project was financially supported by project 1.1 of the “coriolan dragulescu” institute of chemistry, romanian academy. the authors thank to the chemaxon ltd. and openeye ltd. for providing an academic license. и з в о д in silico студије на заглађеном (smoothened) хуманом рецептору и његовим антагонистима у потрази за антитуморским ефектима ana borota, sorin avram, ramona curpan, alina bora, daniela varga, liliana halip и luminita crisan� “coriolan dragulescu” institute of chemistry, romanian academy, 24 mihai viteazul avenue, ro-300223, timisoara, romania у новије време, канцери повезани са ненормалним хеџхог (hh) сигналним путем (hedgehog (hh) pathway signalling) се испитују преко инхибиторима заглађених рецептора (smo, smoothened (smo) receptor inhibitors) који се брзо развијају. међутим, проблеми са познатим инибиторима попут озбиљних споредних ефеката, слабе моћи против солидних тумора или чак стечене отпорности тек треба да се превазиђу развојем нових погодних инхибитора. да би истражили структурне захтеве за антагонисте потребне за инхибицију smo рецептора урадили смо: мапирање фармакофоре, 3d-qsar моделе, претраживање база података и студије докинга. најбоља хипотеза за фармакофору је одабрана, на основу ње је развијен статистички значајан атомски 3d-qsar модел (r 2 = 0,856, q 2 = = 0,611 и pearson-r = 0,817), који је даље подвргнут скринингу, база података, како бу се проценила његова способност да издвоји активна једињења од лажно позитивних. запажена је ефикасност четверопараметарске (four-points) хипотезе фармакофоре (aahr.524) на бази добре метрике процена попут површине испод криве (0,795) и прецизности одмереног просека (0,835) што сугерише да је модел поуздан за предвиђање нових инхибитора за smo рецептор. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {the impact of coal ash and slag dump on the quality of surface and ground waters a case study} j. serb. chem. soc. 84 (5) 527–530 (2019) udc 662.613.11–032.35+66.046.58: jscs–5205 628.1.033+54.061 note 527 note the impact of coal ash and slag dump on the quality of surface and ground waters – a case study ivana ćipranić1, radmila marković2*, stefan đorđievski2, zoran stevanović2 and marija stevanović3 1university of montenegro, faculty of civil engineering, bulevar džordža vašingtona bb, 81000 podgorica, montenegro, 2mining and metallurgy institute bor, zeleni bulevar 35, 19210 bor, serbia and 3jp eps, eps jagodina, 7. jula 62, 35000 jagodina, serbia (received 29 january, accepted 4 february 2019) abstract: this paper presents the assessment of the impact of coal ash and slag from the “maljevac” dump on the quality of water of paleški creek, montenegro. the obtained results confirm the negative influence of surface and groundwater from the dump on the water from the paleški creek. the results obtained by testing of the water samples, collected from paleški creek upstream of the dump, indicate that the water is qualified to be used for drinking, after simple physical treatment and disinfection. the results obtained for the water samples collected from the paleški creek downstream from the dump indicate that the water could be safe for drinking only after the treatment that requires an intensive physical, chemical and biological processing, including some extended treatment. keywords: coal ash; coal slag; water quality. introduction although the material from ash and slag dumps may be used in the construction industry,1 the largest amount of this material is stored in dumps, where leaching of the elements with toxic effects occurs.2 such dumps pose a serious threat to the surrounding soil, surface and groundwater3 as well as to aquatic ecosystems.4 the ash produced from coal combustion is regarded globally as the waste which presents a serious health risk5 and which must be treated in an adequate manner. in order to provide sufficient space for the disposal of coal combustion by-products, ash and slag, generated at pljevlja thermal power plant, the earthen dam “maljevac” was built in 1982 in the riverbed of paleški creek, approx * corresponding author. e-mail: radmila.markovic@irmbor.co.rs https://doi.org/10.2298/jsc190129012c ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 528 ćipranić et al. imately 7 km from the town of pljevlja, montenegro. at the time it was the common practice to dispose of ash and slag waste directly onto the soil surface, without setting up any protection layers. hence, the waste material has been stored directly onto soil in the riverbed of paleški creek for decades. the aim of this paper is to determine the impact of coal ash and slag dump of a thermal power plant on the quality of ground and surface water. the impact evaluation is based on the investigations carried out in the area of the coal ash and slag dump “maljevac” of the thermal power plant in pljevlja (montenegro). results and discussion for the purpose of examining the effects of dumps on the surface water quality, the analysis of water in paleški creek was conducted as the dump directly influences the water of that stream (leachate from the dump is discharged into the stream). the water samples were collected upstream and downstream of the dump. the results of the water samples testing are presented in table i. the sampling points are marked in fig. s-1 of the supplementary material to this paper. table i. the results of the chemical and physical analyses of paleški creek, surface water samples from the dump, and groundwater samples collected from the boreholes on the dam parameter unit sampling point pw-1 pw-2 sw-3 sw-4 gw-5 ph – 7.5–7.8 10.7–10.96 11.8–12.2 7.3–7.45 5.84–6.02 κ µs/cm 390–430 863.5–980 11395–11500 1950–2250 2400–2920 fe mg/l 0.087–0.11 0.047–0.15 0.001–0.121 0.001–0.21 89–229.1 al mg/l 0.017–0.21 1.35–3.72 0.3–0.51 0.014–0.11 2.01–18.34 ca mg/l 85.7–101.2 366.2–400.5 1100–1370 130–284.1 330–447.3 na mg/l 2.08–5 19.67–48 12.3–15.83 36–80 18.35–44.92 zn mg/l 0.007–0.008 0.099–1.35 0.007–0.05 0.006–0.02 0.526–1.38 mg mg/l 6.55–8.32 0.59–2.4 11.89–16.4 68–107.92 216.4–324 mn mg/l 0.004–0.021 0.028–0.045 0.008–0.0135 3.54–0.777 7.67–11.34 p2o5 mg/l 0.24–0.45 0.059–0.09 0.055–0.09 0.006–0.099 0.071–0.15 k2o mg/l 0.78–1.3 239.4–320 24–61.36 16.78 8.3–10.55 pah mg/l < 0.000005 < 0.000005 0.000008 0.00042 0.00091 pcb mg/l < 0.000002 < 0.000002 0.000002 0.000002 0.000002 nitrates mg/l 3.81–8.33 1.99–5.42 5.32–8.29 2.82–3.25 35.2–42 nitrites mg/l 0.008–0.010 0.015–0.199 0.520 0.090 0.077–0.09 sulphates mg/l – 82 18.35–36.3 88–193 363–450 phenols mg/l 0.019 0.0008 0.061 0.0005 0.0005 cd mg/l – – – 0.003–0.02 0.02–0.018 as mg/l – – – – 0.08–0.1 the results obtained by the testing of the water samples collected from paleški creek, upstream of the dump, indicate that the water of the stream is qualified as clear spring water. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. impact of coal ash and slag dump on water 529 paleški creek downstream of the dump is characterized by a large number of the water quality parameters that are above the permissible limits (in montenegro, determined by the directive6): ph value, electrolytic conductivity, the content of sulphates, calcium, sodium, cyanide, etc. the water collected from paleški creek upstream of the dump is classified as “a1” category, while the one collected downstream of the dump is classified as “a3” category. according to the directive6, the category “a1” refers to the waters which can be used for drinking after simple physical treatment and disinfection. on the other hand, the category “a3” encompasses the water that could be safe for drinking only after the treatment requiring an intensive physical, chemical and biological processing, including extended disinfection and chlorination, coagulation, flocculation, decantation, filtration, activated carbon adsorption as well as disinfection with ozone and chlorine. the groundwater samples were surveyed from the two boreholes drilled in the body of the dam and in its plinth. based on the ph values presented in table i, it is noted that the mean value ranges from neutral to mildly acidic. even though the ph value of water near the surface is basic, the obtained mean value results from the fact that the dam was constructed of marly clay aterial considered acidic. electrolytic conductivity is increased. the elevated concentrations of calcium, magnesium, potassium, sodium, cadmium and sulphates are registered. the elevated concentration of ca is a result of calcium oxide dissolution from the coal ash. in addition, there are traces of arsenic within the borehole in the plinth of the dam. the core of the problem is closely related to the solubility of the ash components in water. in terms of the pollution control of ground waters, the so42– are the most significant as it does not bind tightly to the soil. due to this feature, sulphates can be used as “tracers”, i.e., it would be possible to determine the pollution level of ground water from the dump by means of tracking the ions.7 the water samples at the dump were collected from one point at the very surface of the dump. based on the obtained results it is concluded that these samples are characterized by increased the electrolytic conductivity. moreover, the ph value is alkaline. the ph value of the water from boreholes depends on the depth of the borehole. the minimal and the maximal concentrations from table i are similar, which indicates that there is a constant inflow of wastewater. conclusion the impact of the coal ash and the slag dump of the thermal power plant on the quality of surface and groundwater is exceedingly harmful. the quality parameters of the surface water samples (ph value, electrolytic conductivity, the content of sulphates, calcium, sodium, etc.) are above the permissible limits. the groundwater in the vicinity of the dump is characterized by the increased elec________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 530 ćipranić et al. trolytic conductivity, high concentrations of zinc, sulphates, calcium, magnesium, potassium, sodium, cadmium, etc. the harmful effects of the dump on the quality of surface and groundwater should be eliminated in these cases (when the dump was built directly on the ground, without necessary protection) by means of employing the adequate measures of remediation and recultivation. in addition, the material should be removed and used in making concrete and concrete blocks (used in construction industry) as well as for other industrial purposes. и з в о д утицај депоније угљеног пепела и шљаке на квалитет површинских и подземних вода – студија случаја ивана ћипранић1, радмила марковић2, стефан ђорђиевски2, зоран стевановић2 и марија стевановић3 1универзитет црне горе, грађевински факултет, булевар џорџа вашингтона бб, 81000 подгорица, црна гора, 2институт за рударство и металургију бор, зелени булевар 35, 19210 бор и 3јп епс, епс јагодина, 7. јула 62, 35000 јагодина у раду је приказан утицај угљеног пепела и шљаке са депоније маљевац на квалитет воде палешког потока у црној гори. добијени резултати потврђују негативан утицај површинске и подземне воде са одлагалишта на воду палешког потока. резултати добијени испитивањем узорака вода узетих из палешког потока узводно од одлагалишта указују да се вода квалификује као вода која се може користити за пиће након једноставног физичког третмана и дезинфекције. резултати добијени за узорке вода који су сакупљени из палешког потока низводно од одлагалишта указују да би вода могла бити безбедна за пиће само након третмана који захтева интензивну физичку, хемијску и биолошку обраду, укључујући продужени третман. (примљено 29. јануара, прихваћено 4. фебруара 2019) references 1. j. camilleri, m. sammut, f. e. montesin, waste manage. 26 (2006) 853 (https://doi.org/10.1016/j.wasman.2005.11.022) 2. y. s. shim, s. w. rhee, w. k. lee, waste manage. 25 (2005) 473 (https://doi.org/10.1016/j.wasman.2005.03.002) 3. a. popovic, d. s dordevic, energy sources, a 31 (2009) 1553-1560 (https://doi.org/10.1080/15567030802094003) 4. v. pandey, z. usmani, a. chandra, r. k. mishr, v. kumar, chem. ecol. 33 (2017) 777 (https://doi.org/10.1080/02757540.2017.1376663)) 5. b. gottlieb, s. g. gilbert, l.g. evans, coal ash-the toxic threat to our health and environment, a report from physicians for social responsibility and earthјustice, physicians for social responsibility, washington, dc, 2010 (https://www.psr.org/wpcontent/uploads/2018/05/coal-ash.pdf) 6. classification and categorization оf surface and renewable water, in official gazette of montenegro, no.2/07, government of the republic of montenegro, podgorica, 2007 7. m. vemić, r. vemić, in proceedings of iii consultation c ko cigre, 3-16.05.2013, przno, montenegro, 2013. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (dot gain 20%) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /error /compatibilitylevel 1.4 /compressobjects /tags /compresspages true /convertimagestoindexed true /passthroughjpegimages true /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.0000 /colorconversionstrategy /cmyk /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {a survey on the characterization and biological activity of isatin derivatives} j. serb. chem. soc. 85 (8) 979–1000 (2020) udc 547.756:57–188:615.28+542.9+ jscs–5353 547.571’551:539.16+519.677 review 979 review a survey on the characterization and biological activity of isatin derivatives saša ž. drmanić1#, predrag petrović2, dominik r. brkić3, aleksandar d. marinković1# and jasmina b. nikolić1*# 1department of organic chemistry, faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11000 belgrade, serbia, 2department of chemical engineering, faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11000 belgrade, serbia and 3belgrade polytechnic, brankova 17, 11000 belgrade, serbia (received 20 march, accepted 19 april 2020) abstract: the derivatives of isatin have already been known to display a variety of biological activities. therefore, the studies on their activity and its relation to structure have recently become a popular subject for investigation. the examined compounds were synthesized by the reaction of isatin and substituted primary amines and characterized by spectroscopic methods. the investigation of the antimicrobial and antioxidative activity of the synthesized compounds was performed by broth microdilution method. as for the characterization of the investigated isatin based schiff bases, the linear solvation energy relationships (lser) were used to analyze the solvent influence on the uv absorption maxima shifts (νmax), using the well known kamlet–taft model and taking geometrical isomers into consideration when possible. linear free energy relationships (lfer) were used to analyze substituent effect on pka, as well as nmr chemical shifts and νmax values. the antimicrobial activity and characterization were related using both experimental and theoretical methods. keywords: antimicrobial activity; e/z isomers; solvent effects; substituent effects; td-dft; 3d qsar. contents 1. introduction 2. antimicrobial and antioxidative activity of isatin schiff bases 3. solvatochromism of isatin based schiff bases: lser and lfer study 4. detailed structural and quantum chemical study related to antimicrobial activity 5. conclusions * corresponding author. e-mail: jasmina@tmf.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc200320020d ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 980 drmanić et al. 1. introduction the derivatives of isatin (indole-2,3-dione), its schiff and mannich bases, have been reported to show a variety of biological activities, such as antibacterial,1 antifungal2 and anti-hiv3,4 activities. the wide spectrum of isatin derivatives and their various chemical properties have led to their expanded use as precursors for the preparation of many biologically active compounds.5–11 isatin and its metabolites are components of many natural substances and display a wide range of the already mentioned activities such as antiviral, anticancer, antibacterial, antituberculosis, antifungal and anticonvulsants.5–11 our recently published study on the antimicrobial activity, as well as on the antioxidative activity of isatin derivatives showed their moderate to significant antimicrobial activity.12 to the best of our knowledge not much has been reported on the solvatochromic effects of isatin derivatives, therefore the studies were performed by our team recently.13,14 it is already known that the absorption spectra in different solvents are often used for investigation of the solvatochromic effect of organic molecules. when absorption spectra are measured in solvents of different polarity, it is usually found that the positions, intensities, and shape of absorption bands are influenced by the solvent. mostly used spectra which can provide information about solute-solvent are: uv–vis, ir, 1hand 13c-nmr spectra.15 in here presented studies,13,14 uv–vis and nmr data were analysed by the use of lser and lfer models, in order to evaluate the influence of the solvent/ /solute interactions and substituent effects, respectively. quantification of the solvent effects: dipolarity/polarizability and the hydrogen-bonding ability on the uv spectral shifts (νmax), were interpreted by means of the kamlet–taft (lser)16 equation: νmax = ν0 + sπ* + aα + bβ (1) later on, lfer analysis was applied to the uv and nmr data in of studied compounds. the transmission of substituent effects from the substituent r (see fig. 1) to the carbon atoms of interest were studied using equation:17–19 s hρσ= + (2) again, the transmission of substituent effects, i.e., lfer study, was discussed in relation to the geometry of the molecules obtained optimized by density functional theory (dft) calculations.13 furthermore, it is known that schiff bases show e/z isomerisation caused by the presence of imino group, which was analysed in the next part of our investigation.14 for example, β-phenylethylamines in the reaction with isatin form the mixtures of the two stereoisomers e and z, which can be thermodynamically or kinetically controlled, but their ratios may vary, depending on compounds structure and conditions.20–24 it was of interest to find out which of all the mentioned ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. a survey on isatin derivatives 981 characteristics of the isatin derivatives have the impact on their biological activity. fig. 1. the chemical structure of isatin derivatives with the labelling of π-electronic units.13 2. antimicrobial and antioxidative activity of isatin schiff bases the examined schiff bases were synthesized by the reaction of isatin and a corresponding primary amine,12 scheme 1, and the general formula is given in fig. 1.13 the list of synthesized compounds is given in table i. n h o n r n h o o + nh2r ch3oh scheme 1. isatin derivatives synthesis. table i. the synthesized derivatives of isatin12 no. r compound name 2.1 n n s sh 1,3-dihydro-3-[(2,4,5-thiadiazolone-2(3h)-thione)imino]-2h-indol-2-on 2.2 n s 1,3-dihydro-3-[(2-benzothiadiazole) imino]-2h-indol-2-on 2.3 cn 1,3-dihydro-3-[(4-cyanophenyl)imino]-2h-indol-2-one 2.4 n s no2 1,3-dihydro-3-[(5-nitro-2-thiazolyl)imino]-2h-indol-2-one 2.5 n ch3 1,3-dihydro-3-[(4-methyl-2-pyridyl)-2-imino]-2h-indol-2-on 2.6 no2 1,3-dihydro-3-[(4-nitrophenyl)imino]-2h-indol-2-one ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 982 drmanić et al. the compounds 2.1 and 2.5 were not synthesized before.2 compound 2.2 has already been used for metal complexes synthesis,25 compound 2.3* is commercially available, compounds 2.426 and 2.627 have also already been used for research of their antimicrobial activity. the structure of all synthesized compounds (2.1–2.6) from table i was confirmed by melting points, ftir, 1hand 13c-nmr spectra and also by elemental analysis for new compounds (2.1 and 2.5).12 the antimicrobial activity of all synthesized compounds 2.1–2.6 was determined on a wide range of different microorganisms (table ii) by the broth microdilution method.28 table ii. the examined bacteria and fungi types12 no. microorganism atcc no. 1 staphylococcus aureus 6538 2 lysteria monocytogenes 19115 3 enterococcus faecalis 29212 4 shigella sonnei 29930 5 salmonella enteritidis 13076 6 yersinia enterolitica 27729 7 escherichia coli 35150 8 proteus hauseri 13315 9 pseudomonas aeruginosa 27853 10 candida albicans 10259 all examined compounds have shown considerable activity against all tested microorganisms except for compound 2.3, which has shown rather weak activity against e. coli, p. aeruginosa and c. albicans, in the range of investigated concentrations. generally, the examined activity can be described as moderate with some selectivity against gram-positive (g+) or gram-negative (g–) strains of bacteria, or yeast c. albicans. the selectivity to g– bacteria is of importance, as it enables the antibiotic agent based on a g– selective compound to be taken without the support of an agent that recovers the gastrointestinal tract, because it contains g+ natural bacteria. the activity of certain examined isatin derivatives against fungi is also important for they can be applied as antifungal agents. the overall results of the antimicrobial screening are given in table iii. some of the isatin derivatives synthesized in this research have displayed significant activity against various examined bacteria and fungi, while the others were only moderately or even weakly active. compound 2.1 has shown the most prominent overall activity on both g+ (s. aureus and l. monocytogenes) and g– bacterial strains, (y. enterolitica and p. hauseri). the highest activity of this compound was noticed against the s. aureus * scientific exchange, inc., 105 pine river road, p.o. box 918, center ossipee, nh 03814, usa. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. a survey on isatin derivatives 983 (mic value of 0.16 mm) and close to it against l. monocytogenes (mic value of 0.32 mm). however, compound 2.1 has displayed only moderate activity on y. enterolitica, unlike compound 2.5, which was significantly active against the same strain (mic value of 0.33 mm). except the above mentioned, the overall antimicrobial activity of compound 2.5 is not as strong as that of compound 2.1. besides y. enterolitica, it has also shown some moderate activity on s. sonei and somewhat slighter on p. hauseri. compound 2.6 behaved somewhat similarly to compound 2.5 but without any really prominent antimicrobial activity. it has displayed only moderate activity to y. enterolitica and s. sonei. compounds 2.2 and 2.3 have generally shown comparably weak antimicrobial activity. the only observation that can be of interest is the relative selectivity of compound 2.3 to l. monocytogenes (mic value of 0.63 mm). compound 2.4 can be noticed for certain moderate activity against s. aureus, e. faecalis and also s. sonei.12 table iii. antimicrobial activity of examined compounds (mic / mm)12 microorganism compound 2.1 2.2 2.3 2.4 2.5 2.6 1. s. aureus 0.16 2.24 2.53 0.57 5.27 4.68 2. l. monocytogenes 0.32 1.12 0.63 2.28 2.64 1.17 3. e. faecalis 2.56 2.24 2.53 0.57 2.64 4.68 4. s. sonnei 1.28 1.12 5.06 0.57 0.66 0.59 5. s. enteritidis 2.56 1.12 >5.06 1.14 5.27 4.68 6. y. enterolytica 0.64 1.12 2.53 1.14 0.33 0.59 7. e. coli 2.56 1.12 >5.06 1.14 2.64 4.68 8. p.hauseri 0.64 1.12 5.06 1.14 0.99 1.17 9. p.aeruginosa 5.12 2.24 >5.06 2.28 2.64 2.34 10. c. albicans 2.56 2.24 >5.06 2.28 2.64 1.17 furhermore, all the synthesized compounds were screened for antioxidative activity by the diphenylpycrilhydrazyl radical (dpph).29 the results of dpph analysis have shown that the most prominent antioxidative activity displays compound 2.1, while the other investigated specimens, including pure isatin, have shown very slight if any activity. with the increase of the concentration of compound 2.1, the absorbance of dpph was decreased, displaying linear dependence of dpphred in the range of examined concentrations (c / mm), which are described by the following equation:12 dpphred / % = 5.099 + (101.02±5.24)c (3) r= 0.995, s= 3.17, n = 6 dpphred is actually the percent of dpph reduction and c is the concentration of compound 2.1, given in mm. this equation enables the precise determination of the concentration which reduces 50 % of dpph concentration ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 984 drmanić et al. (dc50). compound 2.1 showed prominent antioxidative activity, with dc50 value of 0.444 mm.12 3. solvatochromism of isatin based schiff bases: lser and lfer study another series of eleven isatin derivatives (1,3-dihydro-3-arylimino-2h -indol-2-one), including the compound 2.5 from the previous one (compound 3.7 in this series) was synthesized and the list of compounds is given in table iv.13 the characterisation of the synthesized isatin derivatives is described in the original paper.13 table iv. the list of synthesized isatin derivatives13 compd. compound substituent r 3.1 1,3-dihydro-3-(phenylimino)-2h-indol-2-one 3.2 1,3-dihydro-3-[(2-bromophenyl)imino]-2h-indol-2-one br 3.3 1,3-dihydro-3-[(3-bromophenyl)imino]-2h-indol-2-one br 3.4 1,3-dihydro-3-[(4-bromophenyl)imino]-2h-indol-2-one br 3.5 1,3-dihydro-3-[(3-methyl-2-pyridinyl)imino]-2h-indol-2-one n ch3 3.6 1,3-dihydro-3-[(4-methyl-2-pyridinyl)imino]-2h-indol-2-one n ch3 3.7 1,3-dihydro-3-[(5-methyl-2-pyridinyl)imino]-2h-indol-2-one n ch3 3.8 1,3-dihydro-3-[(6-methyl-2-pyridinyl)imino]-2h-indol-2-one n ch3 3.9 1,3-dihydro-3-(3-quinolinylimino)-2h-indol-2-one n 3.10 1,3-dihydro-3-(6-quinolinylimino)-2h-indol-2-one n 3.11 1,3-dihydro-3[2-(8-hydroxy)quinolinyl imino]-2h-indol-2-one n oh the uv absorption spectra were recorded in the range from 200 to 600 nm in 22 solvents of different polarity, in order to study substituent effect on the ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. a survey on isatin derivatives 985 solvatochromism of the investigated compounds. the absorption spectra of the isatin derivatives 3.1–3.11, indicate the presence of two bands, corresponding to different electronic transition of the investigated compounds. the examples of the uv–vis absorption spectra of compounds 3.1–3.11 in acetone, acetonitrile (acn), benzyl alcohol (bzoh) and n,n-dimethylformamide (dmf) are given in fig. s-1 of the supplementary material to this survey,13 and the complete results are given in the original paper.13 the shifts of νmax in uv absorption spectra showed relatively weak dependence on the both solvent and substituent effects compared to those of the parent compound 3.1. the data13 indicate that the values of absorption frequencies of the investigated compounds depend on the electronic effect of the substituent present on c3 position of indole-2-one core. the introduction of br as a substituent in c4 position of the phenyl ring (compound 3.4) and all quinoline derivatives (compounds 3.9–3.11) bring about the positive solvatochromism, comparing to the unsubstituted compound. the other compounds showed νmax shifting to higher wavelength in 2-chloroethanol, 2-methoxyethanol, dmf, dmac and acn, while in other solvents no rule could be observed (irregular behaviour regarding both substituent and solvent). the highest batochromic shifts was found for compoubd 3.9 in almost all solvents used. the kamlet–taft solvent parameters are taken from literature30 as well as substituent constants used in lser and lfer correlations.19 the presented data confirm that the positions of the uv–vis absorption frequencies depend on the nature of the solvent used and the substituent present at the aryl or phenyl ring at the imino group of the examined isatin derivatives.13 the results are presented in the original paper.13 according to the correlation results, obtained by the use of kamlet–taft equation,13 alternation of solvatochromic coefficients with respect to solvent/substituent effects exists. the positive sign of s and a coefficients for isatin derivatives (compounds 3.1, 3.3–3.5 and 3.9–3.11) indicates a hypsochromic shifts with increasing solvent dipolarity/polarizability and hydrogen-bond donor capability. largest hypsochromic shift of the absorption maxima, regarding coefficient s and a, were found for the compounds 3.5 and 3.9, respectively. the positive values of s and a coefficients suggest better stabilization of the ground state relative to the electronic excited state with the increasing solvent polarity, i.e., higher dipolar properties of the molecule in the ground state, with more pronounced hba (hydrogen bond acceptor) properties of solvated molecules.13 the percentage contribution of solvatochromic parameters for all isatin derivatives, showed the highest pπ value of compound 3.5 (71.01 %), with lowest pα value (2.28 %). oppositely could be noticed for compound 3.1 with highest value for pβ (70.72 %) and lowest pπ value (8.88 %). highest pα value was obtained for comp. 3.4 (51.28 %).13 from the correlation analysis obtained by the kamlet–taft equation, the negative sign of s and a coefficients, except for comp. 3.9 (positive values of ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 986 drmanić et al. 1.57 and 1.42, respectively), were found indicating a batochromic shift with the increasing solvent dipolarity/polarizability.13 this suggests better stabilization of the electronic excited state relative to the ground state, i.e., higher dipolarity/polarizability and hbd (hydrogen bond properties of solvated molecules in the excited state. alternation of the sign of coefficients b, positive values found for compounds 3.3, 3.4, 3.8 and 3.11 indicate better stabilization of the isatin derivatives in the ground state, in other words higher hba solvent interaction with solute molecule was found. for the other compounds the opposite behaviour was noticed. the non-specific solvent effect is a factor of the highest contribution to uv–vis spectral shifts of all the investigated compounds, except for compound 3.9 with the highest contribution of hba solvent effect (45.04 %), and compound 3.11 with the highest contribution of pα (44.15 %). the highest value of pπ was obtained for compound 3.5 (65.41 %). hba solvent effect is of lower contribution and the highest values of solvent hba effect was found for compound 3.9 (–2.45) and exceptionally low values were found for compounds 3.6–3.8 (–0.05, –0.06 and 0.02, respectively). the presented results, obtained using the kamlet–taft model, indicated that the solvent effects on uv–vis absorption spectra of the investigated isatin derivatives are rather complex, due to the diversity of the contribution of both solvent and substituent effect in studied compounds. this also indicated that the electronic behaviour of the nitrogen atoms in indol-2-on moiety is significantly different between derivatives with high contribution of localized hba effect, that originates from the electronaccepting quinoline and the methyl substituted pyridyl groups.13 the lfer concept was applied to the νmax and scs values of isatin derivatives with the aim to get an insight into substituent electronic effect on the absorption maxima shifts and nmr chemical shifts. an analysis, using lfer principles in the form of the eq. (2) was performed. the hammett substituent constants are given in the literature,13 and the obtained correlation results are presented in the original paper.13 the observed ρ values indicate different susceptibilities of the chemical shifts to substituent effects. the correlations were of reasonably good to high quality, which means that the scs values reflect the electronic substituent effects correctly. evidently the chemical shifts of c1’ showed an increased susceptibility and normal substituent effect. the presented results indicate that the contribution of both substituent effects, electron-donating and electron-accepting, have significant influence on electron-density shift in overall investigated molecule. the effectiveness of the transmission of substituent effects was determined by the conformational change of the investigated molecules which originate from the out-of-plane rotation of the aryl substituent, with respect to indole-2-one plane, defined by the torsion angle θ (fig. 1). the reverse substituent effect was observed at h1 and c2 carbon. the existence of these correlations was interpreted as an evidence of sub________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. a survey on isatin derivatives 987 stituent effect on the change of the electronic density over investigated molecule. the presence of an electron-accepting –c=o group at c2 position, and electrondonating amino group (n1-h), as a part of π1-electronic system of amide group, contribute to the intensive electronic interactions which exists in 3-(substituted imino)-2-pyrrolidone moiety.31 the correlations for c2 showed negative values of correlation coefficients ρ. the negative sign of the reaction constant, ρ, indicates a certain reverse behaviour, i.e., the value of scs decreases when the electron-withdrawing ability of the substituents, measured by σ, increases. such finding clearly reflect the behaviour opposite to normal polarization in keto group which comes from the contribution of two opposite effects: electronaccepting c2=o group and aryl substituents with the participation of the electron-donating effect of the indole nitrogen. magnitude of this effect strongly depends on the electron-accepting character of aryl substituents which cause the adequate/proportional reverse π-electron density shift from c2=o keto group. the contribution of the resonance interaction depends on spatial arrangement of the aryl moiety, and thus, the favourable conformation provides the effective transmission of the resonance effect to c2 and c3 carbons. according to that, it is expected that the presence of the electron-donating group support normal polarization of c2=o, and the electron-accepting group exert opposite effect, which is reflected in the reverse polarization. the normal polarization at c3 carbon is a reflection of the contribution of electronic effects in both π-electronic units (fig. 1), and primarily dictated by polarization of c=n imino bond which is more or less disturbed by change of substituent at phenyl (aryl) moiety. it is obvious that the chemical shifts of c1' show an increased susceptibility and normal substituent effect related to the substituent constant.13 the correlation results of uv–vis absorption maxima of the investigated compounds, obtained by the use of lfer principles indicate the influences of both solvent and substituent effect on uv–vis absorption maxima shift. these results indicate that the solvent effects: dipolarity/polarizability, hbd and hba abilities cause the appropriate sensitivity of the position of absorption maxima (νmax) to substituent effect.13 highly dipolar aprotic solvent dmf, and solvent with low hbd effect, acn, as well as those with high hbd effect, like ethanol, contribute to the opposite behaviour of the absorption frequencies shift at higher wavelength with respect to the substituent effects. aprotic solvents do not stabilize anions well, while they usually stabilize larger and more dispersible positive charges better. lower contribution of substituent effects in a solvent with higher relative permittivity can be explained by the fact that highly dipolar surrounding medium suppresses electron density shift inducing lower susceptibility of the absorption maxima shift to electronic substituent effects. similar behaviours were found for acn and ethanol, and it could be observed that solvent dipolarity/polarizability is the most sig________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 988 drmanić et al. nificant effect which contribute to the reverse relationship of νmax to substituent effects. the opposite substituent effect on νmax change was found in thf.13 in the two solvents, which showed hbd properties, namely acn and ethanol, the negative correlation slopes of lower wavelength peak was obtained for the former. somewhat higher sensitivity of νmax to substituent effect was found for acn (first set of solvents), and thf. in the dmf, ethanol and thf negative solvatochromism was obtained, indicating better stabilization of ground state of investigated compounds. somewhat lower values and similar trend of correlation slope was found for ethanol, due to lower contribution of solvent dipolarity/polarizability effect. this result suggests that the transmission of electronic substituent effects significantly depends on the conformation of studied molecules and solvent properties. the presented results showed that the transmission of substituent electronic effects through π-resonance units takes place by balanced contribution of two modes: through localized π-electronic unit and overall conjugated system of investigated compounds. their contribution depends on substitution pattern, as well as solvent properties. this fact implies that the electron density change is of localized/extended delocalization phenomena in compounds with the electron-acceptor substituents. the consequence of this is a lower substituent effect in a solvent with higher hydrogen bond accepting ability.13 an additional analysis of solvent and substituent effects on the measured absorption frequencies and the conformational changes of the studied compounds, needed theoretical calculations, i.e., geometry optimization and therefore the molecular electrostatic potential (mep) analysis were performed.13 the ground state geometries of compounds 3.1–3.11 were fully optimized with dft method. the theoretical absorption spectra of compound 3.1 were calculated in acetone, acetonitrile, ethanol, tetrahydrofuran, dimethylsulfoxide, formamide and toluene with time-dependent density functional theory (td-dft) method.13 geometry optimization of the investigated molecules was performed using b3lyp functional with 6-311g(d,p) basis set.13 the most stable conformations of compounds 3.1–3.11 are presented in fig. s-2 of the supplementary material. the elements of optimized geometries of calculated compounds are given in the original paper. the theoretical absorption spectra of compound 3.113 were calculated in acetone, acetonitrile, ethanol, tetrahydrofuran, dimethylsulfoxide, formamide and toluene by td-dft method, and showed very good agreement with the experimental data. the calculation of the optimal geometry, with the focus on determination of the value of torsion angle θ (fig. 1), gives valuable results required for better understanding of the transmission of substituent effects, i.e., electron density distribution. in the investigated molecules, these values are different and mostly depend on substituent present. somewhat larger deviation of θ was noticed for compounds 3.2, 3.5–3.8 and 3.11, indicating the significance of the extended ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. a survey on isatin derivatives 989 resonance interaction in the electron-donor substituted compounds. oppositely, in electron-acceptor substituted compounds the appropriate contribution of n,π -conjugation (nitrogen lone pair participation) to overall electronic interaction with π-electronic system of pyridone unit causes the perturbation of π-electron density. the geometric elements of the substituted isatin derivatives turned out to be similar to those for the unsubstituted one. the introduction of mainly electronaccepting substituent causes a decrease of c3=n bond length, i.e., a substituent supports normal polarization in the imino group, causing the appropriate shift of electron density to nitrogen. the presence of an electron-donating group attached to a complex structure of aryl moiety contributes to the electron density shift from the phenyl ring to the indol moiety, causing the increased molecule planarization. a decrease of the c2=o lengths is caused by the superposition of two effect: normal polarization in c2=o bond and the opposite effect of the electron -withdrawing group at c3 position; the consequence of this is a slight shifting of π-electron density to carbonyl c atom. therefore the length of n1–c2 gets longer, comparing to compound 3.1 as a result of the suppression of amide type of resonance.13 the larger deviation from the planarity was found for compounds 3.2, 3.7, 3.8 and 3.11 which is associated with ortho-effect of 2-br in compound 3.2, presence of the electron-donating methyl group situated at pyridyl in different position (compounds 3.7 and 3.8) as well as the strong electron-donating hydroxyl group in compound 3.11. the two opposite electron accepting effects operate all in investigated compounds: the electron-accepting aryl substituents and indole-2-one core which influence the appropriate geometrical adjustment of these molecules, as a response to the electronic demand of the electron deficient environment. except for the vicinity of the electron density at c2=o and nitrogen atom in compounds 3.5–3.8 and 3.11, which are also contributing factor to the increased deviation from planarity, due to the repulsion of negative potential present at these molecular fragments.13 the variation of substituent properties clearly indicates that the contributions of both conformations and donor–acceptor character are involved in the electronic transition of investigated compounds. the electron density of the investigated compounds is presented using mep analysis, which was used to evaluate the charge distribution over investigated compounds and to illustrate the three dimensional charge distributions overall the investigated molecules. mep potential, at a point in space around a molecule, gives the information about the net electrostatic effect produced at that point by the total charge distribution (electron + proton) of the molecule and correlates with dipole moments, electro-negativity, partial charges and chemical reactivity of the molecules. an electron density iso-surface mapped with electrostatic potential surface depicts the size, shape, charge density and the site of chemical reactivity of the molecules. mep shown in fig. s-3 of the supplementary material illustrates the three dimensional charge distributions overall the investigated molecules. as it ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 990 drmanić et al. can be seen from the fig. s-3, the different values of the electrostatic potential at the surface are represented by different colours; red represents regions of the most electronegative electrostatic potential, it indicates the region of high electron density, i.e., the sites favourable for electrophilic attack; blue represents regions of the most positive electrostatic potential, i.e., region of low electron density favourable for nucleophilic attack, and green represents regions of zero potential. the potential increases in the order: red<orange<yellow<green<blue. the blue colour indicates the strong attractive potential, the region favourable for hba solvent interaction, while red colour indicates the repulsive potential, and includes the sites favourable for hbd solvent interactions.13 there is an obvious negative potential at the oxygen atom, a part of the keto group (c2=o) in the indole-2-one structure, and nitrogen atom present in the structure of the aryl substituent. thus, most of the hba capabilities of the investigated compounds could be assumed to be from keto group (c2=o) and aryl nitrogen which are electron -acceptor groups and cause the increase of electron density at these two sites, therefore creating a favourable interaction with proton-donating solvents.13 in the molecules with heterocyclic structure the nitrogen present acts as a centre that shows significant proton-acceptor interaction with solvent molecules. non-planarity of the investigated molecules significantly contributes to the electron density distribution and therefore the electron density transfer occurs due to the both solvents and substituent influence. the resonance effect of nitrogen atom increases the substituents negative potential, which causes the significant contribution of hbd solvent effect. in cases where nitrogen heteroatoms are in the ortho position, relative to the c=n bond (compounds 3.6–3.8), some steric hindrance is observed, and increase of the electron density of nitrogen as a result of the obtained conformation of the investigated compounds which results in reduction of the electron density in the rest of the heterocyclic ring. these factors contribute to the increase of the proton-accepting capability with solvent molecules that have pronounced hbd effect.13 the influence of the substituents electronic effects when they are in a different position on the geometry and electron density distribution was observed in compounds 3.2–3.4. the effect of a bromine atom, which shows a negative inductive and a positive resonance effect on the electron density distribution over the compounds 3.2–3.4. significantly depends on the substituent position, i.e., ortho, meta or para-position. the electron-acceptor effect of the bromine atom affects the shifting of the electron density to a substituent which causes significant change in the geometry of the ortho, meta and para-derivatives. the consequences are the differences in electron density distribution and therefore some different interactions with a solvent.13 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. a survey on isatin derivatives 991 4. detailed structural and quantum chemical study related to antimicrobial activity the list of compounds synthesized in this part of the research (the general formula given in fig. 1), namely the third series, and their elemental analysis are given in table v.14 only the compounds 4.1 and 4.16 were used before (labeled as the compound 3.1 the third chapter and the compound 2.6 in the second chapter, respectively). table v. the list of synthesized isatin derivatives14 no. compound substituent 4.1 1,3-dihydro-3-(phenylimino)-2h-indol-2-one h 4.2 1,3-dihydro-3-[(2-hydroxyphenyl)imino]-2h-indol-2-one 2-oh 4.3 1,3-dihydro-3-[(3-hydroxyphenyl)imino]-2h-indol-2-one 3-oh 4.4 1,3-dihydro-3-[(4-hydroxyphenyl)imino]-2h-indol-2-one 4-oh 4.5 1,3-dihydro-3-[(2-methoxyphenyl)imino]-2h-indol-2-one 2-och3 4.6 1,3-dihydro-3-[(3-methoxyphenyl)imino]-2h-indol-2-one 3-och3 4.7 1,3-dihydro-3-[(4-methoxyphenyl)imino]-2h-indol-2-one 4-och3 4.8 1,3-dihydro-3-[(2-chlorophenyl)imino]-2h-indol-2-one 2-cl 4.9 1,3-dihydro-3-[(3-chlorophenyl)imino]-2h-indol-2-one 3-cl 4.10 1,3-dihydro-3-[(4-chlorophenyl)imino]-2h-indol-2-one 4-cl 4.11 1,3-dihydro-3-[(2-iodophenyl)imino]-2h-indol-2-one 2-i 4.12 1,3-dihydro-3-[(3-iodophenyl)imino]-2h-indol-2-one 3-i 4.13 1,3-dihydro-3-[(4-iodophenyl)imino]-2h-indol-2-one 4-i 4.14 1,3-dihydro-3-[(2-nitrophenyl)imino]-2h-indol-2-one 2-no2 4.15 1,3-dihydro-3-[(3-nitrophenyl)imino]-2h-indol-2-one 3-no2 4.16 1,3-dihydro-3-[(4-nitrophenyl)imino]-2h-indol-2-one 4-no2 imino compounds can arrange themselves into eor z-configuration around the c=n double bond and the chemical environment of the azomethine carbon (c3) is different for such isomers. the investigated isatin derivatives showed chemical shift of the azomethine carbon in the range of 150.43–156.72 for e isomer, and 150.90–155.16 for z isomer.14 the stereochemistry of the imines was established using 2d homonuclear (cosy and noesy) and heteronuclear (hsqc and hmbc) nmr techniques.14 as an example, the chemical shifts of compound 4.10 were assigned by a combined use of one-dimensional (1h and 13c proton decoupled)14 and two-dimensional nmr experiments (cosy, noesy, hsqc and hmbc)14. in order to assess conformation of 4.10 in a solution, two-dimensional noesy sequence was applied.14 the e stereochemistry turned out to be the major isomer in dmso-d6 solutions as determined by the noesy experiments that showed proximity of c4-h and c2'-h atoms.14 in addition, the signal for h1 (see fig. 1) of the e-isomer was shifted considerably up-field relative to the h1 signal of the parent compound isatin. more________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 992 drmanić et al. over, the chemical shifts of the proton present in z-isomer showed little difference to those found in parent compound.14 the 7.5:2.5 isomer ratio for compound 4.10 in dmso-d6 was obtained from 1h-nmr spectrum.14 analogously, the analyses of 1h-nmr data of n1–h1 (see fig. 1) proton were used for the calculation of e/z isomer ratio of other studied compounds.14 the obtained results showed that e form dominate in dmso solution, without regard to the electronic influences of substituents. four compounds with 2and 4-no2, and 3-oh and 3-och3 group showed presence of only e isomer. on the other hand, the influence of electron-donating groups causes the stabilization of z isomer. such properties could contribute to the change of physico-chemical characteristics and to the differences in biological activity, within these compounds as a function of varying isomer compositions. as a continuation of the presented work, the determination of acidity constants was carried out using uv–vis spectroscopy. in hydroxy substituted compounds two pka values were obtained corresponding to the dissociation of proton on oxygen (ph 8.00–9.00) and isatin nitrogen (ph 9.68–12.11). acidity constants, ka, ka1 and ka2 values, were calculated according to the literature method.32 the obtained pka, pka1 and pka2 values are given in the original paper.14 ka and ka2 represent the dissociation of the amide n-h hydrogen, while ka1 corresponds to the dissociation of the hydroxy substituted isatins.14 the variation of the values of ionization constants showed the dependence on electronic substituent effects, as well as an appropriate contribution of steric effect in compounds 4.2, 4.5, 4.8 and 4.11. similar pka and pka2 values of compounds 4.15 and 4.16, with respect to 4.3 and 4.4, showed unexpected behaviour, when the transmission mode of substituent effects through π1and π2-units (fig. 1) is considered. the electron-donating capability of hydroxy group in compounds 4.2–4.4 contribute to the slight decrease of pka values. unexpectedly high pka values, vice versa lower acidity, of compounds 4.14–4.16, in comparison to 4.2–4.4, is a result of appropriate degree of the attenuation of the transmission of substituent effect through azomethine bond and competitive π-conjugative interactions in π1 and π2-units.14 on the other hand, the values of ionization constants of compounds 4.5–4.13 behave in a regular fashion indicating that the position-dependent influences of substituent effects are the main contributing factor to changes of pka. the trends of increasing of the pka in compounds 4.5–4.13, are followed by pka values increase as the electron-withdrawing capability of substituent increase.14 in order to understand and quantify the influence of structural effect and ionization constants of prospective sites at investigated molecules lfer analysis, the already mentioned hammett’s ssp method was performed. the obtained correlation results14 clearly pointed out that the relationship pka vs. σ showed a significant influence, of both electron-accepting and electron-donating substi________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. a survey on isatin derivatives 993 tuent effects, on the change of acidity of n-h hydrogen on isatin ring. the reverse substituent effect on pka values change means that the increased electron -accepting power of a substituent cause the increase of pka value, although σ increases. higher sensitivity of pka to substituent effect was found in second series of compounds due to the contribution of both steric and strong electron-accepting character of substituents.14 in order to get some deeper insight into solvent and substituent effects on the conformational changes of the isatin based compounds, the quantum-chemical calculations were performed. geometry optimization and charge density analysis was performed by the use of mp2 method with 6-311g(d,p) basis set. the significant stability of the compounds 4.1–4.3, 4.6–4.9 and 4.11–4.13 in e form were obtained and in addition, the geometries of the investigated molecules were fully optimized by the use of mp2 method, the detailed elements of optimized geometries are given in the original paper.14 non-consistent substituent dependent variation and some noticeable differences in the trend of the elements of the optimized geometries of both forms of isatin derivatives were registered. in general, low/moderate influences of electronic substituent effects could be noticed. small variation of n1–c2 bond length indicates a low influence of substituent effect, i.e., a low contribution of resonance in interaction in amide group (π3-unit), and low cross-interaction with π1-unit. simultaneously, c2–c3 bond length increase in compounds containing strong/moderate electron-donating hydroxy/methoxy substituent in 2and 4-position of e and z isomers, excluding compound 4.5 in e, and 4.2 and 4.5 in z form.14 contrary to that, low increase was observed for meta-substituted derivatives. such trend of c2–c3 bond length change indicates that two opposite electron accepting effects operate in the π2-unit: the electron accepting phenyl substituted ring and c2=o carbonyl groups, as a response to the electronic demand of the electron deficient environment. the normal carbonyl groups polarization is suppressed and causes a slight bond length decrease.14 the introduction of both electron-donor and electron-acceptor substituents causes the increasing of c3=n3 bond length in all e isomers, except compound 4.14 (2-no2). the results are clearly opposite for the compounds in z isomers where the highest increase of bond length were found for 2and 4-oh and for 2 and 4-och3 substituted compounds.14 the opposite trend is displayed when electron-acceptor (such as no2 group) is present. the compounds with ortho-, metaand para-halogen substituent showed similar values for n1-c2 and c3=n3 bond length in derivatives in e isomers, except compound 4.11. presence of a halogen cause the decrease of the n3-ph bond length in all e isomers with the largest effect found for ortho-substituted compounds. this result reflect the effect of the extended conjugation operative in the π1and π2-unit on the π-electron density shifts towards c2=o carbonyl group and supporting n,π-conjugation in ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 994 drmanić et al. the π3-unit.14 a decrease in the n3-ph bond length, which is a part of the aniline π-electronic system (π1-unit), contribute to the greater extent of the n,π-conjugation. this bond is slightly longer in the electron-donor 3-oh/3-och3 substituted derivatives. on the other hand, in z isomer generally, all n3-ph bond length are lower for orthoand para-substituted ones than in compound 4.1, while the bond increase were observed for all nitro substituted compounds. these results implied that the attenuation of the resonance interaction was the process of lower significance in compounds 4.14–4.16 as a result of competitive cross -interactions of π1and π2-units.14 better understanding of the transmission of substituent effects was based on the value of torsion angle θ (fig. 1) which contributes to the extended conjugation. more planar structure provides higher contribution of the extended π-conjugation which in turn produces bathochromic shift in uv spectrum. the values of the torsional angle θ for e isomers are fairly similar, except in 2and 4-substituted compounds, e.g., 2-oh and 2-och3, indicating the significance of the extended resonance interaction in electron-donor substituted compounds. an electron-donor substituent (hydroxy and methoxy) supports the electron density shift from the π1-unit (substituted phenyl ring) to the isatin moiety, causing the whole molecule planarization in a greater extent. also, the deviation from the planarity rises with the increasing steric effect of phenyl substituent, except for compound 4.7 in z isomer (4-och3) with almost planar geometry (θ = 0.01). comparing to the unsubstituted z isomer, larger values of θ were obtained for halogen and nitro-substituted compounds. in these compounds the contribution of n,π-conjugation (nitrogen lone pair participation) to overall electronic interaction cause perturbation of π-electron density in the molecule as a whole. the deviation from the planarity increases with the decreasing electron acceptor ability of the arylidene substituent.14 in the next step, the mechanism of electronic excitations and the electron density distribution in ground and excited states was studied by the calculation of homo/lumo energies (ehomo/elumo) and egap values. td-dft results indicated a significant contribution of a single particle homo to lumo excitations in ground to the first excited state transition, higher than 60 % for all calculated compounds, except for compound 4.15 (47 %) and 4.16 (48 %), for isatins in z form. similar results were obtained for compounds in e form (higher contribution than 59 % for all compounds) except compound 4.11 (40 %).14 a lower egap values were observed for all compounds in z form, except of the compound 4.2. concerning electron-donor substituents, small influences on egap changes could be observed, and the lowest values were found for hydroxy and methoxy-substituted compound, i.e., compounds 4.4 (5.59) and 4.7 (5.60), respectively. according to td-dft analysis, the solvent effects on the change of ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. a survey on isatin derivatives 995 ehomo/elumo and egap showed a little difference between the results obtained for the gas phase and the calculations involving solvent dmso, etoh and acn.14 an illustration of the differences of atomic charges in the excited and in the ground state (δcharge) for the appropriate atoms are given in fig. s-4a (e isomers) and s-4b (z isomers) of the supplementary material.14 the presence of an electron-donor substituent (hydroxy and methoxy) in e isomers (fig. s-4a), cause a decrease of electron density on c1’ (except compound 4.2, 2-oh), imino carbon c3 and imino nitrogen n3, compared to unsubstituted compound 4.1. otherwise, the presence of halogen substituents cause an increase of the amount of charge (δcharge) on c1’ and imino nitrogen n3, while imino carbon c3 has the similar values as compound 4.1. effect of electron-accepting character of nitro group in e isomers induce reduction of the electron density on c1’ as well as imino on nitrogen n3 and increasing electron density on imino carbon c3 relative to compound 4.1.14 relative to z isomer of the unsubstituted compound 4.1 (fig. s-4b) value of the amount of charge (δcharge) on c1’ and imino carbon c3 is decreasing if the hydroxy group is in ortho and para position, as well as for methoxy group in para position. oppositely, the electron density on imino nitrogen n3 in presence of 2-oh and 4-ome, compounds 4.2 and 4.7, increases respectively. all chloro -substituted z isomers, including compound 4.13 (4-i), have a larger amount of charge (δcharge) on c1’, in comparison to compound 4.1. therefore, if the halogens are in ortho position, the imino carbon c3 and the nitrogen n3 gain δcharge. the presence of the electron-accepting nitro group causes the decreasing of electron density on c1’ and imino carbon c3, while opposite is true for the imino nitrogen n3. the electronic charge change upon transition, presented separately for e and z isomers, give a picture on local/overall electron densities shift.14 additionally, the intramolecular charge transfer (ict) was interpreted with the aid of the td-dft calculations related to the distance between two centres of the density depletion and the density increment zones (dct) and amount of transferred charge (qct) in the course of excitation, the detailed results are given in the original paper.14 according to the results, it could be seen that in both forms the distinct ict processes were observed with electron density transfer from the isatin ring to the substituted phenyl in the course of transition. it is observed that the strongest ict take place in molecules 4.14 (2-no2) for z isomers and 4.6 (3-ome) for e isomers, demonstrating the transfer of 0.732 e– over 2.131 ǻ and of 0.776 e– over 2.608 ǻ, respectively. on the other hand, in molecule 4.7 (4-methoxy substituent), the intramolecular charge transfer of 0.703 e– over 0.156 ǻ becomes a local process which occurs within imino bridging group (fig. s-5 of the supplementary material).14 the variation of substituent patterns clearly indicate that the contributions of both conformational arrangement and donor–accepting character are involved in ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 996 drmanić et al. the ict mechanism of the investigated molecules. the decreasing trend, in order from ortho to para position, of the qct in z isomers, for halogen (chloro and iodo) and nitro substituted isatin derivatives, can be observed from the results.14 on the other hand, qct values change of nitro substituted isatin derivatives in e isomers showed the increasing trend.14 for hydroxy and methoxy substituted derivatives in both e and z isomers, and chloroand iodo-substituted compounds in e isomers the value of calculated qct for ortho, meta and para position showed non-linear relationship. the specific behaviour of hydroxy substituted isatin derivatives in e and z forms and methoxy in e form was reflected in higher qct values, which are found for meta substituted compounds. on the other hand, the methoxy substituted compounds in z form and chloro and iodo substituted ones in e form showed the lowest value for meta substituted isatin derivatives.14 in our research,14 as the continuation of the previous paper,12 the isatin derivatives were tested for their antibacterial activity against staphylococcus aureus +, listeria monocytogenes +, shigella sonnei –, yersinia enterocolitica –, escherichia coli –, proteus hauseri –, pseudomonas aeruginosa –, cryptococcus neoformans and candida albicans, by the already used broth microdilution method. the obtained results are presented in table vi. compounds 4.7 (4 -ome), 4.12 (3-i) and 4.13 (4-i) were excluded from the antimicrobial analysis due to poor solubility in 10 % dmso. all the other compounds showed activity on some of the microbial strains used in the study.14 the activity of the investigated isatin derivatives against the same microbial strains is similar in most cases, with few exceptions. however, there is a trend of considerable activity of ortho-substituted hydroxy and chloro derivatives and orthoand para-nitro deritable vi. mic values (μg ml-1) of isatin derivatives against the investigated microbial strains;14 compounds 7, 12 and 13 are excluded from the table vi due to not having antimicrobial activity microorganism compound 1 2 3 4 5 6 8 9 10 11 14 15 16 s. aureus 1250 1250 1250 >1250 1250 1250 1250 1250 >1250 1250 1250>1250 1250 l. monocytegenes 1250 313 >1250>1250 1250 1250 313 1250 1250 1250 313 1250 313 s. sonnei 625 313 1250 625 625 625 313 625 1250 >1250 313 1250 156 y. enterocolytica 1250 156 >1250 313 1250 625 313 625 1250 1250 156 1250 156 e. coli 1250 1250>1250>1250>1250 1250 1250>1250>1250>12501250 1250 1250 p. hauseri 1250 313 >1250 1250 1250 1250 313 1250 1250 156 313 1250 313 p. aeruginosa 625 1250>1250>1250>1250>12501250>1250 1250 1250 625 1250 625 c. neoformans 156 19 39 39 156 >1250 39 156 156 19 39 156 39 c. albicans 1250 313 >1250>1250>1250>1250 313 625 1250 1250 1250 1250 313 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. a survey on isatin derivatives 997 vatives against most of the strains. while former are electron-donors and latter an electron-acceptors, they are all electron-negative groups, which could be an important property for the antimicrobial activity. the difference is most pronounced in the case of compound 4.2 (2-oh) and compound 4.3 (3-oh) activity against y. enterocolitica, which is more than 10 times greater in former (mic = 156 mm for 2-oh and mic > 1250 mm for 3-oh).14 there is no difference in activity against g+ and g– bacteria in general, but some strains are more susceptible, as l. monocytogenes, s. sonnei, y. enterocolitica and p. hauseri.14 as for fungal strains, c. neoformans was shown to be far more sensitive to the tested compounds than c. albicans, while the mic values for c. albicans were similar to those for bacterial strains. the mic values obtained for c. neoformans were, in some cases, even 100 times greater (compound 4.2; 2-oh and compound 4.11; 2-i, mic = 0.019 mm). compounds 4.3 (3-oh), 4.4 (4-oh), 4.8 (2-cl), 4.14 (2-no2), 4.16 (4-no2) also showed prominent antifungal activity against this human pathogen (mic = 0.038 mm).14 the overall activity of the compounds was most pronounced against the fore mentioned c. neoformans. compounds 4.2 (2-oh) and 4.8 (2-cl) showed the best activity against c. albicans (mic = 0.313 mm) of all investigated compounds. the obtained results suggest that the other substituents present on phenyl or heterocyclic ring of the isatin derivatives should be tested.14 in order to explore the structural properties important for the antimicrobial activity of compounds 4.1–4.16, qsar models were generated, taking the mic values for s. sonnei, y. enterocolitica as well as c. neoformans but the statistically significant model was obtained only for the activity of compounds toward c. neoformans.14 principal component analysis (pca) was performed using the whole set of grind-2 descriptors. model with 3 principal components (pc) explained 77.78 % of x sum of squares (ssxacc), and 70.22 % of x variance (varxacc).14 partial least squares regression model (pls) was created in order to correlate he structural features of compounds with antimicrobial potency toward c. neoformans. after filtering of descriptors through 2 cycles of ffd, two latent variables (lv) model with r2 = 0.89, q2 (leave-one-out, loo cross-validation) = = 0.69, and standard deviation of error of prediction (sdep) = 0.29.14 from the pls coefficients plot (fig. s-6 of the supplementary material) several variables important for the activity were identified. the most informative variables in pls model are the one that clearly separate the more potent compounds from the less. structural motifs associated with the most informative variables were found.14 the positively correlated variables are depicted on compound 4.2 as a model for the more potent compounds (fig. s-7 of the supplementary material). the variables with the negative influence on potency are ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 998 drmanić et al. depicted on the least potent compound 4.6 (fig. s-8 of the supplementary material).14 5. conclusions a series of 30 isatin derivatives, some of them new compounds, were synthesised in order to examine their properties and the application potential. their structures were confirmed by spectral methods. the antimicrobial screening was performed, and some compounds showed relative selectivity to certain bacterial strains. in order to test antioxidative activity, dpph reduction test was performed on some of the compounds, which should be continued. from the solvatochromic and characterization point of view uv–vis and nmr data were analysed by the use of lser and lfer principles and it could be concluded that solvent effects have significant influence on the transmission mode of substituent effects. the quantum chemical calculations, performed next, showed that the present substituents significantly change the extent of conjugation. then, the e/z isomer ratios of different investigated isatin compounds were estimated from the point of nmr data, theoretical calculations and uv–vis spectra. the inclusion of solvent effects in the td-dft calculations demonstrated that substituents, depending on their position in molecules, and solvent effects significantly affect the ict character. the testing of antimicrobial activity was continued further on isatin derivatives, with the aim of relating the activity to the compound structure. the most of the investigated isatin derivatives exhibited moderate activity against bacterial strains, but compounds with hydroxy, nitro group and chlorine in ortho position as well as para nitro showed considerable activity. the 3d qsar model created pointed on hydroxyl groups as substituents important for the potency of compounds toward fungal strain c. neoformans. this complex research was performed to study the examined compounds for their potential biological activity as well as to link activity with molecular structure. the aim was to determine compounds that should be submitted to further investigation, since they have a considerable chance for practical use, particularly as active compounds of medicines. acknowledgement. this work was supported by the ministry of education, science and technological development of the republic of serbia (contract no.451-03-68/2020-14/ /200135). supplementary material additional data are available electronically at the pages of journal website: https:// //www.shd-pub.org.rs/index.php/jscs/index, or from the corresponding author on request. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. a survey on isatin derivatives 999 и з в о д преглед карактеризације и биолошке активности деривата исатина саша ж. дрманић1, предраг петровић2, доминик р. бркић3, александар д. маринковић1 и јасмина б. николић1 1катедра за органску хемију, технолошко–металуршки факултет, универзитет у београду, карнегијева 4, 11000 београд, 2катедра за хемијско инжењерство, технолошко–металуршки факултет, универзитет у београду, карнегијева 4, 11000 београд и 3београдска политехника, бранкова 17, 11000 београд деривати исатина су познати по потенцијалној биолошкој активности, па су постали интересантни за проучавање везе између наведене активности и њихових структурних карактеристика. наш тим их је годинама истраживао са оба аспекта и у овом прегледном раду су приказани сви резултати од значаја до којих смо дошли. испитивани деривати исатина су синтетисани реакцијом између исатина и различитих примарних амина, окарактерисани су спектроскопским методама (ftir, nmr) као и елементалном анализом. на њима су извршена и разна теоријска проучавања као и одређивање антимикробне активности, како би се стекла што потпунија слика о вези између њихове структуре и активности и одредила једињења за даља испитивања због њиховог потенцијала за примену. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {study on the removal of no from flue gas by wet scrubbing using naclo3} j. serb. chem. soc. 84 (10) 1183–1192 (2019) udc 66.074+546.172.6:66.08.3+ jscs–5256 546.33’131:66.095.828 original scientific paper 1183 study on the removal of no from flue gas by wet scrubbing using naclo3 deqi shi, guoxin sun and yu cui* school of chemistry and chemical engineering, university of jinan, no. 336, west of nan xinzhuang, jinan, 250022, shangdong, p. r. china (received 5 march, revised and accepted 17 may 2019) abstract: in order to remove nitric oxide (no) from flue gas, from small coal -fired boilers, it is necessary to exploit the cost-effective wet denitration technology. the absorption of no with sodium chlorate solution was studied. the effects of experimental conditions, such as temperature, naclo3 concentration, type of acid, mole ratio of naclo3 to hydrogen ions, on no removal rate were investigated, and the optimal conditions were established. as the effect of temperature on denitration was related to the type of acid used, the temperature required for sulfuric acid was high, and the temperature required for nitric acid was low. the optimal mole ratio between naclo3 and the two types of acids was the same. the reaction products were analyzed by ion chromatography. the reacted solution could be recycled after the removal of sodium chloride. the reaction mechanism and the total chemical reaction equation of naclo3 denitration were deduced. the thermodynamic derivations showed that this oxidation reaction could proceed spontaneously and the reaction was very thorough. naclo3 exhibited high no removal efficiency and its denitration cost was much lower than sodium chlorite. keywords: nitrogen oxides; oxidation; absorption; naclo3; denitration. introduction air pollution caused by the burning of fossil fuels is increasingly serious, and the nox emitted by the fuels can lead to atmospheric ozone depletion, acid rain1,2 and visibility problems, through a series of complex reactions with water and oxygen.3 denitration methods such as selective catalytic reduction (scr) and selective non-catalytic reduction (sncr) have been extensively studied proved to be a great success in large power plants. however, due to the high cost of scr and the low efficiency of sncr, industrial denitration process4 is still immature in * corresponding author. e-mail: chm_cuiy@ujn.edu.cn https://doi.org/10.2298/jsc190305053s ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 1184 shi, sun and cui the practical application of small coal-fired boilers.5,6 in addition, the ammonia denitration efficiency is low due to the large temperature difference of flue gas from small boilers. these technologies can not be applied to the treatment of dusty flue gases in steel, ceramics and cement plants. a large amount of dust in the flue gas can easily block the catalyst channel and cause catalyst poisoning. the development of efficient denitration technology is of great significance to environmental protection in various countries. the wet denitration technology is less affected by temperature in removing dust. it requires less investment and is suitable for coal-fired boilers in oil wells and remote areas. therefore, it is necessary to develop economical and efficient wet denitration technology.7–11 wet denitration technology is one of the most popular technologies in industrial scale in recent years. the method uses a strong oxidant to oxidize no to nitrogen dioxide (no2),12,13 which can be absorbed by water or alkaline solution. up to now, a varity of oxidants, such as clo2,14 kmno4/naoh,15 naclo2,16–18 naclo2/naoh,19–21 naclo2/(nh2)2co,22 h2o223,24 and water-soluble ferrous -chelating agents,25 may be included. among these oxidants, naclo2 showed better oxidation performance and the industrial application test was carried out. however, the treatment cost of the system was too high to be a practical nox control process. in order to reduce the denitration cost and improve the denitration efficiency, naclo3 was used to remove nox from simulated flue gas in this paper. one molecule of naclo3 can oxidize more no than the one of commonly used naclo2, while the price of naclo3 is only one third of naclo2. the reacted liquid could be reused after removing nacl from the solution. therefore, developing the denitration technology with naclo3 has a good prospect for the wet denitration of industrial flue gas. however, there are few basic researches on naclo3 denitration technology. in this research, a new denitration solution made of naclo3 was used for the removal of no in a bubble reactor. various influencing factors on the removal efficiency of no were measured, in order to determine the optimal experimental conditions. the reaction mechanism and the total chemical reaction equation for denitration using naclo3 were deduced. this method might be applied to an industrial scale denitration process. experimental materials the analytical grade reagents used in experiment were h2so4 (95–98 wt. %) and hno3 (65–68 wt. %), obtained from laiyang chemical reagent factory, china. naclo3 (purity > 99 wt. %) was obtained from tianjin chemical reagent factory, china. standard gases, n2 (99 %) and no/n2 (2.02 vol. ‰ no) span gas, were obtained from oxygen co., ltd., china. reverse osmosis water was applied to prepare the solutions. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. removal of no by wet scrubbing 1185 experimental setup the experimental system of this research consists of three parts: a flue gas simulation section, a bubble reactor and a nitrogen oxide analyzer. a schematic diagram of the experimental device is shown in fig. 1. fig. 1. schematic diagram of the experimental setup. nitrogen and no/n2 were obtained from cylinders and metered through mass flow controllers. the total flow of blended gas was kept at 160 ml/min. the simulated flue gas was then introduced into the mixer and no was diluted to 800 ppm. the absorption experiments were performed in a bubbling reactor. the temperature of the absorbing liquid (solution volume = 500 ml; liquid height = 12 cm) was controlled by a water bath. the solution was stirred magnetically and the simulated flue gas entered the bubbling reactor. the concentration of nox was continuously recorded by flue gas analyzer. in order to protect the flue gas analyzer, the gas coming from the reactor passed through a drying tube containing anhydrous calcium chloride and a scrubber with phosphoric acid. data analysis when the simulated flue gas flowed through the aqueous solutions of naclo3, no reacted with the oxidant and was removed. the removal efficiency of no in % was defined as: in out in no nox f no 100 c c e c − = (1) where ef is the efficiency of no removal, cnoin and cnoxout represent the inlet and outlet gas concentrations, respectively. the denitration rate was calculated by the integral method. results and discussion contrast study in different systems naclo3 was used to remove no in a bubble reactor and a series of comparative experiments were carried out under different conditions. the result is shown in fig. 2. no removal efficiency was 29.11 % in water, 28.82 % in h2so4 solution and 29.63 % in hno3 solution. the removal efficiency of no was only 29.52 % by naclo3 solution without the presence of acid, which was just similar to water. however, the removal efficiency of no achieved 84.40 % by the com________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 1186 shi, sun and cui bination of 0.50 % naclo3 and 0.60 mol/l hno3. similarly, the removal efficiency of no in the mixed solution of naclo3 and h2so4 reached 86.17 %. the results showed that naclo3 combined with acid significantly promoted the removal of no. fig. 2. contrast study of denitration in different systems. effect of temperature the reaction temperature was an important factor affecting the denitration effect. experiments were carried out under the conditon of room temperature to 80 °c. it could be seen from fig. 3a that the concentration of outlet nox gradually decreased when the temperature rose from 30 to 80 °c in naclo3/h2so4 system. considering the accelerating evaporation of water with the increase of temperature, the optimal temperature in practical application was chosen to be 80 °c. fig. 3. effect of temperature on no removal efficiency. however, for naclo3/hno3 system, the denitration efficiency decreased rapidly from 85.14 to 54.83 % with the temperature from 40 to 80 °c (fig. 3b), which was contrary to the naclo3/h2so4 system. therefore, 40 °c was selected as the optimal temperature for naclo3/hno3 system. the optimal temperature ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. removal of no by wet scrubbing 1187 was close to the operating temperature of wet flue gas desulfurization (wfgd), which was very conducive to the joint operation of desulfurization and denitration process. effect of naclo3 concentration the effect of naclo3 concentration on no absorption efficiency was carried out to ascertain the optimal naclo3 concentration and the results were shown in fig. 4. the removal efficiency of no in naclo3/h2so4 system increased sharply with an increment of naclo3 concentration at first, and then grew slowly. when the concentration of naclo3 was 0.50 %, the no removal efficiency reached 83.53 %. this can be explained from two aspects: chemical reaction and physical properties. the chemical reaction can improve the removal efficiency of no, but the viscosity of denitration liquid increases with the increment of naclo3 concentration, therefore, the liquid diffusion coefficient and solubility of no decrease. fig. 4. effect of naclo3 concentration on no removal efficiency: h2so4 concentration, 0.30 mol/l, hno3 concentration, 0.60 mol/l. the effect of naclo3 concentration on no absorption efficiency in naclo3/ /hno3 system indicated that no absorption efficiency increased sharply from 35.48 to 91.65 % with naclo3 concentration rising from 0.05 to 0.50 %. but the change was not obvious when naclo3 concentration exceeded 0.50 %. so the optimal naclo3 concentration was chosen to be 0.50 %. effect of naclo3 to h+ mole ratio and type of acid the influence of naclo3 to h+ mole ratio on the removal efficiency of no was studied, and the result was shown in fig. 5. a significant increment of the no removal efficiency from 53.30 to about 87.32 % was observed when the mole ratio of naclo3 to h+ varied from 1:2.13 to 1:8.50. when the mole ratio of naclo3 to h+ was 1:12.76, the maximum absorption efficiency of no was about 91.68 %. this indicated that the oxidizing ability of naclo3 gradually increased with the increase of acid intensity.26,27 with the further increase of molar ratio, the removal efficiency of no did not change much. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 1188 shi, sun and cui fig. 5. effect of molar ratio of naclo3 to h + and type of acid on no removal efficiency. the type of acid was also a factor affecting no absorption. it could be seen from fig. 5 that the absorption efficiency of no by h2so4 was slightly higher than by hno3 at the same molar ratio. analysis of products in solution in order to deduce the reaction mechanism, the results of ion chromatography (ic) analysis of the products are given in table i. nitrites were not found, while the main anions in the absorbent solution were nitrates and chlorides. this indicates that the nox directly oxidize to nitrates. the cl– comes from the reduced chlorate. table i. products of denitration; a1 – 1:12.76 (h2so4); a2 – 1:17.01 (h2so4); a3 – 1:12.76 (hno3); a4 – 1:17.01 (hno3) medium cno3/ mg l -1 ccl/ mg l -1 a1 85.03 24.243 a2 80.12 21.205 a3 – 24.240 a4 – 22.066 denitration reaction mechanism it was worth pointing out that the solution remained clear and transparent rather than yellow-green during the denitration process using naclo3 solution, indicating that no chlorine dioxide (clo2) was generated. this was different from the phenomenon of removing nox from simulated flue gas by acidic naclo2 solution.28,29 clo2 is a very harsh gas and even very small amount of clo2 can be detected. in a mass concentration of 0.025 % naclo2 and 0.12 % h2so4 solution, it could be observed clearly that the solution was yellow-green. however, the solution of a mass concentration of 0.40 % naclo3 and 2.55 % h2so4 remained colourless, proving that no clo2 existed. the process involved numerous ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. removal of no by wet scrubbing 1189 chemical reactions. the mechanism of no absorption by naclo3 may be as follows: naclo3 + h+ ↔ na+ + hclo3 (2) 13no + 6hclo3 + 5h2o → 6hcl + 3no2 + 10hno3 (3) 3no2 + h2o → 2hno3 + no (4) 2no + h2o + hclo3 → hcl + 2hno3 (5) 2no + h2o + naclo3 + h+ → na+ + hcl + 2hno3 (6) naclo3 + 2no + h2o → 2hno3 + nacl (7) the reported denitration mechanism with naclo2 is shown in eqs. (8)–(12), in which clo2 is produced in the reaction process, which possesses a potential hazard for the actual use: 8naclo2 + 8h+ → 6clo2 + cl2 + 4h2o + 8na (8) 5no + 2clo2 + h2o → 2hcl + 5no2 (9) 5no + clo2 + 3h2o → hcl + 5hno3 (10) 5no + 3clo2 + 4h2o → 3hcl + 5hno3 (11) 3naclo2 + 4no + 2h2o → 4hno3 + 3nacl (12) reacting 1.00 kg of no requires 2.26 kg naclo2, but only 1.77 kg naclo3 calculated with eqs. (7) and (12). in addition, the price of naclo3 is one third of naclo2. in other words, the cost of naclo3 process is about a quarter of the current cost of naclo2 wet denitration technology. due to the high rate of denitration and low cost, the denitration technology with naclo3 has a good industrial application prospect for the wet denitration of industrial flue gas. chemical thermodynamics the thermodynamic data of chemical reactions are important for assessing the extent of the reaction (eq. (7)). data calculations of the thermodynamic parameters were conducted and the enthalpy change of reaction (δrhm(t)), gibbs energy change of reaction (δrgm(t)) and chemical reaction equilibrium constant (k ) of eq. (7) were obtained. the thermodynamic data of related substances in this research were given in table ii.30 table ii. standard formation enthalpy, standard formation gibbs function, standard entropy and calorific capacity substance δf hm / kj mol -1 δf g m / kj mol -1 sm / j mol -1 k-1 cp / j mol -1 k-1 hno3 (aq) –207.36 –111.34 146.4 –86.6 naclo3 (aq) –344.09 –269.91 221.3 111.3 nacl (aq) –407.27 –393.17 115.5 –90.0 no (g) 91.29 87.60 210.76 29.85 h2o (l) –285.83 –237.14 69.95 75.35 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 1190 shi, sun and cui δrhm(t) at different temperatures have been calculated based on the eqs. (13) and (14): r m f m f m( ) (products) (reactants)h t h hγ γδ = δ − δ  (13) r m r m r p.m 289.15 ( ) d t h t h c tδ = δ + δ (14) r p.m p.m p.m(products) (reactants)c c cγ γδ = −  (15) the results of eq. (14) were δrhm = –374.65 kj/mol, δrhm (313.15 k) = = –382.29 kj/mol and δrhm (353.15 k) = –402.68 kj/mol. the results show that δrhm, δrhm (313.15 k) and δrhm (353.15 k) values of the chemical reaction are negative, which indicates that the reaction between no and naclo3 is exothermic. therefore, the chemical equilibrium for this reaction is unfavourable at high temperatures from the thermodynamics point of view. however, the reaction rate is generally affected by the temperature, and the reaction rate increases at high temperatures. the effect of temperature on the removal efficiency of no showed there was an optimal reaction temperature at which no could be removed quickly. this was the result of a combination of chemical thermodynamics and kinetics. δrgm(t) at 298.15, 313.15 and 353.15 k was calculated by eqs. (16) and (17): r m f m f m(products) (reactants)g g gγ γδ = δ − δ  (16) r m r m r m r p.m r r m 298.15 ( ) ( ) 298.15 d t m g t h t s t c h t s t t t δ = δ − δ = δ = δ − δ −  (17) the results of eq. (17) were δrgm = –284 kj/mol, δrgm (313.15 k) = = –279.33 kj/mol and δrgm (353.15 k) = –267.21 kj/mol. the chemical reaction equilibrium constant (k ) of eq. (7) was given as follows: ln g rt kδ = − (18) the results of eq. (18) for k are 1049.78, 1046.60 and 1039.52. the results showed that δrgm, δrgm (313.15 k) and δrgm (353.15 k) of the chemical reaction were high negative values, compared to –40 kj/mol, and k values calculated were far greater than 105.31 from these thermodynamic derivations, we can suggest that this oxidation reaction can proceed spontaneously and the reaction is very thorough. in the actual operation, the reason why the denitration rate did not reach 100 % should be that the residence time was short, resulting in insufficient reaction. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. removal of no by wet scrubbing 1191 conclusions naclo3 is a promising absorbent for the removal of no from flue gas. the denitration efficiency increased with temperature from 30 to 80 °c in naclo3/ /h2so4 system. however, for naclo3/hno3 system, the denitration efficiency decreased with temperature from 40 to 80 °c. the no removal efficiency increased with naclo3 and acid concentrations. the optimal mole ratio of naclo3 to h+ was 1:12.76. the no removal efficiency was 91.86 %. the cost of naclo3 process was about a quarter of the naclo2 wet denitration technology. the reaction product was nacl and could be recovered. therefore, naclo3 method may be an alternative way to solving the problem of denitration of dusty flue gas small boilers produced intermittently. acknowledgements. the authors would like to thank talent training program in western shandong province (2017grc5227). и з в о д студија о уклањању no из емисије мокрим скрубингом помоћу naclo3 deqi shi, guoxin sun и yu cui school of chemistry and chemical engineering, university of jinan, no. 336, west of nan xinzhuang, jinan, 250022, shangdong, p. r. china за уклањање азот-моноксида (no) из гасне емисије малих котлова на угаљ, неопходно је употребити економичну технологију влажне денитрације. проучавана је апсорпција no помоћу раствора натријум-хлората. установљени су утицаји експерименталних услова као што су температура, концентрација naclo3, врста киселине, молски однос naclo3 према водоничним јонима – на брзину уклањања no, и утврђени оптимални услови. како је утицај температуре био повезан са врстом киселине, температура потребна за сумпорну киселину (h2so4) била је висока, а температура потребна за азотну киселину (hno3) ниска. оптимални молски однос naclo3 према овим двема киселинама био је исти. продукти реакције анализирани су јонском хроматографијом. реакциони раствор се могао рециклирати након уклањања натријум-хлорида. изведен је закључак о механизму и укупној једначини реакције naclo3 денитрације. добијени термодинамички резултати показали су да је ова оксидациона реакција спонтана и врло темељна. naclo3 је показао високу ефикасност уклањања no, а трошак денитрације је био много нижи него помоћу натријум-хлорита. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {comparison of mixing performances of t, y and arrow-shaped micromixers using villermaux-dushman protocol at low reynolds number} j. serb. chem. soc. 85 (3) 381–394 (2020) udc 53.031.05:539.24+546.151’155:66.01 jscs–5308 original scientific paper 381 comparison of mixing performances of t, y and arrow-shaped micromixers using villermaux–dushman protocol at low reynolds number onyeka s. okwundu1,2*, mohammed fuseini1, ahmed h. el-shazly1,3 and marwa f. elkady1,4 1department of chemical and petrochemical engineering, egypt–japan university of science and technology (e-just), egypt, 2department of chemical engineering, university of benin, nigeria, 3chemical engineering department, alexandria university, alexandria, egypt and 4fabrication technology department, advanced technology and new materials research institute (atnmri), city of scientific research and technology applications, alexandria, egypt (received 28 march; revised 23 july; accepted 3 september 2019) abstract: mixing performance is commonly assessed using the villermaux– –dushman protocol. mixing in passive mixers may need to be at very low reynolds number (re). this study is aimed at comparing the mixing performances of t, y and arrow-shaped micromixers using the villermaux–dushman protocol at re < 100. the mixing performance test was run at flow rates of 500 to 1 ml h-1 with the pressure drop measurement. based on uv absorbance values and experimental mixing times of mixed fluids from the three micromixers, the order of mixing performance at re > 26.5 was y < t < arrow-shaped micromixers. at lower re values, the order of performance based on the absorbance and the experimental mixing times, became inconsistent. however, the comparison of mixing performance based on predicted mixing time gave consistent order of performance at all re values. the instability in uv absorbance of mixed reagents in villermaux–dushman protocol was noted as the major cause of the reported inconsistency at very low re. the inadequacy of villermaux– –dushman protocol at low re was verified via facile solvent extraction test. a more reliable technique should be used to assess the mixing performance of micromixers at very low re and on-line uv measurement should be adopted for the villermaux–dushman experiment. keywords: efficiency of mixers; iodide/iodate test experiment; passive mixers; micro-channel; laminar flow regime; confluence shape. *corresponding author: onyeka.okwundu@eng.uniben.edu https://doi.org/10.2298/jsc190328095o ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 382 okwundu et al. introduction most chemical processes require contacting of different substances to achieve homogeneity, emulsification, dispersion or even chemical reaction as in the case of combination reactions where different molecular species have to contact before conversion. one of the most important methods of enhancing mixing at micro level is using micro scale devices.1 dating back to the early nineties, the micro scale devices such as micromixers (or microreactors; when chemical reaction is involved) have attracted great attention in laboratory as well as on industrial or commercial scale.2,3 the values of reynolds number (re = ρud/μ; where: ρ is the density, u is the flow velocity, d is the characteristic diameter of the micro channel cross section, μ is the dynamic viscosity of fluid; assuming that flow regime laws apply in micro-channels) are usually very low in micro-structured devices due to their small dimensions; the internal cross sectional diameter in the order of 102 μm.4 therefore, the flows in micro channels fall within the laminar regime. but, if the flow in micro-channels is in the laminar regime, how is the fluid mixing possible then? based on the way mixing is achieved, there are two classes of micromixers: the active and the passive micromixers.5,6 only the passive micromixer, for which fluid–fluid interfacial contacting (mixing) is effected by the energy of flowing fluids (no external energy source like mechanical stirring is employed, as in the case of active ones), is implied throughout this study. the reduced cross section of micro-channels leads to the increased rate of fluid shearing and based on the micromixer geometry or the configuration (point of confluence, obstruction, bend or twist), the fluid in laminar flow gets deformed via convection as it flows past such obstructions, bends, twists or as streams meet at point of confluence and that is the mechanism of mixing in passive micromixers.6,7 primarily, there is diffusive mixing at molecular level and this diffusion is enhanced by the relatively reduced diffusion path or cross section of flow.7 in fact, “molecular diffusion is the ultimate and finally the only process really able to mix components of a fluid on the molecular scale”.6 as a result of their small sized channels, the micromixers have often increased fluid surface area to volume ratio and this makes them very useful in high mass transfer and heat transfer applications.5 commenge and falk noted some other interesting applications of micromixers arising from their compact nature, to include: safety of operation in case of highly exothermic and fast reactions involving hazardous chemicals; new and untapped reaction routes for chemical synthesis (such as direct fluorination), which are very dangerous can be accessed and there is improved efficiency in energy utilization by using passive micromixers.3 in addition, the small sized devices save materials and time; since just little volume of fluid is required to operate them and reasonably short residence ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ low reynolds number villermaux-dushman test 383 time is needed. unlike most chemical process equipment that are bulky, micromixers are very portable. consequent to the relevance of mixing and the beneficial nature of micromixers, the assessment of the mixing performance of micromixers is a very important step for their design (making good choices). while it may be so easy to assess the performance of some micro-devices by the comparison of some standard parameters like heat transfer coefficient for heat exchangers, the case of micromixers is quite difficult.2 a number of techniques have been utilized to assess the mixing performance and they are included in order of increasing preference: the visualization of dyes as their colour fades away, due to the dilution by mixing or as their colour changes, in case of ph sensitive dyes, the visualization of reactions that progress with change in colour and most recently, the monitoring of competitive test reaction schemes.1–3,8 amongst the competitive test reaction schemes used for mixing performance assessment, the villermaux–dushman reaction scheme which consists of two parallel reactions: neutralization and redox reactions in competition for proton from acid, is well known and most used.3,7 the neutralization reaction given by eq. (1) is instantaneous (extremely fast) while the redox reaction (eq. (2)) is just fast.5,6 all species in eqs. (1)–(3) are aqueous: h2bo3– + h+  h3bo3 (instantaneous) (1) io3– + 5i+ 6h+  3i2 + 3h2o (just fast) (2) i2 + i–  i3– (3) a strong acid is used to generate the protons in one reactant (acidic) solution while the second (buffer) solution for the mixing contains: io3–, i– and h2bo3–. falk and commenge 6 require that only a critical stoichiometric amount of acid is needed to neutralize the borate ions according to eq. (1) must be used for the test, while fournier et al 9 state that the acid should just be the limiting reagent. the concept of the villermaux–dushman protocol is briefed as follows.3,6 when the mixing is perfect, the stoichiometrically fitted instantaneous reaction (eq. (1)) completely consumes the protons, leaving no room for the slower eq. (2) to proceed. however, when mixing is poor, there is segregation (heterogeneous fluid behaviour) of the two reactant solutions into acidic and buffer segregates (local sites), making the amount of protons in every acidic segregate to be excessively high to just complete the neutralization of the borate ions in the immediate surrounding (boundary/interphase). the excess protons enable the occurrence of the redox reaction to form iodine. the extent of segregation/size of the segregates depends on the mixing efficiency/performance and that determines the amount of iodine to be produced. eq. (3), which utilizes the iodine, is very useful for the quantitative analysis because the amount of i3– produced can be easily measured using uv spectrophotometer at a wavelength of 353 nm. hence, the relatively ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 384 okwundu et al. low absorbance values of mixed products indicate relatively high mixing performance and vice versa. ehrfeld et al2 share similar idea but they utilized acetate in place of borate species in eq. (1). some works2,10 are reported with product absorbance measured at 352 nm, noting that the uv spectrum has a peak at that wavelength, rather than at 353 nm; however, the values are quite close. commenge and falk 3 further noted that the test gives optimum (most sensitive) result when the rates are such that the neutralization occurs in zero seconds (quasi-instantaneous) and the redox reaction occurs at roughly the same time as the mixing time. villermaux–dushman protocol is also known as the iodide/iodate test method. following the villermaux–dushman protocol, the performance of a mixer at specific conditions should be assessed based on the observed absorbance value of the mixed products (the higher the absorbance, the poorer the performance and vice versa). unfortunately, several authors3,6,11 recommend that the mixing performance criteria should not be concentration dependent. mixing time (tm), according to commenge and falk 3 which ought to be adopted by asano et al.11 is given in eq. (4). obviously, the model is concentration dependent, since it includes absorbance and initial concentrations as variables: tm = 0.33odc(h+)0–4.55c(ki)0–1.5c(kio3)05.8c(naoh)0–2c(h3bo3)0–2 (4) where od is optical density which is equivalent to the absorbance of mixed reagents/sample for cuvette of optical density of 1 cm; c(i)0 is the initial molar concentration of chemical species i (in the stock reagent solution). however, satisfactory attempts to express tm as a concentration free parameter (in terms of kinematic viscosity and specific power dissipation rate) are reported somewhere.9 by the analysis of literature results, commenge and falk obtained a relationship between the theoretical mixing time (tmp) in s and the specific power dissipation (ε) in w kg–1 as given in eq. (5). they recommend that the equation should be used when pressure drop data due, to the flow through micromixer, are available. eq. (5) was used to predict the mixing time in this work. the specific power dissipation or specific energy dissipation rate is expressed in terms of volumetric flow rate (q), pressure drop (∆p), density (ρ) and control volume of the micromixer (v) as shown in eq. (6):3 tmp = 0.15ε–0.45 (5) ε = q∆p/(ρv) (6) due to the variations in channel dimensions and fluid flow conditions for different experimental set-ups, the works on micromixer performance are better reported in terms of re values rather than volumetric flow rates or velocity. also, it may not be appropriate to compare the values of performance data from different works because several authors report different reagent concentrations and reagents’ flow rate ratios.11 in the evaluation of mixing performance of t, y and oriented y micromixers with the spatially arranged outlet channel using viller________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ low reynolds number villermaux-dushman test 385 maux–dushman protocol, at minimum flow rate of 10 ml min–1, at average fluid velocity of 0.331 ms–1 (minimum re = 265), the reduction in segregation index (the improvement in mixing performance) with the increasing flow rate is reported.5 from this result, by comparison of segregation index values at common flow rates, the oriented y micromixers (which has arrow-shaped confluence) gave the best mixing performance followed by t and then y-mixer.5 also, the similar result on comparison of mixer performances and the effect of flow rate on mixing performance was earlier reported using an online spectroscopic analysis system (uvmini1240, shimadzu), still at re > 265.7 the information on mixing performance at very low re values is scarce and such information may be very valuable in certain applications like drug delivery and in achieving mixing down to cellular level in biological systems. in consolidation with the works of other researchers, this study aims to compare the mixing performances of three different configurations of micromixers (t, y and arrow-mixers) using the villermaux–dushman test reaction at low re values. to achieve this aim, the villermaux–dushman protocol was carried out for the micromixers with pressure measurements and then the absorbance values of mixed products from each of the micromixers, the experimental and predicted mixing times, were compared at various re values. materials and methods materials t, y and arrow-shaped micromixers made by 3d printing, each with the uniform internal diameter of 0.5 mm and length of 30 mm after confluence, were used in this study. two syringes each of capacity 60 ml and 26.72 mm diameter were used to feed the storage tank. other accessories such as g28 screws, teflon® and small, micro-sized, pipes were used for connection. the consumable chemicals, which include: sodium hydroxide pellets (product of applichem gmbh, germany), 99 % pure potassium iodide (product of chem-lab nv, belgium), boric acid (product of mp biomedicals, llc, france), potassium iodate (rankem™, rfcl limited, india), methanol (>99.9 %, product of fisher scientific, uk) and hydrochloric acid of 37 % purity and specific gravity of 1.18 (product of fisher scientific, uk) were all of analytical grade and accessed from chemical and petrochemical engineering laboratory, e-just, egypt. hedeya cooking oil was purchased from fathalla-gomla market, borg el-arab, alexandria, egypt. dual-syringe infusion pump (model: 115 vac; cole-parmer™, usa), kobold hand -held pressure measuring device for differential pressure for 2 external sensors (hnd-p215; with accuracy of 0.0005 psi), 2 kobold pressure sensors (hnd-ps19) and hitachi uv spectrophotometer (u-3900; for absorbance measurement, with 0.0005 accuracy), equipped with sample cell/cuvette of optical path length = 1cm, were accessed from different laboratories in e-just. methods villermaux–dushman experiment with pressure measurement. one liter each of the acid (0.03 m aqueous hcl) and buffer (0.09 m h3bo3(aq), 0.09 m naoh(aq), 0.006 m kio3(aq) and ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 386 okwundu et al. 0.032 m ki(aq)) solutions for villermaux–dushman reaction were prepared and labelled solution 1 and solution 2, respectively.11,12 two 60 ml syringes were filled with solutions 1 and 2, labelled and clamped onto the dual syringe pump. the y-mixer was connected with two pressure probes (sensors) at the mixer inlet and outlet pipes. the experimental set-up is as shown in fig. 1a and b; it shows details of the micro-channels of the y, t and the arrow -shaped micromixers displaying the confluence shapes and the angular deviation of fluid streams at point of confluence (with flow directions). a) b) fig. 1. a) schematic diagram of the experimental set-up; b) illustrative details of the micro-channels of the mixers showing the confluence shapes and angular deviations (60, 90 and 120°) of fluid streams at point of confluence. the syringe pump was switched on and set to: volume, diameter and flow rate of 60 ml, 26.72 mm and 500 ml hr-1, respectively. afterwards, the pump was run. the pressure readings were taken after the flow had attained the steady state (as indicated by the constant pressure readings), while 2 ml of mixed sample was collected after discarding the first 20 ml that flowed out in order to ensure steady concentration. uv absorbance of the collected sample was measured as fast as possible within the set wavelength range of 320 to 370 nm, and recorded. next, the flow rate was successively changed to 400, 300, 250, 200, 150, 100, 70, 50, 30, 15, 7 and 1 ml hr-1, while repeating the same mixing performance characterization procedure each time. afterwards, the y-mixer was replaced with the t-mixer and then with the ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ low reynolds number villermaux-dushman test 387 arrow-shaped mixer while repeating the same procedure. syringes 1 and 2 were refilled with solutions 1 and 2, respectively, each time their content reduced below 25 ml. all through the experiment, the connected micromixer was fixed at a horizontal position with the pressure probes maintained at the same position to avoid changes in pressure head (variation in hydraulic pressure with height). the upper limit of flow rate used in this study (500 ml h-1) was fixed by the maximum pressure limit (5.8 psi) of the kobold (hnd -ps19) pressure sensors, while the lower limit (1 ml h-1) was fixed by time consideration. finally, the mixing performance of the micromixers was assessed based on the uv absorbance, the predicted mixing time (tmp) and the experimental mixing time (tm). the density and viscosity of the dilute solutions used in the experiment were assumed to be the same as that of water (approximately 1 g cm-3 and 1 cp, respectively). with these solution fluid parameters, together with the micro-channel dimension, it was possible to express flows in dimensionless form as re. results and discussion mixing performance characterization based on uv absorbance values the acid and buffer concentrations used resulted in high absorbance values at flow rates below 150 ml h–1 (re = 26.5). according to falk and commenge,3,6 the absorbance–concentration relationship, otherwise known as the beer– –lambert law, is valid for the tri-iodide ion at the absorbance range below 2.5. the dilution of the mixed samples of high absorbance is discouraged;3 a failed attempt to characterize performance by dilution of mixed samples with high uv absorbance is reported in literature.12 consequently, the concentrations of solutions 1 and 2 were each halved by doubling their volumes with distilled water before proceeding with the experimental runs for flow rates below 150 ml h–1. as a result of this modification, the result (fig. 2) is presented in two parts. a) b) fig. 2. effect of re values on absorbance: at stated reagent concentration for higher flows (a) and with halved reagent concentration for lower flow rates (b). fig. 2a and b show the effect of re on absorbance at the stated reagent concentration for higher flows and halved concentration for lower flows, respectively. fig. 2a clearly shows that the uv absorbance increased progressively for ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 388 okwundu et al. all the micromixers with the decreasing re. by comparison, the arrow-mixer gave the least absorbance (best mixing performance), while the y-mixer gave the highest absorbance (least mixing performance) for flows above the re value of 26.5. at re value of 26.5, the three mixers measured up in absorbance, while it is clear from fig. 2b that there is no consistent comparative trend in absorbance for the micromixers at re < 26.5. in quest for possible reasons for the inconsistency in the absorbance trend at re < 26.5, the uv absorbance of mixed samples were measured with respect to time (age after sampling). it was found that the absorbance of mixed samples for all re values, were not stable; the values reduced with samples’ age. ehrfeld et al.2 noted this instability in absorbance, although they report an increase in the absorbance, using sodium acetate in place of naoh and boric acid. the observed instability could be due to the reversible nature of most of the reactions in the villermaux–dushman reaction scheme. as a result of this, the time lag between samples collection and measurement was kept to the minimum, for greater accuracy. also, the spectrophotometer was set to cover short range of wavelength (320 to 370 nm) to reduce response time in measurement. the observed sharp drop in absorbance at re = 0.2, for all mixers as shown in fig. 2b, could be due to the long time spent in collecting samples at such low flow rate – allowing such a long time lag for mixed samples absorbance to depreciate. substantial reasons for the absorbance instability with samples’ age cannot be given because the kinetics of villermaux–dushman reaction is not well understood.13 again, the mixed samples were quickly collected and analyzed for the uv absorbance at very close time intervals from the on-set of mixing till exhaustion of syringe contents, at different re values. the result showed that the steady absorbance value (with noise of ±0.01 at re < 17.7 and ±0.003 at re ≥ 17.7 was attained after collecting at most 5 ml of mixed solution. the observed noise at steady state (at re < 17.7) is quite high, but too small to account for the observed inconsistency, because the observed disorderliness in absorbance (for fig. 2b) is greater than 0.01. therefore, the noise could have contributed slightly to the inconsistency. this test justified the assumption of the steady state concentration during sampling (discarding the first 20 ml of mixed sample). mixing performance characterization based on mixing times the high mixing performance is characterized by the low mixing time. figs. 3 and 4 show the effect of changing re on the experimental mixing time (tm) and on the predicted mixing time (tmp), respectively. by comparison, as it can be seen from the figures, the values of the experimental mixing time at a given re are more distinct than the predicted ones which are clustered together. again, the range of values of tmp is lower than the experimental mixing time (tm). fig. 3a and b were derived from the absorbance data presented in fig. 2a and b, and the ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ low reynolds number villermaux-dushman test 389 same discussion applies. although fig. 4 is not quite clear due to the clustered points; for all re, tmp was the lowest for the arrow mixer and the highest for the y-mixer. tmp increased for all mixers as re value was decreased. the increment in tmp was gradual, but steeped up at re < 12.4. the similar step-up with decreasing re is also noticeable in fig. 3a; meaning that the sensitivity of mixing performance to the changes in re is higher at low re, than at high re. the maximum value of tmp was achieved at the least flow velocity (re = 0.2), as it can be seen from fig. 4. while from fig. 3b a very low tm (high mixing performance) was obtained, which appears unreasonable because at very low re, mixing is wholly dominated by the molecular diffusion without convective (vortex) enhancement. however, numerical simulations have shown that the reduction of re at certain range could lead to the increase in mixing quality.14–17 but that does not justify the inconsistency in order of performance encountered here, which is not different from an earlier report.12 the predicted mixing time of fig. 4 does not agree with the theoretical results,14–17 because the predicted model (eq. 5)3 is independent of chemical species in the mixed stream. a b fig. 3. effect of re values on experimental mixing time: with normal reagent concentration (a) and with adjusted reagent concentration (b). fig. 4. effect of re values on predicted mixing time. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 390 okwundu et al. effect of the confluence shape on mixing performance can therefore be explained as follows. at a given re > 30, there is improvement in performance, since the confluence shape encourages more fluid-fluid and/or fluid–wall impact when two fluid streams meet, and vice versa. referring back to fig. 1b, two factors are of importance in explaining the order of performance: the impact on collision and the simultaneous angular deviation in fluid flow direction. when the inlet fluid streams approach each other from opposite directions, as in the t-shaped confluence, the fluid-fluid collision impact is “head on” and maximum, compared to the lower impact obtainable when the streams approach each other at an angle (as in the y and arrow-shaped confluences). the effect of bend on fluid mixing along channels18 also plays great role in mixing at confluence points, because with greater deviation at confluence, the enhanced mixing is achievable. the angular deviation in flow direction for t-shaped confluence is just 90° while y-shape offers the least deviation. the arrow-shaped confluence caused the greatest agitation during the collision of streams due to its maximum angular deviation (>90°), coupled with its slight collision impact. t was better than y, because it offers the greatest collision impact and better deviation than y-mixer. in all cases, the mixing is enhanced with increasing re value in the range > 30. fig. 5. effect of re on pressure drop. fig. 6. effect of re on energy dissipation rate. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ low reynolds number villermaux-dushman test 391 variation of pressure drop and specific energy dissipation rate with re the modification of concentration stated under “mixing performance characterization based on uv absorbance values” section did not affect the pressure drop and therefore figs. 4 to 6 are not split into two. the effect of inlet re on pressure drop (∆p) as a result of flow through each micromixer is shown in fig. 5. from the figure, ∆p increased somewhat linearly as re increased. the variation in ∆p among the three micromixers becomes wider (diverges) as one moves from low re to higher re. the similar trend is reported by hossain et al.,8 jafari et al.19 and kockmann et al.15 to understand this divergence, we consider the possible expressions for ∆p, or pressure head, as a function of re or velocity, hence the hagen–poiseuille equation and the darcy’s equation. but, the flows in micromixers are not ideally laminar to be described as hagen–poiseuille flow. based on darcy’s relationship, ∆p is proportional to fu2; where f is a dimensionless parameter (a function of flow velocity and other factors), commonly known as the friction factor and u is the average velocity of flow.20 when the fluid streams (from two identical branches of micromixer) collide/ /meet, mixing takes place at the expense of energy which is felt as a drop of pressure. the effect of re on energy dissipation rate (ε) is shown in fig. 6. from the figure, the variation of ε with respect to re is very similar to the observed trend for ∆p, but is more of a curve than straight line, as a result of the extra velocity factor, as related in eq. (6). it is clear that higher energy is spent at higher re and the consequence of it is the improvement in mixing performance. this is in agreement with the literature.6,19 the order of energy dissipation rate is ar > t > y-mixers. verification of the inadequacy of villermaux–dushman protocol at low re values the simple interphase mass transfer between two immiscible liquids (water and crude biodiesel) was used to verify the observed performance inconsistency in this study.21 briefly, the hydrophobic (crude biodiesel) phase resulting from ultrasonic assisted-methanolysis of fresh cooking hedeya oil in presence of 1 % koh as homogeneous catalyst for 40 min, with methanol to oil mole ratio of 6:1, at 60 °c; was contacted with the alkaline water (0.016 m koh) in the same experimental set-up of fig. 1, at flow rates of 70, 50, 20, 7 and 1 ml h–1, each for t, y and arrow-shaped micromixers, respectively. after micromixing, 5 ml of the resulting aqueous phase was titrated over 0.03 m hcl, using phenolphthalein as an indicator. we are currently working to fully develop this performance characterization technique and the details would be published soon. titrimetry revealed a progressive increase in the alkalinity of biodiesel wash water, for all the micromixers, as the flow rate was increased. table i shows the titrimetric result of aqueous extract phase. also, at every flow rate, the order of mixing performance was arrow > t > y mixer. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 392 okwundu et al. table i. titrimetric result of aqueous extract phase flow rate, ml h-1 volume of hcl used, ml arrow-mixer t-mixer y-mixer 70 3.65 3.50 3.15 50 3.35 3.20 3.00 20 3.10 2.95 2.85 7 2.95 2.90 2.85 1 2.85 2.85 2.80 conclusion the mixing performances of t, y and arrow-shaped micromixers have been characterized by the villermaux–dushman protocol at low re. based on uv absorbance, experimental and predicted mixing times; comparison of mixing performance of the micromixers was made at various re. facile solvent extraction test was performed for the result verification. from the results of this study, it can be concluded that the arrow-shaped confluence offers the best mixing performance, while the y-shape offers the least of the three confluence shapes at re > 30. the villermaux–dushman protocol is not adequate at lower re. increase in re within the range 30 < re < 100, leads to the improvement in mixing performance. mixing performance is more sensitive to changes in re at low re values. improvement in mixing performance, due to the shape of confluence or to the increase in re value occurs at the cost of fluid energy per unit time (power). a more reliable mixing performance characterization method has been used to verify the inadequacy of the villermaux–dushman protocol at very low re. finally, in order to reduce the effect of time lag, on-line (in-situ) absorbance measurement should be adopted. nomenclature re reynolds number ar arrow (micromixer) ρ density of fluid, kg m-3 μ dynamic viscosity of fluid, kg m-1 s-1 u flow velocity of fluid stream, m s-1 d characteristic diameter of flow channel, m uv ultraviolet od optical density or mixed sample absorbance ci,0 initial molar concentration of chemical species i, mol l-1 ε specific power dissipation or specific rate of energy dissipation, w kg-1 or j kg-1 s-1 q volumetric flow rate, ml h-1 δp pressure drop, pa v control volume for mixing, m3 tm experimental mixing time, s tmp predicted mixing time, s f dimensionless friction factor in darcy’s equation ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ low reynolds number villermaux-dushman test 393 acknowledgments. the authors acknowledge the tutelage of dr. k. yoshimoto and dr. y. muranaka of kyoto university, and supervision of dr. m.f. elkady and prof. a.h. el-shazly during their project based learning at egypt–japan university of science and technology (e-just), egypt. the authors also acknowledge scholarship support from ministry of higher education (mohe) and japan international cooperation agency (jica) in e-just, egypt. и з в о д поређење перформанси мешања за t, y и микромиксере облика стрелице коришћењем villermaux–dushman протокола при ниским вредностима re бројева onyeka s. okwundu1,2, mohammed fuseini1, ahmed h. el-shazly1,3 и marwa f. elkady1,4 1 department of chemical and petrochemical engineering, egypt–japan university of science and technology (e-just), egypt, 2 department of chemical engineering, university of benin, nigeria, 3 chemical engineering department, alexandria university, alexandria, egypt и 4 fabrication technology department, advanced technology and new materials research institute (atnmri), city of scientific research and technology applications, alexandria, egypt перформасе мешања се уобичајено процењују коришћењем villermaux–dushman протокола. мешање у пасивним миксерима може бити пожељно при веоме ниским вредностима рејнолдсовог (re) броја. циљ овог истраживања је поређење перформанси мешања за т, y и микромиксере облика стрелице, применом villermaux–dushman протокола, за re < 100. тестови за испитивање перформанси мешања су вршени при протоцима од 200 до 1 ml/h, при чему су вршена мерења пада притиска. на основу вредности uv абсорбанција и експерименталних времена мешања за 3 микромиксера, добијен је следећи редослед перформанси мешања за re > 26,5: y < t < микромиксер облика стрелице. при нижим вредностима re броја, редослед перформанси мешања заснован на абсорбанси и експерименталним вредностима времена мешања, је постајао неконзистентан. међутим, поређење перформанси мешања заснованих на предвиђеном времену мешања је дало конзистентан редослед за све вредности re броја. примећена је нестабилност у мерењима uv абсорбанције помешаних реагенаса у villermaux–dushman протоколу, која представља главни разлог неконзистензног понашања при веома ниским re бројевима. неадекватност виљ villermaux–dushman протокола при ниским re бројевима је потврђена помоћу једноставног теста екстракцијом растварачем. да би се испитале перформансе мешања при веома ниским re бројевима требало би користити поузданију методу, као и онлајн uv мерење које би требало усвојити за villermaux–dushman експеримент. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice effect of an industrial chemical waste on the uptake volume 84 (2019) 1 2019 list of referees editorial board of the journal is grateful to the following referees for reviewing the manuscripts during 2019: nadica abazović, institute of nuclear sciences vinča, belgrade, serbia ahmed abu‐dief, chemistry department, faculty of science, faculty of science, sohag university, sohag, egypt radoslav adžić, chemistry division, brookhaven national laboratory, usa tahmineh akbarzadeh, faculty of pharmacy, tehran university of medical sciences, tehran, iran mara aleksić, faculty of pharmacy, university of belgrade, serbia sellamuthu anbu, centro de química estrutural, universidade de lisboa, portugal ivan anđelković, innovation center of the faculty of chemistry, university of belgrade, serbia katarina anđelković, faculty of chemistry, university of belgrade, serbia ljubica anđelković, institute of chemistry, technology and metallurgy, university of belgrade, serbia nanjangud venkatesh anil kumar, manipal institute of technology, manipal, india jasmina anojčić, faculty of science, university of novi sad, serbia vesna antić, faculty of agriculture, university of belgrade, serbia davor antanasijević, faculty of technology and metallurgy, university of belgrade, serbia ozgur ozgur arar, department of chemistry, ege university, i̇zmir, turkey mohammed arifuddin, institute of pharmaceutical education and research (niper), hyderabad, india stevan armaković, department of physics, faculty of sciences, university of novi sad, serbia ayten ates, cumhuriyet university, sivas, turkey jelena avdalović, institute of chemistry, technology and metallurgy, university of belgrade, serbia md. rabiul awual, japan atomic energy agency, japan fulvio baggi, neuroimmunology and neuromuscular diseases unit, neurological institute 'carlo besta', milan, italy jelena bajat, faculty of technology and metallurgy, university of belgrade, serbia marija baranac-stojanović, faculty of chemistry, university of belgrade, serbia jiří barek, charles university, prague, czech republic giampaolo barone, dipartimento di scienze e tecnologie biologiche, università di palermo, italy ivana n. beara, department of chemistry, biochemistry and environmental protection, faculty of sciences university of novi sad, serbia vladimir beškoski, faculty of chemistry, university of belgrade, serbia dejan bezbradica, faculty of technology and metallurgy, university of belgrade, serbia venkat bhethanabotla, department of chemical and biomolecular engineering, university of south florida, tampa, usa 2 journal of the serbian chemical society siniša bikić, faculty of technical sciencies, university of novi sad, serbia martin bilek, university of hradec kralove, czech republic wesley browne, faculty of science and engineering, faculty of science and engineering, university of groningen, the netherlands dušan bučevac, vinča institute of nuclear sciences, university of belgrade mihael bučko, defence university, military academy, belgrade, serbia elzbieta budzisz, collegium medicum in bydgoszcz, nicholaus copernicus university in torun, poland josef caslavsky, faculty of chemistry, brno university of technology, czech republic paromita chakraborty, institute of science and technology, kattankulathur, tamil nadu, india daniel cozzolino, agri-chemistry group, school of medical and applied sciences, central queensland university (cqu), north rockhampton, australia aurelien crochet, fribourg center for nanomaterials, frimat machine pool responsible, university of fribourg, germany jános csanádi, faculty of science, university of novi sad, serbia agnes csiszar, department of botany and nature conservation university of west hungary, sopron, hungary ilija cvijetić, inovation center of faculty of chemistry, university of belgrade, serbia danica ćujić, institute for the application of nuclear energy, university of belgrade, serbia renato araújo da costa, faculty of computer engineering, federal institute of pará, brazil aleksandra daković, institute for technology of nuclear and other mineral raw materials, belgrade, serbia marko daković, faculty of physical chemistry, university of belgrade, serbia claude daul, department of chemistry, university of fribourg, switzerland césar augusto moraes de abreu, universidade federal de pernambuco, department of chemical engineering, recife, brazil aleksandar dekanski, institute of chemistry, technology and metallurgy, university of belgrade, serbia vesna despotović, faculty of sciences, university of novi sad, serbia aleksandar devečerski, , institute of nuclear sciences vinča, university of belgrade, serbia tomislav došlić, faculty of civil engineering, university of zagreb, croatia ljiljana đekić, faculty of pharmacy, university of belgrade, serbia stojan đokić, department of chemical and materials engineering, university of alberta, edmonton, canada svetlana đogo-mračević, department of analytical chemistry, faculty of pharmacy, university of belgrade, serbia mihal đuriš, institute of chemistry, technology and metallurgy, university of belgrade, serbia enis džunuzović, faculty of technology and metallurgy, university of belgrade, serbia nevenka elezovic, institute for multidisciplinary research, university of belgrade, serbia suzana filipović, institute of technical sciences of sasa, belgrade, serbia boris furtula, department of chemistry, faculty of science, university of kragujevac, serbia slobodan gadžurić, faculty of sciences, dep. of chemistry, university of novi sad gordana gajić, institute for biological research „sinisa stankovic”, university of belgrade, radmila garić-grulović, faculty of technology and metallurgy, university of belgrade, serbia marija gavrović-jankulović, faculty of chemistry, university of belgrade, serbia jasmina glamočlija, institute for biological research siniša stanković, university of belgrade, serbia 2019 list of referees 3 nikola gligorijević, institute for the application of nuclear energy, university of belgrade, serbia biljana dj. glišić, department of chemistry, faculty of science, university of kragujevac, serbia gordana gojgić-cvijović, institute of chemistry, technology and metallurgy, university of belgrade, serbia snežana gojković, faculty of technology and metallurgy, university of belgrade, serbia branimir grgur, faculty of technology and metallurgy, university of belgrade, serbia sanja grgurić šipka, faculty of chemistry, university of belgrade. serbia sonja grubišić, faculty of chemistry, university of belgrade. serbia maja gruden, faculty of chemistry, university of belgrade. serbia amjad hameed, nuclear institute for agriculture and biology (niab), faisalabad, pakistan baki hazer, kapadokya university, faculty of applied sciences, department of aircraft airframe engine maintenance, ürgüp nevşehir, turkey saša horvat, department of chemistry, biochemistry and environmental protection, faculty of science, university of novi sad nenad ignjatović, institute of technical sciences of the serbian academy of sciences and arts, belgrade, serbia ivana ivančev-tumbas, faculty of sciences, university of novi sad, serbia jasna ivanović, faculty of technology and metallurgy, university of belgrade, serbia gorica ivaniš, faculty of technology and metallurgy, university of belgrade, serbia đorđe janaćković, faculty of technology and metallurgy, university of belgrade, serbia biljana jančić stojanović, faculty of pharmacy, university of belgrade, serbia radmila jančić heinemann, faculty of technology and metallurgy, university of belgrade, serbia goran janjić, institute of chemistry, technology and metallurgy, university of belgrade, serbi radomir jasiński, institute of organic chemistry and technology, cracow university of technology, poland stanka jerosimić, faculty of physical chemistry, university of belgrade, serbia sanja jevtić, faculty of technology and metallurgy, university of belgrade, serbia branimir jovančićević, faculty of chemistry, university of belgrade, serbia jelena jovanović, faculty of physical chemistry, university of belgrade, serbia jovan jovanović, faculty of technology and metallurgy, university of belgrade, serbia ljubinko jovanović, educons university, sremska kamenica, serbia milica jovanović krivokuća, institute for application of nuclear energy, belgrade-zemun, serbia predrag jovanović, faculty of pharmacy, university of belgrade, serbia vladislava jovanović, institute of chemistry, technology and metallurgy, university of belgrade, serbia vladimir jović, institute for multidisciplinary research, university of belgrade, serbia aleksandar jovović, faculty of mechanical engineering, university of belgrade, serbia ivan juranić, faculty of chemistry, university of belgrade, serbia goran kaluđerović, department of bioorganic chemistry, leibniz-institute of plant biochemistry, halle (saale), germany tatjana kaluđerović radoičić, faculty of technology and metallurgy, university of belgrade, serbia musawwer khan, department of chemistry, aligarh muslim university, aligarh, india seokmin kim, department of mechanical engineering, chung-ang university, seoul, south korea jakob kljun, faculty of chemistry and chemical technology, university of ljubljana, slovenia. vesna kojić, oncology institute of vojvodina, sremska kamenica, serbia vanja kokol, institute of engineering materials and design, university of maribor, slovenia 4 journal of the serbian chemical society nikola knežević, faculty of technology and metallurgy, university of belgrade, serbia mirjana kostić, faculty of technology and metallurgy, university of belgrade, serbia maja kozarski, institute of food technology and biochemistry, faculty of agriculture, university of belgrade, serbia snežana kravić, faculty of technology, university of novi sad, serbia raviraj kulkarni, centre for nanoscience and nanotechnology, belgaum, india aleksandar lolić, faculty of chemistry, university of belgrade, serbia francisco lopez, departament d'enginyeria química, facultat d'enologia, universitat rovira i virgili, tarragona, spain jelena lozo, faculty of biology, university of belgrade, serbia marija lučić škorić, innovation center of the faculty of technology and metallurgy, belgrade, serbia fiderman machuca martínez, escuela de ingeniería química, universidad del valle, cali, colombia pedro maireles-torres, universidad de malaga, malaga, spain vesna maksimović, vinča nstitute of nuclear sciences, university of belgrade, serbia nedeljko manojlović, department of pharmacy, faculty of science university of kragujevac, serbia aleksandra margetić, institute of chemistry, technology and metallurgy, university of belgrade, serbia bojana marković, institute of chemistry, technology and metallurgy, university of belgrade, serbia smilja marković, institute of technical sciences of the serbian academy of sciences and arts, belgrade, serbia romana masnikosa, faculty of physical chemistry, university of belgrade, serbia branko matović, department of materials science, vinča institute of nuclear sciences, belgrade, serbia srđan matijašević, institute for technology of nuclear and other mineral raw materials, belgrade, serbia ljiljana matović, vinča institute of nuclear sciences, university of belgrade, serbia mihai medeleanu, politechnica university, faculty of chemical and environmental engineering, timisoara, romania katarina mihajlovski, faculty of technology and metallurgy, university of belgrade, serbia nikola milašinović, department of forensics, academy of criminalistic and police studies, belgrade, serbia mirta milić, institute for medical research and occupational health, zagreb, croatia sonja milićević, institute for technology of nuclear and other mineral raw materials, belgrade, serbia milan milivojević, faculty of technology and metallurgy, university of belgrade, serbia simeon minić, faculty of chemistry, university of belgrade, serbia nemanja mirković, faculty of agriculture, university of belgrade, serbia tarek mohamed, department of chemistry, faculty of science al-azhar university, cairo, egypt jelena molnar jazić, faculty of sciences, university of novi sad, serbia agnes moricz, plant protection institute, centre for agricultural research, hungarian academy of sciences, budapest, hungary takeshi nakano, research center for environmental preservation, osaka university, japan maryam nazari, faculty of chemistry, razi university, kermanshah, iran 2019 list of referees 5 bojana nedić vasiljević, faculty of physical chemistry, university of belgrade, serbia olgica nedić, institute for the application of nuclear energy, university of belgrade, serbia mehdi nekoei, deparment of chemistry, shahrood branch, islamic azad university, shahrood, iran biljana nigović, department of pharmaceutical, analysis, faculty of pharmacy and biochemistry, university of zagreb, croatia аndrea nikolić, faculty of sciences, university of novi sad, serbia irena nikolić, faculty of metallurgy and technology, university of podgorica, montenegro jasmina nikolić, faculty of technology and metallurgy, university of belgrade, serbia milan nikolić, faculty of chemistry, university of belgrade, serbia nebojša nikolić, institute of chemistry, technology and metallurgy, department of electrochemistry, university of belgrade, serbia kobra nikoofar, department of chemistry, faculty of physics and chemistry, alzahra university, vanak, tehran, iran slađana novaković, institute of nuclear sciences, laboratory of theoretical physics and condensed matter physics, university of belgrade, serbia verónica núñez, departamento de bioquimica y biologia molecular, facultad de medicina universidad de salamanca, spain aleksandar oklješa, faculty of sciences, university of novi sad, serbia dejan opsenica, faculty of chemistry, university of belgrade, serbia igor opsenica, faculty of chemistry, university of belgrade, serbia bojana ostojić, department of chemistry, institute of chemistry, tehnology and metallurgy, university of belgrade, serbia sibel a. özkan, faculty of pharmacy, ankara university, turkey elena pahontu, faculty of pharmacy, university of medicine and pharmacy "carol davila", bucharest, romania manojit pal, dr. reddy’s institute of life sciences, gachibowli, hyderabad, india nebojša pantelić, faculty of agriculture, university of belgrade, serbia szabolcs pap, environmental research institute, thurso, caithness, scotland, uk christos pappas, geoponiko panepistimion athinon, department of food science and nutrition, athens, greece miomir pavlović, institute of chemistry, tehnology and metallurgy, university of belgrade, serbia nataša pejić, faculty of pharmacy, university of belgrade, serbia maura pellei, school of science and technology, chemistry division, university of camerino, italy franc perdih, faculty of chemistry and chemical technology, ljubljana, slovenia miljenko perić, faculty of physical chemistry, university of belgrade, serbia milena petković, faculty of physical chemistry university of belgrade, serbia dušan petrović, forschungszentrum jülich gmbh predrag petrović, department of chemical engineering, faculty of technology and metallurgy, university of belgrade, serbia rada petrović, faculty of technology and metallurgy, university of belgrade, serbia lato pezo, institute of general and physical chemistry, university of beograd, serbia marijana ponjavić, faculty of technology and metallurgy, university of belgrade, serbia svetlana popović, faculty of technology, university of novi sad, serbia velimir popsavin, faculty of science, university of novi sad, serbia radivoje prodanović, department of biochemistry, faculty of chemistry, university of belgrade, serbia 6 journal of the serbian chemical society ralph puchta, department of chemistry and pharmacy, friedrich alexander university erlangennürnberg, erlangen, germany vladislav rac, faculty of agriculture, university of belgrade, zemun, serbia bojan radak, vinča institute of nuclear sciences, university of belgrade, serbia dušanka radanović, institute of chemistry, technology and metallurgy, university of belgrade, serbia mirjana radanović, faculty of sciences, university of novi sad, serbia slavko radenković, department of chemistry, faculty of science, university of kragujevac, serbia maja radetic, faculty of technology and metallurgy, university of belgrade, serbia jelena radić-perić, faculty of physical chemistry, university of belgrade, serbia ivona radović, faculty of technology and metallurgy, university of belgrade, serbia ahmad raheel, department of chemistry, quaid-i-azam university, islamabad, pakistan vesna rakić, faculty of agriculture, university of belgrade, serbia harry ramanantoanina, university of fribourgh, switzerland slavica ražić, faculty of pharmacy university of belgrade, serbia ana rilak, department of chemistry, faculty of science, university of kragujevac, serbia petar ristivojević, inovacioni centar hemijskog fakulteta, belgrade, serbia dragana robajac, institute for the application of nuclear energy, university of belgrade, serbia marko rodić, faculty of sciences, university of novi sad, serbia anna roglans, department of chemistry, universitat de girona, spain adamo domenico rombolà, department of agricultural sciences, alma mater studiorum, university of bologna, italy tamara rončević, faculty of sciences, university of novi sad, serbia nada savić, faculty of applied ecology – futura, metropolitan university, belgrade, serbia jan schwarzbauer, rwth aachen university, institute of geology and geochemistry of petroleum and coal, aachen, germany milan senćanski, vinča institute of nuclear sciences, university of belgrade, serbia lakshmipathi senthilkumar, bharathiar university, tamilnadu, india mario saparrat, facultad de ciencias agrarias y forestales, universidad nacional de la plata, argentina milica simović, faculty of technology and metallurgy, university of belgrade, serbia marta skrajda-brdak, faculty of food sciences, university of warmia and mazury in olsztyn, olsztyn, poland carmen soto, centro regional de estudios en alimentos saludables (creas), conicyt-regional gore, valparaíso, chile pavle spasojević, innovation centre of faculty of technology and metallurgy, serbia marko stamenić, faculty of technology and metallurgy, university of belgrade, serbia remco stan, technical university of munich, germany vojislav stanić, vinča institute of nuclear sciences, university of belgrade, serbia dragomir stanisavljev, faculty of physical chemistry, university of belgrade, serbia nada stanković, institute of molecular genetics and genetic engineering (imgge), university of belgrade, serbia stepan stepanović, institute of chemistry, technology and metallurgy, department of chemistry, university of belgrade, serbia sanja stevanović, institute of chemistry, technology and metallurgy, department of electrochemistry, university of belgrade, serbia 2019 list of referees 7 zorica stojanović, faculty of technology, university of novi sad, serbia gordana stojanović, department of chemistry, faculty of science and mathematics, university of niš, serbia marina stojanovska, institute of chemistry, faculty of natural science and mathematics, ss cyril and methodius university, skopje, macedonia adam strachota, institute of macromolecular chemistry, academy of sciences of the czech republic, prague, czech republic istvan szilagyi, faculty of science and informatics, department of physical chemistry and materials science, university of szeged, hungary zoran šaponjić, vinča institute of nuclear sciences, belgrade, serbia dragana šeklić, department for biology and ecology, faculty of science, university of kragujevac, serbia marinela šokarda slavić, institute of chemistry, technology and metallurgy department of chemistry, university of belgrade, serbia bogdan šolaja, faculty of chemistry, university of belgrade, serbia tatjana šolević knudsen, institute of chemistry, technology and metallurgy, university of belgrade, serbia lubomir švorc, faculty of chemical and food technology, slovak university of technology, bratislava, slovak republic ibrahim tegin, department of chemistry, faculty of arts and science, siirt university, tyrkey vele tešević, faculty of chemistry, university of belgrade, serbia nadia todorova, national center for scientific research demokritos, institute of materials science, athens, greece tamara todorović, faculty of chemistry, university of belgrade, serbia anđelka tomašević, institut za pesticide i zašitu životne sredine, beograd-zemun, serbia biljana tomasević, faculty of chemistry, university of belgrade, serbia milorad tomič, university of east sarajevo, faculty of technology zvornik, zvornik, republic of srpska, bih jelena tričković, department of chemistry, biochemistry and environmental science, faculty of sciences, university of novi sad, serbia jorge trilleras, facultad de ciencias basicas, universidad del atlantico, puerto colombia, colombia nemanja trišović, faculty of technology and metallurgy, university of belgrade, serbia tijana trtić-petrović, vinča institute of nuclear sciences, belgrade, serbia đenđi vaštag, faculty of sciences, university of novi sad, serbia đorđe veljović, faculty of technology and metallurgy, university of belgrade, serbia zoran višak, centro de química estrutural, instituto superior tecnico, universidade de lisboa, portugal željko vitnik, department of chemistry, institute of chemistry, technology and metallurgy, university of belgrade, serbia dimitra voutsa, school of chemistry, aristotle university of thessaloniki, greece vesna vučić, institute for medical research, university of belgrade, serbia marija vukčević, faculty of technology and metallurgy, university of belgrade, serbia predrag vulić, faculty of mining and geology, university of belgrade, serbia gordana vunjak-novaković, biomedical engineering and medical sciences, department of biomedical engineering, columbia university, new york, ny, usa malcolm watson, faculty of sciences, university of novi sad, serbia 8 journal of the serbian chemical society erdal yabalak, faculty of arts and science, mersin university, mersin, turkey semra yaprak turan, abant izzet baysal üniversitesi, bolu, turkey snežana zarić, faculty of chemistry, university of belgrade, serbia alam zeb, university of malakand, chakdara, pakistan tijana zeremski, institute of field and vegetable crops, novi sad, serbia peng zhou, school of life science and technology, university of electronic science and technology of china, chengdou, sichuan, pr china manfred zinn, university of applied sciences western switzerland (hes-so), institute for life technologies, sion, switzerland matija zlatar, institute of chemistry, technology and metallurgy, university of belgrade, serbia mire zloh, university of hertfordshire, hatfield, uk branka žarković, faculty of agriculture, university of belgrade, serbia irena žižović, faculty of technology and metallurgy, university of belgrade, serbia {influence of electrochemical conditions on the regioand stereoselectivity of selenocyclization of alkenyl hydantoins} j. serb. chem. soc. 86 (6) 585–590 (2021) short communication jscs–5445 585 short communication influence of electrochemical conditions on the regioand stereoselectivity of selenocyclization of alkenyl hydantoins biljana m. šmit1*, petar b. stanić1, ljubinka g. joksović2, darko p. ašanin1 and zoran simić2 1institute for information technologies, university of kragujevac, jovana cvijića bb, kragujevac, serbia and 2faculty of science, university of kragujevac, radoja domanovića 12, kragujevac, serbia (received 22 october 2020, revised 21 february, accepted 23 march 2021) abstract: 5-alkenyl hydantoins and alkenyl spirohydantoins are converted into bicyclic and tricyclic hydantoins, under indirect electrochemical conditions, generating the phenylselenyl cation in situ. the reactions proceeded in good to exelent yields. the influence of electrochemical conditions on regioand diastereoselectivity of the selenocyclization reactions is investigated. keywords: electrosynthesis; constant-current electrolysis; selenylation; ring closure. introduction the use of electrochemical methods in organic synthesis has become increasingly more popular due to simple procedures and laboratory techniques and the use of cleaner and greener solvents. it is worth noting that the outcomes of the reactions can be vastly different, while some reactions can only be carried out under electrochemical conditions.1 the hydantoin core represents an important pharmacophore occurring in many biologically active compounds mostly known due to their antimicrobial, anticancer and anticonvulsant activity.2,3 spirohydantoins4 and fused5 polycyclic hydantoins are the leading compounds in drug discovery, due to their various biological activities. selenocyclization is a convenient and useful tool for the construction of heterocycles.6,7 we described a methodology for the synthesis of bicyclic and tricyclic fused hydantoin scaffold, using selenocyclization for the construction of sterically constrained structures that have potential in peptidomimetic drug design.8,9 in this work we decided to use the electrochemically generated phenylselenyl cation in the cyclization of 5-alkenyl hydantoins and alkenyl spirohydantoins and explore whether these condit * corresponding author. e-mail: biljana.smit@uni.kg.ac.rs https://doi.org/10.2298/jsc201022023s ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 586 šmit et al. ions have an effect on the course of the reaction, especially on regioand stereoselectivity. experimental general. all alkenyl hydantoins used as substrates were synthesized according to the procedures described previously.8 reagent ph2se2 was used as supplied by aldrich. gas– liquid chromatography (glc) was performed by varian instument model 3400. 1h-nmr spectra were run in cdcl3 on a varian gemini 2000 spectrometer at 200 mhz. general procedure for the electrochemical selenocyclization of hydantoins (1a–j). a solution of 1a–j (1 mmol), ph2se2 (156 mg, 0.5 mmol), silica gel (150 mg, 5 mmol) and naclo4 (123.5 mg, 1 mmol) in mecn (10 ml) was placed in an undivided electrolysis cell and electrolyzed under a constant current (10 ma) at room temperature. after completion, the reaction mixture was stirred overnight. the solvent was distilled off, residue dissolved in ch2cl2, washed with sat. nahco3 solution and brine, and dried over anh. na2so4. the solvent was evaporated and the reaction mixture was analyzed by glc and 1h-nmr spectroscopy. results and discussion the alkenyl hydantoins contain a double bond and an internal nitrogen nucleophile, and they are suitable substrates for the intramolecular electrophilic cyclization. over several decades, electrophilic selenium reagents have been proven to be quite useful for this purpose. in some cases, electrochemical selenylations have advantages over other related methods.10,11 the cyclization of the previously synthesized 5-alkenyl hydantoins 1a–g and alkenyl spirohydantoins 1h–j was performed by the means of electrochemically generated phenylselenyl cation, which originates from diphenyldiselenide in a mecn solution of naclo4 (scheme 1). perchlorate in this process serves as a mediator. before that, we tried to perform the reaction of the commercially available product 1d, with other supporting electrolytes (licl, kcl, nabr, ki), solvents and electrodes, but naclo4 in mecn and c–pt electrodes gave the best results (table i, entry 6). scheme 1. selenocyclization of alkenyl hydantoins 1a–g and alkenyl spirohydantoins 1h–j. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ electrocyclization of alkenyl hydantoins 587 table i. optimization of electrochemical conditions for the cyclization reaction of 1d entry anode–cathode supporting electrolyte solvent overall yielda (6/5), % diasteromeric ratioa cis/trans, % 1 pt–pt kcl ch3cn 10 78:21 2 pt–pt nabr ch3cn 6 – 3 pt–pt ki ch3cn 5 75:25 4 pt–pt naclo4 ch3cn 31 62:38 5 pt–pt naclo4 ch3cn 4 – 6 c–pt naclo4 ch3cn 63 (46:54) 6(59:41); 5(69:31) 7 c–cu et4nbr ch3cn 1 – 8 c–cu naclo4 ch3cn 47 (68:32) 6(51:49); 5(7:93) 9 c–cu licl ch3cn traces – 10 c–cu ki ch3cn 34 (42:58) 6(52:48); 5(72:28) 11 c–pt naclo4 ch2cl2:ch3cn=2:1 traces – 12 c–pt naclo4 py:ch3cn=1:1 traces – 13 c–pt naclo4 toluene:ch3cn=2:1 57 (83:17) 6(48:52); 5(84:16) 14 c–pt naclo4 thf undetectable – 15 c–pt naclo4 ethanol undetectable – 16 c–pt naclo4 dmf traces – aratio of regio and diastereoisomers are obtained from glc and 1h-nmr spectra the easy oxidation of perchlorate at the anode provides in situ generation of phse+ able to react with the π-electron system of the substrate. the reaction yields products resulting from the nucleophilic attack of the nitrogen atom to the cyclic seleniranium ion intermediate during the cyclization step. under the chosen reaction conditions, a series of alkenyl hydantoins 1a–j was subjected to electrochemical selenocyclization (table ii). in the previously reported results,8,9 the reaction was regiospecific and 5-membered regioisomers were formed via favorable 5-exo-trig ring closure process which is both kinetically and thermodynamically favoured.12 products where the bridgehead substituent and ch2seph group are in cis positions one to another were formed predominantly. in contrast, when the phenylselenyl cation is electrochemically generated in situ, it was noticed that the regioand diastereoselectivity both depend on the steric hindrance at c-5 of the hydantoin ring and the c-c double bond. 5-exo-trig cyclization products are also obtained regiospecifically in most cases, but with poorer stereoselectivity. the amount of trans-diastereoisomers is increased in comparison to the previous results, implying higher thermodynamic control of the cyclization process. exceptions are the cyclizations of 1c and 1j where the steric hindrance is most pronounced and the kinetic control is favoured, increasing the cis-selectivity. only in the absence of steric hindrance in the starting alkenyl hydantoin, 6-membered regioisomers are formed via 6-endo -trig ring closure process. in the case of 1a, where a methyl group is attached on c-5 and the double bond is unsubstituted, the six-membered regioisomer is obtained almost exclusively and as a trans-isomer stereospecifically. six-mem________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 588 šmit et al. bered product is also obtained in the cyclization of 1d, where a methyl group is attached on c5 and the double bond is terminally disubstituted, but without regioand stereoselectivity. when the double bond is non-terminally substituted, like in 1e, despite having a methyl group attached on c-5, the six-membered regioisomer is not formed at all. it is assumed that the methyl group on the double bond directly hinders the approach of seleniranium cation to the nitrogen atom during cyclization. regardless of the substitution of the double bond, the cyclization of alkenyl spirohydantoins 1h–j depends on the size of the cycloalkyl group. the product of 6-endo-trig ring closure occurs predominantly only in the case of 1h without any stereocontrol, while the minor product of 5-exo-trig ring closure occurs with the reversed stereoselectivity compared to results reported previously.9 the bulkier six-membered ring in 1i and 1j presumably prevents the formation of another six-membered ring. table ii. selenocyclization of alkenyl hydantoins 1a–g and alkenyl spirohydantoins 1h–j substrate yield, % regioisomer ratioa 2:3 dr (cis/trans)a 2 dr (cis/trans)a 3 72 1:99 – 0:100 88 100:0 70:30 – 96 100:0 71:29 – 63 54:46 69:31 59:41 73 100:0 56:44 – 63 100:0 44:56 – 75 100:0 48:52 – 86 35:65 24:76 50:50 ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ electrocyclization of alkenyl hydantoins 589 table ii. continued substrate yield, % regioisomer ratioa 2:3 dr (cis/trans)a 2 dr (cis/trans)a 3 97 100:0 68:32 – nh hn o o 82 100:0 100:0 – aratio of regioand diastereoisomers are obtained from glc and 1h nmr spectra conclusion the influence of electrochemical conditions on selenocyclization reactions of alkenyl hydantoins and spirohydantoins has been explored. various different solvent/mediator systems have been taken into consideration and mecn/naclo4 showed the best results. steric hindrances in the starting alkenyl hydantoins influence the regioand the stereochemical outcome of the reactions and in these conditions thermodynamic control is more present. acknowledgement. this work was supported by the ministry of education, science and technological development of the republic of serbia (cont. no: 451-03-9/2021-14). и з в о д утицај електрохемијских услова на региои стереоселективност селеноциклизације алкенилхидантоина биљана м. шмит1, петар б. станић1, љубинка г. јоксовић2, дарко п. ашанин1 и зоран симић2 институт за информационе технологије, универзитет у крагујевцу, јована цвијића бб, крагујевац и 1природно–математички факултет, универзитет у крагујевцу, радоја домановића 12, крагујевац 5-алкенилхидантоини и алкенилспирохидантоини су преведени у бицикличне и трицикличне хидантоине под индиректним електрохемијским условима при којима се фенилселенил-катјони стварају in situ. реакције су се одигравале у добрим до одличним приносима. испитиван је утицај електрохемијских услова на региои дијастереоселективност реакција селеноциклизације. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {spatial distribution of pahs in riverbed sediments of the danube river in serbia: anthropogenic and natural sources} j. serb. chem. soc. 84 (12) 1439–1453 (2019) udc 628.336.1:547.57+504.4.054+ jscs–5276 556.53(282.243.74) original scientific paper 1439 spatial distribution of pahs in riverbed sediments of the danube river in serbia: anthropogenic and natural sources maja brborić1, branislav vrana2, jelena radonić1#, mirjana vojinović miloradov1 and maja turk sekulić1*# 1university of novi sad, faculty of technical sciences, department of environmental engineering and occupational safety and health, trg dositeja obradovića 6, 21000 novi sad, serbia and 2masaryk university, faculty of sciences, research center for toxic compounds in the environment, recetox, kotlářská 267/2, 611 37 brno, czech republic (received 6 june, revised 14 june, accepted 17 june 2019) abstract: the top layer of riverbed sediments from 10 sites along the danube river in serbia were analysed for the presence of polycyclic aromatic hydrocarbons (pahs). potential sources of pollution were identified using different multivariate techniques. total concentrations of 29 pahs in sediment samples ranged from 128.27 to 676.85 µg/kg dry weight. based on these concentrations and pah patterns, a clear spatial distribution of examined localities was determined. the diagnostic ratios indicated a pyrolytic origin of pahs, except for two sites where pah contamination originated from petrogenic sources. the varimax rotated principal component analysis revealed three main factors, namely coal, wood, and biomass combustion; vehicular emissions; and natural sources; accounting for 92.1 % of the variance in the original datasets. the sites with different pollution load, depending on main sources identified, were arranged by hierarchical cluster analysis. the results were compared with sediment quality guidelines, and the obtained values of equilibrium partitioning sediment benchmarks toxic unit and total toxic equivalent quantities of benzo[a]pyrene suggested that danube sediments do not pose an elevated ecotoxicological risk for benthic organisms. keywords: polycyclic aromatic hydrocarbons; contaminant pattern; aquatic sediment. introduction discharge of partially treated or untreated wastewater from human agglomerations, industry, and agriculture into surface water bodies cause significant pollution of the danube. in serbia, 44 human agglomerations are located in the * corresponding author. e-mail: majaturk@uns.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc190129056b 1440 brborić et al. danube basin, with a total of 1.44 million population equivalent (pe). most agglomerations (70 %) are connected to neither sewage collecting systems nor wastewater treatment plants. a large portion of industrial wastewaters from 326 registered water polluters (flow: 461,315 m3/day, suspended solids: 629 ton/day) is still being discharged without any or with insufficient pre-treatment into the danube and its tributaries. the contribution of organic pollutants from 113 registered agricultural sources is also significant.1 the situation in the vojvodina region is further complicated by the previous armed conflicts. burning or damaging of industrial objects (oil refineries, fuel storages, power plants), infrastructure (bridges, roads), and military objects in the federal republic of yugoslavia during the nato operation allied force led to the release of a large amount of various hydrophobic organic compounds (hocs) into the environment.1 various hocs, including polycyclic aromatic hydrocarbons (pahs), pose a high risk for humans and the environment and represent a significant environmental problem.2 some pahs remain and cycle in the aquatic environment and can be transported as dissolved or sorbed on sediments over vast distances, potentially damaging aquatic ecosystems and consequently impacting water status and human health.3 most of pahs are released from well-known anthropogenic sources4 rather than natural ones like volcanic activity and biosynthesis by bacteria and plants.5 considering that certain number of pahs, as well as their environmental mixtures, are potent immunosuppressants and show strong mutagenicity and carcinogenicity,2 there is a need to continuously collect the monitoring data on these contaminants and information on sources and relevant pathways. sorption of hocs on sediments and suspended particles controls their environmental fate, transport, and effects in aquatic systems. concentration levels of pollutants in aquatic sediments may give elementary information on the contamination status, sources, long-term dynamic changes responding strongly to the hydrological situation, and environmental risks incurred by contaminants in freshwater ecosystems.6 river pollution is distinguished by complex processes of sedimentary deposition that include an archive of past and present anthropogenic activities.5 hydrophobic toxic contaminants tend to accumulate in sediments and, therefore, a contaminant buried in the sediment could be released in the aquatic eco-environment through different processes, posing a risk to biota and human population. in serbia, the available data on the sources and levels of pahs are limited. the primary goal of the paper was to determine the level of riverbed sediment pollution with 29 pahs and, in particular, to determine the composition of pahs, their spatial distribution and contamination sources, as well as to assess environmental and human health risk. the findings of this study can serve as the basis for the remediation initiatives aimed at eradicating pahs from danube sediments. danube sediment contaminations with pahs 1441 experimental details related to sediment sampling and preservation are given in supplementary material to this paper (section s-1). sediment analysis only a brief summary of the analytical procedure is shown here, while an itemized explanation of sample preparation and chemical analysis are presented in section s-2 and fig. s-2 (supplementary material). firstly, the collected sediment samples were freeze-dried and sieved. hot automated soxhlet extraction of spiked sediment samples with internal standards was performed using dichloromethane. activated silica was used for sample clean-up. the sample volume was reduced using the azeotrope principle on kuderna-danish evaporation. quantification was performed using gas chromatography–mass spectrometry (gc–ms). data analysis in order to determine potential sources of pahs in danube sediments, we applied the diagnostic ratios method, principal component analysis (pca), and hierarchical cluster analysis (hca). although the diagnostic ratios method is the most commonly used technique, it does not provide information on the quantitative contribution of different pah sources.8 thus, it was supplemented by pca, which is a helpful tool for the assessment of potential emission sources and their distribution. in this paper, pca was applied to the parameters pertaining to different sites, in order to extract components relative to different factors, as well as to clarify the variance in the observed data. according to liu et al.,9 factor loadings were classified as “strong” (matches the absolute values of > 0.75), “moderate” (0.75–0.50) and “weak” (0.50– –0.30). hca allowed for the grouping of sampling localities depending on the probable sources of pahs in danube sediments based on the factors identified through pca. this approach allowed for a precise point of pollution to be identified as a possible transport route of contamination. statistical analysis was implemented using the ibm spss statistics 22 software (ibm corporation, armonk, new york, usa). results and discussion quantification and composition profiles of pahs pah concentration levels in the sediment samples collected at the studied localities and the toc content are given in table s-ii (supplementary material). the total amount of 29 pahs in sediments ranged from 128.27 (d10) to 676.85 μg/kg dry weight (d.w.), d9, fig. 1, with an average value of 347.61 µg/kg d.w. and median ± sd of 298.46±179.06 μg/kg d.w. the highest level was recorded at site d9, located 10 km downstream of pančevo. during the 1999 nato operations in serbia, the bombing and destruction of the petrochemical plant “hip petrohemija”, the chemical fertilizer plant “hip-azotara”, and the oil refinery “nis petrol” resulted in complete or incomplete combustion. after the air strikes, the united nations environment programme (unep) defined pančevo as one of the key ecological hot spots in the western balkans. this military campaign led to the discharge of toxic pollutants into the air, soil, surface water, and consequently groundwater.10 according to unep,10 the recorded sediment concentrations of 16 pahs in the vicinity of pančevo ranged 1442 brborić et al. between 67 and 460 μg/kg d.w. thirteen years later, within the presented study, for the same group of pahs, we detected the increased values of 520 μg/kg d.w. in our case, the values increasing from 460 μg/kg to 520 μg/kg d.w., without any new registered anthropogenic influences, are a typical example of the pseudo -persistence of pahs. this confirms the fact that the input rate is significantly higher than the output rate. the increase of 60 μg/kg can be attributed to the natural sources of pahs with great certainty. observing the categorization of 26 pahs in sediments, proposed by baumard et al.10 (low: 0 to 100 μg/kg, moderate: 100 to 1000 μg/kg, high: 1000 to 5000 μg/kg, and very high: > 5000 μg/kg), pah levels at all our sampling points can be classified as moderate. fig. 1. concentration levels of pahs in the danube riverbed sediment samples. a differentiation of pah concentration levels in bottom sediments collected from various waterways in serbia and abroad is shown in table i. the concentration levels of pahs obtained in our study were only slightly higher than those observed within the yangzi river delta and guan river estuary, in china11,13 and considerably lower than those in the majority of other analysed aquatic sediments shown in table i. the pah concentration levels detected in our study were similar to those registered in serbian sediment samples reported by crnković et al.14 and sakan et al.14 when interpreting these findings, it should be noted that, in most of the aforementioned studies, the levels of only 16 epa pahs were assessed. the comparison of sedimentary pah data with maximum allowed concentrations, that the serbian and canadian legislation prescribed for sediments, have showed danube sediment contaminations with pahs 1443 that these pollutants are not expected to be a fundamental cause for eco-chemical and ecological degradation in examined sediments in serbia. table i. comparisons of the total pahs concentrations (μg/kg d.w.) in sediments obtained in previously conducted research; n.d. – not detectable (below detection limits) sampling sites country date of sampling number of samples number of pahs compounds σpahs – range µg/kg ref. this study serbia 2012 10 29 128.3 – 676.8 – mansfeld region, saxony-anhalt germany 1998 3 15 5.1×103 – 10.4×103 4 danube serbia 2001 6 16 307 – 1,452 15 upper danube river germany 2002 10 16 n.d. – 26.32 103 16 yangzi river delta china 2005 38 15 91.3 – 614.4 11 danube and sava serbia 2005 2 16 d: 213.1 – 575.4 s:416.2–595.3 14 danube, sava, tisa serbia 2008 3 6 d: < n.d. – 689 s: 240–395 t: 86.1–728 14 mithi river india 2009-11 3 17 1206.0–4735.0 18 danube estuarine coast ukraine 2010 10 16 329.6 – 1,093.3 19 cauca river colombia 2010-11 8 12 n.d. – 3,739.0 3 guan river estuary china 2011 13 21 90 – 218 13 the correlation between total organic carbon (toc) content and pah concentrations in danube sediments was examined. statistical correlation analysis of all obtained results showed a non-significant linear correlation between toc and pah concentration levels in this study (r = 0.60, p < 0.05). this was confirmed with the shapiro–wilk test of normality and its significance of 0.639. pearson correlation coefficient analysis demonstrated that only several individual compounds at selected locations were more significantly positively associated with toc values. locality d7 showed a positive correlation of two pahs with toc: ret (r = 0.75, p 0.05) and per (r = 0.72, p < 0.05). moreover, within our study, the measured toc range was low and very narrow (between 0.3 and 1.3 %, fig. s-3, supplementary material). in such a situation, a very strong correlation between pahs and toc would be more surprising than expected. these conclusions were confirmed with pca scatter plot before and after the normalization to toc values (fig. s-8, supplementary material). pah composition profiles in danube sediments were dominated by 5-ring pahs, as shown in fig. s-4 (supplementary material). in the total pah amount, the 2and 3-ring pahs contributed 15 %, while the remaining 85 % comprised of 4-, 5and 6-ring pahs. these findings indicate a greater presence of pahs introduced into danube sediments by pyrolytic processes sources, that were subsequently introduced in each environmental matrix through dry and wet atmo1444 brborić et al. spheric deposition and soil surface and river runoff. ip shows the highest concentrations in all analysed samples (8.60–80.58 μg/kg d.w.), followed by flu and pyr (6.99-77.82 and 4.96–56.24 μg/kg d.w., respectively,table s-ii, supplementary material). only site d5 is characterized by the predominance of 3-ring pahs, contributing more than 60 % to the total pah amount. at the same point, significantly greater fl concentration was observed (88.37 μg/kg d.w.), almost 60 times higher than at site d8. these results show that this is probably caused by heavy traffic and vicinity of the novi sad oil refinery. possible pahs sources diagnostic ratios. to elucidate the sediment contamination origin, the high molecular weight/low molecular weight (lmw/hmw) ratios were used, where lmw pahs = nap + acy + ace + fl + phe + ant + flu + pyr + b[a]a + chr + bip + ret + b[b]fl + bnt + tph and hmw pahs = b[b]flu + b[k]flu + b[a]p + ip + db[ah]a + b[ghi]p + b[ghi]flu + cp[cd]p + b[j]flu + b[e]p + per + db[ac]ant + ant + cor. as displayed in fig. s-5 (supplementary material), apart from localities d1 and d5 (with the lmw/hmw ratio of 1.57 and 4.66, respectively), all lmw/hmw ratios were lower than 1. these findings indicate that pahs at sampling sites d2–d4 and d6–d10 are pyrolytic, whereas those at sites d1 and d5 are petrogenic in origin. in addition to intensive road and water traffic at both sites, danube goods-transportation centre has an impact on site d1, while oil refinery and traffic hold-ups at the port of novi sad contributed to the pollution at d5. tsymbalyuk et al.19 also showed that pahs with 4 to 6 aromatic rings clearly predominated in riverbed sediments from the estuarine danube area. flu/pyr and phe/ant ratios are calculated by another method for assessing the contamination sources. it is generally agreed that low phe/ant values (< 10) and high flu/pyr values (> 1) are characteristic of pyrolytic pollution.5 fig. s-6 (supplementary material) displays the distribution of phe/ant and flu/pyr ratios for the studied sampling points. the resulting plot reveals that for the analysed sediments phe/ant is < 10 and flu/pyr is > 1, indicating predominantly pyrogenic origin of these pahs. however, diagnostic phe/ant and flu/pyr ratios are usually unreliable and may be lower than those used as emission sources indicators.19 in addition, the pah sources in sediments can also be identified using the following concentration ratios: fl/fl+pyr, ant/178 (ant/ant+phen), ip/ip + b[ghi]p and b[a]a/228 (b[a]a/b[a]a+chr). in this study, the values of these ratios indicate the dominance of pyrolytic emission sources, with the highest values of ratios associated with site d4 (table s-iii, supplementary material). in certain cases, the pah ratios are incompatible, because the origins of pah emissions can be different, random, and irregular.19 to compensate for these discre danube sediment contaminations with pahs 1445 pancies, the total pah index was calculated using eq. (1).19 various sources reported single indexes, normalized by the lower limit of four considered ratios (petrogenic source in table s-iii) were used. [ ] [ ] index ipfl b a aant (ip+b ghi p)(fl+pyr) 178 228totalpahs = + + + 0.4 0.1 0.2 0.2 (1) when the total pahs index > 4, high-temperature processes are investigated as sources of pahs, while values < 4 indicate a predominance of oil sources.19 the values obtained in this study are within the 5.66–7.27 range (table s-iii), confirming that high-temperature processes are dominant sources of pahs. principal component analysis pca with varimax rotation was applied to ascertain the quantitative contribution of different pah sources and provide the most convincing results. three principal components explaining 92.1 % of the variance are presented in fig. s-7 (supplementary material). pc1 explained approximately 70.8 % of the variance, with high loadings of acy, phe, ant, flu, pyr, b[a]a, chr, b[b]flu, b[k]flu, b[a]p, ip, db[ah]a, b[ghi]p, b[b]fl, bnt, b[ghi]flu, cp[cd]p, tph, b[j]flu, b[e]p, db[a]ant, anth and cor. according to irwin et al.,21 acy can be released to the environment through petroleum refining effluents, coal tar distillation, municipal wastewater treatment, and waste incineration processes. on the other hand, ip can be formed during combustion or raised-temperature processes that include different hydrocarbon mixtures. ant, chr, b[a]a, b[b]flu, b[b]flu and b[a]p are considered specific markers for coal combustion, while the typical indicators of wood combustion include phe, ant, pyr, b[a]a and chr.21 based on the pah emissions released from the ignition of coal dust and crumb rubber at 1000 °c (combustion temperature in a coal power plant), phe, flu and pyr were determined as predominant pahs in the emission from furnaces.23 high loading factor for b[k]flu, b[a]p, b[ghi]p, db[ah]a and chr was attributed to coal combustion, while high loading factor for ant, chr, b[b]flu and b[k]flu suggested stationary pah emission sources.24 schauer et al.24 measured the pah emission rates from household combustion of different types of wood (eucalyptus, oak and pine). their results showed presence of ace, acy, fl, phe, ant, ret, flu, pyr, b[ghi]flu, cp[cd]p, chr, tph, b[b]flu, b[k]flu, b[j]flu, b[e]p, b[a]p, per, ip and anth in all samples of emitted smoke. gryglewicz et al.25 examined sulfur -containing polycyclic aromatic compounds in supercritical coal and observed the presence of bnt in coal flame. according to ravindra et al.,26 the increased b[e]p levels in sediments are the consequence of fossil fuel power plants. therefore, it is reasonable to assign pc1 as sources of coal, wood and biomass com1446 brborić et al. bustion, as a result of heating, industrial, and agricultural activities. a significant number of point sources originating from the industrial and individual production of smoked meat, as a significant part of a diet in this part of serbia, also increase the emission levels. pc2 explained around 12.0 % of the variance with high loadings of ace, fl and bip. it also has a moderate loading for nap, phe and ant. higher levels of ace and fl were suggested to indicate vehicular combustion, while the high abundance of nap can be characteristic of exhaust from diesel engines21. according to irwin et al.,21 bip is detected in various petroleum products. moreover, volatilization or spills of petroleum products across the waterway transport contribute to the increased emissions of ace, fl and nap.28 ravindra et al.26 found that engines with diesel exhaust emissions result in phe and ant dominance in pollutant profiles. the danube is characterized by intensive shipping activities, so pc2 is selected to represent vehicular emission sources. pc3 explained about 9.3 % of the variance with high loadings of ret and per and moderate loading for nap. gocht et al.28 claimed that there are strong indications of natural processes that produce per at low temperatures. zakaria et al.28 indicated that perylene can be produced during early diagenesis through in situ conversion of pigment (perylenequinone) or other types of organic matter. the results reported by wen et al.29 suggest that the phytoplankton could be a possible source of ret in sediments and nap and phe are pahs arising from “natural” sources (biological and diffuse).31 thus, factor pc3 was attributed to natural sources. after comparing the pca scatter plots of pah concentrations before and after the normalization to toc, it is evident that there are no significant differences in sediment concentrations across the sites. pca scatter plots before and after the normalization to toc values confirmed a non-significant correlation between pah concentration values and toc. furthermore, these plots confirm a clear separation of d5. site d5 was clearly separated from other sites in terms of pah composition along pc1 and pc2 (fig. s-8a, supplementary material). pc1 was positively loaded with hmw pahs, while pc2 was loaded with lmw pahs, indicating recent sediment pollution, as water solubility of pahs with two and three aromatic rings is much greater, relative to those of higher molecular mass (fig. s-8b, supplementary material). hierarchical cluster analysis (hca) the results of hierarchical cluster analysis are presented graphically, as the cluster tree. the dendrogram of the hca of sampling sites compared to the first factor pc1 is presented in fig. s-9a (supplementary material). the first group includes sampling sites d3 and d4. the vicinity of beočin cement plant probably has a great influence on the elevated concentrations of the above-men danube sediment contaminations with pahs 1447 tioned pahs at these sites. the second group includes sites d6, d7 and d8, where a moderate value of pahs was recorded, most likely due to the vicinity of rural areas where the combustion of coal and wood is the main source of heating, as well as biomass burning on agricultural fields. the proximity of “nis (petroleum industry of serbia) gazprom neft” oil refinery, oil and gas production, and processing facilities, along with transport of petroleum products, as well as the vicinity of the cogeneration system, contribute substantially to the increased pah concentrations at site d6. the third group includes sites d1, d5 and d10. the production processes that include smoking of meat delicacies in the meat factory “neoplanta” (10 km from the sampling point) have an impact on the presence of pahs at site d5. a significant contribution to pahs recorded at d1 arises from individual domestic production and processing of smoked meat. for site d10, the confluence of the great morava into the danube leads to the dilution and lowering of pah concentrations.31 the fourth group represents sites d2 and d9, which are mostly contaminated with pahs emitted from coal, fuel, and wood combustion. these locations are distinguished by the highest concentration levels of pahs, except nap, ace and fl, most likely due to close proximity to areas with intensive agricultural and industrial activities. it is worth mentioning that there are no official data on the quantity of smoked and dried meat from individual households in serbia, or on the quantities of burnt grass and crops. intensive agricultural processes involving wheat and corn are the key activities in the studied area, and the burning of these residues could be an important source of pahs. the dendrogram which corresponds to the second factor pc2 is shown in fig. s-9b (supplementary material). this dendrogram separates into two clusters all the sites at which we detected elevated concentrations of pahs originating from vehicular (mobile) emissions. one cluster includes only site d5, whereas the second one groups all other sites. the highest concentration of ace, fl and bip was measured at sampling site d5 as a result of the proximity of novi sad (the second largest city in serbia with 341,625 inhabitants in 2014). novi sad has one of the largest ports in the region, as well as intensive commercial and tourist activities, due to which the vehicular emissions are much higher than those observed at other sites. the dendrogram of selected sites compared to the third factor pc3 is presented in fig. s-9c (supplementary material). since factor pc3 denotes the natural sources of pahs, four clusters have been singled out, whereby the highest concentrations of ret and per were recorded at d7 in the vicinity of the danubetisa confluence. the tisa river is characterized by diverse desmid flora, such as chlorophyta and zygnematophyceae, bacteria, and various plants that can produce certain pahs through biosynthesis.33 the cluster with medium loading included d2, d3, d4, d6 and d9. however, for these sites, no data on sources of 1448 brborić et al. natural pahs exist; thus, it can be assumed that the thermal waters of the pannonian basin are one of their sources, as these were found to contain light pahs in deeper layers (below 1,300 m). moreover, pahs could occur as a result of naturally accumulated oil and gas in this basin, which is probably the case for sites with detectable (d8 and d10) and low detectable load (d1 and d5). pollution impact of pahs biological effects. in order to estimate the potential of pahs from danube sediments to produce harmful effects on all environmental matrices, wildlife, and human health, concentration levels were compared to the international sediment quality guidelines (sqgs). two sets of sqgs, effects range low–effects range median (erl/erm) and threshold effect level–probable effect level (tel/pel) values, have been used in this research to estimate the environmental toxicity of chosen pahs in danube sediments. moreover, these sets can be applied to recognize three ranges of contaminant concentrations, i.e., 1) low range – probably without the appearance of harmful biological effects (below the tel or erl values); 2) middle range – possible occurrence of harmful biological effects (between the tel or erl and pel or erm values); 3) upper range – probably harmful biological effects will occur (values beyond pel or erm values). erl/ /erm and tel/pel have been established to have a certain predictive capability, while taking into account their chemical bioavailability. comparison of pollutant levels with sqgs (table s-iv, supplementary material) confirmed that the concentration levels of all pahs were less than erm and pel values at all 10 studied sites. fluorene concentrations in riverbed sediment at d5 (88 μg/kg) were in the middle range (erl/tel and erm/pel) and were below erl/tel at the remaining nine sites. considering that site d5 is characterized by high contamination and low content of toc, this finding is expected. only light pahs were recorded at elevated levels and no adverse biological effects are anticipated at this site. in addition, at d9 (677 μg/kg), the total concentration of all quantified pahs exceeded the tel value, while remaining below the erl limit. in the analysed sediment samples, no value exceeds the erm/pel limits. these results are contrary to those shown in an investigation performed in taiwan, where the σpahs were between erl and erm values in five sediment samples (6.3 %), and below erl at the remaining 15 samples (93.7 %).34 pahs are pseudo-persistent in the environment and are characterized by liposoluble and carcinogenic properties. the bioavailability of most pollutants in sediments, according to the us epa,35 could be estimated using equilibrium partitioning sediment benchmarks (epsbs). for epsbs toxic unit (epsbtu) development of pahs in sediments, the normalized value of individual pahs compared with its final chronic value (fcv) was applied. after calculating σepsbtu for all examined pahs, it is necessary to apply a factor of uncertainty. danube sediment contaminations with pahs 1449 pah uncertainty factors were set to 4.14. values of the adjusted σepsbtu of pahs detected in the danube sediment ranged from 0.06 to 0.42. since the obtained values are below 1, it is assumed that the sediment will not be toxic to aquatic life. the reported results are consistent with the fact that pah concentration levels determined in danube sediments cannot be associated with a significant risk to humans and wildlife. potential toxic risk. the estimation of sediment toxicity collected from 10 sites along the danube was accomplished based on the summed concentrations of seven proven/probably carcinogenic individual pahs (pahscarc), containing b[a]a, chr, b[b]flu, b[k]flu, b[a]p, db[ah]a and ip34. the summary concentration values of the seven pahscarc (pahs7carc) in this paper were in the range of 26.54 to 277.54 μg/kg, with an average concentration of 135.31 μg/kg. in addition, the sediments from d9 contained slightly higher levels of pahscarc than those at other sites, while the sediment collected at site d5 had the lowest values. the obtained values were significantly lower than those determined for pahs7carc of superficial sediments from the region of estuarine danube coast (pahs7carc: 130.5−551.9 μg/kg19) and mithi river, india (pahs7carc for three sampling stations: 1,513±238, 1,369±242 and 1,018±130 μg/kg18). considering all probably carcinogenic pahs, b[a]p is the unique determined polyarene for which sufficient published toxicological data exist, in order to allow the calculation of carcinogenic potency factor. therefore, the probable toxicity of danube sediments was estimated by calculating the total toxic benzo[a]pyrene equivalent (teqcarc) for all pahscarc, using the toxic equivalency factor (tefcarc) relative to b[a]p for each pah (table s-v, supplementary material) and using the following equation:8 carc carci itotalteq tef= c (2) with: ci representing the values of particular pahscarc concentration (μg/kg) and total teqcarc in the danube ranged from 6.49 to 74.94 ng teq/g, with an average concentration of 35.20 ng teq/g. in comparison, obtained values of teq7carc were slightly higher than those pertaining to another region examined in the previous investigations, such as mvudi and nzhelele rivers (teq7carc: 24.86 ng teq/g and 18.40 ng teq/g, respectively35), and lower then total teqcarc values in sediments of danube estuary (teq7carc: 34.9 to 195.1 ng teq/g19), and mithi river in india (teq7carc for three sampling stations: 322.6, 255.28 and 188.21 ng teq/g18). the contribution of individual carcinogenic pahs to the total teqcarc in the danube varied based on the following chronology: b[a]p (67.66 %), ip (11.29 %), b[b]flu (7.95 %), db[ah]a (5.39 %), b[a]a (4.40 %), b[k]flu (2.84 %), and chr (0.47 %). the low and considerable values of teq7carc concentrations indicate low carcinogenicity. 1450 brborić et al. conclusion presence, origin and ecological risk of twenty-nine pahs detected and quantified in riverbed layer sediment collected from 10 sites along the danube in serbia were investigated. total 29 pah concentrations in sediment samples were relatively low in comparison with other relevant studies around the world, but still provide highly valuable information on the current status of aquatic systems. different statistical tools were used to provide a more precise picture of pah sources. the origin of pahs, as determined by the diagnostic ratios method, revealed the prevalence of pyrolytic origin at all studied sites, with the exception of d1 and d5, where the petrogenic origin was noted. due to the high presence of “light” pahs in sediment, the site d5 was distinguished as the area with significant recent contamination. analysis of the results using pca confirmed the dominant presence of anthropogenic sources (combustion and vehicular emissions). in addition to the expected potential anthropogenic sources of pah pollution, pca also revealed the presence of natural sources in this area. cluster analysis proves that the sediment sample collected near pančevo (d9), situated downstream of belgrade and industrial agglomeration pančevo, was characterized by the most significant pollution. the outcome of possible toxicity, as well as the evaluations of biological effects, show that danube riverbed sediments have a relatively low level of hazardous pollution. the only exception is site d9, which has a medium level of toxic pollution. the findings contribute to the establishment of a surveillance monitoring programme that would cover the serbian part of danube, with special focus on the impact of extensive areas under heavy agricultural activities. for that purpose and in order to measure the freely dissolved concentrations, the continuous sampling (with time weighted average concentration) should be performed, extremely low levels of organic pollutants should be detected and passive samplers should be used in future investigations. supplementary material additional data are available electronically from http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgements. this investigation was financed by the ministry of education, science and technological development, republic of serbia (iii46009 and tr34014), the czech ministry of education, youth and sports (lm2015051) and the european structural and investment funds, operational programme research, development, education (cz.02.1.01/ /0.0/0.0/16_013/0001761). danube sediment contaminations with pahs 1451 и з в о д просторна дистрибуција полицикличних ароматичних угљоводоника у седименту реке дунава у србији: антропогени и природни извори маја брборић1, бранислав врана2, јелена радонић1, мирјана војиновић милорадов1 и маја турк секулић 1 1 универзитет у новом саду, факултет техничких наука, департман за инжењерство заштите животне средине и заштите на раду, трг доситеја обрадовића 6, нови сад и 2 masaryk university, faculty of sciences, research center for toxic compounds in the environment, recetox, kotlářská 267/2, 611 37 brno, czech republic у раду је анализирано присуство полицикличних ароматичних угљоводоника (pah) у површинском слоју седимената прикупљених на 10 локација дуж реке дунав на територији србије. потенцијални извори загађења су идентификовани коришћењем различитих мултиваријатних техника. укупне концентрације 29 pah у узорцима седимената кретале су се у распону од 128,27 до 676,85 μg/kg. на основу добијених концентрација и pah образаца, утврђена је јасна просторна дистрибуција на испитиваним локалитетима. дијагностички односи указују на присуство пиролитичких pah, осим на два места где је pah контаминација настала из петрогених извора. варимакс ротирана анализа главних компоненти је дефинисала три главна извора (сагоревање угља, дрва и биомасе; емисије из возила; природни извори) који чине 92,1 % варијансе у оригиналним скуповима података. локације са различитим оптерећењима контаминације у зависности од идентификованих главних извора су уређене помоћу кластер анализе. резултати су упоређени са смерницама за квалитет седимента, а добијене вредности токсичних јединица током примене равнотежног партиционисања расподеле седимента и укупне токсичне еквивалентне количине 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {understanding bioplastic materials current state and trends} j. serb. chem. soc. 85 (12) 1507–1538 (2020) udc 577.11:766.092.000.57:620.28:502 jscs–5390 survey 1507 survey understanding bioplastic materials – current state and trends sanja jeremic1, jelena milovanovic1, marija mojicevic2, sanja skaro bogojevic1 and jasmina nikodinovic-runic1* 1institute of molecular genetics and genetic engineering, vojvode stepe 444a, 11042 belgrade 152, serbia and 2athlone institute of technology, dublin road, athlone, co. westmeath, ireland (received 20 july, revised 31 august, accepted 4 september 2020) abstract: plastic pollution is now considered one of the largest environmental threats facing humans and animals globally. development of bioplastic materials may offer part of the solution as bioplastics include both nondegradable and biodegradable materials with both being important for sustainability. bioplastic materials are currently being designed to encompass minimal carbon footprint, high recycling value and complete biodegradability. this review examines recent developments and trends in the field of bioplastic materials. a range of the most utilized bioplastic materials is presented (poly(lactic acid) (pla), polyhydroxyalkanoate (pha), starch, cellulose, bio-based poly(butylene succinate) (bio-pbs) and bio-polyethylene (bio-pe)) including their production, application and degradation options. keywords: bioplastics; biopolymers; biodegradation; poly(lactic acid); sustainability polyhydroxyalkanoates. contents 1. introduction 1.1. bioplastics – definitions 1.2.historic perspective of bioplastics 2. description of important bioplastics 2.1. polylactic acid (pla) 2.2. polyhydroxyalkanoates (pha) 2.3. starch and cellulose 2.4. poly(butylene succinate) (pbs) 2.5. biope 3. (bio)degradation and recycling of bioplastics 4. conclusions * corresponding author. e-mail: jasmina.nikodinovic@imgge.bg.ac.rs https://doi.org/10.2298/jsc200720051j ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1508 jeremic et al. 1. introduction “plastics” as synthetic polymers were introduced about 100 years ago and today they are one of the most produced, used, and versatile materials.1 nevertheless, their biggest advantage, namely durability, turned out to be their biggest problem, as their degradation rate does not match their intended service life and so their build-up in the environment is inevitable. environmental pollution from plastic is a vast issue, given that in 2018 alone the worldwide production of plastics from fossil resources reached almost 360 million tons, while in europe it reached 62 million tones.2 this translates that each person consumes 50 kg of plastic per year in the european union and 68 kg per year in the united states.3 approximately half of the synthetic polymers are used in single-use or shortlived products, which mostly end up on landfills and in oceans, where they become fragmented over time into ‘microplastics’ that harm and kill various organisms, finally ending up on our plates.4 in january 2018, the eu released its vision for a more sustainable plastics industry to be achieved by 2030. the new plastics strategy states that 100 % of plastics should either be reusable or recyclable by 2030.2 environmental concerns over plastic pollution coupled with a growing debate over crude oil dependence and depletion have sparked and fuelled interest in bioplastics. bioplastics include both nondegradable and biodegradable plastics (fig. 1) and both are important for sustainable solutions. the production and use of bioplastics are generally regarded as more sustainable activities when compared with plastic production from petroleum (petrochemical plastics; petroplastics) because they rely less on fossil fuels as the carbon source. they also introduce fewer, net-new greenhouse emissions if they biodegrade.5 bioplastics also significantly reduce hazardous waste caused by oil-derived plastics. however, the manufacture of bioplastic materials is still often reliant on petroleum as a source of energy and materials.6,7 1.1. bioplastics – definitions the term “bioplastic” is often used by the public and in scientific literature. however, the term “bioplastic” refers to either to the bio-based origin of the plastic or the biodegradable character of the plastic. these two aspects of plastic are not synonymous, and therefore, the term ‘bioplastic’ is confusing. in this review, a clear distinction is made between bio-based and biodegradable plastics (fig. 1). according to the international union of pure and applied chemistry (iupac), a bioplastic is derived from ‘‘biomass or monomers derived from biomass and which, at some stage in its processing into finished products, can be shaped by flow’’.8 it is also important to make the distinction between degradable, biodegradable and compostable. these terms are often incorrectly used interchangeably. degradable plastic is a plastic that will undergo some significant structural ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. bioplastics: current state and trends 1509 change in some definite environment. the “environment” may as well be an industrial process instead of some naturally occurring microbial one. according to the iupac, biodegradable polymers are defined as “polymers, susceptible to degradation by biological activity, with the degradation accompanied by a lowering of its mass”.9 some other definitions require a biodegradable material to be mineralized into carbon dioxide, water, and biomass during biodegradation (standard cen/tr 15351:2006).10 fig. 1. not all bio-based plastics are biodegradable, and not all biodegradable plastics are bio-based. pe=polyethylene; pp=polypropylene; pet=poly(ethylene terephthalate); ps=polystyrene; pvc=poly(vinyl chloride); ptt=poly(1,3-propylene terephthalate); pcl=poly(caprolactone); pbat= poly(butylene adipate terephthalate); pbs(a)= =poly(butylene succinate(adipate)); pla=polylactide; pha=polyhydoxyalkanoate. compostable plastic is a plastic “capable of undergoing biological decomposition in a compost site as part of an available program, such that the plastic is not visually distinguishable and breaks down to carbon dioxide, water, inorganic compounds, and biomass, at a rate consistent with known compostable materials (e.g., cellulose) and leaves no toxic residue...” defined by the american society for testing and materials.11 unfortunately, the term “biodegradable plastic” has also been used by producers of specially modified petrochemical-based plastics that appear to biodegrade. this is the case with the plastics to which a degradation initiator has been added to achieve a controlled uv/oxidation disintegration process. this type of plastic may be referred to as degradable plastic or oxy-degradable plastic or photodegradable plastic as the process is not initiated by microbial action. however, plastic bag manufacturers often misrepresent the degradability of their products by misleading the use of the terms biodegradable or compostable. it has been shown that even biodegradable plastics might not biodegrade in every environment.12 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1510 jeremic et al. 1.2. historic perspective of bioplastics natural plastic materials (plant gum, shellac, starch) have been used for thousands of years.1 the first plastics in the modern sense were celluloid and cellophane and they were bio-based. cellophane, commercialized in 1912, is still in use today.13 in the early 1950s, amylomaize (>50 % amylose content corn) was successfully bred and commercial bioplastics applications started to be explored. modern bioplastics started emerging in the 1980s when the environmental effects of plastic waste became obvious. the first bioplastics were blends of starch with conventional polymers so that certain biodegradability and the use of natural feedstock were partly achieved. in 1982, biopol (poly(hydroxybutyrate), phb) was introduced as the first fully biodegradable plastic, but costing nearly 20 times its non-biodegradable competitors. in 2004, nec developed a flame-retardant plastic, poly(lactic acid) (pla), without the employment of halogens and phosphorus compounds. in 2005, fujitsu became one of the first technology companies to make personal computer cases from bioplastics, which were featured in their fmv-biblo nb80k line. in 2007, braskem of brazil announced it had developed a route to manufacture high-density polyethylene (hdpe) using ethylene derived from sugar cane.1,14 there are three ways to produce bioplastics that include: i) using natural bio -based polymers such as starch with partial modifications to meet the requirements; ii) producing monomers by fermentation followed by polymerization (pla, poly(butylene succinate) (pbs), bio-based polyethylene, bio-pe); iii) producing them by bacteria directly (polyhydroxyalkanoates, phas).15 the first generation technologies focused on feedstock such as corn, starch, or rice to produce bioplastics, which were deemed unacceptable in the food vs. fuel debate, so the focus shifted towards various waste streams from wood and paper as well as food industries or to third generations from micro-algae, marine bacteria, sewage sludge).15–17 in the last decade, the production of bioplastics has been accelerated and reached substitution alternatives for major petrochemical plastics (table i). table i. bioplastics alternatives for major petrochemical plastics; source: chemical market resources inc.; substitution potential: +++ high; ++ medium; + low; – not foreseen bioplastic ldpea hdpea ppa psa pvca peta pura plaa + ++ ++ ++ – ++ – phaa ++ +++ +++ ++ + ++ ++ starch blends ++ ++ ++ + – ++ – bio-pe +++ +++ – – – – – aldpe = low-density polyethylene; hdpe = high-density polyethylene; ps = polystyrene; pp = polypropylene; pvc = poly(vinyl chloride); pet = poly(ethylene terephthalate); pur = polyurethane; pla = polylactide; pha = polyhydoxyalkanoate ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. bioplastics: current state and trends 1511 in most cases, they exhibit similar properties and can even exceed the performances of conventional plastics: e.g., pla can replace fossil-based polystyrene (ps), and can be modified to replace conventional pe or polypropylene (pp); pha can gradually substitute pp and low-density polyethylene (ldpe) with similar physicochemical, thermal, and mechanical properties; poly(hydroxybutyrate) (phb) possesses better physical properties than pp for food packaging applications and is completely nontoxic. in addition, bio-based polyesters have the potential to avoid the release of micro-plastics if they are formulated to be biodegradable or compostable. on the other hand, the cost of bioplastics is currently still not competitive with that of petroplastics. bioplastics do not yet reach fossil fuel parity of fossil fuel-derived energy for their manufacture, reducing the cost advantage over petroleum-based plastics.5 2. description of important bioplastics bioplastics contribute less than one percent to the overall plastics production, but the market is growing. about half (44.5 % in 2019) of all bioplastics on the market are bio-based and not biodegradable (fig. 2). bio-pe, bio-based pet (bio-pet) and bio-based ptt (bio-ptt) account for the majority of these so -called drop-in materials. they are more or less equivalent to their fossil-based counterparts, so they are very convenient for the industry. producers and recyclers can drop them into their existing infrastructure, however, they cause the same kind of pollution as petrochemical plastics. besides pla, which accounts for 14 % of the global production capacity for bioplastics (biodegradable and non-biodegradable), mainly starch blends (21 %), other biodegradable polyesters including pbs (4 %) and phas (1 %) are produced on the industrial scale (fig. 2). fig. 2. global production capacities of bioplastics in 2019 (data adapted from european bioplastics18). five commercial bioplastics have been chosen for this review based on the volume of their production and the wide range of applications that they cover (table ii). ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1512 jeremic et al. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. bioplastics: current state and trends 1513 2.1. poly(lactic acid) (pla) pla is a biobased and biodegradable aliphatic polyester that represented 13.9 % of global bioplastic production capacities in 2019 (fig. 220). lactic acid (2-hydroxy propionic acid) is the basic monomer of pla produced either by bacterial fermentation of basic carbohydrates or by chemical synthesis. industrial lactic acid producers, such as natureworks and total corbion, utilize the lactic fermentation process rather than synthesis.21 the use of corn starch, cassava roots, sugar cane, or potato as the carbohydrate source makes the industrial process of lactic acid production sustainable and renewable.22 pla is one of the most efficient biopolymers, yielding 1 kg of polymer from 1.6 kg of the fermented amount of carbohydrate feedstock, while other biopolymers require 2–3 times more feedstock for production. pla and pla-blends generally come in the form of granulates with various properties and are used in the plastics processing industry for the production of foil, moulds, cups, bottles and mulch films used in agricultural fields. on the other hand, due to the relatively low glass transition temperature of pla, pla cups cannot hold hot liquids, so much research is devoted to the development of a heat resistant pla. additional research is also ongoing to find even more eco-friendly and economical methods for producing lactic acid by using crop residues, such as stems, straw, husks, and leaves as carbohydrate sources.23 lactic acid can be used to produce pla of variable molecular weights, however, usually, only high mw pla has major commercial value in various industries. there are three routes for pla production: 1) direct condensation polymerization: esterification of lactic acid monomers and free water removal using progressive vacuum and high temperatures, resulting in a less-desired low mw pla; 2) direct polycondensation in an azeotropic solution: pla production by direct condensation and continuous removal of condensation water by azeotropic distillation; 3) polymerization through lactide formation (ring-opening method): industrially accomplished for high mw pla production using lactide as an intermediate state. this process results in a metal catalyst combining with a lactide to form larger pla molecules. pla has versatile applications in the food-packing industry, medical and cosmetics industry, textile industry, structural applications and 3d printing (table ii). the global pla market was valued at usd 673.88 million in 2018 and it is estimated that it will generate a net revenue of approximately usd 2277.57 million by 2027. the pla market is dominated by 1–5 major players, i.e., natureworks, total corbion, synbra technology bv, futerro and sulzer ltd., with natureworks and total corbion being the major producers, with a ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1514 jeremic et al. capacity of 1.5×105 and 0.75×105 metric t per year, respectively (table s-i of the supplementary material paper to this paper).24 in the past 10 years, the vast majority of patents regarding pla production are related to methods of processing pla into materials for specific applications, such as 3d printing wires, fibbers, sheets, etc., or the modification of available pla in order to improve the properties (cn108705753a, cn106738783a, cn104138310a, us2015337097a1 and cn104356365a). when it comes to patents concerning the production of raw material, they are mainly focused on methods of obtaining high molecular weight pla (us9023953b2, us9062006b2 and cn106750199a). additionally, two patents should be emphasized since they deal with the production of lactic acid using agricultural feedstock and sideline products, thus making pla production process ecologically and environmentally even more viable (us7507561b2, cn104592500a). as mentioned, pla has some significant shortcomings, such as low melt strength, poor processability, high brittleness, low toughness, and slow biodegradation rate, which hamper specific pla applications.25 therefore, pla is often blended/combined with a range of different polymers, additives, and fillers in order to improve material properties, reduce costs and open up new opportunities for pla application.26 some of the most studied pla blends with other polymers are listed below: • the development of pla blends with biobased and biodegradable thermoplastic starch (tps) is of great interest for food packaging applications and could also be considered in biomedical applications, due to achieved improvements in toughness;27 • addition of biobased and biodegradable phb to pla enhances its crystallinity, which is important for food packaging applications in order to increase the barrier performance of pla based materials;28 • pla blends with biodegradable pcl have improved ductility and toughness compared to neat pla, and increased tensile strength compared to neat pcl, and as such, they are utilized in tissue engineering and grafts, as well as in packaging applications with easy-open peelable feature;29 • pla and biodegradable pbs blends exhibit ductile behaviour with gradual losses of strength and modulus during biodegradation, so they have potential in food packaging applications. in addition, pbs/pla blends have excellent mechanical properties and suitability as materials for 3d printing;30,31 • pla and synthetic, non-degradable peg blends have decreased viscoelasticity, and increased hydrophilicity and degradation rate, and often are used for drug delivery and scaffolds. recently, 3d-printed scaffolds have also been produced using pla/peg blends.32 recycling, as one of the possible disposal routes for plastic, has been extensively studied on pla. currently, only 10 % of pla waste is returned to the ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. bioplastics: current state and trends 1515 pla production process.33 pla can undergo mechanical or chemical recycling. an easy and the cost-effective way to recycle post-consumer pla is mechanical recycling, which involves recovering, sorting, regrinding, and reprocessing the waste plastic, has become common practice in the industry.34 however, this method has limitations affecting the pla quality, thus limiting the range of products in which it could be used, and reducing the economic viability.35 an alternative is chemical recycling, during which pla is hydrolyzed at a high temperature to yield lactic acid, which could be readily polymerized to high mw pla. natureworks is an example of successful off-grade ingeo™ recycling using a chemical method, while galactic started a pilot unit to produce recycled pla using a chemical recycling process (loopla). however, chemical recycling is still a complex and expensive process. although pla is potentially recyclable, no separate recycling stream for pla yet exists.36 the presence of pla in the current plastic recycling infrastructure, even at low concentrations, causes contamination of the recycling stream and production of inferior recycled material. thus, it is crucial to remove pla from other plastics and to establish single streams for materials such as pla in order to recycle them. in addition, pla is assigned the resin identification code (ric) number “7-other” (astm d7611), with several other, rather new, polymers, and that is not considered accurate for the identification and (pre)sorting of pla from a waste mixture.35 another end-life scenario for pla and the natural way of recycling is biodegradation. numerous literature data deal with pla degradability in soil and compost. the majority of these studies used industrially produced pla for testing, and polymer weight loss or molecular weight loss as methods to assess degradability. pla degradation upon disposal in the environment is very challenging due to the resilience of pla to the actions of microorganisms in soil or sewage under ambient conditions.21 no significant pla degradation was observed when the polymer was buried in a real soil environment, or in soil under controlled temperatures of 25 and 37 °c, even after 24 months of exposure.37–40 however, pla – including commercially available pla bottles and pla deli containers, could be degraded in the compost after only 45–60 days, but high temperatures of 50–60 °c are required to hydrolyze the polymer into smaller molecules, which are then degraded by microorganisms to co2 and h2o.41,42 a search of patents from the previous decade revealed a large number of patented microorganisms and corresponding enzymes with pla degrading activity (table iii). since pla must first be hydrolyzed at elevated temperatures to reduce the molecular weight before biodegradation can commence,38 it is more accurate to say that pla is compostable, but due to a specific temperature and moisture conditions necessary for proper pla composting, the path to zero waste is complicated and unattainable at this moment. in contrast to soil and compost, only a few studies deal with pla biodegradation in aquatic environments. since after 10 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1516 jeremic et al. weeks no evidence of microbial degradation was found, the authors suggested that marine microbes have a limited ability to degrade pla.43,44 table iii. examples of patented microorganisms for pla degradation patent number year pla-degrading microorganism (enzymes) cn110317762a 2019 pseudomonas sp. lxm88/protease wo2017198786a1 2017 – ca2937569a1 2017 alcanivorax borkumensis, rhodopseudomonas palustris /hydrolase wo2016146540 a1 2016 micromonospora sp./hydrolase wo2016062695a1 2016 actinomadura keratinilytica t16-1/hydrolase au2014325231a1 2016 protease ep2483429a1 2012 ochrobactrum sp. cn102380180a 2012 lentzea waywayandensis, tritirachium album, amycolatopsis orientalis cn102181378 (a) 2011 pseudomonas sp. ds1001 jp4273504b2 2009 aspergillus oryzae, aspergillus soya /serine hydrase 2.2. polyhydroxyalkanoates (pha) phas are a family of natural biodegradable polyesters, usually produced by prokaryotes as cytoplasmatic water-insoluble storage compounds of carbon and energy.45 since the discovery of the simple phb homopolymer by lemoigne in the 1920s, a family of over 150 different aliphatic polyesters of the same general structure was revealed. as a result, the polymer properties demonstrated by this family are very broad.46 although their shape, size, structure, and physical properties depend on the producing organism and cultivation conditions, all polyesters are thermoplastic, water-insoluble, non-toxic, relatively resistant to hydrolytic degradation, biocompatible and biodegradable.47 phas are classified into two major subdivisions by the carbon chain length of their monomeric units: short-chain-length-3-hydroxyalkanoates (scl-3ha) have 3–5 carbon atoms and medium-chain-length-3-hydroxyalkanoates (mcl-3ha) with 6–16 carbon atoms.45 phb was the first bacterial pha identified. the unique properties of phas are recognized as better oxygen barrier than pp and pet, better water vapour barrier (than pp), and fat/odour barrier. such superior physicochemical properties of pha (e.g., in reference to pp) have promoted their usage in various fields, including food packaging.48 however, the poor mechanical performance and melt processing behaviour of phb, i.e., high brittleness, low thermal stability and difficult processing, along with insufficient barrier properties, limits its widespread use.49 several companies have developed pha copolymers in order to improve the properties of phas. the incorporation of secondary different hydroxy acid (ha) monomers other than phb, such as 3-hydroxyvalerate (3hv), 3-hydroxyhexa________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. bioplastics: current state and trends 1517 noate (3hh), 3-hydroxypropionate (3hp) and 4-hydroxybutyrate (4hb), into the polymer chain to form copolymers is a common strategy.47 one of the first copolymers to be manufactured in the industry was biopol®, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (phbv). this copolymer is a thermoplastic and has a melting temperature in the range of 140–180 °c. it has a range of uses, such as packaging, shampoo bottles, disposable razors, disposable cups, surgical stitches, surgical pins, disposable knives and forks, woven medical patches and nappy linen.47 the mole percentage of 3hv is important in determining the properties of the copolymer since the composition of p(3hb-co-3hv) can range from 0 to 30 mol. % 3hv. li et al. found better tensile strength and young’s modulus of p(3hb-co-3hv-co-3hhp) in comparison to p(3hb).50 the p(3hb-co-4hb) copolymer exhibits a broad array of morphologies, such as highly crystalline to elastomeric based on the concentration of 4hb.51 phb has a comparable melting temperature to pla, and thus allows for the blending of both polymers in their molten state.52 blends of phas with natural raw materials from sustainable resources are well discussed.53 the most common are pha blends with starch, lignin and cellulose derivatives.54,55 many years of research efforts have led to the large-scale production of pha and its copolymers. procter and gamble have developed nodax® (table ii), pha polymer consisting of phb and a comparatively small quantity of medium chain length (mcl) monomers. the mcl units used include 3hh, 3-hydroxyoctanoate (3ho) and 3-hydroxydecanoate (3hd). tianan’s biopolymer enmat® is a phbv copolymer containing about 5 % 3hv, which improves the flexibility of the polymer. the usa based company newlight technologies chose methane as the carbon source for fermentation with a planned capacity of 43,000 t per year within the next 20 years. other commercial phas are produced by bio-on (minerv®), metabolix (mvera®, mirel®) and phb industrial (biocycle®) among other manufacturers (table s-ii). worldwide, more than 20 companies are known to be engaged in pha production and applications. china, italy, the usa and brazil represent the main producers of phas using corn, cassava, sugar beet juice and sugarcane.56 pha has been produced with limited success on the market mostly due to the high production costs and instability of thermo-mechanical properties resulting from changing molecular weights and structures, which are related to varying the pha synthase activity.57 the mechanical properties and biocompatibility of pha can also be changed by blending, modifying the surface or combining pha with other polymers, enzymes and inorganic materials, allowing a wider range of applications. although 300 types of natural pha producers have been identified, only a few bacteria have been adopted for commercial production of pha. such bacteria, including alcaligenes latus, bacillus megaterium, cupriavidus necator and ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1518 jeremic et al. pseudomonas oleovorans, are found to convert different kinds of carbon sources into pha.48 the main carbon sources are glucose, fructose and fatty acids, such as lauric acid. in particular, the feedstock cost for pha production represents half of the overall production cost.58 an enormous number of studies have been conducted to find low-cost feedstock for pha production on the industrial scale. although food waste is a good initial feedstock for the production of bioplastics, it must be pre-treated to improve or modify the physicochemical and biological properties.59 gerngross and co-workers reported for the first time the in vitro production of phb.60 this finding brought a new perspective and insight into economical processes for the synthesis of phas using enzymes as a catalyst and will offer the possibility of carrying out reactions in aqueous solution, which is a cheaper and more environmentally benign medium. unusual phas can be synthesized from a wide range of substrates mediated by commercially available enzymes, such as lipases and cutinases. major disadvantages of this approach include high consumption of organic solvents, a progressive increase of medium viscosity over time and relatively low mw of the obtained polymers.61 to commercialize the enzymatic process, the production and purification costs of the enzyme need to be reduced.62 today industries are still using the more robust and conventional way to synthesize phas, which is microbial fermentation. three techniques have been investigated, including mechanical, chemical and biological recycling.63 phas can be mechanically recycled with some loss of molecular weight and mechanical properties.64 recently, phbv was mechanically recycled by zaverl et al.,65 showing that phbv is recyclable for up to five cycles because the reprocessing cycles did not significantly affect the mechanical properties (tensile and flexural testing, and impact toughness). the main disadvantage of mechanical recycling is that it is not applicable for the collected bioplastics from waste, which is expected to be heterogeneous. chemical recycling of phas by thermal degradation has resulted in a transformation of phas into vinyl monomers.66 ariffin and co-workers used alkali earth compound catalysts (cao and mg(oh)2) to enforce smooth and selective degradation of the polymer at a lower temperature. the obtained monomers, crotonic acid and 2-pentenoic acid, were then used as feedstock for the production of poly (crotonic acid-co -acrylic acid) through copolymerization. phas are an appropriate candidate for biological recycling as their biodegradation can occur under both aerobic and anaerobic conditions.67,68 numerous bacteria, streptomycetes, and fungi isolated from different terrestrial and aquatic environments produce extracellular pha depolymerases in order to hydrolyze solid pha into water-soluble monomers or oligomers to be used as nutrients.69 in this sense, many scl-pha depolymerases have been purified and characterized in contrast to a limited number of mcl-pha depolymerases. it is very difficult to ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. bioplastics: current state and trends 1519 compare the activities of phb depolymerases because of the various set of conditions used in the tests. martinez-tobon and co-workers compared the activity of strains with proven and predicted phaz activity for phb film degradation, which helped to identify strains displaying high degradation activity. the strains crupriavidus sp. and comamonas testosteroni 31a showed the highest percentage weight loss after 7 days, 90 and 53 %, respectively.70 there are some examples of highly tolerant enzymes in harsh conditions such as phazs depolymerases from c. testosteroni ym1004, schlegelella sp. kb1a, schlegelella thermodepolymerans, streptomyces sp. in1 and thermus thermophilus hb8. these biocatalysts could be employed in the degradation of polymers from industrial wastes that may require high ph and/or temperatures since polymer solubility is increased and microbial contamination is reduced.71 several factors can influence the time needed for total biodegradation, including polymer characteristics, surrounding conditions, and the type of degrading organisms. the ‟real-life tests” separate composting, soil burial and field-testing.68 in a study by hablot et al., the degradation of pla/pha non-woven mulches was performed using simulated weathering. at 63 °c, the repetition of uv irradiation and the water spray system promoted the degradation of the pla/pha blend and molecular weight loss of 90 % was achieved after 180 days.72 when the phb films were immersed in the sea at a depth of 1.5 m, their weight loss after 4 weeks was about 90 %. on the contrary, the biodegradation in seawater by the laboratory test method for 4 weeks was around 50 %.73 biodegradation tests performed in artificial environments lack transferability to real conditions and, therefore, there is the necessity of environmentally authentic and relevant field-testing conditions. in the patent base for the last 10 years, a great number of innovations related to the advancement of the pha production process have been described (table s-iii). many inventions are focused on downstream feedstock costs (wo2014032633a1, us20190360008a1). the microbial production of pha copolymers from two raw materials (sugar cane and sugar beet) containing sucrose is disclosed (ep2780461a1). isolation of new pha-producing microorganisms and genetic modification of existing ones is also the purpose of many inventions. in wo2012149162, the organisms are engineered to efficiently utilize ethanol (or in some cases xylose) as the carbon source to produce a range of pha copolymers in a cost-effective yield. the process of producing monomers from pha by depolymerisation is the focus of many inventions (cn104328062a, wo2016085396a1). a method for efficiently decomposing pha and converting it to biogas containing methane as the main component, particularly under anaerobic conditions, was disclosed by kaneka (wo2015122190a1). additionally, major pha producers disclosed several patents related to the improvement of the production process, processing, ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1520 jeremic et al. and functional modification of phas (us2017369908a1, wo2018021046a1, and us2020109423a1). 2.3. starch and cellulose the interest in polysaccharides as biomaterials has been increasing continuously during the past decade owing to their applications in pharmaceuticals, biomedical use, food supplements, and cosmetics. the fact that these polymers, such as cellulose and starch, are extracted from natural resources has led to the impression of good biocompatibility and biodegradability.74 starch is a well-known, versatile, inexpensive and primary energy reserve polysaccharide in plants. generally, the native starch isolated from different plants tends to have limited shear resistance, thermal resistance, thermal decomposition, and a high tendency toward retrogradation. additionally, starch dissolves in the aqueous media, and it shows low mechanical and shapes stabilities in liquids. to overcome these limitations, starch can be combined with stable, synthetic, thermoplastic polymers.75 for example, pairing starch with pcl resulted in improved processability of starch, reduced high stiffness, and overcome high moisture sensitivity of starch, which is one of the greatest weaknesses of starch as a biomaterial. on the other hand, starch improves the biodegradability of pcl and, as the cheapest biomaterial on the planet, starch can substantially lower the high cost of the final product.76,77 development of starch blending techniques tends to be more interesting while potential applications are extensive and they can be able to substitute an older material that exhibits the same properties. among its tissue-engineering applications, starch is most famous for its use in generating scaffolds for bone regeneration due to its bone-bonding behaviour when reinforced with hydroxyapatite, good mechanical properties, non-cytotoxic and biocompatible nature, excellent support for cell adhesion, and thermoplastic behaviour when combined with thermoplastic polymers.78,79 the plant material is grounded in water, the debris is filtered from the slurry, and starch granules are obtained after centrifugation from the suspension.80 the content of amylose and amylopectin in starch varies and largely depends on the starch source. typically, the amylose content is between 18–28 %. the ratio of amylose and amylopectin in the starch may affect starch behaviour during processing and the properties of the end product. as the amylose content increases, the crystallinity of starch-based products increases as well, resulting in texture firming.81 several techniques may be applied to develop starch-based biomaterials with improved properties: thermoplasticization, cross-linking, esterification and blending with different polymers. most of the starch produced worldwide is derived from corn but other types, such as cassava, sweet potato, potato and wheat starch, are also produced in large amounts.82 italy-based novamont is a manufacturer of starch-based plastic called mater-bi used to make biobag branded certified compostable bags (table ii). ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. bioplastics: current state and trends 1521 biobag® is the world’s largest brand of certified compostable bags and films made from mater-bi. spudware® was one of the first bioplastic starch-based cutleries brought to the market. it stood out for its ability to withstand high heat. biomass packaging® uses tsp to manufacture water-soluble packing (http:// //www.biomasspackaging.com/education/bioplastics/). some fishing hooks are also made from starch-based polymers. since corn starch can absorb 1000 times its weight in moisture, it is used for disposable diapers (tethistm), fuel filters to remove water (super absorbent co.), and as a treatment for burns (sonett).83 cellulose is the most abundant renewable resource on the planet and the major structural component in plant cell walls. besides plants, some species of bacteria and algae produce cellulose. in general, cellulose, regardless of the source, is a highly crystalline and high molecular weight biopolymer and is, usually, fibrous, tough and hydrophilic but insoluble in water and other common solvents. the chemical structure of cellulose makes it suitable to form hydrogels, which are used in tissue engineering, cartilage modelling, bone implantation, cell culture scaffolds, enhanced drug delivery, heavy metal absorbance and for retaining soil water and efficient fertilizer release for agricultural efficiency. due to its abundance, cellulose can serve as a virtually inexhaustible source of raw material in the production of sustainable bioproducts.84,85 nanocomposites of cellulose have revolutionized the medical field and are being used in tissue engineering, ligament engineering and wound healing. in addition, cellulose-based ethanol production helps to reduce the pressure on conventional energy sources.86,87 as its anhydroglucopyranose unit contains reactive hydroxyl groups, cellulose has potential use in the design of advanced polymeric materials. so far, most of the industrial modifications of cellulose to improve its properties have been cellulose esters, ethers and graft copolymers, obtained by exchange of the hydroxyl groups of the cellulose molecules.88 cellulose can be produced by two means: natural synthesis procedures including plant photosynthesis and microbial synthesis.89 cellulose is present in small quantities in all brown algae, and most of red and golden algae. the most effective producers of bacterial cellulose (bc) are a. xylinum, a. hansenii and a. pasteurianus.90–92 bc is chemically pure, free of lignin, hemicellulose and pectin. current methods of bc production include static culture, submerged fermentation through aerated or agitated cultivation and airlift bioreactors.93 in static cultures, bc is formed at the liquid–air interface as hydrogel-like membranes, with thickness from millimetres to centimetres. the microfibrillar structure of bc is responsible for most of its properties, such as high tensile strength, high crystallinity index and higher degree of polymerization.94,95 the modification of bc can be performed during its biogenesis by the introduction of different substances into the bc-producing growth medium or by modification of bacterial cells. the other approaches for modification of cellulose (plant or bacterial) inc________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1522 jeremic et al. lude the use of physical factors, such as ultrasound irradiation (or sonication), static magnetic field (smf), or rotating magnetic field (rmf) exposure. bc can be modified by cultivation with an agno3 solution, which results with the bc membrane containing silver nanoparticles. the new properties of bc can also be achieved by incorporating reinforcing particles into the membrane structure.96,97 it is challenging to estimate the time that cellulosic material will be available to microbial degradation because of the limited number of microbial organisms able to degrade solid cellulose and due to different crystalline forms of cellulose. as a result, microbial degradation of solid cellulose can often be the rate-limiting step. the deconstruction of cellulose contained in the plant cell wall requires the action of specific enzymes able to release degradation products from this substrate.98 enzymatic degradation of cellulose is generally performed by hydrolases. biodegradation of cellulose wastes by fungal or bacterial enzymatic activities represents a large area of research experiments concerning the influence of different physical and biochemical factors.99 natureflex™ by futamura (japan) is a major source of renewable cellophane film (natural biopolymer made of cellulose from plants). nature flex bags generally degrade in several weeks in a home compost pile or a commercial compost facility. biomass packaging carries cellophane products made with nature works llc, a 100 % cellulose biopolymer with excellent oxygen, grease, oil and moisture barrier characteristics. they are used as packaging films for bakery product wraps, other food wraps, and food-grade transparent bags. bioprocess®, xcell® and biofill® are bacterial cellulose-based products already available commercially for topical application in wound healing,100 http://www.biomasspackaging.com/education/bioplastics/). bc is extensively used in the food industry, packaging and acoustic diaphragms for audio speakers and headphones. large surface area and great absorbance properties enable the use of very low bc concentrations/amounts to form excellent binding, thickening and coating agents. papers that are coated with bc are extremely strong and smooth as the coating protects the underlying fibres from moisture.101 the biodegradation of starch-based materials depends on the starch processing method used as well as on the biodegradability of other components. the main elements in biodegradability testing are the incubation of the sample under conditions conducive to microbial attack and/or their enzymes, and evaluation of the degree of degradation. polyvinyl alcohol (pva) and starch are biodegradable in some microbial environments: they are compatible and their blends present good film properties. several wheat starch/pva/glycerol blends prepared using the solution casting technique under iso 14855 were examined by composting for 45 days. starch and glycerol were absolutely degraded while pva appeared practically intact.102 the biodegradation of co-extruded starch/pla films was studied in liquid, inert solid, and composting media using experimental iso ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. bioplastics: current state and trends 1523 methods. the percentage of mineralization in the compounds was higher than the minimum required 60 % that allows a compound to be classified as biodegradable. researchers found that the addition of starch enhanced the biodegradation of the pla component, especially in liquid media.103 studies also showed that ternary blends composed of pla, pcl, and starch buried in soil degraded rapidly in the first 8 weeks.104 biodegradability of cellulose is dependent on the degree of crystallinity, structure, functional groups, cross-linking and molecular weight of the cellulose.105 two general types of substrates were used to measure the biodegradation of cellulose: relatively unaltered natural substrates (pure crystalline cellulose or biomass) and modified cellulosic substrates (substituted or dyed celluloses). studies showed that phb/bc composite biodegraded at a greater rate and extent than those of phb alone, reaching 80 % degradation after 30 days, whereas phb did not reach this level of degradation until close to 50 days of composting.106,107 even though the biodegradation of some cellulose samples have been studied, there is still a lack of a comprehensive study on the biodegradability of a variety of cellulose-based packaging materials that could potentially replace synthetic packaging films.108 a list of patents related to starch and cellulose is presented in table s-iv of the supplementary material. the process described in wo2017130106a1 relates to the production of starch from microalgae, which is more efficient in comparison to the conventional production. method of producing a starch-based biodegradable polymer, using processed cannabis waste as the carbon source, is described in wo2020037394a1, while the process for producing biodegradable plastic from renewable resource-based agricultural by-products is covered in wo2019155398a1. patent wo2018125897a1 is related to the production of material formed from one or more starches, a plasticizer and water, resulting in improved sustainability, biodegradability and increased strength. cn102585485b relates to the preparation of a composite material (starch combined with thermoplastic polyurethane). this material has the advantages of high mechanical property, low cost, biodegradability and the preparation process is simple. wo2016138593a1 also covers the potential of starch to combine with one or more biodegradable polymers (pcl, pbs, pla, and pha) in order to obtain biocomposites with tailored properties. wo2018041779a1 and wo2020034958a1 describes the application of starch and its modifications in food packaging using low tensile strength products, while wo2017091463a1 provides methods for making coated particles using different kinds of starch. patent wo2019209834a1 relates to new additive materials that are physically blended with polymeric materials, such as starch, to impart biodegradability to polymers that are not otherwise biodegradable. patent wo2020014762a1 relates to the production of a nanocellulosic material based on fractioning a cellulosic pulp stream originating ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1524 jeremic et al. from pre-treatment or mechanical defibrillation. wo2019221535a1 presents a method for the manufacture of crystal nanocellulose and wo2020015884a1 describes a device and a method for producing nanocellulose from natural raw materials. wo2017160218a1 also relates to a method of manufacturing a cellulose product from cellulose fibres. on the other hand, bacterial cellulose production on cassava bagasse as a suitable feedstock and its processing is covered in wo2016029432a1. methods for the production and processing of multi-phase biomaterials based on bacterially synthesized nanocellulose are presented in wo2016113400a1. wo2017192476a1 presents an invention that provides a pulp product with nanocellulose as a tool for improving the strength properties. cellulose derivative with excellent thermoplasticity, waterproofness, elastic modulus and impact strength and its production method are presented in wo2017061190a1. patent wo2018187782a1 provides a method for the modification of the surface of polymer-based materials (bacterial nanocellulose, nanocellulose and a cellulose derivative) and compositions generated thereby. wo2016174104a1 relates to the use of a bacterial nanocellulose composite in chip technology and material engineering, and furthermore, for medical purposes (skin, tissue or neuro transplant). wo2020035734a1 is also connected with medical usage of cellulose nanofibrils describing a method of producing a threedimensional autologous fat graft. 2.4. poly(butylene succinate) (pbs) pbs is an aliphatic biodegradable polyester produced by polycondensation from two raw materials: succinic acid (sa) and 1,4-butanediol (bdo). the mechanical properties of pbs are similar to those of low-density polyethylene (ldpe) or polypropylene (pp)109,110 but it can be biodegraded in contrast to ldpe and pp.111 beside the production of pbs by polycondensation, recently, a lipase-catalysed synthesis of pbs was developed.112 pbs synthesized by lipase has a narrower polydispersity index than that synthesized by polycondensation, but the remaining lipase may cause a problem in the subsequent thermal processing.112 in the beginning, pbs was a biodegradable but petro-based polyester, while currently, large plants for producing renewably sourced sa have been developed and mitsubishi commercializes bio-based pbs (bio-pbs) that is obtained using bio-based sa (bio-sa). furthermore, bio-based bdo (bio-bdo) can also be produced on an industrial scale. nowadays, products of 100 % biopbs can be found on the markets. bio-pbs is produced in various demo and pilot plants, mainly from sugarcane and cassava monomers.113 the production of bio-pbs includes the productions of bdo and sa, and the production of bdo includes the production of bio-ethanol and sa (argonne national laboratory, 2014).113 the production of bio-ethanol using sugarcane is separated into three major stages: the sugarcane plantation, the conversion of sugarcane into molasses by ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. bioplastics: current state and trends 1525 sugar milling and refining, and the processing of molasses into bio-ethanol by fermentation.113 in a more recent study, a novel, nonphosphorylative pathway was used to convert biomass sugars to bdo with a 100 % theoretical maximum molar yield.114 in this pathway, assimilation of sugars into the tca cycle is allowed and afterward built of artificial biosynthetic pathways to bdo using the enzymes 2-ketoacid decarboxylases and alcohol dehydrogenases.114 bio-sa is mainly produced by several microorganisms as a fermentation product. in 2008, bioamber built the first plant that uses escherichia coli as the host microorganism and wheat-derived glucose as the substrate for sa production. four years later, reverdia, a joint venture between dsm and roquette, started production of bio-sa from starch using low-ph yeast technology, and myriant technologies set up a sa plant with a production capacity of 30 million pounds from unrefined sugars as feedstock using e. coli as the host organism.115 derived from natural resources, such as sugarcane, cassava and corn, bio-pbs is compostable into biomass, carbon dioxide and water. there are already several producers with existing commercial capacities for the production of bio-pbs and additional dedicated and non-dedicated capacities are expected to start up in the coming years (table s-v). bio-pbs is currently being employed in a wide range of industries, such as packaging, agriculture, pharmaceutical, consumer goods, electronics and electrical, textile, automotive and interiors.116 it is also utilized to produce bowls, plates, plastic utensils, and diapers.116 amongst others biopolymers, bio-pbs has strengths in flexibility, natural fibre compatibility, heat resistance and biodegradability under specific conditions (according to din en 13432). it was found that pbs polymer was more ductile if some adipic acid/lactic acid or some other monomer was added during the standard polymerization of sa and bdo.117 these results indicate that copolymerization of pbs is essential for improving its characteristics. depending on the monomer and the stereochemical nature of the monomer, different pbs (bio-pbs) copolymers have been registered, such as: pbsa (poly(butylene succinate-co-adipate)), pbst (poly(butylene succinate-co -terephthalate)), pbsf (poly(butylene succinate-co-fumarate)), pbs-dls (poly(butylene-succinate-dilinoleic succinate), p(bs-bms) poly(butylene succinateco-butylene 2-methylsuccinate), pbs-pla blends, etc. amongst the different possibilities for pbs copolymers, pbsa, pbst and pbs-pla blends are the most commonly mentioned in the literature. pbsa copolymer is better degraded than pbs due to its lower crystallinity and glass transition temperature,118 and together with pbs it was manufactured under the trademark bionolle® (series 1000 and 3000, respectively) by showa highpolymer co., ltd. for application as trash bags, plant pots, filaments, bottles, gloves, containers, laminated paper, trays, etc.71 pbst copolymer has a lower crystallinity than pbs, but biodegrades more slowly in contrast to pbsa.111 pbst like other aliphatic-aromatic copolymers may be used as a film or coating in disposable food packaging. pbs–pla ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1526 jeremic et al. blends with low toxicity have potential in food packaging, biomedicine and agricultural markets.119 commercial pbs-pla blends have been utilized for production of food service ware by natureworks llc.64 based on extensive research, pbs-pla blends have been successfully modified and processed into fibres, blown films, flat films, and sheets.25 pbs and its copolymers are biodegradable in lipase solution, soil burial, water, activated sludge and compost.120–122 currently, about 75 % of plastic waste is disposed in sanitary landfills and about 25 % is recycled.123 in general, bioplastics can degrade under anaerobic conditions in landfills releasing methane, but with a proper composting facility, bioplastics can be converted to compost and recycled.124 recycling of plastic waste and bio-based waste, such as bio-pbs, may be realised through several processes: mechanical, chemical and organic processes. chemical recycling of bio-based pbs and its copolymers is environmentally harmful, thus the use of polyester-degrading enzymes is an eco-friendly alternative. many hydrolases from several fungi and bacteria have been discovered and successfully evaluated for their activity against different aliphatic and aromatic pbs polymers and copolymers.71,125 bio-pbs produced by ptt mcc biochem has industrially compostable and home compostable grades, but the current grade used for paper coating is only industrially compostable.126 bio-pbs can be easily separated from paper when soaking under water without damaging the paper pulp, which can be recycled to produce paper again, or as backyard compost, or in the case of a leak to the environment, it will degrade eventually in a limited time.126 testing the degradation phenomena of bio-pbs plastics in the environment can be classified into three categories: field tests, simulation tests, and laboratory tests.127 the performed degradation studies of a commercially available pbs and pbsa copolymer in various environments have shown that the most favourable degradation environment was compost, which contains microorganisms and natural enzymes that support degradation.128 in these studies, degradation after four to six weeks was observed, followed by a strong mass loss in the compost, due to interaction with enzymes. in addition, the strength of polymers was significantly reduced. biodegradation test in soil gave the same results as biodegradation in compost, but the changes in the molecular parameters were less intense because of the lower concentration of microorganisms, enzymes and lower temperature of the process.128 according to the patents base, in the last 10 years, most patents are based on bio-pbs biodegradability. mostly, patents deal with the promotion of methods for preparing new bio-pbs materials having improved mechanical properties, compatibility, control of degradation rate, and effective reduction of the cost of bio-pbs production (cn109608835a, cn103709688a, cn110240788a, cn109867921a, cn106589854b and cn105670248a). bio-pbs materials with improved properties have been used through patents in the last 10 years mainly in ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. bioplastics: current state and trends 1527 the form of coated paper, synthetic fibre, trash bag, coffee capsule cutlery, etc. (us20180058010a1, us20140021574a1, wo2010151798a2 and wo2016105217a1). 2.5. bio-polyethylene (bio-pe) pe consists of long chains of ethylene and it is produced as either hdpe or ldpe, or linear low density polyethylene (lldpe).129 pe is chemically synthesized by polymerization of ethane.129 bio-based pe (bio-pe) is chemically identical to fossil-based pe, it has the same technical properties and is not biodegradable.130 bio-pe is used to produce high value products using low pollution processes and contributing to the global environmental balance. the process involves: pre-treatment, enzymatic scarification, fermentation, dehydration and polymerization.131 the ethanol produced by fermentation from renewable resources can be used as a raw material for polyethylene production. production of bio-pe from renewable resources consists of three steps:130 1) synthesis of ethanol by a fermentation process from sugars, extracted from natural materials, e.g., sugarcane; 2) chemical dehydration reaction transforming of ethanol into ethylene; 3) “classical” reaction of polymerization of ethylene into polyethylene to make the various grades of pe (hdpe, ldpe, lldpe). bio-based pe produced this way is used in packaging applications, such as carrying bags, plastic films and bottles, automotive fuel tanks, injection melded parts, tubes.132 the most used pe copolymer is hdpe. hdpe is a rigid polymer with polymer chains packed closer one to other resulting in greater intermolecular forces. the presence of a strong intermolecular forces results in a dense, highly crystalline material form. it is a relatively tough and resistant polymer that can withstand high temperatures, up to 120 °c, without any effect on the properties of the material. furthermore, hdpe is an extremely resistant material to many chemicals, hence its widespread use in healthcare and laboratory environments. it is resistant to many acids, alcohols, aldehydes, esters, bases and oils. the braskem company is the world-leading supplier of bio-pe (hdpe, ldpe and lldpe) and the current braskem bio-pe grades are mainly targeted towards food packaging, cosmetics, personal care, automotive parts and toys, but there are many other companies in the world producing bio-pe (table s-vi.). bio-pe is a bio-based polymer but it is not biodegradable. it is well known that polyethylene is resistant to degradation due to its chemical and biological inertness, which is the result of its high molecular weight, three-dimensional structure and hydrophobic nature, all of which interfere with polyethylene availability for the action of microorganisms, light, water, etc. according to some research,133,134 the addition of antioxidants and stabilizers protects polyethylene against oxidation at the production stage, which further increases its resistance to degradation. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1528 jeremic et al. the degradation of a commercial environmentally degradable pe was investigated in two stages: firstly, by abiotic oxidation in an air oven to simulate the effect of the compost environment and secondly, in the presence of selected microorganisms.135,136 it was observed that microbial growth occurred in the presence of pe samples that had been compression moulded to thick sections but had not been deliberately pre-oxidized. changes in the molecular weight distribution occurred after preheating in air at 60 °c and not at ambient temperatures, but colonization of microorganisms occurred on all samples.135 also, erosion of the film surface was observed in the vicinity of the microorganisms and the decay of oxidation products on the surface of the polymer film was found to be associated with the formation of protein and polysaccharides, due to the growth of microorganisms (rhodococcus rhodochrous atcc 29672, cladosporium cladosporoides atcc 20251, nocardia asteroides gk 911).135,136 nowadays, bio -pe is accepted for recycling in many recycling centres, especially hdpe. the recycling process consists of the collection and separation of the plastic using sink–float separation or near-infrared radiation (nir) techniques, then homogenization and melting of the plastic, and finally cooling the plastic into pellets that can be reused for the production of, for example, toys, rope, piping etc.137 recycling hdpe has many benefits, such as cost-efficiency of the manufacture of a product from recycled hdpe instead of producing “virgin” plastic. according to the base of patents in the last 10 years, most patents are based on pe biodegradability and recycling. patents are focused on improving pe materials (hdpe, ldpe) in terms of advancing their mechanical properties and production of materials with a simple structure that is easy to recycle. many patents have marketed products, such as blow moulding, injection moulding and foam products, which showed that new materials of excellent quality and biodegradability can be produced from recycled pe materials (cn207467268u, us9593177, us9637626b2 and us2016/0108217a1). analysing the commercial application of patents over the last decade, it has been shown that bio-pe has found application in bags, boxes, bottles, toys, pipes, etc. (table s-vi). 3. (bio)degradation and recycling of bioplastics in january 2018, the eu released its vision for a more sustainable plastics industry to be achieved by 2030. the new plastics strategy states that 100 % of plastics should be either reusable or recyclable by 2030. prevention, re-use, collection and recycling should always be the first choice in efforts to achieve some of these goals. as mentioned, to overcome both oil dependence, price fluctuations and enhance resource efficiency, bioplastic materials constitute an applicable route that has to be explored as a part of the solution. plastics that are currently marketed as “biodegradable” will themselves contribute to plastic pollution if they are lost or littered. they do not break down as quickly and completely ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. bioplastics: current state and trends 1529 in the environment and can thus harm wildlife and ecosystems. to reinforce the positive impact of bioplastics, successful recycling strategies also need to be clearly proposed and applied. in general, the purpose of recycling is to convert the production of postconsumer waste into building blocks for the production of new polymers. the degree of biodegradation varies with temperature, polymer stability, and available oxygen content. consequently, most bioplastics will only degrade in the tightly controlled conditions of industrial composting units. apart from starchbased bioplastics, in compost piles or simply in the soil/water, most bioplastics will not degrade.138 bioplastics are still plastics and being made from plants or having the potential to biodegrade under limited conditions does not make them “planet-safe”. presently, there is a need for more transparency about environmental claims of bioplastic products. a ban on labelling plastic products as “biodegradable” or as “compostable”, unless they meet strict standards, is urgently needed. there are currently few international organizations that have established standards and testing methods for compostability,139 namely: • american society for testing and materials – astm-6400-99, • european standardization committee (cen) – en13432, • international standards organization (iso) – iso14855 (only for biodegradation), • german institute for standardization (din) – din v49000. the astm, cen and din standards specify the criteria for biodegradation, disintegration and eco-toxicity in order for a plastic to be called compostable. biodegradability is determined by measuring the amount of co2 produced over a certain period by the biodegrading plastic. the standards require 60 % conversion of carbon into carbon dioxide within 180 days for resins made from single polymer and 90 % conversion of carbon into carbon dioxide for copolymers or polymer mixes. disintegration is measured by sieving the material to determine the biodegraded size and less than 10 % should remain on a 2 mm screen within 120 days. eco-toxicity is measured by having concentrations of heavy metals below the limits set by the standards and by testing plant growth by mixing the compost with soil in different concentrations and comparing it with controlled compost. the en 13432 industrial standard is arguably the most international in scope and compliance with this standard is required to claim a product be compostable in the european market. in summary, it requires biodegradation of 90 % of the materials in a laboratory within 180 days. the astm-6400 standard is the regulatory framework for the united states and sets a less stringent threshold of 60 % biodegradation within 180 days, again within commercial composting conditions. the most accurate standard test method for anaerobic environments is the astm d5511 – 02 standard test method for determining anaerobic biodeg________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1530 jeremic et al. radation of plastic materials under high-solids anaerobic-digestion conditions. another standard test method for testing in anaerobic environments is the astm d5526 – 94(2002) standard test method for determining anaerobic biodegradation of plastic materials under accelerated landfill conditions. however, this latter test has proven extremely difficult to perform. both of these tests are used for the iso dis 15985 on determining anaerobic biodegradation of plastic materials. it should be born in mind that none of the standards for plastics labelled as biodegradable or compostable today makes them suitable for disposal in the open environment. however, currently, the most likely destination of many biodegradable products is the landfill as numerous composting facilities focus on food scraps and either will not accept biodegradable packaging or will still frequently screen it along with other plastics for shipment to a landfill. the environmental consequences of land filled biodegradable materials have led some researchers and advocates to push for more universal access to compost collection, as well as to the development of altogether new materials.7,140 as mentioned, pla needs industrial composting conditions for biodegradation, including temperatures above 58 °c. it needs to be properly managed and routed to specialized industrial composting or recycling facilities. under the right circumstances, microorganisms can turn the material into carbon dioxide and water within a couple of weeks. however, if it becomes littered or dumped, pla remains for much longer. when pure pla ends up in seawater, it does not seem to biodegrade at all. other kinds of bioplastics are known to better biodegrade in marine environments.141 however, whether that really happens in a specific case, and how long it will take, is highly unpredictable. improved recycling strategies have to be developed for bioplastics. in particular, bio-based polyesters (pla, pha/b and pbs) have demonstrated the potential for either mechanical or biochemical recycling.63 ongoing research includes bio-upcycling efforts142 as well as improvement of chemical recycling processes.143 on the other hand, research is intensive on the side of material design, where molecular triggers are explored.19 4. conclusions bioplastics are a large family of materials with widely varying properties. as with conventional plastics, the end-of-life options depend entirely on the application, the way the product is disposed of by its user, and the available infrastructure in the region where the product is being disposed. therefore, “biodegradable”, a magic word for environmentally minded consumers, is not always a green magic bullet. claims that bioplastics reduce environmental impact still lack sufficient evidence.140 whether bio-based plastics are more sustainable than fossil-based ones, and what contribution they could have as a building block for ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. bioplastics: current state and trends 1531 sustainable development of an innovative bioeconomy, still cannot be answered conclusively due to insufficient data.140 it is evident that there is a lot of branding around biodegradability and that a demand for biodegradables continues to climb – with packaging, take-out containers, even designer jeans and athletic shoes now carrying this designation. it is important that consumers do not allow biodegradable marketing tactics to influence their purchasing decisions and to trade one set of environmental problems with another. it is evident that bioplastics will not solve the plastic waste crisis, so tackling consumption remains the key. abbreviations 3hh 3-hydroxyhexanoate 3hd 3-hydroxydecanoate 3ho 3-hydroxyoctanoate 3hp3 hydroxypropionate 3hv 3-hydroxyvalerate 4hb4 hydroxybutyrate bc bacterial cellulose bdo 1,4-butanediol bio-bdo bio-based 1,4-butanediol bio-pbs bio-based poly(butylene succinate) bio-pe bio-based polyethylene bio-pet bio-based poly(ethylene terephthalate) bio-ptt bio-based poly(1,3-propylene terephthalate) bio-sa bio-based succinic acid hdpe high-density polyethylene iupac international union of pure and applied chemistry ldpe low-density polyethylene lldpe linear low density polyethylene mcl-3ha medium-chain length 3-hydroxyalkanoates nir near-infrared radiation pbat poly(butylene adipate terephthalate) pba poly(butylene adipate) pbs poly(butylene succinate) pbsa poly(butylene succinate-co-adipate) pbst poly(butylene succinate-co-terephthalate) pbsf poly(butylene succinate-co-fumarate) pbs-dls poly(butylene-succinate-dilinoleic succinate) p(bs-bms) poly(butylene succinate-co-butylene 2-methylsuccinate) pcl poly(caprolactone) pe polyethylene pet poly(ethylene terephthalate) pha polyhydoxyalkanoate phb poly(hydroxybutyrate) phbv poly(3-hydroxybutyrate-co-3-hydroxyvalerate) pla poly(lactic acid) pp polypropylene ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1532 jeremic et al. ps polystyrene ptt poly(1,3-propylene terephthalate) pur polyurethane pva poly(vinyl alcohol) pvc poly(vinyl chloride) sa succinic acid scl-3ha short-chain-length 3-hydroxyalkanoates tps thermoplastic starch supplementary material additional data are available electronically at the pages of journal website: https:// //www.shd-pub.org.rs/index.php/jscs/index, or from the corresponding author on request. acknowledgements. this project has received funding from the european union’s horizon 2020 research and innovation programme under grant agreement no. 870292 (bioicep). и з в о д разумевање биопластичних материјала: тренутно стање и трендови сања јеремић1, јелена миловановић1, марија мојићевић2, сања шкаро богојевић1 и јасмина никодиновић-рунић1 1институт за молекуларну генетику и генетичко инжењерство, војводе степе 444а, 11042 београд и 2атлон технолошки институт, dublin road, athlone, co. westmeath, ireland загађење пластиком сада се сматра једном од највећих претњи по животну средину по људе. развој биопoлимерних материјала може бити део решења јер биопластика укључује и неразградиве и биоразградиве материјале, а обе су важне за одрживи развој. биопластични материјали се у овом моменту дизајнирају тако да имају минималан угљенични отисак, високу могућност рециклирања и потпуну биоразградивост. овај преглед приказује недавна дешавања и трендове у области биопластичних материјала. представљен је низ биопластичних материјала који се најчешће користе: (поли-(млечна киселина) (pla), поли(хидроксиалканоат) (pha), скроб, целулоза, поли(бутилен-сукцинат) на бази обновљивих материјала (био-pbs) и полиетилен од обновљивих сировина (био-pe)), укључујући њихову производњу, могућности примене и деградацију. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {properties of humic acids from copper tailings 20 years after reclamation} j. serb. chem. soc. 85 (3) 407–419 (2020) udc 547.992–004.12:546.56+504.53:66.097.38 jscs–5310 original scientific paper 407 properties of humic acids from copper tailings 20 years after reclamation svjetlana b. radmanović1*, mirjana m. marković2, uroš d. jovanović2, maja d. gajić-kvaščev2, djuro m. čokeša2 and jasmina a. lilić3 1faculty of agriculture, university of belgrade, nemanjina 6, 11080 belgrade, serbia, 2chemical dynamics laboratory, vinča institute of nuclear sciences, university of belgrade, p.o. box 522, 11001 belgrade, serbia and 2serbia zijin bor copper, 19210 bor, serbia (received 17 july, revised and accepted 21 october 2019) abstract: part of cu post flotation tailings of serbia zijin bor copper, serbia, was reclaimed by restoration of top soil with arable soils, and revegetation in 1991. humic acids isolated from these technosols were investigated to find out if their properties underwent any changes since reclamation. two groups of control samples were used. elemental composition (chns analysis) falls within the range of average soil humic acids. humic acids belong to the type b pointing out to its lower humification degree (uv–vis). relative abundances of functional groups are ranged as follows: polysaccharide c ≥ aromatic c > carboxyl c > oh group > aliphatic c. aromaticity indexes are low, 1.88– 3.25 (atr-ftir). basic units at ph 10 are in the 11.7–26.8 nm range. pronounced reaggregation (1462-–5218 nm) at ph 3 points out to less expressed humic acid sol stability, as well as to increase in aromatic condensation degree (dynamic light scattering). no significant changes have occurred in technosol humic acids since the recultivation, confirming stability of their properties over time (pca). nevertheless, humic acids from very strongly acidic technosols show higher humifycation degree possibly originating from arable soils used in reclamation, but more likely from low soil ph and low litter input, results of unsuccessful reclamation. keywords: technosols; top soil restoration; revegetation; humification degree. introduction numerous mines exploit ores and, after extraction of economic elements, discharge residual wastes in the environment.1 study of post-mining soils focuses on two important aspects: soil formation processes and soil restoration as a basic precondition of ecosystem recovery. development of these soils is influenced by various environmental conditions and types of land use. rate of pedogenesis and * corresponding author. e-mail: scupac@agrif.bg.ac.rs https://doi.org/10.2298/jsc190717112r ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 408 radmanović et al. degree of soil evolution particularly depend on residence time and nature of parent material. technogenic materials may control functioning and evolution of soils, their potential impact on the surrounding environment and soil suitability for plants growth. since organic matter addition and humification are the key processes in the first steps of soil development, vegetation plays the key role as initial soil forming factor. dominant problems in revegetation are physical, nutritional and/or toxicological properties of mine wastes.2–4 copper mining and processing have a profound impact on the environment producing a huge amount of waste worldwide.5 one of the largest mines in serbia is serbia zijin bor copper. beside other mine wastes, post-flotation tailings (pft) dumps as a result of copper ore processing were formed. the pft dump “polje 2” was definitively abandoned in 1987.6 in addition to degradation of large land areas these mine wastes are sources of pollution to the surrounding environment, especially for the nearby city of bor. pft formed are of very pure soil conditions, predominantly high as and cu concentration, which disable plant growth, and associated soil organic matter (som) accumulation.1,6 therefore recultivation is the only way to recover these degraded areas. an area of pft (about 16 ha) was reclaimed by reconstructing the topsoil with natural arable soil material, an average depth of 40 cm, in 1991. the soil was taken from the southern part of the city of bor where the residential area expanded. one part of the reclaimed area was planted with grass and the other with trees, without any other management practice applied. our previous investigations on pedological characteristics of pft6 showed that there were no spontaneous revegetation and som genesis on non-reclaimed areas due to pure soil characteristics, predominantly high as and cu content, as already mentioned. on reclaimed area of pft formed a horizon (about 45 cm deep) is a heterogeneous mixture of arable soil materials and tailings in various ratios, characterized by degraded structure, low humus content, mostly acid, high as and cu concentrations and low soil microbial activity. according to lilić et al.,6 these soils are classified as spolic technosols (phytotoxic). part of the a horizon is covered with wind-deposited tailings (originating from a non-reclaimed part of pft), soils belonging to the spolic technosols (phytotoxic, areninuvic). according to unfavourable soil characteristics and poor vegetation, it is obvious that this soil reclamation attempt was unsuccessful, as already noticed.7 humic acids (has) from technosols on cu pft about 20 years after reclamation were investigated in this study. it is well known that the main processes in soil formation are the som accumulation and transformation. humic substances, including has, are the most abundant fractions of som. their composition, structure and properties predetermined by conditions of soil formation8 can be used as indicators of pedogenetic processes.9 has are the most reactive compounds in soil, i.e., have the capacity for diverse chemical and physical interact________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ humic acids after tailing reclamation 409 ions in the environment. these interactions are well known to depend on the ha chemical structure and composition.10 according to chaudhuri et al.,11 molecular properties of soil organic carbon (soc) were useful indicators of both soc dynamics and soil quality in mine soil chronosequence. as the a horizon of investigated technosols is a mixture of arable soils and tailings, it can be assumed that the investigated has originate from the arable soils. ha properties (elemental composition, fractions of carbon of aliphatic and aromatic groups and their ratio, optical and other properties) are related to soil genesis environmental conditions and are persistent over time,12 retaining characteristics typical for the given soil type.8,13 therefore, there is a possibility for tehnosol has investigated in this study to retain main properties of has from the arable soils used. however, these has existed under exchanged soil environmental conditions and plant residues entrance during the last 20 years. hence, technosol has were investigated to find out if they underwent any changes in elemental composition, optical properties and reaggregation ability 20 years from recultivation. experimental the study area (fig. s-1 of the supplementary material to this paper) is located in the immediate vicinity of the city of bor, east serbia, at about 400 m above sea level (44º05’ n, 22º06’ e). the climate in the region is of a temperate continental type characterized by short hot summers and a long, cold winters with average annual air temperature of 11 °c, and precipitation of 550 mm. the winds blowing from the northwest are the most frequent and the strongest. on the reclaimed area of pft, birch (betula pendula l.), shrubs (rosa canina l. and rubus caesus l.), and several grass species among which dominant nardus stricta l. and agropyrum repens l. are present, but very sporadically. here and there verbascum phlomoides l. and bryopsida spp sedge appear too. in our previous investigations of pedological characteristics of ptf,6 30 soil samples were taken at equal intervals in the direction of the letter l. to investigate soil ha properties, 6 soil samples (tc1-6) were selected due to the differences in ph (4.17–7.24), c (0.39–1.24 %) and clay content (18.46–27.34 %), table s-i of the supplementary material. five soil samples (tc1–tc5) were taken from the a horizon at a 0–25 cm depth. sixth soil sample (tc6) was collected at a 15–50 cm depth, from the a horizon covered with a wind-deposited 15 cm thin layer originating from non-reclaimed area of post-flotation tailings. to the best of our knowledge, there is no appropriate ha sample which could be used as control (there are no data on tc ha properties immediately after the reclamation as well as of ha properties of arable soils used in the reclamation process). therefore, two groups of ha control samples were chosen for this study. the first group consisted of three has (cb7– –cb9) isolated from natural arable soils used in crop production (0–25 cm depth), originating from the location nearby a new residential area of bor, which surface soil layers were used for reclamation of pft (fig. s-1). the second group of control samples includes two has isolated from soils formed under different environmental conditions. the first ha (cch) was isolated from chernozem (0–25 cm depth), originating from novi banovci (44º57ʹ n, 20º16ʹ e), serbia. the second ha control sample (ces) was ihss standard ha obtained from elliott soil.14,15 detailed description of control samples is given in the supplementary material. soil ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 410 radmanović et al. texture, aggregate stability (mean weight diameter mwd), organic c and ph were determined by common methods.16 ha sample was isolated from approx. 5 kg tc and 2 kg control soil material from each sampling location and used for all measurements in three replicates. ha samples were isolated using a modified ihss method (ha gel was dried at 35 °c, powdered, and sieved using a 0.05 mm sieve).14 the c, h and n contents of ha samples were determined using elemental analyzer (chns 628, leco corporation, usa) after drying the samples over p2o5 under vacuum. their percentages were calculated on an ash-free basis. the oxygen was calculated by the difference. ash content was determined by a dry combustion method (50 mg ha at 750 °c for 8 h). the c/n, o/c, h/c and o/h atomic ratios were calculated by determining the ratio of c to n, o to c, h to c, and c to h contents, respectively. internal oxidation degree (ω) was calculated according to the formula: ω = (2o+3n–h)/c,13 where o, n, h and c are contents (in at. %) of oxygen, nitrogen, hydrogen and carbon, respectively. uv–vis absorption spectra of ha sol (prepared in 0.05 m nahco3 solution to contain 0.1 g dm-3 c, ph 8.3–8.4), in the 200–800 nm range were recorded using a uv–vis spectrophotometer (evolution 60s, thermo fisher scientific, usa) with 1 nm resolution. optical indexes e280 (absorbance at 280 nm/mg c),11 e4/e6 (absorbance ratio a465/a665),8 e3/e5 (a350/a550),17 e2/e3 (a225/a350),18 then ∆logk (loga400–loga600) and rf (15a600/c, c is concentration in mg of c in 1 ml of ha solution)19 were used as indicators of humification degree, most widely used for comparison of ha samples. atr-ftir spectra of has in the 4000–400 cm-1 range were recorded by an alpha spectrometer (bruker, germany, 4 cm-1 resolution, 64 scans). air spectrum was used as background. peak intensities were determined relative to the baseline dependant on the spectral region. baselines in the 3700–1800 and 3000–2800 cm-1 range were used for 3283 and 2920 cm-1 bands, respectively. intensities of 1705, 1620, 1520, 1080 and 1030 cm-1 bands were determined using the baseline between 1830 and 400 cm-1. relative peak intensities of 3283, 2920, 1705, 1620, 1080 and 1030 cm-1 bands were calculated by dividing peak intensity values by that for the 1520 cm-1 band. aromaticity index (i1620/i2920) was calculated by dividing the peak height for the 1620 cm-1 band (determined by using the baseline between 1696 and 1530 cm-1) by previously determined peak height for the 2920 cm-1 band.20 each peak height was calculated as a mean of two replicates. to perform the dynamic light scattering (dls) and zeta potential (zp) measurements, the ha sols (0.02 g dm-3) were prepared using deionized water and their phs were adjusted by adding 0.1 and 1.0 m hcl or naoh solutions. nacl was added to maintain ionic strength constant (0.1 m) within ±10 %, even though the maximum amount of hcl or naoh solution was used. the prepared ha sols were equilibrated for 24 h at 25±2 °c and their phs determined before the measurement. the measurements were performed using a zeta-sizer nano zs with 633 nm he–ne laser (malvern, uk), and the data were analyzed by the zetasizer software version 6.20 (malvern, uk). measurement details are given by jovanović et al.21 absorbances of alkaline and acid sols at 465 and 665 nm were recorded by uv–vis spectroscopy to calculate the e4/e6 index. the ha characteristics were organized in three datasets as follows: the first, technosols and all control soils; the second, technosols and control bor soils; and the third, technosols. the correlation of soils and has data, as well as the pca of has data (auto scaled prior to analysis) were performed using an ibm spss statistics 19 software package. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ humic acids after tailing reclamation 411 results and discussion elemental composition of investigated ha samples (table i) reveals the contents obtained to fall within the range of average values reported for soil ha.22 atomic ratios have been used as indicators of variations in ha properties as a function of ha origin.22,23 generally, high c/n and o/h and low o/c and h/c reflect high degree of aromatic condensation, maturity and stability of has, i.e., the degree of ha humification. besides, ω has been used as an indicator of the progress in plant residue humification.13 positive values of ω indicate welldrained soils with prevailing oxidizing conditions favorable for ha oxidation, while negative values reflect anaerobic soil conditions.24 the atomic ratios appear to be qualitatively useful parameters to compare compositional differences between various humic materials.22 differentiation in the degree of oxidation between the second control has, cch and ces and other has is clearly indicated in table i. at the same time, there is no clear difference between investigated tc has and the first control cb has. elemental composition of tc and cb has point out to their similar origin. h/c atomic ratios were higher than 1, indicating prevalence of aliphatic structures in both tc and cb has,23 while lower o/h ratios reveal lower degree of oxidation and low content of oxygen -containing functional groups.25 according to negative ω values, it can be assumed that tc3 and cb9 has were formed under soil anaerobic conditions.13 table i. elemental composition, ash content, atomic ratios and internal oxidation degree of humic acids (ha) isolated from technosols (tc1–tc6) and control natural soils: arable soils near the city of bor (cb7–cb9), chernozem (cch) and standard elliot soil (ces) ha c h o n content of ash, % c/n o/c h/c o/h ω contenta, wt. % atomic ratio tc1 52.38 5.36 37.23 5.03 0.63 12.15 0.605 1.2281 0.492 0.228 tc2 52.11 5.28 37.83 4.78 0.32 12.70 0.574 1.2160 0.475 0.186 tc3 53.68 5.46 35.81 4.91 0 12.76 0.490 1.2206 0.401 -0.006 tc4 57.97 5.03 32.00 5.00 0.27 13.54 0.467 1.0413 0.448 0.113 tc5 57.48 5.17 32.09 5.26 0.44 12.76 0.447 1.0794 0.414 0.049 tc6 52.86 4.87 37.12 5.15 0 11.97 0.642 1.1053 0.581 0.430 cb7 56.95 5.03 32.62 5.40 0.20 12.31 0.487 1.0599 0.459 0.158 cb8 56.23 5.29 32.84 5.64 0.07 11.63 0.447 1.1288 0.396 0.023 cb9 50.76 5.79 39.01 4.44 0.19 13.35 0.505 1.3624 0.369 -0.134 cch 52.29 4.31 39.57 3.83 0.23 15.95 0.810 0.9888 0.820 0.820 ces 58.13 3.60 34.13 4.14 0.88 16.39 0.770 0.7430 1.037 0.981 aash and moisture-free basis according to the literature data,11,17,18,26 the lower e2/e3, e3/e5, e4/e6 and δlogk and higher e280 and rf indexes, the higher the ha optical density. the obtained e280, e2/e3, e3/e5, e4/e6, δlogk and rf values (<43.31, >1.44, >4.67, 4.89-5.36, > 0.6399 and < 66.6, respectively, table ii) indicate low humification ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 412 radmanović et al. degree of tc has. the e4/e6 ratio slightly higher than 5 suggests a relatively low degree of aromatic polycondensation and/or relatively small particle size and molecular weight of tc has.27 control cb has have very different optical indexes, but lower humification degree than tc has. cch and ces has reveals higher e280 and rf and lower e2/e3, e3/e5, e4/e6 and δlogk indicating higher humification degree in comparison with that of tc and cb has. according to kumada,19 cch and ces belong to type a, tc1–tc6 and cb9 are of type b, while cb7 and cb8 are of type p soil has, meaning that their optical density decreases in the order: cch ≈ ces > tc1–tc6 ≈ cb9 > cb7 ≈ cb8. table ii. optical indexes of humic acids (ha) isolated from technosols (tc1–tc6) and control natural soils: arable soils near the city of bor (cb7–cb9), chernozem (cch) and standard elliot soil (ces), obtained by uv–vis spectroscopy ha e280 e2/e3 e3/e5 e4/e6 δlogk rf tc1 40.76±0.27 1.74±0.07 5.21±0.01 5.12±0.01 0.67401±0.0035 48.1±0.4 tc2 42.34±0.51 1.50±0.03 4.71±0.01 4.89±0.01 0.6399±0.0029 61.6±0.4 tc3 43.31±1.58 1.46±0.04 5.21±0.04 5.36±0.01 0.6794±0.0036 57.9±0.4 tc4 41.26±0.28 1.65±0.03 5.05±0.01 5.06±0.01 0.6650±0.0028 52.2±0.3 tc5 40.69±0.95 1.73±0.04 5.20±0.01 5.19±0.07 0.6776±0.0022 48.3±0.2 tc6 43.24±0.59 1.44±0.03 4.671±0.004 5.12±0.01 0.6442±0.0016 66.6±0.2 cb7 38.94±0.04 1.94±0.05 4.99±0.01 5.15±0.02 0.6572±0.0039 45.6±0.4 cb8 42.21±0.15 1.70±0.07 5.22±0.01 5.33±0.01 0.6844±0.0037 49.1±0.4 cb9 37.32±0.05 2.11±0.09 5.52±0.01 5.79±0.01 0.6969±0.0043 36.5±0.3 cch 47.24±3.58 1.14±0.11 3.94±0.05 4.390±0.003 0.6065±0.0006 105.5±0.1 ces 45.53±2.47 1.24±0.04 4.05±0.08 3.900±0.002 0.5736±0.0006 94.5±0.1 relative band intensities (atr-ftir spectra) have been used to estimate relative abundances of broad classes of functional groups (oh, n–h, i3300; alkyl, i2923; carboxyl, i1702; aromatic c, i1620; carbohydrate-like structures/polysaccharides, i1080 and i1030).11 these relative band intensities, as well as aromaticity index (i1620/i2920) values20,21 have been used to compare ha molecular characteristics. relative band intensities and aromaticity index values obtained in this study are shown in table iii. relative abundances of functional groups present are ranged as follows: polysaccharide c = aromatic c > carboxyl c > oh group > aliphatic c for tc1–tc3 and tc6 has, and polysaccharide c > aromatic c > carboxyl c > oh group > aliphatic c for tc4-5 has. aromaticity index values are low (1.88-3.25). it is obvious from table iii that relative band intensities, as well as aromaticity index values, for cch and ces are different from those of tc and cb has. additionally, tc and cb has are similar, as confirmed by elemental and uv–vis analysis. lower abundances of oxygen-containing groups (i3293, i1705, i1080 and i1030) in tc and cb has are in agreement with lower ω, o/c and o/h. less aromatic and more aliphatic component abundance, as well as lower aromaticity indexes, are consistent with higher h/c ratio and lower optical ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ humic acids after tailing reclamation 413 density obtained by uv–vis indexes, pointing out to their lower humification degree.19,29 table iii. some relative band intensities and aromaticity index values of humic acids (ha) from ttechnosols (tc1–tc6) and control natural soils: arable soils near the city of bor (cb7–cb9), chernozem (cch) and standard elliot soil (ces), obtained by atr-ftir (measurement uncertainty < 5 %) ha i3273a i2923a i1705a i1620a i1080a i1030 a i1620/i2920b tc1 0.71 0.18 0.85 1.31 0.59 0.58 2.50 tc2 0.71 0.21 1.04 1.41 0.70 0.79 2.02 tc3 0.65 0.18 1.00 1.40 0.67 0.66 2.42 tc4 0.76 0.24 1.05 1.44 0.73 0.76 1.88 tc5 0.75 0.23 1.00 1.41 0.72 0.75 1.91 tc6 0.59 0.14 1.08 1.45 0.66 0.63 3.25 cb7 0.76 0.22 0.93 1.36 0.68 0.70 1.98 cb8 0.77 0.20 1.02 1.41 0.69 0.70 2.09 cb9 0.70 0.17 1.00 1.48 0.86 0.89 3.16 cch 0.88 0.12 1.73 1.52 0.75 0.69 4.29 ces 0.90 0.11 1.35 1.74 0.86 0.91 4.24 arelative band intensities (to intensity of c=c aromatic band at 1520 cm-1); baromaticity index humic substances are able to rearrange and restructure themselves in response to environmental changes such as ph, ionic strength, moisture as well as hs concentration.21,30 aggregation properties of has significantly influence their interactions in nature. for example metal complexation31 is the reason why is important to study their colloidal character. in this study, psd and zp were measured in highly alkaline solutions (ph 10), low ha concentration (0.02 g dm–3) and constant ionic strength (0.1 m nacl solution) to obtain ha basic unit size.21 alkaline solutions were acidified by hcl to ph 3 to examine the ha reaggregation ability. since ps is closely related to colloidal stability of humic particles,21,32 zp at both phs was measured. it is obvious (table iv) that zps are negative for all has at both phs (–22.7 to –31.9 mv at ph 10 and –16.3 to –19.4 mv at ph 3). basic units of tc has at ph 10 are in the range 11.7–26.8 nm, except for tc1 (151.4 nm). after acidification, pronounced tc ha reaggregation (1462–5218 nm) is evident, pointing out to less negative zp values, i.e., less expressed ha sol stability. by inspecting ps and zp values in table iv, difference between all control has and tc has is obvious at both phs, but more pronounced at ph 10. pronounced aggregation of cch and ces ha control samples can be related to higher relative atr-ftir band intensities (i1705 and i3272) typical of carboxyl and phenolic (oh) functional groups. on the contrary, aggregation of other control samples (cb7 and cb8, as well as tc5) could not be explained by already mentioned relative intensities likely due to the presence of two competitive intramolecular contraction and intermolecular aggregation processes influencing ha particle size.30 com________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 414 radmanović et al. paring e4/e6 indexes under alkaline and acid conditions, an increase in aromatic condensation degree for tc has can be noticed, opposite to its decrease for bc has and almost no change for cch and ces has. table iv. particle size (ps), zeta potential (zp) and e4/e6 index of humic acids (ha) isolated from technosols (tc1–tc6) and control natural soils: arable soils near the city of bor (cb7–cb9), chernozem (cch) and elliot soil (ces) ha ph 10 ph 3 psa / nm zp / mv e4/e6b psa / nm zp / mv e4/e6 tc1 151.4 –27.5±1.4 4.77 2282 –16.3±1.9 3.86 tc2 26.6 –23.0±2.7 2.59 1977 –18.5±1.2 3.75 tc3 15.0 –31.9±1.4 2.54 3284 –19.4±1.3 3.29 tc4 18.0 –22.7±4.4 2.65 1462 –18.9±1.2 4.52 tc5 26.8 –27.5±2.4 2.49 5218 –19.1±1.2 2.78 tc6 11.7 –29.4±1.9 2.69 3337 –17.4±1.1 3.03 cb7 17.0 –19.1±2.9 5.06 5031 –17.2±1.0 3.70 cb8 14.7 –14.0±2.2 4.69 5363 –17.6±0.7 3.60 cb9 14.3 –22.4±3.0 4.65 491 –13.7±0.7 3.73 cch 10.9 –23.1±2.0 5.07 5359 –16.8±1.1 3.65 ces 10.2 –18.5±4.7 4.85 4658 –16.9±2.0 3.67 ameasurements uncertainty <6 %; bmeasurement uncertainty <2 % according to the literature,33 ha chemical structure and properties significantly influence the efficiency of ha-metal binding. the results obtained in this study have shown changes in tc ha structure and properties with ph, which likely affect their interaction with metals. ha-metal binding is of great importance at cu post-flotation tailings due to extremely high as and cu concentrations6 and this problem should be emphasized in future investigations. pca score plots obtained for ha characteristics for three datasets: tcs, cbs, cch and ces (fig. s-2 of the supplementary material), tcs and cbs (fig. s-3 of the supplementary material) and tcs (fig. s-4 of the supplementary material). the first three principal components (fig. s-2) describe more than 84 % of the total variance (56.6 and 17.0 % for the first (pc1) and the second (pc2) principal component, respectively) enabling very good insight into data structure. similarly, the first three principal components describe nearly 79 % in the second dataset (fig. s-3, pc1 36.0 % and pc2 26.1 %), and nearly 84 % of the total variance in the third dataset (fig. s-4, pc1 40.9 % and pc2 27.2 %). the score plot (fig. s-2) shows that two groups of the first dataset can be considered as separated along the pc2 axis in terms of linear separability and classification. clear separation of cch and ces from tc and cb has can be noticed, as well as from each other, which is expected regarding their origins. obviously, rf, o/h, c/n, ω, o/c, i1620/i2923 (along positive pc1 axis) and e3/e5, δlogk, e4/e6 and h content (along negative pc1 axis) are informative for ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ humic acids after tailing reclamation 415 clear difference between cch and ces has and tc and cb has. also, c and o contents carry the most of variance along the pc2 axis causing separation inside the tc and cb group. tc and cb has had similar properties since 79 % of the total variance has been preserved (fig. s-3), while the content of h, i1080 and i1030 cause separation of cb9 has. considerable heterogeneity noticed in fig. s-4 confirms differences in tc has. tc1 (characterized by ps at ph 10), tc2 and tc6 (rf, e280, carboxyl and aromatic c), tc3 (zp at ph 10, ps at ph 3, e4/e6 and n) as well as tc4 and tc5 has (aliphatic c and polysaccharides), can be considered as separated. tc1–tc3 and tc6 has originate from very strongly to strongly acid soils with low soc content, while tc4 and tc5 has come from slightly acid and neutral soils with slightly higher soc and clay content, as well as higher mwd. dependence of has properties on some basic characteristics of technosols was confirmed by correlation analysis, indicating to dependence of humification process on soil conditions.19 higher tc soil clay content is related to higher abundance of cooh acidic groups, aromatic c, polysaccharides and degree of aromatic condensation (h/c ratio, 0.880*, 0.912*, 0.899*, 0.812* and –0.835*, respectively; *p < 0.05, **p < < 0.01). it is well known that clay minerals absorb humic substances, which makes them less available to microorganisms,8,34 affects the stability of some components such as polysaccharides11 and contributes to the formation of more stable forms.2 tc soil mwd is highly correlated with ha c and o content, as well as o/c and h/c ratio (0.979**, –0.991**, –0.857* and –0.757, respectively) suggesting increase in soil aggregate stability with increasing ha humification degree. the c content, c/n ratio, i3273, i2923, and i1030 of has are in high positive correlation with tc soil ph, while it is highly negatively correlated with o content, o/c and o/h ratio, ω and i1620/i2923 (0.928**, 0.701, 0.722, 0.805, 0.762, –0.950**, –0.936**, –0.732, –0.715 and –0.730, respectively). according to kumada,19 higher ha humification degree in acid to strongly acid soils can be a consequence of autoxidative changes in organic materials accelerated by fe and mn oxides. also, e2/e3, i3273 and i2923 are significantly higher and e280, rf and i1620/i2923 lower with increasing soil organic c (0.891*, 0.932**, 0.844*, –0.887*, –0.933**, and –0.812*, respectively). has have more aliphatic and less aromatic constituents, i.e., lower humification degree with increase in som. higher soil ph influences better vegetation cover and hence higher plant litter input, likely causing lower has humification degree. the highest aromaticity index, oxidation and aromatic condensation degree were obtained for tc6 ha extracted from a 15–50 cm layer covered with wind deposited cu post-flotation tailings and no vegetation. these tc6 ha characteristics might originate from arable soils used in reclamation, but the contribution of very low soil ph and the absence of vegetation (fresh litter input) to the highest ha humification degree in ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 416 radmanović et al. the last 20 years is more likely. decrease in ha humification degree with increasing fresh plant litter input has already been reported.11,13,25 comparison of the results obtained in this study with those for other mine soils is difficult due to different reclamation methods or mine wastes. according to our knowledge, comparable investigations of has from soils formed on cu post-flotation tailings recultivated by top layer reconstruction with arable soils are missing. humified organic c was increased in depleted copper mine soil using organic amendments, on the contrary to tree vegetation.35 although soils on coal-mine spoils are quite different from those on cu post-flotation tailings, results obtained indicate an increase in hs humification degree on reclaimed areas.11 according to abakumov et al.2 caloricity of ha tends to increase with increasing site age in a reclaimed mine soil chronosequence (maximum 7–10 years), while on unreclaimed site no trend was evident, explained by ecosystem heterogeneity. in this study, increase of humification degree is not noticed for all tc has likely due to heterogeneous soil properties. humification degree is probably increased only for has from very strongly acid soils due to influence of autoxidative changes accelerated by fe and mn oxides and absence of fresh litter input. these results confirm that quantity of total organic matter entering the soil, as well as soil conditions11,13,19,25 influence soil humification process. pure soil conditions of technosols studied are likely results of ineffective pft reclamation. soil top reconstruction process was performed by irregular mixing of arable soils and tailing, the consequence of which are strong acid conditions. also, acid tailing was wind-transferred from non-reclaimed to reclaimed pft areas. as already mentioned,7 cu pft remediation is difficult to accomplish, expensive and often unsuccessful. further, residence time is an important factor influencing ha humification.2,11 results of this study have shown that in the 20 years period humification changes occur only in extremely different soil conditions, while has in similar soil conditions were not significantly changed. conclusion results of elemental analysis, uv–vis and atr-ftir spectroscopy point out to lower humification degree of tc has. pronounced tc has reaggregation at ph 3 points out to less negative zp values, as well as to increase in aromatic condensation degree. according to pca, tc and cb has are clearly differentiated from the second ha control group (cch and ces) indicating that tc and cb has are likely of the same origin. tc has are not separated from the first ha control group (cb – arable soils near bor), which probably means that no significant changes have occurred in tc has over the past 20 years since the recultivation, confirming stability of has properties over time. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ humic acids after tailing reclamation 417 nevertheless, it is obvious that has extracted from very strongly acid tc soils show higher aromaticity index, oxidation and aromatic condensation degree compared to has from slightly acid and neutral tc, as well as cb soils. these tc ha characteristics could originate from arable soils used in reclamation, but the contribution of very strongly acid soil conditions and low fresh litter input (bare or absent vegetation) to high humification degree is more likely. unsuccessful reclamation resulted in both very low soil ph and fresh litter input probably causing higher humification degree of has originated from very strongly acid tc soils. also, their aggregation is less pronounced in comparison to other tc and control has studied, which could influence efficiency of ha–metal binding. it was shown that 20 years period is long enough to change tc has humification degree only under extremely changed soil conditions. supplementary material additional data are available electronically at the pages of journal website: http://www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. this study was financially supported by the ministry of education, science and technological development of the republic of serbia (projects no.: 37006 and 45012). и з в о д особине хуминских киселина са јаловишта бакра 20 година након рекултивације свјетлана б. радмановић1, мирјана м. марковић2, урош д. јовановић2, маја д. гајић-квашчев2, ђуро м. чокеша2 и јасмина а. лилић3 1пољопривредни факултет, универзитет у београду, nemanjina 6, 11080 београд, 2лабораторија за динамичку хемију, институт за нуклеарне науке винча, универзитет у београду, п. пр. 522, 11001 београд и 3serbia zijin bor copper, 19210 бор део постфлотационог јаловишта serbia zijin bor copper, бор, је 1991. године рекултивисан реконструкцијом површинског слоја обрадивим земљиштем и ревегетацијом. из формираних техносола су изоловане хуминске киселине које су испитиване са циљем да се утврди да ли је дошло до промене њихових особина 20 година после рекултивације. коришћене су две групе контролних узорака. елементални састав (chns анализа) се налазио унутар опсега карактеристичког за земљишне хуминске киселине. хуминске киселине припадају б типу што указује на њихов нижи степен хумифицираности (uv–vis). редослед релативне заступљености функционалних група је следећи: полисахаридни c ≥ ароматични c > карбоксилни c > oh група > алифатични c. вредности ароматичног индекса су ниске (1,88–3,25, atr-ftir). основне јединице на ph 10 су у опсегу 11,7–26,8 nm. изражена реагрегација (1462–5218 nm) на ph 3 указује на нижу стабилност сола хуминских киселина, као и на повећање степена ароматичности (dls). резултати показују да није дошло до значајних промена особина хуминских киселина техносола 20 година после рекултивације (pca), што указује на њихову стабилност. ипак, хуминске киселине из веома киселих техносола имају већи степен хумифицираности који је можда наслеђен из обрадивог земљишта коришћеног у рекултивацији, али је вероватније последица ниског ph и нижег прилива органских остатака, а који су резултат неуспешне рекултивације. 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {the physicochemical properties of the deep eutectic solvents with triethanolamine as a major component} j. serb. chem. soc. 85 (10) 1303–1315 (2020) udc 547.415.5’262–185+544.35.03:544.3.03 jscs–5376 original scientific paper 1303 the physicochemical properties of the deep eutectic solvents with triethanolamine as a major component biljana s. đorđević1, dragan z. troter1, vlada b. veljković1,3, mirjana lj. kijevčanin2, ivona r. radović2 and zoran b. todorović1* 1university of niš, faculty of technology, bulevar oslobođenja 124, 16000 leskovac, serbia, 2faculty of technology and metallurgy, university of belgrade, karnegijeva 4, 11120 belgrade, serbia and 3serbian academy of sciences and arts, knez mihailova 35, 11000 belgrade, serbia (received 25 april, revised 16 july, accepted 27 august 2020) abstract: different deep eutectic solvents (dess) of triethanolamine (teoa) and oxalic acid (oa), glacial acetic acid (aa), l-(+)-lactic acid (la), oleic acid (ola), glycerol (g), ethylene glycol (eg), propylene glycol (peg), choline chloride (chcl) or 1,3-dimethylurea (dmu) were prepared and characterized regarding their physicochemical (density, viscosity, electrical conductivity, refractive index, coefficient of volume expansion, molecular volume, lattice energy and heat capacity) properties over the temperature range of 293.15–363.15 k at 101.325 kpa. for all tested dess, the density, viscosity and refractive index decreased with rising temperature, while the electrical conductivity increased. the temperature dependence of viscosity and electrical conductivity are described by the vogel–tamman–fulcher equations. the viscosity and molar conductivity, which exhibited a linear behaviour, were correlated by the fractional walden rule. besides, the fourier transform infrared spectroscopy (ftir) was used to study the functional groups of these dess while thermogravimetric analysis (tga) and differential scanning calorimetry (dsc) provided the information about their stability. the tested dess of teoa possess desirable properties for use in various industrial processes, such as extractions, separations, chemical technology and biotechnology. keywords: dess; density; viscosity; conductivity; refractive index. introduction deep eutectic solvents (dess) are a class of two-component or three-component liquid solutions, able to dissolve many other organic and inorganic compounds. their advantages are low cost, easy preparation, biodegradability, nontoxicity, wide liquid range, non-volatility, thermal stability and non-reactivity with water. many different chemicals are used for their preparation, mostly sub * corresponding author. e-mail: ztodorovictfle@yahoo.com https://doi.org/10.2298/jsc200425050d ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2020 scs. 1304 đorđević et al. stituted quaternary ammonium salts (usually choline chloride, chcl) and hydrogen bond donors (hbds) like alcohols, amides, carboxylic acids, esters, ethers and hydrated metal salts of halides, nitrates and acetates, which provide mixtures with a lower melting point than the constituents.1 despite the recommendation for various chemical reactions2,3 and the uses as a „green” solvent in cosmetic formulations, polishes, paints, inks, etc., triethanolamine (teoa) has been rarely used in the preparation of dess. for instance, shekaari et al.4 used chcl:teoa, as well as chcl:monoethanolamine, chcl:diethanolamine, for benzene and thiophene extraction from n-hexane/aromatic mixtures. the amine-based dess, such as chcl:monoethanolamine, chcl:diethanolamine and chcl:methyldiethanolamine, have already been used in co2 capture due to their high co2 dissolution capability5–8 and in the pretreatment of wheat straw for improving enzymatic hydrolysis.9 liquid solutions based on organic solvents, particularly ionically conducting ones, have been applied for the electrolytic capacitors and batteries and in electrolytic processes of cathodic deposition of very electronegative metals.10,11,13 however, to the best of the authors’ knowledge, the physicochemical and thermodynamic properties of teoa-based dess have not been studied yet, except the chcl-teoa des.12,13 in order to involve the teoa-based dess in the future laboratory and industrial processes, it is crucial for chemists and chemical engineers to know, or be able to predict their physicochemical properties. the physical properties, such as density, viscosity, electrical conductivity and refractive index, could provide essential information on the purity of samples and the molecular interactions in the liquid, which will be useful for the design of the contacting equipment and modeling of the process.14 in this study, the physicochemical (density, viscosity, electrical conductivity, refractive index, thermal expansion coefficient, molecular volume, lattice energy, and heat capacity) properties of different dess of teoa as hydrogen bond acceptor (hba) and oxalic acid (oa), glacial acetic acid (aa), l-(+)-lactic acid (la), oleic acid (ola), glycerol (g), ethylene glycol (eg), propylene glycol (peg), chcl or 1,3-dimethylurea (dmu) as hbds were measured in the temperature range of 293.15–363.15 k at 101.325 kpa for the first time. the study was performed in the region of liquid state of binary systems (melting points of tested dess are in the range of 283.15–313.15 k, table s-i, supplementary material to this paper). the fractional walden rule was employed for understanding the relationship between molar conductivity and viscosity of the tested dess. additionally, the functional groups of these dess were studied by the ftir while the tga and dsc analyses were performed in order to get an insight into their stability in the temperature range interesting for their industrial point of view. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ properties of the deep eutectic solvents 1305 experimental materials teoa (99.0 %), chcl, eg and dmu (all ≥ 98.0 %) were obtained from sigma aldrich. peg and g (both ph. eur. grade) were provided from meilab (belgrade, serbia). aa was from zorka (šabac, serbia). ola (99.0 %) was obtained from sigma aldrich. oa and la (both 99.0 %) were purchased from moslab (belgrade, serbia). all chemicals were used as purchased. information on the used chemicals is presented in table s-i. preparation of dess dess were prepared by combining teoa with the selected compound at the desired molar ratio (table s-ii of the supplementary material) on a rotary evaporator at 70 °c, as described elsewhere.15 all weight measurements were performed by a digital balance (adam equipment, model nbl 214e) having an accuracy of ±1×10-4 g. the prepared dess were kept in the well-closed glass bottles and stored in a desiccator containing cacl2 before characterization to prevent moisture absorption. the water content of the dried dess was determined by the karl–fischer method (metrohm 73kf coulometer). a summary of the compositions, molar mass and water content (mass fraction), for the investigated dess, has been presented in table s-ii while their ftir spectra are shown in fig. s-1 of the supplementary material. physicochemical properties of dess all measurements were conducted at 101.325 kpa in the temperature range between 293.15 and 363.15 k after 15 min of thermostatting. density was measured using a dma 4500 anton paar densitometer with the temperature measurement standard uncertainty of ±0.005 k. viscosity was measured by a rotational viscometer (visco basic plus, ver. 0.8, fungilab s.a., barcelona, spain) while a b250 proline conductivity meter was used for the electrical conductivity measurements. an automatic atago refractometer a100 was applied to measure the refractive index. the densitometer was calibrated by measuring the density of double-distilled water and the results were compared to the reference values indicated in the calibration certificate of the standards. the rotational viscometer was calibrated measuring the viscosity of standard silicon oils (fungilab) certified for each used spindle. the refractometer was calibrated measuring the certified refractive index liquids (cargille laboratories) and the refractive index of distilled water. the conductivity meter was calibrated by using the 0.001 m standard solution of kcl (hach). three replicates were carried out for each measurement of density, viscosity, conductivity and refractive index with the estimated standard uncertainty (u) of ±0.5 kg·m-3, 5 % of the measured value, ± 0.0001 s·m-1 and ± 0.00005, respectively. tga and dsc analyses the tga and dsc analyses were done in the temperature range of 298.15–273.15 k only for the liquid dess, namely teoa:g, teoa:eg, teoa:peg, chcl:teoa, as well as the chcl-based dess with the same hydrogen bond donors for comparison. these analyses were performed using a sta449f5 jupiter®, netzsch tga-dsc instrument in an argon atmosphere with a total gas flow of 140 ml/min and a heating rate of 3 k/min. the calibration of the temperature and the enthalpy of the tga-dsc instrument were carried out using the indium standard (purity > 99.999 %, sigma aldrich) following the instrument supplier instructions. ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 1306 đorđević et al. results and discussions effect of temperature on density of dess the dependence of the density of the studied dess on temperature is shown in fig. 1. the experimental density values are given in tables s-iii–s-xi of the supplementary material. fig. 1. temperature dependence of the density (ρ) of the tested dess: teoa:oa – , teoa:aa – , teoa:la – , teoa:ola – , teoa:g – , teoa:eg – , teoa:peg – , chcl:teoa –  and teoa:dmu – . for the temperature range of 293.15–363.15 k, the density of the tested dess were within the range of 1362.6–1042.1 kg∙m–3. at 313.15 k, the densities of the tested dess follow the order: teoa:peg < teoa:ola < teoa:eg < teoa:dmu < chcl:teoa < teoa:aa < teoa:g < teoa:la < teoa:oa. due to the lack of data for most of the tested teoa-based dess, their density was compared with the density of the dess based on the same or similar donors. the density of dess depends directly on the binding ability between their constitutional components. teoa:g has a higher density than n,n-diethylethanol ammonium chloride:g (1:2),16 tetrapropylammonium bromide:g (1:3),17 chcl:g (1:2)18 because teoa has the highest number of –oh groups and makes the stronger intermolecular h-bonds network than the other dess with g.19 for the same reason the density of teoa:eg is higher than the density of the n,n -diethylethanol ammonium chloride:eg des16 and chcl:eg in the mole ratios of 1:1 and 1:213,18,20 but lower than the density of the chcl:triethylene glycol des in the 1:3 mole ratio21 and the density of teoa:oa is higher than that of chcl:oa (1:1)12,13 and n,n-diethylethanol ammonium chloride:oa (3:2).22 although teoa has a higher number of –oh groups than chcl, the density of teoa:peg is lower than the density for chcl:peg (1:2).18 this phenomenon may be explained by the different molecular organization or packing of the des according to the „hole theory”, which assumes that, after melting, the ionic material contains empty spaces arising from thermally generated local density fluctuations.19 ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ properties of the deep eutectic solvents 1307 the density of chcl:teoa is almost the same as those of some chcl-based dess.12,18,21,23,24 the present density values for chcl:teoa are lower than the values reported for chcl:teoa (1:2)12 but higher than that reported for chcl:teoa (1:1),13 chcl:monoethanolamine (1:5–8)6 and chcl:diethanolamine (1:4–6).25 for chcl:teoa, with the same mole ratio, the difference in the density values could be due to the experimental error. a decrease of the chcl:teoa molе ratio reduces the des density because teoa has a strong cohesive energy due to the presence of an important intermolecular h-bonds network and addition of chcl was attributed to the partial rupture of this h-bonds network.19 density was used for calculating the molecular volume (vm), the lattice energy (upot) and the heat capacity (cp) using the already-published equations (see supplementary material);13,26 their values at 313.15 k are listed in table s-xiii. the lattice energy of the dess was like those of the molten salts27 which explained their liquid state at this temperature. the molar volume of the teoa:ola (1:1) des was the highest due to the highest molecular size of ola. the greatest influence of the molecular size of the hbd/hba on the molar volume was previously reported for different dess.28 hence, the teoa:ola (1:1) and chcl:teoa (1:2) dess had the highest heat capacity values among all tested teoa-based dess. effect of temperature on viscosity of dess the viscosity of dess depends strictly on their molecular size and the strength of the hydrogen bonding in their structures. the increased hydrocarbon chain size of a des causes an increase in the viscosity.30 also, the temperature, nature of hbd (alkyl chain length) and salt/hbd molе ratio greatly influence the viscosity of the tested dess. the experimental viscosity values are presented in tables s-iii–s-xi. the temperature dependence of the viscosity for the tested dess, described by the vogel–tamman–fulcher (vtf) equation, is shown in fig. 2. the vtf equations describing the viscosity–temperature relationship for the analyzed dess are shown in supplementary material. as can be seen, the viscosity decreased with increasing the temperature, probably due to the diminution of the intermolecular forces between the molecules as a result of diminishing of the hydrogen bonds in dess. for the temperature range of 293.15–363.15 k, the viscosity of the tested dess lies in the range of 39.76–0.001 pa∙s. at 313.15 k, the viscosity of the tested dess follow the order: teoa:eg < teoa:peg < teoa:g < teoa:oa < teoa:aa < chcl:teoa < teoa:la < teoa:dmu < teoa:ola. as for the density, due to the lack of the published data, the viscosity of the tested teoa-based dess was compared with the viscosity of the dess based on the same or similar donors. the viscosity of teoa:g is lower than those rep________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 1308 đorđević et al. orted for n,n-diethylethanol ammonium chloride:g,16 tetrapropyl ammonium bromide:g (1:2)17 and chcl:g (1:2).18 the viscosity of teoa:eg is almost the same to those of n,n-diethylethanol ammonium chloride:eg16 and chcl:eg in molе ratios of 1:113 and 1:218 while lower than that for chcl:triethylene glycol (1:3).21 the change transfer via h-bonds between peg and chloride anion from chcl is the main reason for the larger viscosity of chcl:peg (1:2) relative to teoa:peg.18 although the teoa:g has a higher density than n,n-diethylethanol ammonium chloride:g (1:2),16 tetrapropylammonium bromide:g (1:3),17 chcl:g (1:2)18 it has lower value for viscosity than above cited dess. this might be due to a different molecular organization of the dess. the viscosity of the des depends on the chemical structure of the hbd. teoa has a higher number of –oh groups than the other hbds, but the factors like the alkyl chain length, the ion size and the electrostatic interactions may have a greater impact on the viscosity. at teoa:eg the influence of these factors is smaller, so that this des has almost the same viscosity as the dess compared above.19 fig. 2. vtf plot of the viscosity (η) for the following dess: teoa:oa – , teoa:aa – , teoa:la – , teoa:ola – , teoa:g – , teoa:eg – , teoa:peg – , chcl:teoa –  and teoa:dmu – . the viscosity of teoa:oa is higher than those of chcl:oa (1:1)12,13 and almost the same to those for n,n-diethylthanol ammonium chloride:oa (3:2).22 the viscosity of chcl:teoa is almost the same to that of chcl:teoa (1:2)12 and chcl:diethanolamine (1:4–6)25 but higher than those of chcl:teoa (1:1)13 and chcl:monoethanolamine (1:5–8).6 the chcl:teoa viscosity is almost the same to those reported for some chcl-based dess21,23 while the viscosities of teoa:dmu and chcl:dmu (1:2) are similar.18 the larger viscosities of the dess with dmu or chcl, compared to the dess with polyols, are the result of the differences in the strengths of hydrogen bonds. two methyl groups in dmu also show a major effect on the viscosity of its dess, as seen with chcl:dmu (1:2).18 similarly, teoa:ola is the most viscous des among all the dess with ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ properties of the deep eutectic solvents 1309 carboxylic acids due to the largest number of methyl groups. dess with longer alkyl chain and with carboxylic groups (teoa:ola; teoa:dmu; teoa:la; teoa:aa; teoa:oa) have larger viscosities than dess with polyols (teoa:g; teoa:peg; teoa:eg) with –oh groups. obviously the presence of one extra carboxylic or longer alkyl chain leads to an increase in the viscosity.19 among the polyol-based dess, teoa:g has the highest viscosity since g forms stronger h-bonds than the other polyol-based dess because it has one more –oh group in the molecule than eg and peg.19 in order to get a more detailed insight into the viscous flow and the thermodynamic functions of activation, the following expression for the viscosity of liquid mixtures is applied:13 * * a δ δ ln ηv h s hn rt r = − (1) where η is the viscosity, v is the molar volume of the eutectic mixture (calculated as the ratio of average mdes and density of the mixture at desired temperature), h is the planck’s constant, na is the avogadro’s number, r is the universal gas constant, t is the absolute temperature, δh* is the activation enthalpy change of the viscous flow and δs* is the entropy change of the viscous flow. in fig. 3, ln (ηv/hna) is shown as a function of t–1, eq. (1), for each of the tested dess. the δh* and δs* values were calculated from the slope and the intercept of the straight lines, respectively. table s-xv of the supplementary material contains the values of the thermodynamic functions of activation at 313.15 k (all tested dess are liquid at this temperature). fig. 3. plots of ln (ηv/hna) versus 1/t for the tested dess: teoa:oa – , teoa:aa – , teoa:la – , teoa:ola – , teoa:g – , teoa:eg – , teoa:peg – , chcl:teoa –  and teoa:dmu – . because the dess with teoa have the properties like the dess with chcl,12,13,18,21,23 they can serve as their suitable replacement in the possible industrial applications and various chemical reactions. however, due to the high ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 1310 đorđević et al. density and viscosity of several dess with teoa at room temperature, it is advisable to apply these dess in the technological processes at temperatures higher than 313.15 k. effect of temperature on the electrical conductivity of dess the experimental electrical conductivity values, as well as the additional considerations, are presented in tables s-iii–s-xi. the electrical conductivity values for the tested dess in the temperature range of 293.15–363.15 k lie in the range of 0.00015-9.003 s·m–1. at 313.15 k, the electrical conductivities of the tested dess are in the following order: teoa:peg < teoa:g < teoa:dmu < teoa:eg < teoa:ola < teoa:la < teoa:aa < teoa:oa < chcl:teoa. teoa:g has less electrical conductivity compared to the literature values for the chcl:g (1:2).18,24 the same case is for the teoa:eg, which electrical conductivity is lower than those reported for chcl:eg, in the mole ratios of 1:113 and 1:220 but higher than that of the chcl:triethylene glycol (1:3) des.21 the electrical conductivity of teoa:peg is lower than that of chcl:peg (1:2).18 the teoa:oa electrical conductivity is lower than that of chcl:oa (1:1)12 and almost the same to that of n,n-diethylethanol ammonium chloride:oa (3:2).22 the electrical conductivity of chcl:teoa is higher than the values reported for chcl:teoa (1:2)12 and chcl:teoa (1:1).13 the electrical conductivity of teoa:dmu is lower than that of chcl:dmu (1:2).18 the conductivity of dess is affected by both the mobility and number of charged species. dess with relatively high viscosities exhibit poor ionic conductivities. in relation to all tested dess teoa:g and teoa:peg had the lowest viscosity, so it is logical that they have the highest conductivity.19 generally, all teoa-based dess have lower conductivity compared to the chcl-based dess because high weight ratio of the salt in chcl contributes to the higher number of charge carriers of the chcl relative to the teoa. also, the larger size of the teoa compared to the chcl results in both reduced ion mobility and lower conductivity.19 the temperature dependence of the electrical conductivity of the tested dess described by the vtf equation is shown in fig. 4. molar conductivity and viscosity relationship the temperature dependence of the molar conductivity of the tested dess described by the vtf equation is shown in fig. 5 (additional considerations are given in supplementary material). the walden rule is very useful for the des categorization. data for the dilute aqueous 0.01 m kcl solution is used for drawing the „ideal” reference line,31 which is representative for the independent ions with no interionic interactions.29 solvents with the ability to form ions are located very close to the ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ properties of the deep eutectic solvents 1311 reference line, while the solvents with this lower ability are further away.21,30 as it can be seen in fig. 6, the tested dess belong to the category of the „subionic liquids”, indicating the non-ionic structure of the dess. fig. 4. vtf plot of the electrical conductivity (κ) for the dess: teoa:oa – , teoa:aa – , teoa:la – , teoa:ola – , teoa:g – , teoa:eg – , teoa:peg – , chcl:teoa –  and teoa:dmu – . fig. 5. vtf plot for the molar conductivity (κ) of the tested dess: teoa:oa – , teoa:aa – , teoa:la – , teoa:ola – , teoa:g – , teoa:eg – , teoa:peg – , chcl:teoa –  and teoa:dmu – . effect of temperature on the refractive index of dess the linear dependence of the refractive index of the studied dess on temperature is shown in fig. 7 (additional considerations are given in supplementary material). as expected, the refractive index of the pure dess decreases linearly with the increase of temperature depending on the structure and the nature of their components that are combined with teoa. when they are heated, the molecules are moving faster, which results in the density and refractive index reduction. the refractive index of the tested dess in the temperature range of 293.15– –363.15 k lies within the range of 1.4991–1.4137. at 313.15 k, the refractive ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 1312 đorđević et al. index of the tested dess follows the order: teoa:ola < teoa:peg < teoa:eg < teoa:g < teoa:aa < teoa:oa < teoa:la < chcl:teoa < teoa:dmu. the refractive index values of these dess agrees to those of the other dess with chcl.14 the experimental refractive index values are represented in tables s-iii–s-xi as well as additional considerations in the supplementary material. fig. 6. walden plots for the tested dess: teoa:oa – , teoa:aa – , teoa:la – , teoa:ola – , teoa:g – , teoa:eg – , teoa:peg – , chcl:teoa –  and teoa:dmu – . fig. 7. temperature dependence of the refractive index (nd) of the tested dess: teoa:oa – , teoa:aa – , teoa:la – , teoa:ola – , teoa:g – , teoa:eg – , teoa:peg – , chcl:teoa –  and teoa:dmu –  the refractive index of teoa:g is almost the same to those of n,n-diethylethanol ammonium chloride:g,16 tetrapropylammoniumbromide:g (1:2)17 and chcl:g (1:2).18 almost the same values of the refractive index of teoa:peg and chcl:peg (1:2),18 and chcl:teoa, chcl:monoethanolamine6 and chcl:diethanolamine (1:4–6)25 are related to their similar molecular structures. the refractive index of teoa:dmu and chcl:dmu (1:2) are almost the same.18 ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ properties of the deep eutectic solvents 1313 tga and dsc analyses the tga and dsc curves of the selected teoaand chcl-based dess with the same donors are shown in fig. s-2 of the supplementary material. the tga and dsc analysis were performed over the temperature range of 298.15– –373.15 k. since the investigated systems are liquids, it is understandable that no melting temperature have been observed in the dsc traces. furthermore, even though the first derivatives of dsc and tga curves show some peaks at certain temperatures, they cannot be related neither to some special changes in the molecular structure of the mixtures, nor to the phase transition. they indicate the stability of the dess within the tested temperature range, which is favourable for their potential application in the industry. conclusions the physicochemical properties (density, viscosity, electrical conductivity and refractive index) of several teoa-based dess were measured in the temperature range of 293.15–363.15 k at 101.325 kpa. the thermal expansion coefficient, the molecular volume, the lattice energy and the heat capacity of these dess were calculated using the density and the viscosity data. the vogel–tamman–fulcher equations were used to characterize the temperature dependence of the viscosity and the electrical conductivity for the investigated dess. while the density, the viscosity the and refractive index of the tested dess decrease with the increasing temperature, their electrical conductivity rises. according to the walden rule, these dess are classified as „subionic liquids”. among these dess, those with polyols and lower carboxylic acids exhibit low viscosities at lower temperatures, so they can serve as solvents in possible industrial applications and various chemical reactions, while it is recommended to heat up dess with oleic acid and dmu before use and apply them at temperatures higher than 313.15 k. the ftir analysis showed no chemical changes occurred during their preparation, while tga and dsc analyses confirmed the stability of the dess within the tested temperature range, which is favourable for their potential application in the industrial processes, such as extractions of different compounds, separations, chemical and biochemical technology. supplementary material additional data and considerations are available electronically from http:// //www.shd.org.rs/jscs/, or from corresponding author on request. acknowledgements. this work has been funded by the ministry of education, science and technological development of the republic of serbia (project assigned to the faculty of technology, leskovac, university of niš, researchers’ group iii 45001, no. 451-03-68/202014/200133) and the serbian academy of sciences and arts (the project f-78). ________________________________________________________________________________________________________________________ (cc) 2020 scs. available on line at www.shd.org.rs/jscs/ 1314 đorđević et al. и з в о д физичко–хемијске и термодинамичке особине еутектичких растварача са триетаноламином биљана с. ђорђевић1, драган з. тротер1, влада б. вељковић1,3, мирјана љ. кијевчанин2, ивона р. радовић2 и зоран б. тодоровић1 1технолошки факултет, универзитет у нишу, булевар ослобођења 124, 16000 лесковац, 2технолошко–металуршки факултет, универзитет у београду, карнегијева 4, 11000 београд и 3српска академија науке и уметности, кнез михајлова 35, 11000 београд у овом раду направљени су еутектички растварачи триетаноламинa sa оксалном киселином, глацијалном сирћетном киселином, млечном киселином, олеинском киселином, глицеролом, етилен гликолом, пропилен гликолом, холин-хлоридом и 1,3-диметилуреом. свим еутектичким растварачима одређене су физичко–хемијске особине (густина, вискозитет, електрична проводљивост и индекс рефракције) и израчунате термодинамичке особине (коефицијент запреминског ширења, моларна запремина, енергија решетке и топлотни капацитет) у функцији од температуре у опсегу 293,15– –363,15 к на 101,325 кpa. код свих припремљених еутектичких растварача густина, вискозитет и индекс рефракције опадају са порастом температуре док електрична проводљивост расте. ове температурне зависности вискозности и електричне проводљивости су описане vogel–tamman–fulcher једначином. вискозитет и моларна проводљивост, који показују линеарно понашање, били су у корелацији са фракционим wаlden правилом. инфрацрвена спектроскопија са фуријеовом (fourier) трансформацијом коришћена је за проучавање функционалних група еутектичких растварача, док су термогравиметријска анализа и диференцијална скенирајућа калориметрија коришћене за одређивање њихових стабилности. утврђено је да анализирани еутектички растварачи поседују својства која им омогућавају примену у различите индустријске процесе попут екстракције, сепарације, хемијске технологије и биотехнологије. 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<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> /enu (use these settings to create adobe pdf documents best suited for high-quality prepress printing. created pdf documents can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {towards edible ionic liquids cholinium taurate} j. serb. chem. soc. 84 (9) 991–1004 (2019) udc 544.275–128:547.932:547.436:663/664 jscs–5241 original scientific paper 991 towards edible ionic liquids – cholinium taurate milan vraneš1*#, aleksandar tot1#, jovana panić1#, snežana papović1, slobodan gadžurić1# and dragana četojević-simin2 1faculty of sciences, department of chemistry, biochemistry and environmental protection, university of novi sad, trg dositeja obradovića 3, 21000 novi sad, serbia and 2oncology institute of vojvodina, institutski put 4, 21204 sremska kamenica, serbia (received 13 april, revised 18 may, accepted 20 may 2019) abstract: in this work, the possibilities and benefits of using an ionic liquid as a potential dietary supplement are presented and discussed for the first time. ionic liquids prevent the development of microorganisms due to high ion concentration and thus, prevent perishability of the food products. thermal stability, structure, as well as the experimental density and viscosity in the temperature range from 20 to 50 °c and at the atmospheric pressure 1×105 pa of newly synthesized cholinium taurate ionic liquid, [chol][tau], are determined. according to the performed physicochemical characterization, it can be concluded that the synthesized ionic liquid is suitable for application in the food industry. the temperature variation of viscosity and density is discussed in terms of processes, packaging, and storage of [chol][tau]. also, the antiproliferative activity of [chol][tau] is determined and compared with those obtained for ascorbic acid and aspirin® as the standards. keywords: ionic liquids; food additive; synergistic effect; choline; taurine; antiproliferative activity. introduction in the last two decades, ionic liquids (ils) have been explored and investigated due to their remarkable properties and potential applications in many industrial and technological areas. because of their high chemical and thermal stability, the ability to dissolve large number of compounds and the fine tuning properties by the proper selection and the introduction of various functional groups in the il components, they are now widely used in organic synthesis, catalysis, liquid–liquid extractions, gas absorption, dissolving of the cellulose, electrodeposition etc. also, ionic liquids are very often attributed as “green solvents” mostly because of their low volatility and flammability.1 this claim has * corresponding author. e-mail: milan.vranes@dh.uns.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc190413047v ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 992 vraneš et al. launched a series of scientific research on their environmental impact, toxicity and biodegradability.2 the results showed that the common and the most studied imidazolium and pyrrolidinium-based ionic liquids are not as benign as much as it was previously thought since they show significant toxicity to the cells and micro-organisms, as well as to plants and animals.3 the biodegradation of the il is often not straightforward, since the product may be more toxic than the starting compound.4 as a consequence, the studies on the possible use of ionic liquids as a dietary supplement are almost non-existent, and ils application in the food industry is mostly limited to use them as novel extractants in food analysis.5,6 on the other hand, there are numerous advantages of ionic liquids that may be applied in food industry such as good solubility in water, better bioavailability, colloidal particles dispersed in ils7–9 and designed lipophilicity, which allows easier transport of the nutrients through the cell membrane and possibility of ils synthesis with synergistic cation and anion performances. recent studies showed that tetraalkylammonium based ils are less toxic than those based on the imidazolium, pyrrolidinium or pyridinium ions.2 also, the toxicity may be significantly decreased by introducing the polar hydroxyl, ether or carboxyl groups in the side alkyl chain of the cation.10 therefore, the possibility of synthesis completely non-toxic ionic liquids, that could be used in the diet as the food additives, can be considered. one of the most promising ils for that purpose are certainly those with choline based cation which contains quaternary ammonium group and the polar oh group in its structure, expecting thus very low toxicity.11 choline is biologically widespread micronutrient which cannot be completely degraded under aerobic conditions.12 in recent years, many choline based ionic liquids were synthesized and characterized showing their low toxicity and excellent biodegradability.13–15 in the case of choline based ils with amino acids as the anions, it was found that toxicity is even lower than the toxicity of the choline chloride, which is widely used as the food additive in the animal diet.12 regarding the use of choline in the human diet, it was officially recognized by the us institute of medicine’s food and nutrition board in 1998, whereby the adequate daily intake for adult women is 425 mg (450 mg for pregnant women and 550 mg for lactating women) and 550 mg for adult men. choline is synthesized in the liver de novo by the methylation of phosphatidylethanolamine (pe) to phosphatidylcholine (pc), but these quantities are insufficient for the organism, and choline intake via food is necessary.16 in the body, choline is essential for the maintenance of structural integrity and signalling functions of the cell membrane, transport of the fats and cholesterol, as well as for the synthesis of the neurotransmitter acetylcholine. in particular, the most crucial role of choline is a donor of the methyl group, because as a precursor of betaine allows methylation of the homocysteine into methionine, and thus prevents hyperhomo________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. edible ionic liquids 993 cysteinemia, which is the cause of cardiovascular disease.17,18 also, choline is essential for the brain development of the fetus and improves the visuomotor performance of healthy humans. lack of choline causes liver and muscle damage.16,19 the usual daily dietary need is relatively easy to meet. through food, choline comes in the form of lipid-soluble phosphatidylcholine (48 % of the total intake), and the most important sources are foods of animal origin, such as eggs, poultry, beef, pork, ham, and salmon. another possibility of intake is the free water-soluble choline (23 %), and in that form can be found in the bread (wheat or rice), milk, bananas, orange juice and peanut butter. however, the research performed on athletes has shown that the free choline concentration decrease during intense exercise and that the supplementation of choline can improve endurance,20,21 and, therefore, choline may be found as an ingredient of many pre-workout sport supplements.22,23 in these products, choline is mainly found in the form of a cholinium tartrate and less frequently as cholinium citrate. it is known that most of the tartrate is destroyed in the intestinal tract by the present microorganisms,24 while for the citrate is not found any positive effect on athletic performance or body composition.25,26 thus, it is justified to replace them with the new non-toxic forms of choline-based salts with anions that exhibit an ergogenic effect. also, the salts in the liquid forms such as ionic liquids can be easily combined with other components in the process of commercial production of dietary and sports supplements, as well as in the food industry in order to increase the nutritional value. therefore, in this work, new choline based ionic liquid with taurate as the anion is synthesized. taurine is one of the sulfur-containing amino acids, does not form proteins and in natural form can be found in high concentrations in skeletal and cardiac muscle and brain tissue, making about 0.1 % of total human body weight. numerous studies have confirmed the positive effect of taurine on sports performances,27–29 it decreases oxidative stress,30 but also it shows an essential effect in the protection of the skeletal muscle and heart.31 consequently, taurine is often added as an active component of energy drinks and other sports supplements.32 for the newly synthesized cholinium taurate ionic liquid, the toxicity test is performed together with the physicochemical and thermal properties study in order to fully characterize it and to discuss the potential application of [chol][tau] as a novel additive to sports supplements. one of the goals is to evaluate cytotoxicity through determining cell growth effects of the newly synthesized ionic liquid, starting compounds of the synthesis (cholinium chloride and taurine) and standards (ascorbic acid and aspirin®) in human fetal lung cell line mrc-5 derived from the healthy tissue.33 multi-endpoint bioassays that are based on the whole-cell response in mammalian cell lines are powerful indicators of metabolic, biochemical, and genetic alterations that arise under the influence ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 994 vraneš et al. of evaluated compounds. they possess predictive power in terms of the risk to higher organisms. experimental synthesis of [chol][tau] the calculated amount of taurine (sigma aldrich, ω ≥ 99 %) is dissolved in water at room temperature, and the obtained solution is titrated by the potentiometric method using the aqueous choline hydroxide (sigma aldrich, ω ≥ 46 %) in the concentration of 1.7524 mol·dm–3. after achieving equivalence point (ph 9.02), the obtained cholinium taurate (fig. 1) is purified. water is removed by the vacuum evaporation for 120 min at 70 °c and the obtained [chol][tau] was kept in the vacuum desiccator over p2o5. after drying, water content in the il was found to be 215 ppm using the karl fisher titration. fig. 1. structure of [chol][tau] ionic liquid. the structure is confirmed by 1h-, 13c-nmr and ftir spectroscopy. the thermal stability is checked applying thermogravimetric (tg) and differential scanning calorimetry (dsc) analysis. physicochemical properties such as density and viscosity are measured in the temperature range from 20 to 50 °c. nmr spectra were recorded in d2o at 25 °c on a bruker advance iii 400 mhz spectrometer. tetramethylsilane was used as an accepted internal standard for calibrating chemical shift for 1h and 13c. ftir measurements were performed using a thermo-nicolet nexus 670 spectrometer, equipped with a universal atr sampling accessory. the measurements were performed with a total of 60 scans, at 25 °c, and a spectral resolution of 2 cm–1 in a range of ν from 750 to 4000 cm–1. the software package omnic, version 6.2, was used in the data acquisition and spectral analysis. the thermal characterization of the sample was performed by thermogravimetric analysis using simultaneous tg/dsc thermal analyzer sdt q600 (ta instruments, usa). sample (≈2.5 mg) was placed in an open platinum pan. measurements were carried out in the nitrogen atmosphere at a flow rate of 100 cm3⋅min–1 up to 500 °c with a heating rate of 10 °c min–1. the instrument was calibrated (temperature and enthalpy) using the indium standard. density and viscosity measurements the vibrating tube densimeter, rudolph research analytical ddm 2911, with automatic viscosity correction, was used for density measurements. the accuracy and precision of the densimeter were ±0.00005 g·cm–3. the instrument was automatically thermostatted (peltier type) within ±0.01 °c with automatic correction of the viscosity and was calibrated at the atmospheric pressure before each series of measurements. the calibration was performed using ambient air and bi-distilled ultrapure water in the temperature range from 20 to 50 °c. viscosity was measured using a brookfield viscosimeter dv ii+ pro connected to the thermostat and filled with about 8 cm3 of tested binary mixtures and pure components. the ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. edible ionic liquids 995 spindle type (sc4-18) was immersed and the rounds per minute (rpm) was set in order to obtain a suitable torque. a viscometer cell protected from moisture with the compartment made by the manufacturer was calibrated using the liquids of different viscosities obtained from the manufacturer. measurements were performed in the temperature range from 20 to 50 °c with the rotation speed of 60rpm for pure ionic liquid. the viscosity data at the corresponding temperatures were recorded automatically on a computer and then processed in origin 2016. cell lines and cytotoxicity tests sulforhodamine b and antibiotic/antimycotic solution were purchased from sigma aldrich, fetal bovine serum and dulbecco’s modified essential medium were supplied from paa laboratories gmbh, trypsin was from serva, and ethylenediaminetetraacetic acid (edta) from laphoma. all substances were diluted in 9 mg⋅cm–3 nacl solution and sterilized using 0.22 μm syringe filters. ionic liquid and standards of cholinium chloride and taurine were studied in the concentration range from 125 to 2000 μg⋅cm–3, while ascorbic acid and aspirin® were investigated in the range from 62.5 to 1000 μg⋅cm–3. cell growth activity was evaluated in vitro in human fetal lung cell line mrc-5 (ecacc 84101801). cells were grown in dulbecco’s modified essential medium with 45 mg⋅cm–3 glucose supplemented with 10 % heat-inactivated fetal bovine serum (biowest, nuaille, france), 100 μg⋅cm–3 of penicillin, 100 μg⋅cm–3 of streptomycin and 0.25 μg⋅cm–3 of amphotericin b (antibiotic/antimycotic solution; sigma-aldrich). cells were cultured in 25 cm3 flasks at 37 °c in the atmosphere of 5 % co2 and high humidity, and sub-cultured twice a week. a single cell suspension was obtained using 0.1 % trypsin with 0.04 % edta. the cell lines were harvested and plated into 96-well microtiter plates at a seeding density of 4×103 cells/well in a volume of 180 µl, and pre-incubated in complete medium supplemented with 5 % fbs at 37 °c for 24 h. serial two-fold dilutions (20 μl) of tested substances were added to achieve final concentrations. an equal volume of solvent was added in control wells. after the addition of dilutions, microplates were incubated at 37 °c for 48 h. the cell growth was evaluated by colorimetric sulforhodamine b assay of skehan et al.34 colour development was measured using multiscan ascent photometer at 540 nm against 620 nm as background. the effect on cell growth was calculated as 100at/ac (%), where at is the absorbance of the test sample and ac of the control. dose-effect (concentration-cell growth) curves were plotted for each treatment, and ic50 values were determined. the results of cell growth activity were obtained in two independent experiments, each performed in quadruplicate (n = 8). results and discussion spectroscopy analysis obtained 1h-nmr, 13c-nmr and ftir spectra of [chol][tau] are presented in figs. s1 and s2 of the supplementary material of this paper. 1h-nmr spectrum (d2o, δ / ppm): 3.09 (m, 4h, nh2ch2ch2so3); 3.28 (bs, 9h, n(ch3)3); 3.60 (t, 2h, ch2n(ch3)3); 4.13 (m, 2h, ch2oh). 13c-nmr spectrum (d2o, δ / ppm): 36.65 (ch2nh2); 53.32 (ch2so3–); 53.87, 53.90, 53.93 (n(ch3)3), 55.60 (ch2oh); 67.44 (ch2n(ch3)3). ftir spectrum of [chol][tau] is recorded in the wavelength range from 500 to 4000 cm–1. ir (neat) 3348 (stretching oh); 3034 (sym. stretching ch, ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 996 vraneš et al. (n(ch3)3)); 1603 (asym. bending nh2); 1480 (rocking ch3); 1360 and 1284 (wagging ch2); 1170 (asym. stretching so3); 1086 (twisted nh2 and twisted ch2); 1031 (sym. stretching so3); 952 (asym. stretching cco); 794 (stretching c–s) ); 605 (out-of-plane deformation γ so3); 581 (out-of-plane deformation γ so3); 520 (in-plane deformation γ so3). thermal analysis of [chol][tau] in order to determine the thermal stability of the ionic liquid [chol][tau], the thermogravimetric analysis was performed. the results of thermal stability for potential food additive are very important, since they indicate which temperature range is suitable for the thermal processing of foods which contains the additive. the obtained thermogravimetric curve is presented in fig. 2. fig. 2. tg (solid line), dsc (short dash) and dta (long dash) analysis of [chol][tau], mt – mass at temperature t, mo – inital mass. it can be seen from fig. 2 that the decomposition is a two-stage process, and begins at the 197 °c. thermo-gravimetric analysis for choline ionic liquids with amino acids containing the carboxyl group instead of sulfonic group showed one -stage thermal decomposition13 between 150–203 °c. based on the weight variation curves, the temperature by 2 and 5 % weight loss (t2 %, t5 %) was specified, and obtained values are 137.4 and 189.2 °c. the high thermal stability of ionic liquid does not require special temperature conditions during warehousing and preservation. also, the presented thermal stability enables high-temperature technological processes during the food preparation. thermal stability of [chol][tau] is lower in comparison with choline chloride35 (300 °c), which is a consequence of weaker interactions between ions in ionic liquid, that originate from the more voluminous and better steric hindrance of taurate anion compared to chloride. on the other hand, higher thermal stability for taurine (328 °c)36 can be explained by ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. edible ionic liquids 997 his zwitterionic structure with strong electrostatic interactions and hydrogen bond formation in crystalline form,37 while in ionic liquid taurine exists in an anionic form with strong repulsive electrostatic interactions. viscosity and density of [chol][tau] essential features of liquid food additives in many areas of food processing are viscosity and density. knowing the physicochemical properties, with particular emphasis on viscosity and its variation with temperature, plays a vital role in design engineering processes in the food industry. the viscosity of food additives must be as low as possible for easier technical and technological processes and transport through the plant, reducing thus the price of operating costs. viscosity is closely associated with the textural and sensory attributes of the food product, such as appearance and flavour of food ingredients. based on the viscosity measurements for a given product the sensory assessment of food texture could be predicted and may be changed in order to produce an additive with textural parameters acceptable to the consumer. viscosity results obtained from the experimental measurements are shown in table s-i of the supplementary material and graphically presented in fig. 3. from fig. 3 can be seen that the viscosity of [chol][tau] decreases with increasing temperature as expected. fig. 3. temperature dependence of [chol][tau] density (d, square) and dynamic viscosity (η, circle). the obtained viscosity results for [chol][tau] are compared with commonly used food ingredients and food additives (fig. 4). it is evident that the food industry uses even more viscous components than [chol][tau], which indicates that [chol][tau] ionic liquid is a promising ingredient and food supplement.38 in fig. 5 are presented and compared the changes of viscosity with temperature for [chol][tau] ionic liquid and honey as the natural representative mix________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 998 vraneš et al. ture of covalent compounds (glucose and fructose). fig. 5 shows that [chol][tau] has about ten times lower viscosity in comparison with honey, while the temperature dependence of viscosity has a similar trend. fig. 4. comparison of [chol][tau] viscosity at 25 °c with commonly used food ingredients and food additives. fig. 5. comparison of dynamic viscosity (η) for [chol][tau] (circle) and honey (square). variation of viscosity with temperature was fitted using the logarithmic form of the arrhenius equation38 (fig. 6): ln η = c + eart (1) where c is the pre-exponential coefficient, ea is the activation energy of viscous flow, and r is the universal gas constant. the activation energy of viscous flow was calculated from the slope of the arrhenius plot and the obtained values amount to 48.83 and 90.85 kj·mol–1 for [chol][tau] and honey, respectively, indicating the easier viscous flow of ionic liquid. for the food manufacturing process optimization, the warehousing in cans and jars of foods being sterilized is partly dependent on their density.39 density is an important physicochemical property, and its temperature dependence is related to volume changes. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. edible ionic liquids 999 fig. 6. variation of ln η viscosity with t–1 for [chol][tau] (circle) and honey (square). the measured density values are also shown in table s-i and fig. 3. the temperature dependence of [chol][tau] density is compared with those obtained for honey and presented in fig. 7. it is evident that the value of [chol][tau] density decreases with the increase of temperature, wherein the density variation with temperature is less pronounced in the case of ionic liquid, in comparison with honey. this trend is a consequence of the ionic liquid specific structure and electrostatic interactions between cation and anion in [chol][tau]. a relatively small variation of ionic liquid density (and volume) with temperature, combined with negligible vapour pressure, makes it suitable for safe storage in sealed containers and non-temperature controlled conditions. fig. 7. variation of density (d) with temperature for [chol][tau] (circle) and honey (square). on the basis of the experimental densities, the thermal expansion coefficients for the of [chol][tau] and honey, αp, can also be calculated: ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 1000 vraneš et al. p p,m 1 d d t α ∂ = −  ∂  (2) the determined thermal expansion coefficient tabulated in table s-i and presented in fig. 8. fig. 8. variation of thermal expansion coefficients with temperature for [chol][tau] (circle) and honey (square). based on these results, it is clear that the volume of ionic liquid slowly expands with an increase of the temperature, which facilitates the process of packaging and storage of [chol][tau] without the risk of sudden expansion in high-temperature storage conditions. cytotoxicity of [chol][tau] in order to consider the potential application of [chol][tau] in the food industry, it is necessary to perform and evaluate the cytotoxicity of the synthesized ionic liquid. antiproliferative activity of [chol][tau] was tested on the human non-tumor cell line (normal fetal lung fibroblasts mrc-5), and the obtained results are shown in table i. mrc-5 is the well characterized human diploid fibroblast (hdf) cell line, derived from the healthy fetal lung tissue that is remarkably stable and retains predominantly normal diploid karyotype of the original tissue. beside cytotoxicity of [chol][tau], the influence of commercial used substances (aspirin®, vitamin c) on cytotoxicity of mrc-5 cell line were examined and the obtained results are presented in table i. the antiproliferative activity was expressed as ic50 value, defined as the dose of a compound that inhibits cell growth by 50 % and represent the effectiveness of a substance in inhibiting a ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. edible ionic liquids 1001 specific biological or biochemical function. according to well-known criteria, substances with ic50 values lower than 20 μg⋅cm–3 had satisfying antiproliferative activity. compounds with ic50 in the interval of 20–100 μg⋅cm–3 can be considered weakly cytotoxic, and substances can be considered inactive if their ic50 values are higher than 100 μg⋅cm–3. table i. cell growth activity of ionic liquids and standards in mrc-5 cell line after 48 h exposition compound ic50 / μg⋅cm –3 [chol][tau] 1426.78±10.83a choline chloride >2000a taurine >2000a ascorbic acid 398.60±24.96b aspirin® >1000b a125 to 2000 μg⋅cm–3 concentration range; b62.5 to 1000 μg⋅cm–3 concentration range as can be seen from table i, the starting compounds choline chloride and taurine can be considered as non-toxic in the investigated concentration range. on the other hand, ascorbic acid shows much higher cytotoxicity in comparison with [chol][tau]. conclusion based on the performed physicochemical characterization, it can be concluded that the synthesized ionic liquid is suitable for application as a food additive. the obtained experimental results indicate that the volume of ionic liquid slowly expands with temperature, which can make the processes of packaging and storage of [chol][tau] easier and without the risk of sudden expansion in high-temperature storage conditions. in combination with negligible vapour pressure and relatively low viscosity of [chol][tau], manipulation and handling during the industrial processes and storage are less complicated. according to the examination of antiproliferative activity, [chol][tau] is less toxic than vitamin c. supplementary material additional data are available electronically from the journal web site: http:// //www.shd.org.rs/jscs/, or from the corresponding author on request. acknowledgement. this work was financially supported by the ministry of education, science and technological development of serbia (project number on172012). ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2019 scs. 1002 vraneš et al. и з в о д холинијум-таурат – пут ка јестивим јонским течностима милан вранеш1, александар тот1, јована панић1, снежана паповић1, слободан гаџурић1 и драгана четојевић-симин2 природно–математички факултет, департман за хемију, биохемију и заштиту животне средине, трг доситеја обрадовића 3, нови сад и 1институт за онкологију војводине, институтски пут 4, 21204 сремска каменица у овом раду су је предочена могућност употребе јонских течности као додатака у исхрани људи, по први пут. јонске течности спречавају развој микроорганизама у прехрамбеним производима, услед могућности постизања високих концентрација електролита у њима. испитана је термичка стабилност, структура и измерене вискозности и густине у температурном опсегу 20 дo 50 °c и на атмoсферском притиску (1×105 pa) синтетисане јонске течности – холинијум-таурата. на основу резултата физичко-хемијских мерења, може се закључити да је испитивана јонска течност погодна за манипулацију у процесима прехрамбене индустрије. испитана је и антипролиферативна активност синтетисане јонске течности, а резултати су упоређени са стандрадним стандардним супстанцама – аспирином и витамином ц. 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {synthesis and assessment of the cytotoxic effect of some of 1,4-dihydropyridine derivatives which contain azole moiety} j. serb. chem. soc. 86 (11) 1013–1021 (2021) original scientific paper jscs–5479 published 1 october 2021 1013 synthesis and assessment of the cytotoxic effect of some of 1,4-dihydropyridine derivatives which contain azole moiety saeed ghorbannejad1, karim akbari dilmaghani1* and abbas nikoo2 1department of chemistry, faculty of science, urmia university, urmia, iran and 2shahid bakeri high education center, urmia university, urmia, iran (received 18 august 2020, revised 5 august, accepted 10 august 2021) abstract: a number of 1,4-dihydropyridine derivatives (9a–d, 10a–d and 11a–d) were designed and synthesized by the reaction of 1,3,4-oxadiazole-5-thiones and 1,2,4-triazole-5-thiones to 2,6-dibromomethyl-3,5-diethoxycarbonyl-4-(3 -nitrophenyl)-1,4-dihydropyridine. the synthesized compounds were characterized using ft-ir, 1h-nmr, 13c-nmr spectral data, esi-ms and elemental analysis. the cytotoxicity of the synthesized compounds was evaluated in human breast cancer (mcf-7) cells based on the results of mtt assay. the results indicated that compound diethyl 4-(3-nitrophenyl)-2,6-bis[((5-(3-nitrophenyl)-1,3,4-oxadiazol-2-yl)thio)methyl]-1,4-dihydro pyridine-3,5-dicarboxylate (9b) with (ic50 = 23±2.32 µm) was the most potent derivative against mcf-7 cells. based on the results, the use of oxadiazole moiety in the c2 and c6 positions of 1,4-dihydropyridine ring system enhanced the cytotoxic potential of these derivatives. therefore, some of the oxadiazole-substituted 1,4-dhps may facilitate further modifications which result in the discovery of potent cytotoxic agents. keywords: dimethylformamide; 3-nitrobenzaldehyde; 1,3,4-oxadiazole; 1,2,4-triazole. introduction 1,4-dihydropyridines (dhps) have assumed considerable importance in the field of organic and medicinal chemistry due to their interesting pharmacological activities. the results of different studies have highlighted the fact that the dhps are highly effective calcium antagonists and are used for treating various cardiovascular system (cvs) activities.1–3 in addition to the cvs activities of dhps, they possess a variety of biological activities including, cytotoxic activities,4–6 anti-proliferative activities,7 multidrug resistance activities8,9 and anti-tumour activities.10–13 however, synthesizing dhps derivatives is an active and ongoing * corresponding author. e-mail: k.adilmaghani@urmia.ac.ir https://doi.org/10.2298/jsc200818064g ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 1014 ghorbannejad, dilmaghani and nikoo research area. chemotherapy is still one of the most effective methods for treating cancer. the potential uses of the dhps scaffolds in the chemotherapy are well-documented and stem from their suitability for reversing the drug resistance in the treatment of cancer.14,15 calcium channel blockers like verapamil16 and nicardipine,17 among others have been reported to successfully overcome drug resistance. notwithstanding, the introduction of calcium channel blockers to clinical use might pose a therapeutic problem which results from their strong vasodilator function.18 consequently, a substance which has a strong capability to overcome anticancer drug resistance and does not lead to calcium antagonistic activity can be of great value in chemotherapy. research has shown that, generally, dhps display more cytotoxicity towards cancer cells in comparison with the non-cancer cells.19 the discovery of new dhp derivatives can encourage the development of novel and effective therapies for diverse pathologies, including cancer. the efficiency of oxadiazole and triazole derivatives has been assessed and proved for a wide range of pharmacological uses. the connection of 1,3,4-oxadiazoles or 1,2,4-triazoles to the 1,4-dhps core has produced a combination scaffold. the dhps can be selectively functionalized in several positions. the synthesis and anticancer activities of bis(1,3,4-oxadiazole-2-thiol) and bis(4-amino1,2,4-triazole-3-thiole) derivatives of dhps in the c3 and c5 have been reported.20 moreover, research has shown the synthesis and biological activities of 1,3,4-oxadiazole derivatives which are linked to n1 of dhp ring system.21 in spite of the highly developed chemistry of the dhps, there is not adequate information about the synthesis of 1,4-dhps bearing-substituents other than hydrogen atoms or alkyl groups in the c2 and c6. in our previous study, we reported the synthesis and antimicrobial assessment of 1,4-dihydropyridines with azole derivatives in the c2 and c6 positions of dhp ring.22 based on the above-mentioned gap in the related literature, the present study focuses on synthesizing the novel 1,4-dihydropyridine derivative, which are linked to triazole and oxadiazole moieties, and evaluating their cytotoxic activities. experimental the chemicals of sigma–aldrich and merck were used to produce the chemicals of this study. the solvents were purified based on the standard procedures before their use in the study. the thin-layer chromatography (tlc) analysis was performed in the case of the recoated silica gel (e-merck kieselgel 60 f254 aluminium sheets) plates. n-bromosuccinimide (nbs) and tetramethylsilane (tms) were purchased from merck. the melting points were determined on open capillaries using a digital melting point apparatus. the ft-ir spectra were recorded as kbr pellets on a thermo nicolet nexus 670 ft-ir. the 1hand 13c-nmr spectra were recorded on the bruker avance aqs 300 mhz spectrometer at 300 and 100 mhz, respectively. the chemical shifts were measured in dimethyl sulfoxide (dmso-d6) as solvent relative to tms as the internal standard. these abbreviations were used to describe the multiplicities of signals in nmr spectra (s = singlet, d = doublet, t = triplet, q = quartet, dd = ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 1,4-dihydropyridines with cytotoxic effect 1015 doublet of doublets and br = broad signal). the mass spectra were recorded on a jeol-jms 600 (fab ms) instrument and the esi-ms spectra were recorded on an agilent technologies 5975c vl msd mass spectrometer which operated at an ionization potential of 70 ev. the chns analysis was performed using chns-932 leco analyser. the 3a–d23,24, 4a–d25, 5a–d26 and 6a–d27 compounds were produced according to the reviewed literature. analytical and spectral data of the synthesized compound are given in supplementary material to this paper. procedure for preparing diethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7) a mixture of ethylacetoacetate (0.02 mol, 2.60 g), ammonium acetate (0.015 mol, 1.16 g) and 3-nitrobenzaldehyde (0.01 mol, 1.51 g) in 50 % ethanol (50 ml) was mixed well under reflux for 12 h. the contests were cooled after the reaction (which was monitored by tlc via n-hexane/etoac (4:1) as eluent. the precipitate was filtered, washed with water and crystallized from ethanol.28,29 procedure for synthesizing diethyl 2,6-bis(bromomethyl)-4-(3-itrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (8) nbs (0.02 mol, 3.56 g) was added to a solution of the diethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7, 0.01 mol, 3.74 g) in methanol (100 ml) portion-wise at ambient temperature. the reaction mixture was stirred at room temperature for 24 h. the pale yellow precipitate was filtered and washed with water. the precipitate was crystallized from ethanol.29 general procedure for synthesizing the 9a–d, 10a–d and 11a–d compounds a mixture of 3a–d, 4a–d or 6a–d (0.02 mol), naoh (0.02 mol, 0.8 g) and dmf/h2o (50/50, 50 ml) was stirred at room temperature for 1 h. moreover, diethyl 2,6-bis(bromomethyl)-4-(3-nitrophenyl)-1,4-dihydropyridine-3, and 5-dicarboxylate (8, 0.01 mol, 5.32 g) were added to it and it was stirred at room temperature for 8–12 h. the reaction mixture was poured into water (100 ml) and the residue was extracted with ch2cl2. the organic layer was washed with water, dried over na2so4 and evaporated and recrystallized from ethanol. biological assessment reagent and chemical. (rpmi-1640) and fetal bovine serum (fbs) were purchased from (gibco, usa). 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (mtt) was obtained from sigma. penicillin/streptomycin was purchased from invitrogen (san diego, ca, usa). dimethyl sulphoxide (dmso) were obtained from merck. cell culture. the human breast cancer cells (mcf-7) were purchased from national cell bank of iran (pasteur, tehran, iran). these cells were cultured in roswell park memorial institute 1640 (rpmi-1640) (gibco, usa) medium which was enriched with 10 % fetal bovine serum (fbs, gibco, usa), 100 unit/ml penicillin and 100 mg/ml streptomycin and was maintained under 37 °c and 5 % co2 conditions. the cells, which reached the 70 % confluence, were sub cultured and were used for conducting the experiments. cell viability assay (mtt). in order to determine the cytotoxic effect of various compounds on the viability of mcf-7 cells, the mtt reduction assay was performed as described previously.30-32 first mcf-7 cells were plated in 96-well microplates at a density of 1×104 cells per well and were maintained overnight at 37 °c to allow them to attach to the bottom of the wells. after cell attachment, the medium was removed and cells were treated with various compounds (9a–d, 10a–d and 11a–d) at the concentrations which ranged from 10 to 100 µm. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 1016 ghorbannejad, dilmaghani and nikoo all of the compounds were dissolved in dmso and were diluted in medium in a way that the maximum concentration of dmso in the wells did not exceed 0.5 %. cells were further incubated for 48 h. thirdly, the fresh medium, which contained 500 µg/ml mtt powder, was added to each well and plates were incubated for another 4 h time at 37 °c. then mtt and media mixtures were removed and formazan crystals which formed by the mitochondrial dehydrogenase activity of vital cell were solubilized in 200 µl dmso and was put on an orbital shaker for 20 min. finally, the absorbance of each plate was measured at 570 nm with the background correction at 620 nm using an elisa plate reader (statefax, usa). effects of the drug cell viability were calculated using cells treated with dmso as control. cell survival was calculated using the formula: survival, % = [(absorbance of treated cells – absorbance of culture medium)/(absorbance of untreated cells – absorbance of culture medium)]×100. the experiment was done in triplicate and the inhibitory concentration (ic) values were calculated from a dose-response curve. graph pad prism software 6.01 was used to calculate ic50 values. ic50 is the concentration in µm required for 50 % inhibition of cell growth as compared to that of the untreated control. ic50 values were determined from the linear portion of the curve by calculating the concentration of agent that reduced absorbance in treated cells, compared to control cells, by 50 %. evaluation is based on mean values from three independent experiments, each comprising at least six micro cultures per concentration level. ic50 values represent the mean of triplicate determination (n = 3) ± sd with (95 %) confidence interval. the cytotoxicity of the synthesized derivatives was not compared to standard drugs.33 results and discussion the 3a–d, 4a–d, 5a–d and 6a–d derivatives were prepared according to the method which was described in the relevant literature.23–27 it is well-known that the thiol–thione tautomeric equilibrium exists in 3a–d, 4a–d and 6a–d compounds. on the basis of 1h-nmr and ft-ir experimental findings, it is argued that the thione tautomer is more stable than thiol in the solution. 1h-nmr spectra of these compounds exhibited the nh signal as a broad peak in the δ 12–14 ppm range which supports the proposed thione structure. the appearance of a c=s absorption peak in the 1248–1278 cm–1 region indicated that the oxadiazoles and triazoles were in their thione form.34 diethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7) was synthesized using the classical hantzsch three-component reaction method. this method includes the cyclocondensation of 3-nitrobenzaldehyde with two equivalents of ethyl acetoacetate in the presence of a nitrogen donor such as ammonia or ammonium acetate (based on the procedure reported in the literature).33 we needed a quick entry into 1,4-dihydropyridine-3,5-dicarboxylic acid diesters in which the 2,6-methyl groups were altered by a range of different groups. allylic bromination is the replacement of a hydrogen on a carbon adjacent to a double bond. allylic bromination in dihydropyridines was performed by nbs. the synthesis of 2,6-dibromomethyl-3,5-diethoxycarbonyl-1,4-dihydropyridine (8) was achieved as a result of the bromination of the corresponding 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (7) by nbs in methanol according to the procedure which was described in the literature.29 the ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 1,4-dihydropyridines with cytotoxic effect 1017 bromine atoms in compound 8 can be replaced with the other substituents (scheme 1). scheme 1. synthesis of compound 8. the replacement of the bromines of compound 8 with 1,2,4-triazole-5-thiones 3a–d, 1,3,4-oxadiazole-5-thiones 4a–d and 4-amino-3-mercapto-1,2,4-triazoles 6a–d was carried out in the presence of sodium hydroxide as a base in dmf in order to afford the corresponding coupled 1,4dhps (9a–d, 10a–d and 11a–d) (scheme 2). scheme 2. the synthesis of compounds 9a–d, 10a–d and 11a–d. ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 1018 ghorbannejad, dilmaghani and nikoo the structures of 9a–d, 10a–d and 11a–d compounds were identified using spectroscopic methods. in the ir spectra the disappearance of the c=s absorption peak in the 1248–1278 cm–1 region and the absence of nh peak at δ 12–14 ppm supported the connection of oxadiazole and triazoles to 1,4-dhp ring. the ch2x protons in the c2 and c6 positions of symmetrically substituted 1,4-dihydropyridine ring became diastereotopic and provided an ab system in the corresponding 1h-nmr spectra. the extent of the observed anisotropy of the methylene protons must have been influenced by the spatial conformation between the ester groups and the formation of a ch…o=c intramolecular hydrogen bonding.35 the increase in concentration resulted in a decrease in the viability of cells for all of the compounds and indicated that the cytotoxicity of all of the compounds depended on the concentration. some of the compounds including 10a and c did not display a high level of cytotoxicity towards mcf-7 cells. nonetheless, a number of the other compounds including 9b and d displayed a high level of cytotoxicity towards these cells at a concentration which was confirmed by their ic50 values. according to the in vitro mtt assay, the ic50 represents the concentration of the newly synthesized compounds that is required for 50 % inhibition of the human breast cancer cell (mcf-7) viability. the ic50 value for each compound was calculated and summarized in table i. as shown in table i, based on the ic50 value, the most cytotoxic compound was 9b. therefore, it can be suggested that, this compound is a potent cytotoxic agent. table i. cytotoxic activity of the synthesized 1,4-dhp derivatives assessed by the mtt assay compound r molecular weight ic50 / µm mcf-7 ic50 / μg ml-1 mcf-7 9a c6h5– 726 45±2.72 32.67 9b 3-no2–c6h4– 816 23±2.32 18.76 9c 2-ho–c6h4– 758 40±4.65 30.32 9d 2-cl–c6h4– 794 33±1.55 26.20 10a c6h5– 876 63±3.10 55.12 10b 3-no2–c6h4– 966 57±4.27 55.06 10c 2-oh–c6h4.– 908 >100 10d 2-cl–c6h4– 944 49±3.10 46.25 11a c6h5– 754 54±3.10 40.71 11b 3-no2–c6h4– 844 44±1.8 36.16 11c 2-ho–c6h4– 786 51±4.6 39.16 11d 2-cl–c6h4– 822 47±4.65 38.63 conclusion in the present study, diethyl-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3 and 5-dicarboxylate derivatives were coupled with 1,3,4-oxadiazole-5-thiones and 1,2,4-triazole-5-thiones in the c2, c6 positions of 1,4-dihydropyridine ring system in order to produce compounds with greater cytotoxicity. the synthesized compounds were characterized using ft-ir, 1h-nmr, 13c-nmr spec________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 1,4-dihydropyridines with cytotoxic effect 1019 tral data, esi mass and elemental analysis. the cytotoxic effects of the new compounds on human breast cancer (mcf-7) cells were investigated using mtt assay. the results of the mtt assay showed that a number of compounds including 9b and d displayed good cytotoxicity at a certain concentration. this finding was confirmed by their ic50 values. the highest potency was observed in the case of mcf-7 cells (9b: ic50 = 23±2.32 μm). based on the results, the 10c compound did not have a cytotoxic effect on the tested cancer cell line due to its bulky scaffold and the steric hindrance in its site of action. these preliminary encouraging results of the biological screening of the tested compounds may offer an excellent framework for the discovery of potent cytotoxic agents in this field. supplementary material additional data and information are available electronically at the pages of journal website: https://www.shd-pub.org.rs/index.php/jscs/article/view/9811, or from the corresponding author on request. acknowledgements. the authors are grateful to urmia university which provided them with a fellowship for the present study. our grateful thanks go to dr. vahid shafiei-irannejad and morteza molaparast (from cellular and molecular medicine institute, urmia university of medical sciences) who investigated the cytotoxicity of the compounds and to prof. dr. joachim thiem (from hamburg university) and prof. dr. abdolali alizadeh (from tarbiat modares university) for the esi-ms. и з в о д синтеза и одређивање цитотоксичног ефекта деривата 1,4-дихидропиридина који садрже азолску структуру saeed ghorbannejad1, karim akbari dilmaghani1 и abbas nikoo2 1department of chemistry, faculty of science, urmia university, urmia, iran и 2shahid bakeri high education center, urmia university, urmia, iran синтетисана је серија деривата 1,4-дихидропиридина (9a–d, 10a–d и 11a–d) реакцијом 1,3,4-оксадиазол-5-тиона или 1,2,4-триазол-5-тиона са 2,6-дибромметил-3,5-диетоксикарбонил-4-(3-нитрофенил)-1,4-дихидропиридином. синтетисана једињења су окарактерисана помоћу ft-ir, 1 h-nmr, 13 c-nmr и esi-ms спектара и елементалном анализом. испитана је цитотоксичност добијених деривата према ћелијама хуманог канцера дојке (mcf-7) мтт есејом. резултати указују да једињење диетил 4-(3-нитрофенил)-2,6-бис[((5-(3-нитрофенил)-1,3,4-оксадиазол-2-ил)тио)метил]-1,4-дихидропиридин-3,5-дикарбоксилат (9b) са (ic50 = 23 ± 2,32 μm) има највећу активност према mcf-7 ћелијама. на основу добијених резултата оксадиазолски део структуре на c2 и c6 положајима 1,4-дихидропиридинског система повећава цитотоксични потенцијал ових деривата. из тога произилази да би неки 1,4-dhp деривати који садрже оксадиазолске супституенте омогућили припрему нових активнијих једињења. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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can be opened with acrobat and adobe reader 5.0 and later.) >> /namespace [ (adobe) (common) (1.0) ] /othernamespaces [ << /asreaderspreads false /cropimagestoframes true /errorcontrol /warnandcontinue /flattenerignorespreadoverrides false /includeguidesgrids false /includenonprinting false /includeslug false /namespace [ (adobe) (indesign) (4.0) ] /omitplacedbitmaps false /omitplacedeps false /omitplacedpdf false /simulateoverprint /legacy >> << /addbleedmarks false /addcolorbars false /addcropmarks false /addpageinfo false /addregmarks false /convertcolors /converttocmyk /destinationprofilename () /destinationprofileselector /documentcmyk /downsample16bitimages true /flattenerpreset << /presetselector /mediumresolution >> /formelements false /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles false /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice {ambient air particles: the use of ion chromatography and multivariate techniques in the analysis of water-soluble substances} j. serb. chem. soc. 86 (7–8) 753–766 (2021) original scientific paper jscs–5459 753 ambient air particles: the use of ion chromatography and multivariate techniques in the analysis of water-soluble substances žaklina n. todorović1, jelena m. radulović1, ivana d. sredović ignjatović2, ljubiša m. ignjatović3*# and antonije e. onjia4 1anahem laboratory, mocartova 10, 11160 belgrade, serbia, 2university of belgrade, faculty of agriculture, nemanjina 6, 11080 belgrade, serbia, 3university of belgrade, faculty of physical chemistry, studentski trg 12–16, 11000 belgrade, serbia and 4university of belgrade, faculty of technology and metallurgy, karnegieva 4, 11000 belgrade, serbia (received 26 august, revised 22 november, accepted 30 november 2020) abstract: seventeen water-soluble substances (of sodium, ammonium, potassium, magnesium, calcium, formate, methanesulfonate, glyoxylate, chloride, nitrite, nitrate, glutarate, succinate, malate, malonate, sulfate and oxalate) in 94 samples of particle matter in the ambient air, collected over ten months, in a suburb of belgrade (serbia), were determined by ion chromatography. to apportion the sources of the air pollution, the log-transformed data were processed by applying multivariate techniques. principal component and factor analysis identified three main factors controlling the data variability: stationary combustion processes with the highest loadings of oxalate, malonate and malate; landfill emission and secondary inorganic aerosol characterized by high levels of ammonium, nitrate and sulfate; a contribution of mineral dust composed of magnesium, calcium and chloride. the hierarchical cluster analysis pointed out a differentiation of the samples into five groups belonging to different variables inputs. for the classification of ambient air samples using nine selected ions, the recognition ability of linear discriminant analysis, k-nearest neighbors, and soft independent modeling of class analogy were 87.0, 94.6, and 97.8 %, respectively. time-series analysis showed that the traffic emission is more pronounced in winter in contrast to the mineral dust influence, while the effect of waste combustion exhibits no trend. keywords: organic acids; pollution sources; pca; emission factors; time-series. introduction over the last few decades, the analysis of ambient air particles by means of different analytical techniques and different receptor modeling methods has received increasing attention.1,2 * corresponding author. e-mail: ljignjatovic@ffh.bg.ac.rs # serbian chemical society member. https://doi.org/10.2298/jsc200826077t ________________________________________________________________________________________________________________________ (cc) 2021 scs. available on line at www.shd.org.rs/jscs/ 754 todorović et al. the complimentary use of inductively coupled plasma mass spectrometry (icp-ms) for trace elements, and gas chromatography–mass spectrometry (gc– –ms) for volatile and semi-volatile organics has been the traditional approach to the chemical analysis of ambient air samples.3,4 of many other analytical techniques that are being used, high-performance liquid chromatography coupled to mass spectrometry (hplc–ms) due to its good sensitivity and resolution power is receiving more and more popularity in trace organics quantification.5 on the other hand, ion chromatography (ic) gained an important role as a technique of choice for the analysis of water-soluble ions in atmospheric air particles.1,2,6–9 since water-soluble substances make up a third or more of the particulate mass of ambient air particles, these ions have been studied intensively in recent years.10–14 those investigations provided essential information on the physicochemical properties of ambient air particles, the mechanism of their formation and source of pollution. the surface acidity and hygroscopicity properties of ambient air particles govern their chemical and optical behaviors. these particles in ambient air mainly consist of inorganic ions. besides the mentioned inorganic matter, an organic component, i.e., a considerable amount of low-molecular weight (lmw) organic acids, are present in the water-soluble part of a typical ambient air particle.15,16 initially, this was proved by means of gc–ms, which is a suitable technique after a derivatization step that has to be performed in order to obtain these acids in their volatile form.17 the derivatization process is often troublesome, and it was shown that the application of ic for lmw organic acids analysis is more advantageous than gc–ms.18 an optimized ic method can readily be applied for the analysis of major cations (sodium, ammonium, potassium, magnesium, calcium) and inorganic anions (chlorides, nitrates, sulfates, oxalates, phosphates, bromides), even if they are present at a very low level in aqueous solution.19–21 here, an isocratic ic in the cation-exchange mode was applied for the analysis of five common cations, whereas twelve inorganic anions and lmw organic acids were simultaneously analyzed by anion-exchange gradient ic. in order to obtain a better insight into ambient air particles at the studied site, the ic results were evaluated by different multivariate pattern recognition methods. multivariate analysis of water-soluble inorganic matter, measured together with pahs, metals and reactive oxygen species, has been extensively studied.2,10,22 as mentioned above, this approach requires the use of several analytical techniques. although there are many studies in which inorganic ions together and lmw organic acids were analyzed, there is a lack of literature on pattern recognition studies on such datasets.6,8–10 in addition, different conventional multivariate techniques produce different results, even when used together.23 in this work, common non-supervised pattern recognition methods: principal ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. ions in ambient air particles 755 component analysis (pca), factor analysis (fa), and hierarchical cluster analysis (hca) were compared, while supervised pattern recognition methods, such as linear discriminant analysis (lda), k-nearest neighbors (k-nn) and soft independent modeling of class analogy (simca), were evaluated for their recognition ability. these methods assign the output values according to some specific machine learning algorithms. the method is supervised, provided that a training dataset is used. on the other hand, in an unsupervised method, it is possible to perform pattern recognition even if no dataset is present.23 experimental the sampling procedure is presented in the supplementary material to this paper. standard solutions. individual stock standard solutions (1000 mg dm-3) were supplied by accustandard, merck, or fluka. for some ions, i.e., most organic acid salts (glyoxylates, methanesulfonates, glutarates, succinates, malonates), there are no commercially available stock standard solution (1000 mg dm-3 or less). in this case, standards were made by dissolving an appropriate amount of an analyte in deionized water. the stock standards were stored at 4 °c, whereas composite working standards at lower analyte concentrations, in a select range similar to the expected analyte concentrations in the samples, were prepared before analysis. the concentrations were calculated from 5 points standard calibration curves using the external standard method. as a quality control measure, duplicates, blanks and spiked samples were also analyzed. anion ic system. a dionex dx-500 ion chromatography system consisting of a gradient pump, ed40 electrochemical detector operating in the conductivity mode (detector current setting: 50 ma), injection valve fitted with 400 μl sample loop, asrs-ultra self-regenerating suppressor (external water mode), an as11 (250 mm×4 mm) analytical column, an ag11 (50 mm×4 mm) guard column, and a peaknet 5.1 chromatography workstation. a mixture of hplc grade methanol and hydroxide ions, at a flow rate of 2.0 ml min-1, was used as the eluent. the following gradient program was applied: 1) time: 0.0 min, koh: 0.175 mm, ch3oh: 5.0 %, 2) time: 6.0 min, koh: 0.575 mm, ch3oh: 5.0 %, 3) time: 26.0 min, koh: 8.575 mm, ch3oh: 17.0 % and 4) time: 30.0 min, koh: 10.175 mm, ch3oh: 5.0 %. cation ic system. a dionex dx-500 ion chromatograph with a conductometric detector, a cs12 (250 mm×4 mm) analytical column, a cg12 (50 mm×4 mm)×guard column, a csrs -ultra self-regenerating suppressor operating in the recycling mode, and a peaknet 5.1 chromatography workstation. the eluent was 20 mm methanesulfonic acid (isocratic). the injection sample loop was 100 μl. the system was operated at room temperature with a flow rate of 1.0 cm3 min-1. data analysis. the statistical data analyses in this work was performed by means of minitab, spss and pls toolbox for matlab software packages. details of pca, fa, hca, k-nn and simca procedure are given elsewhere.23 results and discussion ion chromatography analysis typical ic chromatograms of ambient air particles extract are shown in figs. 1 and 2. in the cation-exchange mode, five common cations (sodium, ammonium, potassium, magnesium and calcium) are easily separated in less than 10 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 756 todorović et al. min. in some samples, lithium-ion was detected at trace level, but it was not quantified in this study. ordinarily, lithium is eluted as the first ion and fairly separated from sodium. fig. 1. typical cation chromatogram of an ambient air particles sample extract. column: dionex cs12. eluent: 20 mm methanesulfonic acid (isocratic). peaks: 1) lithium, 2) sodium, 3) ammonium, 4) potassium, 5) magnesium, 6) calcium. fig. 2. anion chromatogram of the same ambient air particles sample extract, as in fig. 1. column: dionex as11. eluent: see the experimental part for details. peaks: 1) fluoride, 2) lactate, 3) acetate, 4) glycolate, 5) formate, 6) methanesulfonate, 7) glyoxylate, 8) chloride, 9) nitrite, 10) nitrate, 11) glutarate, 12) pimelate, 13) succinate, 14) malate, 15) carbonate, 16) malonate, 17) sulfite, 18) fumarate, 19) sulfate, 20) oxalate, 21) azelate. for any source apportioning method to be applied in an air pollution study, the number of measured chemical substances (variables) should be as high as possible. preferably, there should be no missing values in the data set. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. ions in ambient air particles 757 a few studies have made the apportionment of sources of air pollution even when using only major ions data.13,14,24 although, it is known that not enough tracer information is carried by major ions only. furthermore, lmw organic acids are shown to be important tracers for different particle emission sources.15 in this work, for the separation of anions, a multi-step gradient provided simultaneous separation of both inorganic ions (chloride, nitrite, nitrate, sulfate) and ionic form of lmw organic acids (formate, glyoxylate, methanesulfonate, glutarate, succinate, malate, malonate, oxalate). in addition to these anions, several other anions (bromide, phosphate, sulfite, benzoate, caproate, pimelate, fumarate, phthalate, acetate, lactate, glycolate, pyruvate, tartrate, azelate, etc. that were detected in some samples, are not included in this statistical evaluation. the reason was incomplete measurement results for these rarely detected anions in most samples so that some of these anions were often either below the quantification limit of the applied method (pyruvate, tartarate, bromide, phtalate, caproate, fumarate) or there were co-elution problems (fluoride/lactate/acetate, acetate/ /glycolate) or problem with their stability (sulfite, azelate). thus, 17 ions were quantified in all samples. descriptive statistics after the completion of ic measurements, the final data matrix 94×17 (observations x variables) was formed. in the first step of data examination, the ryan–joiner test was used for testing the hypothesis of normal distribution.23 in all statistical tests, the significance level was 0.05. as shown for oxalate in fig. s-2a (supplementary material), the plotted points do not approximate a straight line. the departures from linearity indicate that the actual data distribution deviates from a normal distribution. in contrast, the log-transformed data (fig. s-2b) appear to be well fitted on the normal probability plot with r and p values well above 0.99 and 0.1, respectively. all other variables follow a similar distribution; however, for the sake of brevity, their graphs are not displayed. for further statistical evaluation, the transformed values were used. the data matrix was tested for outliers by applying the rosner test.23 two samples with high sulfate and ammonia concentrations were discarded as outliers. therefore, explorative multivariate analysis was realized on the final 92×17 data matrix. the arithmetic means, minimum values, maximum values, and standard deviations for the concentrations of all substances in the studied ambient air particles were calculated (table i). it can be seen that the variables could be considered as major ions (nitrate, sulfate, ammonium, sodium, chloride, calcium), trace ions (formate, glyoxylate, methanesulfonate, nitrite, glutarate, succinate, malate, malonate), and those ones at medium to a high level (potassium, magnesium, oxalate). skewness is a measure of the lack of symmetry varied between 0.5 and 2.5, which means that this data distribution is more or less right-skewed. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 758 todorović et al. since uniform distribution for different analytes is very rare, kurtosis values are used to estimate the size of tailings. this dataset has both low and high kurtosis, i.e., ranged from –0.5 to 7.6. table i. descriptive statistics of the data for the studied water-soluble substances in ambient air particles in mirijevo outskirt of belgrade in the period 09.09.2013–25.06.2014 no. variable c / ng m -3 mean median skewness kurtosis st. deviation max. min. 1. sodium 617 581 1.1 1.1 254 1382 229 2. ammonium 2186 1707 1.0 0.4 1598 6700 304 3. potassium 167 154 1.1 1.3 67 408 69 4. magnesium 144 123 1.2 0.9 86 406 27 5. calcium 929 685 1.4 1.8 780 3781 94 6. formate 16.7 14.8 1.0 0.2 8.2 38.0 5.2 7. methanesulfonate 25.5 18.7 1.9 4.1 23.3 118 2.8 8. glyoxylate 9.5 7.9 1.0 0.2 5.7 25.8 1.2 9. chloride 1217 869 1.0 0.3 1030 4282 54.9 10. nitrite 20.9 18.3 0.5 –0.5 11.2 47.7 4.4 11. nitrate 3765 3405 0.8 0.2 2019 9837 634 12. glutarate 7.2 5.4 2.0 5.2 5.8 34.0 0.9 13. succinate 18.0 15.0 2.4 7.6 14.6 86.7 4.0 14. malate 15.8 8.9 2.5 6.4 19.0 94.2 1.6 15. malonate 19.5 9.2 1.8 2.5 25.7 108 0.1 16. sulfate 3230 2873 1.0 0.5 1736 8221 1105 17. oxalate 83.2 79.6 0.7 –0.2 42.7 193 17.1 unsupervised pattern recognition these data were subjected to several common multivariate pattern recognition methods, which are described in detail elsewhere.23,25,26 principal component analysis (pca) is a method of removing the highly inter-correlated nature of variations in atmospheric concentrations. thurston and spengler suggest that the dataset must have many more samples than analytes if stable pca results are to be derived.27 their requirements: n ≥ m + 50 (where n is the number of samples; m is the number of analytes) is fulfilled in this work. when pca was applied to the autoscaled data matrix, three principal components (pcs) appeared to account for 84 % of the variance in the data. the eigenvalue and cumulative variance as a function of the number of pcs are shown in fig. s-3 (supplementary material). in this case, the descriptor space dimensionality is reduced to three since only the first three pcs were retained. these pcs meet the kaiser criterion to have the eigenvalues higher than one.23 all other pcs show eigenvalues lower than one, with an exponential trend in the decrease. in order to highlight the relations between variables, the data set of concentration measurements was subjected to factor analysis (fa). this method was ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. ions in ambient air particles 759 applied with pca extraction of the correlation matrix and varimax orthogonal rotation. the fa method identified three significant factors (table ii): the first (24.6 % of variance) comprises organic ions oxalate, malonate, malate with high loadings and glutarate, succinate, methanesulfonate, formate, nitrite, and sulfate with relatively lower loadings. table ii. varimax rotated factor loadings (values > 0.5 are in bold) no. variable factor 1 factor 2 factor 3 1. sodium 0.257 0.223 0.192 2. ammonium 0.225 0.909 –0.210 3. potassium 0.140 0.146 0.288 4. magnesium –0.255 –0.217 0.871 5. calcium –0.396 –0.183 0.853 6. formate 0.449 0.214 –0.234 7. methanesulfonate 0.501 0.381 –0.199 8. glyoxylate 0.092 0.431 0.138 9. chloride –0.453 0.195 0.687 10. nitrite 0.493 0.323 –0.359 11. nitrate 0.068 0.953 –0.029 12. glutarate 0.585 0.491 –0.299 13. succinate 0.596 0.650 –0.243 14. malate 0.837 0.165 –0.396 15. malonate 0.800 0.241 –0.302 16. sulfate 0.396 0.791 –0.017 17. oxalate 0.842 0.295 –0.295 all organic ions, except succinate and glyoxylate, have higher loadings in this factor than in the second one. oxalate and malonate are markers for combustion processes.8,28 hence, this factor identifies exposure to emissions from fuel combustion. although, other biogenic sources could also contribute to the production of these two acids. mainly composed of ammonium, nitrate, sulfate, and succinate, the second factor accounts for a further 22.7 % of the variance. the association between sulfate and nitrate suggests an anthropogenic origin. waste landfill emissions that occur in the vicinity of the sampling site, as well as a secondary inorganic aerosol, might be responsible for this factor. the third factor with 16.8 % of the variance was composed of magnesium, sodium, chloride, and with a lower loading of potassium. this source profile strongly suggests that their concentrations are affected by mineral dust from both natural and anthropogenic sources. except for potassium (loading = 0.536) and calcium (loading = 0.565), no significant loading value was obtained for any variable on factors 4 and 5, which are responsible for 6.9 and 6.6 % of the total variance, respectively. it is relevant that some ions, such as methanesulfonate, nitrite, glutarate, sulfate, succinate, obtained rather high scores both on the first and second factor, indicating more than one possible significant emission source. ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 760 todorović et al. hierarchical cluster analysis (hca) was made on the variables that had previously been standardized.29 an amalgamation rule employed the ward method to link the objects, while for the measure the proximity between samples, the squared euclidean distance was selected.30 the dendrogram is shown in fig. 3. as a result of applying hca to the factor score matrix, the ambient air particles split into two main groups. furthermore, each group could be subdivided into three or two sub-groups, respectively. there are, therefore, five clusters (c1, c2, c3, c4 and c5), the composition of which depends on the inputs of variables. fig. 3. dendrogram of cluster analysis of ambient air particles. cluster c1 consists of the samples characterized by high score values for the combustion factor, whereas the second group of samples (c2) has high levels in the landfill emission and secondary inorganic aerosol factor. the samples with medium–low scores of both the combustion and waste emission/secondary inorganic aerosol factors (c1 and c2) are grouped in cluster c3. these three groups (c1, c2, and c3) form one of the two main groups that all samples can be split. the fourth cluster (c4) comprises samples with the highest levels of the variables (magnesium, calcium, chloride) for the mineral dust factor, while medium–high score values for the previous factor (c4) are characteristic for the samples in c5. supervised pattern recognition in order to evaluate the possibility of classification of ambient air particles into these five groups, three common supervised pattern recognition techniques were employed, linear discriminant analysis (lda), k-nearest neighbors (k-nn), and soft independent modeling of class analogy (simca).31,32 first, the samples were classified using all variables. then, nine variables with the first three highest loadings in each factor in table ii (malate, malonate, oxalate, ammonium, nitrate, sulfate, magnesium, calcium, chloride) were selected ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. ions in ambient air particles 761 as key features for classification. in all cases, the leave-one-out method of cross -validation was used. linear discriminant analysis (lda) is a very simple supervised classification technique that reduces the dimensionality of the features in a similar way as pca. lda makes projection axes for separating the classes by minimizing the within-class variance and maximizing the between-class variance. in this case, lda produced a classification success rate of 83.7 % for all samples and all variables. the separation of ambient air particles by two discriminant functions is shown in fig. 4. when performing lda with the reduced number of variables, 87.0 % of grouped samples were correctly classified. fig. 4. discriminant analysis of ambient air particles (solid symbols are the group centroids). one of the simplest classifiers is the k-nn algorithm. it classifies an input feature vector by determining the k closest training vectors according to a distance metric. then, this input vector is assigned to a class to which the majority of its k nearest neighbors belong. here, the metric employed was the euclidean distance. as in the case of lda, k-nn and simca were performed using both all and selected variables. the obtained results are given in table iii. using both methods, the recognition ability for the five classes was highly satisfactory; the percentages of classification obtained were 93.5–100 %. table iii. recognition and prediction ability (%) of lda, k-nn and simca to classify ambient air particles; (a) – all variables, (b) – nine selected variables ability for: method lda k-nn simca recognition (a) 83.7 93.5 100 (b) 87.0 94.6 97.8 prediction (a) 52.8 94.6 78.3 (b) 56.5 92.4 73.9 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 762 todorović et al. another approach that can give a more realistic error rate is to split the data into two parts, one part to create the discriminant function or model, and the other part as a testing set. randomly splitting an equal number of samples (46 samples) into these two sets, the k-nn method produces good prediction power (94.6 and 92.4 %), whereas lda (52.8 and 56.5 %) and simca (78.3 and 73.9 %) give a much lower percentage of the correctly classified samples than previously, for both all variables and nine variables, respectively. time-series analysis to examine the trends of the concentration changes from each emission source, over a ten-month period (fall 2013 to summer 2014), the concentrations of indicator ions (with the highest loadings) for various emission sources were plotted by the moving averages for the samples in fig. 5. fig. 5. variation of ambient air particles concentrations of oxalate, nitrate, and calcium in the mirijevo outskirt of belgrade during the period 2013 to 2014. the dot lines indicate the quadratic fit. the time-trends of these variables were also fitted by quadratic curves. variables in factor 1 show an affinity to change with time, increasing from low values in the fall to a maximum during the winter, followed by a slight decrease through the spring in 2014. this trend resembled that reported elsewhere.21,33 ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. ions in ambient air particles 763 the variables in factor 3 showed the opposite trends over the same time period, whereas those in factor 2 have no trend. similar seasonal behavior for the ions comprising the latter two factors was obtained by other authors.22,24,33 a significant fluctuation is present with all analyzed variables. no monthly periodicity was revealed. the reason for seasonal variation could be found in the local climate and weather conditions. hence, dust resuspension (factor 1) is more intense in the summer, with many dry days with no snow coverage. on the other hand, the fuel combustion (factor 3) is more intense in winter days. no seasonal changes in the municipally waste landfill activities was observed, causing no trend in factor 2. generally, these results could be explained by the occurrence of atmospheric transport on a local scale. conclusions the combined use of ion chromatography and multivariate pattern recognition methods can provide valuable information for an ambient air particle research study. some water-soluble substances, such as bromide and phosphate, which are traditionally measured in ambient air particle samples by naa, xrf, pixe, icp-ms, and used as important source markers, can also be quantified by ic, but the applied ic separation in this work should to be additionally optimized. the obtained data chemical set showed the characteristic of log-normal distribution, while multivariate methods revealed a distinctive grouping of variables and observations. of the three common supervised pattern recognition methods (lda, k-nn and simca), the classification prediction ability was only satisfactory when applying k-nn. some variables do not contribute to the classification. the emissions from combustion processes, landfill emission together with secondary inorganic aerosol, and a contribution of mineral dust were identified as the three dominant factors that have an effect on pollutant levels at the studied site. the landfill emission/secondary inorganic aerosol undergoes no change in seasons, whereas the other two other factors show different behavior during winter and summer. in general, a considerable high level of sulfate, nitrate, ammonium, and lmw organic acids in the ambient air particles at the site pointed to a strong anthropogenic influence. supplementary material additional data are available electronically at the pages of journal website: https:// //www.shd-pub.org.rs/index.php/jscs/index, or from the corresponding author on request. acknowledgements. this work was supported by the ministry of education, science and technological development of the republic of serbia (contract no. 451-03-68/2020-14/ /200135). ________________________________________________________________________________________________________________________available on line at www.shd.org.rs/jscs/ (cc) 2021 scs. 764 todorović et al. и з в о д честице у амбијенталном ваздуху: примена јонске хроматографије и мултиваријантних техника анализе водено-растворних супстанци жаклина н. тодоровић1, јелена м. радуловић1, ивана д. средовић игњатовић2, љубиша м. игњатовић3 и антоније е. оњиа4 1aнахем лабораторија, моцартова 10, 11160 београд, 2универзитет у београду, пољопривредни факултет, немањина 6, 11080 београд, 3универзитет у београду, факултет за физичку хемију студентски трг 12–16, 11 000 београд и 4универзитет у београду, технолошко–металуршки факултет, карнегијева 4, 11000 београд методом јонске хроматографије одређен је садржај 17 супстанци растворљивих у води (које садрже натријум, амонијум, калијум, магнезијум, калцијум, формате, метансулфонате, глиоксилате, хлориде, нитрите, нитрате, глутарате, сукцинате, малате, малонате, сулфате и оксалате) у укупно 94 узорка честица у амбијенталном ваздуху, који су узорковани током десет месеци у предграђу београда. да би се одредили извори загађења ваздуха, добијени резултати су најпре трансформисани у њихове логаритме, а потом анализирани применом мултиваријантних техника. анализа главних компоненти и факторска анализа идентификовали су три главна фактора који контролишу варијабилност података: процес стационарног сагоревања са највећим оптерећењем оксалата, малоната и малата, емисија са депоније и секундарни неоргански аеросол које карактеришу високи нивои амонијум-јона, нитрата и сулфата и допринос минералне прашине која се састоји од материја магнезијума, калцијума и хлорида. примена хијерархијске анализе кластера указала је на диференцијацију узорака у пет група које припадају различитим профилима аналита. за класификацију узорака амбијенталног ваздуха помоћу девет одабраних јона, способност препознавања методе линеарне дискриминантне анализе, методе k-најближих суседа и методе меког независног моделовања аналогне класе износили су 87,0, 94,6 и 97,8 %. анализа временских серија показала је да је фактор емисије из саобраћаја у току зиме израженији, за разлику од фактора минералне прашине. ефекат стационарног сагоревања не показује тренд. 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/hrv (za stvaranje adobe pdf dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. stvoreni pdf dokumenti mogu se otvoriti acrobat i adobe reader 5.0 i kasnijim verzijama.) /hun 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/pdfxoutputintentprofileselector /documentcmyk /preserveediting true /untaggedcmykhandling /leaveuntagged /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] >> setdistillerparams << /hwresolution [2400 2400] /pagesize [612.000 792.000] >> setpagedevice